Characterization and regulation of UDP-glucuronosyltransferases in steroid target tissues1

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Abstract

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-3α,17β-diol glucuronide found in the human prostate, breast cyst fluid and ovary follicular fluid suggest that glucuronidation of 5α-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 5α-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.

Introduction

Uridine diphosphate glucuronosyltransferase (UGT) enzymes catalyze the transfer of the glucuronyl group from uridine 5′-diphosphoglucuronic acid to active endogenous and exogenous molecules having functional groups of oxygen, nitrogen, sulphur and carbon. The resulting glucuronide products are more polar, generally water soluble, less toxic and more easily excreted than the substrate molecule. Examples of endogenous substrates that are glucuronidated include bilirubin, bile acids and steroids whereas, xenobiotics such as drugs and pollutants are also detoxified by UGT enzymes1, 2.

It has been proposed by several groups that plasma levels of 5α-reduced C19 steroid glucuronides, namely androsterone glucuronide (ADT-G) and androstane-3α, 17β-diol glucuronide (3α-diol-G), reflect the peripheral tissue conversion of adrenal and gonadal C19 steroids to potent androgens, especially DHT3, 4, 5, 6, 7. For example, castration is known to reduce the level of dihydrotestosterone (DHT) in the prostate by approximately 60% and the plasma concentration of ADT-G and 3α-diol-G is also reduced by the same percentage after castration[8]suggesting a relationship between the production of DHT in extrahepatic tissues and circulating 5α-reduced C19 steroid glucuronides. In addition, the plasma levels of steroid conjugates are increased in several patients having pathologies such as acne and hirsutism which are related to increased 5α-reduced C19 steroid formation[9]. The increase in the plasma concentration of 5α-reduced C19 steroid glucuronides in women with hirsutism is further stimulated when the plasma concentrations of the adrenal precursors dehydroepiandrosterone (DHEA) or its sulfate (DHEAS) are elevated9, 10.

It is widely accepted that the liver is a major site of glucuronidation. However, it is now clear that extrahepatic tissues are also involved in the conjugation of compounds to which these tissues are exposed. Glucuronidation has been demonstrated in the kidney, gut, lung, skin, brain, fat, thymus, prostate and breast[11]. As well, we have demonstrated high levels of 5α-reduced C19 steroid glucuronides in human prostate, breast cyst fluid and ovary follicular fluid suggesting that glucuronidation of 5α-reduced C19 steroids occurs in these tissues[7].

Fig. 1 illustrates the role of UGT in the metabolism of DHT which is an important physiological regulator in the prostate. It is generally believed that the concentration of DHT in the cell is dependant upon (1) the availability of circulating precursor C19 steroids DHEA, DHEA-S, androst-5-ene-3β-17β-diol (5-diol), 5-diol-sulfate and androstenedione (4-dione) from the adrenal and 4-dione and testosterone from the testis and (2) the activity of steroid transforming enzymes 3β-hydroxysteroid dehydrogenase 4-ene-5-ene isomerase, 3α-hydroxysteroid dehydrogenase, 17β-hydroxysteroid dehydrogenase and 5α-reductase. In the concept of intracrinology[12], the expression of enzymes involved in steroid metabolism regulate the level and action of steroid hormones in steroid target tissues. Enzymes that catalyze the conversion and production of steroids are well understood however, enzymes that catalyze the conjugation and catabolism of steroids, including UGT enzymes, have received much less attention. Considering the high circulating levels of glucuronidated DHT metabolites such as ADT and 3α-diol, and the presence of UGT enzymes found in several steroid target tissues, we propose that glucuronidation plays a prominent role in regulating the levels of DHT in these tissues.

To investigate the metabolism of androgens, the prostate tumor cell line LNCaP, an androgen-dependant cell line derived from a lymph node carcinoma of a prostate cancer patient was used. Despite the inconvenience of having a mutated androgen receptor for the study of steroid action, there is now good evidence that LNCaP cells possess all the enzymes necessary to transform C19 steroids by a metabolic pathway identical to that occurring in the human prostate, including the formation of steroid glucuronides. Complete characterization of these metabolites was performed using mass spectrometry and demonstrated the formation of 3-hydroxysteroid and 17-hydroxysteroid glucuronides[13].

In this review, the characterization of two steroid UGT enzymes expressed in LNCaP cells and in extrahepatic steroid target tissues is presented and the mechanisms regulating the expression and activity of these UGTs in LNCaP cells is described.

Section snippets

A specific human UGT enzyme for 3-hydroxy-C19 steroids and 17-hydroxy-C19 steroids: UGT2B17

To isolate the cDNA encoding the C19 steroid UGT isoenzyme, an LNCaP cell and a human prostate cDNA library was screened using a pool of radiolabelled cDNA probes synthesized from the human UGT2B7, UGT2B10 and UGT2B15 cDNAs previously isolated from human liver. The UGT2B17 cDNA isolated is 2107 bases in length and contains an open reading frame of 1590 bases flanked by a 5′-untranslated region of 51 bp and a 3′-untranslated region of 463 bp (Fig. 2). Amino acid sequence alignment shows that

Acknowledgements

This work was supported by the Medical Research Council (MRC) of Canada, the Fonds de la recherche en Santé du Québec, and Endorecherche. Éric Lévesque is holder of a scholarship from the MRC of Canada.

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    UDP-glucuronic acid is the activated donor molecule for glucuronidation. Subsequently, the glucuronic acid is coupled with a steroid hydroxy group leading to the formation of the steroid β-D-glucuronide, whereby the stereochemistry of the steroid in the respective position is preserved (Fig. 5(b)) [173,174]. These reactions are catalyzed by enzymes of the UGT-glucuronosyltransferase superfamily (UGTs), with the UGT1A and UGT2B subfamily catalyzing the glucuronidation of steroids [175].

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1

Proceedings of the 13th International Symposium of the Journal of Steroid Biochemistry & Molecular Biology “Recent Advances in Steroid Biochemistry & Molecular Biology” Monaco, 25–28 May 1997.

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