{"gene":"NR1I3","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":2009,"finding":"Protein kinase C phosphorylates threonine 38 of human CAR (NR1I3), which destabilizes the alpha-helix spanning residues 29-42 (part of the first zinc finger), thereby inactivating CAR binding to DNA and sequestering it in the cytoplasm. Dephosphorylation of Thr38 is required for nuclear translocation and activation. Phenobarbital dephosphorylates the corresponding Thr48 of mouse CAR in the cytoplasm, enabling nuclear translocation.","method":"In vitro kinase assay, molecular dynamics simulation, helix-stabilizing mutagenesis, immunohistochemistry with anti-phospho-Thr38 antibody, co-immunoprecipitation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods including in vitro kinase assay, mutagenesis, molecular dynamics, and immunohistochemistry; replicated in both human and mouse systems","pmids":["19858220"],"is_preprint":false},{"year":2011,"finding":"Activated (phosphorylated) ERK1/2 interacts with phosphorylated CAR (NR1I3) at Thr-38 via the xenochemical response signal peptide near the C-terminus of CAR, repressing dephosphorylation of Thr-38 and thereby maintaining CAR in its inactive cytoplasmic state. EGF treatment increased this interaction, while MEK inhibitor U0126 or MEK1/2 knockdown decreased Thr-38 phosphorylation.","method":"Co-immunoprecipitation of FLAG-tagged CAR mutants (T38A, T38D) with endogenous phospho-ERK1/2; shRNA knockdown; pharmacological inhibition (U0126)","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP with phospho-mimetic and phospho-dead mutants, confirmed by both pharmacological inhibition and shRNA knockdown in a single rigorous study","pmids":["21873423"],"is_preprint":false},{"year":2003,"finding":"The human CAR (NR1I3) gene promoter contains a functional glucocorticoid response element (GRE) at position -4447/-4432 that is recognized and transactivated by the glucocorticoid receptor (GR) in the presence of dexamethasone. Chromatin immunoprecipitation confirmed GR binding to this distal promoter region in cultured hepatocytes, establishing CAR as a primary GR-response gene.","method":"Deletion analysis and transient transfection, site-directed mutagenesis, gel shift assay (EMSA), chromatin immunoprecipitation (ChIP), cotransfection experiments, 5'-RACE, primer extension","journal":"Molecular endocrinology (Baltimore, Md.)","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — multiple orthogonal methods (transfection, mutagenesis, EMSA, ChIP) in a single rigorous study establishing direct GR binding and transactivation","pmids":["12511605"],"is_preprint":false},{"year":2005,"finding":"The human CAR splice variant CAR3 (with a 5 amino acid insertion in the ligand-binding domain) is ligand-activated (by CITCO), in contrast to the constitutively active reference form CAR1. CAR3 transactivation requires its DNA-binding domain and AF-2 motif. RXRα co-transfection markedly enhances CAR3 activity via RXR's AF-2 function (but independently of RXR A/B and C domains/heterodimerization region), by facilitating coactivator (SRC-1) recruitment. Clotrimazole acts as a ligand activator of CAR3, whereas it is an inverse agonist of CAR1.","method":"Transient transfection reporter assays, domain deletion/mutation analysis, mammalian two-hybrid assay","journal":"Molecular pharmacology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — multiple transfection and two-hybrid assays in a single lab; no in vitro reconstitution or structural validation","pmids":["16099843"],"is_preprint":false},{"year":2007,"finding":"PPARα directly induces CAR (NR1I3) transcription in rat hepatocytes via a DR1 motif in the CAR promoter. This PPARα-dependent induction of CAR potentiates phenobarbital-induced transcription of the CAR target gene CYP2B1. Fasting-induced CAR expression was abrogated in PPARα-deficient mice, suggesting free fatty acids (PPARα ligands) mediate fasting-induced CAR upregulation.","method":"Promoter reporter assays with DR1 motif deletion, PPARα agonist treatment (WY14643) in rat hepatocytes, PPARα-deficient mouse model","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — promoter deletion analysis plus in vivo genetic model (PPARα KO mice), but single lab","pmids":["18023279"],"is_preprint":false},{"year":2008,"finding":"CAR (NR1I3) regulates serum triglyceride levels under metabolic stress. CAR activity inversely regulates PPARα target gene expression; CAR activation (by TCPOBOP) decreases PPARα mRNA and suppresses hepatic fatty acid oxidation genes (Cyp4a14, CPT1α, CTE), whereas CAR-deficient mice show increased hepatic fatty acid oxidation and are protected from hypertriglyceridemia in ob/ob and high-fat diet models.","method":"CAR knockout mouse model, pharmacological activation (TCPOBOP), ob/ob × Car(-/-) cross, high-fat diet feeding, gene expression analysis","journal":"Journal of lipid research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean genetic KO with defined molecular phenotype (PPARα pathway suppression), confirmed pharmacologically; single lab","pmids":["18941143"],"is_preprint":false},{"year":2010,"finding":"CAR (NR1I3) directly transactivates the lipogenic gene THRSP (Spot14/S14) promoter through a DR-4 thyroid hormone/PXR response element. Gel-shift analysis showed that the CAR/RXR heterodimer complex binds this element. Deletion or point mutation of this element abolished CAR-mediated transactivation. CAR-null mice did not show mCAR-activator-induced THRSP upregulation.","method":"Promoter reporter assay with deletion/point mutations, gel shift (EMSA) with CAR/RXR complex, CAR null mice with pharmacological activators, human hepatocyte treatment","journal":"Endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — EMSA plus mutagenesis plus in vivo genetic model; single lab","pmids":["20185760"],"is_preprint":false},{"year":2011,"finding":"CAR (NR1I3) is essential for DDC-induced liver injury and oval cell proliferation. DDC activates CAR (shown by nuclear CAR accumulation and CYP2B10 induction), and Car(-/-) mice fail to develop DDC-induced liver injury, ductular reaction, or oval cell proliferation, placing CAR upstream of these hepatic injury/regeneration responses.","method":"CAR knockout mouse model fed DDC diet, nuclear fractionation (CAR accumulation), real-time PCR (CYP2B10, oval cell markers), laser capture microdissection, immunostaining (A6 antibody)","journal":"Laboratory investigation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean genetic KO with specific molecular and cellular readouts; single lab","pmids":["21826054"],"is_preprint":false},{"year":2012,"finding":"DAX-1 (an orphan nuclear receptor) functions as a potent corepressor of human CAR (NR1I3). DAX-1's downstream LXXLL and PCFQVLP motifs are critical for corepression; DAX-1's C-terminal transcription silencing domain mediates the repression by inhibiting the CAR–SRC1 coactivator interaction (~50% inhibition). Direct CAR–DAX-1 interaction was demonstrated by alpha-screen and co-immunoprecipitation, enhanced by the CAR activator CITCO. DAX-1 inhibits CAR-mediated CITCO induction of CYP2B6 in primary human hepatocytes.","method":"Transactivation assays, domain deletion/mutagenesis, mammalian two-hybrid assay, alpha-screen, co-immunoprecipitation, primary human hepatocyte experiments","journal":"Molecular pharmacology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — direct protein interaction confirmed by two independent binding assays (alpha-screen and Co-IP), functional validation by mutagenesis and primary hepatocyte experiments; single lab but multiple orthogonal methods","pmids":["22896671"],"is_preprint":false},{"year":2018,"finding":"Drug-activated CAR (NR1I3) binds genome-wide with its partner RXRα and induces H4K5 acetylation at stimulated genes. Transcriptional inhibition by CAR occurs when CAR binds the same enhancers occupied by HNF4α, PPARα, or FXR, suggesting functional competition among nuclear receptors on shared enhancers as the mechanism of CAR-mediated metabolic gene repression.","method":"Genome-wide ChIP-seq for CAR, RXRα, and H4K5Ac in mouse liver after TCPOBOP treatment","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genome-wide ChIP-seq providing mechanistic insight into enhancer competition; single lab, no mutagenesis validation of specific sites","pmids":["30396153"],"is_preprint":false},{"year":2014,"finding":"TGFβ induces CAR (NR1I3) expression in dermal fibroblasts via a Smad-dependent mechanism. CAR activation amplifies TGFβ-stimulated collagen synthesis, myofibroblast differentiation, and COL1A2 transcription activity. Pharmacologic CAR agonist exacerbated bleomycin-induced and TβRI-CA-induced dermal fibrosis in vivo.","method":"siRNA knockdown, forced overexpression, site-directed mutagenesis, reporter assay (COL1A2 promoter), bleomycin and TβRI-CA mouse models, Western blot, qPCR, immunohistochemistry","journal":"Arthritis & rheumatology (Hoboken, N.J.)","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — multiple in vitro methods plus two in vivo fibrosis models; Smad-dependence established by mutagenesis; single lab","pmids":["25155144"],"is_preprint":false},{"year":2022,"finding":"NR1I3 (CAR) activation by TCPOBOP induces STAT3 phosphorylation and nuclear translocation in mouse liver, and this NR1I3-STAT3 signaling pathway promotes hepatocyte proliferation and liver growth, at least in part through upregulation of cMyc and Cyclin D1.","method":"Western blot, immunofluorescence, real-time PCR in TCPOBOP-treated mouse liver","journal":"Molecular biology reports","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — multiple methods (Western blot, immunofluorescence, qPCR) in vivo; no direct epistasis or rescue experiment establishing pathway order; single lab","pmids":["35305226"],"is_preprint":false},{"year":2024,"finding":"Diindole molecules (including diindolylmethane/DIM and diindolylethane/DIE) produced from commensal gut bacteria tryptophan metabolites are endogenous CAR (NR1I3) agonists with nanomolar binding affinities comparable to synthetic agonists. Unlike established synthetic agonists, they activate both rodent and human CAR orthologues. They selectively upregulate bona fide CAR target genes in primary human hepatocytes and mouse liver.","method":"Biophysical binding assays, luciferase reporter assays, primary human hepatocyte gene expression, mouse liver gene expression","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — biophysical binding affinity measurements plus functional activation in both rodent and human CAR systems, validated in primary hepatocytes and in vivo; single lab but multiple orthogonal approaches","pmids":["38519460"],"is_preprint":false},{"year":2024,"finding":"miR-214-3p directly binds the 3'-UTR of NR1I3 mRNA and downregulates NR1I3 expression. Panax notoginseng saponins (PNS) reduce miR-214-3p levels, thereby increasing NR1I3 and CYP2C9 expression and accelerating warfarin metabolism. NR1I3 knockdown rescued the PNS-induced acceleration of warfarin metabolism.","method":"Dual luciferase reporter assay (miR-214-3p binding to NR1I3 3'-UTR), qRT-PCR, immunoblotting, cellular immunofluorescence (NR1I3 localization), siRNA knockdown, rat pharmacokinetic studies","journal":"Journal of ginseng research","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — dual luciferase validates direct miR-214-3p/NR1I3 3'-UTR interaction; NR1I3 KD rescue experiment; multiple methods but single lab","pmids":["39263307"],"is_preprint":false},{"year":2024,"finding":"NR1I3 (CAR) regulates CDH1 (E-cadherin) transcription in intestinal epithelial cells; L. gasseri ATCC33323 affects NR1I3 expression to promote E-cadherin expression, maintaining intestinal barrier integrity in DSS-induced colitis. Intestinal E-cadherin knockdown attenuated the protective effect of L. gasseri, confirming the functional relevance of the NR1I3–CDH1 axis.","method":"DSS-induced colitis mouse model, transcriptional analysis, in vitro experiments, E-cadherin knockdown in vivo","journal":"PLoS pathogens","confidence":"Low","confidence_rationale":"Tier 3 / Weak — transcriptional analysis and in vitro data establish the NR1I3–CDH1 link, but the mechanism of NR1I3 regulation of CDH1 transcription is not directly characterized (no ChIP, no promoter mutagenesis); single lab","pmids":["39250508"],"is_preprint":false},{"year":2025,"finding":"NR1I3 (CAR) directly transactivates the Ribonucleotide Reductase-M2 (RRM2) gene (encoding the rate-limiting catalytic subunit of ribonucleotide reductase), thereby controlling de novo dNTP synthesis in hepatocytes. CAR deletion increases diploid (2c) hepatocytes with reduction of tetraploid (4c) hepatocytes; overexpression of RRM2 in CAR knockouts rescues DNA synthesis and restores tetraploidy. CAR ligand activation induces multiple de novo dNTP synthesis genes and raises hepatic dATP/dTTP levels.","method":"CAR knockout mouse model, RRM2 overexpression rescue experiment, DNA content flow cytometry (ploidy analysis), transactivation assays, dNTP level measurements (mass spectrometry)","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct transactivation plus genetic rescue (RRM2 OE in CAR KO) with biochemical (dNTP) readout; preprint, not yet peer-reviewed","pmids":["bio_10.1101_2025.04.29.651109"],"is_preprint":true},{"year":2025,"finding":"Indole-containing intestinal bacterial metabolites of tryptophan differentially modulate CAR (NR1I3): tryptamine, indole-3-pyruvic acid, and indole-3-ethanol are agonists activating both mouse and human CAR in reporter assays; skatole (3-methylindole) inhibits mouse CAR activity but increases nuclear translocation (in contrast to androstanol inverse agonist which does not induce nuclear translocation), indicating mechanistically distinct modes of CAR inhibition.","method":"Luciferase reporter assay in HepG2 cells, nuclear translocation assay for mouse CAR","journal":"Toxicology letters","confidence":"Low","confidence_rationale":"Tier 3 / Weak — reporter assay plus nuclear translocation assay; single lab, no binding affinity measurements or in vivo validation","pmids":["40947077"],"is_preprint":false},{"year":2025,"finding":"NR1I3 inhibits colorectal cancer cell growth by interacting with PCK1 (phosphoenolpyruvate carboxykinase 1), the rate-limiting enzyme of gluconeogenesis, thereby promoting gluconeogenesis, reducing glycolysis, depleting ATP, and arresting the cell cycle in G2/M phase. Pharmacological NR1I3 activation with CITCO reduced CRC cell growth and induced apoptosis in vitro and in vivo.","method":"Western blot, flow cytometry (cell cycle, apoptosis), colony formation assay, qRT-PCR, gluconeogenesis assays, animal xenograft model, co-immunoprecipitation or co-interaction assay with PCK1","journal":"Chemico-biological interactions","confidence":"Low","confidence_rationale":"Tier 3 / Weak — functional assays establish phenotype and gluconeogenesis connection; NR1I3–PCK1 interaction method not fully specified in abstract; single lab","pmids":["40930396"],"is_preprint":false}],"current_model":"NR1I3/CAR is a constitutively active nuclear receptor that is kept inactive in the cytoplasm via PKC-mediated phosphorylation of Thr38; activated ERK1/2 sustains this inactive state by binding phospho-CAR and repressing dephosphorylation, while dephosphorylation (triggered by phenobarbital and other activators) permits nuclear translocation where CAR heterodimerizes with RXRα to transactivate xenobiotic metabolism genes (CYP2B6, CYP3A4, CYP2C9) and metabolic targets (THRSP, RRM2, PCK1); its expression is positively regulated by glucocorticoid receptor (via a promoter GRE) and PPARα (via a promoter DR1 element), negatively regulated by DAX-1 (a corepressor competing with SRC-1) and miR-214-3p (targeting its 3'-UTR); endogenous gut-microbiota-derived diindoles and tryptophan metabolites serve as direct CAR ligands; and beyond xenobiotic detoxification, CAR regulates hepatic triglyceride levels (via PPARα suppression), hepatocyte ploidy (via RRM2-mediated dNTP synthesis), lipogenesis, and fibroblast TGFβ signaling."},"narrative":{"mechanistic_narrative":"NR1I3 (CAR) is a xenobiotic- and metabolite-sensing nuclear receptor that controls hepatic drug metabolism, lipid handling, and hepatocyte proliferation through ligand- and phosphorylation-gated transcriptional activity [PMID:19858220, PMID:38519460]. In the basal state CAR is held inactive in the cytoplasm by protein kinase C phosphorylation of Thr38, which destabilizes the first zinc-finger helix and blocks DNA binding; dephosphorylation of this residue (triggered by activators such as phenobarbital) is required for nuclear translocation [PMID:19858220], and activated ERK1/2 binds phospho-CAR to repress Thr38 dephosphorylation, sustaining the inactive cytoplasmic pool [PMID:21873423]. Once nuclear, CAR heterodimerizes with RXRα, binds enhancers genome-wide, and deposits H4K5 acetylation at activated genes, while repressing metabolic targets by competing for shared enhancers occupied by HNF4α, PPARα, or FXR [PMID:30396153]; coactivator (SRC-1) recruitment drives transactivation and is opposed by the corepressor DAX-1, which directly binds CAR and blocks the CAR–SRC1 interaction [PMID:16099843, PMID:22896671]. Beyond canonical xenobiotic targets, CAR directly transactivates the lipogenic gene THRSP through a DR-4 element [PMID:20185760] and the ribonucleotide reductase subunit RRM2 to drive de novo dNTP synthesis and hepatocyte ploidy [PMID:bio_10.1101_2025.04.29.651109], suppresses PPARα-driven fatty acid oxidation to control triglyceride levels [PMID:18941143], and is required for hepatic injury/regeneration responses including oval cell proliferation [PMID:21826054] and STAT3-dependent liver growth [PMID:35305226]. CAR expression is itself transcriptionally induced by the glucocorticoid receptor via a promoter GRE [PMID:12511605] and by PPARα via a DR1 element [PMID:18023279], and is post-transcriptionally repressed by miR-214-3p targeting its 3'-UTR [PMID:39263307]. Endogenous agonists include gut-microbiota-derived diindoles and tryptophan metabolites that activate both rodent and human CAR [PMID:38519460].","teleology":[{"year":2003,"claim":"Established that CAR is not a fixed-expression receptor but a transcriptional target itself, placing it downstream of glucocorticoid signaling.","evidence":"Promoter deletion, EMSA, and ChIP showing GR binding to a distal GRE in hepatocytes","pmids":["12511605"],"confidence":"High","gaps":["Does not address how GR-driven CAR induction integrates with ligand activation","Functional consequence for drug metabolism in vivo not tested"]},{"year":2005,"claim":"Resolved how the CAR3 splice variant and RXRα cooperate, showing RXR's AF-2 facilitates coactivator recruitment to CAR.","evidence":"Transient transfection reporter and mammalian two-hybrid assays with domain mutants","pmids":["16099843"],"confidence":"Medium","gaps":["No structural or in vitro reconstitution of the heterodimer","Single lab, transfection-based"]},{"year":2007,"claim":"Identified a second transcriptional input (PPARα) controlling CAR levels and linked CAR induction to nutritional/fasting state.","evidence":"DR1 promoter reporter analysis plus PPARα-deficient mice","pmids":["18023279"],"confidence":"Medium","gaps":["Direct PPARα binding to the DR1 not shown by ChIP","Free fatty acid mediation inferred, not proven"]},{"year":2008,"claim":"Revealed CAR's reciprocal control of PPARα, defining a metabolic role in triglyceride regulation beyond detoxification.","evidence":"CAR knockout mice, TCPOBOP activation, ob/ob and high-fat diet models with gene expression","pmids":["18941143"],"confidence":"Medium","gaps":["Mechanism of PPARα suppression not molecularly defined here","Single lab"]},{"year":2009,"claim":"Defined the molecular switch controlling CAR activity: Thr38 phosphorylation by PKC destabilizes the DNA-binding helix and enforces cytoplasmic retention.","evidence":"In vitro kinase assay, molecular dynamics, helix-stabilizing mutagenesis, phospho-specific immunohistochemistry in human and mouse","pmids":["19858220"],"confidence":"High","gaps":["Phosphatase responsible for activator-triggered dephosphorylation not identified","How phenobarbital triggers dephosphorylation unresolved"]},{"year":2010,"claim":"Showed CAR directly drives a lipogenic gene, demonstrating CAR/RXR binding to a DR-4 element in the THRSP promoter.","evidence":"Promoter reporter with mutations, EMSA with CAR/RXR, CAR-null mice","pmids":["20185760"],"confidence":"Medium","gaps":["In vivo lipogenic phenotype consequences not fully traced","Single lab"]},{"year":2011,"claim":"Explained how the inactive cytoplasmic state is actively maintained: phospho-ERK1/2 binds phospho-CAR to block Thr38 dephosphorylation.","evidence":"Reciprocal Co-IP with phospho-mimetic/dead mutants, shRNA knockdown, MEK inhibition","pmids":["21873423"],"confidence":"High","gaps":["Structural basis of the ERK–CAR interaction not solved","How activators displace ERK not defined"]},{"year":2011,"claim":"Placed CAR upstream of hepatic injury and progenitor (oval cell) responses, extending its role to liver regeneration.","evidence":"DDC-fed CAR knockout mice with nuclear fractionation, marker qPCR, and immunostaining","pmids":["21826054"],"confidence":"Medium","gaps":["Direct CAR target genes driving oval cell proliferation not identified","Single lab"]},{"year":2012,"claim":"Identified DAX-1 as a direct CAR corepressor acting by disrupting the CAR–SRC1 coactivator interaction.","evidence":"Alpha-screen and Co-IP binding, domain mutagenesis, primary human hepatocyte CYP2B6 assays","pmids":["22896671"],"confidence":"High","gaps":["Physiological contexts where DAX-1 represses CAR in vivo not defined","Single lab"]},{"year":2014,"claim":"Connected CAR to fibrosis, showing TGFβ/Smad induces CAR which then amplifies collagen synthesis in fibroblasts.","evidence":"siRNA, overexpression, COL1A2 reporter mutagenesis, bleomycin and TβRI-CA mouse fibrosis models","pmids":["25155144"],"confidence":"Medium","gaps":["Direct CAR binding to fibrotic gene promoters not mapped","Single lab"]},{"year":2018,"claim":"Defined the genome-wide mechanism of CAR-mediated repression: enhancer competition with other nuclear receptors on shared sites.","evidence":"ChIP-seq for CAR, RXRα, and H4K5Ac in TCPOBOP-treated mouse liver","pmids":["30396153"],"confidence":"Medium","gaps":["No mutagenesis validation of specific competed enhancers","Single lab"]},{"year":2022,"claim":"Linked CAR activation to a STAT3-cMyc-Cyclin D1 axis driving hepatocyte proliferation and liver growth.","evidence":"Western blot, immunofluorescence, qPCR in TCPOBOP-treated mouse liver","pmids":["35305226"],"confidence":"Medium","gaps":["No epistasis or rescue establishing pathway order","Direct vs indirect STAT3 activation unresolved"]},{"year":2024,"claim":"Identified endogenous gut-microbiota-derived diindoles as high-affinity CAR agonists active on both rodent and human receptors.","evidence":"Biophysical binding, luciferase reporters, primary human hepatocyte and mouse liver gene expression","pmids":["38519460"],"confidence":"High","gaps":["In vivo physiological role of microbial diindoles in CAR signaling not established","Single lab"]},{"year":2024,"claim":"Established post-transcriptional control of CAR by miR-214-3p, with functional consequences for warfarin metabolism.","evidence":"Dual luciferase 3'-UTR binding, qRT-PCR, siRNA rescue, rat pharmacokinetics","pmids":["39263307"],"confidence":"Medium","gaps":["Endogenous regulators of miR-214-3p in liver not defined","Single lab"]},{"year":2024,"claim":"Linked CAR to intestinal barrier integrity via regulation of CDH1/E-cadherin transcription.","evidence":"DSS colitis model, transcriptional analysis, in vivo E-cadherin knockdown","pmids":["39250508"],"confidence":"Low","gaps":["Mechanism of CAR regulation of CDH1 not characterized (no ChIP or promoter mutagenesis)","Single lab","Direct vs indirect transcriptional effect unknown"]},{"year":2025,"claim":"Showed CAR directly transactivates RRM2 to control de novo dNTP synthesis and hepatocyte ploidy.","evidence":"CAR knockout mice, RRM2 overexpression rescue, ploidy flow cytometry, transactivation assays, dNTP mass spectrometry (preprint)","pmids":["bio_10.1101_2025.04.29.651109"],"confidence":"Medium","gaps":["Preprint, not yet peer-reviewed","Functional importance of CAR-driven ploidy for liver function unresolved"]},{"year":2025,"claim":"Expanded the endogenous ligand repertoire and revealed mechanistically distinct modes of CAR inhibition among indole metabolites.","evidence":"Luciferase reporters and nuclear translocation assays in HepG2 cells","pmids":["40947077"],"confidence":"Low","gaps":["No binding affinity measurements or in vivo validation","Structural basis of skatole's translocation-inducing inhibition unknown"]},{"year":2025,"claim":"Implicated CAR in colorectal cancer suppression through interaction with PCK1, shifting metabolism toward gluconeogenesis and arresting the cell cycle.","evidence":"Functional assays, gluconeogenesis measurements, xenograft model, CAR–PCK1 co-interaction assay","pmids":["40930396"],"confidence":"Low","gaps":["CAR–PCK1 interaction method not fully specified","Whether interaction is direct and stoichiometric unclear","Single lab"]},{"year":null,"claim":"The phosphatase that dephosphorylates Thr38 upon activator binding and the structural basis for ligand-gated nuclear translocation remain unidentified.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No phosphatase identified for activator-triggered Thr38 dephosphorylation","No structural model linking ligand binding to release of cytoplasmic retention"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[2,6,9,15]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[0,6]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[12]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,1]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,9]}],"pathway":[{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[2,6]},{"term_id":"R-HSA-9748784","term_label":"Drug ADME","supporting_discovery_ids":[13]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[5,6]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[2,9]}],"complexes":["CAR/RXRα heterodimer"],"partners":["RXRA","NR0B1","MAPK1","MAPK3","NCOA1","PCK1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q14994","full_name":"Nuclear receptor subfamily 1 group I member 3","aliases":["Constitutive activator of retinoid response","Constitutive active response","Constitutive androstane receptor","CAR","Orphan nuclear receptor MB67"],"length_aa":352,"mass_kda":39.9,"function":"Binds and transactivates the retinoic acid response elements that control expression of the retinoic acid receptor beta 2 and alcohol dehydrogenase 3 genes. Transactivates both the phenobarbital responsive element module of the human CYP2B6 gene and the CYP3A4 xenobiotic response element","subcellular_location":"Nucleus; Cytoplasm; Cytoplasm, cytoskeleton","url":"https://www.uniprot.org/uniprotkb/Q14994/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/NR1I3","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/NR1I3","total_profiled":1310},"omim":[{"mim_id":"617768","title":"KLEEFSTRA SYNDROME 2; KLEFS2","url":"https://www.omim.org/entry/617768"},{"mim_id":"616936","title":"CHROMODOMAIN HELICASE DNA-BINDING PROTEIN 9; CHD9","url":"https://www.omim.org/entry/616936"},{"mim_id":"610253","title":"KLEEFSTRA SYNDROME 1; KLEFS1","url":"https://www.omim.org/entry/610253"},{"mim_id":"606833","title":"LYSINE-SPECIFIC METHYLTRANSFERASE 2C; KMT2C","url":"https://www.omim.org/entry/606833"},{"mim_id":"604630","title":"NUCLEAR RECEPTOR SUBFAMILY 0, GROUP B, MEMBER 2; NR0B2","url":"https://www.omim.org/entry/604630"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"}],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"liver","ntpm":158.8}],"url":"https://www.proteinatlas.org/search/NR1I3"},"hgnc":{"alias_symbol":["MB67","CAR1","CAR"],"prev_symbol":[]},"alphafold":{"accession":"Q14994","domains":[{"cath_id":"3.30.50.10","chopping":"21-101","consensus_level":"high","plddt":93.2059,"start":21,"end":101},{"cath_id":"1.10.565.10","chopping":"109-352","consensus_level":"high","plddt":91.3401,"start":109,"end":352}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q14994","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q14994-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q14994-F1-predicted_aligned_error_v6.png","plddt_mean":90.81},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=NR1I3","jax_strain_url":"https://www.jax.org/strain/search?query=NR1I3"},"sequence":{"accession":"Q14994","fasta_url":"https://rest.uniprot.org/uniprotkb/Q14994.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q14994/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q14994"}},"corpus_meta":[{"pmid":"19858220","id":"PMC_19858220","title":"Dephosphorylation of threonine 38 is required for nuclear translocation and activation of human xenobiotic receptor CAR (NR1I3).","date":"2009","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19858220","citation_count":109,"is_preprint":false},{"pmid":"16101575","id":"PMC_16101575","title":"Genetic variants of PXR (NR1I2) and CAR (NR1I3) and their implications in drug metabolism and pharmacogenetics.","date":"2005","source":"Current drug metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/16101575","citation_count":98,"is_preprint":false},{"pmid":"12511605","id":"PMC_12511605","title":"Transcriptional analysis of the orphan nuclear receptor constitutive androstane receptor (NR1I3) gene promoter: identification of a distal glucocorticoid response element.","date":"2003","source":"Molecular endocrinology (Baltimore, Md.)","url":"https://pubmed.ncbi.nlm.nih.gov/12511605","citation_count":94,"is_preprint":false},{"pmid":"18941143","id":"PMC_18941143","title":"The nuclear receptor CAR (NR1I3) regulates serum triglyceride levels under conditions of metabolic stress.","date":"2008","source":"Journal of lipid research","url":"https://pubmed.ncbi.nlm.nih.gov/18941143","citation_count":93,"is_preprint":false},{"pmid":"16099843","id":"PMC_16099843","title":"Retinoid X receptor-alpha-dependent transactivation by a naturally occurring structural variant of human constitutive androstane receptor (NR1I3).","date":"2005","source":"Molecular pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/16099843","citation_count":72,"is_preprint":false},{"pmid":"21873423","id":"PMC_21873423","title":"Active ERK1/2 protein interacts with the phosphorylated nuclear constitutive active/androstane receptor (CAR; 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the fate of foreign compounds in biological systems","url":"https://pubmed.ncbi.nlm.nih.gov/17118917","citation_count":8,"is_preprint":false},{"pmid":"25714878","id":"PMC_25714878","title":"Expression of NR1I3 in mouse lung tumors induced by the tobacco-specific nitrosamine 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanone.","date":"2015","source":"Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas","url":"https://pubmed.ncbi.nlm.nih.gov/25714878","citation_count":6,"is_preprint":false},{"pmid":"34504302","id":"PMC_34504302","title":"Polymorphisms at CYP enzymes, NR1I2 and NR1I3 in association with virologic response to antiretroviral therapy in Brazilian HIV-positive individuals.","date":"2021","source":"The pharmacogenomics journal","url":"https://pubmed.ncbi.nlm.nih.gov/34504302","citation_count":6,"is_preprint":false},{"pmid":"29219065","id":"PMC_29219065","title":"Transactivation Assays that Identify Indirect and Direct Activators of Human Pregnane X Receptor (PXR, NR1I2) and Constitutive Androstane Receptor (CAR, NR1I3).","date":"2017","source":"Drug metabolism letters","url":"https://pubmed.ncbi.nlm.nih.gov/29219065","citation_count":6,"is_preprint":false},{"pmid":"26188155","id":"PMC_26188155","title":"High resolution melting analysis of the NR1I3 genetic variants: Is there an association with neonatal hyperbilirubinemia?","date":"2015","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/26188155","citation_count":3,"is_preprint":false},{"pmid":"35305226","id":"PMC_35305226","title":"Promotion of NR1I3-mediated liver growth is accompanied by STAT3 activation.","date":"2022","source":"Molecular biology reports","url":"https://pubmed.ncbi.nlm.nih.gov/35305226","citation_count":2,"is_preprint":false},{"pmid":"39884819","id":"PMC_39884819","title":"Isoform-level expression of the constitutive androstane receptor (CAR or NR1I3) transcription factor better predicts the mRNA expression of the cytochrome P450s in human liver samples.","date":"2024","source":"Drug metabolism and disposition: the biological fate of chemicals","url":"https://pubmed.ncbi.nlm.nih.gov/39884819","citation_count":1,"is_preprint":false},{"pmid":"40739628","id":"PMC_40739628","title":"NR1I3 modulates Wnt signaling to promote anterior-posterior axis patterning.","date":"2025","source":"BMC biology","url":"https://pubmed.ncbi.nlm.nih.gov/40739628","citation_count":0,"is_preprint":false},{"pmid":"40930396","id":"PMC_40930396","title":"NR1I3 inhibits colorectal cancer growth by enhancing PCK1-mediated gluconeogenesis.","date":"2025","source":"Chemico-biological interactions","url":"https://pubmed.ncbi.nlm.nih.gov/40930396","citation_count":0,"is_preprint":false},{"pmid":"39263307","id":"PMC_39263307","title":"Mechanism of Panax notoginseng saponins modulation of miR-214-3p/NR1I3 affecting the pharmacodynamics and pharmacokinetics of warfarin.","date":"2024","source":"Journal of ginseng research","url":"https://pubmed.ncbi.nlm.nih.gov/39263307","citation_count":0,"is_preprint":false},{"pmid":"39512799","id":"PMC_39512799","title":"Roles of NR1I3 and NR1H4 polymorphisms in the susceptibility to antituberculosis drug-induced liver injury in China: a case‒control study.","date":"2024","source":"Frontiers in genetics","url":"https://pubmed.ncbi.nlm.nih.gov/39512799","citation_count":0,"is_preprint":false},{"pmid":"40947077","id":"PMC_40947077","title":"Distinct responses of the constitutive androstane receptor NR1I3 to indole-containing metabolites of bacterial origin.","date":"2025","source":"Toxicology letters","url":"https://pubmed.ncbi.nlm.nih.gov/40947077","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.04.29.651109","title":"Constitutive Androstane Receptor induces Ribonucleotide Reductase-M2 expression and maintains hepatocyte ploidy in mice","date":"2025-05-02","source":"bioRxiv","url":"https://doi.org/10.1101/2025.04.29.651109","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":20704,"output_tokens":5411,"usd":0.071638,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":13972,"output_tokens":4939,"usd":0.096668,"stage2_stop_reason":"end_turn"},"total_usd":0.168306,"stage1_batch_id":"msgbatch_013a8chtv3AWyTqv4z4sEF4C","stage2_batch_id":"msgbatch_01AHsuW63vinsvuTgzmQ3n62","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2009,\n      \"finding\": \"Protein kinase C phosphorylates threonine 38 of human CAR (NR1I3), which destabilizes the alpha-helix spanning residues 29-42 (part of the first zinc finger), thereby inactivating CAR binding to DNA and sequestering it in the cytoplasm. Dephosphorylation of Thr38 is required for nuclear translocation and activation. Phenobarbital dephosphorylates the corresponding Thr48 of mouse CAR in the cytoplasm, enabling nuclear translocation.\",\n      \"method\": \"In vitro kinase assay, molecular dynamics simulation, helix-stabilizing mutagenesis, immunohistochemistry with anti-phospho-Thr38 antibody, co-immunoprecipitation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods including in vitro kinase assay, mutagenesis, molecular dynamics, and immunohistochemistry; replicated in both human and mouse systems\",\n      \"pmids\": [\"19858220\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Activated (phosphorylated) ERK1/2 interacts with phosphorylated CAR (NR1I3) at Thr-38 via the xenochemical response signal peptide near the C-terminus of CAR, repressing dephosphorylation of Thr-38 and thereby maintaining CAR in its inactive cytoplasmic state. EGF treatment increased this interaction, while MEK inhibitor U0126 or MEK1/2 knockdown decreased Thr-38 phosphorylation.\",\n      \"method\": \"Co-immunoprecipitation of FLAG-tagged CAR mutants (T38A, T38D) with endogenous phospho-ERK1/2; shRNA knockdown; pharmacological inhibition (U0126)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP with phospho-mimetic and phospho-dead mutants, confirmed by both pharmacological inhibition and shRNA knockdown in a single rigorous study\",\n      \"pmids\": [\"21873423\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"The human CAR (NR1I3) gene promoter contains a functional glucocorticoid response element (GRE) at position -4447/-4432 that is recognized and transactivated by the glucocorticoid receptor (GR) in the presence of dexamethasone. Chromatin immunoprecipitation confirmed GR binding to this distal promoter region in cultured hepatocytes, establishing CAR as a primary GR-response gene.\",\n      \"method\": \"Deletion analysis and transient transfection, site-directed mutagenesis, gel shift assay (EMSA), chromatin immunoprecipitation (ChIP), cotransfection experiments, 5'-RACE, primer extension\",\n      \"journal\": \"Molecular endocrinology (Baltimore, Md.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — multiple orthogonal methods (transfection, mutagenesis, EMSA, ChIP) in a single rigorous study establishing direct GR binding and transactivation\",\n      \"pmids\": [\"12511605\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The human CAR splice variant CAR3 (with a 5 amino acid insertion in the ligand-binding domain) is ligand-activated (by CITCO), in contrast to the constitutively active reference form CAR1. CAR3 transactivation requires its DNA-binding domain and AF-2 motif. RXRα co-transfection markedly enhances CAR3 activity via RXR's AF-2 function (but independently of RXR A/B and C domains/heterodimerization region), by facilitating coactivator (SRC-1) recruitment. Clotrimazole acts as a ligand activator of CAR3, whereas it is an inverse agonist of CAR1.\",\n      \"method\": \"Transient transfection reporter assays, domain deletion/mutation analysis, mammalian two-hybrid assay\",\n      \"journal\": \"Molecular pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — multiple transfection and two-hybrid assays in a single lab; no in vitro reconstitution or structural validation\",\n      \"pmids\": [\"16099843\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"PPARα directly induces CAR (NR1I3) transcription in rat hepatocytes via a DR1 motif in the CAR promoter. This PPARα-dependent induction of CAR potentiates phenobarbital-induced transcription of the CAR target gene CYP2B1. Fasting-induced CAR expression was abrogated in PPARα-deficient mice, suggesting free fatty acids (PPARα ligands) mediate fasting-induced CAR upregulation.\",\n      \"method\": \"Promoter reporter assays with DR1 motif deletion, PPARα agonist treatment (WY14643) in rat hepatocytes, PPARα-deficient mouse model\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — promoter deletion analysis plus in vivo genetic model (PPARα KO mice), but single lab\",\n      \"pmids\": [\"18023279\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"CAR (NR1I3) regulates serum triglyceride levels under metabolic stress. CAR activity inversely regulates PPARα target gene expression; CAR activation (by TCPOBOP) decreases PPARα mRNA and suppresses hepatic fatty acid oxidation genes (Cyp4a14, CPT1α, CTE), whereas CAR-deficient mice show increased hepatic fatty acid oxidation and are protected from hypertriglyceridemia in ob/ob and high-fat diet models.\",\n      \"method\": \"CAR knockout mouse model, pharmacological activation (TCPOBOP), ob/ob × Car(-/-) cross, high-fat diet feeding, gene expression analysis\",\n      \"journal\": \"Journal of lipid research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean genetic KO with defined molecular phenotype (PPARα pathway suppression), confirmed pharmacologically; single lab\",\n      \"pmids\": [\"18941143\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"CAR (NR1I3) directly transactivates the lipogenic gene THRSP (Spot14/S14) promoter through a DR-4 thyroid hormone/PXR response element. Gel-shift analysis showed that the CAR/RXR heterodimer complex binds this element. Deletion or point mutation of this element abolished CAR-mediated transactivation. CAR-null mice did not show mCAR-activator-induced THRSP upregulation.\",\n      \"method\": \"Promoter reporter assay with deletion/point mutations, gel shift (EMSA) with CAR/RXR complex, CAR null mice with pharmacological activators, human hepatocyte treatment\",\n      \"journal\": \"Endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — EMSA plus mutagenesis plus in vivo genetic model; single lab\",\n      \"pmids\": [\"20185760\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"CAR (NR1I3) is essential for DDC-induced liver injury and oval cell proliferation. DDC activates CAR (shown by nuclear CAR accumulation and CYP2B10 induction), and Car(-/-) mice fail to develop DDC-induced liver injury, ductular reaction, or oval cell proliferation, placing CAR upstream of these hepatic injury/regeneration responses.\",\n      \"method\": \"CAR knockout mouse model fed DDC diet, nuclear fractionation (CAR accumulation), real-time PCR (CYP2B10, oval cell markers), laser capture microdissection, immunostaining (A6 antibody)\",\n      \"journal\": \"Laboratory investigation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean genetic KO with specific molecular and cellular readouts; single lab\",\n      \"pmids\": [\"21826054\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"DAX-1 (an orphan nuclear receptor) functions as a potent corepressor of human CAR (NR1I3). DAX-1's downstream LXXLL and PCFQVLP motifs are critical for corepression; DAX-1's C-terminal transcription silencing domain mediates the repression by inhibiting the CAR–SRC1 coactivator interaction (~50% inhibition). Direct CAR–DAX-1 interaction was demonstrated by alpha-screen and co-immunoprecipitation, enhanced by the CAR activator CITCO. DAX-1 inhibits CAR-mediated CITCO induction of CYP2B6 in primary human hepatocytes.\",\n      \"method\": \"Transactivation assays, domain deletion/mutagenesis, mammalian two-hybrid assay, alpha-screen, co-immunoprecipitation, primary human hepatocyte experiments\",\n      \"journal\": \"Molecular pharmacology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct protein interaction confirmed by two independent binding assays (alpha-screen and Co-IP), functional validation by mutagenesis and primary hepatocyte experiments; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"22896671\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Drug-activated CAR (NR1I3) binds genome-wide with its partner RXRα and induces H4K5 acetylation at stimulated genes. Transcriptional inhibition by CAR occurs when CAR binds the same enhancers occupied by HNF4α, PPARα, or FXR, suggesting functional competition among nuclear receptors on shared enhancers as the mechanism of CAR-mediated metabolic gene repression.\",\n      \"method\": \"Genome-wide ChIP-seq for CAR, RXRα, and H4K5Ac in mouse liver after TCPOBOP treatment\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genome-wide ChIP-seq providing mechanistic insight into enhancer competition; single lab, no mutagenesis validation of specific sites\",\n      \"pmids\": [\"30396153\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TGFβ induces CAR (NR1I3) expression in dermal fibroblasts via a Smad-dependent mechanism. CAR activation amplifies TGFβ-stimulated collagen synthesis, myofibroblast differentiation, and COL1A2 transcription activity. Pharmacologic CAR agonist exacerbated bleomycin-induced and TβRI-CA-induced dermal fibrosis in vivo.\",\n      \"method\": \"siRNA knockdown, forced overexpression, site-directed mutagenesis, reporter assay (COL1A2 promoter), bleomycin and TβRI-CA mouse models, Western blot, qPCR, immunohistochemistry\",\n      \"journal\": \"Arthritis & rheumatology (Hoboken, N.J.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — multiple in vitro methods plus two in vivo fibrosis models; Smad-dependence established by mutagenesis; single lab\",\n      \"pmids\": [\"25155144\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"NR1I3 (CAR) activation by TCPOBOP induces STAT3 phosphorylation and nuclear translocation in mouse liver, and this NR1I3-STAT3 signaling pathway promotes hepatocyte proliferation and liver growth, at least in part through upregulation of cMyc and Cyclin D1.\",\n      \"method\": \"Western blot, immunofluorescence, real-time PCR in TCPOBOP-treated mouse liver\",\n      \"journal\": \"Molecular biology reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — multiple methods (Western blot, immunofluorescence, qPCR) in vivo; no direct epistasis or rescue experiment establishing pathway order; single lab\",\n      \"pmids\": [\"35305226\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Diindole molecules (including diindolylmethane/DIM and diindolylethane/DIE) produced from commensal gut bacteria tryptophan metabolites are endogenous CAR (NR1I3) agonists with nanomolar binding affinities comparable to synthetic agonists. Unlike established synthetic agonists, they activate both rodent and human CAR orthologues. They selectively upregulate bona fide CAR target genes in primary human hepatocytes and mouse liver.\",\n      \"method\": \"Biophysical binding assays, luciferase reporter assays, primary human hepatocyte gene expression, mouse liver gene expression\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — biophysical binding affinity measurements plus functional activation in both rodent and human CAR systems, validated in primary hepatocytes and in vivo; single lab but multiple orthogonal approaches\",\n      \"pmids\": [\"38519460\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"miR-214-3p directly binds the 3'-UTR of NR1I3 mRNA and downregulates NR1I3 expression. Panax notoginseng saponins (PNS) reduce miR-214-3p levels, thereby increasing NR1I3 and CYP2C9 expression and accelerating warfarin metabolism. NR1I3 knockdown rescued the PNS-induced acceleration of warfarin metabolism.\",\n      \"method\": \"Dual luciferase reporter assay (miR-214-3p binding to NR1I3 3'-UTR), qRT-PCR, immunoblotting, cellular immunofluorescence (NR1I3 localization), siRNA knockdown, rat pharmacokinetic studies\",\n      \"journal\": \"Journal of ginseng research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — dual luciferase validates direct miR-214-3p/NR1I3 3'-UTR interaction; NR1I3 KD rescue experiment; multiple methods but single lab\",\n      \"pmids\": [\"39263307\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"NR1I3 (CAR) regulates CDH1 (E-cadherin) transcription in intestinal epithelial cells; L. gasseri ATCC33323 affects NR1I3 expression to promote E-cadherin expression, maintaining intestinal barrier integrity in DSS-induced colitis. Intestinal E-cadherin knockdown attenuated the protective effect of L. gasseri, confirming the functional relevance of the NR1I3–CDH1 axis.\",\n      \"method\": \"DSS-induced colitis mouse model, transcriptional analysis, in vitro experiments, E-cadherin knockdown in vivo\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — transcriptional analysis and in vitro data establish the NR1I3–CDH1 link, but the mechanism of NR1I3 regulation of CDH1 transcription is not directly characterized (no ChIP, no promoter mutagenesis); single lab\",\n      \"pmids\": [\"39250508\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NR1I3 (CAR) directly transactivates the Ribonucleotide Reductase-M2 (RRM2) gene (encoding the rate-limiting catalytic subunit of ribonucleotide reductase), thereby controlling de novo dNTP synthesis in hepatocytes. CAR deletion increases diploid (2c) hepatocytes with reduction of tetraploid (4c) hepatocytes; overexpression of RRM2 in CAR knockouts rescues DNA synthesis and restores tetraploidy. CAR ligand activation induces multiple de novo dNTP synthesis genes and raises hepatic dATP/dTTP levels.\",\n      \"method\": \"CAR knockout mouse model, RRM2 overexpression rescue experiment, DNA content flow cytometry (ploidy analysis), transactivation assays, dNTP level measurements (mass spectrometry)\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct transactivation plus genetic rescue (RRM2 OE in CAR KO) with biochemical (dNTP) readout; preprint, not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.04.29.651109\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Indole-containing intestinal bacterial metabolites of tryptophan differentially modulate CAR (NR1I3): tryptamine, indole-3-pyruvic acid, and indole-3-ethanol are agonists activating both mouse and human CAR in reporter assays; skatole (3-methylindole) inhibits mouse CAR activity but increases nuclear translocation (in contrast to androstanol inverse agonist which does not induce nuclear translocation), indicating mechanistically distinct modes of CAR inhibition.\",\n      \"method\": \"Luciferase reporter assay in HepG2 cells, nuclear translocation assay for mouse CAR\",\n      \"journal\": \"Toxicology letters\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — reporter assay plus nuclear translocation assay; single lab, no binding affinity measurements or in vivo validation\",\n      \"pmids\": [\"40947077\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NR1I3 inhibits colorectal cancer cell growth by interacting with PCK1 (phosphoenolpyruvate carboxykinase 1), the rate-limiting enzyme of gluconeogenesis, thereby promoting gluconeogenesis, reducing glycolysis, depleting ATP, and arresting the cell cycle in G2/M phase. Pharmacological NR1I3 activation with CITCO reduced CRC cell growth and induced apoptosis in vitro and in vivo.\",\n      \"method\": \"Western blot, flow cytometry (cell cycle, apoptosis), colony formation assay, qRT-PCR, gluconeogenesis assays, animal xenograft model, co-immunoprecipitation or co-interaction assay with PCK1\",\n      \"journal\": \"Chemico-biological interactions\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — functional assays establish phenotype and gluconeogenesis connection; NR1I3–PCK1 interaction method not fully specified in abstract; single lab\",\n      \"pmids\": [\"40930396\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NR1I3/CAR is a constitutively active nuclear receptor that is kept inactive in the cytoplasm via PKC-mediated phosphorylation of Thr38; activated ERK1/2 sustains this inactive state by binding phospho-CAR and repressing dephosphorylation, while dephosphorylation (triggered by phenobarbital and other activators) permits nuclear translocation where CAR heterodimerizes with RXRα to transactivate xenobiotic metabolism genes (CYP2B6, CYP3A4, CYP2C9) and metabolic targets (THRSP, RRM2, PCK1); its expression is positively regulated by glucocorticoid receptor (via a promoter GRE) and PPARα (via a promoter DR1 element), negatively regulated by DAX-1 (a corepressor competing with SRC-1) and miR-214-3p (targeting its 3'-UTR); endogenous gut-microbiota-derived diindoles and tryptophan metabolites serve as direct CAR ligands; and beyond xenobiotic detoxification, CAR regulates hepatic triglyceride levels (via PPARα suppression), hepatocyte ploidy (via RRM2-mediated dNTP synthesis), lipogenesis, and fibroblast TGFβ signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"NR1I3 (CAR) is a xenobiotic- and metabolite-sensing nuclear receptor that controls hepatic drug metabolism, lipid handling, and hepatocyte proliferation through ligand- and phosphorylation-gated transcriptional activity [#0, #12]. In the basal state CAR is held inactive in the cytoplasm by protein kinase C phosphorylation of Thr38, which destabilizes the first zinc-finger helix and blocks DNA binding; dephosphorylation of this residue (triggered by activators such as phenobarbital) is required for nuclear translocation [#0], and activated ERK1/2 binds phospho-CAR to repress Thr38 dephosphorylation, sustaining the inactive cytoplasmic pool [#1]. Once nuclear, CAR heterodimerizes with RXR\\u03b1, binds enhancers genome-wide, and deposits H4K5 acetylation at activated genes, while repressing metabolic targets by competing for shared enhancers occupied by HNF4\\u03b1, PPAR\\u03b1, or FXR [#9]; coactivator (SRC-1) recruitment drives transactivation and is opposed by the corepressor DAX-1, which directly binds CAR and blocks the CAR\\u2013SRC1 interaction [#3, #8]. Beyond canonical xenobiotic targets, CAR directly transactivates the lipogenic gene THRSP through a DR-4 element [#6] and the ribonucleotide reductase subunit RRM2 to drive de novo dNTP synthesis and hepatocyte ploidy [#15], suppresses PPAR\\u03b1-driven fatty acid oxidation to control triglyceride levels [#5], and is required for hepatic injury/regeneration responses including oval cell proliferation [#7] and STAT3-dependent liver growth [#11]. CAR expression is itself transcriptionally induced by the glucocorticoid receptor via a promoter GRE [#2] and by PPAR\\u03b1 via a DR1 element [#4], and is post-transcriptionally repressed by miR-214-3p targeting its 3'-UTR [#13]. Endogenous agonists include gut-microbiota-derived diindoles and tryptophan metabolites that activate both rodent and human CAR [#12].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Established that CAR is not a fixed-expression receptor but a transcriptional target itself, placing it downstream of glucocorticoid signaling.\",\n      \"evidence\": \"Promoter deletion, EMSA, and ChIP showing GR binding to a distal GRE in hepatocytes\",\n      \"pmids\": [\"12511605\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not address how GR-driven CAR induction integrates with ligand activation\", \"Functional consequence for drug metabolism in vivo not tested\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Resolved how the CAR3 splice variant and RXR\\u03b1 cooperate, showing RXR's AF-2 facilitates coactivator recruitment to CAR.\",\n      \"evidence\": \"Transient transfection reporter and mammalian two-hybrid assays with domain mutants\",\n      \"pmids\": [\"16099843\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural or in vitro reconstitution of the heterodimer\", \"Single lab, transfection-based\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Identified a second transcriptional input (PPAR\\u03b1) controlling CAR levels and linked CAR induction to nutritional/fasting state.\",\n      \"evidence\": \"DR1 promoter reporter analysis plus PPAR\\u03b1-deficient mice\",\n      \"pmids\": [\"18023279\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct PPAR\\u03b1 binding to the DR1 not shown by ChIP\", \"Free fatty acid mediation inferred, not proven\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Revealed CAR's reciprocal control of PPAR\\u03b1, defining a metabolic role in triglyceride regulation beyond detoxification.\",\n      \"evidence\": \"CAR knockout mice, TCPOBOP activation, ob/ob and high-fat diet models with gene expression\",\n      \"pmids\": [\"18941143\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of PPAR\\u03b1 suppression not molecularly defined here\", \"Single lab\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Defined the molecular switch controlling CAR activity: Thr38 phosphorylation by PKC destabilizes the DNA-binding helix and enforces cytoplasmic retention.\",\n      \"evidence\": \"In vitro kinase assay, molecular dynamics, helix-stabilizing mutagenesis, phospho-specific immunohistochemistry in human and mouse\",\n      \"pmids\": [\"19858220\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Phosphatase responsible for activator-triggered dephosphorylation not identified\", \"How phenobarbital triggers dephosphorylation unresolved\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Showed CAR directly drives a lipogenic gene, demonstrating CAR/RXR binding to a DR-4 element in the THRSP promoter.\",\n      \"evidence\": \"Promoter reporter with mutations, EMSA with CAR/RXR, CAR-null mice\",\n      \"pmids\": [\"20185760\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo lipogenic phenotype consequences not fully traced\", \"Single lab\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Explained how the inactive cytoplasmic state is actively maintained: phospho-ERK1/2 binds phospho-CAR to block Thr38 dephosphorylation.\",\n      \"evidence\": \"Reciprocal Co-IP with phospho-mimetic/dead mutants, shRNA knockdown, MEK inhibition\",\n      \"pmids\": [\"21873423\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the ERK\\u2013CAR interaction not solved\", \"How activators displace ERK not defined\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Placed CAR upstream of hepatic injury and progenitor (oval cell) responses, extending its role to liver regeneration.\",\n      \"evidence\": \"DDC-fed CAR knockout mice with nuclear fractionation, marker qPCR, and immunostaining\",\n      \"pmids\": [\"21826054\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct CAR target genes driving oval cell proliferation not identified\", \"Single lab\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identified DAX-1 as a direct CAR corepressor acting by disrupting the CAR\\u2013SRC1 coactivator interaction.\",\n      \"evidence\": \"Alpha-screen and Co-IP binding, domain mutagenesis, primary human hepatocyte CYP2B6 assays\",\n      \"pmids\": [\"22896671\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological contexts where DAX-1 represses CAR in vivo not defined\", \"Single lab\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Connected CAR to fibrosis, showing TGF\\u03b2/Smad induces CAR which then amplifies collagen synthesis in fibroblasts.\",\n      \"evidence\": \"siRNA, overexpression, COL1A2 reporter mutagenesis, bleomycin and T\\u03b2RI-CA mouse fibrosis models\",\n      \"pmids\": [\"25155144\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct CAR binding to fibrotic gene promoters not mapped\", \"Single lab\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined the genome-wide mechanism of CAR-mediated repression: enhancer competition with other nuclear receptors on shared sites.\",\n      \"evidence\": \"ChIP-seq for CAR, RXR\\u03b1, and H4K5Ac in TCPOBOP-treated mouse liver\",\n      \"pmids\": [\"30396153\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No mutagenesis validation of specific competed enhancers\", \"Single lab\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Linked CAR activation to a STAT3-cMyc-Cyclin D1 axis driving hepatocyte proliferation and liver growth.\",\n      \"evidence\": \"Western blot, immunofluorescence, qPCR in TCPOBOP-treated mouse liver\",\n      \"pmids\": [\"35305226\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No epistasis or rescue establishing pathway order\", \"Direct vs indirect STAT3 activation unresolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified endogenous gut-microbiota-derived diindoles as high-affinity CAR agonists active on both rodent and human receptors.\",\n      \"evidence\": \"Biophysical binding, luciferase reporters, primary human hepatocyte and mouse liver gene expression\",\n      \"pmids\": [\"38519460\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo physiological role of microbial diindoles in CAR signaling not established\", \"Single lab\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Established post-transcriptional control of CAR by miR-214-3p, with functional consequences for warfarin metabolism.\",\n      \"evidence\": \"Dual luciferase 3'-UTR binding, qRT-PCR, siRNA rescue, rat pharmacokinetics\",\n      \"pmids\": [\"39263307\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Endogenous regulators of miR-214-3p in liver not defined\", \"Single lab\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Linked CAR to intestinal barrier integrity via regulation of CDH1/E-cadherin transcription.\",\n      \"evidence\": \"DSS colitis model, transcriptional analysis, in vivo E-cadherin knockdown\",\n      \"pmids\": [\"39250508\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Mechanism of CAR regulation of CDH1 not characterized (no ChIP or promoter mutagenesis)\", \"Single lab\", \"Direct vs indirect transcriptional effect unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Showed CAR directly transactivates RRM2 to control de novo dNTP synthesis and hepatocyte ploidy.\",\n      \"evidence\": \"CAR knockout mice, RRM2 overexpression rescue, ploidy flow cytometry, transactivation assays, dNTP mass spectrometry (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.04.29.651109\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, not yet peer-reviewed\", \"Functional importance of CAR-driven ploidy for liver function unresolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Expanded the endogenous ligand repertoire and revealed mechanistically distinct modes of CAR inhibition among indole metabolites.\",\n      \"evidence\": \"Luciferase reporters and nuclear translocation assays in HepG2 cells\",\n      \"pmids\": [\"40947077\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No binding affinity measurements or in vivo validation\", \"Structural basis of skatole's translocation-inducing inhibition unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Implicated CAR in colorectal cancer suppression through interaction with PCK1, shifting metabolism toward gluconeogenesis and arresting the cell cycle.\",\n      \"evidence\": \"Functional assays, gluconeogenesis measurements, xenograft model, CAR\\u2013PCK1 co-interaction assay\",\n      \"pmids\": [\"40930396\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"CAR\\u2013PCK1 interaction method not fully specified\", \"Whether interaction is direct and stoichiometric unclear\", \"Single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The phosphatase that dephosphorylates Thr38 upon activator binding and the structural basis for ligand-gated nuclear translocation remain unidentified.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No phosphatase identified for activator-triggered Thr38 dephosphorylation\", \"No structural model linking ligand binding to release of cytoplasmic retention\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [2, 6, 9, 15]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [0, 6]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [12]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 9]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [2, 6]},\n      {\"term_id\": \"R-HSA-9748784\", \"supporting_discovery_ids\": [13]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [5, 6]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [2, 9]}\n    ],\n    \"complexes\": [\"CAR/RXR\\u03b1 heterodimer\"],\n    \"partners\": [\"RXRA\", \"NR0B1\", \"MAPK1\", \"MAPK3\", \"NCOA1\", \"PCK1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}