©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Substrate Specificity, Gene Structure, and Tissue-specific Distribution of Multiple Human 3-Hydroxysteroid Dehydrogenases (*)

(Received for publication, March 2, 1995 )

Marilyn Khanna (1) Ke-Nan Qin (1) Regina W. Wang (2) K.-C. Cheng (1)(§)

From the  (1)Department of Pediatrics, Cornell University Medical College, New York, New York 10021 and the (2)Department of Drug Metabolism, Merck, Sharp & Dohme Research Laboratories, Rahway, New Jersey 07065

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

We have expressed in Escherichia coli functionally active proteins encoded by two human cDNAs that were isolated previously by using rat 3alpha-hydroxysteroid dehydrogenase cDNA as the probe. The expressed proteins catalyzed the interconversion between 5alpha-dihydrotestosterone and 5alpha-androstane-3alpha,17beta-diol. Therefore, we name these two enzymes type I and type II 3alpha-hydroxysteroid dehydrogenases. The type I enzyme has a high affinity for dihydrotestosterone, whereas the type II enzyme has a low affinity for the substrate. The tissue-specific distribution of these two enzymes was determined by reverse transcription polymerase chain reaction using gene-specific oligonucleotide primers. The mRNA transcript of the type I enzyme was found only in the liver, whereas that of the type II enzyme appeared in the brain, kidney, liver, lung, placenta, and testis. The structure and sequence of the genes encoding these two 3alpha-hydroxysteroid dehydrogenases were determined by analysis of genomic clones that were isolated from a EMBL3 SP6/T7 library. The genes coding for the type I and type II enzymes were found to span approximately 20 and 16 kilobase pairs, respectively, and to consist of 9 exons of the same sizes and boundaries. The exons range in size from 77 to 223 base pairs (bp), whereas the introns range in size from 375 bp to approximately 6 kilobase pairs. The type I gene contains a TATA box that is located 27 bp upstream of multiple transcription start sites. In contrast, the type II gene contains two tandem AP2 sequences juxtaposed to a single transcription start site.


INTRODUCTION

3alpha-Hydroxysteroid dehydrogenase (3alpha-HSD) (^1)belongs to the aldo-keto reductase family, which includes aldehyde reductase(1) , aldose reductase(2) , dihydrodiol dehydrogenase(3) , bovine prostaglandin F synthase(4) , frog eye lens -crystallin(5) , human chlordecone reductase(6) , and numerous related proteins recently identified in rats and humans in our laboratory(7) . These enzymes catalyze the conversion of aldehydes and ketones to alcohols by utilizing NADH and/or NADPH as the cofactor and exist in cellular cytoplasm as monomeric 34-36-kDa proteins. These enzymes exhibit distinct but overlapping substrate specificities and are inhibited by a number of drugs, such as phenobarbital, pyrazole, chlorpromazine, indomethacin, sodium valproate, quercetin, and ethacrynic acid(8) .

3alpha-HSD may be the most versatile enzyme in the aldo-keto reductase family due to its ability to utilize a large array of substrates. For example, rat 3alpha-HSD also carries dihydrodiol dehydrogenase activity (9) . The importance of dihydrodiol dehydrogenase in the detoxification of polycyclic aromatic hydrocarbons was demonstrated by its ability to reduce benzo(a)pyrene mutagenic activity in the Ames test (10) . 3alpha-HSD also converts bile acid precursors to bile acids and may serve as a bile acid transporter in the liver and intestine(11, 12) .

3alpha-HSD in the liver is responsible for inactivation of steroid hormones and maintenance of the homeostatic balance of circulating steroid hormones. Nevertheless, not all of the 3alpha-HSD metabolites are physiologically inactive. For example, the 3alpha-hydroxy A-ring-reduced pregnane steroids (allopregnanolone) have been shown to exert sedative, hypnotic, and anesthetic effects when they were administered to animals (13) . In addition, these tetrahydrosteroids also produce a number of other behavioral effects, such as anticonflict, anticonvulsant, and analgesic actions(14) . These neurological effects have been linked to the binding of the tetrahydrosteroids to the major inhibitory -aminobutyric acid receptor complex. It was recently shown that the levels of tetrahydrosteroids, such as allopregnanolone and allotetrahydrodeoxycorticosterone, were elevated rapidly and robustly in the brain and plasma of rats after exposure to ambient temperature swimming stress(15) . Administration of progesterone to healthy humans induced changes in fatigue and in delayed verbal recall; these changes correlate with the levels of 3alpha-tetrahydro-metabolites. These studies support the hypothesis that 3alpha-tetrahydrosteroids are endogenous modulators of the gamma-aminobutyric acid receptor(16) .

It was previously suggested (17, 18) that multiple 3alpha-HSD isozymes exist in the human. In this report, we demonstrate that proteins encoded by two of the cDNAs we previously isolated carry 3alpha-HSD activity. In addition, we compare the enzyme characteristics and describe the gene structure of these steroid hormone-metabolizing enzymes.


EXPERIMENTAL PROCEDURES

Materials

Restriction endonucleases and related reagents were purchased from International Biotechnologies. Total cellular RNA samples from human tissues were purchased from Clontech. The Prism sequencing kit, reverse transcriptase, and RNase inhibitor were obtained from Perkin-Elmer. Other reagents were obtained from Sigma.

Expression and Characterization of Type I and Type II 3alpha-Hydroxysteroid Dehydrogenases

Type I and type II 3alpha-HSD cDNAs were amplified by polymerase chain reaction using the following pairs of oligonucleotides: 5`-GTACCCGGGATGGATCCCAAATATCAG-3` with 5`-GTACCCGGGCTAATATTCATCTGAAAA-3` and 5`-GTAGAATTCATGGATTCCAAACACAAG-3` with 5`-GTAGAATTCATGTTAATATTCATCTGA-3`. The amplified DNA was treated with restriction endonuclease (either SmaI or EcoRI) prior to ligation with SmaI- or EcoRI-treated pKK2.7 expression vector(19) . The ligated product was used for transforming Escherichia coli 1899. Ampicillin-resistant clones were selected by plating the bacteria on LB agar plates containing 50 µg/ml ampicillin. Clones expressing the human type I enzyme were analyzed by Western immunoblotting using a monoclonal antibody (9G3) raised against rat 3alpha-hydroxysteroid dehydrogenase, and clones expressing the type II enzyme were analyzed by a polyclonal antibody raised against a purified human 3alpha-HSD(20) .

Bacteria were harvested from a 10-liter batch of overnight culture by centrifugation. The bacterial pellet was resuspended in 1 liter of 10 mM Tris-HCl buffer, pH 7.4, and then subjected to pulse sonication. Bacterial cytosols containing the expressed enzyme were recovered from the sonicated mixture by centrifugation at 10,000 g for 10 min.

Conversion of 5alpha-dihydrotestosterone to 5alpha-androstane-3alpha,17beta-diol by the bacterial cytosol was determined according to a procedure previously established(21) . The reaction mixture in a 1-ml final volume contains 1-20 nmol of dihydrotestosterone, 10 nCi of [^14C]5alpha-dihydrotestosterone, 1 mM NADPH, 300 µg of bacterial lysate, and 100 mM sodium phosphate, pH 7.4. The reaction was performed at 37 °C for 10 min. The metabolite and remaining substrate were extracted with methylene chloride and were separated by thin-layer chromatography using a mixed solvent system (chloroform/ethyl acetate/ethanol (4:1:0.2)). The 3alpha-hydroxyl metabolite was identified based upon the R value previously established(21) .

5alpha-Androstane-3alpha,17beta-diol was converted from 5alpha-dihydrotestosterone by using rat 3alpha-HSD that we had previously expressed in bacteria(19) . Assays of the 3alpha-dehydrogenase activity of the expressed human enzymes were carried out in a reaction mixture that contained 3 nmol of 5alpha-androstane-3alpha,17beta-diol, 10 nCi of 5alpha-androstane-3alpha,17beta-diol, 300 µg of bacterial lysate, 1 mM NADP, and 100 mM sodium phosphate, pH 7.4, in a final volume of 1 ml. Extraction and separation of metabolites were performed according to the procedure described above.

The activity of the expressed human 3alpha-HSD was also determined using the substrates chenodeoxycholic acid, acenaphthenol, and androsterone according to a procedure established previously(19) . The protein concentration was measured by the Lowry procedure(22) .

Library Screening and Sequencing of DNA

A human genomic library in EMBL SP6/T7 (Clontech) was screened using human HAKRa and HAKRb cDNAs that were isolated in our laboratory as the probes (23) . The library was plated out on host E. coli NM539 cells, and lifts were taken on nitrocellulose membranes. Phage DNA attached to the membrane was denatured, neutralized, and cross-linked to the membrane by UV light. Plaque hybridization was performed at 55 °C in a solution containing 6 SSC (1 SSC = 0.15 M NaCl and 0.015 M sodium citrate, pH 7.5), 0.1% SDS, and 5 Denhardt's solution. Washing was performed at 60 °C in a solution containing 2 SSC and 0.1% SDS. Positive plaques were isolated and rescreened after dilution. Phages were isolated by the plate lysate method; DNA was extracted after treatment with phenol/chloroform. Polymerase chain reactions using specific primers were performed to amplify DNA sequences. The PCR products were isolated by electrophoresis in a low-melting-point agarose gel and binding to glass beads. Automated DNA sequencing of the PCR products was performed using a procedure previously described(24) .

Primer Extension

Two synthetic oligonucleotides, 5`-CTCTGCAGGCGCATAGGTGCCAAATCCCAG-3` and 5`-CTCTGGAGGTGCATAGGTGCCAAATCCCAA-3`, complementary to nucleotide positions 55-84 of the type I and type II cDNAs, respectively, were used as extension primers. 4 µg of human liver RNA was co-precipitated with 0.5 pmol of P-5` end-labeled primer, and the pellet was dissolved in a buffer for reverse transcription (25) . 200 units of Moloney murine leukemia virus reverse transcriptase, 20 units of RNase inhibitor, and 0.2 µM of dNTPs were added to the mixture for a final volume of 30 µl. Primer extension was performed at 37 °C for 1 h. Afterward, the sample was phenol/chloroform-extracted, ethanol-precipitated, resuspended in formamide dye, and denatured at 100 °C for 5 min prior to electrophoresis on a 6% polyacrylamide gel.

Reverse Transcription Polymerase Chain Reaction

Reverse transcription polymerase chain reaction (RT-PCR) was performed using gene-specific oligonucleotide primers(26) . The following oligonucleotide primers were synthesized for the reaction: human type I 3alpha-HSD, 5`-GGAACCCAACGACATAAACT-3` and 5`-GGCCAGGACCACAACCCCACGCT-3`; human type II, 5`-GGATCTCAACGAGACAAACG-3` and 5`-GGCCAGGACCACAACCCCACGCT-3`; and beta-actin, 5`-CGCCACATTGATACGGCCTCT-3` and 5`-GGAGATTCACTAACACTTGAC-3`. The single-stranded cDNA was synthesized using reverse transcriptase and random primers that were provided in the RT-PCR kit (Perkin-Elmer). 35 cycles of PCR amplification were routinely performed under the following conditions: 1 min at 94 °C, 1 min at 60 °C, and 2 min at 72 °C. Amplified DNA was resolved on a 2% agarose gel containing 5 µg/ml ethidium bromide and then visualized under UV light.


RESULTS

Expression and Characterization of Human 3alpha-HSDs

We have previously reported the overexpression of functionally active rat 3alpha-HSD in E. coli using a bacterial vector pKK2.7(20) . The same strategy was used here for the expression of human 3alpha-HSD. Expression of both type I and type II human 3alpha-HSDs in bacteria was confirmed by Western immunoblotting (Fig. 1). The expression of the type I 3alpha-HSD was detected by using a monoclonal antibody, 3G6, which was raised against the rat 3alpha-HSD(21) . This monoclonal antibody, however, could not recognize the type II 3alpha-HSD. In our preliminary studies, we have purified a human 3alpha-HSD that did not cross-react with monoclonal antibody 3G6 (data not shown). Using this purified human 3alpha-HSD as an antigen, we prepared a polyclonal antibody that appeared to be specifically against the type II 3alpha-HSD (Fig. 1B).


Figure 1: Western blot analysis of human type I and type II 3alpha-HSDs. A, monoclonal antibody 3G6 was used for the detection of the type I enzyme. B, a polyclonal antibody raised against a purified human 3alpha-HSD was used for the detection of the type II enzyme. Each lane contains 100 µg of protein from the bacterial lysate. The lanes labeled TYPE I and TYPE II contain lysates from bacteria that carry the type I and the type II expression vectors, respectively. The lane labeled CONTROL contains lysates from bacteria that carry the empty expression vector. The positions and molecular masses of protein size markers are shown on the left side of the blots.



Both type I and type II human 3alpha-HSDs could convert 5alpha-dihydrotestosterone to 5alpha-androstane-3alpha,17beta-diol in the presence of NADPH as the cofactor (Fig. 2). The apparent affinities as determined by double-reciprocal plots of the enzyme activities for the substrate appear to be rather different (Fig. 3A). The K of the type I enzyme is approximately 1 uM, whereas that of the type II enzyme is 20 uM (Fig. 3). Both enzymes were found to carry the dehydrogenase activity when 5alpha-androstane-3alpha,17beta-diol was used as the substrate. In addition, both forms of 3alpha-HSD were found to catalyze the 3alpha-dehydrogenation of androsterone (Table 1). These results suggest that human type I and type II enzymes contain both reductase and dehydrogenase activities.


Figure 2: Thin-layer chromatography separation of the substrate (5alpha-dihydrotestosterone) and the 3alpha-HSD product (5alpha-androstane-3alpha,17beta-diol). 300 µg of protein from bacterial lysate was used for the assay of 3alpha-HSD activity as described under ``Experimental Procedures.'' Both the ^14C-labeled substrate and the metabolite in the reaction mixture were extracted with methylene chloride and applied to a thin-layer chromatography plate. Separation of the substrate and the metabolite was performed using a mix-solvent system containing chloroform/ethyl acetate/ethanol (4:1:0.2). The substrate and the metabolite were identified based upon their Rvalues by autoradiography. The lane labeled Control contains the reaction mixture from the lysate of bacteria carrying an empty vector. The lanes labeled Type I and Type II contain the substrate and metabolite generated by the lysates of bacteria carrying the type I and type II expression vectors, respectively.




Figure 3: Conversion of 5alpha-dihydrotestosterone to 5alpha-androstane-3alpha,17beta-diol catalyzed by type I and type II human 3alpha-HSDs. A, double-reciprocal plots of the enzyme activity. B, apparent K and V(max) (mean ± standard error, n = 4) of the type I and type II 3alpha-HSDs. The K and V(max) parameters were determined based upon the double-reciprocal plots using an Enzfit program. Assay conditions were identical to those described in the legend of Fig. 2.





The type I and type II enzymes are catalytically active when 1-acenaphthenol was used as the substrate, suggesting that both enzymes carry dihydrodiol dehydrogenase activity. Much higher activities were found when chenodeoxycholic acid was used as the substrate, indicating that these two enzymes are involved in the production of bile acids in the liver (Table 1).

The Structure of Human 3alpha-HSD Gene

Approximately 500,000 phages from a human genomic library in the EMBL SP6/T7 vector were screened. Six positive clones were isolated when human type I (HAKRa) and type II (HAKRb) 3alpha-HSD cDNAs were used as the probe and showed different but overlapping restriction patterns (Fig. 4). Southern blot analysis further confirmed that three of the clones contained overlapping sequences of the type I gene and the other clones contained the type II sequences (data not shown). The 5`-flanking sequence and introns were identified by PCR using appropriate oligonucleotide primers. The type I 3alpha-HSD gene, which was derived from the overlapping clones, contains 9 exons and spans approximately 20 kb in length. The coding sequence of the type I 3alpha-HSD is identical to that of human chlordecone reductase(18) . The type II gene also contains 9 exons and spans approximately 15 kb. The coding sequences of the type I and the type II 3alpha-HSDs are different but highly homologous. The exon-intron junctions of both genes were identified by comparing the sequences with the cDNA sequences. All of the splice sites contained the GT splice donor and AG splice acceptor. The sequences around the donor and the acceptor conform with the consensus sequences for intronic donor (5`-GT(A/G)AGT-3`) and acceptor (5`-(Y)nN(C/T)AG-3`) splice signals (27) .


Figure 4: Structures of the type I and type II human 3alpha-HSD genes. A, regions of the type I gene contained in the three overlapping clones, KQ8, KQ7 and KQ26, are indicated by solid lines. B, regions of the type II gene contained in the clones, KQ11, KQ13 and KQ18, are also indicated by solid lines. The nine exons are numbered and represented by filled boxes. The size marker is shown on the bottom of the figure.



The exon-intron arrangements of the type I and type II genes appear to be very similar, except that the sizes of some of their corresponding introns vary considerably. The introns of the type I gene range in size from 375 bp to approximately 6 kb, whereas that of the type II gene range from 375 bp to approximately 4 kb. Three of the introns (introns 2, 3, and 5) in the type I gene are larger than that of the type II gene. Only two of the introns (introns 6 and 8) interrupt the coding sequence within codons.

The sizes and boundaries of each of the nine exons in these two genes are identical (Fig. 4). Exon 1 contains 84 bp of the translated sequence and some of the untranslated sequence. Exons 2-9 contain the rest of the coding sequence. In the type I 3alpha-HSD gene, exon 9 contains 42 bp of the translated sequence and 181 bp of the untranslated sequence. The untranslated sequence contains an AATAAA polyadenylation signal. In the type II 3alpha-HSD gene, exon 9 also contains 42 bp of the the translated sequence, but the untranslated sequence was only partially determined. The nine exons range in size from 77 to 223 bp. Fig. 5shows the alignment of the exons and intron-exon junctions of these two genes. Using a DNASTAR sequence alignment program to compare the homology between these two genes, we found high degrees of sequence identity in exons (85%) as well as in introns (60%).


Figure 5: Sequence comparison of the exons and intron-exon junctions of type I and type II human 3alpha-HSd genes. The coding nucleotide sequence is shown in capital letters. The amino acid sequence is displayed under the nucleotide sequence. Base numbering is based upon the the position of the amino acid codon in the coding sequence.



Transcription Start Site

The sizes of the mRNA transcripts of both type I and type II genes as detected by Northern blot analysis were approximately 1200-1400 nucleotides, which included 969 bp of coding sequence and approximately 180 bp of 3`-untranslated sequence(18) . Therefore, the 5`-untranslated sequence contains less than 200 nucleotides in the mRNA transcript. To locate the transcription start site(s), primer extension was performed using an antisense oligodeoxynucleotide primer complementary to part of the exon 1 sequence.

The primer extension experiment showed multiple extended products containing 109-111 nucleotides for the type I gene, indicating that the 5`-untranslated sequence of the gene consists of 25-27 nucleotides (Fig. 6A). For the type II gene, a single extended product containing 152 nucleotides was found, indicating that the 5`-untranslated sequence of the type II gene consists of 68 nucleotides (Fig. 6B).


Figure 6: Primer extension analysis. A synthetic oligonucleotide complementary to the nucleotide position 54 to 84 was used for the primer extension study. A, lane 1 shows the extension products from the type I gene. Lanes 2-5 show the sequence ladder generated with the extension primer on a PCR product. Lane 2, G; lane 3, A; lane 4, T; lane 5, C. B, lane 5 shows the extension product of the type II gene. Lanes 1-4 show the sequence ladder generated with the extension primer on a PCR product. Lane 1, G; lane 2, A; lane 3, T; lane 4, C.



Tissue-specific Distribution of Human 3alpha-HSD

Previously, we have shown by Northern blot hybridization that both type I and type II genes were predominantly expressed in the liver and moderately expressed in other tissues(18) . Because multiple human cDNAs that were found in the liver displayed high degrees of structural similarity to the human 3alpha-HSD, it is likely that these mRNAs will cross-hybridize with different probes. In order to obtain a more definitive result of the tissue distribution, we used gene-specific oligonucleotide primers in the reverse transcription polymerase chain reaction to analyze the presence of their mRNA transcript. These oligonucleotide primers were chosen from the most diverse sequence regions and could amplify only their respective genes(28) . In our preliminary RT-PCR experiments we have performed the PCR reactions for various cycles of amplification (e.g. 30, 35, and 40 cycles). The results of the preliminary study indicated that our RT-PCR reactions were at least semi-quantitative (data not shown). The result of the RT-PCR is shown in Fig. 7. The type I mRNA is expressed only in the liver. On the other hand, the type II mRNA is found in moderate levels in all the tissues that we examined, such as the brain, kidney, liver, lung, placenta, and testis.


Figure 7: RT-PCR of the expression of type I 3alpha-HSD, type II 3alpha-HSD, and beta-actin. RT-PCR was performed using the total RNA isolated from human brain, kidney, liver, lung, placenta, and testis as the template. Oligonucleotide PCR primers used in these three panels were specific for the genes of type I 3alpha-HSD, type II 3alpha-HSD, and beta-actin, respectively. 35 cycles of PCR amplification were performed under the following conditions: 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 2 min. The PCR products were electrophoresed in a 2% agarose gel and visualized after staining with ethidium bromide.



Sequence Comparison of the Human 3alpha-HSD 5`-Upstream Regions

The KQ8 contains approximately 1 kb of the 5`-upstream sequence of the human type I gene (Fig. 4). Sequence analysis of the region revealed several putative binding sites of transcription factors (Fig. 8). A TATA box consensus sequence (TATAAA) was found 27 bp upstream of the transcription start sites. We could not find any typical CAT box sequence (CAAT) upstream of the TATA box. Several AP1 like sequences were found within 400 of the first ATG initiation codon (Fig. 8). The KQ18 contains approximately 4 kb of the 5`-flanking sequence of the type II gene. In contrast to the type I gene, the type II gene does not contain a TATA box or a CAAT box. Two tandem AP2 sequences were found 8 bp upstream of the transcription start site, suggesting that these AP2 sequences may be involved in the control of the transcription.


Figure 8: Comparison of human type I 3alpha-HSD and type II 3alpha-HSD 5`-flanking sequences. Alignment of the two sequences was performed using the ALIGN program (DNASTAR). Identities between these two sequences are indicated by displaying the base in between the two lines of sequences. Dashes are inserted for optimal alignment. Base numbering is based upon the the position of the first ATG start codon as +1. The transcription initiation sites are marked with stars. The first ATG, TATA box, and potential AP1 and AP2 sequences in the genes are underlined.



Fig. 8shows the sequence alignment of the 5`-upstream sequences of the type I and type II genes. A 60% sequence identity was found between the 5`-upstream sequences of these two genes. Several gaps were introduced into the sequences in order to obtain the best alignment. In addition, there were several regions of sequences that displayed no significant homology. These diverse regions of the 5`-upstream sequence may contain tissue-specific promoter activity that causes the difference in the tissue-specific expression of these two genes.


DISCUSSION

In this paper we report the identification of two human 3alpha-hydroxysteroid dehydrogenases and their gene structures, which provide an opportunity for future studies on the regulation of their activity and tissue specificity. The existence of multiple forms of 3alpha-HSD in human liver has been suggested by purification of multiple proteins exhibiting 3alpha-HSD activity (17) and by molecular cloning of multiple human cDNAs encoding proteins structurally related to rat 3alpha-hydroxysteroid dehydrogenase(18) . The type I 3alpha-HSD shows a sequence identical to that of the human chlordecone reductase reported by Winters et al.(6) , except that the cDNA isolated by us contains a full-length coding sequence. As demonstrated here, the protein encoded by this full-length cDNA is shown to contain 3alpha-HSD activity. Recently Lou et al.(29) isolated a cDNA that was identical in sequence to one of the human cDNAs previously identified(18) . The protein encoded by this cDNA, obtained by expression in bacteria, exhibited dihydrodiol dehydrogenase/bile acid-binding protein (BABP) activity but not 3alpha-HSD activity(29) . The type II 3alpha-HSD appears to be different from but similar to the type I enzyme (human chlordecone reductase) and the dihydrodiol dehydrogenase/BABP. The gene structures of both human type I and type II 3alpha-HSD are similar to that of the dihydrodiol dehydrogenase/BABP as previously reported by us (24) and by Lou et al.(29) with the exception that type I 3alpha-HSD gene contains several large introns. Although a high degree of sequence homology was found between the 5`-flanking sequences of the type I and type II 3alpha-HSD, the transcripts of these two genes show a marked difference in the tissue-specific distribution. The type I 3alpha-HSD appears to be a liver-specific enzyme, whereas the type II 3alpha-HSD is constitutively expressed in most tissues. Future investigation into the molecular mechanism underlying the tissue-specificity of the multiple human 3alpha-HSDs and dihydrodiol dehydrogenase/BABP may yield a model system for elucidation of the regulatory element(s) responsible for the tissue-specificity. Our finding that multiple 3alpha-HSDs exist in humans further complicates the putative role of 3alpha-HSD in the production and regulation of neuroactive tetrahydrosteroids in the human brain. It is tempting to speculate that the type I 3alpha-HSD may play an essential role in the liver metabolism of steroid hormones, whereas the type II 3alpha-HSD may be responsible for the production of neuroactive steroids in the brain.

Molowa et al.(30) showed that an increase in the chlordecone reductase activity in gerbils appeared to be due to an increase in the transcriptional activity after the administration of chlordecone. In studies by Ciaccio et al.(31) , transcription of dihydrodiol dehydrogenase was induced by certain xenobiotics, such as ethacrynic acid, dimethyl maleate, and t-butylquinone. It was speculated that the induction of dihydrodiol dehydrogenase may be via the regulatory sequence AP1, because these xenobiotics also induce the DT diaphorase gene, which is controlled by the AP1 sequence within the antioxidant responsive element(32) . Homology search of the 5`-flanking sequence of the type I and type II 3alpha-HSD genes revealed several AP1-like sequences near the putative promoter region. For this reason, we are currently investigating the role of these AP1 sequences on the control of the promoter activity.

The genes encoding two other aldo-keto reductases, human aldose reductase and the mouse major vas deferens protein, have been recently delineated(33, 34) . In contrast to the genes encoding human 3alpha-HSDs and dihydrodiol dehydrogenase/BABP, which contain 9 exons, the aldose reductase and major vas deferens protein genes contain 10 exons. In addition, the exon-intron arrangement of the human aldose reductase and major vas deferens protein genes differ from those of the genes reported here. Much less homology was found in the intron sequences and the 5`-flanking sequences between the genes encoding for type I 3alpha-HSD/dihydrodiol dehydrogenase and dihydrodiol dehydrogenase/BABP and the genes for aldose reductase and major vas deferens protein. Furthermore, aldose reductase is located at chromosome 7q35, whereas both 3alpha-HSD and dihydrodiol dehydrogenase/BABP are located at chromosome 10p14-15(28) . Therefore, it is apparent that 3alpha-HSDs and dihydrodiol dehydrogenase/BABP are more phylogenetically related to each other. The aldo-keto reductase superfamily is involved in the metabolism of endogenous substrates, such as steroid hormones, prostaglandins, and bile acids, and xenobiotics, such as drugs and environmental carcinogens. Future studies in the delineation of structure-function relationships, regulation, and induction of the aldo-keto reductases may shed light on the physiological and pathological functions of this important family of enzymes.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank[GenBank]-L43839[GenBank].

§
Supported in part by National Institutes of Health Grant DK-44177, by the Mellon Teacher-Scientist Award from the Andrew W. Mellon Foundation, and by the Readers Digest Research Fellowship. To whom correspondence should be addressed: Dept. of Pharmacology, MC 1505, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06030. Tel.: 203-679-3704; Fax: 203-679-2473.

(^1)
The abbreviations used are: 3alpha-HSD, 3alpha-hydroxysteroid dehydrogenase; PCR, polymerase chain reaction; RT-PCR, reverse transcription polymerase chain reaction; kb, kilobase pair(s); bp, base pairs; BABP, bile acid-binding protein.


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