A Role for the Mitogen-Activated Protein Kinase in Mediating the Ability of Thyrotropin-Releasing Hormone to Stimulate the Prolactin Promoter

Ying-Hong Wang and Richard A. Maurer

Department of Cell and Developmental Biology Oregon Health Sciences University Portland, Oregon 97201


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The hypothalamic hormone, TRH, stimulates PRL secretion and gene transcription. We have examined the possibility that the mitogen-activated protein kinase (MAPK) may play a role in mediating TRH effects on the PRL gene. TRH was found to stimulate sustained activation of MAPK in PRL-producing, GH3 cells, consistent with a possible role in transcriptional regulation. A kinase-defective, interfering MAPK kinase (MAPKK) mutant reduced TRH induction of the PRL promoter. Treatment with the MAPKK inhibitor, PD98059, blocked TRH-induced activation of MAPK and also reduced TRH induction of a PRL-luciferase reporter gene, confirming that MAPK activation is necessary for TRH effects on PRL gene expression. Previous studies have demonstrated that the PRL promoter contains binding sites for members of the Ets family of transcription factors, which are important for mediating MAPK responsiveness of the PRL promoter. Mutation of specific Ets sites within the PRL promoter reduced responsiveness to both TRH and MAPK. The finding that DNA elements required for MAPK responsiveness of the PRL gene colocalize with DNA elements required for TRH responsiveness further supports a role for MAPK in mediating TRH effects on the PRL gene. We also explored the signaling mechanisms that link the TRH receptor to MAPK induction. Occupancy of the TRH receptor results in activation of protein kinase C (PKC) as well as increases in the concentration of Ca2+ due to release from intracellular stores and entry of Ca2+ through Ca2+ channels. A PKC inhibitor, GF109203X, and an L-type Ca2+ channel blocker, nimodipine, both partially reduced TRH-induced MAPK activation and PRL promoter activity. The effects of the two inhibitors were additive. These studies are consistent with a signaling pathway involving PKC- and Ca2+-dependent activation of MAPK, which leads to phosphorylation of an Ets transcription factor and activation of the PRL promoter.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The ability of TRH to stimulate PRL synthesis and secretion involves the interaction of the hormone with a G protein-coupled receptor at the plasma membrane (1, 2). The TRH receptor couples to Gq to stimulate the activity of phospholipase Cß (3), leading to increased intracellular levels of diacylglycerol and inositol 1,4,5-trisphosphate (4). Diacylglycerol activates protein kinase C (PKC), which is accompanied by redistribution of PKC from a soluble to a particulate subcellular fraction (5). Inositol 1,4,5-trisphospate stimulates the release of Ca2+ from intracellular stores, resulting in a first phase of Ca2+ elevation (6). A second phase of Ca2+ elevation is due to membrane depolarization, which leads to Ca2+ influx through L-type voltage-sensitive Ca2+ channels (7). Increases in intracellular Ca2+ lead to activation of a Ca2+/calmodulin-dependent protein kinase (8). It has also been shown that TRH can activate the mitogen activated protein kinase (MAPK)-signaling pathway, also known as the extracellular regulated kinase (ERK) in GH3 pituitary tumor cells (9). Thus multiple kinase pathways including PKC, Ca2+/calmodulin-dependent kinase, and MAPK are activated by TRH.

The specific signaling pathways that permit TRH to rapidly stimulate PRL gene transcription (10, 11) have not been clearly defined. There is evidence that activation of PKC (12), Ca2+/calmodulin-dependent protein kinases (13, 14), or MAPK (15) can stimulate the PRL promoter. However, the role that these pathways play in mediating TRH effects has not been resolved. There is evidence that PKC may not be required for TRH-induced activation of the PRL promoter (16, 17). Although inhibition of Ca2+/calmodulin-dependent protein kinases was found to reduce the ability of TRH to activate the PRL promoter, the DNA elements that are required for responsiveness to specific Ca2+/calmodulin-dependent protein kinases do not colocalize with TRH-responsive DNA elements (14). Thus, there are questions concerning the role that PKC and Ca2+/calmodulin-dependent protein kinases play in mediating TRH effects on the PRL promoter.

In the present study we have examined a possible role for the MAPK pathway in mediating TRH effects on the PRL promoter. We have used several approaches to determine whether MAPK activity is required for TRH effects on the PRL promoter. We have also assessed the role that PKC activation and Ca2+ signaling play in activating the MAPK pathway in GH3 cells.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Activation of MAPK Can Stimulate PRL Promoter Activity
Previous studies have shown that constitutively active forms of Ras or Raf can activate the PRL promoter, suggesting that activation of the MAPK signaling pathway is sufficient to stimulate PRL transcription (15, 18). While perhaps unlikely, it is possible that Ras or Raf may lead to signaling events other than activation of MAPK (19, 20). To further assess the ability of the MAPK pathway to alter PRL promoter activity, we tested the effects of a constitutively active form of MAPK kinase (MAPKK), also known as MAPK/ERK kinase (MEK). As the MAP kinases, ERK1 and ERK2, are the only known substrates of MEK (21), this approach allows a further test of the ability of MAPK to regulate the PRL promoter. The expression vector for constitutively active MEK produced substantial activation of the PRL reporter gene (Fig. 1AGo) and had little or no effect on the thymidine kinase minimal promoter (Fig. 1BGo). Thus, MAPK activation selectively stimulates PRL promoter activity.



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Figure 1. Activation of the MAPK Pathway Can Stimulate PRL Promoter Activity

Reporter genes containing 255 bp of the proximal region and promoter of the rat PRL gene linked to firefly luciferase (A) or the herpes simplex virus thymidine kinase promoter linked to luciferase (B) were transfected with a control empty vector (control) or an expression vector for constitutively active MEK (Activated MEK). The cells also received a control reporter gene that expresses Renilla luciferase. The cells were collected 48 h after transfection, and firefly and Renilla luciferase activity was determined. The values obtained for Renilla luciferase activity were used to correct for transfection efficiency. Firefly luciferase values are the average ± SE of three separate transfections.

 
TRH Induces Prolonged MAPK Activation
In some systems, sustained rather than transient activation of a signaling pathway is required to induce long-term responses. For instance, in PC12 cells, sustained MAPK activation is associated with differentiation (22). Although previous studies have shown that TRH can rapidly activate MAPK (9), these studies examined very early time points and did not determine whether TRH has more prolonged effects on the kinase activities. To determine whether TRH-induced MAPK activation is a reasonable candidate for mediating long-term transcriptional effects of TRH, the time course of MAPK activation by TRH in GH3 cells was determined (Fig. 2Go). To assess MAPK activation, an immuno-complex assay was used. For this assay, cell lysates were immunoprecipitated with an antibody to ERK2, and then the immunoprecipitated proteins were incubated with [32P]ATP and GST-Elk1 as the kinase substrate. The immuno-complex assay demonstrated that TRH-induced ERK2 activation was maximal at the earliest time point examined, 2.5 min, and declined at later time points (Fig. 2AGo). However, it is important to note that at all TRH time points, including the 75-min treatment, ERK2 activity of TRH-treated samples was greater than that of the untreated control. This activation at later time points has been observed in several different experiments (data not shown). The use of anti-phospho-ERK antibodies to detect the activated form of ERKs also yielded results consistent with sustained activation of MAPK. MAPK is activated by phosphorylation of both threonine and tyrosine residues (23). Antibodies directed specifically against the phosphorylated forms of MAPK detect both ERK1 and ERK2. Increased phosphorylation of ERK1 and ERK2 was detectable for at least 1 h (Fig. 2BGo). Analysis of total, immunoreactive ERK1 and ERK2 indicated that TRH-induced increases in ERK phosphorylation were not due to increases in the amount of these kinases (Fig. 2CGo). These results provide evidence that TRH induces an initial burst of MAPK activation followed by a lower, but clearly detectable, prolonged phase of activation.



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Figure 2. Time Course of TRH-Stimulated MAPK Activation

MAPK activation was determined by an immunocomplex assay (A) or by immunoblotting with an antibody to phospho-ERK (B). GH3 cells were treated with 100 nM TRH for the indicated times, and cell lysates were prepared. For the immunocomplex kinase assay, the cell lysates were immunoprecipitated with agarose bead-conjugated anti-ERK2 antibody. The ERK2 immunoprecipitate was then incubated with [32P]ATP, and a GST-Elk1 fusion protein was used as a substrate. The phosphorylated proteins were then resolved by denaturing gel electrophoresis. For the immunoblot analysis of MAPK activation, cell lysates were resolved by denaturing gel electrophoresis, transferred to a membrane, and phospho-ERK visualized by immunostaining. To assess total levels of MAPK, the blot was reprobed with an antibody that detects both the activated and nonactivated forms of ERK2 and more weakly detects both forms of ERK1.

 
Activation of MAPK has been shown to lead to phosphorylation and activation of Elk1, a member of the Ets family of transcription factors, and a component of the serum response factors (24). To determine whether TRH-induced MAPK activation is sufficient in magnitude and duration to alter a transcriptional event, we examined the activation of a GAL4-Elk1 fusion (Fig. 3Go). TRH stimulated expression of the GAL4-luciferase reporter gene more than 20-fold. Substitution of serine 383 of Elk1 with alanine, which disrupts a critical MAPK phosphorylation site (24, 25), strongly diminished TRH-induced activation of Elk1, suggesting that the transcriptional response likely involves direct phosphorylation of Elk1 by MAPK. These results suggest that TRH treatment of GH3 cells activates MAPK in a manner that is sufficient to modulate a transcriptional response.



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Figure 3. TRH Treatment of GH3 Cells Can Increase the Activity of a MAPK-Responsive Transcription Factor

GH3 cells were transfected with plasmids encoding either GAL4-Elk1 or mutant GAL4-Elk1 in which serine-383, which is part of an important MAPK phosphorylation site, was mutated to alanine (GAL4-ElkS383A). The cells also received a firefly luciferase reporter gene containing five GAL4-binding sites upstream of a minimal promoter as well as a control reporter gene that expresses Renilla luciferase. At 48 h after transfection, the cells were treated with 100 nM TRH or 10 nM EGF and then collected 6 h later for analysis of luciferase activity. Firefly luciferase values are the average ± SE of three separate transfections that have been corrected for transfection efficiency.

 
MAPK Activation Is Necessary for TRH-Induced Increases in PRL Promoter Activity
A kinase-defective, interfering mutant form of MEK was used to investigate the functional role of MAPK. MEK activates MAPK by phosphorylating MAPK at both threonine and tyrosine residues (23). The mutant MEK was made by substituting an alanine for a lysine residue within the ATP-binding site of the enzyme. Although the mutant MEK can be phosphorylated by Raf, it cannot phosphorylate and activate MAPK (26, 27) and therefore interferes with signal transduction. To determine an effective concentration of mutant MEK expression vector, increasing concentrations of the expression vector were tested for their ability to reduce TRH-induced GAL4-Elk activation (Fig. 4AGo). The MEK mutant reduced both basal and TRH-induced activation of Gal4-Elk1 in a dose-dependent manner (Fig. 4AGo), suggesting that the MEK mutant can interfere with endogenous MEK signaling. Based on the titration study, the ability of 2 µg of the mutant MEK expression vector to inhibit TRH effects on PRL-luciferase (Fig. 4BGo), GAL4-Elk (Fig. 4CGo), or the thymidine kinase-luciferase reporter gene (Fig. 4DGo) was compared. This concentration of the mutant MEK vector had similar effects to reduce TRH-induced activation of a PRL-luciferase reporter gene and GAL4-Elk1. In contrast, the mutant MEK had little or no effect on the thymidine kinase promoter (Fig. 4DGo), suggesting that the inhibitory effects on the PRL promoter are not due to nonspecific inhibition of transcription.



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Figure 4. Inhibition of MAPK Reduces TRH-Induced PRL Promoter Activity

GH3 cells were transfected with an expression vector for an interfering, kinase-deficient mutant MEK (MEK mutant) to inhibit activation of MAPK. To determine an effective concentration of mutant MEK expression vector, increasing concentrations of the expression vector were tested for their ability to reduce TRH-induced GAL4-Elk activation (A). Based on the titration study, the ability of 2 µg of the mutant MEK expression vector to inhibit TRH effects on PRL-luciferase (B), GAL4-Elk (C), or thymidine kinase-luciferase reporter gene (D) was examined. Cells were treated with TRH at 48 h after transfection and then collected for analysis of luciferase activity 6 h later. All values are means ± SE for three separate transfections.

 
PD98059 is a selective inhibitor of MEK1 and MEK2 (28, 29). This inhibitor blocks MAPK activation in several cell types and blocks processes such as neurite outgrowth in nerve growth factor-treated PC12 cells (30). We first tested whether this inhibitor was able to block TRH-induced MAPK activation in GH3 cells (Fig. 5Go). Treatment of GH3 cells with 100 µM PD98059 almost completely blocked TRH-induced ERK2 activation. The effects of PD98059 on TRH-induced activation of the PRL promoter and GAL4-Elk were then examined (Fig. 6Go). PD98059 substantially reduced both PRL promoter and Gal4 promoter activation by TRH. The results of studies using an expression vector for an interfering, kinase-defective MEK as well as the PD98059 studies suggest that MAPKs are required for TRH-induced activation of the PRL promoter.



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Figure 5. The MEK Inhibitor, PD98059, Blocks TRH-Induced Activation of MAPK

GH3 cells were untreated or pretreated with 100 µM PD98059 for 1 h and then treated with 100 nM TRH or no additional treatment for 6 h. The cells were lysed, and ERK2 activity was assessed by immunocomplex kinase assay using GST-Elk1 as a substrate.

 


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Figure 6. The MEK Inhibitor, PD98059, Reduces TRH-Induced Activation of the PRL Promoter

GH3 cells were transfected with either the 255PRL-luciferase construct (A) or an expression vector for GAL4-Elk1 plus a reporter gene containing five GAL4- binding sites upstream of a minimal promoter linked to luciferase (B). At 48 h after transfection, the cells were untreated (control) or treated with 100 µM PD98059 as indicated, and after incubation for an additional hour, half of the cultures were treated with 100 nM TRH. Cells were harvested 6 h after TRH treatment and luciferase activity was determined. All values are means ± SE for three separate transfections that have been corrected for transfection efficiency.

 
DNA Elements That Contribute to TRH Responsiveness of the PRL Promoter Colocalize with MAPK-Responsive Elements
Several binding sites for members of the Ets family of transcription factors have previously been identified as important for mediating responses to activation of the MAPK pathway (15, 18). To determine whether Ets binding sites are also important for TRH responsiveness, we prepared reporter genes containing block mutations that disrupt specific Ets-binding sites within the PRL promoter (Fig. 7AGo). The wild-type and mutant PRL reporter constructs were transfected into GH3 cells and compared for responses to TRH (Fig. 7BGo) or an expression vector for activated MEK (Fig. 7CGo). In general, the specific Ets mutations had a similar pattern of effects on responsiveness to TRH and activated MEK. Consistent with previous reports (31), disruption of an Ets site located at -211 to -208 had the greatest effect on reducing responsiveness to MEK and also had the greatest effect on reducing TRH-induced reporter gene activity. The Ets sites that were tested are those that have been reported in previous work to have a possible involvement in regulating the PRL gene. There are other possible Ets-binding sites within the proximal region of the PRL gene. At present, it would be premature to make conclusions about the role of non-Ets factors in mediating responses to TRH and MAPK. Nonetheless, the similar pattern of effects of Ets mutations provides evidence that DNA elements necessary for TRH responsiveness colocalize with DNA elements required for MAPK responsiveness.



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Figure 7. Colocalization of TRH- and MAPK-Responsive DNA Elements in the PRL Promoter

Specific Ets sites (solid boxes) that are adjacent to binding sites for Pit-1 (gray boxes) in PRL promoter were mutated as indicated (A). Reporter genes containing Ets site mutations in the context of the 255PRL-luciferase reporter gene (100 ng) were transfected into GH3 cells and tested for responsiveness to 100 nM TRH treatment for 6 h (B) or transfection of 150 ng of an expression vector encoding a constitutively active form of MEK (C). All values are means ± SE for three separate transfections that have been corrected for variations in transfection efficiency.

 
TRH-Induced Activation of MAPK and the PRL Promoter Is Mediated by PKC and Ca2+ Influx
Previous studies have used chronic treatment with phorbol esters to provide evidence that PKC plays a role in TRH-induced MAPK induction (9). To further test the role of PKC in mediating TRH effects on MAPK activity, we selected the PKC inhibitor, GF109203X (32), and determined ERK2 activity using an immunocomplex assay (Fig. 8Go). As expected, GF109203X substantially blocked phorbol myristate acetate (PMA)-induced ERK2 activation, indicating that the inhibitor successfully blocked PKC-dependent MAPK activation. The inhibitor partially reduced TRH-stimulated ERK2 activation and had little or no effect on the ability of epidermal growth factor (EGF) to activate ERK2. While the ability of GF109230X to reduce TRH-induced MAPK activation is somewhat modest, it has been consistently observed in several experiments (for instance, see also Fig. 9Go). These results suggest that TRH appears to induce MAPK activation through both PKC-dependent and PKC-independent pathways.



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Figure 8. The PKC Inhibitor, GF109203X, Reduces TRH-Induced Activation of MAPK

GH3 cells were untreated or treated with 5 µM GF109203X for 20 min, and subsequently treated with 100 nM TRH for 2.5 min, 10 nM EGF for 5 min, or 100 nM PMA for 10 min. ERK2 activity was assessed by immunocomplex kinase assay.

 


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Figure 9. Analysis of the Time Course of TRH-Induced MAPK Activation after Treatment with the Ca2+ Channel Blocker, Nimodipine, or the PKC Inhibitor, GF109203X

GH3 cells were pretreated with 500 nM nimodipine or 1 µM GF109203X for 20 min and then treated with 100 nM TRH for the indicated time, and cell lysates were prepared. ERK2 activity was determined by an immunocomplex kinase assay.

 
TRH also leads to the entry of extracellular Ca2+ through L-type voltage-sensitive Ca2+ channels resulting in a sustained, plateau-like elevation of intracellular Ca2+ (33). A role for Ca2+ influx in TRH-mediated activation of the PRL promoter has previously been described (34, 35, 36). As Ca2+ influx has been shown to lead to MAPK activation in some cells (37, 38, 39, 40, 41), it seemed possible that Ca2+ influx might contribute to TRH-induced MAPK induction. To explore this possibility, GH3 cells were treated with nimodipine, a blocker of L-type Ca2+ channels (7). Nimodipine slightly reduced ERK2 activation at all time points examined (Fig. 9Go). At the later time points, from 10 min to 60 min, ERK2 activation was approximately half that of control cells. Similar results have been observed in several different experiments (data not shown). The PKC inhibitor, GF109203X, had a different time course of effects on ERK2 phosphorylation. GF109203X substantially reduced TRH-induced ERK2 activation at the earliest time point, 2.5 min, and had relatively little effect at the later time points. The combination of nimodipine plus GF109203X appeared to reduce ERK2 activation more than either treatment alone. Similar results were obtained when ERK1 and ERK2 activation was assessed by immunoblotting with a phospho-ERK antibody (data not shown). These findings suggest that both PKC activation and Ca2+ influx may play a role in TRH-induced MAPK activation. PKC appears to play a major role at early times after TRH stimulation while Ca2+ influx plays a more important role at later time points.

Inhibitor studies were then used to test the role of Ca2+ influx and PKC activation in mediating TRH-induced PRL promoter activation. GH3 cells were pretreated with nimodipine, GF109203X, or a combination of the inhibitors, and the ability of TRH to stimulate the expression of a PRL-luciferase reporter gene was examined (Fig. 10Go). Both GF109203X and nimodipine treatment rather strongly blunted the ability of TRH to stimulate PRL promoter activity, and the combination of the two inhibitors almost completely blocked TRH effects. These experiments provide evidence that TRH-induced PKC activation and Ca2+ influx contribute to MAPK induction and activation of the PRL promoter. To further test the role of Ca2+ influx-induced MAPK activation in modulating PRL gene expression, the effects of the MEK inhibitor, PD98059, on Ca2+ influx-stimulated PRL promoter activity was examined (Fig. 11Go). Control or PD98059-treated GH3 cells were stimulated with the Ca2+ channel agonist, Bay K8644, which has been demonstrated to stimulate PRL promoter activity (35, 42). The ability of PD98059 to substantially reduce Ca2+ influx-stimulated PRL promoter activity provides evidence that MAPK plays a role in mediating Ca2+ effects on transcription in this system.



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Figure 10. TRH-Induced Activation of the PRL Promoter Is Inhibited by Treatment with GF109203X and Nimodipine

GH3 cells were transfected with the 255PRL-luciferase reporter gene. At 48 h after transfection, the cells were treated with 500 nM nimodipine or 1 µM GF109203X for 20 min, and then half of the cultures were treated with 100 nM TRH for 6 h. All values are means ± SE for three separate transfections that have been corrected for transfection efficiency.

 


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Figure 11. Ca2+ Influx-Induced Activation of the PRL Promoter Is Inhibited by Treatment with the MEK Inhibitor, PD98059

GH3 cells were transfected with the 255PRL-luciferase reporter gene. At 48 h after transfection, the cells were treated with 100 µM PD98059 for 1 h, and then half of the cultures were treated with 1 µM Bay K8644 for 6 h. All values are means ± SE for three separate transfections that have been corrected for transfection efficiency.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
These studies provide evidence that TRH-induced MAPK activation likely plays a role in regulating transcription of the PRL gene. Previous studies have shown that activated Ras can stimulate PRL promoter activity (15, 18), implying that activation of the MAPK pathway is sufficient to stimulate PRL gene expression. Our studies demonstrate that an expression vector for constitutively active MEK also increases PRL promoter activity. This result offers additional evidence that it is indeed the MAPK cascade which is sufficient to activate the PRL promoter. Our work also extends previous studies demonstrating that TRH can activate MAPK. Our findings demonstrate that TRH can induce prolonged activation of MAPK. TRH-induced MAPK activation was observed for at least 1 h, consistent with the ability of TRH to stimulate prolonged activation of PRL gene transcription, which persists for hours (10). The finding that TRH can lead to activation of the GAL4-Elk1 transcription factor offers additional evidence that TRH can alter a transcriptional event and also implies that TRH probably induces nuclear localization of MAPK. Importantly, transfection of a kinase-defective, interfering MEK mutant or addition of the MEK inhibitor, PD98059, was found to suppress TRH-induced PRL promoter activity. Our studies thus provide evidence that TRH activates the MAPK pathway and that MAPK activation is sufficient and necessary for activation of the PRL promoter.

The finding that DNA sequences of the PRL promoter that are required for TRH responsiveness colocalize with DNA sequences required for MAPK-responsiveness reinforces the view that MAPK plays a role in mediating transcriptional responses to TRH. Previous studies have provided evidence that binding sites for members of the Ets family of transcription factors are important for MAPK- and multihormonal responsiveness of the PRL promoter (18, 31, 43, 44). Therefore, we tested TRH- and MAPK responsiveness of reporter genes with clustered point mutations that disrupted several consensus Ets-binding sites. Disruption of an Ets site at position -211 to -208 of the PRL promoter had a substantial effect to reduce both TRH- and MAPK-responsiveness. This Ets site was previously found to be important for ras-responsiveness (43). Interestingly, although we previously used multimers of synthetic binding sites to demonstrate that an Ets site at position -162 to -159 is capable of responding to MAPK activation, this element was not required for TRH- or MAPK responsiveness within the context of the normal PRL promoter. It is not clear why the upstream Ets binding site at -211 to -208 plays the predominant role in mediating MAPK respon- siveness.

It is probably important that TRH effects on MAPK persist for at least 1 h. The kinetics of MAPK activation can influence nuclear translocation of MAPK and therefore alter access to nuclear substrates (22). Therefore, the kinetics of MAPK activation can have important effects on physiological responses. For instance, in PC12 cells, NGF causes sustained MAPK activation and nuclear translocation of MAPK and induces differentiation (45). In contrast, EGF results in a transient cytoplasmic activation of MAPK that fails to induce differentiation of PC12 cells (22).

Our studies provide evidence that a PKC-dependent pathway contributes to TRH effects on both MAPK activation and stimulation of the PRL promoter. The PKC inhibitor, GF109203X, partially inhibited TRH-induced activation of the MAPK and PRL promoter activity, providing evidence that both PKC-dependent and PKC-independent pathways mediate TRH effects. Our finding that PKC appears to play a role in mediating TRH effects on PRL gene expression differs from earlier studies, which concluded that PKC was not involved (35). The previous studies used chronic phorbol ester treatment to deplete cells of PKC activity while we used GF109203X. Phorbol ester treatment is a very potent activator of the PRL promoter, which stimulates transcription for more than 24 h, making it difficult to interpret the effects of PKC depletion (17). The use of GF109203X avoids the complication of chronic phorbol ester treatment, which both activates and depletes PKC activity. GF109203X inhibits the activity of most PKC isoforms (32, 46). It is possible that differences in the effects of GF109203X, as compared with chronic PMA treatment, reflect differential inhibition of specific subsets of PKC isozymes.

The present studies also provide evidence that Ca2+ influx plays a role in mediating TRH effects on both MAPK activation and PRL gene expression. TRH treatment stimulates a rapid transient increase in cytosolic Ca2+ as well as a sustained, plateau-like elevation in Ca2+ levels, which is dependent on influx through Ca2+ channels (4). Although previous studies have shown that the L-type Ca2+ channel blockers can inhibit the ability of TRH to activate the PRL promoter (35, 36), the signaling mechanisms that permit Ca2+ influx to regulate transcription have not been identified. Our finding that nimodipine reduces TRH-induced MAPK activation suggests that MAPK is likely at least part of the mechanism mediating Ca2+ responsiveness. Nimodipine effects on TRH-induced MAPK activation were most prominent at later time points, consistent with the known contribution of Ca2+ channels to the later, plateau phase of Ca2+ elevation (4). Furthermore, the MEK inhibitor, PD98059, blunted the ability of Ca2+ influx to increase PRL promoter activity, providing evidence that MAPK plays a role in mediating Ca2+ effects on PRL transcription.

There are many possible mechanisms that may permit Ca2+ influx to activate the MAPK pathway in GH3 cells. In some cells Ca2+ influx leads to tyrosine phosphorylation of the EGF receptor in a ligand-independent manner and subsequent activation of the MAPK by Shc, Grb2, the guanine nucleotide exchange factor Sos1, and Ras (40). The mechanism responsible for ligand-independent tyrosine phosphorylation of the EGF receptor is not well established. A Ca2+-responsive, tyrosine kinase such as PYK2 (38) would be a candidate for mediating this response. However, we have been unable to demonstrate the presence of PYK2 in GH3 cells (Y.-H. Wang, unpublished studies). Src transformation of fibroblasts does lead to tyrosine phosphorylation of the EGF receptor (39). In addition, targeted gene disruption of the Src family member Fyn suggests that the Fyn protein may play a role in calcium-dependent responses in the nervous system, such as synaptic potentiation and memory formation (47). Thus, it remains quite possible that some member of the Src family of tyrosine kinases may contribute to Ca2+ effects on MAPK activation. Another mechanism that might mediate Ca2+ effects on MAPK activation would involve a Ca2+-sensitive guanine nucleotide exchange factor, such as Ras-GRF (37). Finally, Ca2+ influx may activate MAPK through Ca2+/calmodulin-dependent kinases (41).

Our studies suggest a model of PRL gene expression in which TRH generates two different signals, PKC activation and increased intracellular Ca2+ levels, which converge to activate MAPK. MAPK likely influences transcription of the PRL gene through phosphorylation of an Ets transcription factor, probably Ets-1 (31, 48). While this broadly outlines a signaling pathway, there are many aspects of this pathway that have not been identified. In addition, it remains possible that pathways other than MAPK activation also contribute to TRH effects on the PRL promoter. For instance, TRH activates Ca2+/calmodulin-dependent kinase type II in GH3 cells (8), and inhibitor studies using KN-62 suggest that a Ca2+/calmodulin-dependent protein kinase participates in TRH-induced PRL gene expression (14). However, KN-62 has little or no effect on TRH-induced MAPK activation (Y.-H. Wang, unpublished result). Thus, a Ca2+/calmodulin-dependent protein kinase pathway that is independent of the MAPK may also contribute to TRH-induced PRL gene expression. Although an expression vector for a constitutively active form of Ca2+/calmodulin-dependent protein kinase type II can activate PRL reporter genes that contain the distal enhancer region, removal of this region almost completely eliminates responsiveness (14). In contrast, the distal enhancer is not required for TRH responsiveness (35, 42). Although Ca2+/calmodulin-dependent protein kinase type II may not be sufficient to activate the proximal promoter, it remains possible that it acts in concert with the MAPK pathway to modulate the PRL promoter. Thus, the ability of TRH to modulate transcription of the PRL gene may depend on a complex interaction between PKC, MAPK, and perhaps of Ca2+/calmodulin-dependent protein kinase-signaling pathways.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Cell culture media and supplies were purchased from Gibco BRL (Gaithersburg, MD). Radioisotopes and reagents for enhanced chemiluminescent detection of immunocomplexes were purchased from DuPont NEN Nuclear (Boston, MA). Anti-ERK2 antibody, anti-phospho-ERK antibody, agarose beads-conjugated anti-ERK2 antibody, and horseradish peroxidase-coupled anti-mouse IgG antibody were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The MEK inhibitor, PD98059, was purchased from Alexis Corp. (San Diego, CA); PKC inhibitor, GF109203X, from Calbiochem (La Jolla, CA); nimodipine from Research Biochemicals International (Natick, MA); EGF from Boehringer Mannheim (Indianapolis, IN); TRH from Peninsula Laboratories, Inc. (Belmont, CA).

Reporter Genes and Expression Vectors
A reporter gene containing the proximal 255 bp of the 5'-flanking region of the rat PRL gene was prepared by PCR amplification of the appropriate region from the PRL gene (49), which was inserted upstream of the firefly luciferase-coding sequence in the promoterless construct, pLuc-Link (50). PRL reporter genes containing mutations in specific Ets factor-binding sites were prepared by oligonucleotide-directed mutagenesis. The specific mutations involved mutation of the Ets site at -211 to -208 with the sequence TTCC to the sequence TGAA, the Ets site at -162 to -159 from TTCC to AGGC, and the dual Ets site at -75 to -66 from GGAAgaGGAT to GGCCgaTTAT. A luciferase reporter gene containing 5 GAL4 binding sites upstream of a minimal promoter, as well as an expression vector for a Gal4-Elk1 fusion protein, have been described previously (51, 52). A reporter gene, pRL, expressing Renilla luciferase, was purchased from Promega Corp. (Madison, WI).

A mammalian expression vector for kinase-defective MEK1 mutant (53) was generously provided by Dr. Edwin G. Krebs. An expression vector encoding a constitutively active form of MEK1 was generated by replacing Ser 218 and Ser 222 by glutamic acid and cloned into pCDNA3 vector (45).

Culture and Transfections
GH3 cells were cultured in DMEM supplemented with 15% horse serum, 2.5% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. For transient transfection assays, 2.5 x 105 cells per well were planted in six-well plates 1 day before the transfection. DNA was introduced into these cells using lipofectamine (Gibco BRL) in serum-free medium according to a protocol provided by the manufacturer. In each experiment the total amount of transfected DNA per culture dish was constant (usually at 2 µg). After 5 h of treatment with the lipofectamine/DNA mixture, an equal volume of serum-containing culture medium was added. After an additional 18 h, the cells were transferred to serum-free medium. TRH treatments were applied 24 h after transfer to serum-free medium, and the cells were then lysed 5–6 h later for reporter gene analysis. In most experiments, cells were transfected with a firefly luciferase reporter gene and, as an internal standard for transfection efficiency, a control plasmid that expresses Renilla luciferase. Renilla luciferase requires a different substrate than firefly luciferase and can be assayed independently. Cells were lysed, and the activities of firefly and Renilla luciferase were determined using a protocol and reagents from Promega Corp.. Total firefly luciferase light units were normalized to total Renilla luciferase activity. The results of transfection studies are reported as means ± SE of the mean for several separate transfections (individual culture dishes) that were performed as part of the same experiment.

Immunoblotting
For analysis of protein expression, cells were grown to approximately 80% confluency, transferred to serum-free medium, and then treated with inhibitors and agonists as indicated. After treatment, the cells were washed twice with ice-cold 0.15 M NaCl, 0.01 M NaPO4 (pH 7.4). The cells were lysed for 10 min on ice in 20 mM Tris, pH 7.5, 1% Triton X-100, 10% glycerol, 50 mM ß-glycerolphosphate, 2 mM EGTA, and 1 mM dithiothreitol. The lysates were centrifuged for 20 min at 12,000 x g. The supernatants (100 µg protein) were adjusted to contain 1% SDS and 5% 2-mercaptoethanol and heated for 3 min in a boiling water bath before electrophoresis on an SDS-containing polyacrylamide gel (54). Proteins were then transferred by electroblotting to a polyvinylidene difluoride membrane. The membranes were incubated in 0.15 M NaCl, 0.01 M NaPO4 (pH 7.4) containing 3% BSA and 0.01% Tween 20 for 1 h and then incubated with a primary antibody for 1 h. After six washes with 0.15 M NaCl, 0.01 M NaPO4 (pH 7.4), 0.01% Tween 20, horseradish peroxidase-conjugated secondary antibody was incubated with the blot for 30 min. After extensive washing, the proteins were detected by using the enhanced chemiluminescence system (DuPont NEN).

ERK Immunocomplex Kinase Assay
Cells were treated and lysed as described for immunoblotting. Cell extracts were incubated with agarose-conjugated anti-ERK2 antibody for 1–2 h. Immunoprecipitates were washed once in lysis buffer, twice in 500 mM NaCl, 100 mM Tris-HCl, pH 7.5, 0.1% Triton X-100, and 2.5% sucrose and once in kinase assay buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 0.1% Triton X-100, and 2 mM EGTA). In vitro kinase assays were carried out for 25 min at 30 C in 20 µl of kinase assay buffer supplemented with 1 µCi of [{gamma}-32P]ATP and 5 µg glutathione S-transferase fusion protein, which includes the carboxy-terminal transcriptional activation of Elk1 (residues 307–428) including several MAPK phosphorylation sites (24). The GST-Elk1 fusion protein was expressed in Escherichia coli and purified using glutathione-Sepharose beads (55). The kinase reactions were stopped by adding SDS to 1% (wt/vol), the reactions were resolved on a denaturing, 10% polyacrylamide gel, and the phosphorylated proteins were detected by autoradiography.


    ACKNOWLEDGMENTS
 
We thank Denis Glenn for preparing an expression vector for constitutively active MEK, Paul Kievit for preparing some of the PRL reporter constructs used in this study, and Bobbi Maurer for aid in preparing this manuscript.


    FOOTNOTES
 
Address requests for reprints to: Richard A. Maurer, Department of Cell and Developmental Biology, L215, Oregon Health Sciences University, 3181 Southwest Sam Jackson Park Road, Portland, Oregon 97201.

This research was supported by NIH Grant DK-40339 to R. Maurer.

Received for publication February 26, 1999. Revision received April 16, 1999. Accepted for publication April 20, 1999.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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