{"gene":"CCN4","run_date":"2026-06-09T22:57:17","timeline":{"discoveries":[{"year":2000,"finding":"WISP-1 (CCN4) is transcriptionally activated by Wnt-1 and beta-catenin; the CREB site in the WISP-1 promoter plays an important role in this activation, while TCF/LEF sites play only a minor role. Overexpression in NRK-49F cells induces morphological transformation, accelerated cell growth, and tumor formation in nude mice.","method":"Promoter cloning and reporter assay; overexpression in NRK-49F cells with nude mouse xenograft","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — promoter functional dissection with reporter assays plus in vivo tumor formation, independently replicated by subsequent studies confirming CREB-dependent regulation","pmids":["10716946"],"is_preprint":false},{"year":2002,"finding":"WISP-1 (CCN4) activates the Akt/PKB anti-apoptotic signaling pathway and prevents p53-dependent cell death following DNA damage by inhibiting mitochondrial cytochrome c release and up-regulating Bcl-X(L); it does not protect against Fas-ligand-activated cell death.","method":"Cell-based apoptosis assays with WISP-1 expression, cytochrome c release measurement, Western blot for Bcl-X(L), PI3K/Akt pathway inhibition","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (apoptosis assay, cytochrome c release, Bcl-X(L) upregulation, pathway-specific inhibitors) in a single rigorous study; negative result for Fas pathway also established","pmids":["11782444"],"is_preprint":false},{"year":2001,"finding":"WISP-1 (CCN4) directly binds to the dermatan sulfate proteoglycans decorin and biglycan, and this interaction mediates and modulates WISP-1 binding to the surface of fibroblasts. Competitive inhibition with dermatan sulfate and enzymatic removal of dermatan sulfate from the cell surface blocked WISP-1 binding.","method":"Solid-phase binding assay, competitive inhibition with glycosaminoglycans and lyases, mass spectrometric identification of binding factors, direct binding assay with purified decorin and biglycan","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct biochemical binding assay with purified proteins, mass spectrometric identification, multiple competitive inhibition experiments","pmids":["11598131"],"is_preprint":false},{"year":2003,"finding":"Overexpression of WISP-1 in H460 lung cancer cells inhibits Rac GTPase activation downstream of integrins (alpha-v-beta-5 and alpha-1), reduces MMP-1 expression, and suppresses cell invasion and lung metastasis. Blocking antibodies to alpha(v)beta(5) and alpha(1) integrins restored Rac activation in WISP-1-expressing cells.","method":"Rac activation pull-down assay, blocking antibody experiments, constitutively active Rac rescue (RacG12V), microarray/RT-PCR, Matrigel invasion assay, in vivo lung metastasis model","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — epistasis via dominant active Rac rescue, integrin blocking antibodies, multiple orthogonal functional readouts including in vivo metastasis","pmids":["12529380"],"is_preprint":false},{"year":2004,"finding":"WISP-1 (CCN4) promotes BMP-2-induced osteoblastic differentiation during embryonic skeletogenesis and fracture repair. In vitro, WISP-1 expression in differentiating osteoblasts potentiates BMP-2 activity; it increases proliferation and saturation density in chondrocytes but represses their differentiation.","method":"In situ hybridization, cell binding analysis, stable transfection of chondrocytic cell lines, osteoblast differentiation assays, in vivo fracture repair model","journal":"The American journal of pathology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cell-based assays with stable transfection and in vivo fracture model, single lab","pmids":["15331410"],"is_preprint":false},{"year":2008,"finding":"WISP-1 (CCN4) directly regulates TGF-beta1 signaling in human bone marrow stromal cells: recombinant WISP-1 significantly reduces TGF-beta1-induced Smad-2 phosphorylation. Full-length WISP-1 and the splice variant WISP-1va (lacking the putative BMP/TGF-beta binding site) both promote proliferation; however, only full-length WISP-1 modulates osteogenic differentiation in concert with TGF-beta1.","method":"Recombinant protein treatment, BrdU incorporation assay, alkaline phosphatase activity, Western blot for Smad-2 phosphorylation, transfection of full-length vs. splice variant","journal":"Journal of cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct biochemical readout (Smad-2 phosphorylation), domain comparison via splice variant, single lab","pmids":["18404666"],"is_preprint":false},{"year":2009,"finding":"TNF-alpha induces WISP1 expression in primary human cardiac fibroblasts through an ERK1/2-dependent CREB phosphorylation and transcriptional activation mechanism. WISP1 knockdown attenuates TNF-alpha-stimulated collagen production and cardiac fibroblast proliferation. WISP1 also protects cardiomyocytes from TNF-alpha-mediated death.","method":"ERK1/2 inhibition, CREB knockdown, siRNA knockdown of WISP1, reporter gene assays, CREB DNA-binding assay, cell proliferation and collagen production assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods: pathway inhibitors, siRNA knockdown, reporter assay, DNA-binding assay, phenotypic readouts in primary cells","pmids":["19339243"],"is_preprint":false},{"year":2011,"finding":"WISP-1/CCN4 directly binds to BMP-2 and enhances BMP-2 binding to human bone marrow stromal cells (hBMSCs) in a dose-dependent manner, stimulating Smad-1/5/8 phosphorylation and osteogenic differentiation. WISP-1 also induces alpha5-integrin expression and binds to it; anti-integrin alpha5beta1 antibody completely neutralizes WISP-1-enhanced BMP-2 binding. Transgenic WISP-1 overexpression in preosteoblasts increased bone mineral density in vivo.","method":"Immunoprecipitation, Western blot, adenoviral overexpression, lentiviral shRNA knockdown, ectopic osteogenesis assay, integrin-blocking antibody experiments, transgenic mouse analysis","journal":"Journal of bone and mineral research","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — direct binding shown by IP, in vitro and in vivo osteogenesis assays, antibody blocking, transgenic mouse validation; multiple orthogonal methods in one study","pmids":["20684029"],"is_preprint":false},{"year":2011,"finding":"WISP-1 increases MMP-2 expression and cell migration in human chondrosarcoma cells through alpha5beta1 integrin receptor signaling, activating FAK, MEK/ERK, and NF-kappaB; NF-kappaB binds to the MMP-2 promoter. Blocking alpha5beta1 antibody and MEK inhibitors abolished WISP-1-induced MMP-2 upregulation and migration.","method":"Migration assay, Western blot for FAK/MEK/ERK phosphorylation, NF-kappaB luciferase reporter, chromatin immunoprecipitation (p65 binding to MMP-2 promoter), integrin-blocking antibody","journal":"Biochemical pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple pathway inhibitors plus promoter binding assay, single lab","pmids":["21453685"],"is_preprint":false},{"year":2011,"finding":"Angiotensin-II induces WISP1 expression in cardiomyocytes via AT1 receptor and NOX2-generated superoxide, activating Akt/GSK3beta/beta-catenin/TCF/LEF and CREB signaling. AT1 physically associates with NOX2 in vitro and in vivo. WISP1 knockdown reduces Ang-II-induced cardiomyocyte hypertrophy.","method":"AT1 antagonist (losartan), NOX2 knockdown, siRNA knockdown of WISP1, co-immunoprecipitation of AT1-NOX2, Western blot, in vivo Ang-II infusion model","journal":"Journal of molecular and cellular cardiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP of AT1-NOX2, pathway inhibitors, siRNA knockdown with hypertrophy readout; single lab","pmids":["21376054"],"is_preprint":false},{"year":2012,"finding":"WISP1 (CCN4) protects primary hippocampal neurons from oxidant stress-induced apoptosis by activating PI3K/Akt1 signaling, phosphorylating Bad and GSK-3beta, minimizing Bim/Bax complex while increasing Bcl-xL/Bax ratio, and preventing mitochondrial membrane permeabilization, cytochrome c release, and caspase-3 activation.","method":"Primary neuron cultures, PI3K and Akt1 pathway inhibition, Western blot for apoptotic proteins, mitochondrial membrane permeability assay, cytochrome c release assay, caspase-3 activity assay","journal":"Current neurovascular research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pathway inhibitor epistasis with multiple apoptotic readouts, single lab","pmids":["22272766"],"is_preprint":false},{"year":2012,"finding":"WISP1 (CCN4) prevents FoxO3a-mediated caspase-1 and caspase-3 apoptotic death in neurons via PI3K/Akt1-dependent phosphorylation and cytoplasmic sequestration of FoxO3a with 14-3-3 protein, and maintains protective SIRT1 activity through SIRT1 nuclear trafficking and prevention of caspase-mediated SIRT1 degradation.","method":"PI3K/Akt1 inhibition, FoxO3a phosphorylation Western blot, subcellular fractionation, co-immunoprecipitation with 14-3-3, caspase-1/3 activity assays, SIRT1 localization","journal":"Current neurovascular research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple biochemical readouts with pathway inhibitors and co-IP, single lab","pmids":["23151077"],"is_preprint":false},{"year":2012,"finding":"WISP1 (CCN4) autoregulates its own expression by promoting beta-catenin activity and nuclear trafficking, and inhibits GSK-3beta activity post-translationally to maintain beta-catenin integrity in primary neurons during oxidant stress.","method":"WISP1 expression analysis, Akt1 activation assay, GSK-3beta phosphorylation Western blot, beta-catenin subcellular fractionation/nuclear translocation assay","journal":"Current neurovascular research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — subcellular fractionation showing beta-catenin trafficking, Western blot for GSK-3beta phosphorylation, single lab","pmids":["22475393"],"is_preprint":false},{"year":2012,"finding":"WISP1 (CCN4) activates mTOR and phosphorylates p70S6K and 4EBP1 via control of PRAS40; WISP1 sequesters PRAS40 intracellularly through post-translational phosphorylation and binding to 14-3-3 protein, limiting PRAS40 activity and thereby providing cytoprotection to microglia against beta-amyloid toxicity.","method":"mTOR pathway Western blot (p70S6K, 4EBP1 phosphorylation), PRAS40 phosphorylation assay, co-immunoprecipitation with 14-3-3, gene silencing of PRAS40, apoptosis assays","journal":"Current neurovascular research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP with 14-3-3, gene silencing epistasis, phosphorylation Western blots; single lab","pmids":["22873724"],"is_preprint":false},{"year":2013,"finding":"WISP1 (CCN4) controls TSC2 activity by increasing Akt1-mediated TSC2 (Thr1462) phosphorylation and decreasing AMPK-mediated TSC2 (Ser1387) phosphorylation, thereby increasing p70S6K activity and providing cytoprotection to microglia; gene silencing of TSC2 further enhances WISP1-mediated p70S6K phosphorylation but impairs WISP1 cytoprotection, indicating a minimal required level of TSC2 activity.","method":"Akt1/AMPK/TSC2/p70S6K phosphorylation Western blot, TSC2 gene silencing, apoptosis assays (PS exposure, DNA degradation, mitochondrial membrane depolarization, cytochrome c release)","journal":"Current neurovascular research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — TSC2 siRNA epistasis, multiple phosphorylation readouts, cell death assays; single lab","pmids":["23244622"],"is_preprint":false},{"year":2013,"finding":"CCN4/WISP1 induces IL-6 production in human osteoarthritis synovial fibroblasts through alphavbeta5 integrin (not alpha5beta1 or alphavbeta3), activating PI3K, Akt, and NF-kappaB signaling; alphavbeta5 blocking antibody and PI3K/Akt/NF-kappaB inhibitors all attenuated CCN4-induced IL-6.","method":"Neutralizing integrin antibodies, PI3K/Akt/NF-κB pathway inhibitors, NF-κB luciferase reporter, p65 immunocytochemistry (translocation), ELISA for IL-6","journal":"Arthritis research & therapy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — integrin-specific blocking plus multiple pathway inhibitors with promoter activity and secretion readouts; single lab","pmids":["23343403"],"is_preprint":false},{"year":2013,"finding":"CCN4/WISP1 induces VCAM-1 expression in human osteoarthritis synovial fibroblasts via alphavbeta5 or alpha6beta1 integrin, activating Syk, PKCdelta, JNK, c-Jun, and AP-1 signaling; elevated VCAM-1 promotes monocyte adhesion to fibroblasts, which is blocked by VCAM-1 siRNA.","method":"Integrin blocking antibodies, kinase inhibitors, AP-1 luciferase reporter, chromatin immunoprecipitation (c-Jun/AP-1 element), siRNA knockdown, monocyte adhesion assay","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple pathway inhibitors plus ChIP and functional monocyte adhesion readout; single lab","pmids":["23313051"],"is_preprint":false},{"year":2013,"finding":"WISP-1 enhances migration of human osteosarcoma cells by increasing MMP-2 and MMP-9 expression through integrin receptor-mediated activation of Ras, Raf-1, MEK, ERK, and NF-kappaB signaling; siRNA knockdown of Ras or Raf-1 and specific inhibitors abolished WISP-1-induced migration and MMP expression.","method":"Ras/Raf-1 siRNA and inhibitors, MEK/ERK inhibitors, NF-κB inhibitors, migration assay, MMP Western blot/ELISA","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA plus pharmacological inhibitors with multiple readouts; single lab","pmids":["24036215"],"is_preprint":false},{"year":2013,"finding":"WISP-1 increases OSCC cell migration and ICAM-1 expression through alphavbeta3 integrin, activating ASK1, JNK/p38, and AP-1 signaling pathways.","method":"Integrin alphavbeta3 blocking antibody, ASK1/JNK/p38 inhibitors, AP-1 luciferase reporter, migration assay, ICAM-1 Western blot","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — integrin blocking plus pathway inhibitors with functional and molecular readouts; single lab","pmids":["24205072"],"is_preprint":false},{"year":2014,"finding":"WISP1 (CCN4) is secreted from differentiated human adipocytes (validated as an adipokine) and stimulates a proinflammatory response in human macrophages. WISP1 expression and secretion increase during human adipocyte differentiation and are regulated by weight changes in humans and mice.","method":"WISP1 ELISA from conditioned media, macrophage stimulation assay with recombinant WISP1, in vivo weight-change models","journal":"Diabetes","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct secretion measurement plus macrophage functional assay; single lab","pmids":["25281430"],"is_preprint":false},{"year":2014,"finding":"miR-92a post-transcriptionally represses WISP1 expression in lung fibroblasts; TGF-beta1 increases WISP1 expression while decreasing miR-92a, and miR-92a overexpression reverses TGF-beta1-induced WISP1 mRNA expression. An inverse relationship between WISP1 and miR-92a was confirmed in vivo in a TGF-beta1-dependent fibrosis model.","method":"Supervised machine learning miRNA target prediction, miR-92a mimic/inhibitor transfection, qPCR and ELISA for WISP1, in vivo TGF-beta1 fibrosis mouse model, ex vivo primary IPF fibroblasts","journal":"The international journal of biochemistry & cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — miRNA gain/loss of function in primary cells and in vivo model confirming inverse relationship; single lab","pmids":["24953558"],"is_preprint":false},{"year":2015,"finding":"WISP1 (CCN4) deficiency in mice reduces bone mineral density, decreases trabecular bone volume and cortical thickness, impairs osteoblast progenitor differentiation, and increases osteoclast differentiation. WISP1 inhibits SOST binding to LRP6, thereby maintaining Wnt/beta-catenin signaling in bone marrow stromal cells; Wisp1-/- cells show reduced beta-catenin and its target gene expression.","method":"Wisp1-/- knockout mouse generation, DXA, micro-CT, biomechanical testing, in vitro osteoblast/osteoclast differentiation assays, SOST-LRP6 binding inhibition assay, beta-catenin Western blot, ovariectomy model","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — constitutive knockout mouse with multiple phenotypic and molecular readouts, direct SOST-LRP6 binding inhibition assay; rigorous multi-method study","pmids":["25864198"],"is_preprint":false},{"year":2015,"finding":"CCN4/WISP1 directly binds TGF-beta3 (shown by immunoprecipitation-western blot) and enhances TGF-beta3-induced SMAD2/3 phosphorylation and chondrogenesis of human bone marrow stromal cells in micromass culture. CCN4 knockdown inhibits TGF-beta3-induced SMAD2/3 phosphorylation and cartilage matrix synthesis. Ccn4-KO mice show reduced chondrocyte marker expression and fail to repair articular cartilage defects.","method":"Adenoviral overexpression and siRNA knockdown of CCN4, SMAD2/3 phosphorylation Western blot, micromass culture chondrogenesis assay, immunoprecipitation-western blot for CCN4-TGF-beta3 binding, Ccn4-KO mouse cartilage defect model","journal":"Bone","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — direct binding shown by IP, Smad epistasis via OE/KD, in vivo KO with functional defect; multiple orthogonal methods","pmids":["26555637"],"is_preprint":false},{"year":2015,"finding":"WISP-1 promotes angiogenesis in oral squamous cell carcinoma by inducing VEGF-A expression via the integrin alphavbeta3/FAK/c-Src pathway, which transactivates EGFR/ERK/HIF-1alpha signaling. This in turn recruits endothelial progenitor cells and triggers neovascularization in vitro and in vivo.","method":"Integrin alphavbeta3 blocking, FAK/c-Src/EGFR/ERK/HIF-1alpha inhibitors and siRNA, EPC tube formation/migration assay, in vivo xenograft angiogenesis model","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple pathway inhibitors and siRNA with in vivo validation; single lab","pmids":["25738362"],"is_preprint":false},{"year":2016,"finding":"WISP1 is required for TGF-beta1- and TNF-alpha-dependent induction of IL-6 in primary human lung fibroblasts; siRNA-mediated WISP1 knockdown or antibody-mediated neutralization reduces fibroblast proliferation, which is partially rescued by exogenous IL-6, establishing a WISP1-IL-6 pro-proliferative axis.","method":"Luciferase reporter for WISP1 promoter, siRNA knockdown, antibody neutralization, ELISA for IL-6, proliferation assay, IL-6 rescue experiment","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA + neutralizing antibody + IL-6 rescue establishes causal chain; single lab","pmids":["26867691"],"is_preprint":false},{"year":2016,"finding":"WISP-1 promotes VEGF-C expression and lymphangiogenesis in OSCC cells via integrin alphavbeta3/ILK/Akt signaling, and this cascade inhibits miR-300 expression. Conditioned media from WISP-1-treated OSCC cells promotes tube formation and migration in lymphatic endothelial cells.","method":"Integrin alphavbeta3/ILK/Akt inhibitors, VEGF-C ELISA, lymphatic endothelial cell tube formation and migration assay, miR-300 expression analysis","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pathway inhibitors with functional lymphangiogenesis readout and miRNA regulation; single lab","pmids":["26824419"],"is_preprint":false},{"year":2016,"finding":"Wnt2 induces WISP-1 expression in vascular smooth muscle cells (VSMCs) via beta-catenin/TCF signaling; recombinant WISP-1 promotes VSMC migration in an integrin-dependent manner. WISP-1-/- mice show significantly suppressed intimal thickening after carotid artery ligation, and adenoviral elevation of plasma WISP-1 increases intimal thickening in vivo.","method":"siRNA knockdown of Wnt2 and WISP-1, VSMC migration assay, integrin dependence test, WISP-1-/- mouse carotid ligation model, adenoviral WISP-1 overexpression in vivo","journal":"Arteriosclerosis, thrombosis, and vascular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout mouse plus adenoviral gain-of-function in vivo, siRNA knockdown, integrin-dependent migration; multiple orthogonal in vitro and in vivo approaches","pmids":["27199447"],"is_preprint":false},{"year":2017,"finding":"Macrophage-derived IL-10, secreted following intestinal mucosal injury, activates epithelial CREB, which drives WISP-1 synthesis and secretion. WISP-1 then induces epithelial cell proliferation and wound closure. Conditional deletion of IL-10 in CD11c+ macrophages impairs wound closure, establishing an IL-10/CREB/WISP-1 signaling axis.","method":"Conditional CD11c-specific IL-10 knockout mice, Rag1-/- mice, in vitro CREB activation assay, WISP-1 protein measurement, wound closure assay, cell proliferation assay","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional knockout in vivo with mechanistic in vitro validation, multiple genetic models","pmids":["28783045"],"is_preprint":false},{"year":2017,"finding":"WISP-1 promotes VEGF-A expression and angiogenesis in osteosarcoma through FAK/JNK/HIF-1alpha signaling and downregulation of miR-381; FAK, JNK, and HIF-1alpha inhibitors or siRNA abolished WISP-1-induced VEGF-A expression and angiogenesis both in vitro and in vivo.","method":"FAK/JNK/HIF-1alpha inhibitors and siRNA, VEGF-A ELISA, EPC migration/tube formation assay, miR-381 expression analysis, in vivo xenograft angiogenesis model","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple pathway inhibitors and siRNA with in vivo validation; single lab","pmids":["28406476"],"is_preprint":false},{"year":2017,"finding":"WISP1/CCN4 inhibits adipocyte differentiation by repressing PPARgamma transcriptional activity; a direct physical association between WISP1 and PPARgamma was demonstrated, followed by proteasome-dependent degradation of PPARgamma. Silencing WISP1 enhanced adipocyte differentiation.","method":"Overexpression and siRNA knockdown in 3T3-F442A cells, PPARgamma transcriptional activity reporter, co-immunoprecipitation of WISP1 with PPARgamma, proteasome inhibitor treatment, adipogenic marker expression","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct co-IP of WISP1-PPARgamma plus proteasome inhibitor assay and gain/loss of function; single lab","pmids":["28496206"],"is_preprint":false},{"year":2018,"finding":"CCN4/WISP1 stimulates dermal fibroblast migration and proliferation via alpha5beta1 integrin receptors, activating ERK/JNK signaling downstream. CCN4 also counterbalances TNF-alpha-mediated inhibition of COL1A1 and FN and stimulation of MMPs by inhibiting NF-kappaB/p65 signaling. Ccn4-KO mice show delayed wound closure with reduced Col1a1 and fibronectin expression.","method":"Ccn4-KO mice, Boyden chamber migration assay, siRNA knockdown, adenoviral CCN4 transduction, integrin alpha5beta1 blocking antibody, ERK/JNK Western blot, NF-kappaB signaling assay, gene expression analysis","journal":"Matrix biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO mouse plus siRNA knockdown plus adenoviral OE plus integrin blocking; multiple orthogonal approaches across in vivo and in vitro systems","pmids":["29330021"],"is_preprint":false},{"year":2018,"finding":"Osteoblast-derived WISP-1 promotes adherence of prostate cancer cells to osteoblasts via a VCAM-1/integrin alpha4beta1 system: WISP-1 induces VCAM-1 expression in PCa cells and integrin alpha4beta1 expression in osteoblasts via the MAPK pathway; blocking VCAM-1 or integrin alpha4beta1 attenuates cancer cell adhesion. WISP-1 also induces BMP2, BMP4, and osteopontin in osteoblasts and inhibits RANKL-dependent osteoclastogenesis.","method":"VCAM-1 and integrin alpha4beta1 blocking antibodies, MAPK pathway analysis, BMP2/4/OPN expression assay, osteoclastogenesis assay","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — receptor blocking antibodies with adhesion readout and multiple molecular targets; single lab","pmids":["29627497"],"is_preprint":false},{"year":2018,"finding":"WISP1 impairs insulin action in human skeletal muscle cells and mouse hepatocytes by inhibiting phosphorylation of insulin receptor, Akt, GSK-3beta, FOXO1, and p70S6K, and by inhibiting insulin-stimulated glycogen synthesis and suppression of gluconeogenic genes.","method":"Recombinant WISP1 treatment of primary human skeletal muscle cells and AML12 hepatocytes, insulin signaling Western blot, glycogen synthesis assay, gluconeogenic gene expression","journal":"Diabetologia","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct recombinant protein treatment with multiple signaling and functional readouts in two cell types; single lab","pmids":["29754289"],"is_preprint":false},{"year":2018,"finding":"WISP1 contributes to sepsis-induced acute lung injury via a WISP1-alphavbeta3 integrin signaling pathway in macrophages: TLR4 activation induces de novo synthesis of alphavbeta3 via ERK (MyD88/NF-kappaB dependent), enabling WISP1 to stimulate TNF-alpha release. This effect was absent in beta3-/- macrophages.","method":"TLR4-/-, MyD88-/-, TRIF-/-, beta3-/- mouse macrophages, ERK inhibitor, siRNA knockdown, anti-WISP1/anti-integrin beta5 antibodies in vivo two-hit model","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic knockout macrophages identify integrin beta3 requirement, ERK pathway inhibition, in vivo antibody blockade; single lab","pmids":["27349568"],"is_preprint":false},{"year":2018,"finding":"In mechanical ventilation plus sepsis-induced acute lung injury, WISP1 levels are increased intrapulmonarily via TLR4 signaling; WISP1 signals through integrin beta5 to amplify TLR4-mediated inflammation. Neutralizing WISP1 or integrin beta5 antibodies partially inhibited the two-hit ALI phenotype. In macrophages, TLR4 increases integrin beta5 expression in a MyD88/NF-kappaB-dependent manner, and WISP1 requires integrin beta5 for enhanced cytokine release.","method":"TLR4-/-, MyD88-/-, TRIF-/- mice; anti-WISP1 and anti-integrin beta5 antibodies in vivo; siRNA knockdown of TLR4 and integrin beta5 in macrophages; cytokine ELISA","journal":"Critical care","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple genetic knockouts and antibody blockade in vivo plus siRNA in vitro; single lab","pmids":["30445996"],"is_preprint":false},{"year":2019,"finding":"WISP1 (CCN4) is a FAP-derived matricellular signal in the muscle stem cell niche that controls expansion and asymmetric commitment of muscle stem cells (MuSCs) through Akt signaling. Loss of WISP1 from aged fibro-adipogenic progenitors (FAPs) contributes to impaired MuSC function; transplantation of young FAPs or systemic WISP1 treatment rescues myogenic capacity in aged mice.","method":"Transcriptomic profiling, genetic mouse models, FAP transplantation, systemic WISP1 administration, Akt signaling Western blot, MuSC expansion and differentiation assays","journal":"Cell stem cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — FAP transplantation, systemic rescue, Akt pathway mechanism, in vivo muscle regeneration readout; multiple orthogonal approaches","pmids":["30686765"],"is_preprint":false},{"year":2019,"finding":"WISP1 (CCN4) directly binds type I collagen to promote its linearization in vitro (in the absence of cells) and in vivo in tumors, thereby facilitating cancer cell invasion and promoting spontaneous breast cancer metastasis. This collagen linearization is independent of cell-generated mechanical forces.","method":"Cell-free in vitro collagen linearization assay with recombinant WISP1, direct WISP1-collagen binding assay, in vivo tumor collagen imaging, breast cancer metastasis mouse model with WISP1 manipulation","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — cell-free in vitro reconstitution of collagen linearization, direct binding assay, in vivo tumor validation; rigorous mechanistic study","pmids":["31294477"],"is_preprint":false},{"year":2019,"finding":"WISP1 in glioblastoma signals through Integrin alpha6beta1-Akt to maintain glioma stem cells (GSCs) by autocrine mechanism, and promotes M2 tumor-associated macrophage survival by paracrine mechanism. WISP1 is regulated by Wnt/beta-catenin in GSCs, and carnosic acid inhibits this axis to suppress GBM growth.","method":"WISP1 silencing in GSCs, integrin alpha6beta1-Akt pathway analysis, autocrine/paracrine assays, GSC maintenance and TAM survival assays, carnosic acid treatment","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — receptor-pathway identification with silencing plus pharmacological inhibition; single lab","pmids":["32541784"],"is_preprint":false},{"year":2019,"finding":"WISP1 knockout in melanoma cells increases proliferation but represses migration, invasion, and metastasis. WISP1 promotes an EMT-like gene expression signature (E-cadherin repression, fibronectin/N-cadherin induction) by activating AKT and MEK/ERK signaling; WISP1 knockout-associated metastasis repression is reversed by reintroduction of WISP1 or SNAI1.","method":"WISP1 knockout (B16F10, YUMM1.7), transwell invasion/migration assays, gene expression profiling, recombinant WISP1 rescue, SNAI1 reintroduction, AKT/MEK/ERK kinase inhibitors, in vivo metastasis assays in C57BL/6 and NSG mice","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout with molecular rescue, pathway inhibitors, multiple in vivo metastasis models with two cell lines","pmids":["30723155"],"is_preprint":false},{"year":2019,"finding":"WISP-1 promotes EMT in OSCC cells via FAK/ILK/Akt and Snail signaling and downregulates miR-153-3p expression; the miR-153-3p/Snail axis mediates WISP-1-induced EMT.","method":"FAK/ILK/Akt inhibitors, Snail expression analysis, miR-153-3p expression measurement, EMT marker Western blot","journal":"Cancers","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pathway inhibitors plus miRNA-EMT axis characterization; single lab","pmids":["31795469"],"is_preprint":false},{"year":2020,"finding":"Wisp1 promotes proliferation of adult mouse and human pancreatic beta cells in vivo; the beta cell trophic effect is dependent on Akt signaling, as shown using isolated mouse and human islets. Wisp1 is more abundant in pre-weaning than adult mice serum.","method":"In vivo Wisp1 administration, transplanted beta cell proliferation assay, isolated islet culture with Wisp1, Akt pathway inhibition, ELISA for circulating Wisp1","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo and ex vivo beta cell proliferation with Akt inhibition epistasis; single lab but cross-species validation","pmids":["33239617"],"is_preprint":false},{"year":2020,"finding":"FAT10 (a ubiquitin-like protein) degrades WISP1 protein via FAT10ylation (direct post-translational modification), while simultaneously stabilizing beta-catenin to increase WISP1 mRNA, creating a protein/mRNA expression discordance. FAT10 overexpression-induced WISP1 protein decrease promotes hepatocellular carcinoma proliferation.","method":"FAT10 overexpression and knockdown, co-immunoprecipitation for FAT10-WISP1 interaction, proteasome inhibitor experiments, beta-catenin stabilization assay, WISP1 mRNA and protein quantification, in vitro and in vivo HCC proliferation assays","journal":"Hepatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP demonstrating FAT10ylation, proteasome inhibitor validation, in vivo evidence; single lab","pmids":["29790184"],"is_preprint":false},{"year":2021,"finding":"WISP-1 promotes fibrosis and hypertrophy of the ligamentum flavum by activating the Hedgehog-Gli1 signaling pathway in fibroblasts; mechanical stress increases WISP-1 expression, and WISP-1 induces myofibroblast transition via Hedgehog signaling. Cyclopamine (Hedgehog inhibitor) attenuated WISP-1-induced fibrogenesis in vitro and in the in vivo mechanical stress rabbit model.","method":"Mechanical stretch, WISP-1 treatment of LF fibroblasts, Hedgehog-Gli1 pathway Western blot/reporter, cyclopamine inhibition, alpha-SMA expression, in vivo rabbit mechanical stress model","journal":"Experimental & molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pathway inhibitor epistasis in vitro and in vivo; single lab","pmids":["34158608"],"is_preprint":false},{"year":2022,"finding":"WISP1 drives liver fibrosis progression (not onset) through integrin-dependent activation of MRTF (myocardin-related transcription factor), leading to transcriptional reprogramming of myofibroblast cytoskeleton and motility. WISP1-deficient mice are protected against fibrosis progression; a therapeutic anti-WISP1 antibody halted progression of established fibrosis in NASH models.","method":"Wisp1-/- mice, NASH fibrosis mouse models, anti-WISP1 antibody treatment, MRTF pathway analysis, integrin signaling assay, myofibroblast cytoskeleton and motility readouts","journal":"Cell metabolism","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO plus therapeutic antibody in established fibrosis models, MRTF pathway mechanism identified; multiple in vivo models","pmids":["35987202"],"is_preprint":false},{"year":2022,"finding":"Apoptotic cancer cell-derived signals activate Notch1 signaling in cancer-associated fibroblasts (CAFs) via the DLL1/BAI1-Rac1 efferocytosis pathway, inducing WISP-1 secretion which suppresses CAF activation and inhibits cancer cell migration/invasion and lung metastasis. Notch1 inhibition or siRNA blocked WISP-1 production and reversed the antimetastatic effects.","method":"Pharmacological Notch1 inhibition, siRNA Notch1 silencing, WISP-1 immunodepletion from conditioned media, migration/invasion assays, syngeneic mouse lung metastasis model, CAF marker expression assay","journal":"Cellular & molecular immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Notch1 siRNA/inhibitor plus WISP-1 immunodepletion with in vivo metastasis readout; single lab","pmids":["36241874"],"is_preprint":false},{"year":2015,"finding":"WISP-1 full-length protein and C-terminal domain-containing truncations (VWC, TSP, CT domains) promote epithelial cell adhesion in an integrin-dependent manner (partially mediated by alphavbeta5, alphavbeta3, and beta1 integrins). Beta-catenin activation and CXCL3 secretion also reside within the C-terminal domains of WISP-1 but are not blocked by integrin neutralizing antibodies. Native WISP-1 from human lung tissue exists as a high-order oligomer.","method":"Baculovirus expression of truncated WISP-1 proteins, cell adhesion assay, integrin-blocking antibodies, beta-catenin reporter assay, CXCL3 ELISA, size-exclusion analysis of native protein","journal":"Journal of cell communication and signaling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain dissection with recombinant truncations, integrin blocking, beta-catenin reporter; single lab","pmids":["25657057"],"is_preprint":false},{"year":2012,"finding":"Notch1 activation in fibroblasts induces WISP-1 expression in a Wnt11-dependent (but Wnt1-independent) manner; WISP-1 suppresses fibroblast proliferation and mediates Notch1's growth-inhibitory effect, since siRNA blockade of WISP-1 relieves Notch1-induced growth inhibition.","method":"Notch1 knockout MEFs, NICD1 overexpression, DN-MAML-1, Wnt11 knockdown, WISP-1 siRNA, cell growth and motility assays","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic Notch1 knockout and rescue, Wnt11 knockdown epistasis, WISP-1 siRNA epistasis; single lab","pmids":["22715413"],"is_preprint":false},{"year":2018,"finding":"WISP1 (Wisp-1) is a cardiac angiokine whose transcription is regulated by HDACs; HDAC inhibition with SAHA post-MI elevates Wisp-1 expression in the border zone. Human coronary artery endothelial cells produce WISP-1 and respond to autocrine WISP-1 signaling that promotes their pro-angiogenic behavior; altering endogenous WISP-1 in HCAECs directly impacts network density in vitro.","method":"HDAC inhibitor (SAHA) treatment, post-MI mouse model, WISP-1 expression measurement, HCAEC WISP-1 knockdown/overexpression, endothelial network formation assay","journal":"JCI insight","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — HDAC regulation of WISP-1 transcription, WISP-1 gain/loss in endothelial cells with functional angiogenesis readout; single lab","pmids":["29467324"],"is_preprint":false},{"year":2017,"finding":"WISP1 in osteoarthritis promotes expression of MMP3, MMP9, ADAMTS4, and ADAMTS5 in synovial tissue (increasing cartilage catabolism). Wisp1-/- mice show significantly decreased cartilage degeneration in three independent experimental OA models (collagenase injection, DMM, ACLT), with reduced protease expression and decreased aggrecan cleavage (NITEGE neoepitope). Aging-related spontaneous cartilage degeneration was not decreased in Wisp1-/- mice.","method":"Wisp1-/- knockout mice, three independent OA induction models, qRT-PCR for proteases, NITEGE immunohistochemistry, micro-CT, recombinant WISP1 stimulation of human OA synovium","journal":"Osteoarthritis and cartilage","confidence":"High","confidence_rationale":"Tier 2 / Strong — constitutive KO validated in three independent experimental OA models plus direct human tissue stimulation; replicated across models","pmids":["28735021"],"is_preprint":false},{"year":2009,"finding":"Nitric oxide (NO) increases WISP-1 expression in colonic epithelial cells through a beta-catenin- and CREB-dependent, but Wnt-1-independent, pathway. NO-induced WISP-1 then directly induces soluble collagen secretion in colonic fibroblast cells. In iNOS-/- mice, colitis-related WISP-1 increase was significantly attenuated.","method":"NO donor (DETA-NONOate) treatment, beta-catenin and CREB pathway inhibition, WISP-1 mRNA/protein measurement, collagen secretion assay in fibroblasts, iNOS-/- mouse colitis models","journal":"Journal of molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological pathway inhibition with iNOS-/- genetic validation in vivo; single lab","pmids":["19238344"],"is_preprint":false},{"year":2015,"finding":"Wisp1 mediates Bmp3-stimulated mesenchymal stem cell (MSC) proliferation: Bmp3 upregulates Wisp1 expression prior to MSC proliferation, recombinant Wisp1 stimulates MSC proliferation in a concentration-dependent manner, and siRNA-mediated Wisp1 knockdown attenuates Bmp3-induced proliferation.","method":"DNA microarray, recombinant Wisp1 treatment, [3H]-thymidine incorporation, siRNA knockdown of Wisp1, Bmp3 treatment","journal":"Journal of molecular endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA knockdown epistasis, recombinant protein gain-of-function, temporal expression analysis; single lab","pmids":["26489765"],"is_preprint":false},{"year":2007,"finding":"CCN4/WISP1 splice variants are expressed in chondrocytes: full-length WISP1, WISP1v (lacking the second module/VWC domain), and a novel WISP1vx (lacking second, third modules and part of first). WISP1v expression increases dramatically during terminal chondrocyte differentiation and overexpression of WISP1v promotes alkaline phosphatase gene expression (a marker of terminal differentiation), suggesting WISP1v plays a role in endochondral ossification.","method":"RT-PCR characterization of splice variants, primary chondrocyte differentiation culture, WISP1v overexpression, alkaline phosphatase gene expression assay","journal":"The FEBS journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain deletion via natural splice variant with gain-of-function differentiation assay; single lab","pmids":["17381509"],"is_preprint":false},{"year":2018,"finding":"WISP1 promotes non-alcoholic fatty liver disease and skeletal muscle insulin resistance through a TLR4-activated inflammation/JNK signaling pathway; WISP1 increases JNK phosphorylation, NF-kappaB-dependent inflammation, lipogenesis, and triglyceride accumulation in hepatocytes and suppresses insulin signaling in muscle cells. These effects were abrogated by NF-kappaB, JNK, and TLR4 knockdown.","method":"WISP1 treatment of mouse hepatocytes and C2C12 cells, NF-kappaB/JNK/TLR4 siRNA knockdown, Western blot for insulin signaling, triglyceride accumulation assay, in vivo WISP1 knockdown in HFD mice","journal":"Journal of cellular physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA epistasis for three pathway components plus in vivo knockdown; single lab","pmids":["29319180"],"is_preprint":false},{"year":2016,"finding":"WISP1 overexpression stimulates proliferation and migration of human VSMCs via AKT signaling; WISP1 increases GSK3alpha/beta phosphorylation, cyclin D1, and MMP9 expression. AKT inhibitor (AZD5363) and PI3K inhibitor (LY294002) abolished WISP1-induced proliferation and migration and downstream target expression.","method":"Adenoviral WISP1 overexpression, EdU incorporation assay, scratch wound healing, transwell migration, AKT/PI3K inhibition, Western blot for cyclin D1/MMP9/GSK3","journal":"European journal of pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — adenoviral OE plus pharmacological AKT/PI3K inhibition with multiple functional readouts; single lab","pmids":["27321870"],"is_preprint":false}],"current_model":"CCN4/WISP1 is a secreted matricellular protein transcriptionally activated by Wnt/beta-catenin (via a CREB-dependent promoter element) that exerts pleiotropic effects by binding extracellular partners—including decorin, biglycan, BMP-2, TGF-beta3, and type I collagen—and signaling through integrins (alpha5beta1, alphavbeta3, alphavbeta5, alpha6beta1, alpha4beta1) to activate intracellular cascades including PI3K/Akt, ERK/MEK, NF-kappaB, CREB, and MRTF, thereby promoting cell survival (via Bcl-xL/cytochrome c and FoxO3a/PRAS40/mTOR), proliferation, migration, osteoblast/chondrocyte differentiation (by enhancing BMP-2 and TGF-beta3 signaling), muscle stem cell expansion (via Akt), collagen linearization and metastasis, and fibrosis progression (via the WISP1–MRTF axis), while its activity is post-transcriptionally regulated by miR-92a and post-translationally by FAT10ylation-dependent proteasomal degradation."},"narrative":{"mechanistic_narrative":"CCN4 (WISP1) is a secreted matricellular protein, transcriptionally activated downstream of Wnt/beta-catenin through a CREB-dependent promoter element, that integrates extracellular cues into cell-survival, proliferation, differentiation, migration, and fibrotic programs [PMID:10716946, PMID:22475393, PMID:19238344]. It binds extracellular matrix components and growth factors directly—dermatan-sulfate proteoglycans decorin and biglycan that anchor it to the cell surface [PMID:11598131], BMP-2 and TGF-beta3 whose Smad signaling it potentiates to drive osteoblast and chondrocyte differentiation [PMID:20684029, PMID:26555637], and type I collagen, which it linearizes in a cell-force-independent manner to facilitate invasion and breast cancer metastasis [PMID:31294477]. CCN4 signals predominantly through integrins—alpha5beta1, alphavbeta3, alphavbeta5, alpha6beta1, and alpha4beta1—to engage FAK, MEK/ERK, PI3K/Akt, NF-kappaB, and AP-1 cascades, thereby inducing proteases, adhesion molecules, and angiogenic factors that promote tumor cell migration, monocyte/macrophage recruitment, and neovascularization [PMID:21453685, PMID:23343403, PMID:23313051, PMID:25738362, PMID:29627497]. A recurrent pro-survival module operates through PI3K/Akt: CCN4 raises the Bcl-xL/Bax ratio, blocks mitochondrial cytochrome c release, and phosphorylates and sequesters FoxO3a and PRAS40 with 14-3-3 to sustain mTOR signaling, protecting neurons, microglia, and cardiomyocytes from apoptotic and oxidant stress [PMID:11782444, PMID:22272766, PMID:23151077, PMID:22873724]. In tissue homeostasis, CCN4 maintains Wnt signaling in bone by blocking SOST binding to LRP6, supporting bone mineral density [PMID:25864198], and acts as a niche factor that drives muscle stem cell expansion via Akt [PMID:30686765]. Genetic loss and antibody-blockade studies establish CCN4 as a driver of fibrosis progression, signaling through integrin-dependent activation of MRTF to reprogram the myofibroblast cytoskeleton in liver fibrosis and through Hedgehog-Gli1 in ligamentum flavum fibrosis [PMID:35987202, PMID:34158608]. Its expression is post-transcriptionally repressed by miR-92a and its protein is degraded through FAT10ylation-dependent proteasomal turnover [PMID:24953558, PMID:29790184].","teleology":[{"year":2000,"claim":"Established CCN4 as a Wnt/beta-catenin target and a transforming gene, defining the upstream signal that drives its expression and its oncogenic potential.","evidence":"Promoter dissection with reporter assays and overexpression with nude mouse xenograft in NRK-49F cells","pmids":["10716946"],"confidence":"High","gaps":["CREB versus TCF/LEF contribution not resolved at single-element resolution","Did not identify the secreted protein's receptors or extracellular partners"]},{"year":2001,"claim":"Identified the first direct extracellular binding partners, showing CCN4 docks to the cell surface via dermatan sulfate proteoglycans.","evidence":"Solid-phase and direct binding assays with purified decorin and biglycan plus glycosaminoglycan competition and mass spectrometry","pmids":["11598131"],"confidence":"High","gaps":["Functional consequence of proteoglycan binding for downstream signaling not defined","Binding domain on CCN4 not mapped"]},{"year":2002,"claim":"Defined the core pro-survival mechanism, showing CCN4 acts through Akt to block intrinsic (but not Fas-mediated) apoptosis.","evidence":"Apoptosis assays, cytochrome c release, Bcl-xL Western blot and PI3K/Akt inhibition","pmids":["11782444"],"confidence":"High","gaps":["Receptor coupling CCN4 to PI3K/Akt not identified","Whether survival effect is autocrine or paracrine unresolved"]},{"year":2004,"claim":"Connected CCN4 to BMP and skeletal biology, showing it potentiates BMP-2-driven osteoblast differentiation while restraining chondrocyte maturation.","evidence":"In situ hybridization, stable transfection of chondrocytic lines, osteoblast differentiation assays and in vivo fracture repair","pmids":["15331410"],"confidence":"Medium","gaps":["Direct BMP-2 binding not yet demonstrated at this stage","Opposing chondrocyte versus osteoblast effects mechanistically unexplained"]},{"year":2011,"claim":"Demonstrated direct BMP-2 binding and integrin alpha5beta1 dependence, establishing CCN4 as a co-receptor-like enhancer of BMP/Smad osteogenesis in vivo.","evidence":"Immunoprecipitation, adenoviral overexpression, shRNA, integrin-blocking antibody and transgenic mouse bone density analysis","pmids":["20684029"],"confidence":"High","gaps":["Structural basis of the BMP-2/integrin/CCN4 ternary complex unknown"]},{"year":2015,"claim":"Showed CCN4 directly binds TGF-beta3 to drive chondrogenesis and that knockout mice fail cartilage repair, extending its growth-factor-potentiating role to TGF-beta.","evidence":"IP-Western for CCN4-TGF-beta3 binding, OE/KD Smad2/3 epistasis, micromass chondrogenesis and Ccn4-KO cartilage defect model","pmids":["26555637"],"confidence":"High","gaps":["Reconciliation with earlier report of CCN4 suppressing TGF-beta1/Smad2 signaling not addressed"]},{"year":2015,"claim":"Established CCN4 as a positive regulator of bone mass acting upstream of Wnt by blocking the SOST-LRP6 inhibitory interaction.","evidence":"Wisp1-/- mice with DXA, micro-CT, biomechanics, differentiation assays and SOST-LRP6 binding inhibition assay","pmids":["25864198"],"confidence":"High","gaps":["Whether SOST competition is direct or matrix-mediated not fully resolved"]},{"year":2013,"claim":"Mapped the integrin-to-protease signaling logic in cancer cells, linking specific integrin heterodimers to MMP induction, adhesion molecules, and migration.","evidence":"Integrin-blocking antibodies, kinase inhibitors, NF-kappaB/AP-1 reporters, ChIP and migration/adhesion assays across chondrosarcoma, synovial fibroblast and OSCC systems","pmids":["21453685","23343403","23313051","24036215","24205072"],"confidence":"Medium","gaps":["Cell-type basis for integrin heterodimer selectivity unexplained","Most readouts from single labs with overexpression/recombinant protein"]},{"year":2013,"claim":"Detailed the neuronal/microglial cytoprotection module, showing CCN4 sequesters FoxO3a and PRAS40 via 14-3-3 and tunes TSC2 to sustain mTOR-dependent survival.","evidence":"PI3K/Akt inhibition, co-IP with 14-3-3, subcellular fractionation, gene silencing of PRAS40/TSC2 and apoptosis assays in primary neurons and microglia","pmids":["22272766","23151077","22873724","23244622"],"confidence":"Medium","gaps":["Upstream receptor coupling CCN4 to Akt in neural cells not identified","All from a single lab"]},{"year":2019,"claim":"Established cell-force-independent collagen linearization as a direct biophysical CCN4 function driving metastasis.","evidence":"Cell-free in vitro collagen linearization, direct binding assay and in vivo breast cancer metastasis model","pmids":["31294477"],"confidence":"High","gaps":["Molecular mechanism by which CCN4 reorganizes collagen fibrils unresolved"]},{"year":2019,"claim":"Identified CCN4 as a FAP-derived niche signal controlling muscle stem cell expansion via Akt, linking its decline to aging-impaired regeneration.","evidence":"Transcriptomics, genetic mouse models, FAP transplantation, systemic WISP1 rescue and Akt pathway analysis","pmids":["30686765"],"confidence":"High","gaps":["Receptor on muscle stem cells mediating the Akt effect not defined"]},{"year":2022,"claim":"Defined CCN4 as a driver of fibrosis progression acting through integrin-dependent MRTF activation, validated by genetic loss and therapeutic antibody blockade.","evidence":"Wisp1-/- mice, NASH fibrosis models, anti-WISP1 antibody, MRTF and integrin signaling analysis","pmids":["35987202"],"confidence":"High","gaps":["Specific integrin and intracellular link to MRTF not pinpointed","Whether the same axis operates in non-hepatic fibrosis unclear"]},{"year":2018,"claim":"Identified post-transcriptional (miR-92a) and post-translational (FAT10ylation) controls limiting CCN4 abundance, explaining mRNA/protein discordance.","evidence":"miR-92a mimic/inhibitor with fibrosis model, and FAT10 OE/KD with co-IP and proteasome inhibitor experiments","pmids":["24953558","29790184"],"confidence":"Medium","gaps":["FAT10ylation site on CCN4 not mapped","Both from single labs without independent replication"]},{"year":null,"claim":"How CCN4's distinct integrin engagements, growth-factor binding, and direct matrix remodeling are coordinated into context-specific outputs, and what defines its principal signaling receptor, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of CCN4 with any integrin or growth factor","Determinants of integrin heterodimer selectivity across tissues unknown","Reconciliation of pro- versus anti-tumor/fibrotic roles across contexts incomplete"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[5,7,22]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[36]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[8,15,30]},{"term_id":"GO:0048018","term_label":"receptor ligand activity","supporting_discovery_ids":[35,40]}],"localization":[{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[2,19,36]},{"term_id":"GO:0031012","term_label":"extracellular matrix","supporting_discovery_ids":[2,36]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[8,15,30,37]},{"term_id":"R-HSA-1474244","term_label":"Extracellular matrix organization","supporting_discovery_ids":[36,30]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[7,21,22,35]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[1,10,11]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[15,16,33,34]}],"complexes":[],"partners":["DCN","BGN","BMP2","TGFB3","COL1A1","ITGA5","ITGB5","PPARG"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O95388","full_name":"CCN family member 4","aliases":["WNT1-inducible-signaling pathway protein 1","WISP-1","Wnt-1-induced secreted protein"],"length_aa":367,"mass_kda":40.3,"function":"Downstream regulator in the Wnt/Frizzled-signaling pathway. Associated with cell survival. Attenuates p53-mediated apoptosis in response to DNA damage through activation of AKT kinase. Up-regulates the anti-apoptotic Bcl-X(L) protein. Adheres to skin and melanoma fibroblasts. In vitro binding to skin fibroblasts occurs through the proteoglycans, decorin and biglycan","subcellular_location":"Secreted","url":"https://www.uniprot.org/uniprotkb/O95388/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CCN4","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CCN4","total_profiled":1310},"omim":[{"mim_id":"603398","title":"CELLULAR COMMUNICATION NETWORK FACTOR 4; CCN4","url":"https://www.omim.org/entry/603398"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Cytosol","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"ovary","ntpm":18.8}],"url":"https://www.proteinatlas.org/search/CCN4"},"hgnc":{"alias_symbol":["WISP-1"],"prev_symbol":["WISP1-OT1","WISP1-UT1","WISP1"]},"alphafold":{"accession":"O95388","domains":[{"cath_id":"4.10.40.20","chopping":"47-118","consensus_level":"medium","plddt":91.2717,"start":47,"end":118},{"cath_id":"2.10.70","chopping":"122-154","consensus_level":"high","plddt":87.3315,"start":122,"end":154},{"cath_id":"-","chopping":"161-187","consensus_level":"medium","plddt":77.1319,"start":161,"end":187},{"cath_id":"2.10.90","chopping":"231-342","consensus_level":"high","plddt":91.3464,"start":231,"end":342}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O95388","model_url":"https://alphafold.ebi.ac.uk/files/AF-O95388-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O95388-F1-predicted_aligned_error_v6.png","plddt_mean":77.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CCN4","jax_strain_url":"https://www.jax.org/strain/search?query=CCN4"},"sequence":{"accession":"O95388","fasta_url":"https://rest.uniprot.org/uniprotkb/O95388.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O95388/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O95388"}},"corpus_meta":[{"pmid":"10716946","id":"PMC_10716946","title":"WISP-1 is a Wnt-1- and beta-catenin-responsive oncogene.","date":"2000","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/10716946","citation_count":274,"is_preprint":false},{"pmid":"11751417","id":"PMC_11751417","title":"Elevated levels of connective tissue growth factor, WISP-1, and CYR61 in primary breast cancers associated with more advanced features.","date":"2001","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/11751417","citation_count":269,"is_preprint":false},{"pmid":"30686765","id":"PMC_30686765","title":"Aging Disrupts Muscle Stem Cell Function by Impairing Matricellular WISP1 Secretion from Fibro-Adipogenic Progenitors.","date":"2019","source":"Cell stem cell","url":"https://pubmed.ncbi.nlm.nih.gov/30686765","citation_count":255,"is_preprint":false},{"pmid":"28783045","id":"PMC_28783045","title":"Macrophage-derived IL-10 mediates mucosal repair by epithelial WISP-1 signaling.","date":"2017","source":"The Journal of clinical investigation","url":"https://pubmed.ncbi.nlm.nih.gov/28783045","citation_count":168,"is_preprint":false},{"pmid":"32541784","id":"PMC_32541784","title":"Dual Role of WISP1 in maintaining glioma stem cells and tumor-supportive macrophages in glioblastoma.","date":"2020","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/32541784","citation_count":140,"is_preprint":false},{"pmid":"11782444","id":"PMC_11782444","title":"WISP-1 attenuates p53-mediated apoptosis in response to DNA damage through activation of the Akt kinase.","date":"2002","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/11782444","citation_count":134,"is_preprint":false},{"pmid":"15331410","id":"PMC_15331410","title":"WISP-1 is an osteoblastic regulator expressed during skeletal development and fracture repair.","date":"2004","source":"The American journal of pathology","url":"https://pubmed.ncbi.nlm.nih.gov/15331410","citation_count":130,"is_preprint":false},{"pmid":"17579708","id":"PMC_17579708","title":"Expression of Cyr61, CTGF, and WISP-1 correlates with clinical features of lung cancer.","date":"2007","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/17579708","citation_count":125,"is_preprint":false},{"pmid":"20684029","id":"PMC_20684029","title":"WISP-1/CCN4 regulates osteogenesis by enhancing BMP-2 activity.","date":"2011","source":"Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research","url":"https://pubmed.ncbi.nlm.nih.gov/20684029","citation_count":121,"is_preprint":false},{"pmid":"25281430","id":"PMC_25281430","title":"WISP1 is a novel adipokine linked to inflammation in obesity.","date":"2014","source":"Diabetes","url":"https://pubmed.ncbi.nlm.nih.gov/25281430","citation_count":105,"is_preprint":false},{"pmid":"19339243","id":"PMC_19339243","title":"WISP1, a pro-mitogenic, pro-survival factor, mediates tumor necrosis factor-alpha (TNF-alpha)-stimulated cardiac fibroblast proliferation but inhibits TNF-alpha-induced cardiomyocyte death.","date":"2009","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19339243","citation_count":104,"is_preprint":false},{"pmid":"11598131","id":"PMC_11598131","title":"WISP-1 binds to decorin and biglycan.","date":"2001","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11598131","citation_count":101,"is_preprint":false},{"pmid":"21109017","id":"PMC_21109017","title":"WNT1 inducible signaling pathway protein 1 (WISP1): a novel mediator linking development and disease.","date":"2010","source":"The international journal of biochemistry & cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/21109017","citation_count":98,"is_preprint":false},{"pmid":"12529380","id":"PMC_12529380","title":"Overexpression of WISP-1 down-regulated motility and invasion of lung cancer cells through inhibition of Rac activation.","date":"2003","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12529380","citation_count":97,"is_preprint":false},{"pmid":"25219658","id":"PMC_25219658","title":"WISP1: Clinical insights for a proliferative and restorative member of the CCN family.","date":"2014","source":"Current neurovascular research","url":"https://pubmed.ncbi.nlm.nih.gov/25219658","citation_count":82,"is_preprint":false},{"pmid":"25794425","id":"PMC_25794425","title":"CCN4/WISP1 (WNT1 inducible signaling pathway protein 1): a focus on its role in cancer.","date":"2015","source":"The international journal of biochemistry & cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/25794425","citation_count":80,"is_preprint":false},{"pmid":"24953558","id":"PMC_24953558","title":"miR-92a regulates TGF-β1-induced WISP1 expression in pulmonary fibrosis.","date":"2014","source":"The international journal of biochemistry & 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Overexpression in NRK-49F cells induces morphological transformation, accelerated cell growth, and tumor formation in nude mice.\",\n      \"method\": \"Promoter cloning and reporter assay; overexpression in NRK-49F cells with nude mouse xenograft\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — promoter functional dissection with reporter assays plus in vivo tumor formation, independently replicated by subsequent studies confirming CREB-dependent regulation\",\n      \"pmids\": [\"10716946\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"WISP-1 (CCN4) activates the Akt/PKB anti-apoptotic signaling pathway and prevents p53-dependent cell death following DNA damage by inhibiting mitochondrial cytochrome c release and up-regulating Bcl-X(L); it does not protect against Fas-ligand-activated cell death.\",\n      \"method\": \"Cell-based apoptosis assays with WISP-1 expression, cytochrome c release measurement, Western blot for Bcl-X(L), PI3K/Akt pathway inhibition\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (apoptosis assay, cytochrome c release, Bcl-X(L) upregulation, pathway-specific inhibitors) in a single rigorous study; negative result for Fas pathway also established\",\n      \"pmids\": [\"11782444\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"WISP-1 (CCN4) directly binds to the dermatan sulfate proteoglycans decorin and biglycan, and this interaction mediates and modulates WISP-1 binding to the surface of fibroblasts. Competitive inhibition with dermatan sulfate and enzymatic removal of dermatan sulfate from the cell surface blocked WISP-1 binding.\",\n      \"method\": \"Solid-phase binding assay, competitive inhibition with glycosaminoglycans and lyases, mass spectrometric identification of binding factors, direct binding assay with purified decorin and biglycan\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct biochemical binding assay with purified proteins, mass spectrometric identification, multiple competitive inhibition experiments\",\n      \"pmids\": [\"11598131\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Overexpression of WISP-1 in H460 lung cancer cells inhibits Rac GTPase activation downstream of integrins (alpha-v-beta-5 and alpha-1), reduces MMP-1 expression, and suppresses cell invasion and lung metastasis. Blocking antibodies to alpha(v)beta(5) and alpha(1) integrins restored Rac activation in WISP-1-expressing cells.\",\n      \"method\": \"Rac activation pull-down assay, blocking antibody experiments, constitutively active Rac rescue (RacG12V), microarray/RT-PCR, Matrigel invasion assay, in vivo lung metastasis model\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — epistasis via dominant active Rac rescue, integrin blocking antibodies, multiple orthogonal functional readouts including in vivo metastasis\",\n      \"pmids\": [\"12529380\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"WISP-1 (CCN4) promotes BMP-2-induced osteoblastic differentiation during embryonic skeletogenesis and fracture repair. In vitro, WISP-1 expression in differentiating osteoblasts potentiates BMP-2 activity; it increases proliferation and saturation density in chondrocytes but represses their differentiation.\",\n      \"method\": \"In situ hybridization, cell binding analysis, stable transfection of chondrocytic cell lines, osteoblast differentiation assays, in vivo fracture repair model\",\n      \"journal\": \"The American journal of pathology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell-based assays with stable transfection and in vivo fracture model, single lab\",\n      \"pmids\": [\"15331410\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"WISP-1 (CCN4) directly regulates TGF-beta1 signaling in human bone marrow stromal cells: recombinant WISP-1 significantly reduces TGF-beta1-induced Smad-2 phosphorylation. Full-length WISP-1 and the splice variant WISP-1va (lacking the putative BMP/TGF-beta binding site) both promote proliferation; however, only full-length WISP-1 modulates osteogenic differentiation in concert with TGF-beta1.\",\n      \"method\": \"Recombinant protein treatment, BrdU incorporation assay, alkaline phosphatase activity, Western blot for Smad-2 phosphorylation, transfection of full-length vs. splice variant\",\n      \"journal\": \"Journal of cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct biochemical readout (Smad-2 phosphorylation), domain comparison via splice variant, single lab\",\n      \"pmids\": [\"18404666\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"TNF-alpha induces WISP1 expression in primary human cardiac fibroblasts through an ERK1/2-dependent CREB phosphorylation and transcriptional activation mechanism. WISP1 knockdown attenuates TNF-alpha-stimulated collagen production and cardiac fibroblast proliferation. WISP1 also protects cardiomyocytes from TNF-alpha-mediated death.\",\n      \"method\": \"ERK1/2 inhibition, CREB knockdown, siRNA knockdown of WISP1, reporter gene assays, CREB DNA-binding assay, cell proliferation and collagen production assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods: pathway inhibitors, siRNA knockdown, reporter assay, DNA-binding assay, phenotypic readouts in primary cells\",\n      \"pmids\": [\"19339243\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"WISP-1/CCN4 directly binds to BMP-2 and enhances BMP-2 binding to human bone marrow stromal cells (hBMSCs) in a dose-dependent manner, stimulating Smad-1/5/8 phosphorylation and osteogenic differentiation. WISP-1 also induces alpha5-integrin expression and binds to it; anti-integrin alpha5beta1 antibody completely neutralizes WISP-1-enhanced BMP-2 binding. Transgenic WISP-1 overexpression in preosteoblasts increased bone mineral density in vivo.\",\n      \"method\": \"Immunoprecipitation, Western blot, adenoviral overexpression, lentiviral shRNA knockdown, ectopic osteogenesis assay, integrin-blocking antibody experiments, transgenic mouse analysis\",\n      \"journal\": \"Journal of bone and mineral research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — direct binding shown by IP, in vitro and in vivo osteogenesis assays, antibody blocking, transgenic mouse validation; multiple orthogonal methods in one study\",\n      \"pmids\": [\"20684029\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"WISP-1 increases MMP-2 expression and cell migration in human chondrosarcoma cells through alpha5beta1 integrin receptor signaling, activating FAK, MEK/ERK, and NF-kappaB; NF-kappaB binds to the MMP-2 promoter. Blocking alpha5beta1 antibody and MEK inhibitors abolished WISP-1-induced MMP-2 upregulation and migration.\",\n      \"method\": \"Migration assay, Western blot for FAK/MEK/ERK phosphorylation, NF-kappaB luciferase reporter, chromatin immunoprecipitation (p65 binding to MMP-2 promoter), integrin-blocking antibody\",\n      \"journal\": \"Biochemical pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple pathway inhibitors plus promoter binding assay, single lab\",\n      \"pmids\": [\"21453685\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Angiotensin-II induces WISP1 expression in cardiomyocytes via AT1 receptor and NOX2-generated superoxide, activating Akt/GSK3beta/beta-catenin/TCF/LEF and CREB signaling. AT1 physically associates with NOX2 in vitro and in vivo. WISP1 knockdown reduces Ang-II-induced cardiomyocyte hypertrophy.\",\n      \"method\": \"AT1 antagonist (losartan), NOX2 knockdown, siRNA knockdown of WISP1, co-immunoprecipitation of AT1-NOX2, Western blot, in vivo Ang-II infusion model\",\n      \"journal\": \"Journal of molecular and cellular cardiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP of AT1-NOX2, pathway inhibitors, siRNA knockdown with hypertrophy readout; single lab\",\n      \"pmids\": [\"21376054\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"WISP1 (CCN4) protects primary hippocampal neurons from oxidant stress-induced apoptosis by activating PI3K/Akt1 signaling, phosphorylating Bad and GSK-3beta, minimizing Bim/Bax complex while increasing Bcl-xL/Bax ratio, and preventing mitochondrial membrane permeabilization, cytochrome c release, and caspase-3 activation.\",\n      \"method\": \"Primary neuron cultures, PI3K and Akt1 pathway inhibition, Western blot for apoptotic proteins, mitochondrial membrane permeability assay, cytochrome c release assay, caspase-3 activity assay\",\n      \"journal\": \"Current neurovascular research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pathway inhibitor epistasis with multiple apoptotic readouts, single lab\",\n      \"pmids\": [\"22272766\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"WISP1 (CCN4) prevents FoxO3a-mediated caspase-1 and caspase-3 apoptotic death in neurons via PI3K/Akt1-dependent phosphorylation and cytoplasmic sequestration of FoxO3a with 14-3-3 protein, and maintains protective SIRT1 activity through SIRT1 nuclear trafficking and prevention of caspase-mediated SIRT1 degradation.\",\n      \"method\": \"PI3K/Akt1 inhibition, FoxO3a phosphorylation Western blot, subcellular fractionation, co-immunoprecipitation with 14-3-3, caspase-1/3 activity assays, SIRT1 localization\",\n      \"journal\": \"Current neurovascular research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple biochemical readouts with pathway inhibitors and co-IP, single lab\",\n      \"pmids\": [\"23151077\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"WISP1 (CCN4) autoregulates its own expression by promoting beta-catenin activity and nuclear trafficking, and inhibits GSK-3beta activity post-translationally to maintain beta-catenin integrity in primary neurons during oxidant stress.\",\n      \"method\": \"WISP1 expression analysis, Akt1 activation assay, GSK-3beta phosphorylation Western blot, beta-catenin subcellular fractionation/nuclear translocation assay\",\n      \"journal\": \"Current neurovascular research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — subcellular fractionation showing beta-catenin trafficking, Western blot for GSK-3beta phosphorylation, single lab\",\n      \"pmids\": [\"22475393\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"WISP1 (CCN4) activates mTOR and phosphorylates p70S6K and 4EBP1 via control of PRAS40; WISP1 sequesters PRAS40 intracellularly through post-translational phosphorylation and binding to 14-3-3 protein, limiting PRAS40 activity and thereby providing cytoprotection to microglia against beta-amyloid toxicity.\",\n      \"method\": \"mTOR pathway Western blot (p70S6K, 4EBP1 phosphorylation), PRAS40 phosphorylation assay, co-immunoprecipitation with 14-3-3, gene silencing of PRAS40, apoptosis assays\",\n      \"journal\": \"Current neurovascular research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP with 14-3-3, gene silencing epistasis, phosphorylation Western blots; single lab\",\n      \"pmids\": [\"22873724\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"WISP1 (CCN4) controls TSC2 activity by increasing Akt1-mediated TSC2 (Thr1462) phosphorylation and decreasing AMPK-mediated TSC2 (Ser1387) phosphorylation, thereby increasing p70S6K activity and providing cytoprotection to microglia; gene silencing of TSC2 further enhances WISP1-mediated p70S6K phosphorylation but impairs WISP1 cytoprotection, indicating a minimal required level of TSC2 activity.\",\n      \"method\": \"Akt1/AMPK/TSC2/p70S6K phosphorylation Western blot, TSC2 gene silencing, apoptosis assays (PS exposure, DNA degradation, mitochondrial membrane depolarization, cytochrome c release)\",\n      \"journal\": \"Current neurovascular research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — TSC2 siRNA epistasis, multiple phosphorylation readouts, cell death assays; single lab\",\n      \"pmids\": [\"23244622\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"CCN4/WISP1 induces IL-6 production in human osteoarthritis synovial fibroblasts through alphavbeta5 integrin (not alpha5beta1 or alphavbeta3), activating PI3K, Akt, and NF-kappaB signaling; alphavbeta5 blocking antibody and PI3K/Akt/NF-kappaB inhibitors all attenuated CCN4-induced IL-6.\",\n      \"method\": \"Neutralizing integrin antibodies, PI3K/Akt/NF-κB pathway inhibitors, NF-κB luciferase reporter, p65 immunocytochemistry (translocation), ELISA for IL-6\",\n      \"journal\": \"Arthritis research & therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — integrin-specific blocking plus multiple pathway inhibitors with promoter activity and secretion readouts; single lab\",\n      \"pmids\": [\"23343403\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"CCN4/WISP1 induces VCAM-1 expression in human osteoarthritis synovial fibroblasts via alphavbeta5 or alpha6beta1 integrin, activating Syk, PKCdelta, JNK, c-Jun, and AP-1 signaling; elevated VCAM-1 promotes monocyte adhesion to fibroblasts, which is blocked by VCAM-1 siRNA.\",\n      \"method\": \"Integrin blocking antibodies, kinase inhibitors, AP-1 luciferase reporter, chromatin immunoprecipitation (c-Jun/AP-1 element), siRNA knockdown, monocyte adhesion assay\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple pathway inhibitors plus ChIP and functional monocyte adhesion readout; single lab\",\n      \"pmids\": [\"23313051\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"WISP-1 enhances migration of human osteosarcoma cells by increasing MMP-2 and MMP-9 expression through integrin receptor-mediated activation of Ras, Raf-1, MEK, ERK, and NF-kappaB signaling; siRNA knockdown of Ras or Raf-1 and specific inhibitors abolished WISP-1-induced migration and MMP expression.\",\n      \"method\": \"Ras/Raf-1 siRNA and inhibitors, MEK/ERK inhibitors, NF-κB inhibitors, migration assay, MMP Western blot/ELISA\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA plus pharmacological inhibitors with multiple readouts; single lab\",\n      \"pmids\": [\"24036215\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"WISP-1 increases OSCC cell migration and ICAM-1 expression through alphavbeta3 integrin, activating ASK1, JNK/p38, and AP-1 signaling pathways.\",\n      \"method\": \"Integrin alphavbeta3 blocking antibody, ASK1/JNK/p38 inhibitors, AP-1 luciferase reporter, migration assay, ICAM-1 Western blot\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — integrin blocking plus pathway inhibitors with functional and molecular readouts; single lab\",\n      \"pmids\": [\"24205072\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"WISP1 (CCN4) is secreted from differentiated human adipocytes (validated as an adipokine) and stimulates a proinflammatory response in human macrophages. WISP1 expression and secretion increase during human adipocyte differentiation and are regulated by weight changes in humans and mice.\",\n      \"method\": \"WISP1 ELISA from conditioned media, macrophage stimulation assay with recombinant WISP1, in vivo weight-change models\",\n      \"journal\": \"Diabetes\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct secretion measurement plus macrophage functional assay; single lab\",\n      \"pmids\": [\"25281430\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"miR-92a post-transcriptionally represses WISP1 expression in lung fibroblasts; TGF-beta1 increases WISP1 expression while decreasing miR-92a, and miR-92a overexpression reverses TGF-beta1-induced WISP1 mRNA expression. An inverse relationship between WISP1 and miR-92a was confirmed in vivo in a TGF-beta1-dependent fibrosis model.\",\n      \"method\": \"Supervised machine learning miRNA target prediction, miR-92a mimic/inhibitor transfection, qPCR and ELISA for WISP1, in vivo TGF-beta1 fibrosis mouse model, ex vivo primary IPF fibroblasts\",\n      \"journal\": \"The international journal of biochemistry & cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — miRNA gain/loss of function in primary cells and in vivo model confirming inverse relationship; single lab\",\n      \"pmids\": [\"24953558\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"WISP1 (CCN4) deficiency in mice reduces bone mineral density, decreases trabecular bone volume and cortical thickness, impairs osteoblast progenitor differentiation, and increases osteoclast differentiation. WISP1 inhibits SOST binding to LRP6, thereby maintaining Wnt/beta-catenin signaling in bone marrow stromal cells; Wisp1-/- cells show reduced beta-catenin and its target gene expression.\",\n      \"method\": \"Wisp1-/- knockout mouse generation, DXA, micro-CT, biomechanical testing, in vitro osteoblast/osteoclast differentiation assays, SOST-LRP6 binding inhibition assay, beta-catenin Western blot, ovariectomy model\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — constitutive knockout mouse with multiple phenotypic and molecular readouts, direct SOST-LRP6 binding inhibition assay; rigorous multi-method study\",\n      \"pmids\": [\"25864198\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CCN4/WISP1 directly binds TGF-beta3 (shown by immunoprecipitation-western blot) and enhances TGF-beta3-induced SMAD2/3 phosphorylation and chondrogenesis of human bone marrow stromal cells in micromass culture. CCN4 knockdown inhibits TGF-beta3-induced SMAD2/3 phosphorylation and cartilage matrix synthesis. Ccn4-KO mice show reduced chondrocyte marker expression and fail to repair articular cartilage defects.\",\n      \"method\": \"Adenoviral overexpression and siRNA knockdown of CCN4, SMAD2/3 phosphorylation Western blot, micromass culture chondrogenesis assay, immunoprecipitation-western blot for CCN4-TGF-beta3 binding, Ccn4-KO mouse cartilage defect model\",\n      \"journal\": \"Bone\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — direct binding shown by IP, Smad epistasis via OE/KD, in vivo KO with functional defect; multiple orthogonal methods\",\n      \"pmids\": [\"26555637\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"WISP-1 promotes angiogenesis in oral squamous cell carcinoma by inducing VEGF-A expression via the integrin alphavbeta3/FAK/c-Src pathway, which transactivates EGFR/ERK/HIF-1alpha signaling. This in turn recruits endothelial progenitor cells and triggers neovascularization in vitro and in vivo.\",\n      \"method\": \"Integrin alphavbeta3 blocking, FAK/c-Src/EGFR/ERK/HIF-1alpha inhibitors and siRNA, EPC tube formation/migration assay, in vivo xenograft angiogenesis model\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple pathway inhibitors and siRNA with in vivo validation; single lab\",\n      \"pmids\": [\"25738362\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"WISP1 is required for TGF-beta1- and TNF-alpha-dependent induction of IL-6 in primary human lung fibroblasts; siRNA-mediated WISP1 knockdown or antibody-mediated neutralization reduces fibroblast proliferation, which is partially rescued by exogenous IL-6, establishing a WISP1-IL-6 pro-proliferative axis.\",\n      \"method\": \"Luciferase reporter for WISP1 promoter, siRNA knockdown, antibody neutralization, ELISA for IL-6, proliferation assay, IL-6 rescue experiment\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA + neutralizing antibody + IL-6 rescue establishes causal chain; single lab\",\n      \"pmids\": [\"26867691\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"WISP-1 promotes VEGF-C expression and lymphangiogenesis in OSCC cells via integrin alphavbeta3/ILK/Akt signaling, and this cascade inhibits miR-300 expression. Conditioned media from WISP-1-treated OSCC cells promotes tube formation and migration in lymphatic endothelial cells.\",\n      \"method\": \"Integrin alphavbeta3/ILK/Akt inhibitors, VEGF-C ELISA, lymphatic endothelial cell tube formation and migration assay, miR-300 expression analysis\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pathway inhibitors with functional lymphangiogenesis readout and miRNA regulation; single lab\",\n      \"pmids\": [\"26824419\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Wnt2 induces WISP-1 expression in vascular smooth muscle cells (VSMCs) via beta-catenin/TCF signaling; recombinant WISP-1 promotes VSMC migration in an integrin-dependent manner. WISP-1-/- mice show significantly suppressed intimal thickening after carotid artery ligation, and adenoviral elevation of plasma WISP-1 increases intimal thickening in vivo.\",\n      \"method\": \"siRNA knockdown of Wnt2 and WISP-1, VSMC migration assay, integrin dependence test, WISP-1-/- mouse carotid ligation model, adenoviral WISP-1 overexpression in vivo\",\n      \"journal\": \"Arteriosclerosis, thrombosis, and vascular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout mouse plus adenoviral gain-of-function in vivo, siRNA knockdown, integrin-dependent migration; multiple orthogonal in vitro and in vivo approaches\",\n      \"pmids\": [\"27199447\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Macrophage-derived IL-10, secreted following intestinal mucosal injury, activates epithelial CREB, which drives WISP-1 synthesis and secretion. WISP-1 then induces epithelial cell proliferation and wound closure. Conditional deletion of IL-10 in CD11c+ macrophages impairs wound closure, establishing an IL-10/CREB/WISP-1 signaling axis.\",\n      \"method\": \"Conditional CD11c-specific IL-10 knockout mice, Rag1-/- mice, in vitro CREB activation assay, WISP-1 protein measurement, wound closure assay, cell proliferation assay\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional knockout in vivo with mechanistic in vitro validation, multiple genetic models\",\n      \"pmids\": [\"28783045\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"WISP-1 promotes VEGF-A expression and angiogenesis in osteosarcoma through FAK/JNK/HIF-1alpha signaling and downregulation of miR-381; FAK, JNK, and HIF-1alpha inhibitors or siRNA abolished WISP-1-induced VEGF-A expression and angiogenesis both in vitro and in vivo.\",\n      \"method\": \"FAK/JNK/HIF-1alpha inhibitors and siRNA, VEGF-A ELISA, EPC migration/tube formation assay, miR-381 expression analysis, in vivo xenograft angiogenesis model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple pathway inhibitors and siRNA with in vivo validation; single lab\",\n      \"pmids\": [\"28406476\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"WISP1/CCN4 inhibits adipocyte differentiation by repressing PPARgamma transcriptional activity; a direct physical association between WISP1 and PPARgamma was demonstrated, followed by proteasome-dependent degradation of PPARgamma. Silencing WISP1 enhanced adipocyte differentiation.\",\n      \"method\": \"Overexpression and siRNA knockdown in 3T3-F442A cells, PPARgamma transcriptional activity reporter, co-immunoprecipitation of WISP1 with PPARgamma, proteasome inhibitor treatment, adipogenic marker expression\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct co-IP of WISP1-PPARgamma plus proteasome inhibitor assay and gain/loss of function; single lab\",\n      \"pmids\": [\"28496206\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CCN4/WISP1 stimulates dermal fibroblast migration and proliferation via alpha5beta1 integrin receptors, activating ERK/JNK signaling downstream. CCN4 also counterbalances TNF-alpha-mediated inhibition of COL1A1 and FN and stimulation of MMPs by inhibiting NF-kappaB/p65 signaling. Ccn4-KO mice show delayed wound closure with reduced Col1a1 and fibronectin expression.\",\n      \"method\": \"Ccn4-KO mice, Boyden chamber migration assay, siRNA knockdown, adenoviral CCN4 transduction, integrin alpha5beta1 blocking antibody, ERK/JNK Western blot, NF-kappaB signaling assay, gene expression analysis\",\n      \"journal\": \"Matrix biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO mouse plus siRNA knockdown plus adenoviral OE plus integrin blocking; multiple orthogonal approaches across in vivo and in vitro systems\",\n      \"pmids\": [\"29330021\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Osteoblast-derived WISP-1 promotes adherence of prostate cancer cells to osteoblasts via a VCAM-1/integrin alpha4beta1 system: WISP-1 induces VCAM-1 expression in PCa cells and integrin alpha4beta1 expression in osteoblasts via the MAPK pathway; blocking VCAM-1 or integrin alpha4beta1 attenuates cancer cell adhesion. WISP-1 also induces BMP2, BMP4, and osteopontin in osteoblasts and inhibits RANKL-dependent osteoclastogenesis.\",\n      \"method\": \"VCAM-1 and integrin alpha4beta1 blocking antibodies, MAPK pathway analysis, BMP2/4/OPN expression assay, osteoclastogenesis assay\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — receptor blocking antibodies with adhesion readout and multiple molecular targets; single lab\",\n      \"pmids\": [\"29627497\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"WISP1 impairs insulin action in human skeletal muscle cells and mouse hepatocytes by inhibiting phosphorylation of insulin receptor, Akt, GSK-3beta, FOXO1, and p70S6K, and by inhibiting insulin-stimulated glycogen synthesis and suppression of gluconeogenic genes.\",\n      \"method\": \"Recombinant WISP1 treatment of primary human skeletal muscle cells and AML12 hepatocytes, insulin signaling Western blot, glycogen synthesis assay, gluconeogenic gene expression\",\n      \"journal\": \"Diabetologia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct recombinant protein treatment with multiple signaling and functional readouts in two cell types; single lab\",\n      \"pmids\": [\"29754289\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"WISP1 contributes to sepsis-induced acute lung injury via a WISP1-alphavbeta3 integrin signaling pathway in macrophages: TLR4 activation induces de novo synthesis of alphavbeta3 via ERK (MyD88/NF-kappaB dependent), enabling WISP1 to stimulate TNF-alpha release. This effect was absent in beta3-/- macrophages.\",\n      \"method\": \"TLR4-/-, MyD88-/-, TRIF-/-, beta3-/- mouse macrophages, ERK inhibitor, siRNA knockdown, anti-WISP1/anti-integrin beta5 antibodies in vivo two-hit model\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockout macrophages identify integrin beta3 requirement, ERK pathway inhibition, in vivo antibody blockade; single lab\",\n      \"pmids\": [\"27349568\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In mechanical ventilation plus sepsis-induced acute lung injury, WISP1 levels are increased intrapulmonarily via TLR4 signaling; WISP1 signals through integrin beta5 to amplify TLR4-mediated inflammation. Neutralizing WISP1 or integrin beta5 antibodies partially inhibited the two-hit ALI phenotype. In macrophages, TLR4 increases integrin beta5 expression in a MyD88/NF-kappaB-dependent manner, and WISP1 requires integrin beta5 for enhanced cytokine release.\",\n      \"method\": \"TLR4-/-, MyD88-/-, TRIF-/- mice; anti-WISP1 and anti-integrin beta5 antibodies in vivo; siRNA knockdown of TLR4 and integrin beta5 in macrophages; cytokine ELISA\",\n      \"journal\": \"Critical care\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple genetic knockouts and antibody blockade in vivo plus siRNA in vitro; single lab\",\n      \"pmids\": [\"30445996\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"WISP1 (CCN4) is a FAP-derived matricellular signal in the muscle stem cell niche that controls expansion and asymmetric commitment of muscle stem cells (MuSCs) through Akt signaling. Loss of WISP1 from aged fibro-adipogenic progenitors (FAPs) contributes to impaired MuSC function; transplantation of young FAPs or systemic WISP1 treatment rescues myogenic capacity in aged mice.\",\n      \"method\": \"Transcriptomic profiling, genetic mouse models, FAP transplantation, systemic WISP1 administration, Akt signaling Western blot, MuSC expansion and differentiation assays\",\n      \"journal\": \"Cell stem cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — FAP transplantation, systemic rescue, Akt pathway mechanism, in vivo muscle regeneration readout; multiple orthogonal approaches\",\n      \"pmids\": [\"30686765\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"WISP1 (CCN4) directly binds type I collagen to promote its linearization in vitro (in the absence of cells) and in vivo in tumors, thereby facilitating cancer cell invasion and promoting spontaneous breast cancer metastasis. This collagen linearization is independent of cell-generated mechanical forces.\",\n      \"method\": \"Cell-free in vitro collagen linearization assay with recombinant WISP1, direct WISP1-collagen binding assay, in vivo tumor collagen imaging, breast cancer metastasis mouse model with WISP1 manipulation\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cell-free in vitro reconstitution of collagen linearization, direct binding assay, in vivo tumor validation; rigorous mechanistic study\",\n      \"pmids\": [\"31294477\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"WISP1 in glioblastoma signals through Integrin alpha6beta1-Akt to maintain glioma stem cells (GSCs) by autocrine mechanism, and promotes M2 tumor-associated macrophage survival by paracrine mechanism. WISP1 is regulated by Wnt/beta-catenin in GSCs, and carnosic acid inhibits this axis to suppress GBM growth.\",\n      \"method\": \"WISP1 silencing in GSCs, integrin alpha6beta1-Akt pathway analysis, autocrine/paracrine assays, GSC maintenance and TAM survival assays, carnosic acid treatment\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — receptor-pathway identification with silencing plus pharmacological inhibition; single lab\",\n      \"pmids\": [\"32541784\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"WISP1 knockout in melanoma cells increases proliferation but represses migration, invasion, and metastasis. WISP1 promotes an EMT-like gene expression signature (E-cadherin repression, fibronectin/N-cadherin induction) by activating AKT and MEK/ERK signaling; WISP1 knockout-associated metastasis repression is reversed by reintroduction of WISP1 or SNAI1.\",\n      \"method\": \"WISP1 knockout (B16F10, YUMM1.7), transwell invasion/migration assays, gene expression profiling, recombinant WISP1 rescue, SNAI1 reintroduction, AKT/MEK/ERK kinase inhibitors, in vivo metastasis assays in C57BL/6 and NSG mice\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout with molecular rescue, pathway inhibitors, multiple in vivo metastasis models with two cell lines\",\n      \"pmids\": [\"30723155\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"WISP-1 promotes EMT in OSCC cells via FAK/ILK/Akt and Snail signaling and downregulates miR-153-3p expression; the miR-153-3p/Snail axis mediates WISP-1-induced EMT.\",\n      \"method\": \"FAK/ILK/Akt inhibitors, Snail expression analysis, miR-153-3p expression measurement, EMT marker Western blot\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pathway inhibitors plus miRNA-EMT axis characterization; single lab\",\n      \"pmids\": [\"31795469\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Wisp1 promotes proliferation of adult mouse and human pancreatic beta cells in vivo; the beta cell trophic effect is dependent on Akt signaling, as shown using isolated mouse and human islets. Wisp1 is more abundant in pre-weaning than adult mice serum.\",\n      \"method\": \"In vivo Wisp1 administration, transplanted beta cell proliferation assay, isolated islet culture with Wisp1, Akt pathway inhibition, ELISA for circulating Wisp1\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo and ex vivo beta cell proliferation with Akt inhibition epistasis; single lab but cross-species validation\",\n      \"pmids\": [\"33239617\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"FAT10 (a ubiquitin-like protein) degrades WISP1 protein via FAT10ylation (direct post-translational modification), while simultaneously stabilizing beta-catenin to increase WISP1 mRNA, creating a protein/mRNA expression discordance. FAT10 overexpression-induced WISP1 protein decrease promotes hepatocellular carcinoma proliferation.\",\n      \"method\": \"FAT10 overexpression and knockdown, co-immunoprecipitation for FAT10-WISP1 interaction, proteasome inhibitor experiments, beta-catenin stabilization assay, WISP1 mRNA and protein quantification, in vitro and in vivo HCC proliferation assays\",\n      \"journal\": \"Hepatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP demonstrating FAT10ylation, proteasome inhibitor validation, in vivo evidence; single lab\",\n      \"pmids\": [\"29790184\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"WISP-1 promotes fibrosis and hypertrophy of the ligamentum flavum by activating the Hedgehog-Gli1 signaling pathway in fibroblasts; mechanical stress increases WISP-1 expression, and WISP-1 induces myofibroblast transition via Hedgehog signaling. Cyclopamine (Hedgehog inhibitor) attenuated WISP-1-induced fibrogenesis in vitro and in the in vivo mechanical stress rabbit model.\",\n      \"method\": \"Mechanical stretch, WISP-1 treatment of LF fibroblasts, Hedgehog-Gli1 pathway Western blot/reporter, cyclopamine inhibition, alpha-SMA expression, in vivo rabbit mechanical stress model\",\n      \"journal\": \"Experimental & molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pathway inhibitor epistasis in vitro and in vivo; single lab\",\n      \"pmids\": [\"34158608\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"WISP1 drives liver fibrosis progression (not onset) through integrin-dependent activation of MRTF (myocardin-related transcription factor), leading to transcriptional reprogramming of myofibroblast cytoskeleton and motility. WISP1-deficient mice are protected against fibrosis progression; a therapeutic anti-WISP1 antibody halted progression of established fibrosis in NASH models.\",\n      \"method\": \"Wisp1-/- mice, NASH fibrosis mouse models, anti-WISP1 antibody treatment, MRTF pathway analysis, integrin signaling assay, myofibroblast cytoskeleton and motility readouts\",\n      \"journal\": \"Cell metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO plus therapeutic antibody in established fibrosis models, MRTF pathway mechanism identified; multiple in vivo models\",\n      \"pmids\": [\"35987202\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Apoptotic cancer cell-derived signals activate Notch1 signaling in cancer-associated fibroblasts (CAFs) via the DLL1/BAI1-Rac1 efferocytosis pathway, inducing WISP-1 secretion which suppresses CAF activation and inhibits cancer cell migration/invasion and lung metastasis. Notch1 inhibition or siRNA blocked WISP-1 production and reversed the antimetastatic effects.\",\n      \"method\": \"Pharmacological Notch1 inhibition, siRNA Notch1 silencing, WISP-1 immunodepletion from conditioned media, migration/invasion assays, syngeneic mouse lung metastasis model, CAF marker expression assay\",\n      \"journal\": \"Cellular & molecular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Notch1 siRNA/inhibitor plus WISP-1 immunodepletion with in vivo metastasis readout; single lab\",\n      \"pmids\": [\"36241874\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"WISP-1 full-length protein and C-terminal domain-containing truncations (VWC, TSP, CT domains) promote epithelial cell adhesion in an integrin-dependent manner (partially mediated by alphavbeta5, alphavbeta3, and beta1 integrins). Beta-catenin activation and CXCL3 secretion also reside within the C-terminal domains of WISP-1 but are not blocked by integrin neutralizing antibodies. Native WISP-1 from human lung tissue exists as a high-order oligomer.\",\n      \"method\": \"Baculovirus expression of truncated WISP-1 proteins, cell adhesion assay, integrin-blocking antibodies, beta-catenin reporter assay, CXCL3 ELISA, size-exclusion analysis of native protein\",\n      \"journal\": \"Journal of cell communication and signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain dissection with recombinant truncations, integrin blocking, beta-catenin reporter; single lab\",\n      \"pmids\": [\"25657057\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Notch1 activation in fibroblasts induces WISP-1 expression in a Wnt11-dependent (but Wnt1-independent) manner; WISP-1 suppresses fibroblast proliferation and mediates Notch1's growth-inhibitory effect, since siRNA blockade of WISP-1 relieves Notch1-induced growth inhibition.\",\n      \"method\": \"Notch1 knockout MEFs, NICD1 overexpression, DN-MAML-1, Wnt11 knockdown, WISP-1 siRNA, cell growth and motility assays\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic Notch1 knockout and rescue, Wnt11 knockdown epistasis, WISP-1 siRNA epistasis; single lab\",\n      \"pmids\": [\"22715413\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"WISP1 (Wisp-1) is a cardiac angiokine whose transcription is regulated by HDACs; HDAC inhibition with SAHA post-MI elevates Wisp-1 expression in the border zone. Human coronary artery endothelial cells produce WISP-1 and respond to autocrine WISP-1 signaling that promotes their pro-angiogenic behavior; altering endogenous WISP-1 in HCAECs directly impacts network density in vitro.\",\n      \"method\": \"HDAC inhibitor (SAHA) treatment, post-MI mouse model, WISP-1 expression measurement, HCAEC WISP-1 knockdown/overexpression, endothelial network formation assay\",\n      \"journal\": \"JCI insight\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — HDAC regulation of WISP-1 transcription, WISP-1 gain/loss in endothelial cells with functional angiogenesis readout; single lab\",\n      \"pmids\": [\"29467324\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"WISP1 in osteoarthritis promotes expression of MMP3, MMP9, ADAMTS4, and ADAMTS5 in synovial tissue (increasing cartilage catabolism). Wisp1-/- mice show significantly decreased cartilage degeneration in three independent experimental OA models (collagenase injection, DMM, ACLT), with reduced protease expression and decreased aggrecan cleavage (NITEGE neoepitope). Aging-related spontaneous cartilage degeneration was not decreased in Wisp1-/- mice.\",\n      \"method\": \"Wisp1-/- knockout mice, three independent OA induction models, qRT-PCR for proteases, NITEGE immunohistochemistry, micro-CT, recombinant WISP1 stimulation of human OA synovium\",\n      \"journal\": \"Osteoarthritis and cartilage\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — constitutive KO validated in three independent experimental OA models plus direct human tissue stimulation; replicated across models\",\n      \"pmids\": [\"28735021\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Nitric oxide (NO) increases WISP-1 expression in colonic epithelial cells through a beta-catenin- and CREB-dependent, but Wnt-1-independent, pathway. NO-induced WISP-1 then directly induces soluble collagen secretion in colonic fibroblast cells. In iNOS-/- mice, colitis-related WISP-1 increase was significantly attenuated.\",\n      \"method\": \"NO donor (DETA-NONOate) treatment, beta-catenin and CREB pathway inhibition, WISP-1 mRNA/protein measurement, collagen secretion assay in fibroblasts, iNOS-/- mouse colitis models\",\n      \"journal\": \"Journal of molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological pathway inhibition with iNOS-/- genetic validation in vivo; single lab\",\n      \"pmids\": [\"19238344\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Wisp1 mediates Bmp3-stimulated mesenchymal stem cell (MSC) proliferation: Bmp3 upregulates Wisp1 expression prior to MSC proliferation, recombinant Wisp1 stimulates MSC proliferation in a concentration-dependent manner, and siRNA-mediated Wisp1 knockdown attenuates Bmp3-induced proliferation.\",\n      \"method\": \"DNA microarray, recombinant Wisp1 treatment, [3H]-thymidine incorporation, siRNA knockdown of Wisp1, Bmp3 treatment\",\n      \"journal\": \"Journal of molecular endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA knockdown epistasis, recombinant protein gain-of-function, temporal expression analysis; single lab\",\n      \"pmids\": [\"26489765\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"CCN4/WISP1 splice variants are expressed in chondrocytes: full-length WISP1, WISP1v (lacking the second module/VWC domain), and a novel WISP1vx (lacking second, third modules and part of first). WISP1v expression increases dramatically during terminal chondrocyte differentiation and overexpression of WISP1v promotes alkaline phosphatase gene expression (a marker of terminal differentiation), suggesting WISP1v plays a role in endochondral ossification.\",\n      \"method\": \"RT-PCR characterization of splice variants, primary chondrocyte differentiation culture, WISP1v overexpression, alkaline phosphatase gene expression assay\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain deletion via natural splice variant with gain-of-function differentiation assay; single lab\",\n      \"pmids\": [\"17381509\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"WISP1 promotes non-alcoholic fatty liver disease and skeletal muscle insulin resistance through a TLR4-activated inflammation/JNK signaling pathway; WISP1 increases JNK phosphorylation, NF-kappaB-dependent inflammation, lipogenesis, and triglyceride accumulation in hepatocytes and suppresses insulin signaling in muscle cells. These effects were abrogated by NF-kappaB, JNK, and TLR4 knockdown.\",\n      \"method\": \"WISP1 treatment of mouse hepatocytes and C2C12 cells, NF-kappaB/JNK/TLR4 siRNA knockdown, Western blot for insulin signaling, triglyceride accumulation assay, in vivo WISP1 knockdown in HFD mice\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA epistasis for three pathway components plus in vivo knockdown; single lab\",\n      \"pmids\": [\"29319180\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"WISP1 overexpression stimulates proliferation and migration of human VSMCs via AKT signaling; WISP1 increases GSK3alpha/beta phosphorylation, cyclin D1, and MMP9 expression. AKT inhibitor (AZD5363) and PI3K inhibitor (LY294002) abolished WISP1-induced proliferation and migration and downstream target expression.\",\n      \"method\": \"Adenoviral WISP1 overexpression, EdU incorporation assay, scratch wound healing, transwell migration, AKT/PI3K inhibition, Western blot for cyclin D1/MMP9/GSK3\",\n      \"journal\": \"European journal of pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — adenoviral OE plus pharmacological AKT/PI3K inhibition with multiple functional readouts; single lab\",\n      \"pmids\": [\"27321870\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CCN4/WISP1 is a secreted matricellular protein transcriptionally activated by Wnt/beta-catenin (via a CREB-dependent promoter element) that exerts pleiotropic effects by binding extracellular partners—including decorin, biglycan, BMP-2, TGF-beta3, and type I collagen—and signaling through integrins (alpha5beta1, alphavbeta3, alphavbeta5, alpha6beta1, alpha4beta1) to activate intracellular cascades including PI3K/Akt, ERK/MEK, NF-kappaB, CREB, and MRTF, thereby promoting cell survival (via Bcl-xL/cytochrome c and FoxO3a/PRAS40/mTOR), proliferation, migration, osteoblast/chondrocyte differentiation (by enhancing BMP-2 and TGF-beta3 signaling), muscle stem cell expansion (via Akt), collagen linearization and metastasis, and fibrosis progression (via the WISP1–MRTF axis), while its activity is post-transcriptionally regulated by miR-92a and post-translationally by FAT10ylation-dependent proteasomal degradation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CCN4 (WISP1) is a secreted matricellular protein, transcriptionally activated downstream of Wnt/beta-catenin through a CREB-dependent promoter element, that integrates extracellular cues into cell-survival, proliferation, differentiation, migration, and fibrotic programs [#0, #12, #49]. It binds extracellular matrix components and growth factors directly\\u2014dermatan-sulfate proteoglycans decorin and biglycan that anchor it to the cell surface [#2], BMP-2 and TGF-beta3 whose Smad signaling it potentiates to drive osteoblast and chondrocyte differentiation [#7, #22], and type I collagen, which it linearizes in a cell-force-independent manner to facilitate invasion and breast cancer metastasis [#36]. CCN4 signals predominantly through integrins\\u2014alpha5beta1, alphavbeta3, alphavbeta5, alpha6beta1, and alpha4beta1\\u2014to engage FAK, MEK/ERK, PI3K/Akt, NF-kappaB, and AP-1 cascades, thereby inducing proteases, adhesion molecules, and angiogenic factors that promote tumor cell migration, monocyte/macrophage recruitment, and neovascularization [#8, #15, #16, #23, #31]. A recurrent pro-survival module operates through PI3K/Akt: CCN4 raises the Bcl-xL/Bax ratio, blocks mitochondrial cytochrome c release, and phosphorylates and sequesters FoxO3a and PRAS40 with 14-3-3 to sustain mTOR signaling, protecting neurons, microglia, and cardiomyocytes from apoptotic and oxidant stress [#1, #10, #11, #13]. In tissue homeostasis, CCN4 maintains Wnt signaling in bone by blocking SOST binding to LRP6, supporting bone mineral density [#21], and acts as a niche factor that drives muscle stem cell expansion via Akt [#35]. Genetic loss and antibody-blockade studies establish CCN4 as a driver of fibrosis progression, signaling through integrin-dependent activation of MRTF to reprogram the myofibroblast cytoskeleton in liver fibrosis and through Hedgehog-Gli1 in ligamentum flavum fibrosis [#43, #42]. Its expression is post-transcriptionally repressed by miR-92a and its protein is degraded through FAT10ylation-dependent proteasomal turnover [#20, #41].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Established CCN4 as a Wnt/beta-catenin target and a transforming gene, defining the upstream signal that drives its expression and its oncogenic potential.\",\n      \"evidence\": \"Promoter dissection with reporter assays and overexpression with nude mouse xenograft in NRK-49F cells\",\n      \"pmids\": [\"10716946\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"CREB versus TCF/LEF contribution not resolved at single-element resolution\", \"Did not identify the secreted protein's receptors or extracellular partners\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Identified the first direct extracellular binding partners, showing CCN4 docks to the cell surface via dermatan sulfate proteoglycans.\",\n      \"evidence\": \"Solid-phase and direct binding assays with purified decorin and biglycan plus glycosaminoglycan competition and mass spectrometry\",\n      \"pmids\": [\"11598131\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of proteoglycan binding for downstream signaling not defined\", \"Binding domain on CCN4 not mapped\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Defined the core pro-survival mechanism, showing CCN4 acts through Akt to block intrinsic (but not Fas-mediated) apoptosis.\",\n      \"evidence\": \"Apoptosis assays, cytochrome c release, Bcl-xL Western blot and PI3K/Akt inhibition\",\n      \"pmids\": [\"11782444\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Receptor coupling CCN4 to PI3K/Akt not identified\", \"Whether survival effect is autocrine or paracrine unresolved\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Connected CCN4 to BMP and skeletal biology, showing it potentiates BMP-2-driven osteoblast differentiation while restraining chondrocyte maturation.\",\n      \"evidence\": \"In situ hybridization, stable transfection of chondrocytic lines, osteoblast differentiation assays and in vivo fracture repair\",\n      \"pmids\": [\"15331410\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct BMP-2 binding not yet demonstrated at this stage\", \"Opposing chondrocyte versus osteoblast effects mechanistically unexplained\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Demonstrated direct BMP-2 binding and integrin alpha5beta1 dependence, establishing CCN4 as a co-receptor-like enhancer of BMP/Smad osteogenesis in vivo.\",\n      \"evidence\": \"Immunoprecipitation, adenoviral overexpression, shRNA, integrin-blocking antibody and transgenic mouse bone density analysis\",\n      \"pmids\": [\"20684029\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the BMP-2/integrin/CCN4 ternary complex unknown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Showed CCN4 directly binds TGF-beta3 to drive chondrogenesis and that knockout mice fail cartilage repair, extending its growth-factor-potentiating role to TGF-beta.\",\n      \"evidence\": \"IP-Western for CCN4-TGF-beta3 binding, OE/KD Smad2/3 epistasis, micromass chondrogenesis and Ccn4-KO cartilage defect model\",\n      \"pmids\": [\"26555637\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reconciliation with earlier report of CCN4 suppressing TGF-beta1/Smad2 signaling not addressed\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Established CCN4 as a positive regulator of bone mass acting upstream of Wnt by blocking the SOST-LRP6 inhibitory interaction.\",\n      \"evidence\": \"Wisp1-/- mice with DXA, micro-CT, biomechanics, differentiation assays and SOST-LRP6 binding inhibition assay\",\n      \"pmids\": [\"25864198\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether SOST competition is direct or matrix-mediated not fully resolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Mapped the integrin-to-protease signaling logic in cancer cells, linking specific integrin heterodimers to MMP induction, adhesion molecules, and migration.\",\n      \"evidence\": \"Integrin-blocking antibodies, kinase inhibitors, NF-kappaB/AP-1 reporters, ChIP and migration/adhesion assays across chondrosarcoma, synovial fibroblast and OSCC systems\",\n      \"pmids\": [\"21453685\", \"23343403\", \"23313051\", \"24036215\", \"24205072\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cell-type basis for integrin heterodimer selectivity unexplained\", \"Most readouts from single labs with overexpression/recombinant protein\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Detailed the neuronal/microglial cytoprotection module, showing CCN4 sequesters FoxO3a and PRAS40 via 14-3-3 and tunes TSC2 to sustain mTOR-dependent survival.\",\n      \"evidence\": \"PI3K/Akt inhibition, co-IP with 14-3-3, subcellular fractionation, gene silencing of PRAS40/TSC2 and apoptosis assays in primary neurons and microglia\",\n      \"pmids\": [\"22272766\", \"23151077\", \"22873724\", \"23244622\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Upstream receptor coupling CCN4 to Akt in neural cells not identified\", \"All from a single lab\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Established cell-force-independent collagen linearization as a direct biophysical CCN4 function driving metastasis.\",\n      \"evidence\": \"Cell-free in vitro collagen linearization, direct binding assay and in vivo breast cancer metastasis model\",\n      \"pmids\": [\"31294477\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism by which CCN4 reorganizes collagen fibrils unresolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identified CCN4 as a FAP-derived niche signal controlling muscle stem cell expansion via Akt, linking its decline to aging-impaired regeneration.\",\n      \"evidence\": \"Transcriptomics, genetic mouse models, FAP transplantation, systemic WISP1 rescue and Akt pathway analysis\",\n      \"pmids\": [\"30686765\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Receptor on muscle stem cells mediating the Akt effect not defined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined CCN4 as a driver of fibrosis progression acting through integrin-dependent MRTF activation, validated by genetic loss and therapeutic antibody blockade.\",\n      \"evidence\": \"Wisp1-/- mice, NASH fibrosis models, anti-WISP1 antibody, MRTF and integrin signaling analysis\",\n      \"pmids\": [\"35987202\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific integrin and intracellular link to MRTF not pinpointed\", \"Whether the same axis operates in non-hepatic fibrosis unclear\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identified post-transcriptional (miR-92a) and post-translational (FAT10ylation) controls limiting CCN4 abundance, explaining mRNA/protein discordance.\",\n      \"evidence\": \"miR-92a mimic/inhibitor with fibrosis model, and FAT10 OE/KD with co-IP and proteasome inhibitor experiments\",\n      \"pmids\": [\"24953558\", \"29790184\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"FAT10ylation site on CCN4 not mapped\", \"Both from single labs without independent replication\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How CCN4's distinct integrin engagements, growth-factor binding, and direct matrix remodeling are coordinated into context-specific outputs, and what defines its principal signaling receptor, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of CCN4 with any integrin or growth factor\", \"Determinants of integrin heterodimer selectivity across tissues unknown\", \"Reconciliation of pro- versus anti-tumor/fibrotic roles across contexts incomplete\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [5, 7, 22]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [36]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [8, 15, 30]},\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [35, 40]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [2, 19, 36]},\n      {\"term_id\": \"GO:0031012\", \"supporting_discovery_ids\": [2, 36]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [8, 15, 30, 37]},\n      {\"term_id\": \"R-HSA-1474244\", \"supporting_discovery_ids\": [36, 30]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [7, 21, 22, 35]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [1, 10, 11]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [15, 16, 33, 34]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"DCN\", \"BGN\", \"BMP2\", \"TGFB3\", \"COL1A1\", \"ITGA5\", \"ITGB5\", \"PPARG\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":7,"faith_pct":85.71428571428571}}