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Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030
Address all correspondence and requests for reprints to: JoAnne S. Richards, Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. E-mail: joanner{at}bcm.tmc.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Although both FSH and LH bind cognate G protein-coupled receptors that activate adenylyl cyclase, stimulate production of cAMP, and activate protein kinase A (PKA), it remains to be fully understood how each gonadotropin activates distinct patterns of gene expression at defined stages of ovarian differentiation. The cAMP regulatory element (CRE)-binding protein (CREB) is one extensively studied target of cAMP and PKA, but an increasing number of other transcriptional regulators are now recognized targets of gonadotropins. Some of these are PKA dependent; others are regulated by alternative cAMP-mediated actions in granulosa cells (6).
FSH has been shown to regulate the expression of many genes in granulosa cells as small follicles grow and acquire functional characteristics of preovulatory follicles. The FSH-mediated changes in granulosa cell function are both rapid and progressive. Activation of adenylyl cyclase and nuclear transport of the catalytic subunit of PKA is associated with FSH induction of serum and glucocorticoid-regulated kinase (Sgk) via a mechanism dependent on the Sp1 transcription factor (6, 7). In addition, Sgk and its close relative (protein kinase B/Akt) are phosphorylated (8, 9). The rapid appearance and activation of these kinases are related to the induction of cell cycle regulators, cyclin D2 and cyclin E. Immediate early transcription factors such as Jun/Fos family members are also induced by FSH (10). All of these early responses to FSH mediate the progressive differentiation of granulosa cells. Fully differentiated granulosa cells of preovulatory follicles express specific genes including P450aromatase, LH receptor, and inhibin-
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The human chorionic gonadotropin (hCG)/LH-induced processes of ovulation and luteinization involve dramatic restructuring of the follicle and changes in extracellular matrix composition and the function of granulosa and theca cells. During the periovulatory period immediately after the surge of LH, remodeling of the follicle and reprogramming of gene expression are mediated by the transient expression of specific transcriptional regulators. Many genes that are expressed in the preovulatory follicle are turned off by the LH surge, including cyclin D2 and cyclin E (3) and aromatase (12) in granulosa cells and 17-hydroxylase in theca cells (13). Other genes required for ovulation and luteinization are increased in response to the LH surge, including inhibitors of the cell cycle (p21CIP1 and p27KIP1), inflammation-related proteins (cyclooxygenase-2, COX-2), steroidogenic enzymes (steroidogenic acute regulatory protein and P450scc;), and matrix-remodeling proteases [MMP (14), cathepsin L, and a disintegrin and metalloprotease with thrombospondin motifs-1 (ADAMTS-1) (15, 16, 17)]. Transcription factors transiently induced during this periovulatory period include the progesterone receptor (18), CAAT-enhancer binding protein ß (C/EBPß; Refs. 19 and 20), and Jun/Fos family members (10). Targeted disruption of the COX-2 (21), progesterone receptor (22, 23), and C/EBPß (24) genes in mice has confirmed that each are obligatory for normal ovulation.
More recently, we identified the immediate-early transcription factor, early growth response factor (Egr-1; also known as NGFI-A, Krox 24, zif/268, or TIS8) as an LH-regulated gene in the rat ovary by differential display RT-PCR (25). Rapid but transient expression of Egr-1 mRNA was induced in preovulatory follicles by an ovulatory dose of the LH analog hCG. This pattern of expression was remarkably similar to that of the LH-regulated genes that mediate ovulation processes, C/EBPß, PR, and COX-2. That hCG/LH could induce Egr-1 in granulosa cells is of interest since GC-rich enhancer elements that potentially bind Egr-1 are present in many of the above mentioned LH-regulated genes. Additionally, Egr-1 null mice exhibit impaired reproductive functions; production of pituitary LHß in response to GnRH is abrogated and ovarian LH receptor expression is also impaired (26, 27). Thus, Egr-1 appears essential for normal ovarian function.
The present studies were undertaken to determine the specific actions of FSH and LH on Egr-1 expression in granulosa cells at defined stages of differentiation. The promoter of the Egr-1 gene was analyzed to determine which transcription factors bind and regulate its activity in response to gonadotropins, as well as the agonists cAMP and phorbol myristate acetate (PMA). For these studies the expression of Egr-1 and transcription factor binding activity in ovaries of hormonally primed rats, primary rat granulosa cell cultures, and Egr-1 null mice were investigated.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). Ovaries of H rats and H rats treated for 3d with estradiol (HE) contained no detectable Egr-1 mRNA. After iv injection of FSH, Egr-1 mRNA was transiently induced specifically in granulosa cells of growing follicles after 2 h (HEF 2 h). Expression returned to undetectable levels in preovulatory follicles of rats 48 h after FSH treatment (HEF 48 h). When these rats were injected iv with an acute ovulatory dose of hCG, Egr-1 mRNA again increased rapidly in granulosa cells and was expressed transiently. Egr-1 mRNA was first detectable within 2 h, peaked at 4 h, and then declined by 8 h and 12 h, becoming nondetectable in corpora lutea present after 24 h. These results show that Egr-1 can be transiently induced by FSH in granulosa cells of small follicles as well as by hCG/LH in preovulatory follicles.
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). Interestingly, some Egr-1 protein was also detectable in theca cells of FSH- and hCG-stimulated follicles (Fig. 2B, arrowheads).
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). A faster migrating immunoreactive band predominantly seen between 8 and 24 h after hCG treatment may represent a cleavage product generated during rapid turnover of this transiently expressed protein. The bimodal induction of Egr-1 mRNA after hCG treatment was reproduced in Western blot as well as EMSA analyses (see below). In contrast, the related zinc finger transcription factor Sp1 was present in equivalent amounts at all stages of hormonally induced differentiation (Fig. 1C, lower panel).
cAMP-Inducible Transcription Factor Binding Elements Within the Proximal Egr-1 Promoter Mediate Gonadotropin Induction
To analyze the signaling mechanism by which gonadotropins induced transcriptional activation of the Egr-1 gene, granulosa cells from immature rats were differentiated by treatment with hormones or signaling agonists in vitro. Similar to in vivo gonadotropin treatment, Egr-1 protein was transiently induced in immature granulosa cells between 30 and 90 min after treatment with FSH and testosterone (Fig. 2, upper panel). After in vitro maturation of granulosa cells by 48 h of treatment with FSH/T, a dose of forskolin (Fo) designed to mimic adenylyl cyclase activation by LH resulted in a greater and more sustained induction of Egr-1. Levels of Sp1 protein in the same extracts remained uniform throughout all stages of differentiation (Fig. 2, lower panel). Thus, increased intracellular cAMP by FSH or Fo treatment is sufficient to mediate induction of Egr-1 in immature and differentiated granulosa cells. These results show that primary granulosa cell cultures reflect the responses to gonadotropins in vivo and provide a testable system for analyzing Egr-1 promoter function.
In initial reporter assays, similar granulosa cell cultures were transfected with plasmids containing serial 5'-deletions of the rat Egr-1 promoter region fused to the firefly luciferase reporter cassette. Reporter constructs containing -1380 bp, -389 bp, or -164/+33 bp of 5'-Egr-1 promoter sequence in the pXP-2 reporter showed equivalent basal activity and responded with greater than 2.5-fold induction after 4 h of Fo treatment. Truncation at -55/+33bp greatly diminished basal and induced activity (Fig. 3A). These results localized the functional region of the Egr-1 promoter in granulosa cells between -164 and -55 bp. Therefore, this sequence of the rat Egr-1 promoter was studied in more detail to identify transcription factor binding and function of specific elements within this region.
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). We determined the granulosa cell transcription factors binding these elements by EMSA. A GC-rich region from -64 bp/-46 bp of the Egr-1 promoter contains two elements that bound Sp1 in HeLa cells (29). The same GC-box probe formed four complexes in extracts of cultured granulosa cells (Fig. 3C, complexes I–IV). Complexes (I, II, and IV) were identified by supershift analysis as homo- and heterodimers of Sp1 and Sp3 transcription factors. Binding activity of these Sp1/3 complexes was detectable in extracts of unstimulated cells, was increased by Fo and more dramatically increased after Fo/PMA treatment for 4 h. The inhibitor of PKA activity, H89, did not impair Fo-mediated Sp1/3 binding. Rather, inhibition of Erk activation by PD98059 treatment reduced the binding of Sp1/3 in Fo-treated cells. Complex III was present only in Fo- and Fo/PMA-treated extracts and contained Egr-1 as demonstrated by supershift analysis (Fig. 3C). The Fo-mediated induction of Egr-1 binding was sensitive to both H89 and PD98059 inhibitors, suggesting that PKA as well as MAPK pathways may mediate the cAMP induction of Egr-1. The pattern of Egr-1 binding the GC-box from its own promoter in EMSA mimicked the binding observed using a consensus Egr-1 element as probe (data not shown) as well as the amount of Egr-1 observed in Western blot (Fig. 2).
Further analysis of the -164-bp region identified two putative cAMP response elements (CREs) at -131 bp and -62 bp. Each putative CRE bound two complexes in granulosa cell extracts. A constitutively binding, slow migrating complex (Fig. 3D, complex I) was identified by supershift analysis as the CRE binding protein (CREB). Treatment with Fo increased CREB phosphorylation as determined by supershift of the complex with an antibody specific for Ser133-phosphorylated CREB (P-CREB). A faster migrating complex that was induced to bind both CREs in Fo-treated cells (complex II, Fig. 3D; and data not shown) remains to be positively identified. The induction by Fo of complex II, but not CREB binding activity, was sensitive to the PKA inhibitor H89 while neither complex was sensitive to PD98059 (Fig. 3D). Binding of complex II was also reduced in the presence of anti-CREB antibodies, suggesting that it is antigenically related to CREB and is thus likely to be a member of the CREB/CRE modulator (CREM)/activating transcription factor (ATF) family that is recognized by these antibodies. However, complex II was not supershifted by antibodies that specifically recognize the related transcription factors ATF-1 or ATF-2 (data not shown).
A CArG-box or serum response element (SRE) at -74 bp binds a single complex identified as serum response factor (SRF) by supershift analysis. This activity was present in unstimulated cells and not altered by Fo or Fo/PMA treatment of cells nor was binding sensitive to H89 or PD98059 activity (Fig. 3E). The second noncanonical CArG-box at -96 bp bound SRF with more than 10-fold lower efficiency as determined by our competitive EMSA analyses (data not shown).
Transcription Factors Binding the Egr-1 Promoter in Vivo Are Regulated by Gonadotropins
To determine the pattern of gonadotropin-regulated transcription factor binding in vivo, each promoter element was further analyzed using granulosa cell extracts from hormonally primed H rats. In agreement with the GC-box binding activity seen in vitro, Sp1/3 complexes binding the GC-box probe increased after 2 h of FSH treatment, at which time Egr-1 binding activity was also detected (Fig. 4A). FSH treatment for 48 h dramatically increased Sp1 binding, while Egr-1 binding was undetectable. Administration of hCG to HEF-treated rats for 2–4 h resulted in reduced Sp1/3 binding to the GC-box, whereas Egr-1 binding was greatly increased. This reciprocal relationship between Egr-1 and Sp1 binding to the GC-box suggested that Egr-1 may displace Sp1-DNA binding. To test this, consensus element probes that bound only Sp1/3 or Egr-1 were used. Complexes containing Sp1/3 bound the Sp1 consensus probe in all samples in a pattern similar to the binding to the Egr-1 GC-box. The amount of Sp1/3 binding was increased markedly by FSH treatment and subsequently declined after hCG treatment with the exception of animals treated with hCG for 8 h (Fig. 4, A and B). This reduction is not related to changes in Sp1 protein concentration (see Fig. 1). Egr-1 binding to its consensus element mimicked its binding to the GC-box probe and confirms the mRNA and protein expression observed by in situ hybridization and Western blot (Fig. 1) as well as in previous studies (25). The authenticity of Sp1/3 and Egr-1 binding DNA activity was verified by supershift of each complex with specific antibodies (Fig. 4, right panels). These observations demonstrate that Sp1/3 binding activity is modulated in granulosa cells by the actions of FSH and LH and is independent of Egr-1 binding to its overlapping site.
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). SRF binding to SRE-74 was also constitutive in granuolsa cells of H rats as seen in cultured cells treated with gonadotropin agonists (Fig. 4E).
Gonadotropin-Mediated Changes in Sp1/3 Binding Activity Does Not Involve Competitive Displacement by Egr-1
Dynamic gonadotropin modulation of Sp1 and Egr-1 binding to the GC-box-64/-46 element was observed in each model of granulosa cell differentiation. To test more directly whether Egr-1 could alter Sp1 binding to this element, we first sought to express Egr-1 in granulosa cell cultures in the absence of gonadotropin or agonist treatments. Primary cultures of granulosa cells from pregnant mare serum gonadotropin (PMSG)-primed rats were transfected with 0, 2, or 4 µg of the pcDNA3.1 expression vector under the control of the cytomegalovirus (CMV) enhancer containing the rat Egr-1 coding sequence. Binding of Egr-1 to its consensus element in EMSA increased 1.7- and 2.4-fold, respectively, in cells transfected with 2 or 4 µg pcDNA-Egr-1 compared with 4 µg empty vector transfection (Fig. 5B). In the same samples, Egr-1 binding to the GC-box probe increased 1.4- and 1.7-fold, respectively, whereas Sp1 binding in Egr-1-transfected cells remained similar to that in controls (Fig. 5A). Sp1 binding to its consensus element probe was unchanged (Fig. 5C).
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). Similar results were obtained using extracts from individual mice in two additional experiments. These results, along with the changes in binding of Sp1 to a consensus element probe in stimulated HEF rats (Fig. 4B), suggest that a gonadotropin signaling mechanism(s) other than induction of Egr-1 regulates Sp1 DNA binding activity in granulosa cells.
CREB, SRF, and Sp1 Cooperatively Mediate Egr-1 Promoter Activity in Granulosa Cells
We next sought to ascertain the role of each transcription factor binding element in modulating Egr-1 transcriptional activity in granulosa cells. For these studies, -164/+33 and -96/+33 Egr-1 promoter fragments were ligated upstream of the luciferase reporter in the pGL3-basic vector and site-specific mutants of each putative regulatory element were generated (see Fig. 3B). WT and mutant promoter-reporter constructs were transfected into cultured granulosa cells and treated with Fo ± PMA (agonists of LH signaling pathways) or specific signaling antagonists H89 and PD98059.
Basal activity of the -164/+33-Luc construct was 5-fold greater than empty pGL3-basic vector. Mutation of either CRE-131 or SRE-74 increased basal transcription 3-fold, while a GC-box mutation showed 20% reduced activity compared with the -164/+33-Luc control (Fig. 6A). Likewise, basal activity in the -96/+33 construct was increased 8-fold by mutation of SRE-74. Although the GC-box mutation did not alter basal activity, it prevented the increase in activity resulting from mutation of the SRE-74. Transcriptional induction by Fo or Fo+PMA in the -164/+33 construct, was approximately 10- or 40-fold, respectively, and was unaltered by mutation of either the SRE-74 or GC-box. Mutation of CRE-131, however, consistently reduced inducibility by Fo or Fo+PMA 40% (Fig. 6A). Truncation of the Egr-1 5'-regulatory sequence at -96 bp to remove the CRE-131 as well as SRE-96 (which showed little binding activity in EMSA) resulted in a similar loss of inducibility to that caused by CRE-131 mutation (Fig. 6B). Inducibility was further decreased by mutation of the SRE or the SRE and GC-box regions, yielding activity levels similar to empty pGL3-basic vector. Mutation of the GC-box alone resulted in 50% lower inducible activity compared with WT -96/+33-Luc controls (Fig. 6B). These results suggest that the interactions of the CRE-131, SRE-74, and the GC-box involve complex activator and repressor functions. Cooperative activity of each element is necessary for the full transactivation of Egr-1 expression.
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). Although removal of the CRE-131 in the -96 construct diminished overall promoter activity, it did not prevent induciblity by Fo or the reduction by kinase inhibitors. Thus, Fo inducibility of the Egr-1 promoter is dependent on both PKA and MAPK signaling. This result supports the observed reduction of Fo-induced Egr-1 binding in EMSA of cell extracts treated with the same inhibitors (Fig. 3B).
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) compared with cotransfection of the empty expression vector both in unstimulated cells or after treatment with Fo or Fo+PMA. Identical results were obtained when the -164/+33-Luc-reporter was cotransfected with the Egr-1 or empty expression vectors in NIH-3T3 fibroblasts (data not shown). Thus, the potential for positive feedback by Egr-1 to further induce its own expression through the GC-box element is implied. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In these studies we provide several novel observations that relate to the response of granulosa cells to gonadotropins. First that FSH as well as LH induces expression of Egr-1 in granulosa cells in vivo and in vitro, indicating that Egr-1 can impact granulosa cell function in growing follicles. Egr-1 can interact with LH-R gene regulatory sequences (37), which may be a critical event in FSH-mediated expression of LH-R in differentiating granulosa cells. The onset of the LH surge activates a transient periovulatory increase in Egr-1 expression that may mediate ovulatory gene expression. Several genes with potential roles in ovulation have been shown to bind Egr-1: these include membrane prostaglandin E2 synthase [mPGES, (38)] and proteases MT1-MMP (39), cathepsin L (40), and ADAMTS-1 (41). The latter two each posses GC-box elements containing overlapping sites that bind Egr-1 and Sp1 in a similar pattern to that of the Egr-1 promoter (our unpublished observation). In preliminary characterization of Egr-1 null mice, an important specific role for Egr-1 during ovulation is emerging (Russell, D. L., and J. S. Richards, unpublished observations).
Egr-1 expression in response to FSH as well as LH is rapid and transient and dependent on the activation of at least two cellular signaling pathways, PKA and Erk. Activation of PKA and Erk pathways by FSH and LH are documented (42, 43), but genes specifically regulated by their combined actions are less well characterized. We show that Fo and PMA synergize to induce Egr-1 expression and activate the Egr-1 promoter, while inhibitors of PKA and Erk reduce Egr-1 transactivation. Importantly, the induction of Egr-1 in granulosa cells afforded a system in which to study promoter regulation. The proximal region within -164/-55 bp of the Egr-1 gene contained the necessary elements for transactivation of the Egr-1 gene, a similar region to that previously shown necessary for Egr-1 induction by GM-CSF in myeloid cells (31).
Egr-1 is not expressed in granulosa cells in vivo unless they receive an acute gonadotropin stimulus. Similarly, granulosa cells in culture respond vigorously to FSH agonists. Several scenarios are possible to explain hormone-induced transactivation of this gene. Repressors could be modified and/or displaced, transcriptional activators recruited, or activators and cofactors modified to facilitate transcriptional initiation. These possibilities are not mutually exclusive. Our studies in granulosa cells indicate that transcriptional activators and repressors interact via the CRE, SRE, and GC-box to mediate the complex regulation of Egr-1 expression.
A role for the GC-box regulating basal promoter activity in cooperation with SRE-74 was indicated since mutation of this region reduced activity in -164 and -96/+33 promoter contexts and reversed a large increase in basal activity introduced by SRE-74 mutation. Sp1/3 and Egr-1 strongly bound this element in a highly regulated manner. In immature granulosa cells the binding of Sp1/3 and Egr-1 to the GC-box or an independent consensus sequence increased with FSH stimulation, while cellular concentrations of Sp1 protein were uniform. Inhibitors of Erk (but not PKA) reduced binding of Sp1 without altering protein concentrations. Thus, phosphorylation of Sp1 through Erk pathways appears to be one essential first step regulating binding to the GC-box of the Egr-1 promoter in granulosa cells.
After hCG stimulation of mature preovulatory rat ovaries, Egr-1 strongly bound the GC-box, whereas Sp1 binding to this element or a consensus element was rapidly reduced. Binding of Egr-1 to the GC-box or a consensus sequence correlated with cellular Egr-1 levels, suggesting a direct relationship between expression and binding activity; however, protein modification cannot be ruled out. We initially anticipated that Egr-1 binding its own promoter may displace Sp1 and repress its own transactivation. Binding competition between Sp1 and Egr-1 is common among GC-rich elements that exhibit overlapping Sp1- and Egr-1 binding motifs (44, 45, 46). Additionally direct protein-protein interaction whereby Egr-1 inhibits Sp1 DNA binding activity has been reported (47). However, competitive interaction with Egr-1 does not appear to be the mechanism displacing Sp1 binding in periovulatory granulosa cells. Exogenous expression of Egr-1 in cultured granulosa cells without gonadotropin agonist treatment did not displace Sp1 binding, and hCG treatment reduced granulosa cell Sp1 binding equivalently in Egr-1 null mice and WT littermates. Overexpression of Egr-1 in two cell types, granulosa cells and NIH3T3-fibroblasts, increased transactivation and revealed no evidence of auto-repression. Thus the binding of Egr-1 to the GC-box of its own promoter region can have little role in repression during the transient expression pattern although it may be capable of enhancing the maximal promoter function. Alternative mechanisms of down-regulation of expression in granulosa cells may involve cAMP-mediated recruitment of another repressor molecule such as inducible cAMP early repressor (ICER; see below).
Changes in Sp1 binding activity have been shown to involve phosphorylation (48, 49, 50, 51) or glycosylation (52), consistent with the possibility that posttranslational modifications of Sp1 in response to FSH and LH modulate Sp1 binding. Sp1 is a known target for Erks (51, 53), and Sp1 phosphorylation and binding to GC-rich elements is sensitive to Erk inhibition of other genes (53, 54, 55). The level and specific sites of Sp1 phosphorylation as well as potential actions of phosphatases after stimulation with FSH vs. LH will need further investigation to fully understand the effects of gonadotropins on binding. The binding activity of Sp1/3 may also be context specific since binding of Sp1/3 to a GC-rich region of the Sgk gene was uniform at all stages of granulosa cell differentiation (7).
Binding of CREB to the CRE-131 is highly important for Egr-1 induction since mutation of this region alone reduced Fo and Fo/PMA induction by approximately 40%, and removal of CRE-131 in the truncated -96/+33 construct similarly reduced activity. In ß-islet cells Egr-1 expression is similarly dependent on CRE-131 and other unidentified factors within -196 bp of the promoter (33). Likewise, mutation of a similar element in the mouse Egr-1 promoter resulted in 40% lower induction by GnRH in pituitary gonadotropes. Interestingly this required interaction with distal SREs (36). We found constitutive binding of CREB to the CRE in granulosa cells, while phospho-CREB binding was identified only after gonadotropin/cAMP treatment. Phosphorylation of CREB in response to gonadotropins is well described (4, 56) and P-CREB interaction with the coactivator CREB-binding protein stimulates expression of gonadotropin/CRE regulated ovarian genes (6), including P450aromatase (8) and inhibin- (57). However, surprisingly, we also noted that mutation of CRE-131 consistently increased basal activity as did mutation of the proximal SRE-74.
Along with P-CREB, a second complex (complex II) was induced to bind CRE in gonadotropin- or Fo-stimulated cells. Although this complex remains to be positively identified, it may also be important for the regulation of Egr-1. Complex II was immunoreactive to CREB and P-CREB antibodies but not ATF1/2 antibodies This complex may contain a CREB-related protein such as CREM or ICER, a repressor transcribed from an LH-dependent initiation site in the CREM gene (58). Regulation of the ICER protein in periovulatory granulosa cells shows a similar pattern to the CRE binding by complex II in this study, being induced between 2 and 12 h after hCG and then returning to basal levels (59). The induction of this CRE binding complex is potentially involved in down-regulation of Egr-1 expression. Alternatively, this complex may contain another unknown CREB-related transcriptional activator or repressor.
Our data provide the first evidence that SRF is expressed in granulosa cells and binds the Egr-1 promoter. Mutation of SRE-76 clearly increased basal promoter function in each reporter context but did not alter agonist induction in the -164/+33 construct, perhaps because the CRE is present and functional. In the -96 construct, mutation of the SRE reduced Fo and Fo/PMA activation, indicating that it confers gonadotropin inducibility at least in this context. Mutation of the SRE and GC-box in the -96 truncated construct is required for complete loss of basal and induced transactivation above that of the empty pGL3 reporter, indicating disruption of all functional promoter elements. Inhibitory effects of H89 and PD98059 were also observed in the -96 construct containing both the SRE and the GC-box but lacking the CRE. Thus, the SRE as well as the GC region appear to be involved in PKA- and Erk-mediated transactivation. This SRE, along with other more distal SREs, was also found previously to be important for GnRH induction of Egr-1 in gonadotropes (36). Like CREB binding to the CRE, SRF occupies the promoter constitutively, suggesting that transcriptional regulation through this element after gonadotropin stimulation involves SRF phosphorylation, which facilitates docking of transcriptional coactivators such as CREB-binding protein (60, 61). In the c-fos gene, enhancer SRF is also a repressor of basal expression and is necessary for full activation involving changes in phosphorylation state via serum-responsive kinase cascades (62) and recruitment of corepressors (63). Likewise our results suggest that in the absence of agonist stimulation the CRE-131 and neighboring SRE-74 contribute promoter silencing, and reversal of this repression may be a necessary mechanism for gonadotropin-mediated increase of Egr-1 expression.
In conclusion, we have shown Egr-1 expression in response to FSH and LH stimulation of rodent ovaries. Egr-1 presumably interacts with other ovarian gene promoters to mediate transcription of a subset of gonadotropin-induced genes. Numerous factors known to be regulated by Egr-1 are expressed in the ovary with important roles in folliculogenesis and/or ovulation. Among these are transcriptional regulators such as p53 (64, 65), cytokines, and growth factors TNF (66) and TGFß, fibronectin (67), or cellular adhesion factors CD-44 (68). Potential candidates for Egr-1 regulation with particular relevance in the periovulatory period include proteases MT1-MMP (39), cathepsin L (40), and ADAMTS-1 (41). Importantly, LH-R expression and corpus luteum formation may require Egr-1 (26); however, the pattern of LH-R gene expression is not well correlated with Egr-1, because LH-R is rapidly down-regulated in the periovulatory period. These studies provide the basis for future characterization of the phenotype of the Egr-1 null mice by indicating that Egr-1 may play roles in both the formation of preovulatory follicles as well as in the initiation of ovulation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-32P]dCTP was from ICN Biochemicals, Inc. (Cleveland, OH). BenchMark protein molecular size markers were from Life Technologies, Inc. (Gaithersburg, MD). The enhanced chemiluminescence detection system and luciferin substrate were from Amersham Pharmacia Biotech (Arlington Heights, IL). Kodak X-Omat AR film and NTB-2 liquid emulsion were from Eastman Kodak Co. (Rochester, NY). Specific antibodies against Sp1, Sp3, Egr-1, SRF, ATF-1/2, and Sap1a were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies to CREB and P-CREB were from New England Biolabs, Inc. (Beverly, MA); these antibodies detect CREB as well as the closely related CREM and ATF family members. Rat Egr-1 expression construct (pJDM-Egr-1) was generously provided by Dr. Jeffery Milbrandt (Washington University Medical School). For these experiments, Egr-1 coding sequence was subcloned into pcDNA3.1 (Invitrogen, Carlsbad, CA).
Animals
Hypophysectomized (H) rats were purchased from Harlan Industries (Indianapolis, IN), provided food and water ad libitum, and housed under a 12-h light, 12-h dark schedule. Animals were treated in accordance with the NIH Guide for Care and Use of Laboratory Animals. Protocols were approved by the Institutional Animal Care and Use Committee, Baylor College of Medicine (Houston, TX). Ovarian follicle growth and differentiation were stimulated in H rats by hormonal treatment as described previously (69, 70). Commencing 3–4 d after hypophysectomy (H), rats received daily sc injections of 17ß-estradiol (1.5 mg) for three consecutive days (HE). The following day rats received either a single tail-vein injection of 1 µg FSH (HEF 2 h) or were treated for 2 d by sc injection of FSH twice each day (HEF 48 h). Luteinization was induced in HEF rats by tail-vein injection of 10 IU hCG (HEF/hCG 2–48 h). After each treatment animals were euthanized, ovaries were extirpated and granulosa cells were isolated by ovarian puncture for preparation of whole -cell extracts (WCEs). Each treatment group included four rats except for the H group for which 12 animals were used.
Heterozygous mice carrying a null allele for Egr-1 were kindly provided by Dr. Jeffery Milbrandt (27) and established in a breeding colony under a 12 h light, 12-h dark schedule and given rodent chow and water ad libitum. Litters were weaned at 21 d of age, at which time female mice were injected ip with PMSG (4 IU) followed after 44 h with 5 IU hCG. After 4-h hCG treatment, mice were euthanized and granulosa cells were isolated and processed for EMSA as described for rats above.
In Situ Hybridization
Ovaries isolated from H rats stimulated as described above were fixed in 4% paraformaldehyde solution, paraffin-embedded, and sectioned. In situ hybridization was performed as previously described (3). Antisense and sense riboprobes for rat Egr-1 were labeled with [35S]UTP by in vitro transcription and incubated overnight with sections at 55 C in the presence of 50% formamide. After treatment with ribonuclease A and repeated washing to a final stringency of 0.1x SSC, slides were exposed to liquid emulsion autoradiography for 4 d and then counterstained with hematoxylin.
Immunohistochemistry
Rehydrated 7-µm sections of paraffin-embedded ovaries were treated with 3% H2O2 to block endogenous peroxidase activity and then incubated overnight with anti-Egr-1 antiserum (no. 588, Santa Cruz Biotechnology, Inc.) 1:250 dilution in 10% normal goat serum. Slides were washed three times in PBS (pH 7.5) containing 0.025% Tween 20 and then incubated with biotinylated goat antirabbit IgG 1:500 (Vector Laboratories, Inc., Burlingame, CA) for 1 h. After washing with PBS, sections were incubated with streptavidin-conjugated horseradish peroxidase for 1 h, and then washed again and treated with diaminobenzidine substrate (Vector Laboratories, Inc.) for 5 min. Slides were then dehydrated and mounted without counterstaining.
Whole-Cell Extracts
Granulosa cells were isolated from preovulatory ovaries or scraped from culture dishes in PBS and then resuspended in 150–200 µl of 10 mM Tris-buffer containing 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, 400 mM potassium chloride, 1 mM vanadate, and protease inhibitors [WCE buffer (71)]. Cells and nuclei were lysed by three rapid freeze-thaw cycles, centrifuged at 12,000 x g, and protein concentrations of soluble extracts were measured (Bradford method: Bio-Rad Laboratories, Inc., Richmond, CA). Dissected corpora lutea from HEF/hCG 24 h or 48 h were homogenized at 4 C in WCE buffer and then treated as for granulosa cell extracts.
EMSA
EMSAs were performed as described previously (69). Briefly, protein extracts (2.5 µg per lane) were incubated for 30 min at room temperature with 50,000 cpm of end-labeled double-stranded oligonucleotide probe and poly(deoxyinosinic-deoxycytidylic) acid in a final buffer volume of 20 µl containing 15 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM dithiothreitol, 1 mM EDTA, 5 mM MgCl2, and 12% glycerol. For supershift experiments, antibodies were incubated with extracts for 30 min on ice before labeled probe was added. Bound and unbound probe was separated by 5% acrylamide gel electrophoresis, and gels were dried and exposed to x-ray film. The sense strand sequences of oligonucleotide probes with core binding elements underlined were:
Western Blot Analyses
Whole-cell extracts (50 µg) were resolved on 10% acrylamide gels by reducing SDS-PAGE, followed by electrophoretic transfer to polyvinylidine difluoride membrane (Immobilon-P, Millipore Corp. Bedford, MA). Membranes were blocked by 1 h incubation at room temperature with 3% nonfat milk, followed by 1 h incubation with specific primary antibodies in 3% milk and washing in TBST [10 mM Tris (pH 7.5), 150 mM NaCl, and 0.05% Tween 20]. Blots were then incubated with 1:10,000 of horseradish peroxidase-linked antirabbit or antimouse IgG (Amersham Pharmacia Biotech) followed by six 5-min washes with TBST. Enhanced chemiluminescence detection was performed according to the manufacturer’s specifications. Blots were stripped for subsequent reanalysis by washing at 50 C for 30 min in 62.5 mM Tris-HCl (pH 6.7), 100 mM 2-mercaptoethanol, and 2% sodium dodecyl sulfate.
Granulosa Cell Culture and Luciferase Reporter Assays
Granulosa cells were harvested as described above from intact immature (d 23) rats (Harlan) 48 h after stimulation with 10 IU PMSG (pregnant mare serum gonadotropin). Cells were placed in culture in serum-coated plastic wells at a density of 1 x 106 cells/ml of serum-free DMEM-F12 medium as previously described (56). For EMSA and Western blot analyses, cells were cultured overnight in defined medium (0 h) and then treated as described and harvested at indicated times by washing once and then scraping in ice-cold PBS. Cell pellets were resuspended in WCE buffer and extracts were prepared as described above.
For luciferase assays, cells were plated in 1.9 cm2 wells in DMEM-F12 medium containing 5% fetal bovine serum. After 4 h of culture, cells were transfected with 250 ng of the specified Egr-1-promoter-luciferase DNA construct using FuGene 6 transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN) as per the manufacturer’s instructions. Transfected cells were cultured overnight in serum-containing medium, and then washed two times for 15 min in serum-free medium, after which medium was replaced with serum-free DMEM-F12 containing hormone or agonist treatments [Fo (10 µM) or PMA (20 nM) or inhibitors H89 (10 µM) or PD98059 (10 µM)]. After 4 h, cells were washed in PBS and lysates were prepared by scraping in 0.1 M Tris, pH 8.0, containing 0.1% Triton X-100. Luciferase activity was analyzed in 40 µl of lysate on a MLX luminometer (Dynex Technologies, Chantilly, VA). Relative light units were normalized to protein content in each lysate sample. Each experiment was performed in triplicate and repeated at least three times. Data are presented as mean ± SEM from experiments repeated at least three times. Statistical t test analyses were used to compare the activity of mutants with WT reporter constructs in granulosa cells with the same treatment; P < 0.05 was considered statistically significant.
For reporter assays involving exogenous expression of Egr-1, granulosa cells or 3T3-fibroblasts were plated in DMEM-F12 with 5% fetal bovine serum at 2 x 106 cells per well in six-well culture plates. Cells were transfected as described above with 1 µg of the reporter construct along with 4 µg of the CMV-Egr-1 expression construct or empty vector, kindly provided by Dr. J. Milbrandt (Washington University Medical School, Department of Pathology). After overnight culture, cells were washed twice with serum-free DMEM-F12, and treatments were added in serum-free medium. After 4 h of treatment, luciferase assays were performed as described above.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Present address: Departmento de Bioquimica y Biologia Molecular, Facultad de Ciencias de la Salud, Universidad de Las Palmas Gran Canaria, 35016 Las Palmas de Gran Canaria, Spain. 
2 Present address: Unidad de Genética Molecular, Hospital Marqués de Valdecilla, Edificio Escuela Universitaria de Enfermería, Avda. Valdecilla s/n, 39008 Santander, Spain.
Abbreviations: ATF, Activating transcription factor; C/EBPß, CAAT-enhancer binding protein ß; CMV, cytomegalovirus; COX-2, cyclooxygenase 2; CRE, cAMP regulatory element; CREB, CRE-binding protein; CREM, CRE modulator; Egr, early growth response factor; Fo, forskolin; hCG, human chorionic gonadotropin; ICER, inducible cAMP early repressor; LH-R, LH receptor; MMP, matrix-remodeling protease; P-CREB, Ser133-phosphorylated CREB; PKA, protein kinase A; PMA, phorbol myristate acetate; PMSG, pregnant mare’s serum gonadotropin; Sgk, serum and glucocorticoid-regulated kinase; SRE, serum response element; SRF, serum response factor; WCE, whole-cell extract; WT, wild-type.
Received for publication February 8, 2002. Accepted for publication January 15, 2003.
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