Functional Interactions, Phosphorylation, and Levels of 3',5'-Cyclic Adenosine Monophosphate-Regulatory Element Binding Protein and Steroidogenic Factor-1 Mediate Hormone-Regulated and Constitutive Expression of Aromatase in Gonadal Cells

Diana L. Carlone and JoAnne S. Richards

Department of Cell Biology Baylor College of Medicine Houston, Texas 77030


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The proximal promoter of the rat aromatase CYP19 gene contains two functional regions that, by 5'-deletion analyses, have been shown to confer hormone/cAMP inducibility to chimeric genes in primary cultures of rat granulosa cells and constitutive expression in R2C Leydig cells. Promoter region A binds Steroidogenic Factor-1 (SF-1); region B binds cAMP-regulatory element binding protein (CREB) and two other factors (designated X and Y). Mutations were generated within the context of the intact promoter to selectively eliminate the binding of either SF-1, CREB, CREB plus factors X and Y, or all of the above. When expression vectors that failed to bind either CREB alone or CREB plus factors X and Y were transfected into granulosa cells, cAMP-dependent chloramphenicol acetyltransferase (CAT) activity was reduced 65% indicating that CREB alone, and not factors X and Y, mediates the cAMP response of this cAMP response element-like domain. Similarly, cAMP-dependent CAT activity was reduced 50% in constructs that failed to bind SF-1 and was abolished with vectors that were unable to bind either factor. In R2C Leydig cells, the absence of either CREB or SF-1 binding resulted in an almost complete loss in CAT activity. Both immunoreactive CREB and phosphorylated CREB (phosphoCREB) were present in extracts and nuclei of R2C cells. Immunoreactive phosphoCREB was low in granulosa cell extracts and nuclei but increased rapidly (90 min) in response to FSH/cAMP and was highest at 48 h, at a time when SF-1 was also phosphorylated and expression of the endogenous gene was elevated. Although the amount of CREB and SF-1 remained unchanged in response to FSH, LH mediated a rapid decrease in the amount of SF-1 (but not CREB) that is coincident with decreased aromatase mRNA in luteinizing granulosa cells. Taken together, the data indicate that expression of the aromatase gene is dependent on the additive interactions of regions A and B of the aromatase promoter in granulosa cells and the synergistic interactions of these same regions in R2C cells and that these interactions are dependent, in turn, on the phosphorylation of CREB and SF-1 and the content of these factors, as well as the presence of putative coregulatory molecules.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Maturation and differentiation of the ovarian follicle involves not only the presence of pituitary gonadotropins but also the activation of specific ovarian genes in response to these peptide hormones (1, 2). The CYP19 gene encodes the aromatase cytochrome P450 enzyme that converts testosterone to estradiol. Expression of this gene is regulated throughout the life cycle of the rat follicle and corpus luteum by the sequential actions of the gonadotropins, FSH and LH, and by the cytokine PRL (3, 4, 5, 6, 7, 8). In response to FSH, steady state levels of aromatase mRNA increase, with maximal levels detected in granulosa cells of the preovulatory follicles (3). The surge of LH that initiates the onset of ovulation exerts a dramatic negative effect on steady state levels of aromatase message resulting in a complete loss of transcript within 6 h (5, 6). During pregnancy, PRL is responsible for elevated levels of aromatase message and protein in the corpus luteum (4). These changes, which occur in vivo, can be mimicked in granulosa cells cultured sequentially in the presence of FSH and LH (7, 8). In contrast to these sequential changes in aromatase expression that characterize granulosa cell differentiation and luteinization, aromatase mRNA is constitutively expressed at high levels in the rat R2C Leydig cell line (9).

One of the mechanisms by which FSH regulates transcription of the aromatase gene involves stimulation of the cAMP-signaling cascade that activates A-kinases and leads to the phosphorylation of key substrates including specific transcription factors (1, 2). Two regions within the rat aromatase promoter have been identified to mediate cAMP-induced expression in granulosa cells (10, 11, 12). These same two regions are also present in the promoter II of the human aromatase gene (13). The most proximal element (region A) lies between -90 and -66 bp and binds Steroidogenic Factor-1 (SF-1) or adrenal binding protein (Ad4BP), an orphan receptor member of the steroid/thyroid hormone receptor superfamily (14, 15). The hexameric SF-1-binding site [(C/A) AGGTCA] is present within a large number of steroidogenic genes (14, 15) and has been shown to be critical for their transcriptional activation in adrenal and gonadal cells. Targeted deletion of the SF-1 gene results in mice that lack gonads and adrenals and exhibit abnormal gonadotrope development (16), indicating that the expression and activity of SF-1 are critical during early embryonic development and likely involve SF-1-regulated expression of tissue-specific genes in addition to those encoding steroidogenic enzymes (17). In the adult rat ovary, the levels of SF-1 mRNA and protein are relatively constant (10, 11, 18) suggesting that posttranslational modifications as well as transcriptional control may be important for mediating the functional activity of SF-1.

The distal element of the aromatase promoter (region B) lies between -161 and -138 bp and contains a cAMP-response element (CRE)-like sequence (TGCACGTCA) (19, 20). This region has been shown to bind CRE-binding protein (CREB) along with two additional proteins designated as X and Y (12). Because mutations that prevent binding of all three proteins result in a decrease in promoter activity (12), we hypothesized that either CREB alone or that factors X and Y (with putative functions redundant of CREB) were responsible for the transactivation of promoter activity at this site. This hypothesis was based on the known complexity of the CREB/ATF superfamily (21), on the presence of multiple, alternatively spliced forms of CREB in different tissues, and on the fertility of the original CREB knockout mice (21, 22, 23). More recently, Blendy et al. (24) demonstrated that although the original CREB knockout mice did not express CREB {alpha}- or {delta}-isoforms, the levels of CREB ß as well as the CREB/ATF family member, CRE modulator, were increased. Therefore, factors X and Y that bind the aromatase CRE region were considered potential members of the CREB/activating transcription factor (ATF) family.

The functional activity of CREB is dependent upon the phosphorylation of serine 133 (25) that occurs primarily by activation of A-kinase. CREB phosphorylation occurs rapidly (within minutes) after stimulation of cells with hormones or cAMP (25), and the phosphorylation site is antigenic (26). Furthermore, the ability of phosphoCREB to transactivate gene expression involves binding to the coactivator CBP (CREB-binding protein; Refs. 27–29). Although recent reports have also shown that SF-1 can be phosphorylated by A-kinase (30), it is not yet known whether this phosphorylation is obligatory for transcriptional activity. As indicated above, regulation of the aromatase gene in cells is complex. It can be induced in granulosa cells by low levels of FSH/cAMP, rapidly and dramatically down-regulated in these same cells by high levels of LH and cAMP, or expressed at elevated, cAMP-independent levels in an R2C Leydig cell line. These distinct patterns of aromatase expression indicated that several steps and signaling pathways might regulate either the content or activity of factors, such as CREB and SF-1, that bind to regions A and B of the aromatase gene promoter.

Based on the foregoing observations this study was undertaken to delineate whether specific interactions of factors binding to region A and B mediate cAMP-responsive and constitutive activation of the rat aromatase promoter in granulosa cells and R2C Leydig cells, respectively. To accomplish this goal, mutations of region A and region B were generated within the context of an intact promoter. Mutants within the CRE-like sequence were generated to determine the functional role of CREB and whether factors X and Y exerted redundant activities. Mutations within the hexameric SF-1 site were generated to determine whether SF-1 interacted with CREB for transactivation of the aromatase promoter. To determine whether changes in the content and phosphorylation of CREB and SF-1 were related to the activation of the aromatase gene, Western blot analyses, immunocytochemical localization, immunoprecipitation, and electrophorectic mobility shift assays (EMSAs) were performed. Results of these studies indicate that regions A and B interact in an additive manner in granulosa cells and in a synergistic manner in R2C Leydig cells, that levels of phosphoCREB are related to cAMP induction in granulosa cells and constitutive expression in R2C Leydig cells, and that loss of aromatase mRNA is associated with a marked decrease in SF-1 in response to the ovulatory LH surge. Thus, changes in either the phosphorylation state of CREB or the content of SF-1 appear to exert a major impact on transcriptional activation of the endogenous gene, further underscoring the importance of the functional interactions of regions A and B in mediating aromatase gene expression.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Differential Binding of CREB and Additional Proteins to Region B in the Rat Aromatase Promoter
Region B of the rat aromatase promoter forms three distinct DNA/protein complexes, one of which has been shown to be CREB by competitive binding and antibody supershift analyses (Fig. 1Go; Ref.12). To elucidate which nucleotides within the CRE-like region are responsible for the binding of CREB and factors X and Y, mutant oligonucleotides were synthesized (Fig. 1BGo) and tested by EMSAs using nuclear extracts from granulosa cells of preovulatory follicles (HEF; Fig. 2Go), in which expression of aromatase has been shown to be the highest (7). As in previous studies (12), three protein/DNA complexes corresponding to CREB, X, and Y were formed when the intact -161 oligonucleotide was used as a labeled probe (Fig. 2Go, A-C; lane 2). All three complexes were competed by the addition of cold competitor -161 oligonucleotide, whereas only the upper band was competed by an authentic unlabeled CRE oligonucleotide (Ref. 12; Fig. 2AGo; lanes 3 and 4). The faster migrating bands are nonspecific. The binding of all three complexes was also inhibited by the addition of unlabeled -158/-157 mutant (-158 M) oligonucleotides (Fig. 2Go B; lane 4). The -156/-155 mutant oligonucleotide (-156M) failed to compete for the binding of CREB but did compete for the binding of the other two factors, X and Y (Fig. 2BGo; lane 5). Mutations of bases -155/-154 or -154/-153 (-155M and -154M, respectively) rendered these oligonucleotides incapable of competing for binding of CREB, X, or Y (Fig. 2BGo; lanes 6 and 7). EMSAs were also performed using whole cell extracts from R2C Leydig cells and yielded similar results (data not shown). When increasing concentrations (50-, 100-, and 200-fold excess) of the competitor oligonucleotides were added to the reactions, as little as 50-fold excess of -161, -158M, and -156M competed for the binding of factors X and Y (Fig. 2CGo). The binding of CREB was more effectively reduced by -158M than -161 and was not altered by -156M. Collectively, these data show that the binding of CREB can be functionally separated from that of factors X and Y.



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Figure 1. Mutations in Region B of the Rat Aromatase Promoter

A, Schematic of the regulatory elements of the rat aromatase promoter. Region A (-90/-66 bp) binds SF-1 (hatched rectangle). Region B (-161/-138 bp) binds three factors, CREB (solid rectangle), X (open circle) and Y (speckled square). B, Mutant oligonucleotides generated within the CRE-like sequence of region B. -161 represents the intact region B with the CRE-like sequence underlined. -158M, -156M, -155M, and -154M designate the mutations (underlined) at positions -158 and -157, -156 and -155, -155 and -154, -154 and -153, respectively. The -154M has previously been described (12).

 


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Figure 2. Mutations within Region B Generate Distinct Binding Patterns

A, EMSAs were performed using nuclear extracts (5 µg) prepared from preovulatory granulosa cells of hormonally primed (HEF) rats and the -161 (-161/-138) oligonucleotide as probe (200 pg, 13 fmol). Three distinct protein/DNA complexes were formed (CREB, X, and Y). The identity of CREB was determined using 200-fold excess of authentic CRE as shown previously (12). B, EMSAs were performed as described in panel A using 200-fold excess of unlabeled oligonucleotides (described in Fig. 1Go) to compete for protein binding. C, EMSAs were performed as described in panel A except that an increasing amount of unlabeled oligonucleotide (50-, 100-, and 200- fold excess) was used to demonstrate specificity of binding. HEF: H, hypophysectomized; E, estradiol; F, FSH.

 
CREB Binding Required for cAMP-Induced and Constitutive Transactivation of Aromatase CAT Reporter Constructs
The differential binding of CREB and factors X and Y to the various oligonucleotides provided us with a means of testing the ability of X and Y to transactivate the aromatase promoter in the absence of CREB. Therefore, specific mutants of aromatase promoter-reporter CAT constructs were generated and tested in transient transfection assays using granulosa cells and R2C Leydig cells (Fig. 3Go). When granulosa cells were transfected with the -161 arom CAT or -158M arom CAT constructs (which bind all three proteins) a 6- to 8-fold increase in CAT activity was observed in the presence of forskolin (Fig. 3AGo). The -156M arom CAT vector that binds factors X and Y but not CREB exhibited 65% less forskolin-inducible activity than the -161 arom CAT (Fig. 3AGo). Furthermore, the activity of the -156M arom CAT construct was similar to that of constructs that do not bind any of the factors, namely, the -154M arom CAT (Fig. 3AGo) and -155M arom CAT (data not shown). The results of these transfection assays indicate that CREB specifically transactivates cAMP inducibility within region B of the promoter. In the absence of forskolin, all constructs tested yielded CAT activity levels comparable to the negative control construct (pCAT Basic), which contains no promoter sequence.



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Figure 3. Functional Activity of CREB, X, and Y in Both cAMP Induction and Constitutive Expression of the Transgene

A, Histogram of the CAT activity (% conversion) in granulosa cells transfected with 15 µg of various promoter-reporter constructs and cultured for 24 h. Twenty micrograms of protein were assayed for 18 h to determine CAT activity. The data presented are the mean value of duplicate samples in two separate experiments. The error bars represent ± SD. B, Histogram of the CAT activity (% conversion) in R2C Leydig cells transfected with the same constructs and cultured for 48 h. Ten micrograms of protein were assayed for 2 h to determine CAT activity. The data presented are the mean value of duplicate samples in two separate experiments. The error bars represent ± SD. C, pCAT control; B, pCAT basic.

 
Constitutive expression of aromatase by R2C Leydig cells also appears to depend upon the binding of CREB to a functional CRE (Fig. 3BGo). The -161 and -158M constructs exhibited high CAT activity (in the absence of forskolin); whereas, constructs lacking CREB binding (-154M and -156M arom CAT) had minimal CAT activity (7% of -161 arom CAT). These results indicate that CREB binding to region B is also required for high constitutive expression of aromatase in R2C Leydig cells.

Cooperative Interactions Occur between CREB and SF-1 in Both cAMP-Induced and Constitutive Expression of the Aromatase Promoter
SF-1 has previously been shown to be the factor binding to region A of the rat aromatase promoter (Refs. 10 and 11; see Fig. 8Go). To determine whether SF-1 binding in region A affected either cAMP-induced or constitutive expression of aromatase, additional aromatase promoter-CAT constructs were generated. The SF-1-binding site was mutated either in the context of the intact -161 arom promoter that contains the functional CRE or in the -95 arom CAT promoter that lacks the CRE (Fig. 4Go). EMSAs were performed to verify that the mutated hexameric motif (AGGTCA to ATTTCA) was unable to bind SF-1 (data not shown).



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Figure 8. The LH Surge Decreases SF-1 Binding and Protein Content

A, EMSAs were performed using nuclear extracts (1.5 µg) prepared from granulosa cells of preantral (HE), preovulatory (HEF), and ovulatory (HEF plus either 2-h or 10-h hCG) follicles, whole cell extracts from R2C Leydig cells (7.5 µg), and the SF-1 hexameric oligonucleotide (-90/-66) as probe (200 pg, 13 fmol). A single protein/DNA complex corresponding to SF-1 is shown (arrow; Ref. 12). B, The same nuclear extracts (HEF, HEF plus 2- or 10-h hCG; 10 µg protein) and whole cell extracts (R2C Leydig; 50 µg protein) were fractionated on a 10% SDS-PAGE and transferred to nitrocellulose. Blots were incubated with anti-SF-1 (1:10,000). Immunoreactive proteins were visualized using [125I]protein A and exposed to x-ray film. C, EMSA was performed as in panel A using the CRE-like region B of the aromatase promoter as the labeled probe.

 


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Figure 4. Schematic of Mutant Aromatase Promoter-Reporter Constructs

Mutant SF-1-binding constructs were generated in either the context of the intact promoter (-161 vectors) or a deleted promoter containing only region A (-95 vectors). The -161, -156M, and -154M were described in Fig. 1Go. The -161M, -156DM, and -154DM were generated by ligating a mutant region A oligonucleotide into the intact promoter without or with CRE mutant sites. The -95 and -95M were generated by ligating either the normal or mutant region A oligonucleotide to the minimal 50 promoter-reporter construct. Binding of CREB, X, Y, and SF-1 are illustrated by the following symbols: CREB, solid rectangle; X, open circle; Y, speckled square; SF-1, hatched square.

 
These various constructs were transiently transfected into primary granulosa cells and tested in the absence or presence of forskolin (Fig. 5AGo). Maximal activity (35% conversion) was induced by forskolin in cells transfected with the intact -161 arom CAT construct. Mutation of the SF-1-binding site (-161M arom CAT) resulted in a 50% reduction in forskolin-induced CAT activity (Fig. 5AGo). The level of activity was comparable to that of the -156 M and -154M arom CAT constructs, both of which fail to bind CREB. No induction of transgene activity by forskolin was observed in vectors with double mutations, -156DM arom CAT and -154DM arom CAT, which fail to bind CREB and SF-1. The promoter construct containing the SF-1-binding site alone (-95 arom CAT) did exhibit a slight but significant 2-fold induction of CAT activity in response to forskolin. Mutation of the SF-1-binding site (-95M arom CAT) resulted in complete loss of forskolin-induced CAT activity. These results indicate that a cooperative or additive interaction between SF-1 and CREB is required for cAMP activation of aromatase promoter in granulosa cells.



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Figure 5. Functional Interactions of CREB and SF-1-Binding Sites in Granulosa Cells and R2C Leydig Cells

A, Histogram of the CAT activity (% conversion) in granulosa cells transfected with 15 µg of various promoter-reporter constructs and cultured for 24 h. Twenty micrograms of protein were assayed for 18 h to determine CAT activity. The data presented are the mean value of duplicate samples in two separate experiments. The error bars represent ± SD. B, Histogram of the CAT activity (% conversion) in R2C Leydig cells transfected with the same constructs and cultured for 48 h. Ten micrograms of protein were assayed for 2 h to determine CAT activity. The data presented are a representative experiment using duplicate samples. The error bars represent ± SD. C, pCAT control; B, pCAT basic.

 
When these same constructs were transfected into R2C cells, different responses were observed (Fig. 5BGo). Whereas the -161 arom CAT exhibited 32% conversion, the -161M arom CAT construct lacking the SF-1-binding site had only 2% conversion. Thus, mutation of the SF-1-binding site in the presence of a functional CRE within region B resulted in a complete loss of CAT activity. Likewise, vectors lacking a functional CREB-binding site had little activity, and this was not reduced further in the double mutants (DMs), -156DM arom CAT and -154DM arom CAT. The -95 arom CAT had low activity that was reduced by mutation of the SF-1 site. These data indicate that both the CREB- and SF-1-binding sites must be intact and interact in a synergistic manner for transactivation of the aromatase promoter in R2C cells.

Phosphorylation of CREB and SF-1 in Granulosa Cells and R2C Leydig Cells
Although CREB homodimers bind DNA at CRE-like sequences, phosphorylation of CREB at serine 133 is required for it to transactivate promoter activity (25). Serine 133 resides within a substrate consensus site for protein kinase A, but other kinases are also capable of phosphorylating CREB at this same residue (26, 31, 32, 33, 34). To determine whether CREB was phosphorylated in response to increased levels of cAMP in granulosa cells and was constitutively phosphorylated in R2C Leydig cells, Western blot analyses were performed using specific CREB and anti-phosphoCREB antibodies, the latter of which recognizes CREB phosphoserine 133 (Ref. 26; Fig. 6Go). The amount of CREB was essentially constant in granulosa cells cultured in the absence of hormones or in the presence of either forskolin or FSH/T for 0–48 h (Fig. 6AGo; upper left panel). Similar amounts of CREB were also detected in R2C cells (Fig. 6AGo; upper right panel). When these same samples were analyzed for the content of phosphoCREB, the anti-phosphoCREB antibody recognized two distinct immunoreactive proteins (Fig. 6AGo; lower panel). The 43-kDa phosphoprotein (larger arrow; lower band) comigrated with CREB and is considered, therefore, to be authentic CREB protein. The 52-kDa immunoreactive phosphoprotein (smaller arrow; upper band) migrated more slowly than CREB, was not recognized by the specific CREB antibody, and therefore is not CREB. Because the anti-phosphoCREB antibody recognizes a phosphorylation region common to other CREB family members (26), antibodies to other members of the CREB family (CRE modulator, ATF-1) as well as other CREB antibodies were tested, but none recognized the higher molecular weight protein (21, 26). Therefore, this protein is either yet another CREB/ATF family member or is an unrelated protein that has a phosphorylation domain similar to that present in CREB.



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Figure 6. Phosphorylation of CREB in Response to FSH and Forskolin

A, Western blot analysis of CREB and phosphoCREB in granulosa cells and R2C cells. Granulosa cells were cultured for varying times in medium alone or medium containing either forskolin (Fo; 10 µM) or FSH (50 ng/ml) and 10 ng/ml testosterone (24 h and 48 h). R2C cells were cultured for 48 h in medium without hormones. Cell extracts were prepared, fractionated by 10% SDS-PAGE, and transferred to nitrocellulose. Blots were incubated with either anti-CREB (1:1000) or anti-phosphoCREB (1:500) IgG. Immunoreactive proteins (43-kDa CREB, open arrow; 43 kDa phosphoCREB, large solid arrow; 52-kDa protein; small solid arrow) were visualized using [125I]Protein A and exposed to x-ray film. B, Immature granulosa cells were cultured on coverglass slips in the presence (+) or absence(-) of forskolin (Fo) for 48 h as described in panel A. R2C Leydig cells were also cultured for 48 h. Cells were then fixed in 4% paraformaldehyde and incubated overnight with either anti-CREB (top panel) or anti-phosphoCREB (bottom panel) IgG. Localization of these proteins was visualized by fluorescein-labeled anti-rabbit IgG. Original magnification is 63x.

 
In the absence of either forskolin or FSH, the immunoreactive 43-kDa protein identified as phoshoCREB was barely detectable (Fig. 6AGo, lower panel; lane 1, large arrow). The addition of forskolin caused an increase in immunoreactive phosphoCREB at 90 min that decreased at 6 h but was again increased to a higher amount at 48 h. Similar results were observed in response to FSH with the exception that the secondary increase in phosphoCREB occurred as early as 24 h and was maintained at 48 h. Because alterations in the amount of phosphoCREB were not due to changes in the concentration of CREB protein (Fig. 6AGo; upper panel), these results indicate that the stimulation of cAMP production in granulosa cells exerts a biphasic response with respect to CREB phosphorylation and subsequent activation. Immunocytochemical localization studies showed that high levels of CREB were present in nuclei of granulosa cells cultured in the presence or absence of forksolin for 48 h (Fig. 6BGo; top panel) corresponding to Western analysis data (Fig. 6AGo; upper panel). However, granulosa cells cultured for 48 h in the presence of forskolin (+) expressed a higher level of nuclear phosphoCREB as compared with those cultured without forskolin (-) (Fig. 6BGo; lower panel). CREB and phosphoCREB were also detected in cell extracts and nuclei of R2C Leydig cells (Fig. 6Go). Interestingly, the highest levels of phosphoCREB, as detected by Western blotting and indirect immunofluorescent labeling, were observed in cells expressing the greatest amounts of aromatase mRNA (7, 9).

Because FSH/cAMP are necessary for expression of the endogenous gene in granulosa cells (7) and for increased transcription transgenes containing the SF-1 binding site in region A, we sought to determine whether SF-1 was also phosphorylated in granulosa cells that express high levels of aromatase mRNA in response to FSH. Accordingly, in vivo phosphorylation studies were performed using granulosa cells cultured for 48 h in the presence (+) or absence (-) of FSH (Fig. 7Go). A phosphoprotein of approximately 60 kDa corresponding in size to SF-1 (see Fig. 8BGo) was immunoprecipitated using an SF-1-specific antibody from cells cultured in the presence of FSH (Fig. 7Go, + lane). A similar band corresponding to SF-1 was not phosphorylated in cells cultured without FSH (- lane). Thus, SF-1 as well as CREB appear to be an FSH/cAMP-dependent phosphoprotein.



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Figure 7. Phosphorylation of SF-1 in Response to FSH

SF-1 was immunoprecipitated from cellular extracts (50 µg) prepared from granulosa cells cultured in the presence (+) or absence (-) of FSH/t for 48 h and labeled with [32P]orthophosphate as described in Materials and Methods. A 60-kDa protein corresponding to SF-1 was detected in the + lane (arrow).

 
LH Reduces SF-1 in Preovulatory Granulosa Cells
Whereas FSH increases aromatase mRNA in granulosa cells, the LH surge causes a rapid loss of aromatase transcripts in vivo (3) and in vitro (8). This decrease is presumed to be due, at least in part, to changes in aromatase transcription rate (8). If CREB and SF-1 mediate transcription of the endogenous gene, then a change in the expression, binding, or activation (phosphorylation) of these factors might be expected to occur in response to high levels of LH. To investigate these possibilities, two approaches were used. First, EMSAs were performed by incubating oligonucleotides containing the hexameric SF-1 binding site with nuclear extracts from granulosa cells isolated from ovaries of hormonally primed hypophysectomized (H) rats (Fig. 8AGo). A specific SF-1 complex was formed when oligonucleotides with the hexameric motif were incubated with preantral (HE) and preovulatory (HEF) granulosa cell extracts (lanes 2 and 3) and R2C Leydig cell extracts (lane 6). However, when human CG (hCG) was given to proestrous rats to mimic the endogenous LH surge, the SF-1/DNA complex was reduced by approximately 50% (lane 4) and 70% (lane 5) at 2 and 10 h, respectively, after treatment. A similar decline in SF-1 binding was detected when whole cell extracts were prepared from granulosa cells cultured for 48 h with FSH (preovulatory) and then exposed to hCG for 2 or 10 h (data not shown). Second, Western blot analyses were performed to determine whether loss of SF-1 binding was due to changes in SF-1 protein content. A single immunoreactive band corresponding to SF-1 at approximately 60 kDa was observed in nuclear extracts of preovulatory granulosa cells (Fig. 8BGo, lane 1). This immunoreactive SF-1 band was markedly reduced in nuclear extracts from granulosa cells exposed to hCG for either 2 or 10 h (lanes 2 and 3). SF-1 was also detected in R2C Leydig whole cell extracts (lane 4).

When these same granulosa cell and R2C cell extracts were used in EMSAs with a labeled aromatase CRE (-161/-138 bp of region A), three binding complexes were observed with each sample (Fig. 8CGo). Furthermore, no changes in amount of CREB binding (uppermost complex; see Fig. 2AGo) were observed in preovulatory granulosa cells treated with hCG (LH) (Fig. 8CGo). Likewise, the amount of immunoreactive CREB remained constant during this time period (Ref. 35 and data not shown). In addition, the phosphorylation state of CREB also did not vary as a consequence of hCG (data not shown). Taken together, these data indicate that loss of SF-1 but not CREB is associated with the decrease in aromatase whereas increased phosphorylation of both factors is required for induction of the aromatase gene.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
This study documents that different molecular mechanisms mediate hormone/cAMP induction, down-regulation, and constitutive expression of the rat aromatase gene by CREB and SF-1 in gonadal cells. Specifically, the functional interactions of the hexameric (CA)AGGTCA motif that binds SF-1 (region A) and the CRE that binds CREB and two other factors (region B) have been analyzed within the context of the intact promoter. When promoter-reporter constructs were transfected into granulosa cells and R2C cells, only those vectors that could bind CREB exhibited functional activity; namely, responsiveness to cAMP in granulosa cells and high basal activity in R2C cells. These observations eliminated the possibility that factors X and Y might provide redundant functions for CREB, a notion that was entertained at the initiation of these studies because numerous isoforms of CREB had been identified (21, 22) and because CREB knockout mice exhibited a normal phenotype devoid of obvious reproductive abnormalities (23). This phenotype was unexpected given the obligatory roles of FSH and LH in the growth and function of the ovarian follicle and ovulation (1) and the presumed preeminence of cAMP and CREB in mediating the actions of the gonadotropins on specific genes (1, 2, 36, 37). Further analyses of these mice have provided a different explanation. Blendy et al. (24) have recently reported that these animals are not complete CREB knockouts and that CREB ß, which is generally expressed in extremely low amounts, was present at much higher levels.

One intriguing aspect of the results obtained in these experiments is that the molecular mechanisms or pathways involved in cAMP activation of the aromatase promoter in granulosa cells differ from those controlling constitutive expression in R2C cells. By using selected mutants of the CREB and SF-1 binding sites within the context of the intact promoter, we show that CREB and SF-1 interact in an additive manner to confer cAMP responsiveness in granulosa cells, whereas they interact synergistically to confer high constitutive activity in R2C cells. Specifically, within granulosa cells, loss of binding of either factor in the context of the intact promoter reduced transgene activity by at least half, whereas complete loss of activity was achieved only when both CREB and SF-1 binding sites were ablated simultaneously. Although the involvement of SF-1 in the induction of a cAMP-responsive gene has been described elsewhere (30), this is the first study to demonstrate that the hexameric SF-1-binding site is required within the context of the intact promoter to provide maximal cAMP inducibility. In R2C Leydig cells that constitutively express high levels of aromatase, the binding of both CREB and SF-1 are critical for elevated, cAMP-independent expression of the transgenes. Aromatase promoter-CAT reporter constructs containing mutations within either CREB- or SF-1-binding sites or both exhibited little or no activity.

Not only are interactions of regions A and B of the aromatase promoter cell specific, but these interactions are associated with specific changes in the phosphorylation and amount of factors that regulate aromatase induction in the ovary by FSH, its down-regulation by LH, and its constitutive expression in R2C Leydig cells. Phosphorylation of CREB was associated with both cAMP induction of aromatase transgenes in granulosa cells and with their constitutive expression in R2C Leydig cells. Although there is abundant evidence that cAMP can activate A-kinase in granulosa cells (38) and transactivate genes containing CRE sites (36, 37), the kinase(s) responsible for the phosphorylation of CREB in the R2C Leydig cells have not yet been identified. Because forskolin (cAMP) does not regulate expression of the endogenous aromatase gene or the transgenes in R2C Leydig cells (9, 10), it is possible that A-kinase is maximally activated by factors present in the serum in which the cells are cultured. Alternatively, other kinases (e.g. C-kinase, calmodulin (CAM) kinase, RSK-2) that are capable of phosphorylating CREB at the serine 133 residue (31, 32, 33, 34), the site obligatory for transactivation (25) and antibody recognition (26), may be activated in R2C cells. Thus, it is possible that cellular signaling pathways other than, or in addition to, A-kinase phosphorylate and activate CREB in granulosa cells and R2C cells.

Phosphorylation of CREB is also dependent on cell differentiation. One of the novel observations of these studies is that although the amount of CREB remained constant during granulosa cell differentiation (Fig. 6AGo; Refs. 12 and 35), the phosphorylation of CREB was markedly elevated in cells cultured in the presence of FSH or forskolin for 48 h. Thus, the phosphorylation of CREB is not only rapid and transient (25) but is subsequently increased and maintained as the cells differentiate. As a consequence of this biphasic pattern of phosphorylation in response to FSH, the highest levels of phosphoCREB in granulosa cells were coincident with the maximal induction of the endogenous aromatase gene (7) and the acquisition of the preovulatory phenotype (1). Several factors may account for the greater phosphorylation of CREB in the differentiated cells. First, although stimulation of cAMP production by FSH and forskolin is greatest at 60 min in these cultured cells, the nucleotide continues to be synthesized at elevated levels for 24–48 h (7, 39). Second, total A-kinase activity may be increased. Previous studies have shown that the concentrations of the regulatory type IIß (RIIß) subunit of A-kinase is increased dramatically in preovulatory granulosa cells in vivo and in vitro (8, 39, 40, 41). Although we originally thought that the increase in this regulatory subunit might serve as a sponge to reduce the sensitivity of granulosa cells to high cAMP and thereby prevent premature luteinization, it is also possible that these high levels of RIIß reflect a transient increase in total kinase holoenzyme with the rapid dissociation of the subunits and the transport of the activated C subunit to the nucleus. Such a hypothesis would support the observations of McKnight and colleagues (42), who have shown that overexpression of regulatory subunits leads to more holoenzyme. Alternatively, the activity of a specific phosphatase may be decreased as granulosa cells differentiate, thereby leading to increased phosphorylation of CREB as well as other granulosa cell proteins (43). These include RIIß (39, 44), SF-1 (Fig. 7Go), and a protein that is antigenically distinct from CREB but which is recognized by the phosphoCREB antibody (Fig. 6AGo). Whether this protein is a member of the CREB family or an unrelated protein with a similar antigenic phosphorylation site remains to be determined.

SF-1 is known to play a key role in ovarian cell function based on targeted disruption of the SF-1 gene in transgenic mice (16), altered function of granulosa cells cultured in the presence of antisense oligonucleotides (45), and the regulation of transcription of specific transgenes containing the hexameric SF-1-binding site (10, 18, 30). However, little is yet know about the phosphorylation of SF-1 and whether phosphorylation of this factor regulates its transcriptional activity. In the adult ovary and developing follicles, SF-1 mRNA and protein levels appear to be relatively constant (10, 11, 18), suggesting that activation of this orphan receptor occurs through a posttranslational modification. We show herein that granulosa cells cultured with FSH for 48 h contain similar levels of SF-1 but greater amounts of phosphorylated SF-1 than those cultured in the absence of FSH. It is not yet known whether this phosphorylation occurs as a consequence of A-kinase or whether it alters the activity of SF-1 in granulosa cells. However, Zhang and Mellon (30) have recently demonstrated that A-kinase can phosphorylate recombinant SF-1 in vitro on serine and threonine residues, thereby indicating that SF-1 is a putative substrate for A-kinase. Although the levels of SF-1 protein do not exhibit major changes during follicular growth or in response to low levels of FSH in culture (10), elevated levels of LH (high cAMP?) acting on differentiated, preovulatory granulosa cells cause a dramatic decrease in SF-1 protein. This loss of SF-1 protein is associated with the rapid loss of aromatase mRNA in granulosa cells of ovulating follicles. Because the amount of CREB and phosphoCREB did not change during this time (data not shown), the down-regulation of aromatase appears to be associated more with the loss of SF-1 than to any change in CREB. These results provide a physiological basis to support the pharmacological actions of antisense oligonucleotides that decrease SF-1 and aromatase in cultured cells (45).

In summary, these studies provide evidence that the additive interactions of regions A and B of the aromatase promoter in granulosa cells and the synergistic interactions of these same regions in R2C cells are dependent on the phosphorylation of CREB and SF-1, the amounts of these factors, as well as the presence (activation) of additional coregulatory molecules (46, 47, 48), one of which is likely to be such CREB binding protein, CBP.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Animals
Immature (day 22 of age) Holtzman Sprague-Dawley female rats (Harlan, Indianapolis, IN) were housed on a 16-h light, 8-h dark regimen in the Center for Comparative Medicine at Baylor College of Medicine and provided food and water ad libitum. Animals were treated in accordance with the principles and procedures outlined in the NIH Guide for the Care and Use of Laboratory Animals. All procedures were approved by the Institutional Committee on Animal Care and Use, Baylor College of Medicine (Houston, TX).

Promoter Constructs
Construction of the -161 aromatase-CAT reporter construct has been previously described (12). The mutant CRE aromatase promoter-CAT reporter constructs (-158M, -156M, -155M, -154M) were generated by chemical synthesis of oligonucleotides (Genosys, The Woodlands, TX) containing the designated mutations within the -161/-138 region (Fig. 1Go). Annealed oligonucleotides were ligated into the -138 aromatase CAT vector (digested with HindIII and XhoI), previously described (12). Constructs containing the SF-1-binding site mutation within the context of the intact region (-161M) or within the CRE mutant constructs [DMs designated -156DM and -154DM] were generated by chemical synthesis (Genosys) of a mutant oligonucleotide corresponding to region A (-95/-50 bp) in which the SF-1-binding site was mutated from AGGTCA to ATTTCA. The annealed mutant SF-1 oligonucleotide was ligated into the -161, -156M, or -154M aromatase CAT vectors at the DRAII and APOI sites. To generate the -95 and SF-1 mutant (-95M) aromatase CAT vectors, the normal or mutant oligonucleotide (-95/-50 bp) was ligated to the -50/+13 bp fragment of the aromatase promoter and inserted into the pCAT basic vector. The sequence and orientation of each mutant were confirmed by sequence analyses and purified by a CsCl gradient. The pCAT Control vector (Promega, Madison, WI), which contains the Simian Virus-40 promoter and enhancer sequences, served as a positive control for transfection reproducibility. The pCAT basic (Promega), which lacks eukaryotic promoter and enhancer sequences, was used as control for basal vector activity.

Electrophoretic Mobility Shift Assays
To test the ability of factors to bind these mutant oligonucleotides, EMSAs were performed as previously described (48, 49). Briefly, nuclear extracts (NE) were prepared from granulosa cells of hypophysectomized (H) rats treated sequentially with estradiol (HE; preantral), FSH (HEF; preovulatory), and an ovulatory dose of hCG as previously described (12). Oligonucleotides were labeled with [P32]deoxy-CTP (3000 Ci/mmol, ICN, La Mesa, CA) and DNA Polymerase-l (Klenow enyzme; large fragment, Promega, Madison, WI) to a specific activity of 108. For studies investigating the protein/DNA interactions at the CRE (region B) site, 5 µg NE protein from preovulatory (HEF) granulosa cells were incubated with labeled -161/-138 fragment (200 pg, 13 fmol) in the presence of 5.8 µg poly(deoxyinosinic-deoxycytidylic)acid for 30 min at room temperature. Specificity was analyzed by adding increasing concentrations of unlabeled competitor DNA, either the aromatase CRE or a commercial consensus CRE (Promega) as previously described (12). To determine SF-1 binding, 1.5 µg NE protein from granulosa cells of preantral (HE), preovulatory (HEF), and ovulatory (hCG, 2 and 10 h) rats were incubated with the labeled hexameric (-90/-66) motif (10). Protein/DNA complexes were resolved on 5% polyacrylamide gels in 0.5x TBE (0.089 M Tris borate, pH 8.3, and 2 mM EDTA) at 150 V at 22 C after 30 min prerun. The gels were dried and exposed to Kodak X-AR film at -80 C. Quantification of SF-1 binding was determined using a Betascope 603 Blot Analyzer (Betagen Corp., Waltham, MA).

Transfection and CAT Assays
Granulosa cells were isolated from immature female rats (day 23 of age) (36, 39). Cells were cultured in 35-mm multiwell plates in 3 ml DMEM-F-12 media (1:1, GIBCO/BRL, Gaithersburg, MD) supplemented with 20 mM HEPES (Curtain Matheson Scientific, Inc., Houston, TX), 100 IU/ml penicillin, 100 mg/ml streptomycin (Sigma, St. Louis, MO), and 1% FBS (Hyclone, Logan, UT). The cells were transiently transfected 18 h later using 15 µg plasmid DNA and the calcium phosphate precipitation method (50). Four hours later the DNA was removed, the cells were washed with HBSS -Mg-Cl (GIBCO/BRL) and cultured in the presence or absence of 10 µM forskolin (dissolved in ethanol, Calbiochem, San Diego, CA). After 24 h, the cells were lysed by freeze-thaw procedure, and the protein concentrations were determined by the method of Bradford using the Bio-Rad (Richmond, CA) protein assay. CAT activity was analyzed using 20 µg protein for 18 h according to a standard protocol (51). The amount of radioactivity in the substrate and acetylated products was quantified using the Betascope 603 Blot Analyzer.

R2C Leydig cells [American Type Cell Collection (ATCC), Rockville, MD] were grown in Ham’s F-10 (GIBCO/BRL) supplemented with 12.5% equine serum (Hyclone), 2.5% FBS (GIBCO/BRL), and 100 IU/ml penicillin, 100 µg/ml streptomycin. Cells were plated overnight at a density of 500,000 cells per 35-mm multiwell plate and transiently transfected in a manner similar to granulosa cells, with the exception that 4 h after DNA addition, cells were glycerol shocked (20% in HBSS +Mg+Cl, GIBCO/BRL) for 2 min at 37 C, washed with HBSS -Mg-Cl (GIBCO/BRL), and cultured for 48 h in the R2C medium mentioned above. CAT assays were performed using 10 µg protein for 2 h.

Western Blot Analysis
Western blot analyses of CREB and phosphorylated CREB (phosphoCREB) were performed as described previously (26) using specific antibodies that recognized CREB and phosphoCREB (serine 133). Granulosa cells were isolated and cultured for varying amounts of time (see Fig. 6Go legend) in the presence or absence of ovine FSH (50 ng/ml, NIH oFSH-16; National Hormone and Pituitary Program, Baltimore, MD) or forskolin (10 µM) together with testosterone (10 ng/ml, Steraloids, Keene, NH). R2C Leydig cells were cultured as described above. After culture, cells were washed with PBS and lysed with boiling SDS sample buffer [100 mM Tris (pH 6.8), 2% SDS, 20% glycerol, 10% ß-mercaptoethanol, and bromophenol blue, 100 C]. Cell extracts were then boiled (100 C) for 5 min. Samples were electrophoresed on a 10% SDS-polyacrylamide gel by standard methods (52). Proteins were transferred to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH) in a buffer containing 25 mM Tris, 192 mM glycine, and 20% methanol (53) at 12 V for 18 h at 4 C. Blots were rinsed in Tris-buffered saline-0.05% Tween-20 (TBS-T) and blocked with 4% BSA (BSA, Fraction V, United States Biochemical Corp., Cleveland, OH) for 1 h at 22 C. The blots were incubated with rabbit anti-phosphoCREB IgG (1:5,000; generously provided by Dr. David Ginty, Johns Hopkins University, Baltimore, MD) in 4% BSA in TBS-T for 18 h at 4 C, with shaking. To detect CREB, the blots were first blocked with 5% nonfat milk in TBS-T for 1 h at 22 C, shaking. Blots were then incubated with rabbit anti-CREB IgG (1:1000 in TBS-T, Ab 244 generously provided by Dr. Marc Montminy, Salk Institute, La Jolla, CA) at 4 C for 18 h, shaking. After several TBS-T washes, the presence of CREB or phosphoCREB was detected using I125 protein A (1:1000 vol/vol in TBS-T, specific activity 1 mCi/ml, ICN) for 3 h at 22 C with shaking. The blots were washed with TBS-T and exposed to x-ray film.

Western blot analyses of SF-1 were performed as described previously (54). Briefly, nuclear extracts (10 µg) from granulosa cells of preovulatory (HEF) and ovulatory (HEF treated for 2 and 10 h with hCG) follicles, as well as 50 µg of whole cell extracts from R2C Leydig cells, were boiled (100 C) in an SDS-PAGE loading buffer [0.05 M cyclohexylaminoethane sulfonic acid, 2% SDS, 10% glycerol, 2% ß-mercaptoethanol, and bromophenol blue, pH 9.5] and electrophoresed on a 10% SDS-polyacrylamide gel by standard methods (52). Proteins were transferred to nitrocellulose membrane (Schleicher & Schuell) in a buffer containing 25 mM Tris, 192 mM glycine, and 20% methanol (53) at 12 V for 18 h at 4 C. The blots were rinsed in 140 mM NaCl, 10 mM KPO4, pH 7.5, 10 mg/ml BSA for 6 h at 22 C with shaking. The blots were then incubated with rabbit anti-Ad4BP (SF-1) IgG (1:10,000; generously provided by Dr. Ken-ichirou Morohashi, Kyushu University, Fukuoka, Japan) in 140 mM NaCl, 10 mM KPO4, pH 7.5, 10 mg/ml BSA, 0.1% Triton X-100, 0.02% SDS for 18 h at 4 C. The blots were washed in 140 mM NaCl, 10 mM KPO4, pH 7.5, 0.1% Triton X-100, 0.02% SDS at 22 C. After the washes, SF-1 was detected using [125I]Protein A (1:1000 in wash buffer; specific activity, 1 mCi/ml) for 3 h at 22 C with shaking. The blots were washed and exposed to x-ray film.

Immunocytochemistry
Granulosa and R2C Leydig cells were cultured as above on glass coverslips for varying times in the presence or absence of FSH or forskolin. Cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA) in PBS for 30 min at 22 C, washed in 10 mM glycine in PBS, and treated with 0.5% NP-40 in PBS. The cells were blocked with 3% BSA in PBS and incubated with anti-phosphoCREB IgG (1:1000) or anti-CREB IgG (1:500) in 3% BSA in PBS at 4 C for 18 h. After several PBS washes, cells were incubated with fluorescein-labeled goat anti-rabbit IgG (1:20, Pierce, Rockford, IL) in 3% BSA in PBS for 1 h at 22 C. CREB and phosphoCREB were visualized on a Zeiss Axiophot microscope.

Phosphorylation and Immunoprecipitation of SF-1
Phosphorylation of granulosa cell proteins was performed as previously described (39). Briefly, granulosa cells were cultured in serum free DMEM:F12 in the presence or absence of FSH and testosterone for 48 h. Cells were then washed and preincubated for 2 h in phosphate-free DMEM (GIBCO). Fresh medium with FSH and testosterone and 2.5 mCi/ml of [32P]orthophosphate (ICN) were added for 2 h. The cells were then washed with PBS and resuspended in whole cell extract buffer containing phosphatase inhibitors (55). The cells were lysed by freeze-thaw procedure, and the protein concentrations were determined by the method of Bradford using the Bio-Rad protein assay. Immunoprecipitations were performed as follows. Protein A Sepharose CL-4B beads (Sigma) were preswollen in PBS, 0.1% SDS, 0.2% Triton X-100, and incubated with equal volumes of anti-Ad4BP IgG (1:100 in the above buffer) for 7 h at 4 C, with shaking. Unbound antibody was removed by excess washing of the Sepharose beads. An equal volume of the 32P-labeled whole cell extract protein (50 µg) was then incubated with the beads for 18 h at 4 C, with shaking. Beads were washed with excess buffer to remove unbound labeled proteins. Bound 32P-labeled proteins were eluted from the beads by addition of an equal volume of SDS-PAGE loading buffer and boiled at 100 C for 10 min. Samples were electrophoresed on a 10% SDS-polyacrylamide gel by standard methods (52), dried, and exposed to x-ray film.


    ACKNOWLEDGMENTS
 
The authors wish to thank Kristen Williams for her technical assistance and Dr. Marc Montminy (Salk Institute, La Jolla Ca), Dr. David Ginty (Johns Hopkins University, Baltimore MD), and Dr. Ken-ichirou Morohashi (Kyushu University, Japan) for their generous gifts of the CREB, phosphoCREB, and Ad4BP antibodies, respectively.


    FOOTNOTES
 
Address requests for reprints to: JoAnne S. Richards, Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030.

This work was supported by NIH Grants 16272 (to J.S.R.), HD-07165, and NRSA-HD-07991 (to D.L.C.).


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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