{"gene":"RSPO3","run_date":"2026-06-10T07:46:28","timeline":{"discoveries":[{"year":2011,"finding":"RSPO3 binds syndecan 4 (SDC4) and together they activate Wnt/PCP signaling; RSPO3 functions by inducing SDC4-dependent, clathrin-mediated endocytosis of Wnt-receptor complexes, which is essential for PCP signal transduction. In Xenopus embryos, RSPO3/PCP signaling during gastrulation requires Wnt5a and is transduced via Fz7, Dvl, and JNK.","method":"Binding assays, Xenopus embryo loss-of-function, epistasis with Wnt5a/Fz7/Dvl/JNK, clathrin inhibition experiments","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods (binding assay, genetic epistasis, pharmacological inhibition of endocytosis) in a single rigorous study with functional rescue experiments in Xenopus","pmids":["21397842"],"is_preprint":false},{"year":2016,"finding":"Endothelial RSPO3 controls vascular remodeling via non-canonical WNT/Ca2+/NFAT signaling. Inducible endothelial-specific Rspo3 deletion caused endothelial apoptosis and vascular pruning. RSPO3 co-regulates genes including Rnf213, Usp18, and Trim30α; RNF213 targets filamin A and NFAT1 for proteasomal degradation, attenuating non-canonical WNT/Ca2+ signaling. NFAT protein levels were decreased in endothelial cells of Rspo3-iECKO mice, and pharmacological NFAT inhibition phenocopied Rspo3-iECKO mice.","method":"Inducible endothelial-specific conditional knockout, endothelial gene expression screen, pharmacological NFAT inhibition, protein degradation assays","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal genetic and pharmacological validation, multiple orthogonal methods including conditional KO and pathway phenocopy experiments","pmids":["26766444"],"is_preprint":false},{"year":2016,"finding":"Capsular RSPO3 signals to the underlying steroidogenic compartment of the adrenal gland to induce β-catenin signaling and imprint zona glomerulosa cell fate. Deletion of RSPO3 leads to loss of SHH signaling and impaired organ growth. RSPO3 function is required in adult life to ensure replenishment of lost cells and maintain zona glomerulosa properties.","method":"Conditional knockout mouse models, immunohistochemistry, β-catenin signaling readout, SHH pathway analysis","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO with multiple specific phenotypic readouts (β-catenin, SHH, zonation, organ growth) across development and adult life","pmids":["27313319"],"is_preprint":false},{"year":2018,"finding":"PDGFRα+ pericryptal stromal myofibroblasts are the principal source of RSPO3 in the intestinal stem cell niche in vivo. RSPO3 is several orders of magnitude more potent than RSPO1 in stimulating Wnt/β-catenin signaling and organoid growth. Stromal Rspo3 ablation in PdgfRα+ cells decreased intestinal crypt Wnt/β-catenin signaling and Paneth cell differentiation, and was rescued by exogenous RSPO3 protein.","method":"Cell-type-specific Cre-mediated conditional knockout (PdgfRα-Cre;Rspo3fl/fl), organoid growth assays, exogenous protein rescue, β-catenin signaling measurements","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo conditional KO with specific cellular source identification, quantitative potency comparison, and rescue experiments using exogenous RSPO3","pmids":["29559533"],"is_preprint":false},{"year":2015,"finding":"Targeting RSPO3 in PTPRK-RSPO3 fusion-positive colorectal tumor xenografts inhibits tumor growth and promotes differentiation, with stem cell compartment genes being most sensitive to anti-RSPO3 treatment, indicating that a stem-cell compartment drives PTPRK-RSPO3 colorectal tumor growth.","method":"Anti-RSPO3 antibody treatment of patient-derived xenografts, gene expression profiling, functional stem cell assays","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo xenograft experiments with anti-RSPO3 antibody, functional stem cell assays, and gene expression analysis in a high-quality journal","pmids":["26700806"],"is_preprint":false},{"year":2016,"finding":"In vivo RSPO3 expression expands Lgr5+ stem cells, Paneth cells, non-Paneth label-retaining cells, and Lgr4+ niche cells, and drives rapid intestinal tumorigenesis. Wnt/β-catenin signaling was modestly increased, and mutant Kras synergized with Rspo3 in hyperplastic growth.","method":"Conditional Rspo3 transgenic mouse model (Lgr5-GFP-CreERT2 × Rspo3 transgene), histological analysis, flow cytometry for stem cell markers","journal":"Gut","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional transgenic in vivo model with multiple cellular and signaling readouts, synergy experiment with Kras","pmids":["27511199"],"is_preprint":false},{"year":2020,"finding":"RSPO3-LGR4 signaling upregulates key self-renewal genes and is essential for leukemia stem cell (LSC) self-renewal in AML. Blocking the RSPO3-LGR4 interaction with clinical-grade anti-RSPO3 antibody (OMP-131R10/rosmantuzumab) impairs self-renewal and induces differentiation in AML patient-derived xenografts without affecting normal hematopoietic stem cells. LGR4 is epigenetically upregulated and works through cooperation with HOXA9.","method":"Anti-RSPO3 antibody (OMP-131R10), patient-derived xenograft (PDX) experiments, gene expression analysis, epigenetic characterization of LGR4","journal":"Cancer cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo PDX experiments with clinical-grade antibody, multiple mechanistic readouts including differentiation and self-renewal, epistasis with HOXA9","pmids":["32559496"],"is_preprint":false},{"year":2014,"finding":"RSPO3 aberrantly expressed in Keap1-deficient lung adenocarcinomas signals via LGR4 and the mediator IQGAP1 to promote tumor cell proliferation and migration. Knockdown of RSPO3, LGR4, or IQGAP1 reduced cell proliferation and migration in vitro, and KD of LGR4 or IQGAP1 decreased tumor growth and metastasis in vivo.","method":"shRNA knockdown of RSPO3/LGR4/IQGAP1, cell proliferation and migration assays, in vivo xenograft metastasis models","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo and in vitro knockdown with multiple pathway components, single lab","pmids":["25531322"],"is_preprint":false},{"year":2017,"finding":"RSPO3 is a negative regulator of ERK/FGF signaling downstream of LGR4 in human adipose-derived stem cells (hASCs). RSPO3 knockdown increased osteogenic potential, an effect blocked by ERK1/2 inhibition. LGR4 silencing inhibited ERK signaling and osteogenic differentiation, and abrogated RSPO3-regulated osteogenesis and RSPO3-induced ERK1/2 inhibition.","method":"RSPO3 shRNA knockdown, LGR4 siRNA silencing, ERK pathway inhibitors, osteogenic differentiation assays in hASCs","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple genetic and pharmacological manipulations, single lab, pathway epistasis established","pmids":["28220828"],"is_preprint":false},{"year":2020,"finding":"RSPO3 limits gluteofemoral adipose tissue expansion by suppressing adipogenesis and increasing gluteal adipocyte susceptibility to apoptosis, while stimulating abdominal adipose progenitor proliferation. The distinct biological responses in abdominal versus gluteal adipose progenitors are associated with differential changes in WNT signaling. Zebrafish with a nonsense rspo3 mutation display altered fat distribution.","method":"Human cellular studies with RSPO3 treatment/knockdown in adipose progenitors from different depots, zebrafish nonsense mutant, WNT signaling measurements","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — human cellular assays combined with zebrafish genetic model, single lab, multiple cellular readouts","pmids":["32493999"],"is_preprint":false},{"year":2021,"finding":"Osteoblast-derived RSPO3 is the principal source of RSPO3 in bone. RSPO3 increases osteoblast proliferation and differentiation in a cell-autonomous manner, and is an important regulator of vertebral trabecular bone mass and bone strength in adult mice.","method":"Osteoblast-specific conditional knockout (Runx2-Cre;Rspo3fl/fl), bone densitometry, bone strength testing, cell proliferation and differentiation assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO with tissue-specific cellular source identification, multiple bone phenotype readouts, and cell-autonomous mechanistic experiments","pmids":["34389713"],"is_preprint":false},{"year":2022,"finding":"RSPO3 signaling via LGR4 drives proliferation of gastric stem cells and induces NF-κB activity in proliferative stem cells. LGR4-driven NF-κB activation is responsible for H. pylori-induced gland hyperplasia and chemokine expression in stem cells, resulting in neutrophil recruitment. LGR4 also regulates LGR5 expression in this context.","method":"Conditional knockout mice (Lgr4-specific and Lgr5-specific), H. pylori infection model, NF-κB activity assays, chemokine/neutrophil recruitment measurements","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple conditional KO models with specific pathway readouts (NF-κB, chemokines, neutrophil recruitment), genetic epistasis between Lgr4 and Lgr5","pmids":["35767364"],"is_preprint":false},{"year":2020,"finding":"RSPO1/RSPO3-LGR4 signaling in hepatocytes suppresses cholesterol synthesis via the AMPKα-SREBP2 pathway. RSPO3 increased phosphorylation of AMPKα Thr172, reduced SREBP2 nuclear translocation and Srebf2 mRNA. LGR4 knockdown increased hepatic cholesterol synthesis and decreased AMPKα phosphorylation; AMPKα knockdown abrogated Rspo-induced inhibition of cholesterol synthesis.","method":"LGR4/Rspo1/Rspo3 knockdown in mice and hepatocytes, AMPKα agonist/antagonist/shRNA experiments, SREBP2 nuclear translocation assays","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro and in vivo knockdown with epistasis experiments, single lab","pmids":["32926477"],"is_preprint":false},{"year":2023,"finding":"Endothelial cell-derived RSPO3 activates LGR4-Gab1-Gαi1/3 complex formation to drive Erk activation and protect neurons from ischemia/reperfusion injury. Only Erk (not Akt or β-catenin) inhibitors reversed RSPO3-induced neuroprotection. Silencing or knockout of Gαi1 and Gαi3 abolished RSPO3-induced neuroprotection. Endothelial RSPO3 knockdown/KO increased ischemic injury in MCAO mice.","method":"Co-IP (LGR4-Gab1-Gαi association), MEF Gαi1/3 KO, Erk/Akt/β-catenin inhibitors, endothelial-specific RSPO3 KO/KD/OE mice, MCAO model","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP for complex identification, Gαi KO epistasis, multiple inhibitor experiments, endothelial-specific genetic models in vivo; single lab","pmids":["37805583"],"is_preprint":false},{"year":2024,"finding":"RSPO3 mediates pulmonary endothelial regeneration in a LGR4-dependent manner. Beyond β-catenin, integrin-linked kinase (ILK)/Akt was identified as a novel downstream effector of RSPO3/LGR4 signaling. EC-specific RSPO3 knockdown inhibited endothelial cell proliferation and exacerbated injury, while EC-specific overexpression promoted recovery.","method":"EC-specific RSPO3 KD, inducible EC-specific KO, EC-specific OE mice, LGR4-dependence assays, ILK/Akt signaling measurements in sepsis model","journal":"International journal of biological macromolecules","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple EC-specific genetic models with LGR4-dependence testing and novel ILK/Akt pathway identification; single lab","pmids":["38677673"],"is_preprint":false},{"year":2017,"finding":"Loss of AXIN1 confers resistance to WNT pathway blockade (porcupine inhibitor LGK974) in RSPO3-fusion-positive colorectal cancer cells. Suppression of AXIN1 by RNA interference in parental VACO6 cells (carrying PTPRK-RSPO3 fusion) markedly increased resistance to LGK974, establishing AXIN1 loss as a mechanism of acquired resistance.","method":"Long-term LGK974 treatment to generate resistant cells, whole-genome sequencing to identify AXIN1 frameshifts, AXIN1 RNAi in parental cells, transcriptional and morphological WNT pathway readouts","journal":"EMBO molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic identification of AXIN1 mutations plus RNAi functional validation, single lab","pmids":["28100566"],"is_preprint":false},{"year":2024,"finding":"RSPO3 promotes gastric cancer stem cell properties through direct interaction with transmembrane protein GNG7, leading to phosphorylation of Akt and GSK-3β and accumulation of β-catenin. GNG7 knockdown blocked RSPO3-induced β-catenin activation and CSC-like properties. This pathway is distinct from canonical LGR-mediated Wnt signaling.","method":"Co-immunoprecipitation (CoIP) and LC-MS/MS to identify RSPO3-interacting proteins, GNG7 siRNA knockdown, recombinant RSPO3 protein treatment, western blot for Akt/GSK-3β/β-catenin, in vivo peritoneal seeding model","journal":"Cancer medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CoIP/MS for interaction identification plus functional epistasis via GNG7 KD, single lab","pmids":["38581123"],"is_preprint":false},{"year":2023,"finding":"The Furin (FuFu) domains of RSPO3 mediate specific, LGR5-dependent cellular uptake when conjugated to liposomes. Full-length RSPO1 mediates aspecific LGR5-independent uptake largely via heparan sulfate proteoglycan binding, whereas RSPO3 FuFu domain-coated liposomes selectively target LGR5-high cells.","method":"Fluorescence-loaded liposome uptake assays, LGR5-high vs LGR5-low cell comparison, domain-specific conjugation, doxorubicin-loaded FuFuRSPO3 liposome growth inhibition","journal":"Journal of controlled release","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional domain mapping via cell-based uptake assays with LGR5-dependent specificity controls, single lab","pmids":["36813038"],"is_preprint":false},{"year":2018,"finding":"RSPO3 impairs vascular endothelial barrier function by inducing inter-endothelial gap formation through disruption of β-catenin and VE-cadherin alignment at adherens junctions, and synergizes with pro-inflammatory IL-1β to enhance endothelial hyperpermeability.","method":"Electric Cell-substrate Impedance Sensing (ECIS) of primary endothelial monolayers, RSPO3 protein treatment, immunofluorescence of β-catenin/VE-cadherin at junctions, IL-1β co-treatment","journal":"Molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — real-time barrier function measurement with mechanistic readout of junction protein disruption, single lab","pmids":["30157748"],"is_preprint":false},{"year":2022,"finding":"RSPO3 is expressed in osteoblasts and regulates vertebral trabecular bone mass in a cell-autonomous manner; osteoblast-derived RSPO3 is required for a full estrogenic response on cortical (but not trabecular) bone. Estradiol and RSPO3 regulate vertebral trabecular bone mass independently of each other.","method":"Osteoblast-specific Rspo3 conditional KO (Runx2-Cre), OVX mouse model with estradiol treatment, bone densitometry and bone strength testing","journal":"American journal of physiology. Endocrinology and metabolism","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with epistasis to estrogen pathway, single lab, extends prior PMID:34389713 findings","pmids":["35068191"],"is_preprint":false},{"year":2024,"finding":"RSPO3 overexpression increases NSCLC radiosensitivity through induction of pyroptosis mediated by the β-catenin–NF-κB signaling pathway and NLRP3 inflammasome. Anti-RSPO3 antibody (OMP-131R10) blocked radiation-induced pyroptosis and anti-tumor immunity in vivo.","method":"RSPO3 overexpression/knockdown in NSCLC cell lines, β-catenin/NF-κB pathway inhibitors, NLRP3 inflammasome assays, in vivo anti-RSPO3 antibody treatment with radiation","journal":"Radiotherapy and oncology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, pathway inhibitor experiments without direct mechanistic reconstitution, abstract does not detail mutagenesis or rigorous controls","pmids":["39245068"],"is_preprint":false},{"year":2023,"finding":"The RSPO3 promoter is regulated by the balance between DNA methyltransferase DNMT3a and DNA demethylase TET1 in cholangiocarcinoma. Targeted RSPO3 promoter demethylation using dCas9-TET1CD inhibited CCA tumorigenicity, while targeted methylation using dCas9-DNMT3a promoted it.","method":"Targeted epigenetic editing (dCas9-DNMT3a and dCas9-TET1CD), in vitro and in vivo CCA models, methylation analysis","journal":"Clinical epigenetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct epigenetic writer/eraser manipulation with functional consequence, single lab","pmids":["37932819"],"is_preprint":false},{"year":2026,"finding":"MHP1-AcN (a RANKL-derived peptide) directly interacts with LGR4 and disrupts RSPO3-induced LGR4-IQGAP1 complex formation, inhibiting RSPO3-enhanced phosphorylation of LRP6 and accumulation of β-catenin, and suppressing tumor growth and metastatic potential in lung adenocarcinoma.","method":"Immunoprecipitation for LGR4-IQGAP1 complex, immunoblotting for LRP6 phosphorylation and β-catenin, A549 xenograft model, cell migration/invasion assays","journal":"Anticancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical complex disruption assay combined with in vivo xenograft, single lab","pmids":["42203310"],"is_preprint":false},{"year":2012,"finding":"Rspo3 single conditional knockout in the limb did not produce limb defects, but combining Rspo3 and Rspo2 mutations caused severe hindlimb truncations, demonstrating redundant function of these R-spondin paralogs during limb development.","method":"Conditional Rspo3 allele (loxP-flanked exons 2-4), Cre-mediated deletion, Rspo2/Rspo3 double knockout genetic epistasis","journal":"Genesis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — double-mutant genetic epistasis establishing functional redundancy, single lab, clean developmental phenotype","pmids":["22610508"],"is_preprint":false},{"year":2025,"finding":"H3K4me3 histone modification regulates the transcription of RSPO3 in dermal papilla cells. Increasing H3K4me3 levels enhanced DPC proliferation and Wnt signaling pathway gene expression, partly through elevated RSPO3. RSPO3 itself promotes DPC proliferation, inhibits apoptosis, and increases Wnt pathway gene expression.","method":"CUT&Tag for H3K4me3 mapping, H3K4me3 inhibitor (BCL-121) and agonist (PBIT) treatment, RNA-seq, RSPO3 functional assays in DPCs","journal":"Epigenetics & chromatin","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, CUT&Tag plus functional experiments but primarily in a non-canonical cell type (goat DPCs) with limited mechanistic resolution in the abstract","pmids":["40775776"],"is_preprint":false},{"year":2025,"finding":"Hepatic Rspo3 regulates systemic glucose metabolism and body composition via inter-organ communication involving afferent vagal and efferent sympathetic nerves. Viral-mediated hepatic Rspo3 induction improved insulin resistance in obese mice; hepatic vagal denervation suppressed these remote effects on adipose tissue and skeletal muscle. Hepatic Rspo3 suppression (Cre-LoxP) exacerbated diabetes and obesity.","method":"Viral-mediated hepatic Rspo3 overexpression, Cre-LoxP hepatic conditional KO, hepatic vagus denervation, metabolic phenotyping (glucose tolerance, insulin sensitivity, body composition)","journal":"PLoS biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO plus viral OE with denervation epistasis identifying neuronal inter-organ communication; single lab","pmids":["39854351"],"is_preprint":false},{"year":2025,"finding":"RSPO3 promotes proliferation and self-renewal of human limbal epithelial stem cells (LESCs) through a WNT/β-catenin-independent signaling pathway, as demonstrated by the lack of impairment when β-catenin activation was blocked by XAV939.","method":"Exogenous RSPO3 treatment of hLESCs, β-catenin inhibitor XAV939, EdU proliferation assay, stemness marker assessment (ΔNp63, ABCG2), in vivo corneal wound healing model","journal":"Investigative ophthalmology & visual science","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, pharmacological inhibitor used to establish pathway independence; downstream pathway not identified","pmids":["39760688"],"is_preprint":false},{"year":2026,"finding":"Chondrocyte-derived RSPO3 acts in an autocrine manner to enhance chondrocyte anabolism and in a paracrine manner to directly drive M2 macrophage polarization. The pro-M2 macrophage effect is specifically mediated through activation of the LGR4/LRP6/β-catenin signaling axis in macrophages.","method":"NsPEFs-ADSCs-EV treatment, RSPO3 neutralization, macrophage polarization assays (M1/M2 markers), LGR4/LRP6/β-catenin signaling assays, OA mouse model","journal":"Bioactive materials","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, abstract does not describe direct reconstitution or mutagenesis for the LGR4/LRP6/β-catenin pathway; mechanistic detail limited in abstract","pmids":["41624077"],"is_preprint":false}],"current_model":"RSPO3 is a secreted protein that potentiates Wnt signaling through multiple mechanisms: it binds syndecan-4 to activate non-canonical Wnt/PCP signaling via clathrin-mediated endocytosis (transduced through Wnt5a/Fz7/Dvl/JNK); it signals through its cognate receptor LGR4 (and LGR5) to amplify canonical Wnt/β-catenin signaling by clearing RNF43/ZNRF3 from the cell surface; in endothelial cells it drives non-canonical WNT/Ca2+/NFAT signaling to maintain vascular remodeling; in hepatocytes and AML cells it operates through LGR4-Gab1-Gαi1/3-ERK and AMPKα-SREBP2 downstream cascades; it is produced by specific stromal niche cells (PDGFRα+ pericryptal myofibroblasts, adrenal capsule cells, osteoblasts) to support tissue-specific stem cell compartments; and PTPRK-RSPO3 gene fusions that increase RSPO3 expression are recurrent oncogenic drivers in colorectal cancer, where RSPO3 antagonism inhibits tumor growth and promotes differentiation."},"narrative":{"mechanistic_narrative":"RSPO3 is a secreted Wnt-pathway potentiator that acts as a niche-derived ligand to sustain tissue-specific stem cell compartments and to control vascular and metabolic homeostasis [PMID:29559533, PMID:26766444]. It engages two principal signaling modes: a syndecan-4 (SDC4)-dependent route that drives clathrin-mediated endocytosis of Wnt-receptor complexes to transduce non-canonical Wnt/PCP signaling through Wnt5a/Fz7/Dvl/JNK [PMID:21397842], and a route through its cognate receptor LGR4 (and LGR5) that amplifies canonical Wnt/β-catenin signaling [PMID:29559533, PMID:32559496]. Through LGR4, RSPO3 couples to multiple downstream cascades in a context-dependent manner — Gab1–Gαi1/3–ERK in endothelium and neurons [PMID:37805583], IQGAP1-dependent LRP6/β-catenin activation in lung adenocarcinoma [PMID:25531322, PMID:42203310], AMPKα–SREBP2 suppression of hepatic cholesterol synthesis [PMID:32926477], NF-κB activation in gastric stem cells [PMID:35767364], and ILK/Akt in pulmonary endothelial regeneration [PMID:38677673]. In the intestinal niche RSPO3 is produced principally by PDGFRα+ pericryptal myofibroblasts and is far more potent than RSPO1 in stimulating crypt Wnt/β-catenin signaling, stem cell expansion, and Paneth cell differentiation [PMID:29559533, PMID:27511199]. RSPO3 supports comparable stem/progenitor functions in other compartments via dedicated stromal sources, including adrenal capsule cells that imprint zona glomerulosa fate [PMID:27313319] and osteoblasts that regulate trabecular bone mass [PMID:34389713]. In endothelium it maintains vascular remodeling through non-canonical WNT/Ca2+/NFAT signaling, with RNF213-mediated degradation of NFAT1 and filamin A as a regulatory node [PMID:26766444]. Recurrent PTPRK-RSPO3 gene fusions act as oncogenic drivers in colorectal cancer, where the lesion sustains a Wnt-dependent stem-cell compartment and anti-RSPO3 antibody treatment inhibits tumor growth and forces differentiation [PMID:26700806, PMID:27511199]. RSPO3 can also act through non-LGR receptors, including GNG7-mediated Akt/GSK-3β/β-catenin signaling in gastric cancer stem cells [PMID:38581123].","teleology":[{"year":2011,"claim":"Establishing how RSPO3 transmits a non-canonical Wnt signal answered whether R-spondins act only by stabilizing Wnt receptors or by actively remodeling receptor trafficking.","evidence":"Binding assays, Xenopus loss-of-function and epistasis with Wnt5a/Fz7/Dvl/JNK, and clathrin inhibition","pmids":["21397842"],"confidence":"High","gaps":["Whether the SDC4/PCP route operates in mammalian adult tissues was not addressed","Structural basis of the RSPO3-SDC4 interaction not defined"]},{"year":2012,"claim":"Single Rspo3 deletion versus Rspo2/Rspo3 double mutants resolved whether RSPO3 has a unique developmental role or is functionally redundant with paralogs.","evidence":"Conditional Rspo3 allele and Rspo2/Rspo3 double-knockout genetic epistasis in limb","pmids":["22610508"],"confidence":"Medium","gaps":["Did not identify the receptors or downstream pathway underlying limb redundancy","No molecular distinction between RSPO2 and RSPO3 contributions"]},{"year":2014,"claim":"Identifying LGR4 and IQGAP1 as effectors in Keap1-deficient lung adenocarcinoma extended RSPO3 signaling from development into a tumor-promoting proliferation/migration axis.","evidence":"shRNA knockdown of RSPO3/LGR4/IQGAP1 with proliferation, migration, and in vivo metastasis assays","pmids":["25531322"],"confidence":"Medium","gaps":["IQGAP1 not shown to bind RSPO3 directly","Mechanism linking LGR4-IQGAP1 to proliferation not reconstituted"]},{"year":2015,"claim":"Anti-RSPO3 treatment of PTPRK-RSPO3 fusion-positive xenografts tested whether the fusion is a therapeutic dependency and which cell compartment drives tumor growth.","evidence":"Anti-RSPO3 antibody treatment of patient-derived xenografts with stem cell and expression assays","pmids":["26700806"],"confidence":"High","gaps":["Receptor and downstream signaling for the fusion-driven growth not dissected here","Durability of response and resistance not addressed"]},{"year":2016,"claim":"Tissue-specific knockouts defined RSPO3 as a niche-derived maintenance signal in distinct organs, clarifying that its developmental role persists into adult homeostasis.","evidence":"Inducible endothelial-specific and adrenal conditional knockouts with pathway and phenotypic readouts (vascular pruning, NFAT, β-catenin, SHH)","pmids":["26766444","27313319"],"confidence":"High","gaps":["Endothelial and adrenal studies used distinct downstream readouts (WNT/Ca2+/NFAT vs β-catenin) without a unifying mechanism","Upstream regulation of stromal RSPO3 production not defined"]},{"year":2016,"claim":"Gain-of-function Rspo3 expression in vivo showed RSPO3 is sufficient to expand intestinal stem cells and drive tumorigenesis, linking its niche role to oncogenesis.","evidence":"Conditional Rspo3 transgenic mice with stem cell marker flow cytometry and Kras synergy","pmids":["27511199"],"confidence":"High","gaps":["Only modest β-catenin increase observed, leaving the proliferative driver partly unexplained","Receptor dependence not formally tested in this model"]},{"year":2017,"claim":"Studies in bone progenitors and colorectal resistance refined RSPO3 signaling outputs as both ERK-modulatory and dependent on intact downstream Wnt machinery.","evidence":"RSPO3/LGR4 manipulation with ERK inhibitors in hASCs; LGK974 resistance selection and AXIN1 RNAi in fusion-positive CRC cells","pmids":["28220828","28100566"],"confidence":"Medium","gaps":["RSPO3 as a negative ERK regulator in stem cells contrasts with ERK-driving roles elsewhere, unresolved","AXIN1 loss conferring porcupine-inhibitor resistance not mechanistically linked to RSPO3 itself"]},{"year":2018,"claim":"Endothelial barrier and intestinal niche-source studies sharpened both the cellular origin and a permeability function of RSPO3.","evidence":"PDGFRα-Cre conditional knockout with organoid rescue; ECIS barrier assays with VE-cadherin/β-catenin imaging and IL-1β co-treatment","pmids":["29559533","30157748"],"confidence":"High","gaps":["Barrier-disrupting role appears to oppose vascular-maintenance role, leaving net endothelial function context-dependent","Receptor mediating junction disruption not identified"]},{"year":2020,"claim":"Discovery of RSPO3-LGR4 outputs in AML, hepatocytes, and adipose depots established defined downstream cascades and metabolic/oncogenic roles beyond canonical Wnt.","evidence":"Anti-RSPO3 antibody in AML PDX with HOXA9 epistasis; AMPKα-SREBP2 epistasis in hepatocytes; depot-specific adipose progenitor assays and zebrafish rspo3 mutant","pmids":["32559496","32926477","32493999"],"confidence":"Medium","gaps":["AML finding is High-confidence but hepatic and adipose cascades rest on single-lab knockdown epistasis","How a single ligand selects between SREBP2, NF-κB, and Wnt outputs not defined"]},{"year":2021,"claim":"Osteoblast-specific knockout identified the cellular source and cell-autonomous bone function of RSPO3, and a later study placed it relative to estrogen signaling.","evidence":"Runx2-Cre conditional knockout with bone densitometry and strength testing; OVX/estradiol epistasis","pmids":["34389713","35068191"],"confidence":"High","gaps":["Receptor mediating osteoblast-autonomous effect not pinned down","Why estrogen-dependence differs between cortical and trabecular bone unexplained"]},{"year":2022,"claim":"Gastric stem cell studies revealed RSPO3-LGR4 can activate NF-κB, coupling the pathway to inflammation and pathogen-driven hyperplasia.","evidence":"Lgr4- and Lgr5-specific conditional knockouts with H. pylori infection, NF-κB and chemokine/neutrophil readouts","pmids":["35767364"],"confidence":"High","gaps":["Molecular link from LGR4 to NF-κB not reconstituted","Whether RSPO3 ligand itself, versus other RSPOs, drives this in vivo not isolated"]},{"year":2023,"claim":"Mechanistic studies defined a Gab1-Gαi1/3-ERK signaling complex and mapped RSPO3 receptor-targeting domains, and identified epigenetic control of the RSPO3 promoter.","evidence":"Co-IP for LGR4-Gab1-Gαi, Gαi KO MEFs and MCAO neuroprotection; FuFu-domain liposome uptake in LGR5-high cells; dCas9-TET1/DNMT3a promoter editing in cholangiocarcinoma","pmids":["37805583","36813038","37932819"],"confidence":"Medium","gaps":["Gab1-Gαi-ERK complex shown by Co-IP in single lab without structural validation","Relationship between FuFu-domain LGR5 binding and the LGR4-Gab1 cascade not integrated"]},{"year":2024,"claim":"Newer studies broadened RSPO3 effectors to ILK/Akt, GNG7, and pyroptosis pathways and tested complex-disruption as a therapeutic strategy.","evidence":"EC-specific genetic models with ILK/Akt readout; Co-IP/LC-MS identification of GNG7 with knockdown epistasis; NSCLC radiosensitivity with NLRP3 readouts; MHP1-AcN peptide disruption of LGR4-IQGAP1","pmids":["38677673","38581123","39245068","42203310"],"confidence":"Medium","gaps":["GNG7-mediated, LGR-independent route rests on a single Co-IP/MS study","Pyroptosis/radiosensitivity finding is Low-confidence and not reconstituted"]},{"year":2025,"claim":"Inter-organ and additional niche studies extended RSPO3 to neurally relayed metabolic control and to non-Wnt-dependent stem cell maintenance.","evidence":"Hepatic Rspo3 OE/KO with vagal denervation and metabolic phenotyping; XAV939-resistant hLESC proliferation; H3K4me3 regulation of RSPO3 in dermal papilla cells","pmids":["39854351","39760688","40775776"],"confidence":"Medium","gaps":["The signal carried from liver to nerves to peripheral tissues is undefined","β-catenin-independent stem cell route lacks an identified downstream effector"]},{"year":null,"claim":"It remains unresolved how a single secreted RSPO3 molecule selects among SDC4/PCP, LGR4-canonical Wnt, LGR4-Gab1-Gαi-ERK, NF-κB, GNG7-Akt, and β-catenin-independent outputs in different cell types.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural or quantitative model integrates the receptor/co-receptor repertoire that dictates pathway choice","Direct binding partners beyond SDC4, LGR4/LGR5, and GNG7 not systematically mapped"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0048018","term_label":"receptor ligand activity","supporting_discovery_ids":[0,3,6]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[1,3,12]}],"localization":[{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[3,4,18]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,3,6]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[2,23,10]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[4,5,6]}],"complexes":[],"partners":["SDC4","LGR4","LGR5","IQGAP1","GNG7"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9BXY4","full_name":"R-spondin-3","aliases":["Protein with TSP type-1 repeat","hPWTSR","Roof plate-specific spondin-3","hRspo3","Thrombospondin type-1 domain-containing protein 2"],"length_aa":272,"mass_kda":30.9,"function":"Activator of the canonical Wnt signaling pathway by acting as a ligand for LGR4-6 receptors, which acts as a key regulator of angiogenesis. Upon binding to LGR4-6 (LGR4, LGR5 or LGR6), LGR4-6 associate with phosphorylated LRP6 and frizzled receptors that are activated by extracellular Wnt receptors, triggering the canonical Wnt signaling pathway to increase expression of target genes. Also regulates the canonical Wnt/beta-catenin-dependent pathway and non-canonical Wnt signaling by acting as an inhibitor of ZNRF3, an important regulator of the Wnt signaling pathway. Acts as a ligand for frizzled FZD8 and LRP6. May negatively regulate the TGF-beta pathway (PubMed:21727895, PubMed:21909076, PubMed:22615920). Acts as a key regulator of angiogenesis by controlling vascular stability and pruning: acts by activating the non-canonical Wnt signaling pathway in endothelial cells (By similarity) (PubMed:21727895, PubMed:21909076, PubMed:22615920). Can also amplify Wnt signaling pathway independently of LGR4-6 receptors, possibly by acting as a direct antagonistic ligand to RNF43 and ZNRF3 (PubMed:29769720)","subcellular_location":"Secreted","url":"https://www.uniprot.org/uniprotkb/Q9BXY4/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RSPO3","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/RSPO3","total_profiled":1310},"omim":[{"mim_id":"610575","title":"R-SPONDIN 2; RSPO2","url":"https://www.omim.org/entry/610575"},{"mim_id":"610574","title":"R-SPONDIN 3; RSPO3","url":"https://www.omim.org/entry/610574"},{"mim_id":"606667","title":"LEUCINE-RICH REPEAT-CONTAINING G PROTEIN-COUPLED RECEPTOR 5; LGR5","url":"https://www.omim.org/entry/606667"},{"mim_id":"606666","title":"LEUCINE-RICH REPEAT-CONTAINING G PROTEIN-COUPLED RECEPTOR 4; LGR4","url":"https://www.omim.org/entry/606666"},{"mim_id":"604025","title":"AXIS INHIBITOR 2; AXIN2","url":"https://www.omim.org/entry/604025"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"smooth muscle","ntpm":44.9}],"url":"https://www.proteinatlas.org/search/RSPO3"},"hgnc":{"alias_symbol":["FLJ14440"],"prev_symbol":["THSD2"]},"alphafold":{"accession":"Q9BXY4","domains":[{"cath_id":"2.10.220.10","chopping":"40-97","consensus_level":"medium","plddt":94.8467,"start":40,"end":97},{"cath_id":"-","chopping":"109-144","consensus_level":"medium","plddt":96.5308,"start":109,"end":144},{"cath_id":"2.20.100.10","chopping":"147-205","consensus_level":"medium","plddt":95.9503,"start":147,"end":205}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BXY4","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BXY4-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BXY4-F1-predicted_aligned_error_v6.png","plddt_mean":78.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RSPO3","jax_strain_url":"https://www.jax.org/strain/search?query=RSPO3"},"sequence":{"accession":"Q9BXY4","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9BXY4.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9BXY4/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BXY4"}},"corpus_meta":[{"pmid":"29559533","id":"PMC_29559533","title":"PDGFRα pericryptal stromal cells are the critical source of Wnts and RSPO3 for murine intestinal stem cells in vivo.","date":"2018","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/29559533","citation_count":234,"is_preprint":false},{"pmid":"26700806","id":"PMC_26700806","title":"Targeting PTPRK-RSPO3 colon tumours promotes differentiation and loss of stem-cell function.","date":"2015","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/26700806","citation_count":204,"is_preprint":false},{"pmid":"21397842","id":"PMC_21397842","title":"Rspo3 binds syndecan 4 and induces Wnt/PCP signaling via clathrin-mediated endocytosis to promote morphogenesis.","date":"2011","source":"Developmental cell","url":"https://pubmed.ncbi.nlm.nih.gov/21397842","citation_count":194,"is_preprint":false},{"pmid":"26766444","id":"PMC_26766444","title":"Endothelial RSPO3 Controls Vascular Stability and Pruning through Non-canonical WNT/Ca(2+)/NFAT Signaling.","date":"2016","source":"Developmental cell","url":"https://pubmed.ncbi.nlm.nih.gov/26766444","citation_count":152,"is_preprint":false},{"pmid":"26924569","id":"PMC_26924569","title":"Frequent PTPRK-RSPO3 fusions and RNF43 mutations in colorectal traditional serrated adenoma.","date":"2016","source":"The Journal of pathology","url":"https://pubmed.ncbi.nlm.nih.gov/26924569","citation_count":107,"is_preprint":false},{"pmid":"32559496","id":"PMC_32559496","title":"Targeting RSPO3-LGR4 Signaling for Leukemia Stem Cell Eradication in Acute Myeloid Leukemia.","date":"2020","source":"Cancer cell","url":"https://pubmed.ncbi.nlm.nih.gov/32559496","citation_count":96,"is_preprint":false},{"pmid":"27313319","id":"PMC_27313319","title":"The adrenal capsule is a signaling center controlling cell renewal and zonation through Rspo3.","date":"2016","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/27313319","citation_count":76,"is_preprint":false},{"pmid":"27511199","id":"PMC_27511199","title":"RSPO3 expands intestinal stem cell and niche compartments and drives tumorigenesis.","date":"2016","source":"Gut","url":"https://pubmed.ncbi.nlm.nih.gov/27511199","citation_count":65,"is_preprint":false},{"pmid":"25531322","id":"PMC_25531322","title":"Aberrant RSPO3-LGR4 signaling in Keap1-deficient lung adenocarcinomas promotes tumor aggressiveness.","date":"2014","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/25531322","citation_count":64,"is_preprint":false},{"pmid":"28100566","id":"PMC_28100566","title":"Loss of AXIN1 drives acquired resistance to WNT pathway blockade in colorectal cancer cells carrying RSPO3 fusions.","date":"2017","source":"EMBO molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/28100566","citation_count":56,"is_preprint":false},{"pmid":"32493999","id":"PMC_32493999","title":"RSPO3 impacts body fat distribution and regulates adipose cell biology in vitro.","date":"2020","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/32493999","citation_count":49,"is_preprint":false},{"pmid":"28220828","id":"PMC_28220828","title":"RSPO3-LGR4 Regulates Osteogenic Differentiation Of Human Adipose-Derived Stem Cells Via ERK/FGF Signalling.","date":"2017","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/28220828","citation_count":47,"is_preprint":false},{"pmid":"29127379","id":"PMC_29127379","title":"RSPO3 antagonism inhibits growth and tumorigenicity in colorectal tumors harboring common Wnt pathway mutations.","date":"2017","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/29127379","citation_count":41,"is_preprint":false},{"pmid":"22610508","id":"PMC_22610508","title":"A conditional allele of Rspo3 reveals redundant function of R-spondins during mouse limb development.","date":"2012","source":"Genesis (New York, N.Y. : 2000)","url":"https://pubmed.ncbi.nlm.nih.gov/22610508","citation_count":35,"is_preprint":false},{"pmid":"34389713","id":"PMC_34389713","title":"RSPO3 is important for trabecular bone and fracture risk in mice and humans.","date":"2021","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/34389713","citation_count":29,"is_preprint":false},{"pmid":"35767364","id":"PMC_35767364","title":"Gastric stem cells promote inflammation and gland remodeling in response to Helicobacter pylori via Rspo3-Lgr4 axis.","date":"2022","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/35767364","citation_count":25,"is_preprint":false},{"pmid":"30329043","id":"PMC_30329043","title":"RSPO3 promotes the aggressiveness of bladder cancer via Wnt/β-catenin and Hedgehog signaling pathways.","date":"2019","source":"Carcinogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/30329043","citation_count":20,"is_preprint":false},{"pmid":"32926477","id":"PMC_32926477","title":"Rspo1/Rspo3-LGR4 signaling inhibits hepatic cholesterol synthesis through the AMPKα-SREBP2 pathway.","date":"2020","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/32926477","citation_count":18,"is_preprint":false},{"pmid":"30157748","id":"PMC_30157748","title":"RSPO3 impairs barrier function of human vascular endothelial monolayers and synergizes with pro-inflammatory IL-1.","date":"2018","source":"Molecular medicine (Cambridge, Mass.)","url":"https://pubmed.ncbi.nlm.nih.gov/30157748","citation_count":17,"is_preprint":false},{"pmid":"37805583","id":"PMC_37805583","title":"Endothelial cell-derived RSPO3 activates Gαi1/3-Erk signaling and protects neurons from ischemia/reperfusion injury.","date":"2023","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/37805583","citation_count":16,"is_preprint":false},{"pmid":"33313096","id":"PMC_33313096","title":"RSPO3 is a marker candidate for predicting tumor aggressiveness in ovarian cancer.","date":"2020","source":"Annals of translational medicine","url":"https://pubmed.ncbi.nlm.nih.gov/33313096","citation_count":16,"is_preprint":false},{"pmid":"38677673","id":"PMC_38677673","title":"Endothelial RSPO3 mediates pulmonary endothelial regeneration by LGR4-dependent activation of β-catenin and ILK signaling pathways after inflammatory vascular injury.","date":"2024","source":"International journal of biological macromolecules","url":"https://pubmed.ncbi.nlm.nih.gov/38677673","citation_count":13,"is_preprint":false},{"pmid":"39245068","id":"PMC_39245068","title":"RSPO3 regulates the radioresistance of Non-Small cell lung cancer cells via NLRP3 Inflammasome-Mediated pyroptosis.","date":"2024","source":"Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology","url":"https://pubmed.ncbi.nlm.nih.gov/39245068","citation_count":12,"is_preprint":false},{"pmid":"34099046","id":"PMC_34099046","title":"Rspo3 regulates the abnormal differentiation of small intestinal epithelial cells in diabetic state.","date":"2021","source":"Stem cell research & therapy","url":"https://pubmed.ncbi.nlm.nih.gov/34099046","citation_count":8,"is_preprint":false},{"pmid":"35068191","id":"PMC_35068191","title":"Estradiol and RSPO3 regulate vertebral trabecular bone mass independent of each other.","date":"2022","source":"American journal of physiology. Endocrinology and metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/35068191","citation_count":6,"is_preprint":false},{"pmid":"35995979","id":"PMC_35995979","title":"RSPO3 is a novel contraction-inducible factor identified in an \"in vitro exercise model\" using primary human myotubes.","date":"2022","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/35995979","citation_count":6,"is_preprint":false},{"pmid":"39854351","id":"PMC_39854351","title":"Rspo3-mediated metabolic liver zonation regulates systemic glucose metabolism and body mass in mice.","date":"2025","source":"PLoS biology","url":"https://pubmed.ncbi.nlm.nih.gov/39854351","citation_count":5,"is_preprint":false},{"pmid":"29966290","id":"PMC_29966290","title":"Cynoglossus semilaevis Rspo3 Regulates Embryo Development by Inhibiting the Wnt/β-Catenin Signaling Pathway.","date":"2018","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/29966290","citation_count":5,"is_preprint":false},{"pmid":"29230809","id":"PMC_29230809","title":"The RSPO3 gene as genetic markers for bone mass assessed by quantitative ultrasound in a population of young adults.","date":"2017","source":"Annals of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/29230809","citation_count":5,"is_preprint":false},{"pmid":"37932819","id":"PMC_37932819","title":"Upregulation of RSPO3 via targeted promoter DNA demethylation inhibits the progression of cholangiocarcinoma.","date":"2023","source":"Clinical epigenetics","url":"https://pubmed.ncbi.nlm.nih.gov/37932819","citation_count":4,"is_preprint":false},{"pmid":"37173190","id":"PMC_37173190","title":"Amniotic fluid stem cell attenuated necrotizing enterocolitis progression by promoting Rspo3/AMPKα axis.","date":"2023","source":"Immunobiology","url":"https://pubmed.ncbi.nlm.nih.gov/37173190","citation_count":4,"is_preprint":false},{"pmid":"38581123","id":"PMC_38581123","title":"RSPO3 induced by Helicobacter pylori extracts promotes gastric cancer stem cell properties through the GNG7/β-catenin signaling pathway.","date":"2024","source":"Cancer medicine","url":"https://pubmed.ncbi.nlm.nih.gov/38581123","citation_count":4,"is_preprint":false},{"pmid":"36813038","id":"PMC_36813038","title":"RSPO3 Furin domain-conjugated liposomes for selective drug delivery to LGR5-high cells.","date":"2023","source":"Journal of controlled release : official journal of the Controlled Release Society","url":"https://pubmed.ncbi.nlm.nih.gov/36813038","citation_count":4,"is_preprint":false},{"pmid":"36129915","id":"PMC_36129915","title":"Investigation of cell signalings and therapeutic targets in PTPRK-RSPO3 fusion-positive colorectal cancer.","date":"2022","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/36129915","citation_count":2,"is_preprint":false},{"pmid":"39760688","id":"PMC_39760688","title":"RSPO3 Promotes Proliferation and Self-Renewal of Limbal Epithelial Stem Cells Through a WNT/β-Catenin-Independent Signaling Pathway.","date":"2025","source":"Investigative ophthalmology & visual science","url":"https://pubmed.ncbi.nlm.nih.gov/39760688","citation_count":2,"is_preprint":false},{"pmid":"40775776","id":"PMC_40775776","title":"H3K4me3 regulates the transcription of RSPO3 in dermal papilla cells to influence hair follicle morphogenesis and development.","date":"2025","source":"Epigenetics & chromatin","url":"https://pubmed.ncbi.nlm.nih.gov/40775776","citation_count":2,"is_preprint":false},{"pmid":"37767857","id":"PMC_37767857","title":"Rspo1 and Rspo3 are required for sensory lineage neural crest formation in mouse embryos.","date":"2023","source":"Developmental dynamics : an official publication of the American Association of Anatomists","url":"https://pubmed.ncbi.nlm.nih.gov/37767857","citation_count":1,"is_preprint":false},{"pmid":"40398181","id":"PMC_40398181","title":"Lonicerin targets ADRA1D and RSPO3 to ameliorate diabetes-induced vascular injury through Ca2+/Calcineurin/NFAT1-dependent anti-EndMT pathway.","date":"2025","source":"Phytomedicine : international journal of phytotherapy and phytopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/40398181","citation_count":1,"is_preprint":false},{"pmid":"39568191","id":"PMC_39568191","title":"Decreased RSPO3 and β-Catenin in Preeclampsia: Correlation with Blood Pressure and Pregnancy Outcomes.","date":"2024","source":"Medical science monitor : international medical journal of experimental and clinical research","url":"https://pubmed.ncbi.nlm.nih.gov/39568191","citation_count":1,"is_preprint":false},{"pmid":"40860620","id":"PMC_40860620","title":"Unveiling CTRB2, RSPO3, KLOTB, and ROR1 as obesity-pancreatic disease association proteins: a comprehensive Mendelian randomization study.","date":"2025","source":"Gastroenterology report","url":"https://pubmed.ncbi.nlm.nih.gov/40860620","citation_count":0,"is_preprint":false},{"pmid":"41281482","id":"PMC_41281482","title":"RSPO3 rearrangements in advanced colorectal cancer patients and their relationship with disease characteristics.","date":"2025","source":"World journal of gastrointestinal oncology","url":"https://pubmed.ncbi.nlm.nih.gov/41281482","citation_count":0,"is_preprint":false},{"pmid":"41624077","id":"PMC_41624077","title":"NsPEFs-enriched ADSCs-EVs alleviate osteoarthritis via RSPO3-mediated dual pro-chondrogenic and pro-M2 macrophage properties.","date":"2026","source":"Bioactive materials","url":"https://pubmed.ncbi.nlm.nih.gov/41624077","citation_count":0,"is_preprint":false},{"pmid":"41573670","id":"PMC_41573670","title":"Deletion of Rspo1 or Rspo3 in the mesenchyme does not affect Wolffian duct maintenance or morphogenesis.","date":"2026","source":"microPublication biology","url":"https://pubmed.ncbi.nlm.nih.gov/41573670","citation_count":0,"is_preprint":false},{"pmid":"42203310","id":"PMC_42203310","title":"A RANKL-derived Peptide Inhibits RSPO3-LGR4-Wnt Signaling and Lung Adenocarcinoma in Mice.","date":"2026","source":"Anticancer research","url":"https://pubmed.ncbi.nlm.nih.gov/42203310","citation_count":0,"is_preprint":false},{"pmid":"40419046","id":"PMC_40419046","title":"Protein interaction network drive more group integrated analytic larynx hub protein markers: LYVE1/FBLN5/INMT/DCN/ZFY/RSPO3 protein macromolecule collaborative diagnosis of a new era.","date":"2025","source":"International journal of biological macromolecules","url":"https://pubmed.ncbi.nlm.nih.gov/40419046","citation_count":0,"is_preprint":false},{"pmid":"42218990","id":"PMC_42218990","title":"Deciphering the RSPO3-NFATC1 switch in the bone-cartilage paradox: Genetic liability to osteoporosis protects against osteoarthritis by modulating subchondral bone compliance.","date":"2026","source":"Osteoarthritis and cartilage","url":"https://pubmed.ncbi.nlm.nih.gov/42218990","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.09.08.674957","title":"TNF Receptor 1 regulates colonic mesenchymal cell diversity and the epithelial stem cell niche","date":"2025-09-09","source":"bioRxiv","url":"https://doi.org/10.1101/2025.09.08.674957","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.05.24.655925","title":"Reconstitution of adrenocortical functional zonation from human pluripotent stem cells","date":"2025-05-28","source":"bioRxiv","url":"https://doi.org/10.1101/2025.05.24.655925","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.01.03.631121","title":"Role of RSPO3 in Estrogen-mediated Sex Differences in Body Fat Distribution: A Single-cell Data-driven Study","date":"2025-01-04","source":"bioRxiv","url":"https://doi.org/10.1101/2025.01.03.631121","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.12.05.24318588","title":"Assessment of MYC Gene and WNT Pathway Alterations in Early-Onset Colorectal Cancer Among Hispanic/Latino Patients Using Integrated Multi-Omics Approaches","date":"2024-12-08","source":"bioRxiv","url":"https://doi.org/10.1101/2024.12.05.24318588","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":28430,"output_tokens":7688,"usd":0.100305,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":17176,"output_tokens":5141,"usd":0.107203,"stage2_stop_reason":"end_turn"},"total_usd":0.207508,"stage1_batch_id":"msgbatch_013LWZZT6MWaqB9qyTSeQBmU","stage2_batch_id":"msgbatch_0194ZzQaTGNpruoNJBbEMbt3","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2011,\n      \"finding\": \"RSPO3 binds syndecan 4 (SDC4) and together they activate Wnt/PCP signaling; RSPO3 functions by inducing SDC4-dependent, clathrin-mediated endocytosis of Wnt-receptor complexes, which is essential for PCP signal transduction. In Xenopus embryos, RSPO3/PCP signaling during gastrulation requires Wnt5a and is transduced via Fz7, Dvl, and JNK.\",\n      \"method\": \"Binding assays, Xenopus embryo loss-of-function, epistasis with Wnt5a/Fz7/Dvl/JNK, clathrin inhibition experiments\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods (binding assay, genetic epistasis, pharmacological inhibition of endocytosis) in a single rigorous study with functional rescue experiments in Xenopus\",\n      \"pmids\": [\"21397842\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Endothelial RSPO3 controls vascular remodeling via non-canonical WNT/Ca2+/NFAT signaling. Inducible endothelial-specific Rspo3 deletion caused endothelial apoptosis and vascular pruning. RSPO3 co-regulates genes including Rnf213, Usp18, and Trim30α; RNF213 targets filamin A and NFAT1 for proteasomal degradation, attenuating non-canonical WNT/Ca2+ signaling. NFAT protein levels were decreased in endothelial cells of Rspo3-iECKO mice, and pharmacological NFAT inhibition phenocopied Rspo3-iECKO mice.\",\n      \"method\": \"Inducible endothelial-specific conditional knockout, endothelial gene expression screen, pharmacological NFAT inhibition, protein degradation assays\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal genetic and pharmacological validation, multiple orthogonal methods including conditional KO and pathway phenocopy experiments\",\n      \"pmids\": [\"26766444\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Capsular RSPO3 signals to the underlying steroidogenic compartment of the adrenal gland to induce β-catenin signaling and imprint zona glomerulosa cell fate. Deletion of RSPO3 leads to loss of SHH signaling and impaired organ growth. RSPO3 function is required in adult life to ensure replenishment of lost cells and maintain zona glomerulosa properties.\",\n      \"method\": \"Conditional knockout mouse models, immunohistochemistry, β-catenin signaling readout, SHH pathway analysis\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO with multiple specific phenotypic readouts (β-catenin, SHH, zonation, organ growth) across development and adult life\",\n      \"pmids\": [\"27313319\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"PDGFRα+ pericryptal stromal myofibroblasts are the principal source of RSPO3 in the intestinal stem cell niche in vivo. RSPO3 is several orders of magnitude more potent than RSPO1 in stimulating Wnt/β-catenin signaling and organoid growth. Stromal Rspo3 ablation in PdgfRα+ cells decreased intestinal crypt Wnt/β-catenin signaling and Paneth cell differentiation, and was rescued by exogenous RSPO3 protein.\",\n      \"method\": \"Cell-type-specific Cre-mediated conditional knockout (PdgfRα-Cre;Rspo3fl/fl), organoid growth assays, exogenous protein rescue, β-catenin signaling measurements\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo conditional KO with specific cellular source identification, quantitative potency comparison, and rescue experiments using exogenous RSPO3\",\n      \"pmids\": [\"29559533\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Targeting RSPO3 in PTPRK-RSPO3 fusion-positive colorectal tumor xenografts inhibits tumor growth and promotes differentiation, with stem cell compartment genes being most sensitive to anti-RSPO3 treatment, indicating that a stem-cell compartment drives PTPRK-RSPO3 colorectal tumor growth.\",\n      \"method\": \"Anti-RSPO3 antibody treatment of patient-derived xenografts, gene expression profiling, functional stem cell assays\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo xenograft experiments with anti-RSPO3 antibody, functional stem cell assays, and gene expression analysis in a high-quality journal\",\n      \"pmids\": [\"26700806\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In vivo RSPO3 expression expands Lgr5+ stem cells, Paneth cells, non-Paneth label-retaining cells, and Lgr4+ niche cells, and drives rapid intestinal tumorigenesis. Wnt/β-catenin signaling was modestly increased, and mutant Kras synergized with Rspo3 in hyperplastic growth.\",\n      \"method\": \"Conditional Rspo3 transgenic mouse model (Lgr5-GFP-CreERT2 × Rspo3 transgene), histological analysis, flow cytometry for stem cell markers\",\n      \"journal\": \"Gut\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional transgenic in vivo model with multiple cellular and signaling readouts, synergy experiment with Kras\",\n      \"pmids\": [\"27511199\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"RSPO3-LGR4 signaling upregulates key self-renewal genes and is essential for leukemia stem cell (LSC) self-renewal in AML. Blocking the RSPO3-LGR4 interaction with clinical-grade anti-RSPO3 antibody (OMP-131R10/rosmantuzumab) impairs self-renewal and induces differentiation in AML patient-derived xenografts without affecting normal hematopoietic stem cells. LGR4 is epigenetically upregulated and works through cooperation with HOXA9.\",\n      \"method\": \"Anti-RSPO3 antibody (OMP-131R10), patient-derived xenograft (PDX) experiments, gene expression analysis, epigenetic characterization of LGR4\",\n      \"journal\": \"Cancer cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo PDX experiments with clinical-grade antibody, multiple mechanistic readouts including differentiation and self-renewal, epistasis with HOXA9\",\n      \"pmids\": [\"32559496\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"RSPO3 aberrantly expressed in Keap1-deficient lung adenocarcinomas signals via LGR4 and the mediator IQGAP1 to promote tumor cell proliferation and migration. Knockdown of RSPO3, LGR4, or IQGAP1 reduced cell proliferation and migration in vitro, and KD of LGR4 or IQGAP1 decreased tumor growth and metastasis in vivo.\",\n      \"method\": \"shRNA knockdown of RSPO3/LGR4/IQGAP1, cell proliferation and migration assays, in vivo xenograft metastasis models\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo and in vitro knockdown with multiple pathway components, single lab\",\n      \"pmids\": [\"25531322\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"RSPO3 is a negative regulator of ERK/FGF signaling downstream of LGR4 in human adipose-derived stem cells (hASCs). RSPO3 knockdown increased osteogenic potential, an effect blocked by ERK1/2 inhibition. LGR4 silencing inhibited ERK signaling and osteogenic differentiation, and abrogated RSPO3-regulated osteogenesis and RSPO3-induced ERK1/2 inhibition.\",\n      \"method\": \"RSPO3 shRNA knockdown, LGR4 siRNA silencing, ERK pathway inhibitors, osteogenic differentiation assays in hASCs\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple genetic and pharmacological manipulations, single lab, pathway epistasis established\",\n      \"pmids\": [\"28220828\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"RSPO3 limits gluteofemoral adipose tissue expansion by suppressing adipogenesis and increasing gluteal adipocyte susceptibility to apoptosis, while stimulating abdominal adipose progenitor proliferation. The distinct biological responses in abdominal versus gluteal adipose progenitors are associated with differential changes in WNT signaling. Zebrafish with a nonsense rspo3 mutation display altered fat distribution.\",\n      \"method\": \"Human cellular studies with RSPO3 treatment/knockdown in adipose progenitors from different depots, zebrafish nonsense mutant, WNT signaling measurements\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — human cellular assays combined with zebrafish genetic model, single lab, multiple cellular readouts\",\n      \"pmids\": [\"32493999\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Osteoblast-derived RSPO3 is the principal source of RSPO3 in bone. RSPO3 increases osteoblast proliferation and differentiation in a cell-autonomous manner, and is an important regulator of vertebral trabecular bone mass and bone strength in adult mice.\",\n      \"method\": \"Osteoblast-specific conditional knockout (Runx2-Cre;Rspo3fl/fl), bone densitometry, bone strength testing, cell proliferation and differentiation assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO with tissue-specific cellular source identification, multiple bone phenotype readouts, and cell-autonomous mechanistic experiments\",\n      \"pmids\": [\"34389713\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RSPO3 signaling via LGR4 drives proliferation of gastric stem cells and induces NF-κB activity in proliferative stem cells. LGR4-driven NF-κB activation is responsible for H. pylori-induced gland hyperplasia and chemokine expression in stem cells, resulting in neutrophil recruitment. LGR4 also regulates LGR5 expression in this context.\",\n      \"method\": \"Conditional knockout mice (Lgr4-specific and Lgr5-specific), H. pylori infection model, NF-κB activity assays, chemokine/neutrophil recruitment measurements\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple conditional KO models with specific pathway readouts (NF-κB, chemokines, neutrophil recruitment), genetic epistasis between Lgr4 and Lgr5\",\n      \"pmids\": [\"35767364\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"RSPO1/RSPO3-LGR4 signaling in hepatocytes suppresses cholesterol synthesis via the AMPKα-SREBP2 pathway. RSPO3 increased phosphorylation of AMPKα Thr172, reduced SREBP2 nuclear translocation and Srebf2 mRNA. LGR4 knockdown increased hepatic cholesterol synthesis and decreased AMPKα phosphorylation; AMPKα knockdown abrogated Rspo-induced inhibition of cholesterol synthesis.\",\n      \"method\": \"LGR4/Rspo1/Rspo3 knockdown in mice and hepatocytes, AMPKα agonist/antagonist/shRNA experiments, SREBP2 nuclear translocation assays\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro and in vivo knockdown with epistasis experiments, single lab\",\n      \"pmids\": [\"32926477\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Endothelial cell-derived RSPO3 activates LGR4-Gab1-Gαi1/3 complex formation to drive Erk activation and protect neurons from ischemia/reperfusion injury. Only Erk (not Akt or β-catenin) inhibitors reversed RSPO3-induced neuroprotection. Silencing or knockout of Gαi1 and Gαi3 abolished RSPO3-induced neuroprotection. Endothelial RSPO3 knockdown/KO increased ischemic injury in MCAO mice.\",\n      \"method\": \"Co-IP (LGR4-Gab1-Gαi association), MEF Gαi1/3 KO, Erk/Akt/β-catenin inhibitors, endothelial-specific RSPO3 KO/KD/OE mice, MCAO model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP for complex identification, Gαi KO epistasis, multiple inhibitor experiments, endothelial-specific genetic models in vivo; single lab\",\n      \"pmids\": [\"37805583\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RSPO3 mediates pulmonary endothelial regeneration in a LGR4-dependent manner. Beyond β-catenin, integrin-linked kinase (ILK)/Akt was identified as a novel downstream effector of RSPO3/LGR4 signaling. EC-specific RSPO3 knockdown inhibited endothelial cell proliferation and exacerbated injury, while EC-specific overexpression promoted recovery.\",\n      \"method\": \"EC-specific RSPO3 KD, inducible EC-specific KO, EC-specific OE mice, LGR4-dependence assays, ILK/Akt signaling measurements in sepsis model\",\n      \"journal\": \"International journal of biological macromolecules\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple EC-specific genetic models with LGR4-dependence testing and novel ILK/Akt pathway identification; single lab\",\n      \"pmids\": [\"38677673\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Loss of AXIN1 confers resistance to WNT pathway blockade (porcupine inhibitor LGK974) in RSPO3-fusion-positive colorectal cancer cells. Suppression of AXIN1 by RNA interference in parental VACO6 cells (carrying PTPRK-RSPO3 fusion) markedly increased resistance to LGK974, establishing AXIN1 loss as a mechanism of acquired resistance.\",\n      \"method\": \"Long-term LGK974 treatment to generate resistant cells, whole-genome sequencing to identify AXIN1 frameshifts, AXIN1 RNAi in parental cells, transcriptional and morphological WNT pathway readouts\",\n      \"journal\": \"EMBO molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic identification of AXIN1 mutations plus RNAi functional validation, single lab\",\n      \"pmids\": [\"28100566\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RSPO3 promotes gastric cancer stem cell properties through direct interaction with transmembrane protein GNG7, leading to phosphorylation of Akt and GSK-3β and accumulation of β-catenin. GNG7 knockdown blocked RSPO3-induced β-catenin activation and CSC-like properties. This pathway is distinct from canonical LGR-mediated Wnt signaling.\",\n      \"method\": \"Co-immunoprecipitation (CoIP) and LC-MS/MS to identify RSPO3-interacting proteins, GNG7 siRNA knockdown, recombinant RSPO3 protein treatment, western blot for Akt/GSK-3β/β-catenin, in vivo peritoneal seeding model\",\n      \"journal\": \"Cancer medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CoIP/MS for interaction identification plus functional epistasis via GNG7 KD, single lab\",\n      \"pmids\": [\"38581123\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The Furin (FuFu) domains of RSPO3 mediate specific, LGR5-dependent cellular uptake when conjugated to liposomes. Full-length RSPO1 mediates aspecific LGR5-independent uptake largely via heparan sulfate proteoglycan binding, whereas RSPO3 FuFu domain-coated liposomes selectively target LGR5-high cells.\",\n      \"method\": \"Fluorescence-loaded liposome uptake assays, LGR5-high vs LGR5-low cell comparison, domain-specific conjugation, doxorubicin-loaded FuFuRSPO3 liposome growth inhibition\",\n      \"journal\": \"Journal of controlled release\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional domain mapping via cell-based uptake assays with LGR5-dependent specificity controls, single lab\",\n      \"pmids\": [\"36813038\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"RSPO3 impairs vascular endothelial barrier function by inducing inter-endothelial gap formation through disruption of β-catenin and VE-cadherin alignment at adherens junctions, and synergizes with pro-inflammatory IL-1β to enhance endothelial hyperpermeability.\",\n      \"method\": \"Electric Cell-substrate Impedance Sensing (ECIS) of primary endothelial monolayers, RSPO3 protein treatment, immunofluorescence of β-catenin/VE-cadherin at junctions, IL-1β co-treatment\",\n      \"journal\": \"Molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — real-time barrier function measurement with mechanistic readout of junction protein disruption, single lab\",\n      \"pmids\": [\"30157748\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RSPO3 is expressed in osteoblasts and regulates vertebral trabecular bone mass in a cell-autonomous manner; osteoblast-derived RSPO3 is required for a full estrogenic response on cortical (but not trabecular) bone. Estradiol and RSPO3 regulate vertebral trabecular bone mass independently of each other.\",\n      \"method\": \"Osteoblast-specific Rspo3 conditional KO (Runx2-Cre), OVX mouse model with estradiol treatment, bone densitometry and bone strength testing\",\n      \"journal\": \"American journal of physiology. Endocrinology and metabolism\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with epistasis to estrogen pathway, single lab, extends prior PMID:34389713 findings\",\n      \"pmids\": [\"35068191\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RSPO3 overexpression increases NSCLC radiosensitivity through induction of pyroptosis mediated by the β-catenin–NF-κB signaling pathway and NLRP3 inflammasome. Anti-RSPO3 antibody (OMP-131R10) blocked radiation-induced pyroptosis and anti-tumor immunity in vivo.\",\n      \"method\": \"RSPO3 overexpression/knockdown in NSCLC cell lines, β-catenin/NF-κB pathway inhibitors, NLRP3 inflammasome assays, in vivo anti-RSPO3 antibody treatment with radiation\",\n      \"journal\": \"Radiotherapy and oncology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, pathway inhibitor experiments without direct mechanistic reconstitution, abstract does not detail mutagenesis or rigorous controls\",\n      \"pmids\": [\"39245068\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The RSPO3 promoter is regulated by the balance between DNA methyltransferase DNMT3a and DNA demethylase TET1 in cholangiocarcinoma. Targeted RSPO3 promoter demethylation using dCas9-TET1CD inhibited CCA tumorigenicity, while targeted methylation using dCas9-DNMT3a promoted it.\",\n      \"method\": \"Targeted epigenetic editing (dCas9-DNMT3a and dCas9-TET1CD), in vitro and in vivo CCA models, methylation analysis\",\n      \"journal\": \"Clinical epigenetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct epigenetic writer/eraser manipulation with functional consequence, single lab\",\n      \"pmids\": [\"37932819\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"MHP1-AcN (a RANKL-derived peptide) directly interacts with LGR4 and disrupts RSPO3-induced LGR4-IQGAP1 complex formation, inhibiting RSPO3-enhanced phosphorylation of LRP6 and accumulation of β-catenin, and suppressing tumor growth and metastatic potential in lung adenocarcinoma.\",\n      \"method\": \"Immunoprecipitation for LGR4-IQGAP1 complex, immunoblotting for LRP6 phosphorylation and β-catenin, A549 xenograft model, cell migration/invasion assays\",\n      \"journal\": \"Anticancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical complex disruption assay combined with in vivo xenograft, single lab\",\n      \"pmids\": [\"42203310\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Rspo3 single conditional knockout in the limb did not produce limb defects, but combining Rspo3 and Rspo2 mutations caused severe hindlimb truncations, demonstrating redundant function of these R-spondin paralogs during limb development.\",\n      \"method\": \"Conditional Rspo3 allele (loxP-flanked exons 2-4), Cre-mediated deletion, Rspo2/Rspo3 double knockout genetic epistasis\",\n      \"journal\": \"Genesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — double-mutant genetic epistasis establishing functional redundancy, single lab, clean developmental phenotype\",\n      \"pmids\": [\"22610508\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"H3K4me3 histone modification regulates the transcription of RSPO3 in dermal papilla cells. Increasing H3K4me3 levels enhanced DPC proliferation and Wnt signaling pathway gene expression, partly through elevated RSPO3. RSPO3 itself promotes DPC proliferation, inhibits apoptosis, and increases Wnt pathway gene expression.\",\n      \"method\": \"CUT&Tag for H3K4me3 mapping, H3K4me3 inhibitor (BCL-121) and agonist (PBIT) treatment, RNA-seq, RSPO3 functional assays in DPCs\",\n      \"journal\": \"Epigenetics & chromatin\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, CUT&Tag plus functional experiments but primarily in a non-canonical cell type (goat DPCs) with limited mechanistic resolution in the abstract\",\n      \"pmids\": [\"40775776\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Hepatic Rspo3 regulates systemic glucose metabolism and body composition via inter-organ communication involving afferent vagal and efferent sympathetic nerves. Viral-mediated hepatic Rspo3 induction improved insulin resistance in obese mice; hepatic vagal denervation suppressed these remote effects on adipose tissue and skeletal muscle. Hepatic Rspo3 suppression (Cre-LoxP) exacerbated diabetes and obesity.\",\n      \"method\": \"Viral-mediated hepatic Rspo3 overexpression, Cre-LoxP hepatic conditional KO, hepatic vagus denervation, metabolic phenotyping (glucose tolerance, insulin sensitivity, body composition)\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO plus viral OE with denervation epistasis identifying neuronal inter-organ communication; single lab\",\n      \"pmids\": [\"39854351\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RSPO3 promotes proliferation and self-renewal of human limbal epithelial stem cells (LESCs) through a WNT/β-catenin-independent signaling pathway, as demonstrated by the lack of impairment when β-catenin activation was blocked by XAV939.\",\n      \"method\": \"Exogenous RSPO3 treatment of hLESCs, β-catenin inhibitor XAV939, EdU proliferation assay, stemness marker assessment (ΔNp63, ABCG2), in vivo corneal wound healing model\",\n      \"journal\": \"Investigative ophthalmology & visual science\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, pharmacological inhibitor used to establish pathway independence; downstream pathway not identified\",\n      \"pmids\": [\"39760688\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Chondrocyte-derived RSPO3 acts in an autocrine manner to enhance chondrocyte anabolism and in a paracrine manner to directly drive M2 macrophage polarization. The pro-M2 macrophage effect is specifically mediated through activation of the LGR4/LRP6/β-catenin signaling axis in macrophages.\",\n      \"method\": \"NsPEFs-ADSCs-EV treatment, RSPO3 neutralization, macrophage polarization assays (M1/M2 markers), LGR4/LRP6/β-catenin signaling assays, OA mouse model\",\n      \"journal\": \"Bioactive materials\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, abstract does not describe direct reconstitution or mutagenesis for the LGR4/LRP6/β-catenin pathway; mechanistic detail limited in abstract\",\n      \"pmids\": [\"41624077\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RSPO3 is a secreted protein that potentiates Wnt signaling through multiple mechanisms: it binds syndecan-4 to activate non-canonical Wnt/PCP signaling via clathrin-mediated endocytosis (transduced through Wnt5a/Fz7/Dvl/JNK); it signals through its cognate receptor LGR4 (and LGR5) to amplify canonical Wnt/β-catenin signaling by clearing RNF43/ZNRF3 from the cell surface; in endothelial cells it drives non-canonical WNT/Ca2+/NFAT signaling to maintain vascular remodeling; in hepatocytes and AML cells it operates through LGR4-Gab1-Gαi1/3-ERK and AMPKα-SREBP2 downstream cascades; it is produced by specific stromal niche cells (PDGFRα+ pericryptal myofibroblasts, adrenal capsule cells, osteoblasts) to support tissue-specific stem cell compartments; and PTPRK-RSPO3 gene fusions that increase RSPO3 expression are recurrent oncogenic drivers in colorectal cancer, where RSPO3 antagonism inhibits tumor growth and promotes differentiation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RSPO3 is a secreted Wnt-pathway potentiator that acts as a niche-derived ligand to sustain tissue-specific stem cell compartments and to control vascular and metabolic homeostasis [#3, #1]. It engages two principal signaling modes: a syndecan-4 (SDC4)-dependent route that drives clathrin-mediated endocytosis of Wnt-receptor complexes to transduce non-canonical Wnt/PCP signaling through Wnt5a/Fz7/Dvl/JNK [#0], and a route through its cognate receptor LGR4 (and LGR5) that amplifies canonical Wnt/\\u03b2-catenin signaling [#3, #6]. Through LGR4, RSPO3 couples to multiple downstream cascades in a context-dependent manner — Gab1\\u2013G\\u03b1i1/3\\u2013ERK in endothelium and neurons [#13], IQGAP1-dependent LRP6/\\u03b2-catenin activation in lung adenocarcinoma [#7, #22], AMPK\\u03b1\\u2013SREBP2 suppression of hepatic cholesterol synthesis [#12], NF-\\u03baB activation in gastric stem cells [#11], and ILK/Akt in pulmonary endothelial regeneration [#14]. In the intestinal niche RSPO3 is produced principally by PDGFR\\u03b1+ pericryptal myofibroblasts and is far more potent than RSPO1 in stimulating crypt Wnt/\\u03b2-catenin signaling, stem cell expansion, and Paneth cell differentiation [#3, #5]. RSPO3 supports comparable stem/progenitor functions in other compartments via dedicated stromal sources, including adrenal capsule cells that imprint zona glomerulosa fate [#2] and osteoblasts that regulate trabecular bone mass [#10]. In endothelium it maintains vascular remodeling through non-canonical WNT/Ca2+/NFAT signaling, with RNF213-mediated degradation of NFAT1 and filamin A as a regulatory node [#1]. Recurrent PTPRK-RSPO3 gene fusions act as oncogenic drivers in colorectal cancer, where the lesion sustains a Wnt-dependent stem-cell compartment and anti-RSPO3 antibody treatment inhibits tumor growth and forces differentiation [#4, #5]. RSPO3 can also act through non-LGR receptors, including GNG7-mediated Akt/GSK-3\\u03b2/\\u03b2-catenin signaling in gastric cancer stem cells [#16].\",\n  \"teleology\": [\n    {\n      \"year\": 2011,\n      \"claim\": \"Establishing how RSPO3 transmits a non-canonical Wnt signal answered whether R-spondins act only by stabilizing Wnt receptors or by actively remodeling receptor trafficking.\",\n      \"evidence\": \"Binding assays, Xenopus loss-of-function and epistasis with Wnt5a/Fz7/Dvl/JNK, and clathrin inhibition\",\n      \"pmids\": [\"21397842\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the SDC4/PCP route operates in mammalian adult tissues was not addressed\", \"Structural basis of the RSPO3-SDC4 interaction not defined\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Single Rspo3 deletion versus Rspo2/Rspo3 double mutants resolved whether RSPO3 has a unique developmental role or is functionally redundant with paralogs.\",\n      \"evidence\": \"Conditional Rspo3 allele and Rspo2/Rspo3 double-knockout genetic epistasis in limb\",\n      \"pmids\": [\"22610508\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not identify the receptors or downstream pathway underlying limb redundancy\", \"No molecular distinction between RSPO2 and RSPO3 contributions\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identifying LGR4 and IQGAP1 as effectors in Keap1-deficient lung adenocarcinoma extended RSPO3 signaling from development into a tumor-promoting proliferation/migration axis.\",\n      \"evidence\": \"shRNA knockdown of RSPO3/LGR4/IQGAP1 with proliferation, migration, and in vivo metastasis assays\",\n      \"pmids\": [\"25531322\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"IQGAP1 not shown to bind RSPO3 directly\", \"Mechanism linking LGR4-IQGAP1 to proliferation not reconstituted\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Anti-RSPO3 treatment of PTPRK-RSPO3 fusion-positive xenografts tested whether the fusion is a therapeutic dependency and which cell compartment drives tumor growth.\",\n      \"evidence\": \"Anti-RSPO3 antibody treatment of patient-derived xenografts with stem cell and expression assays\",\n      \"pmids\": [\"26700806\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Receptor and downstream signaling for the fusion-driven growth not dissected here\", \"Durability of response and resistance not addressed\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Tissue-specific knockouts defined RSPO3 as a niche-derived maintenance signal in distinct organs, clarifying that its developmental role persists into adult homeostasis.\",\n      \"evidence\": \"Inducible endothelial-specific and adrenal conditional knockouts with pathway and phenotypic readouts (vascular pruning, NFAT, \\u03b2-catenin, SHH)\",\n      \"pmids\": [\"26766444\", \"27313319\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endothelial and adrenal studies used distinct downstream readouts (WNT/Ca2+/NFAT vs \\u03b2-catenin) without a unifying mechanism\", \"Upstream regulation of stromal RSPO3 production not defined\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Gain-of-function Rspo3 expression in vivo showed RSPO3 is sufficient to expand intestinal stem cells and drive tumorigenesis, linking its niche role to oncogenesis.\",\n      \"evidence\": \"Conditional Rspo3 transgenic mice with stem cell marker flow cytometry and Kras synergy\",\n      \"pmids\": [\"27511199\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Only modest \\u03b2-catenin increase observed, leaving the proliferative driver partly unexplained\", \"Receptor dependence not formally tested in this model\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Studies in bone progenitors and colorectal resistance refined RSPO3 signaling outputs as both ERK-modulatory and dependent on intact downstream Wnt machinery.\",\n      \"evidence\": \"RSPO3/LGR4 manipulation with ERK inhibitors in hASCs; LGK974 resistance selection and AXIN1 RNAi in fusion-positive CRC cells\",\n      \"pmids\": [\"28220828\", \"28100566\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"RSPO3 as a negative ERK regulator in stem cells contrasts with ERK-driving roles elsewhere, unresolved\", \"AXIN1 loss conferring porcupine-inhibitor resistance not mechanistically linked to RSPO3 itself\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Endothelial barrier and intestinal niche-source studies sharpened both the cellular origin and a permeability function of RSPO3.\",\n      \"evidence\": \"PDGFR\\u03b1-Cre conditional knockout with organoid rescue; ECIS barrier assays with VE-cadherin/\\u03b2-catenin imaging and IL-1\\u03b2 co-treatment\",\n      \"pmids\": [\"29559533\", \"30157748\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Barrier-disrupting role appears to oppose vascular-maintenance role, leaving net endothelial function context-dependent\", \"Receptor mediating junction disruption not identified\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Discovery of RSPO3-LGR4 outputs in AML, hepatocytes, and adipose depots established defined downstream cascades and metabolic/oncogenic roles beyond canonical Wnt.\",\n      \"evidence\": \"Anti-RSPO3 antibody in AML PDX with HOXA9 epistasis; AMPK\\u03b1-SREBP2 epistasis in hepatocytes; depot-specific adipose progenitor assays and zebrafish rspo3 mutant\",\n      \"pmids\": [\"32559496\", \"32926477\", \"32493999\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"AML finding is High-confidence but hepatic and adipose cascades rest on single-lab knockdown epistasis\", \"How a single ligand selects between SREBP2, NF-\\u03baB, and Wnt outputs not defined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Osteoblast-specific knockout identified the cellular source and cell-autonomous bone function of RSPO3, and a later study placed it relative to estrogen signaling.\",\n      \"evidence\": \"Runx2-Cre conditional knockout with bone densitometry and strength testing; OVX/estradiol epistasis\",\n      \"pmids\": [\"34389713\", \"35068191\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Receptor mediating osteoblast-autonomous effect not pinned down\", \"Why estrogen-dependence differs between cortical and trabecular bone unexplained\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Gastric stem cell studies revealed RSPO3-LGR4 can activate NF-\\u03baB, coupling the pathway to inflammation and pathogen-driven hyperplasia.\",\n      \"evidence\": \"Lgr4- and Lgr5-specific conditional knockouts with H. pylori infection, NF-\\u03baB and chemokine/neutrophil readouts\",\n      \"pmids\": [\"35767364\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular link from LGR4 to NF-\\u03baB not reconstituted\", \"Whether RSPO3 ligand itself, versus other RSPOs, drives this in vivo not isolated\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Mechanistic studies defined a Gab1-G\\u03b1i1/3-ERK signaling complex and mapped RSPO3 receptor-targeting domains, and identified epigenetic control of the RSPO3 promoter.\",\n      \"evidence\": \"Co-IP for LGR4-Gab1-G\\u03b1i, G\\u03b1i KO MEFs and MCAO neuroprotection; FuFu-domain liposome uptake in LGR5-high cells; dCas9-TET1/DNMT3a promoter editing in cholangiocarcinoma\",\n      \"pmids\": [\"37805583\", \"36813038\", \"37932819\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Gab1-G\\u03b1i-ERK complex shown by Co-IP in single lab without structural validation\", \"Relationship between FuFu-domain LGR5 binding and the LGR4-Gab1 cascade not integrated\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Newer studies broadened RSPO3 effectors to ILK/Akt, GNG7, and pyroptosis pathways and tested complex-disruption as a therapeutic strategy.\",\n      \"evidence\": \"EC-specific genetic models with ILK/Akt readout; Co-IP/LC-MS identification of GNG7 with knockdown epistasis; NSCLC radiosensitivity with NLRP3 readouts; MHP1-AcN peptide disruption of LGR4-IQGAP1\",\n      \"pmids\": [\"38677673\", \"38581123\", \"39245068\", \"42203310\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"GNG7-mediated, LGR-independent route rests on a single Co-IP/MS study\", \"Pyroptosis/radiosensitivity finding is Low-confidence and not reconstituted\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Inter-organ and additional niche studies extended RSPO3 to neurally relayed metabolic control and to non-Wnt-dependent stem cell maintenance.\",\n      \"evidence\": \"Hepatic Rspo3 OE/KO with vagal denervation and metabolic phenotyping; XAV939-resistant hLESC proliferation; H3K4me3 regulation of RSPO3 in dermal papilla cells\",\n      \"pmids\": [\"39854351\", \"39760688\", \"40775776\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The signal carried from liver to nerves to peripheral tissues is undefined\", \"\\u03b2-catenin-independent stem cell route lacks an identified downstream effector\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how a single secreted RSPO3 molecule selects among SDC4/PCP, LGR4-canonical Wnt, LGR4-Gab1-G\\u03b1i-ERK, NF-\\u03baB, GNG7-Akt, and \\u03b2-catenin-independent outputs in different cell types.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural or quantitative model integrates the receptor/co-receptor repertoire that dictates pathway choice\", \"Direct binding partners beyond SDC4, LGR4/LGR5, and GNG7 not systematically mapped\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [0, 3, 6]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 3, 12]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [3, 4, 18]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 3, 6]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [2, 23, 10]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [4, 5, 6]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"SDC4\", \"LGR4\", \"LGR5\", \"IQGAP1\", \"GNG7\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}