the journal of biological chemistry vol. no. 23, issue of ... · the journal of biological...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

VOl. 268, No. 23, Issue of August 15, pp, 17404-17412,1993 Printed in U. S. A.

Characterization of Chinese Hamster Ovary Cell Lines Expressing Human Steroid 5a-Reductase Isozymes*

(Received for publication, February 17, 1993)

Anice E. ThigpenS, Kristine M. Cala, and David W. Russell$ From the Department of Molecular Genetics, the University of Texas Southwestern Medical Center, Dallas, Texas 75235

Membrane-bound isozymes of steroid 5ar-reductase, designated 1 and 2, synthesize the potent androgen, dihydrotestosterone. Isozyme 1 has an alkaline pH optimum (7.0-8.5), whereas isozyme 2 has an acidic pH optimum (5.0). To gain insight into this enigmatic dif- ference, Chinese hamster ovarian cell lines expressing the human Sa-reductase isozymes were established. The half-lives of both proteins are >30 h and are not altered by the 4-azasteroid inhibitors finasteride and 17&(N, N,-diethyl)carbamoyl-4-methyl-4-aza-S~- androstan-3-one. Nanomolar concentrations of finasteride block immunoprecipitation of isozyme 2 by antipeptide antibodies, which suggests that drug binding alters protein conformation. In contrast, finasteride (50 pM) has no effect on immunoprecipitation of isozyme 1. Both isozymes are localized to the endoplasmic reticulum by immunocytochemistry and have their carboxyl termini exposed to the cytoplasm. In cell lysates, isozyme 2 exhibits a V,, at pH 5.0 but has a higher substrate affinity at neutral pH. In intact and permeabilized cells, isozyme 2 has an apparent substrate K,,, similar to that determined in cell lysates at neutral pH. The results suggest that isozyme 2 is more efficient at neutral pH and that the acidic pH optimum determined in lysates is a consequence of cell lysis.

The establishment of permanent cell lines that express enzymes of therapeutic interest has facilitated the development of drugs that act on the expressed target. By providing an enriched source of enzyme activity, these cell lines allow the rapid screening of large numbers of potential effectors, the elucidation of structure-activity relationships in lead compounds, and the determination of selectivity. Transfection of cDNAs encoding the target enzyme into host cells that do not express an endogenous activity is frequently used to establish over-expressing cell lines. This approach is especially useful when the enzyme or receptor of interest exists in multiple isozymic forms.

* This research was supported by National Institutes of Health Grant GM-43753 and Robert A. Welch Foundation Grant 1-0971. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Recipient of Postdoctoral Fellowship HD-07504 from the Na- tional Institutes of Health.

§ To whom correspondence should be addressed Dept. of Molecu- lar Genetics, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, Texas 75235-9046.

The enzyme steroid 5a-reductase (5a-reductase)’ is one such therapeutic target that exists in at least two isozymic forms, designated type 1 and type 2 (Andersson et al., 1991). 5a-Reductase catalyzes the conversion of testosterone to dihydrotestosterone (DHT) (Wilson, 1975), and abnormal levels of DHT are associated with several human diseases (Griffin and Wilson, 1989). For example, a genetic deficiency of DHT synthesis caused by mutations in the Sa-reductase type 2 gene leads to birth defects in the male urogenital tract (Thigpen et aL, 1992a, 199213; Wilson et hl., 1993). Conversely, increased levels of DHT have been correlated with benign growth of the prostate in elderly men, hirsutism in females, acne, and male pattern baldness (Metcalf et al., 1989; Tenover, 1991).

The characterization of 5a-reductase isozymes at the biochemical level has been difficult due to the insolubility of this membrane-bound enzyme and to uncertainties in the tissue distribution of isozymes (Wilson, 1975). To circumvent these difficulties, expression cloning strategies have been used to isolate cDNAs that encode two isozymes of 5a-reductase (Andersson et al., 1989a, 1991). When expressed transiently in cultured cells or Xenopus oocytes, type 1 cDNAs from rat and man specify an enzyme with a basic pH optimum that is sensitive to certain steroidal and non-steroidal inhibitors (Andersson et al., 1989a; Andersson and Russell, 1990; Harris et al., 1992; Hirsch et al., 1993). Type 2 cDNAs encode an enzyme with an acidic pH optimum and a distinct pharma- cology from that of the type 1 isozyme (Andersson et al., 1991; Jenkins et al., 1992; Normington and Russell, 1992; Harris et al., 1992).

The biochemical basis for the unusual acidic pH optimum of the type 2 isozyme remains unknown; however, it is known that both the rat and human type 2 isozymes exhibit this property and that mutation of the type 2 isozyme can shift the optimum to a more basic pH (Thigpen et al., 1992a, 1992b). An acidic pH optimum could reflect the localization of the type 2 isozyme in an acidic subcellular compartment or an unusual lipid microenvironment in a target membrane. Inter- estingly, subcellular fractionation studies have reported that the 5a-reductase activity in rat and human prostate is predominantly associated with the nucleus (Moore and Wilson, 1972; Hudson, 1981), while that in rat liver cosediments with a membrane fraction containing marker proteins of the en-

’ The abbreviations used are: Steroid 5a-reductase, NADPH:A‘-3- oxosteroid-501-oxidoreductase; testosterone, 17P-hydroxy-4-andros- ten-3-one; dihydrotestosterone (DHT), 17p-hydroxy-5a-androstan- %one; androstenedione, androst-4-ene,3,17-dione; progesterone, 4-pregnene-3,20-dione; 4-MA, 17P-(N, N,-diethyl)carbamoyl-4- methyl-4-aza-5a-androstan-3-one; finasteride,l7P-(N-t-butyl)carbamoyl-4-aza-5a-androst-l-en-3-one, LY191704,trans-8-chloro-4- methyl-l,2,3,4,4a,5,6,10b- octahydrobenzo[f]quinolin-3-one; CHO, Chinese hamster ovary; NP-40, Nonidet P-40; PBS, phosphate-buffered saline; BiP, immunoglobulin heavy chain binding protein; MOPS, morpholinepropanesulfonic acid; PIPES, 1,4-piperazinedi- ethanesulfonic acid.

17404

CHO Cell Lines Expressing Human 5a-Reductase Isozymes 17405

doplasmic reticulum (Moore and Wilson, 1972). In an effort to gain further insight into this and other

properties of the &-reductase isozymes, permanent Chinese hamster ovary (CHO) cell lines have been established that express the human type 1 and 2 proteins. Utilizing these cell lines, the half-lives, subcellular localization, inhibitor profiles, and pH optima of the 5a-reductase isozymes have been ex- amined.

EXPERIMENTAL PROCEDURES

Materials-Polyclonal antipeptide sera directed against 25 amino acid segments of the type 1 (amino acid residues 232-256) or type 2 (amino acid residues 227-251) isozymes were produced in rabbits using synthetic peptides as described previously (Thigpen and Russell 1992; Thigpen et al., 1992a). 14C-Radiolabeled steroids (-50 Ci/mmol) were purchased from Du Pont-New England Nuclear, and 35S-labeled amino acids (Tran"S-labelTM, -1000 Ci/mmol) were from ICN. K.CAT, a computer program for the analysis of enzyme kinetic data was obtained from BioMetallics, Inc. (Princeton, NJ). Protein A- Sepharose was from Pharmacia LKB Biotechnologies Inc. Fluore- scently labeled secondary antibodies were from Zymed or Vector Laboratories. The rat anti-immunoglobulin heavy chain binding protein (BiP) monoclonal antibody was a generous gift of Dr. David G. Bole, University of Michigan. The 4-azasteroids, 4-MA (17P-(N,N- diethyl)carbamoyl-4-methyl-4-aza-5a-andostan-3-one) and finasteride (17~-(N,t-butyl)carbamoyl-4-aza-5a-androst-l-en-3-one) were supplied by Dr. Gary Rasmusson of Merck, Sharp and Dohme Re- search Laboratories. LY191704 was from Dr. Blake L. Neubauer, Eli Lilly, Corp. Bovine serum albumin, Fraction V fatty acid free, was purchased from Boehringer Manneheim. Affi-Gel active ester agarose and reagents for determining protein concentrations were from Bio- Rad. Detergents were from Calbiochem.

Cell Culture ana' Transfection-5a-Reductase type 1 and 2 cDNAs were subcloned into the pCMV8 and pCMV7 expression vectors (derivatives of pCMV1, Andersson et al., 1989b), respectively. CHO cells, maintained in Dulbecco's modified Eagle's medium/Ham's nu- trient mixture F-12 (l:l), supplemented with 5% fetal calf serum, 50 pg/ml streptomycin, and 50 units/ml penicillin, were cotransfected by calcium phosphate precipitation with a plasmid bearing a G418 resistance gene (pSV3neo) together with one of the &-reductase expression plasmids. Cells were selected for G418 resistance by growth in the presence of 700 pg/ml of drug. Colonies expressing 5a-reductase were identified by enzyme assay and purified by serial dilution. The nomenclature for the transfected cell lines is: CHO-1827 = cells expressing the type 1 isozyme, CHO-1829 = cells expressing the type 2 isozyme, CHO-1880 = cells expressing (3418 resistance.

Enzyme Assays-To assay 5a-reductase activity in intact cells, 14C- labeled steroids were added to the medium at a final concentration of 2 p~ from ethanol stocks. Cells were maintained at 37 "C in a 5% CO, incubator in the presence of I4C-labeled steroid for 10-20 min. Medium was removed and extracted with 10 volumes of methylene chloride. Following isolation and evaporation of the organic phase, steroids were dissolved in 50 pl of chloroform/methanol(2:1), spotted on thin layer chromatographyplates (No. 5748, E. Merck, Darmstadt, Germany), and resolved by development in chloroform/ethyl acetate (3:l). Reaction products were visualized by exposure of the chromato- gram to film, and radiolabeled steroids were quantitated by scintil- lation counting.

To determine the apparent K,,, for steroid substrates in intact cells, CHO-1827 or CHO-1829 cells were plated on day 1 in six-well plates at a density of 30,000 cells/well. On day 3, cells were washed once with 37 "C medium minus supplements. Serial dilutions of "C-labeled steroids were then added to the cells in this medium, and assays were conducted at 37 "C for 5 min with gentle shaking. Enzyme activity was measured as described above.

Selective permeabilization of the cell plasma membrane by digitonin was accomplished by incubating cells in buffer A (10 mM PIPES, pH 7.0, 0.3 M sucrose, 100 mM KCl, 2.5 mM MgC12, 1 mM EDTA, 10 pg/ml digitonin) for 10 min (Roitelman et al., 1992). Prior to assaying 5a-reductase at pH 7.0, cells were washed once with buffer B (buffer A minus digitonin). Prior to assay at pH 5.0, cells were washed once in buffer C (buffer B containing 10 mM MOPS, pH 5.0, in place of PIPES). Assays were performed at 37 "C with gentle shaking in the presence of radiolabeled steroid at pH 7.0 (buffer B) or pH 5.0 (buffer C). The addition of NADPH (1 mM), steroid

concentrations, and incubation times were as indicated in the figure legends.

Perforation of cell membranes by nitrocellulose filter overlay was performed as described by Ktistakis et al. (1991). CHO cells were grown on glass coverslips, rinsed once with non-supplemented medium followed by phosphate-buffered saline (PBS, 5 mM Na2HP04, 1.3 mM KH2P0,, pH 7.4, 200 mM NaC1, 3 mM KCl). Following removal of excess moisture, coverslips were placed cell-side down for 2 min on nitrocellulose filters that had been prewet with PBS. Coverslips were removed from the nitrocellulose and 5a-reductase was assayed at pH 5.0 or 7.0 with ["Cltestosterone and NADPH at final concentrations of 2 p~ and 1 mM, respectively. Incubations were performed at 37 "C for 10 min.

For assay of 5a-reductase activity in cell lysates, cells were harvested from plates in PBS with a rubber policeman, pelleted by centrifugation, and frozen in liquid NB for storage at -80 "C, or lysed and assayed immediately. Cell pellets were homogenized in 10 mM potassium phosphate, pH 7.0, 150 mM KCl, 1 mM EDTA with three short pulses of a Brinkman polytron. 5a-Reductase assays were performed in 0.1 M Tris-citrate buffers at the indicated pH. ["C] Steroid concentrations and incubation times are indicated in the figure legends. Assays were performed at 37 "C with gentle shaking for 10-20 min, contained 1-5 pg of extract protein, and were initiated by the addition of NADPH to a final concentration of 1 mM. The concentration of protein in cell extracts was determined by the method of Bradford (1976) using bovine y-globulin as a standard. Apparent K,,, and Vmax values were derived by the analysis of data with K.CAT, a computer program that fits data to the Michaelis- Menten equation using non-linear regression. Apparent Ki values were derived from secondary replots of K,,, or VmaX values.

Immunoprecipitation-Cells were plated in 100-mm dishes, grown to 50-70% confluence in supplemented media, washed twice in 37 "C pulse media (Dulbecco's modified Eagle's medium lacking cysteine and methionine, supplemented with 350 pg/ml proline, buffered with sodium bicarbonate, and adjusted to pH 7.0). Cells were incubated in 5 ml of pulse media containing 300 pCi of Tran35S-labelTM for the indicated intervals in a 5% COz incubator at 37 "C, washed twice with PBS, and lysed by the addition of 1.5 ml of 4 "C SDS lysis buffer (50 mM Tris-C1, pH 7.5,150 mM NaCl, 4 mM EDTA, 1 mM phenylmeth- ylsulfonyl fluoride, 1% (w/v) SDS, 0.5% (w/v) deoxycholic acid), or Nonidet P-40 (NP-40) lysis buffer (SDS lysis buffer except that 1% (v/v) NP-40 was substituted for SDS). Dishes were kept on ice for 10 min, after which lysates were centrifuged at 10,000 X g for 10 min at 4 "C. Primary antibodies (0.4 mg) directed against 5a-reductase type 1 or 2 were added to the supernatant and incubated overnight at 4 "C with shaking. Protein A-Sepharose beads were added and the incubations were continued for another 1-2 h. The beads were pelleted by 15 s of centrifugation at 10,000 X g and washed three times in buffer 1 (50 mM Tris-C1, pH 7.5, 150 mM NaC1,4 mM EDTA, 1% (v/v) NP- 40), once in buffer 2 (buffer 1 with 500 mM NaCl), and once in buffer 3 (buffer 1 minus NP-40). The last wash was followed by a 1-min centrifugation at 10,000 X g. Sample buffer (62.5 mM Tris-C1, pH 6.8, 2% (w/v) SDS, 10% (v/v) glycerol, 0.75% (w/v) bromphenol blue, 2.5% (v/v) 2-mecaptoethanol) was added to the beads, and the mixture was boiled for 3 min. The beads were pelleted and proteins in the supernatant were fractionated by SDS-polyacrylamide gel electrophoresis (Thigpen and Russell, 1992). Gels were enhanced, dried, and exposed to film as indicated. Quantitation of immunoprecipitated &-reductase was performed on an Ambis Radio Analytic Imaging System 4000.

Immunocytochemistry-Polyclonal antisera specific for 5a-reductase type 1 or 2 were preabsorbed against extracts from mock- transfected cells (CHO-1880). Briefly, cells were grown to confluence, harvested in PBS, and pelleted by centrifugation. Pellets were homogenized in either 0.1 M MOPS, pH 7.5, 0.3 M NaCl, or 0.1 M MOPS, pH 7.5, 80 mM CaClZ, and cell lysate proteins were coupled to Affi-Gel15 or Affi-Gel 10 resins, (Bio-Rad), respectively. Following coupling, the resins were extensively washed, and polyclonal antibodies specific for each &-reductase were applied and incubated at 4 "C with gently shaking for 4 h. Eluates from the CHO-1880 Affi-Gel resins were used as primary antibodies in immunocytochemistry.

For the subcellular localization of 5a-reductase isozymes by indirect immunofluorescence, transfected cells were grown on glass coverslips in supplemented medium, rinsed three times in PBS, and fixed for 30 min at 26 "C in 2% (w/v) paraformaldehyde, 10 mM sodium periodate, 75 mM lysine-HC1,37 mM sodium phosphate, pH 6.2. Cells were washed four times in PBS and permeabilized in Saponin buffer (2.5 mM Tris-C1, pH 7.5, 137 mM NaCl (TBS), 0.1% (w/v) saponin,

17406 CHO Cell Lines Expressing Human Sa-Reductase Isozymes

0.5% (w/v) bovine serum albumin) for 25 min at 26 "C. Polyclonal

diluted as indicated in Saponin buffer, added to cells, and incubated antisera specific for 5a-reductase type 1 or 2, or preimmune sera were

at 37 "C for 1 h. Cells were washed four times in Saponin buffer and incubated at 37 "C for 30 min with fluorescein-labeled goat anti- rabbit antibodies diluted 1:lOO in Saponin buffer. Cells were then washed four times in Saponin buffer, twice in TBS, once in water, and mounted onto slides with 90% glycerol (v/v), PBS, pH 9.0, 2.5% (w/v) 1,4-diazabicyclo(2.2.2)octane (Sigma, D-2522). Fluorescence microscopy was performed with a Zeiss Photo Microscope 111 using a 63X Planapo oil immersion objective and fluorescein and rhodamine filter packages. Photomicrographs were obtained with Fujichrome P1600D film.

To determine the membrane topology of 5a-reductase, transfected cells were grown on glass coverslips in supplemented medium, rinsed two times in PBS and fixed as described above. To selectively permeabilize the plasma membrane, cells were incubated for 10 min at 26 "C in buffer A, washed three times in PBS, and then in PBS, 1% (w/v) bovine serum albumin for 30 min at 26 "C. To permeabilize all membranes, cells were incubated in Saponin buffer for 30 min at 26 "C. Anti-5a-reductase antibodies, diluted as indicated, and rat monoclonal anti-BiP antibody (Bole et al., 1986), diluted 1:25, were mixed, added to the cells, and incubated for 30 min at 26 "C with secondary antibodies (fluorescein-labeled goat anti-rabbit IgG and Texas Red-labeled sheep anti-rat IgG, each diluted 1:lOO). Cells were washed, mounted, and photographed as described above.

RESULTS

Transfection of CHO Cells-CHO cell lines expressing the human type 1 and 2 steroid 5a-reductase cDNAs were established by cotransfection with plasmids encoding resistance to the antibiotic G418. Mock-transfected or non-transfected CHO cell lysates contained very low endogenous levels of hamster 5a-reductase activity ( 4 0 pmol of DHT formed/ min/mg protein). In contrast, cell lysates prepared from the CHO-1827 line transfected with the type 1 cDNA and from the CHO-1829 line transfected with the type 2 cDNA expressed high levels of Sa-reductase activity (2.1-7.6 nmol DHT/min/mg protein, Table I). The apparent K,,, values for steroid substrates as well as for the NADPH cofactor are similar to those previously determined in transiently transfected cells (Anderson et al., 1991). The apparent Ki values for two competitive 4-azasteroid inhibitors (finasteride, 4- MA) and a non-steroidal inhibitor (LY191704) were also comparable to those previously determined (Table I).

Immunoprecipitation of 5a-Reductase Isozymes-As shown in Fig. 1, immunoprecipitation of '%labeled proteins from CHO-1827 cells with a rabbit polyclonal antibody against the type 1 isozyme followed by SDS-polyacrylamide gel electrophoresis yielded a polypeptide with a calculated M, of 23,000. Immunoprecipitation of radiolabeled CHO-1829 cells with an anti-type 2 serum produced a slightly smaller 5a-reductase type 2 protein with a calculated M, of 21,000. Inclusion of

excess peptide antigen in the immunoprecipitation reactions resulted in a loss of these specifically recognized proteins. Immunoprecipitation of mock-transfected cells with immune sera, or of either of the transfected cell lines with preimmune sera, did not produce a specific protein (Fig. 1).

Turnover of Sa-Reductase Isozymes-To study the turnover of the 5a-reductase isozymes, transfected CHO cells were subjected to the pulse chase protocol described under "Exper- imental Procedures." Immunoprecipitation of cell lysates containing the type 1 isozyme yielded the results shown in the left panel of Fig. 2. A radiolabeled protein of 23 kDa was precipitated at zero times of chase, and the intensity of this protein gradually decreased with longer chase times (Fig. 2, inset). The calculated half-life for the type 1 isozyme was approximately 30 h. Experiments with the CHO-1829 cells indicated that the half-life of the type 2 isozyme was similarly about 30 h (Fig. 2, right panel and inset). In two experiments, one of which is shown in Fig. 2, the inclusion of 1.0 p M 4-MA or finasteride in the pulse-chase protocol did not appreciably affect the turnover of either 5a-reductase isozyme.

Finasteride Affects Immunoprecipitation-In the course of measuring the half-lives of the 5a-reductase isozymes, we noted that the presence of finasteride affected the ability to immunoprecipitate the type 2 isozyme but not the type 1 isozyme (Fig. 3). Solubilization of radiolabeled cells followed by lysis with buffers containing the detergent NP-40 and immunoprecipitation yielded the 21-kDa Sa-reductase type 2 protein (Fig. 3, lower panel, lane 2). However, when the concentration of finasteride was increased from 0 to 50 nM in the chase reaction, the ability to immunoprecipitate the type 2 isozyme was lost (lunes 3-7, lower panel). The drug concentration required to affect this transition was sharp (between 0.5 and 5 nM) and was in the range needed to inhibit enzyme activity (Table I). The ability of finasteride to disrupt immunoprecipitation of the type 2 isozyme was not affected by the pH of the immunoprecipitation buffer or by isolation of microsomes prior to carrying out the precipitation reaction (data not shown). If, however SDS, rather than NP-40, was used to solubilize the cells, immunoprecipitation of the type 2 isozyme was not inhibited by finasteride (lune 8, lower panel). Finasteride was unique in its ability to cause a loss of immunoprecipitability in NP-40 cell lysates since the presence of even high concentrations (50 p ~ ) of 4-MA did not affect antibody recognition of the type 2 isozyme (lane 9, lower panel).

Experiments of similar design carried out with CHO cells expressing the type 1 isozyme indicated that recognition of this isozyme was independent of the presence of finasteride or 4-MA (Fig. 3, upper panel).

TABLE I Characterization of human 5a-reductase type 1 and 2 isozymes expressed in CHO cells

CHO cell line isolation, cell homogenate preparation, and enzyme assay were carried out as described under "Experimental Procedures." Human type 1 Human type 2

K , V,, K , K , V-. Ki

P M nmol/min/mg protein nM W nmol/min/mg protein nM

Substrate/cofactor Testosterone 1.7 2.8 Androstenedione 0.3 5.7 Progesterone 1.3 7.6 NADPH 3-5

0.2 2.4 0.2 2.4 0.2 2.1 7-10

Inhibitor Finasteride 325 12

LY191704 3.0 20,000 4-MA 8.0 4.0


CELLS

ANTI-SERUM

PEPTIDE

46 -

30- Y

x

r‘

14-

-1 M CHO-1827 M CHO-1829 CELLS

DETERGENT

FINASTERIDE

4-MA

S S FIG. 1. Immunoprecipitation of Ba-reductase isozymes. The

indicated CHO cell lines were cultured as described under “Experi- mental Procedures.” On day 3 of cell growth, the cells were pulse labeled with 60 pCi/ml of Tran%-labelTM in cysteine/methionine- free medium for 4 h. Immunoprecipitation of 5a-reductase isozymes from SDS-solubilized cell extracts, SDS-polyacrylamide gel electrophoresis, and autoradiography were performed as described under “Experimental Procedures.” Antisera 1 and 2 are directed against the type 1 and 2 isozymes, respectively. Antisera P refers to the corresponding preimmune sera. The symbol + indicates that the immunoprecipitation reaction was carried out in the presence of 7 pg/ml of peptide antigen. The positions of migration of radiolabeled stand- ards are indicated on the left and right of the autoradiogram. The gel was exposed to XAR-5 film for 4 h.

I 4-Azasteroid “\ 1 .o

4-Azasteroid A-A None

H 4.MA - 1 H Finasteride

0 10 20 30 40 0 10 20 30 40 50 Chase (hour)

FIG. 2. Degradation of S6S-radiolabeled 5a-reductase isozymes in CHO cell lines in the presence and absence of 4- azasteroid inhibitors. CHO cell lines expressing the indicated isozyme were pulse labeled for 30 min with 60 pCi/ml of Tran%- labelT“, followed by a chase for the indicated period of time in medium containing 0.1 mM unlabeled cysteine and methionine. Where indicated, 1 PM 4-MA or finasteride were included in the pulse and chase medium. Immunoprecipitation in SDS-containing buffers, SDS-polyacrylamide gel electrophoresis, and autoradiography were carried out as described under “Experimental Procedures.” The gels were exposed to XAR-5 film for 4-5 days. Insets, autoradiograms of immunoprecipitated 5a-reductase isozymes in the absence of 4-azasteroids. Left and right panels, the radioactivity in the bands corresponding to the indicated isozymes was measured by counting in an Ambis 4000. The resulting counts/minute were plotted as a function of time. The “counts/minute X lo-”’ a t zero time represents radioactivity present in the immunoprecipitated 5a-reductase isozyme after 0 h of chase. The results shown are representative of three separate experiments.

pH Optima of 5a-Reductase Isozymes-The type 1 isozyme in lysates of CHO-1827 cells demonstrated an alkaline pH optimum in the range of 7.0 to 8.5, while the type 2 isozyme in lysates of CHO-1829 cells demonstrated an acidic pH optimum in the range of 4.9 to 5.1 (data not shown). These pH optima are identical to those reported using cell lysates from transiently transfected cells and in tissue extracts (And- ersson et al., 1991).

CHO-1827 I

23 kDa - - 1

1 2 3 4 5 6 7 8 9

Type

CELLS

DETERGENT

FINASTERIDE

4-MA

21 kDa- ~. W 7 - W ”.& o (L f Type

2 1 2 3 4 5 6 7 8 9

FIG. 3. Finasteride alters immunoprecipitation of 5a-reductase type 2. The indicated cell line was cultured as described under “Experimental Procedures.” On day 3, cells were pulse-labeled with Tran‘%-IabelTM for 4 h in the presence of the indicated concentrations of 4-azasteroid, and then lysed with buffers containing either 1% NP-40 ( N ) or 1% SDS (S). Immunoprecipitation, SDS-polyacrylamide gel electrophoresis, and autoradiography were carried out as described under “Experimental Procedures.” The gels were exposed to XAR-5 film for 12 h. M, mock-transfected CHO-1880 cells. The positions of migration of the 23-kDa 5a-reductase type 1 isozyme and the 21-kDa 5a-reductase type 2 isozyme are indicated.

CHO-1827 TYPE 1

CHO-1829 TYPE 2

B.

DRUG NH4CI CQ MON - NH4CI CQ MON - FIG. 4. Effect of various drugs that perturb intracellular

pH on 5a-reductase isozyme activity. Panels A and B, on day 3 of culture, the indicated CHO cell line received 3 ml of medium containing either 20 mM NH4Cl, 100 pM chloroquine (CQ), 10 p M monensin ( M O N ) , or no drug (-). After incubation for 3 h, [ “C] testosterone was added to a final concentration of 2 pM, and the incubation was continued for an additional 10 min. Thereafter, the media were extracted with methylene chloride, and the conversion of [“C]testosterone (7‘) to [“CIDHT was determined by thin layer chromatography as described under “Experimental Procedures.”

To determine if the acidic pH optimum of the type 2 isozyme reflected a localization of the protein in an acidic subcellular compartment, transfected CHO cells were treated with compounds known to disrupt the pH of internal organ- elles. The ability of a 5a-reductase isozyme to convert testosterone to DHT was then measured both in treated and in non-treated control cells. Incubation of CHO-1827 and CHO- 1829 cells with 20 mM NH4Cl, 200 p M chloroquine, or 10 pM monensin did not significantly affect the activity of either isozyme (Fig. 4). In these experiments, the pH-dependent ability of lysosomes to sequester acridine orange (2.5 mM) was used as a positive control for drug action. In each experiment, the addition of drug resulted in the failure of acridine orange to sequester in the lysosomal compartment (data not shown).

17408 CHO Cell Lines Expressing Human 5a-Reductase Isozymes

Immunocytochemical Localization of Sa-Reductase Iso- Staining of mock-transfected CHO cells with the type 2 zymes-To determine the subcellular location of each 5a- immune serum or CHO-1829 cells with preimmune sera reductase isozyme, CHO cells were permeabilized with digi- yielded only a very low level of background staining (Figs. 5, tonin (10 pg/ml), fixed with paraformaldehyde, and subjected D and E, respectively). In contrast, incubation of the CHO- to indirect immunofluorescence analysis using preimmune 1829 cells with anti-type 2 immune sera produced an intense and immune antisera. No subcellular localization of immu- fluorescence pattern similar to that seen for the type 1 isozyme nofluorescence was seen when either mock-transfected CHO in the CHO-1827 cells. The subcellular localization of the 5a- cells were stained with immune serum against the type 1 reductase isozymes did not change when cells were incubated isozyme (Fig. 5A) , or when CHO-1827 cells were stained with for 3 h in the presence of 2 mM testosterone, 2 mM DHT, 1 a preimmune serum (Fig. 5B). Incubation of CHO-1827 cells mM 4-MA, or 1 mM finasteride prior to immunocytochemical with the type 1 immune sera produced an intense staining staining (data not shown). pattern consistent with localization to the endoplasmic retic- Topology of 5a-Reductase Isozymes-The antipeptide ant- ulum (Fig. 5 C ) . ibodies directed against the carboxyl termini of the 5a-reduc-

FIG. 5. Intracellular localization of Sa-reductase isozymes by indirect immunofluorescence. CHO cells were stained with rabbit polyclonal antibodies followed by fluorescein-labeled goat anti-rabbit I g G and subsequently photographed as described under “Experimental Procedures.” The CHO cell line and rabbit IgG sera used were: panel A, mock-transfected CHO-1880 cells stained with 5a-reductase type 1 immune serum; panel B, CHO-1827 cells stained with preimmune serum; panel C, CHO-1827 cells stained with type 1 immune sera; panel D, mock-transfected CHO-1880 cells stained with 5a-reductase type 2 immune sera; panel E, CHO-1829 cells stained with preimmune serum; and panel F, CHO-1829 cells stained with type 2 immune sera.


tase isozymes were used in indirect immunofluorescence studies to determine the membrane topology of these epitopes. CHO-1827 cells were treated with the detergent saponin, which permeabilizes all membranes of the cell, and stained with either the anti-type 1 carboxyl-terminal serum or with a rat monoclonal antibody against a lumenal protein of the endoplasmic reticulum (BiP). An identical immunofluorescence pattern was seen with both anti-sera (Figs. 6, A and B ) . This result confirms the endoplasmic reticulum locale of the 5a-reductase isozyme. When the CHO-1827 cells were treated with low concentrations of the less harsh detergent digitonin, which permeabilizes the plasma membrane but leaves the endoplasmic reticulum membrane intact, a reticular pattern of staining was seen after incubation with the anti-type 1 serum (Fig. 6C). As expected, when the same cells were stained with the anti-BiP antibody, no reticular pattern of staining was visualized (Fig. 6D). These results imply that the epitope recognized by the polyclonal 5a-reductase antiserum (i.e. the carboxyl terminus) is on the cytoplasmic side of the endoplasmic reticulum membrane, in contrast to the epitope recognized by the anti-BiP antibody, which is known to reside within the endoplasmic reticulum (Gething and Sambrook, 1992). In experiments not shown, the epitope recognized by the antipeptide antibody against the type 2 isozyme was also localized to the cytoplasmic side of the endoplasmic reticulum membrane.

5a-Reductase Activity in Permeabilized Cells-CHO-1827 cells were incubated in buffers containing or lacking digitonin

and then assayed for 5a-reductase enzyme activity at pH 5.0 or 7.0 (Fig. 7A). In the absence of detergent, enzyme activity was not dependent on the addition of exogenous NADPH and did not show a pH preference. In contrast, in the presence of digitonin, the enzyme activity was NADPH dependent and exhibited the neutral pH optimum characteristic of the type 1 isozyme (Fig. 7A).

Parallel experiments with the CHO-1829 cells yielded quite different results (Fig. 7B). As expected, intact cells incubated in the absence of detergent did not require exogenous NADPH to support 5a-reductase activity, whereas incubation of cells with digitonin-containing buffers yielded a type 2 enzyme activity that was dependent on exogenous NADPH. Surpris- ingly, in digitonin-treated cells, the 5a-reductase activity was similar at pH 5.0 and 7.0 (Fig. 7B). This result indicated that gentle permeabilization of cells with digitonin-containing buffers preserved a pH 7.0 activity in the type 2 isozyme. For unknown reasons, the activity detected at pH 5.0 in intact cells was about two times higher than that detected at pH 7.0.

5a-Reductase Activity in Perforated Celki-CHO-1827 and CHO-1829 cells were perforated by a nitrocellulose overlay method and the pH dependence of the 5a-reductase isozymes was determined. Perforation of CHO-1827 cells yielded a 5a- reductase type 1 activity that exhibited a neutral pH optimum (Fig. 8A). Perforation of CHO-1829 cells resulted in a 5a- reductase type 2 enzyme with low activity at pH 7.0 and high activity at pH 5.0 (Fig. 8B). Thus, cellular perforation was not associated with maintenance of the neutral pH activity of

FIG. 6. Determination of membrane topology of Sa-reductase type 1 carboxyl terminus. 5n-Reductase type 1 and BiP were simultaneously detected in the CHO-1827 cells by staining with rabbit polyclonal anti-5n-reductase sera and rat monoclonal anti-BiP antibody followed by fluorescein-labeled goat anti-rabbit I g G and Texas Red-labeled sheep anti-rat IgG. Subsequent photomicroscopy was carried out as described under “Experimental Procedures.” Panel A, Saponin-permeabilized cells photographed to reveal 5a-reductase type 1 staining. Panel E , same cells as panel A but photographed to reveal BiP staining. Panel C, digitonin-permeabilized cells photographed to reveal 5a-reductase type 1 staining. Panel D, same cells as panel C but photographed to reveal BiP staining.


CHO-1827 CHO-1829 TYPE 1 TYPE 2 6o 1

PH DIGITONIN NADPH

FIG. 7. &-Reductase isozyme activity in CHO cells permeabilized with digitonin. The indicated line of transfected CHO cells was permeabilized with buffers containing 10 pg/ml digitonin and assayed for 5a-reductase enzyme activity at the indicated pH in the presence or absence of 1 mM NADPH. The conversion of [“CC] testosterone (T) into [”CIDHT was determined by thin layer chromatography assay as described under “Experimental Procedures.”

i20[ w p 10

0 0 z

0 CHO-1827 CHO-1829

TYPE 1 TYPE 2

FIG. 8. Sa-Reductase isozyme activity in perforated CHO cells. The indicated cell line (panel A = CHO-1827, panel B = CHO- 1829) was either left intact or perforated by nitrocellulose overlay as described under “Experimental Procedures,” and then assayed for 5a-reductase isozyme activity at the indicated pH. Perforation, enzyme assay, and percent conversion of [“C]testosterone (T) to [“C] DHT were determined as described under “Experimental Proce- dures.”

TABLE I1 pH versus K,, human 5a-reductase type 2

Apparent K,,, and V,, values were derived from triplicate assays carried out as described under “ExDerimental Procedures.”

pH

4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0

K, P M

0.261 0.259 0.063 0.015 0.007 0.004 0.006 0.008

V- nmol DHT/rninlmg

0.06 3.08 1.08 0.41 0.25 0.20 0.18 0.15

0.23 11.9 17.1 27.3 35.7 50.0 30.0 19.4

the type 2 isozyme, as had been seen after permeabilization using digitonin.

Efficiency of Sa-Reductase Type 2 Isozyme at Different pH Values-A series of kinetic studies were carried out in which the apparent Vmax and K,,, of the type 2 isozyme were determined in buffers of different pH (Table 11). A measure of the relative efficiency of the isozyme at each pH was made by plotting V,,,/K,,, uersus pH (Fig. 9). In these experiments, the type 2 isozyme was found to be most efficient at neutral

4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 pH

FIG. 9. &-Reductase type 2 is most efficient at neutral pH. The data from Table 111 are plotted as V,../K, versus pH.

TABLE 111 Apparent testosterone K,,, values ( p ~ ) for Sa-reductase isozymes in

cell lysates, intact cells, and permeabilized cells Apparent K,,, values were measured in duplicate experiments as

described under “Experimental Procedures.”

Isozyme Cell lysates Digitonin-permeabilized cells Intact cells

p H 5.0 p H 7.0 p H 5.0 p H 7.0 Human 1 ND 1.7 ND” 2.3 4.1 Human 2 0.23 0.004 1.1 0.01 0.05 ND, not determined.

pH. The isozyme was equally stable in the presence of NADPH at both acid and neutral pH (data not shown), thus the pH dependence of substrate affinity could not to be attributed to differential stability of the protein.

Kinetic Constants of 5a-Reductase in Whole Cells-The results of Fig. 9 suggest that the type 2 isozyme is more efficient at neutral pH. If the type 2 isozyme is localized to a subcellular compartment with a neutral pH, then the affinity of the isozyme for testosterone in whole cells should be higher than that measured in cell lysates at pH 5.0. To test this hypothesis, the apparent K, values for testosterone of the type 1 and 2 isozymes were determined in intact cells, digitonin-permeabilized cells, and in cell lysates (Table 111). The affinity of the enzyme for testosterone in both intact ( K , = 0.05 PM) and permeabilized cells in pH 7.0 buffers ( K , = 0.01 p M ) was similar to that determined at pH 7.0 in cell lysates (K, = 0.004 p ~ ) . The affinity of the enzyme for substrate in permeabilized cells at pH 5.0 ( K , = 1.1 PM) was similar to that measured in cell lysates at this pH ( K , = 0.23 FM). The affinity of the type 1 isozyme for testosterone was essentially the same with all assay conditions (Table 111). Similar results were obtained when progesterone and androstenedione were used as substrates in these experiments (data not shown).

DISCUSSION

In this paper, we describe the characterization of permanent CHO cell lines that express the human 5a-reductase type 1 and 2 cDNAs. The half-lives of the two 5a-reductase isozymes are long (>30 h) and are not altered by the presence of the competitive 4-azasteroid inhibitors finasteride and 4-MA. Fi- nasteride was found to alter the ability of rabbit polyclonal antibodies to immunoprecipitate the type 2, but not the type 1, isozyme. Both isozymes were localized to the endoplasmic reticulum and shown to have their carboxyl termini exposed to the cytoplasm using immunocytochemistry. In cell lysates, the type 1 isozyme exhibits a Vmax at pH 7.0, whereas the type 2 isozyme exhibits a Vmax at pH 5.0. However, the type 2 isozyme has a lower apparent K, for testosterone at pH 7.0 than at pH 5.0. Experiments with intact and permeabilized cells confirm that the type 2 isozyme has a lower apparent

CHO Cell Lines Expressing Human Sa-Reductase Isozymes 17411

K,,, at neutral pH within the cell. The long (-30 h) half-lives of the 5a-reductases suggest

that these isozymes may not be subject to regulation at the level of protein turnover. In accord with this hypothesis, the inclusion of substrate, product, or competitive inhibitors in the culture media did not affect the half-lives of the isozymes (Fig. 2), as has been found for other proteins with short half- lives (Goldstein and Brown, 1990; Murakami et al., 1992).

A surprising finding from the half-life studies was the ability of finasteride to affect the immunoprecipitation of the type 2 but not the type 1 isozyme (Fig. 3). This effect was observed in cells lysed with the non-ionic detergent NP-40 and not in cells lysed with the ionic detergent SDS. The simplest interpretation of this result is that when finasteride binds to the type 2 isozyme in the presence of NP-40, the conformation of the protein is altered so that it is no longer recognized by the antibody. When cells are lysed with SDS, the isozyme is presumably denatured and so releases the bound finasteride, ultimately allowing recognition by the antibody. The putative ability of finasteride to alter the structure of the type 2 isozyme appears to be an unique property of this compound since immunoprecipitation was not abolished by the equally potent inhibitor, 4-MA. The tight (essentially irreversible) binding of finasteride to the type 2 isozyme may explain in part why this drug is so effective at decreasing serum DHT levels in humans (McConnell et al., 1992; Gorm- ley et al., 1992).

The alkaline and acidic pH optima manifest by 5a-reductase type 1 and 2, respectively, are classic properties that led to the discovery of these two isozymes (Moore and Wilson, 1976). The acidic pH optimum of the type 2 isozyme appears to be an intrinsic feature of the protein, since some substitu- tion mutations result in a more basic pH optimum (Thigpen et al., 1992a, 1992b). This feature is also evolutionarily con- served in that the rat type 2 isozyme has an acidic pH optimum (Normington and Russell, 1992).

We used immunocytochemistry to test the hypothesis that the acidic pH optimum reflected the subcellular localization of the type 2 isozyme within an acidic compartment of the cell (Fig. 5 ) . These experiments revealed that both isozymes reside within the endoplasmic reticulum, a compartment thought to have a largely neutral pH (Anderson and Pathak, 1985). In agreement with this result, compounds known to disrupt the internal pH of acidic subcellular compartments like the endosome and lysosome did not affect the activity of the type 2 isozyme in intact cells (Fig. 4). Taken together, these data suggest that the acidic pH optimum of the type 2 isozyme in cell lysates is not a consequence of the enzyme's subcellular location.

Previous investigations have shown that 5a-reductase activity in the human prostate (predominantly the type 2 isozyme, Jenkins et al., 1992) is associated with the nuclear membrane (Hudson, 1981). The finding that both isozymes are present in the endoplasmic reticulum of transfected CHO cells may reflect cell type differences, species differences, or a consequence of overexpression in the cultured cells, The prostate is an active exocrine gland and has a proliferated endoplasmic reticulum (Cavazos, 1975). Therefore, it seems unlikely that the differences are due to variations in the amounts of endoplasmic reticulum membrane. The endoplasmic reticulum is contiguous with the outer nuclear membrane (Fawcett, 1981), thus these disparate results may reflect differences in the architecture of prostate vers'sus cultured hamster fibroblast cells. Future immunocytochemistry or electron microscopy in prostate cells may shed light on this conundrum.

By differential permeabilization of transfected CHO cell membranes, it was shown that the carboxyl termini of the 5a- reductase isozymes lie on the cytoplasmic side of the endoplasmic reticulum bilayer (Fig. 6). The overall topology of these very hydrophobic proteins is not known, and hydropa- thy plots have not so far revealed distinct transmembrane domains (Andersson et al., 1989a). It is interesting to note that mutation of arginine 246 disrupts NADPH cofactor binding (Thigpen et al., 1992b). This residue lies within the peptide epitope recognized by the type 2 antiserum, thus, localization of the carboxyl termini of the 5a-reductase isozymes may reflect a functional requirement for access to cytoplasmic cofactor. In support of this hypothesis, 5a-reductase enzyme activity in cells with permeabilized plasma membranes but intact endoplasmic reticulum required addition of NADPH (Fig. 7).

Permeabilization of CHO cells by various means was also used to gain insight into why the type 2 isozyme has repeatedly been shown to have an acidic pH optimum. Treatment of CHO-1829 cells with low concentrations of digitonin was associated with a type 2 isozyme whose activity demonstrated both an acidic and a neutral pH optimum (Fig. 7). In contrast, when cells were perforated by nitrocellulose overlay, the type 2 enzyme activity had an acidic pH optimum and very low activity at pH 7.0 (Fig. 8). Finally, an analysis of the relative efficiency of the type 2 isozyme via kinetic methods also pointed to a neutral pH optimum (Fig. 9, Table 111).

These results are consistent with the type 2 isozyme having a neutral pH optimum within the cell, but when cells are disrupted using mechanical methods (scraping, polytroning, and perforation) there is a shift to an acidic pH optimum. This shift may reflect a disruption of the lipid bilayer, an association with or a dissociation from the type 2 isozyme of an allosteric molecule, or a conformational change within the isozyme itself. In an attempt to distinguish these possibilities, CHO-1829 cells and lysates were incubated with a large number of compounds (detergents, kinases, phosphatases, inhibitors of phosphatases and kinases, cytoskeleton disrup- ters, and membrane perturbants, etc.) in an effort to preserve the neutral pH form of the type 2 isozyme. None of the treatments so far tested have accomplished this goal. We are currently attempting to purify the type 1 and 2 isozymes to further explore the biochemical basis of their pH optima.

The human type 2 isozyme in intact or permeabilized CHO cells has an apparent substrate affinity in the nanomolar range, whereas the type 1 isozyme exhibits micromolar affin- ities for steroid substrates (Table 111). Inasmuch as these in vitro measured K, values reflect the behavior in vivo of 5a- reductase type 2, the isozyme would appear well suited to act under conditions of limiting substrate. This situation is characteristic of the embryo, in which the production of testosterone by the fetal testes is low and conversion into DHT is crucial for sexual development (Griffin and Wilson, 1989; Wilson et al., 1993). Conversely, under conditions of saturating substrate concentration, such as occurs in the sexually mature male, a small amount of the type 2 isozyme might be expected to synthesize considerable DHT. This ability may have implications for the treatment of disorders associated with an overproduction of DHT, such as benign prostatic hyperplasia. In this disease, complete inhibition of 5a-reductase type 2 (the predominant isozyme in the human prostate) may be difficult due to the presence of high levels of circulating testosterone and the efficiency of the isozyme, which may explain in part why finasteride therapy is not beneficial in all males with benign prostatic hyperplasia (Gormley et al., 1992).


Acknowledgments-We thank Edith Womack for cell culture advice, Lou Hersch for advice on enzyme kinetics, Richard Anderson and Nick Ktistakis for microscope access and cell biology advice, Gary Rasmusson and Blake Neubauer for inhibitors, Helen H. Hobbs for critical review of the manuscript, and members of the Department of Molecular Genetics for suggestions and comments.

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