{"gene":"BMP6","run_date":"2026-06-09T22:02:44","timeline":{"discoveries":[{"year":2009,"finding":"BMP6 is an endogenous ligand for hemojuvelin (HJV) that regulates hepcidin expression and iron metabolism in vivo. Physical interaction between HJV.Fc and BMP6 was demonstrated, and Bmp6-null mice develop hereditary hemochromatosis-like phenotype with reduced hepcidin expression and tissue iron overload. A neutralizing antibody to BMP6 inhibits hepcidin expression and increases serum iron in vivo.","method":"Co-immunoprecipitation/pulldown (HJV.Fc–BMP6 physical interaction), neutralizing antibody in vivo, Bmp6-/- mouse knockout with phenotypic readout (serum iron, tissue iron, hepcidin mRNA)","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal functional assays (HJV.Fc pulldown, neutralizing antibody, knockout mouse), replicated by independent lab same year (PMID:19252488)","pmids":["19252486"],"is_preprint":false},{"year":2009,"finding":"Targeted disruption of Bmp6 in mice causes massive iron overload in liver, exocrine pancreas, heart, and renal tubules with markedly reduced hepatic phospho-Smad1/5/8, failure of Smad nuclear translocation, and severely reduced hepcidin synthesis, demonstrating BMP6 is functionally non-redundant for activating the BMP/Smad signaling axis that drives hepcidin expression.","method":"Bmp6 knockout mouse; Western blot (phospho-Smad1/5/8); immunohistochemistry (Smad nuclear translocation); quantitative RT-PCR (hepcidin mRNA); tissue iron measurements","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean knockout with multiple orthogonal readouts, independently replicated by companion paper (PMID:19252486)","pmids":["19252488"],"is_preprint":false},{"year":2007,"finding":"Crystal structure of BMP-6 was solved, and surface plasmon resonance showed BMP-6 binds type I and type II serine/threonine kinase receptors. BMP-6 signaling capability served as a reference comparator demonstrating its receptor binding mediates Smad-based signaling in whole-cell luciferase assays.","method":"X-ray crystallography; surface plasmon resonance (BIAcore); Smad-based whole-cell luciferase assay","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure plus biophysical binding assay and cell-based functional validation in single study","pmids":["17924656"],"is_preprint":false},{"year":2011,"finding":"Liver iron content independently and positively correlates with hepatic Bmp6 mRNA expression and Smad1/5/8 signaling, whereas transferrin saturation activates downstream Smad1/5/8 signaling without inducing Bmp6 mRNA, indicating that circulating and tissue iron differentially regulate the BMP6-Smad pathway to control hepcidin. The Erk1/2 MAPK pathway is not activated by iron in vivo.","method":"In vivo acute and chronic iron administration in mice; quantitative RT-PCR (Bmp6 mRNA, Smad7, hepcidin); Western blot (phospho-Smad1/5/8, phospho-Erk1/2); correlation analysis of transferrin saturation vs. liver iron content","journal":"Hepatology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal in vivo methods (gene expression, protein phosphorylation) with careful separation of iron pools, single lab","pmids":["21488083"],"is_preprint":false},{"year":2018,"finding":"Erythroferrone (ERFE) suppresses hepcidin by acting as a ligand trap that preferentially binds BMP5, BMP6, and BMP7, inhibiting hepatic BMP/Smad signaling. Cell-free HTRF competition assays showed BMP5, BMP6, and BMP7 compete with anti-ERFE for binding to ERFE, and recombinant ERFE specifically abrogated hepcidin induction by BMP5, BMP6, and BMP7 but not BMP2, BMP4, or BMP9.","method":"Cell-free homogeneous time-resolved fluorescence (HTRF) competition binding assay; in vitro SMAD1/5/8 phosphorylation assay; in vivo EPO/ERFE mouse experiments; neutralizing anti-ERFE antibody rescue","journal":"Blood","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cell-free binding assay plus in vitro signaling plus in vivo genetic rescue, multiple orthogonal methods in single study","pmids":["30097509"],"is_preprint":false},{"year":2019,"finding":"Nrf2 is activated by iron-induced mitochondria-derived pro-oxidants in liver sinusoidal endothelial cells and drives Bmp6 expression, which in turn increases hepcidin synthesis in neighboring hepatocytes. Nrf2 knockout mice show impaired Bmp6-hepcidin response to iron. Pharmacological Nrf2 activation stimulates the Bmp6-hepcidin axis.","method":"Nrf2 knockout mouse; in vivo iron loading (oral and parenteral); quantitative RT-PCR; pharmacological Nrf2 activation; cell fractionation/oxidative stress measurements","journal":"Nature metabolism","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic knockout with multiple iron loading conditions and pharmacological rescue, single lab","pmids":["31276102"],"is_preprint":false},{"year":2023,"finding":"Liver sinusoidal endothelial cells (LSECs) sense non-transferrin-bound iron (NTBI) and induce Bmp6 expression via Nrf2-mediated oxidative stress, which then acts paracrinally on hepatocytes to induce hepcidin. Endothelial-specific Tfr1 ablation (TfrcTek-Cre mice) showed Tfr1 contributes to iron sensing by LSECs, particularly under low-iron conditions, and single-cell transcriptomics confirmed LSECs as the predominant Bmp6-expressing liver cell type.","method":"Conditional endothelial-specific Tfr1 knockout mouse (TfrcTek-Cre); single-cell transcriptomics; quantitative RT-PCR (Bmp6, Hamp); serum NTBI measurement; correlation analysis","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Moderate — conditional genetic KO plus single-cell transcriptomics plus in vivo iron challenges, multiple orthogonal methods","pmids":["36351237"],"is_preprint":false},{"year":2017,"finding":"BMP2, expressed predominantly in liver endothelial cells, plays a non-redundant role in hepcidin regulation in addition to BMP6. Endothelial-conditional Bmp2 knockout mice have hepcidin deficiency and iron overload. However, BMP6 is absolutely required for hepcidin induction by serum iron (acute oral iron gavage fails to induce hepcidin in Bmp6-/- mice), whereas BMP2 has at least a partially redundant role.","method":"Conditional endothelial Bmp2 knockout mouse; Bmp6-/- mouse; acute oral iron gavage vs. chronic dietary iron loading; neutralizing BMP2/4 antibody; quantitative RT-PCR (hepcidin, Smad targets)","journal":"American journal of hematology","confidence":"High","confidence_rationale":"Tier 2 / Strong — dual conditional KO approaches plus antibody blockade plus dietary challenge, replicated in two companion studies (PMIDs 28815688 and 30478858)","pmids":["28815688","30478858"],"is_preprint":false},{"year":2011,"finding":"BMP6 stimulates TMPRSS6 (matriptase-2) expression at mRNA and protein levels and increases matriptase-2 activity in vitro. Inhibitor of DNA binding 1 (Id1) is identified as the key BMP-Smad pathway element mediating TMPRSS6 regulation by BMP6. In vivo, Tmprss6 mRNA is induced by chronic iron treatment or BMP6 injection and is blocked by anti-BMP6 neutralizing antibody, establishing a negative feedback loop.","method":"In vitro BMP6 treatment (RT-PCR, Western blot, matriptase-2 activity assay); siRNA knockdown of Id1; in vivo BMP6 injection and neutralizing antibody; quantitative RT-PCR","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Moderate — in vitro mechanistic dissection (Id1 siRNA) plus in vivo rescue experiment, multiple orthogonal methods single lab","pmids":["21622652"],"is_preprint":false},{"year":2018,"finding":"Hemojuvelin (HJV) is required for BMP6/Smad signaling to hepcidin; Hjv-/- primary hepatocytes show severely impaired BMP6/Smad signaling (but not IL-6/Stat or BMP2/Smad signaling), abolishing synergism with IL-6/Stat during inflammation. This establishes that Hjv selectively facilitates BMP6 signal transduction.","method":"Hjv-/- mouse; primary murine hepatocyte culture; BMP6 and BMP2 stimulation; Western blot (phospho-Smad, phospho-Stat3); hepcidin mRNA quantification; LPS, FSL1, and E. coli infection in vivo","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Moderate — primary cell mechanistic dissection plus multiple in vivo infection models, single lab with multiple orthogonal methods","pmids":["30213871"],"is_preprint":false},{"year":2017,"finding":"Deletion of Bmp6 in Hjv-/- mice further represses Smad signaling and hepcidin expression in the liver, indicating BMP6 can signal to hepcidin partially independently of HJV in female mice. This genetic epistasis also demonstrated that baseline BMP/Smad signaling determines the level of hepcidin reached after LPS challenge, consistent with synergy between IL-6/Stat3 and BMP/Smad pathways.","method":"Bmp6-/-;Hjv-/- double-knockout mouse (genetic epistasis); quantitative RT-PCR; Western blot (phospho-Smad); LPS challenge; tissue iron measurement","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — rigorous double-KO epistasis with multiple readouts, two independent KO lines","pmids":["29021231"],"is_preprint":false},{"year":2009,"finding":"BMP6/HFE epistasis: Hfe-deficient mice with iron overload show increased Bmp6 mRNA and protein but do NOT show increased phospho-Smad1/5/8 or Id1 (BMP signaling indicators), unlike mice with secondary iron overload. This indicates HFE facilitates signal transduction downstream of the BMP6 ligand, rather than regulating Bmp6 expression.","method":"Hfe-/- mouse; Western blot (phospho-Smad1/5/8); quantitative RT-PCR (Id1, hepcidin, Bmp6); comparison to secondary iron-overload mice","journal":"Blood","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic model with multiple signaling readouts, but epistatic placement relies on negative finding (no pSmad increase) in single lab","pmids":["19622835"],"is_preprint":false},{"year":2013,"finding":"Iron-induced Bmp6 mRNA expression is localized to non-parenchymal liver cells (primarily hepatic stellate cells), not hepatocytes. Iron-saturated transferrin induces Bmp6 mRNA in isolated hepatic stellate cells but not hepatocytes, suggesting non-parenchymal cells are the primary source of BMP6 for paracrine hepcidin regulation.","method":"In vivo dietary iron loading; cell fractionation into hepatocytes and non-parenchymal cells; quantitative RT-PCR; isolated hepatic stellate cell treatment with iron-saturated transferrin","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cell-type fractionation plus in vitro cell treatment, single lab, no full functional rescue","pmids":["23565256"],"is_preprint":false},{"year":2010,"finding":"Iron overload induces Bmp6 mRNA and protein expression in the liver but NOT in the duodenum across three mouse strains, establishing liver as the iron-responsive source of BMP6 for hepcidin regulation.","method":"Iron-enriched diet and Hfe-/- mouse models; quantitative RT-PCR; immunohistochemistry; Western blot for Bmp6 protein in liver and duodenum","journal":"Haematologica","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — three mouse strains and two iron-overload models, single lab, protein and mRNA orthogonal methods","pmids":["20952515"],"is_preprint":false},{"year":1994,"finding":"Recombinant VGR-1/BMP-6 protein is secreted and proteolytically processed to yield the mature molecule. Continuous secretion of BMP-6 from tumor cells in vivo induces surrounding host mesenchymal cells to undergo endochondral bone formation, establishing BMP-6 as a secreted bone-inductive morphogen acting on mesenchymal cells.","method":"CHO cell overexpression of recombinant murine BMP-6; Western blot (secretion and processing); subcutaneous injection in athymic nude mice; histological analysis of cartilage and bone formation","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro secretion/processing confirmed by Western blot plus in vivo bone induction assay, single lab","pmids":["8089189"],"is_preprint":false},{"year":1995,"finding":"VGR-1/BMP-6 overexpression in pluripotent mesenchymal ROB-C26 cells induces osteoblastic differentiation in vitro and the extracellular matrix from these cells induces ectopic bone formation in vivo. This osteoinductive effect requires BMP-6 presentation within the matrix (not soluble BMP-6) and is blocked by neutralizing BMP-6 antibody but not by anti-TGF-β1 antibody.","method":"Stable transfection of ROB-C26 cells; neutralizing antibody blockade (anti-BMP6 vs. anti-TGF-β1); in vivo ectopic bone formation assay; MyoD overexpression competition experiment","journal":"Cell growth & differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain-of-function plus neutralizing antibody specificity control plus in vivo assay, single lab","pmids":["7547504"],"is_preprint":false},{"year":2019,"finding":"Deubiquitinase PSMD14 stabilizes the ALK2 type I BMP receptor by removing K48-linked ubiquitin chains added by Smurf1 E3 ligase, thereby preventing proteasomal degradation of ALK2 and sustaining BMP6 signaling pathway initiation.","method":"Human DUB siRNA library screen; immunoprecipitation; ubiquitination assay; immunoblot; in vitro and in vivo colorectal cancer xenograft model","journal":"EBioMedicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical ubiquitination assay plus in vivo xenograft, single lab","pmids":["31685442"],"is_preprint":false},{"year":2007,"finding":"BMP6-induced myosin-X (Myo10) localizes in filopodia and is required for BMP-dependent filopodial assembly, endothelial cell alignment and directional migration toward BMP6 gradients, and BMP6-dependent Smad activation. BMP6 receptor ALK6 co-localizes with Myo10 in filopodia in a BMP6-dependent manner and both exhibit intrafilopodial motility.","method":"Microarray (Myo10 as BMP6 target); siRNA knockdown of Myo10; co-localization imaging (confocal microscopy); directional migration assay; Western blot (Smad activation)","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA loss-of-function plus co-localization plus functional migration and signaling readouts, single lab","pmids":["18158328"],"is_preprint":false},{"year":2012,"finding":"Estrogen regulates hepcidin expression via GPR30 (7-transmembrane estrogen receptor)-dependent upregulation of BMP6 in hepatocytes. GPR30 siRNA abolishes E2-induced hepcidin and BMP6 expression; GPR30 agonist G1 recapitulates E2 effects on BMP6 and hepcidin in vivo in ovariectomized mice.","method":"Ovariectomized mouse model; HepG2 cell culture; GPR30 siRNA knockdown; GPR30 agonist G1 treatment; quantitative RT-PCR; in vivo estrogen/G1 supplementation","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA plus pharmacological agonist plus in vivo mouse model, single lab","pmids":["22792339"],"is_preprint":false},{"year":2015,"finding":"Heterozygous missense mutations in the BMP6 pro-peptide domain (p.Pro95Ser, p.Leu96Pro, p.Gln113Glu) cause defective BMP6 secretion, reduced SMAD1/5/8 signaling, and loss of hepcidin production in transfected cells, establishing that the BMP6 pro-peptide is essential for proper processing, secretion, and downstream signaling.","method":"Cell transfection with mutant vs. wild-type BMP6; immunofluorescence (secretion); quantitative RT-PCR (hepcidin); immunoblot (phospho-Smad1/5/8); serum hepcidin by LC-MS/MS; liver biopsy immunohistochemistry","journal":"Gastroenterology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional mutagenesis in cells with multiple orthogonal readouts, supported by patient phenotype, single lab","pmids":["26582087"],"is_preprint":false},{"year":2020,"finding":"BMP6 binding to heparin and heparan sulfate is mediated by N-terminal basic residues (R5, R6, R7) and C-terminal basic residues (K126, K127, R129). Mutation of both sites abolished heparin-binding activity of recombinant monomeric BMP6 and binding to membrane heparan sulfate on CHO-K1 cells.","method":"Solid-phase heparin binding assay; microcantilever assay; CHO-K1 cell membrane heparan sulfate binding; site-directed mutagenesis; molecular dynamics simulation","journal":"Biochimica et biophysica acta. General subjects","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — mutagenesis with binding assay plus molecular dynamics, single lab, multiple methods","pmids":["33232799"],"is_preprint":false},{"year":1999,"finding":"BMP-6 functions as an autocrine stimulator of chondrocyte maturation. BMP-6 mRNA expression precedes type X collagen induction during chondrocyte maturation. PTHrP directly inhibits BMP-6 expression, blocking maturation; exogenous BMP-6 overcomes PTHrP inhibition and stimulates Indian hedgehog mRNA, placing BMP-6 upstream of Ihh in the chondrocyte maturation pathway.","method":"Primary embryonic chondrocyte monolayer culture; exogenous BMP-6 treatment; PTHrP inhibition and rescue; RT-PCR (BMP-6, type X collagen, Ihh mRNA); alkaline phosphatase activity assay","journal":"Journal of bone and mineral research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain-of-function with exogenous BMP-6 plus PTHrP inhibition/rescue, multiple functional readouts, single lab","pmids":["10234567"],"is_preprint":false},{"year":2001,"finding":"Bmp6 and Bmp7 are required together for cardiac cushion formation and septation during mouse embryonic development. Bmp6;Bmp7 double mutants show a marked delay in outflow tract endocardial cushion formation, defects in valve morphogenesis and chamber septation, and lethality between 10.5–15.5 dpc, whereas single mutants have no cardiac defect.","method":"Bmp6;Bmp7 double-mutant mouse (genetic epistasis); in situ hybridization; histological analysis","journal":"Developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — rigorous double-mutant genetic approach with histological phenotyping, single lab","pmids":["11437450"],"is_preprint":false},{"year":2005,"finding":"Endogenous BMP2 and BMP6 cooperatively regulate bone formation in vivo. Bmp2+/-;Bmp6-/- compound-deficient mice show reduced trabecular bone volume with suppressed bone formation and impaired endochondral (but not intramembranous) bone formation after fracture, whereas single-deficient animals show no such defect.","method":"Bmp2+/-;Bmp6-/- compound knockout mouse; micro-CT/histomorphometry; fracture healing model; bone marrow cell culture osteogenic differentiation assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — compound knockout genetic epistasis with multiple bone phenotype readouts and in vitro rescue, single lab","pmids":["16109715"],"is_preprint":false},{"year":1998,"finding":"Bmp6-null mice are viable and fertile with only a transient delay in sternal ossification (not other skeletal elements), and Bmp2 and Bmp6 are co-expressed in hypertrophic cartilage, suggesting functional compensation. Sternal defects are exacerbated in Bmp5/Bmp6 double mutants.","method":"Gene-targeted Bmp6 knockout mouse; skeletal analysis (histology, in situ hybridization); Bmp5;Bmp6 double mutant analysis","journal":"Developmental genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean knockout with detailed skeletal phenotyping and double-mutant epistasis, single lab","pmids":["9664685"],"is_preprint":false},{"year":2009,"finding":"BMP-6 inhibits miR-21 transcription in MDA-MB-231 breast cancer cells through repression of deltaEF1 and AP-1 transcription factors. BMP-6 reduces binding of deltaEF1 to the E2-box on the miR-21 promoter (miPPR-21) and decreases c-Fos/c-Jun expression, thereby inhibiting MDA-MB-231 cell invasion via upregulation of PDCD4.","method":"Luciferase reporter assay (miPPR-21 constructs); ChIP assay (deltaEF1 binding to miPPR-21); site-directed mutation; RT-PCR; siRNA knockdown; invasion assay","journal":"Cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP plus luciferase reporter plus mutagenesis plus functional invasion readout, single lab","pmids":["19308091"],"is_preprint":false},{"year":2007,"finding":"BMP-6 promotes E-cadherin expression in breast cancer cells by inhibiting deltaEF1 transcription and physically dislodging deltaEF1 from the E-cadherin proximal promoter, thereby de-repressing E-cadherin transcription. RNAi knockdown of deltaEF1 abolishes E-cadherin transactivation by BMP-6.","method":"Luciferase reporter assay (E-cadherin promoter); ChIP assay (deltaEF1 binding); RNAi knockdown of deltaEF1; Western blot; quantitative RT-PCR","journal":"BMC cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP plus luciferase plus RNAi rescue, single lab","pmids":["17997862"],"is_preprint":false},{"year":2003,"finding":"BMP-6 inhibits proliferation of renal cell carcinoma (RCC) cells through BMP receptor type II. RCC cell lines lacking BMP-RII are resistant to BMP-6-induced growth inhibition. Transfection of BMP-RII restores BMP-6 sensitivity, and constitutively active BMP-RIA or -RIB restores BMP-6 reporter activity in resistant cells.","method":"Adenoviral transduction (constitutively active BMP-RIA, RIB); BMP-RII transfection; BMP-6-responsive luciferase reporter; cell proliferation assay; Northern blot/RT-PCR","journal":"Clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — receptor reconstitution in resistant cells plus reporter assay, single lab","pmids":["14676131"],"is_preprint":false},{"year":2008,"finding":"BMP-6 induction in prostate cancer PC3M cells leads to nuclear translocation of SMADs, activation of Id-1, increased migration and invasion, upregulation of MMP-1 and MMP-9 mRNA, and direct transcriptional activation of MMP-1 and MMP-9 promoters. BMP-6 also activates MAPK phosphorylation.","method":"Doxycycline-inducible BMP-6 expression system; Western blot (SMAD translocation, MAPK phosphorylation); luciferase reporter (MMP-1, MMP-9 promoters); migration/invasion assay; RT-PCR","journal":"The Journal of pathology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — inducible expression system with multiple downstream readouts including promoter luciferase, single lab","pmids":["18072288"],"is_preprint":false},{"year":2018,"finding":"BMP-6 inhibits FSH-induced progesterone production in rat granulosa cells via Smad1/5/8 signaling and Id-1 transcription. Melatonin impairs this BMP-6/Smad signaling by upregulating inhibitory Smad6 (but not Smad7), thereby counteracting BMP-6-mediated inhibition of steroidogenesis.","method":"Primary rat granulosa cell culture; BMP-6 + melatonin co-treatment; Western blot (phospho-Smad1/5/8); RT-PCR (Id-1, Smad6/7, MT1/MT2); progesterone ELISA; cAMP assay","journal":"The Journal of steroid biochemistry and molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — primary cell culture with multiple signaling readouts, single lab","pmids":["24751708"],"is_preprint":false},{"year":2018,"finding":"BMP6 downregulates GDNF expression in human granulosa-lutein cells via ALK2 and ALK3 type I receptors, activating both SMAD1/5 and ERK1/2 (but not AKT or p38) signaling. Combined knockdown of SMAD4 and ERK1/2 inhibition completely reverses BMP6-induced GDNF downregulation, demonstrating both pathways are required.","method":"Immortalized SVOG cells and primary human granulosa-lutein cells; kinase receptor inhibitor plus siRNA dual knockdown of ALK2/ALK3, SMAD1/5/4; ERK1/2 inhibitor; Western blot (phospho-SMAD1/5/8, phospho-ERK1/2, phospho-AKT, phospho-p38); quantitative RT-PCR (GDNF)","journal":"Endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — dual inhibition approach plus multiple signaling readouts, single lab","pmids":["29750278"],"is_preprint":false},{"year":2019,"finding":"BMP6 overexpression in vivo (adenovirus injection) inhibits the telogen-to-anagen transition of hair follicles. BMP6 signaling and Wnt10b signaling mutually inhibit each other in hair follicle skin, and hair follicle stem cell activation is competitively regulated by BMP6 (inhibitor) and Wnt10b (activator).","method":"Adenovirus-mediated BMP6 overexpression in mouse skin; in situ hybridization; immunofluorescence; H&E staining; BrdU tracing","journal":"Cell communication and signaling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo overexpression with histological and molecular readouts, single lab","pmids":["30791955"],"is_preprint":false},{"year":2021,"finding":"The SIN3A-HDAC1/2 repressor complex silences BMP6 expression in melanoma cells, causing increased metastatic dissemination via suppression of BMP6-activated SMAD5 signaling. A downstream effector FAM83G/PAWS1 contributes to metastatic progression through actin-dependent cytoskeletal dynamics. Pharmacological HDAC1/2 inhibition restores BMP6 expression and reduces circulating melanoma cells.","method":"Mouse melanoma metastasis model; pharmacological SIN3A-HDAC1/2 complex inhibition; Western blot (phospho-SMAD5); ChIP (SIN3A-HDAC1/2 at BMP6 locus); siRNA knockdown of FAM83G/PAWS1; cell migration assay; in vivo circulating tumor cell enumeration","journal":"Molecular cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP plus pharmacological inhibition plus in vivo model plus downstream effector identification, single lab","pmids":["34610961"],"is_preprint":false},{"year":2022,"finding":"In Sjögren's syndrome, HSP70 released by lysosomal exocytosis (triggered by LAMP3 overexpression) acts as an endogenous TLR4 ligand that stimulates BMP6 expression in monocytic cells. In vivo LAMP3 overexpression in mice induces BMP6 expression and an SS-like phenotype. RNA ISH identified monocytic lineage cells as the cellular source of BMP6 in SS salivary glands.","method":"RNA ISH on salivary gland sections; in vitro TLR4 signaling assays; single-cell transcriptomics of human PBMCs; siRNA knockdown; LAMP3 overexpression in mice; RT-PCR correlation analysis","journal":"The Journal of clinical investigation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro mechanism plus in vivo mouse model plus single-cell transcriptomics, single lab","pmids":["35113815"],"is_preprint":false},{"year":2022,"finding":"The RNF4 ubiquitin ligase acts upstream of BMP6 and its co-receptor RGMb (Dragon) in a signaling axis required for osteogenic differentiation of human bone marrow mesenchymal stem cells. Knockdown of either RGMb or BMP6 halts osteogenic differentiation, and combined co-addition of purified RGMb and BMP6 proteins to RNF4-deficient cells fully restores differentiation.","method":"RNF4 siRNA in hBMSCs; conditioned media rescue; BMP6 and RGMb knockdown; protein co-addition rescue; transcriptional analysis; xenograft osteosarcoma model","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function plus protein rescue with two factors, single lab","pmids":["36153321"],"is_preprint":false},{"year":2001,"finding":"BMP-6 (and Ca2+) induces terminal differentiation of epidermal keratinocytes with a specific switch in E2F expression from E2F-1/2/3 (undifferentiated) to E2F-5 (differentiated), accompanied by formation of nuclear E2F5·p130·HDAC1 complexes. E2F-5 overexpression inhibits DNA synthesis in an HDAC-dependent manner, linking BMP-6-driven differentiation to permanent cell cycle exit.","method":"Primary murine keratinocyte culture; BMP-6 treatment; co-immunoprecipitation (E2F5·p130·HDAC1 complex); E2F-5 overexpression; HDAC inhibitor; DNA synthesis assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus gain-of-function plus pharmacological rescue, single lab","pmids":["11319226"],"is_preprint":false},{"year":2018,"finding":"HIF-1α directly binds to the hypoxia-response element (HRE) in the BMP6 promoter to repress BMP6 transcription in keratinocytes. HIF-1α overexpression reduces BMP6 expression, promotes proliferation and inhibits terminal differentiation, effects that are mediated via BMP6 suppression.","method":"HIF-1α overexpression in primary human keratinocytes; ChIP assay (HIF-1α binding to HRE in BMP6 promoter); quantitative RT-PCR; cell proliferation assay; differentiation marker expression","journal":"Experimental dermatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP demonstrating direct HIF-1α/BMP6 promoter binding plus functional overexpression readouts, single lab","pmids":["30230035"],"is_preprint":false},{"year":2011,"finding":"Intraocular BMP6 protein injection in mice upregulates retinal hepcidin expression and alters retinal labile iron levels. Bmp6-/- mice develop age-dependent retinal iron accumulation and degeneration, establishing BMP6 as a regulator of retinal iron homeostasis through local hepcidin induction.","method":"Intraocular BMP6 injection in mice; quantitative RT-PCR (hepcidin); calcein-AM labile iron assay; Bmp6-/- mouse with age-dependent retinal phenotyping; cultured RPE cell BMP6 modulation","journal":"The American journal of pathology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo BMP6 injection plus knockout model with mechanistic pathway readout, single lab","pmids":["21703414"],"is_preprint":false},{"year":2015,"finding":"A 190 bp enhancer ~2.5 kb 5' of the Bmp6 gene drives expression in developing teeth and fins; a predicted Smad3 binding site in this enhancer is required for its activity, and pharmacological inhibition of TGFβ signaling abolishes enhancer activity and severely reduces endogenous Bmp6 expression. TALEN-mediated disruption of the enhancer in vivo dramatically reduces Bmp6 expression, identifying a required cis-regulatory element.","method":"Transgenic reporter assays in sticklebacks and zebrafish; site-directed mutagenesis of Smad3 binding site; pharmacological TGFβ inhibition; TALEN enhancer disruption in vivo; quantitative RT-PCR","journal":"Developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis plus pharmacological plus in vivo TALEN disruption, single lab in fish ortholog model","pmids":["25732776"],"is_preprint":false},{"year":2017,"finding":"BMP6 regulates human Sertoli cell proliferation and apoptosis through Smad2/3 phosphorylation and cyclin D1 upregulation, as well as activation of DACH1 and TFAP2A. BMP6 siRNA inhibits Sertoli cell division and increases apoptosis; the signaling operates through a non-canonical Smad2/3 pathway.","method":"Primary human Sertoli cell culture; BMP6 treatment and siRNA knockdown; Western blot (phospho-Smad2/3, cyclin D1, DACH1, TFAP2A); CCK-8 and EDU proliferation assays; Annexin V/PI apoptosis assay; ELISA (SCF)","journal":"Scientific reports","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, single cell type, pathway identification by Western blot without mutagenesis confirmation","pmids":["28387750"],"is_preprint":false},{"year":2020,"finding":"BMP6 modulates VEGF signaling by regulating VEGFR2 expression and acts via the Hippo signaling effector TAZ in endothelial cells. BMP6 induces angiogenesis in a matrigel plug assay in vivo, making BMP6 the first BMP family member demonstrated to directly regulate Hippo signaling and neovessel formation.","method":"C57/Bl6 VEGF gene transfer mouse model; porcine myocardial ischemia model; siRNA knockdown; VEGFR2 expression assay; TAZ reporter; matrigel plug assay in nude mice","journal":"Angiogenesis","confidence":"Low","confidence_rationale":"Tier 3 / Weak — in vivo angiogenesis assay plus in vitro siRNA, but TAZ pathway placement based on limited mechanistic follow-up, single lab","pmids":["33021694"],"is_preprint":false},{"year":2018,"finding":"BMP6 improves glycemia in ob/ob mice and inhibits hepatic gluconeogenesis in rat H4IIE hepatoma cells by downregulating PepCK expression via Smad signaling, antagonizing cAMP-driven glycogenolytic effects. BMP6 acts on pancreatic and liver cells through Alk3, Alk6, and ActRIIA receptors (identified using receptor Fc fusion proteins in luciferase reporter and glucose output assays).","method":"ob/ob mouse BMP6 treatment; oral glucose tolerance test; chromatin immunoprecipitation (PepCK); glucose output assay in H4IIE cells; receptor Fc fusion protein specificity assay; luciferase reporter; quantitative RT-PCR","journal":"Acta diabetologica","confidence":"Low","confidence_rationale":"Tier 3 / Weak — in vivo mouse study plus in vitro mechanistic assays but receptor identification relies on Fc fusion protein competition without mutagenesis, single lab","pmids":["30539233"],"is_preprint":false}],"current_model":"BMP6 is a secreted TGF-β superfamily ligand that functions as the principal endogenous activator of the BMP/Smad1/5/8 signaling pathway in liver sinusoidal endothelial cells and hepatocytes to induce hepcidin expression and maintain systemic iron homeostasis; it binds the co-receptor hemojuvelin (HJV) and acts through ALK2/ALK3 type I receptors, its expression is transcriptionally induced in LSECs by non-transferrin-bound iron via Nrf2-driven oxidative stress, and it additionally regulates mesenchymal differentiation (osteoblastogenesis, chondrogenesis), keratinocyte terminal differentiation, endothelial cell migration via myosin-X/filopodia, and ovarian steroidogenesis via Smad-dependent pathways."},"narrative":{"mechanistic_narrative":"BMP6 is a secreted, proteolytically processed TGF-β superfamily morphogen that serves as the principal iron-responsive ligand of the hepatic BMP/Smad1/5/8 axis controlling systemic iron homeostasis [PMID:19252486, PMID:19252488, PMID:8089189]. Genetic ablation of Bmp6 in mice produces a hereditary hemochromatosis-like phenotype with multi-organ iron overload, loss of hepatic phospho-Smad1/5/8 and Smad nuclear translocation, and severely reduced hepcidin synthesis, establishing BMP6 as functionally non-redundant for activating this pathway [PMID:19252486, PMID:19252488]. BMP6 physically binds the co-receptor hemojuvelin (HJV), which selectively facilitates BMP6 signal transduction to hepcidin, and signals through type I/type II serine-threonine kinase receptors to drive Smad-dependent transcription [PMID:19252486, PMID:17924656, PMID:30213871]. Iron loading induces Bmp6 expression in liver non-parenchymal cells—predominantly sinusoidal endothelial cells—which sense non-transferrin-bound iron through Nrf2-mediated oxidative stress and act paracrinally on hepatocytes, with circulating and tissue iron pools differentially engaging the pathway [PMID:21488083, PMID:31276102, PMID:36351237, PMID:23565256, PMID:20952515]. The pathway is embedded in feedback and regulatory loops: BMP6 induces TMPRSS6/matriptase-2 via Id1 as a negative-feedback brake on hepcidin, while erythroferrone suppresses signaling by sequestering BMP6 as a ligand trap [PMID:30097509, PMID:21622652]. Beyond iron, BMP6 acts as an osteoinductive and chondrogenic morphogen on mesenchymal cells, cooperating with BMP2/BMP5/BMP7 in bone formation and cardiac cushion development [PMID:8089189, PMID:7547504, PMID:11437450, PMID:16109715, PMID:9664685], drives keratinocyte terminal differentiation through an E2F-5/p130/HDAC1 switch [PMID:11319226], directs endothelial filopodial assembly and directional migration via myosin-X [PMID:18158328], and modulates Smad/Id1-dependent programs in ovarian granulosa-cell steroidogenesis and in multiple cancers [PMID:19308091, PMID:18072288, PMID:24751708, PMID:29750278]. Heterozygous missense mutations in the BMP6 pro-peptide domain impair secretion and downstream Smad signaling, causing loss of hepcidin production and iron overload in patients [PMID:26582087].","teleology":[{"year":2007,"claim":"Established the structural and biophysical basis for BMP6 signaling by showing it directly engages type I and type II serine-threonine kinase receptors to drive Smad activation.","evidence":"X-ray crystallography, surface plasmon resonance, and Smad-based luciferase reporter","pmids":["17924656"],"confidence":"High","gaps":["Did not identify the iron-specific receptor configuration in vivo","No co-receptor (HJV) interaction defined at this stage"]},{"year":2009,"claim":"Answered which BMP ligand controls iron homeostasis by identifying BMP6 as the non-redundant endogenous activator of hepatic BMP/Smad signaling that drives hepcidin, with HJV as its binding co-receptor.","evidence":"HJV.Fc–BMP6 pulldown, neutralizing antibody in vivo, and Bmp6-/- mice with iron/hepcidin readouts (two companion papers)","pmids":["19252486","19252488"],"confidence":"High","gaps":["Cellular source of iron-induced BMP6 not resolved","Mechanism of iron-to-Bmp6-transcription coupling unknown"]},{"year":2009,"claim":"Distinguished ligand regulation from signal transduction by showing HFE acts downstream of BMP6 (facilitating signaling), since Hfe-/- mice raise Bmp6 but fail to raise phospho-Smad/Id1.","evidence":"Hfe-/- mouse, phospho-Smad and Id1 readouts versus secondary iron overload","pmids":["19622835"],"confidence":"Medium","gaps":["Epistatic placement rests on a negative pSmad finding","Molecular interaction of HFE with the receptor complex undefined"]},{"year":2010,"claim":"Localized the iron-responsive BMP6 source by demonstrating iron induces Bmp6 in liver but not duodenum across strains.","evidence":"Dietary iron and Hfe-/- models, qRT-PCR, IHC, Western blot","pmids":["20952515"],"confidence":"Medium","gaps":["Specific liver cell type not yet pinpointed","Iron-sensing transducer to Bmp6 promoter unknown"]},{"year":2011,"claim":"Separated circulating from tissue iron signals, showing liver iron correlates with Bmp6 mRNA while transferrin saturation activates Smad without inducing Bmp6, refining how two iron pools tune the pathway.","evidence":"Acute/chronic iron loading in mice, qRT-PCR, phospho-Smad/Erk Western blot, correlation analysis","pmids":["21488083"],"confidence":"High","gaps":["Molecular sensors for the two iron pools not identified","Erk pathway role excluded but alternative non-Smad arms untested"]},{"year":2011,"claim":"Defined a negative-feedback brake by showing BMP6 induces TMPRSS6/matriptase-2 through Id1, limiting hepcidin output.","evidence":"In vitro BMP6 treatment, Id1 siRNA, in vivo BMP6 injection and anti-BMP6 antibody","pmids":["21622652"],"confidence":"High","gaps":["Quantitative contribution of this loop to systemic iron set-point unclear"]},{"year":2013,"claim":"Refined the cellular source to liver non-parenchymal cells (hepatic stellate cells) responding to transferrin-bound iron rather than hepatocytes.","evidence":"Cell fractionation, isolated stellate cell iron treatment, qRT-PCR","pmids":["23565256"],"confidence":"Medium","gaps":["No functional rescue confirming stellate-cell BMP6 drives hepcidin","Conflicts with later endothelial-source data — cell-type attribution unsettled"]},{"year":2017,"claim":"Established cooperative but distinct roles for endothelial BMP2 versus BMP6, with BMP6 absolutely required for the acute serum-iron response.","evidence":"Endothelial-conditional Bmp2 KO, Bmp6-/-, acute gavage versus dietary loading, anti-BMP2/4 antibody (companion studies)","pmids":["28815688","30478858"],"confidence":"High","gaps":["How BMP2 and BMP6 form functional heterodimers/complexes in vivo not defined"]},{"year":2017,"claim":"Used genetic epistasis to show BMP6 can signal to hepcidin partially independently of HJV and that baseline BMP/Smad tone sets the inflammatory hepcidin ceiling.","evidence":"Bmp6-/-;Hjv-/- double-knockout mice, phospho-Smad, qRT-PCR, LPS challenge","pmids":["29021231"],"confidence":"High","gaps":["HJV-independent receptor route not molecularly identified","Sex-specific differences mechanistically unexplained"]},{"year":2018,"claim":"Identified erythroferrone as a ligand trap that preferentially sequesters BMP6 (and BMP5/7), explaining erythroid suppression of hepcidin.","evidence":"Cell-free HTRF competition, in vitro SMAD1/5/8 assay, EPO/ERFE mouse experiments, anti-ERFE rescue","pmids":["30097509"],"confidence":"High","gaps":["Stoichiometry and structural basis of ERFE–BMP6 trapping not resolved"]},{"year":2018,"claim":"Showed HJV selectively facilitates BMP6 (not BMP2 or IL-6/Stat) signaling and is required for BMP6/IL-6 synergy during inflammation.","evidence":"Hjv-/- primary hepatocytes, BMP6/BMP2 stimulation, phospho-Smad/Stat3 Western blot, in vivo infection models","pmids":["30213871"],"confidence":"High","gaps":["Structural basis of HJV's BMP6 selectivity undefined"]},{"year":2019,"claim":"Resolved the iron-sensing-to-transcription link by showing Nrf2, activated by iron-induced mitochondrial pro-oxidants in LSECs, drives Bmp6 expression.","evidence":"Nrf2-/- mice, iron loading, pharmacological Nrf2 activation, oxidative stress measurements","pmids":["31276102"],"confidence":"High","gaps":["Direct Nrf2 binding to the Bmp6 promoter not demonstrated here","Other transcriptional inputs not excluded"]},{"year":2023,"claim":"Pinpointed LSECs as the dominant Bmp6-expressing cell and showed they sense NTBI via Nrf2 with a Tfr1 contribution under low iron, acting paracrinally on hepatocytes.","evidence":"Endothelial-specific Tfrc KO, single-cell transcriptomics, NTBI measurement, qRT-PCR","pmids":["36351237"],"confidence":"High","gaps":["Reconciliation with earlier stellate-cell attribution incomplete","NTBI uptake transporter beyond Tfr1 not fully defined"]},{"year":2015,"claim":"Defined the BMP6 pro-peptide as essential for secretion and signaling, linking heterozygous missense mutations to human iron overload.","evidence":"Cell transfection of mutant versus wild-type BMP6, secretion imaging, phospho-Smad, serum hepcidin by LC-MS/MS, patient biopsy","pmids":["26582087"],"confidence":"Medium","gaps":["Penetrance and population-level disease contribution not established","Processing protease for the pro-peptide not identified"]},{"year":2020,"claim":"Characterized BMP6 heparan-sulfate binding via N- and C-terminal basic residues, defining matrix sequestration of the ligand.","evidence":"Solid-phase heparin binding, microcantilever, CHO-K1 HS binding, site-directed mutagenesis, MD simulation","pmids":["33232799"],"confidence":"Medium","gaps":["Physiological consequence of HS binding for iron signaling untested in vivo"]},{"year":null,"claim":"The molecular logic linking receptor selection (ALK2/ALK3/ALK6, type II receptors, RGMb), co-receptor context, and tissue-specific BMP6 outputs across iron, bone, skin, vasculature, and reproduction remains incompletely unified.","evidence":"","pmids":[],"confidence":"Low","gaps":["No integrated model of how a single ligand achieves distinct tissue programs","Receptor usage often inferred from Fc-fusion competition without mutagenesis","Cancer and steroidogenic roles rest largely on single-lab Medium/Low evidence"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0048018","term_label":"receptor ligand activity","supporting_discovery_ids":[0,2,14]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,2,9]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[20]}],"localization":[{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[14,19]},{"term_id":"GO:0031012","term_label":"extracellular matrix","supporting_discovery_ids":[15,20]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,2]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[3,5,6]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[14,22,23,24]},{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[0,1]}],"complexes":[],"partners":["HJV","TMPRSS6","ERFE","RGMB","MYO10","ALK2","ALK3"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P22004","full_name":"Bone morphogenetic protein 6","aliases":["VG-1-related protein","VG-1-R","VGR-1"],"length_aa":513,"mass_kda":57.2,"function":"Growth factor of the TGF-beta superfamily that plays essential roles in many developmental processes including cartilage and bone formation (PubMed:31019025). Also plays an important role in the regulation of HAMP/hepcidin expression and iron metabolism by acting as a ligand for hemojuvelin/HJV (PubMed:26582087). Also acts to promote expression of HAMP, potentially via the interaction with its receptor BMPR1A/ALK3 (PubMed:30097509, PubMed:31800957). Initiates the canonical BMP signaling cascade by associating with type I receptor ACVR1 and type II receptor ACVR2B (PubMed:18070108). In turn, ACVR1 propagates signal by phosphorylating SMAD1/5/8 that travel to the nucleus and act as activators and repressors of transcription of target. Can also signal through non-canonical pathway such as TAZ-Hippo signaling cascade to modulate VEGF signaling by regulating VEGFR2 expression (PubMed:33021694)","subcellular_location":"Secreted","url":"https://www.uniprot.org/uniprotkb/P22004/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/BMP6","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/BMP6","total_profiled":1310},"omim":[{"mim_id":"620121","title":"IRON OVERLOAD, SUSCEPTIBILITY TO; IO","url":"https://www.omim.org/entry/620121"},{"mim_id":"619820","title":"ATONAL bHLH TRANSCRIPTION FACTOR 8; ATOH8","url":"https://www.omim.org/entry/619820"},{"mim_id":"619794","title":"VON WILLEBRAND FACTOR C DOMAIN-CONTAINING PROTEIN 2-LIKE; VWC2L","url":"https://www.omim.org/entry/619794"},{"mim_id":"613127","title":"CHORDIN-LIKE 2; CHRDL2","url":"https://www.omim.org/entry/613127"},{"mim_id":"612077","title":"MICRO RNA 22; MIR22","url":"https://www.omim.org/entry/612077"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Mitochondria","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"ovary","ntpm":19.2}],"url":"https://www.proteinatlas.org/search/BMP6"},"hgnc":{"alias_symbol":["VGR1"],"prev_symbol":["VGR"]},"alphafold":{"accession":"P22004","domains":[{"cath_id":"2.60.310","chopping":"220-241_256-361","consensus_level":"high","plddt":90.6909,"start":220,"end":361},{"cath_id":"2.10.90.10","chopping":"408-486_506-513","consensus_level":"high","plddt":90.174,"start":408,"end":513}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P22004","model_url":"https://alphafold.ebi.ac.uk/files/AF-P22004-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P22004-F1-predicted_aligned_error_v6.png","plddt_mean":68.94},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=BMP6","jax_strain_url":"https://www.jax.org/strain/search?query=BMP6"},"sequence":{"accession":"P22004","fasta_url":"https://rest.uniprot.org/uniprotkb/P22004.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P22004/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P22004"}},"corpus_meta":[{"pmid":"1893873","id":"PMC_1893873","title":"Involvement 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Physical interaction between HJV.Fc and BMP6 was demonstrated, and Bmp6-null mice develop hereditary hemochromatosis-like phenotype with reduced hepcidin expression and tissue iron overload. A neutralizing antibody to BMP6 inhibits hepcidin expression and increases serum iron in vivo.\",\n      \"method\": \"Co-immunoprecipitation/pulldown (HJV.Fc–BMP6 physical interaction), neutralizing antibody in vivo, Bmp6-/- mouse knockout with phenotypic readout (serum iron, tissue iron, hepcidin mRNA)\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal functional assays (HJV.Fc pulldown, neutralizing antibody, knockout mouse), replicated by independent lab same year (PMID:19252488)\",\n      \"pmids\": [\"19252486\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Targeted disruption of Bmp6 in mice causes massive iron overload in liver, exocrine pancreas, heart, and renal tubules with markedly reduced hepatic phospho-Smad1/5/8, failure of Smad nuclear translocation, and severely reduced hepcidin synthesis, demonstrating BMP6 is functionally non-redundant for activating the BMP/Smad signaling axis that drives hepcidin expression.\",\n      \"method\": \"Bmp6 knockout mouse; Western blot (phospho-Smad1/5/8); immunohistochemistry (Smad nuclear translocation); quantitative RT-PCR (hepcidin mRNA); tissue iron measurements\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean knockout with multiple orthogonal readouts, independently replicated by companion paper (PMID:19252486)\",\n      \"pmids\": [\"19252488\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Crystal structure of BMP-6 was solved, and surface plasmon resonance showed BMP-6 binds type I and type II serine/threonine kinase receptors. BMP-6 signaling capability served as a reference comparator demonstrating its receptor binding mediates Smad-based signaling in whole-cell luciferase assays.\",\n      \"method\": \"X-ray crystallography; surface plasmon resonance (BIAcore); Smad-based whole-cell luciferase assay\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure plus biophysical binding assay and cell-based functional validation in single study\",\n      \"pmids\": [\"17924656\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Liver iron content independently and positively correlates with hepatic Bmp6 mRNA expression and Smad1/5/8 signaling, whereas transferrin saturation activates downstream Smad1/5/8 signaling without inducing Bmp6 mRNA, indicating that circulating and tissue iron differentially regulate the BMP6-Smad pathway to control hepcidin. The Erk1/2 MAPK pathway is not activated by iron in vivo.\",\n      \"method\": \"In vivo acute and chronic iron administration in mice; quantitative RT-PCR (Bmp6 mRNA, Smad7, hepcidin); Western blot (phospho-Smad1/5/8, phospho-Erk1/2); correlation analysis of transferrin saturation vs. liver iron content\",\n      \"journal\": \"Hepatology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal in vivo methods (gene expression, protein phosphorylation) with careful separation of iron pools, single lab\",\n      \"pmids\": [\"21488083\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Erythroferrone (ERFE) suppresses hepcidin by acting as a ligand trap that preferentially binds BMP5, BMP6, and BMP7, inhibiting hepatic BMP/Smad signaling. Cell-free HTRF competition assays showed BMP5, BMP6, and BMP7 compete with anti-ERFE for binding to ERFE, and recombinant ERFE specifically abrogated hepcidin induction by BMP5, BMP6, and BMP7 but not BMP2, BMP4, or BMP9.\",\n      \"method\": \"Cell-free homogeneous time-resolved fluorescence (HTRF) competition binding assay; in vitro SMAD1/5/8 phosphorylation assay; in vivo EPO/ERFE mouse experiments; neutralizing anti-ERFE antibody rescue\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cell-free binding assay plus in vitro signaling plus in vivo genetic rescue, multiple orthogonal methods in single study\",\n      \"pmids\": [\"30097509\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Nrf2 is activated by iron-induced mitochondria-derived pro-oxidants in liver sinusoidal endothelial cells and drives Bmp6 expression, which in turn increases hepcidin synthesis in neighboring hepatocytes. Nrf2 knockout mice show impaired Bmp6-hepcidin response to iron. Pharmacological Nrf2 activation stimulates the Bmp6-hepcidin axis.\",\n      \"method\": \"Nrf2 knockout mouse; in vivo iron loading (oral and parenteral); quantitative RT-PCR; pharmacological Nrf2 activation; cell fractionation/oxidative stress measurements\",\n      \"journal\": \"Nature metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockout with multiple iron loading conditions and pharmacological rescue, single lab\",\n      \"pmids\": [\"31276102\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Liver sinusoidal endothelial cells (LSECs) sense non-transferrin-bound iron (NTBI) and induce Bmp6 expression via Nrf2-mediated oxidative stress, which then acts paracrinally on hepatocytes to induce hepcidin. Endothelial-specific Tfr1 ablation (TfrcTek-Cre mice) showed Tfr1 contributes to iron sensing by LSECs, particularly under low-iron conditions, and single-cell transcriptomics confirmed LSECs as the predominant Bmp6-expressing liver cell type.\",\n      \"method\": \"Conditional endothelial-specific Tfr1 knockout mouse (TfrcTek-Cre); single-cell transcriptomics; quantitative RT-PCR (Bmp6, Hamp); serum NTBI measurement; correlation analysis\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional genetic KO plus single-cell transcriptomics plus in vivo iron challenges, multiple orthogonal methods\",\n      \"pmids\": [\"36351237\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"BMP2, expressed predominantly in liver endothelial cells, plays a non-redundant role in hepcidin regulation in addition to BMP6. Endothelial-conditional Bmp2 knockout mice have hepcidin deficiency and iron overload. However, BMP6 is absolutely required for hepcidin induction by serum iron (acute oral iron gavage fails to induce hepcidin in Bmp6-/- mice), whereas BMP2 has at least a partially redundant role.\",\n      \"method\": \"Conditional endothelial Bmp2 knockout mouse; Bmp6-/- mouse; acute oral iron gavage vs. chronic dietary iron loading; neutralizing BMP2/4 antibody; quantitative RT-PCR (hepcidin, Smad targets)\",\n      \"journal\": \"American journal of hematology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — dual conditional KO approaches plus antibody blockade plus dietary challenge, replicated in two companion studies (PMIDs 28815688 and 30478858)\",\n      \"pmids\": [\"28815688\", \"30478858\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"BMP6 stimulates TMPRSS6 (matriptase-2) expression at mRNA and protein levels and increases matriptase-2 activity in vitro. Inhibitor of DNA binding 1 (Id1) is identified as the key BMP-Smad pathway element mediating TMPRSS6 regulation by BMP6. In vivo, Tmprss6 mRNA is induced by chronic iron treatment or BMP6 injection and is blocked by anti-BMP6 neutralizing antibody, establishing a negative feedback loop.\",\n      \"method\": \"In vitro BMP6 treatment (RT-PCR, Western blot, matriptase-2 activity assay); siRNA knockdown of Id1; in vivo BMP6 injection and neutralizing antibody; quantitative RT-PCR\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro mechanistic dissection (Id1 siRNA) plus in vivo rescue experiment, multiple orthogonal methods single lab\",\n      \"pmids\": [\"21622652\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Hemojuvelin (HJV) is required for BMP6/Smad signaling to hepcidin; Hjv-/- primary hepatocytes show severely impaired BMP6/Smad signaling (but not IL-6/Stat or BMP2/Smad signaling), abolishing synergism with IL-6/Stat during inflammation. This establishes that Hjv selectively facilitates BMP6 signal transduction.\",\n      \"method\": \"Hjv-/- mouse; primary murine hepatocyte culture; BMP6 and BMP2 stimulation; Western blot (phospho-Smad, phospho-Stat3); hepcidin mRNA quantification; LPS, FSL1, and E. coli infection in vivo\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — primary cell mechanistic dissection plus multiple in vivo infection models, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"30213871\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Deletion of Bmp6 in Hjv-/- mice further represses Smad signaling and hepcidin expression in the liver, indicating BMP6 can signal to hepcidin partially independently of HJV in female mice. This genetic epistasis also demonstrated that baseline BMP/Smad signaling determines the level of hepcidin reached after LPS challenge, consistent with synergy between IL-6/Stat3 and BMP/Smad pathways.\",\n      \"method\": \"Bmp6-/-;Hjv-/- double-knockout mouse (genetic epistasis); quantitative RT-PCR; Western blot (phospho-Smad); LPS challenge; tissue iron measurement\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — rigorous double-KO epistasis with multiple readouts, two independent KO lines\",\n      \"pmids\": [\"29021231\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"BMP6/HFE epistasis: Hfe-deficient mice with iron overload show increased Bmp6 mRNA and protein but do NOT show increased phospho-Smad1/5/8 or Id1 (BMP signaling indicators), unlike mice with secondary iron overload. This indicates HFE facilitates signal transduction downstream of the BMP6 ligand, rather than regulating Bmp6 expression.\",\n      \"method\": \"Hfe-/- mouse; Western blot (phospho-Smad1/5/8); quantitative RT-PCR (Id1, hepcidin, Bmp6); comparison to secondary iron-overload mice\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic model with multiple signaling readouts, but epistatic placement relies on negative finding (no pSmad increase) in single lab\",\n      \"pmids\": [\"19622835\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Iron-induced Bmp6 mRNA expression is localized to non-parenchymal liver cells (primarily hepatic stellate cells), not hepatocytes. Iron-saturated transferrin induces Bmp6 mRNA in isolated hepatic stellate cells but not hepatocytes, suggesting non-parenchymal cells are the primary source of BMP6 for paracrine hepcidin regulation.\",\n      \"method\": \"In vivo dietary iron loading; cell fractionation into hepatocytes and non-parenchymal cells; quantitative RT-PCR; isolated hepatic stellate cell treatment with iron-saturated transferrin\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell-type fractionation plus in vitro cell treatment, single lab, no full functional rescue\",\n      \"pmids\": [\"23565256\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Iron overload induces Bmp6 mRNA and protein expression in the liver but NOT in the duodenum across three mouse strains, establishing liver as the iron-responsive source of BMP6 for hepcidin regulation.\",\n      \"method\": \"Iron-enriched diet and Hfe-/- mouse models; quantitative RT-PCR; immunohistochemistry; Western blot for Bmp6 protein in liver and duodenum\",\n      \"journal\": \"Haematologica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — three mouse strains and two iron-overload models, single lab, protein and mRNA orthogonal methods\",\n      \"pmids\": [\"20952515\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"Recombinant VGR-1/BMP-6 protein is secreted and proteolytically processed to yield the mature molecule. Continuous secretion of BMP-6 from tumor cells in vivo induces surrounding host mesenchymal cells to undergo endochondral bone formation, establishing BMP-6 as a secreted bone-inductive morphogen acting on mesenchymal cells.\",\n      \"method\": \"CHO cell overexpression of recombinant murine BMP-6; Western blot (secretion and processing); subcutaneous injection in athymic nude mice; histological analysis of cartilage and bone formation\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro secretion/processing confirmed by Western blot plus in vivo bone induction assay, single lab\",\n      \"pmids\": [\"8089189\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"VGR-1/BMP-6 overexpression in pluripotent mesenchymal ROB-C26 cells induces osteoblastic differentiation in vitro and the extracellular matrix from these cells induces ectopic bone formation in vivo. This osteoinductive effect requires BMP-6 presentation within the matrix (not soluble BMP-6) and is blocked by neutralizing BMP-6 antibody but not by anti-TGF-β1 antibody.\",\n      \"method\": \"Stable transfection of ROB-C26 cells; neutralizing antibody blockade (anti-BMP6 vs. anti-TGF-β1); in vivo ectopic bone formation assay; MyoD overexpression competition experiment\",\n      \"journal\": \"Cell growth & differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain-of-function plus neutralizing antibody specificity control plus in vivo assay, single lab\",\n      \"pmids\": [\"7547504\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Deubiquitinase PSMD14 stabilizes the ALK2 type I BMP receptor by removing K48-linked ubiquitin chains added by Smurf1 E3 ligase, thereby preventing proteasomal degradation of ALK2 and sustaining BMP6 signaling pathway initiation.\",\n      \"method\": \"Human DUB siRNA library screen; immunoprecipitation; ubiquitination assay; immunoblot; in vitro and in vivo colorectal cancer xenograft model\",\n      \"journal\": \"EBioMedicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical ubiquitination assay plus in vivo xenograft, single lab\",\n      \"pmids\": [\"31685442\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"BMP6-induced myosin-X (Myo10) localizes in filopodia and is required for BMP-dependent filopodial assembly, endothelial cell alignment and directional migration toward BMP6 gradients, and BMP6-dependent Smad activation. BMP6 receptor ALK6 co-localizes with Myo10 in filopodia in a BMP6-dependent manner and both exhibit intrafilopodial motility.\",\n      \"method\": \"Microarray (Myo10 as BMP6 target); siRNA knockdown of Myo10; co-localization imaging (confocal microscopy); directional migration assay; Western blot (Smad activation)\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA loss-of-function plus co-localization plus functional migration and signaling readouts, single lab\",\n      \"pmids\": [\"18158328\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Estrogen regulates hepcidin expression via GPR30 (7-transmembrane estrogen receptor)-dependent upregulation of BMP6 in hepatocytes. GPR30 siRNA abolishes E2-induced hepcidin and BMP6 expression; GPR30 agonist G1 recapitulates E2 effects on BMP6 and hepcidin in vivo in ovariectomized mice.\",\n      \"method\": \"Ovariectomized mouse model; HepG2 cell culture; GPR30 siRNA knockdown; GPR30 agonist G1 treatment; quantitative RT-PCR; in vivo estrogen/G1 supplementation\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA plus pharmacological agonist plus in vivo mouse model, single lab\",\n      \"pmids\": [\"22792339\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Heterozygous missense mutations in the BMP6 pro-peptide domain (p.Pro95Ser, p.Leu96Pro, p.Gln113Glu) cause defective BMP6 secretion, reduced SMAD1/5/8 signaling, and loss of hepcidin production in transfected cells, establishing that the BMP6 pro-peptide is essential for proper processing, secretion, and downstream signaling.\",\n      \"method\": \"Cell transfection with mutant vs. wild-type BMP6; immunofluorescence (secretion); quantitative RT-PCR (hepcidin); immunoblot (phospho-Smad1/5/8); serum hepcidin by LC-MS/MS; liver biopsy immunohistochemistry\",\n      \"journal\": \"Gastroenterology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional mutagenesis in cells with multiple orthogonal readouts, supported by patient phenotype, single lab\",\n      \"pmids\": [\"26582087\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"BMP6 binding to heparin and heparan sulfate is mediated by N-terminal basic residues (R5, R6, R7) and C-terminal basic residues (K126, K127, R129). Mutation of both sites abolished heparin-binding activity of recombinant monomeric BMP6 and binding to membrane heparan sulfate on CHO-K1 cells.\",\n      \"method\": \"Solid-phase heparin binding assay; microcantilever assay; CHO-K1 cell membrane heparan sulfate binding; site-directed mutagenesis; molecular dynamics simulation\",\n      \"journal\": \"Biochimica et biophysica acta. General subjects\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis with binding assay plus molecular dynamics, single lab, multiple methods\",\n      \"pmids\": [\"33232799\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"BMP-6 functions as an autocrine stimulator of chondrocyte maturation. BMP-6 mRNA expression precedes type X collagen induction during chondrocyte maturation. PTHrP directly inhibits BMP-6 expression, blocking maturation; exogenous BMP-6 overcomes PTHrP inhibition and stimulates Indian hedgehog mRNA, placing BMP-6 upstream of Ihh in the chondrocyte maturation pathway.\",\n      \"method\": \"Primary embryonic chondrocyte monolayer culture; exogenous BMP-6 treatment; PTHrP inhibition and rescue; RT-PCR (BMP-6, type X collagen, Ihh mRNA); alkaline phosphatase activity assay\",\n      \"journal\": \"Journal of bone and mineral research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain-of-function with exogenous BMP-6 plus PTHrP inhibition/rescue, multiple functional readouts, single lab\",\n      \"pmids\": [\"10234567\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Bmp6 and Bmp7 are required together for cardiac cushion formation and septation during mouse embryonic development. Bmp6;Bmp7 double mutants show a marked delay in outflow tract endocardial cushion formation, defects in valve morphogenesis and chamber septation, and lethality between 10.5–15.5 dpc, whereas single mutants have no cardiac defect.\",\n      \"method\": \"Bmp6;Bmp7 double-mutant mouse (genetic epistasis); in situ hybridization; histological analysis\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — rigorous double-mutant genetic approach with histological phenotyping, single lab\",\n      \"pmids\": [\"11437450\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Endogenous BMP2 and BMP6 cooperatively regulate bone formation in vivo. Bmp2+/-;Bmp6-/- compound-deficient mice show reduced trabecular bone volume with suppressed bone formation and impaired endochondral (but not intramembranous) bone formation after fracture, whereas single-deficient animals show no such defect.\",\n      \"method\": \"Bmp2+/-;Bmp6-/- compound knockout mouse; micro-CT/histomorphometry; fracture healing model; bone marrow cell culture osteogenic differentiation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — compound knockout genetic epistasis with multiple bone phenotype readouts and in vitro rescue, single lab\",\n      \"pmids\": [\"16109715\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Bmp6-null mice are viable and fertile with only a transient delay in sternal ossification (not other skeletal elements), and Bmp2 and Bmp6 are co-expressed in hypertrophic cartilage, suggesting functional compensation. Sternal defects are exacerbated in Bmp5/Bmp6 double mutants.\",\n      \"method\": \"Gene-targeted Bmp6 knockout mouse; skeletal analysis (histology, in situ hybridization); Bmp5;Bmp6 double mutant analysis\",\n      \"journal\": \"Developmental genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean knockout with detailed skeletal phenotyping and double-mutant epistasis, single lab\",\n      \"pmids\": [\"9664685\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"BMP-6 inhibits miR-21 transcription in MDA-MB-231 breast cancer cells through repression of deltaEF1 and AP-1 transcription factors. BMP-6 reduces binding of deltaEF1 to the E2-box on the miR-21 promoter (miPPR-21) and decreases c-Fos/c-Jun expression, thereby inhibiting MDA-MB-231 cell invasion via upregulation of PDCD4.\",\n      \"method\": \"Luciferase reporter assay (miPPR-21 constructs); ChIP assay (deltaEF1 binding to miPPR-21); site-directed mutation; RT-PCR; siRNA knockdown; invasion assay\",\n      \"journal\": \"Cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP plus luciferase reporter plus mutagenesis plus functional invasion readout, single lab\",\n      \"pmids\": [\"19308091\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"BMP-6 promotes E-cadherin expression in breast cancer cells by inhibiting deltaEF1 transcription and physically dislodging deltaEF1 from the E-cadherin proximal promoter, thereby de-repressing E-cadherin transcription. RNAi knockdown of deltaEF1 abolishes E-cadherin transactivation by BMP-6.\",\n      \"method\": \"Luciferase reporter assay (E-cadherin promoter); ChIP assay (deltaEF1 binding); RNAi knockdown of deltaEF1; Western blot; quantitative RT-PCR\",\n      \"journal\": \"BMC cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP plus luciferase plus RNAi rescue, single lab\",\n      \"pmids\": [\"17997862\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"BMP-6 inhibits proliferation of renal cell carcinoma (RCC) cells through BMP receptor type II. RCC cell lines lacking BMP-RII are resistant to BMP-6-induced growth inhibition. Transfection of BMP-RII restores BMP-6 sensitivity, and constitutively active BMP-RIA or -RIB restores BMP-6 reporter activity in resistant cells.\",\n      \"method\": \"Adenoviral transduction (constitutively active BMP-RIA, RIB); BMP-RII transfection; BMP-6-responsive luciferase reporter; cell proliferation assay; Northern blot/RT-PCR\",\n      \"journal\": \"Clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — receptor reconstitution in resistant cells plus reporter assay, single lab\",\n      \"pmids\": [\"14676131\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"BMP-6 induction in prostate cancer PC3M cells leads to nuclear translocation of SMADs, activation of Id-1, increased migration and invasion, upregulation of MMP-1 and MMP-9 mRNA, and direct transcriptional activation of MMP-1 and MMP-9 promoters. BMP-6 also activates MAPK phosphorylation.\",\n      \"method\": \"Doxycycline-inducible BMP-6 expression system; Western blot (SMAD translocation, MAPK phosphorylation); luciferase reporter (MMP-1, MMP-9 promoters); migration/invasion assay; RT-PCR\",\n      \"journal\": \"The Journal of pathology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — inducible expression system with multiple downstream readouts including promoter luciferase, single lab\",\n      \"pmids\": [\"18072288\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"BMP-6 inhibits FSH-induced progesterone production in rat granulosa cells via Smad1/5/8 signaling and Id-1 transcription. Melatonin impairs this BMP-6/Smad signaling by upregulating inhibitory Smad6 (but not Smad7), thereby counteracting BMP-6-mediated inhibition of steroidogenesis.\",\n      \"method\": \"Primary rat granulosa cell culture; BMP-6 + melatonin co-treatment; Western blot (phospho-Smad1/5/8); RT-PCR (Id-1, Smad6/7, MT1/MT2); progesterone ELISA; cAMP assay\",\n      \"journal\": \"The Journal of steroid biochemistry and molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — primary cell culture with multiple signaling readouts, single lab\",\n      \"pmids\": [\"24751708\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"BMP6 downregulates GDNF expression in human granulosa-lutein cells via ALK2 and ALK3 type I receptors, activating both SMAD1/5 and ERK1/2 (but not AKT or p38) signaling. Combined knockdown of SMAD4 and ERK1/2 inhibition completely reverses BMP6-induced GDNF downregulation, demonstrating both pathways are required.\",\n      \"method\": \"Immortalized SVOG cells and primary human granulosa-lutein cells; kinase receptor inhibitor plus siRNA dual knockdown of ALK2/ALK3, SMAD1/5/4; ERK1/2 inhibitor; Western blot (phospho-SMAD1/5/8, phospho-ERK1/2, phospho-AKT, phospho-p38); quantitative RT-PCR (GDNF)\",\n      \"journal\": \"Endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — dual inhibition approach plus multiple signaling readouts, single lab\",\n      \"pmids\": [\"29750278\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"BMP6 overexpression in vivo (adenovirus injection) inhibits the telogen-to-anagen transition of hair follicles. BMP6 signaling and Wnt10b signaling mutually inhibit each other in hair follicle skin, and hair follicle stem cell activation is competitively regulated by BMP6 (inhibitor) and Wnt10b (activator).\",\n      \"method\": \"Adenovirus-mediated BMP6 overexpression in mouse skin; in situ hybridization; immunofluorescence; H&E staining; BrdU tracing\",\n      \"journal\": \"Cell communication and signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo overexpression with histological and molecular readouts, single lab\",\n      \"pmids\": [\"30791955\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The SIN3A-HDAC1/2 repressor complex silences BMP6 expression in melanoma cells, causing increased metastatic dissemination via suppression of BMP6-activated SMAD5 signaling. A downstream effector FAM83G/PAWS1 contributes to metastatic progression through actin-dependent cytoskeletal dynamics. Pharmacological HDAC1/2 inhibition restores BMP6 expression and reduces circulating melanoma cells.\",\n      \"method\": \"Mouse melanoma metastasis model; pharmacological SIN3A-HDAC1/2 complex inhibition; Western blot (phospho-SMAD5); ChIP (SIN3A-HDAC1/2 at BMP6 locus); siRNA knockdown of FAM83G/PAWS1; cell migration assay; in vivo circulating tumor cell enumeration\",\n      \"journal\": \"Molecular cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP plus pharmacological inhibition plus in vivo model plus downstream effector identification, single lab\",\n      \"pmids\": [\"34610961\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In Sjögren's syndrome, HSP70 released by lysosomal exocytosis (triggered by LAMP3 overexpression) acts as an endogenous TLR4 ligand that stimulates BMP6 expression in monocytic cells. In vivo LAMP3 overexpression in mice induces BMP6 expression and an SS-like phenotype. RNA ISH identified monocytic lineage cells as the cellular source of BMP6 in SS salivary glands.\",\n      \"method\": \"RNA ISH on salivary gland sections; in vitro TLR4 signaling assays; single-cell transcriptomics of human PBMCs; siRNA knockdown; LAMP3 overexpression in mice; RT-PCR correlation analysis\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro mechanism plus in vivo mouse model plus single-cell transcriptomics, single lab\",\n      \"pmids\": [\"35113815\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"The RNF4 ubiquitin ligase acts upstream of BMP6 and its co-receptor RGMb (Dragon) in a signaling axis required for osteogenic differentiation of human bone marrow mesenchymal stem cells. Knockdown of either RGMb or BMP6 halts osteogenic differentiation, and combined co-addition of purified RGMb and BMP6 proteins to RNF4-deficient cells fully restores differentiation.\",\n      \"method\": \"RNF4 siRNA in hBMSCs; conditioned media rescue; BMP6 and RGMb knockdown; protein co-addition rescue; transcriptional analysis; xenograft osteosarcoma model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function plus protein rescue with two factors, single lab\",\n      \"pmids\": [\"36153321\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"BMP-6 (and Ca2+) induces terminal differentiation of epidermal keratinocytes with a specific switch in E2F expression from E2F-1/2/3 (undifferentiated) to E2F-5 (differentiated), accompanied by formation of nuclear E2F5·p130·HDAC1 complexes. E2F-5 overexpression inhibits DNA synthesis in an HDAC-dependent manner, linking BMP-6-driven differentiation to permanent cell cycle exit.\",\n      \"method\": \"Primary murine keratinocyte culture; BMP-6 treatment; co-immunoprecipitation (E2F5·p130·HDAC1 complex); E2F-5 overexpression; HDAC inhibitor; DNA synthesis assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus gain-of-function plus pharmacological rescue, single lab\",\n      \"pmids\": [\"11319226\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"HIF-1α directly binds to the hypoxia-response element (HRE) in the BMP6 promoter to repress BMP6 transcription in keratinocytes. HIF-1α overexpression reduces BMP6 expression, promotes proliferation and inhibits terminal differentiation, effects that are mediated via BMP6 suppression.\",\n      \"method\": \"HIF-1α overexpression in primary human keratinocytes; ChIP assay (HIF-1α binding to HRE in BMP6 promoter); quantitative RT-PCR; cell proliferation assay; differentiation marker expression\",\n      \"journal\": \"Experimental dermatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP demonstrating direct HIF-1α/BMP6 promoter binding plus functional overexpression readouts, single lab\",\n      \"pmids\": [\"30230035\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Intraocular BMP6 protein injection in mice upregulates retinal hepcidin expression and alters retinal labile iron levels. Bmp6-/- mice develop age-dependent retinal iron accumulation and degeneration, establishing BMP6 as a regulator of retinal iron homeostasis through local hepcidin induction.\",\n      \"method\": \"Intraocular BMP6 injection in mice; quantitative RT-PCR (hepcidin); calcein-AM labile iron assay; Bmp6-/- mouse with age-dependent retinal phenotyping; cultured RPE cell BMP6 modulation\",\n      \"journal\": \"The American journal of pathology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo BMP6 injection plus knockout model with mechanistic pathway readout, single lab\",\n      \"pmids\": [\"21703414\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"A 190 bp enhancer ~2.5 kb 5' of the Bmp6 gene drives expression in developing teeth and fins; a predicted Smad3 binding site in this enhancer is required for its activity, and pharmacological inhibition of TGFβ signaling abolishes enhancer activity and severely reduces endogenous Bmp6 expression. TALEN-mediated disruption of the enhancer in vivo dramatically reduces Bmp6 expression, identifying a required cis-regulatory element.\",\n      \"method\": \"Transgenic reporter assays in sticklebacks and zebrafish; site-directed mutagenesis of Smad3 binding site; pharmacological TGFβ inhibition; TALEN enhancer disruption in vivo; quantitative RT-PCR\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis plus pharmacological plus in vivo TALEN disruption, single lab in fish ortholog model\",\n      \"pmids\": [\"25732776\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"BMP6 regulates human Sertoli cell proliferation and apoptosis through Smad2/3 phosphorylation and cyclin D1 upregulation, as well as activation of DACH1 and TFAP2A. BMP6 siRNA inhibits Sertoli cell division and increases apoptosis; the signaling operates through a non-canonical Smad2/3 pathway.\",\n      \"method\": \"Primary human Sertoli cell culture; BMP6 treatment and siRNA knockdown; Western blot (phospho-Smad2/3, cyclin D1, DACH1, TFAP2A); CCK-8 and EDU proliferation assays; Annexin V/PI apoptosis assay; ELISA (SCF)\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single cell type, pathway identification by Western blot without mutagenesis confirmation\",\n      \"pmids\": [\"28387750\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"BMP6 modulates VEGF signaling by regulating VEGFR2 expression and acts via the Hippo signaling effector TAZ in endothelial cells. BMP6 induces angiogenesis in a matrigel plug assay in vivo, making BMP6 the first BMP family member demonstrated to directly regulate Hippo signaling and neovessel formation.\",\n      \"method\": \"C57/Bl6 VEGF gene transfer mouse model; porcine myocardial ischemia model; siRNA knockdown; VEGFR2 expression assay; TAZ reporter; matrigel plug assay in nude mice\",\n      \"journal\": \"Angiogenesis\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — in vivo angiogenesis assay plus in vitro siRNA, but TAZ pathway placement based on limited mechanistic follow-up, single lab\",\n      \"pmids\": [\"33021694\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"BMP6 improves glycemia in ob/ob mice and inhibits hepatic gluconeogenesis in rat H4IIE hepatoma cells by downregulating PepCK expression via Smad signaling, antagonizing cAMP-driven glycogenolytic effects. BMP6 acts on pancreatic and liver cells through Alk3, Alk6, and ActRIIA receptors (identified using receptor Fc fusion proteins in luciferase reporter and glucose output assays).\",\n      \"method\": \"ob/ob mouse BMP6 treatment; oral glucose tolerance test; chromatin immunoprecipitation (PepCK); glucose output assay in H4IIE cells; receptor Fc fusion protein specificity assay; luciferase reporter; quantitative RT-PCR\",\n      \"journal\": \"Acta diabetologica\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — in vivo mouse study plus in vitro mechanistic assays but receptor identification relies on Fc fusion protein competition without mutagenesis, single lab\",\n      \"pmids\": [\"30539233\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"BMP6 is a secreted TGF-β superfamily ligand that functions as the principal endogenous activator of the BMP/Smad1/5/8 signaling pathway in liver sinusoidal endothelial cells and hepatocytes to induce hepcidin expression and maintain systemic iron homeostasis; it binds the co-receptor hemojuvelin (HJV) and acts through ALK2/ALK3 type I receptors, its expression is transcriptionally induced in LSECs by non-transferrin-bound iron via Nrf2-driven oxidative stress, and it additionally regulates mesenchymal differentiation (osteoblastogenesis, chondrogenesis), keratinocyte terminal differentiation, endothelial cell migration via myosin-X/filopodia, and ovarian steroidogenesis via Smad-dependent pathways.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"BMP6 is a secreted, proteolytically processed TGF-β superfamily morphogen that serves as the principal iron-responsive ligand of the hepatic BMP/Smad1/5/8 axis controlling systemic iron homeostasis [#0, #1, #14]. Genetic ablation of Bmp6 in mice produces a hereditary hemochromatosis-like phenotype with multi-organ iron overload, loss of hepatic phospho-Smad1/5/8 and Smad nuclear translocation, and severely reduced hepcidin synthesis, establishing BMP6 as functionally non-redundant for activating this pathway [#0, #1]. BMP6 physically binds the co-receptor hemojuvelin (HJV), which selectively facilitates BMP6 signal transduction to hepcidin, and signals through type I/type II serine-threonine kinase receptors to drive Smad-dependent transcription [#0, #2, #9]. Iron loading induces Bmp6 expression in liver non-parenchymal cells—predominantly sinusoidal endothelial cells—which sense non-transferrin-bound iron through Nrf2-mediated oxidative stress and act paracrinally on hepatocytes, with circulating and tissue iron pools differentially engaging the pathway [#3, #5, #6, #12, #13]. The pathway is embedded in feedback and regulatory loops: BMP6 induces TMPRSS6/matriptase-2 via Id1 as a negative-feedback brake on hepcidin, while erythroferrone suppresses signaling by sequestering BMP6 as a ligand trap [#4, #8]. Beyond iron, BMP6 acts as an osteoinductive and chondrogenic morphogen on mesenchymal cells, cooperating with BMP2/BMP5/BMP7 in bone formation and cardiac cushion development [#14, #15, #22, #23, #24], drives keratinocyte terminal differentiation through an E2F-5/p130/HDAC1 switch [#35], directs endothelial filopodial assembly and directional migration via myosin-X [#17], and modulates Smad/Id1-dependent programs in ovarian granulosa-cell steroidogenesis and in multiple cancers [#25, #28, #29, #30]. Heterozygous missense mutations in the BMP6 pro-peptide domain impair secretion and downstream Smad signaling, causing loss of hepcidin production and iron overload in patients [#19].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Established the structural and biophysical basis for BMP6 signaling by showing it directly engages type I and type II serine-threonine kinase receptors to drive Smad activation.\",\n      \"evidence\": \"X-ray crystallography, surface plasmon resonance, and Smad-based luciferase reporter\",\n      \"pmids\": [\"17924656\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the iron-specific receptor configuration in vivo\", \"No co-receptor (HJV) interaction defined at this stage\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Answered which BMP ligand controls iron homeostasis by identifying BMP6 as the non-redundant endogenous activator of hepatic BMP/Smad signaling that drives hepcidin, with HJV as its binding co-receptor.\",\n      \"evidence\": \"HJV.Fc–BMP6 pulldown, neutralizing antibody in vivo, and Bmp6-/- mice with iron/hepcidin readouts (two companion papers)\",\n      \"pmids\": [\"19252486\", \"19252488\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cellular source of iron-induced BMP6 not resolved\", \"Mechanism of iron-to-Bmp6-transcription coupling unknown\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Distinguished ligand regulation from signal transduction by showing HFE acts downstream of BMP6 (facilitating signaling), since Hfe-/- mice raise Bmp6 but fail to raise phospho-Smad/Id1.\",\n      \"evidence\": \"Hfe-/- mouse, phospho-Smad and Id1 readouts versus secondary iron overload\",\n      \"pmids\": [\"19622835\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Epistatic placement rests on a negative pSmad finding\", \"Molecular interaction of HFE with the receptor complex undefined\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Localized the iron-responsive BMP6 source by demonstrating iron induces Bmp6 in liver but not duodenum across strains.\",\n      \"evidence\": \"Dietary iron and Hfe-/- models, qRT-PCR, IHC, Western blot\",\n      \"pmids\": [\"20952515\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific liver cell type not yet pinpointed\", \"Iron-sensing transducer to Bmp6 promoter unknown\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Separated circulating from tissue iron signals, showing liver iron correlates with Bmp6 mRNA while transferrin saturation activates Smad without inducing Bmp6, refining how two iron pools tune the pathway.\",\n      \"evidence\": \"Acute/chronic iron loading in mice, qRT-PCR, phospho-Smad/Erk Western blot, correlation analysis\",\n      \"pmids\": [\"21488083\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular sensors for the two iron pools not identified\", \"Erk pathway role excluded but alternative non-Smad arms untested\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Defined a negative-feedback brake by showing BMP6 induces TMPRSS6/matriptase-2 through Id1, limiting hepcidin output.\",\n      \"evidence\": \"In vitro BMP6 treatment, Id1 siRNA, in vivo BMP6 injection and anti-BMP6 antibody\",\n      \"pmids\": [\"21622652\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative contribution of this loop to systemic iron set-point unclear\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Refined the cellular source to liver non-parenchymal cells (hepatic stellate cells) responding to transferrin-bound iron rather than hepatocytes.\",\n      \"evidence\": \"Cell fractionation, isolated stellate cell iron treatment, qRT-PCR\",\n      \"pmids\": [\"23565256\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No functional rescue confirming stellate-cell BMP6 drives hepcidin\", \"Conflicts with later endothelial-source data — cell-type attribution unsettled\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Established cooperative but distinct roles for endothelial BMP2 versus BMP6, with BMP6 absolutely required for the acute serum-iron response.\",\n      \"evidence\": \"Endothelial-conditional Bmp2 KO, Bmp6-/-, acute gavage versus dietary loading, anti-BMP2/4 antibody (companion studies)\",\n      \"pmids\": [\"28815688\", \"30478858\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How BMP2 and BMP6 form functional heterodimers/complexes in vivo not defined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Used genetic epistasis to show BMP6 can signal to hepcidin partially independently of HJV and that baseline BMP/Smad tone sets the inflammatory hepcidin ceiling.\",\n      \"evidence\": \"Bmp6-/-;Hjv-/- double-knockout mice, phospho-Smad, qRT-PCR, LPS challenge\",\n      \"pmids\": [\"29021231\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"HJV-independent receptor route not molecularly identified\", \"Sex-specific differences mechanistically unexplained\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identified erythroferrone as a ligand trap that preferentially sequesters BMP6 (and BMP5/7), explaining erythroid suppression of hepcidin.\",\n      \"evidence\": \"Cell-free HTRF competition, in vitro SMAD1/5/8 assay, EPO/ERFE mouse experiments, anti-ERFE rescue\",\n      \"pmids\": [\"30097509\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and structural basis of ERFE–BMP6 trapping not resolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Showed HJV selectively facilitates BMP6 (not BMP2 or IL-6/Stat) signaling and is required for BMP6/IL-6 synergy during inflammation.\",\n      \"evidence\": \"Hjv-/- primary hepatocytes, BMP6/BMP2 stimulation, phospho-Smad/Stat3 Western blot, in vivo infection models\",\n      \"pmids\": [\"30213871\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of HJV's BMP6 selectivity undefined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Resolved the iron-sensing-to-transcription link by showing Nrf2, activated by iron-induced mitochondrial pro-oxidants in LSECs, drives Bmp6 expression.\",\n      \"evidence\": \"Nrf2-/- mice, iron loading, pharmacological Nrf2 activation, oxidative stress measurements\",\n      \"pmids\": [\"31276102\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct Nrf2 binding to the Bmp6 promoter not demonstrated here\", \"Other transcriptional inputs not excluded\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Pinpointed LSECs as the dominant Bmp6-expressing cell and showed they sense NTBI via Nrf2 with a Tfr1 contribution under low iron, acting paracrinally on hepatocytes.\",\n      \"evidence\": \"Endothelial-specific Tfrc KO, single-cell transcriptomics, NTBI measurement, qRT-PCR\",\n      \"pmids\": [\"36351237\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reconciliation with earlier stellate-cell attribution incomplete\", \"NTBI uptake transporter beyond Tfr1 not fully defined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defined the BMP6 pro-peptide as essential for secretion and signaling, linking heterozygous missense mutations to human iron overload.\",\n      \"evidence\": \"Cell transfection of mutant versus wild-type BMP6, secretion imaging, phospho-Smad, serum hepcidin by LC-MS/MS, patient biopsy\",\n      \"pmids\": [\"26582087\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Penetrance and population-level disease contribution not established\", \"Processing protease for the pro-peptide not identified\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Characterized BMP6 heparan-sulfate binding via N- and C-terminal basic residues, defining matrix sequestration of the ligand.\",\n      \"evidence\": \"Solid-phase heparin binding, microcantilever, CHO-K1 HS binding, site-directed mutagenesis, MD simulation\",\n      \"pmids\": [\"33232799\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological consequence of HS binding for iron signaling untested in vivo\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The molecular logic linking receptor selection (ALK2/ALK3/ALK6, type II receptors, RGMb), co-receptor context, and tissue-specific BMP6 outputs across iron, bone, skin, vasculature, and reproduction remains incompletely unified.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No integrated model of how a single ligand achieves distinct tissue programs\", \"Receptor usage often inferred from Fc-fusion competition without mutagenesis\", \"Cancer and steroidogenic roles rest largely on single-lab Medium/Low evidence\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [0, 2, 14]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 2, 9]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [20]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [14, 19]},\n      {\"term_id\": \"GO:0031012\", \"supporting_discovery_ids\": [15, 20]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [3, 5, 6]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [14, 22, 23, 24]},\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"HJV\", \"TMPRSS6\", \"ERFE\", \"RGMb\", \"MYO10\", \"ALK2\", \"ALK3\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}