Analysis of the Role of the Mitogen-Activated Protein Kinase in Mediating Cyclic-Adenosine 3',5'-Monophosphate Effects on Prolactin Promoter Activity

Paul Kievit, Jeffrey D. Lauten 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 mechanisms mediating cAMP effects to stimulate transcription of the PRL gene have been examined. Treatments that elevate intracellular cAMP concentrations were found to stimulate the mitogen-activated protein kinase (MAPK) in GH3 cells. Elevated cAMP was also found to stimulate activation of the GTP-binding protein, Rap1. Rap1GAP1 reduced cAMP-induced phosphorylation of MAPK, offering evidence that Rap1 may play a role in mediating activation of MAPK. Treatment of GH3 cells with PD98059, an inhibitor of the MAPK pathway, reduced the ability of forskolin to activate a PRL reporter gene, providing evidence that MAPK contributes to cAMP-mediated effects on the PRL promoter. As previous studies have implicated Ets factor binding sites within the PRL promoter in mediating responses to MAPK, we expected that the Ets sites would also play a role in cAMP responsiveness. Surprisingly, mutation of all of the consensus Ets factor binding sites in the proximal PRL promoter greatly reduced responsiveness to epidermal growth factor (EGF) and TRH but did not reduce cAMP responsiveness. Experiments using an expression vector for adenovirus 12S E1a provided evidence that the coactivators, CREB binding protein and/or p300, probably play a role in cAMP responsiveness of the PRL promoter. Interestingly, the ability of a GAL4-p300 fusion protein to enhance reporter gene activity was stimulated by cAMP in a MAPK-dependent manner. These findings provide evidence for a model for cAMP-induced PRL transcription involving Rap1-induced MAPK activity leading to stimulation of the transcriptional coactivators, CBP and p300.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Although it has been known for some time that cAMP can stimulate PRL gene expression (1, 2), it has been difficult to determine the mechanisms mediating this response. Studies utilizing expression vectors for the catalytic subunit of the cAMP-dependent protein kinase [protein kinase A (PKA)] or the heat-stable inhibitor of PKA (PKI) have provided evidence that PKA is sufficient and necessary for cAMP-induced activation of the PRL gene (3, 4). However, the events downstream of PKA have been elusive. Unlike many PKA-regulated genes, stimulation of PRL gene expression by cAMP probably does not involve CREB (5, 6). Several studies have provided evidence that the pituitary-specific POU transcription factor, Pit-1, may play a role in mediating the effects of cAMP (6, 7, 8) as well as other signaling pathways (9, 10) on PRL transcription. The finding that Pit-1 is phosphorylated in response to elevated cAMP levels in GH3 cells (11, 12) rather strongly supported the view that Pit-1 may function as a cAMP-regulated transcription factor. However, studies using Pit-1 mutants in which the phosphorylation sites are removed (13, 14) have provided evidence that cAMP-induced activation of the PRL promoter probably does not require phosphorylation of Pit-1. Although this does not exclude Pit-1 from contributing to cAMP-mediated effects on PRL transcription, it is by no means clear that phosphorylation of Pit-1 mediates cAMP effects, and other mechanisms need to be considered. We have obtained evidence that cAMP can activate the mitogen-activated protein kinase (MAPK)-responsive transcription factor, Elk1, providing indirect evidence that cAMP may stimulate MAPK in GH3 cells (12), raising the possibility that the MAPK pathway may be involved in mediating effects of cAMP on transcription of the PRL gene. We have also recently obtained evidence that TRH effects on PRL transcription may be mediated, at least in part, through activation of MAPK (15). Thus it is possible that both the TRH and cAMP signaling pathways may utilize the MAPK cascade to stimulate PRL gene expression.

In the present study we have examined the ability of cAMP to stimulate MAPK activity and tested the role that cAMP-induced MAPK activation plays in modulating PRL transcription. We have found that cAMP can activate MAPK in GH3 cells and that the increase in MAPK is required for full effects of cAMP on PRL transcription. Analysis of DNA sequences of the PRL gene, which are required for cAMP responsiveness, reveals both similarities and differences in the role of specific Ets factor binding sites for TRH and cAMP responsiveness. We have also assessed the possible role that the transcriptional coactivators, CREB binding protein (CBP) and p300, play in mediating cAMP-induced activation of the PRL promoter.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Elevation of cAMP Induces MAPK Activity in GH3 Cells
Previous studies from this laboratory used an indirect assay involving transcriptional activation of a GAL4-Elk1 fusion protein to test for cAMP effects on MAPK activation (16). To directly assess MAPK activation, we used an immunocomplex assay (Fig. 1Go, A, B, and C). For this assay, cell lysates were immunoprecipitated with an antibody to a specific MAPK family member after which the immunoprecipitated proteins were incubated with [{gamma}-32P]ATP and glutathione-S-transferase (GST)-Elk1 as a substrate. This assay demonstrated that treatment with chlorophenylthio-cAMP stimulated the activity of MAPK family members, extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2) (Fig. 1Go, A and B), with only a minor effect on the activity of the stress-activated MAPK, JNK1 (c-Jun N-terminal protein kinase-1) (Fig. 1CGo). ERK1 and ERK2 are activated by phosphorylation of specific tyrosine and threonine residues (17), and an antibody that can detect this phosphorylation was also used to assess ERK activation (Fig. 1DGo). Treatment of cells with forskolin, an agent that increases intracellular cAMP levels, resulted in the rapid stimulation of the phosphorylation of ERK1 and ERK2 (Fig. 1DGo). There was little or no change in the level of total immunoreactive ERK1 and ERK2, suggesting that the increased phosphorylation is not due to a change in the total amount of these kinases (Fig. 1EGo). Experiments were then performed to compare the time course of ERK activation by forskolin and epidermal growth factor (EGF). Cells were treated with either forskolin or EGF for varying times, and ERK activation was determined using a phospho-specific antibody and the results quantitated using a Lumi-Imager system (Roche Molecular Biochemicals, Indianapolis, IN) (Fig. 1Go, F and G). EGF effects on ERK phosphorylation appeared to reach maximal levels more quickly and also appeared to decline somewhat more rapidly. Several different experiments supported the view that EGF effects on ERK activity reach a maximum more quickly than cAMP effects, but decay more rapidly (data not shown). Overall, the analysis of cAMP effects on MAPK confirm our earlier results (16) and studies from Jacob et al. (18), which demonstrated cAMP-induced activation of MAPK in the GH4 cell line. The present studies also provide information about the time course and specificity of MAPK activation.



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Figure 1. Time Course of cAMP-Induced MAPK Activation

A–C, Immunocomplex kinase assays of MAPK activation. GH3 cells were treated with 0.5 mM CPT-cAMP for various periods of time. ERK1 (A), ERK2 (B), or JNK1 (C) was immunoprecipitated from 500 µg of cell lysates using antibodies conjugated to protein A/G agarose. MAPK activity was assessed by incubating the immunoprecipitate with [32P]-{gamma}-ATP and a GST-Elk1 fusion protein as substrate. The phosphorylated proteins were resolved on a denaturing polyacrylamide gel and detected by autoradiography. EGF (10 nM) and anisomycin (10 ng/ml) were used as positive controls for activity of ERK and JNK, respectively. D–G, Immunoblot analysis of ERK activation. GH3 cells were cultured in serum-free medium for 24 h and treated with 10 µM forskolin or 10 nM EGF for the indicated times. Cell lysates (50 µg) were resolved on a denaturing polyacrylamide gel, transferred to a membrane, and phosphorylated ERK1 and ERK2 were detected with a phosphorylation-specific antibody (D). To determine the total amount of ERK present, the membrane was stripped and reprobed with an antibody directed against ERK-1 protein (E). This antibody also detects ERK-2, although to a lesser extent. In a separate experiment GH3 cells were treated with 10 µM forskolin or 10 nM EGF for varying times and ERK activation was detected by a phosphorylation-specific antibody and then quantitated by Lumi-Imager analysis (F and G).

 
The major signaling pathway that mediates responses to cAMP involves activation of the cAMP-dependent protein kinase (PKA). An expression vector for PKI, the heat-stable inhibitor of PKA (4), was used to determine whether PKA is required for cAMP-induced activation of MAPK. For this study, MAPK induction was assessed indirectly through analysis of the activation of a GAL4-Elk1 fusion protein (19). GH3 cells were transfected with a GAL4-Elk1 expression vector and a GAL4-dependent luciferase reporter gene either in the presence or absence of a PKI expression vector (Fig. 2AGo). PKI strongly reduced the ability of forskolin to stimulate GAL4-Elk1 activity as measured by the reporter gene assay. Although PKI reduced EGF-stimulated GAL4-Elk1 activation, PKI also had an effect on basal activity in the absence of hormonal treatment, and the fold activation remained similar. The pharmacological inhibitor H-89 was also used to determine whether inhibition of PKA led to a decrease in MAPK activation (Fig. 2Go, B and C). GH3 cells were treated with H-89, after which forskolin was added for 5 min and the cells were collected. Activation of MAPK was determined by immunological detection of phosphorylated ERK1 and ERK2 as described above. Addition of H-89 substantially blocked the ability of forskolin to activate ERK1 and ERK2, whereas EGF-induced phosphorylation was essentially unchanged. In addition, the tyrosine kinase inhibitor genistein had relatively little effect on the induction of ERK phosphorylation by forskolin, but greatly reduced EGF-induced ERK phosphorylation. The ability of PKA inhibitors to attenuate both cAMP-induced activation of GAL4-Elk1 and phosphorylation of ERK1 and ERK2 suggests that PKA is required for cAMP-induced stimulation of MAPK activity.



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Figure 2. The cAMP-Dependent Protein Kinase Is Required for Full cAMP-Induced ERK1 and ERK2 Activation

A, Effects of inhibition of PKA by PKI on GAL4-Elk1 activity. GH3 cells were transfected with a GAL4-dependent luciferase reporter gene and an expression vector encoding a GAL4-Elk1 fusion protein. The cells were also transfected with either an empty vector (control), or a vector encoding PKI. The cells were treated with EGF (10 nM) or forskolin (10 µM) for 6 h and collected for analysis of luciferase activity. Reporter gene activity is reported as luciferase activity from three independent transfections ± SEM normalized to ß-galactosidase activity. B and C, Effects of inhibition of PKA or tyrosine kinases on MAPK activation. GH3 cells were treated with 20 µM of the PKA inhibitor H-89 or 40 µM of the tyrosine kinase inhibitor, genistein, for 30 min. Cells were stimulated for 5 min with forskolin or EGF and harvested. Cell lysates (50 µg) were separated on a denaturing polyacrylamide gel and transferred to a membrane. Activated ERK was detected with the antibody directed against the phosphorylated form of ERK1 and ERK2 (B). To test for equal loading, the membrane was stripped and probed for total amount of immunoreactive ERK1 and ERK2 (C).

 
Analysis of a Possible Role of the GTP Binding Protein, Rap1, in Mediating Responses to cAMP
Recent studies have provided new insights into cAMP signal transduction and demonstrated that, in specific cell types, Rap1 can play a crucial role in mediating cAMP-induced MAPK activation (20). These studies have shown that cAMP can stimulate Rap1 activation leading to activation of B-Raf, MEK1, and MAPK. To determine whether this pathway might be present in GH3 cells, cAMP-induced activation of Rap1 was investigated (Fig. 3Go). This assay is based on the ability of RalGDS to interact with activated, GTP-bound Rap1, but not with inactive, GDP-bound Rap1 (21). Lysates from forskolin-treated GH3 cells were incubated with the Rap1-binding domain of RalGDS and then bound Rap1 was detected immunologically (Fig. 3AGo). Activation of Rap1 was detected after 1 min, the earliest tested time point, and maximum activation was achieved in 5–10 min. In the same experiment, the time course of MAPK activation was also examined using antibodies for phospho-ERK (Fig. 3CGo). There appear to be some differences in the time course of Rap1 and ERK activation. At the 2-min time point, Rap1 was only modestly activated, whereas ERK phosphorylation appears to be near maximal. This finding suggests that mechanisms other than Rap1 activation may contribute to the ability of cAMP to stimulate MAPK activity. Alternatively, the activation of Rap1 in the early time points may be sufficient for maximal ERK activation. In any case, the ability of cAMP to stimulate Rap1 activation suggests a possible role in at least partially mediating effects on MAPK.



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Figure 3. cAMP Activates Rap1 in GH3 Cells

GH3 cells were cultured in serum-free medium for 24 h before treatment with 10 µM forskolin. Cells were lysed, and activated Rap1 was isolated with a GST-RalGDS fusion protein bound to beads. The Rap1-RalGDS complex was washed, and immobilized proteins were resolved on a denaturing polyacrylamide gel. The proteins were transferred to a membrane, and bound Rap1 was visualized with a polyclonal antibody (A). To verify that equal amounts of Rap1 were present in the lysates, 2% of the input was separated on a denaturing polyacrylamide gel and transferred to a membrane (B). Rap1 was detected using the polyclonal antibody. For the ERK phosphorylation time course, 50 µg of the GH3 lysate was resolved on a denaturing polyacrylamide gel, and phosphorylated ERK1 and ERK2 were detected using a phosphorylation-specific antibody (C). Equal loading was determined by stripping the blot and reprobing with an ERK1 antibody (D).

 
To test whether the activation of Rap1 is necessary for MAPK activation, an expression vector for the Rap1 GTPase activating protein (Rap1GAP1) was used (22). Forced expression of Rap1GAP1 should maintain Rap1 in the inactive, GDP-bound state, and thus act as an antagonist to this signaling pathway. Therefore, a Rap1GAP1 expression vector was transfected with an expression vector for FLAG-tagged ERK2 (Fig. 4Go). Tagged ERK2 was immunoprecipitated, and activated ERK2 was detected immunologically using a phospho-specific antibody (Fig. 4AGo). Rap1GAP1 reduced both basal and forskolin-induced levels of phosphorylated ERK2 at both time points tested. The ability of Rap1GAP1 to reduce ERK2 phosphorylation offers evidence that Rap1 may contribute to cAMP-induced MAPK activation in GH3 cells. To determine whether Rap1 contributes to cAMP-mediated gene expression, GH3 cells were transfected with the Rap1GAP1 expression vector and a PRL reporter gene (Fig. 4DGo). At the higher tested concentrations, Rap1GAP1 was capable of reducing both cAMP-induced and basal PRL reporter gene activity. As was observed with the effects of Rap1GAP1 on cAMP-induced ERK2 phosphorylation, the effects of cAMP were not completely blocked. It is not clear whether the remaining cAMP response is a reflection of limited efficacy of the transfected Rap1GAP1 expression vector or if it indicates that a component of the cAMP response involves a Rap1-independent pathway.



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Figure 4. Rap1 Is Required for Full Effects of cAMP to Activate MAPK and Gene Expression

A–C, Analysis of the effects of Rap1GAP1 on ERK activation in GH3 cells. GH3 cells were transfected with FLAG-tagged ERK2 (4 µg) and either FLAG-tagged Rap1GAP1 (6 µg) or empty vector (6 µg) as indicated. After serum deprivation, 10 µM forskolin was added for the indicated times. FLAG-tagged proteins were precipitated from the cell lysates (1 mg), and phospho-ERK2 was detected with a phosphorylation-specific polyclonal antibody (A). Expression levels of Rap1GAP1 (B) and ERK2 (C) were verified by immunoblot analysis of cell lysates (100 µg) using the FLAG monoclonal antibody. D, Analysis of the effects of Rap1GAP1 expression on PRL reporter gene activity. GH3 cells were transfected with increasing amounts of the FLAG-Rap1GAP1 construct and a reporter construct containing the -255 to +34 region of the PRL promoter. The total amount of transfected DNA was normalized with empty expression vector (pcDNA3). At 24 h after transfection, the cells were treated with 10 µM forskolin for 6 h and collected for analysis of luciferase activity. Reporter gene activity is reported as luciferase activity from three independent transfections ± SEM normalized to ß-galactosidase activity.

 
We also tested the ability of a different reagent to interfere with the Rap1 signaling pathway. Vossler et al. (20) found that Rap1-N17 can function as a dominant negative mutant to block cAMP-induced ERK and GAL4-Elk1 activation in PC12 cells. Therefore, we tested the effects of Rap1-N17 on ERK activation in GH3 cells. Surprisingly, we found that Rap1-N17 had no detectable effect on cAMP-induced ERK activation or GAL4-Elk1 activation (data not shown). Another group has also failed to detect an effect of Rap1-N17 on cAMP-induced ERK activation in a different cell type (23). It is possible that the differing ability of Rap1GAP1 and Rap1-N17 to block cAMP-induced ERK activation reflects a different efficacy of these reagents in blocking endogenous Rap1 signaling. Alternatively, this observation may indicate that the Rap1GAP1 and Rap1-N17 interfere with different signaling pathways. We cannot exclude the possibility that Rap1GAP1 may block a signaling pathway other than Rap1, perhaps a related small GTPase.

MAPK Activation Is Required for cAMP-Stimulated Transcriptional Activity
To determine whether cAMP-stimulated MAPK activity is required for specific gene expression, we used the MEK1 inhibitor, PD98059. PD98059 has a relatively high affinity for MEK1 and can block the ability of MEK1 to activate ERK1 and ERK2 (24). GH3 cells were pretreated with 100 µM PD98059 for 30 min and then treated with forskolin for 5 min and assayed for ERK phosphorylation (Fig. 5AGo). PD98059 treatment greatly reduced cAMP-stimulated phosphorylation of ERK1 and ERK2.



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Figure 5. MAPK Activation Is Required for Full Effects of cAMP on PRL Gene Expression

A, Analysis of the effects of the MAPK inhibitor, PD98059, on forskolin-induced ERK activity in GH3 cells. GH3 cells were cultured in serum-free medium for 24 h, pretreated for 30 min with the vehicle, dimethylsulfoxide (DMSO) (control), or 100 µM PD98059 followed by 10 µM forskolin treatment for 5 min. Cell lysates were resolved on a denaturing polyacrylamide gel, transferred to a membrane, and probed with an antibody specific to the phosphorylated form of ERK. B and C, Analysis of the effects of the MAPK inhibitor, PD98059, on forskolin-induced reporter gene activity. GH3 cells were transfected with either a GAL4-dependent luciferase reporter gene and an expression vector encoding a GAL4-Elk1 fusion protein (B) or a PRL promoter containing the proximal 255 bp fused to the luciferase reporter gene (C). At 24 h after transfection, GH3 cells were pretreated for 30 min with either the vehicle, DMSO (control), or 100 µM PD98059. The cells were then treated with either 10 µM forskolin or 10 nM EGF for 6 h, after which time the cells were lysed and analyzed for luciferase activity. Reporter gene activity is reported as light units from three independent transfections ± SEM normalized to ß-galactosidase activity.

 
The ability to PD98059 to block cAMP-induced transcriptional activation was then tested (Fig. 5Go, B and C). PD98059 strongly reduced the ability of forskolin to stimulate GAL4-Elk1 activity as assessed by a GAL4-dependent reporter gene (Fig. 5BGo). This finding is consistent with our observation that cAMP induces MAPK activity and the known ability of MAPK to induce Elk1 activity (19). PD98059 reduced, but did not completely block, forskolin effects to induce a PRL reporter gene (Fig. 5CGo). Similar results were obtained when a kinase-defective MEK1 was used to inhibit activation of the MAPK pathway (data not shown). These findings provide evidence that cAMP-induced activation of MAPK plays a key role in stimulating Elk1 transcriptional activation and also contributes to cAMP-induced enhancement of PRL transcription.

Analysis of the Role of Ets Binding Sites in Mediating cAMP-Induced Activation of the PRL Promoter
The proximal region of the PRL gene contains a number of consensus sites for members of the Ets family of transcription factors (Fig. 6AGo). As MAPK has been shown to stimulate the transcriptional activity of several members of the Ets family (19, 25), the Ets sites appear to be excellent candidates for mediating the transcriptional response to cAMP-induced MAPK activation. Indeed, previous studies have shown that disruption of some of the Ets sites of the PRL gene can reduce transcriptional responses to Ras (26) or TRH (15). To determine whether any of the Ets sites play a role in cAMP-dependent PRL gene expression, PRL reporter constructs were tested for cAMP, TRH, and EGF responsiveness (Fig. 6BGo). Some of the Ets site mutants were previously described (15), and several Ets site mutants were newly prepared for this study. The only mutation that reduced cAMP, TRH, and EGF responsiveness to less than 50% of wild type was mutation of the Ets site located at position -211, a site that previously has been shown to be important for MAPK responsiveness (15, 26). Disruption of other Ets sites either reduced responsiveness only modestly or, in some cases, stimulated reporter gene activation in response to cAMP, TRH, or EGF.



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Figure 6. Mutational Analysis of Putative Ets Factor Binding Sites in the Proximal PRL Promoter

Specific Ets factor binding sites within the proximal region of the PRL gene were disrupted as indicated by the schematic diagram for the PRL promoter (A) where Ets sites are indicated by black rectangles and Pit-1 binding sites are indicated by gray rectangles. GH3 cells were transfected with PRL promoter reporter constructs in which individual Ets factor binding sites were disrupted (B) or a reporter gene in which all of the consensus Ets sites were disrupted (C). After serum deprivation, the cells were treated with 10 µM forskolin, 100 nM TRH, or 10 nM EGF for 6 h. Cells were collected and assayed for luciferase activity. The basal levels for the various promoters were as follows (in arbitrary light units): wild type, 446 ± 93.1; mut1, 617 ± 66.1; mut2, 2,096 ± 253; mut3, 444 ± 36.7; mut4, 798 ± 78.9; mut5, 293 ± 20.6. The fold induction of the wild-type promoter by forskolin, TRH, and EGF was 13, 3.2, and 8.7, respectively. Reporter gene activity is reported as the light units from three independent transfections ± SEM normalized to ß-galactosidase activity.

 
As there are multiple Ets sites in the proximal region of the PRL gene, it seemed possible that the modest effects of mutating single Ets sites reflected some redundancy in their ability to confer MAPK responsiveness. To assess the overall contribution of Ets sites within the proximal region of the PRL gene, a reporter construct was created in which all the consensus Ets sites were mutated. This construct was tested for its ability to be activated by the activators forskolin, EGF, and TRH (Fig. 6CGo). Surprisingly, the construct in which all of the Ets sites were mutated was more responsive to forskolin treatment than the wild-type PRL promoter. In contrast, and as expected, reporter gene responses to TRH and EGF were reduced in the Ets mutant. Thus, although cAMP, TRH, and EGF all lead to activation of MAPK, the requirements for downstream transcription factor targets of these signaling pathways appear to differ.

Elevation of cAMP Enhances p300 Transcriptional Activity in a MAPK-Dependent Manner
As the preceding studies suggest that Ets factors are probably not required for transcriptional responses to cAMP, we considered a possible role for other factors. Recent studies have provided evidence that the ability of cAMP to stimulate PRL promoter activity in heterologous cells probably involves the closely related coactivator proteins, CBP and p300 (27, 28). To test the role of endogenous CBP and/or p300 in mediating responsiveness of the PRL promoter to cAMP, we used an expression vector for the adenovirus 12S E1a protein, which blocks CBP and p300 activity (29, 30). We found that increasing amounts of the E1a expression vector substantially reduced the ability of forskolin to stimulate PRL promoter activity in GH3 cells (Fig. 7AGo). To test for possible nonspecific effects of E1a, we compared the wild-type E1a to an E1a mutant ({Delta}2–35) that is defective for interaction with CBP/p300 (31). The mutant E1a had much smaller effects on cAMP- and EGF-stimulated PRL gene expression (Fig. 7BGo). These findings offer support for the view that CBP/p300 plays a role in transcriptional regulation of the PRL gene. To further explore this topic, we elected to test the ability of cAMP to stimulate the transcriptional activity of p300. We prepared an expression vector for a GAL4-p300 fusion that was cotransfected with a GAL4-dependent reporter gene. The results demonstrate that GAL4-p300 activity is substantially increased by treatment with forskolin (Fig. 7CGo). Interestingly, treatment with the MEK1 inhibitor, PD98059, almost completely blocked the ability of forskolin to enhance GAL4-p300 activity. These findings offer evidence that in GH3 cells, cAMP stimulates p300 activity in a MAPK-dependent manner.



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Figure 7. Analysis of a Possible Role for the Coactivator CBP/p300 in cAMP-Stimulated Activation of the PRL Promoter

A, Analysis of the effects of increasing amounts of an adenovirus E1a expression vector on forskolin- and EGF-induced PRL reporter gene activity. GH3 cells were transfected with a PRL reporter construct and either an empty vector (pcDNA3) or increasing amounts of E1a expression vector as indicated. The cells were treated with 10 µM forskolin or 10 nM EGF for 6 h after which the cells were collected and assayed for luciferase activity. B, Comparison of wild-type and mutant E1a on activation of the PRL promoter. GH3 cells were transfected with a PRL reporter construct and 1 µg of an expression vector for either the wild-type E1a or mutant E1a in which residues 2–35 have been deleted ({Delta}2–35). The cells were treated with 10 µM forskolin or 10 nM EGF for 6 h, and the cells were then collected and assayed for luciferase activity. C, Analysis of PD98059 effects on forskolin-induced activation of a GAL4-p300 fusion protein. GH3 cells were transfected with an expression vector encoding a GAL4-p300 fusion protein and a reporter construct containing five GAL4 binding sites. The cells were incubated in serum-free medium for 24 h and then treated with 100 µM PD98059 for 30 min. Forskolin (10 µM) was added and cells were collected and assayed 6 h later. Reporter gene activity is reported as light units from three independent transfections ± SEM normalized to ß-galactosidase activity.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
These studies provide evidence that cAMP can activate the MAPK signaling pathway in GH3 cells and that MAPK activation contributes to the ability of cAMP to stimulate PRL promoter activity. Analysis of ERK phosphorylation as well as immunocomplex assay of ERK activity have provided substantial evidence that elevated cAMP can stimulate MAPK activity. Inhibition of the MAPK pathway was found to reduce the ability of cAMP to activate the PRL promoter and a GAL4-Elk1 fusion protein. These findings as well as previous studies (15, 16, 18, 32) suggest that the cAMP, EGF, and TRH signaling pathways all converge in GH3 cells to stimulate MAPK activity, which then plays a role in stimulating PRL gene expression.

The signaling pathway that mediates cAMP effects on MAPK in GH3 cells may involve the small GTP-binding protein, Rap1, although other pathways may be involved. Rap1 has been shown to play an important role in mediating cAMP-induced MAPK activation in PC12 cells (20). Rap1 can respond to cAMP through two different mechanisms. One mechanism involves phosphorylation of Rap1 by PKA, leading to changes in activity (33). The other mechanism involves direct binding of cAMP to the Rap1 GDP exchange protein, Epac (34). We found that elevation of cAMP in GH3 cells leads to rapid activation of Rap1. An expression vector for Rap1GAP1, which should inactivate Rap1, reduced cAMP-induced MAPK activation and PRL promoter activity. On the other hand, Rap1-N17, which should also block the Rap1 pathway (20), did not reduce cAMP effects on MAPK or the PRL promoter. It is possible that Rap1-N17 is simply not as effective in blocking signaling through the endogenous Rap1 pathway as Rap1GAP1. However, we cannot rule out the possibility that Rap1GAP1 may have effects on signaling pathways other than Rap1, perhaps blocking the activity of a related, small GTPase. In some cells, cAMP can modulate MAPK activity through activation of Ras (35). However, cAMP has little if any ability to activate Ras in GH3 cells, and a dominant negative form of Ras did not block cAMP induction of MAPK in GH3 cells (P. Kievit, unpublished observations). Another mechanism that might mediate cAMP effects on MAPK would involve modulation of a protein phosphatase. It has been shown that PKA can phosphorylate the protein tyrosine phosphatase, PTP-SL, leading to decreased interaction between the phosphatase and ERK1 and ERK2, thus allowing for an increase in ERK phosphorylation and activation (36). We have not tested a possible role for PTP-SL in mediating PKA effects in GH3 cells.

Concerning the events downstream of MAPK, it seemed likely that members of the Ets family of transcription factors would be involved in mediating transcriptional regulation of the PRL gene. Previous studies have led to a model in which Ras, TRH, or EGF can stimulate MAPK activity leading to Ets factor phosphorylation and transcriptional activation involving several specific DNA elements in the PRL gene (15, 16, 26, 37, 38). As the present studies demonstrate that cAMP can stimulate MAPK activity, it seemed probable that Ets sites would also play a role in mediating transcriptional responses to cAMP. To test this possibility, Ets binding sites within the proximal region of the PRL promoter were mutated, including several Ets sites that have not previously been studied. Similar to previous studies examining Ras- or TRH-responsiveness (15, 38), mutation of the Ets site at position -211 decreased cAMP responsiveness to less than 50% of wild-type activity. Interestingly, this region of the PRL gene has been shown to interact with the LIM homeodomain transcription factor, Lhx3 (39), and Lhx3 has been shown to enhance Ras responsiveness of the PRL promoter (40). Mutation of other single Ets sites either had smaller effects or actually stimulated cAMP responsiveness. The increases in responsiveness that were observed at some sites may be due to disruption of the binding of a repressor. It has been reported that some Ets factors can function as repressors, and one has been shown to inhibit PRL promoter activity (41, 42). To further explore the overall role of Ets sites, a PRL promoter reporter gene construct in which all of the consensus Ets sites were disrupted was prepared. Surprisingly, the promoter construct with all Ets sites disrupted was even more responsive to cAMP than the wild-type reporter gene. In contrast, disruption of all of the consensus Ets sites substantially reduced EGF responsiveness and partially reduced TRH responsiveness. Thus, it appears that there is a differential requirement for Ets sites in mediating regulation of the PRL promoter. The consensus Ets sites are not required for cAMP responsiveness, but are necessary for full responses to EGF or TRH. This observation is somewhat surprising in view of the fact that all of these signaling pathways appear to converge to activate MAPK. One explanation may involve differing kinetics of MAPK activation. We found that cAMP- and EGF-induced activation of ERKs have somewhat different time courses. The effects of cAMP were observed to have a slower onset and appeared to persist somewhat longer than EGF effects on MAPK activation. Studies in other systems have provided evidence that the kinetics of MAPK induction may have a profound effect on the induced response. For instance, in PC12 cells, transient MAPK activation results in proliferation while sustained MAPK activation is associated with differentiation (43). Alternatively, the differential requirement for Ets sites may result from differences in the combined action of several signaling pathways. The ability of cAMP to stimulate both PKA and MAPK may lead to activation of different transcription factors than occurs after activation of MAPK alone or MAPK combined with other signaling pathways.

The ability of the PRL promoter to respond to cAMP appears to involve the transcriptional coactivators, CBP and p300. Previous studies have used antibody blocking experiments to provide evidence that CBP and p300 play a role in mediating the ability of PRL promoter to respond to cAMP (27, 28). We have used an expression vector for adenovirus E1a as a CBP/p300 antagonist and confirmed that these coactivators appear to be required for full responsiveness to cAMP. As CBP and p300 can bind to either Pit-1 or Ets-1 in a constitutive manner (27, 28, 44, 45), both transcription factors may constitutively recruit CBP/p300 to the PRL promoter. Our studies with a GAL4-p300 fusion provide evidence that the transcriptional activity of p300 can be stimulated by elevated cAMP in a MAPK-dependent manner. Thus, it is possible that CBP/p300 is constitutively present at the PRL promoter and that the transcriptional activity of CBP/p300 is modulated by several signaling pathways, including cAMP, that converge on the MAPK pathway. Alternatively, Xu et al. (27) have suggested that activation of growth factor or cAMP pathways leads to recruitment of CBP/p300 to the PRL promoter. Additional studies assessing the recruitment of CBP/p300 to the PRL promoter in GH3 cells are required to distinguish between these two models. The recent development of chromatin immunoprecipitation assays should provide an appropriate technology to address this question.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Tissue culture reagents and media were purchased from Life Technologies, Inc. (Gaithersburg, MD). Antibodies to Rap1, ERK1, ERK2, phosphorylated ERK, antimouse and antirabbit horseradish-peroxidase-conjugated IgG and immunoprecipitation reagents were acquired from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). A polyclonal antibody to phosphorylated ERK was purchased from New England Biolabs, Inc. (Beverly, MA). Antibodies to the FLAG epitope and anti-FLAG M2 affinity resin were purchased from Sigma (St. Louis, MO). The chemical inhibitor PD98059 was obtained from Alexis Corp. (San Diego, CA), and H-89 and genistein were purchased from Calbiochem (La Jolla, CA). Forskolin and chlorophenylthio-cAMP (CPT-cAMP) were purchased from Sigma. EGF was from Roche Molecular Biochemicals (Indianapolis, IN). TRH was purchased from Peninsula Laboratories, Inc. (San Carlos, CA). Radioisotopes and chemiluminescence reagents were obtained from Dupont-NEN Life Science Products (Boston, MA).

Reporter Genes and Expression Vectors
A PRL reporter construct containing sequences representing the -255 to +34 region of the rat PRL gene (46) was obtained by PCR and inserted upstream of the firefly luciferase coding sequence in the pLuc-Link vector (47). Individual consensus Ets factor binding sites in the PRL 5'-flanking region were disrupted by oligonucleotide-directed mutagenesis using PCR. The specific mutations for each site were as follows: the Ets sites at -211 to -208 and -183 to -180 were mutated from TTCC to TGAA, the site at -162 to -159 from TTCC to GGCC, the site at -95 to -92 from GGAA to GTTC, and the site at -75 to -66 from GGAAGAGGAT to GGCCGATTAT.

A GAL4-p300 construct was generated by in-frame subcloning of the full coding sequence of p300 into a vector containing the GAL4 (1–147) DNA binding domain downstream of the cytomegalovirus promoter (48). The RSV-PKI vector (4), RSV-Pit-1 vector (8), GAL4-Elk1 (49) construct, and the luciferase reporter containing five GAL4 binding sites (48) have been described previously. Dr Philip Stork kindly provided GST-RalGDS protein and the FLAG-Rap1GAP1 and FLAG-ERK2 expression vectors. The E1a deletion mutant, {Delta}2–35 (50), was obtained from Dr. James Lundblad.

Cell Culture and Transfection
GH3 cells were cultured in DMEM supplemented with 2.5% FBS and 15% equine serum. For transfection, GH3 cells were seeded into six-well dishes 1 day before transfection at an approximate density of 500,000 cells per well. DNA (1–3 µg total) was transfected using the Lipofectamine reagent (Life Technologies, Inc.) according to the manufacturer’s protocol. After overnight incubation, the cells were treated with agonists for 6 h, after which the cells were washed, lysed, and analyzed for luciferase activity. All experiments with the PD98059 compound were performed in DMEM supplemented with BSA (6.6 mg/ml) to prevent precipitation of PD98059. Cells were transfected with CMV-ß-galactosidase expression vector as an internal standard (51).

Immuno-Complex Kinase Assay
MAPK immunoprecipitation assays were performed as previously described (49). Briefly, GH3 cells were treated with CPT-cAMP for the indicated time intervals, washed twice with PBS, and resuspended in lysis buffer (20 mM HEPES-KOH, pH 7.4, 2 mM EGTA, 50 mM ß-glycerophosphate, 10% glycerol, 1% Triton X-100, 1 mM EDTA, 2 mM sodium vanadate). ERK1, ERK2, and JNK1 were precipitated using the appropriate antibody linked to protein A/G agarose, and MAPK activity was determined by incubating the precipitates with a bacterially expressed glutathione-S-transferase-Elk1 fusion protein in the presence of 1 µCi [{gamma}-32P]-ATP. The reactions were stopped with loading buffer, resolved on a 10% polyacrylamide, denaturing gel, and phosphorylated proteins were detected by autoradiography.

Immunoblot Analysis
For immunoblot analysis, GH3 cells were grown to approximately 80% confluency, cultured in serum-free medium for 24 h, and treated with agonist for specific times. Cells were lysed in BOS buffer (50 mM Tris-HCl, pH 8.0, 10% glycerol, 1% Nonidet-P40, 200 mM NaCl, 2.5 mM MgCl2, 2 mM sodium vanadate) supplemented with a mixture of protease inhibitors (Complete Proteinase Inhibitor, Roche Molecular Biochemicals). Equal amounts of protein (50 µg) were loaded on a 10% polyacrylamide, denaturing gel. Proteins were transferred to a polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA) before incubation with antibodies to detect specific proteins. In many cases, membranes were treated to remove bound antibody to permit immunoblot analysis of a second protein. The membrane was incubated for 30 min in stripping buffer (100 mM ß-mercaptoethanol, 1% SDS, 62.5 mM Tris-HCl, pH 6.8), washed in TBS-T (50 mM Tris, pH 8.0, 150 mM NaCl, 0.1% Tween 20), and reprobed with the appropriate antibody. All antibodies were used at a concentration of 1:1,000 in TBS-T, 5% nonfat milk, except the FLAG antibody was used at a dilution of 1:10,000. The proteins were visualized on radiography film using a secondary antibody conjugated to horseradish peroxidase and a chemiluminescence detection kit (NEN Life Science Products, Boston, MA). Quantitation of immunoblot analysis was performed using the Lumi-Imager F-1 system (Roche Molecular Biochemicals).

Affinity Assay for Rap1 Activation
Rap1 activation assays were performed according to Franke et al. (21). GH3 cells were treated with forskolin for the indicated times and harvested in BOS buffer supplemented with complete protease inhibitor. Cell lysates were cleared by brief centrifugation and adjusted with lysis buffer to a final concentration of 1 mg/ml. Equal amounts of protein were incubated for 1 h in the presence of GST-RalGDS Rap1 binding domain precoupled to glutathione beads. Precipitates were washed three times with lysis buffer before separation on a denaturing polyacrylamide gel and immunoblot analysis as described.


    ACKNOWLEDGMENTS
 
We thank Drs. Philip Stork and James Lundblad for helpful advice and generous gifts of a number of reagents. We thank B. Maurer for aid with these studies.


    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. E-mail: maurerr{at}ohsu.edu

This research was supported by Public Health Service Grant DK-40339 to R.A.M.

Received for publication August 8, 2000. Revision received November 17, 2000. Accepted for publication December 6, 2000.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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