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• W2912347823 abstract "All-trans retinoic acid (atRA) is used to treat certain cancers and dermatologic diseases. A common adverse effect of atRA is hypercholesterolemia; cytochrome P450 (CYP) 7A repression is suggested as a driver. However, the underlying molecular mechanisms remain unclear. We investigated CYP7A1 expression in the presence of atRA in human hepatocytes and hepatic cell lines. In HepaRG cells, atRA increased cholesterol levels dose-dependently alongside dramatic decreases in CYP7A1 expression. Lentiviral-mediated CYP7A1 overexpression reversed atRA-induced cholesterol accumulation, suggesting that CYP7A1 repression mediated cholesterol accumulation. In CYP7A1 promoter reporter assays and gene-knockdown studies, altered binding of hepatocyte nuclear factor 4 α (HNF4α) to the proximal promoter was essential for atRA-mediated CYP7A1 repression. Pharmacologic inhibition of c-Jun N-terminal kinase (JNK) and ERK pathways attenuated atRA-mediated CYP7A1 repression and cholesterol accumulation. Overexpression of AP-1 (c-Jun/c-Fos), a downstream target of JNK and ERK, repressed CYP7A1 expression. In DNA pull-down and chromatin immunoprecipitation assays, AP-1 exhibited sequence-specific binding to the proximal CYP7A1 promoter region overlapping the HNF4α binding site, and atRA increased AP-1 but decreased HNF4α recruitment to the promoter. Collectively, these results indicate that atRA activates JNK and ERK pathways and the downstream target AP-1 represses HNF4α transactivation of the CYP7A1 promoter, potentially responsible for hypercholesterolemia. All-trans retinoic acid (atRA) is used to treat certain cancers and dermatologic diseases. A common adverse effect of atRA is hypercholesterolemia; cytochrome P450 (CYP) 7A repression is suggested as a driver. However, the underlying molecular mechanisms remain unclear. We investigated CYP7A1 expression in the presence of atRA in human hepatocytes and hepatic cell lines. In HepaRG cells, atRA increased cholesterol levels dose-dependently alongside dramatic decreases in CYP7A1 expression. Lentiviral-mediated CYP7A1 overexpression reversed atRA-induced cholesterol accumulation, suggesting that CYP7A1 repression mediated cholesterol accumulation. In CYP7A1 promoter reporter assays and gene-knockdown studies, altered binding of hepatocyte nuclear factor 4 α (HNF4α) to the proximal promoter was essential for atRA-mediated CYP7A1 repression. Pharmacologic inhibition of c-Jun N-terminal kinase (JNK) and ERK pathways attenuated atRA-mediated CYP7A1 repression and cholesterol accumulation. Overexpression of AP-1 (c-Jun/c-Fos), a downstream target of JNK and ERK, repressed CYP7A1 expression. In DNA pull-down and chromatin immunoprecipitation assays, AP-1 exhibited sequence-specific binding to the proximal CYP7A1 promoter region overlapping the HNF4α binding site, and atRA increased AP-1 but decreased HNF4α recruitment to the promoter. Collectively, these results indicate that atRA activates JNK and ERK pathways and the downstream target AP-1 represses HNF4α transactivation of the CYP7A1 promoter, potentially responsible for hypercholesterolemia. Retinoids (vitamin A and its derivatives) modulate physiological processes including proliferation, differentiation, and apoptosis, via regulation of multiple genes (1Clagett-Dame M. Knutson D. Vitamin A in reproduction and development.Nutrients. 2011; 3: 385-428Crossref PubMed Scopus (250) Google Scholar, 2Mark M. Ghyselinck N.B. Chambon P. Function of retinoic acid receptors during embryonic development.Nucl. Recept. Signal. 2009; 7: e002Crossref PubMed Scopus (279) Google Scholar). These regulatory effects of retinoids are mediated mainly by retinoic acids (RAs), the bioactive metabolites of retinoids. All-trans RA (atRA) is the most biologically active RA and is used effectively for the treatment of cancers and dermatological disorders (3Schlenk R.F. Dohner K. Kneba M. Gotze K. Hartmann F. Del Valle F. Kirchen H. Koller E. Fischer J.T. Bullinger L. et al.Gene mutations and response to treatment with all-trans retinoic acid in elderly patients with acute myeloid leukemia. Results from the AMLSG Trial AML HD98B.Haematologica. 2009; 94: 54-60Crossref PubMed Scopus (163) Google Scholar, 4Schlenk R.F. Frohling S. Hartmann F. Fischer J.T. Glasmacher A. del Valle F. Grimminger W. Gotze K. Waterhouse C. Schoch R. et al.Phase III study of all-trans retinoic acid in previously untreated patients 61 years or older with acute myeloid leukemia.Leukemia. 2004; 18: 1798-1803Crossref PubMed Scopus (146) Google Scholar, 5Venditti A. Stasi R. Del Poeta G. Buccisano F. Aronica G. Bruno A. Pisani F. Caravita T. Masi M. Tribalto M. et al.All-trans retinoic acid and low-dose cytosine arabinoside for the treatment of ‘poor prognosis’ acute myeloid leukemia.Leukemia. 1995; 9: 1121-1125PubMed Google Scholar, 6Zane L.T. Leyden W.A. Marqueling A.L. Manos M.M. A population-based analysis of laboratory abnormalities during isotretinoin therapy for acne vulgaris.Arch. Dermatol. 2006; 142: 1016-1022Crossref PubMed Scopus (127) Google Scholar). Of note, one of the most prevalent side effects of retinoid drug therapy is hypercholesterolemia (affecting 31% of patients) that potentially promotes atherosclerosis (6Zane L.T. Leyden W.A. Marqueling A.L. Manos M.M. A population-based analysis of laboratory abnormalities during isotretinoin therapy for acne vulgaris.Arch. Dermatol. 2006; 142: 1016-1022Crossref PubMed Scopus (127) Google Scholar, 7Bershad S. Rubinstein A. Paterniti J.R. Le N.A. Poliak S.C. Heller B. Ginsberg H.N. Fleischmajer R. Brown W.V. Changes in plasma lipids and lipoproteins during isotretinoin therapy for acne.N. Engl. J. Med. 1985; 313: 981-985Crossref PubMed Scopus (176) Google Scholar, 8Brelsford M. Beute T.C. Preventing and managing the side effects of isotretinoin.Semin. Cutan. Med. Surg. 2008; 27: 197-206Crossref PubMed Scopus (117) Google Scholar). Cholesterol homeostasis is regulated mainly by the rates of cholesterol synthesis and elimination in the liver. These processes are modulated by the levels of the enzymes catalyzing the rate-limiting steps: the conversion of HMG-CoA to mevalonate by HMG-CoA reductase (HMGCR) for cholesterol synthesis (9Goldstein J.L. Brown M.S. Regulation of the mevalonate pathway.Nature. 1990; 343: 425-430Crossref PubMed Scopus (4542) Google Scholar) and cytochrome P450 7A1 (CYP7A1)-mediated conversion of cholesterol to bile acids for elimination (10Vlahcevic Z.R. Pandak W.M. Stravitz R.T. Regulation of bile acid biosynthesis.Gastroenterol. Clin. North Am. 1999; 28: 1-25Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Previous studies reported that atRA represses CYP7A1 expression in HepG2 cells and human hepatocytes, as well as in mouse liver (11Cai S.Y. He H. Nguyen T. Mennone A. Boyer J.L. Retinoic acid represses CYP7A1 expression in human hepatocytes and HepG2 cells by FXR/RXR-dependent and independent mechanisms.J. Lipid Res. 2010; 51: 2265-2274Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 12Schmidt D.R. Holmstrom S.R. Fon Tacer K. Bookout A.L. Kliewer S.A. Mangelsdorf D.J. Regulation of bile acid synthesis by fat-soluble vitamins A and D.J. Biol. Chem. 2010; 285: 14486-14494Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), but the detailed molecular mechanism of how atRA leads to CYP7A1 repression is unclear. Furthermore, the effects of retinoids on HMGCR expression/activity in the liver remain unknown. The expression of CYP7A1 is tightly controlled at transcriptional and posttranscriptional levels (13Crestani M. Sadeghpour A. Stroup D. Galli G. Chiang J.Y. Transcriptional activation of the cholesterol 7alpha-hydroxylase gene (CYP7A) by nuclear hormone receptors.J. Lipid Res. 1998; 39: 2192-2200Abstract Full Text Full Text PDF PubMed Google Scholar, 14Crestani M. Stroup D. Chiang J.Y. Hormonal regulation of the cholesterol 7 alpha-hydroxylase gene (CYP7).J. Lipid Res. 1995; 36: 2419-2432Abstract Full Text PDF PubMed Google Scholar, 15Hylemon P.B. Gurley E.C. Stravitz R.T. Litz J.S. Pandak W.M. Chiang J.Y. Vlahcevic Z.R. Hormonal regulation of cholesterol 7 alpha-hydroxylase mRNA levels and transcriptional activity in primary rat hepatocyte cultures.J. Biol. Chem. 1992; 267: 16866-16871Abstract Full Text PDF PubMed Google Scholar, 16Pandak W.M. Li Y.C. Chiang J.Y. Studer E.J. Gurley E.C. Heuman D.M. Vlahcevic Z.R. Hylemon P.B. Regulation of cholesterol 7 alpha-hydroxylase mRNA and transcriptional activity by taurocholate and cholesterol in the chronic biliary diverted rat.J. Biol. Chem. 1991; 266: 3416-3421Abstract Full Text PDF PubMed Google Scholar, 17Stravitz R.T. Hylemon P.B. Heuman D.M. Hagey L.R. Schteingart C.D. Ton-Nu H.T. Hofmann A.F. Vlahcevic Z.R. Transcriptional regulation of cholesterol 7 alpha-hydroxylase mRNA by conjugated bile acids in primary cultures of rat hepatocytes.J. Biol. Chem. 1993; 268: 13987-13993Abstract Full Text PDF PubMed Google Scholar, 18Wang D.P. Stroup D. Marrapodi M. Crestani M. Galli G. Chiang J.Y. Transcriptional regulation of the human cholesterol 7 alpha-hydroxylase gene (CYP7A) in HepG2 cells.J. Lipid Res. 1996; 37: 1831-1841Abstract Full Text PDF PubMed Google Scholar). Multiple microRNAs are known to decrease mRNA stability by targeting sequences in the 3′-untranslated region of CYP7A1 mRNA (19Song K.H. Li T. Owsley E. Chiang J.Y. A putative role of micro RNA in regulation of cholesterol 7alpha-hydroxylase expression in human hepatocytes.J. Lipid Res. 2010; 51: 2223-2233Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The promoter of CYP7A1 contains conserved response elements for multiple transcription factors with different functionality (20Stroup D. Crestani M. Chiang J.Y. Identification of a bile acid response element in the cholesterol 7 alpha-hydroxylase gene CYP7A.Am. J. Physiol. 1997; 273: G508-G517PubMed Google Scholar). For example, pregnane X receptor (PXR) represses the CYP7A1 promoter (21Bhalla S. Ozalp C. Fang S. Xiang L. Kemper J.K. Ligand-activated pregnane X receptor interferes with HNF-4 signaling by targeting a common coactivator PGC-1alpha. Functional implications in hepatic cholesterol and glucose metabolism.J. Biol. Chem. 2004; 279: 45139-45147Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 22Li T. Chiang J.Y. Mechanism of rifampicin and pregnane X receptor inhibition of human cholesterol 7 alpha-hydroxylase gene transcription.Am. J. Physiol. Gastrointest. Liver Physiol. 2005; 288: G74-G84Crossref PubMed Scopus (178) Google Scholar), whereas hepatocyte nuclear factor 4α (HNF4α) and liver receptor homolog-1 (LRH-1) activates the promoter (13Crestani M. Sadeghpour A. Stroup D. Galli G. Chiang J.Y. Transcriptional activation of the cholesterol 7alpha-hydroxylase gene (CYP7A) by nuclear hormone receptors.J. Lipid Res. 1998; 39: 2192-2200Abstract Full Text Full Text PDF PubMed Google Scholar, 23De Fabiani E. Mitro N. Anzulovich A.C. Pinelli A. Galli G. Crestani M. The negative effects of bile acids and tumor necrosis factor-alpha on the transcription of cholesterol 7alpha-hydroxylase gene (CYP7A1) converge to hepatic nuclear factor-4: a novel mechanism of feedback regulation of bile acid synthesis mediated by nuclear receptors.J. Biol. Chem. 2001; 276: 30708-30716Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 24Kir S. Zhang Y. Gerard R.D. Kliewer S.A. Mangelsdorf D.J. Nuclear receptors HNF4alpha and LRH-1 cooperate in regulating Cyp7a1 in vivo.J. Biol. Chem. 2012; 287: 41334-41341Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 25Lu T.T. Makishima M. Repa J.J. Schoonjans K. Kerr T.A. Auwerx J. Mangelsdorf D.J. Molecular basis for feedback regulation of bile acid synthesis by nuclear receptors.Mol. Cell. 2000; 6: 507-515Abstract Full Text Full Text PDF PubMed Scopus (1226) Google Scholar). HNF4α is functionally modulated by multiple mechanisms, including intracellular signaling and protein-protein interactions. For example, activation of MAPKs, such as c-Jun N-terminal kinases (JNKs), ERKs, and p38, can inhibit HNF4α activity (23De Fabiani E. Mitro N. Anzulovich A.C. Pinelli A. Galli G. Crestani M. The negative effects of bile acids and tumor necrosis factor-alpha on the transcription of cholesterol 7alpha-hydroxylase gene (CYP7A1) converge to hepatic nuclear factor-4: a novel mechanism of feedback regulation of bile acid synthesis mediated by nuclear receptors.J. Biol. Chem. 2001; 276: 30708-30716Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 26Guo H. Gao C. Mi Z. Zhang J. Kuo P.C. Characterization of the PC4 binding domain and its interactions with HNF4alpha.J. Biochem. 2007; 141: 635-640Crossref PubMed Scopus (13) Google Scholar, 27Vető B. Bojcsuk D. Bacquet C. Kiss J. Sipeki S. Martin L. Buday L. Bálint B.L. Aranyi T. The transcriptional activity of hepatocyte nuclear factor 4 alpha is inhibited via phosphorylation by ERK1/2.PLoS One. 2017; 12: e0172020Crossref PubMed Scopus (25) Google Scholar). The AP-1 protein family members serve as downstream effectors of ERK and JNK signaling pathways. Upon activation, these proteins form homodimers or heterodimers to regulate the expression of their target genes. Bile acids are known to activate c-Jun, a member of the AP-1 protein family, that interacts with HNF4α, leading to CYP7A1 repression (28Li T. Jahan A. Chiang J.Y. Bile acids and cytokines inhibit the human cholesterol 7 alpha-hydroxylase gene via the JNK/c-jun pathway in human liver cells.Hepatology. 2006; 43: 1202-1210Crossref PubMed Scopus (115) Google Scholar). Additionally, transcriptional activity of HNF4α can be inhibited by its interaction with corepressors, such as small heterodimer partner (SHP) (29Lee Y.K. Dell H. Dowhan D.H. Hadzopoulou-Cladaras M. Moore D.D. The orphan nuclear receptor SHP inhibits hepatocyte nuclear factor 4 and retinoid X receptor transactivation: two mechanisms for repression.Mol. Cell. Biol. 2000; 20: 187-195Crossref PubMed Scopus (265) Google Scholar, 30Shimamoto Y. Ishida J. Yamagata K. Saito T. Kato H. Matsuoka T. Hirota K. Daitoku H. Nangaku M. Yamagata K. et al.Inhibitory effect of the small heterodimer partner on hepatocyte nuclear factor-4 mediates bile acid-induced repression of the human angiotensinogen gene.J. Biol. Chem. 2004; 279: 7770-7776Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 31Zhou T. Zhang Y. Macchiarulo A. Yang Z. Cellanetti M. Coto E. Xu P. Pellicciari R. Wang L. Novel polymorphisms of nuclear receptor SHP associated with functional and structural changes.J. Biol. Chem. 2010; 285: 24871-24881Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Of note, atRA is known to induce SHP expression in human hepatocytes (11Cai S.Y. He H. Nguyen T. Mennone A. Boyer J.L. Retinoic acid represses CYP7A1 expression in human hepatocytes and HepG2 cells by FXR/RXR-dependent and independent mechanisms.J. Lipid Res. 2010; 51: 2265-2274Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) and activate MAPK in multiple tissues, including breast cancer and intestinal cells (32Namachivayam K. MohanKumar K. Arbach D. Jagadeeswaran R. Jain S.K. Natarajan V. Mehta D. Jankov R.P. Maheshwari A. All-trans retinoic acid induces TGF-beta2 in intestinal epithelial cells via RhoA- and p38alpha MAPK-mediated activation of the transcription factor ATF2.PLoS One. 2015; 10: e0134003Crossref PubMed Scopus (18) Google Scholar, 33Wang Q. Wieder R. All-trans retinoic acid potentiates Taxotere-induced cell death mediated by Jun N-terminal kinase in breast cancer cells.Oncogene. 2004; 23: 426-433Crossref PubMed Scopus (53) Google Scholar). Retinoids can regulate gene transcription by binding to their cognate receptors, RA receptors (RARs) and retinoid X receptors (RXRs). The complex subsequently binds to the RAR response element (RARE), two direct repeats of hexameric sequences (AGGTCA-like) with 5 base pair spacers (i.e., DR5), and modulates the promoter activities of target genes (34de Thé H. Vivanco-Ruiz M.M. Tiollais P. Stunnenberg H. Dejean A. Identification of a retinoic acid responsive element in the retinoic acid receptor beta gene.Nature. 1990; 343: 177-180Crossref PubMed Scopus (845) Google Scholar, 35O'Reilly K. Bailey S.J. Lane M.A. Retinoid-mediated regulation of mood: possible cellular mechanisms.Exp. Biol. Med. (Maywood). 2008; 233: 251-258Crossref PubMed Scopus (69) Google Scholar). Functional RARE was previously identified in the Cyp7a1 promoter of rodents (13Crestani M. Sadeghpour A. Stroup D. Galli G. Chiang J.Y. Transcriptional activation of the cholesterol 7alpha-hydroxylase gene (CYP7A) by nuclear hormone receptors.J. Lipid Res. 1998; 39: 2192-2200Abstract Full Text Full Text PDF PubMed Google Scholar), but it is unknown whether the respective sequences in the human CYP7A1 promoter are functional. RXR binding of retinoids can also lead to activation of its permissive binding partners, including PXR (36Pérez E. Bourguet W. Gronemeyer H. de Lera A.R. Modulation of RXR function through ligand design.Biochim. Biophys. Acta. 2012; 1821: 57-69Crossref PubMed Scopus (135) Google Scholar) and farnesoid X receptor (FXR). FXR transactivates the SHP promoter, and SHP, in turn, can repress HNF4α transactivation of the CYP7A1 promoter (24Kir S. Zhang Y. Gerard R.D. Kliewer S.A. Mangelsdorf D.J. Nuclear receptors HNF4alpha and LRH-1 cooperate in regulating Cyp7a1 in vivo.J. Biol. Chem. 2012; 287: 41334-41341Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 25Lu T.T. Makishima M. Repa J.J. Schoonjans K. Kerr T.A. Auwerx J. Mangelsdorf D.J. Molecular basis for feedback regulation of bile acid synthesis by nuclear receptors.Mol. Cell. 2000; 6: 507-515Abstract Full Text Full Text PDF PubMed Scopus (1226) Google Scholar, 37Goodwin B. Jones S.A. Price R.R. Watson M.A. McKee D.D. Moore L.B. Galardi C. Wilson J.G. Lewis M.C. Roth M.E. et al.A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis.Mol. Cell. 2000; 6: 517-526Abstract Full Text Full Text PDF PubMed Scopus (1510) Google Scholar). In this study, we report that atRA increases cholesterol levels, potentially by reducing CYP7A1 expression. We found that atRA activates the MAPK/AP-1 signaling pathway and inhibits the recruitment of HNF4α to the CYP7A1 promoter, leading to CYP7A1 repression. Results from transient transfection and promoter reporter assays also suggest that the involvement of multiple nuclear receptors (i.e., PXR, FXR, LRH-1, RAR, RXR, and SHP) in the atRA action on CYP7A1 expression is unlikely. HepaRG cells were purchased from Biopredic International (Saint Grégoire, France) and cultured in Williams' medium E supplemented with 10% FBS (Gemini, West Sacramento, CA), 5 µg/ml insulin, 2 mM l-glutamine, and 50 µM hydrocortisone hemisuccinate for 2 weeks. Confluent HepaRG cells were cultured in the same medium containing 2% DMSO (differentiation medium) for another 2 weeks. Medium was replaced every 2–3 days. Fully differentiated HepaRG cells were used as a liver model. Primary human hepatocytes were obtained from the Liver Tissue Cell Distribution System (Pittsburgh, PA; funded by National Institutes of Health Contract HHSN276201200017C). Upon receipt, medium was replaced with Williams' medium E supplemented with 0.1 µM dexamethasone, 2 mM l-glutamine, and 1% ITS solution (catalog no. I3146, Sigma). After stabilization for 24 h, cells were used for experiments. HEK293T and HepG2 cells were obtained from ATCC (Manassas, VA) and cultured in DMEM supplemented with 10% FBS and 2 mM l-glutamine. All cells were maintained at 37°C in a humidified incubator containing 5% CO2. A stock solution of atRA (catalog no. PHR1187, Sigma) was prepared at a concentration of 10 mM in DMSO. Cells were starved in FBS-free medium overnight before atRA treatment. The final DMSO concentrations in medium were 2.01% or 0.01% (2.01% for differentiated HepaRG cells, 0.01% for human hepatocytes and HepG2 cells). The media containing atRA were replaced every 24 h. Total cholesterol levels were determined using the cholesterol assay kit (Cell Biolabs, San Diego, CA), according to the manufacturer's instructions. Briefly, cells were washed with ice-cold PBS and homogenized in a mixture of chloroform, isopropanol, and NP-40 (7:11:0.1). After centrifugation at 15,000 g, organic phase was transferred to a new tube and dried until the solvents were removed. The dried pellets were dissolved in the assay diluent that contains cholesterol esterase and incubated with the cholesterol reaction reagent for 45 min at 37°C. Absorbance was measured at 540 nm. Total RNA was extracted using Trizol reagent (Thermo Fisher) and was reverse-transcribed into cDNA using a High-Capacity cDNA reverse-transcription kit (Thermo Fisher). Quantitative PCR (qPCR) was performed using the StepOnePlusTM real-time PCR system with PrimeTime probes (Integrated DNA Technologies, Coralville, IA). The following probes were used: CYP7A1 (Hs.PT.58.21408221), HMGCR (Hs.PT.58.41105492), HNF4A (Hs.PT.58.22303533), SHP (Hs.PT.58.38586840), ABCA1 (Hs.PT.58.27452429), ABCG5 (Hs.PT.58.40909601), ABCG8 (Hs.PT.58.40210561), and GAPDH (Hs.PT.39a.22214836). The values were expressed as mRNA levels normalized to those of GAPDH (2-ΔΔct method). Cells were lysed in RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.25% deoxycholic acid, 1% NP-40, and 1 mM EDTA) containing protease/phosphatase inhibitor cocktails (Roche, Mannheim, Germany). Proteins were separated by SDS-PAGE and transferred to a PVDF membrane. Membranes were incubated with 5% skim milk in TBS containing 0.1% Tween 20 (TBST) for 1 h and incubated with primary Abs against CYP7A1 (catalog no. ab79847, Abcam), HNF4α (catalog no. PP-H1415-00, R&D Systems), SHP (catalog no. sc-271511, Santa Cruz), p-P38 [catalog no. 4511, Cell Signaling Technology (CST)], P38 (catalog no. 8690, CST), p-JNK (catalog no. 4668, CST), JNK (catalog no. 9252, CST), p-ERK (catalog no. 4370, CST), ERK (catalog no. 4695, CST), p-MAPKAPK-2 (p-MK2; catalog no. 3007, CST), MAPKAPK-2 (MK2; catalog no. 3042, CST), p-c-Jun (catalog no. 2361, CST), c-Jun (catalog no. 9165, CST), c-Fos (catalog no. 2250, CST), and RARα (catalog no. sc-773, Santa Cruz) at 4°C overnight. Membranes were washed with TBST and incubated with secondary Ab for 1 h. Proteins were visualized with chemiluminescent substrate (Thermo Fisher). Cells were fixed in medium containing 1% paraformaldehyde at room temperature for 10 min and incubated in 125 mM glycine solution for 5 min to quench cross-links. Cells were washed with PBS and lysed in hypotonic buffer solution (20 mM Tris, pH 7.5, 1.5 mM MgCl2, 10 mM KCl, 0.5% NP-40, and protease and phosphatase inhibitor cocktails; Roche) for 10 min. After centrifugation at 10,000 g at 4°C for 1 min, supernatants were discarded, and nuclear pellets were rinsed with PBS and sonicated in RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.25% deoxycholic acid, 1% NP-40, 1 mM EDTA, and protease and phosphatase inhibitor cocktails) to shear DNA to the length ranging from 200 to 1,000 bp. After centrifugation at 16,000 g at 4°C for 10 min, supernatants were incubated with Ab-conjugated Dynabeads (Thermo Fisher) overnight. Chromatin immunoprecipitation (ChIP)-grade Abs against HNF4α (catalog no. PP-H1415-00, R&D Systems), c-Jun (catalog no. 9165, CST), and c-Fos (catalog no. 2250, CST) were conjugated to Dynabeads. The beads were washed with low-salt buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 1% Triton X-100, and 1 mM EDTA), high-salt buffer (20 mM Tris pH 8.0, 500 mM NaCl, 1% Triton X-100, and 1 mM EDTA), and TE buffer (10 mM Tris, pH 8.0, and 1 mM EDTA). Immune complexes were eluted in elution buffer (1% SDS and 0.1 M NaHCO3), and cross-linking was reversed by adding NaCl and heating at 65°C, followed by protease K treatment. DNA fragments were recovered using a Qiaquick PCR kit (Qiagen) and quantified by qPCR. The following PrimeTime probes were used to detect CYP7A1 promoter region: 5′-ggtctctgattgctttggaac-3′ (forward), 5′-catagtatccagatccattaacttgag-3′ (reverse), and 5′-acctgtggacttagttcaaggcca-3′ (probe). The signal was normalized by that from the amplified fragment without immunoprecipitation and expressed as percent input. Cells were washed with PBS and lysed in cytoplasmic extract buffer (20 mM Tris, pH 7.5, 1.5 mM MgCl2, 10 mM KCl, 0.5% NP-40, and protease/phosphatase inhibitor cocktails) for 10 min. The cytosolic fraction was isolated by centrifugation at 10,000 g at 4°C for 1 min. The pellets were rinsed with ice-cold PBS and lysed in nuclear extract buffer (20 mM Tris, pH 7.5, 1.5 mM MgCl2, 0.42 M NaCl, 0.2 mM EDTA, 25% glycerol, and protease/phosphatase inhibitor cocktails) at 4°C for 1 h. The nuclear fraction was isolated by centrifugation at 16,000 g at 4°C for 10 min. The supernatants were used in the DNA pull-down assay. The 5′ biotin-labeled oligonucleotides and nonlabeled oligonucleotides (competitor) were synthesized by Integrated DNA Technologies. The oligonucleotide sequences were as follows: WT, 5′-cctgtggacttagttcaaggccagttactacc-3′; m1, cctgccacattagttcaaggccagttactacc-3′; and m2, 5′-cctgtggacttctcccaaggccagttactacc-3′. Duplex oligonucleotides (4 μg)-conjugated Dynabeads (M-280 Streptavidin, Thermo Fisher) were incubated with nuclear extracts (200 μg) at room temperature for 2 h. The beads were washed with PBS, and proteins were eluted by boiling in 1× sample buffer (50 mM Tris, pH 6.8, 10% glycerol, 5% 2-mercaptoethanol, 2% SDS, and 0.2% bromophenol blue). The proteins were separated by SDS-PAGE followed by Western blot analysis. The insert DNA of the CYP7A1 promoter between −200 and +50 was synthesized by Genscript (Piscataway, NJ) and cloned into pGL3 vector to generate pGL3-CYP7A1 (−200/+50) plasmid. Deletion fragments of the CYP7A1 promoter were PCR-amplified using pGL3-CYP7A1 (−200/+50) as a template and cloned into pGL3 vector to generate pGL3-CYP7A1 (−100/+50 and −50/+50) plasmids. The mutations in pGL3-CYP7A1 constructs were made by Genscript. Expression vectors for c-Jun and c-Fos were purchased from Genscript. pcDNA3-HNF4A plasmid was received from Frances M. Sladek (University of California, Riverside, CA). HepG2 cells were transfected with promoter reporter constructs with or without expression vectors using FuGENE HD reagent (Promega) for 24 h and treated with atRA for 24 h. Luciferase activity was measured using GloMax luminometer (Promega) and normalized to the Renilla luciferase activity. Flag-tagged CYP7A1 clone was purchased from Genscript. Coding region of Flag-CYP7A1 was PCR-amplified and cloned into VVPW lentiviral expression vector (gift of G. L. Gusella, Mount Sinai Hospital, New York) to create VVPW-Flag-CYP7A1 plasmid. HEK293T cells were cotransfected with VVPW-Flag-CYP7A1, pCMV-VSVG, and psPAX2 plasmids (38Kistler A.D. Singh G. Altintas M.M. Yu H. Fernandez I.C. Gu C. Wilson C. Srivastava S.K. Dietrich A. Walz K. et al.Transient receptor potential channel 6 (TRPC6) protects podocytes during complement-mediated glomerular disease.J. Biol. Chem. 2013; 288: 36598-36609Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 39Ren G. Tardi N.J. Matsuda F. Koh K.H. Ruiz P. Wei C. Altintas M.M. Ploegh H. Reiser J. Podocytes exhibit a specialized protein quality control employing derlin-2 in kidney disease.Am. J. Physiol. Renal Physiol. 2018; 314: F471-F482Crossref PubMed Scopus (8) Google Scholar) in a ratio of 3:2:1 using FuGENE HD reagent according to the manufacturer's instructions to produce virus harboring Flag-CYP7A1. Control virus was produced using empty VVPW vector together with pCMV-VSVG and psPAX2. After 12 h transfection, medium was replaced, and viruses were produced for 48 h. Medium containing virus particles was filtered through a 0.45 µm filter, concentrated by Lenti-X Concentrator (Clontech). The viral particles were titrated by a Lenti-X titration kit (Takara). HepaRG cells and human hepatocytes were transduced with equal amounts of lentiviral particles in the presence of polybrene (2 µg/ml). Gene expression was silenced by siRNA transfection using Dharmafect 1 (Dharmacon, Lafayette, CO), according to the manufacturer's instruction. The following siRNAs (Dharmacon) were used: control (catalog no. D-001206-14), HNF4A (catalog no. M-003406-02), and SHP (catalog no. M-003410-01). All values were presented as means ± SD. For comparison of two groups, statistical differences were determined by Student's t-test. For statistical testing of multiple groups, one-way ANOVA followed by posthoc Tukey's test was performed. We first examined whether atRA affects total cholesterol level in human liver cells. Primary hepatocytes and differentiated HepaRG cells have been used to recapitulate the processes of the intact liver (40Kanebratt K.P. Andersson T.B. Evaluation of HepaRG cells as an in vitro model for human drug metabolism studies.Drug Metab. 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