Prolactin and Estrogen Enhance the Activity of Activating Protein 1 in Breast Cancer Cells: Role of Extracellularly Regulated Kinase 1/2-Mediated Signals to c-fos

Jennifer H. Gutzman, Sarah E. Nikolai, Debra E. Rugowski, Jyoti J. Watters and Linda A. Schuler

Molecular and Environmental Toxicology Program (J.H.G., L.A.S.) and Department of Comparative Biosciences (J.H.G., S.E.N., D.E.R., J.J.W., L.A.S.), University of Wisconsin-Madison, Madison, Wisconsin 53706

Address all correspondence and requests for reprints to: L.A. Schuler, Department of Comparative Biosciences, University of Wisconsin, 2015 Linden Drive, Madison, Wisconsin 53706. E-mail: schulerl{at}svm.vetmed.wisc.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Despite the important roles of both prolactin (PRL) and 17ß-estradiol (E2) in normal mammary development as well as in breast cancer, and coexpression of the estrogen receptor (ER) and PRL receptor in many mammary tumors, the interactions between PRL and E2 in breast cancer have not been well studied. The activating protein 1 (AP-1) transcription factor, a known regulator of processes essential for normal growth and development as well as carcinogenesis, is a potential site for cross-talk between these hormones in breast cancer cells. Here we demonstrate that PRL and E2 cooperatively enhance the activity of AP-1 in MCF-7-derived cells. In addition to the acute PRL-induced ERK1/2 activation, PRL and E2 also individually elicited delayed, sustained rises in levels of phosphorylated p38 and especially ERK1/2. Together, these hormones increased the dynamic phosphorylation of ERK1/2 and c-Fos, and induced c-fos promoter activity. Synergistic activation of the transcription factor, Elk-1, reflected the PRL-E2 interaction at ERK1/2 and is a likely mechanism for activation of the c-fos promoter via the serum response element. The enhanced AP-1 activity resulting from the interaction of these hormones may increase expression of many target genes that are critical for oncogenesis and may contribute to neoplastic progression.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE IMPORTANCE OF mammogenic hormones such as prolactin (PRL) and 17ß-estradiol (E2) in normal mammary growth and development (reviewed in Refs. 1, 2, 3, 4, 5) has led to investigation of their roles in the development and progression of breast cancer. E2 has been clearly defined as a mammary mitogen, and antiestrogen therapy is the most successful treatment for patients with estrogen receptor-{alpha} (ER{alpha})-positive breast tumors (reviewed in Refs. 6, 7, 8, 9). A role for PRL has been more controversial. However, several separate lines of evidence support a role for this hormone in breast cancer as well (reviewed in Refs. 10, 11, 12). These include recent large epidemiological studies correlating circulating PRL levels with breast cancer incidence and preneoplastic lesions associated with high breast cancer risk, reports of high levels of PRL receptor (PRLR) expression in tumors, and mammary PRL synthesis permitting autocrine/paracrine activity. Although the relationships between E2 and PRL in physiological activities of the mammary gland are only beginning to be understood, ER{alpha} and PRLR are coexpressed in many breast tumors (13, 14, 15), and PRL can increase ER{alpha} levels in breast cancer cells (16, 17). However, surprisingly little is known about how PRL and E2 may interact in mammary carcinogenesis, particularly regarding signaling cross-talk within target cells.

The transcription factor, activating protein 1 (AP-1), is a likely target for interaction between PRL and E2. AP-1 is a dimeric complex of basic leucine-zipper proteins, including the most studied protein families, Jun (c-Jun, JunB, JunD) and Fos (c-Fos, FosB, Fra-1, Fra-2). Jun and Fos specifically bind as homo- or heterodimers to AP-1-responsive elements [also known as 12-O-tetradecanoyl-13-acetate (TPA)] response elements), and regulate gene transcription. The activity of AP-1 components is regulated at multiple levels, including transcription, mRNA stability, and protein phosphorylation and stability (reviewed in Ref. 18). MAPKs, including the c-Jun N-terminal kinases (JNKs), ERKs, and p38 MAPKs, play critical roles at each of these regulatory levels (reviewed in Refs. 18 and 19).

Since its discovery, the multifaceted role of AP-1 in normal cellular functions including cell proliferation, survival, and differentiation, as well as in processes important in carcinogenesis, such as transformation, invasion, and angiogenesis, is increasingly appreciated (reviewed in Refs. 18, 19, 20, 21, 22). Overexpression of AP-1 proteins in vitro can induce cell transformation and proliferation, and in transgenic models, can induce tumorigenesis. AP-1 has been implicated in many types of human cancer, including breast cancer. Jun/Fos activity and/or expression correlated with tumor grade, cell cycle-regulatory protein expression, estrogen receptor (ER) expression, and/or tamoxifen resistance and metastases in several studies (23, 24, 25, 26, 27), demonstrating the importance of AP-1 in clinical disease.

We have recently shown that PRL rapidly activates AP-1 in breast cancer cells (28). PRL directs a complex signaling network to this transcription factor involving multiple proximal pathways. Janus kinase (Jak) 2 is required for the PRL response, although c-Src kinase, phosphatidylinositol 3'-kinase (PI3K), and protein kinase C, also contribute to this activity. ERK1/2 are the primary downstream activators of c-Jun, JunB, and c-Fos in this system, resulting in increased protein levels, posttranslational modifications, and DNA binding of these components. These pathways provide multiple opportunities to interact with estrogen signaling in mammary carcinogenesis in vivo.

E2 activates AP-1 elements via a mechanism distinct from the classical estrogen response element (ERE). In the model proposed by Kushner et al. (29), the ER acts as a coactivator by protein-protein interactions with AP-1 family members to enhance transcription of genes whose promoters contain AP-1 sites (reviewed in Ref. 29). This mechanism does not require binding of ER to DNA and is dependent not only on ER isoform and ligand-specific activation of the AF-1 and AF-2 regions of the ER, but also on cell context (29, 30, 31).

The importance of E2 in our current understanding of mammary carcinogenesis and therapeutic approaches, and our growing awareness of the contributions of PRL to this disease, point to the importance of studying the interactions between PRL and E2 in breast cancer. We have developed a derivative of the well-studied, ER{alpha}-positive breast cancer cell line, MCF-7, which does not express PRL endogenously and is therefore more sensitive to exogenous PRL (32). In this MCF-7-derived cell model, we determined that PRL and E2 interact to enhance AP-1 activity by increasing the delayed phosphorylation of ERK1/2, levels of phosphorylated c-Fos, and c-fos promoter activity.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PRL and E2 Interact to Enhance the Activation of AP-1 in Breast Cancer Cells
To investigate PRL and E2 interactions in stimulation of AP-1 activity, we transiently transfected PRL-deficient MCF-7 cells (32) with an AP-1 reporter gene construct containing four AP-1 consensus responsive elements (4XAP-1-luc) (33), as well as additional long PRLR (lPRLR) to maximize signal. The lPRLR is the most abundant isoform in the normal mammary gland (34, 35), as well as in several breast cancer cell lines including MCF-7 cells (Brockman, J. L., and L. A. Schuler, unpublished observations) and contains the largest cytoplasmic domain of the alternatively spliced PRLR isoforms (10).

As seen in Fig. 1AGo, treatment with PRL increased AP-1 activity after 6 h as we have shown previously (28); E2 treatment alone or in combination with PRL had no effect at this time. However, after 24 h, E2 treatment had induced AP-1 activity 2- to 3-fold, similar to previous reports (29), and PRL-stimulated AP-1 activity remained elevated (28). Interestingly, at this time, both hormones together further increased AP-1 activity, suggesting cross-talk between PRL and E2 signaling pathways to this transcription factor complex. Experiments conducted in the presence of the cell cycle inhibitor, hydroxyurea, did not alter the AP-1 induction by PRL, E2, or both hormones together, suggesting that this activation was not a result of increased DNA synthesis (data not shown). The pXP2-luc enhancerless parent vector was not activated in the presence of the lPRLR or hormone treatment (data not shown).



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Fig. 1. PRL and E2 Interact to Enhance AP-1 Activity at 24 h

A, Effect of time on E2 activation of AP-1 and interaction with PRL. Cells were cotransfected with 4XAP-1-luc, lPRLR, and ß-galactosidase. After transfection, cells were washed and treated with or without 4 nM PRL and/or 1 nM E2 for 6 or 24 h. Cell lysates were harvested and assayed for luciferase and ß-galactosidase activity as described in Materials and Methods. B, Continued presence of PRL is required for AP-1 activation. Cells were cotransfected with 4XAP-1-luc, lPRLR, and ß-galactosidase. After transfection, cells were treated with or without 4 nM PRL and/or 1 nM E2. Incubation in hormone(s) is indicated by the solid arrow, followed by washout (time shown); continued incubation in serum-free conditions is indicated by the dashed arrow. All cell lysates were harvested 24 h after the initial treatment. Lysates were assayed as in panel A. In panels A and B, relative activity represents the mean of the corrected luciferase activity from at least three independent experiments, represented as mean fold change relative to the vehicle-treated control transfection ± SEM. For statistical analyses, hormone treatment groups harvested at the same times (panel A), or washed at the same times (panel B), were compared using one-way ANOVA, followed by Student-Newman-Keuls multiple comparison test. Different letters denote significant differences within these groups (P < 0.05). In panel A, the asterisk denotes a significant difference between the same treatment groups harvested at 6 and 24 h using Student’s t test (P < 0.005).

 
To determine whether the prolonged activation of AP-1 by PRL and the interaction between PRL and E2 were dependent on the continual presence of hormone, cells were treated for variable times, after which the hormonal treatments were washed away at the times indicated, and the incubations were continued in serum free media until harvest at 24 h after the beginning of treatment (Fig. 1BGo). This removal of hormones in the media did not diminish E2 activation of AP-1, which is not surprising because E2 is retained in the nucleus (36). Interestingly, however, when the E2-containing media were removed after 18 h, followed by harvest and analysis 6 h later, AP-1 activity was nearly double that of cells that had been incubated undisturbed with E2 for the full 24 h. This may indicate activation of stress-induced pathways by the washing procedure that potentiate E2 signals to this enhancer or reflect the actual time course of E2 action. In contrast, no PRL-induced activity was detected if PRL was removed at 18 h before assay 6 h later, suggesting that PRL-induced effects require the continued presence of the hormone. PRL also did not enhance E2 activity at this time, possibly reflecting a similar requirement for prolonged exposure to hormone, although we cannot rule out that E2 had already maximized the response.

Proximal Pathways for PRL and E2 Signaling to AP-1
We employed E2-conjugated BSA to determine whether the E2 effect resulted from activation of a membrane ER. E2-BSA has been shown to bind and activate the membrane ER, but is not capable of entering the cell (37, 38). As seen in Fig. 2AGo, left, E2-BSA alone did not activate AP-1 or interact with PRL to signal to AP-1, suggesting that the E2 effects observed here require intracellular E2. Because antiestrogens can activate AP-1 in certain cell contexts, in contrast to classical EREs (29), we examined the effects of two of these compounds. As shown in Fig. 2BGo, ICI 182,780 modestly reduced AP-1 activity, but did not alter the ability of PRL to increase activity above these levels. In contrast, tamoxifen did not alter unstimulated activity (data not shown).



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Fig. 2. Proximal Signaling Pathways for PRL and E2 Induction of AP-1 Activity at 24 h

A, E2-BSA, which activates only membrane ER, does not activate AP-1 alone or enhance PRL induction. Left, Cells were cotransfected with 4XAP-1-luc, lPRLR, and ß-galactosidase. After transfection, cells were treated with 4 nM PRL, 1 nM E2, 1 nM E2-BSA, or 100 nM ICI 182,780 as indicated. Lysates were harvested 24 h after treatment and assayed for luciferase and ß-galactosidase activity. Relative activity represents the mean of the corrected luciferase activity from at least three independent experiments, represented as mean fold change relative to the vehicle-treated control transfection ± SEM. For statistical analysis, groups were compared using one-way ANOVA, followed by Student-Newman-Keuls multiple comparison test; different letters denote significant differences (P < 0.05). Right, To confirm that E2-BSA cannot activate nuclear ER, cells were cotransfected with oxytocin receptor (OT)-ERE-luc, lPRLR, and ß-galactosidase. Cells were treated with vehicle, 1 nM E2, 1 nM E2-BSA, or 10 nM E2-BSA for 24 h and assayed as above. Representative experiment with each data point in triplicate ± SD and graphed as relative luciferase units (RLU). B, ICI 182,780 slightly reduces unstimulated AP-1 activity but does not alter the ability of PRL to activate AP-1. Cells were transfected and treated with or without PRL, with or without ICI 182,780 as above, and activation of the luciferase reporter was determined as above. Results are presented and analyzed as for panel A. C, Importance of Jak2 in PRL and E2 activation of AP-1. Cells were cotransfected with 4XAP-1-luc, lPRLR, and ß-galactosidase, as well as dominant negative (DN) Jak2, where indicated. After transfection, cells were washed and treated with or without 4 nM PRL and/or 1 nM E2 in serum-free media. Lysates were harvested 24 h after treatment and assayed as in panel A. Relative activity represents the mean of the corrected luciferase activity from at least three independent experiments, represented as mean fold change relative to the vehicle-treated control transfection ± SEM. For statistical analysis, hormone treatment groups transfected with the same constructs were compared using one-way ANOVA, followed by Student-Newman-Keuls multiple comparison test; different letters denote significant differences within these groups (P < 0.05). The asterisk denotes a significant difference in fold activation between the control group and the DN Jak2 group receiving the same hormonal treatment using Student’s t test (P < 0.005).

 
PRL is able to activate multiple kinase cascades in its target cells (reviewed in Ref. 10), and we demonstrated that several of these are important in acute signaling to AP-1 in breast cancer cells (28). Because of the well-established role of Jak2 in PRL signaling in the mammary gland (39, 40), we investigated the importance of this kinase in the sustained PRL-induced activity and interactions with E2. Cotransfection with dominant negative Jak2 completely blocked PRL-induced AP-1 activity, without altering E2 action (Fig. 2CGo). However, the response to both hormones together was only partially reduced (6-fold to 4-fold), suggesting that at least one component of the PRL enhancement of E2 signaling was not Jak2 dependent. Similar results were found with the Jak-selective inhibitor AG490 (data not shown). Despite evidence for Src in PRL signaling to AP-1 at 6 h (28) and the ability of E2 to activate c-Src in MCF-7 cells (41), neither dominant negative c-Src nor the Src family kinase inhibitor PP1 reduced hormone-induced activity (data not shown). Because PI3K was important in the acute AP-1 response to PRL, we examined the effect of the PI3K inhibitors, LY294002 and wortmannin. Although LY294002 reduced hormone-induced AP-1 activity, wortmannin did not alter the responses at concentrations that blocked the phosphorylation of Akt, suggesting that LY294002 had other nonspecific effects on AP-1 signaling pathways after treatment for this extended time (data not shown). These data indicate that PRL and E2 initiate distinct signaling pathways leading to their cross-talk at AP-1, and that Jak2 is essential for PRL signaling to AP-1, although the interaction between PRL and E2 is only partially dependent on this kinase.

The Role of ERK1/2 in PRL and E2 Activation of AP-1
Both PRL and E2 have been shown to activate MAPKs (28, 42, 43), which in turn activate AP-1 proteins by increasing their synthesis and transcriptional activity (reviewed in Ref. 19). To investigate their role in PRL and E2 signaling to AP-1, we examined PRL- and E2-induced ERK1/2 phosphorylation after acute exposure to hormone (15 min). PRL strongly increased ERK1/2 phosphorylation (Fig. 3AGo), similar to our previous reports (28, 44). In contrast, E2 treatment did not detectably increase ERK1/2 phosphorylation at 15 min. However, more prolonged treatment (12 and 24 h) with both hormones individually increased phosphorylated ERK1/2 (Fig. 3BGo), which became quite striking upon treatment with PRL and E2 together, suggesting that this pathway may play a functional role in the hormonal interaction. We therefore investigated the effect of the MAPK kinase 1/2 chemical inhibitor, U0126, on hormonal signaling to the AP-1 enhancer. U0126 has been employed extensively to inhibit ERK1/2 activation (reviewed in Ref. 45), and we have previously shown that it selectively blocks PRL-induced ERK1/2 activation in this model system (28). As shown in Fig. 3CGo, U0126 slightly raised unstimulated AP-1 activity, but blocked further induction by these hormones. U0126 also ablated the increase in cell number induced by these hormones alone and together (Fig. 3DGo). These data suggest that the delayed increases in ERK1/2 phosphorylation play critical roles in mediating PRL and E2 signals to AP-1, and increases in proliferation and/or decreases in apoptosis in these cells.



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Fig. 3. ERK1/2 Is Important in PRL and E2 Activation of AP-1 at 24 h

A and B, Phosphorylation of ERK1/2 after PRL and/or E2 treatment. PRL-deficient cells were transfected with the lPRLR isoform as described in Materials and Methods. Cells were treated with 4 nM PRL and/or 1 nM E2 for the times indicated. Cell lysates were harvested, and equal amounts of protein were examined by Western analysis for phosphorylation of ERK1/2, as well as levels of total ERK1/2 (representative experiments). C, Effect of chemical inhibition of ERK1/2 on PRL and E2 activation of AP-1. Cells were cotransfected with 4XAP-1-luc, lPRLR, and ß-galactosidase. After transfection, cells were washed and pretreated for 1 h with 10 µM U0126 and then treated with or without 4 nM PRL and/or 1 nM E2 in the presence of inhibitor for 24 h. Relative activity represents the mean of luciferase values from at least three independent experiments, represented as mean fold change relative to the vehicle-treated control transfection ± SEM. For statistical analysis, hormone treatment groups treated with the vehicle or inhibitor were compared separately using one-way ANOVA, followed by Student-Newman-Keuls multiple comparison test; different letters denote significant differences within these groups (P < 0.05). D, Effect of chemical inhibition of ERK1/2 on PRL- and E2-induced increases in cell number. Cells were cultured in serum-free media for 24 h before treatment with vehicle, 4 nM PRL, and/or 1 nM E2 with or without 10 µM U0126 for an additional 48 h, when total viable cells were counted using a hemocytometer. Results are expressed as percent of the number of cells present before treatment ± SD (representative experiment). For statistical analysis, the effect of hormone treatment was assessed as for panel C. The asterisk indicates a significant difference from the nonhormonally treated samples at time zero. DMSO, Dimethylsulfoxide.

 
Secreted Factors Do Not Mediate the Extended ERK1/2 Phosphorylation or the AP-1 Response to PRL and/or E2
Our data indicated that E2 did not activate AP-1 from a membrane receptor (Fig. 2AGo); however, E2 has been shown to increase the synthesis/ release of secreted factors, such as epidermal growth factor family members (46, 47, 48), which may contribute to E2-induced phosphorylation of ERK1/2 and AP-1 activity in our cells. Therefore, we treated cells with conditioned media (CM) from cells that had been treated with hormone for 24 h. Both hormones retain full activity over this time frame (our unpublished observations), so that measured activity is the sum of exogenous hormonal treatments as well as any factors secreted during that period. As shown in Fig. 4AGo, cells treated with PRL CM for 15 min responded with dramatically increased ERK1/2 phosphorylation, as expected (see Fig. 3AGo). Surprisingly, a similar exposure to E2 CM did not stimulate ERK1/2 phosphorylation, nor did E2 augment the effect of PRL, suggesting that the activation of ERK1/2 induced by E2 after 12 h and 24 h is not the result of E2-induced secretion of other growth factors.



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Fig. 4. Role of Secreted Factors in PRL and E2 Activation of AP-1

A, CM do not contain additional factors that increase ERK1/2 phosphorylation. Cells transfected with lPRLR, as described in Materials and Methods, were treated for 24 h with vehicle, PRL, E2, or both hormones together. After 24 h, cell media were harvested and used to treat untransfected cells for 15 min. Cell lysates were harvested, and phosphorylated ERK1/2 and total ERK1/2 protein was examined by Western analysis (representative experiment). B, CM do not contain factors that further stimulate AP-1 activity. Left, Cells were transfected and treated as in Fig. 1AGo. Right, Media from cells treated for 24 h as in Fig. 4AGo were used to treat cells similarly transfected. AP-1 activity was determined 6 h after treatment. Relative activity represents the mean of the corrected luciferase activity from at least three independent experiments, represented as mean fold change relative to the vehicle-treated control transfection ± SEM. For statistical analyses, control and CM treatment groups were compared using one-way ANOVA, followed by Student-Newman-Keuls multiple comparison test. Different letters denote significant differences within these groups (P < 0.05).

 
To further investigate this possibility, we examined the ability of PRL and E2 CM to stimulate AP-1 activity. Because the E2 response is evident only after a prolonged exposure to hormone (24 h), we treated cells for only 6 h with CM (harvested from cells treated for 24 h) to determine whether secreted factors could more rapidly induce an E2 response or enhance PRL activity. As shown in Fig. 4BGo, CM elicited the same response at this time as the normal PRL and E2 treatments. Together, these results suggest that the E2 activation of AP-1, as well as the interaction between PRL and E2, is not mediated by secondary factors secreted into the cell culture media.

Role of p38 MAPK in PRL and E2 Activation of AP-1
Like ERK1/2, p38 is also able to induce AP-1 activity, and we have shown that PRL treatment can induce acute p38 phosphorylation in these cells (28). Activation of these kinases typically is reported to occur rapidly and transiently, similar to the induction of transcription of AP-1 proteins (reviewed in Ref. 18). However, because PRL and E2 were able to stimulate ERK1/2 activity after a relatively long latency, we investigated the effect of longer hormonal treatments on p38 phosphorylation. As shown in Fig. 5AGo, both PRL and E2 stimulated phosphorylation of p38 after 12 h and 24 h of treatment, implicating this MAPK in the hormonal signals to AP-1 as well.



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Fig. 5. Role for p38 in PRL and E2 Activation of AP-1

A, Phosphorylation of p38 after PRL and/or E2 treatment. Cells were plated, transfected with the lPRLR isoform as described in Materials and Methods, and treated with 4 nM PRL and/or 1 nM E2 for the times indicated. Cell lysates were harvested, and equal amounts of protein were examined by Western analysis for phosphorylation of p38, as well as levels of total p38 (representative experiment). B, Effect of chemical inhibition of p38 on PRL and E2 activation of AP-1. Cells were cotransfected with 4XAP-1-luc, lPRLR, and ß-galactosidase. After transfection, cells were washed and pretreated for 1 h with vehicle or 20 µM SB202190 and then treated with or without 4 nM PRL and/or 1 nM E2 in the continued presence of inhibitor for 24 h. Lysates were harvested and analyzed. Relative activity represents the mean of luciferase values from at least three independent experiments, represented as mean fold change relative to the vehicle-treated control transfection ± SEM. For statistical analysis, hormone treatment groups treated with the same inhibitor were compared using one-way ANOVA, followed by Student-Newman-Keuls multiple comparison test; different letters denote significant differences within these groups (P < 0.05). DMSO, Dimethylsulfoxide.

 
We therefore employed the p38 inhibitor, SB202190 (49), to investigate the importance of this kinase in our observations. Like U0126, SB202190 elevated unstimulated activity and prevented further increases in response to both PRL and E2 alone (Fig. 5BGo). However, this inhibitor only partially reduced the ability of both hormones together to raise activity above baseline. These data suggest that p38 is important for both PRL and E2 signaling to AP-1 at 24 h, but is less critical than ERK1/2 for the PRL and E2 interaction.

JNK and c-Jun in PRL and E2 Activation of AP-1
We have previously shown that JNK1/2 activity is an important determinant of basal AP-1 activity, but it does not play a major role in the PRL-induced AP-1 activity assessed after 6 h of hormone treatment (28), despite acute PRL induction of phosphorylation of these MAPKs in these cells (28, 50, 51). Therefore, we investigated the ability of PRL and E2 to induce phosphorylation of JNK1/2 after prolonged hormone treatment. As shown in Fig. 6AGo, after 12 h, both hormones modestly increased JNK1/2 phosphorylation. After 24 h, phosphorylated JNK1/2 was elevated in the vehicle-treated control, which may be a stress response from the extended time in serum free media, and neither PRL nor E2 had further effects. Consistent with elevation of phospho-JNK1/2 at 12 h, E2, in particular, increased levels of phosphorylated c-Jun (Ser63), a JNK target (18). Like phosphorylated JNK, levels of phospho-c-Jun in unstimulated cells were elevated at 24 h, and hormone treatment had little additional effect. Total levels of c-Jun were slightly increased by both hormones individually, with no further increase by both hormones together after both 12 and 24 h of treatment. In contrast, treatment with PRL and E2 did not alter levels of JunB at these times (data not shown).



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Fig. 6. Role for JNK1/2 and c-Jun in PRL and E2 Activation of AP-1 at 24 h

A, Phosphorylation of JNK1/2 and c-Jun after PRL and/or E2 treatment. Cells were plated and transfected with the lPRLR isoform as described in Materials and Methods. Cells were treated with 4 nM PRL and/or 1 nM E2 for the times indicated. Cell lysates were harvested, and equal amounts of protein were examined by Western analysis for activation of JNK1/2, total JNK1/2, phosphorylation of c-Jun, and total c-Jun (representative experiments). B, Effect of JIP-1 and TAM-67 on PRL- and E2-induced AP-1 activity. Cells were cotransfected with 4XAP-1-luc, lPRLR, and ß-galactosidase, as well as JIP-1 or TAM-67 where indicated. After transfection, cells were washed and treated with or without 4 nM PRL and/or 1 nM E2 for 24 h. Lysates were harvested and analyzed for luciferase and ß-galactosidase activity. Relative activity represents the mean of the corrected luciferase activity from at least three independent experiments, represented as mean fold change relative to the vehicle-treated control transfection ± SEM.

 
To quantitatively address the role of this pathway in PRL and E2 signaling to AP-1 in these cells, we cotransfected JNK-interacting protein (JIP-1), which binds to JNK in the cytoplasm and thereby inhibits its action (52). As shown in Fig. 6BGo, cotransfection with JIP-1 dramatically reduced unstimulated activity, but did not alter the ability of PRL or E2 to increase AP-1 activity above the unstimulated levels. Transfection of higher amounts of JIP-1 DNA produced a dose-dependent decrease in unstimulated AP-1 activity, with no change in the fold stimulation by hormones (data not shown). A similar effect was observed in the presence of the dominant negative c-Jun construct, TAM-67, which lacks a transactivation domain (53). Together, these results indicate that JNK and one of its downstream targets, c-Jun, are important in determining the basal and maximal levels of AP-1 activity in the cell, although they are not major players in PRL or E2 signaling to this transcription factor in this system.

Role for c-Fos in PRL- and E2-Induced AP-1 Activity
We have previously shown that PRL induces phosphorylation of c-Fos in these breast cancer cells after 1 h of treatment, primarily via the ERK1/2 pathway (28). To determine whether this event also might be involved in the hormonally induced AP-1 activity at later times, we examined the effects of PRL and E2 on c-Fos by Western analysis. Longer autoradiographic exposures revealed increased c-Fos modifications after hormonal treatment, including signals of reduced mobilities (arrowhead) (Fig. 7AGo), which we have previously demonstrated to be phosphorylated c-Fos (28). These modifications were more prominent in cells treated with PRL and E2 together for 12 h. They were also evident in cells treated with the hormones individually as well as together for 24 h, despite a modest increase in these modifications in unstimulated cells at this later time. We did not detect a change in total c-Fos protein levels during this period (Fig. 7AGo, shorter exposure). However, because c-Fos undergoes rapid turnover, it is possible that steady state levels do not reflect effects on synthesis, particularly because activated c-Fos has been shown to undergo increased degradation (54). Levels of FosB were not altered (data not shown); Fra-1 is not expressed in MCF-7 cells (55).



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Fig. 7. PRL and E2 Activation of c-Fos

A, Effect of PRL and E2 treatment on c-Fos. Cells were plated and transfected with the lPRLR isoform as described in Materials and Methods. Cells were treated with 4 nM PRL and/or 1 nM E2 for the times indicated. Cell lysates were harvested, and equal amounts of protein were examined by Western analysis for c-Fos. Arrowhead indicates phosphorylated c-Fos (representative experiment). B, Effect of PRL and E2 on activation of the c-Fos promoter. Cells were cotransfected with c-Fos promoter-luciferase reporter construct (56 ), lPRLR, and ß-galactosidase. After transfection, cells were washed and treated with or without 4 nM PRL and/or 1 nM E2 for 6 or 24 h. Lysates were harvested and analyzed for luciferase and ß-galactosidase activity. C, Effect of inhibition of ERK1/2 on PRL and E2 activation of the c-Fos promoter. Cells were cotransfected with c-Fos promoter reporter construct, lPRLR, and ß-galactosidase. After transfection, cells were washed and pretreated for 1 h with vehicle or 10 µM U0126, treated with or without 4 nM PRL and/or 1 nM E2 for 24 h, and then harvested and analyzed as in panel B. D, Effect of PRL and E2 on activation of Elk-1. Cells were cotransfected with the pFR-luciferase reporter construct containing five Gal4 binding sites, the pFA2-Elk-1 construct containing the Elk-1 transcriptional activator fused to the Gal4 DNA-binding domain, lPRLR, and ß-galactosidase. For panels B, C, and D, relative activity represents the mean of the corrected luciferase activity from at least three independent experiments, represented as mean fold change relative to the vehicle-treated control transfection ± SEM. For statistical analysis in panels B and C, hormone treatment groups treated under the same conditions were compared using one-way ANOVA, followed by Student-Newman-Keuls multiple comparison test; different letters denote significant differences within these groups (P < 0.05). In panel D, asterisks denote significant differences between the vehicle-treated control and the indicated hormone treatment using Student’s t test, (*, P = 0.027; **, P = 0.0012; ***, P = 0.0002).

 
Because transcriptional regulation is also a major site of control of total c-Fos activity, we examined the effects of these hormones on the c-fos promoter, taking advantage of the relatively short half-life of luciferase to evaluate events in a temporal context. We employed a proximal promoter construct (56) that includes the serum response element (SRE), cAMP-response elements, and a sis-inducible element recognized by signal transducers and activators of transcription (57), but contains neither the more distal imperfect palindromic estrogen-responsive element nor the GC-rich sequence recognized by Sp1, both of which also have been implicated in responses to estrogen (58, 59). As shown in Fig. 7BGo, PRL and E2 modulate c-fos promoter activity in these cells in a manner that is strikingly similar to their effects on AP-1 activity (compare Fig. 1AGo with Fig. 7BGo). After 6 h of hormone treatment, PRL, but not E2, elevated c-fos promoter activity. After 12 h, E2 remained unable to detectably affect promoter activity, but in combination with PRL, modestly elevated activity over PRL alone (~40%; data not shown). By 24 h, both PRL and E2 individually increased promoter activity, and, together, they further augmented activity.

Because of the prominent roles of ERK1/2 in hormonally induced AP-1 activity (Fig. 3BGo), we used the selective inhibitor, U0126, to examine the contribution of these MAPKs to PRL and E2 signals to the c-fos promoter. As shown in Fig. 7CGo, U0126 reduced unstimulated levels, and blocked the response of both hormones individually, similar to its effects on AP-1 activity. However, PRL and E2 together were able to modestly increase c-fos promoter activity above the vehicle-treated control, although the fold activation was reduced. In contrast, neither inhibition of p38 with SB202190, nor inhibition of JNK1/2 with cotransfected JIP-1, altered hormone signaling to this promoter (data not shown). Activity of the SRE in this promoter is regulated by MAPK activation of ternary complex factors, including Elk-1 (reviewed in Refs. 60 and 61), suggesting a likely mechanism for the interactions we observed. As shown in Fig. 7DGo, both PRL and E2 individually significantly stimulated Elk-1 activity and, in combination, dramatically synergized to activate Gal4-Elk-1. Together, these data suggest that c-Fos plays a key role in PRL and E2 induction of AP-1 activity via ERK1/2-mediated activation of Elk-1 to activate the c-fos promoter.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Physiological events in vivo are regulated by a complex interplay of signals, including multiple growth factors and hormones. However, much remains unknown about these interactions and their role in pathogenic processes. Despite the importance of both PRL and E2 in normal mammary development as well as in breast cancer (reviewed in Refs. 1, 2, 3, 4, 5), and coexpression of ER and PRLR in many mammary tumors (13, 14, 15), relatively little is known about the cross-talk between these hormones. Here we have shown that PRL and E2 cooperatively activate the transcription factor AP-1 in breast cancer cells. This enhanced AP-1 activity could increase expression of many target genes that are critical for oncogenesis, including those influencing cell survival, proliferation, differentiation, angiogenesis, and invasion, which may be important for neoplastic progression.

Despite the extensive network regulating AP-1 activity, our data demonstrate that the hormonal cross-talk between PRL and E2 described here is initiated primarily by a rise in levels of phosphorylated ERK1/2 that occurs relatively late after exposure to both hormones and stimulates c-fos promoter activity. PRL, but not E2, stimulates an early, transient rise in ERK1/2 activity in these cells as well as other cell types (reviewed in Ref. 10), which attenuates over about 2 h (28). However, both PRL and E2 elicit a delayed, sustained phosphorylation of ERK1/2, beginning considerably later than has been examined in most other studies. This is a site of interaction between these hormones. The importance of signal duration in dictating the appropriate outcome is increasingly recognized for diverse functions such as proliferation, apoptosis, and T cell activation (62, 63, 64). The duration of the early wave of ERK1/2 phosphorylation has been linked to stabilization of c-Fos protein by growth factors (62); we show here that the subsequent rise in phosphorylated ERK1/2 increases c-fos transcription as well. These events are likely to contribute to proliferation; inhibition of MAPK kinase 1/2 blocked hormonally induced increases in cell number, consistent with reports of PRL-induced proliferation in our system (44), as well as E2-induced growth of other breast cancer cell lines (43, 65). This later rise in ERK1/2 activity may be due to secondary effects moderating hormonal responsiveness (in the case of PRL), intermediary signals, and/or changes in phosphatase activity (reviewed in Ref. 66). Further experiments are required to distinguish these possibilities.

The temporal pattern of activity of the proximal c-fos promoter induced by these hormones individually and together mirrors their effects on AP-1 activity. Transcription of c-fos is regulated by multiple cis-acting elements that are highly responsive to mitogens. Many extracellular mitogenic stimuli increase the activity of the SRE enhancer via ERK1/2 phosphorylation of ternary complex factors, including Elk-1 (reviewed in Refs. 60 and 61). The inhibition of most of the hormonal responses in the current study with U0126, and the striking synergy between these hormones to activate Elk-1 activity, implicate this pathway in PRL and E2 signaling in the current studies as well. The resulting hormonally sustained c-Fos activity contrasts with many other reports of transient induction of c-Fos, which is then succeeded by other Fos family members in cycling cells (reviewed in Ref. 18). Whether this is characteristic of PRL and E2, or is particular to the phenotype of these cells, remains to be determined.

Our studies with dominant negative constructs and chemical inhibitors showed that other PRL- and E2-activated MAPKs, p38 and JNK1/2, also contribute to AP-1 activity. Interestingly, the E2-induced increase in phosphorylation of c-Jun differs from the inhibition reported at other targets (67, 68, 69). Although these kinases were not implicated in PRL/E2 activation of the c-fos promoter, like ERK1/2, both p38 and JNK1/2 can phosphorylate both c-Fos as well as c-Jun, which would enhance their transactivation potential (reviewed in Ref. 18). Furthermore, p38 has been shown to stabilize labile mRNAs, including transcripts for these AP-1 components (70). However, although both PRL and E2 slightly increased JNK1/2 phosphorylation and consequently phosphorylation of c-Jun, and c-Jun was a critical determinant of basal AP-1 activity, this cascade does not appear to be rate limiting in either PRL or E2 responses as evaluated by change relative to basal levels.

Nonclassical E2 signaling to AP-1 has been shown to play a key role in normal development of the reproductive system, as well as the mammary gland in vivo (71). The ability of the ER to function as a coactivator in this complex has been identified as one underlying mechanism (29, 31, 72). Modulation of the level and activity of ER{alpha} by ERK1/2 and p38 (Refs. 43 and 73 and references therein) may contribute to the effects of inhibitors of these kinases on unstimulated and E2-induced signals to AP-1 in the current study, as well as account for some component of the interactions with PRL. However, the inability of E2 alone to detectably increase AP-1 activity or augment PRL signals before 24 h argues against this as the major mechanism for E2 action in the present study. The relatively long latency of the effects of E2 alone on both AP-1 and the c-fos promoter, and the close temporal relationship to E2-induced phosphorylation of ERK1/2, are consistent with indirect signals. E2 has been reported to activate the SRE in the c-fos promoter via ERK1/2-mediated phosphorylation of Elk-1 (74). However, the temporal pattern of enhancer activity and relationship to ERK1/2 phosphorylation was not closely examined. The present studies also indicate a role for p38, which, like phosphorylation of ERK1/2, appears to require a relatively long exposure to hormone. p38 has been reported to be a mediator of estrogenic compounds in several cell types including breast cancer cells (75, 76), although the time course of p38 phosphorylation in these studies was not described. The mechanism(s) whereby E2 activates these MAPKs remains unclear. Our data do not support a role for either membrane-associated ER or E2-stimulated release of secreted factors. An interesting recent report showed that mutant ER restricted to the cytoplasm can activate AP-1 in an ERK1/2-dependent fashion in HC11 cells (77). E2 may employ different mechanisms to signal to AP-1 in cells of distinct phenotypes, which may contribute to the disparities in reports even among breast cancer cell lines (55, 59, 78, 79, 80). These events at AP-1 sites in situ would be further complicated by other direct and indirect E2/ER-initiated actions elsewhere in the promoter. For example, E2 directs dynamic changes in local transcriptional activators and repressors near the distal AP-1 site of the cyclin D1 promoter (81).

In contrast to the complex signaling network mediating acute PRL activation of MAPKs and, subsequently, AP-1 in these cells (28), the sustained activation of AP-1 in response to this hormone is mediated predominantly by Jak2, consistent with the central role for this kinase in PRL actions in the mammary gland in vivo (39, 40). Whereas PRL-phosphorylated Jak2 can rapidly activate the Ras-Raf-ERK cascade (42), PRL may initiate the later increases in ERK1/2 and p38 phosphorylation via indirect mechanisms as discussed above. However, our washout studies demonstrated that prolonged exposure to PRL nonetheless is required for the sustained AP-1 response. The underlying mechanisms and relationship to signal attenuation and desensitization events are under investigation. Maximal PRL potentiation of the E2 response at AP-1 also requires Jak2/p38-independent pathways not identified in the current studies. PRL activation of multiple other proximal kinases (reviewed in Ref. 10), and their potential to mediate effects on other transcriptional coactivators and corepressors similar to that reported for other growth factors (82, 83, 84), may contribute to the interaction with E2.

Our studies demonstrate that PRL and E2 can cooperatively enhance prolonged activation of AP-1, which may promote many carcinogenic processes. Differences in AP-1 responses to both estrogenic ligands (30, 31) as well as PRL (Autzman, J. H., L. M. Avendt, D. E. Rugowski, S. E. Nikolai, H. Rui, and L. A. Schuler, manuscript in preparation) in different breast cancer cell lines emphasize the importance of cell context in determining the sensitivity of this pathway to hormonal regulation, which may be altered with disease progression. Investigation of hormonal cross-talk in normal and neoplastic mammary cancer cells will increase our understanding of the interactions that occur at different stages of tumorigenesis and potentially lead to improved treatment modalities for this disease.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
The following antibodies were used for Western analyses: c-Jun, phospho-c-Jun, ERK1/2, phospho-ERK1/2, p38, phospho-p38, phospho-JNK1/2, and JNK1/2 from Cell Signaling Technology (Beverly, MA); c-Fos (sc-52X), from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Recombinant hPRL (lot AFP795) was obtained through the National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases, and Dr. Parlow. The following inhibitors were used in some experiments: U0126 from Promega Corp. (Madison, WI); ICI 182,780 from Tocris Laboratories (Bristol, UK); and SB 202190 from Calbiochem (San Diego, CA). All other reagents were obtained from Sigma-Aldrich Corp. (St. Louis, MO) unless otherwise noted.

Plasmids
The AP-1-luciferase construct (4XAP-1-luc) contains four GCN4 consensus AP-1-responsive elements located upstream of a luciferase reporter (33). The control parent luciferase vector (pXP2-luc) does not contain the AP-1 elements. The ERE-luciferase construct (OT-ERE-luc) contains three oxytocin receptor estrogen-responsive elements located upstream of a luciferase reporter (85). The c-Fos promoter construct contains 465 bp of the proximal c-Fos promoter upstream of a luciferase reporter (56). The PathDetect Elk-1 trans-reporting system was obtained from Stratagene (La Jolla, CA). Expression constructs employed were: dominant negative Jak2-{Delta}829 (dominant negative Jak2) from D. M. Wojchowski (86); JIP-1 from M. Dickens (52); dominant negative c-Jun (TAM-67) from M. J. Birrer (53); and lPRLR isoform from C. Clevenger (35).

Cell Culture
PRL-deficient MCF-7 cells were maintained as previously described (32). Before all experiments (4–5 d) cells were grown in phenol red-free RPMI 1640 containing 5% three times charcoal-stripped fetal bovine serum, penicillin, and streptomycin. All treatments were done in serum-free, phenol red-free RPMI 1640. The effect of hormones on cell number was assessed as described elsewhere (32). In brief, cells were cultured in serum-free media for 24 h before the addition of hormones in the presence or absence of 10 µM U0126 for an additional 48 h, when total viable cells were counted using a hemocytometer.

Transient Transfections
PRL-deficient MCF-7 cells were plated into 12-well tissue culture plates at 3 x 105 cells per well and allowed to adhere overnight. Cells were then serum starved for 24 h and transiently transfected using SuperFect (QIAGEN, Valencia, CA) as previously described (28). Within each experiment, total amount of transfected DNA was equalized with vector DNA. After 4 h, the transfection complex was replaced with serum-free media with or without 4 nM PRL and/or 1 nM E2 for the time indicated. Cell lysates were harvested and analyzed for luciferase, and ß-galactosidase activity and luciferase values were corrected for transfection efficiency as described (87). Transfections with the PathDetect Elk-1 trans-reporting system (Stratagene) were conducted as described above using DNA concentrations suggested by the manufacturer’s protocol.

For washout experiments, cells were plated, transfected, and treated as above. At the times indicated, cells were washed once with serum-free media, and then the incubation was continued with hormones in serum-free, phenol red-free RPMI 1640 media. All lysates were harvested 24 h after the initiation of hormone treatment, and luciferase and ß-galactosidase activities were analyzed as above. Relative activity is the mean of at least three independent experiments represented as fold change relative to the vehicle control.

Immunoblotting
For Western analyses, after estrogen withdrawal, cells were plated at 8 x 106 cells per 100-mm plate and allowed to adhere overnight. Cells were then serum starved for 24 h and transfected with the lPRLR isoform using SuperFect according to the manufacturer’s protocol. To normalize for transfection efficiency among treatment groups, cells were replated at 106 cells per 60-mm dish in phenol red-free RPMI 1640 containing 5% three times charcoal-stripped fetal bovine serum, penicillin, and streptomycin and allowed to recover overnight. The next day cells were treated in serum-free media with or without 4 nM PRL and/or 1 nM E2 for the times indicated. Cell lysates were harvested and analyzed as previously described (32). Primary antibody concentrations were as follows: phospho-ERK1/2, 1:5,000; ERK1/2, 1:1,000; phospho-p38, 1:1,000; p38, 1:1,000; phospho-JNK, 1:1,000, JNK1/2, 1:1,000; c-Jun, 1:1,000; phospho-c-Jun, 1:1,000; and c-Fos, 1:10,000.

For some experiments, CM were harvested from cells that had been transfected as described above, and treated for 24 h with PRL and/or E2. Media were added directly to cells before analysis of ERK1/2 phosphorylation or AP-1 activity.


    ACKNOWLEDGMENTS
 
We thank Drs. Jack Gorski, Margaret Shupnik, and Fern Murdoch for helpful discussions and suggestions, and Drs. Jennifer Brockman and Kathy O’Leary for critically reading the manuscript.


    FOOTNOTES
 
This work was supported by National Institutes of Health Grants R01 CA 78312 and DAMD17-01-1-0460.

First Published Online March 3, 2005

Abbreviations: AP-1, Activating protein-1; CM, conditioned media; E2, 17ß-estradiol; ER{alpha}, estrogen receptor {alpha}; ERE, estrogen response element; Jak2, Janus kinase 2; JIP, JNK-interacting protein; JNK, c-Jun N-terminal kinase; lPRLR, long PRLR; PI3K, phosphatidyl inositol 3'-kinase; PRL, prolactin; PRLR, PRL receptor; SRE, serum response element.

Received for publication August 30, 2004. Accepted for publication February 22, 2005.


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