{"gene":"FSTL1","run_date":"2026-06-09T23:54:44","timeline":{"discoveries":[{"year":2010,"finding":"DIP2A was identified as a receptor for FSTL1 on endothelial cells. Co-immunoprecipitation demonstrated direct physical interaction between FSTL1 and DIP2A. Knockdown of DIP2A by siRNA reduced FSTL1 binding to cells, diminished FSTL1-stimulated endothelial cell survival, migration, and tube formation, and inhibited FSTL1-induced Akt phosphorylation. In cardiac myocytes, DIP2A ablation reduced FSTL1-mediated protection against hypoxia/reoxygenation-induced apoptosis and suppressed FSTL1-induced Akt phosphorylation.","method":"Co-immunoprecipitation, siRNA knockdown, cell survival/migration/differentiation assays, Western blot for Akt phosphorylation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP establishing direct interaction, siRNA knockdown with multiple orthogonal functional readouts (survival, migration, tube formation, Akt phosphorylation) in two cell types","pmids":["20054002"],"is_preprint":false},{"year":2011,"finding":"Fstl1 directly interacts with BMP4 and negatively regulates BMP4/Smad1/5/8 signaling during lung development. Fstl1-deficient mice showed elevated pSmad1/5/8 activity, impaired alveolar epithelial differentiation, and insufficient surfactant production. Reducing BMP signaling with Noggin rescued pulmonary atelectasis in Fstl1-deficient mice, placing Fstl1 as a BMP4 antagonist in lung morphogenesis.","method":"Genetic knockout mouse model, direct binding assay (Fstl1-BMP4 interaction), Smad1/5/8 phosphorylation assays, Noggin rescue epistasis experiment, in vitro BMP4-induced surfactant gene expression assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — direct binding demonstrated in vitro, genetic epistasis rescue with Noggin, multiple orthogonal methods in vivo and in vitro","pmids":["21482757"],"is_preprint":false},{"year":2012,"finding":"Fstl1 antagonizes BMP signaling in the developing ureter. Fstl1-null mice showed elevated pSmad1/5/8 in ureters. In vitro, Fstl1 was shown to directly bind to ALK6, a BMP receptor specifically expressed in ureteric epithelial cells, providing a mechanism for Fstl1-mediated BMP pathway suppression during ureter development.","method":"Genetic knockout mouse model, in vitro binding assay (Fstl1-ALK6 interaction), pSmad1/5/8 immunostaining","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding shown in vitro plus in vivo genetic KO with signaling readout, single lab","pmids":["22485132"],"is_preprint":false},{"year":2013,"finding":"The miR-198/FSTL1 switch is controlled post-transcriptionally: TGF-β signaling downregulates KSRP (KHSRP), which is required for miR-198 processing from the FSTL1 primary transcript. When KSRP is inhibited, the transcript is redirected toward FSTL1 protein translation. FSTL1 protein expression promotes keratinocyte migration, while miR-198 inhibits migration by targeting DIAPH1, PLAU, and LAMC2.","method":"Human ex vivo organ culture wound model, TGF-β signaling manipulation, KSRP binding/knockdown experiments, luciferase reporter assays, keratinocyte migration assays","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (organ culture, siRNA knockdown, reporter assays, migration assays), mechanistic pathway dissection in human tissue","pmids":["23395958"],"is_preprint":false},{"year":2015,"finding":"Epicardial FSTL1 protein promotes cardiomyocyte cell cycle entry and division. Epicardial FSTL1 declines after myocardial infarction and is replaced by myocardial expression. Myocardial FSTL1 does not promote regeneration. Application of human FSTL1 protein via an epicardial patch stimulates cardiomyocyte proliferation and improves cardiac function in mouse and swine MI models.","method":"Epicardial patch delivery of recombinant human FSTL1 protein, cardiomyocyte cell cycle entry and division assays, mouse and swine MI models, transgenic FSTL1 overexpression","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — protein reconstitution via epicardial patch, replicated in two animal species (mouse and swine), multiple functional readouts including cell division","pmids":["26375005"],"is_preprint":false},{"year":2006,"finding":"MyoD directly activates expression of miR-206, which targets sequences in the Fstl1 3'UTR and is sufficient to suppress Fstl1 expression during skeletal muscle differentiation.","method":"Fibroblast-to-myoblast conversion by MyoD overexpression, luciferase reporter assays with Fstl1 3'UTR, miR-206 gain-of-function experiments","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct reporter assay establishing miR-206 targeting of Fstl1 3'UTR, functional sufficiency demonstrated, mechanistically placed downstream of MyoD","pmids":["17030984"],"is_preprint":false},{"year":2017,"finding":"TGF-β1 upregulates Fstl1 expression in lung fibroblasts via the Smad3-c-Jun pathway. While TGF-β1 activates Smad, MAPK, and Akt pathways, only Smad2/3 inhibition eliminated TGF-β1-induced Fstl1 expression. A functional c-Jun transcription site in the Fstl1 promoter was identified by luciferase reporter analysis.","method":"Mouse pulmonary fibroblast cultures, pharmacological pathway inhibitors, luciferase reporter assays with Fstl1 promoter constructs, qRT-PCR and Western blot","journal":"American journal of physiology. Lung cellular and molecular physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — promoter reporter assay plus pathway inhibitor panel, single lab, multiple orthogonal methods","pmids":["28495857"],"is_preprint":false},{"year":2018,"finding":"Fstl1 promotes temozolomide resistance in glioblastoma by competitively binding DIP2A and blocking DIP2A nuclear translocation. DIP2A normally cooperates with the HDAC2-DMAP1 complex to enhance H3K9Ac deacetylation and prevent MGMT transcription, increasing temozolomide sensitivity. FSTL1 binding to DIP2A prevents this, leading to increased promoter H3K9Ac and MGMT expression. DIP2A depletion abolished the effects of Fstl1 on MGMT expression and temozolomide resistance.","method":"Co-immunoprecipitation (Fstl1-DIP2A interaction), siRNA knockdown, chromatin immunoprecipitation (H3K9Ac), gene expression assays, in vivo xenograft models","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP establishing direct interaction, epistasis via DIP2A depletion, chromatin mark measurement, in vitro and in vivo validation with multiple orthogonal methods","pmids":["30542120"],"is_preprint":false},{"year":2019,"finding":"Crystal structure of the FK domain of murine Fstl1 was solved at high resolution, revealing that the FK domain forms a stable dimer in both solution and crystal. The FK domain was found to be indispensable for proper Fstl1 function during TGF-β signaling transduction. The potential for Fstl1 to function as a dimer during interaction with TGF-β (which itself forms dimers) was proposed based on structural data.","method":"X-ray crystallography of FK domain, solution studies (dimerization), functional assays of FK domain mutants in TGF-β signaling","journal":"Protein science : a publication of the Protein Society","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure solved plus functional validation of FK domain in TGF-β signaling, single lab","pmids":["31351024"],"is_preprint":false},{"year":2022,"finding":"Macrophage FSTL1 promotes liver fibrosis by binding directly to PKM2 via its FK domain, promoting PKM2 phosphorylation and nuclear translocation, reducing PKM2 ubiquitination, enhancing PKM2-dependent glycolysis, and increasing M1 macrophage polarization via NF-κB pathway activation. Myeloid-specific FSTL1 knockout attenuated liver fibrosis and reduced M1 polarization.","method":"Myeloid-specific FSTL1 knockout mice, Co-IP (FSTL1-PKM2 direct binding via FK domain), Western blot for PKM2 phosphorylation and nuclear translocation, ubiquitination assays, glycolysis measurements, in vitro macrophage polarization assays, pharmacological PKM2 activator (DASA-58)","journal":"Gut","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — direct binding via Co-IP, domain-specific interaction identified, myeloid-specific KO with clear phenotype, multiple orthogonal mechanistic readouts, pharmacological rescue","pmids":["35140065"],"is_preprint":false},{"year":2022,"finding":"FSTL1 interacts with Wnt ligands (specifically Wnt3a) and Frizzled receptors (specifically FZD4) but not with the co-receptor LRP6. FSTL1 interacts with Wnt3a through its extracellular calcium-binding (EC) domain and VWC domain, and with FZD4 through its EC domain. FSTL1 increased the association of Wnt3a with FZD4 and thereby enhanced Wnt/β-catenin signaling and fibrogenesis in obstructed kidneys.","method":"Co-immunoprecipitation (FSTL1-Wnt3a, FSTL1-FZD4 interactions), domain deletion analysis, FSTL1 overexpression/inhibition in obstructed mouse kidneys, Wnt/β-catenin reporter assays, single-cell RNA-Seq for expression localization","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP with domain mapping, in vivo genetic models, multiple signaling pathway readouts, single lab with multiple orthogonal methods","pmids":["35525270"],"is_preprint":false},{"year":2021,"finding":"FSTL1 secreted by activated fibroblasts binds to TLR4 on hepatocellular carcinoma cells, resulting in activation of AKT/mTOR/4EBP1 signaling, promoting HCC growth, metastasis, and maintenance of tumor-initiating cells.","method":"Recombinant FSTL1 treatment of HCC cells and 3D organoids, receptor binding assay (TLR4 identified as receptor), Western blot for AKT/mTOR/4EBP1 signaling, conditioned medium experiments, preclinical mouse models with FSTL1 blockade","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — receptor identification with functional downstream signaling, in vivo models, single lab","pmids":["34551961"],"is_preprint":false},{"year":2020,"finding":"Dynamic resistance exercise stimulates skeletal muscle FSTL1 secretion. FSTL1 binds receptor DIP2A on endothelial cells and activates Smad2/3 signaling to promote cardiac angiogenesis. TGFβR1 inhibitor reduced pSmad2/3 and VEGF-A expression but did not affect FSTL1-DIP2A direct Smad2/3 activation, demonstrating that the FSTL1-DIP2A-Smad2/3 axis is independent of TGFβR1.","method":"Rat MI model with resistance exercise, AAV-FSTL1 injection, recombinant FSTL1 treatment of HUVECs, TGFβR1 inhibitor pharmacological epistasis, Western blot for DIP2A and pSmad2/3, immunofluorescence, tubule assay","journal":"Journal of sport and health science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological epistasis with TGFβR1 inhibitor defining pathway independence, multiple in vitro and in vivo methods, single lab","pmids":["33246164"],"is_preprint":false},{"year":2018,"finding":"FSTL1 promotes cardiac angiogenesis and myocardial energy substrate metabolism normalization in heart failure via AMPK activation. FSTL1 stimulated oxygen consumption through AMPK activation in primary cardiac and skeletal muscle myocytes in vitro.","method":"Conscious dog HF model with acute and chronic FSTL1 infusion, radiolabeled substrate tracking (3H-oleate, 14C-glucose), in vitro primary myocyte assays with AMPK pathway analysis","journal":"Circulation. Heart failure","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo infusion with metabolic substrate tracking plus in vitro AMPK mechanistic validation, single lab, large animal model","pmids":["29317401"],"is_preprint":false},{"year":2017,"finding":"In glioblastoma, Fstl1 interacts with BMP4 but not with BMPR2, competitively inhibiting BMP4-BMPR2 association. Fstl1 overexpression suppressed BMP4/Smad1/5/8 signaling pathway activation, promoting glioma cell proliferation, while BMP4 overexpression reversed this effect.","method":"Co-immunoprecipitation (Fstl1-BMP4 and Fstl1-BMPR2 interactions), Western blot for pSmad1/5/8, cell proliferation and colony formation assays, orthotopic xenograft, BMP4 rescue experiment","journal":"Cellular physiology and biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP showing selective binding to BMP4 but not BMPR2, BMP4 overexpression rescue epistasis, multiple functional readouts, single lab","pmids":["29212066"],"is_preprint":false},{"year":2017,"finding":"FSTL1 blocks Wnt7a-mediated repression of ERK phosphorylation, enabling MMP9 production that degrades the extracellular matrix and facilitates metastasis. Separately, EGF hijacks the miR-198/FSTL1 switch to sustain FSTL1 translation, driving metastasis through parallel DIAPH1 and FSTL1 pathways.","method":"Head and neck squamous cell carcinoma cell lines, Wnt7a pathway manipulation, ERK phosphorylation assays, MMP9 expression and activity assays, FSTL1 gain/loss-of-function, migration/invasion assays","journal":"The Journal of experimental medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional pathway experiments with signaling readouts, single lab, multiple cell line models","pmids":["28827448"],"is_preprint":false},{"year":2017,"finding":"Macrophage-derived Fstl1 induces oncostatin M (OSM) expression, promoting asthmatic airway remodeling. Macrophage-specific Fstl1 knockout (Lys-Cre/Fstl1Δ/Δ) reduced airway remodeling and OSM levels. Exogenous Fstl1 induced airway remodeling and increased OSM, while anti-OSM antibody blocked Fstl1-induced remodeling, eosinophilic inflammation, and airway hyperresponsiveness.","method":"Macrophage-specific conditional Fstl1 knockout mice, allergen challenge model, recombinant Fstl1 administration, anti-OSM antibody blockade, airway remodeling and inflammation readouts","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — macrophage-specific conditional KO with clear phenotype, exogenous protein rescue, antibody blockade epistasis, multiple readouts identifying Fstl1→OSM pathway","pmids":["26355153"],"is_preprint":false},{"year":2017,"finding":"Knockdown of Fstl1 in hepatic stellate cells suppressed proliferation and reduced α-SMA and collagen I expression in TGF-β1-treated HSCs. Mechanistically, Fstl1 knockdown decreased Smad3 phosphorylation in TGF-β1-induced HSCs, placing Fstl1 as a positive regulator of TGF-β1/Smad3 signaling in liver fibrosis.","method":"siRNA knockdown of Fstl1 in hepatic stellate cells, Western blot for pSmad3, α-SMA, collagen I, cell proliferation assay","journal":"Molecular medicine reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with defined signaling readout (pSmad3), single lab, single method","pmids":["28901425"],"is_preprint":false},{"year":2017,"finding":"Fstl1 is essential for lung airway and vascular smooth muscle formation. Fstl1 was localized to lung smooth muscle cells. Fstl1 knockout impaired airway smooth muscle differentiation, associated with decreased myocardin/SRF transcription factors. Fstl1 knockout also caused hyperplasia of pulmonary artery vascular smooth muscle.","method":"Fstl1-lacZ reporter mouse, Fstl1 knockout allele, histological analysis of trachea/bronchi/pulmonary artery, immunostaining for myocardin/SRF","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with cell-type-specific localization and transcription factor readouts, single lab","pmids":["28574994"],"is_preprint":false},{"year":2017,"finding":"Fstl1 deletion from the endocardial/endothelial lineage (Tie2-Cre) causes sustained BMP and TGFβ signaling after birth, resulting in ongoing endocardial-to-mesenchymal transition, deformed mitral valves, cardiac hypertrophy, and heart failure. This shows that endocardial FSTL1 normally restrains BMP/TGFβ signaling to maintain valve homeostasis.","method":"Conditional knockout (Tie2-Cre; Fstl1 flox), echocardiography, electrocardiography, histology, BMP/TGFβ signaling markers","journal":"Arteriosclerosis, thrombosis, and vascular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — lineage-specific conditional KO with clear cardiac phenotype and signaling pathway readouts, single lab","pmids":["28705792"],"is_preprint":false},{"year":2020,"finding":"Endothelial cell-specific FSTL1 knockout led to increased pSMAD3 in vascular mural cells colocalizing with αSMA in vein walls, increased collagen deposition, and cardiac/vascular fibrosis. TGFβ pathway inhibitor treatment reduced the αSMA abnormalities, demonstrating that endothelial FSTL1 normally suppresses TGFβ/SMAD3 signaling in vascular mural cells.","method":"Conditional endothelial FSTL1 knockout mouse (vs. smooth muscle and hematopoietic cell KOs as controls), pSMAD3 immunostaining, TGFβ inhibitor rescue, collagen deposition assay","journal":"Arteriosclerosis, thrombosis, and vascular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cell-type-specific conditional KO with multiple controls, pharmacological rescue of signaling phenotype, single lab","pmids":["32078339"],"is_preprint":false},{"year":2023,"finding":"Skeletal muscle IRF4 transcriptionally regulates FSTL1 (dual luciferase assay confirmed IRF4 binding to FSTL1 promoter). FSTL1 mediates inter-organ crosstalk between skeletal muscle and the liver in NASH via different receptors (DIP2A and CD14) on different liver cell types. Restoring FSTL1 in muscle of F4MKO mice was sufficient to restore liver pathology.","method":"Muscle-specific IRF4 knockout mice, proteomics, dual luciferase reporter assay (IRF4-FSTL1 promoter), co-culture experiments with liver cells, AAV-mediated FSTL1 rescue","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — dual luciferase assay for transcriptional regulation, muscle-specific KO with rescue experiment, receptor identification by co-culture, single lab","pmids":["37770480"],"is_preprint":false},{"year":2019,"finding":"FSTL1 directly increases expression of MMP-1, MMP-13, iNOS, COX-2, IL-1β, TNF-α, and IL-6 in chondrocytes in a dose-dependent manner, and activates NF-κB and promotes p65 phosphorylation, establishing NF-κB as the signaling pathway mediating FSTL1 pro-inflammatory effects in chondrocytes.","method":"Recombinant FSTL1 treatment of rat chondrocytes, PCR, ELISA, Western blot for NF-κB pathway components (p65 phosphorylation)","journal":"Journal of cellular and molecular medicine","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct protein treatment with dose-response and multiple signaling readouts, single lab, no pathway inhibitor rescue","pmids":["30644158"],"is_preprint":false},{"year":2020,"finding":"FSTL1 promotes nitric oxide-induced chondrocyte apoptosis by activating the SAPK/JNK/Caspase3 signaling pathway. FSTL1 overexpression increased apoptosis in SNP-treated chondrocytes, upregulated MMP1/3/9 and Bax, and reduced Bcl-2 and collagen. The caspase inhibitor Ac-DEVD-FMK impaired FSTL1-induced chondrocyte apoptosis.","method":"FSTL1 overexpression plasmid transfection in chondrocytes, flow cytometry for apoptosis, Western blot for SAPK/JNK/Caspase3 pathway, caspase inhibitor rescue","journal":"Gene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — caspase inhibitor rescue confirms pathway, multiple readouts, single lab","pmids":["31927008"],"is_preprint":false},{"year":2017,"finding":"FSTL1 knockdown in airway smooth muscle cells inhibited PDGF-BB-induced proliferation, arrested cell cycle at G2/M, and reduced migration. Mechanistically, FSTL1 knockdown downregulated PDGF-BB-induced phosphorylation of ERK and AKT in ASM cells.","method":"siRNA knockdown of FSTL1 in human ASM cells, cell proliferation assay, cell cycle analysis by flow cytometry, migration assay, Western blot for p-ERK and p-AKT","journal":"Molecular medicine reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with defined signaling readouts (ERK and AKT phosphorylation), single lab","pmids":["28393245"],"is_preprint":false},{"year":2017,"finding":"FSTL1 activates Wnt/β-catenin signaling through integrin β3 in breast cancer cells, promoting stemness and chemoresistance. Luciferase assays demonstrated that miR-137 reduces FSTL1 mRNA and protein levels, identifying FSTL1 as a direct miR-137 target.","method":"TOP/FOP flash Wnt reporter assay, colony and tumor sphere formation, luciferase assay for miR-137 targeting of FSTL1, Western blot for pathway components","journal":"Cancer biology & therapy","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — reporter assays for both Wnt signaling and miRNA targeting, functional rescue experiments, single lab","pmids":["30336071"],"is_preprint":false},{"year":2015,"finding":"Fstl1 is expressed in brain by pia mater but not in the ventricular zone; FSTL1 from pia mater is required for radial glial cell morphology. Conditional Fstl1 ablation in both expression domains disrupted RGC basal process organization and caused mislocalization of upper-layer projection neurons. VZ-only Fstl1 deletion did not affect RGC morphology. BMP, AKT/PKB, Cdc42, GSK3β, integrin and reelin signaling were unchanged, indicating a unique mechanism.","method":"Conditional Fstl1 knockout mice (EIIa-Cre and Emx1-IREScre lines), cortical histology, immunostaining for RGC markers and signaling pathway components","journal":"Molecular brain","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two conditional KO lines with spatial dissection of FSTL1 source, genetic negative results for known pathways, single lab","pmids":["26382033"],"is_preprint":false},{"year":2025,"finding":"Host FSTL1 enhances rapid recycling of CCR2 to the plasma membrane via activation of the CD14/TLR4/NF-κB/ATP6V1G2 axis, leading to early recruitment of Ly6C+ monocytes/macrophages. This early inflammatory macrophage recruitment is required for MSC-mediated antifibrotic effects in liver cirrhosis.","method":"Fstl1-deficient mice, recombinant FSTL1 rescue, mechanistic dissection of CD14/TLR4/NF-κB/ATP6V1G2 pathway, CCR2 membrane recycling assays, macrophage depletion/tracking experiments, MSC infusion models","journal":"Signal transduction and targeted therapy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with recombinant protein rescue, mechanistic pathway dissection with multiple components, single lab","pmids":["40050288"],"is_preprint":false},{"year":2019,"finding":"FSTL1 promotes alveolar epithelial cell senescence by enhancing TGF-β1 signaling, and this enhancement is dependent on SENP1-mediated deSUMOylation. TGF-β1-induced FSTL1 upregulates SENP1 expression in senescent AECs, and interfering with SENP1 inhibited FSTL1-dependent promotion of AEC senescence and improved pulmonary fibrosis.","method":"TGF-β1 treatment of AECs, SENP1 siRNA knockdown, senescence assays, Western blot for FSTL1 and SENP1, in vivo bleomycin mouse model","journal":"Cell biology international","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistasis via SENP1 siRNA knockdown with defined senescence phenotype, in vitro and in vivo, single lab","pmids":["37369969"],"is_preprint":false},{"year":2021,"finding":"FSTL1 activates the NLRP3/IL-1β signaling pathway in macrophages, contributing to asthmatic airway inflammation. Pretreatment with MCC950 (NLRP3 inhibitor) significantly reduced NLRP3 and IL-1β production induced by FSTL1 in mice and in alveolar macrophage MH-S cells.","method":"Fstl1 heterozygous knockout mice, OVA asthma model, recombinant FSTL1 injection, MCC950 pharmacological inhibitor, siFSTL1, Western blot and ELISA for NLRP3/IL-1β","journal":"Inflammation research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological rescue with MCC950, genetic heterozygous KO, recombinant protein gain-of-function, multiple readouts, single lab","pmids":["34076707"],"is_preprint":false},{"year":2020,"finding":"FSTL-1 attenuation in hypomorphic mice causes spontaneous emphysema independent of smoke. Recombinant FSTL-1 treatment of macrophages attenuated NF-κB p65 phosphorylation in an Nr4a1-dependent manner, identifying a FSTL-1→Nr4a1→NF-κB signaling axis in lung macrophage immune tolerance.","method":"FSTL-1 hypomorphic mice, lung morphometry and pulmonary function, RNA-seq identifying Nr4a1, in vitro macrophage recombinant FSTL-1 treatment with NF-κB p65 phosphorylation assay, Nr4a1 dependence testing","journal":"American journal of respiratory and critical care medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNA-seq pathway discovery plus in vitro mechanistic validation with Nr4a1 dependence, hypomorphic mouse model, single lab","pmids":["31834999"],"is_preprint":false},{"year":2013,"finding":"FSTL1 secreted by Snail-positive tumor cells promotes bone metastasis through two mechanisms: direct mediation of tumor cell invasion and bone tropism, and expansion of CD45−ALCAM+ pluripotent mesenchymal stem-like cells from bone marrow, which both directly induce bone metastasis and generate CD8low T cells with weak CTL activity. RNAi-mediated FSTL1 attenuation prevented bone metastasis and reversed these immune dysfunctions.","method":"RNAi knockdown of FSTL1 in tumor cells, flow cytometry for ALCAM+ cell expansion and CD8 T cell characterization, in vivo bone metastasis models, in vitro CTL assays","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNAi loss-of-function with mechanistic cellular readouts (ALCAM+ expansion, CD8 phenotyping), in vivo and in vitro, single lab","pmids":["23966294"],"is_preprint":false},{"year":2018,"finding":"FSTL1 interacts with VIM (vimentin) in colorectal cancer cells. This interaction was identified by co-immunoprecipitation and mediates FSTL1's role in activating focal adhesion signaling and cytoskeleton rearrangement to promote CRC metastasis. TGFβ1-Smad2/3 signaling (via Smad3 transcription factor) was identified as an upstream regulator of FSTL1 protein expression.","method":"Co-immunoprecipitation (FSTL1-VIM), focal adhesion signaling pathway analysis, Smad3 ChIP/reporter for FSTL1 transcriptional regulation, in vitro migration/invasion assays, in vivo liver metastasis model","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP identifying VIM interaction, pathway analysis, in vivo metastasis model, single lab","pmids":["29844309"],"is_preprint":false},{"year":2017,"finding":"Fstl1 promotes glioma stem cell self-renewal through autocrine FSTL1 interacting with TLR2, which inhibits EGFR endocytosis-lysosomal degradation, resulting in activation of the PI3K-AKT signaling pathway. FSTL1 also promotes M2 macrophage polarization via TLR2 signaling.","method":"FSTL1 knockout in GSC cell lines, TLR2 receptor identification, EGFR endocytosis assays, PI3K-AKT pathway Western blot, mouse GBM models, macrophage polarization assays","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — receptor identification (TLR2) with downstream mechanistic dissection (EGFR endocytosis, PI3K-AKT), FSTL1 KO with rescue, in vivo model, single lab","pmids":["39722404"],"is_preprint":false},{"year":2021,"finding":"FSTL1 promotes myocardial fibrosis via a USP10/Notch1 signaling axis. FSTL1 activation of USP10 (a Notch1 deubiquitinase) stabilizes NICD1 (Notch1 intracellular domain), which suppresses myocardial fibrosis. Pharmacological inhibition of USP10 (spautin-1) or Notch signaling (LY3039478) abolished the protective effects of FSTL1 in diabetic MI mice.","method":"AAV9-FSTL1 intracardiac delivery in T2DM-MI mice, USP10 inhibitor (spautin-1) and Notch inhibitor (LY3039478) epistasis experiments, cardiac fibrosis markers, Western blot for USP10/Notch1/NICD1","journal":"Frontiers in cell and developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological epistasis with two inhibitors defining pathway, in vivo gene delivery, defined molecular readouts, single lab","pmids":["34957094"],"is_preprint":false},{"year":2023,"finding":"FSTL1 initiates angiogenesis in endothelial cells by opening intercellular junctions via activation of the Src kinase pathway. FSTL1 increased Src phosphorylation and VEGFR2 phosphorylation, decreased VE-Cadherin, Occludin, Connexin-43, and Claudin-5 expression, and increased endothelial permeability. Src inhibitor (but not VEGFR2 inhibitor) blocked FSTL1-induced effects. H2S upregulated FSTL1 in skeletal muscle by increasing HuR levels, which stabilized FSTL1 transcript.","method":"Recombinant FSTL1 treatment of HUVECs, Src and VEGFR2 pharmacological inhibitors, immunostaining for junction proteins, wound-healing migration assay, permeability assay, HuR siRNA knockdown, mouse hindlimb ischemia model","journal":"American journal of physiology. Cell physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological epistasis distinguishing Src vs VEGFR2, multiple junction protein readouts, HuR mechanism for FSTL1 regulation, in vitro and in vivo, single lab","pmids":["37694287"],"is_preprint":false},{"year":2019,"finding":"miR-29a in mesenchymal stem cells suppresses FSTL1 expression and secretion (validated by dual luciferase reporter assay). This reduces FSTL1 in conditioned medium, which in turn inhibits the JAK2/STAT3 pathway in cardiac myocytes and promotes myocyte apoptosis after hypoxia-reoxygenation injury.","method":"Dual luciferase reporter assay (miR-29a targeting FSTL1), miR-29a overexpression in MSCs, FSTL1 measurement by ELISA in conditioned medium, JAK2/STAT3 Western blot in H9c2 cells, flow cytometry for apoptosis","journal":"Cardiovascular pathology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — luciferase reporter validates direct miRNA-mRNA interaction, conditioned medium experiment links MSC FSTL1 secretion to cardiomyocyte JAK2/STAT3, single lab","pmids":["31945680"],"is_preprint":false},{"year":2024,"finding":"FSTL1 promotes nucleus pulposus cell senescence via TLR4/NF-κB signaling. Recombinant FSTL1 upregulated p16 and p21, increased SA-β-gal-positive cells, induced SASP, and disrupted ECM balance. TLR4 inhibition partly reversed these effects. FSTL1 siRNA in a rabbit puncture IVDD model reduced disc degeneration.","method":"Recombinant FSTL1 treatment of NPCs, TLR4 inhibitor rescue, FSTL1 siRNA in vitro and in vivo (rabbit IVDD model), senescence assays (SA-β-gal, p16/p21), Western blot for TLR4/NF-κB","journal":"Inflammation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — TLR4 inhibitor epistasis, in vitro and in vivo (rabbit model) with multiple senescence readouts, single lab","pmids":["38316670"],"is_preprint":false},{"year":2017,"finding":"BBS4 regulates FSTL1 mRNA levels and also independently modulates FSTL1 secretion. FSTL1 functions as a novel regulator of ciliogenesis, creating a regulatory loop between FSTL1 and cilia. BBS4, cilia, and FSTL1 are coordinated during 3T3-L1 differentiation, with FSTL1 influencing this process at least partly by modulating ciliogenesis.","method":"BBS4 knockdown/knockout in cells, FSTL1 mRNA quantification, FSTL1 secretion assay, ciliogenesis assays (cilia length/frequency), 3T3-L1 differentiation with FSTL1 manipulation","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — BBS4-FSTL1 functional interaction established through loss-of-function, bidirectional FSTL1-cilia regulatory loop demonstrated, single lab","pmids":["28852127"],"is_preprint":false},{"year":2010,"finding":"Fstl1 is a 'preadipokine' that is highly expressed in 3T3-L1 preadipocytes and dramatically downregulated early in differentiation to adipocytes. The Fstl1 protein is secreted by preadipocytes. Negative transcriptional regulation of Fstl1 is mediated by Kruppel-like factor 15 (KLF15), as identified by luciferase reporter assays with Fstl1 5' flanking region constructs.","method":"Northern blot, Western blot of conditioned media, luciferase reporter assays with Fstl1 5' flanking region constructs, KLF15 expression, multiple adipogenesis model time courses","journal":"Mechanisms of development","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — luciferase reporter identifies KLF15 as negative transcriptional regulator, protein secretion confirmed, multiple adipogenesis models, single lab","pmids":["20043993"],"is_preprint":false}],"current_model":"FSTL1 is a secreted, glycosylated matricellular protein that acts as a context-dependent extracellular signaling molecule: it antagonizes BMP signaling by directly binding BMP4 (via its FK domain, which forms a stable dimer) and blocking BMP receptor engagement; activates protective cardiovascular signaling through its receptor DIP2A, triggering Smad2/3 and Akt/AMPK pathways to promote cardiomyocyte proliferation, angiogenesis, and metabolic normalization; promotes inflammation and fibrosis through TLR4/NF-κB, NLRP3/IL-1β, and TGF-β/Smad3 signaling; enhances Wnt/β-catenin signaling by bridging Wnt3a to Frizzled receptors; modulates tumor biology via BMP4 sequestration, integrin β3/Wnt, TLR2/PI3K-AKT, and TLR4/AKT/mTOR pathways; regulates macrophage polarization including through intracellular PKM2 reprogramming; and is transcriptionally induced by TGF-β1 via the Smad3-c-Jun pathway and suppressed by miR-206 (downstream of MyoD) and KLF15."},"narrative":{"mechanistic_narrative":"FSTL1 is a secreted, glycosylated signaling protein that operates as a context-dependent extracellular regulator of BMP, TGF-β, Wnt, and innate-immune signaling, governing tissue morphogenesis, cardiovascular protection, fibrosis, and tumor biology [PMID:21482757, PMID:20054002, PMID:35525270]. In its morphogenetic role, FSTL1 directly binds BMP4 and the BMP receptor ALK6 to antagonize BMP/Smad1/5/8 signaling, a function required for alveolar epithelial differentiation and surfactant production in the lung and for ureter patterning, since Noggin rescues the atelectasis of Fstl1-deficient lungs [PMID:21482757, PMID:22485132]. Its dimerization-competent FK domain is structurally indispensable for engagement of TGF-β-family signaling [PMID:31351024]. On the protective cardiovascular axis, FSTL1 acts through the receptor DIP2A to drive endothelial survival, migration, and tube formation and to protect cardiomyocytes via Akt and a TGFβR1-independent Smad2/3 pathway, while epicardial FSTL1 stimulates cardiomyocyte cell-cycle entry and improves function after myocardial infarction [PMID:20054002, PMID:33246164, PMID:26375005]. FSTL1 also normalizes myocardial energy metabolism through AMPK activation and opens endothelial junctions to initiate angiogenesis via Src/VEGFR2 [PMID:29317401, PMID:37694287]. Paradoxically, FSTL1 is strongly pro-fibrotic and pro-inflammatory in other settings: it potentiates TGF-β1/Smad3 signaling in hepatic stellate cells and alveolar epithelium, bridges Wnt3a to FZD4 through its EC and VWC domains to amplify Wnt/β-catenin-driven renal fibrosis, and engages TLR4/CD14/NF-κB and NLRP3/IL-1β programs that drive macrophage activation, asthmatic airway remodeling, and tissue senescence [PMID:28901425, PMID:35525270, PMID:40050288, PMID:34076707]. Within macrophages it binds PKM2 via its FK domain to reprogram glycolysis and M1 polarization in liver fibrosis [PMID:35140065]. In cancer, FSTL1 promotes glioma proliferation through BMP4 sequestration and temozolomide resistance by blocking DIP2A nuclear translocation and derepressing MGMT, and drives tumor growth and metastasis via TLR4/AKT/mTOR, TLR2/PI3K-AKT, and integrin-β3/Wnt routes [PMID:29212066, PMID:30542120, PMID:34551961, PMID:39722404]. FSTL1 expression is induced by TGF-β1 through Smad3-c-Jun and constrained by miR-206 downstream of MyoD and by KLF15 [PMID:28495857, PMID:17030984, PMID:20043993].","teleology":[{"year":2006,"claim":"Established the first direct transcriptional brake on Fstl1, explaining how its expression is extinguished during a differentiation program.","evidence":"MyoD-driven myoblast conversion with luciferase 3'UTR reporters showing miR-206 targets Fstl1","pmids":["17030984"],"confidence":"High","gaps":["Does not address FSTL1 protein function in muscle","Limited to skeletal muscle context"]},{"year":2010,"claim":"Identified DIP2A as a cell-surface receptor for FSTL1, providing the first molecular handle on its protective signaling and a downstream effector (Akt).","evidence":"Reciprocal Co-IP plus DIP2A siRNA knockdown with survival, migration, tube-formation and Akt readouts in endothelial cells and cardiomyocytes","pmids":["20054002"],"confidence":"High","gaps":["DIP2A signaling mechanism downstream of receptor binding not resolved","How one receptor reconciles protective vs pro-fibrotic FSTL1 outputs unclear"]},{"year":2010,"claim":"Characterized FSTL1 as a secreted preadipokine under negative transcriptional control, broadening its regulatory inputs beyond miRNA.","evidence":"Conditioned-media Western blots and KLF15 luciferase reporter assays across adipogenesis models","pmids":["20043993"],"confidence":"Medium","gaps":["Functional role of FSTL1 in adipocyte biology not defined","Direct KLF15 promoter occupancy not shown"]},{"year":2011,"claim":"Defined FSTL1 as a direct BMP4 antagonist required for lung morphogenesis, establishing its core developmental mechanism via genetic epistasis.","evidence":"Fstl1 knockout mice, in vitro Fstl1-BMP4 binding, pSmad1/5/8 readouts, and Noggin rescue of atelectasis","pmids":["21482757"],"confidence":"High","gaps":["Stoichiometry of FSTL1-BMP4 complex not determined","Whether antagonism is by ligand sequestration or receptor competition not distinguished here"]},{"year":2012,"claim":"Extended BMP antagonism to direct receptor binding, showing FSTL1 can engage a BMP receptor (ALK6) rather than only the ligand.","evidence":"Fstl1-null ureter phenotype with elevated pSmad1/5/8 and in vitro Fstl1-ALK6 binding","pmids":["22485132"],"confidence":"Medium","gaps":["Relative contribution of ligand vs receptor binding to antagonism unresolved","Single-lab finding"]},{"year":2013,"claim":"Revealed a post-transcriptional switch coupling FSTL1 protein production to miR-198, linking TGF-β/KSRP control of a shared transcript to keratinocyte migration.","evidence":"Human ex vivo wound organ culture, KSRP knockdown, luciferase reporters, and migration assays","pmids":["23395958"],"confidence":"High","gaps":["Receptor through which FSTL1 protein drives keratinocyte migration not identified","Generality of the switch beyond skin uncertain"]},{"year":2015,"claim":"Demonstrated that the tissue source of FSTL1 determines its regenerative competence, showing epicardial protein drives cardiomyocyte division.","evidence":"Epicardial patch delivery of recombinant human FSTL1 with cell-cycle and functional readouts in mouse and swine MI","pmids":["26375005"],"confidence":"High","gaps":["Why myocardial FSTL1 lacks regenerative activity (glycosylation? receptor?) not resolved","Receptor mediating cardiomyocyte proliferation not defined here"]},{"year":2017,"claim":"Defined the transcriptional induction arm of FSTL1, placing it downstream of TGF-β1 via Smad3 and c-Jun in fibroblasts.","evidence":"Pathway inhibitor panel and Fstl1 promoter luciferase reporters in lung fibroblasts","pmids":["28495857"],"confidence":"Medium","gaps":["Direct c-Jun promoter occupancy not shown by ChIP here","Single lab"]},{"year":2017,"claim":"Established FSTL1 as a positive amplifier of TGF-β1/Smad3 signaling in fibrosis, contrasting with its BMP-antagonist role.","evidence":"siRNA knockdown in hepatic stellate cells with pSmad3, α-SMA and collagen I readouts","pmids":["28901425"],"confidence":"Medium","gaps":["Mechanism by which FSTL1 enhances Smad3 phosphorylation unknown","No receptor identified in this context"]},{"year":2017,"claim":"Mapped tissue-protective FSTL1 functions in development, including smooth muscle differentiation, valve homeostasis, and cortical radial glia organization, frequently by restraining BMP/TGFβ.","evidence":"Multiple conditional and lineage-specific Fstl1 knockouts with histology and signaling-marker analysis","pmids":["28574994","28705792","26382033"],"confidence":"Medium","gaps":["The BMP/TGFβ-independent cortical mechanism (idx 26) remains undefined","Cell-autonomous vs paracrine contributions not fully separated"]},{"year":2017,"claim":"Identified TLR2 and downstream EGFR/PI3K-AKT as a tumor-promoting FSTL1 axis and linked FSTL1 to macrophage polarization.","evidence":"FSTL1 KO in glioma stem cells, TLR2 receptor identification, EGFR endocytosis and PI3K-AKT assays, in vivo GBM models","pmids":["39722404"],"confidence":"Medium","gaps":["Direct FSTL1-TLR2 binding not biochemically confirmed here","Single lab"]},{"year":2017,"claim":"Demonstrated FSTL1 promotes pro-inflammatory and remodeling programs, including a macrophage Fstl1→OSM axis in airway disease.","evidence":"Macrophage-specific conditional Fstl1 knockout, recombinant protein rescue, and anti-OSM antibody blockade in allergen challenge","pmids":["26355153"],"confidence":"High","gaps":["Receptor on macrophages driving OSM induction not identified here","Link to systemic inflammation untested"]},{"year":2018,"claim":"Uncovered an intracellular/chromatin mechanism in cancer: FSTL1 blocks DIP2A nuclear translocation to derepress MGMT and confer chemoresistance.","evidence":"Co-IP, DIP2A depletion epistasis, H3K9Ac ChIP, and xenograft models in glioblastoma","pmids":["30542120"],"confidence":"High","gaps":["How secreted FSTL1 accesses cytoplasmic DIP2A trafficking not resolved","Generalizability to other DIP2A-dependent tumors unknown"]},{"year":2018,"claim":"Linked FSTL1 to cytoskeletal and metastatic machinery via a vimentin interaction, with Smad3-driven transcriptional control upstream.","evidence":"Co-IP of FSTL1-VIM, focal adhesion pathway analysis, and liver metastasis model in colorectal cancer","pmids":["29844309"],"confidence":"Medium","gaps":["Whether FSTL1-VIM interaction is intracellular or extracellular not clarified","Reciprocal validation limited"]},{"year":2019,"claim":"Provided the structural basis for FSTL1 function, showing the FK domain dimerizes and is required for TGF-β signaling transduction.","evidence":"X-ray crystallography of the murine FK domain plus functional FK mutant assays","pmids":["31351024"],"confidence":"High","gaps":["No co-structure with any ligand or receptor","Functional consequence of dimerization for ligand engagement inferred, not directly shown"]},{"year":2019,"claim":"Established direct pro-inflammatory and pro-apoptotic FSTL1 signaling in chondrocytes via NF-κB and SAPK/JNK/Caspase3.","evidence":"Recombinant FSTL1 dose-response, p65 phosphorylation, and caspase inhibitor rescue in chondrocytes","pmids":["30644158","31927008"],"confidence":"Medium","gaps":["Receptor mediating chondrocyte responses not identified","No NF-κB inhibitor rescue in idx 22"]},{"year":2020,"claim":"Resolved a TGFβR1-independent DIP2A-Smad2/3 angiogenic axis and linked it to exercise-induced muscle FSTL1 secretion.","evidence":"Rat MI exercise model, AAV-FSTL1, HUVEC assays, and TGFβR1 inhibitor pharmacological epistasis","pmids":["33246164"],"confidence":"Medium","gaps":["How DIP2A activates Smad2/3 without TGFβR1 mechanistically unclear","Single lab"]},{"year":2020,"claim":"Showed endothelial FSTL1 normally suppresses TGFβ/SMAD3 in vascular mural cells, and its loss causes emphysema via a macrophage Nr4a1-NF-κB tolerance axis.","evidence":"Endothelial-specific knockout with TGFβ inhibitor rescue, and hypomorphic mice with RNA-seq-guided Nr4a1 dependence testing","pmids":["32078339","31834999"],"confidence":"Medium","gaps":["Reconciling FSTL1 suppression vs amplification of TGFβ/Smad3 across tissues unresolved","Receptor mediating Nr4a1 induction not defined"]},{"year":2022,"claim":"Defined an intracellular macrophage mechanism in which FSTL1 binds PKM2 via its FK domain to reprogram glycolysis and M1 polarization driving liver fibrosis.","evidence":"Myeloid-specific FSTL1 knockout, Co-IP with FK-domain mapping, ubiquitination/glycolysis assays, and DASA-58 pharmacological rescue","pmids":["35140065"],"confidence":"High","gaps":["How extracellular/secreted FSTL1 reaches cytoplasmic PKM2 not resolved","Whether FK-PKM2 binding occurs intracellularly before secretion unknown"]},{"year":2022,"claim":"Identified FSTL1 as a Wnt co-factor that bridges Wnt3a to FZD4 through defined domains, amplifying Wnt/β-catenin-driven fibrosis.","evidence":"Reciprocal Co-IP with domain-deletion mapping, in vivo obstructed kidney models, and Wnt reporter assays","pmids":["35525270"],"confidence":"High","gaps":["Structural basis of the FSTL1-Wnt3a-FZD4 ternary complex not solved","Whether this mechanism operates in non-renal fibrosis untested here"]},{"year":2021,"claim":"Established TLR4-driven oncogenic and fibrotic FSTL1 signaling through AKT/mTOR and NLRP3/IL-1β programs.","evidence":"Recombinant FSTL1 and TLR4 receptor identification with AKT/mTOR readouts in HCC, plus MCC950 NLRP3 inhibitor epistasis in asthma","pmids":["34551961","34076707"],"confidence":"Medium","gaps":["Direct FSTL1-TLR4 binding affinity/structure not characterized","Cofactor requirements (CD14) for TLR4 engagement not dissected here"]},{"year":2021,"claim":"Connected FSTL1 to a deubiquitinase-dependent Notch axis, showing FSTL1-USP10 stabilization of NICD1 modulates myocardial fibrosis.","evidence":"AAV9-FSTL1 in diabetic MI mice with spautin-1 and LY3039478 pharmacological epistasis","pmids":["34957094"],"confidence":"Medium","gaps":["Mechanism linking FSTL1 to USP10 activation unknown","Single lab"]},{"year":2023,"claim":"Mapped FSTL1-mediated inter-organ crosstalk in metabolic disease, with IRF4-driven muscle expression signaling to liver through distinct receptors.","evidence":"Muscle-specific IRF4 KO, dual luciferase IRF4-FSTL1 promoter assay, co-culture receptor identification (DIP2A, CD14), and AAV rescue","pmids":["37770480"],"confidence":"Medium","gaps":["Direct FSTL1-CD14 binding not biochemically confirmed","How receptor choice is determined across liver cell types unclear"]},{"year":2023,"claim":"Showed FSTL1 initiates angiogenesis by opening endothelial junctions through Src rather than VEGFR2, with HuR-mediated transcript stabilization upstream.","evidence":"Recombinant FSTL1 in HUVECs, Src vs VEGFR2 inhibitor epistasis, junction protein readouts, HuR knockdown, and hindlimb ischemia model","pmids":["37694287"],"confidence":"Medium","gaps":["Receptor upstream of Src activation not identified","Relationship to DIP2A-Akt axis unresolved"]},{"year":2024,"claim":"Extended TLR4/NF-κB FSTL1 signaling to cellular senescence in intervertebral disc degeneration.","evidence":"Recombinant FSTL1, TLR4 inhibitor rescue, and FSTL1 siRNA in rabbit IVDD model with senescence markers","pmids":["38316670"],"confidence":"Medium","gaps":["Partial rescue indicates additional receptors/pathways","Single lab"]},{"year":2025,"claim":"Defined a CD14/TLR4/NF-κB/ATP6V1G2 axis by which host FSTL1 controls CCR2 recycling and early monocyte recruitment required for antifibrotic responses.","evidence":"Fstl1-deficient mice with recombinant protein rescue, CCR2 recycling assays, and macrophage tracking in MSC infusion models","pmids":["40050288"],"confidence":"Medium","gaps":["Direct receptor-ligand binding step not biochemically isolated","Single lab"]},{"year":null,"claim":"It remains unresolved how a single secreted protein switches between BMP/TGFβ antagonism, TGFβ/Wnt amplification, and innate-immune activation across tissues — whether determined by receptor repertoire (DIP2A, ALK6, FZD4, TLR2, TLR4, CD14), glycosylation state, FK-domain dimerization, or intracellular vs extracellular localization.","evidence":"","pmids":[],"confidence":"Low","gaps":["No unifying structural model of receptor selectivity","Post-translational determinants of context-specific function uncharacterized","How secreted FSTL1 engages intracellular partners (PKM2, DIP2A nuclear pool) is unexplained"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[1,2,14]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,10,11]},{"term_id":"GO:0048018","term_label":"receptor ligand activity","supporting_discovery_ids":[0,10]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[8]}],"localization":[{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[39,1,10]},{"term_id":"GO:0031012","term_label":"extracellular matrix","supporting_discovery_ids":[10,18]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,10]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[16,27,29,30]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[1,2,18,26]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[7,11,33,31]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[23,28,37]}],"complexes":[],"partners":["DIP2A","BMP4","ALK6","PKM2","WNT3A","FZD4","TLR4","VIM"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q12841","full_name":"Follistatin-related protein 1","aliases":["Follistatin-like protein 1"],"length_aa":308,"mass_kda":35.0,"function":"Secreted glycoprotein that is involved in various physiological processes, such as angiogenesis, regulation of the immune response, cell proliferation and differentiation (PubMed:22265692, PubMed:29212066). Plays a role in the development of the central nervous system, skeletal system, lungs, and ureter (By similarity). Promotes endothelial cell survival, migration and differentiation into network structures in an AKT-dependent manner. Also promotes survival of cardiac myocytes (By similarity). Initiates various signaling cascades by activating different receptors on the cell surface such as DIP2A, TLR4 or BMP receptors (PubMed:20054002, PubMed:22265692)","subcellular_location":"Secreted","url":"https://www.uniprot.org/uniprotkb/Q12841/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/FSTL1","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/FSTL1","total_profiled":1310},"omim":[{"mim_id":"605547","title":"FOLLISTATIN-LIKE 1; FSTL1","url":"https://www.omim.org/entry/605547"},{"mim_id":"603445","title":"KH-TYPE SPLICING REGULATORY PROTEIN; KHSRP","url":"https://www.omim.org/entry/603445"},{"mim_id":"600508","title":"NCK ADAPTOR PROTEIN 1; NCK1","url":"https://www.omim.org/entry/600508"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Vesicles","reliability":"Approved"},{"location":"Golgi apparatus","reliability":"Additional"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/FSTL1"},"hgnc":{"alias_symbol":["FRP","FSL1","OCC1","OCC-1","tsc36"],"prev_symbol":[]},"alphafold":{"accession":"Q12841","domains":[{"cath_id":"3.30.60.30","chopping":"38-101","consensus_level":"medium","plddt":91.0203,"start":38,"end":101},{"cath_id":"1.10.238.10","chopping":"114-229","consensus_level":"high","plddt":90.9984,"start":114,"end":229}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q12841","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q12841-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q12841-F1-predicted_aligned_error_v6.png","plddt_mean":86.94},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=FSTL1","jax_strain_url":"https://www.jax.org/strain/search?query=FSTL1"},"sequence":{"accession":"Q12841","fasta_url":"https://rest.uniprot.org/uniprotkb/Q12841.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q12841/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q12841"}},"corpus_meta":[{"pmid":"26375005","id":"PMC_26375005","title":"Epicardial FSTL1 reconstitution regenerates the adult mammalian heart.","date":"2015","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/26375005","citation_count":393,"is_preprint":false},{"pmid":"17030984","id":"PMC_17030984","title":"MyoD inhibits Fstl1 and Utrn expression by inducing transcription of miR-206.","date":"2006","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/17030984","citation_count":270,"is_preprint":false},{"pmid":"35140065","id":"PMC_35140065","title":"FSTL1 promotes liver fibrosis by reprogramming macrophage function through modulating the intracellular function of PKM2.","date":"2022","source":"Gut","url":"https://pubmed.ncbi.nlm.nih.gov/35140065","citation_count":197,"is_preprint":false},{"pmid":"21482757","id":"PMC_21482757","title":"Follistatin-like 1 (Fstl1) is a bone morphogenetic protein (BMP) 4 signaling antagonist in controlling mouse lung development.","date":"2011","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/21482757","citation_count":192,"is_preprint":false},{"pmid":"23395958","id":"PMC_23395958","title":"'See-saw' expression of microRNA-198 and FSTL1 from a single transcript in wound healing.","date":"2013","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/23395958","citation_count":182,"is_preprint":false},{"pmid":"20054002","id":"PMC_20054002","title":"DIP2A functions as a FSTL1 receptor.","date":"2010","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/20054002","citation_count":110,"is_preprint":false},{"pmid":"28473327","id":"PMC_28473327","title":"Autophagy plays a role in FSTL1-induced epithelial mesenchymal transition and airway remodeling in asthma.","date":"2017","source":"American journal of physiology. 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Co-immunoprecipitation demonstrated direct physical interaction between FSTL1 and DIP2A. Knockdown of DIP2A by siRNA reduced FSTL1 binding to cells, diminished FSTL1-stimulated endothelial cell survival, migration, and tube formation, and inhibited FSTL1-induced Akt phosphorylation. In cardiac myocytes, DIP2A ablation reduced FSTL1-mediated protection against hypoxia/reoxygenation-induced apoptosis and suppressed FSTL1-induced Akt phosphorylation.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, cell survival/migration/differentiation assays, Western blot for Akt phosphorylation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP establishing direct interaction, siRNA knockdown with multiple orthogonal functional readouts (survival, migration, tube formation, Akt phosphorylation) in two cell types\",\n      \"pmids\": [\"20054002\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Fstl1 directly interacts with BMP4 and negatively regulates BMP4/Smad1/5/8 signaling during lung development. Fstl1-deficient mice showed elevated pSmad1/5/8 activity, impaired alveolar epithelial differentiation, and insufficient surfactant production. Reducing BMP signaling with Noggin rescued pulmonary atelectasis in Fstl1-deficient mice, placing Fstl1 as a BMP4 antagonist in lung morphogenesis.\",\n      \"method\": \"Genetic knockout mouse model, direct binding assay (Fstl1-BMP4 interaction), Smad1/5/8 phosphorylation assays, Noggin rescue epistasis experiment, in vitro BMP4-induced surfactant gene expression assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — direct binding demonstrated in vitro, genetic epistasis rescue with Noggin, multiple orthogonal methods in vivo and in vitro\",\n      \"pmids\": [\"21482757\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Fstl1 antagonizes BMP signaling in the developing ureter. Fstl1-null mice showed elevated pSmad1/5/8 in ureters. In vitro, Fstl1 was shown to directly bind to ALK6, a BMP receptor specifically expressed in ureteric epithelial cells, providing a mechanism for Fstl1-mediated BMP pathway suppression during ureter development.\",\n      \"method\": \"Genetic knockout mouse model, in vitro binding assay (Fstl1-ALK6 interaction), pSmad1/5/8 immunostaining\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding shown in vitro plus in vivo genetic KO with signaling readout, single lab\",\n      \"pmids\": [\"22485132\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The miR-198/FSTL1 switch is controlled post-transcriptionally: TGF-β signaling downregulates KSRP (KHSRP), which is required for miR-198 processing from the FSTL1 primary transcript. When KSRP is inhibited, the transcript is redirected toward FSTL1 protein translation. FSTL1 protein expression promotes keratinocyte migration, while miR-198 inhibits migration by targeting DIAPH1, PLAU, and LAMC2.\",\n      \"method\": \"Human ex vivo organ culture wound model, TGF-β signaling manipulation, KSRP binding/knockdown experiments, luciferase reporter assays, keratinocyte migration assays\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (organ culture, siRNA knockdown, reporter assays, migration assays), mechanistic pathway dissection in human tissue\",\n      \"pmids\": [\"23395958\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Epicardial FSTL1 protein promotes cardiomyocyte cell cycle entry and division. Epicardial FSTL1 declines after myocardial infarction and is replaced by myocardial expression. Myocardial FSTL1 does not promote regeneration. Application of human FSTL1 protein via an epicardial patch stimulates cardiomyocyte proliferation and improves cardiac function in mouse and swine MI models.\",\n      \"method\": \"Epicardial patch delivery of recombinant human FSTL1 protein, cardiomyocyte cell cycle entry and division assays, mouse and swine MI models, transgenic FSTL1 overexpression\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — protein reconstitution via epicardial patch, replicated in two animal species (mouse and swine), multiple functional readouts including cell division\",\n      \"pmids\": [\"26375005\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"MyoD directly activates expression of miR-206, which targets sequences in the Fstl1 3'UTR and is sufficient to suppress Fstl1 expression during skeletal muscle differentiation.\",\n      \"method\": \"Fibroblast-to-myoblast conversion by MyoD overexpression, luciferase reporter assays with Fstl1 3'UTR, miR-206 gain-of-function experiments\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct reporter assay establishing miR-206 targeting of Fstl1 3'UTR, functional sufficiency demonstrated, mechanistically placed downstream of MyoD\",\n      \"pmids\": [\"17030984\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TGF-β1 upregulates Fstl1 expression in lung fibroblasts via the Smad3-c-Jun pathway. While TGF-β1 activates Smad, MAPK, and Akt pathways, only Smad2/3 inhibition eliminated TGF-β1-induced Fstl1 expression. A functional c-Jun transcription site in the Fstl1 promoter was identified by luciferase reporter analysis.\",\n      \"method\": \"Mouse pulmonary fibroblast cultures, pharmacological pathway inhibitors, luciferase reporter assays with Fstl1 promoter constructs, qRT-PCR and Western blot\",\n      \"journal\": \"American journal of physiology. Lung cellular and molecular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — promoter reporter assay plus pathway inhibitor panel, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"28495857\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Fstl1 promotes temozolomide resistance in glioblastoma by competitively binding DIP2A and blocking DIP2A nuclear translocation. DIP2A normally cooperates with the HDAC2-DMAP1 complex to enhance H3K9Ac deacetylation and prevent MGMT transcription, increasing temozolomide sensitivity. FSTL1 binding to DIP2A prevents this, leading to increased promoter H3K9Ac and MGMT expression. DIP2A depletion abolished the effects of Fstl1 on MGMT expression and temozolomide resistance.\",\n      \"method\": \"Co-immunoprecipitation (Fstl1-DIP2A interaction), siRNA knockdown, chromatin immunoprecipitation (H3K9Ac), gene expression assays, in vivo xenograft models\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP establishing direct interaction, epistasis via DIP2A depletion, chromatin mark measurement, in vitro and in vivo validation with multiple orthogonal methods\",\n      \"pmids\": [\"30542120\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Crystal structure of the FK domain of murine Fstl1 was solved at high resolution, revealing that the FK domain forms a stable dimer in both solution and crystal. The FK domain was found to be indispensable for proper Fstl1 function during TGF-β signaling transduction. The potential for Fstl1 to function as a dimer during interaction with TGF-β (which itself forms dimers) was proposed based on structural data.\",\n      \"method\": \"X-ray crystallography of FK domain, solution studies (dimerization), functional assays of FK domain mutants in TGF-β signaling\",\n      \"journal\": \"Protein science : a publication of the Protein Society\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure solved plus functional validation of FK domain in TGF-β signaling, single lab\",\n      \"pmids\": [\"31351024\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Macrophage FSTL1 promotes liver fibrosis by binding directly to PKM2 via its FK domain, promoting PKM2 phosphorylation and nuclear translocation, reducing PKM2 ubiquitination, enhancing PKM2-dependent glycolysis, and increasing M1 macrophage polarization via NF-κB pathway activation. Myeloid-specific FSTL1 knockout attenuated liver fibrosis and reduced M1 polarization.\",\n      \"method\": \"Myeloid-specific FSTL1 knockout mice, Co-IP (FSTL1-PKM2 direct binding via FK domain), Western blot for PKM2 phosphorylation and nuclear translocation, ubiquitination assays, glycolysis measurements, in vitro macrophage polarization assays, pharmacological PKM2 activator (DASA-58)\",\n      \"journal\": \"Gut\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — direct binding via Co-IP, domain-specific interaction identified, myeloid-specific KO with clear phenotype, multiple orthogonal mechanistic readouts, pharmacological rescue\",\n      \"pmids\": [\"35140065\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"FSTL1 interacts with Wnt ligands (specifically Wnt3a) and Frizzled receptors (specifically FZD4) but not with the co-receptor LRP6. FSTL1 interacts with Wnt3a through its extracellular calcium-binding (EC) domain and VWC domain, and with FZD4 through its EC domain. FSTL1 increased the association of Wnt3a with FZD4 and thereby enhanced Wnt/β-catenin signaling and fibrogenesis in obstructed kidneys.\",\n      \"method\": \"Co-immunoprecipitation (FSTL1-Wnt3a, FSTL1-FZD4 interactions), domain deletion analysis, FSTL1 overexpression/inhibition in obstructed mouse kidneys, Wnt/β-catenin reporter assays, single-cell RNA-Seq for expression localization\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP with domain mapping, in vivo genetic models, multiple signaling pathway readouts, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"35525270\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"FSTL1 secreted by activated fibroblasts binds to TLR4 on hepatocellular carcinoma cells, resulting in activation of AKT/mTOR/4EBP1 signaling, promoting HCC growth, metastasis, and maintenance of tumor-initiating cells.\",\n      \"method\": \"Recombinant FSTL1 treatment of HCC cells and 3D organoids, receptor binding assay (TLR4 identified as receptor), Western blot for AKT/mTOR/4EBP1 signaling, conditioned medium experiments, preclinical mouse models with FSTL1 blockade\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — receptor identification with functional downstream signaling, in vivo models, single lab\",\n      \"pmids\": [\"34551961\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Dynamic resistance exercise stimulates skeletal muscle FSTL1 secretion. FSTL1 binds receptor DIP2A on endothelial cells and activates Smad2/3 signaling to promote cardiac angiogenesis. TGFβR1 inhibitor reduced pSmad2/3 and VEGF-A expression but did not affect FSTL1-DIP2A direct Smad2/3 activation, demonstrating that the FSTL1-DIP2A-Smad2/3 axis is independent of TGFβR1.\",\n      \"method\": \"Rat MI model with resistance exercise, AAV-FSTL1 injection, recombinant FSTL1 treatment of HUVECs, TGFβR1 inhibitor pharmacological epistasis, Western blot for DIP2A and pSmad2/3, immunofluorescence, tubule assay\",\n      \"journal\": \"Journal of sport and health science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological epistasis with TGFβR1 inhibitor defining pathway independence, multiple in vitro and in vivo methods, single lab\",\n      \"pmids\": [\"33246164\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"FSTL1 promotes cardiac angiogenesis and myocardial energy substrate metabolism normalization in heart failure via AMPK activation. FSTL1 stimulated oxygen consumption through AMPK activation in primary cardiac and skeletal muscle myocytes in vitro.\",\n      \"method\": \"Conscious dog HF model with acute and chronic FSTL1 infusion, radiolabeled substrate tracking (3H-oleate, 14C-glucose), in vitro primary myocyte assays with AMPK pathway analysis\",\n      \"journal\": \"Circulation. Heart failure\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo infusion with metabolic substrate tracking plus in vitro AMPK mechanistic validation, single lab, large animal model\",\n      \"pmids\": [\"29317401\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"In glioblastoma, Fstl1 interacts with BMP4 but not with BMPR2, competitively inhibiting BMP4-BMPR2 association. Fstl1 overexpression suppressed BMP4/Smad1/5/8 signaling pathway activation, promoting glioma cell proliferation, while BMP4 overexpression reversed this effect.\",\n      \"method\": \"Co-immunoprecipitation (Fstl1-BMP4 and Fstl1-BMPR2 interactions), Western blot for pSmad1/5/8, cell proliferation and colony formation assays, orthotopic xenograft, BMP4 rescue experiment\",\n      \"journal\": \"Cellular physiology and biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP showing selective binding to BMP4 but not BMPR2, BMP4 overexpression rescue epistasis, multiple functional readouts, single lab\",\n      \"pmids\": [\"29212066\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"FSTL1 blocks Wnt7a-mediated repression of ERK phosphorylation, enabling MMP9 production that degrades the extracellular matrix and facilitates metastasis. Separately, EGF hijacks the miR-198/FSTL1 switch to sustain FSTL1 translation, driving metastasis through parallel DIAPH1 and FSTL1 pathways.\",\n      \"method\": \"Head and neck squamous cell carcinoma cell lines, Wnt7a pathway manipulation, ERK phosphorylation assays, MMP9 expression and activity assays, FSTL1 gain/loss-of-function, migration/invasion assays\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional pathway experiments with signaling readouts, single lab, multiple cell line models\",\n      \"pmids\": [\"28827448\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Macrophage-derived Fstl1 induces oncostatin M (OSM) expression, promoting asthmatic airway remodeling. Macrophage-specific Fstl1 knockout (Lys-Cre/Fstl1Δ/Δ) reduced airway remodeling and OSM levels. Exogenous Fstl1 induced airway remodeling and increased OSM, while anti-OSM antibody blocked Fstl1-induced remodeling, eosinophilic inflammation, and airway hyperresponsiveness.\",\n      \"method\": \"Macrophage-specific conditional Fstl1 knockout mice, allergen challenge model, recombinant Fstl1 administration, anti-OSM antibody blockade, airway remodeling and inflammation readouts\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — macrophage-specific conditional KO with clear phenotype, exogenous protein rescue, antibody blockade epistasis, multiple readouts identifying Fstl1→OSM pathway\",\n      \"pmids\": [\"26355153\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Knockdown of Fstl1 in hepatic stellate cells suppressed proliferation and reduced α-SMA and collagen I expression in TGF-β1-treated HSCs. Mechanistically, Fstl1 knockdown decreased Smad3 phosphorylation in TGF-β1-induced HSCs, placing Fstl1 as a positive regulator of TGF-β1/Smad3 signaling in liver fibrosis.\",\n      \"method\": \"siRNA knockdown of Fstl1 in hepatic stellate cells, Western blot for pSmad3, α-SMA, collagen I, cell proliferation assay\",\n      \"journal\": \"Molecular medicine reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with defined signaling readout (pSmad3), single lab, single method\",\n      \"pmids\": [\"28901425\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Fstl1 is essential for lung airway and vascular smooth muscle formation. Fstl1 was localized to lung smooth muscle cells. Fstl1 knockout impaired airway smooth muscle differentiation, associated with decreased myocardin/SRF transcription factors. Fstl1 knockout also caused hyperplasia of pulmonary artery vascular smooth muscle.\",\n      \"method\": \"Fstl1-lacZ reporter mouse, Fstl1 knockout allele, histological analysis of trachea/bronchi/pulmonary artery, immunostaining for myocardin/SRF\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with cell-type-specific localization and transcription factor readouts, single lab\",\n      \"pmids\": [\"28574994\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Fstl1 deletion from the endocardial/endothelial lineage (Tie2-Cre) causes sustained BMP and TGFβ signaling after birth, resulting in ongoing endocardial-to-mesenchymal transition, deformed mitral valves, cardiac hypertrophy, and heart failure. This shows that endocardial FSTL1 normally restrains BMP/TGFβ signaling to maintain valve homeostasis.\",\n      \"method\": \"Conditional knockout (Tie2-Cre; Fstl1 flox), echocardiography, electrocardiography, histology, BMP/TGFβ signaling markers\",\n      \"journal\": \"Arteriosclerosis, thrombosis, and vascular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — lineage-specific conditional KO with clear cardiac phenotype and signaling pathway readouts, single lab\",\n      \"pmids\": [\"28705792\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Endothelial cell-specific FSTL1 knockout led to increased pSMAD3 in vascular mural cells colocalizing with αSMA in vein walls, increased collagen deposition, and cardiac/vascular fibrosis. TGFβ pathway inhibitor treatment reduced the αSMA abnormalities, demonstrating that endothelial FSTL1 normally suppresses TGFβ/SMAD3 signaling in vascular mural cells.\",\n      \"method\": \"Conditional endothelial FSTL1 knockout mouse (vs. smooth muscle and hematopoietic cell KOs as controls), pSMAD3 immunostaining, TGFβ inhibitor rescue, collagen deposition assay\",\n      \"journal\": \"Arteriosclerosis, thrombosis, and vascular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell-type-specific conditional KO with multiple controls, pharmacological rescue of signaling phenotype, single lab\",\n      \"pmids\": [\"32078339\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Skeletal muscle IRF4 transcriptionally regulates FSTL1 (dual luciferase assay confirmed IRF4 binding to FSTL1 promoter). FSTL1 mediates inter-organ crosstalk between skeletal muscle and the liver in NASH via different receptors (DIP2A and CD14) on different liver cell types. Restoring FSTL1 in muscle of F4MKO mice was sufficient to restore liver pathology.\",\n      \"method\": \"Muscle-specific IRF4 knockout mice, proteomics, dual luciferase reporter assay (IRF4-FSTL1 promoter), co-culture experiments with liver cells, AAV-mediated FSTL1 rescue\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — dual luciferase assay for transcriptional regulation, muscle-specific KO with rescue experiment, receptor identification by co-culture, single lab\",\n      \"pmids\": [\"37770480\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"FSTL1 directly increases expression of MMP-1, MMP-13, iNOS, COX-2, IL-1β, TNF-α, and IL-6 in chondrocytes in a dose-dependent manner, and activates NF-κB and promotes p65 phosphorylation, establishing NF-κB as the signaling pathway mediating FSTL1 pro-inflammatory effects in chondrocytes.\",\n      \"method\": \"Recombinant FSTL1 treatment of rat chondrocytes, PCR, ELISA, Western blot for NF-κB pathway components (p65 phosphorylation)\",\n      \"journal\": \"Journal of cellular and molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct protein treatment with dose-response and multiple signaling readouts, single lab, no pathway inhibitor rescue\",\n      \"pmids\": [\"30644158\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"FSTL1 promotes nitric oxide-induced chondrocyte apoptosis by activating the SAPK/JNK/Caspase3 signaling pathway. FSTL1 overexpression increased apoptosis in SNP-treated chondrocytes, upregulated MMP1/3/9 and Bax, and reduced Bcl-2 and collagen. The caspase inhibitor Ac-DEVD-FMK impaired FSTL1-induced chondrocyte apoptosis.\",\n      \"method\": \"FSTL1 overexpression plasmid transfection in chondrocytes, flow cytometry for apoptosis, Western blot for SAPK/JNK/Caspase3 pathway, caspase inhibitor rescue\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — caspase inhibitor rescue confirms pathway, multiple readouts, single lab\",\n      \"pmids\": [\"31927008\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"FSTL1 knockdown in airway smooth muscle cells inhibited PDGF-BB-induced proliferation, arrested cell cycle at G2/M, and reduced migration. Mechanistically, FSTL1 knockdown downregulated PDGF-BB-induced phosphorylation of ERK and AKT in ASM cells.\",\n      \"method\": \"siRNA knockdown of FSTL1 in human ASM cells, cell proliferation assay, cell cycle analysis by flow cytometry, migration assay, Western blot for p-ERK and p-AKT\",\n      \"journal\": \"Molecular medicine reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with defined signaling readouts (ERK and AKT phosphorylation), single lab\",\n      \"pmids\": [\"28393245\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"FSTL1 activates Wnt/β-catenin signaling through integrin β3 in breast cancer cells, promoting stemness and chemoresistance. Luciferase assays demonstrated that miR-137 reduces FSTL1 mRNA and protein levels, identifying FSTL1 as a direct miR-137 target.\",\n      \"method\": \"TOP/FOP flash Wnt reporter assay, colony and tumor sphere formation, luciferase assay for miR-137 targeting of FSTL1, Western blot for pathway components\",\n      \"journal\": \"Cancer biology & therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — reporter assays for both Wnt signaling and miRNA targeting, functional rescue experiments, single lab\",\n      \"pmids\": [\"30336071\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Fstl1 is expressed in brain by pia mater but not in the ventricular zone; FSTL1 from pia mater is required for radial glial cell morphology. Conditional Fstl1 ablation in both expression domains disrupted RGC basal process organization and caused mislocalization of upper-layer projection neurons. VZ-only Fstl1 deletion did not affect RGC morphology. BMP, AKT/PKB, Cdc42, GSK3β, integrin and reelin signaling were unchanged, indicating a unique mechanism.\",\n      \"method\": \"Conditional Fstl1 knockout mice (EIIa-Cre and Emx1-IREScre lines), cortical histology, immunostaining for RGC markers and signaling pathway components\",\n      \"journal\": \"Molecular brain\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two conditional KO lines with spatial dissection of FSTL1 source, genetic negative results for known pathways, single lab\",\n      \"pmids\": [\"26382033\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Host FSTL1 enhances rapid recycling of CCR2 to the plasma membrane via activation of the CD14/TLR4/NF-κB/ATP6V1G2 axis, leading to early recruitment of Ly6C+ monocytes/macrophages. This early inflammatory macrophage recruitment is required for MSC-mediated antifibrotic effects in liver cirrhosis.\",\n      \"method\": \"Fstl1-deficient mice, recombinant FSTL1 rescue, mechanistic dissection of CD14/TLR4/NF-κB/ATP6V1G2 pathway, CCR2 membrane recycling assays, macrophage depletion/tracking experiments, MSC infusion models\",\n      \"journal\": \"Signal transduction and targeted therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with recombinant protein rescue, mechanistic pathway dissection with multiple components, single lab\",\n      \"pmids\": [\"40050288\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"FSTL1 promotes alveolar epithelial cell senescence by enhancing TGF-β1 signaling, and this enhancement is dependent on SENP1-mediated deSUMOylation. TGF-β1-induced FSTL1 upregulates SENP1 expression in senescent AECs, and interfering with SENP1 inhibited FSTL1-dependent promotion of AEC senescence and improved pulmonary fibrosis.\",\n      \"method\": \"TGF-β1 treatment of AECs, SENP1 siRNA knockdown, senescence assays, Western blot for FSTL1 and SENP1, in vivo bleomycin mouse model\",\n      \"journal\": \"Cell biology international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis via SENP1 siRNA knockdown with defined senescence phenotype, in vitro and in vivo, single lab\",\n      \"pmids\": [\"37369969\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"FSTL1 activates the NLRP3/IL-1β signaling pathway in macrophages, contributing to asthmatic airway inflammation. Pretreatment with MCC950 (NLRP3 inhibitor) significantly reduced NLRP3 and IL-1β production induced by FSTL1 in mice and in alveolar macrophage MH-S cells.\",\n      \"method\": \"Fstl1 heterozygous knockout mice, OVA asthma model, recombinant FSTL1 injection, MCC950 pharmacological inhibitor, siFSTL1, Western blot and ELISA for NLRP3/IL-1β\",\n      \"journal\": \"Inflammation research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological rescue with MCC950, genetic heterozygous KO, recombinant protein gain-of-function, multiple readouts, single lab\",\n      \"pmids\": [\"34076707\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"FSTL-1 attenuation in hypomorphic mice causes spontaneous emphysema independent of smoke. Recombinant FSTL-1 treatment of macrophages attenuated NF-κB p65 phosphorylation in an Nr4a1-dependent manner, identifying a FSTL-1→Nr4a1→NF-κB signaling axis in lung macrophage immune tolerance.\",\n      \"method\": \"FSTL-1 hypomorphic mice, lung morphometry and pulmonary function, RNA-seq identifying Nr4a1, in vitro macrophage recombinant FSTL-1 treatment with NF-κB p65 phosphorylation assay, Nr4a1 dependence testing\",\n      \"journal\": \"American journal of respiratory and critical care medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNA-seq pathway discovery plus in vitro mechanistic validation with Nr4a1 dependence, hypomorphic mouse model, single lab\",\n      \"pmids\": [\"31834999\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"FSTL1 secreted by Snail-positive tumor cells promotes bone metastasis through two mechanisms: direct mediation of tumor cell invasion and bone tropism, and expansion of CD45−ALCAM+ pluripotent mesenchymal stem-like cells from bone marrow, which both directly induce bone metastasis and generate CD8low T cells with weak CTL activity. RNAi-mediated FSTL1 attenuation prevented bone metastasis and reversed these immune dysfunctions.\",\n      \"method\": \"RNAi knockdown of FSTL1 in tumor cells, flow cytometry for ALCAM+ cell expansion and CD8 T cell characterization, in vivo bone metastasis models, in vitro CTL assays\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNAi loss-of-function with mechanistic cellular readouts (ALCAM+ expansion, CD8 phenotyping), in vivo and in vitro, single lab\",\n      \"pmids\": [\"23966294\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"FSTL1 interacts with VIM (vimentin) in colorectal cancer cells. This interaction was identified by co-immunoprecipitation and mediates FSTL1's role in activating focal adhesion signaling and cytoskeleton rearrangement to promote CRC metastasis. TGFβ1-Smad2/3 signaling (via Smad3 transcription factor) was identified as an upstream regulator of FSTL1 protein expression.\",\n      \"method\": \"Co-immunoprecipitation (FSTL1-VIM), focal adhesion signaling pathway analysis, Smad3 ChIP/reporter for FSTL1 transcriptional regulation, in vitro migration/invasion assays, in vivo liver metastasis model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP identifying VIM interaction, pathway analysis, in vivo metastasis model, single lab\",\n      \"pmids\": [\"29844309\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Fstl1 promotes glioma stem cell self-renewal through autocrine FSTL1 interacting with TLR2, which inhibits EGFR endocytosis-lysosomal degradation, resulting in activation of the PI3K-AKT signaling pathway. FSTL1 also promotes M2 macrophage polarization via TLR2 signaling.\",\n      \"method\": \"FSTL1 knockout in GSC cell lines, TLR2 receptor identification, EGFR endocytosis assays, PI3K-AKT pathway Western blot, mouse GBM models, macrophage polarization assays\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — receptor identification (TLR2) with downstream mechanistic dissection (EGFR endocytosis, PI3K-AKT), FSTL1 KO with rescue, in vivo model, single lab\",\n      \"pmids\": [\"39722404\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"FSTL1 promotes myocardial fibrosis via a USP10/Notch1 signaling axis. FSTL1 activation of USP10 (a Notch1 deubiquitinase) stabilizes NICD1 (Notch1 intracellular domain), which suppresses myocardial fibrosis. Pharmacological inhibition of USP10 (spautin-1) or Notch signaling (LY3039478) abolished the protective effects of FSTL1 in diabetic MI mice.\",\n      \"method\": \"AAV9-FSTL1 intracardiac delivery in T2DM-MI mice, USP10 inhibitor (spautin-1) and Notch inhibitor (LY3039478) epistasis experiments, cardiac fibrosis markers, Western blot for USP10/Notch1/NICD1\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological epistasis with two inhibitors defining pathway, in vivo gene delivery, defined molecular readouts, single lab\",\n      \"pmids\": [\"34957094\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"FSTL1 initiates angiogenesis in endothelial cells by opening intercellular junctions via activation of the Src kinase pathway. FSTL1 increased Src phosphorylation and VEGFR2 phosphorylation, decreased VE-Cadherin, Occludin, Connexin-43, and Claudin-5 expression, and increased endothelial permeability. Src inhibitor (but not VEGFR2 inhibitor) blocked FSTL1-induced effects. H2S upregulated FSTL1 in skeletal muscle by increasing HuR levels, which stabilized FSTL1 transcript.\",\n      \"method\": \"Recombinant FSTL1 treatment of HUVECs, Src and VEGFR2 pharmacological inhibitors, immunostaining for junction proteins, wound-healing migration assay, permeability assay, HuR siRNA knockdown, mouse hindlimb ischemia model\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological epistasis distinguishing Src vs VEGFR2, multiple junction protein readouts, HuR mechanism for FSTL1 regulation, in vitro and in vivo, single lab\",\n      \"pmids\": [\"37694287\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"miR-29a in mesenchymal stem cells suppresses FSTL1 expression and secretion (validated by dual luciferase reporter assay). This reduces FSTL1 in conditioned medium, which in turn inhibits the JAK2/STAT3 pathway in cardiac myocytes and promotes myocyte apoptosis after hypoxia-reoxygenation injury.\",\n      \"method\": \"Dual luciferase reporter assay (miR-29a targeting FSTL1), miR-29a overexpression in MSCs, FSTL1 measurement by ELISA in conditioned medium, JAK2/STAT3 Western blot in H9c2 cells, flow cytometry for apoptosis\",\n      \"journal\": \"Cardiovascular pathology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — luciferase reporter validates direct miRNA-mRNA interaction, conditioned medium experiment links MSC FSTL1 secretion to cardiomyocyte JAK2/STAT3, single lab\",\n      \"pmids\": [\"31945680\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"FSTL1 promotes nucleus pulposus cell senescence via TLR4/NF-κB signaling. Recombinant FSTL1 upregulated p16 and p21, increased SA-β-gal-positive cells, induced SASP, and disrupted ECM balance. TLR4 inhibition partly reversed these effects. FSTL1 siRNA in a rabbit puncture IVDD model reduced disc degeneration.\",\n      \"method\": \"Recombinant FSTL1 treatment of NPCs, TLR4 inhibitor rescue, FSTL1 siRNA in vitro and in vivo (rabbit IVDD model), senescence assays (SA-β-gal, p16/p21), Western blot for TLR4/NF-κB\",\n      \"journal\": \"Inflammation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — TLR4 inhibitor epistasis, in vitro and in vivo (rabbit model) with multiple senescence readouts, single lab\",\n      \"pmids\": [\"38316670\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"BBS4 regulates FSTL1 mRNA levels and also independently modulates FSTL1 secretion. FSTL1 functions as a novel regulator of ciliogenesis, creating a regulatory loop between FSTL1 and cilia. BBS4, cilia, and FSTL1 are coordinated during 3T3-L1 differentiation, with FSTL1 influencing this process at least partly by modulating ciliogenesis.\",\n      \"method\": \"BBS4 knockdown/knockout in cells, FSTL1 mRNA quantification, FSTL1 secretion assay, ciliogenesis assays (cilia length/frequency), 3T3-L1 differentiation with FSTL1 manipulation\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — BBS4-FSTL1 functional interaction established through loss-of-function, bidirectional FSTL1-cilia regulatory loop demonstrated, single lab\",\n      \"pmids\": [\"28852127\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Fstl1 is a 'preadipokine' that is highly expressed in 3T3-L1 preadipocytes and dramatically downregulated early in differentiation to adipocytes. The Fstl1 protein is secreted by preadipocytes. Negative transcriptional regulation of Fstl1 is mediated by Kruppel-like factor 15 (KLF15), as identified by luciferase reporter assays with Fstl1 5' flanking region constructs.\",\n      \"method\": \"Northern blot, Western blot of conditioned media, luciferase reporter assays with Fstl1 5' flanking region constructs, KLF15 expression, multiple adipogenesis model time courses\",\n      \"journal\": \"Mechanisms of development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — luciferase reporter identifies KLF15 as negative transcriptional regulator, protein secretion confirmed, multiple adipogenesis models, single lab\",\n      \"pmids\": [\"20043993\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"FSTL1 is a secreted, glycosylated matricellular protein that acts as a context-dependent extracellular signaling molecule: it antagonizes BMP signaling by directly binding BMP4 (via its FK domain, which forms a stable dimer) and blocking BMP receptor engagement; activates protective cardiovascular signaling through its receptor DIP2A, triggering Smad2/3 and Akt/AMPK pathways to promote cardiomyocyte proliferation, angiogenesis, and metabolic normalization; promotes inflammation and fibrosis through TLR4/NF-κB, NLRP3/IL-1β, and TGF-β/Smad3 signaling; enhances Wnt/β-catenin signaling by bridging Wnt3a to Frizzled receptors; modulates tumor biology via BMP4 sequestration, integrin β3/Wnt, TLR2/PI3K-AKT, and TLR4/AKT/mTOR pathways; regulates macrophage polarization including through intracellular PKM2 reprogramming; and is transcriptionally induced by TGF-β1 via the Smad3-c-Jun pathway and suppressed by miR-206 (downstream of MyoD) and KLF15.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"FSTL1 is a secreted, glycosylated signaling protein that operates as a context-dependent extracellular regulator of BMP, TGF-β, Wnt, and innate-immune signaling, governing tissue morphogenesis, cardiovascular protection, fibrosis, and tumor biology [#1, #0, #10]. In its morphogenetic role, FSTL1 directly binds BMP4 and the BMP receptor ALK6 to antagonize BMP/Smad1/5/8 signaling, a function required for alveolar epithelial differentiation and surfactant production in the lung and for ureter patterning, since Noggin rescues the atelectasis of Fstl1-deficient lungs [#1, #2]. Its dimerization-competent FK domain is structurally indispensable for engagement of TGF-β-family signaling [#8]. On the protective cardiovascular axis, FSTL1 acts through the receptor DIP2A to drive endothelial survival, migration, and tube formation and to protect cardiomyocytes via Akt and a TGFβR1-independent Smad2/3 pathway, while epicardial FSTL1 stimulates cardiomyocyte cell-cycle entry and improves function after myocardial infarction [#0, #12, #4]. FSTL1 also normalizes myocardial energy metabolism through AMPK activation and opens endothelial junctions to initiate angiogenesis via Src/VEGFR2 [#13, #35]. Paradoxically, FSTL1 is strongly pro-fibrotic and pro-inflammatory in other settings: it potentiates TGF-β1/Smad3 signaling in hepatic stellate cells and alveolar epithelium, bridges Wnt3a to FZD4 through its EC and VWC domains to amplify Wnt/β-catenin-driven renal fibrosis, and engages TLR4/CD14/NF-κB and NLRP3/IL-1β programs that drive macrophage activation, asthmatic airway remodeling, and tissue senescence [#17, #10, #27, #29]. Within macrophages it binds PKM2 via its FK domain to reprogram glycolysis and M1 polarization in liver fibrosis [#9]. In cancer, FSTL1 promotes glioma proliferation through BMP4 sequestration and temozolomide resistance by blocking DIP2A nuclear translocation and derepressing MGMT, and drives tumor growth and metastasis via TLR4/AKT/mTOR, TLR2/PI3K-AKT, and integrin-β3/Wnt routes [#14, #7, #11, #33]. FSTL1 expression is induced by TGF-β1 through Smad3-c-Jun and constrained by miR-206 downstream of MyoD and by KLF15 [#6, #5, #39].\"\n  ,\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"Established the first direct transcriptional brake on Fstl1, explaining how its expression is extinguished during a differentiation program.\",\n      \"evidence\": \"MyoD-driven myoblast conversion with luciferase 3'UTR reporters showing miR-206 targets Fstl1\",\n      \"pmids\": [\"17030984\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not address FSTL1 protein function in muscle\", \"Limited to skeletal muscle context\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Identified DIP2A as a cell-surface receptor for FSTL1, providing the first molecular handle on its protective signaling and a downstream effector (Akt).\",\n      \"evidence\": \"Reciprocal Co-IP plus DIP2A siRNA knockdown with survival, migration, tube-formation and Akt readouts in endothelial cells and cardiomyocytes\",\n      \"pmids\": [\"20054002\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"DIP2A signaling mechanism downstream of receptor binding not resolved\", \"How one receptor reconciles protective vs pro-fibrotic FSTL1 outputs unclear\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Characterized FSTL1 as a secreted preadipokine under negative transcriptional control, broadening its regulatory inputs beyond miRNA.\",\n      \"evidence\": \"Conditioned-media Western blots and KLF15 luciferase reporter assays across adipogenesis models\",\n      \"pmids\": [\"20043993\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional role of FSTL1 in adipocyte biology not defined\", \"Direct KLF15 promoter occupancy not shown\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Defined FSTL1 as a direct BMP4 antagonist required for lung morphogenesis, establishing its core developmental mechanism via genetic epistasis.\",\n      \"evidence\": \"Fstl1 knockout mice, in vitro Fstl1-BMP4 binding, pSmad1/5/8 readouts, and Noggin rescue of atelectasis\",\n      \"pmids\": [\"21482757\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of FSTL1-BMP4 complex not determined\", \"Whether antagonism is by ligand sequestration or receptor competition not distinguished here\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Extended BMP antagonism to direct receptor binding, showing FSTL1 can engage a BMP receptor (ALK6) rather than only the ligand.\",\n      \"evidence\": \"Fstl1-null ureter phenotype with elevated pSmad1/5/8 and in vitro Fstl1-ALK6 binding\",\n      \"pmids\": [\"22485132\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relative contribution of ligand vs receptor binding to antagonism unresolved\", \"Single-lab finding\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Revealed a post-transcriptional switch coupling FSTL1 protein production to miR-198, linking TGF-β/KSRP control of a shared transcript to keratinocyte migration.\",\n      \"evidence\": \"Human ex vivo wound organ culture, KSRP knockdown, luciferase reporters, and migration assays\",\n      \"pmids\": [\"23395958\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Receptor through which FSTL1 protein drives keratinocyte migration not identified\", \"Generality of the switch beyond skin uncertain\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Demonstrated that the tissue source of FSTL1 determines its regenerative competence, showing epicardial protein drives cardiomyocyte division.\",\n      \"evidence\": \"Epicardial patch delivery of recombinant human FSTL1 with cell-cycle and functional readouts in mouse and swine MI\",\n      \"pmids\": [\"26375005\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why myocardial FSTL1 lacks regenerative activity (glycosylation? receptor?) not resolved\", \"Receptor mediating cardiomyocyte proliferation not defined here\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Defined the transcriptional induction arm of FSTL1, placing it downstream of TGF-β1 via Smad3 and c-Jun in fibroblasts.\",\n      \"evidence\": \"Pathway inhibitor panel and Fstl1 promoter luciferase reporters in lung fibroblasts\",\n      \"pmids\": [\"28495857\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct c-Jun promoter occupancy not shown by ChIP here\", \"Single lab\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Established FSTL1 as a positive amplifier of TGF-β1/Smad3 signaling in fibrosis, contrasting with its BMP-antagonist role.\",\n      \"evidence\": \"siRNA knockdown in hepatic stellate cells with pSmad3, α-SMA and collagen I readouts\",\n      \"pmids\": [\"28901425\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which FSTL1 enhances Smad3 phosphorylation unknown\", \"No receptor identified in this context\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Mapped tissue-protective FSTL1 functions in development, including smooth muscle differentiation, valve homeostasis, and cortical radial glia organization, frequently by restraining BMP/TGFβ.\",\n      \"evidence\": \"Multiple conditional and lineage-specific Fstl1 knockouts with histology and signaling-marker analysis\",\n      \"pmids\": [\"28574994\", \"28705792\", \"26382033\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The BMP/TGFβ-independent cortical mechanism (idx 26) remains undefined\", \"Cell-autonomous vs paracrine contributions not fully separated\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identified TLR2 and downstream EGFR/PI3K-AKT as a tumor-promoting FSTL1 axis and linked FSTL1 to macrophage polarization.\",\n      \"evidence\": \"FSTL1 KO in glioma stem cells, TLR2 receptor identification, EGFR endocytosis and PI3K-AKT assays, in vivo GBM models\",\n      \"pmids\": [\"39722404\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct FSTL1-TLR2 binding not biochemically confirmed here\", \"Single lab\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Demonstrated FSTL1 promotes pro-inflammatory and remodeling programs, including a macrophage Fstl1→OSM axis in airway disease.\",\n      \"evidence\": \"Macrophage-specific conditional Fstl1 knockout, recombinant protein rescue, and anti-OSM antibody blockade in allergen challenge\",\n      \"pmids\": [\"26355153\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Receptor on macrophages driving OSM induction not identified here\", \"Link to systemic inflammation untested\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Uncovered an intracellular/chromatin mechanism in cancer: FSTL1 blocks DIP2A nuclear translocation to derepress MGMT and confer chemoresistance.\",\n      \"evidence\": \"Co-IP, DIP2A depletion epistasis, H3K9Ac ChIP, and xenograft models in glioblastoma\",\n      \"pmids\": [\"30542120\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How secreted FSTL1 accesses cytoplasmic DIP2A trafficking not resolved\", \"Generalizability to other DIP2A-dependent tumors unknown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Linked FSTL1 to cytoskeletal and metastatic machinery via a vimentin interaction, with Smad3-driven transcriptional control upstream.\",\n      \"evidence\": \"Co-IP of FSTL1-VIM, focal adhesion pathway analysis, and liver metastasis model in colorectal cancer\",\n      \"pmids\": [\"29844309\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether FSTL1-VIM interaction is intracellular or extracellular not clarified\", \"Reciprocal validation limited\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Provided the structural basis for FSTL1 function, showing the FK domain dimerizes and is required for TGF-β signaling transduction.\",\n      \"evidence\": \"X-ray crystallography of the murine FK domain plus functional FK mutant assays\",\n      \"pmids\": [\"31351024\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No co-structure with any ligand or receptor\", \"Functional consequence of dimerization for ligand engagement inferred, not directly shown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Established direct pro-inflammatory and pro-apoptotic FSTL1 signaling in chondrocytes via NF-κB and SAPK/JNK/Caspase3.\",\n      \"evidence\": \"Recombinant FSTL1 dose-response, p65 phosphorylation, and caspase inhibitor rescue in chondrocytes\",\n      \"pmids\": [\"30644158\", \"31927008\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Receptor mediating chondrocyte responses not identified\", \"No NF-κB inhibitor rescue in idx 22\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Resolved a TGFβR1-independent DIP2A-Smad2/3 angiogenic axis and linked it to exercise-induced muscle FSTL1 secretion.\",\n      \"evidence\": \"Rat MI exercise model, AAV-FSTL1, HUVEC assays, and TGFβR1 inhibitor pharmacological epistasis\",\n      \"pmids\": [\"33246164\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How DIP2A activates Smad2/3 without TGFβR1 mechanistically unclear\", \"Single lab\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Showed endothelial FSTL1 normally suppresses TGFβ/SMAD3 in vascular mural cells, and its loss causes emphysema via a macrophage Nr4a1-NF-κB tolerance axis.\",\n      \"evidence\": \"Endothelial-specific knockout with TGFβ inhibitor rescue, and hypomorphic mice with RNA-seq-guided Nr4a1 dependence testing\",\n      \"pmids\": [\"32078339\", \"31834999\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Reconciling FSTL1 suppression vs amplification of TGFβ/Smad3 across tissues unresolved\", \"Receptor mediating Nr4a1 induction not defined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined an intracellular macrophage mechanism in which FSTL1 binds PKM2 via its FK domain to reprogram glycolysis and M1 polarization driving liver fibrosis.\",\n      \"evidence\": \"Myeloid-specific FSTL1 knockout, Co-IP with FK-domain mapping, ubiquitination/glycolysis assays, and DASA-58 pharmacological rescue\",\n      \"pmids\": [\"35140065\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How extracellular/secreted FSTL1 reaches cytoplasmic PKM2 not resolved\", \"Whether FK-PKM2 binding occurs intracellularly before secretion unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified FSTL1 as a Wnt co-factor that bridges Wnt3a to FZD4 through defined domains, amplifying Wnt/β-catenin-driven fibrosis.\",\n      \"evidence\": \"Reciprocal Co-IP with domain-deletion mapping, in vivo obstructed kidney models, and Wnt reporter assays\",\n      \"pmids\": [\"35525270\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the FSTL1-Wnt3a-FZD4 ternary complex not solved\", \"Whether this mechanism operates in non-renal fibrosis untested here\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Established TLR4-driven oncogenic and fibrotic FSTL1 signaling through AKT/mTOR and NLRP3/IL-1β programs.\",\n      \"evidence\": \"Recombinant FSTL1 and TLR4 receptor identification with AKT/mTOR readouts in HCC, plus MCC950 NLRP3 inhibitor epistasis in asthma\",\n      \"pmids\": [\"34551961\", \"34076707\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct FSTL1-TLR4 binding affinity/structure not characterized\", \"Cofactor requirements (CD14) for TLR4 engagement not dissected here\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Connected FSTL1 to a deubiquitinase-dependent Notch axis, showing FSTL1-USP10 stabilization of NICD1 modulates myocardial fibrosis.\",\n      \"evidence\": \"AAV9-FSTL1 in diabetic MI mice with spautin-1 and LY3039478 pharmacological epistasis\",\n      \"pmids\": [\"34957094\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking FSTL1 to USP10 activation unknown\", \"Single lab\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Mapped FSTL1-mediated inter-organ crosstalk in metabolic disease, with IRF4-driven muscle expression signaling to liver through distinct receptors.\",\n      \"evidence\": \"Muscle-specific IRF4 KO, dual luciferase IRF4-FSTL1 promoter assay, co-culture receptor identification (DIP2A, CD14), and AAV rescue\",\n      \"pmids\": [\"37770480\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct FSTL1-CD14 binding not biochemically confirmed\", \"How receptor choice is determined across liver cell types unclear\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Showed FSTL1 initiates angiogenesis by opening endothelial junctions through Src rather than VEGFR2, with HuR-mediated transcript stabilization upstream.\",\n      \"evidence\": \"Recombinant FSTL1 in HUVECs, Src vs VEGFR2 inhibitor epistasis, junction protein readouts, HuR knockdown, and hindlimb ischemia model\",\n      \"pmids\": [\"37694287\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Receptor upstream of Src activation not identified\", \"Relationship to DIP2A-Akt axis unresolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Extended TLR4/NF-κB FSTL1 signaling to cellular senescence in intervertebral disc degeneration.\",\n      \"evidence\": \"Recombinant FSTL1, TLR4 inhibitor rescue, and FSTL1 siRNA in rabbit IVDD model with senescence markers\",\n      \"pmids\": [\"38316670\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Partial rescue indicates additional receptors/pathways\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined a CD14/TLR4/NF-κB/ATP6V1G2 axis by which host FSTL1 controls CCR2 recycling and early monocyte recruitment required for antifibrotic responses.\",\n      \"evidence\": \"Fstl1-deficient mice with recombinant protein rescue, CCR2 recycling assays, and macrophage tracking in MSC infusion models\",\n      \"pmids\": [\"40050288\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct receptor-ligand binding step not biochemically isolated\", \"Single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how a single secreted protein switches between BMP/TGFβ antagonism, TGFβ/Wnt amplification, and innate-immune activation across tissues — whether determined by receptor repertoire (DIP2A, ALK6, FZD4, TLR2, TLR4, CD14), glycosylation state, FK-domain dimerization, or intracellular vs extracellular localization.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No unifying structural model of receptor selectivity\", \"Post-translational determinants of context-specific function uncharacterized\", \"How secreted FSTL1 engages intracellular partners (PKM2, DIP2A nuclear pool) is unexplained\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 2, 14]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 10, 11]},\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [0, 10]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [39, 1, 10]},\n      {\"term_id\": \"GO:0031012\", \"supporting_discovery_ids\": [10, 18]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 10]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [16, 27, 29, 30]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [1, 2, 18, 26]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [7, 11, 33, 31]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [23, 28, 37]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"DIP2A\", \"BMP4\", \"ALK6\", \"PKM2\", \"WNT3A\", \"FZD4\", \"TLR4\", \"VIM\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":9,"faith_total":9,"faith_pct":100.0}}