{"gene":"RSPO3","run_date":"2026-04-28T20:42:06","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, which is essential for PCP signal transduction via Fz7, Dvl, and JNK during Xenopus gastrulation and head cartilage morphogenesis","method":"Co-immunoprecipitation, loss-of-function in Xenopus embryos, pharmacological inhibition of clathrin-mediated endocytosis, epistasis with Wnt5a/Fz7/Dvl/JNK pathway components","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal methods (binding assay, in vivo loss-of-function, pathway epistasis) in a single rigorous study","pmids":["21397842"],"is_preprint":false},{"year":2016,"finding":"Endothelial RSPO3 controls vascular remodeling through non-canonical WNT/Ca2+/NFAT signaling; inducible endothelial Rspo3 deletion causes apoptosis and vascular pruning; RSPO3 and EVI/WLS co-regulate RNF213, USP18, and TRIM30α, which target NFAT1 for proteasomal degradation, and NFAT protein levels are decreased in Rspo3-iECKO endothelial cells","method":"Inducible endothelial-specific Rspo3 knockout mice, genetic epistasis with Evi/Wls knockout, endothelial transcriptomic screen, pharmacological NFAT inhibition phenocopy","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 — clean conditional KO with defined cellular phenotype, epistasis, pharmacological phenocopy, multiple orthogonal methods","pmids":["26766444"],"is_preprint":false},{"year":2018,"finding":"PDGFRα+ pericryptal stromal myofibroblasts are the critical in vivo source of RSPO3 and Wnts for intestinal stem cells; RSPO3 from these stromal cells is required for intestinal crypt Wnt/β-catenin signaling and Paneth cell differentiation, and is several orders of magnitude more potent than RSPO1 in stimulating organoid growth","method":"Cell-type-specific Cre-mediated Rspo3 excision in PdgfRα+ cells, organoid rescue assay with exogenous RSPO3 protein, DSS stress model","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — in vivo conditional knockout with defined cellular and molecular phenotypes, ex vivo rescue experiment","pmids":["29559533"],"is_preprint":false},{"year":2016,"finding":"Capsular RSPO3 signals to the underlying steroidogenic compartment to induce β-catenin signaling and imprint glomerulosa cell fate in the adrenal cortex; deletion of RSPO3 leads to loss of SHH signaling and impaired organ growth, and RSPO3 is required in adult life for replenishment of lost cells and maintenance of zona glomerulosa identity","method":"Conditional Rspo3 knockout in adrenal capsule, immunofluorescence, β-catenin and SHH pathway readouts","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 — conditional KO with multiple defined pathway readouts and functional phenotypes","pmids":["27313319"],"is_preprint":false},{"year":2015,"finding":"Targeting RSPO3 in PTPRK-RSPO3 fusion-positive colorectal xenografts inhibits tumor growth and promotes differentiation, with stem-cell-compartment genes being most sensitive; functional assays establish that RSPO3 drives stem-cell properties in these tumors","method":"Anti-RSPO3 antibody treatment of patient-derived xenografts, gene expression profiling, functional stem cell assays","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 — in vivo xenograft KO with defined molecular and cellular phenotypes, multiple orthogonal assays","pmids":["26700806"],"is_preprint":false},{"year":2016,"finding":"Conditional in vivo RSPO3 overexpression in Lgr5+ intestinal stem cells drives rapid development of hyperplastic, adenomatous, and adenocarcinomatous lesions, expanding Lgr5+ stem cells, Paneth cells, and niche compartments via increased Wnt/β-catenin signaling; mutant Kras synergizes with RSPO3 in hyperplastic growth","method":"Conditional Rspo3 transgenic mouse model crossed with Lgr5-GFP-CreERT2, histopathology, β-catenin signaling readouts","journal":"Gut","confidence":"High","confidence_rationale":"Tier 2 — in vivo gain-of-function with defined cellular and pathway phenotypes, genetic interaction with Kras","pmids":["27511199"],"is_preprint":false},{"year":2020,"finding":"RSPO3 acts through LGR4 to upregulate self-renewal genes and is essential for leukemia stem cell self-renewal in a subset of AML; blocking the RSPO3-LGR4 interaction with anti-RSPO3 antibody (rosmantuzumab) impairs self-renewal and induces differentiation in AML patient-derived xenografts without affecting normal hematopoietic stem cells; LGR4 is epigenetically upregulated and cooperates with HOXA9","method":"Anti-RSPO3 antibody (OMP-131R10) treatment, patient-derived xenografts, LGR4 epigenetic upregulation analysis, HOXA9 cooperation assay","journal":"Cancer cell","confidence":"High","confidence_rationale":"Tier 2 — in vivo PDX model with defined mechanistic target (LGR4), genetic cooperativity, and functional self-renewal readouts","pmids":["32559496"],"is_preprint":false},{"year":2021,"finding":"Osteoblast-derived RSPO3 is the principal source of RSPO3 in bone and in a cell-autonomous manner increases osteoblast proliferation and differentiation to regulate vertebral trabecular bone mass and bone strength","method":"Osteoblast-specific Rspo3 conditional knockout (Runx2-cre), bone mass and strength measurements, cell proliferation and differentiation assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — cell-type-specific conditional KO with defined cellular mechanism and physiological phenotype","pmids":["34389713"],"is_preprint":false},{"year":2014,"finding":"RSPO3 signals through LGR4 (with IQGAP1 as signaling mediator) to promote proliferation and migration of lung cancer cells; knockdown of RSPO3, LGR4, or IQGAP1 reduces cell proliferation and migration in vitro and tumor growth/metastasis in vivo","method":"shRNA knockdown, in vitro proliferation and migration assays, in vivo tumor and metastasis models","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2-3 — defined LGR4/IQGAP1 pathway placement with in vivo validation, single lab","pmids":["25531322"],"is_preprint":false},{"year":2017,"finding":"RSPO3-LGR4 signaling inhibits osteogenic differentiation of human adipose-derived stem cells via suppression of ERK/FGF signaling; LGR4 silencing inhibits ERK signaling and osteogenic differentiation, and loss of LGR4 abrogates RSPO3-regulated osteogenesis and RSPO3-induced ERK1/2 signaling inhibition","method":"shRNA knockdown of RSPO3 and LGR4, ERK pathway inhibitors, osteogenic differentiation assays","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2-3 — genetic and pharmacological epistasis establishing LGR4-ERK pathway, single lab","pmids":["28220828"],"is_preprint":false},{"year":2020,"finding":"RSPO3 (with RSPO1) signals through LGR4 to suppress hepatic cholesterol synthesis via activation of AMPKα (phosphorylation at Thr172) and consequent reduction of SREBP2 nuclear translocation; hepatic LGR4 knockdown increases cholesterol synthesis and decreases AMPKα phosphorylation","method":"In vivo Lgr4 and Rspo1/3 knockdown mice, AMPKα agonist/antagonist/shRNA epistasis, SREBP2 nuclear translocation assay","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 — epistasis with AMPKα confirmed by pharmacology and genetics, in vivo and in vitro, single lab","pmids":["32926477"],"is_preprint":false},{"year":2018,"finding":"RSPO3 impairs barrier function of human vascular endothelial monolayers by inducing inter-endothelial gap formation through disruption of β-catenin and VE-cadherin alignment at adherens junctions, and synergizes with IL-1β to enhance permeability","method":"Electric Cell-substrate Impedance Sensing, immunofluorescence of β-catenin and VE-cadherin, primary human endothelial monolayers from multiple vascular beds","journal":"Molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2-3 — direct functional assay with defined molecular readouts in multiple cell types, single lab","pmids":["30157748"],"is_preprint":false},{"year":2022,"finding":"RSPO3 signaling via LGR4 (not LGR5) drives proliferation of gastric stem cells and induces NF-κB activity in proliferative stem cells; upon H. pylori exposure, LGR4-driven NF-κB activation causes gland hyperplasia and chemokine expression leading to neutrophil recruitment","method":"Conditional Lgr4 and Lgr5 knockout mice, H. pylori infection model, NF-κB reporter assays, histology","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 — conditional KO distinguishing LGR4 vs LGR5, defined NF-κB pathway placement, in vivo infection model","pmids":["35767364"],"is_preprint":false},{"year":2023,"finding":"RSPO3 activates LGR4-Gab1-Gαi1/3 complex formation, leading to Erk activation and neuroprotection against ischemia/reperfusion injury; silencing or knockout of Gαi1/Gαi3 abolishes RSPO3-induced neuroprotection and Erk activation; endothelial RSPO3 protects neurons via this Gαi1/3-Erk axis in vivo","method":"Co-immunoprecipitation (LGR4-Gab1-Gαi1/3 complex), siRNA/KO of Gαi1/Gαi3, endothelial-specific RSPO3 KD/KO/overexpression mice, MCAO model, Erk pathway inhibitors","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 2 — Co-IP establishing novel complex, genetic KO rescue epistasis, in vivo endothelial-specific models with defined pathway","pmids":["37805583"],"is_preprint":false},{"year":2024,"finding":"RSPO3 mediates pulmonary endothelial regeneration in a LGR4-dependent manner; downstream effectors include both β-catenin and integrin-linked kinase (ILK)/Akt; EC-specific RSPO3 deletion inhibits endothelial proliferation and exacerbates injury, while overexpression promotes recovery","method":"EC-specific RSPO3 KD, inducible EC-specific KO, EC-specific overexpression mice, LPS endotoxemia model, ILK/Akt and β-catenin pathway assays","journal":"International journal of biological macromolecules","confidence":"Medium","confidence_rationale":"Tier 2 — multiple conditional mouse models with defined molecular pathway (LGR4/ILK/β-catenin), single lab","pmids":["38677673"],"is_preprint":false},{"year":2024,"finding":"RSPO3 interacts directly with GNG7 (G protein subunit gamma 7) as identified by Co-IP and LC-MS/MS; RSPO3 increases phosphorylation of Akt and GSK-3β and β-catenin expression in gastric cancer cells via GNG7, promoting cancer stem cell properties; GNG7 knockdown abolishes RSPO3-induced β-catenin activation","method":"Co-immunoprecipitation, LC-MS/MS proteomics, siRNA knockdown of GNG7, recombinant RSPO3 protein treatment, in vivo peritoneal seeding model","journal":"Cancer medicine","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP plus MS identified novel binding partner (GNG7), epistasis confirmed by knockdown, in vivo validation, single lab","pmids":["38581123"],"is_preprint":false},{"year":2023,"finding":"The furin (FuFu) domains of RSPO3 mediate highly specific, LGR5-dependent cellular uptake when conjugated to liposomes, distinct from full-length RSPO1 which causes LGR5-independent uptake via heparan sulfate proteoglycans","method":"Liposome conjugation with RSPO3 furin domains vs full-length RSPO1, fluorescence uptake assays in LGR5-high vs LGR5-low cells, doxorubicin delivery functional assay","journal":"Journal of controlled release","confidence":"Medium","confidence_rationale":"Tier 2-3 — domain-specific functional mapping of receptor binding with pharmacological validation, single lab","pmids":["36813038"],"is_preprint":false},{"year":2024,"finding":"RSPO3 regulates NSCLC radioresistance by promoting pyroptosis via the β-catenin-NF-κB signaling pathway and NLRP3 inflammasome; anti-RSPO3 antibody blocks radiation-induced pyroptosis and anti-tumor immunity in vivo","method":"RSPO3 overexpression/knockdown, β-catenin-NF-κB pathway assays, NLRP3 inflammasome readouts, anti-RSPO3 antibody in vivo","journal":"Radiotherapy and oncology","confidence":"Low","confidence_rationale":"Tier 3 — single lab, pathway placement via overexpression/KD without reconstitution or structural validation","pmids":["39245068"],"is_preprint":false},{"year":2020,"finding":"RSPO3 expressed in subcutaneous adipocytes limits gluteofemoral adipose tissue expansion by suppressing adipogenesis and increasing gluteal adipocyte susceptibility to apoptosis, and promotes upper-body fat distribution by stimulating abdominal adipose progenitor proliferation; distinct responses are associated with differential WNT signaling; zebrafish with nonsense rspo3 mutation display altered fat distribution","method":"Human adipose progenitor cell culture assays, zebrafish rspo3 nonsense mutant, WNT signaling pathway readouts","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2-3 — human cellular functional assays plus genetic model organism KO, defined pathway placement, single lab","pmids":["32493999"],"is_preprint":false},{"year":2025,"finding":"Hepatic Rspo3 regulates metabolic liver zonation (pericentral zone) and systemic glucose metabolism; viral induction of Rspo3 in obese mice improves insulin resistance and prevents body weight gain via neuronal inter-organ communication (hepatic vagus nerve); hepatic Rspo3 suppression via Cre-LoxP causes glucose intolerance, insulin resistance, fatty liver, and obesity","method":"Viral-mediated Rspo3 induction, Cre-LoxP conditional suppression, hepatic vagal denervation, metabolic phenotyping","journal":"PLoS biology","confidence":"Medium","confidence_rationale":"Tier 2 — multiple genetic models with defined metabolic pathway placement and inter-organ communication via denervation, single lab","pmids":["39854351"],"is_preprint":false},{"year":2022,"finding":"Osteoblast-derived RSPO3 is required for a full estrogenic effect on cortical (but not trabecular) bone; estradiol and RSPO3 regulate vertebral trabecular bone mass independently of each other, established by epistasis using osteoblast-specific RSPO3 knockout mice","method":"Osteoblast-specific Rspo3 KO (Runx2-cre), ovariectomy + estradiol rescue, bone mass and strength measurements","journal":"American journal of physiology. Endocrinology and metabolism","confidence":"Medium","confidence_rationale":"Tier 2 — genetic epistasis between RSPO3 and estrogen pathways using conditional KO, defined skeletal phenotypes","pmids":["35068191"],"is_preprint":false},{"year":2017,"finding":"Loss of AXIN1 (via frameshift deletions) confers resistance to WNT pathway blockade (porcupine inhibitor LGK974) in RSPO3-fusion-positive colorectal cancer cells; AXIN1 suppression by RNAi in parental VACO6 cells (PTPRK-RSPO3 fusion) recapitulates resistance","method":"Long-term drug selection in RSPO3-fusion CRC cell lines, whole exome sequencing of resistant clones, RNAi validation","journal":"EMBO molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 — mechanistic resistance identified by sequencing + RNAi validation, defines AXIN1 as downstream pathway component","pmids":["28100566"],"is_preprint":false},{"year":2023,"finding":"Rspo1 and Rspo3 are co-expressed with Wnt1/Wnt3a in the roof plate and are required for specification of Ngn2-positive sensory lineage neural crest cells; Rspo1/Rspo3 double knockout partially phenocopies Wnt1/Wnt3a double knockout for this specific neural crest subtype","method":"Rspo1/Rspo3 double conditional knockout mice, Ngn2 lineage tracing, Wnt/β-catenin reporter assay, comparison with Wnt1/Wnt3a dKO","journal":"Developmental dynamics","confidence":"Medium","confidence_rationale":"Tier 2 — genetic epistasis with Wnt pathway in defined developmental context, double KO phenotype","pmids":["37767857"],"is_preprint":false}],"current_model":"RSPO3 is a secreted protein that amplifies Wnt signaling through multiple mechanisms: it binds SDC4 to activate non-canonical Wnt/PCP signaling via clathrin-mediated endocytosis; signals through LGR4 (and LGR5) receptors to potentiate canonical Wnt/β-catenin signaling in intestinal, adrenal, gastric, bone, and hematopoietic stem cell niches; engages a LGR4-Gab1-Gαi1/3-Erk axis for neuroprotection; activates LGR4/ILK/β-catenin and LGR4/AMPKα-SREBP2 signaling in endothelial and hepatic contexts respectively; and in the intestine is supplied predominantly by PDGFRα+ pericryptal stromal myofibroblasts to maintain the crypt stem cell niche, with PTPRK-RSPO3 gene fusions causing oncogenic Wnt pathway hyperactivation in colorectal cancer."},"narrative":{"teleology":[{"year":2011,"claim":"The first mechanistic role for RSPO3 was established in non-canonical Wnt signaling: RSPO3 binds SDC4 and activates Wnt/PCP signaling via clathrin-mediated endocytosis, resolving how R-spondins engage non-canonical pathways during gastrulation.","evidence":"Co-immunoprecipitation, clathrin inhibition, and epistasis with Fz7/Dvl/JNK in Xenopus embryos","pmids":["21397842"],"confidence":"High","gaps":["Whether SDC4-dependent PCP activation operates in mammalian tissues","Structural basis of the RSPO3–SDC4 interaction","Relationship between SDC4 and LGR receptor pathways"]},{"year":2014,"claim":"RSPO3 was placed upstream of an LGR4–IQGAP1 signaling axis in cancer cell proliferation and migration, establishing LGR4 as a functional RSPO3 receptor in a disease-relevant context.","evidence":"shRNA knockdown of RSPO3/LGR4/IQGAP1 with in vitro and in vivo tumor/metastasis assays in lung cancer cells","pmids":["25531322"],"confidence":"Medium","gaps":["IQGAP1's direct binding mode to the RSPO3–LGR4 complex is undefined","Whether IQGAP1 dependency is tissue-specific"]},{"year":2015,"claim":"PTPRK-RSPO3 fusions were shown to be oncogenic drivers in colorectal cancer, with anti-RSPO3 antibody treatment shrinking patient-derived xenografts and inducing differentiation of the Lgr5+ stem cell compartment.","evidence":"Anti-RSPO3 antibody treatment of PTPRK-RSPO3 fusion-positive CRC patient-derived xenografts with gene expression and stem cell assays","pmids":["26700806"],"confidence":"High","gaps":["Full spectrum of RSPO3 fusion partners in CRC","Whether antibody effects are purely through Wnt suppression or additional mechanisms"]},{"year":2016,"claim":"Three independent studies established RSPO3 as a tissue-autonomous niche signal: it controls adrenal zona glomerulosa identity via β-catenin/SHH, drives intestinal tumorigenesis when overexpressed in Lgr5+ cells, and regulates vascular remodeling through a non-canonical WNT/Ca²⁺/NFAT axis in endothelium.","evidence":"Conditional Rspo3 KO in adrenal capsule; conditional Rspo3 transgenic in Lgr5+ intestinal cells with Kras epistasis; inducible endothelial-specific Rspo3 KO with Evi/Wls epistasis and NFAT pharmacological phenocopy","pmids":["27313319","27511199","26766444"],"confidence":"High","gaps":["Whether adrenal and vascular roles are LGR4-dependent","Identity of endothelial RSPO3 receptor(s)","Relative contributions of canonical versus non-canonical pathways in each tissue"]},{"year":2017,"claim":"RSPO3–LGR4 signaling was shown to modulate ERK/FGF signaling to inhibit osteogenic differentiation, and AXIN1 loss was identified as a mechanism of resistance to Wnt pathway blockade in RSPO3-fusion CRC, defining downstream pathway requirements.","evidence":"shRNA knockdown of RSPO3/LGR4 with ERK inhibitors in human adipose-derived stem cells; whole-exome sequencing and RNAi of AXIN1 in PTPRK-RSPO3 fusion CRC cells","pmids":["28220828","28100566"],"confidence":"Medium","gaps":["Whether ERK/FGF suppression operates in vivo bone context","Whether other Wnt destruction complex components also confer resistance"]},{"year":2018,"claim":"PDGFRα+ pericryptal stromal myofibroblasts were identified as the critical in vivo source of RSPO3 for the intestinal stem cell niche, and RSPO3 was shown to disrupt endothelial barrier function via β-catenin/VE-cadherin junctional disorganization.","evidence":"Cell-type-specific Cre-mediated Rspo3 excision in PDGFRα+ cells with organoid rescue; impedance sensing and immunofluorescence in human endothelial monolayers","pmids":["29559533","30157748"],"confidence":"High","gaps":["Whether other stromal sources compensate long-term","The receptor mediating RSPO3's junctional effects on endothelium"]},{"year":2020,"claim":"RSPO3's roles expanded to leukemia stem cell maintenance via LGR4, hepatic cholesterol metabolism via LGR4–AMPKα–SREBP2, and depot-specific adipose tissue distribution through differential Wnt activation, revealing metabolic and hematopoietic functions beyond epithelial stem cell niches.","evidence":"Anti-RSPO3 antibody in AML patient-derived xenografts with HOXA9 cooperativity; Lgr4/Rspo3 knockdown mice with AMPKα epistasis; human adipose progenitor assays and zebrafish rspo3 mutant","pmids":["32559496","32926477","32493999"],"confidence":"High","gaps":["Whether AML sensitivity is predictable by LGR4 expression alone","The direct binding partner mediating AMPKα activation","Mechanism of depot-specific differential Wnt signaling"]},{"year":2021,"claim":"Osteoblast-derived RSPO3 was established as the principal source of RSPO3 in bone, acting cell-autonomously to regulate vertebral trabecular bone mass and strength through osteoblast proliferation and differentiation.","evidence":"Osteoblast-specific Rspo3 conditional knockout (Runx2-cre) with bone densitometry and cellular assays","pmids":["34389713"],"confidence":"High","gaps":["The downstream Wnt pathway branch mediating bone effects","Whether RSPO3 also acts on osteoclasts"]},{"year":2022,"claim":"LGR4 (not LGR5) was definitively identified as the RSPO3 receptor driving gastric stem cell proliferation and NF-κB-dependent inflammatory responses to H. pylori, and osteoblast-specific RSPO3 was shown to be required for full estrogenic effects on cortical bone.","evidence":"Conditional Lgr4 vs Lgr5 KO with H. pylori infection model and NF-κB reporters; osteoblast-specific Rspo3 KO with ovariectomy/estradiol rescue","pmids":["35767364","35068191"],"confidence":"High","gaps":["Whether NF-κB activation is direct or secondary to Wnt signaling","Mechanism of RSPO3–estrogen interplay in cortical bone"]},{"year":2023,"claim":"A novel LGR4–Gab1–Gαi1/3 complex was identified as the effector of RSPO3-mediated Erk activation and neuroprotection, and RSPO3 was shown (with RSPO1) to be required for Ngn2+ sensory neural crest specification downstream of roof plate Wnt signaling.","evidence":"Co-IP of LGR4–Gab1–Gαi1/3, Gαi1/3 KO abolishing neuroprotection in MCAO model; Rspo1/Rspo3 double conditional KO with Ngn2 lineage tracing","pmids":["37805583","37767857"],"confidence":"High","gaps":["Whether Gαi coupling occurs at other LGR4-expressing tissues","Redundancy between RSPO1 and RSPO3 in neural crest beyond Ngn2 lineage","Structural basis of LGR4–Gαi interaction"]},{"year":2024,"claim":"RSPO3 was linked to pulmonary endothelial regeneration via LGR4/ILK/β-catenin, a direct GNG7 interaction activating Akt/GSK-3β/β-catenin in gastric cancer, and hepatic metabolic zonation with inter-organ glucose regulation through the hepatic vagus nerve.","evidence":"EC-specific RSPO3 KO/overexpression in LPS model with ILK pathway assays; Co-IP/LC-MS/MS identifying GNG7 with siRNA epistasis; viral Rspo3 induction and Cre-LoxP suppression with vagal denervation in obese mice","pmids":["38677673","38581123","39854351"],"confidence":"Medium","gaps":["Whether ILK pathway is distinct from β-catenin canonical signaling or convergent","GNG7 interaction requires independent replication","Neural circuit downstream of hepatic vagus is not mapped"]},{"year":null,"claim":"Key open questions include: (1) structural basis of RSPO3's selective engagement of different receptor complexes (SDC4, LGR4, LGR5, GNG7); (2) how tissue-specific cofactors determine canonical vs non-canonical pathway selection; (3) whether Gαi-coupled signaling is a general feature of RSPO3–LGR4 interactions beyond neurons.","evidence":"","pmids":[],"confidence":"Low","gaps":["No high-resolution structure of RSPO3 in complex with any receptor","No systematic comparison of downstream pathway activation across tissues","Relative in vivo contributions of ZNRF3/RNF43 clearance vs direct signaling remain unresolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0048018","term_label":"receptor ligand activity","supporting_discovery_ids":[0,4,6,7,13,14]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,2,5,10,12]}],"localization":[{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[0,2,4,7,11,18]}],"pathway":[{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,6,12,13]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,3,5,6,10,12,13,14]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[0,3,22]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[4,5,6,8,17]},{"term_id":"R-HSA-1500931","term_label":"Cell-Cell communication","supporting_discovery_ids":[2,3,11]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[10,19]}],"complexes":[],"partners":["LGR4","SDC4","LGR5","IQGAP1","GNG7","GNAI1","GAB1"],"other_free_text":[]},"mechanistic_narrative":"RSPO3 is a secreted potentiator of Wnt signaling that operates through multiple receptor systems to regulate stem cell self-renewal, tissue homeostasis, and organ morphogenesis across diverse developmental and adult contexts. In canonical Wnt/β-catenin signaling, RSPO3 acts principally through LGR4 (and LGR5) receptors to maintain intestinal crypt stem cells—where it is supplied by PDGFRα+ pericryptal stromal myofibroblasts—and to govern adrenal zona glomerulosa identity, gastric stem cell proliferation, osteoblast-mediated bone formation, hepatic metabolic zonation, and leukemia stem cell self-renewal [PMID:29559533, PMID:27313319, PMID:35767364, PMID:34389713, PMID:32559496, PMID:39854351]. RSPO3 also engages non-canonical Wnt pathways: it binds syndecan 4 to activate Wnt/PCP signaling via clathrin-mediated endocytosis during embryonic gastrulation, signals through a WNT/Ca²⁺/NFAT axis to control vascular remodeling, and assembles an LGR4–Gab1–Gαi1/3 complex that activates Erk for neuroprotection [PMID:21397842, PMID:26766444, PMID:37805583]. PTPRK-RSPO3 gene fusions drive Wnt-dependent colorectal tumorigenesis by expanding Lgr5+ stem cell compartments, and therapeutic anti-RSPO3 antibodies inhibit tumor growth and promote differentiation in these cancers [PMID:26700806, PMID:27511199]."},"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 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inhibition of clathrin-mediated endocytosis, epistasis with Wnt5a/Fz7/Dvl/JNK pathway components\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods (binding assay, in vivo loss-of-function, pathway epistasis) in a single rigorous study\",\n      \"pmids\": [\"21397842\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Endothelial RSPO3 controls vascular remodeling through non-canonical WNT/Ca2+/NFAT signaling; inducible endothelial Rspo3 deletion causes apoptosis and vascular pruning; RSPO3 and EVI/WLS co-regulate RNF213, USP18, and TRIM30α, which target NFAT1 for proteasomal degradation, and NFAT protein levels are decreased in Rspo3-iECKO endothelial cells\",\n      \"method\": \"Inducible endothelial-specific Rspo3 knockout mice, genetic epistasis with Evi/Wls knockout, endothelial transcriptomic screen, pharmacological NFAT inhibition phenocopy\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean conditional KO with defined cellular phenotype, epistasis, pharmacological phenocopy, multiple orthogonal methods\",\n      \"pmids\": [\"26766444\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"PDGFRα+ pericryptal stromal myofibroblasts are the critical in vivo source of RSPO3 and Wnts for intestinal stem cells; RSPO3 from these stromal cells is required for intestinal crypt Wnt/β-catenin signaling and Paneth cell differentiation, and is several orders of magnitude more potent than RSPO1 in stimulating organoid growth\",\n      \"method\": \"Cell-type-specific Cre-mediated Rspo3 excision in PdgfRα+ cells, organoid rescue assay with exogenous RSPO3 protein, DSS stress model\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo conditional knockout with defined cellular and molecular phenotypes, ex vivo rescue experiment\",\n      \"pmids\": [\"29559533\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Capsular RSPO3 signals to the underlying steroidogenic compartment to induce β-catenin signaling and imprint glomerulosa cell fate in the adrenal cortex; deletion of RSPO3 leads to loss of SHH signaling and impaired organ growth, and RSPO3 is required in adult life for replenishment of lost cells and maintenance of zona glomerulosa identity\",\n      \"method\": \"Conditional Rspo3 knockout in adrenal capsule, immunofluorescence, β-catenin and SHH pathway readouts\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO with multiple defined pathway readouts and functional phenotypes\",\n      \"pmids\": [\"27313319\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Targeting RSPO3 in PTPRK-RSPO3 fusion-positive colorectal xenografts inhibits tumor growth and promotes differentiation, with stem-cell-compartment genes being most sensitive; functional assays establish that RSPO3 drives stem-cell properties in these tumors\",\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 — in vivo xenograft KO with defined molecular and cellular phenotypes, multiple orthogonal assays\",\n      \"pmids\": [\"26700806\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Conditional in vivo RSPO3 overexpression in Lgr5+ intestinal stem cells drives rapid development of hyperplastic, adenomatous, and adenocarcinomatous lesions, expanding Lgr5+ stem cells, Paneth cells, and niche compartments via increased Wnt/β-catenin signaling; mutant Kras synergizes with RSPO3 in hyperplastic growth\",\n      \"method\": \"Conditional Rspo3 transgenic mouse model crossed with Lgr5-GFP-CreERT2, histopathology, β-catenin signaling readouts\",\n      \"journal\": \"Gut\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo gain-of-function with defined cellular and pathway phenotypes, genetic interaction with Kras\",\n      \"pmids\": [\"27511199\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"RSPO3 acts through LGR4 to upregulate self-renewal genes and is essential for leukemia stem cell self-renewal in a subset of AML; blocking the RSPO3-LGR4 interaction with anti-RSPO3 antibody (rosmantuzumab) impairs self-renewal and induces differentiation in AML patient-derived xenografts without affecting normal hematopoietic stem cells; LGR4 is epigenetically upregulated and cooperates with HOXA9\",\n      \"method\": \"Anti-RSPO3 antibody (OMP-131R10) treatment, patient-derived xenografts, LGR4 epigenetic upregulation analysis, HOXA9 cooperation assay\",\n      \"journal\": \"Cancer cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo PDX model with defined mechanistic target (LGR4), genetic cooperativity, and functional self-renewal readouts\",\n      \"pmids\": [\"32559496\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Osteoblast-derived RSPO3 is the principal source of RSPO3 in bone and in a cell-autonomous manner increases osteoblast proliferation and differentiation to regulate vertebral trabecular bone mass and bone strength\",\n      \"method\": \"Osteoblast-specific Rspo3 conditional knockout (Runx2-cre), bone mass and strength measurements, cell proliferation and differentiation assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — cell-type-specific conditional KO with defined cellular mechanism and physiological phenotype\",\n      \"pmids\": [\"34389713\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"RSPO3 signals through LGR4 (with IQGAP1 as signaling mediator) to promote proliferation and migration of lung cancer cells; knockdown of RSPO3, LGR4, or IQGAP1 reduces cell proliferation and migration in vitro and tumor growth/metastasis in vivo\",\n      \"method\": \"shRNA knockdown, in vitro proliferation and migration assays, in vivo tumor and metastasis models\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — defined LGR4/IQGAP1 pathway placement with in vivo validation, single lab\",\n      \"pmids\": [\"25531322\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"RSPO3-LGR4 signaling inhibits osteogenic differentiation of human adipose-derived stem cells via suppression of ERK/FGF signaling; LGR4 silencing inhibits ERK signaling and osteogenic differentiation, and loss of LGR4 abrogates RSPO3-regulated osteogenesis and RSPO3-induced ERK1/2 signaling inhibition\",\n      \"method\": \"shRNA knockdown of RSPO3 and LGR4, ERK pathway inhibitors, osteogenic differentiation assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — genetic and pharmacological epistasis establishing LGR4-ERK pathway, single lab\",\n      \"pmids\": [\"28220828\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"RSPO3 (with RSPO1) signals through LGR4 to suppress hepatic cholesterol synthesis via activation of AMPKα (phosphorylation at Thr172) and consequent reduction of SREBP2 nuclear translocation; hepatic LGR4 knockdown increases cholesterol synthesis and decreases AMPKα phosphorylation\",\n      \"method\": \"In vivo Lgr4 and Rspo1/3 knockdown mice, AMPKα agonist/antagonist/shRNA epistasis, SREBP2 nuclear translocation assay\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — epistasis with AMPKα confirmed by pharmacology and genetics, in vivo and in vitro, single lab\",\n      \"pmids\": [\"32926477\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"RSPO3 impairs barrier function of human vascular endothelial monolayers by inducing inter-endothelial gap formation through disruption of β-catenin and VE-cadherin alignment at adherens junctions, and synergizes with IL-1β to enhance permeability\",\n      \"method\": \"Electric Cell-substrate Impedance Sensing, immunofluorescence of β-catenin and VE-cadherin, primary human endothelial monolayers from multiple vascular beds\",\n      \"journal\": \"Molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — direct functional assay with defined molecular readouts in multiple cell types, single lab\",\n      \"pmids\": [\"30157748\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RSPO3 signaling via LGR4 (not LGR5) drives proliferation of gastric stem cells and induces NF-κB activity in proliferative stem cells; upon H. pylori exposure, LGR4-driven NF-κB activation causes gland hyperplasia and chemokine expression leading to neutrophil recruitment\",\n      \"method\": \"Conditional Lgr4 and Lgr5 knockout mice, H. pylori infection model, NF-κB reporter assays, histology\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO distinguishing LGR4 vs LGR5, defined NF-κB pathway placement, in vivo infection model\",\n      \"pmids\": [\"35767364\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"RSPO3 activates LGR4-Gab1-Gαi1/3 complex formation, leading to Erk activation and neuroprotection against ischemia/reperfusion injury; silencing or knockout of Gαi1/Gαi3 abolishes RSPO3-induced neuroprotection and Erk activation; endothelial RSPO3 protects neurons via this Gαi1/3-Erk axis in vivo\",\n      \"method\": \"Co-immunoprecipitation (LGR4-Gab1-Gαi1/3 complex), siRNA/KO of Gαi1/Gαi3, endothelial-specific RSPO3 KD/KO/overexpression mice, MCAO model, Erk pathway inhibitors\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP establishing novel complex, genetic KO rescue epistasis, in vivo endothelial-specific models with defined pathway\",\n      \"pmids\": [\"37805583\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RSPO3 mediates pulmonary endothelial regeneration in a LGR4-dependent manner; downstream effectors include both β-catenin and integrin-linked kinase (ILK)/Akt; EC-specific RSPO3 deletion inhibits endothelial proliferation and exacerbates injury, while overexpression promotes recovery\",\n      \"method\": \"EC-specific RSPO3 KD, inducible EC-specific KO, EC-specific overexpression mice, LPS endotoxemia model, ILK/Akt and β-catenin pathway assays\",\n      \"journal\": \"International journal of biological macromolecules\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple conditional mouse models with defined molecular pathway (LGR4/ILK/β-catenin), single lab\",\n      \"pmids\": [\"38677673\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RSPO3 interacts directly with GNG7 (G protein subunit gamma 7) as identified by Co-IP and LC-MS/MS; RSPO3 increases phosphorylation of Akt and GSK-3β and β-catenin expression in gastric cancer cells via GNG7, promoting cancer stem cell properties; GNG7 knockdown abolishes RSPO3-induced β-catenin activation\",\n      \"method\": \"Co-immunoprecipitation, LC-MS/MS proteomics, siRNA knockdown of GNG7, recombinant RSPO3 protein treatment, in vivo peritoneal seeding model\",\n      \"journal\": \"Cancer medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus MS identified novel binding partner (GNG7), epistasis confirmed by knockdown, in vivo validation, single lab\",\n      \"pmids\": [\"38581123\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The furin (FuFu) domains of RSPO3 mediate highly specific, LGR5-dependent cellular uptake when conjugated to liposomes, distinct from full-length RSPO1 which causes LGR5-independent uptake via heparan sulfate proteoglycans\",\n      \"method\": \"Liposome conjugation with RSPO3 furin domains vs full-length RSPO1, fluorescence uptake assays in LGR5-high vs LGR5-low cells, doxorubicin delivery functional assay\",\n      \"journal\": \"Journal of controlled release\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — domain-specific functional mapping of receptor binding with pharmacological validation, single lab\",\n      \"pmids\": [\"36813038\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RSPO3 regulates NSCLC radioresistance by promoting pyroptosis via the β-catenin-NF-κB signaling pathway and NLRP3 inflammasome; anti-RSPO3 antibody blocks radiation-induced pyroptosis and anti-tumor immunity in vivo\",\n      \"method\": \"RSPO3 overexpression/knockdown, β-catenin-NF-κB pathway assays, NLRP3 inflammasome readouts, anti-RSPO3 antibody in vivo\",\n      \"journal\": \"Radiotherapy and oncology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single lab, pathway placement via overexpression/KD without reconstitution or structural validation\",\n      \"pmids\": [\"39245068\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"RSPO3 expressed in subcutaneous adipocytes limits gluteofemoral adipose tissue expansion by suppressing adipogenesis and increasing gluteal adipocyte susceptibility to apoptosis, and promotes upper-body fat distribution by stimulating abdominal adipose progenitor proliferation; distinct responses are associated with differential WNT signaling; zebrafish with nonsense rspo3 mutation display altered fat distribution\",\n      \"method\": \"Human adipose progenitor cell culture assays, zebrafish rspo3 nonsense mutant, WNT signaling pathway readouts\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — human cellular functional assays plus genetic model organism KO, defined pathway placement, single lab\",\n      \"pmids\": [\"32493999\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Hepatic Rspo3 regulates metabolic liver zonation (pericentral zone) and systemic glucose metabolism; viral induction of Rspo3 in obese mice improves insulin resistance and prevents body weight gain via neuronal inter-organ communication (hepatic vagus nerve); hepatic Rspo3 suppression via Cre-LoxP causes glucose intolerance, insulin resistance, fatty liver, and obesity\",\n      \"method\": \"Viral-mediated Rspo3 induction, Cre-LoxP conditional suppression, hepatic vagal denervation, metabolic phenotyping\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple genetic models with defined metabolic pathway placement and inter-organ communication via denervation, single lab\",\n      \"pmids\": [\"39854351\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Osteoblast-derived RSPO3 is required for a full estrogenic effect on cortical (but not trabecular) bone; estradiol and RSPO3 regulate vertebral trabecular bone mass independently of each other, established by epistasis using osteoblast-specific RSPO3 knockout mice\",\n      \"method\": \"Osteoblast-specific Rspo3 KO (Runx2-cre), ovariectomy + estradiol rescue, bone mass and strength measurements\",\n      \"journal\": \"American journal of physiology. Endocrinology and metabolism\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis between RSPO3 and estrogen pathways using conditional KO, defined skeletal phenotypes\",\n      \"pmids\": [\"35068191\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Loss of AXIN1 (via frameshift deletions) confers resistance to WNT pathway blockade (porcupine inhibitor LGK974) in RSPO3-fusion-positive colorectal cancer cells; AXIN1 suppression by RNAi in parental VACO6 cells (PTPRK-RSPO3 fusion) recapitulates resistance\",\n      \"method\": \"Long-term drug selection in RSPO3-fusion CRC cell lines, whole exome sequencing of resistant clones, RNAi validation\",\n      \"journal\": \"EMBO molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic resistance identified by sequencing + RNAi validation, defines AXIN1 as downstream pathway component\",\n      \"pmids\": [\"28100566\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Rspo1 and Rspo3 are co-expressed with Wnt1/Wnt3a in the roof plate and are required for specification of Ngn2-positive sensory lineage neural crest cells; Rspo1/Rspo3 double knockout partially phenocopies Wnt1/Wnt3a double knockout for this specific neural crest subtype\",\n      \"method\": \"Rspo1/Rspo3 double conditional knockout mice, Ngn2 lineage tracing, Wnt/β-catenin reporter assay, comparison with Wnt1/Wnt3a dKO\",\n      \"journal\": \"Developmental dynamics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with Wnt pathway in defined developmental context, double KO phenotype\",\n      \"pmids\": [\"37767857\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RSPO3 is a secreted protein that amplifies Wnt signaling through multiple mechanisms: it binds SDC4 to activate non-canonical Wnt/PCP signaling via clathrin-mediated endocytosis; signals through LGR4 (and LGR5) receptors to potentiate canonical Wnt/β-catenin signaling in intestinal, adrenal, gastric, bone, and hematopoietic stem cell niches; engages a LGR4-Gab1-Gαi1/3-Erk axis for neuroprotection; activates LGR4/ILK/β-catenin and LGR4/AMPKα-SREBP2 signaling in endothelial and hepatic contexts respectively; and in the intestine is supplied predominantly by PDGFRα+ pericryptal stromal myofibroblasts to maintain the crypt stem cell niche, with PTPRK-RSPO3 gene fusions causing oncogenic Wnt pathway hyperactivation in colorectal cancer.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"RSPO3 is a secreted potentiator of Wnt signaling that operates through multiple receptor systems to regulate stem cell self-renewal, tissue homeostasis, and organ morphogenesis across diverse developmental and adult contexts. In canonical Wnt/β-catenin signaling, RSPO3 acts principally through LGR4 (and LGR5) receptors to maintain intestinal crypt stem cells—where it is supplied by PDGFRα+ pericryptal stromal myofibroblasts—and to govern adrenal zona glomerulosa identity, gastric stem cell proliferation, osteoblast-mediated bone formation, hepatic metabolic zonation, and leukemia stem cell self-renewal [PMID:29559533, PMID:27313319, PMID:35767364, PMID:34389713, PMID:32559496, PMID:39854351]. RSPO3 also engages non-canonical Wnt pathways: it binds syndecan 4 to activate Wnt/PCP signaling via clathrin-mediated endocytosis during embryonic gastrulation, signals through a WNT/Ca²⁺/NFAT axis to control vascular remodeling, and assembles an LGR4–Gab1–Gαi1/3 complex that activates Erk for neuroprotection [PMID:21397842, PMID:26766444, PMID:37805583]. PTPRK-RSPO3 gene fusions drive Wnt-dependent colorectal tumorigenesis by expanding Lgr5+ stem cell compartments, and therapeutic anti-RSPO3 antibodies inhibit tumor growth and promote differentiation in these cancers [PMID:26700806, PMID:27511199].\",\n  \"teleology\": [\n    {\n      \"year\": 2011,\n      \"claim\": \"The first mechanistic role for RSPO3 was established in non-canonical Wnt signaling: RSPO3 binds SDC4 and activates Wnt/PCP signaling via clathrin-mediated endocytosis, resolving how R-spondins engage non-canonical pathways during gastrulation.\",\n      \"evidence\": \"Co-immunoprecipitation, clathrin inhibition, and epistasis with Fz7/Dvl/JNK in Xenopus embryos\",\n      \"pmids\": [\"21397842\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether SDC4-dependent PCP activation operates in mammalian tissues\", \"Structural basis of the RSPO3–SDC4 interaction\", \"Relationship between SDC4 and LGR receptor pathways\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"RSPO3 was placed upstream of an LGR4–IQGAP1 signaling axis in cancer cell proliferation and migration, establishing LGR4 as a functional RSPO3 receptor in a disease-relevant context.\",\n      \"evidence\": \"shRNA knockdown of RSPO3/LGR4/IQGAP1 with in vitro and in vivo tumor/metastasis assays in lung cancer cells\",\n      \"pmids\": [\"25531322\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"IQGAP1's direct binding mode to the RSPO3–LGR4 complex is undefined\", \"Whether IQGAP1 dependency is tissue-specific\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"PTPRK-RSPO3 fusions were shown to be oncogenic drivers in colorectal cancer, with anti-RSPO3 antibody treatment shrinking patient-derived xenografts and inducing differentiation of the Lgr5+ stem cell compartment.\",\n      \"evidence\": \"Anti-RSPO3 antibody treatment of PTPRK-RSPO3 fusion-positive CRC patient-derived xenografts with gene expression and stem cell assays\",\n      \"pmids\": [\"26700806\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full spectrum of RSPO3 fusion partners in CRC\", \"Whether antibody effects are purely through Wnt suppression or additional mechanisms\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Three independent studies established RSPO3 as a tissue-autonomous niche signal: it controls adrenal zona glomerulosa identity via β-catenin/SHH, drives intestinal tumorigenesis when overexpressed in Lgr5+ cells, and regulates vascular remodeling through a non-canonical WNT/Ca²⁺/NFAT axis in endothelium.\",\n      \"evidence\": \"Conditional Rspo3 KO in adrenal capsule; conditional Rspo3 transgenic in Lgr5+ intestinal cells with Kras epistasis; inducible endothelial-specific Rspo3 KO with Evi/Wls epistasis and NFAT pharmacological phenocopy\",\n      \"pmids\": [\"27313319\", \"27511199\", \"26766444\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether adrenal and vascular roles are LGR4-dependent\", \"Identity of endothelial RSPO3 receptor(s)\", \"Relative contributions of canonical versus non-canonical pathways in each tissue\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"RSPO3–LGR4 signaling was shown to modulate ERK/FGF signaling to inhibit osteogenic differentiation, and AXIN1 loss was identified as a mechanism of resistance to Wnt pathway blockade in RSPO3-fusion CRC, defining downstream pathway requirements.\",\n      \"evidence\": \"shRNA knockdown of RSPO3/LGR4 with ERK inhibitors in human adipose-derived stem cells; whole-exome sequencing and RNAi of AXIN1 in PTPRK-RSPO3 fusion CRC cells\",\n      \"pmids\": [\"28220828\", \"28100566\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether ERK/FGF suppression operates in vivo bone context\", \"Whether other Wnt destruction complex components also confer resistance\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"PDGFRα+ pericryptal stromal myofibroblasts were identified as the critical in vivo source of RSPO3 for the intestinal stem cell niche, and RSPO3 was shown to disrupt endothelial barrier function via β-catenin/VE-cadherin junctional disorganization.\",\n      \"evidence\": \"Cell-type-specific Cre-mediated Rspo3 excision in PDGFRα+ cells with organoid rescue; impedance sensing and immunofluorescence in human endothelial monolayers\",\n      \"pmids\": [\"29559533\", \"30157748\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether other stromal sources compensate long-term\", \"The receptor mediating RSPO3's junctional effects on endothelium\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"RSPO3's roles expanded to leukemia stem cell maintenance via LGR4, hepatic cholesterol metabolism via LGR4–AMPKα–SREBP2, and depot-specific adipose tissue distribution through differential Wnt activation, revealing metabolic and hematopoietic functions beyond epithelial stem cell niches.\",\n      \"evidence\": \"Anti-RSPO3 antibody in AML patient-derived xenografts with HOXA9 cooperativity; Lgr4/Rspo3 knockdown mice with AMPKα epistasis; human adipose progenitor assays and zebrafish rspo3 mutant\",\n      \"pmids\": [\"32559496\", \"32926477\", \"32493999\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether AML sensitivity is predictable by LGR4 expression alone\", \"The direct binding partner mediating AMPKα activation\", \"Mechanism of depot-specific differential Wnt signaling\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Osteoblast-derived RSPO3 was established as the principal source of RSPO3 in bone, acting cell-autonomously to regulate vertebral trabecular bone mass and strength through osteoblast proliferation and differentiation.\",\n      \"evidence\": \"Osteoblast-specific Rspo3 conditional knockout (Runx2-cre) with bone densitometry and cellular assays\",\n      \"pmids\": [\"34389713\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The downstream Wnt pathway branch mediating bone effects\", \"Whether RSPO3 also acts on osteoclasts\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"LGR4 (not LGR5) was definitively identified as the RSPO3 receptor driving gastric stem cell proliferation and NF-κB-dependent inflammatory responses to H. pylori, and osteoblast-specific RSPO3 was shown to be required for full estrogenic effects on cortical bone.\",\n      \"evidence\": \"Conditional Lgr4 vs Lgr5 KO with H. pylori infection model and NF-κB reporters; osteoblast-specific Rspo3 KO with ovariectomy/estradiol rescue\",\n      \"pmids\": [\"35767364\", \"35068191\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether NF-κB activation is direct or secondary to Wnt signaling\", \"Mechanism of RSPO3–estrogen interplay in cortical bone\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"A novel LGR4–Gab1–Gαi1/3 complex was identified as the effector of RSPO3-mediated Erk activation and neuroprotection, and RSPO3 was shown (with RSPO1) to be required for Ngn2+ sensory neural crest specification downstream of roof plate Wnt signaling.\",\n      \"evidence\": \"Co-IP of LGR4–Gab1–Gαi1/3, Gαi1/3 KO abolishing neuroprotection in MCAO model; Rspo1/Rspo3 double conditional KO with Ngn2 lineage tracing\",\n      \"pmids\": [\"37805583\", \"37767857\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Gαi coupling occurs at other LGR4-expressing tissues\", \"Redundancy between RSPO1 and RSPO3 in neural crest beyond Ngn2 lineage\", \"Structural basis of LGR4–Gαi interaction\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"RSPO3 was linked to pulmonary endothelial regeneration via LGR4/ILK/β-catenin, a direct GNG7 interaction activating Akt/GSK-3β/β-catenin in gastric cancer, and hepatic metabolic zonation with inter-organ glucose regulation through the hepatic vagus nerve.\",\n      \"evidence\": \"EC-specific RSPO3 KO/overexpression in LPS model with ILK pathway assays; Co-IP/LC-MS/MS identifying GNG7 with siRNA epistasis; viral Rspo3 induction and Cre-LoxP suppression with vagal denervation in obese mice\",\n      \"pmids\": [\"38677673\", \"38581123\", \"39854351\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether ILK pathway is distinct from β-catenin canonical signaling or convergent\", \"GNG7 interaction requires independent replication\", \"Neural circuit downstream of hepatic vagus is not mapped\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key open questions include: (1) structural basis of RSPO3's selective engagement of different receptor complexes (SDC4, LGR4, LGR5, GNG7); (2) how tissue-specific cofactors determine canonical vs non-canonical pathway selection; (3) whether Gαi-coupled signaling is a general feature of RSPO3–LGR4 interactions beyond neurons.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No high-resolution structure of RSPO3 in complex with any receptor\", \"No systematic comparison of downstream pathway activation across tissues\", \"Relative in vivo contributions of ZNRF3/RNF43 clearance vs direct signaling remain unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [0, 4, 6, 7, 13, 14]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 2, 5, 10, 12]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [0, 2, 4, 7, 11, 18]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 6, 12, 13]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 3, 5, 6, 10, 12, 13, 14]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [0, 3, 22]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [4, 5, 6, 8, 17]},\n      {\"term_id\": \"R-HSA-1500931\", \"supporting_discovery_ids\": [2, 3, 11]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [10, 19]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"LGR4\",\n      \"SDC4\",\n      \"LGR5\",\n      \"IQGAP1\",\n      \"GNG7\",\n      \"GNAI1\",\n      \"GAB1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}