Activation of Human Liver 3a-Hydroxysteroid Dehydrogenase by Clofibrate Derivatives

The NADP-dependent dehydrogenase activity of a predominant isoenzyme of human liver 3a-hydroxysteroid dehydrogenase was activated by antihyperlipidemic drugs, such as bezafibrate and clinofibrate, and by clofibric acid and fenofibric acid (active metabolites of clofibrate and fenofibrate, respectively). The optimal pH of the activation by the drugs was about 7.5, and the concentrations giving maximum stimulation (1.8to 2.4-fold) were 100, 50, 400 and 50 mM for bezafibrate, clinofibrate, clofibric acid and fenofibric acid, respectively. Clofibrate and fenofibrate acted as weak inhibitors, and the clofibric acid derivatives that lack the chloro group, methyl group on the a-carbon or carboxyl group greatly decreased the stimulatory effects. The activation by the drugs increased both Km and kcat (turnover number) values for the coenzyme and substrates. Kinetic analysis with respect to NADP showed that bezafibrate, clinofibrate, clofibric acid and fenofibric acid were nonessential activators showing dissociation constants of 32, 6, 103 and 11 mM, respectively. The combined activators experiments with one of the above drugs and sulfobromophthalein, a known activator specific for this enzyme, and comparison of their effects on the activities of mutant enzymes (with Met replacing Lys-270 or Arg-276) indicated that sulfobromophthalein and the drugs bind to an identical site on the enzyme. These results suggest that the long-term therapy with the antihyperlipidemic drugs influences the metabolism of steroid hormones, bile acids and several ketone-containing drugs mediated by the enzyme. 3aHSD distributes in mammalian tissues, and is involved in the biosynthesis and inactivation of steroid molecules and the regulation of steroid hormone action (Penning et al., 1996). For example, the enzyme has been reported to work in concert with 5a-reductase to regulate the intracellular concentration of 5a-dihydrotestosterone, 5a-androstane-3a,17bdiol and 3a-hydroxy-5a-dihydroprogesterone that play key roles in prostate growth (Lombardo et al., 1992), parturition (Mahendroo et al., 1996) and modulation of the activity of g-aminobutyric acid receptor (Majewska et al., 1986; Martini et al., 1993). In the liver 3aHSD inactivates circulating steroid hormones and plays a role in the bile acid synthesis (Tomkins, 1956; Usui and Okuda, 1986). The outstanding feature of mammalian hepatic 3aHSD is its broad specificity for prostaglandins, drug ketones and trans-dihydrodiols of aromatic hydrocarbons, as well as the steroids (Wörner and Oesch, 1984; Penning et al., 1986; Hara et al., 1988; Ohara et al., 1994, 1995), and the enzyme has the ability to bind bile acids (Stolz et al., 1987). These findings have suggested that this enzyme also acts as prostaglandin oxidoreductase, carbonyl reductase, dihydrodiol dehydrogenase and bile acidbinding protein in the tissue. In human liver, 3aHSD with dihydrodiol dehydrogenase activity exists in multiple forms (Hara et al., 1990; Takikawa et al., 1992), and at least four types of cDNAs for the enzyme have been cloned from human liver cDNA library (Qin et al., 1993; Stolz et al., 1993; Deyashiki et al., 1994). The 3aHSD isoforms reveal 83 to 98% sequence identities, belong to the AKR superfamily, and have been systematically named as AKR 1C1, 1C2, 1C3 and 1C4 (Jez et al., 1997). AKR 1C1, the cDNA for which was originally cloned as that encoding hepatic bile acid-binding protein (Stolz et al., 1993), is identified with human liver dihydrodiol dehydrogenase isoform 1 that exhibits high 20a-hydroxysteroid dehydrogenase and very low 3aHSD activities (Hara et al., 1990, 1996). AKR 1C2 has been shown to be dihydrodiol dehydrogenase isoform 2 and bile acid-binding protein of human liver (Hara et al., 1996), and AKR 1C4 is identical with hepatic dihydrodiol dehydrogenase isoform 4 (Deyashiki et al., 1995) and chlordecone reductase (Winters et al., 1990). Although recombinant AKR 1C3 possesses low 3aHSD activity for limited 3a-hydroxyReceived for publication August 29, 1997. 1 This study was supported by a grant from the Ministry of Education, Science, Sports and Culture of Japan and by Suzuken Memorial Foundation. ABBREVIATIONS: 3aHSD, 3a-hydroxysteroid dehydrogenase; AKR, aldo-keto reductase; K270M, AKR 1C4 with Met replacing Lys-270; R276M, AKR 1C4 with Met replacing Arg-276. 0022-3565/98/2853-1096$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 285, No. 3 Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 285:1096–1103, 1998 1096 at A PE T Jornals on O cber 8, 2017 jpet.asjournals.org D ow nladed from steroids (Khanna et al., 1995), its protein has not been identified in human tissues. Purification of the human liver 3aHSD shows that its predominant form is the AKR 1C4, which has high activity for various 3a-hydroxysteroids including bile acids, in contrast to low activity of AKR 1C1 and 1C2 for some androgens and progestins (Hara et al., 1990; Deyashiki et al., 1992). From pharmaceutical and pharmacological points of view, our previous studies focused to elucidate the effects of drugs on the activities of the human liver 3aHSD isoforms involved in the metabolism of endogenous and xenobiotic compounds, and showed the inhibition of AKR 1C2 and 1C4 by antiinflammatory drugs (Deyashiki et al., 1992), and the activation of AKR 1C4 by sulfobromophthalein (Matsuura et al., 1996). We have found that of the four 3aHSD isoforms AKR 1C4 was activated by antihyperlipidemic clofibrate derivatives that are therapeutically administered for a long period, and show the specificity in terms of the structure of activating molecule and binding site of the enzyme, and the effect on the kinetic properties.