Inducible expression of protein kinase Calpha suppresses steroidogenesis in Y-1 adrenocortical cells

Mary E. Reyland1, David L. Williams2, and Elizabeth K. White1

1 Department of Basic Science and Oral Research, School of Dentistry, University of Colorado, Health Sciences Center, Denver, Colorado 80262; and 2 Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have previously shown that protein kinase C (PKC) suppresses steroidogenesis in Y-1 adrenocortical cells. To ask directly if the PKCalpha isoform mediates this suppression, we have developed Y-1 cell lines in which PKCalpha is expressed from a tetracycline-regulated promoter. Induction of PKCalpha expression in these cell lines results in decreased P450 cholesterol side-chain cleavage enzyme (P450-SCC) activity as judged by the conversion of hydroxycholesterol to pregnenolone. Transcription of a P450-SCC promoter-luciferase construct is also reduced when PKCalpha expression is increased. However, expression of PKCalpha has no effect on 8-bromo-cAMP induction of steroidogenesis, indicating that these pathways function independently to regulate steroidogenesis. To determine the relationship between endogenous PKC activity and steroidogenesis, we examined 12 Y-1 subclones that were isolated by limited dilution cloning. In each of these subclones, steroid production correlates inversely with total PKC activity and with the expression of PKCalpha but not PKCepsilon or PKCzeta . These studies define for the first time the role of a specific PKC isoform (PKCalpha ) in regulating steroidogenesis and P450-SCC activity in adrenocortical cells.

cholesterol side-chain cleavage; transcription; adenosine 3',5'-cyclic monophosphate regulation

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

PREVIOUS WORK FROM OUR laboratory and others suggests that the steady-state level of steroid hormone production in adrenocortical cells reflects a balance between stimulation by the cAMP-dependent protein kinase (PKA) pathway and suppression by the protein kinase C (PKC) pathway (11, 16, 17, 23, 24, 30, 36, 37, 39, 43). Activation of the PKA pathway by adrenocorticotropic hormone (ACTH) results in an acute increase in steroid output due to an increased availability of free cholesterol and a sustained increase due to increased expression of the steroid hydroxylase genes, such as P450 cholesterol side-chain cleavage (P450-SCC) (36, 41). In contrast, the primary effect of PKC on steroidogenesis appears to be suppressive. This is supported by two lines of evidence. First, in ovarian granulosa, testicular leydig, and adrenocortical cells, 12-O-tetradecanoylphorbol-13-acetate (TPA) suppresses the induction of steroidogenesis by trophic hormones and cAMP (16, 23, 24, 30). Second, studies using inhibitors of PKC show that PKC acts as a tonic suppressor of basal steroidogenesis in Y-1 adrenocortical cells and MA-10 leydig cells (22, 37, 39).

We have previously reported that treatment of Y-1 cells with inhibitors of PKC results in a dose-dependent increase in steroid production (37). This is accompanied by a comparable increase in the expression of mRNAs for the steroid synthetic enzymes P450-SCC, P450-11beta -hydroxylase (P450-11beta -OH), and 3beta -hydroxysteroid dehydrogenase (3beta -HSD). Furthermore, in Y-1 cells in which the human apolipoprotein E gene is overexpressed, PKCalpha expression is increased, whereas steroid hydroxylase gene expression and steroidogenesis are suppressed. Treatment of these cells with inhibitors of PKC results in recovery of steroidogenesis and steroid hydroxylase gene expression (38, 39). These results suggest that PKC may regulate basal steroidogenesis primarily by determining the steady-state levels of expression of the steroid hydroxylase genes.

The PKC isoform family consists of at least 11 members that differ in their sensitivity to Ca2+ and in their activation by lipid cofactors (32, 33). The subset of PKC isoforms expressed varies with cell type. In Y-1 cells, we have detected PKCalpha , PKCepsilon , and PKCzeta (Reyland and White, unpublished data), whereas PKCalpha and PKCepsilon are the only isoforms detected in rat adrenal glomerulosa cells (31). To demonstrate definitively that PKCalpha regulates adrenal steroidogenesis, we have created Y-1 cell lines in which the expression of PKCalpha is driven by a tetracycline (tet)-controlled promoter (14). When PKCalpha expression is induced in these cell lines, their ability to synthesize steroids is impaired. These studies thus identify a specific PKC isoform, PKCalpha , as an important regulator of steroidogenesis in adrenocortical cells.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Generation of Tetracycline-Regulated Y1PKCalpha Cell Lines

Cell culture. The Y-1 adrenal cell line was obtained from American Type Culture Collection. Cells were maintained in Ham's F-10 medium supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), 2 mM L-glutamine, 12.5% (vol/vol) heat-inactivated horse serum, and 2.5% (vol/vol) heat-inactivated FCS ("complete medium"). Tet and hygromycin B were obtained from Calbiochem. All additional cell culture reagents were obtained from GIBCO.

Plasmid constructs. pUHD15-1, pUHD10-3, and pUHD13-3 were a generous gift of H. Bujard (14). The expression vector, pUHD/PKCalpha , was made by cloning the mouse PKCalpha cDNA from pLTRPKCalpha (26) into the EcoR I site of pUHD10-3, which places it under the control of the tet-regulated promoter.

Transfection. Generation of stable lines using the tet-regulated system requires two rounds of transfection. First, Y-1 cells were cotransfected with pUHD15-1, which encodes the tet transactivator (tTA) gene, and pSV2neo, which encodes resistance to G418 sulfate, at a ratio of 9:1, by calcium phosphate-mediated gene transfer essentially as described (37). Cell clones were selected in complete medium containing 200 µg/ml G418 sulfate (Geneticin, GIBCO) and screened for expression of the tTA gene by transient transfection of a tet-responsive promoter linked to a luciferase reporter gene. Using this approach, we selected four clonal Y-1 cell lines that express the transfected tTA gene. One of these, Y1UHD/7, was then secondarily transfected with pUHD/PKCalpha together with pCMV hygromycin at a ratio of 9:1. To make control cell lines, Y1UHD/7 cells were transfected with the empty pUHD10-3 vector together with pCMV hygromycin. Hygromycin-resistant cell clones (Y1PKCalpha or Y1Con cells) were selected in 200 µg/ml hygromycin B (active form). Tet (2 µg/ml) was included during selection to suppress expression of PKCalpha . The Y1PKCalpha and Y1Con cell lines were maintained in complete medium containing 100 µg/ml each of G418 sulfate and hygromycin B and 2 µg/ml tet. For most experiments, cells were treated as follows. On day 0, the medium was replaced with complete medium containing either 0 or 0.2 µg/ml (or 2 µg/ml in some cases) tet. The medium was replaced with fresh medium of the same composition on days 2 and 4, and cells were typically used on day 6.

Analysis of Steroid Production

We discovered after the subclones were made that the Y1UHD/7 clone selected for the second stage of transfection lacks expression of the 3beta -HSD and the P450-11beta -OH mRNAs. Consequently, steroid production in the Y1PKCalpha and Y1Con lines derived from the Y1UHD/7 clone was measured as pregnenolone accumulation using an RIA assay kit from ICN. Authentic pregnenolone was used as a standard. Nutridoma SP was purchased from Boehringer Mannheim and 22(R)- and 25-hydroxycholesterol were purchased from Sigma Biochemicals. Values were normalized to total cell protein measured using a Bradford assay kit purchased from Bio-Rad (8). Total secreted steroids in the Y1/DW cell clones were measured by the fluorometric assay of Kowal and Fiedler (18) previously described.

PKC Activity in Cell Homogenates

DEAE-cellulose-purified whole cell, cytosol, and particulate fraction homogenates were prepared as previously described (39). PKC activity in vitro was assayed as described using a kit purchased from BRL-Life Technologies (39). Cell protein was determined by the Bradford method using a kit purchased from Bio-Rad (8).

Immunoblot Analysis of PKC Isoforms

Detection of PKC protein was done as previously described (39). DEAE-cellulose-purified cell homogenates (50 µg for PKCalpha and 40 µg for PKCepsilon and PKCzeta ) were separated by 10% SDS-PAGE and transferred to Immobilon (Millipore). Primary antibodies were as follows: for PKCalpha , rabbit anti-PKCalpha (BRL-Life Technologies); for PKCepsilon , rabbit anti-PKCepsilon (C-15, Santa Cruz); for PKCzeta , rabbit anti-PKCzeta (C-20, Santa Cruz). All primary antibodies were used at a dilution of 1:500. Donkey anti-rabbit antiserum (Amersham), diluted 1:5,000, was used as a secondary antibody. Enhanced chemiluminescence (Amersham) followed by autoradiography was used to detect the signal.

Transient Expression From the P450-SCC Promoter

To make the plasmid P450-SCCluc, the mouse 1.5-kb P450-SCC promoter from p-1500P450-SCChGH (40) was PCR amplified and subcloned into the Bgl II, Sal I sites of pXP2 (34) upstream of the luciferase reporter gene. Transient transfections were done as previously described using the calcium phosphate method (37). The DNA-calcium phosphate mixture was left on overnight. The following morning, the cells were washed with PBS and the medium was replaced with complete medium with or without tet. Cells were harvested for luciferase, protein, and beta -galactosidase determination after an additional 30 h. Luciferase was assayed as described (12), and the relative light units (RLU) were normalized to total cell protein. In some experiments, RLU were normalized to beta -galactosidase expression that was assayed as described (2).

Northern Blot Analysis

RNA was harvested using RNA Stat-60 (Tel Test) and prepared according to the manufacturer's directions. Ten micrograms of total RNA were separated on a 1.2% agarose gel containing 2.2 M formaldehyde, transferred to Nytran (Schleicher & Schuell), cross-linked with ultraviolet light (Stratalinker), and hybridized to the indicated cDNA probe. cDNA probes were prepared by random priming in the presence of [alpha -32P]dCTP (Amersham; 800 Ci/mmol). The pSCC-1.8 cDNA was a generous gift of K. Parker (Duke University). The rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was a gift of K. Marcu (State University of New York at Stony Brook). P450-SCC and GAPDH mRNA expression was quantified by PhosphorImager analysis (Molecular Dynamics).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Regulation of PKCalpha Expression From a Tetracycline-Responsive Promoter

Previous studies that demonstrate that PKC suppresses steroidogenesis and steroid hydroxylase gene expression have relied on chemical inhibitors or activators of PKC. These agents show a broad spectrum of activities, inhibiting some or all PKC isoforms, albeit with different sensitivities. Hence, they do not yield information about the role of specific PKC isoforms. We have previously shown that, in Y-1 cells that express the apolipoprotein E gene, PKCalpha expression is increased, whereas steroidogenesis and steroid hydroxylase gene expression are decreased (38). To test specifically the function of PKCalpha in Y-1 cells, we have developed clonal Y-1 cell lines in which PKCalpha is expressed from a tet-regulated promoter. This approach allows us to study the effect of changes in PKCalpha expression on steroidogenesis within a single cell clone and hence eliminates clonal variations inherent in selecting individual cell lines. With the tet-regulated system, PKCalpha expression is suppressed in the presence of tet and induced following tet removal from the medium. To analyze the Y1PKCalpha cell lines for tet-regulated PKCalpha protein expression, cells were withdrawn from tet for 6 days, after which PKC activity was assayed in vitro. This assay is not specific for PKCalpha but instead measures total "activatable" PKC protein in the cell. As seen in Fig. 1, we identified six clonal Y-1 cell lines in which PKC activity is regulated by tet, indicating expression of the transfected PKCalpha cDNA. Induction of PKC activity in the Y1PKCalpha cell lines on withdrawal from tet ranges from 2- to >20-fold. However, total PKC activity can be increased up to 40-fold over the level seen in the Y1Con lines (compare PKC4/12 "minus" tet to Con1/2 and Con1/6). In most of the cell lines, PKC activity in the presence of 2 µg/ml tet is equal to, or less than, that observed in the Y1Con cell lines, indicating that expression of the transfected gene is virtually shut off. In Fig. 2, PKCalpha -specific protein expression with or without tet was examined by immunoblot analysis. As seen in Fig. 2, PKCalpha protein expression correlates well with PKC activity measurements in vitro.


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Fig. 1.   Induction of protein kinase C (PKC) activity in Y1PKCalpha cell lines on withdrawal of tetracycline (tet). Y1PKCalpha cell lines (PKC4/3, 4/5, 4/7, 4/9, 4/10, and 4/12) or Y1Con cell lines (Con1/2 and 1/6) were grown in the absence of tet to induce PKCalpha protein expression (solid bars) or in the presence of 2 µg/ml tet to repress PKCalpha expression (open bars) for 6 days. Cell homogenates were prepared as described in MATERIALS AND METHODS and partially purified by DEAE-cellulose chromatography. PKC activity is expressed as pmol 32P transferred to a myelin basic protein peptide substrate per mg of cell protein. Activity in the absence of 12-O-tetradecanoylphorbol-13-acetate (TPA) and phosphatidylserine was subtracted from the values shown. Numbers represent duplicate measurements within a single representative experiment. These measurements were repeated 2-7 times for these cell lines with similar results.


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Fig. 2.   Expression of PKCalpha protein in Y1PKCalpha cells. PKC4/5, 4/12, and 4/3 cells and Con1/2 cells were grown in the absence (-) or presence (+) of 2 µg/ml tet to repress PKCalpha expression or in the absence of tet to induce PKCalpha protein expression for 6 days as indicated. PKCalpha -specific protein was analyzed by immunoblot analysis as described in MATERIALS AND METHODS. M, migration of molecular mass standards. This experiment was repeated at least 3 times for each cell line with similar results.

With the use of the tet-regulated expression system, it should be possible to modulate expression of the transfected gene by titrating the amount of tet included in the culture medium. Figure 3A shows that the level of PKCalpha protein expression in PKC4/12 cells increases as the tet concentration in the culture medium decreases. In the presence of 2.0 or 0.2 µg/ml tet, very little PKCalpha expression is detected, indicating nearly complete suppression of the transfected gene. However, as the tet concentration is decreased, expression of PKCalpha protein is increased. When PKCalpha protein abundance was quantified by densitometry, PKCalpha protein was shown to increase 4-fold when cells were maintained in 0.02 µg/ml tet, 11-fold in 0.002 µg/ml tet, 23-fold in 0.0002 µg/ml tet, and a maximum of 27-fold in the absence of tet.


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Fig. 3.   PKC expression in Y1PKCalpha cells: regulation by tet. A: to determine the effect of tet dose on PKCalpha expression, PKC4/12 cells were grown in indicated concentrations of tet for 7 days, after which PKCalpha protein was analyzed by immunoblot analysis as described in MATERIALS AND METHODS. B: to determine the time course of induction of PKCalpha expression when tet is withdrawn, PKCalpha protein was analyzed in PKC4/12 cells grown in 0.2 µg/ml tet and after withdrawal from tet for indicated no. of days. M, migration of molecular mass standards. Each experiment was repeated 3 or more times with similar results.

The kinetics of PKCalpha induction following withdrawal of tet from PKC4/12 cells is shown in Fig. 3B. PKCalpha protein is maximally induced 4 days after withdrawal of tet, and expression is stable at least through day 10. Measurements of PKC activity in vitro indicate that PKC activity increases as early as 24 h, is maximal by 4 days, and remains increased at least through day 14 (data not shown).

We have previously shown that, in Y-1 cells, a fraction of the endogenous PKC pool is in an activated form and that this pool functions to tonically suppress steroidogenesis (37). However, increased PKCalpha protein expression in the Y1PKCalpha cell lines may not necessarily correlate with an increase in the abundance of activated enzyme in vivo, since activators such as diacylglycerol could be limiting. In vivo activation of PKC typically involves translocation of the cytosolic protein to the membrane, where the intracellular pool of activated PKC is thought to be located (6). Thus measurement of the relative distribution of PKC between the membrane and cytosol gives an indirect measurement of the total amount of activated PKC in the cell. To determine the subcellular distribution of PKC in the Y1PKCalpha cell lines, we analyzed the distribution of PKC activity in the particulate (membrane) and cytosolic cellular fractions. PKC activity in the presence of tet presumably reflects primarily endogenous PKC activity. As can be seen in Table 1, the distribution of endogenous PKC between the particulate and cytosolic fraction is characteristic for each cell line, with PKC4/5 cells having only 3% associated with the particulate fraction, whereas in PKC4/3 and 4/12 cells 17% and 9%, respectively, of the total PKC activity are associated with the particulate fraction. The range of PKC activity distribution between these fractions is similar to what we have observed previously for Y-1 cells (39). When Y1PKCalpha cells are withdrawn from tet, the multiples of increase in PKC activity is similar in both the membrane and cytosolic fractions for all cell lines, indicating that the relative distribution of PKC between the two fractions remains fairly constant. However, the total amount of PKC activity in the particulate fraction increases 3- to 15-fold, suggesting that overexpression of PKCalpha results in a proportional increase in the amount of activated PKC in the Y1PKCalpha cells.

                              
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Table 1.   Subcellular distribution of PKC activity in Y1PKCalpha cells

Increased Expression of PKCalpha Reduces Basal Steroidogenesis in the Y1PKCalpha Cell Lines

Steroidogenesis is a complex process that is regulated both by the supply of cholesterol substrate and by the expression level of P450-SCC, which converts cholesterol to pregnenolone. In the parent Y-1 cell line, pregnenolone is subsequentially converted by 3beta -HSD and P450-11beta -OH to 11beta ,20alpha -dihydroprogesterone. However, with the Y1PKCalpha and Y1Con cell lines used in the present study, pregnenolone production is used as a measure of steroid production because the 3beta -HSD and P450-11beta -OH mRNAs are not expressed in these cell lines (see MATERIALS AND METHODS). When serum lipoproteins provide cholesterol substrate, both transport of lipoprotein-derived cholesterol to the inner mitochondrial membrane and expression of P450-SCC determine the rate of steroid production. Side-chain-hydroxylated cholesterol derivatives bypass the intracellular transport steps and have free access to the P450-SCC complex on the inner mitochondrial membrane. Hence, under these conditions, the rate-limiting step is cholesterol side-chain cleavage, and pregnenolone production becomes a functional assay for P450-SCC activity. To determine whether increased expression of PKCalpha in Y-1 cells decreases pregnenolone production, Y1PKCalpha cells were grown for 6 days in the presence or absence of tet and then provided with a hydroxycholesterol substrate. Figure 4 shows the accumulation of pregnenolone over a 24-h period for Y1PKCalpha and Y1Con cell lines provided with 25-hydroxycholesterol (A) or 22(R)-cholesterol (B) as a substrate. As seen in Fig. 4, the Con1/6 cells show little or no tet-dependent difference in pregnenolone production when provided with either substrate. However, in the Y1PKCalpha cells, removal of tet results in a 50-70% decrease in pregnenolone production with both cholesterol substrates. These results indicate that increased expression of PKCalpha in Y-1 cells results in a decreased ability to synthesize pregnenolone, which presumably reflects a decrease in P450-SCC enzyme activity.


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Fig. 4.   Expression of PKCalpha in Y-1 cells suppresses basal steroidogenesis. Con1/6, PKC4/7, or PKC4/12 cells were grown for 6 days in the presence (solid bars) or absence (open bars) of 2 µg/ml tet. On day 7, medium was replaced with Ham's F-10 containing 1% Nutridoma SP and 2 µg/ml tet. On day 8, medium was replaced with the same medium containing 15 µg/ml 25-hydroxycholesterol (A) or 15 µg/ml 22(R)-cholesterol (B). After an additional 24 h, medium was harvested and assayed for pregnenolone as described in MATERIALS AND METHODS. Pregnenolone production in the presence of tet (solid bars) was set at 100%. Pregnenolone production in the absence of tet (open bars) is expressed as a percentage of that produced in the presence of tet. Values represent average of triplicate samples from a single representative experiment that had been repeated 3 times with similar results. Variation between the samples was <20%.

To determine if there is a dose-response relationship between the level of PKCalpha expression and P450-SCC activity in the Y1PKCalpha cells, the tet concentration was varied to provide a range of PKCalpha expression levels, and pregnenolone synthesis was analyzed under each condition. As shown in Fig. 5, pregnenolone production showed little dependence on the tet concentration in the Con1/2 line but showed a clear concentration dependence in the PKC4/12 cell line. This indicates that the magnitude of suppression of pregnenolone synthesis in the PKCalpha cells is dependent on the level of PKCalpha protein that is expressed.


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Fig. 5.   Suppression of steroidogenesis in Y1PKCalpha cells correlates with tet dose. PKC4/12 cells or Con1/2 cells were grown for 8 days in complete medium containing 0.2 (solid bars), 0.002 (dark gray bars), 0.0002 (light gray bars), or 0 µg/ml tet (open bars). On day 9, medium was removed and replaced with Ham's F-10 containing 1% Nutridoma SP and tet as indicated. On day 10, medium was removed and replaced with the same medium containing 15 µg/ml 25-hydroxycholesterol. After an additional 24 h, medium was harvested and assayed for pregnenolone as described in MATERIALS AND METHODS. Pregnenolone production in the presence of 0.2 µg/ml tet (solid bars) was set at 100%. Pregnenolone production in the presence of 0.02, 0.002, and 0 µg/ml tet is expressed as a percentage of that produced in the presence of 0.2 µg/ml tet. Values represent average of triplicate samples from a single representative experiment that had been repeated 3 times with similar results. Variation between the samples was <20%.

PKCalpha Expression Inhibits P450-SCC Transcription in the Y1PKCalpha Cell Lines

To determine if the functional decrease in P450-SCC activity reflects a decrease in P450-SCC gene transcription, we studied the relative expression of p-1500SCCluc, in which luciferase expression is driven by 1.5 kb of the mouse P450-SCC promoter, in Y1PKCalpha and Y1Con cells. The cell lines were grown in the presence or absence of tet and then transiently transfected with p-1500SCCluc. As seen in Fig. 6, withdrawal of tet from the Y1PKCalpha lines (PKC4/7 and PKC4/12) results in a 40% decrease in the expression of p-1500SCCluc, whereas expression of p-1500SCCluc is increased slightly in the Y1Con lines. This indicates that transcription from the P450-SCC promoter is one level at which PKCalpha regulates P450-SCC activity. However, it should be noted that suppression of pregnenolone synthesis in the Y1PKCalpha cell lines is in the range of 50-70% (see Figs. 4 and 5), whereas suppression of P450-SCC transcription is ~40%. Hence, decreased transcription of P450-SCC does not account for all of the suppression in pregnenolone production that we observed in the Y1PKCalpha cells.


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Fig. 6.   Expression of PKCalpha inhibits transcription of the P450-SCC promoter in Y1PKCalpha cells. PKC4/7 and 4/12 cells or Con1/6 and 1/9 cells were grown for 6 days in the presence or absence of 0.2 µg/ml tet before transfection with p-1500SCCluc. Values were normalized to total cell protein and are expressed as the ratio of relative light units of luciferase (RLU) in cells grown without tet to the RLU in cells grown with tet. In some experiments cells were cotransfected with pCMVbeta gal as a control for transfection efficiency. Normalization to total cell protein or beta -galactosidase expression gave essentially the same values. Expression of the pXP2 promoterless construct was less than twice background. Numbers represent triplicate measurements within a single representative experiment ± SD. These experiments were repeated 2-5 times with similar results.

We have also examined the effect of PKCalpha expression on endogenous P450-SCC mRNA levels. Figure 7 shows a Northern blot of P450-SCC mRNA expression in Con1/2, PKC4/5, and PKC4/12 cells cultured in F-10 medium containing 1% Nutridoma SP or in complete medium, with or without the addition of tet. The blot was stripped and reprobed for expression of GAPDH mRNA as a control. Little or no consistent decrease in P450-SCC mRNA was seen on induction of PKCalpha expression when cells were grown under either condition. In fact, removing tet from the culture medium actually increased P450-SCC and GAPDH mRNA expression to some extent in all the cell lines. P450-SCC and GAPDH mRNA expression was quantified by PhosphorImager analysis. After normalization to GAPDH mRNA, the P450-SCC mRNA ratio, without tet to with tet, is 0.9 for Con1/2 cells, 0.8 for PKC4/5 cells, and 1.0 for PKC4/12 cells grown in complete medium. We have also examined changes in P450-SCC protein abundance by Western blot and likewise saw little or no suppression of P450-SCC protein expression under conditions in which PKCalpha expression is induced (data not shown). Our inability to detect changes in P450-SCC mRNA and protein expression may be due to technical difficulties inherent in quantitating such relatively small differences.


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Fig. 7.   Expression of PKCalpha does not suppress endogenous P450-SCC mRNA expression. Con1/2, PKC4/5, and PKC4/12 cells were grown for 5 days in the presence or absence of 2 µg/ml tet. On day 5, medium was replaced with F-10 containing 1% Nutridoma SP (N) or complete media (CM) with or without the addition of tet, as indicated. After an additional 24 h, cells were harvested and RNA prepared as described in MATERIALS AND METHODS. Expression of P450-SCC mRNA was analyzed as described in MATERIALS AND METHODS. Blot was stripped and reprobed for expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. This experiment was repeated 4 or more times with similar results.

PKCalpha Expression Does Not Inhibit cAMP Induction of Steroidogenesis in Y-1 Cells

To determine if expression of PKCalpha suppresses 8-bromo-cAMP (8-BrcAMP) induction of steroidogenesis, PKCalpha 4/12 cells were grown in the presence or absence of tet for 6 days and then stimulated with 8-BrcAMP. Medium was collected and assayed for pregnenolone production after 4 h to measure the acute response to cAMP or after 24 h to measure the sustained response. The acute response is chiefly dependent on cholesterol mobilization, whereas the sustained response reflects increased expression of the steroid hydroxylase genes. As shown in Fig. 8, despite the fact that PKCalpha induction reduces basal pregnenolone production in the PKC4/12 cells, there is clearly a robust cAMP response at both 4 and 24 h. The multiples of increase by cAMP over the basal level are unchanged following PKCalpha induction. This result indicates that the suppression of P450-SCC activity by PKC does not compromise responsiveness to the PKA pathway, confirming the results obtained previously in the parental Y-1 cell line (37). Likewise, PKCalpha expression in the PKC4/12 cells has no effect on 8-BrcAMP induction of p-1500SCCluc transcription (data not shown). These results support our previous findings that PKC and cAMP are reciprocal yet independent regulators of P450-SCC in Y-1 cells (37).


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Fig. 8.   Expression of PKCalpha does not suppress stimulation of steroidogenesis by 8-BrcAMP. PKC4/12 cells were grown for 7 days in the absence or presence of 0.2 µg/ml tet as indicated. On day 8, medium was replaced with complete medium or complete medium containing 1 mM 8-BrcAMP. After an additional 4 h (solid bars) or 24 h (open bars), medium was harvested and assayed for pregnenolone as described in MATERIALS AND METHODS. Values are expressed as ng pregnenolone/mg cell protein. Values shown are average of duplicate measurements of triplicate samples ± SD from a single experiment that had been repeated twice with similar results. Values differ as follows as judged by a one-tailed t-test: * P < 0.007; + P < 0.004; ** P < 0.009; ++ P < 0.03.

Elevated PKC Expression In Vivo Correlates With Decreased Steroidogenesis in Y-1 Cells

To determine if PKC negatively correlates with the ability to synthesize steroids in wild-type Y-1 cells, we examined the relationship between PKC expression and steroidogenesis in 12 Y-1 subclonal lines (Y1/DW cell lines) that were isolated from the Y-1 parent cell line by limited dilution cloning. PKC activity in vitro, which measures total cellular activatable PKC, varied up to 9-fold between the subclones, whereas steroid production varied up to 50-fold. Figure 9 plots steroid production against total PKC activity for the Y1/DW subclones and demonstrates that PKC activity is inversely correlated with the ability to synthesize steroids in these cell clones.


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Fig. 9.   Total PKC activity inversely correlates with steroid production in wild-type Y-1 cells. Twelve Y-1 subclones (referred to as Y1/DW cell lines) were isolated from the parent cell line by limited dilution cloning. Total PKC activity was measured in vitro after partial purification on DEAE-cellulose as described in MATERIALS AND METHODS. Values represent means of 4 measurements and are expressed as pmol of 32P transferred to a myelin basic protein peptide substrate per mg of cell protein. Activity in the absence of TPA and phosphatidylserine was subtracted from the values shown. Steroid production represents mean of medium steroids accumulated in 24 h in duplicate plates and were measured using a fluorometric assay as described in MATERIALS AND METHODS. Values were normalized to total cell protein. Linear regression analysis yields a correlation coefficient (r2) = 0.54. Slope of the regression line differs from 0, P = 0.006. These measurements were repeated twice with similar results.

Y-1 cells appear to express a limited set of PKC isoforms; screening in our laboratory has detected PKCalpha , PKCepsilon , and PKCzeta expression (data not shown). To determine if variations in PKC activity in the Y1/DW cell lines correlate with changes in the expression of specific PKC isoforms, we analyzed expression of PKCalpha , PKCepsilon , and PKCzeta by immunoblot. As seen in Fig. 10A, PKCalpha expression varies dramatically between the subclones. The amount of PKCalpha protein expressed in the individual clones is consistent with the amount of total PKC activity (see legend to Fig. 10); i.e., the clones that express the most PKCalpha protein (DW2, DW4, DW15, DW18, and DW25) have the highest level of PKC activity. Conversely, in clones DW6, DW11, DW14, DW19, DW24, and DW31, PKCalpha protein expression and PKC activity are both low. Typical PKC activity measurements for the parental Y-1 cell line range from 60 to 75 pmol · mg-1 · min-1 (39). Figure 10, B and C, shows immunoblots in which the Y1/DW subclones were analyzed for expression of PKCepsilon and PKCzeta , respectively. As seen in Fig. 10, B and C, PKCepsilon expression varied somewhat between the Y1/DW subclones, but there is no apparent correlation between PKCepsilon expression and total PKC activity or steroid production. In contrast, PKCzeta expression varied little between the cell clones. These data demonstrate that, in vivo, increased PKCalpha expression correlates with a decreased capacity to synthesize steroids and suggests that PKCalpha may function to regulate some aspect of steroidogenesis in Y-1 cells.


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Fig. 10.   Expression of PKC isoforms in the Y1/DW cell lines. PKCalpha , PKCepsilon , and PKCzeta protein expression was analyzed by immunobloting as described in MATERIALS AND METHODS. Clone numbers refer to the Y1/DW cell lines. A: PKCalpha -specific protein expression. Fifty micrograms of DEAE-cellulose-purified cell protein were loaded in each lane. B: PKCepsilon -specific protein expression. Forty micrograms of DEAE-cellulose-purified cell protein were loaded in each lane. In the lane marked +, 20 µg of total cell protein from a GH4 rat pituitary cell line that overexpresses PKCepsilon were run as a positive control (1). C: PKCzeta -specific protein expression. Forty micrograms of DEAE-cellulose-purified cell protein were loaded in each lane. In the lane marked +, 40 µg of total HeLa cell protein were run as a positive control. M, migration of molecular mass standards. These measurements were repeated twice with similar results. Total in vitro PKC activity (pmol · mg-1 · min-1) for the Y1/DW subclones are as follows: DW2, 144; DW4, 228; DW6, 31; DW11, 54; DW14, 64; DW15, 186; DW18, 219; DW19, 67; DW24, 22; DW25, 172; and DW31, 8.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Previous reports, which have used chemical modulators to manipulate PKC activity, suggest that PKC functions as a negative regulator of steroidogenesis in many cell types (16, 22-24, 30, 37). However, to date, direct proof of a role for PKC in the regulation of steroidogenesis and identification of the functional PKC isoform has been lacking. To address these questions, we have generated novel Y-1 cell lines in which the expression and activity of a specific PKC isoform (PKCalpha ) can be acutely regulated in the absence of exogenous inhibitors or activators. The use of a regulatable expression system is particularly desirable in this context, since there is significant variation between Y-1 clonal cell lines in PKC isoform expression and basal steroidogenesis. Using this approach, we demonstrate directly that PKC functions as a suppressor of steroidogenesis and identify PKCalpha as the isoform that mediates this suppression.

The PKC isoform family consists of at least 11 members that differ in their sensitivity to Ca2+ and in their activation by lipid cofactors (32, 33). Recent studies have linked extracellular signals in a variety of cell types to the activation of specific PKC isoforms, e.g., tumor necrosis factor-alpha and PKCzeta activation (29), platelet-derived growth factor and PKClambda , PKCepsilon and PKCdelta activation (28), and nerve growth factor and PKCepsilon activation (15). In addition, in cotransfection experiments, different PKC isoforms vary in their ability to transactivate TPA-responsive genes.

The role of PKC in regulating steroidogenic cell functions has been studied primarily using the PKC activator, TPA. Studies with TPA suggest that activation of PKC suppresses cAMP-mediated induction of steroidogenesis and steroid hydroxylase gene expression in adrenocortical cells (10, 11). Previous work in our laboratory has extended observations gleaned from studying TPA activation of PKC by utilizing specific in vivo inhibitors of PKC to investigate the role of the constitutively activated PKC pool in regulating steroidogenesis in adrenocortical cells (37). The Y-1 cell line is appropriate to address this question because Y-1 cells have an appreciable level of basal steroidogenesis in the absence of exogenous activators of PKA. Our results showed that treatment of Y-1 cells with either staurosporine or calphostin C increases both basal steroid production and the level of expression of mRNAs for P450-SCC, as well as other steroid synthetic enzymes more distal in the pathway (37). Induction of steroid production was accompanied by increased P450-SCC transcription as monitored in transient transfection assays (37). On the basis of these experiments, we hypothesized that a constitutively active pool of PKC functions to tonically regulate steroidogenesis in Y-1 cells (37). Our current results show that inducible expression of the PKCalpha isoform results in an increase in the constitutive pool of active (membrane-associated) PKC in Y-1 cells, which presumably mediates suppression of steroidogenesis.

It has long been observed that treatment of steroidogenic cells with phorbol ester (TPA) blocks stimulation of steroidogenesis by physiological regulators of cAMP, such as luteinizing hormone and ACTH (16, 23, 24, 30). In the H295 human adrenal cell line, activation of PKC via the physiological regulator, ANG II, can likewise suppress forskolin-induced expression of P450-SCC mRNA (7). However, previous work from our laboratory has shown that the PKC and PKA pathways function as reciprocal but independent regulators of steroidogenesis and P450-SCC mRNA expression in Y-1 cells (37). These studies showed that PKC functions as a suppressor of steroidogenesis and P450-SCC mRNA expression even in the absence of a functional PKA pathway, indicating that PKC does not function by suppressing cAMP induction of steroidogenesis (37). Our current studies support this hypothesis, since expression of PKCalpha has no effect on either the acute or chronic stimulation of steroidogenesis by 8-BrcAMP. Because PKCalpha expression has no effect on the acute response to cAMP, it is unlikely that PKCalpha regulates cholesterol mobilization to the mitochondria, but of course this does not rule out the possibility that another PKC isoform may regulate these processes.

The data reported here indicate that increased expression of PKCalpha in Y-1 cells suppresses transcription from the mouse P450-SCC promoter. However, decreased transcription of P450-SCC does not appear to account for the entire reduction in P450-SCC activity we observe. Although our data are consistent with the conclusion that PKCalpha decreases P450-SCC expression, it is also possible that P450-SCC activity is controlled at additional points such as protein turnover or posttranslational modification of the P450-SCC enzyme. In this regard, Vilgrain et al. (42) have reported that PKC can phosphorylate P450-SCC in vitro.

Thus one mechanism by which PKCalpha decreases P450-SCC activity is transcriptional repression. Although there are many examples of genes that are positively regulated by PKC, suppression of transcription by PKC has been reported in only a few instances, such as regulation of the phosphoenolpyruvate carboxykinase and gonadotropin-releasing hormone promoters (9, 35). How PKC and other regulators suppress transcription is not known. Attempts to map the TPA-responsive region of the P450-SCC promoter have yielded conflicting results. Lauber et al. (19) have shown that cAMP response elements in the bovine P450-SCC promoter are sufficient for TPA-mediated repression of cAMP-induced expression, indicating that the cAMP and PKC regulatory regions of the P450-SCC promoter colocalize. Moore et al. (27) have reported that regions of the human P450-P450-SCC promoter responsible for TPA regulation map to the same area as elements required for basal regulation. Thus PKC may regulate expression of P450-SCC by modification of a protein required for basal regulation. In this regard, Leers-Sucheta et al. (20) have reported that TPA activates transcription of the 3beta -HSD type II promoter via steroidogenic factor 1, a transcription factor that is also required for basal expression of type II 3beta -HSD. Another possibility is that PKC inhibits transcription via the transactivator complex, activator protein-1 (AP-1), which mediates gene induction by TPA (3, 4). There is an AP-1 consensus site in the mouse P450-SCC promoter at -319. AP-1 is composed of homo- and heterodimers of members of the Fos and Jun families, and overexpression of c-Jun has been shown to inhibit expression of several tissue-specific genes including creatine kinase (21), atrial natriuretric factor (25), and prolactin (13), suggesting that c-Jun may inhibit tissue-specific gene expression. The availability of Y-1 cell lines in which intracellular PKCalpha activity can be acutely altered will facilitate studies on the mechanism of PKC suppression of P450-SCC transcription.

In this report, using a system in which we can regulate PKCalpha expression, we provide direct evidence that PKCalpha suppresses P450-SCC activity and basal steroidogenesis in Y-1 cells. Furthermore, we show that in the Y-1 subclonal cell lines isolated from the Y-1 parent cell line (Y1/DW cell lines), the ability to synthesize steroid hormones is inversely correlated with expression of PKCalpha . It is of interest that the degree of suppression of steroidogenesis in the Y-1 subclones is larger than would be predicted from the Y1PKCalpha cell lines. This suggests that PKCalpha may function in Y-1 cells to regulate other aspects of steroidogenesis in addition to P450-SCC activity. Because in the Y1/DW cell lines we measure steroidogenesis by production of 20alpha -dihydroprogesterone and its derivatives, the greater decrease in steroid synthesis in these cells may reflect additional changes in enzymatic steps downstream of P450-SCC. Finally, it should be noted that PKC clearly has other regulatory functions in adrenal cells that are distinct from its role as a chronic regulator of steroidogenesis. For example, in adrenocortical glomerulosa cells, binding of ANG II to the ANG II receptor activates PKC and induces mineralocortical hormone synthesis (5). Regulation of steroidogenesis by ACTH has also been proposed to involve activation of PKC (11). These divergent functions may be mediated by distinct PKC isoforms and may reflect the multiple levels of activation and regulation of these signaling pathways by phospholipids and other metabolic regulators.

    ACKNOWLEDGEMENTS

The expert technical assistance of Lisa Huerta, Penny Strockbine, and Wen Zhu is gratefully acknowledged. We are also indebted to Drs. S. Anderson, R. Evans, S. Diamond, and A. Gutierrez-Hartman for helpful discussions throughout the course of this work.

    FOOTNOTES

This work was supported by a National American Heart Grant-in-Aid and a Colorado American Heart Affiliate Grant-in-Aid to M. E. Reyland and by National Heart, Lung, and Blood Institute Grant HL-32868 to D. L. Williams.

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

Address for reprint requests: M. E. Reyland, Dept. of Basic Sciences and Oral Research, Box C286, Univ. of Colorado Health Science Center, Denver, CO 80262.

Received 12 January 1998; accepted in final form 27 May 1998.

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Abstract
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Discussion
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Am J Physiol Cell Physiol 275(3):C780-C789
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