Dean W. Hum

Bile acid-activated nuclear receptor FXR suppresses apolipoprotein A-I transcription via a negative FXR response element

Serum levels of HDL are inversely correlated with the risk of coronary heart disease. The anti-atherogenic effect of HDL is partially mediated by its major protein constituent apoA-I. In this study, we identify bile acids that are activators of the nuclear receptor farnesoid X receptor (FXR) as negative regulators of human apoA-I expression. Intrahepatocellular accumulation of bile acids, as seen in patients with progressive familial intrahepatic cholestasis and biliary atresia, was associated with diminished apoA-I serum levels. In human apoA-I transgenic mice, treatment with the FXR agonist taurocholic acid strongly decreased serum concentrations and liver mRNA levels of human apoA-I, which was associated with reduced serum HDL levels. Incubation of human primary hepatocytes and hepatoblastoma HepG2 cells with bile acids resulted in a dose-dependent downregulation of apoA-I expression. Promoter mutation analysis and gel-shift experiments in HepG2 cells demonstrated that bile acid-activated FXR decreases human apoA-I promoter activity by a negative FXR response element mapped to the C site. FXR bound this site and repressed transcription in a manner independent of retinoid X receptor. The nonsteroidal synthetic FXR agonist GW4064 likewise decreased apoA-I mRNA levels and promoter activity in HepG2 cells.

Isolation and characterization of UGT2B15 (Y85) : a UDP-glucuronosyltransferase encoded by a polymorphic gene

Genetic polymorphisms occur in many of the drug metabolizing enzymes. However, the effect of polymorphisms in the genes encoding phase II drug metabolizing UDP-glucuronosyltransferases is still undescribed, despite the many reported cases of variations in glucuronidation activities. Characterization of the UGT2B15(Y85) cDNA, which was isolated from human prostate and LNCaP cell cDNA libraries, revealed 20 nucleotide differences between UGT2B15(Y85) and the previously characterized UGT2B15 protein UGT2B15(D85). However, only one of the two variations in the coding region leads to an amino acid change from aspartic acid to a tyrosine residue at position 85. The genomic DNA of 27 subjects were analysed by direct sequencing of polymerase chain reaction (PCR) products and demonstrated that UGT2B15(D85) and UGT2B15(Y85) are encoded by variant alleles prevalent in the Caucasian population. Expression of UGT2B15(D85) and UGT2B15(Y85) in HK293 cells demonstrated similar substrate specificities. Of the 65 potential substrates tested for activity, the proteins were active on phenolic compounds, coumarins, flavonoids, drugs and steroid hormones. Both proteins displayed similar Km values of 2.2 and 2.4 microM for androstane-3alpha,17beta-diol and dihydrotestosterone, respectively. However, results suggest that UGT2B15(Y85) has a higher Vmax than UGT2B15(D85). Specific reverse transcriptase (RT)-PCR analysis revealed expression of the UGT2B15 gene in a wide range of extrahepatic tissues including the human liver, kidney, testis, mammary gland, placenta, adipose, skin, uterus, prostate and lung. The wide expression of UGT2B15 in many tissues indicates that it is a major glucuronidation enzyme in humans.

Hepatoprotective effects of the dual peroxisome proliferator-activated receptor alpha/delta agonist, GFT505, in rodent models of nonalcoholic fatty liver disease/nonalcoholic steatohepatitis

Nonalcoholic fatty liver disease (NAFLD) covers a spectrum of liver damage ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), fibrosis, and cirrhosis. To date, no pharmacological treatment is approved for NAFLD/NASH. Here, we report on preclinical and clinical data with GFT505, a novel dual peroxisome proliferator‐activated receptor alpha/delta (PPAR‐α/δ) agonist. In the rat, GFT505 concentrated in the liver with limited extrahepatic exposure and underwent extensive enterohepatic cycling. The efficacy of GFT505 was assessed in animal models of NAFLD/NASH and liver fibrosis (Western diet [WD]‐fed human apolipoprotein E2 [hApoE2] transgenic mice, methionine‐ and choline‐deficient diet‐fed db/db mice, and CCl4‐induced fibrosis in rats). GFT505 demonstrated liver‐protective effects on steatosis, inflammation, and fibrosis. In addition, GFT505 improved liver dysfunction markers, decreased hepatic lipid accumulation, and inhibited proinflammatory (interleukin‐1 beta, tumor necrosis factor alpha, and F4/80) and profibrotic (transforming growth factor beta, tissue inhibitor of metalloproteinase 2, collagen type I, alpha 1, and collagen type I, alpha 2) gene expression. To determine the role of PPAR‐α‐independent mechanisms, the effect of GFT505 was assessed in hApoE2 knock‐in/PPAR‐α knockout mice. In these mice, GFT505 also prevented WD‐induced liver steatosis and inflammation, indicating a contribution of PPAR‐α‐independent mechanisms. Finally, the effect of GFT505 on liver dysfunction markers was assessed in a combined analysis of four phase II clinical studies in metabolic syndrome patients. GFT505 treatment decreased plasma concentrations of alanine aminotransferase, gamma‐glutamyl transpeptidase, and alkaline phosphatase. Conclusion: The dual PPAR‐α/δ agonist, GFT505, is a promising liver‐targeted drug for treatment of NAFLD/NASH. In animals, its protective effects are mediated by both PPAR‐α‐dependent and ‐independent mechanisms. (Hepatology 2013; 58:1941–1952)

Characterization of UDP-glucuronosyltransferases active on steroid hormones ☆

In recent years, the enzymes which are involved in the formation of DHT in steroid target tissues have been well investigated, however, enzymes responsible for the catabolism and elimination of steroids in these tissues, in particular the uridine diphospho-glucuronosyltransferase (UGT) family of enzymes, have received much less attention. We have recently demonstrated that human and monkey are unique in having high plasma levels of C19 steroid glucuronides. These circulating conjugates have been proposed to reflect the peripheral conversion of adrenal and gonadal C19 steroids to potent androgens, especially DHT. In humans, the presence of steroid UGT activities is found in the liver and several extrahepatic tissues including the prostate, mammary gland and ovary. In addition, UGT activities were observed in breast and prostate tumor cell lines such as MCF-7 and LNCaP, respectively. In agreement with the presence of steroid conjugating enzymes in extrahepatic tissues, UGT cDNA clones, which encode steroid conjugating proteins, have been isolated from libraries constructed from human and monkey prostate mRNA. The presence of UGT transcripts and proteins in extrahepatic tissues in both species, as determined by Northern blot, ribonuclease protection, specific RT-PCR, in situ hybridization, Western blot and immunocytochemistry analysis, indicate the relevance of steroid glucuronidation in tissues other than the liver. Knowing that both the human prostate and the human prostate cancer LNCaP cell line express steroid metabolizing proteins, including UGT enzymes, regulation of UGT mRNA and protein levels, as well as promoter activity was studied in these cells. The results demonstrate a differential regulation between the two highly related isoforms UGT2B15 and UGT2B17, where only the expression of UGT2B17 was affected following treatments of LNCaP cells with androgens, growth factors or cytokines. Steroid conjugation by UGT enzymes is potentially involved in hormone inactivation in steroid target tissues, thus modifications in UGT expression levels may influence hormonal responses.

Characterization and regulation of UDP-glucuronosyltransferases in steroid target tissues

Conjugation of compounds by glucuronidation is a pathway found in all vertebrates studied to date. Although, it is widely recognized that the liver is a major site of glucuronidation, it is now clear that extrahepatic tissues are also involved in the conjugation of compounds to which these tissues are exposed. High levels of androsterone glucuronide and androstane-3alpha,17beta-diol glucuronide found in the human prostate, breast cyst fluid and ovary follicular fluid suggest that glucuronidation of 5alpha-reduced C19 steroids occurs in these tissues. Recently, we have reported the tissue distribution of UGT2B15, which can conjugate steroids in several human extrahepatic steroid target tissues including the skin, breast and prostate. We have also isolated a new UGT2B cDNA encoding UGT2B17, that conjugates ADT which is the major 5alpha-reduced C19 steroid glucuronide in the circulation of humans. UGT2B17 is also widely distributed in several human steroid target tissues. This gene was mapped to human chromosome 4q13 and has an exon/intron structure similar to that of rat UGT2B1 and UGT2B2. Both UGT2B15 and UGT2B17, which are able to catalyze the glucuronidation of DHT, are expressed in LNCaP cells. Interestingly, glucuronidation of steroids is markedly regulated by several factors including androgens and growth factors. Treatment of LNCaP cells with dihydrotestosterone (DHT) and epidermal growth factor (EGF) caused a decrease of DHT glucuronidation and UGT2B mRNA levels. RNase protection assays showed a specific decrease of UGT2B17 transcript in LNCaP cells treated with DHT and EGF however, the level of UGT2B15 mRNA was not affected. As well, Western blot analysis demonstrated a diminution of UGT2B17 protein level in response to DHT and EGF. These results demonstrate a differential regulation of different isoforms of steroid conjugating UGTs present in human prostate LNCaP cells. In addition, UGT2B17 was shown to be more labile than UGT2B15 indicating that regulation of UGT2B17 expression would lead to a more rapid change in the level of glucuronidated steroids. Expression of exogenous UGT2B17 in LNCaP cells by gene transfer led to a significant decrease in the androgen response. This result indicates the ability of UGT enzymes to regulate the androgen response by conjugating androgens which abolishes their interaction with their receptor and facilitates their clearance from the cell. The glucuronidation of steroids by UGT enzymes is an important mechanism by which the levels of steroids is regulated in steroid target tissues.

THE MONKEY AND HUMAN URIDINE DIPHOSPHATE-GLUCURONOSYLTRANSFERASE UGT1A9, EXPRESSED IN STEROID TARGET TISSUES, ARE ESTROGEN-CONJUGATING ENZYMES

Considering the physiologic importance of the steroid response, which is regulated in part by steroid levels in a given tissue, relatively little is known about steroid glucuronidation, which is widely accepted as a major pathway involved in the catabolism and elimination of steroid hormones from the human body. In a previous study, it was ascertained that the monkey may be the most appropriate model in which to examine the role of steroid glucuronidation. Northern blot analysis of simian RNA, hybridized with human UGT complementary DNA (cDNA) probes demonstrate the similarity of the transcripts. The simian UGT1A09 cDNA isolated from a liver library is 2396 bp and contains an open reading frame encoding 530 amino acids. The predicted primary structure is most homologous to the human UGT1A9 (hUGT1A9) enzyme, which share 93% identity. Stable transfection of the monkey UGT1A09 (monUGT1A09) cDNA into HK293 cells, expresses a microsomal protein with an apparent molecular mass of 55 kDa. Of the more than 30 end...

Differential regulation of two uridine diphospho-glucuronosyltransferases, UGT2B15 and UGT2B17, in human prostate LNCaP cells.

Although androgens are important regulators in the prostate, other effectors such as growth factors may also act to maintain normal function of the gland. Human prostate and human prostate cancer LNCaP cells express steroid conjugating uridine diphospho-glucuronosyltransferase (UGT) enzymes, and it was shown that the level of UGT activities and transcripts is down-regulated by androgens, especially dihydrotestosterone (DHT). In the present study, we examined the interaction between androgen, epidermal growth factor (EGF), and steroid UGT enzymes. The formation of DHT glucuronide (DHT-G) was inhibited by 47% when LNCaP cells were treated for 6 days with 10 ng/ml of EGF. Northern blot analysis also demonstrated a decrease in the steady-state level of UGT2B transcripts. Treatment with both DHT (0.5 nm) and EGF (10 ng/ml) caused a greater decrease of DHT glucuronidation and UGT2B messenger RNA levels than when the cells were treated with either compound alone. RNase protection assays showed that treatment wit...

The farnesoid X receptor induces very low density lipoprotein receptor gene expression

The farnesoid X receptor (FXR) is a nuclear receptor activated by bile acids (BAs). In response to ligand‐binding, FXR regulates many genes involved in BA, lipid, and lipoprotein metabolism. To identify new FXR target genes, microarray technology was used to profile total RNA extracted from HepG2 cells treated with the natural FXR agonist chenodeoxycholic acid (CDCA). Interestingly, a significant increase of transcript level of the very low density lipoprotein receptor (VLDLR) was observed. Our data, resulting from selective FXR activation, FXR RNA silencing and FXR‐deficient mice, clearly demonstrate that BAs up‐regulate VLDLR transcript levels via a FXR‐dependent mechanism in vitro in human and in vivo in mouse liver cells.

Phosphorylation of Farnesoid X Receptor by Protein Kinase C Promotes Its Transcriptional Activity

The farnesoid X receptor (FXR, NR1H4) belongs to the nuclear receptor superfamily and is activated by bile acids such as chenodeoxycholic acid, or synthetic ligands such as GW4064. FXR is implicated in the regulation of bile acid, lipid, and carbohydrate metabolism. Posttranslational modifications regulating its activity have not been investigated yet. Here, we demonstrate that calcium-dependent protein kinase C (PKC) inhibition impairs ligand-mediated regulation of FXR target genes. Moreover, in a transactivation assay, we show that FXR transcriptional activity is modulated by PKC. Furthermore, phorbol 12-myristate 13-acetate , a PKC activator, induces the phosphorylation of endogenous FXR in HepG2 cells and PKCalpha phosphorylates in vitro FXR in its DNA-binding domain on S135 and S154. Mutation of S135 and S154 to alanine residues reduces in cell FXR phosphorylation. In contrast to wild-type FXR, mutant FXRS135AS154A displays an impaired PKCalpha-induced transactivation and a decreased ligand-dependent FXR transactivation. Finally, phosphorylation of FXR by PKC promotes the recruitment of peroxisomal proliferator-activated receptor gamma coactivator 1alpha. In conclusion, these findings show that the phosphorylation of FXR induced by PKCalpha directly modulates the ability of agonists to activate FXR.

Transcriptional Regulation of Apolipoprotein A5 Gene Expression by the Nuclear Receptor RORα

Objective—The newly identified apolipoprotein A5 (APOA5), selectively expressed in the liver, is a crucial determinant of plasma triglyceride levels. Because elevated plasma triglyceride concentrations constitute an independent risk factor for cardiovascular diseases, it is important to understand how the expression of this gene is regulated. In the present study, we identified the retinoic acid receptor-related orphan receptor-α (RORα) as a regulator of human APOA5 gene expression. Methods and Results—Using electromobility shift assays, we first demonstrated that RORα1 and RORα4 proteins can bind specifically to a direct repeat 1 site present at the position −272/−260 in the APOA5 gene promoter. In addition, using transient cotransfection experiments in HepG2 and HuH7 cells, we demonstrated that both RORα1 and RORα4 strongly increase APOA5 promoter transcriptional activity in a dose-dependent manner. Finally, adenoviral overexpression of hRORα in HepG2 cells led to enhanced hAPOA5 mRNA accumulation. We show that the homologous region in mouse apoa5 promoter is not functional. Moreover, we show that in staggerer mice, apoa5 gene is not affected by RORα. Conclusions—These findings identify RORα1 and RORα4 as transcriptional activators of human APOA5 gene expression. These data suggest an additional important physiological role for RORα in the regulation of genes involved in lipid homeostasis and probably in the development of atherosclerosis.