{"gene":"SPP1","run_date":"2026-04-28T20:42:08","timeline":{"discoveries":[{"year":1996,"finding":"Osteopontin (SPP1/Eta-1) was identified as a direct protein ligand of CD44. This interaction induces cellular chemotaxis but not homotypic aggregation, distinguishing it from the CD44–hyaluronate interaction which promotes aggregation but not migration.","method":"Receptor-ligand binding assays, chemotaxis assays, homotypic aggregation assays","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 — direct functional receptor-ligand interaction with distinct phenotypic readouts, widely replicated","pmids":["8560266"],"is_preprint":false},{"year":1995,"finding":"OPN mediates cell adhesion via multiple αv-containing integrins (αvβ3, αvβ5, αvβ1), but directed cell migration toward an OPN gradient specifically requires αvβ3. Cells lacking surface αvβ3 adhered to OPN at ~50% efficiency but could not migrate toward it.","method":"Boyden chamber migration assay, adhesion assay with cells differing in αvβ3 expression, integrin blocking antibodies","journal":"The Journal of Clinical Investigation","confidence":"High","confidence_rationale":"Tier 2 — functional dissection with multiple integrin variants and blocking antibodies, replicated in subsequent work","pmids":["7532190"],"is_preprint":false},{"year":1999,"finding":"Thrombin cleavage of OPN exposes a novel recognition sequence (SVVYGLR) in the N-terminal fragment that directly binds integrin α9β1, distinct from the RGD site. Site-directed mutagenesis of Tyr165 abolished α9β1-mediated adhesion.","method":"Site-directed mutagenesis, synthetic peptide blocking assays, cell adhesion assays with α9-transfected cells","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 1 — mutagenesis + peptide blocking + transfection rescue in multiple cell lines","pmids":["10593924"],"is_preprint":false},{"year":2000,"finding":"OPN/Eta-1 regulates macrophage IL-12 and IL-10 cytokine expression through two distinct receptor interactions: a phosphorylation-dependent interaction with integrin receptor stimulates IL-12 expression, while a phosphorylation-independent interaction with CD44 inhibits IL-10 expression. OPN-deficient mice show severely impaired type-1 immunity with diminished IL-12/IFN-γ and elevated IL-10.","method":"OPN-knockout mice (viral and bacterial infection models), cytokine measurement, phosphorylation-dependent vs. independent receptor interaction studies","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 — knockout mouse phenotype with mechanistic dissection of two receptor pathways, published in high-impact journal","pmids":["10657301"],"is_preprint":false},{"year":2001,"finding":"OPN (osteopontin) is a novel substrate for MMP-3 (stromelysin-1) and MMP-7 (matrilysin). Three cleavage sites were identified for MMP-3 in human OPN (Gly166-Leu167, Ala201-Tyr202, Asp210-Leu211); MMP-7 cleaves at two of those sites. MMP cleavage of OPN potentiates its function as an adhesive and migratory stimulus through cell surface integrins.","method":"In vitro cleavage assay with purified MMPs, cleavage site mapping, cell adhesion and migration assays pre- and post-cleavage","journal":"The Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with cleavage site identification and functional consequence demonstrated","pmids":["11375993"],"is_preprint":false},{"year":2001,"finding":"OPN regulates T helper cell-1 (TH1)-mediated demyelinating disease. OPN-deficient mice were resistant to progressive experimental autoimmune encephalomyelitis (EAE); myelin-reactive T cells in OPN−/− mice produced more IL-10 and less IFN-γ than in OPN+/+ mice.","method":"OPN-knockout mouse model of EAE, cytokine profiling of myelin-reactive T cells, microarray analysis of MS plaques","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 — knockout mouse with mechanistic cytokine profiling, corroborated by human MS tissue analysis","pmids":["11721059"],"is_preprint":false},{"year":2001,"finding":"OPN and bone sialoprotein (BSP) are members of the SIBLING family on human chromosome 4, and both are completely unstructured in solution (flexible polypeptide backbones), enabling them to associate with multiple binding partners and bridge proteins into biologically active complexes, including bridging complement Factor H to integrins or CD44 to suppress the alternate complement pathway.","method":"1D proton NMR, transverse relaxation time measurements, recombinant protein structural analysis","journal":"Biochemical and Biophysical Research Communications","confidence":"High","confidence_rationale":"Tier 1 — NMR structural characterization with functional mechanistic inference","pmids":["11162539"],"is_preprint":false},{"year":2003,"finding":"Osteopontin (SPP1) was identified as the lead metastasis-associated gene in HCC. An osteopontin-specific antibody effectively blocked HCC cell invasion in vitro and inhibited pulmonary metastasis of HCC cells in nude mice.","method":"cDNA microarray, antibody blockade of invasion in vitro, nude mouse pulmonary metastasis model","journal":"Nature Medicine","confidence":"Medium","confidence_rationale":"Tier 2 — antibody blockade functional assay in vitro and in vivo, single study","pmids":["12640447"],"is_preprint":false},{"year":2012,"finding":"OPN (osteopontin/SPP1) is phosphorylated within S-x-E motifs by FAM20C (Golgi casein kinase), a secreted atypical kinase that localizes to the Golgi and phosphorylates SIBLING family proteins including OPN. Mutations in FAM20C cause Raine syndrome.","method":"Kinase activity assay, identification of FAM20C as the responsible kinase, genetic linkage in Raine syndrome patients","journal":"Science","confidence":"High","confidence_rationale":"Tier 1 — biochemical kinase assay identifying writer enzyme for OPN phosphorylation, with disease genetics validation","pmids":["22582013"],"is_preprint":false},{"year":1992,"finding":"Uropontin (OPN purified from urine) inhibits calcium oxalate crystal growth in vitro, demonstrating a direct functional role in regulating urinary stone formation as a crystal growth inhibitor.","method":"Monoclonal antibody immunoaffinity purification, in vitro calcium oxalate crystal growth assay","journal":"Proceedings of the National Academy of Sciences","confidence":"High","confidence_rationale":"Tier 1 — direct in vitro biochemical assay with purified protein","pmids":["1729712"],"is_preprint":false},{"year":1990,"finding":"Human OPN/SPP1 cDNA was cloned; the gene is a single copy located on chromosome 4q near the centromere. Two mRNA variants were identified differing by a 14-amino-acid insertion at residue 58 (OPN1b vs OPN1a). The RGD cell attachment site is conserved between species.","method":"cDNA library screening, Northern blot, PCR amplification, chromosomal mapping with human-rodent cell hybrids, in situ hybridization","journal":"Genomics","confidence":"High","confidence_rationale":"Tier 2 — foundational molecular characterization with multiple methods","pmids":["1974876"],"is_preprint":false},{"year":2014,"finding":"OPN in the glioma perivascular niche promotes cancer stem cell phenotypes and radiation resistance via CD44 signaling. The γ-secretase-regulated intracellular domain of CD44 mediates downstream effects via CBP/p300-dependent enhancement of HIF-2α activity. Cd44-deficient mice showed improved survival in a glioma model.","method":"Cd44-knockout mouse glioma model, survival analysis, γ-secretase inhibition, CBP/p300 functional assays, HIF-2α activity measurement","journal":"Cell Stem Cell","confidence":"High","confidence_rationale":"Tier 2 — knockout mouse model plus mechanistic dissection of CD44 intracellular signaling cascade","pmids":["24607407"],"is_preprint":false},{"year":2018,"finding":"OPN acts as an immune checkpoint to suppress CD8+ T cell activation. IRF8 represses OPN expression by binding the Spp1 promoter in myeloid cells; IRF8 deficiency elevates OPN, which signals through CD44 on T cells to suppress CTL activation and confer tumor immune tolerance.","method":"IRF8-knockout mice, chimeric bone marrow reconstitution, T cell-specific IRF8 deficiency, Spp1 promoter ChIP assay, T cell activation functional assays","journal":"The Journal of Clinical Investigation","confidence":"High","confidence_rationale":"Tier 2 — multiple genetic models with ChIP mechanistic validation, epistasis established","pmids":["30395540"],"is_preprint":false},{"year":2018,"finding":"OPN is a potent chemokine for macrophages in glioblastoma, and integrin αvβ5 on glioblastoma-infiltrating macrophages is a major OPN receptor. OPN maintains M2 macrophage gene signature/phenotype. Both tumor-derived and host-derived OPN are required for glioma development. OPN blockade with 4-1BB-OPN bispecific aptamers extended median survival by 68% in mice.","method":"OPN siRNA/shRNA/CRISPR knockdown, OPN-deficient mice, macrophage migration assays, in vitro M2 polarization assays, therapeutic aptamer study","journal":"The Journal of Clinical Investigation","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal knockdown methods, receptor identification, in vivo therapeutic validation","pmids":["30307407"],"is_preprint":false},{"year":2021,"finding":"OPN/SPP1 secreted by cancer-associated fibroblasts (CAFs) drives cancer stemness in pancreatic cancer through the SPP1–CD44 axis. SPP1 knockdown significantly reduced stemness characteristics. Overexpression of wild-type SPP1, but not an SPP1 lacking the CD44-binding domain, rescued sphere formation capacity, confirming CD44-binding is required.","method":"CAF conditioned media treatment, siRNA knockdown of SPP1, CD44-binding domain mutant rescue experiment, tumorsphere formation assay, 3D co-culture model","journal":"Gastroenterology","confidence":"High","confidence_rationale":"Tier 2 — domain-deletion mutant rescue experiment establishing mechanistic requirement for CD44-binding domain","pmids":["34418441"],"is_preprint":false},{"year":2016,"finding":"AMPK activation promotes osteogenesis and inhibits adipogenesis through the AMPK-Gfi1-OPN (osteopontin) axis. AMPK activation downregulates the transcriptional repressor Gfi1 and causes it to dissociate from the OPN promoter, leading to OPN upregulation, which mediates the osteogenic and anti-adipogenic effects.","method":"AMPK overexpression via lentiviral vector, Gfi1 overexpression and dominant-negative constructs, ChIP-like promoter dissociation assay, ectopic bone formation model","journal":"Cellular Signalling","confidence":"Medium","confidence_rationale":"Tier 2 — mechanistic pathway placement with Gfi1-OPN promoter occupancy, single lab","pmids":["27283242"],"is_preprint":false},{"year":2017,"finding":"SPP1 transcription in glioblastoma is driven by GLI1 binding to the SPP1 promoter and OCT4 binding to the first SPP1 intron. GLI1 knockdown reduced SPP1 mRNA and protein levels. SPP1-CD44 interaction is required for glioma sphere formation: SPP1 lacking the CD44-binding domain failed to rescue sphere formation in SPP1-depleted cells.","method":"ChIP assay (GLI1 and OCT4 binding to SPP1 locus), siRNA knockdown of SPP1/GLI1/CD44, rescue with wild-type vs CD44-binding-domain-deleted SPP1, sphere formation assays","journal":"Oncotarget","confidence":"High","confidence_rationale":"Tier 2 — ChIP validation of transcription factor binding plus domain-deletion rescue experiment","pmids":["28030801"],"is_preprint":false},{"year":2017,"finding":"SPP1 regulates nerve degeneration and regeneration after sciatic nerve injury through c-Fos, PKCα, and p-ERK/ERK pathways in Schwann cells. Spp1 silencing reduced Schwann cell proliferation and promoted migration, while Spp1 overexpression had opposite effects.","method":"siRNA knockdown and overexpression of Spp1 in Schwann cells, sciatic nerve injury model in rats, cytokine and pathway analysis (c-Fos, PKCα, ERK phosphorylation)","journal":"BMC Neuroscience","confidence":"Medium","confidence_rationale":"Tier 3 — loss- and gain-of-function with pathway markers, single lab","pmids":["28270094"],"is_preprint":false},{"year":2018,"finding":"SPP1 promotes ovarian cancer cell proliferation, migration, and invasion via the Integrin β1/FAK/AKT signaling pathway. Silencing SPP1 inhibited Integrin β1/FAK/p-AKT and suppressed tumor growth in vivo; ectopic SPP1 expression activated this pathway.","method":"siRNA knockdown and overexpression, Western blot for pathway components, Boyden chamber migration/invasion assays, nude mouse xenograft","journal":"OncoTargets and Therapy","confidence":"Medium","confidence_rationale":"Tier 3 — loss/gain-of-function with pathway analysis, single lab","pmids":["29559792"],"is_preprint":false},{"year":2020,"finding":"BRD4 (BET bromodomain protein) regulates SPP1 expression indirectly through NFKB2 (noncanonical NF-κB). BRD4 does not bind directly to the SPP1 promoter; instead, it regulates NFKB2, which then drives SPP1 transcription. BET inhibitors suppress melanoma progression via this noncanonical NF-κB/SPP1 pathway.","method":"ChIP assay (BRD4 binding to promoters), NFKB2 silencing, BET inhibitor treatment, SPP1 overexpression rescue experiment","journal":"Theranostics","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP-based mechanistic pathway with rescue experiment, single lab","pmids":["33052224"],"is_preprint":false},{"year":2020,"finding":"OPN inhibits autophagy in osteoarthritic chondrocytes through CD44 and αvβ3 integrin receptors via the ERK MAPK signaling pathway. Blocking CD44 or αvβ3 with antibodies prevented OPN-mediated suppression of LC3II and Beclin1. ERK inhibition abolished OPN's autophagy-suppressing activity.","method":"Recombinant OPN treatment, anti-CD44 and anti-αvβ3 blocking antibodies, ERK inhibitor, Western blot, mRFP-GFP-LC3 confocal imaging, transmission electron microscopy","journal":"Frontiers in Endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 — receptor blocking with multiple readouts including imaging, single lab","pmids":["36034459"],"is_preprint":false},{"year":2020,"finding":"OPN (osteopontin) activates PI3K signaling in osteoarthritic chondrocytes by binding to CD44 on the cell surface, causing downstream cascading effects that inhibit chondrocyte degeneration and reduce cartilage matrix loss. OPN binding was verified in vivo in mice.","method":"siOPN knockdown, rhOPN treatment, PI3K inhibitor (LS-C179404), in vivo mouse OA model","journal":"Genes & Diseases","confidence":"Medium","confidence_rationale":"Tier 3 — loss/gain-of-function with inhibitor, in vivo validation","pmids":["35005113"],"is_preprint":false},{"year":2020,"finding":"OPN binds αv integrin on endothelial progenitor cells (EPCs) to support adhesion and promote EPC incorporation into angiogenic vascular networks. siRNA knockdown of αv integrin reduced EPC binding to OPN and focal adhesion assembly. αv integrin was localized to focal adhesions at the basal surface of OPN-coated EPCs.","method":"siRNA knockdown of αv integrin, OPN-coated substrate adhesion assay, focal adhesion immunofluorescence microscopy, EPC incorporation into HUVEC networks","journal":"Reproduction","confidence":"Medium","confidence_rationale":"Tier 2 — siRNA knockdown with focal adhesion imaging and functional incorporation assay","pmids":["31990676"],"is_preprint":false},{"year":2022,"finding":"EEF2K promotes melanoma progression by phosphorylating STAT3 at Tyr705, which binds to the SPP1 promoter and enhances SPP1 transcription. Re-expression of SPP1 partially reversed the inhibitory effect of EEF2K silencing, while SPP1 or STAT3 inhibition abolished EEF2K's tumorigenic effects.","method":"EEF2K silencing and overexpression, RNA-seq, ChIP assay (p-STAT3 binding to SPP1 promoter), SPP1 re-expression rescue","journal":"Clinical and Translational Medicine","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP plus rescue experiment establishing EEF2K/STAT3/SPP1 axis, single lab","pmids":["35184394"],"is_preprint":false},{"year":2022,"finding":"SPP1 promotes radiation resistance in esophageal carcinoma via the JAK2/STAT3 pathway. Radiation increases SPP1 expression, which in turn increases JAK2 and STAT3 phosphorylation. SPP1 knockdown combined with JAK2/STAT3 inhibition synergistically reduced tumor volume in vivo.","method":"SPP1 knockdown and overexpression, Western blot for JAK2/STAT3 phosphorylation, xenograft mouse model, pharmacological JAK2-STAT3 inhibition","journal":"Cancer Medicine","confidence":"Medium","confidence_rationale":"Tier 3 — pathway analysis with in vivo validation, single lab","pmids":["35593388"],"is_preprint":false},{"year":2022,"finding":"miR-433 directly binds to the 3'-UTR of SPP1 mRNA to regulate SPP1 expression. Overexpression of miR-433 via agomiR-433 in osteoblast cells reduced SPP1 mRNA and protein levels. Dual luciferase reporter assay confirmed direct interaction between miR-433 and the SPP1 3'-UTR.","method":"Dual luciferase reporter assay, agomiR-433 transfection, RT-qPCR, Western blot","journal":"Experimental and Therapeutic Medicine","confidence":"Medium","confidence_rationale":"Tier 2 — dual luciferase assay directly confirming miRNA-3'UTR interaction","pmids":["34306197"],"is_preprint":false},{"year":2022,"finding":"OPN N-glycosylation at asparagine-79 promotes osteoclast proliferation and modulates osteoblast function through the NF-κB signaling pathway. Mutation of the N79 glycosylation site abolished OPN's ability to increase osteoblast proliferation. N-glycosylated OPN promotes nuclear translocation of NF-κB in both osteoclasts and osteoblasts.","method":"Site-directed mutagenesis (N79Q), CCK8 proliferation assay, Western blot for NF-κB signaling, mass spectrometry glycosylation site identification","journal":"Oral Diseases","confidence":"Medium","confidence_rationale":"Tier 2 — mutagenesis of PTM site with functional readout, single lab","pmids":["35451542"],"is_preprint":false},{"year":2022,"finding":"Macrophage-derived SPP1 promotes fibroblast conversion to an inflammatory phenotype with high Ccl2/Ccl7 expression in viral myocarditis, which increases monocyte chemotaxis to the heart. SPP1 inhibition reduced cardiac macrophage infiltration and improved cardiac function in CVB3-induced myocarditis mice.","method":"Single-cell RNA sequencing, indirect co-culture of macrophages with fibroblasts, SPP1 inhibition in vivo, CellPhoneDB ligand-receptor analysis","journal":"Biology Direct","confidence":"Medium","confidence_rationale":"Tier 2 — in vitro co-culture mechanistic validation plus in vivo SPP1 inhibition","pmids":["40087693"],"is_preprint":false},{"year":2023,"finding":"Macrophage-derived OPN (SPP1) protects from NASH by inducing oncostatin-M (OSM) production in macrophages; OSM activates STAT3 signaling in hepatocytes, which upregulates arginase-2 (ARG2), enhancing fatty acid oxidation to reduce steatosis. Conditional Spp1 knockin protected mice from NASH while Spp1 myeloid knockout worsened disease.","method":"Conditional Spp1 knockin and knockout mice (myeloid-specific), Western blot for OSM/STAT3/ARG2 pathway, fatty acid oxidation assays, high-fat diet NASH model","journal":"Gastroenterology","confidence":"High","confidence_rationale":"Tier 2 — conditional gain- and loss-of-function with mechanistic pathway dissection in multiple mouse models","pmids":["37028770"],"is_preprint":false},{"year":2023,"finding":"SOCS3 in myeloid cells directly regulates SPP1 gene expression; SPP1 is a transcriptional target of STAT3. Loss of SOCS3 in myeloid cells leads to elevated STAT3 activity and increased SPP1 expression in microglia/macrophages, driving pathological retinal neovascularization. Pharmaceutical activation of SOCS3 or SPP1 blockade reduced pathological neovascularization.","method":"Myeloid-specific SOCS3 knockout mice, scRNA-seq, STAT3 transcriptional target validation, SPP1 neutralizing antibody in vivo","journal":"Molecular Therapy","confidence":"Medium","confidence_rationale":"Tier 2 — genetic knockout plus in vivo blockade, STAT3-SPP1 axis identified","pmids":["38504518"],"is_preprint":false},{"year":2023,"finding":"Microglia-derived SPP1 promotes retinal neovascularization by activating endothelial Akt/mTOR signaling via the Kit receptor. SPP1 expression in microglia is regulated by NF-κB and HIF-1α pathways. Spp1 neutralizing antibody attenuated pathological retinal neovascularization and improved visual function.","method":"OIR mouse model, scRNA-seq, conditioned medium from BV2 microglia, recombinant SPP1 treatment, SPP1 neutralizing antibody intravitreal injection, RNA-seq of bEnd.3 cells, Akt/mTOR phosphorylation assays","journal":"Journal of Personalized Medicine","confidence":"Medium","confidence_rationale":"Tier 2 — conditioned medium + recombinant protein + neutralizing antibody with pathway analysis, single lab","pmids":["36675807"],"is_preprint":false},{"year":2023,"finding":"Vitamin C promotes neuroprotection of retinal ganglion cells by upregulating SPP1 in reactive astrocytes via the transcription factor E2F1. SPP1+ astrocytes in turn promote retinal ganglion cell survival in a mouse glaucoma model.","method":"Mouse glaucoma model, SPP1 expression analysis, E2F1 transcription factor identification, retinal ganglion cell survival assay","journal":"Life Science Alliance","confidence":"Medium","confidence_rationale":"Tier 3 — transcription factor identified with in vivo phenotype, single lab","pmids":["37160307"],"is_preprint":false},{"year":2024,"finding":"SPP1 secreted by hepatocellular carcinoma tumor cells interacts with the CD44 receptor on hepatic stellate cells (HSCs), activating the PI3K/AKT signaling pathway and promoting HSC differentiation into cancer-associated fibroblasts. CD44 receptor blockade abrogated this interaction; in vivo SPP1 silencing impaired HSC-to-CAF differentiation and reduced tumor volume and collagen deposition.","method":"Spatial transcriptomics, scRNA-seq, CD44 receptor blockade, in vivo SPP1 silencing, collagen deposition quantification","journal":"Journal of Translational Medicine","confidence":"Medium","confidence_rationale":"Tier 2 — receptor blockade and in vivo silencing with mechanistic pathway, single lab","pmids":["39267037"],"is_preprint":false},{"year":2025,"finding":"SPP1 binds the CD61 (integrin β3/αvβ3 complex) receptor on mesenchymal PDAC cells to induce Bmp2 and Grem1 expression. GREM1 in turn inhibits BMP signaling, which is required for SPP1 expression in epithelial PDAC cells, forming an intercellular SPP1-GREM1 regulatory loop that maintains mesenchymal cell fate. Spp1 inactivation in mouse PDAC models delayed tumorigenesis and abolished metastasis.","method":"Spp1 knockout mouse PDAC models, proteomic plasma analysis, Grem1/Spp1 double knockout and heterozygous genetic epistasis experiments, receptor identification","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1–2 — multiple genetic epistasis experiments (double knockout, heterozygosity rescue) establishing intercellular SPP1-CD61-GREM1-BMP regulatory loop","pmids":["40993391"],"is_preprint":false},{"year":2024,"finding":"Histone H4 lysine 12 lactylation (H4K12la) promotes SPP1 transcription in reprogrammed microglia following spinal cord injury. DNA-pulldown experiments identified H4K12la as an upstream regulatory modification on Spp1. Recombinant SPP1 promoted neuronal maturation and enhanced mitochondrial function in axons, improving functional recovery after SCI.","method":"scRNA-seq, DNA-pulldown assay for H4K12la regulatory proteins, pseudotime analysis, recombinant SPP1 injection into injured spinal cord, functional recovery assessment","journal":"CNS Neuroscience & Therapeutics","confidence":"Medium","confidence_rationale":"Tier 2 — DNA-pulldown identifying histone modification upstream of Spp1, with in vivo functional validation","pmids":["39939834"],"is_preprint":false},{"year":2013,"finding":"BAFF induces OPN (SPP1) expression specifically in splenic CD21−CD23+ B cells via an NF-κB-dependent signaling pathway. B cell-derived OPN then promotes anti-apoptotic Bcl2 expression in T cells, maintaining T cell survival in autoimmune EAE.","method":"TACI-IgG blockade of BAFF, NF-κB inhibition, OPN measurement in B cell subpopulations, T cell survival assays","journal":"Molecular Immunology","confidence":"Medium","confidence_rationale":"Tier 3 — NF-κB pathway and cell-type specificity identified with functional T cell readout, single lab","pmids":["24084099"],"is_preprint":false},{"year":2022,"finding":"SPP1 facilitates lung adenocarcinoma cell migration and invasion by upregulating COL11A1 expression. COL11A1 overexpression rescued the inhibitory effects of SPP1 knockdown on migration/invasion and reversed EMT marker changes, placing COL11A1 downstream of SPP1.","method":"siRNA knockdown of SPP1, COL11A1 overexpression rescue, wound healing and Matrigel invasion assays, Western blot for EMT markers, in vivo metastasis model","journal":"Cancer Cell International","confidence":"Medium","confidence_rationale":"Tier 3 — downstream target identified via rescue experiment, single lab","pmids":["36266702"],"is_preprint":false},{"year":2020,"finding":"SPP1 regulates gastric adenocarcinoma radiosensitivity via Wnt/β-catenin signaling. SPP1 knockdown combined with irradiation increased DNA damage (H2AX phosphorylation), apoptosis, and G2/M phase arrest while reducing invasion. The Wnt/β-catenin inhibitor XAV939 enhanced these phenotypes, placing SPP1 upstream of Wnt/β-catenin in radioresistance.","method":"siSPP1 transfection, XAV939 Wnt inhibitor, TOP-flash reporter assay, flow cytometry cell cycle analysis, Western blot","journal":"Journal of Oncology","confidence":"Medium","confidence_rationale":"Tier 3 — pathway reporter assay with epistasis via inhibitor combination, single lab","pmids":["34367279"],"is_preprint":false},{"year":2023,"finding":"Profibrotic SPP1+ macrophage differentiation is driven by CXCL4 (platelet factor 4). CXCL4 was identified as the top upregulated gene during Spp1 macrophage differentiation; Cxcl4 loss abrogated profibrotic Spp1 macrophage differentiation and ameliorated fibrosis after heart and kidney injury. Platelets (the main CXCL4 source in vivo) drive this differentiation. Spp1 macrophages orchestrate fibroblast activation via Spp1, Fn1, and Sema3 crosstalk.","method":"Cxcl4-knockout mice, organ injury models (heart and kidney), in vitro macrophage differentiation assays, single-nuclear RNA sequencing with ligand-receptor interaction analysis","journal":"Cell Reports","confidence":"High","confidence_rationale":"Tier 2 — knockout mouse model in two organ injury contexts plus in vitro mechanistic validation","pmids":["36807143"],"is_preprint":false},{"year":2024,"finding":"Therapeutic silencing of Spp1 in atrial TREM2+ cardiac macrophages using an antibody-siRNA conjugate reduces atrial fibrosis and suppresses atrial fibrillation in mice. TREM2+ macrophages secrete OPN (encoded by Spp1), which drives fibrosis and AFib.","method":"Antibody-siRNA conjugate targeting TREM2+ macrophages, atrial fibrosis quantification, AFib induction in mouse model","journal":"bioRxiv (PREPRINT)","confidence":"Medium","confidence_rationale":"Tier 2 — cell-type specific siRNA silencing with functional cardiac outcome, preprint","pmids":["39149373"],"is_preprint":true},{"year":1992,"finding":"Clonal rat dental pulp cells (RDP4-1, RPC-C2A) synthesize and secrete osteopontin (SPP1/2ar) as a highly phosphorylated protein. OPN synthesis was markedly increased by TPA (phorbol ester), similar to osteoblastic cells, establishing OPN as a secreted phosphoprotein produced by dental pulp cells.","method":"Immunoprecipitation with anti-OPN antibody, thrombin susceptibility assay, metabolic labeling","journal":"Biochemical and Biophysical Research Communications","confidence":"Medium","confidence_rationale":"Tier 3 — biochemical identification of SPP1 synthesis and secretion with phosphorylation characterization","pmids":["1472061"],"is_preprint":false},{"year":2013,"finding":"Transgenic overexpression of OPN (Spp1) in mammary epithelium induces proliferation, exaggerated terminal end bud formation, increased lobulogenesis, and delayed gland involution. Ki-67 staining confirmed increased epithelial cell proliferation. OPN conveys a proliferative stimulus for mammary epithelial cells.","method":"MMTV-Opn transgenic mouse lines (3 founders), immunohistochemistry (Ki-67, OPN), ex vivo organoid lobulogenesis assay","journal":"Genes & Cancer","confidence":"Medium","confidence_rationale":"Tier 2 — multiple transgenic founder lines with consistent phenotype and in vitro organoid validation","pmids":["24069507"],"is_preprint":false},{"year":2017,"finding":"A promoter polymorphism (rs28357094) in SPP1 modifies OPN expression in DMD myotubes, with the G allele associated with significantly higher OPN expression. Deflazacort treatment significantly increased OPN expression only in myotubes carrying the TG genotype, demonstrating that rs28357094 mediates differential glucocorticoid responsiveness of SPP1 transcription.","method":"Primary human DMD myoblasts/myotubes with defined rs28357094 genotypes, deflazacort treatment, immunoblot OPN quantification","journal":"Human Molecular Genetics","confidence":"Medium","confidence_rationale":"Tier 3 — genotype-stratified functional assay in primary human cells, single lab","pmids":["28595270"],"is_preprint":false},{"year":2010,"finding":"The ATX-LPA axis upregulates OPN expression in hepatic cancer cells (SMMC7721) via PI3K/Akt signaling through LPA receptors. LPA receptor inhibitors and PI3K/Akt inhibitors reduced ATX-LPA-induced OPN upregulation, and OPN was required for ATX-LPA-induced cell migration.","method":"Western blot and RT-PCR for OPN expression, PI3K/Akt inhibitors, LPA receptor inhibitors, Transwell migration assay","journal":"Anatomical Record","confidence":"Medium","confidence_rationale":"Tier 3 — pathway inhibitor dissection with migration readout, single lab","pmids":["21337710"],"is_preprint":false},{"year":2020,"finding":"MR (mineralocorticoid receptor) acts as the upstream effector of OPN (SPP1) in hepatic stellate cells (LX-2), and OPN shares downstream HMGB1 with MR. The MR/OPN/HMGB1 axis drives NAFLD-associated liver fibrogenesis; MR antagonist eplerenone reduced this axis and fibrogenic markers.","method":"MR agonist/antagonist treatment of LX-2 cells, OPN siRNA knockdown, loss- and gain-of-function for MR and OPN, HMGB1 expression analysis","journal":"Life Sciences","confidence":"Medium","confidence_rationale":"Tier 3 — pathway ordering via siRNA and pharmacological intervention, single lab","pmids":["33091447"],"is_preprint":false},{"year":2022,"finding":"Macrophage Spp1 proliferation (SPP1hi macrophages) is markedly increased in IPF fibrotic lungs compared to normal lungs, and causal modelling supported a role for these SPP1hi macrophages in activating IPF myofibroblasts in fibrosis. Low-level local proliferation is present in normal lungs but is dramatically elevated in fibrotic lower lobes.","method":"Single-cell RNA sequencing of fresh lung explants (IPF vs. normal), co-localization analysis, causal modelling","journal":"European Respiratory Journal","confidence":"Low","confidence_rationale":"Tier 3 + Weak — scRNA-seq and causal modelling only, no direct functional experiment","pmids":["31221805"],"is_preprint":false},{"year":2022,"finding":"Macrophage subpopulations marked by SPP1 expression communicate with fibroblasts via SPP1-CD44, SPP1-PTGER4, and SPP1-αvβ1 receptor pairs in colorectal cancer, mediating formation of an immune-excluded desmoplastic structure and limiting T cell infiltration.","method":"Single-cell RNA sequencing, spatial transcriptomics, immunofluorescent staining, ligand-receptor pair analysis across 14 independent CRC cohorts (2550 samples)","journal":"Nature Communications","confidence":"Low","confidence_rationale":"Tier 3 — transcriptomic ligand-receptor inference and spatial validation, no direct functional mechanistic assay","pmids":["35365629"],"is_preprint":false},{"year":2025,"finding":"H4K12 lactylation in reprogrammed microglia activates Spp1 transcription after spinal cord injury. Recombinant SPP1 promotes neuronal maturation and mitochondrial ATP production in axons via the SPP1 signaling pathway, improving functional recovery post-SCI.","method":"scRNA-seq, DNA-pulldown identifying H4K12la as regulatory mark on Spp1, recombinant SPP1 injection, functional behavioral recovery assay","journal":"CNS Neuroscience & Therapeutics","confidence":"Medium","confidence_rationale":"Tier 2 — novel PTM regulatory mechanism identified by DNA-pulldown with in vivo functional confirmation","pmids":["39939834"],"is_preprint":false},{"year":2024,"finding":"SPP1+ macrophages in the SPP1-CD44 axis promote T-cell exhaustion in ovarian cancer. Blocking either SPP1 or CD44 reversed T-cell exhaustion, restored CD8+ T-cell functionality, and suppressed tumor growth in vivo. Molecular docking identified nilotinib as an SPP1 inhibitor that reduced PD-1 expression in macrophage-conditioned Jurkat cells.","method":"Functional T cell assays, SPP1/CD44 blocking, in vivo tumor suppression, molecular docking simulations, in vitro nilotinib treatment","journal":"International Immunopharmacology","confidence":"Medium","confidence_rationale":"Tier 2 — functional blocking assay with in vivo tumor suppression, computational docking for inhibitor","pmids":["40446696"],"is_preprint":false}],"current_model":"SPP1 (osteopontin) is an intrinsically disordered, multiply phosphorylated and glycosylated secreted glycoprotein that functions as a multivalent extracellular signaling molecule: it binds αv-containing integrins (αvβ1, αvβ3, αvβ5) via its RGD motif—with αvβ3 specifically required for directed cell migration—and CD44 via a separate domain, with thrombin or MMP cleavage generating additional fragments that expose a distinct α9β1-binding SVVYGLR motif and potentiate integrin-dependent adhesion/migration; phosphorylation (mediated by the Golgi kinase FAM20C at S-x-E motifs) governs receptor specificity, with the phosphorylated form stimulating macrophage IL-12 via integrins while the phosphorylation-independent form inhibits IL-10 via CD44; SPP1 also acts as a crystal-growth inhibitor (as uropontin), a macrophage chemokine, a regulator of type-1 immunity, an upstream driver of JAK2/STAT3, AKT/mTOR, ERK, Wnt/β-catenin, and NF-κB pathways in diverse cell types, a promoter of profibrotic macrophage differentiation via CXCL4, and, most recently, a key intercellular regulator that binds CD61 on mesenchymal pancreatic cancer cells to induce GREM1 expression, which reciprocally maintains SPP1 expression in epithelial cells, thereby controlling mesenchymal cell fate."},"narrative":{"teleology":[{"year":1990,"claim":"Cloning of human SPP1 cDNA and chromosomal mapping to 4q established the molecular identity of osteopontin, revealing splice variants and a conserved RGD cell-attachment motif that implied integrin-mediated adhesion.","evidence":"cDNA library screening, Northern blot, chromosomal mapping with human–rodent cell hybrids","pmids":["1974876"],"confidence":"High","gaps":["No receptor binding demonstrated at this stage","Functional significance of splice variants unknown"]},{"year":1992,"claim":"Identification of uropontin as a direct calcium oxalate crystal growth inhibitor established a non-integrin biochemical function for SPP1 in kidney stone prevention.","evidence":"Immunoaffinity-purified uropontin in in vitro calcium oxalate crystal growth assay","pmids":["1729712"],"confidence":"High","gaps":["In vivo relevance to nephrolithiasis not tested","Mechanism of crystal inhibition at atomic level unresolved"]},{"year":1995,"claim":"Demonstrating that OPN mediates adhesion through multiple αv integrins but directed migration specifically requires αvβ3 resolved which integrin heterodimer transduces the migratory signal.","evidence":"Boyden chamber migration assay with cells differing in αvβ3 expression and integrin-blocking antibodies","pmids":["7532190"],"confidence":"High","gaps":["Downstream intracellular signaling from αvβ3 not characterized","In vivo migration role not established"]},{"year":1996,"claim":"Discovery that SPP1 is a protein ligand for CD44 that induces chemotaxis—but not homotypic aggregation—defined a second major receptor axis distinct from integrin engagement.","evidence":"Receptor-ligand binding assays and chemotaxis versus aggregation functional comparisons","pmids":["8560266"],"confidence":"High","gaps":["CD44-binding domain not mapped","Relative contribution of CD44 vs integrin pathways in vivo unknown"]},{"year":1999,"claim":"Identification of the cryptic SVVYGLR motif exposed by thrombin cleavage as an α9β1-binding site explained how proteolytic processing generates new receptor specificities from a single SPP1 polypeptide.","evidence":"Site-directed mutagenesis of Tyr165, synthetic peptide blocking, adhesion assay with α9-transfected cells","pmids":["10593924"],"confidence":"High","gaps":["In vivo relevance of thrombin-cleaved OPN fragments not established","Structural basis of α9β1 recognition unresolved"]},{"year":2000,"claim":"Knockout mouse studies revealed that SPP1 is a bifunctional immune regulator: phosphorylation-dependent integrin engagement stimulates IL-12, while phosphorylation-independent CD44 engagement suppresses IL-10, establishing SPP1 as a master switch for type-1 immunity.","evidence":"OPN-knockout mice challenged with viral and bacterial pathogens, cytokine measurement, receptor-pathway dissection","pmids":["10657301"],"confidence":"High","gaps":["Identity of the kinase responsible for immunomodulatory phosphorylation not yet known (resolved in 2012)","Whether this dual-receptor mechanism operates in all tissue macrophages unclear"]},{"year":2001,"claim":"Parallel discoveries established SPP1 as an MMP substrate whose cleavage potentiates adhesion/migration, an intrinsically disordered protein enabling multivalent binding, and a critical regulator of TH1-mediated autoimmune demyelination.","evidence":"In vitro MMP-3/MMP-7 cleavage site mapping; NMR structural analysis confirming complete disorder; OPN-knockout EAE model with cytokine profiling","pmids":["11375993","11162539","11721059"],"confidence":"High","gaps":["Relative contributions of individual MMP cleavage fragments in vivo not dissected","How disorder enables simultaneous multi-receptor engagement structurally undefined"]},{"year":2003,"claim":"Identification of SPP1 as the leading metastasis-associated gene in hepatocellular carcinoma, with anti-OPN antibody blocking invasion and pulmonary metastasis, established SPP1 as a pro-metastatic effector.","evidence":"cDNA microarray of HCC, antibody blockade in vitro invasion and nude mouse metastasis model","pmids":["12640447"],"confidence":"Medium","gaps":["Receptor through which OPN drives HCC metastasis not identified","Antibody specificity concerns in single-study blockade"]},{"year":2012,"claim":"Discovery that FAM20C is the Golgi kinase phosphorylating SPP1 at S-x-E motifs resolved the long-standing question of which kinase controls SPP1's phosphorylation-dependent receptor specificity and immune functions.","evidence":"Biochemical kinase assay identifying FAM20C, genetic validation via Raine syndrome mutations","pmids":["22582013"],"confidence":"High","gaps":["Whether all functionally relevant SPP1 phosphosites are FAM20C substrates unclear","Quantitative relationship between phosphorylation stoichiometry and integrin vs CD44 selectivity not defined"]},{"year":2014,"claim":"Demonstration that OPN in the perivascular niche activates CD44 signaling through γ-secretase-released CD44-ICD/CBP/p300/HIF-2α to promote glioma stem cell phenotypes revealed the intracellular signaling cascade downstream of SPP1–CD44.","evidence":"Cd44-knockout mouse glioma model, γ-secretase inhibition, CBP/p300 and HIF-2α functional assays","pmids":["24607407"],"confidence":"High","gaps":["Whether this CD44-ICD cascade operates outside the glioma niche unknown","Direct SPP1 binding triggering γ-secretase cleavage not biochemically demonstrated"]},{"year":2017,"claim":"Mapping of SPP1 transcriptional regulation to GLI1 and OCT4 binding at the SPP1 locus, combined with CD44-binding-domain deletion rescue experiments, linked hedgehog/stemness pathways to SPP1 expression and confirmed CD44-binding is required for glioma sphere formation.","evidence":"ChIP assay for GLI1/OCT4, siRNA knockdown, rescue with WT vs CD44-binding-domain-deleted SPP1","pmids":["28030801"],"confidence":"High","gaps":["Whether GLI1/OCT4 regulation is tissue-specific or general not tested","Structural basis of SPP1 CD44-binding domain not defined"]},{"year":2018,"claim":"Two concurrent studies established SPP1 as both a macrophage chemokine in glioblastoma (acting via αvβ5 to maintain M2 polarization) and an immune checkpoint suppressing CD8⁺ T-cell activation via CD44 (regulated by IRF8 at the Spp1 promoter), unifying SPP1's roles in tumor immune evasion.","evidence":"CRISPR/siRNA/shRNA knockdown, OPN-deficient mice, macrophage polarization assays; IRF8-knockout mice, ChIP for IRF8-Spp1 promoter, T-cell activation assays","pmids":["30307407","30395540"],"confidence":"High","gaps":["Relative importance of integrin vs CD44 axis in immune suppression across tumor types not resolved","Whether IRF8-mediated Spp1 repression occurs in non-myeloid cells unknown"]},{"year":2020,"claim":"Multiple studies converged on SPP1 signaling through CD44 and integrins to activate PI3K/AKT, ERK, and Wnt/β-catenin pathways in diverse contexts including chondrocytes, endothelial progenitors, and gastric cancer, broadening the downstream effector landscape.","evidence":"Receptor-blocking antibodies, ERK/PI3K inhibitors, Wnt reporter assays, focal adhesion imaging across multiple cell types","pmids":["36034459","35005113","31990676","34367279"],"confidence":"Medium","gaps":["Which downstream pathway is dominant in a given tissue context remains unclear","Direct biochemical link between SPP1-receptor binding and specific kinase activation not reconstituted"]},{"year":2022,"claim":"N-glycosylation at Asn79 was shown to be required for SPP1's osteoclast proliferative activity via NF-κB, while miR-433 was identified as a direct post-transcriptional regulator of SPP1 mRNA, expanding the known post-translational and post-transcriptional regulatory layers.","evidence":"N79Q site-directed mutagenesis with proliferation assay; dual luciferase reporter assay confirming miR-433 binding to SPP1 3′-UTR","pmids":["35451542","34306197"],"confidence":"Medium","gaps":["Full glycosylation map and functional consequences of each site not established","In vivo relevance of miR-433 regulation of SPP1 not tested"]},{"year":2023,"claim":"CXCL4 was identified as the driver of profibrotic SPP1⁺ macrophage differentiation, with CXCL4-knockout abolishing this population and ameliorating fibrosis in heart and kidney injury, placing SPP1⁺ macrophages as a central cellular effector of organ fibrosis.","evidence":"Cxcl4-knockout mice in cardiac and renal injury models, single-nuclear RNA-seq, in vitro macrophage differentiation","pmids":["36807143"],"confidence":"High","gaps":["Whether CXCL4 acts directly on macrophages or via intermediate cells not fully resolved","SPP1-independent functions of SPP1⁺ macrophages not dissected"]},{"year":2023,"claim":"Macrophage-derived SPP1 was shown to protect from NASH by inducing oncostatin-M and activating hepatocyte STAT3/ARG2 to enhance fatty acid oxidation, revealing a tissue-protective metabolic function distinct from SPP1's pro-fibrotic roles.","evidence":"Conditional Spp1 knockin and myeloid-specific knockout mice on high-fat diet, OSM/STAT3/ARG2 pathway dissection","pmids":["37028770"],"confidence":"High","gaps":["Whether hepatoprotective vs profibrotic SPP1 functions are dose-dependent or context-specific not resolved","Receptor mediating SPP1-induced OSM production in macrophages not identified"]},{"year":2025,"claim":"Genetic epistasis in pancreatic cancer models revealed an intercellular SPP1–CD61–GREM1–BMP loop in which epithelial-derived SPP1 binds CD61 on mesenchymal cells to induce GREM1, which reciprocally sustains SPP1 expression and mesenchymal fate, with Spp1 inactivation abolishing metastasis.","evidence":"Spp1 and Grem1 single/double knockout PDAC mouse models, proteomic plasma analysis, heterozygosity rescue","pmids":["40993391"],"confidence":"High","gaps":["Whether the SPP1-GREM1 loop operates in non-pancreatic cancers unknown","Direct structural basis of SPP1-CD61 binding not determined"]},{"year":null,"claim":"Key unresolved questions include the structural basis of SPP1's multivalent receptor engagement as a disordered protein, how phosphorylation stoichiometry quantitatively tunes integrin vs CD44 selectivity in vivo, and what determines whether SPP1⁺ macrophages adopt profibrotic versus tissue-protective functions in different organs.","evidence":"","pmids":[],"confidence":"High","gaps":["No high-resolution structure of SPP1 in complex with any receptor","Quantitative phosphorylation-receptor selectivity relationship undefined","Tissue-specific determinants of SPP1⁺ macrophage functional polarity unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098631","term_label":"cell adhesion mediator activity","supporting_discovery_ids":[1,2,22]},{"term_id":"GO:0048018","term_label":"receptor ligand activity","supporting_discovery_ids":[0,3,13,14,33]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[3,12,28]}],"localization":[{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[0,1,9,40,33]},{"term_id":"GO:0031012","term_label":"extracellular matrix","supporting_discovery_ids":[6,46]}],"pathway":[{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[8]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[3,5,12,13,48]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,11,18,20,30,33]},{"term_id":"R-HSA-1474244","term_label":"Extracellular matrix organization","supporting_discovery_ids":[38,27]},{"term_id":"R-HSA-1500931","term_label":"Cell-Cell communication","supporting_discovery_ids":[0,14,46]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[7,24,33,48]}],"complexes":[],"partners":["CD44","ITGAV","ITGB3","ITGA9","ITGB5","ITGB1","FAM20C","GREM1"],"other_free_text":[]},"mechanistic_narrative":"SPP1 (osteopontin) is a secreted, intrinsically disordered phosphoglycoprotein that functions as a multivalent extracellular signaling molecule integrating cell adhesion, migration, immune regulation, and tissue remodeling by engaging αv-containing integrins (αvβ3, αvβ5, αvβ1) via its RGD motif and CD44 via a separate domain [PMID:7532190, PMID:8560266]. Thrombin and MMP-3/MMP-7 cleavage generates fragments that expose a cryptic SVVYGLR motif binding α9β1 integrin, potentiating adhesion and migration [PMID:10593924, PMID:11375993]; phosphorylation by the Golgi kinase FAM20C at S-x-E motifs governs receptor specificity, with the phosphorylated form stimulating macrophage IL-12 via integrins and the phosphorylation-independent form inhibiting IL-10 via CD44, thereby tuning type-1 immunity [PMID:10657301, PMID:22582013]. SPP1 acts as a macrophage chemokine driving profibrotic SPP1⁺ macrophage differentiation via CXCL4, promotes cancer stemness and immune evasion through CD44- and integrin-dependent activation of PI3K/AKT, JAK2/STAT3, ERK, and NF-κB pathways, suppresses CD8⁺ T-cell activation as an immune checkpoint, and establishes an intercellular SPP1–CD61–GREM1–BMP regulatory loop that maintains mesenchymal cell fate in pancreatic cancer [PMID:36807143, PMID:30395540, PMID:30307407, PMID:40993391]."},"prefetch_data":{"uniprot":{"accession":"P10451","full_name":"Osteopontin","aliases":["Bone sialoprotein 1","Nephropontin","Secreted phosphoprotein 1","SPP-1","Urinary stone protein","Uropontin"],"length_aa":314,"mass_kda":35.4,"function":"Major non-collagenous bone protein that binds tightly to hydroxyapatite. Appears to form an integral part of the mineralized matrix. Probably important to cell-matrix interaction Acts as a cytokine involved in enhancing production of interferon-gamma and interleukin-12 and reducing production of interleukin-10 and is essential in the pathway that leads to type I immunity","subcellular_location":"Secreted","url":"https://www.uniprot.org/uniprotkb/P10451/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SPP1","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/SPP1","total_profiled":1310},"omim":[{"mim_id":"621529","title":"TRANSMEMBRANE PROTEIN 168; TMEM168","url":"https://www.omim.org/entry/621529"},{"mim_id":"618111","title":"ZINC FINGER PROTEIN 64; ZFP64","url":"https://www.omim.org/entry/618111"},{"mim_id":"612826","title":"SPHINGOSINE-1-PHOSPHATE PHOSPHATASE 1; SGPP1","url":"https://www.omim.org/entry/612826"},{"mim_id":"611539","title":"FORKHEAD BOX D3; FOXD3","url":"https://www.omim.org/entry/611539"},{"mim_id":"609566","title":"PARIETAL FORAMINA 3; PFM3","url":"https://www.omim.org/entry/609566"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Golgi apparatus","reliability":"Enhanced"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"brain","ntpm":1840.1},{"tissue":"gallbladder","ntpm":2283.6},{"tissue":"kidney","ntpm":3270.9},{"tissue":"placenta","ntpm":2626.4}],"url":"https://www.proteinatlas.org/search/SPP1"},"hgnc":{"alias_symbol":["BSPI","ETA-1","lnc-PKD2-2-3"],"prev_symbol":["BNSP","OPN"]},"alphafold":{"accession":"Q9BX95","domains":[{"cath_id":"-","chopping":"129-344_354-439","consensus_level":"medium","plddt":91.9557,"start":129,"end":439}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BX95","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BX95-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BX95-F1-predicted_aligned_error_v6.png","plddt_mean":80.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SPP1","jax_strain_url":"https://www.jax.org/strain/search?query=SPP1"},"sequence":{"accession":"Q9BX95","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9BX95.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9BX95/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BX95"}},"corpus_meta":[{"pmid":"35365629","id":"PMC_35365629","title":"Single-cell 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Role of alpha v beta 3 in smooth muscle cell migration to osteopontin in vitro.","date":"1995","source":"The Journal of clinical investigation","url":"https://pubmed.ncbi.nlm.nih.gov/7532190","citation_count":473,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"25910212","id":"PMC_25910212","title":"Widespread macromolecular interaction perturbations in human genetic disorders.","date":"2015","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/25910212","citation_count":454,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"15489334","id":"PMC_15489334","title":"The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).","date":"2004","source":"Genome research","url":"https://pubmed.ncbi.nlm.nih.gov/15489334","citation_count":438,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"24607407","id":"PMC_24607407","title":"Osteopontin-CD44 signaling in the glioma perivascular niche enhances cancer stem cell 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Inactivation of Spp1 abolished metastasis and converted mesenchymal to epithelial PDAC cells.\",\n      \"method\": \"Mouse PDAC genetic models (Spp1 knockout, Grem1 heterozygous/knockout), proteomic analysis of patient plasma, mechanistic rescue experiments with concomitant Grem1 inactivation\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple genetic epistasis experiments in vivo with orthogonal rescue, receptor identification, and mechanistic pathway validation\",\n      \"pmids\": [\"40993391\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SPP1 (osteopontin) secreted by cancer-associated fibroblasts activates cancer stem cell features in pancreatic cancer cells via the SPP1-CD44 axis; SPP1 knockdown significantly reduced stemness characteristics in CAF-conditioned media-treated PC cells, and overexpression of SPP1 lacking a CD44-binding domain failed to rescue sphere formation.\",\n      \"method\": \"Gene knockdown (siRNA), 2D/3D co-culture models, tumorsphere assays, immunohistochemistry in mouse models and human tumors, PCR array\",\n      \"journal\": \"Gastroenterology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including KD with specific phenotypic readout and domain-deletion rescue experiment\",\n      \"pmids\": [\"34418441\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"SPP1 promotes ovarian cancer cell proliferation, migration, and invasion by activating the Integrin β1/FAK/AKT signaling pathway; SPP1 silencing inhibited this pathway and reduced tumor growth in vivo, while ectopic SPP1 expression activated the pathway.\",\n      \"method\": \"siRNA knockdown, ectopic overexpression, Western blotting, xenograft mouse model, CCK-8/wound healing/Matrigel invasion assays\",\n      \"journal\": \"OncoTargets and therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — KD/OE with defined pathway readout; single lab, multiple assays\",\n      \"pmids\": [\"29559792\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"BET inhibitor (targeting BRD4) suppresses SPP1 expression in melanoma via the noncanonical NF-κB pathway; BRD4 does not bind directly to the SPP1 promoter but regulates SPP1 expression through NFKB2. Silencing NFKB2 phenocopied BET inhibitor treatment and SPP1 silencing.\",\n      \"method\": \"ChIP assay, RT-PCR, Western blotting, siRNA knockdown, xenograft mouse model, drug screen\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP assay identifies transcriptional mechanism; genetic epistasis via NFKB2 knockdown; single lab\",\n      \"pmids\": [\"33052224\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"EEF2K upregulates phosphorylation of STAT3 at Tyr705, which binds to the SPP1 promoter and enhances SPP1 transcription, thereby facilitating melanoma progression. Re-expression of SPP1 partially negated the inhibitory effects of EEF2K silencing.\",\n      \"method\": \"RNA-seq, ChIP assay, siRNA/overexpression, in vitro and in vivo models\",\n      \"journal\": \"Clinical and translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP establishes direct STAT3-SPP1 promoter binding; functional epistasis confirmed; single lab\",\n      \"pmids\": [\"35184394\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SPP1 promotes radiation resistance in esophageal carcinoma by facilitating DNA damage repair and activating the JAK2/STAT3 signaling pathway; radiation increases SPP1 expression, which in turn increases JAK2 and STAT3 phosphorylation. SPP1 knockdown combined with JAK2-STAT3 inhibition synergistically reduced tumor volume.\",\n      \"method\": \"siRNA knockdown/overexpression, Western blotting, xenograft mouse model, flow cytometry, immunofluorescence\",\n      \"journal\": \"Cancer medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — KD/OE with defined pathway readout in vitro and in vivo; single lab\",\n      \"pmids\": [\"35593388\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Macrophage-derived SPP1/OPN protects from nonalcoholic steatohepatitis by upregulating oncostatin-M (OSM) in macrophages, which activates STAT3 signaling in hepatocytes to upregulate arginase-2 (ARG2), thereby enhancing fatty acid oxidation. Conditional Spp1 knockout in myeloid cells worsened NASH.\",\n      \"method\": \"Conditional knockin/knockout mouse models (Spp1KI Mye, Spp1KI LvMF, Spp1ΔMye), high-fat/fructose/cholesterol diet NASH model, transcriptomic analysis, pathway validation\",\n      \"journal\": \"Gastroenterology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — conditional genetic models with specific molecular pathway (OPN→OSM→STAT3→ARG2→FAO) validated in vivo; multiple orthogonal approaches\",\n      \"pmids\": [\"37028770\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"SPP1 mediates crosstalk between hepatocellular carcinoma cells and macrophages via SPP1-CD44 and SPP1-PTGER4 receptor-ligand pairs, and triggers polarization of macrophages to M2-phenotype tumor-associated macrophages.\",\n      \"method\": \"Single-cell RNA-seq receptor-ligand analysis, in vitro macrophage polarization experiments\",\n      \"journal\": \"Cancer immunology, immunotherapy : CII\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — computational receptor-ligand identification confirmed by in vitro assay; single lab\",\n      \"pmids\": [\"34028567\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"SPP1 secreted by tumor cells in hepatocellular carcinoma interacts with the CD44 receptor on hepatic stellate cells (HSCs), activating the PI3K/AKT signaling pathway to promote HSC differentiation into cancer-associated fibroblasts. Blockade of CD44 or silencing SPP1 in tumor cells abrogated this interaction and reduced tumor volume and collagen deposition.\",\n      \"method\": \"Spatial transcriptomics, single-cell transcriptomics, SPP1 silencing in vivo, CD44 receptor blockade, in vivo tumor models\",\n      \"journal\": \"Journal of translational medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — SPP1-CD44-PI3K/AKT axis validated with receptor blockade and in vivo SPP1 silencing with specific functional readouts; orthogonal spatial and single-cell evidence\",\n      \"pmids\": [\"39267037\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"SOCS3 in myeloid cells negatively regulates SPP1 expression; STAT3 directly transcribes the Spp1 gene, and pharmaceutical activation of SOCS3 or blocking of SPP1 reduces pathological retinal neovascularization. The SOCS3/STAT3/SPP1 axis controls pathological angiogenesis.\",\n      \"method\": \"Myeloid-specific SOCS3 knockout mice, scRNA-seq, in vitro STAT3/SOCS3 manipulation, pharmacological SPP1 blockade in angiogenesis mouse models\",\n      \"journal\": \"Molecular therapy : the journal of the American Society of Gene Therapy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO plus pharmacological intervention, STAT3 identified as direct transcriptional regulator of Spp1; multiple orthogonal methods\",\n      \"pmids\": [\"38504518\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Microglia-derived SPP1 promotes pathological retinal neovascularization by activating endothelial Kit/Akt/mTOR signaling; recombinant SPP1 enhanced endothelial cell proliferation and migration, SPP1 neutralization blocked these effects, and intravitreal SPP1 neutralizing antibody attenuated pathological neovascularization and improved visual function.\",\n      \"method\": \"scRNA-seq, recombinant SPP1 treatment, SPP1 neutralizing antibody (in vitro and intravitreal injection), RNA-seq of endothelial cells, BV2/bEnd.3 co-culture, OIR mouse model\",\n      \"journal\": \"Journal of personalized medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — SPP1-Kit/Akt/mTOR axis identified with multiple assays and in vivo neutralization; single lab\",\n      \"pmids\": [\"36675807\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"SPP1 (osteopontin) in glioblastoma is transcriptionally regulated by GLI1 binding to the promoter and OCT4 binding to the first intron. GLI1 knockdown reduced SPP1 mRNA and protein. SPP1-CD44 interaction is required for glioma sphere formation, as overexpression of SPP1 lacking the CD44-binding domain failed to rescue sphere formation.\",\n      \"method\": \"ChIP (GLI1, OCT4), siRNA knockdown, stable SPP1 depletion, sphere formation assays, domain-deletion rescue experiments\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP establishes transcriptional mechanism; functional domain-deletion confirms SPP1-CD44 requirement; single lab\",\n      \"pmids\": [\"28030801\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"SPP1 in Schwann cells regulates nerve degeneration and regeneration after sciatic nerve injury through c-Fos, PKCα, and p-ERK/ERK pathways. Spp1 silencing reduced Schwann cell proliferation and promoted migration; overexpression had the opposite effect.\",\n      \"method\": \"siRNA knockdown, overexpression, in vitro Schwann cell assays, in vivo sciatic nerve injury model, Western blotting, mRNA expression\",\n      \"journal\": \"BMC neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — loss-of-function with defined cellular phenotype and pathway identification; single lab, multiple assays\",\n      \"pmids\": [\"28270094\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"AMPK promotes osteogenesis by activating the AMPK-Gfi1-OPN (SPP1) axis: AMPK activation downregulates the transcriptional repressor Gfi1, which dissociates from the OPN promoter, resulting in OPN upregulation. Gfi1 overexpression suppresses OPN-mediated osteogenesis.\",\n      \"method\": \"Lentiviral AMPK overexpression, wild-type/dominant-negative Gfi1 overexpression, OPN promoter-Gfi1 dissociation analysis, ectopic bone formation assay\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — genetic epistasis via Gfi1 manipulation with OPN promoter-binding data; single lab\",\n      \"pmids\": [\"27283242\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SPP1 macrophages in viral myocarditis promote fibroblast conversion to an inflammatory phenotype with high Ccl2/Ccl7 expression via SPP1 signaling, which increases monocyte chemotaxis to the heart, creating a self-recruitment loop. SPP1 inhibition reduced cardiac macrophage infiltration and improved cardiac function.\",\n      \"method\": \"scRNA-seq, indirect co-culture experiments, CellPhoneDB ligand-receptor analysis, macrophage depletion, SPP1 inhibition in CVB3 VMC mouse model\",\n      \"journal\": \"Biology direct\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — in vitro co-culture plus in vivo pharmacological inhibition with defined mechanistic outcome; single lab\",\n      \"pmids\": [\"40087693\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"SPP1 macrophages drive myofibroblast activation in organ fibrosis via CXCL4-dependent differentiation; platelets (the most abundant CXCL4 source in vivo) drive profibrotic Spp1 macrophage differentiation, and Cxcl4 loss abrogates this process. Macrophages orchestrate fibroblast activation via Spp1, Fn1, and Sema3 crosstalk.\",\n      \"method\": \"Cxcl4 knockout mice, heart and kidney injury models, ligand-receptor interaction analysis (snRNA-seq), in vitro and in vivo Cxcl4/Spp1 loss-of-function studies\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with defined fibrotic phenotype and identification of CXCL4 as upstream driver of Spp1 macrophage differentiation; multiple orthogonal approaches in two organ systems\",\n      \"pmids\": [\"36807143\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Macrophage-derived Spp1 accelerates prostatic intraepithelial neoplasia (PIN) cell proliferation via CD44, integrin αv, integrin β1, and integrin β3 receptors, activating Akt and JNK signaling. Blockade of Akt and JNK completely abolished macrophage Spp1-induced PIN cell proliferation.\",\n      \"method\": \"3D culture, recombinant Spp1 treatment, receptor expression analysis, pharmacological Akt/JNK inhibition, proliferation assays\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — receptor identification and pharmacological pathway blockade with defined functional readout; single lab\",\n      \"pmids\": [\"35457063\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"OPN/SPP1 inhibits autophagy in osteoarthritic chondrocytes through CD44 and αvβ3 integrin receptors via the ERK MAPK signaling pathway. Blocking CD44 or αvβ3 integrin reduced LC3II and Beclin1 suppression, and ERK inhibitor blocked OPN-mediated autophagy suppression.\",\n      \"method\": \"Recombinant human OPN treatment, anti-CD44/anti-CD51/61 blocking antibodies, ERK inhibitor, Western blotting, mRFP-GFP-LC3 confocal imaging, TEM\",\n      \"journal\": \"Frontiers in endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — receptor blocking plus pathway inhibition with multiple autophagy readouts; single lab\",\n      \"pmids\": [\"36034459\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"OPN/SPP1 binds the αv integrin subunit on endothelial progenitor cells (EPCs) to support EPC adhesion and promote their incorporation into angiogenic vascular networks; silencing αv integrin reduced EPC binding to OPN and focal adhesion assembly.\",\n      \"method\": \"siRNA knockdown of αv integrin, OPN-coated slides, immunofluorescence of focal adhesions, in vitro HUVEC network incorporation assay, in situ hybridization/IHC\",\n      \"journal\": \"Reproduction (Cambridge, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — siRNA knockdown with specific adhesion and network incorporation readouts; receptor-ligand interaction validated\",\n      \"pmids\": [\"31990676\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"OPN/SPP1 inhibits osteoarthritis progression via the OPN/CD44/PI3K signal axis; OPN directly inhibits PI3K expression in OA chondrocytes by binding CD44, and siOPN or CD44 blockade reversed this effect in mice.\",\n      \"method\": \"siOPN knockdown, recombinant OPN, CD44 inhibitor (LS-C179404), in vivo mouse OA model, histomorphological analysis, Western blotting\",\n      \"journal\": \"Genes & diseases\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — receptor identification with both KD and pharmacological inhibition; in vivo model; single lab\",\n      \"pmids\": [\"35005113\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"BAFF induces OPN (SPP1) expression specifically in splenic CD21−CD23+ B cells via an NF-κB dependent signaling pathway, and B cell-derived OPN in turn induces Bcl2 expression in T cells to promote T cell survival.\",\n      \"method\": \"TACI-IgG BAFF blockade in EAE model, in vitro B cell stimulation, NF-κB pathway analysis, in vivo T cell survival assays\",\n      \"journal\": \"Molecular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — pharmacological BAFF blockade with defined OPN/Bcl2 mechanistic readout; pathway identified; single lab\",\n      \"pmids\": [\"24084099\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The ATX-LPA axis upregulates OPN/SPP1 expression in hepatic cancer cells via activation of Akt through LPA receptors, and OPN is required for ATX-LPA-induced cell migration.\",\n      \"method\": \"Western blotting, real-time PCR, Transwell migration assay, PI3K/Akt inhibitors, LPA receptor inhibitors\",\n      \"journal\": \"Anatomical record (Hoboken, N.J. : 2007)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — pharmacological inhibition identifies pathway; single lab, single cell line\",\n      \"pmids\": [\"21337710\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"The SPP1 promoter polymorphism rs28357094 (TG genotype) increases OPN expression in DMD myotubes, and deflazacort treatment further increases OPN only in TG-genotype myotubes, demonstrating that this SNP modifies glucocorticoid response of the SPP1 gene in a cell-type and differentiation-stage dependent manner.\",\n      \"method\": \"Primary human myoblasts/myotubes with defined genotypes, immunoblot, mRNA quantification, deflazacort treatment\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — defined genotype groups with quantified protein and mRNA; functional consequence of polymorphism on glucocorticoid response established; single lab\",\n      \"pmids\": [\"28595270\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"miR-433 directly binds to the 3'-UTR of SPP1 mRNA and negatively regulates SPP1 expression; reduced miR-433 in patients with tibial fracture combined with brain injury corresponded to elevated SPP1 protein and enhanced callus/heterotopic ossification formation.\",\n      \"method\": \"Dual luciferase reporter assay, agomiR-433 transfection in osteoblasts, Western blotting, RT-qPCR, human tissue samples\",\n      \"journal\": \"Experimental and therapeutic medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — dual luciferase reporter establishes direct miR-433/SPP1 3'-UTR interaction; functional consequences confirmed; single lab\",\n      \"pmids\": [\"34306197\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SPP1 facilitates lung adenocarcinoma cell migration and invasion by upregulating COL11A1 expression; COL11A1 overexpression reversed the inhibitory effects of SPP1 knockdown on migration/invasion and EMT marker changes.\",\n      \"method\": \"siRNA knockdown, Western blotting, migration/invasion assays, in vivo xenograft, COL11A1 overexpression rescue\",\n      \"journal\": \"Cancer cell international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — KD with COL11A1 rescue defines epistatic relationship; multiple assays; single lab\",\n      \"pmids\": [\"36266702\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Vitamin C promotes neuroprotection in reactive astrocytes via upregulation of SPP1 through the transcription factor E2F1; SPP1+ astrocytes then promote retinal ganglion cell survival in a mouse model of glaucoma.\",\n      \"method\": \"In vitro astrocyte treatment, E2F1 transcription factor analysis, mouse glaucoma model, gene expression studies\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — transcription factor (E2F1) identified as regulator of SPP1 in astrocytes with functional in vivo consequences; single lab\",\n      \"pmids\": [\"37160307\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"SPP1+ macrophages in liver cancer drive tumor stemness via a vitronectin (VTN)-dependent paracrine mechanism activating integrin αvβ5/AMPK/YAP1/SOX4 signaling in tumor cells; conversely, CCL15 from cancer cells polarizes M0 macrophages to SPP1+ phenotype, forming a positive feedback loop. Inhibiting integrin αvβ5/YAP1 sensitized liver cancer cells to chemotherapy.\",\n      \"method\": \"scRNA-seq, mechanistic in vitro paracrine assays, integrin αvβ5/YAP1 inhibition, chemosensitization assays\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — paracrine mechanism with defined receptor-signaling pathway and pharmacological inhibition; single lab\",\n      \"pmids\": [\"39216547\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"H4K12 lactylation (H4K12la) in reprogrammed microglia after spinal cord injury promotes SPP1 transcription; recombinant SPP1 promotes neuronal maturation and enhances mitochondrial function in axons, improving functional recovery. SPP1 signaling is primarily localized in microglia post-SCI.\",\n      \"method\": \"scRNA-seq, pseudotime analysis, DNA-pulldown for upstream regulators, rSPP1 injection in vivo, in vitro neuronal maturation assays\",\n      \"journal\": \"CNS neuroscience & therapeutics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — H4K12la upstream regulation identified by DNA-pulldown; functional rSPP1 in vivo rescue; single lab\",\n      \"pmids\": [\"39939834\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"SPP1 secreted by TREM2+ cardiac macrophages drives atrial fibrosis and atrial fibrillation; silencing Spp1 in TREM2+ macrophages using an antibody-siRNA conjugate reduces atrial fibrosis and suppresses AFib in mice.\",\n      \"method\": \"Antibody-siRNA conjugate targeting macrophage Spp1, mouse AFib model, atrial fibrosis quantification\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — cell-type-specific KD with defined fibrotic and electrophysiological readout in vivo; single lab; preprint\",\n      \"pmids\": [\"39149373\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"OPN N-glycosylation at asparagine-79 promotes osteoclast proliferation and activates the NF-κB signaling pathway in both osteoclasts and osteoblasts, promoting nuclear translocation of NF-κB; mutant N79 OPN cannot increase osteoblast proliferation.\",\n      \"method\": \"Site-directed mutagenesis (N79Q), plasmid transfection in osteoblasts/osteoclasts, CCK8 proliferation assay, Western blotting, mass spectrometry for N-glycosylation site identification\",\n      \"journal\": \"Oral diseases\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 — mutagenesis of glycosylation site with functional readout; MS identifies modification site; single lab\",\n      \"pmids\": [\"35451542\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"Dental pulp cells synthesize and secrete osteopontin (SPP1/2ar) as a major highly phosphorylated protein; OPN synthesis markedly increases upon TPA (PKC activator) treatment, indicating PKC-mediated upregulation of SPP1 in these cells.\",\n      \"method\": \"Immunoprecipitation with osteopontin/2ar antibody, thrombin susceptibility assay, TPA treatment, secretion assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single pulldown/immunoprecipitation identifying SPP1 secretion and PKC-mediated regulation; single early study\",\n      \"pmids\": [\"1472061\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Monocytic differentiation of HL-60 cells (via PMA) coordinately induces OPN/SPP1 mRNA and secretion alongside increased surface expression of its receptors CD44 and integrin subunits αv, β3, and β1, suggesting coordinate regulation of SPP1 and its receptors during monocyte differentiation.\",\n      \"method\": \"RT-PCR, ELISA for OPN secretion, flow cytometry for surface receptor expression, comparison to granulocytic differentiation (retinoic acid)\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — correlative expression during differentiation without direct mechanistic perturbation; single lab\",\n      \"pmids\": [\"9525482\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SPP1 (osteopontin) is a secreted, multifunctional glycoprotein that signals through cell-surface receptors including CD44, integrins (αvβ3, αvβ5, α4β1, β1, β3), CD61, and PTGER4 to activate downstream pathways (PI3K/AKT, FAK/AKT, JAK2/STAT3, ERK MAPK, Wnt/β-catenin, Kit/Akt/mTOR) governing cell adhesion, migration, invasion, stemness, fibrosis, immune modulation, and angiogenesis; its transcription is directly regulated by STAT3 (downstream of SOCS3/cytokine signals), GLI1/OCT4 (in glioma), BRD4/NFKB2 (noncanonical NF-κB), E2F1, β-catenin/TCF-4, and Gfi1 (downstream of AMPK), and post-translationally it undergoes N-glycosylation (at Asn-79) and phosphorylation that modulate its activity; in the tumor microenvironment, SPP1+ macrophages interact with fibroblasts via SPP1-CD44 and related axes to promote desmoplasia and immunosuppression, while in pancreatic cancer SPP1 forms an intercellular regulatory loop with CD61/BMP2/GREM1 signaling to maintain mesenchymal cell fate and drive metastasis.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\nI'll classify each paper before extracting discoveries.\n\n**CLASSIFICATION NOTES:**\n\nPapers about bacteriophage SPP1 (Bacillus subtilis phage): EXCLUDE — symbol collision with non-orthologous organism (phage, not the mammalian gene SPP1/osteopontin)\n- PMIDs: 23419885, 12697751, 34027814 (phage), 12034814 (G40P helicase), 6292508 (plasmid transduction), 23268446 (phage replication), 30544981 (phage genome), 106091 (phage transduction), 6094718 (phage homology), 4627475 (phage transcription)\n\nPapers about yeast Spp1 (COMPASS subunit): EXCLUDE — this is the Saccharomyces cerevisiae SET1 complex subunit, a completely different protein from mammalian SPP1/osteopontin. Different function, domain architecture, cellular context.\n- PMIDs: 23160953, 23511748, 19398585\n\nPapers about snake venom BnSP-7/BnSP-6 (Bothrops phospholipases): EXCLUDE — alias collision, completely different proteins\n- PMIDs: 10860537, 26519876, 33486049, 28751219\n\nPaper about BspI (Brucella effector): EXCLUDE — symbol collision\n- PMID: 35840160\n\nPaper about BSPI-C3G (food emulsion): EXCLUDE — symbol collision\n- PMID: 36203951\n\nAll other papers about mammalian SPP1/osteopontin: KEEP\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1996,\n      \"finding\": \"Osteopontin (SPP1/Eta-1) was identified as a direct protein ligand of CD44. This interaction induces cellular chemotaxis but not homotypic aggregation, distinguishing it from the CD44–hyaluronate interaction which promotes aggregation but not migration.\",\n      \"method\": \"Receptor-ligand binding assays, chemotaxis assays, homotypic aggregation assays\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct functional receptor-ligand interaction with distinct phenotypic readouts, widely replicated\",\n      \"pmids\": [\"8560266\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"OPN mediates cell adhesion via multiple αv-containing integrins (αvβ3, αvβ5, αvβ1), but directed cell migration toward an OPN gradient specifically requires αvβ3. Cells lacking surface αvβ3 adhered to OPN at ~50% efficiency but could not migrate toward it.\",\n      \"method\": \"Boyden chamber migration assay, adhesion assay with cells differing in αvβ3 expression, integrin blocking antibodies\",\n      \"journal\": \"The Journal of Clinical Investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — functional dissection with multiple integrin variants and blocking antibodies, replicated in subsequent work\",\n      \"pmids\": [\"7532190\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Thrombin cleavage of OPN exposes a novel recognition sequence (SVVYGLR) in the N-terminal fragment that directly binds integrin α9β1, distinct from the RGD site. Site-directed mutagenesis of Tyr165 abolished α9β1-mediated adhesion.\",\n      \"method\": \"Site-directed mutagenesis, synthetic peptide blocking assays, cell adhesion assays with α9-transfected cells\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis + peptide blocking + transfection rescue in multiple cell lines\",\n      \"pmids\": [\"10593924\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"OPN/Eta-1 regulates macrophage IL-12 and IL-10 cytokine expression through two distinct receptor interactions: a phosphorylation-dependent interaction with integrin receptor stimulates IL-12 expression, while a phosphorylation-independent interaction with CD44 inhibits IL-10 expression. OPN-deficient mice show severely impaired type-1 immunity with diminished IL-12/IFN-γ and elevated IL-10.\",\n      \"method\": \"OPN-knockout mice (viral and bacterial infection models), cytokine measurement, phosphorylation-dependent vs. independent receptor interaction studies\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — knockout mouse phenotype with mechanistic dissection of two receptor pathways, published in high-impact journal\",\n      \"pmids\": [\"10657301\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"OPN (osteopontin) is a novel substrate for MMP-3 (stromelysin-1) and MMP-7 (matrilysin). Three cleavage sites were identified for MMP-3 in human OPN (Gly166-Leu167, Ala201-Tyr202, Asp210-Leu211); MMP-7 cleaves at two of those sites. MMP cleavage of OPN potentiates its function as an adhesive and migratory stimulus through cell surface integrins.\",\n      \"method\": \"In vitro cleavage assay with purified MMPs, cleavage site mapping, cell adhesion and migration assays pre- and post-cleavage\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with cleavage site identification and functional consequence demonstrated\",\n      \"pmids\": [\"11375993\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"OPN regulates T helper cell-1 (TH1)-mediated demyelinating disease. OPN-deficient mice were resistant to progressive experimental autoimmune encephalomyelitis (EAE); myelin-reactive T cells in OPN−/− mice produced more IL-10 and less IFN-γ than in OPN+/+ mice.\",\n      \"method\": \"OPN-knockout mouse model of EAE, cytokine profiling of myelin-reactive T cells, microarray analysis of MS plaques\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — knockout mouse with mechanistic cytokine profiling, corroborated by human MS tissue analysis\",\n      \"pmids\": [\"11721059\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"OPN and bone sialoprotein (BSP) are members of the SIBLING family on human chromosome 4, and both are completely unstructured in solution (flexible polypeptide backbones), enabling them to associate with multiple binding partners and bridge proteins into biologically active complexes, including bridging complement Factor H to integrins or CD44 to suppress the alternate complement pathway.\",\n      \"method\": \"1D proton NMR, transverse relaxation time measurements, recombinant protein structural analysis\",\n      \"journal\": \"Biochemical and Biophysical Research Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — NMR structural characterization with functional mechanistic inference\",\n      \"pmids\": [\"11162539\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Osteopontin (SPP1) was identified as the lead metastasis-associated gene in HCC. An osteopontin-specific antibody effectively blocked HCC cell invasion in vitro and inhibited pulmonary metastasis of HCC cells in nude mice.\",\n      \"method\": \"cDNA microarray, antibody blockade of invasion in vitro, nude mouse pulmonary metastasis model\",\n      \"journal\": \"Nature Medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — antibody blockade functional assay in vitro and in vivo, single study\",\n      \"pmids\": [\"12640447\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"OPN (osteopontin/SPP1) is phosphorylated within S-x-E motifs by FAM20C (Golgi casein kinase), a secreted atypical kinase that localizes to the Golgi and phosphorylates SIBLING family proteins including OPN. Mutations in FAM20C cause Raine syndrome.\",\n      \"method\": \"Kinase activity assay, identification of FAM20C as the responsible kinase, genetic linkage in Raine syndrome patients\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — biochemical kinase assay identifying writer enzyme for OPN phosphorylation, with disease genetics validation\",\n      \"pmids\": [\"22582013\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"Uropontin (OPN purified from urine) inhibits calcium oxalate crystal growth in vitro, demonstrating a direct functional role in regulating urinary stone formation as a crystal growth inhibitor.\",\n      \"method\": \"Monoclonal antibody immunoaffinity purification, in vitro calcium oxalate crystal growth assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct in vitro biochemical assay with purified protein\",\n      \"pmids\": [\"1729712\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1990,\n      \"finding\": \"Human OPN/SPP1 cDNA was cloned; the gene is a single copy located on chromosome 4q near the centromere. Two mRNA variants were identified differing by a 14-amino-acid insertion at residue 58 (OPN1b vs OPN1a). The RGD cell attachment site is conserved between species.\",\n      \"method\": \"cDNA library screening, Northern blot, PCR amplification, chromosomal mapping with human-rodent cell hybrids, in situ hybridization\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — foundational molecular characterization with multiple methods\",\n      \"pmids\": [\"1974876\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"OPN in the glioma perivascular niche promotes cancer stem cell phenotypes and radiation resistance via CD44 signaling. The γ-secretase-regulated intracellular domain of CD44 mediates downstream effects via CBP/p300-dependent enhancement of HIF-2α activity. Cd44-deficient mice showed improved survival in a glioma model.\",\n      \"method\": \"Cd44-knockout mouse glioma model, survival analysis, γ-secretase inhibition, CBP/p300 functional assays, HIF-2α activity measurement\",\n      \"journal\": \"Cell Stem Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — knockout mouse model plus mechanistic dissection of CD44 intracellular signaling cascade\",\n      \"pmids\": [\"24607407\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"OPN acts as an immune checkpoint to suppress CD8+ T cell activation. IRF8 represses OPN expression by binding the Spp1 promoter in myeloid cells; IRF8 deficiency elevates OPN, which signals through CD44 on T cells to suppress CTL activation and confer tumor immune tolerance.\",\n      \"method\": \"IRF8-knockout mice, chimeric bone marrow reconstitution, T cell-specific IRF8 deficiency, Spp1 promoter ChIP assay, T cell activation functional assays\",\n      \"journal\": \"The Journal of Clinical Investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple genetic models with ChIP mechanistic validation, epistasis established\",\n      \"pmids\": [\"30395540\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"OPN is a potent chemokine for macrophages in glioblastoma, and integrin αvβ5 on glioblastoma-infiltrating macrophages is a major OPN receptor. OPN maintains M2 macrophage gene signature/phenotype. Both tumor-derived and host-derived OPN are required for glioma development. OPN blockade with 4-1BB-OPN bispecific aptamers extended median survival by 68% in mice.\",\n      \"method\": \"OPN siRNA/shRNA/CRISPR knockdown, OPN-deficient mice, macrophage migration assays, in vitro M2 polarization assays, therapeutic aptamer study\",\n      \"journal\": \"The Journal of Clinical Investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal knockdown methods, receptor identification, in vivo therapeutic validation\",\n      \"pmids\": [\"30307407\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"OPN/SPP1 secreted by cancer-associated fibroblasts (CAFs) drives cancer stemness in pancreatic cancer through the SPP1–CD44 axis. SPP1 knockdown significantly reduced stemness characteristics. Overexpression of wild-type SPP1, but not an SPP1 lacking the CD44-binding domain, rescued sphere formation capacity, confirming CD44-binding is required.\",\n      \"method\": \"CAF conditioned media treatment, siRNA knockdown of SPP1, CD44-binding domain mutant rescue experiment, tumorsphere formation assay, 3D co-culture model\",\n      \"journal\": \"Gastroenterology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — domain-deletion mutant rescue experiment establishing mechanistic requirement for CD44-binding domain\",\n      \"pmids\": [\"34418441\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"AMPK activation promotes osteogenesis and inhibits adipogenesis through the AMPK-Gfi1-OPN (osteopontin) axis. AMPK activation downregulates the transcriptional repressor Gfi1 and causes it to dissociate from the OPN promoter, leading to OPN upregulation, which mediates the osteogenic and anti-adipogenic effects.\",\n      \"method\": \"AMPK overexpression via lentiviral vector, Gfi1 overexpression and dominant-negative constructs, ChIP-like promoter dissociation assay, ectopic bone formation model\",\n      \"journal\": \"Cellular Signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic pathway placement with Gfi1-OPN promoter occupancy, single lab\",\n      \"pmids\": [\"27283242\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"SPP1 transcription in glioblastoma is driven by GLI1 binding to the SPP1 promoter and OCT4 binding to the first SPP1 intron. GLI1 knockdown reduced SPP1 mRNA and protein levels. SPP1-CD44 interaction is required for glioma sphere formation: SPP1 lacking the CD44-binding domain failed to rescue sphere formation in SPP1-depleted cells.\",\n      \"method\": \"ChIP assay (GLI1 and OCT4 binding to SPP1 locus), siRNA knockdown of SPP1/GLI1/CD44, rescue with wild-type vs CD44-binding-domain-deleted SPP1, sphere formation assays\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — ChIP validation of transcription factor binding plus domain-deletion rescue experiment\",\n      \"pmids\": [\"28030801\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"SPP1 regulates nerve degeneration and regeneration after sciatic nerve injury through c-Fos, PKCα, and p-ERK/ERK pathways in Schwann cells. Spp1 silencing reduced Schwann cell proliferation and promoted migration, while Spp1 overexpression had opposite effects.\",\n      \"method\": \"siRNA knockdown and overexpression of Spp1 in Schwann cells, sciatic nerve injury model in rats, cytokine and pathway analysis (c-Fos, PKCα, ERK phosphorylation)\",\n      \"journal\": \"BMC Neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — loss- and gain-of-function with pathway markers, single lab\",\n      \"pmids\": [\"28270094\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"SPP1 promotes ovarian cancer cell proliferation, migration, and invasion via the Integrin β1/FAK/AKT signaling pathway. Silencing SPP1 inhibited Integrin β1/FAK/p-AKT and suppressed tumor growth in vivo; ectopic SPP1 expression activated this pathway.\",\n      \"method\": \"siRNA knockdown and overexpression, Western blot for pathway components, Boyden chamber migration/invasion assays, nude mouse xenograft\",\n      \"journal\": \"OncoTargets and Therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — loss/gain-of-function with pathway analysis, single lab\",\n      \"pmids\": [\"29559792\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"BRD4 (BET bromodomain protein) regulates SPP1 expression indirectly through NFKB2 (noncanonical NF-κB). BRD4 does not bind directly to the SPP1 promoter; instead, it regulates NFKB2, which then drives SPP1 transcription. BET inhibitors suppress melanoma progression via this noncanonical NF-κB/SPP1 pathway.\",\n      \"method\": \"ChIP assay (BRD4 binding to promoters), NFKB2 silencing, BET inhibitor treatment, SPP1 overexpression rescue experiment\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP-based mechanistic pathway with rescue experiment, single lab\",\n      \"pmids\": [\"33052224\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"OPN inhibits autophagy in osteoarthritic chondrocytes through CD44 and αvβ3 integrin receptors via the ERK MAPK signaling pathway. Blocking CD44 or αvβ3 with antibodies prevented OPN-mediated suppression of LC3II and Beclin1. ERK inhibition abolished OPN's autophagy-suppressing activity.\",\n      \"method\": \"Recombinant OPN treatment, anti-CD44 and anti-αvβ3 blocking antibodies, ERK inhibitor, Western blot, mRFP-GFP-LC3 confocal imaging, transmission electron microscopy\",\n      \"journal\": \"Frontiers in Endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — receptor blocking with multiple readouts including imaging, single lab\",\n      \"pmids\": [\"36034459\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"OPN (osteopontin) activates PI3K signaling in osteoarthritic chondrocytes by binding to CD44 on the cell surface, causing downstream cascading effects that inhibit chondrocyte degeneration and reduce cartilage matrix loss. OPN binding was verified in vivo in mice.\",\n      \"method\": \"siOPN knockdown, rhOPN treatment, PI3K inhibitor (LS-C179404), in vivo mouse OA model\",\n      \"journal\": \"Genes & Diseases\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — loss/gain-of-function with inhibitor, in vivo validation\",\n      \"pmids\": [\"35005113\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"OPN binds αv integrin on endothelial progenitor cells (EPCs) to support adhesion and promote EPC incorporation into angiogenic vascular networks. siRNA knockdown of αv integrin reduced EPC binding to OPN and focal adhesion assembly. αv integrin was localized to focal adhesions at the basal surface of OPN-coated EPCs.\",\n      \"method\": \"siRNA knockdown of αv integrin, OPN-coated substrate adhesion assay, focal adhesion immunofluorescence microscopy, EPC incorporation into HUVEC networks\",\n      \"journal\": \"Reproduction\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — siRNA knockdown with focal adhesion imaging and functional incorporation assay\",\n      \"pmids\": [\"31990676\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"EEF2K promotes melanoma progression by phosphorylating STAT3 at Tyr705, which binds to the SPP1 promoter and enhances SPP1 transcription. Re-expression of SPP1 partially reversed the inhibitory effect of EEF2K silencing, while SPP1 or STAT3 inhibition abolished EEF2K's tumorigenic effects.\",\n      \"method\": \"EEF2K silencing and overexpression, RNA-seq, ChIP assay (p-STAT3 binding to SPP1 promoter), SPP1 re-expression rescue\",\n      \"journal\": \"Clinical and Translational Medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP plus rescue experiment establishing EEF2K/STAT3/SPP1 axis, single lab\",\n      \"pmids\": [\"35184394\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SPP1 promotes radiation resistance in esophageal carcinoma via the JAK2/STAT3 pathway. Radiation increases SPP1 expression, which in turn increases JAK2 and STAT3 phosphorylation. SPP1 knockdown combined with JAK2/STAT3 inhibition synergistically reduced tumor volume in vivo.\",\n      \"method\": \"SPP1 knockdown and overexpression, Western blot for JAK2/STAT3 phosphorylation, xenograft mouse model, pharmacological JAK2-STAT3 inhibition\",\n      \"journal\": \"Cancer Medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — pathway analysis with in vivo validation, single lab\",\n      \"pmids\": [\"35593388\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"miR-433 directly binds to the 3'-UTR of SPP1 mRNA to regulate SPP1 expression. Overexpression of miR-433 via agomiR-433 in osteoblast cells reduced SPP1 mRNA and protein levels. Dual luciferase reporter assay confirmed direct interaction between miR-433 and the SPP1 3'-UTR.\",\n      \"method\": \"Dual luciferase reporter assay, agomiR-433 transfection, RT-qPCR, Western blot\",\n      \"journal\": \"Experimental and Therapeutic Medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — dual luciferase assay directly confirming miRNA-3'UTR interaction\",\n      \"pmids\": [\"34306197\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"OPN N-glycosylation at asparagine-79 promotes osteoclast proliferation and modulates osteoblast function through the NF-κB signaling pathway. Mutation of the N79 glycosylation site abolished OPN's ability to increase osteoblast proliferation. N-glycosylated OPN promotes nuclear translocation of NF-κB in both osteoclasts and osteoblasts.\",\n      \"method\": \"Site-directed mutagenesis (N79Q), CCK8 proliferation assay, Western blot for NF-κB signaling, mass spectrometry glycosylation site identification\",\n      \"journal\": \"Oral Diseases\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mutagenesis of PTM site with functional readout, single lab\",\n      \"pmids\": [\"35451542\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Macrophage-derived SPP1 promotes fibroblast conversion to an inflammatory phenotype with high Ccl2/Ccl7 expression in viral myocarditis, which increases monocyte chemotaxis to the heart. SPP1 inhibition reduced cardiac macrophage infiltration and improved cardiac function in CVB3-induced myocarditis mice.\",\n      \"method\": \"Single-cell RNA sequencing, indirect co-culture of macrophages with fibroblasts, SPP1 inhibition in vivo, CellPhoneDB ligand-receptor analysis\",\n      \"journal\": \"Biology Direct\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vitro co-culture mechanistic validation plus in vivo SPP1 inhibition\",\n      \"pmids\": [\"40087693\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Macrophage-derived OPN (SPP1) protects from NASH by inducing oncostatin-M (OSM) production in macrophages; OSM activates STAT3 signaling in hepatocytes, which upregulates arginase-2 (ARG2), enhancing fatty acid oxidation to reduce steatosis. Conditional Spp1 knockin protected mice from NASH while Spp1 myeloid knockout worsened disease.\",\n      \"method\": \"Conditional Spp1 knockin and knockout mice (myeloid-specific), Western blot for OSM/STAT3/ARG2 pathway, fatty acid oxidation assays, high-fat diet NASH model\",\n      \"journal\": \"Gastroenterology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional gain- and loss-of-function with mechanistic pathway dissection in multiple mouse models\",\n      \"pmids\": [\"37028770\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"SOCS3 in myeloid cells directly regulates SPP1 gene expression; SPP1 is a transcriptional target of STAT3. Loss of SOCS3 in myeloid cells leads to elevated STAT3 activity and increased SPP1 expression in microglia/macrophages, driving pathological retinal neovascularization. Pharmaceutical activation of SOCS3 or SPP1 blockade reduced pathological neovascularization.\",\n      \"method\": \"Myeloid-specific SOCS3 knockout mice, scRNA-seq, STAT3 transcriptional target validation, SPP1 neutralizing antibody in vivo\",\n      \"journal\": \"Molecular Therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic knockout plus in vivo blockade, STAT3-SPP1 axis identified\",\n      \"pmids\": [\"38504518\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Microglia-derived SPP1 promotes retinal neovascularization by activating endothelial Akt/mTOR signaling via the Kit receptor. SPP1 expression in microglia is regulated by NF-κB and HIF-1α pathways. Spp1 neutralizing antibody attenuated pathological retinal neovascularization and improved visual function.\",\n      \"method\": \"OIR mouse model, scRNA-seq, conditioned medium from BV2 microglia, recombinant SPP1 treatment, SPP1 neutralizing antibody intravitreal injection, RNA-seq of bEnd.3 cells, Akt/mTOR phosphorylation assays\",\n      \"journal\": \"Journal of Personalized Medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — conditioned medium + recombinant protein + neutralizing antibody with pathway analysis, single lab\",\n      \"pmids\": [\"36675807\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Vitamin C promotes neuroprotection of retinal ganglion cells by upregulating SPP1 in reactive astrocytes via the transcription factor E2F1. SPP1+ astrocytes in turn promote retinal ganglion cell survival in a mouse glaucoma model.\",\n      \"method\": \"Mouse glaucoma model, SPP1 expression analysis, E2F1 transcription factor identification, retinal ganglion cell survival assay\",\n      \"journal\": \"Life Science Alliance\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — transcription factor identified with in vivo phenotype, single lab\",\n      \"pmids\": [\"37160307\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"SPP1 secreted by hepatocellular carcinoma tumor cells interacts with the CD44 receptor on hepatic stellate cells (HSCs), activating the PI3K/AKT signaling pathway and promoting HSC differentiation into cancer-associated fibroblasts. CD44 receptor blockade abrogated this interaction; in vivo SPP1 silencing impaired HSC-to-CAF differentiation and reduced tumor volume and collagen deposition.\",\n      \"method\": \"Spatial transcriptomics, scRNA-seq, CD44 receptor blockade, in vivo SPP1 silencing, collagen deposition quantification\",\n      \"journal\": \"Journal of Translational Medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — receptor blockade and in vivo silencing with mechanistic pathway, single lab\",\n      \"pmids\": [\"39267037\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SPP1 binds the CD61 (integrin β3/αvβ3 complex) receptor on mesenchymal PDAC cells to induce Bmp2 and Grem1 expression. GREM1 in turn inhibits BMP signaling, which is required for SPP1 expression in epithelial PDAC cells, forming an intercellular SPP1-GREM1 regulatory loop that maintains mesenchymal cell fate. Spp1 inactivation in mouse PDAC models delayed tumorigenesis and abolished metastasis.\",\n      \"method\": \"Spp1 knockout mouse PDAC models, proteomic plasma analysis, Grem1/Spp1 double knockout and heterozygous genetic epistasis experiments, receptor identification\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple genetic epistasis experiments (double knockout, heterozygosity rescue) establishing intercellular SPP1-CD61-GREM1-BMP regulatory loop\",\n      \"pmids\": [\"40993391\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Histone H4 lysine 12 lactylation (H4K12la) promotes SPP1 transcription in reprogrammed microglia following spinal cord injury. DNA-pulldown experiments identified H4K12la as an upstream regulatory modification on Spp1. Recombinant SPP1 promoted neuronal maturation and enhanced mitochondrial function in axons, improving functional recovery after SCI.\",\n      \"method\": \"scRNA-seq, DNA-pulldown assay for H4K12la regulatory proteins, pseudotime analysis, recombinant SPP1 injection into injured spinal cord, functional recovery assessment\",\n      \"journal\": \"CNS Neuroscience & Therapeutics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — DNA-pulldown identifying histone modification upstream of Spp1, with in vivo functional validation\",\n      \"pmids\": [\"39939834\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"BAFF induces OPN (SPP1) expression specifically in splenic CD21−CD23+ B cells via an NF-κB-dependent signaling pathway. B cell-derived OPN then promotes anti-apoptotic Bcl2 expression in T cells, maintaining T cell survival in autoimmune EAE.\",\n      \"method\": \"TACI-IgG blockade of BAFF, NF-κB inhibition, OPN measurement in B cell subpopulations, T cell survival assays\",\n      \"journal\": \"Molecular Immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — NF-κB pathway and cell-type specificity identified with functional T cell readout, single lab\",\n      \"pmids\": [\"24084099\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SPP1 facilitates lung adenocarcinoma cell migration and invasion by upregulating COL11A1 expression. COL11A1 overexpression rescued the inhibitory effects of SPP1 knockdown on migration/invasion and reversed EMT marker changes, placing COL11A1 downstream of SPP1.\",\n      \"method\": \"siRNA knockdown of SPP1, COL11A1 overexpression rescue, wound healing and Matrigel invasion assays, Western blot for EMT markers, in vivo metastasis model\",\n      \"journal\": \"Cancer Cell International\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — downstream target identified via rescue experiment, single lab\",\n      \"pmids\": [\"36266702\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SPP1 regulates gastric adenocarcinoma radiosensitivity via Wnt/β-catenin signaling. SPP1 knockdown combined with irradiation increased DNA damage (H2AX phosphorylation), apoptosis, and G2/M phase arrest while reducing invasion. The Wnt/β-catenin inhibitor XAV939 enhanced these phenotypes, placing SPP1 upstream of Wnt/β-catenin in radioresistance.\",\n      \"method\": \"siSPP1 transfection, XAV939 Wnt inhibitor, TOP-flash reporter assay, flow cytometry cell cycle analysis, Western blot\",\n      \"journal\": \"Journal of Oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — pathway reporter assay with epistasis via inhibitor combination, single lab\",\n      \"pmids\": [\"34367279\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Profibrotic SPP1+ macrophage differentiation is driven by CXCL4 (platelet factor 4). CXCL4 was identified as the top upregulated gene during Spp1 macrophage differentiation; Cxcl4 loss abrogated profibrotic Spp1 macrophage differentiation and ameliorated fibrosis after heart and kidney injury. Platelets (the main CXCL4 source in vivo) drive this differentiation. Spp1 macrophages orchestrate fibroblast activation via Spp1, Fn1, and Sema3 crosstalk.\",\n      \"method\": \"Cxcl4-knockout mice, organ injury models (heart and kidney), in vitro macrophage differentiation assays, single-nuclear RNA sequencing with ligand-receptor interaction analysis\",\n      \"journal\": \"Cell Reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — knockout mouse model in two organ injury contexts plus in vitro mechanistic validation\",\n      \"pmids\": [\"36807143\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Therapeutic silencing of Spp1 in atrial TREM2+ cardiac macrophages using an antibody-siRNA conjugate reduces atrial fibrosis and suppresses atrial fibrillation in mice. TREM2+ macrophages secrete OPN (encoded by Spp1), which drives fibrosis and AFib.\",\n      \"method\": \"Antibody-siRNA conjugate targeting TREM2+ macrophages, atrial fibrosis quantification, AFib induction in mouse model\",\n      \"journal\": \"bioRxiv (PREPRINT)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — cell-type specific siRNA silencing with functional cardiac outcome, preprint\",\n      \"pmids\": [\"39149373\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"Clonal rat dental pulp cells (RDP4-1, RPC-C2A) synthesize and secrete osteopontin (SPP1/2ar) as a highly phosphorylated protein. OPN synthesis was markedly increased by TPA (phorbol ester), similar to osteoblastic cells, establishing OPN as a secreted phosphoprotein produced by dental pulp cells.\",\n      \"method\": \"Immunoprecipitation with anti-OPN antibody, thrombin susceptibility assay, metabolic labeling\",\n      \"journal\": \"Biochemical and Biophysical Research Communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — biochemical identification of SPP1 synthesis and secretion with phosphorylation characterization\",\n      \"pmids\": [\"1472061\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Transgenic overexpression of OPN (Spp1) in mammary epithelium induces proliferation, exaggerated terminal end bud formation, increased lobulogenesis, and delayed gland involution. Ki-67 staining confirmed increased epithelial cell proliferation. OPN conveys a proliferative stimulus for mammary epithelial cells.\",\n      \"method\": \"MMTV-Opn transgenic mouse lines (3 founders), immunohistochemistry (Ki-67, OPN), ex vivo organoid lobulogenesis assay\",\n      \"journal\": \"Genes & Cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple transgenic founder lines with consistent phenotype and in vitro organoid validation\",\n      \"pmids\": [\"24069507\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"A promoter polymorphism (rs28357094) in SPP1 modifies OPN expression in DMD myotubes, with the G allele associated with significantly higher OPN expression. Deflazacort treatment significantly increased OPN expression only in myotubes carrying the TG genotype, demonstrating that rs28357094 mediates differential glucocorticoid responsiveness of SPP1 transcription.\",\n      \"method\": \"Primary human DMD myoblasts/myotubes with defined rs28357094 genotypes, deflazacort treatment, immunoblot OPN quantification\",\n      \"journal\": \"Human Molecular Genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — genotype-stratified functional assay in primary human cells, single lab\",\n      \"pmids\": [\"28595270\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The ATX-LPA axis upregulates OPN expression in hepatic cancer cells (SMMC7721) via PI3K/Akt signaling through LPA receptors. LPA receptor inhibitors and PI3K/Akt inhibitors reduced ATX-LPA-induced OPN upregulation, and OPN was required for ATX-LPA-induced cell migration.\",\n      \"method\": \"Western blot and RT-PCR for OPN expression, PI3K/Akt inhibitors, LPA receptor inhibitors, Transwell migration assay\",\n      \"journal\": \"Anatomical Record\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — pathway inhibitor dissection with migration readout, single lab\",\n      \"pmids\": [\"21337710\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MR (mineralocorticoid receptor) acts as the upstream effector of OPN (SPP1) in hepatic stellate cells (LX-2), and OPN shares downstream HMGB1 with MR. The MR/OPN/HMGB1 axis drives NAFLD-associated liver fibrogenesis; MR antagonist eplerenone reduced this axis and fibrogenic markers.\",\n      \"method\": \"MR agonist/antagonist treatment of LX-2 cells, OPN siRNA knockdown, loss- and gain-of-function for MR and OPN, HMGB1 expression analysis\",\n      \"journal\": \"Life Sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — pathway ordering via siRNA and pharmacological intervention, single lab\",\n      \"pmids\": [\"33091447\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Macrophage Spp1 proliferation (SPP1hi macrophages) is markedly increased in IPF fibrotic lungs compared to normal lungs, and causal modelling supported a role for these SPP1hi macrophages in activating IPF myofibroblasts in fibrosis. Low-level local proliferation is present in normal lungs but is dramatically elevated in fibrotic lower lobes.\",\n      \"method\": \"Single-cell RNA sequencing of fresh lung explants (IPF vs. normal), co-localization analysis, causal modelling\",\n      \"journal\": \"European Respiratory Journal\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 + Weak — scRNA-seq and causal modelling only, no direct functional experiment\",\n      \"pmids\": [\"31221805\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Macrophage subpopulations marked by SPP1 expression communicate with fibroblasts via SPP1-CD44, SPP1-PTGER4, and SPP1-αvβ1 receptor pairs in colorectal cancer, mediating formation of an immune-excluded desmoplastic structure and limiting T cell infiltration.\",\n      \"method\": \"Single-cell RNA sequencing, spatial transcriptomics, immunofluorescent staining, ligand-receptor pair analysis across 14 independent CRC cohorts (2550 samples)\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — transcriptomic ligand-receptor inference and spatial validation, no direct functional mechanistic assay\",\n      \"pmids\": [\"35365629\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"H4K12 lactylation in reprogrammed microglia activates Spp1 transcription after spinal cord injury. Recombinant SPP1 promotes neuronal maturation and mitochondrial ATP production in axons via the SPP1 signaling pathway, improving functional recovery post-SCI.\",\n      \"method\": \"scRNA-seq, DNA-pulldown identifying H4K12la as regulatory mark on Spp1, recombinant SPP1 injection, functional behavioral recovery assay\",\n      \"journal\": \"CNS Neuroscience & Therapeutics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — novel PTM regulatory mechanism identified by DNA-pulldown with in vivo functional confirmation\",\n      \"pmids\": [\"39939834\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"SPP1+ macrophages in the SPP1-CD44 axis promote T-cell exhaustion in ovarian cancer. Blocking either SPP1 or CD44 reversed T-cell exhaustion, restored CD8+ T-cell functionality, and suppressed tumor growth in vivo. Molecular docking identified nilotinib as an SPP1 inhibitor that reduced PD-1 expression in macrophage-conditioned Jurkat cells.\",\n      \"method\": \"Functional T cell assays, SPP1/CD44 blocking, in vivo tumor suppression, molecular docking simulations, in vitro nilotinib treatment\",\n      \"journal\": \"International Immunopharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional blocking assay with in vivo tumor suppression, computational docking for inhibitor\",\n      \"pmids\": [\"40446696\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SPP1 (osteopontin) is an intrinsically disordered, multiply phosphorylated and glycosylated secreted glycoprotein that functions as a multivalent extracellular signaling molecule: it binds αv-containing integrins (αvβ1, αvβ3, αvβ5) via its RGD motif—with αvβ3 specifically required for directed cell migration—and CD44 via a separate domain, with thrombin or MMP cleavage generating additional fragments that expose a distinct α9β1-binding SVVYGLR motif and potentiate integrin-dependent adhesion/migration; phosphorylation (mediated by the Golgi kinase FAM20C at S-x-E motifs) governs receptor specificity, with the phosphorylated form stimulating macrophage IL-12 via integrins while the phosphorylation-independent form inhibits IL-10 via CD44; SPP1 also acts as a crystal-growth inhibitor (as uropontin), a macrophage chemokine, a regulator of type-1 immunity, an upstream driver of JAK2/STAT3, AKT/mTOR, ERK, Wnt/β-catenin, and NF-κB pathways in diverse cell types, a promoter of profibrotic macrophage differentiation via CXCL4, and, most recently, a key intercellular regulator that binds CD61 on mesenchymal pancreatic cancer cells to induce GREM1 expression, which reciprocally maintains SPP1 expression in epithelial cells, thereby controlling mesenchymal cell fate.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"SPP1 (osteopontin) is a secreted multifunctional glycoprotein that orchestrates cell adhesion, migration, stemness, fibrosis, angiogenesis, and immune modulation by engaging cell-surface receptors including CD44, integrins (αvβ3, αvβ5, αv, β1, β3), and PTGER4 to activate downstream PI3K/AKT, FAK/AKT, JAK2/STAT3, ERK MAPK, Kit/Akt/mTOR, and NF-κB signaling cascades [PMID:29559792, PMID:35593388, PMID:36675807, PMID:35457063, PMID:35005113]. Its transcription is directly regulated by STAT3 (downstream of SOCS3 and EEF2K), BRD4/NFKB2 (noncanonical NF-κB), GLI1/OCT4, E2F1, Gfi1 (repressor relieved by AMPK), and histone H4K12 lactylation, while post-transcriptionally miR-433 targets the SPP1 3′-UTR, and post-translationally N-glycosylation at Asn-79 is required for its osteoclast-proliferative and NF-κB-activating functions [PMID:38504518, PMID:35184394, PMID:33052224, PMID:28030801, PMID:27283242, PMID:34306197, PMID:35451542]. In the tumor and fibrotic microenvironments, SPP1+ macrophages engage fibroblasts and stellate cells via SPP1–CD44 signaling to drive desmoplasia, cancer-associated fibroblast differentiation, and immunosuppressive macrophage polarization, while in pancreatic cancer SPP1 signals through CD61/integrin β3 on mesenchymal tumor cells to induce a BMP2/GREM1 intercellular loop that maintains mesenchymal cell fate and is essential for metastasis [PMID:39267037, PMID:36807143, PMID:34418441, PMID:40993391]. Beyond cancer and fibrosis, macrophage-derived SPP1 protects against nonalcoholic steatohepatitis through an OSM/STAT3/ARG2 hepatocyte fatty-acid oxidation axis, promotes pathological retinal neovascularization, and supports nerve regeneration and neuronal maturation after injury [PMID:37028770, PMID:38504518, PMID:28270094, PMID:39939834].\",\n  \"teleology\": [\n    {\n      \"year\": 1992,\n      \"claim\": \"Identification that SPP1/osteopontin is actively synthesized as a secreted, highly phosphorylated protein by dental pulp cells established its identity as a phosphoprotein whose expression is induced by PKC activation, setting the stage for understanding its regulation and extracellular functions.\",\n      \"evidence\": \"Immunoprecipitation and TPA treatment of dental pulp cell cultures\",\n      \"pmids\": [\"1472061\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single cell type with antibody pulldown only; no genetic perturbation\", \"Phosphorylation sites not mapped\", \"No receptor or functional consequence identified\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"The coordinate upregulation of SPP1 and its receptors (CD44, αv, β3, β1 integrins) during monocyte differentiation suggested autocrine/paracrine signaling through these receptors, foreshadowing the SPP1–receptor axes later validated functionally.\",\n      \"evidence\": \"RT-PCR, ELISA, and flow cytometry during PMA-induced monocytic differentiation of HL-60 cells\",\n      \"pmids\": [\"9525482\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Correlative expression data without direct functional perturbation\", \"No receptor blocking or knockdown performed\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Demonstrating that the ATX-LPA axis upregulates SPP1 via Akt in hepatic cancer cells, with SPP1 required for LPA-induced migration, connected SPP1 to lipid-mediated signaling upstream and cell migration downstream.\",\n      \"evidence\": \"PI3K/Akt and LPA receptor inhibitors in hepatic cancer cell migration assays\",\n      \"pmids\": [\"21337710\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single cell line; pharmacological inhibitors only without genetic validation\", \"Receptor through which SPP1 acts on migration not identified\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Establishing that BAFF induces SPP1 in splenic B cells via NF-κB, and that B cell–derived SPP1 in turn induces Bcl2 to promote T cell survival, revealed SPP1 as a mediator of immune cell crosstalk in autoimmune contexts.\",\n      \"evidence\": \"TACI-IgG BAFF blockade in EAE model with in vitro B cell stimulation and T cell survival assays\",\n      \"pmids\": [\"24084099\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single autoimmune model (EAE)\", \"SPP1 receptor on T cells not identified\", \"No SPP1-specific genetic loss-of-function in B cells\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identification of the AMPK–Gfi1–SPP1 transcriptional axis, where AMPK relieves Gfi1 repression of the SPP1 promoter to drive osteogenesis, established a direct transcriptional regulatory mechanism linking metabolic sensing to SPP1-dependent bone formation.\",\n      \"evidence\": \"Lentiviral AMPK overexpression, Gfi1 wild-type/dominant-negative constructs, promoter dissociation analysis, ectopic bone formation assay\",\n      \"pmids\": [\"27283242\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Gfi1 binding site on SPP1 promoter not mapped precisely\", \"In vivo osteogenesis shown only via ectopic assay\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"ChIP-based identification of GLI1 binding the SPP1 promoter and OCT4 binding its first intron in glioblastoma, combined with domain-deletion showing SPP1's CD44-binding domain is required for sphere formation, established that SPP1 is a Hedgehog/stemness-regulated gene whose CD44 interaction drives glioma stem cell maintenance.\",\n      \"evidence\": \"ChIP for GLI1/OCT4, siRNA knockdown, CD44-binding-domain deletion rescue in sphere formation assays\",\n      \"pmids\": [\"28030801\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"OCT4 functional perturbation on SPP1 not tested by KD\", \"Downstream pathway activated by SPP1–CD44 in glioma not defined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Demonstrating that SPP1 promoter polymorphism rs28357094 modifies glucocorticoid-induced SPP1 expression in DMD myotubes in a genotype- and differentiation-stage-dependent manner identified SPP1 as a pharmacogenomic modifier in Duchenne muscular dystrophy.\",\n      \"evidence\": \"Primary human myoblasts/myotubes of defined genotypes treated with deflazacort; immunoblot and mRNA quantification\",\n      \"pmids\": [\"28595270\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which the SNP alters glucocorticoid responsiveness not resolved\", \"No direct transcription factor binding data at the polymorphic site\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Functional validation that SPP1 activates integrin β1/FAK/AKT signaling in ovarian cancer to drive proliferation, migration, and invasion established a canonical receptor-proximal pathway through which secreted SPP1 promotes tumor progression.\",\n      \"evidence\": \"siRNA knockdown and ectopic overexpression with Western blot pathway readouts and xenograft tumor models\",\n      \"pmids\": [\"29559792\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single cancer type\", \"Direct integrin β1 engagement not shown by receptor blockade\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Multiple studies converged to define SPP1's receptor specificity: SPP1 binds αv integrin on endothelial progenitor cells for adhesion and vascular incorporation, signals through CD44 and αvβ3 integrin to suppress autophagy via ERK MAPK in chondrocytes, and inhibits PI3K via CD44 in osteoarthritis, demonstrating context-dependent receptor usage and downstream pathway selection.\",\n      \"evidence\": \"siRNA knockdown of αv integrin on EPCs; anti-CD44/anti-CD51/61 blocking antibodies plus ERK inhibitor in chondrocytes; siOPN and CD44 inhibitor in mouse OA model\",\n      \"pmids\": [\"31990676\", \"36034459\", \"35005113\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How receptor context determines pathway choice (ERK vs PI3K) not resolved\", \"Relative contribution of different receptors in the same tissue not compared side by side\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identifying that BRD4 regulates SPP1 expression indirectly through NFKB2 (noncanonical NF-κB) rather than by direct promoter binding revealed a druggable epigenetic–transcriptional axis controlling SPP1 in melanoma.\",\n      \"evidence\": \"ChIP showing no BRD4 at SPP1 promoter; NFKB2 siRNA phenocopying BET inhibitor effects; xenograft model\",\n      \"pmids\": [\"33052224\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether NFKB2 binds the SPP1 promoter directly not shown by ChIP\", \"BET inhibitor effects on SPP1 in non-melanoma contexts untested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Demonstrating that cancer-associated fibroblast–secreted SPP1 activates cancer stem cell properties via CD44 in pancreatic cancer—with domain-deletion proving the CD44-binding domain is essential—established SPP1 as a paracrine stemness factor in the tumor stroma.\",\n      \"evidence\": \"siRNA knockdown, 3D co-culture tumorsphere assays, CD44-binding-domain deletion rescue\",\n      \"pmids\": [\"34418441\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream signaling pathway in cancer cells not fully dissected\", \"Whether other SPP1 receptors contribute to stemness not excluded\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Direct targeting of the SPP1 3′-UTR by miR-433 was established as a post-transcriptional regulatory mechanism, with reduced miR-433 correlating with elevated SPP1 and enhanced ossification in traumatic brain injury–associated fractures.\",\n      \"evidence\": \"Dual luciferase reporter assay, agomiR-433 transfection in osteoblasts, human tissue correlation\",\n      \"pmids\": [\"34306197\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether miR-433 regulation of SPP1 operates in non-bone tissues unknown\", \"Upstream regulators of miR-433 not identified\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Convergent studies established STAT3 as a direct transcriptional activator of SPP1: EEF2K phosphorylates STAT3 at Tyr705 which binds the SPP1 promoter in melanoma, and separately SPP1 itself activates JAK2/STAT3 in esophageal carcinoma, revealing a potential positive feedback loop.\",\n      \"evidence\": \"ChIP showing STAT3–SPP1 promoter binding; siRNA/overexpression epistasis; xenograft models for both melanoma and esophageal carcinoma\",\n      \"pmids\": [\"35184394\", \"35593388\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether STAT3-mediated SPP1 transcription and SPP1-induced STAT3 activation co-occur in the same cell to form a true autocrine loop not tested\", \"STAT3 binding site on SPP1 promoter not precisely mapped\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Site-directed mutagenesis of Asn-79 demonstrated that N-glycosylation at this residue is required for SPP1's ability to stimulate osteoclast proliferation and activate NF-κB nuclear translocation, establishing a functionally important post-translational modification.\",\n      \"evidence\": \"N79Q mutagenesis, CCK8 proliferation assays, Western blot for NF-κB, MS identification of glycosylation site\",\n      \"pmids\": [\"35451542\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether N79 glycosylation affects receptor binding specificity not tested\", \"Other glycosylation sites not functionally characterized\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Macrophage-derived SPP1 was shown to signal through multiple receptors (CD44, αv, β1, β3 integrins) on prostate neoplasia cells activating Akt and JNK, with pharmacological blockade of both kinases completely abolishing SPP1-induced proliferation, establishing a multi-receptor signaling model.\",\n      \"evidence\": \"Recombinant SPP1 treatment, receptor expression analysis, Akt/JNK inhibitors in 3D PIN cell cultures\",\n      \"pmids\": [\"35457063\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relative contribution of individual receptors not dissected by single-receptor blockade\", \"In vivo validation lacking\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Conditional genetic models revealed that macrophage-derived SPP1 protects against NASH by upregulating oncostatin-M in macrophages, which activates STAT3 in hepatocytes to induce ARG2 and enhance fatty acid oxidation—establishing a non-oncogenic, metabolically protective role for SPP1.\",\n      \"evidence\": \"Myeloid-specific Spp1 conditional knockin/knockout mice on HFFC diet; transcriptomic and pathway validation\",\n      \"pmids\": [\"37028770\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the OPN→OSM→STAT3→ARG2 axis operates in human NASH not confirmed\", \"SPP1 receptor mediating macrophage autocrine OSM induction not identified\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"The identification of CXCL4 (from platelets) as an upstream driver of SPP1+ macrophage differentiation in organ fibrosis, with Cxcl4 knockout abrogating profibrotic Spp1 macrophage generation, placed SPP1+ macrophages as a central hub connecting platelet activation to fibroblast activation across heart and kidney injury models.\",\n      \"evidence\": \"Cxcl4 knockout mice, heart and kidney injury models, snRNA-seq ligand-receptor analysis\",\n      \"pmids\": [\"36807143\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which CXCL4 induces SPP1 transcription in macrophages not defined\", \"Whether blocking SPP1 downstream of CXCL4 is sufficient to prevent fibrosis not directly tested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"The SOCS3/STAT3/SPP1 axis in myeloid cells was shown to control pathological retinal neovascularization: SOCS3 loss derepresses STAT3, which directly transcribes Spp1, and microglia-derived SPP1 then activates endothelial Kit/Akt/mTOR signaling to drive aberrant vessel growth.\",\n      \"evidence\": \"Myeloid-specific SOCS3 KO mice, scRNA-seq, rSPP1 treatment, SPP1 neutralizing antibody in OIR mouse model\",\n      \"pmids\": [\"38504518\", \"36675807\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"SPP1 receptor on endothelial cells mediating Kit activation not identified\", \"Whether SPP1 neutralization affects physiological angiogenesis not assessed\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"SPP1 was shown to mediate intercellular crosstalk between hepatocellular carcinoma cells and macrophages via SPP1–CD44 and SPP1–PTGER4 ligand-receptor pairs, driving M2 macrophage polarization and identifying PTGER4 as an additional SPP1 receptor in immunosuppression.\",\n      \"evidence\": \"Single-cell RNA-seq receptor-ligand analysis with in vitro macrophage polarization validation\",\n      \"pmids\": [\"34028567\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"SPP1–PTGER4 interaction not validated by receptor blockade or binding assay\", \"Downstream signaling from PTGER4 upon SPP1 engagement not characterized\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"SPP1 secreted by tumor cells was shown to interact with CD44 on hepatic stellate cells to activate PI3K/AKT and drive their differentiation into cancer-associated fibroblasts, establishing a tumor–stroma paracrine axis that promotes desmoplasia in hepatocellular carcinoma.\",\n      \"evidence\": \"Spatial and single-cell transcriptomics, SPP1 silencing in vivo, CD44 receptor blockade, tumor volume and collagen quantification\",\n      \"pmids\": [\"39267037\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether SPP1–CD44 axis on stellate cells is specific to HCC or generalizable to other liver pathologies not tested\", \"Additional stellate cell receptors for SPP1 not excluded\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identification that SPP1+ macrophages in liver cancer secrete vitronectin to activate integrin αvβ5/AMPK/YAP1/SOX4 signaling in tumor cells—while CCL15 from cancer cells reciprocally polarizes macrophages to the SPP1+ state—revealed a bidirectional positive feedback loop maintaining tumor stemness and chemoresistance.\",\n      \"evidence\": \"scRNA-seq, mechanistic in vitro paracrine assays, integrin αvβ5/YAP1 pharmacological inhibition\",\n      \"pmids\": [\"39216547\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether VTN is the sole mediator or SPP1 itself also contributes not distinguished\", \"In vivo genetic validation of this feedback loop not performed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"In pancreatic cancer, SPP1 was shown to bind CD61 (integrin β3) on mesenchymal tumor cells to induce BMP2 and GREM1 expression; GREM1 then inhibits BMP signaling in epithelial cells required for their own SPP1 production, forming an intercellular regulatory loop essential for metastasis and mesenchymal cell fate maintenance.\",\n      \"evidence\": \"Spp1 knockout, Grem1 heterozygous/knockout mouse PDAC models, proteomic analysis of patient plasma, rescue experiments\",\n      \"pmids\": [\"40993391\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this SPP1–CD61–BMP2/GREM1 loop operates in non-pancreatic cancers not tested\", \"Structural basis of SPP1–CD61 interaction not defined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"H4K12 lactylation was identified as an epigenetic mark that promotes SPP1 transcription in reprogrammed microglia after spinal cord injury, with recombinant SPP1 promoting neuronal maturation and mitochondrial function in axons, extending SPP1's role to neuroregenerative contexts.\",\n      \"evidence\": \"scRNA-seq, DNA-pulldown for upstream regulators, rSPP1 injection in vivo, in vitro neuronal maturation assays\",\n      \"pmids\": [\"39939834\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Transcription factor binding downstream of H4K12la at the SPP1 locus not identified\", \"SPP1 receptor mediating neuronal maturation not determined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include: how SPP1 receptor selectivity (CD44 vs. various integrins vs. PTGER4) is determined in different cellular contexts; the structural basis for receptor engagement; whether the multiple transcriptional inputs (STAT3, NF-κB, GLI1, E2F1, Gfi1, H4K12la) converge on shared cis-regulatory elements; and whether the protective metabolic role of macrophage-derived SPP1 (NASH) and its deleterious profibrotic/pro-tumorigenic roles can be mechanistically uncoupled for therapeutic targeting.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model of SPP1–receptor complexes available\", \"Integrated cis-regulatory map of the SPP1 promoter/enhancers across cell types not established\", \"Therapeutic window between beneficial and pathological SPP1 signaling undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [0, 1, 2, 6, 8, 10, 14, 16]},\n      {\"term_id\": \"GO:0098631\", \"supporting_discovery_ids\": [18]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [0, 1, 2, 6, 8, 10, 14, 30]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0162582\", \"supporting_discovery_ids\": [2, 5, 8, 10, 16, 26]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 5, 8, 10, 16, 26]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [7, 15, 20]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [0, 1, 3, 4, 5, 24, 26]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"CD44\",\n      \"ITGB3\",\n      \"ITGB1\",\n      \"ITGAV\",\n      \"ITGB5\",\n      \"PTGER4\",\n      \"STAT3\",\n      \"NFKB2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"SPP1 (osteopontin) is a secreted, intrinsically disordered phosphoglycoprotein that functions as a multivalent extracellular signaling molecule integrating cell adhesion, migration, immune regulation, and tissue remodeling by engaging αv-containing integrins (αvβ3, αvβ5, αvβ1) via its RGD motif and CD44 via a separate domain [PMID:7532190, PMID:8560266]. Thrombin and MMP-3/MMP-7 cleavage generates fragments that expose a cryptic SVVYGLR motif binding α9β1 integrin, potentiating adhesion and migration [PMID:10593924, PMID:11375993]; phosphorylation by the Golgi kinase FAM20C at S-x-E motifs governs receptor specificity, with the phosphorylated form stimulating macrophage IL-12 via integrins and the phosphorylation-independent form inhibiting IL-10 via CD44, thereby tuning type-1 immunity [PMID:10657301, PMID:22582013]. SPP1 acts as a macrophage chemokine driving profibrotic SPP1⁺ macrophage differentiation via CXCL4, promotes cancer stemness and immune evasion through CD44- and integrin-dependent activation of PI3K/AKT, JAK2/STAT3, ERK, and NF-κB pathways, suppresses CD8⁺ T-cell activation as an immune checkpoint, and establishes an intercellular SPP1–CD61–GREM1–BMP regulatory loop that maintains mesenchymal cell fate in pancreatic cancer [PMID:36807143, PMID:30395540, PMID:30307407, PMID:40993391].\",\n  \"teleology\": [\n    {\n      \"year\": 1990,\n      \"claim\": \"Cloning of human SPP1 cDNA and chromosomal mapping to 4q established the molecular identity of osteopontin, revealing splice variants and a conserved RGD cell-attachment motif that implied integrin-mediated adhesion.\",\n      \"evidence\": \"cDNA library screening, Northern blot, chromosomal mapping with human–rodent cell hybrids\",\n      \"pmids\": [\"1974876\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No receptor binding demonstrated at this stage\", \"Functional significance of splice variants unknown\"]\n    },\n    {\n      \"year\": 1992,\n      \"claim\": \"Identification of uropontin as a direct calcium oxalate crystal growth inhibitor established a non-integrin biochemical function for SPP1 in kidney stone prevention.\",\n      \"evidence\": \"Immunoaffinity-purified uropontin in in vitro calcium oxalate crystal growth assay\",\n      \"pmids\": [\"1729712\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance to nephrolithiasis not tested\", \"Mechanism of crystal inhibition at atomic level unresolved\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"Demonstrating that OPN mediates adhesion through multiple αv integrins but directed migration specifically requires αvβ3 resolved which integrin heterodimer transduces the migratory signal.\",\n      \"evidence\": \"Boyden chamber migration assay with cells differing in αvβ3 expression and integrin-blocking antibodies\",\n      \"pmids\": [\"7532190\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream intracellular signaling from αvβ3 not characterized\", \"In vivo migration role not established\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Discovery that SPP1 is a protein ligand for CD44 that induces chemotaxis—but not homotypic aggregation—defined a second major receptor axis distinct from integrin engagement.\",\n      \"evidence\": \"Receptor-ligand binding assays and chemotaxis versus aggregation functional comparisons\",\n      \"pmids\": [\"8560266\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"CD44-binding domain not mapped\", \"Relative contribution of CD44 vs integrin pathways in vivo unknown\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Identification of the cryptic SVVYGLR motif exposed by thrombin cleavage as an α9β1-binding site explained how proteolytic processing generates new receptor specificities from a single SPP1 polypeptide.\",\n      \"evidence\": \"Site-directed mutagenesis of Tyr165, synthetic peptide blocking, adhesion assay with α9-transfected cells\",\n      \"pmids\": [\"10593924\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance of thrombin-cleaved OPN fragments not established\", \"Structural basis of α9β1 recognition unresolved\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Knockout mouse studies revealed that SPP1 is a bifunctional immune regulator: phosphorylation-dependent integrin engagement stimulates IL-12, while phosphorylation-independent CD44 engagement suppresses IL-10, establishing SPP1 as a master switch for type-1 immunity.\",\n      \"evidence\": \"OPN-knockout mice challenged with viral and bacterial pathogens, cytokine measurement, receptor-pathway dissection\",\n      \"pmids\": [\"10657301\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the kinase responsible for immunomodulatory phosphorylation not yet known (resolved in 2012)\", \"Whether this dual-receptor mechanism operates in all tissue macrophages unclear\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Parallel discoveries established SPP1 as an MMP substrate whose cleavage potentiates adhesion/migration, an intrinsically disordered protein enabling multivalent binding, and a critical regulator of TH1-mediated autoimmune demyelination.\",\n      \"evidence\": \"In vitro MMP-3/MMP-7 cleavage site mapping; NMR structural analysis confirming complete disorder; OPN-knockout EAE model with cytokine profiling\",\n      \"pmids\": [\"11375993\", \"11162539\", \"11721059\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contributions of individual MMP cleavage fragments in vivo not dissected\", \"How disorder enables simultaneous multi-receptor engagement structurally undefined\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Identification of SPP1 as the leading metastasis-associated gene in hepatocellular carcinoma, with anti-OPN antibody blocking invasion and pulmonary metastasis, established SPP1 as a pro-metastatic effector.\",\n      \"evidence\": \"cDNA microarray of HCC, antibody blockade in vitro invasion and nude mouse metastasis model\",\n      \"pmids\": [\"12640447\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Receptor through which OPN drives HCC metastasis not identified\", \"Antibody specificity concerns in single-study blockade\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Discovery that FAM20C is the Golgi kinase phosphorylating SPP1 at S-x-E motifs resolved the long-standing question of which kinase controls SPP1's phosphorylation-dependent receptor specificity and immune functions.\",\n      \"evidence\": \"Biochemical kinase assay identifying FAM20C, genetic validation via Raine syndrome mutations\",\n      \"pmids\": [\"22582013\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether all functionally relevant SPP1 phosphosites are FAM20C substrates unclear\", \"Quantitative relationship between phosphorylation stoichiometry and integrin vs CD44 selectivity not defined\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Demonstration that OPN in the perivascular niche activates CD44 signaling through γ-secretase-released CD44-ICD/CBP/p300/HIF-2α to promote glioma stem cell phenotypes revealed the intracellular signaling cascade downstream of SPP1–CD44.\",\n      \"evidence\": \"Cd44-knockout mouse glioma model, γ-secretase inhibition, CBP/p300 and HIF-2α functional assays\",\n      \"pmids\": [\"24607407\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this CD44-ICD cascade operates outside the glioma niche unknown\", \"Direct SPP1 binding triggering γ-secretase cleavage not biochemically demonstrated\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Mapping of SPP1 transcriptional regulation to GLI1 and OCT4 binding at the SPP1 locus, combined with CD44-binding-domain deletion rescue experiments, linked hedgehog/stemness pathways to SPP1 expression and confirmed CD44-binding is required for glioma sphere formation.\",\n      \"evidence\": \"ChIP assay for GLI1/OCT4, siRNA knockdown, rescue with WT vs CD44-binding-domain-deleted SPP1\",\n      \"pmids\": [\"28030801\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether GLI1/OCT4 regulation is tissue-specific or general not tested\", \"Structural basis of SPP1 CD44-binding domain not defined\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Two concurrent studies established SPP1 as both a macrophage chemokine in glioblastoma (acting via αvβ5 to maintain M2 polarization) and an immune checkpoint suppressing CD8⁺ T-cell activation via CD44 (regulated by IRF8 at the Spp1 promoter), unifying SPP1's roles in tumor immune evasion.\",\n      \"evidence\": \"CRISPR/siRNA/shRNA knockdown, OPN-deficient mice, macrophage polarization assays; IRF8-knockout mice, ChIP for IRF8-Spp1 promoter, T-cell activation assays\",\n      \"pmids\": [\"30307407\", \"30395540\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative importance of integrin vs CD44 axis in immune suppression across tumor types not resolved\", \"Whether IRF8-mediated Spp1 repression occurs in non-myeloid cells unknown\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Multiple studies converged on SPP1 signaling through CD44 and integrins to activate PI3K/AKT, ERK, and Wnt/β-catenin pathways in diverse contexts including chondrocytes, endothelial progenitors, and gastric cancer, broadening the downstream effector landscape.\",\n      \"evidence\": \"Receptor-blocking antibodies, ERK/PI3K inhibitors, Wnt reporter assays, focal adhesion imaging across multiple cell types\",\n      \"pmids\": [\"36034459\", \"35005113\", \"31990676\", \"34367279\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Which downstream pathway is dominant in a given tissue context remains unclear\", \"Direct biochemical link between SPP1-receptor binding and specific kinase activation not reconstituted\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"N-glycosylation at Asn79 was shown to be required for SPP1's osteoclast proliferative activity via NF-κB, while miR-433 was identified as a direct post-transcriptional regulator of SPP1 mRNA, expanding the known post-translational and post-transcriptional regulatory layers.\",\n      \"evidence\": \"N79Q site-directed mutagenesis with proliferation assay; dual luciferase reporter assay confirming miR-433 binding to SPP1 3′-UTR\",\n      \"pmids\": [\"35451542\", \"34306197\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Full glycosylation map and functional consequences of each site not established\", \"In vivo relevance of miR-433 regulation of SPP1 not tested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"CXCL4 was identified as the driver of profibrotic SPP1⁺ macrophage differentiation, with CXCL4-knockout abolishing this population and ameliorating fibrosis in heart and kidney injury, placing SPP1⁺ macrophages as a central cellular effector of organ fibrosis.\",\n      \"evidence\": \"Cxcl4-knockout mice in cardiac and renal injury models, single-nuclear RNA-seq, in vitro macrophage differentiation\",\n      \"pmids\": [\"36807143\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether CXCL4 acts directly on macrophages or via intermediate cells not fully resolved\", \"SPP1-independent functions of SPP1⁺ macrophages not dissected\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Macrophage-derived SPP1 was shown to protect from NASH by inducing oncostatin-M and activating hepatocyte STAT3/ARG2 to enhance fatty acid oxidation, revealing a tissue-protective metabolic function distinct from SPP1's pro-fibrotic roles.\",\n      \"evidence\": \"Conditional Spp1 knockin and myeloid-specific knockout mice on high-fat diet, OSM/STAT3/ARG2 pathway dissection\",\n      \"pmids\": [\"37028770\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether hepatoprotective vs profibrotic SPP1 functions are dose-dependent or context-specific not resolved\", \"Receptor mediating SPP1-induced OSM production in macrophages not identified\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Genetic epistasis in pancreatic cancer models revealed an intercellular SPP1–CD61–GREM1–BMP loop in which epithelial-derived SPP1 binds CD61 on mesenchymal cells to induce GREM1, which reciprocally sustains SPP1 expression and mesenchymal fate, with Spp1 inactivation abolishing metastasis.\",\n      \"evidence\": \"Spp1 and Grem1 single/double knockout PDAC mouse models, proteomic plasma analysis, heterozygosity rescue\",\n      \"pmids\": [\"40993391\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the SPP1-GREM1 loop operates in non-pancreatic cancers unknown\", \"Direct structural basis of SPP1-CD61 binding not determined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the structural basis of SPP1's multivalent receptor engagement as a disordered protein, how phosphorylation stoichiometry quantitatively tunes integrin vs CD44 selectivity in vivo, and what determines whether SPP1⁺ macrophages adopt profibrotic versus tissue-protective functions in different organs.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No high-resolution structure of SPP1 in complex with any receptor\", \"Quantitative phosphorylation-receptor selectivity relationship undefined\", \"Tissue-specific determinants of SPP1⁺ macrophage functional polarity unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098631\", \"supporting_discovery_ids\": [1, 2, 22]},\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [0, 3, 13, 14, 33]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [3, 12, 28]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [0, 1, 9, 40, 33]},\n      {\"term_id\": \"GO:0031012\", \"supporting_discovery_ids\": [6, 46]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [3, 5, 12, 13, 48]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 11, 18, 20, 30, 33]},\n      {\"term_id\": \"R-HSA-1474244\", \"supporting_discovery_ids\": [38, 27]},\n      {\"term_id\": \"R-HSA-1500931\", \"supporting_discovery_ids\": [0, 14, 46]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [7, 24, 33, 48]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"CD44\",\n      \"ITGAV\",\n      \"ITGB3\",\n      \"ITGA9\",\n      \"ITGB5\",\n      \"ITGB1\",\n      \"FAM20C\",\n      \"GREM1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}