{"gene":"RCN3","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":2023,"finding":"RCN3 promotes fibroblast activation by maintaining persistent TGFβ1 signaling through a positive feedback loop: TGFβ1 upregulates RCN3, which then physically interacts with EZH2 (an epigenetic methyltransferase) and sequesters it in the cytoplasm, thereby releasing EZH2-H3K27me3 epigenetic repression of TGFBR1, leading to persistent TGFBR1 expression and sustained TGFβ1 signaling.","method":"BioID protein interaction assay, co-immunoprecipitation, epigenetic analysis, fibroblast-selective Rcn3 knockdown mouse model with bleomycin-induced lung fibrosis","journal":"Respiratory research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal protein interaction assays (BioID) plus in vivo genetic model, single lab with multiple orthogonal methods","pmids":["37710230"],"is_preprint":false},{"year":2025,"finding":"RCN3's EF-hand domains 3 and 4 interact with GRP78, promoting GRP78 translocation to the cellular membrane, which induces ER stress through elevated intracellular calcium levels and activates the PI3K-AKT-mTOR-S6 signaling pathway by enhancing phosphorylation of key proteins in colorectal cancer cells.","method":"Functional assays (proliferation, migration, invasion in vitro and in vivo), Western blotting for pathway phosphorylation, domain-mapping interaction studies","journal":"Oncogene","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, domain-interaction claim from abstract without explicit mutagenesis/reconstitution details, single study","pmids":["40849588"],"is_preprint":false},{"year":2021,"finding":"Selective ablation of Rcn3 in type II alveolar epithelial cells (AECIIs) significantly alleviated PPE-induced emphysema in mice, establishing that Rcn3 in AECIIs plays a functional role in the lung injury-repair process.","method":"Conditional knockout (CKO) mice with AECII-specific Rcn3 deletion, PPE-induced emphysema model, Western blot, IHC, qPCR","journal":"International journal of chronic obstructive pulmonary disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cell-type-specific genetic knockout with defined in vivo phenotypic readout, single lab","pmids":["33531801"],"is_preprint":false},{"year":2024,"finding":"In gastric cancer, RCN3 protein in cancer-associated fibroblasts regulates PCSK6 expression, and PCSK6 in turn regulates macrophage polarization through STAT1, defining an RCN3/PCSK6/STAT1 axis linking CAFs to immune modulation.","method":"Western blot, qPCR, chromatin immunoprecipitation (ChIP), macrophage M1/M2 polarization marker detection, cellular co-culture experiments","journal":"Frontiers in bioscience (Landmark edition)","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, ChIP and expression assays without full mechanistic reconstitution, single study","pmids":["39206893"],"is_preprint":false},{"year":2026,"finding":"RCN3 interacts with MMP10 through its EF-hand domains 5–6, promoting secretion of MMP10 and activating the PI3K/Akt pathway, thereby facilitating renal cell carcinoma lung metastasis; promoter hypomethylation drives RCN3 upregulation in metastatic RCC.","method":"Co-immunoprecipitation/interaction assays with domain mapping (EF-hand 5–6), in vitro functional assays, in vivo lung metastasis model, bisulfite sequencing, transcriptome sequencing","journal":"Cellular signalling","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, domain-interaction claim from abstract without explicit reconstitution or mutagenesis details confirmed","pmids":["41966439"],"is_preprint":false},{"year":2026,"finding":"RCN3 functions as a stress-responsive intracellular gatekeeper in pulmonary epithelial cells limiting POVPC-induced ferroptosis: RCN3 knockdown aggravated lipid peroxidation, iron accumulation, and GSH/GPX4/SLC7A11 depletion, while RCN3 overexpression attenuated these effects; AECII-specific RCN3 deletion sensitized mice to POVPC-induced ALI, and AAV6-mediated AECII-targeted RCN3 delivery alleviated injury.","method":"AECII-specific RCN3 knockout mice, AAV6-RCN3 overexpression, in vitro ferroptosis assays (iron, lipid peroxidation, GSH/GPX4/SLC7A11 measurement), ferrostatin-1 and deferoxamine rescue","journal":"Free radical biology & medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cell-type-specific genetic KO plus AAV rescue in vivo with multiple orthogonal ferroptosis readouts, single lab","pmids":["41881199"],"is_preprint":false},{"year":2026,"finding":"Secreted RCN3 acts as an early epithelial paracrine mediator activating fibroblasts: RCN3 is secreted via an N140-glycosylation–dependent ER-Golgi pathway, binds TGFβR1 on fibroblasts, and activates canonical Smad2/3 signaling; RCN3 also upregulates TGFβ1 and TGFβR1/2 in a Smad3-dependent manner. Intratracheal exogenous RCN3 alone was sufficient to trigger pulmonary fibrosis in vivo, and early-phase RCN3 neutralization after LPS-ALI attenuated fibrotic remodeling.","method":"Secretomics of LPS-treated pulmonary epithelial cells, N-glycosylation site mutagenesis (N140), recombinant RCN3 intratracheal administration, neutralizing antibody treatment in vivo, Smad2/3 phosphorylation assays, clinical BALF measurements","journal":"Cell communication and signaling : CCS","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis of glycosylation site, in vivo gain-of-function and neutralization with defined signaling readouts, single lab with multiple orthogonal methods","pmids":["41664195"],"is_preprint":false},{"year":2025,"finding":"In colorectal cancer, TGF-β induces RCN3 expression through RELB, and RCN3-positive CAFs exhibit hyper-activated TGF-β signaling, enhance epithelial-mesenchymal transition, invasion, and M2-like macrophage polarization in coculture and xenograft models, creating a positive TGF-β/RCN3 feedback loop.","method":"Multi-omics profiling (bulk/single-cell/spatial transcriptomics, proteomics), in vitro co-culture, xenograft in vivo models, machine-learning signature with SHAP interpretation, in vitro/in vivo validation","journal":"International journal of surgery (London, England)","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, mechanistic claims (RELB→RCN3) stated but experimental details of RELB regulation of RCN3 not fully elaborated in abstract","pmids":["41186513"],"is_preprint":false}],"current_model":"RCN3 is an ER-lumen calcium-binding protein (reticulocalbin) that acts as a multifunctional stress-responsive mediator: intracellularly, it sequesters EZH2 in the cytoplasm to derepress TGFBR1 expression and sustain TGFβ1 signaling in fibroblasts, protects pulmonary epithelial cells from ferroptosis by limiting lipid peroxidation, and interacts with GRP78 (via EF-hand domains 3–4) and MMP10 (via EF-hand domains 5–6) to modulate calcium homeostasis and matrix metalloproteinase secretion; extracellularly, glycosylation-dependent secreted RCN3 acts as an early paracrine mediator that directly engages TGFβR1 on fibroblasts to activate Smad2/3 signaling and drive pulmonary fibrosis."},"narrative":{"mechanistic_narrative":"RCN3 is a calcium-binding reticulocalbin of the ER lumen that functions as a stress-responsive regulator of fibroblast activation, epithelial injury responses, and TGF-β signaling [PMID:37710230, PMID:41664195]. In fibroblasts, TGF-β1 induces RCN3, which then sustains the pathway through a positive feedback loop: RCN3 physically binds the methyltransferase EZH2 and sequesters it in the cytoplasm, relieving EZH2/H3K27me3 repression of TGFBR1 and thereby maintaining persistent TGFBR1 expression and TGF-β1 signaling that drives fibroblast activation in bleomycin-induced lung fibrosis [PMID:37710230]. RCN3 also acts non-cell-autonomously: it is secreted from pulmonary epithelial cells via an N140-glycosylation–dependent ER–Golgi route, binds TGFβR1 on fibroblasts, and activates canonical Smad2/3 signaling, with intratracheal recombinant RCN3 alone sufficient to trigger pulmonary fibrosis and early RCN3 neutralization attenuating fibrotic remodeling [PMID:41664195]. Within type II alveolar epithelial cells, RCN3 supports the injury-repair process and limits POVPC-induced ferroptosis by restraining lipid peroxidation and iron accumulation and preserving the GSH/GPX4/SLC7A11 axis [PMID:33531801, PMID:41881199]. Its EF-hand domains mediate distinct partner interactions—domains 3–4 engage GRP78 and domains 5–6 engage MMP10—coupling RCN3 to calcium handling, ER stress, and metalloproteinase secretion [PMID:40849588, PMID:41966439].","teleology":[{"year":2021,"claim":"Whether RCN3 in alveolar epithelium has a functional role in lung injury was unknown; cell-type-specific deletion established RCN3 in AECIIs as a participant in the injury-repair process.","evidence":"AECII-specific conditional Rcn3 knockout in a PPE-induced emphysema mouse model","pmids":["33531801"],"confidence":"Medium","gaps":["Molecular mechanism by which RCN3 affects AECII injury was not defined","No interaction partners identified in this model"]},{"year":2023,"claim":"It was unclear how RCN3 sustains fibroblast activation; the work showed RCN3 sequesters EZH2 in the cytoplasm to derepress TGFBR1, defining a TGF-β1/RCN3 feedback loop.","evidence":"BioID and co-IP interaction assays, epigenetic (H3K27me3) analysis, and fibroblast-selective Rcn3 knockdown in bleomycin lung fibrosis","pmids":["37710230"],"confidence":"Medium","gaps":["Structural basis of EZH2 binding not resolved","Whether cytoplasmic EZH2 sequestration occurs in other cell types untested"]},{"year":2024,"claim":"The role of fibroblast RCN3 in the tumor immune microenvironment was unknown; an RCN3/PCSK6/STAT1 axis was proposed linking CAFs to macrophage polarization in gastric cancer.","evidence":"ChIP, expression assays, and macrophage M1/M2 polarization co-culture in gastric cancer","pmids":["39206893"],"confidence":"Low","gaps":["Single study without full mechanistic reconstitution","Direct molecular link from RCN3 to PCSK6 not established","Whether the axis operates in vivo not shown"]},{"year":2025,"claim":"Partner interactions and upstream regulation of RCN3 in cancer were uncharacterized; EF-hand 3–4 binding to GRP78 (with ER stress and PI3K-AKT-mTOR activation) and RELB-driven RCN3 induction by TGF-β were reported.","evidence":"Domain-mapping interaction studies and pathway Western blotting in colorectal cancer; multi-omics and co-culture/xenograft for the RELB/RCN3 feedback loop","pmids":["40849588","41186513"],"confidence":"Low","gaps":["EF-hand 3–4/GRP78 interaction lacks explicit mutagenesis confirmation","RELB→RCN3 regulation not fully elaborated experimentally","Single lab per claim"]},{"year":2026,"claim":"Whether RCN3 acts intracellularly versus as a secreted ligand, and how it controls epithelial fate, were open; studies showed RCN3 limits ferroptosis in AECIIs and that glycosylation-dependent secreted RCN3 binds TGFβR1 to activate Smad2/3 and drive fibrosis, while EF-hand 5–6 binds MMP10 to promote metastasis.","evidence":"AECII-specific KO with AAV6 rescue and ferroptosis readouts; secretomics with N140-glycosylation mutagenesis, recombinant RCN3 and neutralizing antibody in vivo; co-IP domain mapping with lung metastasis model","pmids":["41881199","41664195","41966439"],"confidence":"Medium","gaps":["Mechanism by which RCN3 restrains lipid peroxidation not molecularly defined","Receptor-level details of secreted RCN3/TGFβR1 binding not structurally resolved","EF-hand 5–6/MMP10 interaction from abstract without confirmed reconstitution"]},{"year":null,"claim":"How RCN3's distinct EF-hand-mediated partner interactions, its calcium-binding activity, and its dual intracellular/secreted functions are integrated within a single mechanistic framework remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural model of RCN3 EF-hand engagement of distinct partners","Quantitative role of calcium binding in each function not established","Determinants directing RCN3 to intracellular vs secreted roles unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0048018","term_label":"receptor ligand activity","supporting_discovery_ids":[6]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,1,4]}],"localization":[{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[1,6]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0]},{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[6]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,6]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[5]}],"complexes":[],"partners":["EZH2","GRP78","MMP10","TGFBR1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q96D15","full_name":"Reticulocalbin-3","aliases":["EF-hand calcium-binding protein RLP49"],"length_aa":328,"mass_kda":37.5,"function":"Probable molecular chaperone assisting protein biosynthesis and transport in the endoplasmic reticulum (PubMed:16433634, PubMed:28939891). Required for the proper biosynthesis and transport of pulmonary surfactant-associated protein A/SP-A, pulmonary surfactant-associated protein D/SP-D and the lipid transporter ABCA3 (By similarity). By regulating both the proper expression and the degradation through the endoplasmic reticulum-associated protein degradation pathway of these proteins plays a crucial role in pulmonary surfactant homeostasis (By similarity). Has an anti-fibrotic activity by negatively regulating the secretion of type I and type III collagens (PubMed:28939891). This calcium-binding protein also transiently associates with immature PCSK6 and regulates its secretion (PubMed:16433634)","subcellular_location":"Endoplasmic reticulum lumen","url":"https://www.uniprot.org/uniprotkb/Q96D15/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RCN3","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RCN3","total_profiled":1310},"omim":[{"mim_id":"619032","title":"RETICULOCALBIN 3; RCN3","url":"https://www.omim.org/entry/619032"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Endoplasmic reticulum","reliability":"Supported"},{"location":"Vesicles","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/RCN3"},"hgnc":{"alias_symbol":["RLP49"],"prev_symbol":[]},"alphafold":{"accession":"Q96D15","domains":[{"cath_id":"-","chopping":"48-220_263-317","consensus_level":"medium","plddt":91.7882,"start":48,"end":317},{"cath_id":"-","chopping":"221-262","consensus_level":"medium","plddt":89.3981,"start":221,"end":262}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96D15","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96D15-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96D15-F1-predicted_aligned_error_v6.png","plddt_mean":82.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RCN3","jax_strain_url":"https://www.jax.org/strain/search?query=RCN3"},"sequence":{"accession":"Q96D15","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96D15.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96D15/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96D15"}},"corpus_meta":[{"pmid":"37710230","id":"PMC_37710230","title":"TGFβ1-RCN3-TGFBR1 loop facilitates pulmonary fibrosis by orchestrating fibroblast activation.","date":"2023","source":"Respiratory research","url":"https://pubmed.ncbi.nlm.nih.gov/37710230","citation_count":22,"is_preprint":false},{"pmid":"40849588","id":"PMC_40849588","title":"RCN3 functions as a tumor promoter in colorectal cancer by modulating the GRP78-PI3K-AKT signaling pathway.","date":"2025","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/40849588","citation_count":4,"is_preprint":false},{"pmid":"33531801","id":"PMC_33531801","title":"Rcn3 Suppression Was Responsible for Partial Relief of Emphysema as Shown by Specific Type II Alveolar Epithelial Cell Rcn3 CKO Mouse Model.","date":"2021","source":"International journal of chronic obstructive pulmonary disease","url":"https://pubmed.ncbi.nlm.nih.gov/33531801","citation_count":4,"is_preprint":false},{"pmid":"34753797","id":"PMC_34753797","title":"An Expanded Genome-Wide Association Study of Fructosamine Levels Identifies RCN3 as a Replicating Locus and Implicates FCGRT as the Effector Transcript.","date":"2022","source":"Diabetes","url":"https://pubmed.ncbi.nlm.nih.gov/34753797","citation_count":4,"is_preprint":false},{"pmid":"39206893","id":"PMC_39206893","title":"Cancer-Associated Fibroblasts in Gastric Cancer Regulate Macrophage Polarization through RCN3 Pathway.","date":"2024","source":"Frontiers in bioscience (Landmark edition)","url":"https://pubmed.ncbi.nlm.nih.gov/39206893","citation_count":3,"is_preprint":false},{"pmid":"41186513","id":"PMC_41186513","title":"Identification of RCN3-positive cancer-associated fibroblasts as novel drivers of colorectal cancer through a TGF-β pathway-based risk signature.","date":"2025","source":"International journal of surgery (London, England)","url":"https://pubmed.ncbi.nlm.nih.gov/41186513","citation_count":0,"is_preprint":false},{"pmid":"41966439","id":"PMC_41966439","title":"Analysis of paired primary-metastatic renal cancer lesions reveals RCN3-MMP10 axis facilitates lung metastasis.","date":"2026","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/41966439","citation_count":0,"is_preprint":false},{"pmid":"41881199","id":"PMC_41881199","title":"Endogenous oxidized phospholipid POVPC triggers pulmonary epithelial ferroptosis in acute lung injury and is restrained by RCN3.","date":"2026","source":"Free radical biology & medicine","url":"https://pubmed.ncbi.nlm.nih.gov/41881199","citation_count":0,"is_preprint":false},{"pmid":"41664195","id":"PMC_41664195","title":"Secreted RCN3 acts as an early epithelial-fibroblast mediator via TGFβR1-Smad signaling in post-ALI pulmonary fibrosis.","date":"2026","source":"Cell communication and signaling : CCS","url":"https://pubmed.ncbi.nlm.nih.gov/41664195","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7421,"output_tokens":2440,"usd":0.029431,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9500,"output_tokens":2562,"usd":0.055775,"stage2_stop_reason":"end_turn"},"total_usd":0.085206,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2023,\n      \"finding\": \"RCN3 promotes fibroblast activation by maintaining persistent TGFβ1 signaling through a positive feedback loop: TGFβ1 upregulates RCN3, which then physically interacts with EZH2 (an epigenetic methyltransferase) and sequesters it in the cytoplasm, thereby releasing EZH2-H3K27me3 epigenetic repression of TGFBR1, leading to persistent TGFBR1 expression and sustained TGFβ1 signaling.\",\n      \"method\": \"BioID protein interaction assay, co-immunoprecipitation, epigenetic analysis, fibroblast-selective Rcn3 knockdown mouse model with bleomycin-induced lung fibrosis\",\n      \"journal\": \"Respiratory research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal protein interaction assays (BioID) plus in vivo genetic model, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"37710230\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RCN3's EF-hand domains 3 and 4 interact with GRP78, promoting GRP78 translocation to the cellular membrane, which induces ER stress through elevated intracellular calcium levels and activates the PI3K-AKT-mTOR-S6 signaling pathway by enhancing phosphorylation of key proteins in colorectal cancer cells.\",\n      \"method\": \"Functional assays (proliferation, migration, invasion in vitro and in vivo), Western blotting for pathway phosphorylation, domain-mapping interaction studies\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, domain-interaction claim from abstract without explicit mutagenesis/reconstitution details, single study\",\n      \"pmids\": [\"40849588\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Selective ablation of Rcn3 in type II alveolar epithelial cells (AECIIs) significantly alleviated PPE-induced emphysema in mice, establishing that Rcn3 in AECIIs plays a functional role in the lung injury-repair process.\",\n      \"method\": \"Conditional knockout (CKO) mice with AECII-specific Rcn3 deletion, PPE-induced emphysema model, Western blot, IHC, qPCR\",\n      \"journal\": \"International journal of chronic obstructive pulmonary disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell-type-specific genetic knockout with defined in vivo phenotypic readout, single lab\",\n      \"pmids\": [\"33531801\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In gastric cancer, RCN3 protein in cancer-associated fibroblasts regulates PCSK6 expression, and PCSK6 in turn regulates macrophage polarization through STAT1, defining an RCN3/PCSK6/STAT1 axis linking CAFs to immune modulation.\",\n      \"method\": \"Western blot, qPCR, chromatin immunoprecipitation (ChIP), macrophage M1/M2 polarization marker detection, cellular co-culture experiments\",\n      \"journal\": \"Frontiers in bioscience (Landmark edition)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, ChIP and expression assays without full mechanistic reconstitution, single study\",\n      \"pmids\": [\"39206893\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"RCN3 interacts with MMP10 through its EF-hand domains 5–6, promoting secretion of MMP10 and activating the PI3K/Akt pathway, thereby facilitating renal cell carcinoma lung metastasis; promoter hypomethylation drives RCN3 upregulation in metastatic RCC.\",\n      \"method\": \"Co-immunoprecipitation/interaction assays with domain mapping (EF-hand 5–6), in vitro functional assays, in vivo lung metastasis model, bisulfite sequencing, transcriptome sequencing\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, domain-interaction claim from abstract without explicit reconstitution or mutagenesis details confirmed\",\n      \"pmids\": [\"41966439\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"RCN3 functions as a stress-responsive intracellular gatekeeper in pulmonary epithelial cells limiting POVPC-induced ferroptosis: RCN3 knockdown aggravated lipid peroxidation, iron accumulation, and GSH/GPX4/SLC7A11 depletion, while RCN3 overexpression attenuated these effects; AECII-specific RCN3 deletion sensitized mice to POVPC-induced ALI, and AAV6-mediated AECII-targeted RCN3 delivery alleviated injury.\",\n      \"method\": \"AECII-specific RCN3 knockout mice, AAV6-RCN3 overexpression, in vitro ferroptosis assays (iron, lipid peroxidation, GSH/GPX4/SLC7A11 measurement), ferrostatin-1 and deferoxamine rescue\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell-type-specific genetic KO plus AAV rescue in vivo with multiple orthogonal ferroptosis readouts, single lab\",\n      \"pmids\": [\"41881199\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Secreted RCN3 acts as an early epithelial paracrine mediator activating fibroblasts: RCN3 is secreted via an N140-glycosylation–dependent ER-Golgi pathway, binds TGFβR1 on fibroblasts, and activates canonical Smad2/3 signaling; RCN3 also upregulates TGFβ1 and TGFβR1/2 in a Smad3-dependent manner. Intratracheal exogenous RCN3 alone was sufficient to trigger pulmonary fibrosis in vivo, and early-phase RCN3 neutralization after LPS-ALI attenuated fibrotic remodeling.\",\n      \"method\": \"Secretomics of LPS-treated pulmonary epithelial cells, N-glycosylation site mutagenesis (N140), recombinant RCN3 intratracheal administration, neutralizing antibody treatment in vivo, Smad2/3 phosphorylation assays, clinical BALF measurements\",\n      \"journal\": \"Cell communication and signaling : CCS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis of glycosylation site, in vivo gain-of-function and neutralization with defined signaling readouts, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"41664195\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In colorectal cancer, TGF-β induces RCN3 expression through RELB, and RCN3-positive CAFs exhibit hyper-activated TGF-β signaling, enhance epithelial-mesenchymal transition, invasion, and M2-like macrophage polarization in coculture and xenograft models, creating a positive TGF-β/RCN3 feedback loop.\",\n      \"method\": \"Multi-omics profiling (bulk/single-cell/spatial transcriptomics, proteomics), in vitro co-culture, xenograft in vivo models, machine-learning signature with SHAP interpretation, in vitro/in vivo validation\",\n      \"journal\": \"International journal of surgery (London, England)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, mechanistic claims (RELB→RCN3) stated but experimental details of RELB regulation of RCN3 not fully elaborated in abstract\",\n      \"pmids\": [\"41186513\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RCN3 is an ER-lumen calcium-binding protein (reticulocalbin) that acts as a multifunctional stress-responsive mediator: intracellularly, it sequesters EZH2 in the cytoplasm to derepress TGFBR1 expression and sustain TGFβ1 signaling in fibroblasts, protects pulmonary epithelial cells from ferroptosis by limiting lipid peroxidation, and interacts with GRP78 (via EF-hand domains 3–4) and MMP10 (via EF-hand domains 5–6) to modulate calcium homeostasis and matrix metalloproteinase secretion; extracellularly, glycosylation-dependent secreted RCN3 acts as an early paracrine mediator that directly engages TGFβR1 on fibroblasts to activate Smad2/3 signaling and drive pulmonary fibrosis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RCN3 is a calcium-binding reticulocalbin of the ER lumen that functions as a stress-responsive regulator of fibroblast activation, epithelial injury responses, and TGF-β signaling [#0, #6]. In fibroblasts, TGF-β1 induces RCN3, which then sustains the pathway through a positive feedback loop: RCN3 physically binds the methyltransferase EZH2 and sequesters it in the cytoplasm, relieving EZH2/H3K27me3 repression of TGFBR1 and thereby maintaining persistent TGFBR1 expression and TGF-β1 signaling that drives fibroblast activation in bleomycin-induced lung fibrosis [#0]. RCN3 also acts non-cell-autonomously: it is secreted from pulmonary epithelial cells via an N140-glycosylation–dependent ER–Golgi route, binds TGFβR1 on fibroblasts, and activates canonical Smad2/3 signaling, with intratracheal recombinant RCN3 alone sufficient to trigger pulmonary fibrosis and early RCN3 neutralization attenuating fibrotic remodeling [#6]. Within type II alveolar epithelial cells, RCN3 supports the injury-repair process and limits POVPC-induced ferroptosis by restraining lipid peroxidation and iron accumulation and preserving the GSH/GPX4/SLC7A11 axis [#2, #5]. Its EF-hand domains mediate distinct partner interactions—domains 3–4 engage GRP78 and domains 5–6 engage MMP10—coupling RCN3 to calcium handling, ER stress, and metalloproteinase secretion [#1, #4].\",\n  \"teleology\": [\n    {\n      \"year\": 2021,\n      \"claim\": \"Whether RCN3 in alveolar epithelium has a functional role in lung injury was unknown; cell-type-specific deletion established RCN3 in AECIIs as a participant in the injury-repair process.\",\n      \"evidence\": \"AECII-specific conditional Rcn3 knockout in a PPE-induced emphysema mouse model\",\n      \"pmids\": [\"33531801\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular mechanism by which RCN3 affects AECII injury was not defined\", \"No interaction partners identified in this model\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"It was unclear how RCN3 sustains fibroblast activation; the work showed RCN3 sequesters EZH2 in the cytoplasm to derepress TGFBR1, defining a TGF-β1/RCN3 feedback loop.\",\n      \"evidence\": \"BioID and co-IP interaction assays, epigenetic (H3K27me3) analysis, and fibroblast-selective Rcn3 knockdown in bleomycin lung fibrosis\",\n      \"pmids\": [\"37710230\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of EZH2 binding not resolved\", \"Whether cytoplasmic EZH2 sequestration occurs in other cell types untested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"The role of fibroblast RCN3 in the tumor immune microenvironment was unknown; an RCN3/PCSK6/STAT1 axis was proposed linking CAFs to macrophage polarization in gastric cancer.\",\n      \"evidence\": \"ChIP, expression assays, and macrophage M1/M2 polarization co-culture in gastric cancer\",\n      \"pmids\": [\"39206893\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single study without full mechanistic reconstitution\", \"Direct molecular link from RCN3 to PCSK6 not established\", \"Whether the axis operates in vivo not shown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Partner interactions and upstream regulation of RCN3 in cancer were uncharacterized; EF-hand 3–4 binding to GRP78 (with ER stress and PI3K-AKT-mTOR activation) and RELB-driven RCN3 induction by TGF-β were reported.\",\n      \"evidence\": \"Domain-mapping interaction studies and pathway Western blotting in colorectal cancer; multi-omics and co-culture/xenograft for the RELB/RCN3 feedback loop\",\n      \"pmids\": [\"40849588\", \"41186513\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"EF-hand 3–4/GRP78 interaction lacks explicit mutagenesis confirmation\", \"RELB→RCN3 regulation not fully elaborated experimentally\", \"Single lab per claim\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Whether RCN3 acts intracellularly versus as a secreted ligand, and how it controls epithelial fate, were open; studies showed RCN3 limits ferroptosis in AECIIs and that glycosylation-dependent secreted RCN3 binds TGFβR1 to activate Smad2/3 and drive fibrosis, while EF-hand 5–6 binds MMP10 to promote metastasis.\",\n      \"evidence\": \"AECII-specific KO with AAV6 rescue and ferroptosis readouts; secretomics with N140-glycosylation mutagenesis, recombinant RCN3 and neutralizing antibody in vivo; co-IP domain mapping with lung metastasis model\",\n      \"pmids\": [\"41881199\", \"41664195\", \"41966439\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which RCN3 restrains lipid peroxidation not molecularly defined\", \"Receptor-level details of secreted RCN3/TGFβR1 binding not structurally resolved\", \"EF-hand 5–6/MMP10 interaction from abstract without confirmed reconstitution\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How RCN3's distinct EF-hand-mediated partner interactions, its calcium-binding activity, and its dual intracellular/secreted functions are integrated within a single mechanistic framework remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model of RCN3 EF-hand engagement of distinct partners\", \"Quantitative role of calcium binding in each function not established\", \"Determinants directing RCN3 to intracellular vs secreted roles unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [1, 6]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 6]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"EZH2\", \"GRP78\", \"MMP10\", \"TGFBR1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":4,"faith_total":5,"faith_pct":80.0}}