Volume 134, Issue 2, Pages 556-567.e1 (February 2008) Hepatic Fatty Acid Transporter Cd36 Is a Common Target of LXR, PXR, and PPARγ in Promoting Steatosis  Jie Zhou, Maria Febbraio, Taira Wada, Yonggong Zhai, Ramalinga Kuruba, Jinhan He, Jung Hoon Lee, Shaheen Khadem, Songrong Ren, Song Li, Roy L. Silverstein, Wen Xie  Gastroenterology  Volume 134, Issue 2, Pages 556-567.e1 (February 2008) DOI: 10.1053/j.gastro.2007.11.037 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 Activation of LXR induced the expression of CD36 in a liver-specific manner. (A and B) Real-time RT-PCR analysis on the hepatic expression of Cd36, PPARγ, Fae, and Scd-1 in untreated transgenic mice (A) or wild-type mice treated with 22(R)-hydroxycholesterol (22(R)) (single IP dose of 40 mg/kg) (B). All mice shown are males. (C) Human hepatocytes from 3 patients (all of white ethnicity, including a 45-year-old male, a 67-year-old female, an 84-year-old male) were individually mock treated or treated with 22(R) (10 μmol/L) for 24 hours before RNA harvesting and real-time PCR analysis. Results represent the averages and standard deviation. (D) Hepatic, but not intestinal, expression of Cd36 was induced in VP-LXR transgenic mice shown by Northern blot analysis. Lanes represent individual mice with 2 mice in each genotype/sex. (E) CD36 was not induced in 22(R)-treated THP-1 human macrophages (left panel) or primary mouse macrophages (right panel). The PPARγ ligand troglitazone (TRO; 5 μmol/L) was included as a positive control of Cd36 inducer. (F) PPARγ was induced in the liver, but not intestine, of VP-LXR transgenic mice. Cd36 and CD36 indicate the mouse and human genes, respectively. Fae, long-chain free fatty acid elongase; Scd-1, stearoyl CoA desaturase-1; Wt, wild type; Tg, transgenic. Results in A, B, and F represent the averages and standard deviation from 5 mice per group. *P < .05; **P < .01; #P > .05, compared with the wild-type (A and F) or vehicle (B, C, and E) mice. Gastroenterology 2008 134, 556-567.e1DOI: (10.1053/j.gastro.2007.11.037) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 Cd36 is a transcriptional target of LXRα. (A) Partial sequences of the mouse Cd36 gene promoter. The putative LXRE and previously reported PPRE and PXRE are in bold. (B) (Upper panel) VP-LXRα/RXR heterodimers bound to Cd36/DR-7 as revealed by EMSA. Sequences of the wild-type Cd36/DR-7 and its mutant are shown with the mutated nucleotides underlined. The binding was efficiently competed by unlabeled Cd36/DR-7 or Srebp/DR-4. (Lower panel) When radiolabeled Srebp/DR-4 probe was used, the cold DR-7 can compete for the binding but not as efficiently as DR-4. (C) ChIP assay to show the recruitment of LXRα onto the Cd36 gene promoter. HA-LXRα or HA vector control was hydrodynamically transfected into mouse liver. Transfected mice were treated with DMSO or TO1317 (20 mg/kg) for 8 hours before death and ChIP assay using an anti-HA antibody. PCR for a distal region of Cd36 promoter was included as a negative control. Srebp-1c was included as a positive control of LXR target gene. Lanes represent individual mice, and each group has 3 mice. (D) The expression of HA-LXRα protein in transfected livers was confirmed by Western blot analysis using an anti-HA antibody. The β-actin blot was included as a loading control. (E) Transfection and reporter gene assays to show that LXRα activated the synthetic report (tk-Cd36/DR-7) (left panel) and natural mouse Cd36 promoter (pGL-Cd36) (right panel), but not their DR-7 mutant variants, in the presence of GW3965. (F) LXRα mediates activation of the natural human CD36 promoter (pGL-CD36) by 22(R) or GW3965. Transfections in E and F were performed on HepG2 cells, and results shown are fold induction over the DMSO control (set as 1) and represent the averages and standard error from triplicate assays. The concentration for all ligands is 10 μmol/L. Gastroenterology 2008 134, 556-567.e1DOI: (10.1053/j.gastro.2007.11.037) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 The lipogenic effect of LXR agonists was inhibited in Cd36 null mice. (A) Oil-Red O staining on liver frozen sections from the wild-type (Wt) or Cd36 null mice gavaged with vehicle (VEH), 22(R)-hydroxycholesterol (22(R), 40 mg/kg), or GW3965 (20 mg/kg) for 3 days. (B–E) Levels of triglyceride (B and C) or free fatty acid (FFA) (D and E) in the liver (B and D) or plasma (C and E) of the Wt and Cd36 null mice. (F) Gene expression measured by real-time PCR analysis. All mice shown are males. Results in B–F represent the averages and standard deviation from 5 mice per group. *P < .05; **P < .01; #P > .05, all compared with VEH control of the same genotype, or the comparisons are as labeled in F. Gastroenterology 2008 134, 556-567.e1DOI: (10.1053/j.gastro.2007.11.037) Copyright © 2008 AGA Institute Terms and Conditions

Figure 4 Activation of LXR and PXR in transgenic mice cooperated to promote hepatic steatosis. (A) Expression of transgenes in the livers of wild-type (Wt) or transgenic mice detected by Northern blot analysis. The membranes were hybridized with cDNA probes of hPXR (detects VP-PXR only), mLXRα (detects both VP-LXR and endogenous LXRα), mPXR (detects endogenous PXR only), and Gapdh for a loading control. Lanes represent RNA samples pooled from 3 mice. (B) Transgene expression measured by real-time PCR. Results represent averages and standard deviation from 3 mice per group. UD, undetectable. (C) Gross appearance of the liver. (D) Liver weights presented as percentage of the total body weight. (E) Oil-Red O staining on liver frozen sections. (F) Liver levels of triglyceride and cholesterol. Results in D and F represent the averages and standard deviation from 6 mice per group. All mice shown, except those labeled in D, are males. Wt, wild type; Tg, transgenic; DTg, double transgenic. *P < .05; **P < .01, all compared with the wild-type mice. Gastroenterology 2008 134, 556-567.e1DOI: (10.1053/j.gastro.2007.11.037) Copyright © 2008 AGA Institute Terms and Conditions

Figure 5 PXR regulates PPARγ expression in a liver-specific manner, and PPARγ2 is a transcriptional target of PXR. (A) Liver-specific activation of PPARγ in the FABP-VP-PXR transgenic mice as revealed by real-time PCR. Results represent the averages and standard deviation from 5 mice per group. **P < .01, compared with the wild-type (Wt) mice. (B) Partial sequences of the mouse PPARγ2 gene promoter with the 2 DR-3 elements capitalized. The mutant variants are shown with the mutated nucleotides underlined. (C) hPXR/RXR heterodimers bound to radiolabeled PPARγ2/DR-3 sites as shown by EMSA. The bindings were efficiently competed by their respective unlabeled wild-type binding sites but not their mutant variants. (D) ChIP assay to show the recruitment of PXR onto the PPARγ2 gene promoter. HA-mPXR or HA vector control was transfected into mouse liver. Transfected mice were treated with DMSO or PCN (40 mg/kg) for 8 hours before death and ChIP assay using an anti-HA antibody. Cyp3a11 was included as a positive control for PXR target gene. Lanes represent individual mice, and each group has 3 mice. (E) mPXR activated the PPARγ2 promoter reporter gene in a PCN- and DR-3-dependent manner. Gastroenterology 2008 134, 556-567.e1DOI: (10.1053/j.gastro.2007.11.037) Copyright © 2008 AGA Institute Terms and Conditions

Figure 6 LXR and PXR cooperated to activate the Cd36 gene promoter. (A and B) Specific activation of LXRα (A) and mPXR (B) by GW3965 and PCN, respectively. tk-LXRE and tk-PXRE contain LXR response element (LXRE) Srebp-1c/DR-4 and PXR response element (PXRE) Cyp3a23/DR-3, respectively. (C) LXR and PXR cooperated to activate Cd36 promoter. Cd36 promoter reporter gene was cotransfected with expression vectors for mPXR and/or mLXRα into HepG2 cells in the presence of DMSO or indicated ligands. (D) Cotransfection of PPARγ siRNA had little effect on LXR- and/or PXR-mediated Cd36 promoter activation. Transfections were the same as in C, except that cells were cotransfected with control scrambled siRNA or PPARγ siRNA. The concentration of ligands is 10 μmol/L. Gastroenterology 2008 134, 556-567.e1DOI: (10.1053/j.gastro.2007.11.037) Copyright © 2008 AGA Institute Terms and Conditions

Figure 7 Proposed model of LXR, PXR, and PPARγ-controlled network of Cd36 regulation and steatosis. LXR, PXR, and PPARγ converge to regulate Cd36 and promote free fatty acid (FFA) uptake and steatosis. The regulation of Cd36 by LXR and PXR, as well as the regulation of PPARγ by PXR, are liver-specific. References of the original reports are labeled. Asterisks indicate findings of the current study. Gastroenterology 2008 134, 556-567.e1DOI: (10.1053/j.gastro.2007.11.037) Copyright © 2008 AGA Institute Terms and Conditions