Molecular structure of rat hepatic 3 alpha-hydroxysteroid dehydrogenase. A member of the oxidoreductase gene family.

3-alpha-Hydroxysteroid dehydrogenase (3 alpha-HSD) (EC 1.1.1.50) is an important multifunctional oxidoreductase capable of metabolizing steroid hormones, polycyclic aromatic hydrocarbons, and prostaglandins. 3 alpha-HSD is also required for bile acid synthesis and has been suggested to play an important role in net bile acid transport across the hepatocyte (Stolz, A., Takikawa, H., Ookhtens, M., and Kaplowitz, N. (1989) Annu. Rev. Physiol. 51, 166-177). In order to characterize molecular forms and begin to determine its regulation, we now report the nucleotide sequence, tissue distribution, and homology to other members of the oxidoreductase superfamily. Rat hepatic 3 alpha-HSD cDNA encodes for a 322-amino acid protein with a predicted molecular weight of 37,022 expressed in a 2.4-kilobase (kb) message size. Northern blot analysis of total RNA revealed equivalent steady-state levels in liver and intestine in male rats with lower levels of expression in the colon and minimal expression in stomach, lung, and testis. Female liver contained approximately 2-3-fold greater steady-state levels of mRNA as compared to the male liver with equivalent intestinal expression. Two hybridizing bands, 2.4 and 1.4 kb, were identified in total RNA from the ovary. 3 alpha-HSD exhibits 75% amino acid sequence homology with bovine lung prostaglandin F synthetase and 50% homology with human aldose reductases. Amino acid sequence analysis with short chain alcohol dehydrogenases identified a possible NADP(H) cofactor-binding site at the amino terminus. The significant homology of 3 alpha-HSD with both prostaglandin F synthetase and aldose reductases suggest a subdivision of monomeric, NADPH reductases within the larger oxidoreductases superfamily.

3-a-Hydroxysteroid dehydrogenase (3a-HSD) (EC 1.1.1.50) is an important multifunctional oxidoreductase capable of metabolizing steroid hormones, polycyclic aromatic hydrocarbons, and prostaglandins. 3a-HSD is also required for bile acid synthesis and has been suggested to play an important role in net bile acid transport across the hepatocyte (Stolz, A., Takikawa, H., Ookhtens, M., and Kaplowitz, N. (1989) Annu. Rev. Physiol. 51,[166][167][168][169][170][171][172][173][174][175][176][177]. In order to characterize molecular forms and begin to determine its regulation, we now report the nucleotide sequence, tissue distribution, and homology to other members of the oxidoreductase superfamily. Rat hepatic 3a-HSD cDNA encodes for a 322-amino acid protein with a predicted molecular weight of 37,022 expressed in a 2.4-kilobase (kb) message size. Northern blot analysis of total RNA revealed equivalent steady-state levels in liver and intestine in male rats with lower levels of expression in the colon and minimal expression in stomach, lung, and testis. Female liver contained approximately 2-3-fold greater steady-state levels of mRNA as compared to the male liver with equivalent intestinal expression. Two hybridizing bands, 2.4 and 1.4 kb, were identified in total RNA from the ovary. 3a-HSD exhibits 75% amino acid sequence homology with bovine lung prostaglandin F synthetase and 50% homology with human aldose reductases. Amino acid sequence analysis with short chain alcohol dehydrogenases identified a possible NADP(H) cofactor-binding site at the amino terminus. The significant homology of 3ar-HSD with both prostaglandin F synthetase and aldose reductases suggest a subdivision of monomeric, NADPH reductases within the larger oxidoreductases superfamily.
3-a-Hydroxyskroid dehydrogenase (3a-HSD)' (EC 1.1.1.50) is an important, multifunctional reductase which metabolizes steroid hormones and polycyclic aromatic hydrocarbon carcinogens (1,2). 3a-HSD catalyzes the stereospecific reduction of cortisol, progesterone, and testosterone by preferentially utilizing the cofactor NADPH. Reduction of all these hormones is rapidly followed by conjugation at either the 3 or 17 position with sulfates or glucuronides leading to their ultimate elimination. In addition to its role in steroid hormone metabolism, 3a-HSD serves a dual function in rat liver being required for both bile acid synthesis and the efficient intercellular transport of bile acids (3)(4)(5)(6)(7). 3a-HSD stereospecifically reduces the bile acid precursors, 7-a,5-P-cholestane-3one and 7-a,l2-a-dihydroxyl-5-~-cholestane-3-one formed during the synthesis of the primary bile acids. We have previously demonstrated that bile acids binding to the cytosolic 3a-HSD plays an important role in efficient intercellular transport of bile acids from the sinusoidal to the canalicular pole of the rat hepatocyte (4).
Overlapping substrate specificities of the 3a-HSD with other reductases and purification of only a few forms has hindered precise knowledge about isoforms, detailed mechanism of enzymatic catalysis, and gene regulation. In order to further characterize the rat hepatic Sa-HSD at both the structural level and begin to analyze its regulation, we have identified and sequenced the rat hepatic 3a-HSD cDNA. This cDNA sequence encodes for a protein of 322 amino acids with a molecular mass of 37,022 daltons contained in a 2.4-kb message. We now report the deduced cDNA sequence, tissue distribution, and significant homology to other members of the oxidoreductase superfamily.

DISCUSSION
The organ distribution of 3a-HSD mRNA expression concurs with the previously determined immunoreactivity for the Portions of this paper (including "Materials and Methods," "Results," Figs. 1, 2, 4-7, and Table 1) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

Molecular Cloning of Rat Hepatic 3a-Hydroxysteroid
Dehydrogenase copurified Y' bile acid binder and for indomethacin inhibitable 3a-HSD activity ( Fig. 4) (44,45). In contrast, rat dihydrodiol dehydrogenase activity is greatest in the liver and lung followed by heart and intestine (45). Only liver and intestinal dihydrodiol dehydrogenase activity is significantly inhibited by 6-medroxyprogesterone acetate suggesting that the intestinal dihydrodiol dehydrogenase activity is catalyzed by the hepatic form of the 3a-HSD. Our finding of significant hepatic 3a-HSD mRNA in the small intestine is consistent with these prior findings. Purification and sequence analysis of rat di-

C C C C C C T C C C T C T G T T A C C C A T G T T A T G G T A T A A A T T C A G C C T T G G T A A A~T 2242
FIG. 3. Nucleotide sequence and deduced amino acid sequence for rat hepatic 3a-HSD cDNA. The single line represents location of amino acid sequences corresponding to peptides in Table  I  hydrodiol dehydrogenase activity in non-hepatic and intestinal tissues will be required to determine the precise relationship between the hepatic 3a-HSD and dihydrodiol dehydrogenase activity in these other tissues. The predominant localization of 3a-HSD in the liver and intestine suggests a role in xenobiotic metabolism.
The localization of 3a-HSD activity in rat brain and ovary suggests an important role in the metabolism of progesterone and dihydrotestosterone. 3a-HSD activity has also been identified in non-neuronal cells of the olfactory tubercles, pituitary cytosol, and has been purified to homogeneity from total rat brain (46-49). This purified enzyme is potently inhibited by 6-medroxyprogesterone as is the hepatic 3a-HSD activity. In the pituitary, 3a-HSD has been implicated in regulation of both ovulation and gonadotropins by reduction of 5a-pregane-3,20-dione. The speculated role of the 3a-HSD in the olfactory tubercle is to metabolize dihydrotestosterone which may mediate gonadotropin-releasing hormone release. The failure to identify 3a-HSD message in total brain RNA may be due to either low or localized levels of 3a-HSD mRNA or that the 3a-HSD activity reflects the product of another gene.
Ovarian 3a-HSD activity has been implicated in playing an important role in ovarian follicular development by its capacity to metabolize androgens. Dihydrotestosterone inhibits aromatase activity which is critical for follicular development. Ontogeny studies and selective irradiation of granulosa cells implicate the granulosa cells as the site of 3a-HSD activity (50-52). Ovarian Sa-HSD activity fluctuates with estrus and is regulated by gonadotrophins or gonadotrophin-induced steroids indicating a complex mode of regulation. Studies are presently underway to define the cellular localization of the 3a-HSD and to determine the significance of the 1.4-kb hybridizing mRNA.
3a-HSD exhibits striking amino acid homology to both bovine lung prostaglandin F synthetase and human aldose reductases. All these proteins are members of the aldo-keto reductase superfamily, a large family of proteins capable of reducing carbonyl groups on a wide variety of compounds, including sugars, steroid hormones, ketones, and xenobiotic aldehyde. Prostaglandin F synthetase is capable of metabolizing some of the same xenobiotics as the rat hepatic 3a-HSD, but inefficiently reduces dihydrotestosterone (35). The competitive inhibition of 3a-HSD by indomethacin and its capacity to oxidize hydroxyprostaglandins confirms the close homology between these two proteins (2, 8). Comparison of these monomeric reductases to the dimeric dehydrogenase gene family identifies the NADP(H) cofactor-binding site to the amino terminus, whereas this binding site resides in the middle of the dimeric proteins (39).
In conclusion, we have identified the cDNA for the rat hepatic 3a-HSD. The predominant location of the 3a-HSD in the liver and intestine is consistent with its role in xenobiotic metabolism and participation in the intracellular transport of bile acids. Future studies will require the cDNA analysis of other isoforms of 3a-HSD and dihydrodiol dehydrogenase activity to determine their relationship among themselves and to both the dimeric short/long chain alcohol/ poly01 and aldo-keto reductase superfamilies. Significant homology is presumed to exist in the genomic organization of these various genes and may provide insight into mechanism of gene evolution in these large protein superfamilies.

Material and Methods:
Purrhed 30-HSD war prepared as previously reported from male rat livers (7). 33 01 66 ug of protein was lyophilized, underwent reduclion and alkylanon (16) and was then deralled by reverse phase HPLC chromatography on a Vydac C 4 (250 mm 3, 2. I and 0.5 ug of total RNA from male and female lwer, intewnes and colon adjusted 10 equivaicnt amounts with "an-hybridimmg total rat kidney RNA were blotted onto Nytran membrane, hybridized as descnbed above and exposed to Kodak XAR film soalysis using an LKB densitometer (Pharmacia, Piscataway. NJ I.

Results:
cDNA cloning: for both designing oligonucleotide probes for screening cDNA libraries and for verification of the isolated eDNA. Figure  I illustrates one of the two 30-HSD peptide maps. Five peptide fragments were !solated from lhlr peptide map and sequenced for these objectives. The initial cDNA cloning of lhc rat hepatic 30-HSD was performed utilizing a "on-degenerate oligonucleotide probe based on peptide sequence data which was 87% identical to the actual <DNA sequence. Figure 2 illustrates the oligonucleotide probe used for inins1 library screening. restriction map and cloning suategy utilized for the cDNA sequencing. Figure 3 contains the nucleotide sequence with its deduced amino acid sequence.
The carboxyl terminal is clearly identified by the presence  Table  I). Drrpite multiple attempts. no amino terminus amino acid sequence was obtained. Three lines of evidence support 'he location of the initial methionine in Figure 3.
First. this ATG is the only sequence capable of maintaining the proper reading frame so that all Seven pcptide sequences may be identified. Second. nucleotide sequence sunounding this ATC is homologous IO the eukaryoue c o n~e n s u~ sequence of (AICjCCATG identified by at position + 4 (28). Third. predicted molecular weight of 31 kD is similar to the Kozack with the most highly conserved features of a purine at position -3 and guanine observed migration of 33 kD on SDS-polyacrylamide gel electrophoresis (7). Table I Identifies the amino acids sequence of all seven peptides determined by microsequencc analysis with their codon and nucleotide position on the cDNA. Amino acid sequence analysis of peptides identifled one or two additional amino aclds for a given position and may represent microhetcrogeneity or the presence of a contaminating peptide. The correlation of all amino acid sequences 10 the eDNA confirms that the cDNA Clone corresponds to the 30-HSD protein. The RNA message size of 2.4 kB also agrees with the cDNA clone identified.
cDNA cloning of the 3a-HSD gene was dependent on amino acld sequence analysis

Tmwa Diswihulion:
Tissue distribution of rat hepatic 30-HSD was investigated by northern blot analysis. 10 ug of total cellular RNA was probed with the proxnmal 5' Eco RI fragment different organs from adult male and female Sprague-Dawley rats. In the male rat.
of the cDNA clone (135 L1. Figure 2) . Figure 4 illustrarcr the northern blot analysis of 10 liver and inlestine divided into three equal parts expressed equivalent amounts where as the colon expressed reduced levels. After prolonged exposure. minimal message was detected in lung. stomach. hcan and testis. After a 10 day exposure. a barely visiblc 2.4 kB message was observed in total RNA from male brain (data not shown). In female ram. liver expressed greater levels then intestine and colon which were equivalent 10 level$ found in the male. Afler prolangcd exposure. 30-HSD message was also identified in lung and heart. Inlerestmgly,

Hnrnnlop? Strrdics:
PASTA algorithm of Pearron to explore the rclationship of the 3m-llSD to athcr oxidoreductases (30). Significant homology w w found to Bovinc lung proslsglandm P rynthetasc OiC I.I.I.IX) IPGFSI and lluman aldose reductases (EC 1.1.1.21) IALDRI. two othcr NADPH depcndent. monomeric carbonyl reductases. and to Epsilon-cryslnllinc lens protein of the European common frog 01-84). 70% amino acid idcntity was found with via endoperoxidare activity and converting PGD2 to 9o-llB-PCF2 via Il.keto reductase Bovine lung prostaglandin F synthemsc. an cnlymc capable of reducing PGH2 to PFG2o activity (35). Wantanabc has previously demonstrated that rat prostaglandin F synthetase activity is mrrimal in cytosol of lung. followed hy decrcascd l c v e l~ i n different than hepatic 3a-HSD sene expression. Human aldose reductases shams 47% stomach and heart with less activity in kidney and liver (36). This patlern is markedly amino acid identity with 3o.tISD protein. Figure 6 illustrates the alignment. homology and consensus sequcncc of these two reduclares with Ihc newly idenufted 3a-lISD.
Significant homology i s apparent i n a11 three proteins. 3o-HSD also sharer 50% homology wilh the partially sequenced epsilon crystalline lens proteins from common frog (data crysr3llines of vertebrates and invcrtebrales ha$ k e n well described (37.38). Thc