Chapter I Introduction

Home , CYP27A1, CYP2B6, CYP2C19, CYP2C8, CYP2C9, Pregnane X receptor

LI, TIANGANG, Ph.D., May, 2006 BIOMEDICAL SCIENCES

PREGNANE X RECEPTOR REGULATION OF BILE ACID METABOLISM AND

CHOLESTEROL HOMEOSTASIS (189 PP.)

Director of Dissertation: John Y. L. Chiang, Ph.D

The nuclear receptor pregnane X receptor (PXR) is activated by bile acids, steroids and

drugs and regulates a network of genes in lipid and drug mechanisms. The goal of this

study is to investigate the role of PXR in the coordinate regulation of bile acid synthetic

and detoxification genes and its implications in cholestatic liver diseases and treatments.

Cholesterol 7α hydroxylase (CYP7A1) catalyzes the rate-limiting step in the classic bile acids synthetic pathway and plays a key role in controlling bile acids homeostasis.

Quantitative real-time PCR (Q-PCR) showed human PXR agonist rifampicin inhibited

CYP7A1 mRNA expression in primary human hepatocytes. Mammalian two-hybrid

assays, co-immunoprecipitation (co-IP) assays and chromatin immunoprecipitation

(ChIP) assay revealed that ligand-activated PXR strongly interacted with HNF4α, the key

activator of human CYP7A1, and blocked HNF4α interaction with co-activator PGC-1α,

thus resulted in inhibition of CYP7A1. CYP3A4 is the most abundant cytochrome P450 monooxygenase expressed in human liver and intestine. Bile acids and drugs-activated

PXR induces CYP3A4, which converts toxic bile acids to non-toxic metabolites for excretion. Studies using Q-PCR, reporter assays, GST pull-down assays and ChIP assays revealed that PXR strongly induced CYP3A4 gene transcription by interacting with

HNF4α, SRC-1 and PGC-1α. SHP, a negative nuclear receptor, reduced PXR

recruitment of HNF4α and SRC-1 to the CYP3A4 chromatin and inhibited CYP3A4.

Interestingly, PXR concomitantly inhibited SHP gene transcription and maximized the

PXR induction of CYP3A4. Taken together, PXR inhibits CYP7A1 to reduce bile acid

synthesis and induces CYP3A4 to detoxify bile acid. Thus, PXR may play a protective

role against cholestasis. Drugs targeted to PXR may be developed for treating cholestatic

liver diseases induced by bile acids and drugs.

Mitochondrial sterol 27-hydroxylase (CYP27A1) catalyzes the side-chain cleavage

reaction in bile acid synthetic pathways and 27-hydroxylation of cholesterol mainly in the

peripheral tissues. Q-PCR revealed that rifampicin induced CYP27A1 mRNA expression

in the intestine-derived Caco2 cells, but not in primary human hepatocytes and HepG2

cells. Rifampicin stimulated CYP27A1 gene transcription in cholesterol laden Caco2 cells

and increased intracellular 27HOC, which stimulates cholesterol efflux by induction of

cholesterol efflux transporters ABCA1 and ABCG1. Mutagenesis analysis,

electrophoretic mobility shift assay and ChIP assays identified a functional PXR binding

site in the human CYP27A1 gene. These data suggest that 27-hydroxycholesterol is an

endogenous LXRα ligand and the PXR/CYP27A1/LXRα signaling pathway regulate

cholesterol efflux in intestine cells. Results revealed an intestine-specific regulation of

human CYP27A1 gene by PXR and suggested a novel role for PXR in cholesterol metabolism and detoxification in the intestine.

PREGNANE X RECEPTOR REGULATION OF BILE ACID METABOLISM

AND CHOLESTEROL HOMEOSTASIS

A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Tiangang Li

May, 2006

Dissertation written by

Tiangang Li

B.S., Jilin University, 1999

Ph.D., Kent State University, 2006

Approved by

Director, Doctoral Dissertation Committee

, John Y. L. Chiang, Professor

Members, Doctoral Dissertation Committee

______, Amy Milsted, Professor

______, Dean E. Dluzen, Professor

______, James P. Hardwick, Associate Professor

______, John R. D. Stalvey, Professor

______, Philip W. Westerman, Professor

Accepted by

______, Director, School of Biomedical Sciences

______, Dean, College of Arts and Sciences

TABLE OF CONTENTS

LIST OF FIGURES...... v ACKNOWLEDGEMENTS ...... vii CHAPTER I. INTRODUCTION ...... 1 A. BILE ACID BIOSYNTHESIS ...... 1 1. Chemistry...... 1 2. Physiological function ...... 5 3. Bile acid biosynthesis pathways ...... 5 3.1. The classic or neutral pathway...... 8 3.2. Acidic or alternative pathway ...... 8 4. Bile acid transport...... 9 4.1. Enterohepatic circulation ...... 9 4.2. Hepatic bile acid transport ...... 12 4.2.1. hepatic bile acid uptake...... 12 4.2.2. canalicular bile acid excretion ...... 13 4.3. Intestinal bile acid transport...... 14 4.3.1. apical bile acid uptake into the enterocytes ...... 14 4.3.2. basolateral efflux of bile acids from enterocytes ...... 15 5. Cholestasis ...... 16 B. CHOLESTEROL HOMEOSTASIS ...... 18 C. REGULATION OF BILE ACID BIOSYNTHESIS AND CHOLESTEROL HOMEOSTASIS...... 22 1. Nuclear hormone receptor...... 22 2. Regulation of bile acid synthesis ...... 28 2.1. Feedforward regulation of CYP7A1...... 29 2.2. Feedback inhibition of CYP7A1...... 32 2.2.1. FXR/SHP dependent pathway ...... 32 2.2.2. FXR/SHP independent pathways...... 35 3. PXR and bile acid metabolism...... 37 3.1. The nuclear receptor PXR...... 37 3.2. PXR and xenobiotic metabolism ...... 38 3.3. PXR and bile acid metabolism...... 42 4. LXR and cholesterol homeostasis...... 43 D. HYPOTHESIS...... 45 CHAPTER II. EXPERIMENTAL PROCEDURES...... 49 A. CELL CULTURE ...... 49 B. LUCIFERASE REPORTER ASSAY ...... 49 1. Calcium-phosphate-DNA co-precipitation transfection method ...... 50

2. Lipofectamin2000 transfection method ...... 51 3. Luciferase and β-gal assays ...... 52 C. MAMMALIAN HYBRID ASSAYS...... 52 D. SITE-DIRECTED MUTAGENESIS...... 55 E. QUANTITATIVE REAL-TIME PCR...... 56 1. RNA isolation ...... 56 2. Reverse transcription PCR...... 57 3. Real time PCR...... 57 F. ELECTROPHORETIC MOBILITY SHIFT ASSAY (EMSA)...... 58 G. CHROMATIN IMMUNOPRECIPITATION (CHIP) ASSAY ...... 60 H. WESTERN IMMUNOBLOTTING ANALYSIS ...... 63 I. CO-IMMUNOPRECIPITATION ASSAY...... 64 J. GST PULL-DOWN ASSAY...... 65 1. GST fusion protein expression...... 65 2. GST pull-down assay...... 68 K. 27 HYDROXYCHOLESTEROL MEASUREMENT...... 69 CHAPTER III. PXR REGULATION OF HUMAN CYP7A1 ...... 71 A. SIGNIFICANCE AND APPROACHES ...... 71 B. RESULTS...... 73 C. SUMMARY ...... 99 CHAPTER IV. PXR REGULATION OF HUMAN CYP3A4...... 100 A. SIGNIFICANCE AND APPROACH ...... 100 B. RESULTS...... 102 C. SUMMARY ...... 132 CHAPTER V. PXR REGULATION OF HUMAN CYP27A1 ...... 133 A. SIGNIFICANCE AND APPROACHES ...... 133 B. RESULTS...... 134 C. SUMMARY ...... 153 CHAPTER VI. DISCUSSION...... 156 APPENDIX A. ABBREVIATIONS ...... 174 REFERENCES...... 178

LIST OF FIGURES

Figure 1. Cholesterol and bile acid structure...... 4 Figure 2. Bile acid biosynthesis pathways in liver...... 7 Figure 3. Enterohepatic circulation...... 11 Figure 4. cholesterol homeostasis in liver...... 20 Figure 5. Structure of nuclear hormone receptors and their binding sites...... 25 Figure 6. Bile acid responsive elements (BAREs) on human and rat CYP7A1...... 31 Figure 7. Bile acid feedback inhibition of CYP7A1 gene transcription...... 34 Figure 8. PXR regulation of xenobiotic metabolism...... 41 Figure 9. CYP27A1 and cholesterol homeostasis in macrophages...... 48 Figure 10. Mammalian two hybrid assay...... 54 Figure 11. Chromatin immunoprecpitation (ChIP) assay...... 62 Figure 12. GST pull down assay...... 67 Figure 13. Rifampicin-activated PXR suppresses the CYP7A1 reporter activity in transient transfection assays in HepG2 cells...... 77 Figure 14. Effect of rifampicin on human CYP7A1 deletion constructs...... 80 Figure 15. Electrophoretic mobility shift assay of PXR/RXR binding to human and rat BARE-I probes...... 83 Figure 16. Effects of HNF4α, PXR/RXRα and PGC-1α on human and rat CYP7A1 promoter activities...... 87 Figure 17. Mammalian two hybrid assays of PXR interaction with HNF4α...... 89 Figure 18. Rifampicin-activated PXR disrupts HNF4α interaction with PGC-1α...... 92 Figure 19. rifampicin impairs the recruitment of PGC-1α to CYP7A1 chromatin...... 95 Figure 20. Co-immunoprecipitation assay of HNF4α interaction with PXR and PGC-1α...... 98 Figure 21. Effect of HNF4α on PXR regulation of CYP3A4 promoter activities...... 107 Figure 22. GST pull-down assays of PXR interactions with HNF4α,SRC-1 and PGC-1α...... 110 Figure 23. Effect of rifampicin on the bindings of PXR, HNF4α, SRC-1 and PGC-1α to CYP3A4 chromatin...... 113 Figure 24. Competitions between HNF4α, SRC-1 and PGC-1α binding to PXR...... 115 Figure 25. Effect of SHP on PXR induction of CYP3A4 promoter activities...... 118 Figure 26. Effect of SHP on the PXR interaction with HNF4α, SRC-1 and PGC-1α .. 120 Figure 27. GST pull-down assays of SHP competition with HNF4α for PXR binding. 123 Figure 28. Effect of SHP on the bindings of PXR, HNF4α, SRC-1 and PGC-1α to CYP3A4 chromatin...... 126 Figure 29. Effect of HNF4α and PGC-1α on the promoter activities of SHP, CYP7A1, PEPCK and CYP3A4...... 128

Figure 30. Liganded PXR suppressed HNF4α/PGC-1α activation of SHP promoter activity...... 131 Figure 31. Effect of rifampicin on CYP27A1 mRNA expression...... 137 Figure 32. Assay of 27-hydroxycholesterol in Caco2 cells...... 139 Figure 33. Effect of 27-hydroxycholesterol and cholesterol loading on ABCA1 and ABCG1 mRNA expressions...... 142 Figure 34. Effect of rifampicin on ABCA1 and ABCG1 mRNA expression...... 144 Figure 35. Effect of rifampicin on human CYP27A1 promoter activities...... 147 Figure 36. EMSA and ChIP assays of PXR binding to human CYP27A1 gene...... 152 Figure 37. Deletion and mutation analysis of PXR binding sites in the human CYP27A1 promoter...... 155 Figure 38. A model for rifampicin and PXR regulation of CYP7A1 gene transcription.160 Figure 39. PXR regulation of drugs, bile acids and cholesterol metabolism in the intestine...... 169

ACKNOWLEDGEMENTS

The completion of this dissertation was made possible through the support and cooperation of many individuals. I would like to take this opportunity to thank my advisor Dr. John Y.L. Chiang for his excellent guidance and strong support through my

Ph.D. study. Thanks to all the committee members: Dr. Stalvey, Dr. Westerman, Dr,

Hardwick, Dr. Dluzen, Dr. Milsted and Dr. Simmons for their generous assistance and valuable comments on my dissertation work.

CHAPTER I

INTRODUCTION

Bile acids are the end products of cholesterol catabolism in the liver [1]. As the key components of bile formation, bile acids are essential in eliminating cholesterol and various lipid metabolites from the liver and absorbing dietary nutrients and fat soluble vitamins in the intestine. Bile acids also serve as signaling molecules that play important roles in the regulation of whole body lipid homeostasis. However, toxic bile acids accumulated at high levels during certain pathological conditions result in severe liver damage, which may lead to chronic liver diseases such as cholestasis or cirrhosis. Thus, studies on the regulation of bile acid biosynthesis and detoxification become crucial in understanding the molecular basis of bile acid and cholesterol homeostasis and developing potential drug therapies to treat chronic liver diseases. This study investigated the potential role of pregnane X receptor (PXR), a bile acid and drug activated nuclear receptor, in the regulation of bile acid synthesis and detoxification genes including cholesterol 7α hydroxylase (CYP7A1), cytochrome P450 3A4 (CYP3A4) and oxysterol

27-hydroxylase (CYP27A1) and their implications in the bile acid metabolism and cholesterol homeostasis.

A. Bile acid biosynthesis

1. Chemistry

As illustrated in Fig.1, the common structure of bile acids includes a saturated 19-

carbon sterol nucleus with a β-oriented hydrogen at position 5 and a saturated 5-carbon

side chain terminating in carboxylic acid. The hydroxylation reaction of the sterol ring

also results in α-oriented hydroxyl groups at several positions. Bile acids are classified as

primary and secondary bile acids. Primary bile acids are synthesized directly from

cholesterol in the liver. In human, primary bile acids include cholic acid (CA) and

chenodeoxycholic acid (CDCA). Secondary bile acids deoxycholic acid (DCA) and

lithocholic acid (LCA) are formed from cholic acid and cheodeoxycholic acid,

respectively, in the intestine when bacteria enzymes remove a hydroxyl group from the 7

position on the sterol ring. Majority of the bile acids in human are conjugated with

taurine or glycine, Fig.2 shows the structures of taurine or glycine conjugated cholic acid as examples. Under physiological pH, bile acids are present as sodium salts and usually referred to as bile salts [1, 2].

In aqueous phase, bile acids can interact with lipid molecules to form micelles with

their hydrophobic sides facing the lipid core and hydroxyl groups facing the hydrophilic

environment. The biological properties of the bile acids rely on their relative

hydrophobicity. The hydrophobicity of each bile acid is usually determined by the

number and orientation of the hydroxyl groups on the sterol ring and is evaluated by

measuring the partition coefficient between polar and non-polar solvents or the retention

time on a reverse phase HPLC. Hydrophobic bile acids are more efficient in facilitating

cholesterol and lipid absorptions, and also exhibit higher cytotoxicity [3].

Fig.1

Fig. 1. Cholesterol and bile acid structure.

Bile acids are synthesized from cholesterol in the liver. Their common structure includes

a saturated 19-carbon sterol nucleus with a β-oriented hydrogen at position 5 and a

saturated 5-carbon side chain terminating in carboxylic acid. The hydroxylation reaction

of the sterol ring also results in α-oriented hydroxyl groups at several positions. In

humans, primary bile acids include cholic acid and chenodeoxycholic acid. In the

intestine, cholic acid and chenodeoxycholic acid are converted to secondary bile acid

deoxycholic acid and lithocholic acid, respectively. Most bile acids are conjugated with taurine or glycine once produced and presented as sodium salt, which are usually referred to as bile salt.

2. Physiological function

Bile acids play important roles in several physiological aspects. In the liver, bile acids

participate in both cholesterol elimination pathways mentioned previously. Conversion

from cholesterol into bile acids accounts for about 50% of cholesterol disposal from the

body. Further more, bile acids also facilitate biliary secretion of free cholesterol which is then disposed of in the feces. As a key component for the bile formation, bile acids are important in maintaining normal bile flow, which transport various organic compounds, endogenous metabolites and exogenous xenobiotics out of the liver. In the intestine, bile acids are detergents that help absorb dietary cholesterol and fat soluble vitamins from the intestinal lumen. Finally, bile acids involve in the regulation of several biological pathways by binding to nuclear receptors or activation of intracellular signaling pathways

[2].

3. Bile acid biosynthesis pathways

Primary bile acids are synthesized from cholesterol exclusively in the liver through two

general pathways, the classic or neutral pathway and the acidic or alternative pathway [1,

4]. Fig.2 shows important enzymes and intermediates in these two pathways. Enzymes

that catalyze these multi-step reactions are located in the endoplasmic reticulum,

mitochondria, cytosol and peroxisome. The classic pathway is initiated by cholesterol 7α

hydroxylase (CYP7A1) and is thought to be the major bile acid biosynthesis pathway in normal physiology. The acidic pathway is initiated by mitochondrial sterol 27-

Fig.2

Chiang, Endocrine Reviews, 2002

Fig. 2. Bile acid biosynthesis pathways in liver.

Bile acids are synthesized from cholesterol in the liver. Two major bile acid synthesis pathways are classic pathway and acidic pathway. CYP7A1 initiates the classic pathway whereas CYP27A1 initiates acidic pathway. Primary bile acids, cholic acid and chenodeoxycholic acid are the end products of these pathways (see text for details).

Hydroxylase (CYP27A1). Under normal conditions, it accounts for less than 18% of the

total hepatic bile acid production in humans and is considered as a minor bile acid

synthesis pathway.

3.1. The classic or neutral pathway

The classic pathway is also known as the neutral pathway for most of its intermediates

are neutral sterols. In human, this pathway produces CA and CDCA in approximately

equal amounts. CYP7A1, a microsomal cytochrome p450 enzyme, catalyzes the first and

rate-limiting step in the classic pathway to convert cholesterol into 7α hydroxycholesterol

[5]. Microsomal 3β-hydroxy-27-steroid dehydrogenase/isomerase (3β-HSD) then

converts 7α-hydroxycholesterol into 7α-hydroxy-4-cholestene-3-one, which is the

common precursor for both CA and CDCA. 7α-hydroxy-4-cholestene-3-one can be hydroxylated at C-12 position by microsomal sterol 12α-hydroxylase (CYP8B1) and

modified by other cytosolic enzymes and eventually converted to CA [2]. Alternatively,

7α-hydroxy-4-cholestene-3-one can also undergo several enzymatic reactions which

finally lead to the formation of CDCA. Mitochondrial CYP27A1 mediates the steroid

side chain oxidation and cleavage to give carboxyl groups in both CA and CDCA

synthesis [6].

3.2. Acidic or alternative pathway

Alternative pathway was originally revealed by the identification of several acidic

intermediates which are not present in the classic pathway [7, 8]. Alternative pathway

mainly produces CDCA. CYP27A1 catalyzes the first two steps and converts cholesterol

into 27-hydroxycholesterol and then 3β-dihydroxy-5-cholestionic acid[9]. Oxysterol 7α-

hydroxylase (CYP7B1) then catalyzes the hydroxylation reaction at C-7 position of these

two intermediates which are subsequently converted to CDCA by the same enzymes in

classic pathway.

4. Bile acid transport

4.1. Enterohepatic circulation

Bile acids, once produced in the liver, are transported into the bile and stored in the gallbladder. After each meal, bile acids are released into the intestine lumen, where they are efficiently reabsorbed by the enterocytes and transported back to the liver via portal blood for re-excretion into the bile. This process is referred to as enterohepatic circulation of the bile (Fig.3) [2]. Bile acid transporters play important roles in this transport process

(see later) [10]. The canalicular excretion of bile acids into bile against concentration gradient is the major driving force of normal bile flow. The bile acid pool size is defined as the total amount of bile acids circulating in the enterohepatic circulation. In humans, bile acid pool consists of CA, CDCA, DCA and LCA in an approximate 40:40:20:<1 ratio, with a mass of around 2.5-3 gm. After reaching the small intestine, 95% of the bile acids are reabsorbed and only 5% is lost into the feces. The daily loss of bile acids is compensated by de novo synthesis in the liver and thus, a constant bile acid pool is

maintained [2].

Fig.3

Fig. 3. Enterohepatic circulation.

Bile acids, once produced in the liver, are transported into the bile by bile salt export

pump (BSEP). Cholesterol transported by ABCG5/G8 and phospholipids transported by

MDR3 are also essential for normal bile formation. After reaching the intestine lumen,

bile acids are efficiently reabsorbed by the enterocytes via apical sodium-dependent bile

salt transporter (ASBT) and excreted into the portal blood by MDR related protein-

3(MRP3) and t-ASBT. Na+-dependent taurocholate transporter (NTCP) and organic

anion transporters (OATPs) located at basolateral membrane uptake bile acids into the hepatocytes for re-excretion. This process is referred to as enterohepatic circulation of the

bile. 95% of the bile acids are reabsorbed from intestine and only 5% is lost via feces,

which is compensated by de novo synthesis in the liver.

BS- : bile salt; OA-: organic anion; C: cholesterol; PL: phospholipids.

4.2. Hepatic bile acid transport

4.2.1. hepatic bile acid uptake

Hepatocytes are polarized epithelial cells with basolateral (sinusoidal) and apical

(canalicular) membrane domains. Hepatocytes take up bile acids through the basolateral

membrane, which is in direct contact with the portal blood plasma, and excrete bile acid

at the canalicular membrane into the bile [11]. Unconjugated bile acids are uncharged

molecules under physiological PH and can pass the cell membrane via passive diffusion.

However, majority of the bile acids are conjugated with taurine or glycine and present as bile salts. They can not cross the cell membrane and need active transport mechanisms for cellular uptake [12]. Two bile acid transporters, Na+-dependent taurocholate

transporter (NTCP) and organic anion transporters (OATPs) are responsible for

basolateral bile acid transport into the hepatocytes. The Na+-dependent taurocholate

transporter co-transports two Na+ down its gradient into the hepatocytes along with one molecule of conjugated bile acid. The trans-membrane Na+ gradient is in turn maintained

by the Na+- K+-ATPase [13]. Na+ -dependent bile salt uptake pathway accounts for 80%

of the total taurocholate uptake and is considered as the major bile acid transport system

located at the basolateral membrane[14]. The Na+ -independent bile salt uptake is

mediated by several members of OATP family. These transporters have wide substrate

preference. Besides conjugated and unconjugated bile acids, many amphipathic organic

compounds such as bilirubin, selected organic cations and numerous drugs are also taken

up by these transporters[15]. In rat liver, Oatp-1, -2 and -4 account for the bulk Na+ -

independent bile acid uptake while OATP-C is the most relevant isoform in humans[16-

19].

4.2.2. canalicular bile acid excretion

Since the biliary constituents concentration is about100 to 1000 fold higher in the bile

than in the hepatocytes, canalicular bile acid transport represent the rate-limiting step in

bile formation. Several members of the ATP-binding cassette (ABC) transporter familiy,

driven by ATP hydrolysis, are responsible for transporting bile acids and other organic

compounds across the canalicular membrane against their concentration gradients.

The bile salt export pump (BSEP, ABCB11), originally identified as the sister of P-

glycoprotein (SPGP), is mainly responsible for monovalent bile acid (eg.taurocholate)

transport at the canalicular membrane[20]. Mutations in BSEP were first identified in patients with progressive familial intrahepatic cholestasis subtype 2(PFIC-2). The absence of functional BSEP in the hepatic canalicular membrane and less than 1% of normal biliary bile acid concentration found in these patients suggested that BSEP is the major canalicular bile acid transport system[21].

Divalent bile acids with two negative charges such as sulfated taurocholates are

transported across the canalicualr membrane by another ABC transporter called

multidrug–resistant associated protein MRP2 (ABCC2)[22]. Since it also mediates the

excretion of a broad range of divalent organic anions such as glutathione conjugates or

sulfate conjugates, MRP2 is also known functionally as the canalicular multispecific

organic anion transporter (MOAT)[23, 24]. Bilirubin is transported into the bile mainly

via MRP2. Defects in MRP2 gene result in Dubin-Johnson syndrome characterized by

hyperbilirubinaemia and hepatic accumulation of various endogenous and exogenous

organic metabolites [25].

After bile acids are pumped into the bile, they stimulate phospholipids and cholesterol

secretions into the bile, followed by passive inflow of water [26]. Phospholipids are

excreted via the phospholipid flippase MDR3 (ABCB4), and the major phospholipid in

the bile is phosphatidylcholine[27, 28]. Bile formation is the most important pathway for

hepatic free cholesterol elimination. Increased biliary cholesterol secretion in transgenic mice overexpressing ABCG5/G8 and decreased biliary cholesterol concentration in

Abcg5/g8 -/- mice suggested that these ABC half transporters might play essential roles in biliary cholesterol secretion [29]. Bile acids, phospholipids and cholesterol are three major organic solutes of the bile and once secreted, they form mixed micelles to reduce their toxicity to the bile duct. Normal bile formation depends largely on balanced secretion of these constituents. Impaired secretions will disrupt the bile flow and result in cholestasis or gallstone disease.

4.3. Intestinal bile acid transport

4.3.1. apical bile acid uptake into the enterocytes

Once secreted into the intestine lumen, bile acids undergo reabsorption mostly at the

terminal ileum. Like the hepatic basolateral uptake system, intestinal bile acid uptake is

also mediated by Na+ -dependent and independent pathways. The Na+-dependent uptake

is mediated by the ileal bile salt transporter (ISBT), which is also known as the apical

sodium-dependent bile salt transporter (ASBT) [30]. This transporter has substrate

specificity for both primary and secondary conjugated and unconjugated bile acids.

Unlike some hepatic bile acid transporters that also mediate the secretion of other organic

compounds, the substrates for ASBT is strictly limited to bile acids[31]. In rodent, Oatp-3

is thought to play a role in mediating Na+-independent bile salts transport. It has ~80%

homology and similar transport characteristics to hepatic Oatp-2[32]. However, the

importance of this transporter remains to be clarified. Human OATP-A has been detected

in human intestine and proposed as the rodent Oatp-3 ortholog[14]. ASBT is still thought

to be the most efficient pathway in the intestinal bile acid uptake.

4.3.2. basolateral efflux of bile acids from enterocytes

Once absorbed into the enterocytes, bile acids are transported intracellularly by the intestinal bile acid binding protein (I-BABP) to the basolateral membrane to be excreted into the portal blood [33]. A splicing variant of Asbt (t-Asbt) has been indicated as a basolateral bile acid transporter in intestine [34]. MRP3, an ATP-dependent bile salt pump, has substrate specificity for both monovalent and divalent bile acid conjugates.

High expression at the terminal ileum suggests MRP3 is another candidate for basolateral secretion of bile acids across the basolateral membrane[35]. However, the physiological functions of tASBT and MRP3 are still uncertain. One recent study identified the heteromeric organic solute transporter Ostα-Ostβ as a potential basolateral bile acid transporter [36].The Ostα-Ostβ expression is localized at the basolateral surface of ileal

enterocytes in mouse and overexpression of Ostα-Ostβ enhances basolateral efflux of

trauocholate. Future studies may further demonstrate the important role of Ostα-Ostβ in the basolateral efflux of bile acid in the intestine.

5. Cholestasis

Cholestasis is a disease state characterized by impairment or cessation of bile flow.

Patients with this disease accumulate toxic bile acids and other lipid metabolites in the liver and circulation, which eventually leads to liver failure. Liver transplantation is

usually required in such conditions.

Three genetic forms of cholestasis have been identified and named progressive familial

intrahepatic cholestasis type 1, 2 and 3 (PFIC-1, 2 and 3) [37]. PFIC-1, formerly called

Byler’s disease, is characterized by high bile acid concentration in the plasma and very

low bile acid concentration in the bile. The genetic defect has been located on the FIC1

gene encoding ATP8B1, a P-type ATPase[38]. ATP8B1 is an amino phospholipids

flipase. However, FIC1 expression is very low in the liver and high in the intestine, thus,

how the absence of this transporter leads to intrahepatic cholestasis remains to be

explained. Mutations in BESP has been identified in patients with PFIC-2[21]. It is

reported that PFIC-2 is also associated with mutations and low expressions of FXR, a key

regulator of BSEP gene expression. Patients develop similar phenotypes as PFIC-1. The

virtual absence of bile acid in the bile suggests that BESP is the major hepatic bile salt

pump located at the canalicular membrane. Compared to PFIC-1 and 2, patients with

PFIC-3 usually show severe hepatomelgly, jaundice and pruritus which eventually lead to

liver failure if untreated. Studies of murine orthologue Mdr2 has led to the identification of mutant MDR3 genes in PFIC-3 patients [39]. Phospholipids transported into the bile by

MDR3 is essential for normal bile formation and prevention of bile acid toxicities to the cell membrane. Since the high level of bile acid is the major cause of hepatic damage in

PFIC-3, treatment of non-toxic hydrophilic bile acid ursodeoxycholic acid shows beneficial effect in most patients[40].

Besides the genetic defects, cholestasis also develops during certain pathological

conditions. Liver inflammation and sepsis caused by drugs or infection are often

associated with cholestasis [41]. Injection of lipopolyssacride (LPS), a bacteria cell wall

component, induces inflammation and cholestasis in rodents. These animals showed

reduced Mrp2 expression and hyperbilirubinaemia, indicating downregulation of hepatic

transporters during inflammation might be the cause of cholestasis in these animals [41].

Extensive studies have made it clear that nuclear receptors are key regulators for the

expression of these transporter genes. For examples, PXR regulates several hepatic

transporters such as OATP2, MDR1 and MRP2, which are crucial for normal bile

formation. Canalicular bile acid transporter BSEP is strongly induced by FXR in

response to increased bile acid concentrations [42]. Studies from several groups have

shown that some nuclear receptor expressions are decreased dramatically by cytokines

produced during inflammation, which is suggested to be the major cause of reduced

expression of certain hepatic transporters[43-46]. Further studies in patients with hepatic

inflammation and sepsis may be required to demonstrate the contribution of nuclear

receptor down regulation to cholestasis. Another acquired cholestasis is intrahepatic

cholestasis of pregnancy (ICP). The cause of ICP is currently not clear and is

hypothesized to result from the altered hormone status during the last trimester. Several

hormones including estrogen and progesterones have been reported to show inhibitory

effect on canalicular transporters[47].This form of intrahepatic cholestasis is reversible

and is usually rapidly resolved after delivery [47].

B. Cholesterol homeostasis

Cholesterol is not only an essential cell membrane component for maintaining normal

cell functions, but also the precursor to all steroid hormones, bile acids and oxysterols,

which are important regulators in diverse metabolic pathways. However, high

intracellular cholesterol is toxic to the cells, and high serum cholesterol built up in the

arterial walls will lead to the plaque formation, one of the initial steps to develop

atherosclerosis. Hypercholesterolemia is considered as the leading cause of many

cardiovascular diseases and has become a major health concern in developed countries.

Liver is the major organ that regulates cholesterol homeostasis by balancing several

input and output pathways [2]. As illustrated in Fig.4, Liver acquires cholesterol mainly

from the following sources: 1, de novo synthesis of cholesterol; 2, dietary cholesterol

carried by chylomicron remnants (CMR); 3, receptor mediated endocytosis of low

density lipoproteins (LDL); 4, reverse cholesterol transport from peripheral tissues by

high density lipoprotein (HDL). Hepatic cholesterol synthesis from acetyl CoA involves multiple enzymatic reactions. The 3-hydroxy-3-methyl glutaryl-CoA reductase (HMG-

CoA reductase) catalyzes the rate-limiting step in this process [48]. Chylomicrons (CM)

Fig.4

Fig. 4. cholesterol homeostasis in liver.

Block arrows, cholesterol uptake pathways: 1, de novo synthesis of cholesterol from acetyl CoA; 2, dietary cholesterol carried by chylomicron remnants (CMR); 3, receptor mediated endocytosis of low density lipoproteins (LDL); 4, reverse cholesterol transport from peripheral tissues by high density lipoprotein (HDL). Black arrows, cholesterol elimination pathways: 1, conversion of cholesterol into bile acids; 2, bile acid facilitated biliary cholesterol secretion. C: cholesterol; CE: cholesterol ester; HMG CoA R: HMG

CoA reductase; CEH: cholesterol ester hydrolase; ACAT2: Acyl CoA: cholesterol acyl transferase 2; OxLDL: oxidized LDL; LDLR: LDL receptor; LRP: LDLR related protein.

are triglyceride-rich particles synthesized in the intestine and transported into the

circulation. Dietary free cholesterol is converted into cholesterol ester (CE) by Acyl CoA:

cholesterol acyl transferase 2(ACAT2) in the intestine and incorporated into chylomicroms. Lipoprotein lipase (LPL) in the capillary of the adipose and muscle

tissues extracts triglyceride from chylomicron and converts it into chylomicron remnant,

which is then taken up by the liver via LDL receptor relate protein (LRP). In the liver,

Cholesterol esters from CMR are hydrolyzed by cholesterol ester hydrolase (CEH). Free

cholesterol is then converted into cholesterol ester by hepatic ACAT2 and assembled into

very low density lipoprotein (VLDL), which is secreted into the circulation. VLDL

particles become intermediate density lipoprotein (IDL) and LDL after triglyceride is extracted by LPL into the peripheral tissues. IDL and LDL can be taken up by LDL receptors located in both liver and peripheral tissues. LDL is often referred to as “bad”

cholesterol in the circulation. Oxidized LDL taken up by macrophages in the artery wall

via scavenger receptor A1 (SR-A1) or CD36 contributes largely to the foam cell

formation and atherosclerosis development [49]. In contrast, HDL carrying cholesterol

from the peripheral tissues back to the liver via scavenger receptor B-I (SR-BI) for bile

acid synthesis or biliary cholesterol excretion is known as “good” cholesterol. It helps

remove excess cholesterol from peripheral tissues and prevent intracellular cholesterol

accumulations [50].

Liver eliminates cholesterol from the body through two major pathways: 1, conversion

of cholesterol into bile acids and 2, bile acid facilitated biliary cholesterol secretion. Bile

acids biosynthesis is tightly regulated in human. About 5 % of the bile acid is lost

through feces everyday and is compensated by de novo synthesis from cholesterol in the

liver. Conversion of cholesterol into bile acids accounts for 50% of daily cholesterol

disposal in human. Bile acids facilitated cholesterol secretion into the bile is another

significant pathway to eliminate cholesterol from the body. Biliary cholesterol is

eventually transported into the intestine and excreted through feces. The two pathways

account for about 90% of the total daily cholesterol disposal in human. The other 10% of

cholesterol is used for synthesis of steroid hormones[1].

C. Regulation of bile acid biosynthesis and cholesterol homeostasis

Bile acid synthetic rate is tightly controlled by the bile acid returning to the liver via

enterohepatic circulation. When bile acid level increases, it feedback inhibits several key

enzymes such as CYP7A1, CYP8B1 and CYP27A1 in the bile acid synthetic pathways to

reduce bile acid output. Thus, a constant bile acid pool size is maintained. Several nuclear

hormone receptors have been identified as bile acid sensors and play key roles in

mediating bile acid feedback regulation of bile acid and cholesterol homeostasis.

1. Nuclear hormone receptor

Nuclear hormone receptors are a family of ligand- activated transcription factors that

regulate genes in cell growth, differentiation and metabolism [51-53]. Forty-eight nuclear receptor genes have been identified in human genome. Nuclear receptor has a typical modular structure that contains a highly conserved DNA binding domain (DBD) located within the N-terminal region and a less conserved ligand binding domain (LBD) located

23 within the C-terminal region. Ligand-independent activation function-1 domain (AF-1) and ligand-dependent activation function-2 domain (AF-2) are located at the N-terminal and C-terminal, respectively. A hinge region links the DBD and the LBD (Fig.5).

A typical nuclear hormone receptor DBD contains two Cys2/Cys2 Zinc finger motifs where four cystienes within the consensus Cys-X2-Cys-X1-3-Cys-X2-Cys sequence coordinate with a zinc ion to form a loop structure. This motif recognizes short DNA sequences and is essential for nuclear receptor DNA binding. Classic steroid nuclear receptors such as glucocorticoid receptor (GR) or androgen receptor (AR) bind to the palindromic sequence AGAACA-N3-TGTTCT, whereas non-steroid hormone receptors recognize AG(G/T)TCA-like repeats separated by several nucleotides. Most non-steroid hormone nuclear receptors form either heterodimers with retinoic X receptor (RXR) such as liver X receptor (LXR), farnesoid X receptor (FXR) or pregnane X receptor (PXR) or homodimers with itself such as hepatic nuclear factor 4α (HNF4α) and bind to direct repeat (DR), inverted repeat (IR) or everted repeat (ER). The orientation and nucleotide spacing between two half sites determine their DNA binding specificity. Human α fetoprotein transcription factor (FTF) and mouse liver-related homolog (LRH) bind to

TCAAAGGTCA site as a monomer, while small heterodimer partner (SHP) does not bind to DNA due to the lack of a DBD. The consensus DNA binding sites for important nuclear receptors in lipid metabolism are shown in Fig.5.

The LBD of a nuclear receptor folds and forms a hydrophobic pocket into which ligands can bind. A classic model suggests the nuclear receptor LBD binds to co-

Fig.5

Fig. 5. Structure of nuclear hormone receptors and their binding sites.

Classic steroid receptors bind to palindromic consensus sequences. Typical nuclear hormone receptors consist of 6 domains designated as A,B,C,D,E and F. A/B: ligand- independent activation function domain 1(AF-1); C: DNA binding domain (DBD); D: hinge region; E: Ligand binding domain (LBD); E: ligand-dependent activation function domain 2 (AF-2). Typical nuclear hormone receptors form homodimer or heterodimer with RXR and recognize direct repeat (DR), everted repeat (ER) or inverted repeat (IR).

FTF (LRH) binds to a AGGTCA half site as a monomer. SHP is a unique nuclear receptor which lacks a DNA binding domain.

repressors in the absence of ligands and can not activate gene transcription. Ligand

binding changes the conformation of the LBD and exposes a specific co-activator binding

domain within the AF-2 domain, which allows the nuclear receptor to interact with the

LXXLL motif located on the co-activators. The nuclear receptor/co-activator complex

then activates gene transcription through chromatin remodeling and interaction with

general transcriptional machinery. The naturally occurring ligands for many nuclear

receptors have been identified. For example, LXR is activated by oxysterols and regulates

cholesterol homeostasis whereas FXR binds to bile acids and involves in bile acid

metabolism. Nuclear receptor without an identified ligand such as HNF4α is referred to

as orphan receptor.

Nuclear hormone receptors have been identified to regulate genes in diverse biological

pathways. Nuclear receptors that have been identified to regulate bile acid and cholesterol

metabolism are briefly discussed below and in section III. B, C & D.

HNF4α (NR2A1): HNF4α is highly expressed in the liver. It forms a homodimer and binds to DR1 site in its target genes. HNF4α is constitutively active in gene transactivation. Fatty acids have been suggested as the endogenous ligands of HNF4α[54].

HNF4α regulates bile acid synthetic genes CYP7A1, CYP8B1 and CYP27A1 and bile acid

transporter NTCP[55-58]. HNF4α also regulates the hepatic expressions of several

lipoproteins including ApoAI, ApoB and ApoCIII and plays an important role in lipid

homeostasis[59, 60]. Recent studies show PEPCK is highly induced by HNF4α,

suggesting a role of HNF4α in the glucose metabolism. HNF4α is essential for early liver

development and differentiation. Disruption of HNF4α in mice is embryonic lethal.

However, studies from liver- specific HNF4α knockout mice have demonstrated the

importance of HNF4α in the regulation of lipid homeostasis [61]. These animals showed

increased hepatic accumulation of lipid and reduced serum cholesterol and triglyceride,

which was thought to mainly result from decreased bile acid synthesis and apolipoprotein

expressions. Due to the decreased NTCP levels, these mice also accumulated high levels

of bile acid in the serum.

FTF (NR5A2): human FTF and its mouse homolog LRH belong to the Fushi-tarazu

factor-1(Ftz-F1) family of monomeric nuclear receptors. FTF is expressed in liver,

intestine and pancreas. Its DNA binding site has been identified in SHP, HNF1α and

HNF4α. FTF is a relatively weak transcription factor. LRH and LXR bind to mouse

Cyp7a1 and synergistically stimulate its gene transcription. The binding sites of FTF on

CYP7A1 and CYP8B1 are overlapping with HNF4α binding site, and FTF has been

suggested as a negative regulator of human CYP7A1 and CYP8B1 by interfering with

HNF4α binding to these genes. Interestingly, bile acids induce FTF transcription in rat

liver and HepG2 cells. Thus, bile acid induced FTF may directly inhibit CYP7A1 and

CYP8B1 by blocking HNF4α binding to these genes[62, 63].

FXR(NR1H4): FXR is highly expressed in liver, intestine, adrenal and kidney[64].

FXR has recently been identified as a bile acid receptor [65]. Among bile acids, the hydrophobic bile acid CDCA, with an EC50 of about 10-20μM, is the most potent

activator of FXR, where as secondary bile acid DCA and LCA are less effective. FXR is

an important regulator in both bile acid synthesis and transport. FXR mediates the bile

acid feedback inhibition of bile acid synthetic genes CYP7A1, CYP8B1 and CYP27A1 via an indirect mechanism [55, 58, 66](see later). The FXR binding site has been identified in

BSEP[42]. Bile acid activated FXR dramatically induces BSEP to excrete bile acid from the liver into the bile. FXR also stimulates IBABP in response to bile acid to facilitate bile acid transport in enterocytes[67]. FXR has also been shown to regulate phospholipid transport protein(PLTP) and ApoCIII in the lipoprotein metabolism[68, 69]. Fxr knockout mice show increased serum bile acid, cholesterol and triglycerides levels, suggesting a critical role of FXR in the lipid metabolism[70].

SHP (NR0B2): SHP is a unique orphan nuclear receptor that lacks a conserved DNA

binding domain. It contains a receptor interacting domain and a repressor domain, which allows it to interact with many nuclear receptors and transcription factors and inhibit their transcriptional activities [71]. So far, SHP has been found to interact with HNF4α, FTF,

Foxa, Foxo1, LXRα and PXR and exert negative effects on many biological aspects including bile acid synthesis, glucose metabolism, drug metabolism and cholesterol homeostasis [55, 72-74]. SHP transcription is tightly regulated by several nuclear receptors. FTF and FXR bind to SHP promoter and stimulate its gene transcription[75].

Bile acid activated FXR induces SHP, which interacts with FTF and suppresses CYP7A1 and bile acid biosynthesis (see later). SHP also feedback inhibits its own transcription by interacting with FTF, which may provide a mechanism to tightly control intracellular

SHP levels and prevent its inhibitory effect on other biological pathways[62].

2. Regulation of bile acid synthesis

2.1. Feedforward regulation of CYP7A1

The tight regulation of bile acid biosynthesis is mainly achieved through the

transcriptional regulation of CYP7A1, the rate-limiting enzyme in the classic pathway.

The importance of CYP7A1 in the hepatic bile acid biosynthesis has been demonstrated

by gene knockout studies. Genetic disruption of Cyp7a1 in mice resulted in a marked

decrease in bile acid synthesis, malabsorption of nutrient and postnatal lethality[76, 77].

Defects of CYP7A1 in human patients have been recently identified. Although the

residual CYP7A1 activity was sufficient for survival, these patients developed

hypercholesterolemia, gallstone diseases and premature atherosclerosis[78].

It has been very well studied that various members of the nuclear receptors are key

regulators of CYP7A1 gene expression [66, 79, 80]. Two bile acid responsive elements

(BAREs) have been identified on the CYP7A1 promoter [81, 82]. The sequences of these

two elements, highly conserved among species, contain several AGGTCA-like

sequences, which create binding sites for many nuclear hormone receptors (Fig.6).

LXRα/RXR heterodimer binds to the DR4 site in mouse Cyp7a1 promoter BARE-I

region. Upon high cholesterol loading, LXRα is activated by oxysterols and stimulates the transcription of Cyp7a1, which converts excess cholesterol into bile acids[83]. Wild

type mice fed on high cholesterol diet did not accumulate cholesterol in the liver, while

mice lacking LXRα expression were not able to respond to high dietary cholesterol and

developed rapid cholesterol accumulation in the liver[84]. However, in other species such

as human and hamster, the DR4 site on CYP7A1 BARE-I is not well conserved and

CYP7A1 is not stimulated by high cholesterol due to the lack of LXR binding[80].

Fig.6

Fig. 6. Bile acid responsive elements (BAREs) on human and rat CYP7A1.

Rat BARE-I contains a conserved DR4 sequence which binds to LXR/RXR heterodimer and COUP-TFII. This DR4 is not conserved in human BARE-I and factors bind to human

BARE-I have not been identified. BARE-II is well conserved between rat and human.

HNF4α binds to a DR1 site as homodimer. FTF binds to a site overlapping with HNF4α binding site.

Cholesterol feeding suppresses human CYP7A1 mRNA in transgenic mice expressing human CYP7A1 gene [85]. Thus, cholesterol regulation of CYP7A1 is different across species and cholesterol feedforward activation of Cyp7a1 is a unique in mice and rats.

2.2. Feedback inhibition of CYP7A1

2.2.1. FXR/SHP dependent pathway

Under physiological conditions, CYP7A1 is usually under the suppression of bile acids returning to the liver via portal blood. Bile acids are detergents and high levels of bile acids show great cytotoxicity. Bile acid feedback inhibition of its own synthesis provides an important mechanism to maintain constant bile acid pool size and also prevent tissue damages in certain pathological conditions where bile acid levels increase. Several bile acid feedback inhibition pathways are illustrated in Fig.7. The FXR/SHP dependent pathway was the first bile acid feedback inhibition pathway identified. Nuclear receptor

LRH binds to BARE II and stimulates Cyp7a1 gene expression. Bile acid-activated FXR induces the negative nuclear receptor SHP, which interacts with LRH and blocks its interaction with co-activators, and thus results in down regulation of Cyp7a1 gene transcription [66, 86]. Cyp7a1 was strongly suppressed by synthetic FXR agonist

GW4064. However, in Fxr null mice, synthetic FXR agonist GW4064 failed to induce

SHP to inhibit Cyp7a1[70]. Human CYP7A1 is also subject to FXR/SHP suppression in cell based reporter assays [87]. However, the physiological importance of this pathway in humans still needs to be further tested.

Fig.7

Fig. 7. Bile acid feedback inhibition of CYP7A1 gene transcription.

In FXR/SHP dependent pathway, bile acids activated FXR induces SHP, which inhibits

Cyp7a1 by interacting with FTF. In FXR/SHP independent pathways, bile acids can

activate JNK signaling pathway either via activation of PKC or via induction of

inflammation cytokines from hepatic kupffer cells. Bile acids also activate FXR, which

transcriptionally stimulates FGF19. FGF19 activates JNK through its cell surface receptor FGFR4. HNF4α, a key regulator of CYP7A1, is the major downstream target of

JNK. JNK reduces HNF4α protein levels and also inhibits HNF4α transactivating activity

by phosphorylation and thus results in inhibition of CYP7A1. JNK: cjun N-terminal kinase; FGFR4: fibroblast growth factor receptor 4. FGF19: fibroblast growth factor 19;

PKC: protein kinase C.

2.2.2. FXR/SHP independent pathways

In Shp null mice, Cyp7a1 lost its response to the FXR agonist GW4064 compound, but

responded to bile acid suppression, which suggests that besides FXR/SHP pathway, other

SHP independent mechanisms also exist to mediate the bile acid feedback inhibition of

its own synthesis[88, 89]. Later on, SHP independent pathways including PKC/JNK

pathway, cytokine mediated pathways and fibroblast growth factor receptor 4(FGFR4) mediated pathways are suggested by several studies.

Kupffer cells are resident macrophages that reside within the liver at the interface

between the circulation and parenchymal cells. This allows them to activate innate immunity in response to the entrance of bacteria and bacterial products from the intestinal tract. Cytokines produced by Kupffer cells play crucial roles in the regulation of liver functions, such as cytochrome P450s expressions, lipid metabolism, inflammation, viral propagation, and regeneration[90, 91]. Recent studies suggest that Kupffer cells can also function as bile acid sensors [92]. Bile acids induced inflammatory cytokines, such as

tumor necrosis factor α(TNFα) and interleukin 1β (IL-1β), from Kupffer cells will

suppress CYP7A1 mRNA expression via their high affinity receptors on the

hepatocytes[93, 94]. This cytokine mediated bile acid feedback inhibition is supported by

several findings in animal and cell culture studies. Upon a bile acid-containing diet feeding, mice display a rapid induction of TNFα and IL-1β mRNA expression by Kupffer

cells and a concomitant repression of CYP7A1, while treatment of rosiglitazone, a known

inhibitor of cytokine activation, blocks both the induction of hepatic TNFα and IL-1β

mRNA expression and suppression of CYP7A1 mRNA by dietary bile acid feeding[92].

Several evidence indicate that c-jun N-terminal kinase (JNK) is involved in the

cytokine-mediated suppression of CYP7A1. Bile acid or cytokine activation of JNK leads

to the suppression of CYP7A1 while such suppression is blocked by the over-expression of a dominant negative form of JNK. It has also been shown that bile acids can activate protein kinase C (PKC), which suppress CYP7A1 via either JNK or MAPK [95, 96].

These studies clearly indicate that cytokine activated JNK signaling pathways play an important role in bile acid feedback suppression of CYP7A1. Recently, studies from our lab showed IL-1β inhibited HNF4α mRNA and protein levels in human primary hepatocytes. Furthermore, IL-1β also led to the phosphorylation of HNF4α, which reduced its DNA binding activity. These effects were blocked by a specific JNK inhibitor

[97]. Thus, bile acid induced cytokines activate JNK signaling pathway, which suppresses CYP7A1 by inhibiting HNF4α protein expression and trans-activating

activity.

Recent studies revealed that fibroblast growth factor receptor 4 (FGFR4), a cell surface

tyrosine kinase receptor, was involved in mediating bile acid suppression of CYP7A1

[98]. Members of the FGFR family are regulators of embryonic development, wound healing

and adult organ homeostasis [99-101]. Among the four FGFR receptor kinase members,

only FGFR4 is highly expressed in mature hepatocytes [102]. Studies in primary cultures

of human hepatocytes showed bile acid activated FXR transcriptionally stimulated

fibroblast growth factor 19 (FGF19), which activated JNK signaling via its cell surface

recetpor FGFR4. FGF19 treatment of cultured hepatocytes also resulted in the activation

of JNK and suppression of CYP7A1. Mice lacking FGFR4 showed increased Cyp7a1

expression and bile acid pool size, which further supported the role of FGFR4 in the bile

acid feedback inhibition of bile acid biosynthesis[103]. However, since FGF family signaling is known to largely depend on cell to cell communication and its effects are restricted to immediate local environment, FGFR4 is proposed to be a sensor to local infections or damages caused by bile acids or xenobiotics rather than a systemic regulator of cholesterol or bile acid homeostasis.

3. PXR and bile acid metabolism

3.1. The nuclear receptor PXR

PXR (NR1I2) belongs to the nuclear receptor NR1I subfamily, a group that includes

vitamin D receptor (VDR) and constitutive androstane receptor (CAR) [104]. Mouse

PXR was first identified based on the sequence homology with other nuclear receptors

and named PXR based on its activation by various natural and synthetic pregnanes [105].

So far, PXR has been cloned from human, monkey, dog, rabbit, rat, chicken and fish

[106]. The DBD is highly conserved among mammalian PXRs and they share more than

95% sequence homology [107]. PXR forms a heterodimer with RXR and recognizes DR3,

DR4, DR5, ER6 and ER8 DNA binding motifs on its target genes. PXR is mainly

expressed in liver, intestine and colon, where most of the drug metabolizing enzymes are also highly expressed and regulated [108]. Various compounds have been shown to bind and activate PXR in cell-based reporter assays. Classic CYP3A inducer PCN and dexamethasone were shown to be potent mouse PXR agonists, which were also the first

evidence that PXR might play a role in the regulation of CYP3A and drug metabolism.

Interestingly, further studies revealed PXR ligand activation profile varied largely across species. For example, the antibiotic rifampicin is the most potent human PXR agonist, but shows no activity on rodent PXRs, in contrast, PCN does not activate human PXR efficiently. The three-dimensional crystal structure study of PXR LBD has shed light on the underlying basis of PXR’s wide ligand binding specificity and cross-species difference of activation profile [109]. The PXR LBD is folded into a wide spherical ligand binding pocket with a flexible loop structure, which is able to accommodate ligand of different sizes. Twenty hydrophobic amino acids lining evenly around the pocket allow the pocket to form hydrogen bonds with compounds in different orientations. The cross-species activation profile difference is explained by the less conserved LBD and replacement of amino acids in the ligand binding pocket. Mutations of nonconserved amino acids in mouse PXR LBD into human PXR sequence allow the hybrid receptor to respond to activation profile similar to human PXR.

3.2. PXR and xenobiotic metabolism

The body must defend itself against the toxicity of various xenobiotics that are derived from the environment or endobiotics that are synthesized by the body itself. The cytochrome p450 family of heme containing monooxygenases are often induced by such chemicals, which are then oxidized by these enzymes [110]. By doing so, the body converts toxic lipophilic chemicals into non-toxic hydrophilic metabolites which are ready for excretion. CYP3A4 is the most abundant CYP isoform expressed in human

liver, while its rodent counterparts are known as mouse Cyp3a11 and rat Cyp3a23. Now,

it is certain that the nuclear receptor PXR plays a key role in the regulation of CYP3A

[105, 108, 111]. PXR binding sites have been identified on the promoters of CYP3A isoforms in different species. Activation of PXR by steroids or drugs transcriptionally induces CYP3A expressions. The role of PXR in the CYP3A induction was clearly demonstrated by studies in mice lacking Pxr (Pxr-/-) [112]. These animals showed no overt phenotype changes. Serum analysis showed normal cholesterol, triglyceride, glucose and liver enzymes levels, which suggest PXR may not be essential for normal development or physiology. However, PCN treatment failed to induce Cyp3a11 expression and resulted in severe liver damages in these mice. These effects were not seen in wild type mice. Later on, a list of drug metabolizing genes are revealed to be direct PXR target genes. Besides CYP3A family, genes involved in phase I oxidation (eg.

CYP2B6, CYP2C8, CYP2C9 and CYP2C19), phase II conjugation (eg. glutathione S transferase, sulotransferase, UDP glucoronosyltransferase and carboxylesterase) and phase III transport (eg. OATP2, MDR1 and MRP2) are also up-regulated by PXR [106,

113, 114]. Thus, PXR coordinately regulates a network of genes to facilitate the detoxification and elimination of xenobiotics and drugs (see Fig.8).

Since CYP3A4 is responsible for metabolizing more than 50% of the current

prescription drugs, activation of PXR also provides a basis for potential drug-drug

interactions in patients taking multiple prescriptions, whereas one drug accelerates the

metabolism of a second drug[108]. For example, hyperforin, the active ingredient in the

over-the–counter anti-depression and inflammation drug, St. John’s Wort (SJW), has been

Fig.8

Fig. 8. PXR regulation of xenobiotic metabolism.

PXR is activated by a variety of xenobiotics and endotoxins. Upon ligand activation,

PXR transcriptionally induces genes in phase I oxidation, phase II conjugation and phase

III transport to detoxify and facilitate eliminations of xenobiotics and their metabolites from the body. MDR1: multidrug resistance protein1; MRP2: multidrug resistance related protein 2.

found to activate PXR and induce CYP3A4, which enhances the metabolism of several prescription drugs such as the immunosuppressant cyclosporin, the HIV protease inhibitor indinavir and the anticoagulant warfarin[115-118]. Due to the discoveries of

PXR mediated drug-drug interactions, new drugs are now screened for PXR activities and potential candidates are modified, replaced or withdrawn from the market for drug safety.

3.3. PXR and bile acid metabolism

Rifampicin has been used for over a decade to treat cholestatic patients [119, 120]. The beneficial effects of rifampicin treatment is currently not clear, and is thought to be partly due to the activation of PXR, which induces CYP3A4 and other enzymes and transporters involved in detoxification and transport of toxic bile acid and other lipid metabolites.

Recent reports that PXR can be activated by LCA further support a role of PXR in such defensive mechanism. LCA is a particular toxic bile acid that is accumulated to high levels during cholestasis. In the liver and intestine, CYP3A4 converts LCA into none- toxic hyocholic acid (HCA), which is subsequently secreted by MRP2. Thus, activation of PXR promotes the detoxification of LCA and prevents its hepatic toxicity. For many years, it has been known that Cyp7a1 transcription is suppressed in mice treated with

PCN and this suppression is abolished in Pxr null mice[121, 122]. These findings indicated PXR might also play a role in reducing bile acid output during cholestasis [113,

114, 122, 123].

4. LXR and cholesterol homeostasis

Considerable evidence has shown that the nuclear receptor LXR (NR1H3) acts as a

whole body cholesterol sensor. Both LXRα and LXRβ are activated by physiological

concentrations of oxysterols such as 22(R)-hydroxycholesterol, 24(S)-

hydroxycholesterol, 24(S),25-epoxycholesterol and 27-hydroxycholesterol[83, 124, 125].

LXRα is mainly expressed in liver, intestine, adipose and macrophages, whereas LXRβ is universally expressed. Besides its role in the up-regulation of Cyp7a1 in rodent to convert excess cholesterol into bile acids and prevents hepatic cholesterol accumulation, LXR also plays a key role in reverse cholesterol transport and lipoprotein metabolism.

Synthetic LXR agonists raise serum HDL cholesterol and inhibited intestinal cholesterol absorption [126, 127]. This is thought to result from the activation of a list of ABC transporters by LXR. In intestine and macrophages, LXR induces ABCA1 and ABCG1, which transport cholesterol to Apo-AI and HDL, respectively. Cholesterol efflux to Apo-

A1 is the initial step in reverse cholesterol transport. The critical role of ABCA1 for cholesterol efflux has been demonstrated by studies in patients with Tangier diseases

[128, 129]. Defects in ABCA1 in these patients led to the absence of serum HDL and accumulation of cholesterol in peripheral tissues. Recently, ABCG1 has also been demonstrated to efflux cholesterol directly to HDL and play an important role in reverse cholesterol transport[130, 131]. ABCG5/G8 half transporters have been identified as direct LXR target genes [132]. As mentioned before, ABCG5/G8 half transporters are expressed at canalicular membrane of the hepatocytes and facilitate cholesterol excretion

into the bile. In the intestine, ABCG5/G8 transport dietary cholesterol along with toxic

plant sterols back to the intestine lumen and limit cholesterol uptake in humans. Patients

with defects in ABCG5 or ABCG8 genes develop sitosterolemia, a disease characterized

by increased dietary cholesterol and plant sterol absorption and hypercholesterolemia.

ApoE is a crucial component of chylomicrons, chylomicron remnants, VLDL and IDL.

ApoE is recognized by LDL receptor and mediates hepatic uptake of these particles[133].

Mice lacking ApoE expression develop hypercholesterolemia and atherosclerosis on a

normal chow diet and have been widely used as models to study cholesterol metabolism.

ApoE was the first gene identified to be regulated by LXR in a tissue specific manner

[134]. LXR up-regulates ApoE in adipose and macrophages but not in the liver. Later on, apolipoproteins ApoCI, ApoCII and ApoCIV were identified as LXR target genes. All these apolipoproteins turn out to be the acceptors of ABC transporter-mediated cholesterol efflux [135]. Finally, cholesterol ester transfer protein (CETP), which is mainly bound to HDL and mediates lipid transfer between HDL and apoB containing particles, is also regulated by LXR[136]. Thus, LXR is a master regulator of cholesterol homeostasis by controlling a net work of genes in cholesterol efflux, transport and elimination pathways.

Recent studies suggested that CYP27A1 may play a role in defense against

atherosclerosis by converting cholesterol in macrophages to 27-hydroxycholesterol

(27HOC), which is transported to hepatocytes and converted to bile acids [137]. This

process is similar to the reverse cholesterol transport from peripheral tissues and

macrophages to the liver. Cholesterol-laden macrophages infiltrate into the endothelium

of arterial wall, causing production of pro-inflammatory cytokines, smooth muscle

proliferation, foam cell formation, endothelium dysfunction and atherosclerosis [138]. 27

HOC is the most abundant oxysterol in circulation. It has been reported that macrophages

loaded with cholesterol produce a high level of 27HOC that is sufficient for activation of

LXR and induction of ABCA1 and ABCG1[139, 140]. Peroxisome proliferator activated

receptor γ (PPARγ) agonist stimulates CYP27A1 activity and production of 27HOC,

which induces ABCA1 and ABCG1 in macrophages. These studies suggest that

CYP27A1 plays a key regulatory role in control of cholesterol efflux and homeostasis in macrophages (Fig.9). The importance of CYP27A1 in control of cholesterol homeostasis

is strongly supported by the findings that CYP27A1 gene mutations in cerebrotendinous

xanthomatosis (CTX) patients cause accumulation of high levels of cholesterol in the peripheral tissues and pre-mature atherosclerosis[141].

D. Hypothesis

Increasing evidence has indicated that activation of nuclear receptor PXR by bile acids

or drugs significantly increases bile flow and decreases bile acids induced hepatic

toxicities in cholestatic patients. Studies of PXR regulation of bile acid synthetic and

detoxifying genes may provide better understandings of the molecular mechanisms of

PXR action and help develop clinical treatments for bile acids and drug induced

cholestatic diseases. The aim of this study is to investigate the role of PXR in the

transcriptional regulation of CYP7A1, CYP3A4 and CYP27A1 genes and how the

coordinate regulation of these genes by PXR may affect bile acids metabolism and

cholesterol homeostasis. Based on the current scientific reports and clinical findings, I hypothesize that ligand-activated PXR may reduce hepatic bile acid synthesis via feedback inhibition of CYP7A1 and increase bile acid detoxification via feedforward stimulation of CYP3A4, and thus reduce bile acids heaptotoxicities during cholestasis.

Furthermore, PXR may also induce CYP27A1 in the liver or intestine and play a role in the cholesterol homeostasis by regulating oxysterol production and therefore, reverse cholesterol transport. The significance, hypothesis and experimental approaches for studying each individual gene are described in detail in the first section of chapter III, IV

or V.

Fig.9

Fig. 9. CYP27A1 and cholesterol homeostasis in macrophages.

Oxidized LDL is taken up by macrophages via SR-AI (CD36). PPARγ agonists or RAR

and RXR agonists stimulate CYP27A1, which converts cholesterol into 27-

hydroxycholesterol. 27-hydroxcholesterol activates LXR to induce ABCA1 and ABCG1,

which transport cholesterol to ApoAI or HDL particles, respectively. HDL transport

cholesterol to the liver via SR-BI for bile acid synthesis or excretion into the bile. 27-

hydroxycholesterol can also be directly transported back to the liver via portal blood for bile acid synthesis. ABCA1 and ABCG1 mediated cholesterol efflux is an important pathway in macrophages to prevent cholesterol accumulation, foam cell formation and atherosclerosis. SR-AI: scavenger receptor AI; SR-BI: scavenger receptor BI

CHAPTER II

EXPERIMENTAL PROCEDURES

A. Cell culture

The human hepatoblastoma cell line HepG2, human embryonic kidney cell line

HEK293 and human adenocarcinoma cell line Caco2 were obtained from the American

Type Culture Collection (Manassas, VA). Cells were grown at 37 degrees in a 1:1

mixture of Dulbecco's modified Eagle's medium and Ham’s F-12 (50:50), (Life

Technologies, Inc.) supplemented with 100 units/ml penicillin G/streptomycin sulfate

(Celox Corp., Hopkins, MN) and 10% (v/v) heat-inactivated fetal calf serum (Irvine

Scientific, Santa Ana, CA). Caco2 cells were cultured in media containing 20% heat-

inactivated fetal calf serum. Primary human hepatocytes were isolated from human

donors (HH1088, 2 yr female; HH1089, 52 yr male; HH1106, 73 year female; HH1115,

22 yr male; HH1117, 68 yr female; HH1118, 73 yr female; HH1119, 29 yr female;

HH1122, 46 yr female; HH1148, 60 yr male) and were obtained through the Liver

Tissue Procurement and Distribution System (LTPADS) of NIH (S. Strom, University

of Pittsburgh, Pittsburgh, PA). Cells were maintained at 37 degrees in HMM modified

Williams E medium (Clonetics, US) supplemented with 10-7 M of insulin and

dexamethasone.

B. Luciferase reporter assay

Luciferase reporter assays allow one to study the transcriptional regulation of a specific

promoter in mammalian cells. Luciferase reporter plasmid is usually constructed by

linking the DNA fragment of a specific gene promoter upstream of the coding sequence

of a firefly luciferase gene. When such constructs are introduced into cell lines by

transfection, the luciferase gene is expressed under the control of the upstream promoter.

The intracellular luciferase levels can be determined by measuring the light production when the cell lysates are incubated with the luciferase substrate, luciferin. A pCMVβ-

galactosidase plasmid constitutively expressing bacterial β-galactosidase (β-gal), under the

control of a cytomegalovirus (CMV) promoter, is also introduced into the cells as an

internal control. The transfection efficiency can be normalized by dividing the luciferase

activity by β-galactosidase activity and the relative luciferase activity represents the

transcriptional activity of the promoter that drives the luciferase gene. In reporter assays,

expression plasmids of transcription factors or certain treatments are usually used to test

their effects on the transcriptional activity of the promoter.

1. Calcium-phosphate-DNA co-precipitation transfection method

Cells were grown to ~80% confluent in 24-well culture plates. One hr prior to the

transfection, medium was removed and replaced with fresh serum-containing medium.

The promoter/luciferase reporter plasmid (1 μg), pCMVβ-galactosidase plasmid (0.2 μg) and co-DNA (0.5μg) were mixed with 3.14 μl 2M CaCl2 and water was added to bring

the volume to 25 μl. An equal volume of 2X HBS (50mM HEPES pH7.1, 280mM NaCl,

10mM KCl, 12mM Dextrose, 1.5 mM Na2HPO4) was then added drop-wise while

vortexing and the mixture was incubated at room temperature for 15-20 min. The mixture

was then vortexed briefly and added into the cell culture media. The cells were then

incubated at 37 degrees for 4 hr.

For HepG2 cells and Caco2 cells, glycerol shock was performed 4 hr after transfection

to improve transfection efficiency. The glycerol shocking solution was prepared by

adding 15% glycerol in 1XHBS solution and warmed to 37 degrees before use. Medium

was carefully removed by aspiration and cells were incubated in 0.5ml glycerol shock solution for 90 sec at room temperature. Shocking solution was then quickly removed and cells were washed twice with 1X PBS. 1 ml of serum-free medium was added to the cells and treatment was added if necessary. Cells were then returned to a 37 degree incubator.

Glycerol shock was not necessary for transfecting HEK293 cells. Medium was simply removed 4 hr after transfection, and cells were washed twice with 1X PBS and 1 ml serum-free medium was added before cells were incubated at 37 degrees.

2. Lipofectamin2000 transfection method

HepG2, Caco2 or 293 cells were cultured to 80% confluence in serum-containing

medium without antibiotic supplements. Then, 0.2μg of reporter plasmid, 0.05 μg of

pCMVβ-galactosidase plasmid and 0.1μg of co-DNA was mixed with 50 μl of Opti-

MEM solution (Invitrogene). 2μl lipofectamin2000 reagent (invitrogene) was mixed with

50μl Opti-MEM solution in another microtube. The two solutions were mixed, vortexed briefly and incubated at room temperature for 20 min. 100μl of the mixture was added to

the cells and the cells were returned to 37 degree incubator. Treatment was added into the

medium 4 hr after transfection as indicated in each individual experiment.

3. Luciferase and β-gal assays

After certain incubation period (usually 48 hr after transfection), medium was removed

and cells were washed once in 1XPBS and lysed in 100 μl lysis buffer (40mM tricine pH7.8, 50mM NaCl, 2mM EDTA, 1mM MgSO4, 5mM DTT, 1% Triton X-100) at room

temperature for 20 min. For luciferase assay, 20 μl of the cell lysate was mixed with luciferase substrate (luciferin) solution and light production was measured by Lumat LB

9501 luminometer (Berthold Systems, Inc., Pittsburgh, PA). For β-gal assay, 50 μl cell lysate was mixed with 50μl 2Xβ-gal solution in 96 well plates. Mixture was incubated at

37 degree until yellowish color developed. Absorbance at 405nM was measured by

Spectra Max 250 microplate reader. Luciferase activity was normalized by dividing the relative light units (RLU) by β-galactosidase activity and expressed as relative luciferase activity. Each assay was done in triplicate and individual experiments were repeated at least three times. Data are plotted as means ± standard deviation. Statistical analyses of treated vs. untreated controls (vehicle or empty plasmids) were performed using

Student’s t-test. A p<0.05 is considered statistically significant.

C. Mammalian hybrid assays

Mammalian hybrid assays are cell-based luciferase reporter assays for studying protein:

protein interactions (Fig10). The reporter used in these assays is 5XUAS-TK-Luc, which

Fig.10

Fig. 10. Mammalian two hybrid assay.

In order to study the interaction between protein X and protein Y, X is fused with Gal4

DBD, which recognize its DNA binding site UAS on the lucifease reporter, and Y is fused with VP16 AD, which has strong transcriptional activating activity. When protein

X and Y interact, VP16-AD is brought to the promoter vicinity and interacts with general transcription machinery and stimulates reporter activity (A). If a third protein factor Z interacts with X, the interaction between X and Y is blocked, and the reporter activity is suppressed (B). Gal4 DBD: Gal4 DNA binding domain; VP16 AD: VP16 activation domain.

contains 5 copies of the upstream activating sequence (UAS) fused upstream of a

thymidine kinase minimum promoter (TK) and the luciferase reporter gene. To study the

possible interaction between protein factor X and Y, protein X is fused with yeast Gal4

DBD and the fusion protein (Gal4-X) is able to bind to the Gal4 binding sites on the reporter. Protein Y is fused with yeast VP16 activating domain (VP16 AD) and the fusion protein VP16-Y shows strong transcriptional activating activity by interacting with general transcription factors. The reporter construct and fusion protein expression

plasmids are introduced into the cells by transfection. If protein X and Y interact, VP16-

AD is recruited to the vicinity of the artificial promoter, and the luciferase reporter is

stimulated over the control (Gal4-X+ VP16). In mammalian one-hybrid assays, plasmids

expressing wild type co-regulators (co-activators and co-repressors) are used instead of

VP16 fusion plasmids. Since the activating or repressing activities of these co-regulators are independent of VP16-AD, the relative strength of the co-activating or co-repressing

activities can be tested and compared between different co-regulators.

D. Site-directed mutagenesis

Site-direct mutagenesis, as performed here, is a PCR based method to introduce base

mutations into certain region of a DNA plasmid. In this study, mutations were introduced

into promoter/ luciferase reporter constructs using PCR-based Quick Change Site-

directed Mutagenesis Kit (Stratagene, La Jolla, CA). Two complementary

oligonucleotides containing the mutations were used as PCR primers. Wild type plasmid

purified from bacteria E. coli was used as template. PCR reactions were set up according

to the manufacturer's instruction using 50 ng of template DNA and 125 ng of primers.

PCR cycling parameters were set as following: denaturing at 95 degree for 2 min,

followed by 18 cycles at 95 degree for 30 sec, 55 degree for 1 min, and 68 degree for 18

min. The PCR reaction mixture was digested by Dpn I for 2 h at 37 degree to remove the

template DNA. The mutant plasmids resulting from the PCR reaction were not

methylated, and thus were not recognized and digested by Dpn I. One μl of the PCR reaction mixture was transformed into XL1-Blue super competent cells (Stratagene) for selection of mutant clones. Mutations in each clone were confirmed by DNA sequencing.

E. Quantitative real-time PCR

1. RNA isolation

Cells were cultured in 6-well plates. Total RNA was isolated from the cells using Tri-

Reagent (Sigma) according to the manufacturer’s protocol. Cells were collected into microtubes in 1 ml Tri-Reagent. After 5 min incubation at room temperature, 0.2 ml chloroform was added and the mixture was vortexed vigorously for 30 sec and kept at room temperature for 15-20 min. The tube was centrifuged at 4 degrees at 10,000x g for

15 min, and upper aqueous phase was carefully transferred to another tube. 0.5 ml isopropanol was then added and RNA was precipitated by centrifugation at 4 degree at

10000x g for 10 min. The RNA pellet was washed with 1 ml ice cold 75% ETOH in

DEPC water and centrifuged at 4 degrees at 10,000x g for 10 min. The supernatant was

discarded, and the pellet was air dried for 15 min at room temperature and dissolved in 50

μl DEPC water.

2. Reverse transcription PCR

Reverse transcription reactions were performed using RETROscript kit according to

manufacturer’s instructions (Ambion Inc., Austin, TX). 2 μg RNA and 2 μl oligo dT primer were mixed with nuclease-free water to a final volume of 12 μl in a PCR reaction tube. The tube was incubated at 78 degrees for 5 min and cooled down on ice for 2 min.

4μl dNTP, 2 μl 10X RT buffer, 1μl RNase inhibitor and 1μl MMLV-reverse transcriptase were then added and the tube was incubated at 42 degrees for 1.5 hr, heated at 95 degrees for 10 min, and stored at -20 degrees.

3. Real time PCR

For real time PCR, samples were prepared according to the PCR Taqman Universal

Master Mix 2X protocol. The reverse transcription reaction mixture containing cDNA

was diluted 10 fold in water. 5 μl cDNA, 12.5 μl Taqman Universal Master Mix 2X,

1.25μl Taqman primers/probe mix and 6.25 μl water were mixed in 96 well PCR plate and PCR was performed on ABI 7500 real time PCR system. A universal PCR cycle perimeters was set for all the reactions as following: step1 (1 cycle): 2 min at 50 degree, step2 (1 repeat): 10 min at 95 degree, step 3 (40 cycles): 15 sec at 95 degree followed by

1 min at 60 degree. Human highly basic protein (HBP) or ubiquitin C (UBC) was used in the same reactions as an internal reference gene. Assay-on-Demand PCR primers and

Taqman MGB probe mix were obtained from Applied Biosystems (Foster City, CA).

Each sample was measured in triplicate. Each experiment was repeated 3 times.

Quantitative PCR analysis was conducted on the ABI Prism 7700 Sequence Detection

System. Relative mRNA expression was quantified using the comparative CT (ΔΔCt) method according to the ABI manual. Ct values were obtained after each real time PCR experiment and relative mRNA expression was calculated as following:

Ct: the cycle number at which the PCR amplified product reaches a fixed threshold.

∆Ct = Ct of gene of interest – Ct of reference gene (HBP or UBC).

∆∆Ct of control = ∆Ct of control - ∆Ct of control (which always equals to zero).

∆∆Ct of treated = ∆Ct of treated - ∆Ct of control

2 2 S.D. ∆Ct = Square Root [(S.D. of Ct of reference gene) + (S.D. of gene of interest) ]

Relative expression: 2-∆∆Ct

High range: 2-(∆∆Ct-S.D.ΔCt)

Low range: 2-(∆∆Ct+S.D.ΔCt)

Since ∆∆Ct of control was set to zero, the relative expression of control is always set as

1 (20=1). Two-fold increase is expressed as 21 =2 and 50% suppression is expressed as 2-1

=0.5. When plotted as bar graphs, the upper end of error bar represents high range minus

relative expression and lower end of error bar represents relative expression minus low

range.

F. Electrophoretic mobility shift assay (EMSA)

EMSA detects protein: DNA interaction in vitro. In vitro synthesized protein factors

and 32P end labeled double strand DNA probes are incubated and loaded on a

polyacrylamide gel for electrophoresis. Since protein bound probes have lower mobilities

than unbound probes, a DNA:protein interaction is indicated by a shifted band when gel

is auto-radiographed.

Nuclear receptor proteins were synthesized in vitro using Quick-coupled Transcription/

Translation Systems (Promega) programmed with receptor expression plasmids

according to the manufacturer's instruction. Double-stranded synthetic probes were

prepared by heating equal molar amounts of complementary oligonucleotides to 95

degrees in 2X SSC (0.5 M NaCl, 15 mM sodium citrate, pH 7.0) and then slowly cooled to room temperature. The resulting double-stranded fragments were labeled with [α32P] dCTP by filling in reaction using the Klenow fragment of DNA polymerase I. Labeled double-stranded probes were purified through G-50 spin columns. For each binding reaction, 50,000 cpm of labeled probe and 5 μl of in vitro translated protein were incubated at room temperature for 20 min in 20 μl of binding buffer containing 12 mM

HEPES, pH 7.9, 50 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, and 15% glycerol and 2

μg of poly (dI-dC). Samples were loaded on a five percent polyacrylamine gel and electrophoresis was performed at room temperature at constant 200 V for 1.5 h. The gel was dried and auto-radiographed using Phosphor Imager 445Si (Molecular Dynamics,

Sunnyvale, CA).

G. Chromatin immunoprecipitation (ChIP) assay

ChIP assay detects protein: DNA interaction at chromatin levels (Fig.11). DNA: protein complexes were crosslinked by incubating intact cells with 1% formaldehyde. The chromatin was then sheared into 0.2 –2 kb fragments by sonication. DNA: protein complex was then immunoprecipitated by specific antibodies against the protein of interest. If the protein binds to a putative DNA binding site, the DNA fragment will be co-precipitated with the protein and can be detected by PCR using primers designed to amplify the specific DNA region. Compared to EMSA, ChIP assay is considered as an in vivo protein: DNA binding assay.

Cells were usually grown in 100 mm culture dishes to 80% confluence. As indicated in

some experiments, 10 μg of nuclear receptor expression plasmids were transfected to

increase the intracellular expression. ChIP assays were performed using ChIP Assay kit

(Upstate Biotec, NY) according to manufacturer’s protocol. Briefly, cells were cross-

linked in 1% formaldehyde for 10 min and washed with ice-cold PBS containing protease

inhibitors (1mM PMSF, 1 μg/ml aprotinin and 1 μg/ml pepstanin A, Sigma) twice. Cells

were scraped and incubated in 1% SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM

Tris-HCl, pH 8.1) for 30 min. on ice and sonicated using a Branson sonifier 250 with a

microtip setting 6 for 15 s pulses at 40% output for a total of 1.5 min to break the DNA

into 0.2 to 2 kb fragments. Cell lysates were collected by centrifugation and diluted 10-

fold in ChIP dilution buffer (0.01% SDS, 1.1 % Triton X-100, 1.2 mM EDTA, 16.7 mM

Tris-HCl, pH 8.1, 167 mM NaCl). Ten percent of the cell lysates were saved and used as

the “input” for immuno-precipitation. After pre-clearing the diluted cell lysate with

Fig.11

Fig. 11. Chromatin immunoprecpitation (ChIP) assay.

To detect protein factor binding to the native chromatin, protein/DNA complex is crosslinked by formaldehyde. Chromatin is then sheared into 0.2-2 kb fragments by soncation. DNA/protein complex containing target protein factor is immunoprecipitated by specific antibodies. Crosslinking is reversed and DNA fragments in the precipitated complex are extracted and subject to PCR amplification. If the protein factor binds to the putative DNA region, the DNA fragments should be co-immunoprecipitated and the PCR product should be detected on an agrose gel.

protein A-agarose, DNA-protein complexes were precipitated by incubating the cell lysates with 10 μg of antibodies (Santa Cruz Biotec. Inc, Santa Cruz, CA) against target proteins overnight followed by a 3 h incubation with protein A or G-agarose beads.

Normal IgG was added as a non-immuno-precipitation control. The beads were washed with low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl) once, high salt wash buffer (0.1 % SDS, 1 % Triton X-100, 2 mM

EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl) once, LiCl wash buffer (0.25 M LiCl,

1% NP40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1) once and TE buffer

(10 mM Tris-HCl, 1 mM EDTA, pH 8.0) twice. Samples were eluted twice with 250 μl of ChIP elution buffer (1% SDS, 0.1 M NaHCO3) and the eluates were combined. The

cross-links were reversed by adjusting NaCl concentration to 200 mM and incubating the

eluates at 65 ° for 4 h followed by a 1 hr incubation in 0.04 μg/μl proteinase K. DNA was

extracted using phenol/chloroform and precipitated using isopropanol. DNA was then

dissolved in 50μl water and 5μl was used as template and PCR amplified for 30 cycles.

PCR product was analyzed on a 2% agrose gel.

H. Western immunoblotting analysis

Cells were cultured to 80% confluence. Cells were collected in 1X SDS lysis buffer

(62.5 mM Tris-HCL, 2mM EDTA, 2.3% SDS. 10% glycerol) containing protease inhibitors (1 mM EGTA, 1 mM PMSF, 2 μg/ml each of leupeptin, pepstatin and aprotinin, Sigma) and homogenized by sonication. Protein concentrations were measured using the BCA protein assay kit (Pierce) according to manufacturer’s instruction and

same amount of total protein in each sample were loaded on a SDS-PAGE for electrophoresis at 100V. Samples were transferred onto a nitrocellulose membrane and

the membrane was blocked in 1XTTBS solution (100mM Tris-HCL, 0.9% NaCl, 1%

Tween-20) containing 5% dry milk for 2 hours at room temperature. Membranes were

then blotted with specific antibodies against target proteins (Santa Cruz Biotechnology,

CA) in 1XTTBS solution overnight at 4 degree. Membranes were then washed 4 times in

1XTTBS and blotted with secondary antibodies (Santa Cruz, CA) for an additional 2

hours at room temperature. Membranes were washed for 4 times in 1XTTBS, incubated

in ECL western blotting detection solution for 2 min (Amersham Biosciences, UK) and

exposed to X-ray film.

I. Co-immunoprecipitation assay

Co-immunoprecipitation assay detects protein: protein interactions in intact cells. Cells

were usually lysed in mild detergent without disrupting interactions between protein

factors. Specific antibodies against a particular protein factor were used to precipitate this

protein factor from the whole cell lysates. Other protein factors that interact with this

protein factor were also co-precipitated from the cell lysates and can be detected with

specific antibodies in western blotting analysis.

Cells were cultured in T150 flasks until 80% confluence. Cells were washed twice with ice cold 1XPBS, collected by scraping and incubated in 500 μl modified RIPA buffer (50 mM Tris-HCl, 1% NP40, 0.25%-deoxycholate, 150 mM NaCl, 1 mM EDTA) containing protease inhibitors (1 mM PMSF, 1 μg/ml aprotinin, 1μg/ml pepstanin, Sigma) for 30

min. Cleared cell lyates were collected by centrifugation at 10,000 X g at 4 degree for 15

min. Clear lysates were pre-cleared by adding 60μl protein G beads slurry and incubated

at 4 degree for 30 min with rotation. The lysates were then centrifuged at 2000rpm for 2

min to precipitate the protein G beads. Supernatant was transferred to a new microtube

and incubated with 10 μg of antibody (Santa Cruz Biotec. Inc.) at 4 degree with rotation

overnight, followed by an additional incubation for 2 h with protein G beads. ProteinG

beads alone were used as negative immunoprecipitation controls. The beads were washed

3 times with RIPA buffer and were boiled in 2X protein loading buffer for 5 min. same amounts of samples were loaded on SDS PAGE gels for western immunoblot analysis using specific antibodies.

J. GST pull-down assay

GST pull-down assays detect protein: protein interactions in vitro (Fig.12). Bacterial

expressed GST fusion protein and in vitro 35S labeled protein factor are incubated in protein binding buffer, and GST-fusion protein is precipitated by immobilized glutathione beads. The 35S labeled protein factor will also be co-precipitated if it interacts

with the GST fusion protein and can be detected on a SDS-PAGE following auto-

radiography.

1. GST fusion protein expression

GST or GST fusion protein expression plasmids were transformed into E. coli BL21 cells (Amersham Pharmacia Biotech). Cells were grown at 37 degree to an O.D. of about

0.8 and induced with 1 mM IPTG (Amersham Pharmacia Biotech) for an additional 2 h at

Fig.12

Fig. 12. GST pull down assay.

To study the possible interaction between protein X and Y, X is synthesized by in vitro translation and labeled with 35S. Y is expressed as a GST tagged fusion protein in bacteria

E.coli. Glutathione is immobilized on agrose beads and incubated with GST-Y and 35S X.

Glutathione is a substrate for GST, thus, GST will bind to glutathione and GST-Y will be precipitated. If protein X interacts with Y, X will also be co-precipitated and detected on a SDS-PAGE by phosphor imager. If X does not interact with Y, X will be lost during the washing steps.

27 degrees. 1 L bacteria cell culture was centrifuged at 4 degree at 5000 rpm and the

bacterial pellets were resuspended in 10 ml Triton X100 lysis buffer (0.05 M Tris-HCl, 5

mM EDTA, 0.05 M NaCl, 0.1% Triton X100, 10% glycerol) containing the protease

inhibitors (1 mM PMSF, 1 μg/ml aprotinin and 1 μg/ml pepstanin A, Sigma) and sonicated 10 times in pulses of 30 sec each. Cell lysate was clarified by centrifugation at

10,000 x g for 10 min at 4°C. 0.5 ml cell lysate was incubated with immobilized

glutathione beads (Amersham Pharmacia Biotech) at 4 ° for 2 h. The beads were washed

four times in the same lysis buffer and the protein bound to the glutathione beads was

eluted with 20 μl elution buffer (50 mM Tris-HCl pH 8.0) containing 10 mM reduced

glutathione. The protein samples, mixed with 20 μl 2x protein loading buffer were boiled

at 95 degree for 5 min and loaded on an SDS PAGE. The gel was then stained with

Coomassie Blue stain (Biorad) to confirm the correct protein expressions.

2. GST pull-down assay

The GST and GST fusion proteins were each incubated with immobilized glutathione

beads (Amersham Pharmacia Biotech) at 4 °C for 2 h. The beads were washed four times

in the Triton X100 lysis buffer and the proteins bound to the glutathione beads were

stored as a 50% slurry in 1XPBS. Typically each reaction had approximately 2μg of the

protein. The 35S labeled proteins were generated using their respective expression

plasmid in the TNT rabbit reticulocyte system (Promega) in the presence of 35S labeled

Methionine. 20μl of the GST or GST-fusion proteins (~1μg) bound to the glutathione

beads was added to 5μl of the 35S labeled proteins and incubated with rotation in GST

69 incubation buffer (50mM KCl, 40mM HEPES pH 7.5, 5mM β-mercaptoethanol, 0.1%

Tween-20. 1% non fat dry milk) at 4oC for 4h. In competition assays, 50μl of non-radio labeled TNT lysates incubated with pcDNA3 or indicated expression plasmids was added to the mixture. Vehicle (DMSO) or PXR agonist rifampicin was also added to a final concentration of 10μM. After incubation the beads were washed three times with the

GST incubation buffer and boiled in 1X protein loading buffer for 5 min. The beads were pelleted by centrifugation and the supernatant from each sample was loaded on an SDS

PAGE gel. One μl of 35S labeled protein was loaded as “Input”. The gel was dried and subjected to autoradiography.

K. 27 hydroxycholesterol measurement

Cells were cultured in T175 flasks untill ~ 80% confluence and treated with cholesterol or rifampicin for 72 hours. Water soluble cholesterol was prepared by dissolving cholesterol in (2-hydroxypropyl)-β-cyclodextran solution (45% w/v in water) (Sigma) and was sonicated each time before treatment. Cells were then washed with ice cold 1X

PBS twice and collected by centrifugation at 5000 rpm for 5 min. Cells were resuspended in 1 ml 1X reaction buffer (0.1 M phosphate pH7.5, 1mM EDTA, 2mM DTT) and lysed by sonication. 100 μl of 20μM 20α-hydroxychoelsterol was added in each reaction as an internal standard. Two units of cholesterol oxidase (Sigma) were added to each tube and tubes were incubated at 37 degree for 30 min to generate α, β unsaturated ketones.

Oxidized cholesterol and oxysterol products can then be detected at 260 nm by HPLC method. The reaction mixtures were extracted twice with 4ml hexane (Sigma) and the

organic layers were evaporated under gentle nitrogen flow at 60 degree to complete dryness. The samples were dissolved in 100μl sample buffer (5% isopropanol in dodecane, Sigma) and analyzed on a normal phase HPLC column (pinnacle II sillica

5μM 4.6X250 mm, Restek, PA) using an isocratic mobile phase (96.5% Hexane, 2.5%

isopropanol, 1% Glacial acetic acid) at a flow rate of 1ml/min. The retention time for

cholesterol, 27-hydroxycholesterol and 20α-hydroxycholesterol was determined by

loading purified compounds into HPLC. To obtain a standard curve for 27-

hydroxycholesterol, samples containing increasing amount of 27-hydroxycholesterol

were loaded onto HPLC. 100 μl of 20μM 20α-hydroxycholesterol was also added into each sample as internal controls. The relative amount of 27-hydroxycholesterol was

normalized to 20α-hydroxycholesterol. A linear relationship between the amount of 27-

hydroxycholesterol loaded and relative 27-hydroxycholesterol amount detected by HPLC

was obtained. The amount of 27-hydroxycholesterol in cell lysate was derived from the

standard curve and divided by the volume of cell pellet to obtain intracellular 27-

hydroxycholesterol concentration. Each experiment was repeated three times and data was and plotted as means ± standard deviation. Statistical analyses of treated vs. untreated controls were performed using Student’s t-test. A value of p<0.05 is considered statistically significant.

CHAPTER III

PXR REGULATION OF HUMAN CYP7A1

A. Significance and approaches

During cholestasis, toxic bile acids are accumulated in the liver and circulation. The body protects against bile acids toxicity by increasing detoxification and excretion of bile acids and decreasing hepatic bile acid biosynthesis. PXR is known to be activated during cholestasis and plays a central role in the protective mechanisms by inducing genes in bile acid detoxification, conjugation and transport. Suppression of Cyp7a1 mRNA expression by PCN in wild type mice and loss of PCN suppression of Cyp7a1 in Pxr null mice suggest ligand activated PXR may also reduce bile acid toxicity by inhibiting

Cyp7a1 and bile acid biosynthesis. The recent finding that PXR is activated by the most toxic bile acid LCA further suggests the existence of a PXR mediated inhibition of

Cyp7a1 pathway [122]. The human PXR agonist rifampicin has been used for decades to treat cholestasis patients and showed beneficial effects. It is necessary to study if PXR regulation of CYP7A1 also exists in human.

Based on these findings, I hypothesized that bile acid and drug activated PXR might

inhibit human CYP7A1 in hepatocytes to reduce bile acid biosynthesis and thus prevent

their hepatic toxicities during cholestasis. To test this hypothesis, I will focuse my study

on the following specific aims:

1. To test how PXR regulates human CYP7A1 gene expression. Primary human

hepatocytes and HepG2 cells will be used as model systems. Rifampicin, a potent

and specific human PXR agonist, will be used to study PXR effect on human

CYP7A1 gene expression. Quantitative real-time PCR will be performed to test if

rifampicin inhibits human CYP7A1 gene transcription in primary human

hepatocytes. I will measure SHP mRNA expression to see if SHP is induced by

rifampicin and inhibits CYP7A1 gene. Finally, I will perform reporter assays in

HepG2 cells to confirm rifampicin effects on human CYP7A1.

2. To identify PXR responsive element on human CYP7A1 promoter. CYP7A1

reporter deletion constructs will be used in transfection assays to map the

promoter region that mediates rifampicin effect on CYP7A1. After identification

of the putative PXR binding sites in the CYP7A1 promoter sequence, I will

perform in vitro EMSA assays and in vivo ChIP assays in primary human

hepatocytes and HepG2 cells to test PXR binding to these sites.

3. To elucidate the mechanism of PXR regulation of human CYP7A1 gene.

HNF4α and PGC-1α have been reported to be the key regulators of human

CYP7A1 [56]. To test if PXR interferes with these factors to inhibit CYP7A1, I

will first do transfection assays to test the effects of PXR, HNF4α and PGC-1α on

CYP7A1 reporter activities. Mammalian two hybrid assays will then be performed

to identify the possible interactions between these factors. Finally, I will use

mammalian two hybrid assay, ChIP assay and co-IP assay to delineate the

mechanism of how PXR, HNF4α and PGC-1α coordinately regulate human

CYP7A1 gene.

B. Results

Rifampicin inhibits CYP7A1 mRNA expression in human primary hepatocytes –

Previously, mRNA expression levels of CYP7A1 and several transcription factors known to regulate CYP7A1 transcription (SHP, PXR, FTF, COUP-TFII, FXR, PGC-1α, HNF4α and Prox1) were measured by real-time PCR in human primary hepatocytes of 7 donors

(HH1088, HH1089, HH1115, HH1117, HH1118, HH1119, and HH1122). Results revealed that human primary hepatocytes expressed relatively low levels of CYP7A1 but abundant nuclear receptors and co-activators known to regulate CYP7A1 gene expression, which suggests that the human primary hepatocyte is suitable for studying CYP7A1 gene regulation by nuclear receptors.

To test if human CYP7A1 transcription was subject to PXR regulation, real-time PCR was used to assay the effect of rifampicin, a human PXR ligand, and CDCA, an FXR ligand, on CYP7A1 mRNA expression levels in human primary hepatocytes. Results from hepatocyte preparation HH1106 are shown in Table 1. After 24 h treatments, CDCA

(30 μM) markedly reduced CYP7A1 mRNA expression by 95% and rifampicin (10 μM) reduced CYP7A1 mRNA expression by about 80%. Since rifampicin is a very specific

PXR agonist, this inhibition is most likely mediated by PXR. SHP mRNA levels were also determined. As expected, CDCA induced SHP mRNA by 6 fold. The observed inverse relationship of CYP7A1 and SHP mRNA expression levels by bile acid is

Table 1. Q-PCR analysis of CYP7A1 mRNA expression in human primary hepatocytes (HH1106).

Treatment Vehicle CDCA(30μM) Rifampicin(10μM) CYP7A1 Relative Expression 1 (0.78-1.28) 0.052 (0.04-0.069) 0.223 (0.145-0.346)

ΔCt ± SDΔCt 10.36±0.36 8.91±0.33 12.53±0.63 SHP Relative Expression 1 (0.79-1.27) 6.19 (5.31-7.21) 0.002 (0.0007-0.0068)

ΔCt ± SDΔCt 8.91±0.33 6.25±0.21 17.71±1.61 CYP3A4 Relative Expression N.D. N.D 56.89 (44.94-72.0)

ΔCt ± SDΔCt 18.53±0.24 18.24±0.106 12.70±0.34

Ct: the threshold cycle at which the amount of amplified target reaches a fixed threshold.

Relative expression: 2-∆∆Ct; high range: 2-(∆∆Ct-SDΔCt), low range: 2-(∆∆Ct+SDΔCt)

∆Ct = Ct of gene of interest - Ct of reference gene (HBP)

2 2 S.D. ∆Ct = Square Root [(S.D. of Ct of reference gene) + (S.D. of gene of interest) ]

N.D.: None detectable, Ct=40.

consistent with the hypothesis that FXR/SHP mechanism mediates bile acid inhibition of

CYP7A1 gene transcription. Interestingly, rifampicin dramatically reduced SHP mRNA expression. As a positive control of PXR induction, CYP3A4 mRNA expression levels

were measured in human primary hepatocytes. The CYP3A4 mRNA levels were too low to be detected in vehicle and CDCA-treated human hepatocytes (Ct = 40), which reflected great variations in CYP3A4 expression between each individual. When the relative expression of control was set at 1 using Ct value of 40, CYP3A4 mRNA was induced by 57-fold. The lack of an inverse relationship between CYP7A1 and SHP mRNA expression in rifampicin-treated hepatocytes provided strong evidence that SHP might not be involved in PXR inhibition of CYP7A1. These results may suggest that ligand-activated PXR reduced SHP gene transcription in human livers.

Rifampicin inhibits CYP7A1 reporter activity in HepG2 cells – I then studied the effect

of human PXR and rifampicin on human CYP7A1 gene transcription by transient

transfection assay in HepG2 cells using a human CYP7A1 promoter/luciferase reporter

construct ph-298/Luc, which contained the human CYP7A1 promoter region -298/+24

linked to a luciferase reporter gene. Rifampicin (10 μM) slightly inhibited CYP7A1

reporter activity assayed in HepG2 cells (Fig. 13A). When human PXR and RXR were

co-transfected in HepG2 cells, rifampicin strongly reduced the reporter activity by 80%

(Fig.13B). These results confirmed that PXR is involved in rifampicin inhibition of

CYP7A1 gene.

Identification of a negative PXR response element - To localize the promoter region that

confers negative rifampicin and PXR effect on human CYP7A1 gene, 5’-deletion

Fig.13

Fig. 13. Rifampicin-activated PXR suppresses the CYP7A1 reporter activity in

transient transfection assays in HepG2 cells.

HepG2 cells were cultured to about 80% confluence. Human CYP7A1/luciferase reporter

(ph-298/Luc) (1 μg) was transiently transfected into HepG2 cells. Cells were treated with

vehicle (DMSO), or rifampicin for 40 h and harvested for luciferase activity assays as

described under Experimental Procedures. A. Effect of rifampicin (10 μM) on human

CYP7A1/Luc reporter activity in HepG2 cells. Data are plotted as means ±S.D. N=1, *, p<0.001, rifampicin-treated vs vehicle. B. Effects of PXR ligands (10 μM) on human

CYP7A1/Luc reporter activity in HepG2 cells transfected with PXR/RXRα expression plasmids (0.5 μg) or empty vector, pcDNA3, and PXR ligands were added as indicated.

The error bars represent the standard deviation from the mean of triplicate assays of an

individual experiment. n=1, *, p<0.0001, rifampicin-treated vs. vehicle. Each individual

experiment was repeated at least 3 times.

constructs of human CYP7A1/Luc reporter containing sequences from –1887/+24, -

298/+24, and -150/+24 were assayed in HepG2 cells transfected with PXR and RXR.

Reporter activities of all three constructs were inhibited about 80% by rifampicin (10μM)

(Fig.14). It was concluded that the PXR response element was located in the region downstream of –150, which contains both BARE-I and BARE-II.

Since BARE-I and BARE-II contain several AGGTCA-like direct repeats, I performed

EMSA using oligonucleotide probes designed based on the nucleotide sequences in the

BARE-I and BARE-II to test if PXR binds to these regions. Sequences of the probes are

shown in Fig.15A. When human BARE-I probe was used for EMSA, a mixture of in

vitro synthesized PXR and RXR shifted a single band (Fig. 15B, lane 4). Neither PXR

nor RXR alone bound the human BARE-I probe (Fig. 15B, lane 3 &4). A probe that

contains a strong PXR binding site ER6 in the human CYP3A4 gene was used as a

positive control for PXR binding (Fig. 15B, lane 1). Excess un-labeled BARE-I or un-

labeled CYP3A4 oligonucleotide completely competed out PXR/RXR binding to the

labeled human BARE-I probes(Fig.15B, lane 5&6). A mutant BARE-I probe, which has

mutations introduced to alter the AGCTCA half-site sequence, did not bind

PXR/RXR(Fig.15B, lane 7). Fig. 15C showed that un-labeled CYP3A4 oligonucleotide

could completely compete out PXR binding to the labeled BARE-I probe at 100-fold

excess. On the other hand, 200-fold excess of un-labeled BARE-I was required to

completely complete out PXR binding to the labeled 3A4 probe. PXR is known to form

heterodimer with RXR and bind to motifs including DR3, DR4, DR5, ER6 and ER8.

Analysis of the nucleotide sequences of human BARE-I revealed a putative DR3 motif

Fig.14

Fig. 14. Effect of rifampicin on human CYP7A1 deletion constructs.

HepG2 cells were cultured to about 80% confluence. Human CYP7A1/luciferase reporter

constructs ph-1887/Luc, ph-298/Luc and ph-150/Luc (1 μg) were transiently transfected

into HepG2 cells. PXR/RXRα expression plasmids (0.5 μg) were co-transfected. Cells

were treated with vehicle (DMSO), or rifampicin (10 μM) for 40 h and harvested for luciferase activity assays as described under Experimental Procedures. Data are plotted as means ±S.D. n=1, *, p<0.001, rifampicin-treated vs vehicle. Each individual experiment

was repeated at least 3 times.

Fig.15

Fig. 15. Electrophoretic mobility shift assay of PXR/RXR binding to human and rat

BARE-I probes.

A. Nucleotide sequences of the probes used in the EMSA assays. Arrows above

sequences indicate hormone response element half-site. DR, direct repeat, ER, everted-

repeat. Lower cases indicate mutations. B. PXR/RXRα binding to human BARE-I

probes. α-P32-labeled probes (5 x 104 cpm) and 5 μl of in vitro synthesized proteins (TNT lysate) were mixed and applied to each lane as indicated. 100-fold excess of unlabeled probes were used as cold competitors. Probe CYP3A4 contains a PXR/RXRα binding

site (ER6) of human CYP3A4 gene. Probe Mut contains a mutant human BARE-I as shown in A. C. Cold competition of PXR/RXRα binding to human BARE-I probe and

CYP3A4 probe. Increasing amount of excess unlabeled probes and 1 μl α-P32-labeled

probes (5 x 104 cpm) were incubated with 5 μl of in vitro synthesized proteins (TNT

lysate) for 20 min and applied to each lane as indicated. D. PXR/RXRα binding to rat

BARE-I probes.

(Fig. 15A). EMSA also revealed that PXR/RXR heterodimer bound to rat BARE-I

probes, which contained a DR4 motif also known to bind LXR/RXR heterodimer

(Fig.15D). PXR did not bind to human or rat BARE-II sequences in EMSA experiments

(data not shown).

Rifampicin inhibits HNF4α and PGC-1α stimulation of human CYP7A1 gene – I then

studied the effect of rifampicin on CYP7A1 reporter activity affected by HNF4α, PXR

and PGC-1α. As shown in Fig. 16A (open bars), HNF4α stimulated human CYP7A1

reporter activity by 5-fold (lane 2). PGC-1α alone stimulated human CYP7A1 reporter

activity by 3-fold (lane 3). Combination of HNF4α and PGC-1α stimulated CYP7A1

reporter activity by 7-fold (lane 5). Interestingly, PXR did not stimulate human CYP7A1

reporter activity in the absence of rifampicin (lane 4). It was remarkable that addition of

rifampicin (shaded bars) strongly inhibited reporter activity in HepG2 cells transfected

with PXR and RXR (lanes 4, 6, 7, and 8). These data revealed that PXR was a strong

repressor of CYP7A1 when activated by a ligand, whereas HNF4α and PGC-1α are

positive regulators of the human CYP7A1 gene. Similar experiment was also performed

using rat Cyp7a1 reporter construct (Fig.16B). Combination of HNF4α and PGC-1α

stimulated rat Cyp7a1 reporter by ~ 30-fold. Rifampicin strongly inhibited rat Cyp7a1

reporter activity when PXR and RXR were co-transfected. It was noted that in the

absence of rifampicin, PXR and RXR stimulated rat Cyp7a1 reporter activity by 4-fold

(Fig. 16B, lane 4). This was consistent with the report that Cyp7a1 expression was

decreased in Pxr null mice, which suggested that in the absence of a ligand, PXR may

bind to BARE-I and stimulate Cyp7a1 transcription. However, PXR and RXR did not

85 stimulate human CYP7A1 in the absence of rifampicin and may not be important for basal expression of human CYP7A1.

PXR interacts with HNF4α and blocks HNF4α recruitment of PGC-1α – It has been reported previously by our lab that HNF4α bound to the BARE-II and COUP-TFII bound to the BARE-I synergistically stimulated rat Cyp7a1 gene transcription. A model was proposed that factors bound to BARE-I and BARE-II interacted and regulated CYP7A1 gene transcription. I therefore wanted to test the hypothesis if PXR, which binds to

BARE-I, might also interact with factors bound to BARE-II to regulate the human

CYP7A1 gene. I performed mammalian two-hybrid assay in HepG2 cells to study PXR interaction with HNF4α and FTF, two factors that bind to human BARE-II. Gal4-PXR-

LBD (a.a. 107-434) containing PXR LBD fused to Gal4 DBD and VP16-HNF4α

(D+LBD, a.a. 125-364) containing HNF4α LBD fused to VP16-AD were used. PXR did not interact with HNF4α in the absence of rifampicin, however, 10 μM rifampicin induced a strong interaction between PXR and HNF4α (37 fold stimulation of the reporter over control). In contrast, PXR interacted very weakly with VP16-FTF

(Fig.17A). I then tested PXR/HNF4α interaction in mammalian two hybrid assay using

VP16-PXR-LBD and Gal4 fusion plasmids containing HNF4α full length, LBD or DBD.

Fig. 17B showed that HNF4α full-length and PXR interacted weakly without addition of a PXR ligand (4-fold of the background). Addition of rifampicin enhanced the interaction. The interactions between Gal4-HNF4α and VP16-PXR fusion proteins were much weaker than between Gal4-PXR/VP16-HNF4α fusions (Fig 17A). HNF4α N-

Fig.16

Fig. 16. Effects of HNF4α, PXR/RXRα and PGC-1α on human and rat CYP7A1 promoter activities.

HepG2 cells were cultured to 80% confluence. Human CYP7A1/luciferase reporter (ph-

298/Luc) (1 μg) (A) or rat CYP7A1/luciferase reporter(-334/Luc) (1 μg) (B) was

transiently transfected into HepG2 cells. 0.5μg of each expression plasmid was cotransfected as indicated. Cells were incubated in serum free media for 40 hours and harvested for luciferase activity assays as described under Experimental Procedures. The error bars represent the standard deviation from the mean of triplicate assays of an individual experiment; n=1, *, p<0.005, rifampicin vs. vehicle. Each individual experiment was repeated at least 3 times.

Fig.17

Fig. 17. Mammalian two hybrid assays of PXR interaction with HNF4α.

A. The fusion plasmids (0.5 μg) Gal4-PXR-LBD, VP16-empty vector, VP16-FTF-LBD

or VP16-HNF4α (0.5 μg) were cotransfected with 5XUAS-TK-LUC reporter plasmid (1

μg) into HepG2 cells. Cells were treated with vehicle (DMSO) or 10 μM rifampicin for

40 h and harvested for luciferase activity assays as described under Experimental

Procedures. The error bars represent the standard deviation from the mean of triplicate

assays of an individual experiment; n=1, *, p< 0.005, rifampicin vs vehicle. B. The Gal4

fusion plasmids (0. 5 μg) containing a full-length, N-terminal A/B+C (1 to 129 a.a.), and

C-terminal D+E+F (130-455 a.a.) of HNF4α, and VP16-PXR-LBD (0.5 μg) were cotransfected with 5XUAS-TK-LUC reporter plasmid (1 μg) into HepG2 cells as indicated. Empty vector VP16 was used as a negative control. Cells were treated with vehicle (DMSA) or 10 μM rifampicin for 40 h and harvested for luciferase activity assays as described in Experimental Procedures. The error bars represent the standard error of the mean of triplicate assays of an individual experiment; n=1, *, p< 0.005, rifampicin- treated vs. vehicle. Each individual experiment was repeated at least 3 times.

terminal region containing DBD (a.a. 1-129) did not interact with PXR, whereas the C-

terminal region containing LBD (a.a. 130-455) interacted with PXR. Taken together,

these data suggested that ligand-activated PXR strongly interacted with HNF4α, and the

interaction involved LBD regions of both PXR and HNF4α.

PGC-1α was originally identified as a co-activator of peroxisome proliferators-

activated receptor-γ (PPARγ). Since then, several other nuclear receptors including

HNF4α, LXRα, RXRα and ERα, and ER-related receptor (ERRα) have been found to

interact with PGC-1α[142, 143]. Recent reports found that HNF4α and PGC-1α are key

regulators of CYP7A1[56]. HNF4α binds to BARE-II and recruits co-activator PGC-1α

to stimulate CYP7A1 transcription. Bile acids inhibited CYP7A1 by reducing PGC-1α interaction with HNF4α. To test if ligand-activated PXR can block PGC-1α recruitment

by interacting with HNF4α, I performed mammalian two hybrid assays. In this assay,

Gal4-PGC-1α and VP-16-HNF4α will interact and stimulate the 5XUAS reporter. If PXR

can block this interaction, co-transfection of PXR expression plasmid will inhibit the

reporter activity (Fig.10B). Fig. 18 showed that Gal4-PGC-1α interacted strongly with

VP-16-HNF4α (500-fold stimulation of 5XUAS reporter). Rifampicin did not have any

effect on the interaction since it is not a ligand of HNF4α. When PXR expression

plasmid was co-transfected, addition of rifampicin strongly inhibited HNF4α and PGC-

1α interaction. Without rifampicin, overexpression of PXR did not affect HNF4α/PGC-

1α interaction. These results suggested that rifampicin promoted PXR interaction with

HNF4α and prevented PGC-1α interaction with HNF4α.

Fig.18

Fig. 18. Rifampicin-activated PXR disrupts HNF4α interaction with PGC-1α.

The fusion plasmids (0.5 μg) VP16-PXR-LBD, Gal4-empty vector or Gal4-PGC-1α and

expression plasimd pSG5-hPXR were cotransfected with 5XUAS-TK-Luc reporter

plasmid into HepG2 cells. The error bars represent the standard deviation from the mean

of triplicate assays of an individual experiment; n=1, *, p<0.005, rifampicin-treated vs. vehicle. Each individual experiment was repeated at least 3 times.

Rifampicin reduces PGC-1α recruitment to the native CYP7A1 chromatin - To further

test the binding and interaction of HNF4α, PXR and PGC-1α to the CYP7A1 gene in

vivo, I performed ChIP assays using human primary hepatocytes and HepG2 cells. PCR

primers were designed to amplify a 391 bp fragment (-432/-41) that contains both BARE-

I and BARE-II sequences. Fig. 19A showed that antibodies against PXR or HNF4α

precipitated the CYP7A1 chromatins in primary human hepatocytes. For ChIP assay

using HepG2 cells, PGC-1α, HNF4α and PXR/RXR were over-expressed by transfection to increase receptor expression levels. Fig. 19B showed that anti-PXR or anti-HNF4α

antibodies precipitated CYP7A1 chromatin. These results confirmed that PXR and

HNF4α bound to the native CYP7A1 chromatin. PGC-1α antibodies also precipitated

the CYP7A1 chromatin (Fig. 19B). Since PGC-1α is a co-activator that lacks a DNA

binding domain, PGC-1α must interact with other factors such as HNF4α in the CYP7A1

chromatin. As a positive control, I also did ChIP assay using antibodies against FTF and

confirmed that FTF bound to the BARE-II. I then studied the effect of rifampicin on the

binding of these transcription factors to the CYP7A1 chromatin. Rifampicin treatments did not affect the binding of HNF4α or PXR to CYP7A1 chromatin, but dramatically reduced PGC-1α association with CYP7A1 chromatin (Fig. 19B). ChIP assays also showed that rifampicin did not alter FTF binding to the chromatin. These data are consistent with our mammalian two-hybrid assay data, and suggested that rifampicin

induced interaction between PXR and HNF4α, which impaired PGC-1α recruitment.

Fig.19

Fig. 19. rifampicin impairs the recruitment of PGC-1α to CYP7A1 chromatin.

A. ChIP assay of PXR and HNF4α binding to human CYP7A1 gene in primary human

hepatocytes. Anti-PXR or anti-HNF4α antibody was used to precipitate the DNA-protein complex in primary human hepatocytes (HH1119). A 391 bp fragment containing BARE-

I & BARE-II was PCR amplified and analyzed on a 2% agarose gel. Protein G was used alone as non-immuno-precipitation control. 10 % of the total cell lysate was used as the

“input”. B. ChIP assay of HNF4α, PXR, FTF and PGC-1α binding to human CYP7A1

chromatin using HepG2 cells. HepG2 cells were transfected with 10 μg of PXR, RXRα,

HNF4α, HA-PGC-1α or FTF expression plasmids and treated with vehicle (DMSO) or

10 μM rifampicin for 40 h. ChIP assays were performed as described under Experimental

Procedures. Anti-PXR, anti-HNF4α, anti-HA or anti-CPF antibodies were used to precipitate the DNA-protein complexes. A 391 bp fragment containing BARE-I and

BARE-II was PCR amplified and analyzed on a 2% agarose gel. Each individual

experiment was repeated at least 3 times.

Rifampicin impairs PGC-1α interaction with HNF4α in hepatocytes – To further

confirm the physical interaction between HNF4α and PXR in intact cells, anti-HNF4α

antibodies were used to immunoprecipitate HNF4α from cell extracts isolated from

HepG2 and human primary hepatocytes. If PXR interacted with HNF4α in these cells,

PXR should be co-immunoprecipitated by anti-HNF4α antibodies in protein complex and

detected by western immunoblot using antibodies against PXR. PXR expression plasmids

were transfected into HepG2 cells to increase PXR protein level and immunoprecipitation

efficiency. Fig. 20A showed that western blotting analysis detected small amount of PXR

co-precipitated with HNF4α. Rifampicin treatment markedly increased PXR protein

amount in the precipitated complex. Anti-HNF4α antibody detected equal amounts of

HNF4α in vehicle and rifampicin-treated IP samples, which confirmed a similar IP

efficiency between each sample. I also did co-IP assay in human primary hepatocytes to

precipitate protein complex with anti-HNF4α antibodies (Fig.20B). Western blotting

analysis detected small amount of PXR protein while rifampicin markedly increased the

amount of PXR associated with HNF4α. Anti-PGC-1α antibody detected PGC-1α

protein in the immnoprecipitates, and rifampicin markedly reduced the amounts of PGC-

1α in the protein complex. These results are consistent with the findings from two-hybrid

assays (Fig. 18) and ChIP assays (Fig. 19B) that rifampicin promoted PXR interaction with HNF4α and excluded PGC-1α from interaction with HNF4α.

Fig.20

Fig. 20. Co-immunoprecipitation assay of HNF4α interaction with PXR and PGC-

1α.

A. Co-immunoprecipitation assay using HepG2 cell extracts. HepG2 cells were transfected with PXR expression plasmid and treated with either vehicle (DMSO) or 10

μM rifampicin for 40 h. Rabbit anti-HNF4α antibody was added to immunoprecipitate the cell lysates as described under Experimental Procedures. Western immunoblots were performed using goat anti-PXR or anti-HNF4α antibody to detect PXR and HNF4α in the precipitates. B. Co-immunoprecipitation assay using human primary hepatocytes.

Human primary hepatoctyes (HH1148) were treated with DMSO or 10 μM rifampicin for

40 h. Rabbit anti-HNF4α antibody was added to cells lysates as described under

Experimental Procedures. Western immunoblots were performed using goat anti-

HNF4α, PXR or PGC-1α antibody to detect these proteins in the immunoprecipitates.

Protein G was added as a negative control.

C. Summary

Here, I studied the mechanism of PXR regulation of human CYP7A1 gene transcription.

CYP7A1 mRNA expression is strongly suppressed by the specific PXR agonist rifampicin in primary human hepatocytes. Further studies show that HNF4α and PGC-1α are key regulators of human CYP7A1. HNF4α binds to BARE-II and recruits co-activator PGC-

1α and stimulates CYP7A1 gene transcription. PXR heterodimerizes with RXR and binds to BARE-I. Rifampicin induces PXR interaction with HNF4α, which blocks PGC-1α recruitment, and results in inhibition of CYP7A1. During cholestasis, PXR is activated by bile acids and other toxic lipid metabolites. On one hand, PXR activates CYP3A4 and transporters to detoxify and eliminate bile acid from the body; on the other hand, PXR inhibits CYP7A1 and reduces bile acid production in the liver.

CHAPTER IV

PXR REGULATION OF HUMAN CYP3A4

A. Significance and approach

Extensive studies have confirmed that PXR is the master regulator of CYP3A4 in

response to endotoxins and xenobiotics. Upon ligand activation, PXR recruits co-

activators to stimulate CYP3A4 gene transcription. However, several recent studies have

indicated that other transcriptional factors may also be important in the regulation of

CYP3A4. First, Foxo1 interacts with PXR as a co-activator and enhances CYP3A4

expression. Insulin leads to the inactivation of Foxo1 and thus results in the downregulation of CYP3A4 [144]. Second, SHP interacts with PXR in a ligand-dependent manner, and suppressed PXR mediates activation of CYP3A4 in vitro[145]. Third, overexpression of HNF4α enhances PXR mediated induction of CYP3A4 in reporter assays, and liver specific knockout of HNF4α impairs PCN induction of an exogenous CYP3A4 reporter gene in mice[146].

These interesting studies have led me to hypothesize that factors including HNF4α,

PGC-1α, SRC-1 and SHP may interact with PXR to coordinately regulate CYP3A4 gene

expression and induction. Differential expressions and regulations of these factors in

response to various physiological and environmental conditions may affect CYP3A4 gene

101 expression and thus, its drug metabolizing activity during certain disease states. The following experimental approaches were designed to test this hypothesis:

1. To determine the roles of HNF4α, PGC-1α, SRC-1 on PXR induction of

CYP3A4. I will first study rifampicin effects on the mRNA expression of

CYP3A4, CYP7A1, PEPCK and SHP in primary human hepatocytes by real-time

PCR. Transfection assays and mammalian hybrid assays will then be performed in

HepG2 cells to study the effects of HNF4α, PGC-1α, SRC-1 on PXR induction of

CYP3A4 reporter activity. I will also test rifampicin effect on PXR interaction

with HNF4α, PGC-1α, SRC-1 by in vitro GST pull down assays. Finally, the

dynamic recruitment of these factors to CYP3A4 chromatin will be studied by in

vivo ChIP assays in HepG2 cells.

2. To reveal the mechanism of SHP inhibition of PXR induction of CYP3A4.

First, a CYP3A4 reporter construct will be used in transfection assays to study the

effect of SHP on the rifampicin induction of CYP3A4. Mammalian hybrid assays

will then be performed to investigate if SHP interferes with PXR DNA binding

property or trans-activating activity. Next, I will use mammalian two hybrid

assays to test if SHP can block PXR interaction with its co-activators HNF4α,

PGC-1α, SRC-1. Finally, the competitions will be confirmed by in vitro GST

pull-down assays and in vivo ChIP assays in HepG2 cells.

3. To study the mechanism of rifampicin inhibition of SHP gene expression.

Real-time PCR has shown that SHP mRNA expression is suppressed by

rifampicin treatment in primary human hepatocytes. HNF4α is known to bind to

102

SHP promoter and stimulate its gene transcription. To test if HNF4α and PGC-1α

are key regulators of SHP, I will perform reporter assays in HepG2 cells to test

their stimulatory effect on a human SHP promoter/luciferase construct. To see if

PXR inhibits SHP and CYP7A1 by the same mechanism and if this is a general

suppression mechanism on HNF4α target genes, I will test the PXR inhibition

effect on SHP, CYP7A1, PEPCK and CYP3A4 by reporter assays.

B. Results

Rifampicin induced CYP3A4 but inhibited SHP mRNA expression in primary human

hepatocytes – I first assayed the effect of rifampicin on CYP3A4, SHP, CYP7A1 and

PEPCK mRNA expression in primary human hepatocytes (Table 2). Rifampicin dose-

dependently induced CYP3A4 mRNA expression by almost 150-fold at 10 μM. In

contrast, rifampicin strongly reduced SHP mRNA expression at very low concentration

(1 μM). These data suggested rifampicin activated PXR induced CYP3A4 but inhibited

SHP gene expression. Inhibition of SHP expression may contribute to the dramatic

induction of CYP3A4 by PXR. The previously observed SHP inhibition of PXR induction

of CYP3A4 in vitro may not be significant in vivo due to the suppressed SHP expression

by PXR in human hepatocytes. The suppression of PEPCK mRNA by rifampicin was

consistent with the model that PXR inhibited HNF4α regulated genes.

Induction of CYP3A4 by PXR required HNF4α co-activation – To further investigate

the role of HNF4α on the PXR transcriptional regulation of CYP3A4 in transient transfection assays. I used a CYP3A4 promoter/luciferase construct, which contained a distal XREM (dXREM) and a proximal ER6 (pER6), a typical CYP3A4 reporter

103

Table 2. Q-PCR analysis of mRNA expressions in human primary hepatocytes (HH1119).

Rifampicin 1μM 5μM 10μM CYP3A4 Relative 6.04(5.82-6.26) 86.44 (83.17-89.84) 147.58(104.08-209.25) Expression 15.27 ±0.052 11.43±0.056 10.65±0.5 ΔCt ± SDΔCt SHP Relative 0.207(0.203-0.212) 0.069(0.065-0.073) 0.13(0.12-0.14) Expression 4.57±0.028 6.16±0.083 5.24±0.116 ΔCt ± SDΔCt CYP7A1 Relative 0.38(0.36-0.4) 0.32(0.31-0.33) 0.093(0.069-0.128) Expression 11.18±0.08 11.42±0.057 13.19±0.45 ΔCt ± SDΔCt PEPCK Relative 0.83(0.81-0.85) 0.27(0.26-0.29) 0.27(0.25-0.3) Expression 4.56±0.03 6.18±0.08 6.18±0.14 ΔCt ± SDΔCt

Ct: the threshold cycle at which the amount of amplified target reaches a fixed threshold.

Relative expression: 2-∆∆Ct; high range: 2-(∆∆Ct-SDΔCt), low range: 2-(∆∆Ct+SDΔCt)

∆Ct = Ct of gene of interest - Ct of reference gene (UBC)

2 2 S.D. ∆Ct = Square Root [(S.D. of Ct of reference gene) + (S.D. of gene of interest) ]

104

construct used in many studies[147]. The distal XREM contained one HNF4α (DR1) and two PXR (DR3, ER6) binding sites, and the proximal ER6 contained one PXR binding site (ER6). In HepG2 cells, without co-transfection of PXR and RXR, rifampicin did not stimulate CYP3A4 reporter activity, which was consistent with several reports that endogenous PXR was not enough to mediate its ligand effect in transient transfection assays[144, 148, 149]. When PXR/RXR were co-transfected, rifampicin stimulated the

reporter activity by ~3.8 fold. Interestingly, when HNF4α was co-transfected, rifampicin

further induced the reporter by up to ~7.2 fold (Fig.21A). Similar results were also seen

when the same experiment was performed in intestinal Caco2 cells (Fig.21B).

Interestingly, in both HepG2 and Caco2 cells, co-transfection of HNF4α alone failed to

stimulate the reporter activity, despite the presence of an HNF4α binding site in the distal

XREM. To test if HNF4α co-activation of CYP3A4 required HNF4α binding to the DR1 site, we used a reporter construct 5XUAS, which contained 5 copies of the Gal4-DBD

binding sites. When Gal4-PXR-LBD fusion plasmid was co-transfected, rifampicin stimulated the reporter by ~20 fold. When HNF4α was overexpressed, rifampicin further

induced the reporter activity by ~35 fold (Fig.21C). Since there was no HNF4α binding site in this reporter construct, it is most likely that HNF4α acted as a co-activator and enhanced PXR induction of CYP3A4 via its direct interaction with PXR.

Since PXR induction of CYP3A4 has been thought to be the result of ligand-dependent

recruitment of co-activators[108], I therefore tested the effect of SRC-1, PGC-1α and

HNF4α on the PXR induction of CYP3A4. Fig.21D showed that when SRC-1 or PGC-1α

was co-transfected alone or in combination, both the basal and rifampicin-induced

105

Fig.21

106

107

Fig. 21. Effect of HNF4α on PXR regulation of CYP3A4 promoter activities.

HepG2 cells (A) or Caco2 cells (B) were cultured to about 80% confluence. CYP3A4

luciferase reporter p(3A4-5’dDR3/dER6/pER6) (0.2 μg) was co-transfected with

PXR/RXR or HNF4α (0.1 μg) as indicated. C. 5XUAS reporter (0.2 μg) was co- transfected into HepG2 cells with Gal4-hPXR-LBD or HNF4α (0.1 μg) as indicated. D.

CYP3A4 luciferase reporter p(3A4-5’dDR3/dER6/pER6) (0.2 μg) was co-transfected with PXR, RXR,SRC-1,PGC-1α and HNF4α (0.1 μg) as indicated. Cells were treated with vehicle (DMSO), or 10 μM rifampicin for 40 h and harvested for luciferase activity assays as described under Experimental Procedures. The error bars represent the standard deviation from the mean of triplicate assays of an individual experiment, n=1, *, p<

0.005, rifampicin-treated vs. vehicle. Each individual experiment was repeated at least 3 times.

108 reporter activities were increased, however, the relative fold induction was not altered significantly. The relative fold induction was only enhanced when HNF4α was also co- transfected (lane 5, 7, 8). A total 134 fold stimulation by rifampicin over the basal reporter activity was observed when all three factors were co-transfected. These data suggested that all three factors were required for the maximum expression of CYP3A4, however, SRC-1 and PGC-1α were more important in the constitutive expression of

CYP3A4 while HNF4α was more important in the ligand-dependent induction.

Next, I studied PXR interactions with HNF4α, PGC-1α and SRC-1 by in vitro GST pull-down assays. GST-HNF4α fusion protein containing full length HNF4α or GST- hPXR fusion protein containing human PXR LBD was used in the GST pull-down experiments. Results showed that a strong interaction between PXR and HNF4α required the ligand activation of PXR, without rifampicin PXR barely interacted with

HNF4α (Fig.22A&B). The PXR/SRC-1 interaction was partially ligand-dependent. SRC-

1 was able to interact with unliganded PXR while rifampicin further increased their interaction (Fig.22C). In contrast, PXR interacted with PGC-1α strongly in the absence of rifampicin, and addition of rifampicin did not further enhance their interaction

(Fig.22D). The ligand dependency of these co-factors to interact with PXR may provide a possible explanation for their distinct activation properties on CYP3A4 promoter activity shown in Fig.21D.

GST pull-down assays are considered as in vitro experiments and are usually independent of the interference from other factors. However, PXR can interact with

HNF4α, PGC-1α and SRC-1, furthermore, HNF4α can also interact with SRC-1 and

109

Fig.22

110

Fig. 22. GST pull-down assays of PXR interactions with HNF4α,SRC-1 and PGC-

1α.

Bacterial expressed GST, GST-HNF4α(full length) fusion or GST-hPXR (LBD) fusion was incubated with 10μl of 35S labeled in vitro translated PXR, HNF4α, SRC-1 or PGC-

1α as indicated. DMSO or 10μM rifampicin was added to the incubation mixture. 1μl of the in vitro translated protein was used as “input”. GST pull-down assays were performed as described under Experimental Procedures.

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PGC-1α[56]. If these factors can form a protein complex, then in vitro GST pull-down

assays that dissect such protein complex and only study the interaction between the two

proteins of interest may not reflect the in vivo condition. Therefore, I performed in vivo

ChIP assays in HepG2 cells to study the effect of rifampicin on the recruitment of these

factors to the CYP3A4 chromatin. Since our real-time PCR revealed that PGC-1α

expression was usually low in HepG2 cells under normal culture conditions, I over-

expressed PGC-1α in HepG2 cells by transfection. PCR primers were designed to

amplify the DNA fragments containing either the distal XREM or the proximal ER6. As

shown in Fig.23A&B, PXR bindings were not altered by rifampicin treatment, while

HNF4α and SRC-1 recruitment were strongly increased by rifampicin. Interestingly, the association of PGC-1α with CYP3A4 chromatin was reduced upon rifampicin treatment, likely due to the competition by other factors that were recruited to PXR upon rifampicin treatment. Our mammalian two hybrid assays showed no competition between SRC-1 and HNF4α for the binding of PXR, suggesting SRC-1 and HNF4α can bind to PXR simultaneously without interfering with each other, while HNF4α did compete out PGC-

1α from PXR (Fig.24A&B).

SHP interacted with PXR and impaired its induction of CYP3A4 – The small

heterodimer partner SHP has been reported to interact with PXR ligand-dependently and

interfered with its stimulation of CYP3A4[145]. To study the mechanism of SHP

inhibition of CYP3A4, I first performed transient transfection assays in HepG2 cells.

When PXR/RXR were co-transfected, CYP3A4 reporter activity was strongly stimulated

by rifampicin. Addition of SHP suppressed this induction to the basal level. Without

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Fig.23

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Fig. 23. Effect of rifampicin on the bindings of PXR, HNF4α, SRC-1 and PGC-1α to

CYP3A4 chromatin.

ChIP assay of PXR, HNF4α, SRC-1 and PGC-1α binding to CYP3A4 chromatin using

HepG2 cells. HepG2 cells were transfected with 10 μg HA-PGC-1α expression plasmids

and treated with vehicle (DMSO) or 10 μM rifampicin for 40 h. ChIP assays were performed as described under Experimental Procedures. Anti-PXR, anti-HNF4α, anti-

PGC-1α or anti-SRC-1 antibodies were used to precipitate the DNA-protein complexes.

DNA fragments containing dXREM (A) or pER6 (B) was PCR amplified and analyzed on a 2% agarose gel. Normal IgG was used alone as non-immuno control. 10% of the total cell lysate was used as “input”.

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Fig.24

115

Fig. 24. Competitions between HNF4α, SRC-1 and PGC-1α binding to PXR.

HepG2 cells were transfected with 5XUAS reporter (0.2 μg) and 0.1 μg expression plasmids: Gal4-PXR(LBD), VP16, VP16-SRC-1, VP16-PXR(LBD), Gal4, Gal4-PGC-

1α(full length) and HNF4α as indicated. Cells were treated with vehicle (DMSO), or 10

μM rifampicin for 40 h and harvested for luciferase activity assays as described under

Experimental Procedures. The error bars represent the standard deviation from the mean of triplicate assays of an individual experiment. n=1, *, p< 0.005. Each individual experiment was repeated at least 3 times.

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rifampicin, SHP did not inhibit the reporter activity, apparently due to the lack of

interaction between SHP and PXR (Fig.25A). Overexpression of HNF4α did not reverse

SHP inhibitory effect on CYP3A4(Fig.25B). To test if SHP impaired PXR activity by

preventing its DNA binding, I used the 5XUAS reporter construct and the plasmid

expressing Gal4 DBD and PXR LBD fusion protein. As expected, the reporter activity

was dramatically increased by rifampicin when Gal4-PXR-LBD and HNF4α were co-

transfected (Fig.25C). Addition of SHP completely blocked rifampicin induction of the

reporter activity. Since the Gal4 DNA binding was not likely to be affected by SHP in

this experiment, this data indicated that SHP might suppress PXR trans-activating activity

by interfering with PXR LBD but not the DNA binding property.

Since HNF4α, SRC-1, PGC-1α and SHP all interacted with PXR via its LBD, SHP might block the recruitment of these co-factors and thus abolish the PXR induction of

CYP3A4. To test this hypothesis, I performed mammalian two-hybrid assays in HepG2 cells to study the possible competitions between SHP and other co-factors for the binding to PXR. When I co-transfected Gal4-PXR-LBD and VP16–HNF4α expression plasmids into HepG2 cells, the 5 X UAS reporter activity was strongly increased upon rifampicin treatment, which indicated a ligand-dependent interaction between PXR and HNF4α.

Addition of SHP reduced such stimulation by ~80%, suggesting SHP can interrupt the

interaction between PXR and HNF4α (Fig.26A). This result was further confirmed by

transfecting Gal4-SHP/VP16-PXR-LBD expression plasmids and using HNF4α as a competitor (Fig.26B). Although SHP can also interact with HNF4α, but such interaction does not require a ligand, the interruption we observed here largely depended on the

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Fig.25

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Fig. 25. Effect of SHP on PXR induction of CYP3A4 promoter activities.

A&B, HepG2 cells were transfected with CYP3A4 luciferase reporter p(3A4-

5’dDR3/dER6/pER6) (0.2 μg) and PXR, RXR, HNF4α or SHP(0.1 μg) as indicated. C,

HepG2 cells were transfected with 5XUAS reporter (0.2 μg) and Gal4, Gal4-PXR-LBD,

HNF4α or SHP (0.1 μg) as indicated. Cells were treated with vehicle (DMSO), or 10 μM

rifampicin for 40 h and harvested for luciferase activity assays as described under

Experimental Procedures. The error bars represent the standard deviation from the mean of triplicate assays of an individual experiment. n=1, *, p< 0.005, rifampicin-treated vs. vehicle. Each individual experiment was repeated at least 3 times.

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Fig.26

120

Fig. 26. Effect of SHP on the PXR interaction with HNF4α, SRC-1 and PGC-1α .

HepG2 cells were transfected with 5XUAS reporter (0.2 μg) and 0.1 μg expression

plasmids: Gal4, Gal4-PXR(LBD), GAL4-SHP (full length), Gal4-PGC-1α(full length),VP16, VP16-HNF4α(D+E),VP16-PXR(LBD), VP16-SRC1(RID), SHP, HNF4α,

SRC-1 and PGC-1α, as indicated. Cells were treated with vehicle (DMSO), or 10 μM rifampicin for 40 h and harvested for luciferase activity assays as described under

Experimental Procedures. The error bars represent the standard deviation from the mean of triplicate assays of an individual experiment. n= 1, *, p< 0.005, rifampicin-treated vs. vehicle. Each individual experiment was repeated at least 3 times.

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addition of PXR ligand rifampicin and was most likely the result of SHP binding to PXR.

To test if such competitions also exist between SHP and SRC-1, in one set of

experiments, I transfected Gal4-SHP/VP16-PXR-LBD fusion plasmids into HepG2 cells

and used SRC-1 expression plasmid as a competitor, in the other set of experiments, I

transfected Gal4-PXR-LBD/VP16-SRC-1 fusion plasmids and used SHP expression

plasmid as a competitor. Results showed there were also competitions between SRC-1

and SHP for the binding of PXR. However, such competitions were much weaker

compared to the competition between SHP and HNF4α (Fig.26C&D). Interestingly,

when the same competition experiments were performed to test SHP and PGC-1α, no

competitions between these two factors were observed, suggesting SHP and PGC-1α

may interact with PXR at different regions (Fig.26E&F). It should be noted that the

competitions between SHP and HNF4α or SRC-1 were reversible and might also depend

on the intracellular ratio of the two factors.

So far, my data has indicated that HNF4α was important in PXR induction of CYP3A4,

and SHP strongly suppressed PXR trans-activating activity mainly by blocking HNF4α binding to PXR. To further test this hypothesis, I next performed GST pull-down assays.

As expected, SHP showed a ligand-dependent interaction with PXR (Fig.27A). The interaction between SHP and HNF4α was not affected by rifampicin (Fig.27B). When we added excess amount of in vitro translated SHP into the pull-down reaction, the amount of PXR that bound to GST-HNF4α was decreased (Fig.27C). Similar result was also obtained when excess PXR was used to compete out SHP from binding to GST-HNF4α

(Fig.27D). Since the amount of in vitro translated protein we used as competitors was

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Fig.27

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Fig. 27. GST pull-down assays of SHP competition with HNF4α for PXR binding.

A&B, Bacterial expressed GST, GST-SHP (full length) fusion or GST-HNF4α (full

length) fusion was incubated with 5μl of 35S labeled in vitro translated PXR or SHP as

indicated. DMSO or 10μM rifampicin was added to the incubation mixture as indicated.

C, GST or GST-HNF4α (full length) fusion was incubated with 5μl of 35S labeled in vitro translated PXR. 50μl of none radio labeled in vitro translated SHP was added as competitor. DMSO or 10μM rifampicin was added to the incubation mixture as indicated.

D. GST or GST-HNF4α (full length) fusion was incubated with 5μl of 35S labeled in vitro translated SHP. 50μl of none radio labeled in vitro translated PXR was added as competitor. DMSO or 10μM rifampicin was added to the incubation mixture as indicated.

1μl of the in vitro translated protein was used as “input”. GST pull-down assays were performed as described under Experimental Procedures.

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limited in the TNT lysates, the competitions we observed here were much weaker when

compared to the mammalian two-hybrid assays results. To further test our hypothesis in

vivo, I then performed ChIP assays in HepG2 cells. I transfected PGC-1α expression plasmid into HepG2 cells to increase its expression levels. I also over-expressed SHP to test its effect on the bindings of PXR, HNF4α, SRC-1 and PGC-1α to CYP3A4 chromatin. Western blotting analysis showed that transfection of 10μg pcDNA3-HA-

SHP expression plasmids dramatically increased intracellular SHP levels(Fig.28A). In

ChIP assays, when 10μg SHP was transfected into HepG2 cells, PXR binding to neither

distal XREM nor pER6 was affected. However, HNF4α and SRC-1 recruitments to these

chromatins were largely reduced, while PGC-1α recruitment was not altered, which

were consistent with our mammalian two hybrid assay and GST pull-down assay results

(Fig.27B&C).

PXR suppresses SHP gene transcription – I next investigated the mechanism by which

ligand-activated PXR suppressed SHP. It has been reported that HNF4α bound to human

SHP promoter and stimulated SHP gene expression[97, 150]. To test if PXR can interact

with HNF4α and suppress SHP gene transcription, I transfected human SHP

promoter/luciferase reporter into 293 cells, a cell line lacking endogenous HNF4α

expression. Without the interference by endogenous HNF4α, SHP reporter activity was

strongly stimulated by 44 fold upon HNF4α and PGC-1α overexpression (Fig.29A). As

expected, human CYP7A1 and human PEPCK reporter activities were also stimulated by

17 fold and 15 fold, respectively, by HNF4α and PGC-1α (Fig.29B&C). However,

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Fig.28

126

Fig. 28. Effect of SHP on the bindings of PXR, HNF4α, SRC-1 and PGC-1α to

CYP3A4 chromatin.

A. 10μg pcDNA3 and 5 or 10 μg pcDNA3-HA-SHP expression plasmid was transfected

into HepG2 cells. 40 h later, cells were collected and protein samples were prepared as described in Experimental procedure. HA specific antibodies were used to detect the exogenous SHP expressions. B&C. ChIP assay of PXR, HNF4α, SRC-1 and PGC-1α binding to CYP3A4 chromatin in HepG2 cells. HepG2 cells were transfected with 10 μg

HA-PGC-1α expression plasmids. 10μg pcDNA3 or pcDNA3-HA-SHP expression plasmid was also transfected as indicated. Cells were treated with 10 μM rifampicin for

40 h. ChIP assays were performed as described under Experimental Procedures. Anti-

PXR, anti-HNF4α, anti-PGC-1α or anti-SRC-1 antibodies were used to precipitate the

DNA-protein complexes. DNA fragments containing dXREM (B) or pER6 (C) was PCR amplified and analyzed on a 2% agarose gel. Normal IgG was used alone as non-immuno control. 10% of the total cell lysate was used as “input”.

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Fig.29

128

Fig. 29. Effect of HNF4α and PGC-1α on the promoter activities of SHP, CYP7A1,

PEPCK and CYP3A4.

HEK293 cells were cultured to about 80% confluence. Human SHP(A), CYP7A1(B),

PEPCK(C) or CYP3A4(D) luciferase reporter (0.2 μg) was co-transfected with HNF4α or

PGC-1α (0.1 μg) as indicated. After 40 h cells were harvested for luciferase activity

assays as described under Experimental Procedures. The error bars represent the standard

deviation from the mean of triplicate assays of an individual experiment. n=1, *, p<

0.005, HNF4α and/or PGC-1α vs. control. Each individual experiment was repeated at least 3 times.

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CYP3A4 reporter was only stimulated by ~ 2 fold by HNF4α and PGC-1α, which again

suggested that the HNF4α binding site may be a weak HNF4α response element in terms

of CYP3A4 activation (Fig.29D). Having confirmed that HNF4α and PGC-1α were strong regulators of human SHP, I performed transient transfection assays using the same

human SHP promoter in HepG2 cells. The reporter activity was stimulated by HNF4α

and PGC-1α to a less extend due to high endogenous HNF4α expressions in this cell line.

When PXR was co-transfected, without addition of rifampicin, the HNF4α/PGC-1α

activation was not affected, however, when 10 μM rifampicin was added, the reporter

activity was strongly suppressed by ~70% (Fig.30A). As controls, human CYP7A1 and

PEPCK were also stimulated by HNF4α/PGC-1α and suppressed by ligand-activated

PXR (Fig.30B&C)[56, 149]. When CYP3A4 reporter was used in such transfection

assays, HNF4α and PGC-1α failed to stimulate the reporter activity, and furthermore,

instead of decreasing the reporter activity, addition of PXR increased basal reporter

activity by about 6 fold, and rifampicin further induced the reporter activity by an

additional 5 fold (Fig.30D). These data indicated that HNF4α bound to SHP, CYP7A1

and PEPCK as a central regulator and recruited PGC-1α to stimulate gene expressions,

PXR acted as a co-repressor of HNF4α and blocked PGC-1α recruitment, thus resulted in

gene suppression; however, on CYP3A4 promoter, PXR was the key regulator and

HNF4α, along with SRC-1 and PGC-1α, acted as co-activators of PXR and stimulated

CYP3A4 gene transcription.

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Fig.30

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Fig. 30. Liganded PXR suppressed HNF4α/PGC-1α activation of SHP promoter activity.

HepG2 cells were cultured to about 80% confluence. Human SHP (A), CYP7A1(B),

PEPCK(C) or CYP3A4(D) luciferase reporter(0.2 μg) was co-transfected with HNF4α,

PGC-1α or PXR(0.1 μg) as indicated. Cells were treated with vehicle (DMSO), or 10 μM

rifampicin for 40 h and harvested for luciferase activity assays as described under

Experimental Procedures. The error bars represent the standard deviation from the mean of triplicate assays of an individual experiment. n=1, *, p< 0.005, rifampicin vs. vehicle.

Each individual experiment was repeated at least 3 times.

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C. Summary

In this study, I investigated the PXR regulation of CYP3A4. Results have revealed that

HNF4α is an important co-activator of PXR. Ligand dependent recruitment of HNF4α by

PXR is a crucial step for CYP3A4 gene activation. Maximal activation of CYP3A4 also requires co-activator PGC-1α and SRC-1. In addition, rifampicin and PXR suppress

HNF4α target genes such as SHP, CYP7A1 and PEPCK by blocking PGC-1α recruitment to their promoters. The concomitant inhibition of SHP by PXR minimizes

SHP inhibition effect of PXR induction on CYP3A4.

PXR REGULATION OF HUMAN CYP27A1

A. Significance and approaches

CYP27A1 has been shown to play a crucial role in regulation of cholesterol

homeostasis in macrophages by converting cholesterol into 27HOC. PPARγ agonists

stimulate CYP27A1 and production of 27HOC, which activates LXR target genes ABCA1

and ABCG1 to efflux cholesterol from macrophages[140]. PXR has recently been

indicated in the regulation of cholesterol homeostasis. Several studies show that

oxysterols and cholesterol metabolites are able to activate PXR and induce Cyp3a in rat and mouse hepatocytes[151, 152]. A recent study reports that PXR null mice fed high cholesterol and cholic acid diet develop acute hepatorenal failure, and suggests that PXR may play a role in detoxification of cholesterol metabolites[153]. PXR is mainly expressed in liver and intestine, two organs that are usually exposed to high levels of cholesterol and are also active in cholesterol metabolism. Thus, I hypothesized that PXR might also regulate CYP27A1 in the liver or intestine and play a role in regulation of cholesterol homeostasis. To test this hypothesis, experiments were designed accordingly to provide answers for the following questions:

1. Is CYP27A1 regulated by PXR? I will use human primary hepatocytes, liver

derived HepG2 cells and intestine derived Caco2 cells as models to study the

PXR regulation of CYP27A1. Quantitative real-time PCR will be performed to

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test the effect of PXR agonist rifampicin on CYP27A1 mRNA expression. I will

then use CYP27A1 promoter/reporter in transfection assays to study PXR

regulation of CYP27A1 in HepG2 cells and Caco2 cells. I will also use HPLC

method to test if changes in CYP27A1 gene expression will affect 27HOC

production.

2. What is the effect of 27-HOC on LXR signaling and cholesterol homeostasis?

Here, I will focus on 27HOC effects on two important intestinal cholesterol efflux

genes, ABCA1 and ABCG1. I will perform quantitative real-time PCR to test how

ABCA1 and ABCG1 mRNA expressions are regulated by cholesterol, 27HOC

and rifampicin.

3. Where is the PXR responsive element on CYP27A1 promoter? I will use

CYP27A1 promoter deletion constructs to locate the promoter region that

mediates PXR effect. After identification of putative PXR binding sites, I will

perform EMSA and ChIP assays to confirm PXR bindings to these sites. Finally,

mutagenesis analysis will be used to confirm functional PXR responsive element

on CYP27A1 promoter.

B. Results

Rifampicin stimulated human CYP27A1 mRNA expression in intestine cells but not human liver cells –To test if CYP27A1 is regulated by PXR in liver or intestine, quantitative real-time PCR was performed to study the effect of rifampicin, a potent human PXR ligand, on CYP27A1 mRNA expression in human primary hepatocytes,

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human hepatocellular carcinoma HepG2 cells, and human colon cancer epithelial Caco2 cells. Cells were treated with rifampicin (20 μM) for 24 h and mRNA expressions of

CYP27A1 and CYP3A4 were measured. Rifampicin induced CYP27A1 mRNA expression by 3.3-fold in Caco2 cells (Fig 31A), but did not affect CYP27A1 mRNA

levels in primary human hepatocytes or HepG2 cells (Fig.31 B&C). As a positive

control, rifampicin strongly induced CYP3A4 mRNA expression by 7-fold in Caco2

cells, 20-fold in HepG2 cells, and 90-fold in human primary hepatocytes. These results

indicated that rifampicin induction of CYP27A1 mRNA expression is intestine-specific.

Cholesterol and rifampicin increased intracellular 27-hydroxycholesterol levels in

Caco2 cells – Since rifampicin induced CYP27A1 mRNA expression in Caco2 cells, I next measured 27HOC by HPLC in Caco2 cells loaded with cholesterol and treated with rifampicin to see if induction of CYP27A1 will lead to increased 27HOC production. Fig

32 shows that addition of soluble cholesterol (20 μg/ml, dissolved in β-cyclodextran) to

culture media strongly increased 27HOC levels by 18-fold in Caco2 cells. The

intracellular 27HOC concentrations were about 1 μM in Caco2 cells cultured in media

without cholesterol and about 18 μM in cholesterol loaded Caco2 cells. Addition of rifampicin (20 μM) to cholesterol-loaded Caco2 cells further increased 27HOC

concentration by 2-fold (Fig 32). These results suggest that cholesterol loading activates

CYP27A1 activity and dramatically increases 27HOC production in Caco2 cells, and that

rifampicin further increases 27HOC production by inducing CYP27A1 gene expression.

27-hydroxycholesterol induced LXR target genes ABCA1 and ABCG1 in Caco2 cells—

To test if rifampicin or cholesterol induced 27HOC will activate LXR and its target

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Fig.31

137

Fig. 31. Effect of rifampicin on CYP27A1 mRNA expression.

Caco2 cells (A), HepG2 cells (B), or primary human hepatocytes (HH1119) (C) were

cultured in 6 well plates. Cells were treated with either vehicle (DMSO) or 20 μM

rifampicin and used to isolate mRNA for Q-PCR analysis of CYP27A1 and CYp3A4

mRNA expressions. Error bars represent the standard deviation from the mean of triplicate assays of an individual experiment. n=1, *, p<0.005, rifampicin vs. vehicle.

Each individual experiment was repeated at least 3 times.

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Fig. 32

139

Fig. 32. Assay of 27-hydroxycholesterol in Caco2 cells.

Caco2 cells were cultured in T175 flasks untill 80% confluence. Cells were treated with vehicle (molecusol), 20 μg/ml cholesterol or 20 µM rifampicin as indicated for 72 hours.

Cells were lysed and 27-hydroxycholesterol concentrations were determined as described under Exiperimental Procedure. The error bars represent the standard deviation from the mean of three individual assays . n=3,*, p<0.005, treated vs. control. The experiment was repeated 3 times.

140 genes, I first studied the effect of cholesterol and 27HOC on mRNA expressions of

ABCA1 and ABCG1 in Caco2 cells. Quantitative real-time PCR analyses in Fig. 33 A &

33B showed that cholesterol induced ABCA1 mRNA expression by 2 fold at 10 μg/ml and 3 fold at 20 μg/ml. Cholesterol induced ABCG1 mRNA by 3 fold at 10 μg/ml and 6 fold at 20 μg/ml. Fig. 33C & 33D show that ABCA1 and ABCG1 mRNA expression levels were induced by 27HOC in a dose-dependent manner in Caco2 cells. 27HOC significantly induced mRNA expression of these two cholesterol efflux transporters at a concentration as low as 0.1 μM. The maximum induction of ABCG1 by 27HOC was achieved at 10 μM, whereas ABCA1 at 50 μM.

Next, I tested if rifampicin treatment could further increase the mRNA expression of

ABCA1 and ABCG1 in cholesterol-loaded Caco2 cells. As shown in Fig 34A, rifampicin induced CYP27A1 mRNA expression by about 2.5 fold in Caco2 cells loaded with 20

μM cholesterol. Rifampicin also significantly increased ABCA1 and ABCG1 mRNA expression in cholesterol-load Caco2 cells (Figs 34B and C). It should be noted that rifampicin did not induce ABCA1 and ABCG1 mRNA expressions in Caco2 cells without loading of cholesterol (data not shown). This suggests that PXR does not directly induce ABCA1 and ABCG1 gene expression and its effects on ABCA1 and ABCG1 are mediated through activation of CYP27A1. I also tested the effect of 9-cis retinoic acid, a

RAR and RXR ligand, on CYP27A1, ABCA1 and ABCG1 mRNA expression in cholesterol-loaded Caco2 cells. Figs 34A, 4B and 4C showed that 9-cis retinoic acid induced CYP27A1, ABCA1 and ABCG1 mRNA levels rather weakly. This was

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Fig.33

142

Fig. 33. Effect of 27-hydroxycholesterol and cholesterol loading on ABCA1 and

ABCG1 mRNA expressions.

Caco2 cells were cultured to about 80% confluence in 6 well plates. Cells were treated

with either increasing amount of cholesterol (A&B) or 27-hydroxycholesterol (C&D), and the relative ABCA1 and ABCG1 mRNA expression (to untreated control as 1) were determined by quantitative real-time PCR. The error bars represent the standard deviation

from the mean of triplicate assays of an individual experiment. n=1, *, p<0.005, treated

vs. control. Each individual experiment was repeated at least 3 times.

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Fig.34

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Fig. 34. Effect of rifampicin on ABCA1 and ABCG1 mRNA expression.

Caco2 cells were cultured to about 80% confluence in 6 well plates. Cells were treated with vehicle (ethanol), 20 μM rifampicin (RIF), 1 μM 9-cis-retinoic acid (9-cis RA), 10

μM 27-hydroxycholesterol (27HOC) or 1 μM TO901317 for 40 h in media containing 20

μg/ml cholesterol. Relative mRNA expression levels were determined by quantitative real-time PCR. The error bars represent the standard deviation from the mean of triplicate assays of an individual experiment. n=1, *, p<0.005, treated vs. control. Each individual experiment was repeated at least 3 times.

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consistent with previous report that retinoid induced CYP27A1 expression in

macrophages.

A potent LXR agonist, TO901317 (1 μM) did not induce CYP27A1 mRNA expression, but strongly induced both ABCA1 and ABCG1 mRNA levels by 3 and 80-fold, respectively. These data suggests that CYP27A1 is a PXR target gene but not a LXR target gene, whereas ABCA1 and ABCG1 are LXR target genes but not a direct PXR

target genes. The potent LXR agonist TO901317 induced ABCG1 much stronger than

ABCA1 in Caco2 cells.

PXR specifically stimulated human CYP27A1 reporter activity in Caco2 cells - I then

performed reporter assays to study PXR regulation of human CYP27A1 gene. The human

CYP27A1 promoter/luciferase reporter (ph-1774CYP27/luc) activity was not stimulated by rifampicin without overexpressing PXR in Caco2 cells (Fig 35A, open bars). In Caco2 cells overexpressing PXR and RXR, rifampicin dose-dependently stimulated CYP27A1

reporter activity (Fig 35A, solid bars). When reporter assay was performed in HepG2

cells, rifampicin did not stimulate CYP27A1 reporter activity even when PXR and RXR

were overexpressed (Fig.35B). As a positive control, CYP3A4 reporter activity was

strongly induced by rifampicin in both Caco2 and HepG2 cells over expressing PXR and

RXR (Fig. 35C&D). These results were consistent with the quantitative real-time PCR

analysis that PXR induced CYP27A1 gene transcription in intestine cells but not liver

cells.

Identification of a functional PXR binding site in the human CYP27A1 promoter –To

identify functional PXR response elements in human CYP27A1 promoter, I performed

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Fig.35

147

Fig. 35. Effect of rifampicin on human CYP27A1 promoter activities.

Caco2 (A & C) or HepG2 cells (B & D) were cultured to about 80% confluence. A

human CYP27A1/luciferase reporter, ph-1774CYP27/Luc (A & B), or human

CYP3A4/luciferase reporter, p (3A4-5’dDR3/dER6/pER6) (C & D) was transiently transfected with or without PXR and RXRα expression plasmids. Cells were treated with vehicle (DMSO), or rifampicin at concentrations indicated and harvested for luciferase activity assays as described under Experimental Procedures. The error bars represent the standard deviation from the mean of triplicate assays of an individual experiment. n=1, *, p<0.005, treated vs. control. Each individual experiment was repeated at least 3 times.

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EMSA, ChIP, and mutagenesis analyses of putative PXR binding sites. PXR/RXR

heterodimer has binding activity to various direct repeats (DR3, DR4, and DR5) and

everted repeats (ER6 and ER8) sequences. Three potential PXR binding sites containing either DR4 or DR5 motif were identified and designated PXRE-A, PXRE-B and PXRE-C

(Fig.36A). A canonical PXR/RXR binding site (ER6) from the human CYP3A4 gene promoter was used as a positive control. Mutant PXR binding sites (MutA, MutB

&MutC) were designed to test the binding specificity (Fig.36A). As shown in Fig. 36B, C

& D, PXR/RXR heterodimer was able to bind to all three probes. Neither PXR nor RXR

alone was able to bind to these probes. Excess un-labeled 3A4 probes competed out

PXR/RXR heterodimer binding to labeled probes. Mutations introduced into these probes

abolished PXR/RXR bindings.

To further confirm that PXR binds to these putative PXR binding sites in CYP27A1

chromatin in vivo, ChIP assays were performed. Caco2 cell lysates were incubated with

antibodies against PXR to immunoprecipitate CYP27A1 chromatin. The fragments

containing PXR binding sties PXRE-A, PXRE-B, and PXRE-C were PCR-amplified and

analyzed on agarose gels. Fig 36E shows that PXR bound to all three putative PXREs in

CYP27A1 chromatin, and rifampicin treatment did not significantly increase these

bindings.

To test if these PXR/RXR binding sites are functional in transactivating CYP27A1

reporter activity, I first used several CYP27A1 promoter/luciferase deletion constructs

containing one, two or three PXRE in transient transfection assays in Caco2 cells over-

expressing PXR and RXR. As shown in Fig.37A, rifampicin stimulated reporter activities

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Fig.36

150

151

152

Fig. 36. EMSA and ChIP assays of PXR binding to human CYP27A1 gene.

A. Nucleotide sequences of the CYP27A1 probes (CYP27-A, CYP27-B, and CYP27-C)

used for the EMSA assays are illustrated. Arrows indicate hormone response element

half-site. DR, direct repeat, ER, everted-repeat. Mutant probes with nucleotide mutations

(lower cases) of the PXR binding sites (MutA, MutB and MutC) are indicated under each

probe. B. EMSA of CYP27-A probe. C. EMSA of CYP27-B probe. D. EMSA of CYP27-

C probe. α-P32-labeled probes (5 x 104 cpm) and 5 μl of in vitro synthesized proteins

(TNT lysate) were mixed and applied to each lane as indicated. Probe 3A4 contains a

PXR/RXRα binding site (ER6) of human CYP3A4 gene. Excess unlabeled CYP3A4 probes or mutant probes were used as cold competitors. E. ChIP assay. Caco2 cells were treated with vehicle DMSO (V) or 20 μM rifampicin (RIF) for 40 h. ChIP assays were performed as described under Experimental Procedures. Anti-PXR antibodies were used to precipitate the DNA-protein complexes. DNA fragment containing each PXR binding site was PCR amplified and analyzed on a 2% agarose gel. Non-immune IgG was used as controls (IgG).

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of the CYP27A1 reporter constructs that contained the PXRE-B (ph-1774CYP27/luc and

ph-992CYP27/luc), but not the reporters without PXRE-B (ph-552/luc, ph-223/luc and

ph-147/luc). Deletion of the PXRE-C located upstream of the PXRE-B did not affect the

stimulatory effect of rifampicin. These data suggest that the site B is necessary and

sufficient for mediating rifampicin stimulation. To further confirm the function of these

PXR-binding sites in regulation of the CYP27A1 gene, I introduced the same mutations that abolished PXR bindings in EMSA into the ph-1774 CYP27/luc construct for reporter assays. Fig.37B showed that all three mutant reporters showed reduced basal activities in

comparison with the wild-type reporter. Mutations of the PXRE-B, but not PXRE-A and

PXRE-C, abolished its responsiveness to rifampicin. Taken together, these data suggest that PXR/RXR heterodimer bind strongly to all three putative PXR binding sites, but only

the PXRE-B is responsive to the rifampicin induction of human CYP27A1 gene

transcription.

C. Summary

This study revealed a novel role of PXR in the regulation of cholesterol homeostasis.

PXR stimulates human CYP27A1 in intestinal cells, but not in hepatocytes. EMSA,

ChIP assay and mutagenesis analysis identified a functional PXRE on human CYP27A1 promoter. Rifampicin and cholesterol activation of CYP27A1 leads to increased intracellular 27HOC production. As a potent LXR agonist, 27HOC binds to LXR and activates ABCA1 and ABCG1, which facilitate cholesterol efflux and promote reverse cholesterol transport.

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Fig.37

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Fig. 37. Deletion and mutation analysis of PXR binding sites in the human CYP27A1

promoter.

A. Human CYP27A1/luciferase reporters containing different length of human CYP27A1

promoter regions were transiently transfected into Caco2 cells with PXR/RXR co-

transfection. Cells were treated with vehicle (DMSO) or 20 μM rifampicin for 40 h and harvested for luciferase activity assays as described under Experimental Procedures.

Boxes labeled A, B, and C represent the putative PXR binding sites shown in Fig. 6A.

Error bars represent the standard deviation from the mean of triplicate assays of an individual experiment. B. Wild type (WT) ph-1774CYP27/luc reporter or mutant reporters with mutation in each PXR/RXR binding site (MutA, MutB, and MutC, indicated by a cross) were transient transfected into Caco2 cells co-transfected with

PXR/RXR expression plasmids. Cells were treated with vehicle (DMSO) or 20 μM rifampicin and harvested for luciferase activity assays as described under Experimental

Procedures. The error bars represent the standard deviation from the mean of triplicate

assays of an individual experiment. n=1, *, p<0.005, treated vs. control. Each individual

experiment was repeated at least 3 times.

CHAPTER VI

DISCUSSION

PXR was originally identified as the key regulator of CYP3A4. However, it has become clear now that PXR regulates an entire net work of genes in the liver and intestine that are involved in the metabolism and elimination of potential toxic chemicals from the body.

The most important feature of PXR is its activation by a remarkably diverse set of chemicals, including both xenobiotics derived from the environment and endobiotics produced by the body itself. Thus, in contrast to most other nuclear receptors that are specialized to recognize particular set of physiological ligands and regulate certain biological pathways, PXR has evolved to serve as a whole body sensor for a broad range of structurally diverse chemicals. The different activation profiles of PXR across species are also suggested to result from differences in diet or xenobiotic exposures or endogenous chemical compositions. As we know, the ligands of a nuclear receptor are usually the intermediates or end products of a biological pathway the nuclear receptor also regulates. For examples, the nuclear receptor FXR is activated by bile acids and participates in the regulation of bile acids biosynthesis and transport; LXR responds to oxysterols and play a key role in cholesterol homeostasis. Since PXR is also known to be activated by bile acids, oxysterols and other cholesterol metabolites, then the questions rise that does PXR regulate bile acid biosynthesis or cholesterol homeostasis, and what other biological pathways could be regulated by PXR? Finally, can PXR be exploited as a

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target to treat human diseases? In order to provide answers to these questions, this study

focused on the PXR regulation of three important genes in bile acid and cholesterol

metabolism, CYP7A1, CYP3A4 and CYP27A1.

My studies first demonstrated a role of PXR in the regulation of human CYP7A1 and bile acid biosynthesis. In this study, human primary hepatocyte was established as a suitable model for studying CYP7A1 gene regulation by nuclear receptors. Quantitative real time PCR analysis showed that CDCA strongly reduced CYP7A1 but induced SHP mRNA expression in human primary hepatocytes. This inverse relationship of CDCA effect on CYP7A1 and SHP expression is consistent with the FXR/SHP mechanism of bile acid inhibition of CYP7A1 gene transcription. In contrast, rifampicin strongly reduced both CYP7A1 and SHP mRNA expression in human primary hepatocytes. This result provides strong evidence that CYP7A1 is subjected to PXR inhibition, and SHP is not involved in this inhibition mechanism. Further studies show that rifampicin-activated

PXR inhibits human SHP gene expression (see discussion later).

In bile acid or drug-induced cholestasis, PXR may be activated to induce CYP3A4,

which catalyzes 6-hydroxylation of bile acids for renal excretion. PXR also induces

DHEA sulfotransferase (STD) that excretes sulfate-conjugated bile acids. The findings of this study suggest that PXR inhibition of CYP7A1 gene transcription may be an additional mechanism for reducing intra-hepatic bile acid concentrations and further protect the liver from toxic effects of bile acids and xenobiotics.

Cell-based mammalian two-hybrid assay, co-IP, and ChIP assays all confirmed the interactions of PXR with HNF4α. The interaction between PXR and HNF4α is novel and

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is consistent with our model that factors bound to the BARE-I and BARE-II interact and

coordinately regulate basal transcription of the CYP7A1 gene. Both two-hybrid assay and

co-IP assay performed in HepG2 and primary hepatocytes showed that rifampicin

markedly enhanced PXR interaction with HNF4α. HNF4α is known to play a central role

in the regulation of bile acid synthesis. Many studies have shown that PGC-1α is a

crucial co-activator of HNF4α. Rifampicin enhances PXR interaction with HNF4α and

dissociates PGC-1α from CYP7A1 chromatin, and results in CYP7A1 inhibition. These

data suggest PXR may function as a co-repressor of HNF4α to regulate gene expressions.

There are reports that HNF4α regulates PXR expression and xenobiotic response during

fetal liver development and HNF4α is required for PXR-mediated xenobiotic induction

of Cyp3a in mice. Therefore, the PXR and HNF4α interaction may be physiologically

important in the coordinated regulation of xenobiotic and bile acid metabolism.

The results of this study suggest a model for PXR, HNF4α and PGC-1α regulation of

the human CYP7A1 gene (Fig. 38). HNF4α and PGC-1α are the most important trans-

activators of the human CYP7A1 gene[56]. HNF4α binds to the BARE-II and recruits

PGC-1α to activate human CYP7A1 gene under normal conditions. PXR binds to BARE-

I but might not be important in CYP7A1 basal expression. Ubiquitous co-activators, SRC-

1 and CBP, are known to interact with PXR or HNF4α. Nuclear receptor/co-activator complexes may facilitate the recruitment of histone acetytransferase (HAT) to remodel the chromatin and allow the recruitment of other co-activator/mediator complexes to assemble the general transcriptional machinery that regulates RNA polymerase II

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Fig.38

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Fig. 38. A model for rifampicin and PXR regulation of CYP7A1 gene transcription.

Upper figure: HNF4α and PGC-1α are the most important activators of human CYP7A1 gene. PGC-1α interacts with HNF4α, which binds to the BARE-II and transactivates the human CYP7A1 gene. According to this study, PXR ia able to bind to the BARE-I. Co- activators, SRC-1 and CBP (cAMP response element binding protein (CEBP) binding protein), and histone acetytransferase (HAT) may be also recruited to remodel chromatin and recruit other co-activator complexes to induce the basal transcription of the CYP7A1 gene by RNA polymerase II. Bottom figure: Rifampicin activates PXR to promote its interaction with HNF4α. This interaction may disrupt the interaction between PGC-1α and HNF4α (and may be also PXR) and result in suppression of CYP7A1 gene transcription. Co-repressors and histone deacetylase (HDAC) may be also recruited to inhibit CYP7A1 gene transcription.

161 activity. Activation of PXR by rifampicin induces PXR/HNF4α interaction and excludes

PGC-1α from interacting with HNF4α, resulting in suppression of the CYP7A1 gene transcription.

Human CYP7A1 is not regulated by LXR and humans lack a feed-forward mechanism to stimulate the conversion of cholesterol into bile acids. Instead, human CYP7A1 may be constantly suppressed by bile acid returning to the liver. In humans, bile acid biosynthesis is mainly regulated at two different levels. Under normal conditions, bile acid biosynthesis is tightly controlled under enterohepatic circulation to provide a constant bile acid pool size. During disease states where bile acid levels increase, bile acid production is further suppressed to reduce bile acid toxicities. So far, several bile acid synthesis inhibition pathways have been suggested. However, they are somewhat functionally distinct from each other.

Primary bile acid are poor PXR ligands[154]. PXR is activated only by secondary bile acid LCA at high concentrations (EC50 ~10μM). During normal conditions, trace amount of LCA is efficiently sulfated and eliminated through bile. LCA might be accumulated to high levels only during cholestasis. Thus, PXR mediated inhibition of

CYP7A1 may not be a very significant feedback inhibition pathway to control bile acid homeostasis under normal physiology. Instead, it may become activated in response to toxic chemical insults. Unlike other bile acid receptors, the broad activation profile also allows PXR to inhibit CYP7A1 in response to various toxic lipid metabolites that are also accumulated during cholestasis. Thus, PXR mediated pathway may be the dominant pathway to reduce bile acid synthesis during cholestasis and other chronicle liver

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diseases. Most important of all, PXR has been proven to serve as an excellent target for

cholestasis treatment by drugs.

In contrast, FXR is strongly activated by primary bile acids returning to the liver via

enterohepatic circulation. FXR/SHP pathway might be constitutively active to inhibit

CYP7A1 and thus might be a major pathway for mediating bile acid feedback inhibition

of its own synthesis under normal physiology. This is supported by low CYP7A1 levels

and high SHP levels found in the liver by many in vivo studies. Like PXR agonist rifampicin and hyperforin (active ingredient in St. John’s Wort), FXR agonist GW4064 is also shown to reduce bile acid synthesis genes such as CYP7A1 and CYP8B1 and increase

lipid transport genes such as BSEP, MRP2 and MDR2 in rodent bile duct ligation model of cholestasis, and showed protective roles in these animals[155, 156]. These studies

suggested a role of FXR as a potential target for drug development to treat intrahepatic

cholestasis.

Cytokines are released from kupffer cells in response to liver inflammation or increased bile acid levels. Cytokine mediated inhibition of CYP7A1 through signaling pathway

provides a third pathway for hepatocytes to shut down bile acid output during cholestasis

or liver injury. However, overproduction of cytokines leads to a general decrease in

nuclear receptor expressions, which cause decreased expression of bile acid and lipid

transporters and impaired FXR and PXR dependent detoxification pathways.

Furthermore, cytokines also increase inflammatory cell infiltration and promote liver

damage.[43, 45, 46, 157] Although inhibition of CYP7A1 and certain other hepatic bile

acid transporters such as NTCP may provide an adaptive response to inflammation to

163 reduce hepatic bile acid accumulation and their toxicities, bile acid and inflammation induced cytokines rather promote the development of cholestasis.

In summary, this study provides the first evidence that PXR is able to interact with

HNF4α, and plays a role in mediating rifampicin and bile acid inhibition of CYP7A1 gene transcription. The PXR pathway may be important for preventing bile acid and drug-induced cholestasis by inhibiting bile acid synthesis, as well as stimulating bile acid transport, excretion and detoxification. Thus, PXR plays a central role in coordinated regulation of bile acid homeostasis and drug metabolism. This may be one of the beneficial effects of using rifampicin and steroids to treat pruritus of intrahepatic cholestasis and primary biliary cirrhosis. Efficacious PXR agonists may be developed for treating these chronic liver diseases.

Of equal importance is the catabolism and detoxification of bile acids and lipid metabolites by CYP3A4. The ability of CYP3A4 to be strongly induced by a large variety of structurally unrelated compounds allows it to efficiently respond to the environmental toxins and protect the body against the toxicities of such chemicals. Over the past decade, extensive studies have provided convincing evidence that PXR is the key regulator of CYP3A4 gene expression. PXR is a species-specific xenosensor and most studies on PXR regulation of CYP3A4 are performed in mouse models. This study used human primary hepatocytes and HepG2 cells as models to study the coordinate regulation of human CYP3A4 by several factors including PXR, HNF4α, SRC-1, PGC-1α and SHP.

Real time PCR revealed a remarkable 150-fold induction of CYP3A4 mRNA expression

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by rifampicin in human primary hepatocytes. In contrast, rifampicin treatment reduced

SHP, CYP7A1 and PEPCK mRNA expression. Further studies revealed that interaction

of PXR with co-activator HNF4α, PGC-1α and SRC-1 contributes to the strong induction of CYP3A4 by rifampicin and that concomitant inhibition of SHP gene expression by

PXR minimizes the inhibitory effect of SHP and maximizes PXR induction of CYP3A4.

Promoter studies showed that over-expression of HNF4α in HepG2 cells strongly

enhanced PXR induction of CYP3A4, and ChIP assays showed HNF4α recruitment to

CYP3A4 chromatin was strongly increased by rifampicin treatment. Upon ligand binding, PXR may recruit many co-factors to form a protein complex, which then interact with the general transcriptional machinery to stimulate CYP3A4 gene transcription. This study demonstrated for the first time that recruitment of nuclear receptor HNF4α to this

protein complex is a crucial step for PXR-dependent induction of CYP3A4. The involvement of nuclear receptor HNF4α in the CYP3A4 gene regulation was first reported in 2003[146]. Recently, HNF4α and PXR were also found to synergistically stimulate CYP2C8 and CYP2C9, which further demonstrated its important role in the regulation of drug metabolizing genes[158, 159]. This study herein provided a novel molecular mechanism of HNF4α co-activation of drug metabolizing gene expressions.

The small heterodimer partner SHP was first identified as a negative regulator that

interacted with LRH1. Induction of SHP by CDCA provided a feedback cascade to

inhibit Cyp7a1 and reduce bile acid biosynthesis in the liver [66, 160]. Later on, SHP was

found to interact with many other factors such as HNF4α, Foxa, Foxo1 and LXRα and

exert negative effects on many biological aspects including bile acid synthesis, glucose

165 metabolism and cholesterol homeostasis[55, 72-74]. The results of the study provided a molecular mechanism of the SHP suppression effect on PXR, which was by blocking co- activator recruitment. This mechanism is supported by recently identified LXXLL motif on SHP, which allowed SHP to interact strongly with FTF and block FTF interaction with its co-activators[86]. The strong suppression of SHP mRNA expression in human primary hepatocytes by rifampicin suggested that the SHP/PXR interaction might not be physiologically significant in vivo. And PXR inhibition of SHP may itself be a part of

PXR protection mechanism, since SHP could interfere with PXR dependent activation of the detoxification network.

The CYP3A4 expression is determined by genetic polymorphism, hormone status, cholestatic liver diseases and exposure to drugs or xenobiotics. The induction of CYP3A4 varies widely in human and may account for the different drug metabolizing activities and drug-drug interactions observed in each individual. This study herein suggests that

CYP3A4 gene expression depend on the coordinate regulation of several factors including

PXR, HNF4α, PGC-1α, SRC-1 and SHP. Other factors such as CEBP α and CEBPβ also regulate the CYP3A4 gene. Future studies may identify more factors that are involved in

CYP3A4 gene regulation. Differential expressions and regulations of these factors in response to various physiological and environmental agents may alter CYP3A4 gene expression, and thus, its drug metabolizing ability.

This study also revealed a general mechanism for PXR inhibition of HNF4α activated gene transcription. Previous studies suggest that PXR inhibit CYP7A1 by blocking

HNF4α recruitment of PGC-1α. This study revealed the same mechanism for PXR

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inhibition of human SHP and PEPCK genes. Recently, PXR was also found to be able to

interact with Foxo1 to regulate gluconeogenic genes[144]. The forkhead transcription

factor Foxo1 is an important activator of gluconeogenic genes. Its DNA binding site has been identified in PEPCK and G6Pase promoter. Foxo 1 interacts with PGC-1α to induce

gene transcription[161]. Insulin binds to its cell surface receptor and activates

AKT(Protein kinase B), which phosphorylates Foxo 1. Phosphorylated Foxo 1 is

excluded from the nucleus and results in inhibition of PEPCK and G6Pase [161-166].

PXR ligand dependently interacts with Foxo1 as a co-repressor and suppresses Foxo1

mediated gene activation. On the other hand, Foxo1 binds to PXR or CAR as a co-

activator and enhances CYP3A4 expression. Insulin treatment leads to the inactivation of

Foxo1 and thus results in the down-regulation of CYP3A4[144]. Therefore, PXR may

inhibit HNF4α and Foxo 1 recruitment of PGC-1 α and results in inhibition of PEPCK. It

is significant that rifampicin activated PXR inhibits both CYP7A1 and SHP expression,

whereas bile acid activated FXR induces SHP to inhibit CYP7A1. It is unlikely that these

two pathways will be activated in vivo at the same time because bile acid activated FXR

strongly induces SHP, which counteracts PXR induction of CYP3A4. Furthermore, these

two pathways do respond to distinct cellular signals: FXR is a receptor strictly for

primary bile acids, while PXR is mainly activated by the metabolites of bile acid,

cholesterol and other lipids.

The study of PXR regulation of the human CYP27A1 gene revealed a novel role of

PXR in cholesterol homeostasis in the intestine. Cholesterol loading activated CYP27A1

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activity and markedly stimulated 27HOC production in Caco2 cells. Rifampicin-activated

PXR induced CYP27A1 gene transcription, increased intracellular 27HOC levels, and

induced ABCA1 and ABCG1 expression in Caco2 cells. Cholesterol (20 μg/ml) induced the intracellular 27HOC level in Caco2 cells by 18-fold to about 18 μM, which is similar to 12 to 15 μM observed in macrophages loaded with cholesterol [140]. Treatment with rifampicin further increased 27HOC to about 36 μM. These concentrations of 27HOC are sufficient for activating LXRα (EC50 for 27HOC = ~100 to 200 nM) [140, 166]. This

study also suggests that 27-hydroxycholesterol is likely the most relevant endogenous

LXRα ligand in intestine cells.

It is interesting to note that cholesterol, 27HOC, rifampicin, 9-cis retinoic acid, and

TO901317 have stronger stimulatory effect on ABCG1 than ABCA1 expression in Caco2

cells. Previous studies also show stronger induction of ABCG1 than ABCA1 by LXRα

agonists in human macrophages [167, 168]. Also induction of ABCG1 by LXRα

agonists in human cells is much stronger than in mouse cells and mouse tissues in vivo

[167, 169]. These data suggest that ABCG1 plays a prominent role in cholesterol efflux and HDL metabolism in human liver and intestine. This study established Caco2 cells as a suitable model for studying human CYP27A1 gene expression and regulation.

Based on this study, I propose that the major role of CYP27A1 in intestine may be to

produce oxysterols that activate LXRα and regulate intestinal cholesterol efflux and

HDL-mediated reverse cholesterol transport. Fig. 39 illustrates the central role of

CYP27A1 in activation of the LXR signaling and cholesterol homeostasis in the intestine.

In brush border membrane, Niemann- Pick C1 Like protein 1 (NPC1L1) absorbs dietary

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Fig.39

169

Fig. 39. PXR regulation of drugs, bile acids and cholesterol metabolism in the intestine.

Left panel: Enterocytes. Intestine cholesterol absorption transporter, NPC1L1, transport dietary cholesterol to enterocytes. Cholesterol activates CYP27A1 to convert cholesterol to 27HOC. LCA or rifampicin-activated PXR induces CYP3A4 which converts LCA to hyodeoxycholic acid (HDCA) for renal excretion. PXR induces MRP3, which excretes drugs and toxic metabolites. PXR also induces CYP27A1. 27HOC activates LXRα, which induces ABCA1 and ABCG1 located in the sinusoidal membrane and efflux cholesterol to ApoA1 and HDL, respectively. LXRα also induces ABCG5/ABCG8 transporters located in the apical membrane that efflux sitosterol and cholesterol to lumen. Right panel: macrophages. LDL and oxidized-LDL are taken up to macrophages by LDL receptor and CD36, respectively. The oxidized LDL carries 9- hydroxyoctadienoic acid (9-HODE), an endogenous PPARγ ligand. PPARγ and LXRα induce LXRα expression. 9-cis retinoic acid (9-cis-RA) activated-RAR/RXR and LXRα induce CYP27A1 expression and 27HOC production, which activates LXRα. LXRα induces cholesterol efflux transporters ABCA1 and ABCG1, and ApoE, ApoCII and lipoprotein lipase (LPL) involved in lipoprotein metabolism. Reverse cholesterol transport mediated by HDL transports cholesterol to liver for catabolism to bile acids.

170

cholesterol into enterocytes [170]. Cholesterol homeostasis is maintained by three

pathways in enterocytes: approximately 70-80% of the dietary cholesterol is converted to cholesterol esters by ACAT2 and assembled into chylomicrons (CM) for delivery dietary lipids to the liver; free cholesterol is effluxed via ABCA1 to ApoA1 and via ABCG1 to

HDL particles, which is subsequently transported to the liver for further degradation or elimination; ABCG5/G8 excretes dietary cholesterol and plant sterol (sitosterol) back to the lumen and limits intestinal cholesterol absorption. The latter two pathways are all stimulated by LXRα [171]. Bile acids, drugs, and cholesterol metabolites activate PXR to induce CYP3A4, which detoxifies bile acids, xenobiotics, and cholesterol [106]. PXR also induces CYP27A1 to metabolize cholesterol to 27HOC, which activates the LXRα signaling pathway and induces cholesterol efflux via activation of ABCA1 and ABCG1.

PXR induction of CYP27A1 may be an adaptive response to a high cholesterol diet to

protect intestine against accumulating toxic cholesterol metabolites.

The PXR/CYP27A1/LXRα signaling in intestine resembles the retinoid-

PPARγ/CYP27A1/LXRα signaling that is activated during monocytes differentiation to

macrophages and in human macrophage-rich atherosclerotic lesions [140]. In

macrophages, LDL receptor and scavenger receptor CD36 mediate the uptake of LDL-

cholesterol and oxidized-LDL, respectively. The endogenous PPARγ ligand, 9-

Hydroxyoctadienoic acid (9-HODE) is a major component of the oxidized-LDL.

Retinoids (9-cis retinoic acid and all-trans-retinoic acid) and 9-HODE synergistically

activates PPARγ/RXR and RXR/RAR to induce CYP27A1 gene transcription [139, 140].

171

LXRα is known to induce ApoE, ApoCII, and lipoprotein lipase (LPL) involved in lipoprotein metabolism. In this study, we found that 9-cis-retinoic acid significantly induced CYP27A1, ABCA1 and ABCG1, but the PPARγ agonist did not have any effect on these genes in Caco2 cells. It appears that different nuclear receptor signaling pathways regulate cholesterol efflux in the macrophage and intestine.

It is interesting but somewhat surprising that PXR induces CYP27A1 in intestine, but

not in liver cells. Some intestine-specific transcription factors or co-activators may be

required for PXR to induce CYP27A1. Also the availability of free cholesterol for

activation of CYP27A1 may be different in liver and intestine. In hepatocytes free

cholesterol is readily available for cholesterol 7α-hydroxylase (CYP7A1) located in the

endoplasmic reticulum. CYP27A1 is located in the inner mitochondria membrane, which

lacks cholesterol. To activate the cholesterol 27-hydroxylase activity of CYP27A1,

cholesterol transport to mitochondria needs to be activated [172]. The alternative bile

acid biosynthesis pathway initiated by CYP27A1 is not regulated much by physiological agents [173], and plays a minor role in bile acid synthesis in healthy humans [174]. In contrast, the intestine is exposed to a high level of cholesterol and CYP7A1 is not expressed. The free cholesterol concentration may be high enough to activate CYP27A1.

Therefore, the main function of CYP27A1 in intestine is to hydroxylate cholesterol and induce the LXRα signaling pathway.

Several bile acid and cholesterol metabolites produced in the bile acid synthesis and

cholesterol/oxysterol synthesis pathways are potent endogenous PXR ligands [151, 152,

175, 176]. Activation of PXR and CYP27A1 may be an adaptive response to a high

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cholesterol diet to remove excess un-esterified cholesterol in the intestine. The intestine is

also exposed to high levels of bile acids, bacteria, and drugs. These agents are known to

cause inflammation and acute phase responses that increase cholesterol synthesis. The

intestine also plays a major role in HDL synthesis and reverse cholesterol transport. It has

been reported recently that induction of CYP3A is correlated to increased plasma HDL

and ApoA1 levels, and rifampicin and other PXR agonists increase ApoA1 expression

and serum HDL-cholesterol levels in rodents [177]. In contrast, rifampicin and LCA

down regulate ABCA1 and scavenger receptor B1 (a HDL receptor) in rat primary

hepatocytes and HepG2 cells, suggesting that PXR inhibits both cholesterol influx and

efflux to prevent accumulation of cholesterol in hepatocytes [178]. It has also been

reported that expression of apical Na+-dependent bile salt transporter (ASBT) in Caco2

cells is inhibited by cholesterol suggesting that cholesterol inhibition of intestinal bile acid transport may be an adaptive response to a high cholesterol diet [179]. Reducing bile acid feedback would stimulate bile acid synthesis, and contribute to reducing serum cholesterol levels.

A recent study reports that a high cholesterol and cholic acid diet cause acute lethality

in Pxr null mice but not in wild type mice [153]. It was suggested that PXR might play a

role in cholesterol detoxification [153]. However, the exact cause of hepatorenal failure is

not known and the cholesterol metabolites that are suspected to accumulate and cause

acute lethality in Pxr null mice have not been identified. Our current study provides a

mechanism by which PXR induces both CYP3A4 and CYP27A1 to detoxify cholesterol

173

metabolites in the intestine, and thus protect liver and kidney from the toxicity of

cholesterol metabolites.

In summary, this study reveals for the first time that PXR is a positive regulator of

human CYP27A1 in the intestinal cells and suggests that PXR may have a possible role in cholesterol detoxification in the intestine. Activation of PXR by endogenous cholesterol

metabolites and drugs may feed forward activate CYP3A4 and CYP27A1 to detoxify bile acids and cholesterol metabolites, and also promote cholesterol efflux and HDL synthesis.

APPENDIX A

ABBREVIATIONS

27HOC: 27-hydroxychoelsterol 3β-HSD: 3β-hydroxy-27-steroid dehydrogenase/isomerase β-gal: β-galactosidase ABCA1: ATP binding cassette transporter AI ABCB4: ATP binding cassette transporter B4 ABCB11: ATP binding cassette transporter B11 ABCC2: ATP binding cassette transporter C2 ABCG1: ATP binding cassette transporter AI ABCG5: ATP binding cassette transporter G5 ABCG8: ATP binding cassette transporter G8 ACAT2: Acyl CoA: cholesterol acyl transferase 2 AF-1: activation function-1 domain AF-2: activation function-2 domain AR: androgen receptor ASBT: apical sodium-dependent bile salt transporter BAREI: bile acid responsive element I BAREII: bile acid responsive element II BSEP: bile salt export pump CA: cholic acid CAR: constitutive androstane receptor CDCA: chenodeoxycholic acid CEBP: CCAAT enhancer binding protein CEH: cholesterol ester hydrolase CETP: cholesterol ester transfer protein

175

ChIP: chromatin immunoprecipitation CMV: cytomegalovirus CM: chylomicron CMR: chylomicron remnant CTX: cerebrotendinous xanthomatosis CYP3A4: cytochrome p450 3A4 CYP7A1: cholesterol 7α hydroxylase CYP7B1: oxysterol 7α-hydroxylase CYP8B1: sterol 12α-hydroxylase CYP27A1: mitochondrial sterol 27-hydroxylase DBD: DNA binding domain DCA: deoxycholic acid DR: direct repeat EMSA: electrophoretic mobility shift assay ER: estrogen receptor ER: everted repeat ERR: ER-related receptor FGF: fibroblast growth factor FGF19: fibroblast growth factor 19 FGFR4: fibroblast growth factor receptor 4 FPIC: progressive familial intrahepatic cholestasis FTF: α fetoprotein transcription factor FXR: farnesoid X receptor G-6-Pase: glucose 6 phosphatase GR: glucocorticoid receptor GST: glutathione S transferase HAT: histone acetyltransferase HBP: Human highly basic protein HCA: hyocholic acid

176

HDL: high density lipoprotein HMG-CoA R: HMG CoA reductase HNF4α: hepatic nuclear factor 4α IBAP: intestinal bile acid binding protein IDL: intermediate density lipoprotein IL-1β: interleukin 1β IP: immunoprecipitation IR: inverted repeat ISBT: ileal bile salt transporter JNK: c-jun N-terminal kinase LBD: ligand binding domain LDL: low density lipoprotein LPL: lipoprotein lipase LPS: lipopolyssacride LCA: lithocholic acid LRH: liver-related homolog LRP: LDL receptor related protein LXR: liver X receptor MDR2: multidrug–resistant protein 2 MOAT: multispecific organic anion transporter MRP2: multidrug–resistant associated protein-2 MRP3: multidrug–resistant associated protein-3 NPC1L1: Niemann- Pick C1 Like protein 1 NTCP: sodium dependent taurocholate transporter OATP-1: organic anion transporter-1 OATP2: organic anion transporters-2 OATP3: organic anion transporters-3 OATP4: organic anion transporters-4 OATP-A: organic anion transporters-A

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OATP-C: organic anion transporters-C OSTα: organic solute transporter α OSTβ: organic solute transporter β PGC-1α: PPARγ coactivator 1α PKC: protein kinase C PPARγ: Peroxisome proliferator activated receptor γ PXR: pregnane X receptor RXR: retinoic X receptor SDS PAGE: SDS polyacrylamide gel electrophoresis SHP: small heterodimer partner SPGP: sister of P-glycoprotein SRA-I: scavenger receptor A1 SRB-I: scavenger receptor B1 SRC-1:steroid receptor coactivator 1 TK: thymidine kinase TNFα: tumor necrosis factor α UAS: upstream activating sequence UBC: ubiquitin C VDR: vitamin D receptor VLDL: very low density lipoprotein XREM: xenobiotic response enhancer module

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