Mélanie Verreault

Human UDP‐glucuronosyltransferase (UGT)1A3 enzyme conjugates chenodeoxycholic acid in the liver

Chenodeoxycholic acid (CDCA) is a liver‐formed detergent and plays an important role in the control of cholesterol homeostasis. During cholestasis, toxic bile acids (BA) accumulate in hepatocytes causing damage and consequent impairment of their function. Glucuronidation, a conjugation reaction catalyzed by UDP‐glucuronosyltransferase (UGT) enzymes, is considered an important metabolic pathway for hepatic BA. This study identifies the human UGT1A3 enzyme as the major enzyme responsible for the hepatic formation of the acyl CDCA‐24glucuronide (CDCA‐24G). Kinetic analyses revealed that human liver and UGT1A3 catalyze the formation of CDCA‐24G with similar Km values of 10.6 to 18.6 μmol/L, respectively. In addition, electrophoretic mobility shift assays and transient transfection experiments revealed that glucuronidation reduces the ability of CDCA to act as an activator of the nuclear farnesoid X‐receptor (FXR). Finally, we observed that treatment of human hepatocytes with fibrates increases the expression and activity of UGT1A3, whereas CDCA has no effect. In conclusion, UGT1A3 is the main UGT enzyme for the hepatic formation of CDCA‐24G and glucuronidation inhibits the ability of CDCA to act as an FXR activator. In vitro data also suggest that fibrates may favor the formation of bile acid glucuronides in cholestatic patients. (HEPATOLOGY 2006;44:1158–1170.)

The liver X‐receptor alpha controls hepatic expression of the human bile acid–glucuronidating UGT1A3 enzyme in human cells and transgenic mice

Glucuronidation, an important bile acid detoxification pathway, is catalyzed by enzymes belonging to the UDP‐glucuronosyltransferase (UGT) family. Among UGT enzymes, UGT1A3 is considered the major human enzyme for the hepatic C24‐glucuronidation of the primary chenodeoxycholic (CDCA) and secondary lithocholic (LCA) bile acids. We identify UGT1A3 as a positively regulated target gene of the oxysterol‐activated nuclear receptor liver X‐receptor alpha (LXRα). In human hepatic cells and human UGT1A transgenic mice, LXRα activators induce UGT1A3 mRNA levels and the formation of CDCA‐24glucuronide (24G) and LCA‐24G. Furthermore, a functional LXR response element (LXRE) was identified in the UGT1A3 promoter by site‐directed mutagenesis, electrophoretic mobility shift assays and chromatin immunoprecipitation experiment. In addition, LXRα is found to interact with the SRC‐1α and NCoR cofactors to regulate the UGT1A3 gene, but not with PGC‐1β. In conclusion, these observations establish LXRα as a crucial regulator of bile acid glucuronidation in humans and suggest that accumulation of oxysterols in hepatocytes during cholestasis favors bile acid detoxification as glucuronide conjugates. LXR agonists may be useful for stimulating both bile acid detoxification and cholesterol removal in cholestatic or hypercholesterolemic patients, respectively. (HEPATOLOGY 2006;44:368–378.)

Lipid-activated transcription factors control bile acid glucuronidation

Bile acids subserve important physiological functions in the control of cholesterol homeostasis. Indeed, hepatic bile acid synthesis and biliary excretion constitute the main route for cholesterol removal from the human body. On the other hand, bile acids serve as natural detergents for the intestinal absorption of dietary cholesterol. However, due to their detergent properties, bile acids are inherently cytotoxic, and their cellular level may be tightly controlled to avoid pathological situations such as cholestasis. Recent investigations have illustrated the crucial roles that a series of ligand-activated transcription factors has in the control of hepatic bile acids synthesis, transport and metabolism. Thus, the lipid-activated nuclear receptors, farnesoid X-receptor (FXR), liver X-receptor (LXR), pregnane X-receptor (PXR) and peroxisome proliferator-activated receptor alpha (PPARα), modulate the expression and activity of genes controlling bile acid homeostasis in the liver. Several members of the UDP-glucuronosyltransferase (UGT) enzymes family are among the bile acid metabolizing enzymes regulated by these receptors. UGTs catalyze glucuronidation, a major phase II metabolic reaction, which converts hydrophobic bile acids into polar and urinary excretable metabolites. This article summarizes our recent observations on the regulation of bile acid conjugating UGTs upon pharmacological activation of lipid-activated receptors, with a particular interest for the role of PPARα and LXRα in controlling human UGT1A3 expression.

Activators of the farnesoid X receptor negatively regulate androgen glucuronidation in human prostate cancer LNCAP cells

Androgens are major regulators of prostate cell growth and physiology. In the human prostate, androgens are inactivated in the form of hydrophilic glucuronide conjugates. These metabolites are formed by the two human UGT2B15 [UGT (UDP-glucuronosyltransferase) 2B15] and UGT2B17 enzymes. The FXR (farnesoid X receptor) is a bile acid sensor controlling hepatic and/or intestinal cholesterol, lipid and glucose metabolism. In the present study, we report the expression of FXR in normal and cancer prostate epithelial cells, and we demonstrate that its activation by chenodeoxycholic acid or GW4064 negatively interferes with the levels of UGT2B15 and UGT2B17 mRNA and protein in prostate cancer LNCaP cells. FXR activation also causes a drastic reduction of androgen glucuronidation in these cells. These results point out activators of FXR as negative regulators of androgen-conjugating UGT expression in the prostate. Finally, the androgen metabolite androsterone, which is also an activator of FXR, dose-dependently reduces the glucuronidation of androgens catalysed by UGT2B15 and UGT2B17 in an FXR-dependent manner in LNCaP cells. In conclusion, the present study identifies for the first time the activators of FXR as important regulators of androgen metabolism in human prostate cancer cells.

Calcitrol (1α,25-dihydroxyvitamin D3) inhibits androgen glucuronidation in prostate cancer cells

Calcitriol (1α,25-dihydroxyvitamin D3), the active metabolite of vitamin D, has recently emerged as a promising therapeutic agent in the treatment of prostate cancer, the second most common cause of cancer death in American males. In the present study, we have analyzed the effects of calcitriol treatment on the expression and activity of the UDP-glucuronosyltransferase (UGT) 2B15 and 2B17 in prostate cancer LNCaP and 22Rv1 cells. These two enzymes share a crucial role in the inactivation of androgens in the human prostate. We report that calcitriol treatment results in lower glucuronide conjugation of the active androgen dihydrotestosterone and its reduced metabolites androstane-3α-diol and androsterone in LNCaP cells. The same treatment also drastically decreased the mRNA and protein levels of UGT2B15 and UGT2B17 in LNCaP and 22Rv1 cells. Using casodex, an androgen receptor (AR) antagonist, and AR-specific small interfering RNA probes, we show that calcitriol requires a functional AR to inhibit the expression of the UGT2B17 gene in LNCaP cells. By contrast, transient transfection and site-directed mutagenesis experiments revealed that calcitriol down-regulates UGT2B15 promoter activity through a responsive region between positions -171 and -113 bp. In conclusion, the present study identifies the vitamin D receptor activator calcitriol as a negative regulator of the UGT2B15- and UGT2B17-dependent inactivation of androgens in prostate cancer LNCaP cells. Androgens promote prostate cancer cell proliferation; thus, the reduction of their inactivation could have a limiting effect of the calcitriol antiproliferative properties in prostate cancer cells. [Mol Cancer Ther 2008;7(2):380–90]

PPARα L162V polymorphism alters the potential of n‐3 fatty acids to increase lipoprotein lipase activity

Omega-3 fatty acids (FAs) may accelerate plasma triglyceride (TG) clearance by altering lipoprotein lipase (LPL) activity. Yet, the ability of n-3 FAs to increase LPL activity is dependent on transcription factors such as peroxisome proliferator-activated receptor alpha (PPARalpha). The objective was to examine the effects of n-3 FAs on LPL activity considering the occurrence of PPARalpha L162V polymorphism. First, 14 pairs of men either L162 homozygotes or carriers of the V162 allele were supplemented with n-3 FAs. Second, transient transfections in HepG2 cells, for the L162- and V162-PPARalpha variants with the peroxisome proliferator-response element from the human LPL gene, were transactivated with n-3 FAs. In vivo results demonstrate that the LPL activity increased non-significantly by 14.4% in L162 homozygotes compared with 6.6% in carriers of the PPARalpha-V162 allele, after n-3 FA supplementation. Additionally, the L162 homozygotes tended towards an inverse correlation between LPL activities and plasma TG levels. Conversely, carriers of the V162 allele showed no such relationship. In vitro data demonstrates that transcription rates of LPL tended to be higher for the L162-PPARalpha than V162-PPARalpha after n-3 FAs activation. Overall, these results indicate that n-3 FA supplementation increases the transcription rate of LPL to a greater extent in L162-PPARalpha than V162-PPARalpha.

Differential expression of the androgen-conjugating UGT2B15 and UGT2B17 enzymes in prostate tumor cells during cancer progression.

CONTEXT Androgens play major roles in prostate cancer initiation and development. In prostate cells, the human uridine diphosphate-glucuronosyltransferase (UGT)2B15 and UGT2B17 enzymes inactivate androgens. OBJECTIVE We investigated in vivo how UGT2B15 and UGT2B17 expressions are affected during prostate cancer development. DESIGN We conducted an observational study of the UGT2B15 and UGT2B17 mRNA and protein levels. SETTING The study was conducted at Laval University (Québec, Canada) and at the University of British Columbia (Vancouver, Canada). PATIENTS/PARTICIPANTS Participants were from a cohort of prostate cancer patients from the Hôtel-Dieu de Québec hospital (Québec; mRNA analyses) and from the Vancouver Prostate Centre tissue bank (Vancouver; tissue microarray experiments). MAIN OUTCOME MEASURES UGT mRNA and protein levels were determined using real-time PCR and immunohistochemical analyses, respectively. RESULTS Both UGT2B15 and UGT2B17 mRNA and protein levels were not significantly associated with Gleason score stratification. However, when protein levels were compared to benign prostatic hyperplasia, UGT2B17 was significantly more abundant in all Gleason-scored tumors. By contrast, UGT2B15 levels were significantly reduced in naive and castration-resistant tumors and undetectable in lymph node metastases. Finally, UGT2B17 proteins were 5-fold more abundant in metastases than in benign samples. CONCLUSIONS The current study reveals that UGT2B15 and UGT2B17 are differentially regulated during prostate cancer progression. Furthermore, this study also identifies the UGT2B15 gene as a negatively regulated target gene in castration-resistant prostate cancer and lymph node metastases.

Profiling Serum Bile Acid Glucuronides in Humans: Gender Divergences, Genetic Determinants, and Response to Fenofibrate

Glucuronidation, catalyzed by uridine 5′‐diphospho‐glucuronosyltransferase (UGT) enzymes, detoxifies cholestatic bile acids (BAs). We aimed to (i) characterize the circulating BA‐glucuronide (BA‐G) pool composition in humans, (ii) determine how sex and UGT polymorphisms influence this composition, and (iii) analyze the effects of the lipid‐lowering drug fenofibrate on the circulating BA‐G profile in 300 volunteers and 5 cholestatic patients. Eleven BA‐Gs were determined in pre‐ and postfenofibrate samples. Men exhibited higher BA‐G concentrations, and various genotype/BA‐G associations were discovered in relevant UGT genes. The chenodeoxycholic acid‐3G (CDCA‐3G) concentration was associated with the UGT2B7 802C>T polymorphism. Glucuronidation assays confirmed the predominant role of UGT2B7 and UGT1A4 in CDCA‐3G formation. Fenofibrate exposure increased the serum levels of five BA‐G species, including CDCA‐3G, and upregulated expression of UGT1A4, but not UGT2B7, in hepatic cells. This study demonstrated that fenofibrate stimulates BA glucuronidation in humans and thus reduces BA toxicity in the liver.

Regulation of endobiotics glucuronidation by ligand-activated transcription factors: physiological function and therapeutic potential.

Recent progresses in molecular pharmacology approaches have allowed the identification and characterization of a series of nuclear receptors (NR) which efficiently control the level UDP-glucuronosyltransferase (UGT) genes expression. These regulatory processes ensure optimized UGT expression in response to specific endogenous and/or exogenous stimuli. Interestingly, numerous endogenous activators of these NRs are conjugated by the UGT enzymes they regulate. In such a case, the NR-dependent regulation of UGT genes corresponds to a feedforward/feedback mechanism by which a bioactive molecule controls its own concentrations. In the present review, we will discuss i) how bilirubin reduces its circulating levels by activating AhR in the liver; ii) how bile acids modulate their hepatic glucuronidation via PXR- and FXR-dependent processes in enterohepatic tissues; and iii) how androgens inhibit their cellular metabolism in prostate cancer cells through an AR-dependent mechanism. Subsequently, with further discussion of the same examples (bilirubin and bile acids), we will illustrate how NR-dependent regulation of UGT enzymes may contribute to the beneficial effects of pharmacological activators of nuclear receptors, such as CAR and PPARa.

The Human UGT1A3 Enzyme Conjugates Norursodeoxycholic Acid into a C23-ester Glucuronide in the Liver

Norursodeoxycholic acid (norUDCA) exhibits efficient anti-cholestatic properties in an animal model of sclerosing cholangitis. norUDCA is eliminated as a C23-ester glucuronide (norUDCA-23G) in humans. The present study aimed at identifying the human UDP-glucuronosyltransferase (UGT) enzyme(s) involved in hepatic norUDCA glucuronidation and at evaluating the consequences of single nucleotide polymorphisms in the coding region of UGT genes on norUDCA-23G formation. The effects of norUDCA on the formation of the cholestatic lithocholic acid-glucuronide derivative and of rifampicin on hepatic norUDCA glucuronidation were also explored. In vitro glucuronidation assays were performed with microsomes from human tissues (liver and intestine) and HEK293 cells expressing human UGT enzymes and variant allozymes. UGT1A3 was identified as the major hepatic UGT enzyme catalyzing the formation of norUDCA-23G. Correlation studies using samples from a human liver bank (n = 16) indicated that the level of UGT1A3 protein is a strong determinant of in vitro norUDCA glucuronidation. Analyses of the norUDCA-conjugating activity by 11 UGT1A3 variant allozymes identified three phenotypes with high, low, and intermediate capacity. norUDCA is also identified as a competitive inhibitor for the hepatic formation of the pro-cholestatic lithocholic acid-glucuronide derivative, whereas norUDCA glucuronidation is weakly stimulated by rifampicin. This study identifies human UGT1A3 as the major enzyme for the hepatic norUDCA glucuronidation and supports that some coding polymorphisms affecting the conjugating activity of UGT1A3 in vitro may alter the pharmacokinetic properties of norUDCA in cholestasis treatment.