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Pravastatin activates the expression of farnesoid X receptor and liver X receptor alpha in Hep3B cells
iliaryand LXRα in Hep3B cells Pravastatin activates Original Article / BFXR
Pravastatin activates the expression of farnesoid X receptor and liver X receptor alpha in Hep3B cells Hyun Woo Byun, Eun Mi Hong, Soo Hee Park, Dong Hee Koh, Min Ho Choi, Hyun Joo Jang, Sea Hyub Kae and Jin Lee Hwasung, Republic of Korea
BACKGROUND: Statins are suggested to preserve gallbladder function by suppressing pro-inflammatory cytokines and preventing cholesterol accumulation in gallbladder epithelial cells. They also affect cross-talk among the nuclear hormone receptors that regulate cholesterol-bile acid metabolism in the nuclei of hepatocytes. However, there is controversy over whether or how statins change the expression of peroxisome proliferator-activated receptor (PPAR)α, PPARγ, liver X receptor α (LXRα), farnesoid X receptor (FXR), ABCG5, ABCG8, and 7α-hydroxylase (CYP7A1) which are directly involved in the cholesterol saturation index in bile. METHODS: Human Hep3B cells were cultured on dishes. MTT assays were performed to determine the appropriate concentrations of reagents to be used. The protein expression of PPARα and PPARγ was measured by Western blotting analysis, and the mRNA expression of LXRα, FXR, ABCG5, ABCG8 and CYP7A1 was estimated by RT-PCR.
and CYP7A1 in human hepatocytes. (Hepatobiliary Pancreat Dis Int 2014;13:65-73) KEY WORDS: pravastatin; PPARγ; liver X receptor α; farnesoid X receptor; gallstone disease
Introduction
T
he cholesterol saturation index (CSI) of bile produced by hepatocytes is a major determinant of cholesterol gallstone formation and cholesterolosis in the gallbladder (GB).[1, 2] Representative nuclear hormone receptors regulate cholesterol-bile acid RESULTS: In cultured Hep3B cells, pravastatin activated PPARα metabolism. These receptors include liver X receptor α and PPARγ protein expression, induced stronger expression of (LXRα), farnesoid X receptor (FXR), and peroxisome PPARγ than that of PPARα, increased LXRα mRNA expression, proliferator-activated receptor (PPAR), and they activated ABCG5 and ABCG8 mRNA expression mediated by influence the regulatory targets of one another.[3-5] FXR as well as LXRα, enhanced FXR mRNA expression, and LXRα in hepatocytes works as a heterodimer with increased CYP7A1 mRNA expression mediated by the PPARγ retinoid X receptor (RXR) to lower the cholesterol and LXRα pathways, together or independently. concentration in response to oxycholesterol. Activation CONCLUSION: Our data suggested that pravastatin prevents of LXRα in hepatocytes induces the transcription of cholesterol gallstone diseases via the increase of FXR, LXRα 7α-hydroxylase (CYP7A1), the rate-limiting enzyme in hepatic bile acid synthesis, and leads to conversion of cholesterol to bile acid.[6] In addition, activated LXRα in hepatocytes induces ABCG5/ABCG8 located at the Author Affiliations: Division of Gastroenterology, Department of Internal canalicular membrane of cells, and transports excessive Medicine, Hallym University College of Medicine, Dongtan Sacred Heart intracellular cholesterol into bile. Thus, sustained Hospital, 40 Seokwoo-dong, Hwasung, Kyungki-Do 445-170, Republic of Korea (Byun HW, Hong EM, Park SH, Koh DH, Choi MH, Jang HJ, Kae SH activation of LXRα-mediated ABCG5/ABCG8 produces and Lee J) supersaturated bile, resulting in the formation of [7-10] However, a recent study[11] Corresponding Author: Jin Lee, MD, Division of Gastroenterology, cholesterol gallstones. Department of Internal Medicine, Hallym University College of Medicine, revealed that the LXR response element (LXRE) is not Dongtan Sacred Heart Hospital, 40 Seokwoo-dong, Hwasung, Kyungki-Do conserved in the human CYP7A1 promoter. Accordingly, 445-170, Republic of Korea (Tel: 82-31-8086-2015; Fax: 82-31-8086-2029; there is controversy over whether LXR agonist activates Email: [email protected]) CYP7A1 in human hepatocytes.[12, 13] © 2014, Hepatobiliary Pancreat Dis Int. All rights reserved. FXR in hepatocytes also works as a heterodimer doi: 10.1016/S1499-3872(14)60009-6 Hepatobiliary Pancreat Dis Int，Vol 13，No 1 • February 15，2014 • www.hbpdint.com • 65
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with RXR to maintain adequate concentrations of bile acid. An increased concentration of bile acid in hepatocytes activates FXR, inducing intracellular CYP7A1 and sterol-12-hydroxylase (CyP8B1) in another pathway of bile acid synthesis, resulting in decreased synthesis of bile acid.[14-16] Additionally, activated FXR induces bile salt exporter pump (BSEP, ABCB11), which secretes intracellular bile acid into bile, and multi-drug resistance gene 3 (MDR3, ABCB4), which secretes intracellular phospholipids into bile, thereby maintaining the solubility of cholesterol in hepatic bile.[8] Recently, it was suggested that activation of FXR induces hepatic expression of ABCG5/ABCG8, which may play a more important role in maintaining hepatic cholesterol homeostasis.[13, 17] PPARs, upon heterodimerization with RXR, function as transcriptional regulators of glucose and lipid metabolism.[18, 19] Some studies[20, 21] have indicated that stimulation of PPARα and PPARγ increases LXRα expression, and that a PPAR response element (PPRE) exists in the promoter of LXRα and functions as a PPARselective response element. In addition, our previous study[22] in GB epithelial cells (GBECs) documented that PPARα and PPARγ ligands can block the NF-κB pathway, thus modulating the inflammatory reaction, and that PPARα and PPARγ agonists also induce LXRαmediated ABCA1 activation. These results suggest that PPAR agonist can preserve GB function. Interestingly, fibrates known as PPARα agonists, contribute to cholesterol gallstone formation because they not only suppress the synthesis of bile acid by PPARα-mediated down regulation of CYP7A1 but also stimulate LXRαABCG5/ABCG8, which promotes cholesterol excretion into bile, leading to cholesterol supersaturation of bile.[23, 24] Accordingly, PPARα agonist cannot be used to prevent gallstones, despite the ability of PPARα to preserve GB function. Statin, an hydroxy-methl-glutaryl CoA (HMG-CoA) reductase inhibitor that is safely used for the treatment of dyslipidemia, inhibits the synthesis of cholesterol in the liver. Clinical evidence on the preventive role of statin in gallstone formation is conflicting. Population-based case-control studies, however, recently reported that long-term sustained statin use decreases the risk of gallstone diseases and reduces the risk of surgery for gallstone diseases.[25, 26] Most studies suggest that statin reduces the CSI of bile, or at least does not worsen the biliary lipid composition,[27, 28] despite PPARα induction. We also demonstrated that in GBECs, statins activate PPARα and PPARγ, modulate the inflammatory response by blocking lipopolysaccharide-induced TNF-α production, and eliminate excessive cholesterol via the LXRα/RXR-
ABCA1-mediated cholesterol efflux system.[29] Little is known about the mechanisms of how statin affects the homeostasis of hepatic cholesterol-bile acid metabolism, which is associated with the CSI of bile. The present study was to investigate the effects of statin on various regulatory factors, including PPARα, PPARγ, LXRα, FXR, ABCG5/ABCG8, and CYP7A1, in human hepatocytes.
Methods Materials Dulbecco's modified Eagle's medium (DMEM) was from Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS) and penicillin/streptomycin were from Hyclone (South Logan, UT, USA). Trypsin/ethylene diamine tetraacetic acid (EDTA), dimethyl sulfoxide (DMSO), 3-[4, 5-dimethylthiazol-2-yl] diphenyl tetrazolium bromide (MTT), 22(R)-hydroxycholesterol, and ursodeoxycholic acid (UDCA) were from Sigma Chemicals (St. Louis, MO, USA). Pravastatin, WY-14643, and troglitazone were from Cayman Chemicals (Ann Arbor, MI, USA). The peroxidase-conjugated anti-rabbit IgG antibody was from Amersham Biosciences (Buckinghamshire, UK). The rabbit polyclonal anti-human β-actin antibody was from Cell Signaling Technology (Danvers, MA, USA). The Western blot detection kit (Visualizer) was from Upstate (Lake Placid, NY, USA). Rabbit polyclonal anti-mouse PPARα antibody (a synthetic peptide corresponding to N-terminal amino acids 1-18 of mouse PPARα (NP_038482.3), which has 100% homology with human PPARα) and rabbit polyclonal PPARγ anti-human antibody were purchased from Abcam (Cambridge, UK). Cell culture Hep3B cells, a hepatoma cell line, were used for our experiments. Stock cultures were grown on 100-mm dishes in DMEM (including 4.5 g/L glucose) supp lemented with 10% FBS, 2 mmol/L L-glutamine, 1.5 g/L sodium bicarbonate, 100 IU/mL penicillin, and 100 μg/ mL streptomycin. The medium was changed twice a week, and the cells were maintained at 37 ℃ in an incubator with 5% CO2. The cells were passaged when confluent (every 5 to 7 days) using trypsin (2.5 g/L) and EDTA (1.0 g/L). For experiments, the cells were grown on 60-mm dishes, and the medium was changed to serum free medium (SFM) containing 0.2% bovine serum albumin (BSA) (Sigma, St. Louis, MO, USA). MTT The loading dose of chemicals or reagents used in
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Pravastatin activates FXR and LXRα in Hep3B cells
the experiments described below was determined by MTT assays as the maximum concentration that did not affect the proliferation of cells for 24 hours. Cells were plated at a density of 5×104 cells/mL in 24-well plates and cultured to 60% confluence in serum-containing regular medium. They were then incubated with or without various concentrations of reagents (pravastatin, WY-14643, and troglitazone) in SFM for 24 hours. MTT (0.5 mg/mL) was then added to each well, and the cells were incubated for another 4 hours at 37 ℃. After decanting the medium, 500 μL of DMSO was added to each well. After 10 minutes of constant and gentle shaking, the color intensity (proportional to the number of live cells) was assessed with an ELX800 (Biotek, Winooski, VT, USA) at a 570 nm wavelength.
RNA extraction and RT-PCR analysis Hep3B cells were cultured to confluence on 60-mm dishes. The cells were then incubated in SFM containing 0.2% BSA with pravastatin (1, 5, 10, 20 μmol/l), WY-14643 (100 μmol/L), troglitazone (10 μmol/L), UDCA (150 μmol/L), or 22(R)-hydroxycholesterol (10 μmol/L) for 24 hours as indicated. The cells were harvested, and RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RT-PCR was performed with the ampiRivert 1-step RT-PCR system according to the manufacturer's instructions (Gendepot, Barker, TX, USA). The sequences of primers are described in Table. The total reaction was performed in 50 μL mixtures containing total RNA (1 μg), 20 pmol primer, RT-PCR enzyme mix (5 U AMV transcriptase, 1.25 U Taq DNA polymerase), and 2× RT-PCR buffer mix (2× reaction buffer, 0.4 mmol/L dNTP, and 3.5 mmol/L MgCl2). The RT reaction was set at 45 ℃ for 45 minutes and then Table. Primer sequences used for RT-PCR Primers Direction Sequence (5' to 3') LXRα GAPDH ABCG5 ABCG8 FXR CYP7A1
Foward Reverse Foward Reverse Foward Reverse Foward Reverse Foward Reverse Foward Reverse
GTT CCC ACG GAT GCT AAT AAT GTT TGC CCT TCT CAG TC GGA GTC AAC GGA TTT GGT C GGA AGA TGG TGA TGG GAT T GGT TCT CAC CAT TCA CCA G GGT TTC TAT TTC CCG TTC C CAG ATT TCC AAC GAC TTC C CC GTC TTC CAG TTC ATA G CCT GGA TTT CTT CTG GAC AT TGT ATT GCG AGT ATG GTT CC CAA CGT ATC ATG AGA CCT CCA GTC CAG CTT CAA ACA TCA CTC GGT AG
Tm Size (℃) (bp) 57 231 57
225
53
220
58
240
57
251
57
102
at 95 ℃ for 3 minutes. The PCR conditions were as follows: denaturation at 94 ℃ for 30 seconds, annealing at 53-58 ℃ for 30 seconds, and extension at 72 ℃ for 30 seconds. The reaction was ended with an additional extension at 72 ℃ for 7 minutes, and then samples were chilled to 4 ℃. The mRNA was amplified by 26 cycles for LXRα, 40 cycles for ABCG5 and CYP7A1, 35 cycles for ABCG8, and 25 cycles for FXR. The PCR products were fractionated by electrophoresis on 2% agarose gels containing ethidium bromide.
Protein extraction and Western blotting analysis Hep3B cells were cultured to confluence on 60-mm dishes with regular media. The cells were treated with pravastatin (5, 10, 20 μmol/L), WY14643 (100 μmol/L), or troglitazone (10 μmol/L) in SFM containing 0.2% BSA for 24 hours as indicated. They were then washed with PBS and harvested with lysis buffer [50 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 1 mmol EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 μmol/L phenylmethylsulfonyl fluoride (PMSF), 5 μg/ mL aprotinin, and 5 μg/mL leupeptin]. Proteins were quantified by the Bradford assay (Sigma St. Louis, Mo, USA). SDS-PAGE was performed with a 4% stacking gel and 10% resolving gel, followed by transfer to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The membranes were blocked overnight at 4 ℃ in a blocking solution [5% skim milk in Tris-buffer with Tween-20 (TBS-T): 200 mmol/L Tris, 500 mmol/L NaCl, pH 7.5, and 0.05% v/v Tween-20]. The membranes were then incubated with rabbit polyclonal PPARα antibody or rabbit polyclonal PPARγ antibody for 1 hour at room temperature. At the same time, the rabbit polyclonal β-actin antibody was used to detect the β-actin housekeeping protein to normalize the amount of protein. The membranes were washed with TBS-T and incubated with peroxidase-conjugated anti-rabbit IgG for 1 hour at room temperature. The membrane was washed and incubated using a Visualizer Western blot detection kit for 5 minutes. Autoradiography was then performed. The signal intensities for specific bands on the Western blotting were quantified using NIH Image J density analysis software (Version 1.20). Statistical analysis Results from each experiment are expressed as the mean±SD of duplicate cultures, and all results described are representative of at least three separate experiments. One-way analysis of variance (ANOVA) for three or more unpaired groups or Student's t test for two unpaired groups was used, and P<0.05 was considered statistically significant.
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Results Pravastatin activates the expression of PPARα and PPARγ in Hep3B cells The expression of PPARα and PPARγ in response to pravastatin (a hydrophilic HMG-CoA reductase inhibitor) was studied in human Hep3B cells. We used 100 μmol/L WY-14643 as a positive control for PPARα and 10 μmol/L troglitazone as a positive control for PPARγ. PPARα expression was increased significantly by a high concentration of pravastatin (P<0.05, versus no-treatment control), but was increased only slightly by low concentrations, 5 or 10 μmol/L of pravastatin (Fig. 1A). PPARγ expression was increased significantly by low concentrations of pravastatin (P<0.01, versus notreatment control) as well as by a high concentration (P<0.001, versus no-treatment control) in a concentration dependent manner (Fig. 1B). These findings suggest that pravastatin has a greater effect on the expression of PPARγ than on the expression of PPARα. Pravastatin activates the expression of LXRα mRNA in Hep3B cells To assess whether statins stimulate the expression of LXRα mRNA, which is induced by activated PPAR, cultured Hep3B cells were incubated with various concentrations of pravastatin, 10 μmol/L 22(R)hydroxycholesterol as an LXRα positive control, and no-treatment as a negative control. When the treatment concentration of pravastatin was 5 μmol/L or more, the expression of LXRα mRNA was significantly increased, compared with the no-treatment control (P<0.05), to a level similar to that observed with 22(R)hydroxycholesterol treatment (Fig. 2). Pravastatin activates FXR mRNA expression in Hep3B cells As a bile acid sensor, FXR is important for the regulation of cholesterol homeostasis. In order to evaluate whether statin stimulates the expression of FXR mRNA, cultured Hep3B cells were incubated with various concentrations of pravastatin, 150 μmol/ L UDCA as a positive control, and no-treatment as a negative control. Pravastatin increased the expression of FXR mRNA significantly in a concentration dependent manner (P<0.01, Fig. 3). Pravastatin induces the expression of ABCG5/ABCG8 mRNA via FXR and LXRα in Hep3B cells We found that pravastatin induced the expression of PPARα, PPARγ, LXRα, and FXR. Because no LXREs have been identified in the promoters of ABCG5 or ABCG8,
whether a potent LXR agonist can induce ABCG5 and ABCG8 in human hepatocytes is controversial.[11] On the other hand, activation of FXR may induce hepatic expression of ABCG5/ABCG8 through a common FXR response element, which promotes biliary-free cholesterol secretions.[13, 17] Accordingly, RT-PCR for ABCG5/ ABCG8 was performed using the same conditions as for LXRα to evaluate whether pravastatin induces the expression of ABCG5 and ABCG8 mRNA. The increased expression of ABCG5 mRNA was significantly dependent on the concentration of pravastatin (Fig. 4A). A high concentration of pravastatin (10 or 20 μmol/L) induced the expression of ABCG8 mRNA, though not as much as 22(R)-hydroxycholesterol (Fig. 4B). We
Fig. 1. Pravastatin activates PPARα and PPARγ expression in Hep3B cells. A: Western blotting analysis of PPARα in Hep3B cells after treatment with pravastatin (PS, 5, 10, 20 μmol/L or PPARα ligand [positive control, WY-14643 (WY), 100 μmol/L] for 24 hours; B: Western blotting analysis of PPARγ in Hep3B cells after treatment with pravastatin (PS, 5, 10, 20 μmol/L) or PPARγ ligand [positive control, troglitazone (TR), 10 μmol/L] for 24 hours. *: P<0.05, versus no-treatment control (Cont); **: P<0.001, versus Cont and P<0.01, versus pravastatin 5 μmol/L (PS-5) and 10 μmol/ L (PS-10) treatment; #: P<0.01, versus Cont. The relative expression was calculated with adjustment of β-actin expression.
Fig. 2. Pravastatin increases LXRα mRNA expression in Hep3B cells. RT-PCR assay for LXRα was performed in the presence of pravastatin (PS, 1, 5, 10, 20 μmol/L) or 22(R)-hydroxycholesterol (positive control, OH-C, 10 μmol/L) for 24 hours. *: P<0.05, versus no-treatment control (Cont). The relative expression was calculated with adjustment of GAPDH expression.
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Fig. 3. Pravastatin increases FXR mRNA expression in Hep3B cells. RT-PCR assay for FXR mRNA was performed in the presence of pravastatin (PS, 1, 5, 10, 20 μmol/L) or UDCA (positive control, 150 μmol/L) for 24 hours. *: P<0.01, versus no-treatment control (Cont); **: P<0.001, versus Cont.
Fig. 4. Pravastatin increases ABCG5/ABCG8 mRNA expression in Hep3B cells. A: RT-PCR assay for ABCG5 mRNA was performed in the presence of pravastatin (PS, 1, 5, 10, 20 μmol/L) or 22(R)hydroxycholesterol (positive control, OH-C, 10 μmol/L) for 24 hours; B: RT-PCR assay for ABCG8 mRNA was performed in the presence of pravastatin (PS, 1, 5, 10, 20 μmol/L) or 22(R)hydroxycholesterol (positive control, OH-C, 10 μmol/L) for 24 hours. *: P<0.05, versus no-treatment control (Cont); **: P<0.01, versus Cont; #: P<0.001, versus Cont. The relative expression was calculated with adjustment of GAPDH expression.
Fig. 5. FXR ligand as well as LXRα ligand increases ABCG5/ ABCG8 mRNA expression in Hep3B cells. RT-PCR assays for ABCG5 (A) and ABCG8 (B) mRNA were performed in the presence of 22-(R)-hydroxycholesterol (OH-C, 10 μmol/L), UDCA (150 μmol/L), and pravastatin (PS, 10 μmol/L) for 24 hours. *: P<0.05, versus no-treatment control (Cont); **: P<0.01, versus Cont and P<0.05 versus UDCA treatment. The relative expression was calculated with adjustment of GAPDH expression.
Fig. 6. Pravastatin increases CYP7A1 mRNA expression mediated by the PPARγ-LXRα pathway in Hep3B cells. A: RT-PCR assay for CyP7A1 mRNA was performed in the presence of pravastatin (PS, 1, 5, 10, 20 μmol/L) for 24 hours; B: RT-PCR assay for CYP7A1 mRNA was performed in the presence of 22(R)-hydroxycholesterol (OH-C, 10 μmol/L), UDCA (150 μmol/L), WY-14643 (100 μmol/ L), and troglitazone (10 μmol) for 24 hours. *: P<0.05, versus notreatment control (Cont); **: P<0.01, versus Cont. The relative expression was calculated with adjustment of GAPDH expression.
also examined whether FXR induces the expression of ABCG5 and ABCG8 mRNA in Hep3B cells. The expression of ABCG5/ABCG8 mRNA was significantly of CYP7A1, which is activated by LXRα and inhibited by increased by UDCA, an FXR ligand, as well as by LXRα FXR via negative feedback. To determine this, RT-PCR ligand. However, LXRα stimulation induced a stronger was performed on Hep3B cells stimulated by pravastatin. expression of ABCG5 and ABCG8 mRNA, compared As shown in Fig. 6A, pravastatin significantly enhanced with FXR stimulation by UDCA (P<0.05) (Fig. 5). the expression of CYP7A1 mRNA in a concentration dependent manner in Hep3B cells. The results in Fig. 6B Pravastatin increases CYP7A1 mRNA expression via indicate that activation of CYP7A1 mRNA by pravastatin PPARγ-LXRα pathway in Hep3B cells is mediated by the PPARγ and LXRα pathways, together In this study, pravastatin appeared to stimulate the or independently. expression of FXR mRNA and LXRα mRNA in cultured Hep3B cells. However, the LXRE is not conserved in the human CYP7A1 promoter,[11] and LXR agonists repress CYP7A1 in human hepatocytes.[12] Therefore, we Discussion wondered whether pravastatin stimulates the expression In this study, statin affected the cross-talk between Hepatobiliary Pancreat Dis Int，Vol 13，No 1 • February 15，2014 • www.hbpdint.com • 69
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nuclear hormone receptors, including PPARα/PPARγ, LXRα, and FXR, associated with cholesterol-bile acid-phospholipid balance in hepatic bile, probably contributing to the prevention of cholesterol gallstones. Additionally, the current study documented possible molecular mechanisms by which statin prevents gallstones formation. The present study demonstrated in hepatocytes that statin stimulates PPARα, PPARγ, and LXRα expression, and that PPARγ expression changes much more than PPARα expression, even in low concentrations of pravastatin. Considering that PPARγ agonist blocks the repression of CYP7A1 mRNA[30] and has a more powerful anti-inflammatory effect than fibrates,[22] the stronger activation of PPARγ induced by statin may play a preventive role in cholesterol gallstones formation. Some studies have suggested probable mechanisms by which statins activate PPARγ: statins enhance peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) activity by suppressing Akt phosphorylation,[31] and PPARγ also interacts with PGC1α.[32] On the other hand, PGC-1α plays a role in the prevention of cholelithiasis via interaction with the bile acid sensor, FXR,[33] and PPARγ may prevent cholesterol accumulation in GBECs by LXRα-mediated ABCA1 activation.[22] ABCG5 and ABCG8 are expressed predominantly on the basolateral (bile-contacting) side of hepatocytes and on the apical (luminal) side of enterocytes in the proximal small intestine of humans and mice. They play a pivotal role in transporting excessive cholesterol from hepatocytes into bile or from enterocytes into the lumen.[10] Accordingly, overexpression of ABCG5 and ABCG8 in hepatocytes may induce supersaturation of bile, resulting in the formation of gallstones.[8] LXRα, a nuclear receptor that plays a key role in regulating genes involved in cholesterol trafficking, is required for the increased expression of ABCG5 and ABCG8 in response to cholesterol or oxycholesterol.[10, 34] However, it was reported that a potent LXR agonist or cholesterol treatment failed to induce ABCG5 and ABCG8 in primary human hepatocytes.[13] In contrast, another study[35] indicated that the expression of both ABCG5 and ABCG8 increases substantially upon LXR activation in human enterocytes. In addition, even though no LXREs have been identified in the promoters of ABCG5 or ABCG8, the intergenic region was found to act as a bidirectional promoter and be partially responsive to treatment with LXR agonists.[36] Results from studies investigating whether statin activates the expression of LXRα, ABCA1, or ABCG5/ABCG8 are inconsistent.[37-41] On the other hand, the hepatic expression of ABCG5/
ABCG8 is induced by activation of FXR through a common FXRE, which promotes cholesterol secretions, and the FXR-ABCG5/ABCG8 pathway may play a more important role in maintaining hepatic cholesterol homeostasis than the LXRα-ABCG5/ABCG8 pathway in human hepatocytes.[13, 17] The present study showed that pravastatin stimulated the expression of ABCG5/ABCG8 mRNA in Hep3B cells through the activation of LXRα and FXR. Although there might be differences between cancer cells and normal hepatocytes, the result suggests that statin mediates cholesterol secretion into bile by inducing ABCG5/ABCG8 expression via both LXRα and FXR activation in hepatocytes. Although statin activates PPARα and LXRα-ABCG5/ ABCG8, which are associated with supersaturated bile, no studies have reported that statin induces gallstones formation. Thus, we considered other potential reasons for increased cholesterol solubility in bile, including CYP7A1 and FXR participation in bile acid homeostasis. Controversy exists over the effects of statins on FXR expression in hepatocytes.[42, 43] Our study demonstrated that statin enhances FXR expression in a concentration dependent manner in hepatocytes. Enhanced FXR expression induced by pravastatin, along with other possible mechanisms described in the next paragraph, presumably contributes to the maintenance or increase of cholesterol solubility in bile. On the other hand, PGC1α, PPARγ, and hepatocyte nuclear factor-4α (HNF-4α) mRNAs are induced after a prolonged stimulus such as fasting, and PGC-1α coactivates PPARγ and/or HNF4α bound to a direct repent-1 (DR-1) element in the FXR promoter, to induce FXR mRNA expression.[44] In addition, statin induces PGC-1α, PPARγ, and HNF4α.[31, 45] Taken together, statin probably induces FXR through PGC-1α, PPARγ, and HNF-4α activation. CYP7A1 is mainly regulated at the gene transcription level by bile acid, cholesterol, amino acid taurine, cytokines, hormones, and transcriptional regulators.[11, 46] The LXRE does not exist in the human CYP7A1 promoter,[11] and some studies[14, 47, 48] reported that LXR agonists repress CYP7A1 in human hepatocytes.[12] Furthermore, FXR inhibits CYP7A1, and PPARα agonist represses CYP7A1.[23, 24] Accordingly, we expected CYP7A1 mRNA levels in Hep3B cells treated with pravastatin to decrease or remain unchanged. However, contrary to our expectations, pravastatin enhanced CYP7A1 expression. Possible mechanisms responsible for the enhanced expression of CYP7A1 by statin are as follows: 1) Although no LXREs have been identified in the promoter of CYP7A1, the intergenic region may partially respond to LXR agonists like ABCG5 and ABCG8.[36] In this experiment,
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enhanced expression of CYP7A1 induced by 22(R)hydroxycholesterol supports this theory. 2) We observed that PPARγ agonist increased the expression of CYP7A1. PGC-1α activates CYP7A1 transcription together with HNF-4α by increasing HNF-4αmediated transactivation of CYP7A1,[49] and PPARγ also interacts with PGC-1α.[44] Therefore, we suggest that the statin-PPARγ-PGC-1α-HNF-4α pathway may be involved. Additional study is needed to substantiate this hypothesis. This result does indicate that statin promotes hepatic bile acid synthesis by inducing CYP7A1 and maintains cholesterol solubility in bile. We used Hep3B cells in this study in view of the technical advantages of hepatocyte culture, because Hep3B and HepG2 hepatoma cell lines have cellular characteristics similar to normal hepatocytes. Many studies on enzymes, receptors, and transporters, such as CYP7A1,[42, 46, 50] LXRα,[51, 52] FXR,[43] and PPAR,[53] associated with cholesterol metabolism in hepatocytes have been performed with HepG2 or Hep3B cells. In addition, if functional protein studies or over/underexpression studies for specific gene targets were performed, they would clearly demonstrate the crosstalk between nuclear hormone receptors. However, the primary objectives of the study were to demonstrate the change in the expression of the proteins or mRNAs of specific gene related to lipid solubility in bile, and our previous study in hamsters have already documented that pravastatin contributes to the prevention of gallstones formation.[54] Moreover, we agree that the nucleation and growth of cholesterol gallstones in humans is a complex phenomenon which is associated with not only cholesterol liver metabolism but also gallbladder parietal factors such as mucins, gallbladder motility, and calcium bilirubinate precipitation.[55, 56] Accordingly, a preventive effect on gallstones disease could be achieved by acting also on the other factors involved in gallstones formation. A study[57] indicated that the expression of FXR and its target gene small heterodimer partner (SHP) was strongly down-regulated in human hepatocellular carcinoma cells, which is consistent with a tumor suppressive role of FXR.[58] Therefore, Hep3B cells treated with pravastatin might show some different response compared with primary hepatocytes in expressional change of FXR mRNA, which may be another limitation of this study. In an aspect, our results showed a possibility that statin may have anti-neoplastic effect because statin enhanced expression of FXR mRNA in Hep3B cells. In conclusion, in human hepatocytes, pravastatin induces the expression of PPARγ, more so than that
of PPARα, and activates the expression of FXR as well as LXRα. In addition, pravastatin activates ABCG5/ ABCG8 expression, which is mediated by both LXRα and FXR, and increases CYP7A1 expression, which is induced by activation of LXRα and PPARγ, together or independently. We postulate that statin increases cholesterol solubility in bile by simultaneous activation of FXR which promotes bile acid excretion and CYP7A1 which enhances hepatic bile acid synthesis,[8] although statin also activates LXRα-ABCG5/ABCG8 which excretes cholesterol into bile.[7-10] We thus suggest that statin-induced increased expression of both FXR and LXRα and enhancement of CYP7A1 expression in human hepatocytes, with the known action of statin to preserve GB function, can play a preventive role in cholesterol gallstones diseases. Contributors: LJ proposed the study. BHW and LJ performed research, wrote the first draft and analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. LJ is the guarantor. Funding: None. Ethical approval: Not needed. Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.
References 1 Jacyna MR, Ross PE, Bakar MA, Hopwood D, Bouchier IA. Characteristics of cholesterol absorption by human gall bladder: relevance to cholesterolosis. J Clin Pathol 1987;40: 524-529. 2 Corradini SG, Elisei W, Giovannelli L, Ripani C, Della Guardia P, Corsi A, et al. Impaired human gallbladder lipid absorption in cholesterol gallstone disease and its effect on cholesterol solubility in bile. Gastroenterology 2000;118: 912-920. 3 Trauner M, Halilbasic E. Nuclear receptors as new perspective for the management of liver diseases. Gastroenterology 2011; 140:1120-1125. 4 Repa JJ, Mangelsdorf DJ. Nuclear receptor regulation of cholesterol and bile acid metabolism. Curr Opin Biotechnol 1999;10:557-563. 5 Russell DW. Nuclear orphan receptors control cholesterol catabolism. Cell 1999;97:539-542. 6 Peet DJ, Janowski BA, Mangelsdorf DJ. The LXRs: a new class of oxysterol receptors. Curr Opin Genet Dev 1998;8:571-575. 7 Moschetta A, Bookout AL, Mangelsdorf DJ. Prevention of cholesterol gallstone disease by FXR agonists in a mouse model. Nat Med 2004;10:1352-1358. 8 Wittenburg H, Lyons MA, Li R, Churchill GA, Carey MC, Paigen B. FXR and ABCG5/ABCG8 as determinants of cholesterol gallstone formation from quantitative trait locus mapping in mice. Gastroenterology 2003;125:868-881. 9 Yu L, York J, von Bergmann K, Lutjohann D, Cohen JC, Hobbs HH. Stimulation of cholesterol excretion by the liver X
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receptor agonist requires ATP-binding cassette transporters G5 and G8. J Biol Chem 2003;278:15565-15570. 10 Repa JJ, Berge KE, Pomajzl C, Richardson JA, Hobbs H, Mangelsdorf DJ. Regulation of ATP-binding cassette sterol transporters ABCG5 and ABCG8 by the liver X receptors alpha and beta. J Biol Chem 2002;277:18793-18800. 11 Chiang JY, Kimmel R, Stroup D. Regulation of cholesterol 7alpha-hydroxylase gene (CYP7A1) transcription by the liver orphan receptor (LXRalpha). Gene 2001;262:257-265. 12 Goodwin B, Watson MA, Kim H, Miao J, Kemper JK, Kliewer SA. Differential regulation of rat and human CYP7A1 by the nuclear oxysterol receptor liver X receptor-alpha. Mol Endocrinol 2003;17:386-394. 13 Li T, Matozel M, Boehme S, Kong B, Nilsson LM, Guo G, et al. Overexpression of cholesterol 7α-hydroxylase promotes hepatic bile acid synthesis and secretion and maintains cholesterol homeostasis. Hepatology 2011;53:996-1006. 14 Makishima M, Okamoto AY, Repa JJ, Tu H, Learned RM, Luk A, et al. Identification of a nuclear receptor for bile acids. Science 1999;284:1362-1365. 15 Wang H, Chen J, Hollister K, Sowers LC, Forman BM. Endogenous bile acids are ligands for the nuclear receptor FXR/BAR. Mol Cell 1999;3:543-553. 16 Seol W, Choi HS, Moore DD. An orphan nuclear hormone receptor that lacks a DNA binding domain and heterodimerizes with other receptors. Science 1996;272:13361339. 17 Li T, Chiang JY. Bile Acid signaling in liver metabolism and diseases. J Lipids 2012;2012:754067. 18 Hirakata M, Tozawa R, Imura Y, Sugiyama Y. Comparison of the effects of pioglitazone and rosiglitazone on macrophage foam cell formation. Biochem Biophys Res Commun 2004; 323:782-788. 19 Mangelsdorf DJ, Evans RM. The RXR heterodimers and orphan receptors. Cell 1995;83:841-850. 20 Chinetti G, Lestavel S, Bocher V, Remaley AT, Neve B, Torra IP, et al. PPAR-alpha and PPAR-gamma activators induce cholesterol removal from human macrophage foam cells through stimulation of the ABCA1 pathway. Nat Med 2001;7: 53-58. 21 Chawla A, Boisvert WA, Lee CH, Laffitte BA, Barak Y, Joseph SB, et al. A PPAR gamma-LXR-ABCA1 pathway in macrophages is involved in cholesterol efflux and atherogenesis. Mol Cell 2001;7:161-171. 22 Lee J, Hong EM, Byun HW, Choi MH, Jang HJ, Eun CS, et al. The effect of PPARalpha and PPARgamma ligands on inflammation and ABCA1 expression in cultured gallbladder epithelial cells. Dig Dis Sci 2008;53:1707-1715. 23 Post SM, Duez H, Gervois PP, Staels B, Kuipers F, Princen HM. Fibrates suppress bile acid synthesis via peroxisome proliferator-activated receptor-alpha-mediated downregulation of cholesterol 7alpha-hydroxylase and sterol 27-hydroxylase expression. Arterioscler Thromb Vasc Biol 2001;21:1840-1845. 24 Kosters A, Frijters RJ, Schaap FG, Vink E, Plösch T, Ottenhoff R, et al. Relation between hepatic expression of ATP-binding cassette transporters G5 and G8 and biliary cholesterol secretion in mice. J Hepatol 2003;38:710-716. 25 Erichsen R, Frøslev T, Lash TL, Pedersen L, Sørensen HT. Long-term statin use and the risk of gallstone disease: A population-based case-control study. Am J Epidemiol 2011;
173:162-170. 26 Merzon E, Weiss NS, Lustman AJ, Elhayani A, Dresner J, Vinker S. Statin administration and risk of cholecystectomy: a population-based case-control study. Expert Opin Drug Saf 2010;9:539-543. 27 Smith JL, Riottot M, Nathanson LK. Effect of statins on biliary lipid and cholesterol galltones. J Kardiol 2002;9:295298. 28 Smith JL, Roach PD, Wittenberg LN, Riottot M, Pillay SP, Nestel PJ, et al. Effects of simvastatin on hepatic cholesterol metabolism, bile lithogenicity and bile acid hydrophobicity in patients with gallstones. J Gastroenterol Hepatol 2000;15: 871-879. 29 Lee J, Hong EM, Koh DH, Choi MH, Jang HJ, Kae SH, et al. HMG-CoA reductase inhibitors (statins) activate expression of PPARalpha/PPARgamma and ABCA1 in cultured gallbladder epithelial cells. Dig Dis Sci 2010;55:292-299. 30 Miyake JH, Wang SL, Davis RA. Bile acid induction of cytokine expression by macrophages correlates with repression of hepatic cholesterol 7alpha-hydroxylase. J Biol Chem 2000;275:21805-21808. 31 Wang W, Wong CW. Statins enhance peroxisome proliferator-activated receptor gamma coactivator-1alpha activity to regulate energy metabolism. J Mol Med (Berl) 2010;88:309-317. 32 Madrazo JA, Kelly DP. The PPAR trio: regulators of myocardial energy metabolism in health and disease. J Mol Cell Cardiol 2008;44:968-975. 33 Bertolotti M, Gabbi C, Anzivino C, Mitro N, Godio C, De Fabiani E, et al. Decreased hepatic expression of PPARgamma coactivator-1 in cholesterol cholelithiasis. Eur J Clin Invest 2006;36:170-175. 34 van der Veen JN, van Dijk TH, Vrins CL, van Meer H, Havinga R, Bijsterveld K, et al. Activation of the liver X receptor stimulates trans-intestinal excretion of plasma cholesterol. J Biol Chem 2009;284:19211-19219. 35 Field FJ, Born E, Mathur SN. LXR/RXR ligand activation enhances basolateral efflux of beta-sitosterol in CaCo-2 cells. J Lipid Res 2004;45:905-913. 36 Remaley AT, Bark S, Walts AD, Freeman L, Shulenin S, Annilo T, et al. Comparative genome analysis of potential regulatory elements in the ABCG5-ABCG8 gene cluster. Biochem Biophys Res Commun 2002;295:276-282. 37 Zanotti I, Potì F, Favari E, Steffensen KR, Gustafsson JA, Bernini F. Pitavastatin effect on ATP binding cassette A1mediated lipid efflux from macrophages: evidence for liver X receptor (LXR)-dependent and LXR-independent mechanisms of activation by cAMP. J Pharmacol Exp Ther 2006;317:395-401. 38 Zhu Y, Wang HJ, Chen LF, Fang Q, Yan XW. Study of ATPbinding cassette transporter A1 (ABCA1)-mediated cellular cholesterol efflux in diabetic golden hamsters. J Int Med Res 2007;35:508-516. 39 Wong J, Quinn CM, Brown AJ. Statins inhibit synthesis of an oxysterol ligand for the liver x receptor in human macrophages with consequences for cholesterol flux. Arterioscler Thromb Vasc Biol 2004;24:2365-2371. 40 Ando H, Tsuruoka S, Yamamoto H, Takamura T, Kaneko S, Fujimura A. Effects of pravastatin on the expression of ATP-binding cassette transporter A1. J Pharmacol Exp Ther 2004;311:420-425.
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41 Sone H, Shimano H, Shu M, Nakakuki M, Takahashi A, Sakai M, et al. Statins downregulate ATP-binding-cassette transporter A1 gene expression in macrophages. Biochem Biophys Res Commun 2004;316:790-794. 42 Fan P, Zhang B, Kuroki S, Saku K. Pitavastatin, a potent hydroxymethylglutaryl coenzyme a reductase inhibitor, increases cholesterol 7 alpha-hydroxylase gene expression in HepG2 cells. Circ J 2004;68:1061-1066. 43 Habeos I, Ziros PG, Psyrogiannis A, Vagenakis AG, Papavassiliou AG. Statins and transcriptional regulation: the FXR connection. Biochem Biophys Res Commun 2005;334: 601-605. 44 Zhang Y, Castellani LW, Sinal CJ, Gonzalez FJ, Edwards PA. Peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC-1alpha) regulates triglyceride metabolism by activation of the nuclear receptor FXR. Genes Dev 2004;18:157-169. 45 Seo M, Inoue I, Ikeda M, Nakano T, Takahashi S, Katayama S, et al. Statins activate human PPARalpha promoter and increase PPARalpha mRNA expression and activation in HepG2 cells. PPAR Res 2008;2008:316306. 46 Li T, Chiang JY. A novel role of transforming growth factor beta1 in transcriptional repression of human cholesterol 7alpha-hydroxylase gene. Gastroenterology 2007;133:16601669. 47 Parks DJ, Blanchard SG, Bledsoe RK, Chandra G, Consler TG, Kliewer SA, et al. Bile acids: natural ligands for an orphan nuclear receptor. Science 1999;284:1365-1368. 48 Lee FY, Lee H, Hubbert ML, Edwards PA, Zhang Y. FXR, a multipurpose nuclear receptor. Trends Biochem Sci 2006;31: 572-580. 49 Abrahamsson A, Gustafsson U, Ellis E, Nilsson LM, Sahlin S, Björkhem I, et al. Feedback regulation of bile acid synthesis in human liver: importance of HNF-4alpha for regulation of CYP7A1. Biochem Biophys Res Commun 2005;330:395-399. 50 Lee MS, Park JY, Freake H, Kwun IS, Kim Y. Green tea catechin enhances cholesterol 7alpha-hydroxylase gene
expression in HepG2 cells. Br J Nutr 2008;99:1182-1185. 51 Shang Q, Pan L, Saumoy M, Chiang JY, Tint GS, Salen G, et al. The stimulatory effect of LXRalpha is blocked by SHP despite the presence of a LXRalpha binding site in the rabbit CYP7A1 promoter. J Lipid Res 2006;47:997-1004. 52 Jiang W, Miyamoto T, Kakizawa T, Nishio SI, Oiwa A, Takeda T, et al. Inhibition of LXRalpha signaling by vitamin D receptor: possible role of VDR in bile acid synthesis. Biochem Biophys Res Commun 2006;351:176-184. 53 Martínez-Jiménez CP, Castell JV, Gómez-Lechón MJ, Jover R. Transcriptional activation of CYP2C9, CYP1A1, and CYP1A2 by hepatocyte nuclear factor 4alpha requires coactivators peroxisomal proliferator activated receptor-gamma coactivator 1alpha and steroid receptor coactivator 1. Mol Pharmacol 2006;70:1681-1692. 54 Dong SH, Lee J, Koh DH, Choi MH, Jang HJ, Kae SH. Pravastatin activates PPARalpha/PPARgamma expression in the liver and gallbladder epithelium of hamsters. Hepatobiliary Pancreat Dis Int 2011;10:185-190. 55 Cariati A, Piromalli E. Ultrastructural basis of the failure of oral dissolution therapy with bile salts and/or statin for cholesterol gallstones. Expert Opin Pharmacother 2012;13: 1387-1388. 56 Cariati A, Piromalli E. Limits and perspective of oral therapy with statins and aspirin for the prevention of symptomatic cholesterol gallstone disease. Expert Opin Pharmacother 2012;13:1223-1227. 57 Zhang W, Chen P, Zhao Y, Lou G. Farnesoid X receptor expression is reduced in human hepatocellular carcinoma. J Med College PLA 2012:27:1-9. 58 Yang F, Huang X, Yi T, Yen Y, Moore DD, Huang W. Spontaneous development of liver tumors in the absence of the bile acid receptor farnesoid X receptor. Cancer Res 2007;67:863-867. Received November 7, 2012 Accepted after revision July 24, 2013
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Pravastatin activates the expression of farnesoid X receptor and liver X receptor alpha in Hep3B cells Hyun Woo Byun, Eun Mi Hong, Soo Hee Park, Dong Hee Koh, Min Ho Choi, Hyun Joo Jang, Sea Hyub Kae and Jin Lee Hwasung, Republic of Korea
BACKGROUND: Statins are suggested to preserve gallbladder function by suppressing pro-inflammatory cytokines and preventing cholesterol accumulation in gallbladder epithelial cells. They also affect cross-talk among the nuclear hormone receptors that regulate cholesterol-bile acid metabolism in the nuclei of hepatocytes. However, there is controversy over whether or how statins change the expression of peroxisome proliferator-activated receptor (PPAR)α, PPARγ, liver X receptor α (LXRα), farnesoid X receptor (FXR), ABCG5, ABCG8, and 7α-hydroxylase (CYP7A1) which are directly involved in the cholesterol saturation index in bile. METHODS: Human Hep3B cells were cultured on dishes. MTT assays were performed to determine the appropriate concentrations of reagents to be used. The protein expression of PPARα and PPARγ was measured by Western blotting analysis, and the mRNA expression of LXRα, FXR, ABCG5, ABCG8 and CYP7A1 was estimated by RT-PCR.
and CYP7A1 in human hepatocytes. (Hepatobiliary Pancreat Dis Int 2014;13:65-73) KEY WORDS: pravastatin; PPARγ; liver X receptor α; farnesoid X receptor; gallstone disease
Introduction
T
he cholesterol saturation index (CSI) of bile produced by hepatocytes is a major determinant of cholesterol gallstone formation and cholesterolosis in the gallbladder (GB).[1, 2] Representative nuclear hormone receptors regulate cholesterol-bile acid RESULTS: In cultured Hep3B cells, pravastatin activated PPARα metabolism. These receptors include liver X receptor α and PPARγ protein expression, induced stronger expression of (LXRα), farnesoid X receptor (FXR), and peroxisome PPARγ than that of PPARα, increased LXRα mRNA expression, proliferator-activated receptor (PPAR), and they activated ABCG5 and ABCG8 mRNA expression mediated by influence the regulatory targets of one another.[3-5] FXR as well as LXRα, enhanced FXR mRNA expression, and LXRα in hepatocytes works as a heterodimer with increased CYP7A1 mRNA expression mediated by the PPARγ retinoid X receptor (RXR) to lower the cholesterol and LXRα pathways, together or independently. concentration in response to oxycholesterol. Activation CONCLUSION: Our data suggested that pravastatin prevents of LXRα in hepatocytes induces the transcription of cholesterol gallstone diseases via the increase of FXR, LXRα 7α-hydroxylase (CYP7A1), the rate-limiting enzyme in hepatic bile acid synthesis, and leads to conversion of cholesterol to bile acid.[6] In addition, activated LXRα in hepatocytes induces ABCG5/ABCG8 located at the Author Affiliations: Division of Gastroenterology, Department of Internal canalicular membrane of cells, and transports excessive Medicine, Hallym University College of Medicine, Dongtan Sacred Heart intracellular cholesterol into bile. Thus, sustained Hospital, 40 Seokwoo-dong, Hwasung, Kyungki-Do 445-170, Republic of Korea (Byun HW, Hong EM, Park SH, Koh DH, Choi MH, Jang HJ, Kae SH activation of LXRα-mediated ABCG5/ABCG8 produces and Lee J) supersaturated bile, resulting in the formation of [7-10] However, a recent study[11] Corresponding Author: Jin Lee, MD, Division of Gastroenterology, cholesterol gallstones. Department of Internal Medicine, Hallym University College of Medicine, revealed that the LXR response element (LXRE) is not Dongtan Sacred Heart Hospital, 40 Seokwoo-dong, Hwasung, Kyungki-Do conserved in the human CYP7A1 promoter. Accordingly, 445-170, Republic of Korea (Tel: 82-31-8086-2015; Fax: 82-31-8086-2029; there is controversy over whether LXR agonist activates Email: [email protected]) CYP7A1 in human hepatocytes.[12, 13] © 2014, Hepatobiliary Pancreat Dis Int. All rights reserved. FXR in hepatocytes also works as a heterodimer doi: 10.1016/S1499-3872(14)60009-6 Hepatobiliary Pancreat Dis Int，Vol 13，No 1 • February 15，2014 • www.hbpdint.com • 65
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with RXR to maintain adequate concentrations of bile acid. An increased concentration of bile acid in hepatocytes activates FXR, inducing intracellular CYP7A1 and sterol-12-hydroxylase (CyP8B1) in another pathway of bile acid synthesis, resulting in decreased synthesis of bile acid.[14-16] Additionally, activated FXR induces bile salt exporter pump (BSEP, ABCB11), which secretes intracellular bile acid into bile, and multi-drug resistance gene 3 (MDR3, ABCB4), which secretes intracellular phospholipids into bile, thereby maintaining the solubility of cholesterol in hepatic bile.[8] Recently, it was suggested that activation of FXR induces hepatic expression of ABCG5/ABCG8, which may play a more important role in maintaining hepatic cholesterol homeostasis.[13, 17] PPARs, upon heterodimerization with RXR, function as transcriptional regulators of glucose and lipid metabolism.[18, 19] Some studies[20, 21] have indicated that stimulation of PPARα and PPARγ increases LXRα expression, and that a PPAR response element (PPRE) exists in the promoter of LXRα and functions as a PPARselective response element. In addition, our previous study[22] in GB epithelial cells (GBECs) documented that PPARα and PPARγ ligands can block the NF-κB pathway, thus modulating the inflammatory reaction, and that PPARα and PPARγ agonists also induce LXRαmediated ABCA1 activation. These results suggest that PPAR agonist can preserve GB function. Interestingly, fibrates known as PPARα agonists, contribute to cholesterol gallstone formation because they not only suppress the synthesis of bile acid by PPARα-mediated down regulation of CYP7A1 but also stimulate LXRαABCG5/ABCG8, which promotes cholesterol excretion into bile, leading to cholesterol supersaturation of bile.[23, 24] Accordingly, PPARα agonist cannot be used to prevent gallstones, despite the ability of PPARα to preserve GB function. Statin, an hydroxy-methl-glutaryl CoA (HMG-CoA) reductase inhibitor that is safely used for the treatment of dyslipidemia, inhibits the synthesis of cholesterol in the liver. Clinical evidence on the preventive role of statin in gallstone formation is conflicting. Population-based case-control studies, however, recently reported that long-term sustained statin use decreases the risk of gallstone diseases and reduces the risk of surgery for gallstone diseases.[25, 26] Most studies suggest that statin reduces the CSI of bile, or at least does not worsen the biliary lipid composition,[27, 28] despite PPARα induction. We also demonstrated that in GBECs, statins activate PPARα and PPARγ, modulate the inflammatory response by blocking lipopolysaccharide-induced TNF-α production, and eliminate excessive cholesterol via the LXRα/RXR-
ABCA1-mediated cholesterol efflux system.[29] Little is known about the mechanisms of how statin affects the homeostasis of hepatic cholesterol-bile acid metabolism, which is associated with the CSI of bile. The present study was to investigate the effects of statin on various regulatory factors, including PPARα, PPARγ, LXRα, FXR, ABCG5/ABCG8, and CYP7A1, in human hepatocytes.
Methods Materials Dulbecco's modified Eagle's medium (DMEM) was from Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS) and penicillin/streptomycin were from Hyclone (South Logan, UT, USA). Trypsin/ethylene diamine tetraacetic acid (EDTA), dimethyl sulfoxide (DMSO), 3-[4, 5-dimethylthiazol-2-yl] diphenyl tetrazolium bromide (MTT), 22(R)-hydroxycholesterol, and ursodeoxycholic acid (UDCA) were from Sigma Chemicals (St. Louis, MO, USA). Pravastatin, WY-14643, and troglitazone were from Cayman Chemicals (Ann Arbor, MI, USA). The peroxidase-conjugated anti-rabbit IgG antibody was from Amersham Biosciences (Buckinghamshire, UK). The rabbit polyclonal anti-human β-actin antibody was from Cell Signaling Technology (Danvers, MA, USA). The Western blot detection kit (Visualizer) was from Upstate (Lake Placid, NY, USA). Rabbit polyclonal anti-mouse PPARα antibody (a synthetic peptide corresponding to N-terminal amino acids 1-18 of mouse PPARα (NP_038482.3), which has 100% homology with human PPARα) and rabbit polyclonal PPARγ anti-human antibody were purchased from Abcam (Cambridge, UK). Cell culture Hep3B cells, a hepatoma cell line, were used for our experiments. Stock cultures were grown on 100-mm dishes in DMEM (including 4.5 g/L glucose) supp lemented with 10% FBS, 2 mmol/L L-glutamine, 1.5 g/L sodium bicarbonate, 100 IU/mL penicillin, and 100 μg/ mL streptomycin. The medium was changed twice a week, and the cells were maintained at 37 ℃ in an incubator with 5% CO2. The cells were passaged when confluent (every 5 to 7 days) using trypsin (2.5 g/L) and EDTA (1.0 g/L). For experiments, the cells were grown on 60-mm dishes, and the medium was changed to serum free medium (SFM) containing 0.2% bovine serum albumin (BSA) (Sigma, St. Louis, MO, USA). MTT The loading dose of chemicals or reagents used in
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the experiments described below was determined by MTT assays as the maximum concentration that did not affect the proliferation of cells for 24 hours. Cells were plated at a density of 5×104 cells/mL in 24-well plates and cultured to 60% confluence in serum-containing regular medium. They were then incubated with or without various concentrations of reagents (pravastatin, WY-14643, and troglitazone) in SFM for 24 hours. MTT (0.5 mg/mL) was then added to each well, and the cells were incubated for another 4 hours at 37 ℃. After decanting the medium, 500 μL of DMSO was added to each well. After 10 minutes of constant and gentle shaking, the color intensity (proportional to the number of live cells) was assessed with an ELX800 (Biotek, Winooski, VT, USA) at a 570 nm wavelength.
RNA extraction and RT-PCR analysis Hep3B cells were cultured to confluence on 60-mm dishes. The cells were then incubated in SFM containing 0.2% BSA with pravastatin (1, 5, 10, 20 μmol/l), WY-14643 (100 μmol/L), troglitazone (10 μmol/L), UDCA (150 μmol/L), or 22(R)-hydroxycholesterol (10 μmol/L) for 24 hours as indicated. The cells were harvested, and RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RT-PCR was performed with the ampiRivert 1-step RT-PCR system according to the manufacturer's instructions (Gendepot, Barker, TX, USA). The sequences of primers are described in Table. The total reaction was performed in 50 μL mixtures containing total RNA (1 μg), 20 pmol primer, RT-PCR enzyme mix (5 U AMV transcriptase, 1.25 U Taq DNA polymerase), and 2× RT-PCR buffer mix (2× reaction buffer, 0.4 mmol/L dNTP, and 3.5 mmol/L MgCl2). The RT reaction was set at 45 ℃ for 45 minutes and then Table. Primer sequences used for RT-PCR Primers Direction Sequence (5' to 3') LXRα GAPDH ABCG5 ABCG8 FXR CYP7A1
Foward Reverse Foward Reverse Foward Reverse Foward Reverse Foward Reverse Foward Reverse
GTT CCC ACG GAT GCT AAT AAT GTT TGC CCT TCT CAG TC GGA GTC AAC GGA TTT GGT C GGA AGA TGG TGA TGG GAT T GGT TCT CAC CAT TCA CCA G GGT TTC TAT TTC CCG TTC C CAG ATT TCC AAC GAC TTC C CC GTC TTC CAG TTC ATA G CCT GGA TTT CTT CTG GAC AT TGT ATT GCG AGT ATG GTT CC CAA CGT ATC ATG AGA CCT CCA GTC CAG CTT CAA ACA TCA CTC GGT AG
Tm Size (℃) (bp) 57 231 57
225
53
220
58
240
57
251
57
102
at 95 ℃ for 3 minutes. The PCR conditions were as follows: denaturation at 94 ℃ for 30 seconds, annealing at 53-58 ℃ for 30 seconds, and extension at 72 ℃ for 30 seconds. The reaction was ended with an additional extension at 72 ℃ for 7 minutes, and then samples were chilled to 4 ℃. The mRNA was amplified by 26 cycles for LXRα, 40 cycles for ABCG5 and CYP7A1, 35 cycles for ABCG8, and 25 cycles for FXR. The PCR products were fractionated by electrophoresis on 2% agarose gels containing ethidium bromide.
Protein extraction and Western blotting analysis Hep3B cells were cultured to confluence on 60-mm dishes with regular media. The cells were treated with pravastatin (5, 10, 20 μmol/L), WY14643 (100 μmol/L), or troglitazone (10 μmol/L) in SFM containing 0.2% BSA for 24 hours as indicated. They were then washed with PBS and harvested with lysis buffer [50 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 1 mmol EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 μmol/L phenylmethylsulfonyl fluoride (PMSF), 5 μg/ mL aprotinin, and 5 μg/mL leupeptin]. Proteins were quantified by the Bradford assay (Sigma St. Louis, Mo, USA). SDS-PAGE was performed with a 4% stacking gel and 10% resolving gel, followed by transfer to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The membranes were blocked overnight at 4 ℃ in a blocking solution [5% skim milk in Tris-buffer with Tween-20 (TBS-T): 200 mmol/L Tris, 500 mmol/L NaCl, pH 7.5, and 0.05% v/v Tween-20]. The membranes were then incubated with rabbit polyclonal PPARα antibody or rabbit polyclonal PPARγ antibody for 1 hour at room temperature. At the same time, the rabbit polyclonal β-actin antibody was used to detect the β-actin housekeeping protein to normalize the amount of protein. The membranes were washed with TBS-T and incubated with peroxidase-conjugated anti-rabbit IgG for 1 hour at room temperature. The membrane was washed and incubated using a Visualizer Western blot detection kit for 5 minutes. Autoradiography was then performed. The signal intensities for specific bands on the Western blotting were quantified using NIH Image J density analysis software (Version 1.20). Statistical analysis Results from each experiment are expressed as the mean±SD of duplicate cultures, and all results described are representative of at least three separate experiments. One-way analysis of variance (ANOVA) for three or more unpaired groups or Student's t test for two unpaired groups was used, and P<0.05 was considered statistically significant.
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Results Pravastatin activates the expression of PPARα and PPARγ in Hep3B cells The expression of PPARα and PPARγ in response to pravastatin (a hydrophilic HMG-CoA reductase inhibitor) was studied in human Hep3B cells. We used 100 μmol/L WY-14643 as a positive control for PPARα and 10 μmol/L troglitazone as a positive control for PPARγ. PPARα expression was increased significantly by a high concentration of pravastatin (P<0.05, versus no-treatment control), but was increased only slightly by low concentrations, 5 or 10 μmol/L of pravastatin (Fig. 1A). PPARγ expression was increased significantly by low concentrations of pravastatin (P<0.01, versus notreatment control) as well as by a high concentration (P<0.001, versus no-treatment control) in a concentration dependent manner (Fig. 1B). These findings suggest that pravastatin has a greater effect on the expression of PPARγ than on the expression of PPARα. Pravastatin activates the expression of LXRα mRNA in Hep3B cells To assess whether statins stimulate the expression of LXRα mRNA, which is induced by activated PPAR, cultured Hep3B cells were incubated with various concentrations of pravastatin, 10 μmol/L 22(R)hydroxycholesterol as an LXRα positive control, and no-treatment as a negative control. When the treatment concentration of pravastatin was 5 μmol/L or more, the expression of LXRα mRNA was significantly increased, compared with the no-treatment control (P<0.05), to a level similar to that observed with 22(R)hydroxycholesterol treatment (Fig. 2). Pravastatin activates FXR mRNA expression in Hep3B cells As a bile acid sensor, FXR is important for the regulation of cholesterol homeostasis. In order to evaluate whether statin stimulates the expression of FXR mRNA, cultured Hep3B cells were incubated with various concentrations of pravastatin, 150 μmol/ L UDCA as a positive control, and no-treatment as a negative control. Pravastatin increased the expression of FXR mRNA significantly in a concentration dependent manner (P<0.01, Fig. 3). Pravastatin induces the expression of ABCG5/ABCG8 mRNA via FXR and LXRα in Hep3B cells We found that pravastatin induced the expression of PPARα, PPARγ, LXRα, and FXR. Because no LXREs have been identified in the promoters of ABCG5 or ABCG8,
whether a potent LXR agonist can induce ABCG5 and ABCG8 in human hepatocytes is controversial.[11] On the other hand, activation of FXR may induce hepatic expression of ABCG5/ABCG8 through a common FXR response element, which promotes biliary-free cholesterol secretions.[13, 17] Accordingly, RT-PCR for ABCG5/ ABCG8 was performed using the same conditions as for LXRα to evaluate whether pravastatin induces the expression of ABCG5 and ABCG8 mRNA. The increased expression of ABCG5 mRNA was significantly dependent on the concentration of pravastatin (Fig. 4A). A high concentration of pravastatin (10 or 20 μmol/L) induced the expression of ABCG8 mRNA, though not as much as 22(R)-hydroxycholesterol (Fig. 4B). We
Fig. 1. Pravastatin activates PPARα and PPARγ expression in Hep3B cells. A: Western blotting analysis of PPARα in Hep3B cells after treatment with pravastatin (PS, 5, 10, 20 μmol/L or PPARα ligand [positive control, WY-14643 (WY), 100 μmol/L] for 24 hours; B: Western blotting analysis of PPARγ in Hep3B cells after treatment with pravastatin (PS, 5, 10, 20 μmol/L) or PPARγ ligand [positive control, troglitazone (TR), 10 μmol/L] for 24 hours. *: P<0.05, versus no-treatment control (Cont); **: P<0.001, versus Cont and P<0.01, versus pravastatin 5 μmol/L (PS-5) and 10 μmol/ L (PS-10) treatment; #: P<0.01, versus Cont. The relative expression was calculated with adjustment of β-actin expression.
Fig. 2. Pravastatin increases LXRα mRNA expression in Hep3B cells. RT-PCR assay for LXRα was performed in the presence of pravastatin (PS, 1, 5, 10, 20 μmol/L) or 22(R)-hydroxycholesterol (positive control, OH-C, 10 μmol/L) for 24 hours. *: P<0.05, versus no-treatment control (Cont). The relative expression was calculated with adjustment of GAPDH expression.
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Fig. 3. Pravastatin increases FXR mRNA expression in Hep3B cells. RT-PCR assay for FXR mRNA was performed in the presence of pravastatin (PS, 1, 5, 10, 20 μmol/L) or UDCA (positive control, 150 μmol/L) for 24 hours. *: P<0.01, versus no-treatment control (Cont); **: P<0.001, versus Cont.
Fig. 4. Pravastatin increases ABCG5/ABCG8 mRNA expression in Hep3B cells. A: RT-PCR assay for ABCG5 mRNA was performed in the presence of pravastatin (PS, 1, 5, 10, 20 μmol/L) or 22(R)hydroxycholesterol (positive control, OH-C, 10 μmol/L) for 24 hours; B: RT-PCR assay for ABCG8 mRNA was performed in the presence of pravastatin (PS, 1, 5, 10, 20 μmol/L) or 22(R)hydroxycholesterol (positive control, OH-C, 10 μmol/L) for 24 hours. *: P<0.05, versus no-treatment control (Cont); **: P<0.01, versus Cont; #: P<0.001, versus Cont. The relative expression was calculated with adjustment of GAPDH expression.
Fig. 5. FXR ligand as well as LXRα ligand increases ABCG5/ ABCG8 mRNA expression in Hep3B cells. RT-PCR assays for ABCG5 (A) and ABCG8 (B) mRNA were performed in the presence of 22-(R)-hydroxycholesterol (OH-C, 10 μmol/L), UDCA (150 μmol/L), and pravastatin (PS, 10 μmol/L) for 24 hours. *: P<0.05, versus no-treatment control (Cont); **: P<0.01, versus Cont and P<0.05 versus UDCA treatment. The relative expression was calculated with adjustment of GAPDH expression.
Fig. 6. Pravastatin increases CYP7A1 mRNA expression mediated by the PPARγ-LXRα pathway in Hep3B cells. A: RT-PCR assay for CyP7A1 mRNA was performed in the presence of pravastatin (PS, 1, 5, 10, 20 μmol/L) for 24 hours; B: RT-PCR assay for CYP7A1 mRNA was performed in the presence of 22(R)-hydroxycholesterol (OH-C, 10 μmol/L), UDCA (150 μmol/L), WY-14643 (100 μmol/ L), and troglitazone (10 μmol) for 24 hours. *: P<0.05, versus notreatment control (Cont); **: P<0.01, versus Cont. The relative expression was calculated with adjustment of GAPDH expression.
also examined whether FXR induces the expression of ABCG5 and ABCG8 mRNA in Hep3B cells. The expression of ABCG5/ABCG8 mRNA was significantly of CYP7A1, which is activated by LXRα and inhibited by increased by UDCA, an FXR ligand, as well as by LXRα FXR via negative feedback. To determine this, RT-PCR ligand. However, LXRα stimulation induced a stronger was performed on Hep3B cells stimulated by pravastatin. expression of ABCG5 and ABCG8 mRNA, compared As shown in Fig. 6A, pravastatin significantly enhanced with FXR stimulation by UDCA (P<0.05) (Fig. 5). the expression of CYP7A1 mRNA in a concentration dependent manner in Hep3B cells. The results in Fig. 6B Pravastatin increases CYP7A1 mRNA expression via indicate that activation of CYP7A1 mRNA by pravastatin PPARγ-LXRα pathway in Hep3B cells is mediated by the PPARγ and LXRα pathways, together In this study, pravastatin appeared to stimulate the or independently. expression of FXR mRNA and LXRα mRNA in cultured Hep3B cells. However, the LXRE is not conserved in the human CYP7A1 promoter,[11] and LXR agonists repress CYP7A1 in human hepatocytes.[12] Therefore, we Discussion wondered whether pravastatin stimulates the expression In this study, statin affected the cross-talk between Hepatobiliary Pancreat Dis Int，Vol 13，No 1 • February 15，2014 • www.hbpdint.com • 69
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nuclear hormone receptors, including PPARα/PPARγ, LXRα, and FXR, associated with cholesterol-bile acid-phospholipid balance in hepatic bile, probably contributing to the prevention of cholesterol gallstones. Additionally, the current study documented possible molecular mechanisms by which statin prevents gallstones formation. The present study demonstrated in hepatocytes that statin stimulates PPARα, PPARγ, and LXRα expression, and that PPARγ expression changes much more than PPARα expression, even in low concentrations of pravastatin. Considering that PPARγ agonist blocks the repression of CYP7A1 mRNA[30] and has a more powerful anti-inflammatory effect than fibrates,[22] the stronger activation of PPARγ induced by statin may play a preventive role in cholesterol gallstones formation. Some studies have suggested probable mechanisms by which statins activate PPARγ: statins enhance peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) activity by suppressing Akt phosphorylation,[31] and PPARγ also interacts with PGC1α.[32] On the other hand, PGC-1α plays a role in the prevention of cholelithiasis via interaction with the bile acid sensor, FXR,[33] and PPARγ may prevent cholesterol accumulation in GBECs by LXRα-mediated ABCA1 activation.[22] ABCG5 and ABCG8 are expressed predominantly on the basolateral (bile-contacting) side of hepatocytes and on the apical (luminal) side of enterocytes in the proximal small intestine of humans and mice. They play a pivotal role in transporting excessive cholesterol from hepatocytes into bile or from enterocytes into the lumen.[10] Accordingly, overexpression of ABCG5 and ABCG8 in hepatocytes may induce supersaturation of bile, resulting in the formation of gallstones.[8] LXRα, a nuclear receptor that plays a key role in regulating genes involved in cholesterol trafficking, is required for the increased expression of ABCG5 and ABCG8 in response to cholesterol or oxycholesterol.[10, 34] However, it was reported that a potent LXR agonist or cholesterol treatment failed to induce ABCG5 and ABCG8 in primary human hepatocytes.[13] In contrast, another study[35] indicated that the expression of both ABCG5 and ABCG8 increases substantially upon LXR activation in human enterocytes. In addition, even though no LXREs have been identified in the promoters of ABCG5 or ABCG8, the intergenic region was found to act as a bidirectional promoter and be partially responsive to treatment with LXR agonists.[36] Results from studies investigating whether statin activates the expression of LXRα, ABCA1, or ABCG5/ABCG8 are inconsistent.[37-41] On the other hand, the hepatic expression of ABCG5/
ABCG8 is induced by activation of FXR through a common FXRE, which promotes cholesterol secretions, and the FXR-ABCG5/ABCG8 pathway may play a more important role in maintaining hepatic cholesterol homeostasis than the LXRα-ABCG5/ABCG8 pathway in human hepatocytes.[13, 17] The present study showed that pravastatin stimulated the expression of ABCG5/ABCG8 mRNA in Hep3B cells through the activation of LXRα and FXR. Although there might be differences between cancer cells and normal hepatocytes, the result suggests that statin mediates cholesterol secretion into bile by inducing ABCG5/ABCG8 expression via both LXRα and FXR activation in hepatocytes. Although statin activates PPARα and LXRα-ABCG5/ ABCG8, which are associated with supersaturated bile, no studies have reported that statin induces gallstones formation. Thus, we considered other potential reasons for increased cholesterol solubility in bile, including CYP7A1 and FXR participation in bile acid homeostasis. Controversy exists over the effects of statins on FXR expression in hepatocytes.[42, 43] Our study demonstrated that statin enhances FXR expression in a concentration dependent manner in hepatocytes. Enhanced FXR expression induced by pravastatin, along with other possible mechanisms described in the next paragraph, presumably contributes to the maintenance or increase of cholesterol solubility in bile. On the other hand, PGC1α, PPARγ, and hepatocyte nuclear factor-4α (HNF-4α) mRNAs are induced after a prolonged stimulus such as fasting, and PGC-1α coactivates PPARγ and/or HNF4α bound to a direct repent-1 (DR-1) element in the FXR promoter, to induce FXR mRNA expression.[44] In addition, statin induces PGC-1α, PPARγ, and HNF4α.[31, 45] Taken together, statin probably induces FXR through PGC-1α, PPARγ, and HNF-4α activation. CYP7A1 is mainly regulated at the gene transcription level by bile acid, cholesterol, amino acid taurine, cytokines, hormones, and transcriptional regulators.[11, 46] The LXRE does not exist in the human CYP7A1 promoter,[11] and some studies[14, 47, 48] reported that LXR agonists repress CYP7A1 in human hepatocytes.[12] Furthermore, FXR inhibits CYP7A1, and PPARα agonist represses CYP7A1.[23, 24] Accordingly, we expected CYP7A1 mRNA levels in Hep3B cells treated with pravastatin to decrease or remain unchanged. However, contrary to our expectations, pravastatin enhanced CYP7A1 expression. Possible mechanisms responsible for the enhanced expression of CYP7A1 by statin are as follows: 1) Although no LXREs have been identified in the promoter of CYP7A1, the intergenic region may partially respond to LXR agonists like ABCG5 and ABCG8.[36] In this experiment,
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enhanced expression of CYP7A1 induced by 22(R)hydroxycholesterol supports this theory. 2) We observed that PPARγ agonist increased the expression of CYP7A1. PGC-1α activates CYP7A1 transcription together with HNF-4α by increasing HNF-4αmediated transactivation of CYP7A1,[49] and PPARγ also interacts with PGC-1α.[44] Therefore, we suggest that the statin-PPARγ-PGC-1α-HNF-4α pathway may be involved. Additional study is needed to substantiate this hypothesis. This result does indicate that statin promotes hepatic bile acid synthesis by inducing CYP7A1 and maintains cholesterol solubility in bile. We used Hep3B cells in this study in view of the technical advantages of hepatocyte culture, because Hep3B and HepG2 hepatoma cell lines have cellular characteristics similar to normal hepatocytes. Many studies on enzymes, receptors, and transporters, such as CYP7A1,[42, 46, 50] LXRα,[51, 52] FXR,[43] and PPAR,[53] associated with cholesterol metabolism in hepatocytes have been performed with HepG2 or Hep3B cells. In addition, if functional protein studies or over/underexpression studies for specific gene targets were performed, they would clearly demonstrate the crosstalk between nuclear hormone receptors. However, the primary objectives of the study were to demonstrate the change in the expression of the proteins or mRNAs of specific gene related to lipid solubility in bile, and our previous study in hamsters have already documented that pravastatin contributes to the prevention of gallstones formation.[54] Moreover, we agree that the nucleation and growth of cholesterol gallstones in humans is a complex phenomenon which is associated with not only cholesterol liver metabolism but also gallbladder parietal factors such as mucins, gallbladder motility, and calcium bilirubinate precipitation.[55, 56] Accordingly, a preventive effect on gallstones disease could be achieved by acting also on the other factors involved in gallstones formation. A study[57] indicated that the expression of FXR and its target gene small heterodimer partner (SHP) was strongly down-regulated in human hepatocellular carcinoma cells, which is consistent with a tumor suppressive role of FXR.[58] Therefore, Hep3B cells treated with pravastatin might show some different response compared with primary hepatocytes in expressional change of FXR mRNA, which may be another limitation of this study. In an aspect, our results showed a possibility that statin may have anti-neoplastic effect because statin enhanced expression of FXR mRNA in Hep3B cells. In conclusion, in human hepatocytes, pravastatin induces the expression of PPARγ, more so than that
of PPARα, and activates the expression of FXR as well as LXRα. In addition, pravastatin activates ABCG5/ ABCG8 expression, which is mediated by both LXRα and FXR, and increases CYP7A1 expression, which is induced by activation of LXRα and PPARγ, together or independently. We postulate that statin increases cholesterol solubility in bile by simultaneous activation of FXR which promotes bile acid excretion and CYP7A1 which enhances hepatic bile acid synthesis,[8] although statin also activates LXRα-ABCG5/ABCG8 which excretes cholesterol into bile.[7-10] We thus suggest that statin-induced increased expression of both FXR and LXRα and enhancement of CYP7A1 expression in human hepatocytes, with the known action of statin to preserve GB function, can play a preventive role in cholesterol gallstones diseases. Contributors: LJ proposed the study. BHW and LJ performed research, wrote the first draft and analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. LJ is the guarantor. Funding: None. Ethical approval: Not needed. Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.
References 1 Jacyna MR, Ross PE, Bakar MA, Hopwood D, Bouchier IA. Characteristics of cholesterol absorption by human gall bladder: relevance to cholesterolosis. J Clin Pathol 1987;40: 524-529. 2 Corradini SG, Elisei W, Giovannelli L, Ripani C, Della Guardia P, Corsi A, et al. Impaired human gallbladder lipid absorption in cholesterol gallstone disease and its effect on cholesterol solubility in bile. Gastroenterology 2000;118: 912-920. 3 Trauner M, Halilbasic E. Nuclear receptors as new perspective for the management of liver diseases. Gastroenterology 2011; 140:1120-1125. 4 Repa JJ, Mangelsdorf DJ. Nuclear receptor regulation of cholesterol and bile acid metabolism. Curr Opin Biotechnol 1999;10:557-563. 5 Russell DW. Nuclear orphan receptors control cholesterol catabolism. Cell 1999;97:539-542. 6 Peet DJ, Janowski BA, Mangelsdorf DJ. The LXRs: a new class of oxysterol receptors. Curr Opin Genet Dev 1998;8:571-575. 7 Moschetta A, Bookout AL, Mangelsdorf DJ. Prevention of cholesterol gallstone disease by FXR agonists in a mouse model. Nat Med 2004;10:1352-1358. 8 Wittenburg H, Lyons MA, Li R, Churchill GA, Carey MC, Paigen B. FXR and ABCG5/ABCG8 as determinants of cholesterol gallstone formation from quantitative trait locus mapping in mice. Gastroenterology 2003;125:868-881. 9 Yu L, York J, von Bergmann K, Lutjohann D, Cohen JC, Hobbs HH. Stimulation of cholesterol excretion by the liver X
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receptor agonist requires ATP-binding cassette transporters G5 and G8. J Biol Chem 2003;278:15565-15570. 10 Repa JJ, Berge KE, Pomajzl C, Richardson JA, Hobbs H, Mangelsdorf DJ. Regulation of ATP-binding cassette sterol transporters ABCG5 and ABCG8 by the liver X receptors alpha and beta. J Biol Chem 2002;277:18793-18800. 11 Chiang JY, Kimmel R, Stroup D. Regulation of cholesterol 7alpha-hydroxylase gene (CYP7A1) transcription by the liver orphan receptor (LXRalpha). Gene 2001;262:257-265. 12 Goodwin B, Watson MA, Kim H, Miao J, Kemper JK, Kliewer SA. Differential regulation of rat and human CYP7A1 by the nuclear oxysterol receptor liver X receptor-alpha. Mol Endocrinol 2003;17:386-394. 13 Li T, Matozel M, Boehme S, Kong B, Nilsson LM, Guo G, et al. Overexpression of cholesterol 7α-hydroxylase promotes hepatic bile acid synthesis and secretion and maintains cholesterol homeostasis. Hepatology 2011;53:996-1006. 14 Makishima M, Okamoto AY, Repa JJ, Tu H, Learned RM, Luk A, et al. Identification of a nuclear receptor for bile acids. Science 1999;284:1362-1365. 15 Wang H, Chen J, Hollister K, Sowers LC, Forman BM. Endogenous bile acids are ligands for the nuclear receptor FXR/BAR. Mol Cell 1999;3:543-553. 16 Seol W, Choi HS, Moore DD. An orphan nuclear hormone receptor that lacks a DNA binding domain and heterodimerizes with other receptors. Science 1996;272:13361339. 17 Li T, Chiang JY. Bile Acid signaling in liver metabolism and diseases. J Lipids 2012;2012:754067. 18 Hirakata M, Tozawa R, Imura Y, Sugiyama Y. Comparison of the effects of pioglitazone and rosiglitazone on macrophage foam cell formation. Biochem Biophys Res Commun 2004; 323:782-788. 19 Mangelsdorf DJ, Evans RM. The RXR heterodimers and orphan receptors. Cell 1995;83:841-850. 20 Chinetti G, Lestavel S, Bocher V, Remaley AT, Neve B, Torra IP, et al. PPAR-alpha and PPAR-gamma activators induce cholesterol removal from human macrophage foam cells through stimulation of the ABCA1 pathway. Nat Med 2001;7: 53-58. 21 Chawla A, Boisvert WA, Lee CH, Laffitte BA, Barak Y, Joseph SB, et al. A PPAR gamma-LXR-ABCA1 pathway in macrophages is involved in cholesterol efflux and atherogenesis. Mol Cell 2001;7:161-171. 22 Lee J, Hong EM, Byun HW, Choi MH, Jang HJ, Eun CS, et al. The effect of PPARalpha and PPARgamma ligands on inflammation and ABCA1 expression in cultured gallbladder epithelial cells. Dig Dis Sci 2008;53:1707-1715. 23 Post SM, Duez H, Gervois PP, Staels B, Kuipers F, Princen HM. Fibrates suppress bile acid synthesis via peroxisome proliferator-activated receptor-alpha-mediated downregulation of cholesterol 7alpha-hydroxylase and sterol 27-hydroxylase expression. Arterioscler Thromb Vasc Biol 2001;21:1840-1845. 24 Kosters A, Frijters RJ, Schaap FG, Vink E, Plösch T, Ottenhoff R, et al. Relation between hepatic expression of ATP-binding cassette transporters G5 and G8 and biliary cholesterol secretion in mice. J Hepatol 2003;38:710-716. 25 Erichsen R, Frøslev T, Lash TL, Pedersen L, Sørensen HT. Long-term statin use and the risk of gallstone disease: A population-based case-control study. Am J Epidemiol 2011;
173:162-170. 26 Merzon E, Weiss NS, Lustman AJ, Elhayani A, Dresner J, Vinker S. Statin administration and risk of cholecystectomy: a population-based case-control study. Expert Opin Drug Saf 2010;9:539-543. 27 Smith JL, Riottot M, Nathanson LK. Effect of statins on biliary lipid and cholesterol galltones. J Kardiol 2002;9:295298. 28 Smith JL, Roach PD, Wittenberg LN, Riottot M, Pillay SP, Nestel PJ, et al. Effects of simvastatin on hepatic cholesterol metabolism, bile lithogenicity and bile acid hydrophobicity in patients with gallstones. J Gastroenterol Hepatol 2000;15: 871-879. 29 Lee J, Hong EM, Koh DH, Choi MH, Jang HJ, Kae SH, et al. HMG-CoA reductase inhibitors (statins) activate expression of PPARalpha/PPARgamma and ABCA1 in cultured gallbladder epithelial cells. Dig Dis Sci 2010;55:292-299. 30 Miyake JH, Wang SL, Davis RA. Bile acid induction of cytokine expression by macrophages correlates with repression of hepatic cholesterol 7alpha-hydroxylase. J Biol Chem 2000;275:21805-21808. 31 Wang W, Wong CW. Statins enhance peroxisome proliferator-activated receptor gamma coactivator-1alpha activity to regulate energy metabolism. J Mol Med (Berl) 2010;88:309-317. 32 Madrazo JA, Kelly DP. The PPAR trio: regulators of myocardial energy metabolism in health and disease. J Mol Cell Cardiol 2008;44:968-975. 33 Bertolotti M, Gabbi C, Anzivino C, Mitro N, Godio C, De Fabiani E, et al. Decreased hepatic expression of PPARgamma coactivator-1 in cholesterol cholelithiasis. Eur J Clin Invest 2006;36:170-175. 34 van der Veen JN, van Dijk TH, Vrins CL, van Meer H, Havinga R, Bijsterveld K, et al. Activation of the liver X receptor stimulates trans-intestinal excretion of plasma cholesterol. J Biol Chem 2009;284:19211-19219. 35 Field FJ, Born E, Mathur SN. LXR/RXR ligand activation enhances basolateral efflux of beta-sitosterol in CaCo-2 cells. J Lipid Res 2004;45:905-913. 36 Remaley AT, Bark S, Walts AD, Freeman L, Shulenin S, Annilo T, et al. Comparative genome analysis of potential regulatory elements in the ABCG5-ABCG8 gene cluster. Biochem Biophys Res Commun 2002;295:276-282. 37 Zanotti I, Potì F, Favari E, Steffensen KR, Gustafsson JA, Bernini F. Pitavastatin effect on ATP binding cassette A1mediated lipid efflux from macrophages: evidence for liver X receptor (LXR)-dependent and LXR-independent mechanisms of activation by cAMP. J Pharmacol Exp Ther 2006;317:395-401. 38 Zhu Y, Wang HJ, Chen LF, Fang Q, Yan XW. Study of ATPbinding cassette transporter A1 (ABCA1)-mediated cellular cholesterol efflux in diabetic golden hamsters. J Int Med Res 2007;35:508-516. 39 Wong J, Quinn CM, Brown AJ. Statins inhibit synthesis of an oxysterol ligand for the liver x receptor in human macrophages with consequences for cholesterol flux. Arterioscler Thromb Vasc Biol 2004;24:2365-2371. 40 Ando H, Tsuruoka S, Yamamoto H, Takamura T, Kaneko S, Fujimura A. Effects of pravastatin on the expression of ATP-binding cassette transporter A1. J Pharmacol Exp Ther 2004;311:420-425.
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41 Sone H, Shimano H, Shu M, Nakakuki M, Takahashi A, Sakai M, et al. Statins downregulate ATP-binding-cassette transporter A1 gene expression in macrophages. Biochem Biophys Res Commun 2004;316:790-794. 42 Fan P, Zhang B, Kuroki S, Saku K. Pitavastatin, a potent hydroxymethylglutaryl coenzyme a reductase inhibitor, increases cholesterol 7 alpha-hydroxylase gene expression in HepG2 cells. Circ J 2004;68:1061-1066. 43 Habeos I, Ziros PG, Psyrogiannis A, Vagenakis AG, Papavassiliou AG. Statins and transcriptional regulation: the FXR connection. Biochem Biophys Res Commun 2005;334: 601-605. 44 Zhang Y, Castellani LW, Sinal CJ, Gonzalez FJ, Edwards PA. Peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC-1alpha) regulates triglyceride metabolism by activation of the nuclear receptor FXR. Genes Dev 2004;18:157-169. 45 Seo M, Inoue I, Ikeda M, Nakano T, Takahashi S, Katayama S, et al. Statins activate human PPARalpha promoter and increase PPARalpha mRNA expression and activation in HepG2 cells. PPAR Res 2008;2008:316306. 46 Li T, Chiang JY. A novel role of transforming growth factor beta1 in transcriptional repression of human cholesterol 7alpha-hydroxylase gene. Gastroenterology 2007;133:16601669. 47 Parks DJ, Blanchard SG, Bledsoe RK, Chandra G, Consler TG, Kliewer SA, et al. Bile acids: natural ligands for an orphan nuclear receptor. Science 1999;284:1365-1368. 48 Lee FY, Lee H, Hubbert ML, Edwards PA, Zhang Y. FXR, a multipurpose nuclear receptor. Trends Biochem Sci 2006;31: 572-580. 49 Abrahamsson A, Gustafsson U, Ellis E, Nilsson LM, Sahlin S, Björkhem I, et al. Feedback regulation of bile acid synthesis in human liver: importance of HNF-4alpha for regulation of CYP7A1. Biochem Biophys Res Commun 2005;330:395-399. 50 Lee MS, Park JY, Freake H, Kwun IS, Kim Y. Green tea catechin enhances cholesterol 7alpha-hydroxylase gene
expression in HepG2 cells. Br J Nutr 2008;99:1182-1185. 51 Shang Q, Pan L, Saumoy M, Chiang JY, Tint GS, Salen G, et al. The stimulatory effect of LXRalpha is blocked by SHP despite the presence of a LXRalpha binding site in the rabbit CYP7A1 promoter. J Lipid Res 2006;47:997-1004. 52 Jiang W, Miyamoto T, Kakizawa T, Nishio SI, Oiwa A, Takeda T, et al. Inhibition of LXRalpha signaling by vitamin D receptor: possible role of VDR in bile acid synthesis. Biochem Biophys Res Commun 2006;351:176-184. 53 Martínez-Jiménez CP, Castell JV, Gómez-Lechón MJ, Jover R. Transcriptional activation of CYP2C9, CYP1A1, and CYP1A2 by hepatocyte nuclear factor 4alpha requires coactivators peroxisomal proliferator activated receptor-gamma coactivator 1alpha and steroid receptor coactivator 1. Mol Pharmacol 2006;70:1681-1692. 54 Dong SH, Lee J, Koh DH, Choi MH, Jang HJ, Kae SH. Pravastatin activates PPARalpha/PPARgamma expression in the liver and gallbladder epithelium of hamsters. Hepatobiliary Pancreat Dis Int 2011;10:185-190. 55 Cariati A, Piromalli E. Ultrastructural basis of the failure of oral dissolution therapy with bile salts and/or statin for cholesterol gallstones. Expert Opin Pharmacother 2012;13: 1387-1388. 56 Cariati A, Piromalli E. Limits and perspective of oral therapy with statins and aspirin for the prevention of symptomatic cholesterol gallstone disease. Expert Opin Pharmacother 2012;13:1223-1227. 57 Zhang W, Chen P, Zhao Y, Lou G. Farnesoid X receptor expression is reduced in human hepatocellular carcinoma. J Med College PLA 2012:27:1-9. 58 Yang F, Huang X, Yi T, Yen Y, Moore DD, Huang W. Spontaneous development of liver tumors in the absence of the bile acid receptor farnesoid X receptor. Cancer Res 2007;67:863-867. Received November 7, 2012 Accepted after revision July 24, 2013
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