Estrogen Modulation of Prolactin Gene Expression Requires an Intact Mitogen-Activated Protein Kinase Signal Transduction Pathway in Cultured Rat Pituitary Cells

Jyoti J. Watters, Tae-Yon Chun, Yong-Nyun Kim, Paul J. Bertics and Jack Gorski

Department of Biochemistry (J.J.W., T.-Y.C., J.G.) and Department of Biomolecular Chemistry (Y.-N.K., P.J.B.) University of Wisconsin Madison, Wisconsin 53706


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression of the PRL gene is regulated by many factors, including cAMP, estradiol (E2), phorbol esters, epidermal growth factor (EGF), and TRH. The promoter region of the rat PRL gene has been shown to contain DNA sequences that are thought to support the direct interaction of estrogen receptors (ERs) with DNA. It is by this direct ER/DNA interaction that estrogen is thought to modulate expression of PRL. We report here that estrogen-induced PRL expression requires an intact mitogen-activated protein kinase (MAPK) signal transduction pathway in cultured rat pituitary cells (PR1 lactotroph and GH3 somatolactotroph cell lines). Interfering with the MAPK signaling cascade by inhibiting the activity of MAPK kinase (MEK) ablates the ability of estrogen to induce PRL mRNA and protein. In these cell lines, estrogen activates extracellular regulated protein kinases ERK-1 and ERK-2 enzyme activities maximally within 10 min of 1 nM E2 treatment. This activity is blocked by pretreatment of the cells with the MEK inhibitors PD98059 and UO126. The mechanism by which ERKs-1 and -2 are activated by estrogen appears to be independent of c-Src since the effects of estrogen on PRL gene expression are not affected by herbimycin A or PP1 administration. c-Raf-1 may be involved in the effects of E2 because estrogen causes the rapid and transient tyrosine phosphorylation of c-Raf-1. The ER antagonist ICI 182,780 blocks both ERK-1 and ERK-2 activation in addition to PRL protein and mRNA, implying a central role for the classical ER in the activation of the MAPK pathway resulting in PRL gene expression.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogen controls the expression of PRL in pituitary lactotrophs, primarily by a transcription-dependent mechanism (1). Classically, estrogen acts by binding to its nuclear receptors that, in turn, interact with specific sequences of DNA (estrogen response elements, EREs) present in the promoters of many genes to modulate their expression. The PRL promoter contains potential EREs (1, 2, 3, 4, 5, 6, 7, 8, 9) although thus far, only large regions of PRL promoter DNA have been shown to confer estrogen responsiveness in reporter gene assays (5, 10). The estrogen response of the PRL promoter is also complicated by the presence of Pit-1 binding sites that have been shown to be required for both basal PRL expression as well as estrogeninduced PRL expression (11, 12, 13, 14, 15).

There are increasing reports of signal transduction cascades that seem to be activated by estrogen in various tissues and cell lines. The mitogen-activated protein kinase (MAPK) pathway is one such pathway, although an exact biochemical mechanism and the endogenous, physiologically relevant consequences of this activation remain unclear. Upon expression of the human estrogen receptor (ER) in the non-ERexpressing COS cells, Migliaccio et al. (16) showed that estrogen activates the tyrosine kinase c-Src, and by this means, transiently activates the MAPKs ERK-1 and -2 (extracellular regulated protein kinases 1 and 2). They showed that estrogen also activates MAPKs in MCF-7 cells. The physical interaction between c-Src and the ER were performed in vitro using ER purified from MCF-7 cells and columns to which c-Src was immobilized. Under these conditions it was shown that the ER interacts with c-Src, and presumably, this interaction results in the estrogen-induced activation of the MAPKs. A physiological consequence of estrogen activation of this pathway, cellular proliferation, has since been shown both in breast cancer cells and in transfected cell systems (17, 18). Similarly, other groups have described the rapid and transient activation of ERKs 1 and 2 in various other cell lines and tissues (cardiomyocytes, colon carcinoma cells, bone cells, and breast carcinoma cells) (19, 20, 21).

We describe here, in a naturally estrogen-responsive, intact cell system, the relationship between estrogen activation of ERKs -1 and -2 and PRL gene expression in pituitary cells. The PRL secreting pituitary cell line, PR1, used in these studies was derived from a diethylstilbestrol-treated Fischer 344 female rat. These cells have been described previously (22, 23) and have been shown to respond to estrogens by increased PRL synthesis and cell proliferation. We have studied the signal transduction pathways activated by estrogen in both PR1 cells and in the more commonly used GH3 pituitary cell line and have used the endogenously expressed and estrogen-regulated PRL gene as a physiological measure and pharmacological readout of the activation of these pathways. The primary function of pituitary lactotrophs is to synthesize and secrete PRL. Our results indicate that the rapid and transient activation of ERKs-1 and -2 by estrogen in these cells results in PRL gene transcription since pretreatment of the cells with MAPK kinase (MEK) inhibitors blocks the ability of estrogen to promote PRL transcription. Estrogen treatment of these cells also appears to result in the rapid tyrosine phosphorylation of c-Raf-1, an event that is thought to be important in enzymatic activation and biological function of the c-Raf-1 enzyme (24). The mechanism of c-Raf tyrosine phosphorylation/activation by estrogen remains unclear; however, it appears to be performed by a herbimycin- and PP1-insensitive tyrosine kinase, i.e. by an enzyme(s) other than those of c-Src family of soluble tyrosine kinases.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Effects of Estrogen and MEK Inhibitors on PRL Protein Production
Treatment of PR1 cells with 17ß-estradiol for 24 h results in a dose-dependent increase in PRL protein production (Fig. 1AGo, top panel). Pretreatment of the cells with the MEK inhibitor PD98059 interferes with the ability of estrogen to promote PRL protein accumulation, while the inhibitor alone had no effect. Intracellular PRL protein accumulation (Fig. 1AGo, lower panel) is difficult to detect in this cell line (22); however, the highest dose of estrogen (1 nM) increased intracellular levels of PRL detectably within 24 h. And as seen with secreted PRL in the top panel, administration of the MEK inhibitor also reduced PRL accumulation in the intracellular pools, indicating that the effects of the MEK inhibitor are not solely an effect on secretion in these cells. Figure 1BGo graphically presents the optical densities of bands measured from Western blots of secreted PRL from cultured PR1 cells after 24 h of treatment with the agents used in Fig. 1AGo. Data represent the mean ± SEM graphed as fold induction over vehicle-treated control cells (n >= 4). The MEK inhibitor had no effect on cell viability as assessed by trypan blue staining, MTT assay (OD490, vehicle, 0.218 ± 0.029; PD98059, 0.219 ± .014), [3H]methionine incorporation into protein (vehicle, 26,842 ± 829 cpm; PD98059, 26,457 ± 857 cpm), or on DNA synthesis as measured by [3H]thymidine incorporation (vehicle, 7,843 ± 940 cpm; PD98059 8519 ± 397 cpm) (mean ± SEM). To more specifically control for the effects of the MEK inhibitor on the expression of other genes, another estrogen-responsive gene, cyclin D3, was evaluated. Cyclin D1 has been shown to be transcriptionally responsive to estrogen (25, 26), most likely by virtue of the presence of an ERE in its promoter (25). Interestingly, cyclin D1 is not expressed at detectable levels in pituitary cells. However, a related gene, cyclin D3, is the predominant cyclin expressed in pituitary cells (Ref. 27 and J. J. Watters and J. Gorski, unpublished observations). We show here that cyclin D3 is also regulated by estrogen (Fig. 1CGo, third panel), in a manner that is dependent upon the function of the ER, since the pure antiestrogen ICI 182,780 ablates the increase in cyclin D3 protein accumulation (Fig. 1CGo, second panel). As indicated in the lowest panel of Fig. 1CGo, in the presence of the MEK inhibitor PD98059, the ER retains the capacity to promote cyclin D3 protein increases, suggesting that the MEK inhibitor is not affecting all transcriptional functions of the ER in these cells but, rather, that it is specific for the effects of estrogen on the PRL gene.



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Figure 1. Effect of Estrogen and a MEK Inhibitor on PRL Synthesis in PR1

Cells PR1 cells were treated for 24 h with ethanol vehicle, 1 nM, 10 pM, or 10 fM E2. Medium was removed and used for analysis of secreted PRL (PRLs). Whole cell lysates were prepared and analyzed for intracellular PRL (PRLi) protein content as described in Materials and Methods. A, Western blot of PR1 cell medium (upper panel) and whole cell lysates (lower panel). Estrogen dose dependently increased the amount of PRL produced by these cells. Administration of the MEK inhibitor PD98059 (50 µM) dramatically reduced the ability of estrogen to promote PRL synthesis. B, Graphic representation of the quantification of optical densities obtained from the immunoblots described above (n >= 4). C, Cyclin D3 immunoblot of PR1 cells grown in medium containing DCCSS and ethanol vehicle (upper panel); medium same as control, but containing 100 nM ICI 182,780 for the times indicated (second panel); medium same as control, but containing 1 nM E2 (third panel); and medium same as control, containing E2 at the indicated concentrations and 50 µM PD98059 for 24 h (fourth panel).

 
Involvement of Tyrosine Kinases and MEK in Estrogen-Stimulated PRL Protein Synthesis
GH3 cells, another rat pituitary cell line, shows the same response to estrogen in terms of PRL production as the PR1 cells (Fig. 2AGo). Also, as seen previously in PR1 cells, the effect of estrogen on induced PRL gene expression is attenuated by administration of the MEK inhibitor PD98059. Another MEK inhibitor, UO126, with a mechanism of action distinct from that of the PD98059 compound, also blocks the ability of estrogen to promote PRL gene transcription. Interestingly, another rapid estrogen response, the down-regulation of ER{alpha}, was not affected by the MEK inhibitors (E. T. Alarid, J. J. Watters and J. Gorski, unpublished observations) providing further evidence that the inhibitors are not interfering with all estrogen responses in these cells. Because it is clear that MEK is involved in the estrogen stimulation of PRL in these lactotroph cell lines, we undertook to determine the identity of the kinase whose activity was induced by estrogen and which might be an upstream activator (either directly or indirectly) of MEK. To do so, we used the broad specificity tyrosine kinase inhibitor, herbimycin A. Herbimycin A is known to inhibit tyrosine kinases of the Src family, among others. When administered at 880 nM or at 4.4 µM, herbimycin A failed to interfere with the ability of estrogen to promote PRL expression in GH3 cells, suggesting that the tyrosine kinases whose activities are sensitive to herbimycin treatment are not involved in the effects of estrogen investigated here (Fig. 2AGo). This is unlike what has been reported previously (16, 21, 27), in the human breast cancer cell line MCF-7 and in the human colon cancer cell line Caco-2. These groups report that the tyrosine kinases c-Src and c-Yes are involved in transducing the mitogenic estrogen signal to the MAPKs. Genistein is a broad specificity tyrosine kinase inhibitor with effects on the c-Src family of enzymes. It is also a compound with estrogenic attributes when used at high concentrations (high µM range). When genistein was used at concentrations normally used to inhibit tyrosine kinase activity (low nM range), it too failed to inhibit the increases in PRL gene expression stimulated by E2 treatment (data not shown). These data support the notion that the c-Src family of enzymes may not be involved in the effects of estrogen on PRL gene expression in pituitary cells. To test the specificity of the MEK inhibitor effects we observed, epidermal growth factor (EGF) was used as a positive control for the activation of the MAPK cascade. Additionally, a pharmacological inhibitor of protein kinase A (PKA) (H89) and a negative control for the PD compound (SB202474) were used (Fig. 2BGo). H89 had no effect on estrogen- or EGF-induced PRL synthesis, while PD98059 diminished the effects of both agents. The negative control SB202474 (a structural analog of PD98059) (28) had no effect on PRL expression in response to treatment with either E2 or EGF. The pure anti-estrogen ICI 182,780 reduced PRL levels stimulated both by estrogen and EGF down to those observed with vehicle treatment (Fig. 2BGo), implying a role for estrogen receptor {alpha} (ER{alpha}) both in the actions of estrogen and in those of EGF. The ability of the pure antiestrogen to decrease EGF-induced PRL gene expression is interesting and may be explained by several possible mechanisms. The ER mediates many of the growth responses of EGF in tissues such as the uterus, and EGF receptor activation can induce ligand-independent transcriptional activation of the ER. This may be one explanation for the antiestrogen blockade of EGF-induced PRL gene expression, although this explanation cannot be all of the answer. If it were, then some of the EGF-responsive elements in the PRL promoter would have mapped to the estrogen-responsive sites. But this does not appear to be the case, with what is known of the PRL promoter at this time.



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Figure 2. Effect of Estrogen and Pharmacological Inhibitors of MEK and Tyrosine Kinases on PRL Production in GH3 Cells

A, Cells were treated for 24 h with ethanol vehicle or 1 nM E2 in the presence or absence of the MEK inhibitors PD98059 (50 µM) or UO126 (10 µM) or the tyrosine kinase inhibitor herbimycin A (880 nM and 4.4 µM). PRL secreted into the tissue culture medium was immunoblotted (representative blot from at least four separate experiments). B, Cells were treated with ethanol vehicle, 1 nM E2, or 50 ng/ml EGF in the presence or absence of the PKA inhibitor H89 (5 µM), the MEK inhibitor PD98059 (50 µM), the inactive structural analog of PD98059, SB202474 (50 µM), or the pure antiestrogen ICI 182,780 (100 nM). Secreted PRL was measured by immunoblotting (representative blot from at least three separate experiments).

 
Signaling Enzymes Involved in Estrogen Effects on PRL Gene Expression
As shown previously, estrogen stimulation of PRL expression is decreased by administration of the MEK inhibitors PD98059 and UO126 (Fig. 3AGo). Because it had previously been reported in MCF-7 cells that the initiating enzyme of the estrogen-induced MAPK cascade was c-Src, and we saw no effect on estrogen-stimulated PRL gene expression using the broad specificity tyrosine kinase inhibitors herbimycin and genistein (Fig. 2AGo), we used a more specific inhibitor of the c-Src family of soluble protein tyrosine kinases, PP1. As shown in Fig. 3AGo, PP1 fails to interfere with the ability of estrogen to induce PRL gene expression. PP1 was used at several concentrations, two of which are shown in Fig. 3AGo. The doses used here were the same as those empirically determined to inhibit whole-cell tyrosine phosphorylation in GH3 cells stimulated by EGF (data not shown). These data suggest that MEK activity is involved in the mechanism by which estrogen increases PRL expression, while tyrosine kinases such as c-Src may not be. To further investigate the role of c-Src in the actions of estrogen, we treated GH3 cells with 1 nM E2, immunoprecipitated c-Src, electrophoresed the proteins by SDS-PAGE, and immunoblotted with antiphosphotyrosine-specific antibodies. The c-Src tyrosine kinase enzyme is activated by tyrosine phosphorylation on either of its two tyrosine residues (reviewed in Refs. 29, 30). If estrogen were stimulating Src tyrosine kinase activity, then an increase in the overall tyrosine phosphorylation status of the c-Src protein would be observed. As shown in Fig. 3BGo, the overall tyrosine phosphorylation of the c-Src protein was unchanged in response to estrogen treatment, but was slightly decreased in the presence of PP1, the Src kinase inhibitor, indicating that the PP1 compound had the capacity to affect basal Src kinase activity in these experiments. The lower panel shows the above blot, stripped and reprobed with c-Src antibodies, to show that fairly equal amounts of protein were immunoprecipitated. Since MEK activity appears important for estrogen-induced PRL expression, and the enzyme c-Raf-1 is well known to phosphorylate and activate MEK, we evaluated the ability of estrogen to activate c-Raf-1. This was done utilizing Western blotting techniques and a phosphospecific antibody directed against the dually phosphorylated tyrosine resides 340 and 341 of the c-Raf-1 protein. Phosphorylation of these residues is believed to correlate with enzymatic activation (24). As shown in Fig. 3CGo, estrogen rapidly and transiently results in the tyrosine phosphorylation of c-Raf-1 in PR1 cells (Fig. 3CGo, upper panel), while having no effect on total c-Raf-1 protein (Fig. 3CGo, lower panel). Investigation of the enzymes that follow Raf and MEK in the MAPK signaling cascade, the MAPKs ERK-1 and ERK-2, indicated that they too were activated in a rapid and transient manner by estrogen in PR1 cells. The upper panel of Fig. 3DGo shows an immunoblot of the enzymatically active ERK-1 and -2 enzymes, using an antibody that specifically recognizes the dually phosphorylated and hence enzymatically active MAPKs. Estrogen appears to activate these enzymes maximally within 10 min of treatment. Activation of c-Raf-1 appeared maximal within 5 min of estradiol treatment, a time frame that precedes that of ERK activation, suggesting that Raf activation is upstream of ERK activation. Pretreatment of cells with the MEK inhibitor PD98059 blocks the ability of estrogen to activate the MAPKs, implying that the activation of MEK by estrogen is involved in ERK activation. The lower panel of Fig. 3DGo shows the results of a reprobing of the blot in the upper panel, with an antibody that recognizes total ERK-1 and -2 proteins (both active and inactive). This blot shows that while there is not detectable activated ERK isoform activity in all lanes of the upper panel, there are abundant and approximately equal amounts of total ERK protein in each lane.



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Figure 3. Effect of Estrogen on PRL Synthesis in the Presence of the c-Src Inhibitor PP1 in GH3 Cells

Effect of estrogen on c-Raf-1 and ERK 1 and ERK 2 activation in PR1 cells. A, GH3 cells were treated with ethanol vehicle or 1 nM E2 for 24 h in the presence or absence of the MEK inhibitors PD98059 (50 µM and 100 µM) and UO126 (10 µM and 30 µM) or the c-Src family of tyrosine kinase inhibitors PP1 (1 µM and 2 µM). Secreted PRL was evaluated by immunoblotting techniques (representative blot from at least three separate experiments). B, Phosphotyrosine immunoblot (i.b.) of c-Src immunoprecipitation (ip) from GH3 cells treated with ethanol vehicle or 1 nM E2 for the indicated times or the c-Src inhibitor PP1 (2 µM). C, PR1 cells were treated with 1 nM E2 for 5, 10,15, 30, 60, or 120 min. Control cells (0) were treated with ethanol vehicle. Supernatants (100,000 x g) were collected as described in Materials and Methods and were immunoblotted using an antibody that specifically recognizes the phosphorylation of the c-Raf-1 protein at tyrosine residues 340 and 341 (upper panel). The lower panel shows the blot from the upper, stripped and reprobed with an antibody that recognizes total c-Raf-1 protein, illustrating that total c-Raf-1 protein is unchanged by estrogen treatment. D, PR1 cells were treated with 1 nM E2 for 5, 10,15, 30, 60, or 120 min. Control cells (0) were treated with ethanol vehicle. Supernatants (100,000 x g) were immunoblotted using an antibody to the dually phosphorylated and enzymatically active MAPKs ERK-1 and ERK-2 (upper panel). The blot was stripped and reprobed with an antibody that recognizes both ERK 1 and ERK 2 total protein. (Blots shown are representative blots from at least five separate experiments.)

 
Estrogen Dose-Response and Steroid Specificity of MAPK Activation
Active ERK-1 and -2 levels in GH3 cells were determined by immunoblotting with the antibodies described above. Optical densities were measured by a Molecular Dynamics, Inc. densitometer as described in Materials and Methods. Data obtained in this way are shown graphically in Fig. 4Go, expressed as the ratio between the active (phospho-ERK) level and that of total ERK in arbitrary units. Each graph is representative of at least three separate experiments. Figure 4AGo illustrates a dose-response curve of estrogen on MAPK activation; 1 nM E2 activated both ERK-1 and ERK-2 activities as shown in Fig. 3Go; 10 pM E2 also appeared to stimulate the MAPKs although to a lesser degree than the higher dose of E2. The lowest dose of estrogen tested, 10 fM, did not have measurable activity by this method. Figure 4BGo shows the results of progesterone, both at 1 nM and 10 pM. Progesterone, 1 nM 17{alpha}-estradiol, and testosterone (Fig. 3CGo) lacked the ability to induce ERK-1 and ERK-2 activation, illustrating the specificity of this activation by estrogen. These hormones also had little capacity to promote PRL gene expression in this cell system (data not shown). The pure ER antagonist ICI 182,780 blocked the ability of estrogen to promote activation of the MAPKs ERK-1 and -2. These results are in agreement with our observations that the ICI compound decreases the effects of E2 on PRL protein expression.



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Figure 4. Steroid Specificity and Estrogen Dose-Response Curves of ERK1 and ERK2 Activation

GH3 cells were treated with A: ethanol vehicle (0) or 1 nM (—•—), 10 pM ({vdots} {square}{vdots}) or 10 fM (- - -{diamond}- - -) E2; B: 1 nM E2 (—•—), 1 nM progesterone ({vdots} {square} {vdots}) or 10 nM progesterone (- - - {diamond} - - -); C: 1 nM E2 (—•—), 1 nM 17{alpha}-estradiol ({vdots} {square} {vdots}), 1 nM testosterone (- - - {diamond} - - -), or 1 nM E2 + 100 nM ICI 182,780 ({vdots} {Delta} {vdots}) for 5–30 min as indicated in the figure. Data are presented as graphic representations of the optical densities obtained from immunoblotting using anti-active ERK antibodies as described in Materials and Methods and as shown in Fig. 3CGo above. Results are plotted as the ratio of phosphospecific ERK-1 to total ERK-1 immunoreactivity (left panel) and the ratio of phosphospecific ERK-2 to total ERK-2 immunoreactivity (right panel) in arbitrary units. Data shown are representative of at least three separate experiments.

 
Effect of Estrogen and MEK Inhibitors on PRL mRNA
Total RNA was isolated from PR1 cells and subjected to standard agarose-formaldehyde electrophoresis as described in Materials and Methods. Cells were treated for the times indicated, and Northern blot analyses were performed. Blots were hybridized with a radioactively labeled mouse PRL cDNA probe and imaged on a Phosphorimager (Molecular Dynamics, Inc., Sunnyvale, CA). Blots were stripped and reprobed with CHOB (Fig. 5Go, A and B, lower panels), a ribosomal protein RNA as a control for equal loading. There was a measurable increase in PRL mRNA in response to E2 treatment for 12 h (Fig. 5AGo, upper panel). This effect was blocked by administration of the MEK inhibitor PD98059. The increase in PRL mRNA elicited by estrogen treatment was also diminished by administration of the ICI compound (Fig. 5BGo, upper panel). The PD compound was able to inhibit the effects of estrogen at all time points tested (Fig. 5Go, A and B, upper panels). These data support the contention that the effects of the MEK inhibitors are occurring not only at the level of the protein itself, but also at the level of the RNA. Since it is widely believed that the effects of estrogen on PRL gene expression are primarily transcriptional (1, 2, 3, 4, 5, 6, 7, 8, 9, 31, 32, 33), and the MEK inhibitor can block the increases in estrogen-induced PRL mRNA, it is likely that MEK (or its downstream kinases) exert their effects transcriptionally on the PRL gene.



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Figure 5. Effect of Estrogen, MEK Inhibitor, and Antiestrogen on PRL mRNA in PR1 Cells

A, Time course of 1 nM E2 on PRL mRNA induction (upper panel) in the presence or absence of the MEK inhibitor PD98059 (50 µM). PR1 cells were treated as specified in Fig. 5AGo for 3, 6, 12, or 18 h and then total RNA harvested and electrophoresed as indicated in Materials and Methods. The blot was stripped and reprobed with a cDNA to CHOB, a ribosomal RNA protein as a control for loading. B, PRL mRNA expression in PR1 cells after 1 nM E2, 50 µM PD98059, or 100 nM ICI 182,780 treatment for 18 or 24 h.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The data reported here strongly imply a role for the MAPK pathway in the actions of estrogen on PRL gene expression. Estrogen has been known since 1995 to activate the MAPK pathway in certain cell types. We show for the first time, that the estrogen-induced activation of PRL gene expression in pituitary lactotroph cells requires an intact MAPK cascade. Interference with this pathway by utilizing inhibitors of MEK ablates the ability of estrogen to increase PRL synthesis (Fig. 1Go). Additionally, we have shown that estrogen activates the MAPKs ERK-1 and ERK-2 in pituitary lactotroph cell lines (Figs. 2Go and 3Go). Fischer 344 rats are a strain highly sensitive to the estrogen induction of lactotroph tumors. When treated with diethylstilbestrol for 1–21 days, these animals show an increase in MAPK activity in whole pituitary extracts (J. J. Watters and J. Gorski, unpublished observations). These data indicate that estrogen activation of MAPKs in lactotrophs may also play a role in pituitary function in vivo.

The identity of the tyrosine kinase activated by estrogen in pituitary lactotrophs that induces the tyrosine phosphorylation of c-Raf-1 remains unknown. Based on our observations using the pharmacological inhibitors herbimycin A and PP1, the c-Src family of soluble tyrosine kinases are likely not involved in mediating the effects of estrogen on the PRL gene, although the c-Src family kinases have been found to be involved in the estrogen activation of tyrosine phosphorylation and MAPK activation in MCF-7 cells (16, 27). The involvement of p21 Ras in this pathway in our cell system is also not yet clear. The activation of rapid tyrosine phosphorylation and subsequent MAPK pathway activation is generally associated with the actions of a membrane-localized receptor related to those of the growth factor/cytokine receptor family. There have been several reports of membrane ERs (34, 35, 36, 37). In fact, GH3 cells have recently been shown to express ERs on their plasma membranes (38, 39). The presence of a receptor for estradiol on the cell surface may allow for interactions with proteins that result in the tyrosine phosphorylation of Raf family kinases and subsequent MAPK pathway activation.

The promoter of the PRL gene has been extensively studied in terms of its ability to respond to various hormones and growth factors. TRH, EGF, cAMP, phorbol esters, and estrogen can regulate expression of the PRL gene. It is believed that the means by which estrogen modulates the expression of this gene is by the direct binding of activated ERs to specific DNA sequences located in the promoter of the PRL gene (5, 6, 7, 8, 9). This promoter also contains binding sites for additional transcription factors some of whose functions are regulated by protein kinases activated by the MAPK and/or PKA pathways. Pit-1 protein function has also been shown to be required for estrogen activation of the PRL promoter (11, 13). Recently, it has been shown that ERs and Pit-1 proteins physically interact in the GH3 and PR1 cell lines used in our studies here, in an estrogen-dependent manner (40). However, it is not yet known whether this interaction with the ER is dependent upon the phosphorylation status of Pit-1 or the ER.

It is also possible that estrogen-induced activation of the MAPK pathway results in the functional modification of a MAPK-responsive transcription factor that controls the expression of PRL, which in concert with the DNA sequences to which ERs bind, results in the cooperative activation of PRL gene expression. It might be hypothesized that independent MAPK activation of a transcription factor that interacts with the PRL promoter would result in the estrogen-responsive sites of the PRL promoter mapping to some elements that are common to those which respond to growth factors and other kinases. However, the activation of signal transduction cascades, although the enzymes themselves are common to different stimuli, are in reality quite specific for the agonist. Therefore, the fact that estrogen-responsive elements in the PRL promoter may not at this time be known to coincide with other transcription factor binding sites known to be responsive to activation of this pathway by other agonists does not necessarily negate the possibility that estrogen may be stimulating the direct phosphorylation of some MAPK-responsive transcription factor.

Another possible explanation for the observed effects with the MEK inhibitors might be related to the function of the ER itself. The ER{alpha} is phosphorylated on ser167 by pp90rsk1 (a downstream target enzyme of the ERKs) (41), and on ser118 by the MAPKs themselves (42). These residues appear important in the recruitment of transcription coactivators such as p68 (43) since the mutation of either or both of these sites results in significant inhibition of ER transcriptional activity (41). Therefore, another possible mechanism for the effects we have observed may involve the estrogen-stimulated MAPK activity having effects on the phosphorylation status of the ER, which in turn results in a further transcriptional enhancement of ER function. Thus, estrogen would serve a dual function: to bind the receptor and stimulate its transcriptional activity while simultaneously interacting with the MAPKs to further increase its function. At this time, there are several possibilities that could explain our observations. There is likely not one single mechanism at work at the PRL promoter, but rather a complex interaction between estrogen, the ER, and other factors involved in the MAPK pathway.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Reagents
PD98059 and PP1 were obtained from Calbiochem (La Jolla, CA). 17ß-Estradiol, 17{alpha}-estradiol, testosterone, progesterone, and herbimycin A were acquired from Sigma (St. Louis, MO). UO126 was purchased from Promega Corp. (Madison, WI).

Tissue Culture
PR1 and GH3 cells were maintained in 100-mm Falcon plates (Becton Dickinson and Co., Franklin Lakes, NJ) in phenol red-free DMEM (MediaTech, Herndon, VA) containing 100 U/ml Penicillin G sulfate, 100 µg/ml Streptomycin sulfate, 0.25 µg/ml Amphotericin, and 2 mM L-Glutamine (Life Technologies, Inc., Gaithersburg, MD) in the presence of 10% FBS. Culture medium was changed every 2 days until cells reached confluency. For experimentation with steroids, cells were washed twice with HBSS and the growth medium was replaced with DMEM containing the above supplements and 10% 3X dextran-coated charcoal-stripped FBS (DCCSS) (44). In addition, cells were treated with 100 nM ICI 182,780 (generously provided by Zeneca Pharmaceuticals, Macclesfield, UK) for 2–3 days to eliminate endogenous estrogenic activity present in the FBS used to propagate the cell line. After this time, cells were washed twice with HBSS and replaced with fresh DMEM/DCCSS for 1 day before stimulation with E2, EGF (Upstate Biotechnology, Inc., Lake Placid, NY), or other hormones as indicated in the figure legends.

PRL, Cyclin D3, and c-Src Protein Analyses
PR1 and GH3 cells were propagated as described above. Cells were washed and replated in DMEM/DCCSS in six-well dishes (Falcon) at a density of 7.5 x 105 to 1.0 x 106 in 2 ml culture medium. The next day, the medium was replaced and the cells were treated with hormones at the concentrations indicated in the figure legends. Twenty four hours after treatment, equal volumes of medium were removed from the wells and mixed with 2X SDS/PAGE sample buffer and assayed by Western blot for the presence of secreted PRL. The cells were lysed in RIPA buffer (1X PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 µg/ml phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 2 µg/ml pepstatin, 1 mM sodium orthovanadate) on ice for 30 min. DNA was sheared by passing the sample through a 22-gauge needle and centrifuged at 15,000 rpm for 20 min. Protein content in the supernatant was assayed by Bradford Protein Assay (Bio-Rad Laboratories, Inc. Hercules, CA), and equal amounts of protein were used for c-Src immunoprecipitations or were loaded per lane and separated by a 10% SDS/PAGE gel (45). Proteins were transferred to Immobilon polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA) and blocked overnight in 10% nonfat milk in PBST (PBS with 0.5% Tween-20). Membranes were incubated with rabbit polyclonal anti-rat PRL antibodies (provided by NIDDK- National Hormone and Pituitary Program, Torrance, CA) or cyclin D3 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) antibodies at a dilution of 1:1000 for 2 h at room temperature in PBST/5% milk. After washing, horseradish peroxidase-linked secondary antibodies were added and target protein bands were detected using the Renaissance enhanced chemiluminescence detection system (NEN Life Science Products, Boston, MA). Relative band intensities were quantified using a Densitometer and Image-Quant software (Molecular Dynamics, Inc.). For c-Src immunoprecipitations, RIPA supernatants were precleared and treated as previously described (46). Briefly, supernatants were precleared by incubating with Protein A-conjugated agarose beads at 4 C for 1 h, followed by centrifugation at 4 C for 1 min at 14,000 rpm. The supernatants were then subjected to immunoprecipitation using 2 µg of polyclonal anti-Src antibody (SC-18) (Santa Cruz Biotechnology, Inc.), or nonspecific IgG overnight at 4 C. This was followed by an incubation with Protein A-agarose beads (Santa Cruz Biotechnology, Inc.) for 2 h at 4 C. After washing the beads three times with ice-cold RIPA buffer, the bound proteins were eluted with SDS-PAGE sample buffer and subjected to electrophoresis and immunoblot analysis using a cocktail of antiphosphotyrosine antibodies 4G10 and PY-20 (Upstate Biotechnology, Inc., Lake Placid, NY, and Transduction Laboratories, Inc., Lexington, KY, respectively) or a monoclonal anti-c-Src antibody (clone GD11) (Upstate Biotechnology, Inc.).

PRL mRNA Analysis
PR1 cell total RNA was isolated using Tri-Reagent (Molecular Research Center, Inc., Cincinnati, OH). RNA (20 µg) was electrophoresed in 1.2% agarose-formaldehyde gels in MOPS buffer (0.04 M MOPS, 0.01 M sodium acetate, 1 mM EDTA, pH 7.0). Gels were rinsed in diethylpyrocarbonate-treated water to remove residual formaldehyde and transferred to Hybond-N nylon membrane (Amersham Pharmacia Biotech, Piscataway, NJ) by capillary transfer overnight in 20x SSC (3 M sodium chloride, 0.3 M sodium citrate). Membranes were briefly washed in 6x SSC after transfer and UV cross-linked using the UV Stratalinker 1800 (Stratagene, La Jolla, CA) and air dried. The mouse PRL cDNA was excised from the pGEM2 plasmid (kindly provided by Michael Karin, UCSD) using HindIII and EcoRI, and gel purified. PRL cDNA probe was randomly primed using the Prime-It II Random Primer Labeling kit (Stratagene) and 32P {alpha}-dATP. Incorporated probe was separated from unincorporated nucleotides using Quick Spin G-50 Sephadex columns (Roche Molecular Biochemicals, Indianapolis, IN). Membranes were hybridized in QuikHyb hybridization solution (Stratagene) containing 1 x 106 cpm/ml radiolabeled PRL cDNA probe for 4 h at 50 C. Membranes were washed once in 2x SSC/0.5% SDS for 15 min at room temperature, once in 1x SSC/0.1%SDS for 15 min at room temperature, and then once in 1x SSC/0.1%SDS for 20 min at 65 C. Washed membranes were apposed to a phosphorimage screen and read by a Phosphorimager (Molecular Dynamics, Inc., Sunnyvale, CA).

ERK and c-Raf Kinase Activation
PR1 and GH3 cells were grown as above in 100-mm plates. They were serum starved (phenol red-free DMEM with supplements) for 16 h before stimulation with steroids or growth factor. Plates were washed twice with ice-cold PBS and twice with ice- cold harvesting buffer H (50 mM ß-glycerophosphate, 1.5 mM EGTA, 0.1 mM sodium orthovanadate, 1 mM DTT, 10 µg/ml aprotinin, 2 µg/ml pepstatin, 10 µg/ml leupeptin, 1 mM benzamidine, and 0.2 mM phenylmethylsulfonyl fluoride). Cells were scraped off the plate in 500 µl harvesting buffer H, sonicated, and subjected to centrifugation at 100,000 x g for 20 min. Supernatants were removed and assayed for protein content as described above. Cytosolic extract was loaded (50 µg per lane) and subjected to 10% SDS-PAGE. Transfer was done as described above. Membranes were blocked overnight at 4 C in 1% BSA/TBST (200 mM Tris, 1.37 M NaCl, 1% Tween-20, pH 7.6). Phosphospecific antibodies against ERKs-1 and -2 (Promega Corp.) were diluted 1:5000 in 0.1% BSA/TBST and incubated at room temperature for 2 h. Blots were washed and incubated in horseradish peroxidase-linked secondary antibody and visualized as described above. Antiphosphotyrosine (340/341)-specific c-Raf-1 antibodies (Biosource International, Camarillo, CA) were used at 1:5000. To control for equal protein loading, blots were stripped (in 100 mM 2mercaptoethanol, 2% SDS, 62.5 mM Tris, pH 6.7) at 50 C for 30 min, blocked at room temperature for 30 min in 10% nonfat milk/PBST, and then reprobed with an ERK-1 antibody (Santa Cruz Biotechnology, Inc.) at 1:5000 in 5% milk/PBST or with an anti-c-Raf-1 antibody in 5% milk/TBST.


    ACKNOWLEDGMENTS
 
We would like to thank Ms. Maria Boardman and Mr. Cris VanHout for their technical assistance and Dr. Elaine Alarid for her helpful comments and suggestions.


    FOOTNOTES
 
Address requests for reprints to: Dr. Jack Gorski, Department of Biochemistry, 433 Babcock Drive, Madison, Wisconsin 53706. E-mail: gorski{at}biochem.wisc.edu

This work was supported by NIH Grants HD-08192 and HH-07259 (J.G.) and National Research Service Award Grant F32CA81733 (J.J.W.).

Received for publication December 20, 1999. Revision received July 27, 2000. Accepted for publication August 4, 2000.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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