The Protein Kinase C System Acts through the Early Growth Response Protein 1 to Increase LHß Gene Expression in Synergy with Steroidogenic Factor-1

Lisa M. Halvorson, Ursula B. Kaiser and William W. Chin

Division of Genetics (L.M.H., W.W.C.) and Endocrine-Hypertension Division (U.B.K.) Department of Medicine Brigham and Women’s Hospital and Harvard Medical School Boston, Massachusetts 02115
Department of Obstetrics and Gynecology (L.M.H.) Tufts University School of Medicine Boston, Massachusetts 02111


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression of the LHß gene has been shown to be modulated by both the orphan nuclear receptor, steroidogenic factor-1 (SF-1), and the early growth response protein 1, Egr-1. It is also well known that LHß mRNA levels are increased after hormonal activation of the protein kinase C (PKC) signaling system, for example by GnRH; however, the mechanisms by which the PKC system exerts this effect has not been fully characterized. By transient transfection of the GH3 cell line, we demonstrate that activation of the PKC system with the phorbol ester, phorbol 12-myristate 13-acetate (PMA), increases activity of region -207/+5 of the rat LHß gene promoter (~2-fold) and markedly augments SF-1-induced stimulation (95-fold in the presence of both factors vs. 13-fold for SF-1 alone). Mutation of the two previously identified Egr-1 sites not only prevents Egr-1 effects on the LHß gene promoter, but also eliminates the synergistic response to PMA and SF-1 together, findings that were confirmed in a longer construct spanning region -797/+5. In the gonadotrope-derived cell line, {alpha}T3–1, these mutations eliminate the GnRH responsiveness of the -207/+5 LHß promoter construct. We next show that PMA treatment (GH3 and {alpha}T3–1 cells) or GnRH treatment ({alpha}T3–1 cells) induces expression of Egr-1, as detected by Egr-1 interaction with Egr-1 DNA-binding sites in the rat LHß gene promoter sequence. Furthermore, we demonstrate that PMA increases steady-state Egr-1 mRNA levels via increased Egr-1 transcription. We conclude that PMA-induced stimulation of LHß gene expression is achieved, at least in part, by induction of Egr-1 expression.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Gonadotropin gene expression is regulated by the complex interaction of multiple factors originating from the hypothalamus, pituitary, and gonads. The hypothalamic decapeptide, GnRH, is believed to exert its effects, at least in part, by activation of the protein kinase C (PKC) second messenger-signaling system in the gonadotrope. GnRH has been shown to increase PKC activity based on the measurement of endogenous PKC activators, such as diacylglycerol, and by kinase activity (1, 2, 3, 4). Increased PKC activity, in turn, up-regulates steady-state LHß mRNA levels and LHß polypeptide biosynthesis, as demonstrated by treatment of rat pituitary cells with phorbol esters (5, 6, 7). As phorbol 12-myristate 13-acetate (PMA)-induced increases in LHß mRNA levels are suppressed by the addition of the transcriptional inhibitor, actinomycin D, the PKC response has been attributed to an increase in LHß mRNA biosynthesis, rather than to an alteration in mRNA stability (8). This result suggests the presence of PMA-stimulated DNA-regulatory element(s) in the LHß gene promoter.

Recent attempts to identify transcriptional regulators of LHß gene expression have identified two transcription factors — steroidogenic factor-1 (SF-1) and the early growth response gene 1 product (Egr-1) — with profound effects on LHß gene promoter activity. SF-1, an orphan member of the nuclear hormone receptor superfamily, is selectively expressed in the gonadotrope subpopulation of the pituitary gland, as well as in the adrenal gland and gonads (9). SF-1 binds to a DNA promoter region called the gonadotrope-specific element, or GSE, as defined in the common glycoprotein {alpha}-subunit by Barnhart and Mellon (10). Variations of this sequence, alternatively called the Ad4 response element, are present in a wide range of genes that play a role in steroidogenesis, sexual differentiation, and adult reproductive function (9, 11). Our studies of the rat LHß gene promoter have characterized two functional GSE sites, located at positions -127 and -59 relative to the transcriptional start site in the rat (12, 13).

In vivo model systems have confirmed the importance of SF-1 in the regulation of LHß gene expression. In addition to other abnormalities, transgenic mice null for the Ftz-F1 gene, which encodes SF-1, lack detectable LHß mRNA levels (9). In a second transgenic model, mutation of the 5'-GSE site of the bovine LHß gene promoter markedly decreased expression of a reporter gene relative to expression levels in the presence of the wild-type promoter (14).

In studies of other members of the steroid receptor superfamily, receptor function has been shown to be profoundly altered by posttranslational modifications, such as phosphorylation, that modulate receptor stability, DNA-binding affinity, and/or transcriptional efficiency (15, 16, 17, 18). SF-1 is known to exist as a phosphoprotein. Carlone and Richards (19) and Zhang and Mellon (20) have demonstrated that SF-1 can be phosphorylated by the catalytic subunit of protein kinase A, resulting in decreased binding to the SF-1-binding site in the rat P450c17 gene promoter. Interestingly, in the type II 3ß-hydroxysteroid dehydrogenase gene promoter, the maximal PMA response requires the presence of an intact GSE site, suggesting that PMA may act through modulation of SF-1 (21). Although it is not known whether SF-1 is phosphorylated by PKC, these observations raise the intriguing possibility that the PKC system may increase LHß gene promoter activity by modulating SF-1 effects.

In vivo and in vitro data have also implicated the early growth response protein 1, Egr-1, in the transcriptional regulation of LHß gene expression (12, 22, 23). Egr-1, also known as zif/268, Krox-24, and NGFI-A, is a member of the immediate early gene family whose members contain a zinc finger domain with a Cys2-His2 motif that recognizes GC-rich nucleotide sequences (24, 25, 26, 27). Within the pituitary gland, Egr-1 expression is limited to the gonadotrope and somatotrope subpopulations based on colocalization of LHß-subunit protein and X-gal staining that is conferred by a lacZ transgene inserted 3' to the endogenous Egr-1 (Krox-24) gene promoter (22). Interestingly, two separate transgenic models have demonstrated specific loss of LHß gene expression in Egr-1-deficient mice with maintenance of normal FSHß gene expression (22, 23). In transient transfection experiments, the addition of Egr-1 has been shown to increase LHß gene promoter activity, an effect attributed to Egr-1 binding sites located at positions -112 and -50 in the rat sequence (12, 23). In a variety of nonreproductive systems, Egr-1 gene expression has been shown to be induced by a wide range of stimuli, including growth factors and phorbol esters (28). Therefore, it is also possible that the observed PMA response of the LHß gene is mediated via induction of Egr-1 expression in the gonadotrope.

In the results reported here, we determine that activation of the PKC system with the phorbol ester, PMA, increases rat LHß promoter activity, both alone and in conjunction with SF-1. Furthermore, we investigate the role of SF-1 and Egr-1 and their cognate DNA-binding sites in the generation of this response.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PMA Acts Alone and in Conjunction with SF-1 to Increase LHß Gene Expression
In previous investigations, steady-state LHß mRNA levels had been shown to be increased by activation of the PKC-signaling system with the phorbol ester, PMA (5, 6). Nevertheless, it had not been definitively determined that this PMA response was due to an increase in transcription rate, rather than altered mRNA stability. We therefore used transient transfection-luciferase reporter assays to determine whether PMA could alter LHß gene promoter activity and whether this effect, if present, acted in concert with the previously described SF-1 response.

While ideally we would have used a gonadotrope-derived cell line for these functional studies, the only cell line available for study at the time these experiments were performed was the {alpha}T3–1 cell line. Although {alpha}T3–1 cells express the endogenous gonadotropin {alpha}-subunit gene, they are unlike normal gonadotropes in that they do not express either of the ß-subunit genes (28). In addition, it has proven technically difficult to obtain high levels of expression of LHß promoter constructs in this cell line, limiting its usefulness for these studies. We therefore performed these experiments in an alternative pituitary-derived cell line, the rat somatolactotrope GH3 cell line. This cell line lacks endogenous SF-1, based on the failure of GH3 nuclear extracts to produce specific protein-DNA complexes with a GSE-containing oligonucleotide probe on electrophoretic mobility shift assay (EMSA) (data not shown). Use of an SF-1-deficient cell line allowed investigation of PMA effects on basal as well as SF-1-stimulated LHß gene promoter activity.

GH3 cells were transfected with a reporter construct containing region -207 to +5 of the rat LHß gene promoter. Treatment with PMA (100 ng/ml) for 4–6 h increased luciferase activity by 2.8-fold, demonstrating that the proximal rat LHß gene promoter can confer PMA responsiveness (Fig. 1AGo). Consistent with previously published results, cotransfection with the cytomegalovirus (CMV)-driven SF-1 expression vector resulted in a 13-fold increase in the luciferase activity of a reporter construct containing region -207 to +5 of the rat LHß gene promoter (13). Interestingly, marked synergy was observed in the presence of both SF-1 and PMA, with a 95-fold increase in luciferase activity in the presence of both factors compared with a 13-fold response to SF-1 alone.



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Figure 1. SF-1 and PMA Act Both Alone and Synergistically to Increase LHß Gene Promoter Activity

A, GH3 cells were transiently transfected with a construct containing region -207/+5 of the rat LHß gene promoter linked to a luciferase reporter construct, pXP2. Cells were cotransfected with a CMV-driven SF-1 expression vector and with an RSV-ß-galactosidase expression vector. Cells were treated with 100 ng/ml PMA or dimethylsulfoxide for 4–6 h starting approximately 40 h after transfection. Luciferase activity was normalized to ß-galactosidase activity, and promoter activity was calculated as fold-change over expression in the presence of the untreated control expression vector. Results are shown as the mean ± SEM of 30 samples in 10 independent experiments. B, GH3 cells were transiently transfected with a construct containing the CMV promoter linked to the cDNA encoding ß-galactosidase and treated as described above. ß-Galactosidase activity was measured and expressed as fold-change over expression in the untreated cells. Results are shown as the mean ± SEM of nine samples in three independent experiments. *, P < 0.05 vs. vehicle-treated control.

 
As seen in Fig. 1BGo, PMA did not increase expression of a construct containing the CMV promoter and the cDNA that encodes ß-galactosidase. This result suggested that the observed interaction between PMA and SF-1 was not due to increased transcription of SF-1 through stimulation of the CMV promoter.

The GSE Sites of the LHß Gene Promoter Do Not Confer a PMA Response
SF-1 transactivation efficiency could also be modified through mechanisms other than increased SF-1 protein levels, such as alterations in posttranslational processing. We reasoned that if PMA was directly altering SF-1 functional activity, a PMA response should be observed in the presence of SF-1 DNA response elements (GSEs). Therefore, four copies of the rat LHß 5'-GSE sequence (position -127) were inserted upstream of the GH minimal promoter, GH50, in the luciferase reporter construct, pXP1. As seen in Fig. 2AGo, these sequences conferred an SF-1 response, but failed to confer a response to PMA, either alone or in conjunction with SF-1. These results suggested that the PMA effect was generated by sequences outside of the GSE sites and was therefore not likely to be due to direct effects on the transcription factor, SF-1, which is known to bind to these regulatory elements.



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Figure 2. The PMA Response Is Not Due to the Presence of GSE Sites in the Proximal LHß Gene Promoter

A, PMA effects in a heterologous GSE-promoter construct. GH3 cells were transiently transfected with a construct containing the GH minimal promoter, GH50, linked to a luciferase reporter construct, pXP1, or with the addition of four copies of the 5'-GSE site. B, Stimulation of LHß gene promoter activity with loss of the GSE sites by site-directed mutagenesis. GH3 cells were transiently transfected with a promoterless luciferase reporter construct, pXP2, or with the same construct plus region -207/+5 of the rat LHß gene promoter present as either the wild-type sequence or with mutations in both GSE sites. Cells were treated (PMA 100 ng/ml x 4–6 h) and the results calculated as described in Fig. 1AGo. Results are shown as the mean ± SEM of nine samples in three independent experiments. n.s., P >= 0.05. *, P < 0.05 vs. vehicle-treated control.

 
This conclusion was confirmed by transfecting cells with a construct containing point mutations in both GSE sites within the context of the native rat LHß gene promoter. These mutations have been shown to eliminate binding by SF-1 on EMSA (12, 13). Despite marked blunting of the SF-1 and SF-1/PMA responses, the response to PMA alone was conserved in the face of these mutations (2.6-fold vs. 2.8-fold in the wild-type construct) (Fig. 2BGo). Note that the empty reporter construct did not respond to either factor, confirming that the observed responses to SF-1 and/or PMA were due to the presence of the rat LHß gene promoter sequences.

PMA Acts through the Egr-1-Binding Sites to Increase LHß Gene Expression
Taken together, the results shown in Fig. 2Go indicated the presence of a non-GSE, PMA-responsive regulatory region in the proximal LHß gene promoter. Coincident with these experiments, our laboratory had identified two functional response elements for the immediate early gene product, Egr-1, a high-affinity site at position -50 and a lower affinity site at -112 in the rat LHß gene promoter. In these studies, we further demonstrated that LHß gene promoter activity was synergistically increased in the presence of both SF-1 and Egr-1 (12), confirming a previous report by Lee et al. (23).

In a number of nonreproductive systems, activation of the PKC system has been shown to induce Egr-1 expression (29, 30). Hypothesizing that PMA-induced stimulation of LHß gene promoter activity may be due to effects on Egr-1, we cotransfected the wild-type rat LHß gene promoter-luciferase reporter construct with SF-1 and/or Egr-1 in the presence or absence of PMA (Fig. 3AGo). As observed previously (Fig. 1Go), PMA and SF-1 together produced a synergistic increase in LHß gene promoter activity. In contrast, PMA did not significantly increase the response to Egr-1 alone or SF-1 and Egr-1. The lack of further stimulation by PMA in the presence of both SF-1 and Egr-1 suggested a mechanism by which PMA induces Egr-1 expression. Egr-1, in turn, would interact with SF-1 to increase LHß promoter activity. In this model, PMA would not provide further stimulation in the presence of maximally effective levels of exogenously introduced Egr-1.



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Figure 3. Mutation of Both Egr-1-Binding Sites Eliminates PMA-Induced Increases in -207/+5 LHß Gene Promoter Activity

A, Stimulation of wild-type LHß gene promoter activity by PMA, SF-1, and/or Egr-1. GH3 cells were transiently transfected with region -207/+5 of the rat LHß gene promoter in a luciferase reporter construct, as well as with CMV-driven expression vectors for SF-1 and/or Egr-1, as indicated. Cells were cotransfected with an RSV-ß-galactosidase expression vector. Cells were treated with 100 ng/ml PMA or dimethylsulfoxide for 4–6 h starting approximately 40 h after transfection. Luciferase activity was normalized to ß-galactosidase activity, and promoter activity was calculated as fold- change over expression in the presence of the untreated control expression vector. *, P < 0.05 relative to vehicle-treated wells. n.s., P >= 0.05. B, Loss of PMA effect with mutation of Egr-1 sites. GH3 cells were transiently transfected with a luciferase reporter construct containing region -207/+5 of the rat LHß gene promoter present as either the wild-type sequence or with mutation of both Egr-1-binding sites, as indicated. *, P < 0.05 between the PMA response of the mutated vs. wild-type construct. C, Mutation of the Egr-1-binding sites eliminates the PMA interaction with SF-1. Cotransfections, PMA treatments, and data calculations for panels B and C were performed as in panel A. Results are shown as the mean ± SEM of at least nine samples in three independent experiments.

 
To confirm the importance of Egr-1 to the PMA response, we next transfected cells with a reporter construct containing mutations in both of the Egr-1 DNA-binding sites. We have previously demonstrated that in vitro translated Egr-1 is unable to bind to an oligonucleotide probe containing these mutations (12). The presence of these Egr-1 site mutations eliminated the ability of PMA to stimulate LHß gene promoter activity (Fig. 3BGo). Furthermore, PMA treatment no longer augmented SF-1-stimulated luciferase activity (Fig. 3CGo).

In the context of a longer region of the rat LHß gene 5'-flanking sequence (-797/+5), synergy between SF-1 and PMA was observed with a 68-fold increase in the presence of both factors compared with an approximately 2-fold increase in the presence of either factor alone (Fig. 4Go, upper panel). This synergy was lost with mutation of the Egr-1 binding sites; however, in contrast to the shorter construct, the PMA alone response was maintained (Fig. 4Go, lower panel).



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Figure 4. Loss of PMA-SF-1 Synergy with Mutation of the Egr-1-Binding Sites in the Context of Region -797/+5 of the Rat LHß Gene Promoter

GH3 cells were transiently transfected with a luciferase reporter construct containing region -797/+5 of the rat LHß gene promoter, present as either the wild-type or with mutation of both Egr-1 DNA-binding sites. Where indicated, cells were cotransfected with a CMV-driven SF-1 expression vector. All cells received an RSV-ß-galactosidase expression vector. Luciferase activity was normalized to ß-galactosidase activity, and promoter activity was calculated as fold-change over expression in the presence of the untreated control expression vector. Results are shown as the mean ± SEM of nine samples in three independent experiments.

 
PMA Induces Egr-1 DNA Binding in Nuclear Extracts
The studies shown in Figs. 3Go and 4Go suggested that PMA acts through Egr-1 to stimulate LHß gene expression. This observed relationship is physiologically relevant only if PMA is able to increase Egr-1 gene expression and/or functional activity in the gonadotrope. While known to be widely distributed, only recently has Egr-1 expression been identified in the gonadotrope subpopulation of the pituitary (22). Interestingly, in preliminary experiments, we had been unable to detect Egr-1 binding to the 3'-Egr site (position -50) using nuclear extracts from either our model cell line, GH3, or the gonadotrope-derived cell lines, {alpha}T3–1 and LßT2 (data not shown).

We, therefore, attempted to induce Egr-1 expression in GH3 cells and in the gonadotrope-derived cell line, {alpha}T3–1, by treatment with PMA and/or a GnRH analog. As shown in Fig. 5AGo, nuclear extracts from untreated GH3 cells formed a single dominant band in the presence of an oligonucleotide probe that contains both the 3'GSE and 3'Egr-1 DNA-regulatory sites (lane 1). Addition of specific antibodies demonstrated the presence of Sp1 (lane 3), but not Egr-1 (lane 2) in this extract. In contrast, nuclear extracts obtained from GH3 cells that had been treated with PMA for 1 h demonstrated induction of a protein-DNA complex that contains Egr-1 (lanes 4 and 5). The relative mobilities of in vitro translated Egr-1 and a purified Sp1 preparation were consistent with the presence of Sp1 and Egr-1 in the extracts (lanes 7 and 8). Similar results were obtained in the gonadotrope-derived {alpha}T3–1 cell line (Fig. 5BGo, lanes 1–4) and LßT2 cell line (data not shown). The induction of Egr-1 binding could also be detected using an oligonucleotide probe that spans the lower affinity 5'-Egr-1 site (data not shown). EMSA did not indicate PMA induction of any proteins other than Egr-1.



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Figure 5. PMA Induces Egr-1 DNA Binding in Nuclear Extracts from GH3 and {alpha}T3–1 Cell Lines

A, EMSA was performed using nuclear extracts from the rat somatolactotrope cell line, GH3 (lanes 1–6), in vitro translated Egr-1 (lane 7), or purified Sp1 (lane 8) protein. Nuclear extracts were derived from cells maintained in the absence or presence of PMA (100 ng/ml for 1 h). Region -67/-35 of the rat LHß gene promoter, which contains the 3'Egr-1-binding site, was [32P]-labeled and used as a probe. Where indicated, antiserum specific to Egr-1 (lanes 2 and 5) or Sp1 (lanes 3 and 6) was added 2 h before electrophoresis. B, DNA-protein complexes formed by nuclear extracts from the mouse gonadotrope-derived cell line, {alpha}T3–1, maintained in control medium (lanes 1 and 5) or in the presence of 100 ng/ml PMA (lanes 2–4) or 100 nM GnRH analog (lanes 6–8) for 1 h before harvest. Antiserum-specific to Egr-1 (lanes 3 and 7) or Sp1 (lanes 4 and 8) was added as indicated. NE, nuclear extract.

 
Noting that GnRH effects are partially transduced through activation of the PKC system, we tested the ability of a GnRH analog to increase Egr-1 gene expression. As shown in Fig. 5BGo (lanes 5–8), GnRH induces detectable Egr-1 binding in the gonadotrope-derived {alpha}T3–1 cell line.

Egr-1 mRNA Levels Are Increased By PMA Treatment
Northern analysis was performed to determine whether the PMA-induced increase in Egr-1-DNA complex formation was due to an increase in Egr-1 biosynthesis as opposed to increased DNA-binding affinity. As shown in Fig. 6Go, A and B, treatment of GH3 cells or {alpha}T3–1 cells with PMA markedly increased steady-state Egr-1 mRNA levels by 16-fold and 34-fold, respectively. Egr-1 mRNA levels in {alpha}T3–1 cells were also increased after exposure to a GnRH analog (50-fold, Fig. 6BGo).



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Figure 6. Northern Analysis Demonstrating Induction of Egr-1 mRNA Expression by PMA and GnRH Analog

A, Total RNA (10 µg) from GH3 cells was subjected to Northern analysis using a full-length mouse Egr-1 cDNA [32P]-labeled probe. The same blot was reprobed with cyclophilin to control for differences in RNA loading. Cells were cultured in control medium (lanes 1 and 2) or in the presence of 100 ng/ml PMA (lanes 3 and 4) for 1 h before harvest. B, Total RNA (7 µg) from gonadotrope-derived {alpha}T3–1 cells was probed with the mouse Egr-1 probe and with cyclophilin. Before harvesting, cells were cultured for 1 h in medium with the appropriate vehicle (lanes 1, 2, 5, and 6), 100 ng/ml PMA (lanes 3 and 4), or 100 nM GnRH analog (lanes 7 and 8). C, PMA increases Egr-1 gene promoter activity. GH3 cells were transiently transfected with a construct containing 1.2 kb of the 5'-flanking region of the mouse Egr-1 gene linked to the luciferase reporter plasmid, pXP2. An RSV-ß-galactosidase vector was cotransfected to control for transfection efficiency. Cells were treated with vehicle or 100 ng/ml PMA for 4 h before harvesting approximately 48 h after transfection. After normalization to ß-galactosidase activity, promoter activity was calculated as fold-change in luciferase activity in treated vs. untreated wells. Results are shown as the mean ± SEM of nine samples from three independent experiments.

 
Steady-state Egr-1 mRNA levels could potentially be altered by changes in either transcription rate and/or stability. To distinguish between these two alternatives, GH3 cells were transiently transfected with a luciferase reporter construct containing 1.2 kb of the Egr-1 gene promoter. PMA treatment increased luciferase activity in this construct by approximately 50-fold, suggesting that PMA up-regulates Egr-1 mRNA levels, at least in part, via increases in transcription of the Egr-1 gene (Fig. 6CGo).

Loss of GnRH Responsiveness with Mutation of the Egr-1 Sites in the LHß Gene Promoter
The PKC system is known to be a major signaling system for GnRH (1, 3). Furthermore, in the present studies, we have demonstrated that GnRH induces Egr-1 mRNA levels and DNA binding in the gonadotrope-like {alpha}T3–1 cell line (Figs. 5Go and 6Go). We, therefore, investigated the role of the Egr-1-binding sites in conferring GnRH responsiveness by transfecting {alpha}T3–1 cells with the -207/+5 LHß promoter construct containing mutations in both of the Egr-1-binding sites. As shown in Fig. 7Go, induction of LHß gene promoter activity by GnRH is lost in this mutated construct.



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Figure 7. Fig. 7. Loss of the GnRH and PMA Response with Mutation of Both Egr-1-Binding Sites in the {alpha}T3–1 Cell Line

{alpha}T3–1 cells were transiently transfected with luciferase reporter constructs containing region -207/+5 of the rat LHß gene promoter present as the wild-type sequence (upper panel) or with mutation of both Egr-1-binding sites (lower panel). Cells were treated for 4–6 h with PMA (100 ng/ml), GnRH analog (100 nM), or appropriate vehicle starting approximately 20 h after transfection. Results are shown as the mean ± SEM of nine samples in three independent experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Normal reproductive function is dependent upon the precise regulation of gonadotropin gene expression by numerous factors that activate an array of signaling systems, including the PKC system. Prior studies by our laboratory and by others have shown that SF-1 and Egr-1 act cooperatively to increase LHß gene promoter activity (12, 23). In the results reported here, activation of the PKC system with the phorbol ester PMA is shown to increase activity of the rat LHß gene promoter, both alone and in synergy with SF-1. We then demonstrate that PMA induces Egr-1 biosynthesis in gonadotrope-like cells and that intact Egr-1-binding sites are required for the synergistic effects of PMA and SF-1 on the LHß gene promoter. Taken together, these results strongly suggest that PMA activation of the PKC-signaling system may increase LHß gene expression via increases in Egr-1 expression.

Mutation of the Egr-1-binding sites eliminated the ability of PMA to stimulate the -207/+5 region of the LHß gene promoter, supporting the role of Egr-1 in mediating the PMA response (Fig. 3Go). Surprisingly, mutation of these sites did not alter the PMA effect in the longer construct, perhaps suggesting the presence of additional PMA-responsive sequences (Fig. 4Go). Nonetheless, this result does not undermine the importance of the Egr-1- binding sites for PMA-induced activation in the presence of SF-1 in both constructs.

Results utilizing an Egr-1 promoter construct indicate that PMA induces Egr-1 gene expression by increasing the transcription rate of this gene (Fig. 6CGo). Nevertheless, our data do not exclude the possibility that mRNA stability and/or posttranslational modifications further modulate Egr-1 functional activity.

Our data also suggest that neither SF-1 nor the GSE sites are required for the PMA response in the LHß gene promoter. This result differs from that observed in the type II 3ß-HSD (3ß-hydroxysteroid dehydrogenase) gene in which mutation of the GSE site blunts PMA-induced increases in promoter activity, even in the absence of SF-1. Interestingly, 5'-deletion of the GSE site restores the PMA response, further complicating interpretation of these results (21). It should be noted that our results do not exclude effects of the PKC system on SF-1. Based on amino acid sequence, SF-1 contains multiple potential PKC phosphorylation sites, and GnRH has been shown to increase SF-1 mRNA levels in vivo (31, 32). Nevertheless, while SF-1 expression may be modulated by PMA, any functional effect, if present, appears to be overwhelmed by the effects of Egr-1 and the Egr-1 DNA-binding sites.

For the functional studies reported here, we used a heterologous system in which a reporter construct containing LHß gene promoter sequences and expression vector(s) for SF-1 and/or Egr-1 were transiently transfected into a pituitary-derived somatolactotrope cell line, GH3. While use of a gonadotrope cell line would be preferable, there is precedent for the use of GH3 cells as a model system for study of the LHß gene. GH3 cells have been shown to support transcription initiation from the authentic start site of the LHß gene and to allow cAMP-mediated increases in LHß gene promoter activity (33). Furthermore, when transfected with the GnRH receptor, GH3 cells demonstrate GnRH-mediated regulation of gonadotropin subunit promoter activity that closely parallels the regulation observed in primary pituitary cells (34).

To confirm the results obtained in GH3 cells, we have repeated selected studies using the gonadotrope-derived cell line, {alpha}T3–1 (28). These cells resemble normal gonadotropes as they express endogenous SF-1 and the glycoprotein {alpha}-subunit; however, neither the LHß nor the FSHß subunit genes are expressed. Results in this cell line confirm that LHß gene promoter activity is increased by PMA and is dependent on the presence of intact Egr-1-binding sites (Fig. 7Go).

A number of laboratories have documented that treatment of pituitary cells with phorbol esters mimics GnRH effects on LHß biosynthesis and secretion (4). Our results in the {alpha}T3–1 cell line clearly demonstrate that treatment with a GnRH analog increases Egr-1 DNA binding, Egr-1 mRNA levels, and Egr-1-dependent LHß promoter activity, paralleling the observed effects of PMA in this cell line and in GH3 cells. Thus, our results are consistent with a mechanism in which GnRH stimulates LHß gene transcription via PKC induction of Egr-1 gene expression.

Of note, GnRH-induced activity of a longer region of the LHß gene 5'-flanking sequence (-797/+5) exceeds the response of the shorter segment (35). We have identified a GnRH-responsive region from -490 to -352 in the rat LHß gene promoter and have implicated the transcription factor Sp1 in the generation of this response (36). Interestingly, the nucleotide regions that include the Egr-1-binding sites have also been found to bind Sp1 on EMSA (12). However, unlike Egr-1, Sp1 binding is not altered by PMA or GnRH treatment (Fig. 5Go).

The signaling mechanism(s) by which GnRH exerts its effects is currently an area of active investigation. Earlier studies in primary pituitary cells indicated a role for the PKC system in modulating LHß mRNA levels (1, 5). Recent work in our laboratory confirmed the importance of this signaling system in a GH3-derived cell line (37). In contrast, using a variety of cell types, Weck et al. (38) concluded that induction of the LHß gene is dependent on calcium influx, rather than the PKC pathway. Further studies will be required to resolve the apparent discrepancy of these results. The data presented here clearly imply a critical role for Egr-1 in transduction of the PMA response and support a role for the PKC system in the mediation of GnRH effects. Nevertheless, these data do not exclude a contribution by calcium-signaling pathways. Full GnRH responsiveness is likely to involve complex interplay among a myriad of sites, including those binding SF-1, Sp1, and Egr-1. Some of these may respond to PKC, whereas others may respond to non-PKC-signaling systems.

In conclusion, our results demonstrate that PMA acts synergistically with SF-1 to increase LHß gene promoter activity and that this effect is mediated via the immediate early gene product, Egr-1. We propose that the magnitude of the PMA-SF-1 cooperative effect and the rapidity of the PMA-induced increase in Egr-1 gene expression may provide one mechanism by which dynamic regulation of LHß gene expression is achieved.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Oligonucleotides
The oligonucleotides used for mutagenesis, EMSAs, and PCRs are shown in Table 1Go. The nucleotide sequence of the rat LHß gene promoter is based on newly obtained sequencing data (available at GenBank accession number AF020505) that differs slightly from that of Jameson et al. (39). The -797LH-S and -207LH-S primers introduced BamHI restriction sites, while the +5LH-AS primer introduced a HindIII site (restriction sites not shown).


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Table 1. Oligonucleotides Used for Generation of Rat LHß Promoter Constructs

 
In Vitro Translated Proteins and Antisera Used in EMSA
In vitro translated Egr-1 was generated from a plasmid containing 3.2 kb of the mouse Egr-1 cDNA (provided by D. Nathans, Johns Hopkins University, Baltimore, MD) using the TNT Coupled Reticulocyte Lysate System (Promega, Madison, WI). The resultant product was determined to be of appropriate size by comparison with [35S]methionine-labeled protein markers by SDS-PAGE. Human Sp1 was purchased from Promega (Madison, WI). Egr-1 and Sp1 polyclonal antisera were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

EMSAs
A double stranded oligonucleotide spanning region -67/-35 of the rat LHß gene promoter was produced by annealing the sense oligonucleotide indicated in Table 1Go with the corresponding antisense oligonucleotide (not shown). Probes were created by T4 polynucleotide kinase end-labeling with [{gamma}-32P]ATP followed by purification over a NICK column (Pharmacia Biotech, Uppsala, Sweden).

Protein samples were incubated with 50,000 cpm of oligonucleotide probe in DNA-binding buffer [20 mM HEPES (pH 7.9), 60 mM KCl, 5 mM MgCl2, 10 mM phenylmethylsulfonylfluoride, 10 mM dithiothreitol, 1 mg/ml BSA, and 5% (vol/vol) glycerol] for 30 min on ice. Where indicated, antiserum (1 µl) was added 30 min after the addition of probe and the incubation continued for 2 h. Protein-DNA complexes were resolved on a 5% nondenaturing polyacrylamide gel in 0.5x Tris-borate-EDTA buffer and subjected to autoradiography and/or quantification using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Plasmids Used in Transfection Studies
The wild-type reporter constructs used for these studies contain either 797 or 207 bp of the 5'-flanking sequence of the rat LHß gene and the first 5 bp of the 5'-untranslated region. These constructs were created by subcloning the PCR product generated by primers +5LH-AS and either -797LH-S or -207LH-S into the pXP2 vector using BamHI/HindIII restriction sites that were introduced by the primers (40).

Mutations in the LHß gene promoter reporter constructs were introduced using the Transformer Site-Directed Mutagenesis Kit (CLONTECH Laboratories, Inc., Palo Alto, CA). Generation of multiple mutations was performed sequentially and, therefore, required the use of two selection primers, one that converted a unique HindIII restriction site to a unique MluI site (pXP2) and the other that reversed this mutation (pXP2-rev). The 5'-GSE mutagenic primer eliminated a TthIII1 restriction site in addition to introducing the desired mutation, as described previously (13). The mutagenic primers for the 3'-GSE, 5'-Egr, and 3'-Egr sites introduce EcoRI, ScaI, and PstI restriction sites, respectively. All reporter constructs were confirmed by dideoxysequencing.

The SF-1 expression vector contained 2.1 kb of the mouse SF-1 cDNA driven by cytomegalovirus promoter sequences in the vector, pCMV5 (provided by K. L. Parker, Southwestern University School of Medicine, Dallas, TX)(41). The Egr-1 expression vector was created by cloning 3.2 kb of the mouse Egr-1 cDNA into pCMV5 at BamHI and HindIII restriction sites (Egr-1 cDNA provided by D. Nathans, Johns Hopkins University) (25). The pCMV5-ßgal construct was obtained by transferring the EcoRI/SalI ß-galactosidase cDNA fragment from pNASSß (CLONTECH, Palo Alto, CA) into the empty pCMV5 vector. The Egr-luc reporter construct contained 1.2 kb of the mouse Egr-1 gene promoter sequence cloned into the SalI site of pXP2 (V. Sukhatme, Harvard Medical School, Boston, MA) (42).

Transfection Experiments
Rat somatolactotrope GH3 cells or mouse gonadotrope-derived {alpha}T3–1 cells were cultured to 50–70% confluence in DMEM supplemented with 10% FCS. For the GH3 cell line, approximately 5 x 106 cells were suspended in 0.4 ml of Dulbecco’s PBS plus 5 mM glucose with the DNA to be transfected. The cells received a single electrical pulse of 240 V at a total capacitance of 1000 µFarads using an Invitrogen Electroporator II apparatus (Invitrogen, San Diego, CA). GH3 cells received 1.5 µg/well of the reporter constructs. The {alpha}T3–1 cell line was transfected using the calcium phosphate precipitation method and 2 µg/well of reporter construct. Where appropriate, cells also received SF-1 and/or Egr-1 expression vectors or an equivalent amount of the empty pCMV5 expression vector in amounts of 1 µg/well (GH3) or 0.1 µg/well ({alpha}T3–1). For both cell lines, cotransfection with an rous sarcoma virus (RSV)-ß-galactosidase plasmid (1 µg/well) allowed correction for differences in transfection efficiency between wells in all experiments except Fig. 1BGo, in which ß-galactosidase activity was used as the primary reporter. Cells were treated with vehicle, PMA (100 ng/ml) (LC Laboratories, San Diego, CA), or GnRH agonist des-Gly10, [D-Ala6]-GnRH ethylamide (100 nM) (Sigma, St. Louis, MO) for 4–6 h starting approximately 40 h (GH3) or 20 h ({alpha}T3–1) after transfection. Cells were then harvested and the cell extracts were analyzed for luciferase and/or ß-galactosidase activities (43, 44). Luciferase activity was normalized to the level of ß-galactosidase activity, where appropriate. Results were then calculated as fold-change relative to expression in the presence of the vehicle-treated control wells. Data are shown as the mean ± SEM.

Northern Blot Analysis
Total RNA was prepared from GH3 or {alpha}T3–1 cells at approximately 50% confluence using the RNeasy MiniKit (Qiagen Inc., Chatsworth, CA). Before extraction, cells were treated for 1 h with vehicle, PMA (100 ng/ml), or the GnRH agonist (100 nM). Seven micrograms ({alpha}T3–1 cells) or 10 µg (GH3 cells) of total RNA were separated by electrophoresis in denaturing agarose gels (2.2 M formaldehyde and 1.5% agarose), transferred to nylon membranes by diffusion (Nytran NY 12 N, Schleicher & Shuell, Dassel, Germany), and cross-linked by UV irradiation. Hybridizations were performed under high stringency conditions [42 C, 16 h: in 50% formamide, 0.5% SDS, 100 mg salmon DNA, 0.9 M NaCl, 12 mM EDTA, and 0.09 M sodium phosphate (pH 7.4)] with 50 ng cDNA fragments randomly labeled with [32P]dCTP. The following cDNA fragments were used as probes: a 3.1-kb insert of the mouse Egr-1 cDNA released with EcoRI from pUC13 (provided by V. Sukhatme, Harvard Medical School) and, as a standard, a 0.7-kb fragment of the rat cDNA encoding cyclophilin (42, 45). The membranes were washed at a final stringency of 0.2 x SSPE-0.3% SDS at 42 C [0.2 x SSPE = 30 mM NaCl, 2 mM sodium phosphate, and 0.2 mM EDTA (pH 7.4)] and then subjected to autoradiography and quantification using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Statistical Analysis
Data were combined across transfection experiments to determine the mean ± SEM of the corrected luciferase activity. Two-way analysis of variance followed by comparisons with Student’s t test were used to assess whether promoter activity was statistically different between the indicated groups. Statistical significance was set at the P < 0.05 level.


    FOOTNOTES
 
Address requests for reprints to: Lisa M. Halvorson, M.D., Division of Reproductive Endocrinology, Box 36, New England Medical Center, 750 Washington Street, Boston, Massachusetts 02111. E-mail: lisa.halvorson{at}es.nemc.org

This work was supported in part by NIH Grant R03-HD-34692 (L.M.H.), R29-HD-33001 (U.B.K.), and R01-HD-19938 (W.W.C.), as well as an American Society for Reproductive Medicine-Ortho Pharmaceutical Research Grant in Reproduction (L.M.H.) and an American Society for Reproductive Medicine-Serono Research Grant (U.B.K.).

Received for publication February 23, 1998. Revision received June 16, 1998. Accepted for publication September 22, 1998.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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