Relative enzymatic activity, protein stability, and tissue distribution ofhuman steroid-metabolizing UGT2B subfamily members

Androgens and estrogens play major roles in cell differentiation, cell grow th, and peptide secretion in steroid target tissues. In addition to the bin ding of these hormones to their receptors, formation and metabolism are imp ortant in the action of steroids. Metabolism of the potent steroid hormones includes glucuronidation, a major pathway of steroid elimination in liver and several steroid target tissues. Glucuronidation is catalyzed by UDP-glu curonosyltransferases (UGTs), which transfer the polar moiety from UDP-gluc uronic acid to a wide variety of endogenous compounds, including steroid ho rmones. The UGT superfamily of enzymes is subdivided into two families, UGT 1 and UGT2, on the basis of sequence homology. To date, six UGT2B proteins have been isolated, namely UGT2B4, UGT2B7, UGT2B10, UGT2B11, UGT2B15, and U GT2B17, all of which have been demonstrated to be active on steroid molecul es, except for UGT2B10 and UGT2B11, for which no substrate was found. The r elative activity of these enzymes on steroidal compounds remains unknown du e to variable levels of UGT2B expression in different in vitro cell line mo dels and various conditions of the enzymatic assays. Comparison of the gluc uronidation rates of these enzymes requires a unique system for UGT2B prote in expression, protein normalization, and enzymatic assays. In this study w e have stably expressed UGT2B4, UGT2B7, UGT2B15, and UGT2B17 in the HK293 c ell line, which is devoid of steroid UGT activity; characterized their kine tic properties relative to UGT protein expression; determined their transcr ipt and protein stabilities; and established extensively their tissular dis tributions. UGT2B7 was demonstrated to glucuronidate estrogens, catechol es trogens, and androstane-3 alpha ,17 beta -diol more efficiently than any ot her human UGTB isoform. UGT2B15 and UGT2B17 showed similar glucuronidation activity for androstane-3 alpha ,17 beta -diol (30% lower than that of UGT2 B7), whereas UGT2B17 demonstrated the highest activity for androsterone, te stosterone, and dihydrotestosterone. UGT2B4 demonstrates reactivity toward 5 alpha -reduced androgens and catechol estrogens, but at a significantly l ower level than UGT2B7, 2B15, and 2B17. Cycloheximide treatment of stably t ransfected HK293 cells demonstrated that the UGT2B17 protein is more labile than the other enzymes; the protein levels decrease after 1 h of treatment , whereas other UGT2B proteins were stable for at least 12 h. Treatment of stable cells with actinomycin D reveals that UGT2B transcripts are stable f or 12 h, except for the UGT2B4 transcript, which was decreased by 50% after the 12-h incubation period. Tissue distribution of the UGT2B enzymes demon strated that UGT2B isoforms are expressed in the liver as well as in severa l extrahepatic steroid target tissues, namely, kidney, breast, lung, and pr ostate. This study clearly demonstrates the relative activities and the maj or substrates of human steroid-metabolizing UGT2B enzymes, which are expres sed in a wide variety of steroid target tissues.