Expression and Characterization of Recombinant Type 2 3{alpha}-Hydroxysteroid Dehydrogenase (HSD) from Human Prostate: Demonstration of Bifunctional 3{alpha}/17ß-HSD Activity and Cellular Distribution

Hsueh-Kung Lin1, Joseph M. Jez1, Brian P. Schlegel, Donna M. Peehl, Jonathan A. Pachter and Trevor M. Penning

Departments of Pharmacology, Biochemistry, and Biophysics (H.K.L., J.M.J., B.P.S., T.M.P) University of Pennsylvania School of Medicine Philadelphia, Pennsylvania 19104
Department of Urology (D.M.P) Stanford University Medical School Stanford, California 94305
Schering-Plough Research Institute (J.A.P.) Kenilworth, New Jersey 07033


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In androgen target tissues, 3{alpha}-hydroxysteroid dehydrogenase (3{alpha}-HSD) may regulate occupancy of the androgen receptor (AR) by catalyzing the interconversion of 5{alpha}-dihydrotestosterone (5{alpha}-DHT) (a potent androgen) and 3{alpha}-androstanediol (a weak androgen). In this study, a 3{alpha}-HSD cDNA (1170 bp) was isolated from a human prostate cDNA library. The human prostatic 3{alpha}-HSD cDNA encodes a 323-amino acid protein with 69.9%, 84.1%, 99.4%, and 87.9% sequence identity to rat liver 3{alpha}-HSD and human type 1, type 2, and type 3 3{alpha}-HSDs, respectively, and is a member of the aldo-keto reductase superfamily. The close homology with human type 2 3{alpha}-HSD suggests that it is either identical to this enzyme or a structural allele. Surprisingly, when the recombinant protein was expressed and purified from Escherichia coli, the enzyme did not oxidize androsterone when measured spectrophotometrically, an activity previously assigned to recombinant type 2 3{alpha}-HSD using this assay. Complete kinetic characterization of the purified protein using spectrophotometric, fluorometric, and radiometric assays showed that the catalytic efficiency favored 3{alpha}-androstanediol oxidation over 5{alpha}-DHT reduction. Using [14C]-5{alpha}-DHT as substrate, TLC analysis confirmed that the reaction product was [14C]-3{alpha}-androstanediol. However, in the reverse reaction, [3H]-3{alpha}-androstanediol was oxidized first to [3H]-androsterone and then to [3H]-androstanedione, revealing that the expressed protein possessed both 3{alpha}- and 17ß-HSD activities. The 17ß-HSD activity accounted for the higher catalytic efficiency observed with 3{alpha}-androstanediol. These findings indicate that, in the prostate, type 2 3{alpha}-HSD does not interconvert 5{alpha}-DHT and 3{alpha}-androstanediol but inactivates 5{alpha}-DHT through its 3-ketosteroid reductase activity. Levels of 3{alpha}-HSD mRNA were measured in primary cultures of human prostatic cells and were higher in epithelial cells than stromal cells. In addition, elevated levels of 3{alpha}-HSD mRNA were observed in epithelial cells derived from benign prostatic hyperplasia and prostate carcinoma tissues. Expression of 3{alpha}-HSD was not prostate specific, since high levels of mRNA were also found in liver, small intestine, colon, lung, and kidney. This study is the first complete characterization of recombinant type 2 3{alpha}-HSD demonstrating dual activity and cellular distribution in the human prostate.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Testosterone, after entering prostatic cells, is translocated to the nucleus and converted to 5{alpha}-dihydrotestosterone (5{alpha}-DHT) by 3-oxo-5{alpha}-steroid-4-dehy-drogenase (5{alpha}-reductase) (1, 2). 5{alpha}-DHT2 is a more potent androgen than testosterone in stimulating prostate growth (3, 4) and preferentially binds to the androgen receptor [AR; dissociation constant (Kd) for the AR of 10-11 M] (5, 6). Elevation of 5{alpha}-DHT content in prostate has been associated with benign prostatic hyperplasia (BPH) in humans and canines (5, 6, 7, 8, 9) and with human prostate carcinoma (PCA) (10, 11). The action of 5{alpha}-DHT may be terminated by 3{alpha}-HSD, which catalyzes the formation of 3{alpha}-androstanediol (a weak androgen; Kd for the AR of 10-6 M) (Fig. 1Go) (12). It has also been proposed that, by catalyzing the reverse reaction, 3{alpha}-HSD may function as a molecular switch and, in this manner, may regulate the amount of 5{alpha}-DHT available for AR binding and activation.



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Figure 1. Androgen Metabolism by 5{alpha}-Reductase, 3{alpha}-HSD, and 17ß-HSD in the Human Prostate

 
Detailed studies of 3{alpha}-HSD in prostate are required to understand its role in androgen metabolism and action. First, controversy exists over the directionality of the 3{alpha}-HSD reaction in the prostate. Studies in dog (13, 14) and rat (15) prostate indicate that the dehydrogenase activity of 3{alpha}-HSD is favored over the reductase activity; others suggest that the opposite occurs in the rat (16, 17), dog (18), and human (19) prostate. Kinetic characterization of recombinant 3{alpha}-HSD obtained from human prostate should help clarify these issues. Second, it is uncertain whether 3{alpha}-HSD is localized to the same cells as 5{alpha}-reductase and the AR in the prostate, and this may impact on its ability to regulate occupancy of the AR.

Attempts to characterize 3{alpha}-HSD from rat, dog, and human prostate have been hampered by difficulties in obtaining pure protein. Partially purified 3{alpha}-HSD from rat prostate shares properties in common with the rat liver enzyme: it functions as an oxidoreductase with dual pyridine nucleotide specificity acting on both 5{alpha}- and 5ß-reduced steroids (16, 17). In dog and human prostate, cytosolic and microsomal 3{alpha}-HSD activities have been described (20, 21, 22). Recently, 3{alpha}-HSD from human hyperplastic prostate has been partially purified (23). However, no homogeneous 3{alpha}-HSD expressed in human prostate has been obtained in significant amounts from any source.

3{alpha}-HSD of the human prostate may be similar to the extensively studied 3{alpha}-HSD from rat liver (24). The rat liver 3{alpha}-HSD (AKR1C9)3 cDNA has been cloned (26) and is a member of the aldo-keto reductase (AKR) superfamily that includes human type 1 (AKR1C4), type 2 (AKR1C3), and type 3 (AKR1C2) 3{alpha}-HSDs (27, 28). Recombinant rat liver 3{alpha}-HSD has been overexpressed in E. coli and shown to be kinetically identical to protein purified directly from rat liver (29). Also, the three-dimensional structures of rat liver 3{alpha}-HSD apoenzyme, the E•NADP+ binary complex, and E · NADP+ · testosterone ternary complex have been solved by x-ray crystallography (30, 31, 32) and site-directed mutagenesis studies performed (29, 33).

To investigate the potential role of 3{alpha}-HSD in the regulation of androgen levels in human prostate, we have cloned a cDNA virtually identical to type 2 3{alpha}-HSD from a human prostate library. This work represents the first complete kinetic characterization of a homogenous 3{alpha}-HSD expressed in human prostate. Our results demonstrate that this protein is better described as a 3{alpha}/17ß-HSD, and that this enzyme may remove 5{alpha}-DHT from the prostatic androgen pool through its 3-ketosteroid reductase activity. Examination of mRNA levels in primary cultures of prostatic cells derived from normal, BPH, and PCA tissues reveals that the transcript is expressed predominantly in epithelial cells [where the AR is found in the absence of type 2 5{alpha}-reductase (34)] suggesting a possible role for 3{alpha}-HSD in terminating androgen action. Importantly, 3{alpha}-HSD mRNA expression may be elevated in both BPH and PCA.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cloning and Sequencing of a cDNA Encoding Type 2 3{alpha}-HSD from Human Prostate
A human colon dihydrodiol dehydrogenase (DD) 1 (AKR1C1) cDNA probe was used to screen a human prostate cDNA library. Although DD1 is predominantly a 20{alpha}-HSD, it differs from DD2 (type 3 3{alpha}-HSD and bile acid-binding protein) by only seven amino acids (35, 36). The initial screen detected 13 positive clones. Partial sequences derived from these clones shared similar, but not identical, nucleotide sequences (data not shown). The complete sequence of one of these clones that possessed the highest sequence identity with the probe was acquired, but there was no ATG translation start codon, indicating that the clone was not full length. Subsequent rapid amplification of 5'-cDNA ends (5'-RACE) identified the ATG start site and two missing codons. The 5'-RACE sequence is shown in Fig. 2Go, and the complete sequence of the cDNA is shown in Fig. 3Go. The cDNA clone of 1170 bp encodes a protein of 323 amino acids with a calculated molecular mass of 36,815 daltons and contained a 3'-untranslated region (UTR) and a polyadenylation signal.



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Figure 2. Identification of the 5'-UTR Region of Human Type 2 3{alpha}-HSD Using 5'-RACE

A, Gel electrophoresis of 5'-RACE product. The amplified DNA product was separated on a 1.2% agarose gel and stained with ethidium bromide. One single band with size of 650 bp was preferentially amplified. B, Nucleotide sequence of the amplified DNA product from three randomly selected plasmids. All three extended products contained identical sequence in the coding region, but the length of their 5'-UTRs differed. This may result from either different individuals that are present in the pooled poly(A)+ RNA template, alternate transcription start sites for type 2 3{alpha}-HSD, or an artifact of immature reverse transcription. Sequences upstream of the ATG start sites are shown; sequences downstream from the ATG start site are underlined.

 


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Figure 3. Nucleotide Sequence and the Predicted Amino Acid Sequence of the Human Prostate 3{alpha}-HSD Clone

The open reading frame contains 969 nucleotides and encodes a protein of 323 amino acids. The polyadenylation site is underlined.

 
Comparison of the predicted amino acid sequence of the cDNA clone with members of the AKR superfamily showed that the protein was 99.4% identical to human type 2 3{alpha}-HSD (AKR1C3; Ref.27) differing only at residues 75 and 175. In addition, this clone has an identical 5'-UTR and shares 94.7% identity with the 3'-UTR of human type 2 3{alpha}-HSD. On this basis it was assumed that the human prostatic 3{alpha}-HSD is type 2 3{alpha}-HSD. The coding region of the prostatic HSD clone also shared 69.9%, 84.1%, 87.9%, and 87.0% amino acid identity with rat liver 3{alpha}-HSD (AKR1C9; Ref.29), human type 1 3{alpha}-HSD (AKR 1C4; Ref.27), human type 3 3{alpha}-HSD (AKR1C2; Ref.28), and human DD1 (AKR1C1; Refs. 35 and 36), respectively (Fig. 4Go).



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Figure 4. Comparison of the Amino Acid Sequences of Mammalian 3{alpha}-HSDs

Only amino acids differing from the human prostate type 2 3{alpha}(17ß)-HSD sequence are shown. Human type 1 3{alpha}-HSD is also chlordecone reductase and DD4 (27, 37, 38); human type 3 3{alpha}-HSD is also bile acid-binding protein (28, 35–37); human 20{alpha}(3{alpha})-HSD is also DD1 (35, 36); x, no amino acid in this position.

 
Overexpression and Purification of Human Recombinant Type 2 3{alpha}-HSD
To identify the enzymatic activity of the protein encoded by the cDNA clone, a prokaryotic expression construct was generated and used to transform E. coli cells. Surprisingly, E. coli sonicates expressing the recombinant protein failed to oxidize androsterone using a spectrophotometric assay, an activity previously assigned to recombinant type 2 3{alpha}-HSD using this assay (27). This result questioned either the identity of the clone or the substrate specificity of human type 2 3{alpha}-HSD. To completely characterize the enzyme activity of the recombinant protein, it was purified to homogeneity in a three-step process to yield 10–15 mg. Since the enzyme was unable to oxidize androsterone, the purification was followed by SDS-PAGE (Fig. 5AGo). The homogeneous enzyme gave a single band on SDS-PAGE similar in size to the 3{alpha}-HSD partially purified from human and rat prostate (16, 23). The protein was also immunoreactive to a rabbit anti-rat liver 3{alpha}-HSD antiserum (Fig. 5BGo).



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Figure 5. Expression and Purification of Recombinant Prostatic Type 2 3{alpha}-HSD from E. coli Cells

A, SDS/PAGE analysis: lane 1, 5 µg purified recombinant rat liver 3{alpha}-HSD; lane 2, 30 µg bacterial cell sonicate; lane 3, 15 µg of the peak fraction from a DE52 cellulose column; lane 4, 5 µg of the peak fraction from a Blue Sepharose affinity column (final step). B, Western blot analysis: lane 1, 0.3 µg purified recombinant rat liver 3{alpha}-HSD; lane 2, 5 µg bacterial cell sonicate; lane 3, 1 µg of the cellulose column peak fraction; lane 4, 0.2 µg of the Blue Sepharose purified protein. Molecular mass markers for panels A and B are noted in kilodaltons.

 
To confirm proper folding of the overexpressed human type 2 3{alpha}-HSD, the binding of NADPH was measured by fluorescence titration. Based on the three-dimensional structure of rat liver 3{alpha}-HSD complexed with cofactor (31, 32), the nicotinamide-binding pocket contains 13 different amino acids encompassing a large portion of the ({alpha}/ß)8-barrel structure and binds NADPH in an unusual extended conformation with a Kd = 143 nM (29). Any significant alterations in folding of the overexpressed protein would alter the binding affinity for NADPH. In type 2 3{alpha}-HSD, nine of these residues are invariant, and the remaining four substitutions are conservative ones. Titration of type 2 3{alpha}-HSD with NADPH yielded a calculated Kd of 107 ± 8 nM, indicating that the purified protein was properly folded. Therefore, the inability to oxidize androsterone can not be attributed to either poor protein expression or the expression of improperly folded protein.

Kinetic Characterization and Product Identification of Recombinant Human Type 2 3{alpha}-HSD
Characterization of the homogeneous recombinant protein initially focused on obtaining specific activities for a variety of substrates at different pH values using NAD(H) and NADP(H) as cofactors. Our results suggested that the purified protein had 3{alpha}-HSD activity, oxidizing 3{alpha}-androstanediol and reducing 5{alpha}-DHT and androstanedione (Table 1Go). The recombinant enzyme displayed no detectable 17ß- or 20{alpha}-HSD activity using the standard spectrophotometric method. As in other HSDs of the AKR superfamily, dual pyridine-nucleotide specificity was observed. Also, the purified protein had similar pH preferences as rat liver 3{alpha}-HSD: at pH 6.0, the reduction reaction rate increased; and at pH 9.0, the oxidation reaction rate increased.


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Table 1. Summary of Specific Activities for Human Type 2 3{alpha}-HSD

 
Kinetic constants for the physiological substrates 5{alpha}-DHT, 3{alpha}-androstanediol, and androstanedione were determined spectrophotometrically. Based on these assays, comparison of kcat/Km values suggested that type 2 3{alpha}-HSD favors oxidation of 3{alpha}-androstanediol over reduction of 5{alpha}-DHT (Table 2Go). In contrast, the reduction of androstanedione is more efficient than oxidation of androsterone, which was barely detectable (Table 2Go). For comparative purposes, we also used compounds (i.e. 9,10-phenanthrenequinone and 4-nitrobenzaldehyde) that are common substrates for other members of the AKR superfamily. These ubiquitous AKR substrates, while efficiently turned over, are nonphysiological. Human prostatic type 2 3{alpha}-HSD had a 20-fold higher Km and a slightly lower Vmax for 9,10-phenanthrenequinone than the rat liver enzyme, whereas the human prostate and rat liver enzyme gave similar kcat/Km values for 4-nitrobenzaldehyde (Table 2Go).


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Table 2. Summary of Kinetic Constants for Type 2 3{alpha}-HSD Obtained by Spectrophotometric Assays

 
The low turnover numbers for endogenous androgen substrates necessitated the use of fluorometric and radiometric assays. By monitoring the fluorescence emission of NADPH at 450 nm or the disappearance of radioactive steroid by TLC, these methods increased sensitivity 100- or 2500-fold, respectively, over the standard spectrophotometric assay. The kinetic constants obtained by these methods were in good agreement with values estimated by the spectrophotometric assays (Table 3Go).


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Table 3. Summary of Kinetic Constants for Type 2 3{alpha}-HSD Obtained by Radiometric and Fluorometric Assays

 
The radiometric assay showed that, as expected, [14C]-5{alpha}-DHT was converted into [14C]-3{alpha}-androstanediol by type 2 3{alpha}-HSD, confirming its ability to reduce 3-ketosteroids (Fig. 6AGo). However, incubation of type 2 3{alpha}-HSD with [3H]-3{alpha}-androstanediol did not produce [3H]-5{alpha}-DHT. Instead, androsterone was produced that was subsequently converted to androstanedione (Fig. 6BGo). These results reveal for the first time that type 2 3{alpha}-HSD also displays 17ß-HSD activity.



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Figure 6. Radiochemical Assay and Product Identification

A, Conversion of 5{alpha}-DHT into 3{alpha}-androstanediol: lane 1, incubation of 5{alpha}-DHT without enzyme; lane 2, incubation with recombinant rat liver 3{alpha}-HSD (15 min with 3 µg); lane 3, incubation with recombinant human type 2 3{alpha}-HSD (60 min with 15 µg). B, Conversion of 3{alpha}-androstanediol into androsterone and androstanedione: lane 1, incubation of 3{alpha}-androstanediol without enzyme; lane 2, incubation with recombinant rat liver 3{alpha}-HSD (15 min with 3 µg); lanes 3, 4, 5, and 6, incubation with recombinant human type 2 3{alpha}-HSD (15 µg) for 15, 30, 45, and 60 min, respectively; lane 7, incubation of androsterone without enzyme; lane 8, incubation of androsterone with recombinant rat liver 3{alpha}-HSD (15 min with 3 µg).

 
Since androsterone was formed before androstanedione appeared, it seems that the 17ß-HSD activity is favored over 3{alpha}-HSD activity in the oxidation direction. Further characterization of the 17ß-HSD activity used [14C]-5{alpha}-DHT and [14C]-testosterone as substrates for oxidation (Table 3Go). Comparison of the catalytic efficiencies for these reactions with that for [3H]-androsterone oxidation indicated that type 2 3{alpha}-HSD preferentially oxidizes 17ß-hydroxysteroids. Similarly, comparison of the catalytic efficiency for the oxidation of [3H]-3{alpha}-androstanediol with that for [3H]-androsterone shows that the 17ß-alcohol is preferentially oxidized over the 3{alpha}-alcohol of the diol. Thus, the higher catalytic efficiency observed with 3{alpha}-androstanediol over 5{alpha}-DHT is primarily due to the 17ß-HSD activity of the enzyme. These data suggest that, in the prostate, type 2 3{alpha}-HSD does not interconvert 5{alpha}-DHT with 3{alpha}-androstanediol but functions to inactivate 5{alpha}-DHT as a result of its 3-ketosteroid reductase activity.

Northern Blot Analysis of 3{alpha}-HSD in Primary Cultures of Human Prostatic Cells
Since type 2 3{alpha}-HSD can inactivate the potent androgen 5{alpha}-DHT, the distribution of 3{alpha}-HSD mRNA was examined within the prostate. Northern analysis was used to detect the levels of mRNA in primary cultures of epithelial and stromal cells derived from normal, BPH, and PCA prostate tissues. A 1.2-kb 3{alpha}-HSD transcript was predominantly expressed in epithelial cells (Fig. 7Go). Remarkably, there was evidence for elevated expression in the epithelial cells derived from areas of the prostate with either BPH and PCA histology. The gels contained similar amounts of RNA as judged by levels of 28S and 18S rRNA, and the transfer was equivalent across the lanes based on hybridization with a probe for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Since type 2 3{alpha}-HSD shares high sequence homology with other AKR superfamily members, which may affect the specificity of hybridization, the Northern blots were stripped and reprobed with the 3'-UTR whose sequence is closely related only to other 3{alpha}-HSD isoforms of the AKR superfamily. Using the 3'-UTR probe, the presence of 3{alpha}-HSD mRNAs was primarily limited to epithelial cells and was elevated in cells from diseased prostatic tissues. Quantification of the Northern analysis using the 3'-UTR probe showed that the ratios of 3{alpha}-HSD:GAPDH mRNA were 0.48 ± 0.08, 0.77 ± 0.25, and 1.60 ± 0.57 for epithelial cells derived from normal, BPH, and PCA tissues, respectively. Based on Student’s t test in which P < 0.05 indicates significance, the difference in expression levels was statistically significant between cells derived from normal and PCA tissues.



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Figure 7. Examination of 3{alpha}-HSD mRNA Expression by Northern Blot Analysis in Primary Cultures of Normal, BPH, and PCA Prostate Cells

Northern blots using an 3{alpha}-HSD cDNA fragment (+1 to +854 bp) excised from pBluescript II ks-vector and radiolabeled with [{alpha}-32P]-dATP by random priming. The same blots were rehybridized with [{alpha}-32P]-dATP-labeled type 2 3{alpha}-HSD 3'-UTR (+995 to +1170 bp). Human GAPDH was used as a control to standardize transfer and loading of the RNA.

 
Tissue-Specific Expression
Since type 2 3{alpha}-HSD was originally cloned from human liver (27), its expression is not prostate specific. To examine the distribution of this enzyme, Northern analysis was performed on RNA from 16 different human tissues (Fig. 8Go). In each tissue, a single 1.2-kb mRNA species was observed using the 3'-UTR for type 2 3{alpha}-HSD as a probe (see above). The levels of 3{alpha}-HSD mRNA were highest in liver, colon, and small intestine. The spleen, placenta, and brain had the lowest levels of 3{alpha}-HSD transcript. Due to the specificity of the probe, the levels of mRNA detected probably reflect the total pool of 3{alpha}-HSD isoforms in each tissue.



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Figure 8. Tissue Distribution of 3{alpha}-HSD in Human Tissues

Northern blots containing RNA from 16 different human tissues were hybridized with a randomly primed probe corresponding to human type 2 3{alpha}-HSD 3'-UTR (+995 to +1170 bp) and then reprobed with a randomly primed probe to human GAPDH.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have described the cloning, expression, and characterization of a 3{alpha}-HSD obtained from a human prostate cDNA library. This cDNA clone appears to be virtually identical to type 2 3{alpha}-HSD originally cloned from human liver (27). The recombinant protein was purified to homogeneity from E. coli and kinetically characterized. Our study is the first documentation of the properties of a 3{alpha}-HSD expressed in human prostate and the first detailed study of the properties of homogenous type 2 3{alpha}-HSD. Kinetic analysis demonstrates that this protein is more accurately described as a 3{alpha}-/17ß-HSD. The distribution of AKR-related 3{alpha}-HSD mRNA across human tissues and within prostate cell types is described. We have shown that type 2 3{alpha}-HSD will inactivate 5{alpha}-DHT by forming 3{alpha}-androstanediol but fails to work as a molecular switch by converting 3{alpha}-androstanediol to 5{alpha}-DHT.

Our cloning indicates that the human prostate contains multiple members of the AKR superfamily. HSDs of this superfamily can be isoforms of 3{alpha}-, 17ß-, or 20{alpha}-HSDs. Khanna et al. (27) cloned type 1 and 2 3{alpha}-HSDs from human liver and showed that type 1 3{alpha}-HSD was present only in the liver and is identical in sequence to DD4 (37) and chlordecone reductase (38). It was subsequently shown that type 1 3{alpha}-HSD was not found in the prostate (39). Type 2 3{alpha}-HSD had a broader expression pattern and was detected in the brain, kidney, liver, lung, placenta, and testis (27). The presence of type 2 3{alpha}-HSD in prostate was not documented. Our tissue-specific Northern analysis confirms the previously reported distribution of 3{alpha}-HSD mRNA and indicates that transcripts similar in size and sequence to type 2 3{alpha}-HSD are found in the prostate, blood leukocytes, colon, small intestine, ovary, thymus, spleen, pancreas, skeletal muscle, and heart. The Northern analysis shown here is not specific for type 2 3{alpha}-HSD and probably detects all the 3{alpha}-HSD isoforms expressed. The high mRNA levels observed in liver, kidney, and intestine are consistent with the known function of 3{alpha}-HSD in these tissues where the different isoforms have roles in bile acid biosynthesis (liver) and xenobiotic metabolism (liver, kidney, and small intestine) (24, 36). Recently, another cDNA clone designated type 3 3{alpha}-HSD (28), which is nearly identical to human liver bile acid-binding protein/DD2 (35, 36, 37), has been isolated from a human prostate cDNA library and shown to display 3{alpha}-HSD activity when transiently expressed in human embryonal kidney (293) cells.

By overexpressing and purifying recombinant type 2 3{alpha}-HSD, we have shown that the protein is similar to the 3{alpha}-HSDs partially purified from rat and human prostate (16, 17, 23). Although previously reported kinetic parameters for prostatic 3{alpha}-HSD activity were determined with crude tissue homogenates, cellular fractions, or partially purified enzymes (16, 18, 20, 21, 22), our characterization yielded comparable results with these earlier studies. For example, Taurog et al. (16) reported Km values for 5{alpha}-DHT and 3{alpha}-androstanediol in the low micromolar range with the partially purified rat prostatic enzyme, and it preferentially used NADPH over NADH as cofactor. The ability of human type 2 3{alpha}-HSD to oxidize androsterone when expressed in E. coli sonicates has been reported (27). In the present study, we were unable to detect this activity using the same spectrophotometric assay in E. coli sonicates. However, we could detect androsterone oxidation with a radiometric assay using purified homogeneous protein. The predicted amino acid sequence we describe here differs from the previously reported sequence for human type 2 3{alpha}-HSD by only two residues. Based on the three-dimensional structures of rat liver 3{alpha}-HSD (30, 31, 32), the two substituted amino acids are not in the vicinity of the active site or the substrate-binding pocket, they are on the periphery of the structure and are unlikely to affect function. Therefore, the differences in the specific activity for androsterone oxidation catalyzed by the two recombinant proteins cannot be explained by differences in sequence.

The directionality of 3{alpha}-HSD activity in the prostate has been a point of debate. Studies of dog (13, 14) and rat (15) prostate suggest that the dehydrogenase activity of 3{alpha}-HSD is favored over the reductase activity, whereas other investigations of dog (18), rat (16, 17), and human (19) prostate indicate that the reduction of 5{alpha}-DHT is preferred.

Based on kinetic characterization of purified recombinant type 2 3{alpha}-HSD, this protein can reduce 5{alpha}-DHT into 3{alpha}-androstanediol but does not catalyze the reverse reaction. When 3{alpha}-androstanediol is used as substrate, the 17ß-alcohol is oxidized preferentially over the 3{alpha}-alcohol to generate androsterone which is converted to androstanedione over time demonstrating that type 2 3{alpha}-HSD is also a 17ß-HSD. This dual activity is not unprecedented. Several HSDs of the AKR superfamily demonstrate this phenomenon. For example, although human liver DD1 is predominantly a 20{alpha}-HSD, it also has 3{alpha}-HSD activity (40). Likewise, type 3 3{alpha}-HSD, which is virtually identical to human bile acid-binding protein, displays mainly a 3{alpha}-HSD activity but exhibits a slight 20{alpha}-HSD activity (28). Additionally, Labrie et al. (41) have reported the cloning of a type 5 17ß-HSD, which shares 99.1% sequence identity to type 2 3{alpha}-HSD. However, the sequence of type 5 17ß-HSD has not been reported. The ability of HSDs in the AKR superfamily to be positionally selective and stereospecific on the one hand (e.g. rat liver 3{alpha}-HSD) and to be promiscuous on the other (e.g. 3{alpha}-/20{alpha}- and 3{alpha}-/17ß-HSDs) raises issues of steroid pocket architecture. This is particularly intriguing with regard to 3{alpha}-/17ß-HSD activity, since in this instance steroid substrates would bind backward (D-ring enters the active site first instead of the A-ring) and upside down (ß-face of the steroid in the {alpha}-face orientation) to maintain stereochemistry of hydride transfer.

Previous studies on the directionality of 3{alpha}-HSD activity in the prostate have never considered the interplay of multiple 3{alpha}-HSD isoforms with subtle differences in substrate specificity. It is possible that differential expression of such isoforms would affect the balance between 5{alpha}-DHT and 3{alpha}-androstanediol within the prostate. As described above, at least two 3{alpha}-HSD isoforms have been detected in the human prostate. While type 2 3{alpha}-HSD may not function as the molecular switch between potent and weak androgens, it is well suited to decreasing 5{alpha}-DHT levels in the prostate by forming 3{alpha}-androstanediol. Considering the fact that human bile acid-binding protein/DD2 also functions as a 3{alpha}-HSD (35), type 3 3{alpha}-HSD may be a better candidate to catalyze the formation of 5{alpha}-DHT from 3{alpha}-androstanediol. However, complete characterization of the substrate specificity of type 3 3{alpha}-HSD for androgens has not been performed.

The colocalization of 3{alpha}-HSD, 5{alpha}-reductase, and the AR within the prostate may also influence androgen metabolism and androgen action. It has been suggested that the stroma is metabolically active and mediates the effect of androgens on the epithelium through epithelial-stromal interactions (42, 43). Northern analysis on primary cultures of epithelial and stromal cells derived from normal, BPH, and PCA tissues using the cDNA 3'-UTR as a probe indicated that 3{alpha}-HSD mRNA transcripts were found predominantly in epithelial derived cell cultures. This distribution compares favorably with the reported localization of 3{alpha}-HSD enzyme activity in epithelial cells (19, 44). Immunohistochemical studies of adult human prostate tissues showed that type 2 5{alpha}-reductase was predominantly expressed in stromal cells, specifically in fibroblasts, with little expression in basal or luminal epithelial cells. In contrast, type 1 5{alpha}-reductase and the AR were found in both stromal and epithelial cells (45). The colocalization of 3{alpha}-HSD with the AR in epithelial cells (which are devoid of type 2 5{alpha}-reductase) and our kinetic studies suggests that type 2 3{alpha}-HSD may protect the AR from binding potent androgens. The higher levels of 3{alpha}-HSD in primary cultures from diseased prostate may reflect either a protective measure to decrease the effect of androgens on abnormal growth of the prostate or may represent a movement toward androgen independent growth of the gland. In this regard it will be interesting to determine whether growth factors modulate type 2 or type 3 3{alpha}-HSD expression.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
All prostate samples were obtained from human patients with their informed consent, and the study was approved by the Institutional Review Board of Stanford University. The human prostate cDNA library and human prostatic poly(A)+ mRNA were obtained from CLONTECH Laboratories (Palo Alto, CA). The pBluescript II ks-vector was from Stratagene (La Jolla, CA), the pET-16b prokaryotic expression vector was from Novagen (Madison, WI), and the pCRII PCR cloning vector was purchased from Invitrogen (San Diego, CA). Blots containing poly(A)+ mRNA from multiple human tissues were purchased from CLONTECH Laboratories. Radiolabeled [4-14C]-5{alpha}-DHT (58.3 mCi/mmol), [4-14C]testosterone (57.3 mCi/mmol), [9,11-3H(N)]-3{alpha}-androstanediol (40.0 Ci/mmol), [9,11-3H(N)]androsterone (57.0 Ci/mmol), and [{alpha}32P]dATP (3,000 Ci/mmol) were obtained from NEN/DuPont (Boston, MA). All other steroids were purchased from Steraloids (Wilton, NH). Nucleotide cofactors were obtained from Boehringer-Mannheim (Indianapolis, IN). All other compounds were ACS grade or better from Sigma Chemical Co. (St. Louis, MO).

Screening of the cDNA Library and DNA Sequencing
A total of 1.2 x 106 plaque forming units were screened from a prostate cDNA library in {lambda}gt10 obtained from a 22-yr-old normal Caucasian male. Screening was performed using random primed [{alpha}-32P]dATP-labeled human colon DD1 (46) as the probe. DD1 is a member of the AKR superfamily with 20{alpha}(3{alpha})-HSD activity (40). Positive clones were plaque purified, and a partial sequence was obtained using an insert screening amplimer set (CLONTECH) flanking both ends of the EcoRI insertion site of {lambda}gt10 vector. The 5'-end insert screening amplimer (5'-AGCAAGTTCAGCCTGGTTAAG-3') was 8 bp upstream from the EcoRI site, and the 3'-end insert screening amplimer (5'-GGGACCTTCTTTATGAGTATT-3') was 22 bp downstream of the EcoRI site. A clone containing a nearly full-length cDNA with the highest nucleotide sequence identity to the human colon DD1 cDNA was excised from the {lambda}gt10 vector using EcoRI partial digestion (an internal EcoRI site was present at 854 bp of the cDNA) to retrieve the entire cDNA. A single 1.2-kb cDNA was purified from the partial digests by agarose gel electrophoresis and subcloned into the EcoRI site of pBluescript II ks-vector for dideoxysequencing. Both strands of the cDNA were sequenced using discrete 5'- and 3'-primers.

Since the cDNA obtained did not contain an ATG translation start codon, the 5'-cDNA was regenerated from a PCR using the template obtained from the 5'-RACE using the Marathon cDNA Amplification kit (CLONTECH). Double-stranded cDNA was synthesized from human prostatic poly(A)+ mRNA (CLONTECH) using oligo (dT)12–18 primers followed by adapter ligation. The 5'-RACE was amplified from the double-stranded cDNA using an adapter primer and a 3'-primer complementary to +630 to +610 bp (5'-ATCTTTCGACTTGCAGAAATC-3') of the cloned 3{alpha}-HSD cDNA and Vent DNA polymerase (New England Biolabs, Beverly, MA). The amplified product was purified from an agarose gel and subcloned into the pCR II vector (pCRII-5'-3{alpha}-HSD).

Expression Vector Construction, Overexpression, and Purification of Recombinant Protein
To establish a prokaryotic expression construct, the full-length cDNA was generated from the 5'-RACE product and the cDNA clone obtained from library screening. The 5'-end of the cDNA amplified from pCRII-5'-3{alpha}-HSD using a mutated 5'-primer (5'-GTGACAGGCCATGGATTCCAA-3', -10 to +11 bp) and a 3'-primer (+630 to +610 bp) where the underline indicates the changes used to generate an NcoI site. The PCR-generated 5'-end of the cDNA was digested with NcoI at both the 5'-end and at an internal NcoI site at +450 bp to generate sticky ends. The 3'-end of the cDNA was excised from the pBluescript II ks- vector by digesting with NcoI and BamHI. The digested 5'- and 3'-fragments were ligated into pET-16b vector linearized with NcoI plus BamHI to generate the complete pET16b-3{alpha}-HSD expression construct. The orientation of the 5'-fragment was confirmed by dideoxysequencing. The construct was transformed into competent E. coli strain DE3(BL21) cells under the control of the lacUV5 promoter. Cells were grown overnight at 37 C in the presence of 100 µg/ml ampicillin. Aliquots of the overnight culture were transferred to 2-liter cultures and growth was monitored spectrophotometrically until A600 nm = 0.6. Protein expression was then induced by addition of 1 mM isopropyl-ß-D-thiogalactopyranoside. After 4 h of incubation, cells were pelleted by centrifugation for lysis by sonication. Overexpressed protein was purified from E. coli sonicates using DE-52 cellulose anion-exchange and Blue Sepharose affinity column chromatography with protein quantification, SDS-PAGE, and Western blot analysis performed as previously described (33).

Kinetic Characterization
Specific activities of pure recombinant type 2 3{alpha}-HSD were measured using a Beckman DU-640 spectrophotometer (Beckman Instruments, Fullerton, CA) by monitoring the change in absorbance of pyridine nucleotide at 340 nm. Specific activities were determined in a 1-ml assay system containing 100 mM potassium phosphate (pH 6.0 or 7.0) or 50 mM glycine/NaOH (pH 9.0) with 4% acetonitrile as cosolvent at 25 C. Cofactor concentrations were 2.3 mM for NAD+ and NADP+ and 200 µM for NADH and NADPH. Substrate concentrations were 75 µM androsterone; 35 µM 5{alpha}-DHT, 3{alpha}-androstanediol, and androstanedione; and 50 µM estrone, 17ß-estradiol, 4-pregnene-3,20-dione, and 4-pregnen-20{alpha}-ol-3-one. The fluorescence titration method used to determine the Kd for NADPH has been described elsewhere (33).

Vmax, kcat, and Km values for reduction of the following substrates were obtained spectrophotometrically using the above assay system at pH 7.0 by varying the substrate concentration as indicated and by maintaining a constant NADPH concentration (200 µM): 5{alpha}-DHT (3.5–52.5 µM), androstanedione (1.85–56.25 µM), 9,10-phenanthrenequinone (5.0–200 µM), and 4-nitrobenzaldehyde (0.050–1.5 mM). Measurements of 3{alpha}-androstanediol (1.75–35 µM) oxidation were performed spectrophotometrically using the same assay system with 2.3 mM NADP+. Kinetic constants were calculated using the ENZFITTER (BioSoft, Cambridge, U.K.) nonlinear regression analysis program (47) to fit untransformed data with a hyperbolic function, as originally described by Wilkinson (48), yielding estimated values and their associated SEs.

For increased sensitivity, a fluorometric assay was used to determine the kinetic constants for oxidation of 3{alpha}-androstanediol. This assay monitors the fluorescence emission of NADPH at 450 nm (slit width 10 nm) with excitation at 340 nm (slit width 5 nm) on a Perkin-Elmer (Norwalk, CT) model 650–10 M fluorometer at 25 C. Each cuvette contained a 1-ml reaction mixture consisting of 100 mM potassium phosphate, pH 7.0, 2.3 mM NADP+, and 3{alpha}-androstanediol (2.0–40 µM) with 4% acetonitrile as cosolvent. The data were analyzed as above.

A radiochemical assay was used to determine the kinetic constants for the reduction of 5{alpha}-DHT. This assay provides increased sensitivity and enabled product identification. Assay systems contained 100 mM potassium phosphate buffer, pH 7.0, 2.3 mM NADPH, and 40,000 cpm of [14C]-5{alpha}-DHT in a 100 µl reaction volume. Vmax, kcat, and Km values for 5{alpha}-DHT reduction were obtained by varying the steroid concentration (3.8–38.2 µM). All reactions were incubated for 30 min at 37 C with aliquots taken every 10 min to calculate initial velocities. Over this time course, the reaction rate was linear. Reactions were quenched by the addition of 400 µl of ethyl acetate, and the resulting extracts were evaporated to dryness and re-dissolved in 40 µl methanol and applied to LK6D Silica TLC plates. Chromatograms were developed in chloroform-ethyl acetate (4:1, vol/vol). The positions of the substrate (5{alpha}-DHT with RF value 0.44) and product (3{alpha}-androstanediol with RF value 0.25) were detected by reference to standards and were visualized by spraying with a 1:1 methanol/H2SO4 solution and heating. The amounts of substrate and product were quantitated by scrapping the corresponding sections of the TLC plate into a toluene-based scintillation fluid, detecting [14C]-radioactivity with a scintillation counter, and converting the corrected counts per min into nanomoles of product using the final specific radioactivity of [14C]-5{alpha}-DHT. Recovery of the radioactive steroid was greater than 90% and varied by less than 5% between samples.

Kinetic constants for the oxidation of androsterone, 3{alpha}-androstanediol, 5{alpha}-DHT, and testosterone were determined using similar radiometric assays. Reactions contained 100,000 cpm of either [3H]-androsterone (3.75–75 µM) or [3H]-3{alpha}-androstanediol (1.5–30 µM) or 40,000 cpm of either [14C]-5{alpha}-DHT (3.8–38.2 µM) or [14C]-testosterone (2.5–50 µM). All reactions were performed with 2.3 mM NADP+. Separation and identification of products used the same solvent system and method as above.

Tissue Distribution of 3{alpha}-HSD mRNA
Blots containing poly(A)+ RNA from multiple human tissues were hybridized to a 176-bp DNA probe corresponding to 3{alpha}-HSD 3'-untranslated region (3'-UTR; 995-1170 bp), which was released from pBluescript II ks- vector (AccI and XhoI double digestion) and random primed with radiolabeled [{alpha}-32P]-dATP with final specific activities of greater than 109 cpm/µg DNA. The 3{alpha}-HSD 3'-UTR sequence matches only 3{alpha}-HSD/DD sequences from GenBank and EMBL using BLAST (49). Hybridization was performed at 42 C for 18 h, and blots were washed with 0.1 x standard saline citrate plus 1% SDS at 60 C for 30 min. The blots were subjected to autoradiography at -70 C. The blots were then stripped and reprobed with a random primed human GAPDH cDNA probe.

Northern Blot Analysis of 3{alpha}-HSD mRNA in Primary Cultures of Human Prostate Cells
Primary cultures of stromal and epithelial cells were prepared from small wedges of tissues dissected from radical or open prostatectomy specimens. After inking to mark areas of tissue removal, prostate specimens were fixed and serially sectioned, and the histology of each area of origin was determined. Cells derived from BPH and PCA tissue were surrounded by cells of similar histology. Tissues were minced and digested with collagenase. Epithelial and stromal cell cultures were established as previously described (50). Cellular RNA was extracted from these cultures by phenol at acidic pH. Total RNA (10 µg) was separated on 1% agarose/formaldehyde gels and transferred to Durolon-UV membranes (Stratagene). Transferred RNA was fixed to the membrane using a UV cross-linker (Stratalinker, Stratagene) followed by hybridization with [{alpha}-32P]-dATP random primed 3{alpha}-HSD cDNA probe (+1 to +854 bp) or a probe corresponding to the 3{alpha}-HSD 3'-UTR (see above). Hybridization conditions and reprobing with human GAPDH were conducted as described. After autoradiography, the results were quantitated using a video densitometer and the UNISCAN software (Analtech, Newark, DE).


    FOOTNOTES
 
Address requests for reprints to: Trevor M. Penning, Department of Pharmacology, University of Pennsylvania School of Medicine, 3620 Hamilton Walk, Philadelphia, Pennsylvania 19104-6084.

This work was supported by NIH Grants DK-47015, CA-39504, and CA-55711 (to T.M.P.).

1 Both of these authors have contributed equally to this work. Back

2 Abbreviations and trivial names used are: 3{alpha}-androstanediol, 5{alpha}-androstane-3{alpha},17ß-diol; androstanedione, 5{alpha}-androstane-3,17-dione; androsterone, 5{alpha}-androstan-3{alpha}-ol-17-one; DD, dihydrodiol dehydrogenase (EC 1.3.1.20); 5{alpha}-DHT, 5{alpha}-dihydrotestosterone (5{alpha}-androstane-17ß-diol-3-one); 3{alpha}-HSD, 3{alpha}-hydroxysteroid dehydrogenase (EC 1.1.1.213, formerly EC 1.1.1.50 renamed due to A-face stereospecificity); 5{alpha}-reductase, 3-oxo-5{alpha}-steroid-4-dehydrogenase (EC 1.3.99.5); testosterone, 4-androsten-17ß-ol-3-one. Back

3 The nomenclature of the aldo-keto reductase superfamily was recommended by the 8th International Symposium on Enzymology and Molecular Biology of Carbonyl Metabolism, held in Deadwood, South Dakota, on June 29–July 3, 1996 (25 ). Back

Received for publication June 6, 1997. Revision received August 22, 1997. Accepted for publication September 2, 1997.


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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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