PRL Activates the Cyclin D1 Promoter Via the Jak2/Stat Pathway
Jennifer L. Brockman,
Matthew D. Schroeder and
Linda A. Schuler
Department of Comparative Biosciences, University of Wisconsin-Madison, Madison, Wisconsin 53706
Address 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.
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ABSTRACT
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PRL promotes cell growth and differentiation in the mammary gland, which has implications for breast cancer as well as normal development. Our data demonstrate that PRL significantly increases proliferation of mammary carcinoma cells. PRL also increases cyclin D1 levels 2-fold, which can be inhibited by actinomycin D, suggesting that transcriptional increases in cyclin D1 are important. Using a defined Chinese hamster ovary cell model system, we demonstrate that the activity of a cyclin D1 promoter-luciferase construct increases after PRL treatment. Furthermore, this increase in promoter activity is predominantly mediated by the Jak2/Stat5 signaling pathway.
The cyclin D1 promoter contains two consensus sequences for PRL-induced Stat binding (GAS sites). Disruption of Stat binding to the distal GAS site destroys PRL-induced promoter activity, whereas disruption of the proximal site has no effect. We have shown by EMSA that PRL induces Stat5a and 5b to bind to the distal GAS site, and immunoprecipitation and subsequent Western analysis of nuclear extracts from PRL-treated cells indicate that Stat5a and 5b can interact as a heterodimer in this system. These data suggest that cyclin D1 may be a target gene for PRL in normal lobuloalveolar development, as well as in the development and/or progression of mammary cancer.
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INTRODUCTION
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PRL PROMOTES cell growth and differentiation in a number of tissues, including the mammary gland. The role of PRL in proliferation of the alveolar cells during pregnancy suggests a potential role in development or progression of mammary cancer. Despite the importance of PRL in rodent models of breast cancer, though, studies in humans have not consistently established pituitary PRL as a factor in human mammary carcinoma (reviewed in Refs. 1, 2, 3, 4). However, documentation of PRL synthesized within mammary epithelial cells (5, 6), which is controlled by different mechanisms than pituitary PRL, and increased expression of PRL receptor (PRLR) in human mammary tumors (6, 7) suggest that this system may be complex, and deserves further study.
The target genes that mediate the action of PRL in the mammary gland under normal and pathological conditions are poorly understood. One target for PRL action in mammary cells may be cyclin D1 because the mammary phenotype of cyclin D1 knockout mice is similar to that of mice deficient in the expression of PRL or PRL receptor (8, 9, 10). D-type cyclins are active in G1 phase of the cell cycle, where they complex with cyclin-dependent kinases (cdks) to catalyze the transition from G1 to S phase of the cell cycle. Cyclin D1 is critical for lobuloalveolar proliferation in the mammary gland during pregnancy. However, overexpression of cyclin D1, as is seen in approximately 50% of human mammary tumors (11, 12), leads to a shortening of the G1 phase, and thus an increase in mitogenesis in mammary tumor cells in culture (13). This can also be seen in transgenic mice, where targeted overexpression of cyclin D1 in mammary epithelial cells leads to tumor formation (14). These observations suggest that strict control of cyclin D1 expression in mammary epithelial cells is necessary to separate normal development from oncogenesis. PRL may contribute to one level of control by modulating the transcriptional activity of the cyclin D1 promoter.
PRL signaling occurs through PRL receptors (PRLR), which belong to the cytokine receptor superfamily (reviewed in Refs. 15, 16, 17). These receptors are composed of an extracellular ligand binding domain, a transmembrane region, and distinct intracellular regions that lack intrinsic catalytic activity. Multiple isoforms of PRLR exist that differ in their cytoplasmic domains and signaling capabilities. All target cells, including mammary epithelial cells, express more than one form of PRLR. Thus, regulation of the complement of PRLR isoforms expressed in mammary cells may modulate the response of these cells to PRL. PRL binding at the PRLR induces the formation of a complex between the receptor and other intracellular proteins. The most well-studied PRL signaling pathway involves Jak2 tyrosine kinase and signal transducers and activators of transcription (Stats). PRLR signaling can activate Stat 1, 3, or 5, which then form homo- or heterodimers that translocate to the nucleus where they bind to specific response elements (GAS sites) in the promoters of target genes. In some systems, Stats can also bind as tetramers to tandemly linked GAS sites (18), which adds further complexity to transcriptional control. Stat proteins can either stimulate or inhibit gene transcription, depending on promoter context (19). Potential PRL-responsive GAS elements are located at -457 and at -224 in the human cyclin D1 promoter.
In addition to the Jak/Stat pathway, PRL has been shown to utilize other pathways in some systems. PRL can activate the Src family of kinases (20, 21) and also MAPKs through activation of Ras (22, 23), and Raf kinase, which stimulates the ERK pathway (24). The ERKs, in turn, can stimulate proliferation by enhancing activator protein-1 (AP-1) activity, which leads to cyclin D1 induction (25). PRL also can activate the JNK group of MAPKs, which is important for its mitogenic signaling and suppression of apoptosis in some cell types (26, 27). Both Jak-Stat and MAPK pathways are activated upon PRL treatment in several breast tumor cell lines, and may act in parallel, or may converge at some point in the signaling pathway (23, 28, 29).
In this paper, we show that PRL enhances the proliferation of mammary epithelial cells, and that PRL-induced increases in cyclin D1 levels are, in part, due to increased transcription. We have examined control of cyclin D1 promoter activity by PRL in a defined CHO cell system. We have demonstrated that: 1) PRL predominantly utilizes the Jak-Stat pathway to transduce its signal to this promoter; 2) the distal GAS site in the cyclin D1 promoter is important for PRL-induced promoter activity; and 3) Stat5a and 5b can bind to the distal GAS site and interact as a heterodimer in this system. These findings contribute to our understanding of the role that PRL plays in mammary gland development, as well as human mammary cancer, and may ultimately improve diagnostic procedures and breast cancer therapies.
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RESULTS
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PRL Treatment Enhances Proliferation of MCF7 Human Breast Cancer Cells
The importance of PRL in growth of lobuloalveoli during pregnancy and the evidence for PRL signaling in mammary tumors suggests that PRL may contribute to the development or progression of mammary cancer, in part, by increasing mitogenesis. Multiple studies have demonstrated a role for PRL, especially that produced endogenously, in proliferation of mammary epithelial tumor cell lines (reviewed in Ref. 4). As shown in Fig. 1A
, exogenous PRL stimulates proliferation of the MCF7-derived cell line, PRE-1. We have previously observed that PRL increases expression of cyclin D1, but not cyclin D3, in these cells (30). As shown in Fig. 1B
, after 6 h of PRL treatment, levels of cyclin D1 are more than doubled (2.36 ± 0.58-fold, mean ± SEM, n = 3). We have previously shown using antisense oligonucleotides, that this magnitude of increase in cyclin D1 is necessary and sufficient for PRL-induced proliferation (30). Pretreatment of cells with actinomycin D to block transcription, results in a decrease in basal cyclin D1 protein levels consistent with the rapid turnover of this cell cycle regulator. Actinomycin D treatment also prevents the PRL-induced increase in cyclin D1 protein levels (untreated control = 1; PRL treatment = 0.95 ± 0.11), which is consistent with an effect at the level of transcription. In contrast, levels of ß-actin are not altered by PRL treatment, and the greater stability of this protein is reflected in the lack of effect of actinomycin D at the 6-h time point.

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Figure 1. Growth of Mammary Epithelial Cells in Response to PRL, and Importance of Transcriptional Control of Cyclin D1 Expression
A, Representative experiment. PRE-1 cells were cultured in serum-free media for 48 h, and then treated with ( , dashed line) or without ( , solid line) 4 nM PRL. Numbers of cells were counted at each time point as described in Materials and Methods. Results are expressed as the mean ± SEM of triplicate plates. B, Representative Western blot analysis of cyclin D1 and ß-actin in cellular lysates. Cells were incubated ± 10 µg actinomycin D for 1 h, then treated ± 4 nM human PRL for an additional 6 h. Replicate experiments showed similar results.
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The Cyclin D1 Promoter as a Target for PRL Action
The effect of PRL on the transcriptional regulation of cyclin D1 was examined using a human cyclin D1 promoter-luciferase construct, D1
-944, which contains 944 bp of proximal cyclin D1 promoter sequence upstream of a luciferase reporter (31). This complex promoter contains binding sites for a number of transcription factors, some of which are diagrammed in Fig. 2
. The consensus GAS sites (TTCNNNGAA) at -457 and -224 were of particular interest for these studies because GAS sites have previously been shown to be sites for cytokine action mediated by Stats in other systems. The sequence of the distal GAS1 site is identical to the canonical Stat5 binding site (PRE) identified in the ß-casein promoter (32), whereas the proximal GAS2 sequence differs in the three internal nucleotides that typically vary among GAS sequences and is not similar to consensus sequences identified for Stats 1 and 3.

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Figure 2. Diagrammatic Representation of the Cyclin D1 Promoter
The human cyclin D1 promoter contains multiple regulatory elements. Two GAS consensus sites are located at -457 and -224, which are potential regulatory elements for Stats.
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Because of endogenously produced PRL, and a complex complement of PRLR isoforms in most breast cancer cell lines, studies relating to promoter activity were done in Chinese hamster ovary (CHO) cells. These cells produce negligible levels of endogenous PRL and only very low levels of PRLR. However, when transfected with PRLR, they are able to mediate PRL signaling to a variety of promoters. As shown in Fig. 3A
, CHO cells cotransfected with PRLR and the strongly responsive PRE3-luciferase construct, increase luciferase expression 6.5-fold in response to PRL. Cotransfection of CHO cells with the more complex D1
-944 reporter construct results in a significant increase in luciferase expression at 8.5 h after addition of PRL. At maximal activation, a 2-fold increase in luciferase expression is seen in response to PRL (Fig. 3B
), indicating that PRL positively regulates the activity of the cyclin D1 promoter.

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Figure 3. PRL Stimulates Cyclin D1 Promoter Activity
A, CHO cells were transiently transfected with the PRE3-Luciferase, PRLR, and ß-galactosidase constructs as described in Materials and Methods, and treated with (solid bar) or without (open bar) 10 nM PRL in serum-free media for 24 h. Cell lysates were assayed for luciferase activity and activities were corrected for transfection efficiencies using ß-galactosidase protein (RLU, relative light units). B, CHO cells were transiently transfected as described in A, except that D1 -944 replaced PRE3-Luciferase. Cells were treated with ( , dashed line) or without ( , solid line) 10 nM PRL in serum-free media for a time course of 048 h. Data represent the mean of at least three separate experiments, ± SEM. Asterisks indicate a statistically significant increase (P < 0.05) in PRL-treated promoter activity compared with non-PRL-treated control.
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The Jak/Stat Pathway Is Important for PRL Signal Transduction to the Cyclin D1 Promoter
Because PRL has been shown to use both the Jak/Stat and MAPK pathways for signaling in other systems, we have examined the role of these pathways in PRL signaling to the cyclin D1 promoter in this system. The Jak2dn mutant, Jak2829, lacks all of the kinase domain after amino acid 829, cannot autophosphorylate, and inhibits autophosphorylation of wild-type Jak2 (33). It also blocks PRL stimulation of transcription via the PRE in CHO cells (34). The effect of Jak2 constructs in the promoter-reporter gene assay (Fig. 4
), shows that overexpression of wild-type Jak2 results in higher basal promoter activity, and no further increase with PRL stimulation. This increase in basal promoter activity may be due to nonspecific autoactivation of Jak2 due to overexpression of the kinase, as is seen in other systems (17). The Jak2dn construct abolishes PRL induction of D1
-944 but does not affect basal promoter activity, as compared with the D1
-944 control without addition of exogenous Jak2. These results indicate that Jak2 is an important component of PRL signal transduction to cyclin D1 in CHO cells.

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Figure 4. JAK2 Is Required for PRL Signaling to the Cyclin D1 Promoter in CHO Cells
Cells were transiently transfected with D1 -944, PRLR, ß-galactosidase, and either JAK2 wt construct or JAK2 dn (JAK2829), then cultured in serum-free media with (solid bar) or without (open bar) 10 nM PRL. After 24 h, samples were assayed for luciferase activity, and ß-galactosidase activity was used to correct for transfection efficiencies. Activity is presented relative to untreated D1 -944-transfected cells. Data represent the mean of five separate experiments, ± SEM. Different letters denote significant differences in PRL-induced activity (P < 0.05).
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The PRLR has been shown to signal through Stats 1, 3, and 5 in various cell systems (35), and these Stats have been shown to be important in growth control (reviewed in Ref. 36). Therefore, the effect of overexpression of wild-type and dominant-negative Stats on PRL signaling to this promoter was examined. Overexpression of wild-type Stat1 results in PRL-induced promoter activity similar to the control (Fig. 5
); however, expression of dominant-negative Stat1 results in a loss of PRL induction. Cotransfection with a wild-type Stat3 construct results in significantly higher PRL-induced promoter activity than controls without exogenous Stats; however, the unstimulated activity is also somewhat higher so that the fold increase in response to PRL remains the same. Expression of dominant-negative Stat3 results in a loss of PRL induction; basal promoter activity is unaffected. Overexpression of both wild-type Stat5a and Stat5b markedly increase PRL-induced promoter activity without altering unstimulated levels, suggesting that the concentration of Stat5 may be limiting in these cells. Transfection of higher levels of Stat5a or Stat5b does not further increase the PRL response (data not shown), nor does cotransfection of Stats 5a and 5b augment the response over that observed with a single Stat5. Expression of dominant negative Stat5a results in both decreased basal and PRL-induced promoter activity. This construct acts as a dominant negative mutant for both Stat5a and Stat5b by forming inactive heterodimers with wild-type Stat proteins (37). Taken together, these data indicate that Stats 1, 3, and 5 are important components of the PRL signal transduction pathway in CHO cells, and that Stat5, independent of Jak2, is also important in basic mechanisms for CHO cell growth.

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Figure 5. Stat 1, 3, and 5 Are Involved in PRL Signaling to the Cyclin D1 Promoter
CHO cells were transiently transfected with D1 -944, PRLR, ß-galactosidase, and either wild-type or dominant negative Stat1, 3, or 5 constructs. Transfected cells were cultured in serum-free media with (solid bar) or without (open bar) 10 nM PRL. After 24 h, samples were assayed for luciferase activity. ß-Galactosidase activity was used to correct for transfection efficiencies, and activity is presented relative to untreated D1 -944-transfected cells. Data represent the mean of at least three separate experiments, ± SEM. An asterisk indicates a significant increase in PRL-induced promoter activity compared with non-PRL-treated cells, and different letters denote significant differences in PRL-induced activity (P < 0.05).
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To determine the involvement of the MAPK pathways in PRL signal transduction, inhibitors directed toward components in this pathway were used. PD98059, an MEK (MAPK kinase) inhibitor, prevents the activation and phosphorylation of MEK1 and MEK2 in vitro and in vivo, whereas SB203580 is an inhibitor of p38/HOG1, and can also inhibit JNK at higher concentrations (27). Treatment of D1
-944-transfected cells with 20 µM of the MEK inhibitor PD98059 has no effect on PRL-induced promoter activity (Fig. 6
), although this concentration of PD98059 inhibits ERK phosphorylation (data not shown), indicating that p42/44 MAPKs are not involved in this PRL response in these cells under these conditions. Treatment of cells with 10 µM SB203580, however, slightly but significantly reduces the extent of PRL induction. This indicates that p38 MAPKs, or possibly JNK, may play a minor role in PRL signal transduction to the cyclin D1 promoter in this system. Treatment of D1
-944-transfected CHO cells with higher concentrations of either SB203580 or PD98059 (50 µM) showed no significant change from the results seen with lower inhibitor concentrations (data not shown).

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Figure 6. Role of MAPKs in Cyclin D1 Promoter Activity
CHO cells were transiently transfected with D1 -944, PRLR, and ß-galactosidase, then treated with either vehicle control, 20 µM PD98059, or 10 µM SB203580 for 2 h. Cells were treated with (solid bar) or without (open bar) 10 nM PRL in the presence of inhibitors for an additional 24 h, and then cells were harvested and assayed for luciferase activity. ß-Galactosidase activity was used to correct for transfection efficiencies, and activity was presented relative to untreated D1 -944-transfected cells. Data represent the mean of three separate experiments, ± SEM. An asterisk indicates a significant increase in PRL-induced promoter activity compared with non-PRL-treated cells, and different letters denote significant differences in PRL-induced activity (P < 0.05).
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Mutational Analysis of the Cyclin D1 Promoter
The GAS consensus sites in the cyclin D1 promoter, and the role of Stats in the PRL response, suggest that these GAS sites may be involved in PRL control of cyclin D1 expression. To assess the involvement of these sites, we mutated the GAS sites to sequences that prevent Stat binding in the context of the intact promoter (38). The promoter containing a mutated GAS1 site is unable to respond to PRL (Fig. 7
), indicating that this site is important for PRL signaling. In contrast, mutation of the GAS2 site in the D1
-944 construct shows no effect on basal or PRL-induced promoter activity, indicating that this site is not important for PRL induction of promoter activity in this system.

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Figure 7. Mutational Analysis of GAS Sites in the Cyclin D1 Promoter
CHO cells were transiently transfected with PRLR, ß-galactosidase, and either D1 -944, or a promoter construct containing a mutated GAS1 or GAS2 site, as described in Materials and Methods. Transfected cells were cultured in serum-free media with (solid bar) or without (open bar) 10 nM PRL. After 24 h, samples were assayed for luciferase activity. ß-Galactosidase activity was used to correct for transfection efficiencies, and activity was presented relative to untreated D1 -944-transfected cells. Data represent the mean of three separate experiments, ± SEM. Different letters denote significant differences in PRL-induced activity (P < 0.05).
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PRL Induces Binding of CHO Nuclear Proteins to GAS1
PRL induces binding of nuclear proteins to GAS1, as demonstrated by EMSA, which is consistent with its role in mediating PRL signals (Fig. 8A
). Extracts from CHO-D6 cells treated with PRL for 30 min show lower mobility complexes (1 and 2) compared with extracts from untreated cells. Unlabeled GAS1 is able to compete for formation of these complexes, but an unlabeled oligonucleotide containing mutated GAS1, or unrelated (Oct-1) oligonucleotide is unable to compete for complex formation. PRL-induced complex formation at the GAS1 site reaches a maximum at 30 min, is decreased approximately 60% after 1 h, and remains stable through 6 h. After 24 h, it has returned to the levels of the untreated control (Fig. 8B
). No additional complexes other than those originally seen at 30 min (1 and 2) form at later times after PRL treatment. Treatment of the nuclear extracts with Stat1 or Stat3 antibodies does not change the mobility of the induced complexes (Fig. 8A
). Stat5b antibody shifted all of both complexes (complex 1 and 2 shifted to complex 3 and 4), whereas anti-Stat5a shifted all of complex 2, and most of complex 1. Additional Stat5a antibody did not shift the remainder of complex 1 (data not shown). While the antibody to Stat5a is made to the unique C terminus and is consequently specific for this Stat, the antibody to Stat5b can cross-react with Stat5a depending on the application and conditions (39). Therefore, we examined nuclear complexes induced by PRL in COS-7 cells, which contain very low levels of endogenous Stats, and therefore can be used to distinguish these factors (Fig. 8C
). In COS-7 cells transfected with Stat5a or Stat5b and treated with PRL, complexes of a mobility similar to those in CHO cells bound GAS1, although both complexes 1 and 2 in Stat5a-transfected cells displayed slightly lower mobility as compared with either CHO cells or Stat5b-transfected COS-7 cells. This is consistent with the somewhat higher mass of Stat5a, compared with Stat5b. In these experiments, the Stat 5a antibody shifts the mobilities of both PRL-induced complexes 1 and 2 in Stat5a-transfected COS-7 cells (COS7/5a), but not in Stat5b-transfected cells (COS7/5b). In contrast, the Stat5b antibody can shift all of both complexes in both cells, indicating that for EMSA under these conditions, anti-Stat5b cross-reacts with Stat5a. Other investigators have reported similar results (40). However, the supershifted complexes formed with Stat5b antibody in COS7/5b cells differ in mobility from those formed in COS7/5a cells, as well as those formed with Stat5a antibody in COS7/5a cells. This may reflect differences in the position of cross-reacting epitopes in the Stat5a and Stat5b complexes.

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Figure 8. PRL Induces Binding of CHO Nuclear Proteins to GAS1 by EMSA
A, CHO cells stably transfected with PRLR were treated for 30 min ± 10 nM PRL, nuclear extracts were prepared, and then were subjected to EMSA with a labeled probe specific for the GAS1 site. Some nuclear extracts were pretreated with unlabeled GAS1 probe, a mutated GAS1 probe, or unlabeled nonspecific (Oct-1) probe. Others were pretreated with antibodies to Stats 1, 3, 5a, or 5b to supershift PRL-induced complexes. B, CHO cells stably transfected with PRLR were treated with 10 nM PRL for a time course of 024 h, nuclear extracts were prepared, and then subjected to EMSA with a labeled probe specific for the GAS1 site. C, COS-7 cells transiently transfected with PRLR and either Stat5a or Stat5b were treated for 30 min ± 10 nM PRL, nuclear extracts were prepared, and then were subjected to EMSA with a labeled probe specific for the GAS1 site. Some nuclear extracts were pretreated with antibodies to Stat5a or 5b to supershift PRL-induced complexes.
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Immunoprecipitation of Stats from Nuclear Extracts
To further understand the composition of the complexes that bind to GAS1, we investigated whether Stat5a and 5b interact as homodimers or heterodimers in response to PRL in this system using immunoprecipitation and Western analysis. Under the conditions used here for Western blotting, the Stat5a and 5b antibodies are specific for the appropriate Stat5 (Fig. 9B
). Western analysis of CHO cell nuclear extracts show that both Stat5a and 5b are present in PRL-treated cells (Fig. 9A
, left and right, NE). The Stat5b antibody detects multiple bands in these extracts, which is consistent with differentially phosphorylated species of Stat5b (41). Nuclear extracts of PRL-treated CHO cells immunoprecipitated with Stat5a antibody, and then immunoblotted with Stat5b antibody (left panel), show that Stat5a and 5b coprecipitate in this system, and that it is the most highly phosphorylated species of Stat5b that associates with Stat5a. Similar results are seen in the reciprocal experiment (right panel).

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Figure 9. Immunoprecipitation of Stat5a and 5b from CHO Cell Nuclear Extracts
A, Representative Western blot of PRL-treated CHO cell nuclear extract (NE), and nuclear extract immunoprecipitated with Stat5 antibodies (IP). Left panel, Immunoprecipitation with Stat5a antibodies and Western blot with Stat5b antibodies. Right panel, Immunoprecipitation with Stat5b antibodies and Western blot with Stat5a antibodies. B, Representative Western blot of PRL-treated COS-7 cell nuclear extracts that had been transiently transfected with Stat5a (COS7/5a) or Stat5b (COS7/5b). Blot was incubated with Stat5b antibody (left panel) or Stat5a antibody (right panel).
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Taken together, these results indicate that activated Stat5a and 5b homodimers are both able to form a predominant (band 1) and lesser (band 2) complex with the GAS1 site (Fig. 8C
). In CHO cells with endogenous Stat5a and 5b, all of the lower mobility complex (band 2), and most of the fastest mobility complex (band 1) contain Stat5a (Fig. 8A
). The data from immunoprecipitation and Western analysis suggest that a large number of Stat5 complexes exist as heterodimers. Complex 1 that remains after supershift with Stat5a antibody may contain Stat5b homodimers, or may contain Stat5a that is not readily accessible to the Stat5a antibody. Nevertheless, as seen in Fig. 8C
, differences in the Stat5 content of the complexes do not account for their differences in mobility. This may indicate the presence of other factors that are recruited to the GAS1 site by PRL.
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DISCUSSION
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Our studies have shown that PRL-induced proliferation of mammary epithelial cells is associated with an increase in the expression of the cell cycle regulator, cyclin D1. The increase in cyclin D1 expression seen here appears to occur at the level of transcription because treatment of cells with actinomycin D blocks the PRL-induced increase in cyclin D1 expression. Our data showing that the activity of a cyclin D1 promoter-luciferase reporter construct is enhanced by PRL treatment in the defined CHO cell system provides additional evidence that PRL can modulate the transcriptional activity of cyclin D1. This is an important step in understanding the mitogenic response to PRL during normal mammary gland development, as well as a potential role in breast cancer, because recent studies have shown that cyclin D1-deficient mice are resistant to mammary cancer induced by some oncogenic processes, including ras and neu, but not myc or Wnt-1 (11).
In this system, JAK2 appears to be essential for the PRL response at this promoter, and Stat5 is the primary mediator of this response, based on the ability of the dominant negative construct to block PRL signaling through endogenous Stat5, and overexpression of wild-type Stat5 to augment PRL-induced promoter activity. This involvement of Stat5 in PRL-induced proliferation is consistent with results seen in a number of cell types, which show that Stat5 is important in cellular proliferation and protection from apoptosis (reviewed in Refs. 36 and 42). Similar results are seen in the mammary gland in vivo, where genetic deletion of Stat5a reduces proliferation of alveolar cells in mammary epithelium (43).
Mutational analysis of the two GAS sites within the cyclin D1 promoter reveals that the distal GAS1 site is required for PRL-induced promoter activity, whereas the proximal GAS2 site is not involved. Another cytokine, IL-3, also activates the cyclin D1 promoter through Stat5 binding to the distal GAS site in hematopoietic cells (44). In our CHO cell system, Stat5 binds to the GAS1 site as well, and the results from immunoprecipitation and EMSA studies in both CHO and COS-7 cells indicate that Stat5a and Stat 5b can bind as homodimers or heterodimers. The two complexes in our EMSA also indicate that Stat5 may complex with other cellular components at the GAS1 site, which can include other DNA binding proteins that interact with coactivators to modulate transcription initiation. Stat5a and 5b can act interchangeably in our system because both can activate the cyclin D1 promoter-luciferase construct, and both bind to the GAS1 site by EMSA. Similar results were described for the ß-casein promoter (45). However, differences in the mammary phenotype of the Stat5a and Stat5b knockout animals (46, 47), and distinct responses to some mediators such as src (48), suggest that there may be differences in the activity of these Stats.
PRL can also activate Stat1 and Stat3 in a number of cell types, including lymphoid, myeloid, and mammary epithelial cells (35). Relatively little is known about the target genes for these Stats in the PRL response, although PRL has been shown to direct binding of Stat1 to a GAS site in the interferon regulatory factor-1 promoter, resulting in transcriptional activation of the interferon regulatory factor-1 gene (49, 50). In our studies, dominant-negative Stat1 and Stat3 significantly decreased PRL-induced cyclin D1 promoter activity, although the mechanism(s) by which they contribute to promoter activity and signaling by PRL are not apparent. Stat1 or Stat3 were not detected by EMSA in complexes at the GAS1 site, as reported here, or at the GAS2 site (data not shown), suggesting that they do not directly bind DNA in this promoter. However, these negative data may be confounded by a low strength of association for these Stats, or masked antibody epitopes, although both antibodies have been successfully used for EMSAs in other systems. Alternatively, Stat1 or Stat3 may associate with other transcriptional regulators outside the GAS site, as has been shown in the C/EBPß promoter, where Stat3 interacts with a CRE-like site to direct transcriptional activity without binding directly to the DNA (51). Stats 1 and 3 may also indirectly contribute to cyclin D1 expression via synthesis of another transcription factor. Like Stat5, both Stats 1 and 3 have been shown to play important roles in growth control in multiple systems (36, 42, 52, 53).
Many growth factors and cytokines, such as IL-3 (44), increase transcription of cyclin D1 by activation of Ras, which signals through a number of downstream targets, including MAPKs. ERK1 and 2 increase cyclin D1 expression in many systems (54, 55). However, our results with the PD98059 inhibitor suggest that ERKs 1 and 2 are not involved in the PRL response in CHO cells, despite the fact that PRL is able to activate these kinases in these cells (56). The slight inhibition of PRL-induced cyclin D1 promoter activity that is seen with the SB203580 inhibitor indicates that some MAPKs may play a small role in promoter activation in this system. At the concentration used here (10 µM), SB203580 can inhibit both p38 MAPKs and JNK (27). PRL has been shown to increase mitogenesis via JNK in Nb2, PC12, and bovine mammary gland epithelial cells (26, 27, 57). In CHO cells, PRL may also activate JNK, which could modulate binding of transcription factors to the AP-1 site in the distal region of the cyclin D1 promoter. Similarly, PRL may activate p38 MAPK, as has been reported for GH (58), leading to activation of transcription factors such as activating transcription factor (ATF) and cAMP response element binding protein (CREB). These potential signaling pathways appear to be secondary to the Jak/Stat pathway in CHO cells. However, in light of cell specificity in other systems, PRL may utilize a broader range of pathways in other cells. For instance, PRL treatment activates Src, and the related kinase Fyn, in some cells (20, 21). v-Src has been shown to induce cyclin D1 expression in a Stat3-dependent manner at the transcriptional level (59), although it is not known whether Stat3 binds directly to the cyclin D1 promoter. Furthermore, in mammary epithelial cells, v-src activation of the cyclin D1 promoter also requires MAPKs, which induce binding of ATF-2/CREB to the ATF/CRE site in the proximal region of the promoter (60). Further experiments will be necessary to examine PRL activation of Src family members in our system.
Our studies examined PRL signaling to the cyclin D1 promoter in a very defined system. In a more complex in vivo system with multiple mitogens, the cyclin D1 promoter would be regulated by a network of signaling cascades. It remains to be seen whether PRL can cooperate or synergize with other signaling components to further enhance cyclin D1 promoter activity because the promoter contains cis-regulatory regions that are targets for multiple signaling pathways. In particular, PRL activation of MAPKs may alter the composition and activity of complexes at both the proximal ATF/CRE site, as well as the more distal AP-1 site of the cyclin D1 promoter. In MCF-7 cells, estrogen induces binding of ATF-2/c-Jun heterodimers to the ATF/CRE site (61), while v-src increases ATF-2/CREB complexes (60), suggesting some mitogen specificity. In addition to the MAPKs, PRL might further modulate activity at this site through STAT 3 (59). Similarly, PRL modification of AP-1 composition at the distal AP-1 site, a target for both Ras- and estrogen-dependent signaling (58, 62), may also influence cyclin D1-dependent growth (58, 63).
Understanding the signaling mechanisms that PRL utilizes in mammary cells, although they appear to be complex, will provide a mechanism to exploit for development of new drug therapies, and will ultimately aid in developing novel breast cancer therapies.
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MATERIALS AND METHODS
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Materials
The cyclin D1 antibody (MS-210-P1) was purchased from Neomarkers (Fremont, CA). Stat antibodies (Stat1, sc-592X; Stat3, sc-7179; Stat5a, sc-1081X; Stat5b, sc-835), control rabbit IgG (sc-2027), and protein A-agarose (sc-2001) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
-32P-ATP was purchased from Amersham Pharmacia Biotech (Arlington Heights, IL), and the inhibitor PD98059 was from Calbiochem (La Jolla, CA). Bovine PRL, Lot AFP7170E, was obtained through NHPP, NIDDK and Dr. A. F. Parlow. The ß-actin antibody (Clone AC-40), the SB203580 inhibitor, and all remaining reagents were obtained from Sigma-Aldrich Corp. (St. Louis, MO).
Plasmid Constructs
The D1
-944 construct was generously provided by Dr. Rolf Muller at the IMT in Marburg, Germany (31). Point mutations of the D1
-944 construct were made with the MORPH Site-specific Plasmid DNA Mutagenesis Kit (5'
3', Inc., Boulder, CO). The GAS1 site (TTCTTGGAA) was mutated to CCCTTGGTA, and the GAS2 site (TTCTATGAA) was mutated to GTCTATGGG to prevent Stat binding (38). Mutations were confirmed by sequence analysis. The PRE3 control plasmid consists of three copies of the consensus sequence for the Stat5 binding site (TTCTTGGAA) from the ß-casein promoter (PRL response element, PRE), upstream of a luciferase reporter (30). The long form of the bovine PRLR was expressed in pcDNA3 (Invitrogen, Carlsbad, CA) (64). Jak2 constructs were provided by Dr. D. Wojchowski (33), Stat1 and 3 constructs were provided by Dr. J. Darnell (65, 66), Stat5a and 5b wild-type constructs were provided by Dr. J. Rosen (49, 67), and Stat5a dominant-negative construct was from Dr. P. Bertics, University of Wisconsin. The cytomegalovirus-ß-galactosidase construct was obtained from Dr. C. Caskey (68).
Mammary Cell Proliferation and Cyclin D1 Protein Expression
MCF7-derived cell line PRE-1 (30) was plated at 5 x 105 cells per 60-mm tissue culture dish. After seeding, the cells were washed once with PBS and cultured in serum-free Roswell Park Memorial Institute 1640 medium for 48 h, before treatment with vehicle or 4 nM PRL. The cells were harvested with trypsin at the indicated times, stained with trypan blue and counted using a hemocytometer. For Western analyses, cells were plated at 2 x 106 cells per 100-mm tissue culture dish, and cultured in serum-free media for 48 h. Cells were treated with vehicle or 10 µg/ml actinomycin D for 1 h, and then treated with vehicle or 4 nM PRL for an additional 6 h. Cells were lysed, 30 µg protein fractionated by SDS-PAGE, and cyclin D1 and ß-actin were detected as described (30). Quantification of the signals was performed using a Molecular Dynamics, Inc. (Sunnyvale, CA) Densitometer and ImageQuant, version 4.2a software.
Cell Culture and Transient Transfection
Chinese hamster ovary (CHO-K1) cells (ATCC no. CCL-61), and COS-7 cells (ATCC no. CRL-1651), were maintained in DMEM/F12 containing 5% FBS and penicillin/streptomycin (Life Technologies, Inc., Gaithersburg, MD). For transfection, CHO cells were plated into 12-well tissue culture plates at 1 x 105 cells/well and incubated 24 h at 37 C in a humidified 5% CO2 incubator. Cells were washed once with serum-free DMEM-F12, placed in serum-free media containing 1 mM hydroxyurea, and incubated 1618 h at 37 C to arrest the cells. Media was replaced with fresh serum-free media and cells were transfected using SuperFect (QIAGEN Inc., Valencia, CA). DNA (1.5 µg total DNA/well) was mixed with 3 µl SuperFect reagent and incubated 10 min at 25 C. The DNA/SuperFect complex was added to the cells in serum-free media and incubated 4 h at 37 C. The transfection complex was then replaced with fresh serum-free media with or without 10 nM bovine PRL and cells were incubated 24 h at 37 C. For harvesting, cells were washed once with PBS and 55 µl of 1x cell culture lysis reagent (Promega Corp., Madison, WI) was added to each well. Cells were scraped from the dish, the lysates were cleared by centrifugation, and the supernatants were assayed immediately or stored at -80 C.
Reporter Gene Assays
Luciferase activity of cell lysates was determined by adding 25 µl lysate to 100 µl of luciferase substrate in a Turner Designs Model 20/20 luminometer (Turner Designs, Sunnyvale, CA). ß-Galactosidase activity was measured by the Galacto Light Plus kit (Tropix Inc., Bedford, MA). Luciferase values were corrected for transfection efficiency by determining the ratio of luciferase activity/µl to ß-galactosidase activity/µl and expressed as relative luciferase units.
Preparation of Nuclear Extracts
CHO cells that were stably transfected with PRLR (CHO-D6) (34) were plated at a density of 2.5 x 106 cells/10 cm plate in DMEM-F12 media with 5% FBS and 0.5 mg/ml geneticin, and incubated at 37 C overnight. Cells were placed in serum-free DMEM-F12 for approximately 18 h at 37 C, and then stimulated with 10 nM PRL for various times before harvesting nuclear extracts as described previously (69). COS-7 cells were plated as described above in DMEM-F12 media with 5% FBS, incubated 2 d at 37 C, then transiently transfected with PRLR and either Stat5a or Stat5b wild-type constructs, using SuperFect reagent. Cells were placed in FBS-containing media for 24 h at 37 C, then placed in serum-free DMEM-F12 for 18 h at 37 C. Cells were stimulated with 10 nM PRL for 30 min at 37 C before harvesting nuclear extracts. Protein concentrations of nuclear extracts were determined by the BCA protein assay (Pierce Chemical Co., Rockford, IL).
EMSA
EMSA was performed as described previously (69). Dried gels were visualized and quantitated on a Storm Phosphoimaging System. The GAS1 probe consisted of a synthetic double-stranded oligonucleotide (Integrated DNA Technologies, Coralville, IA) corresponding to the sequences 5'-CGTGGAGTTCTTGGAAATGCGCC-3'. To assay for competition of GAS1 binding, a mutant double-stranded GAS1 oligonucleotide (5'-GTGCCCTCGTGGAGCCCTTGGAAATGCGC-3') and a double-stranded nonspecific Oct-1 oligonucleotide (5'-AGAGGATCCATGCAAATGGACGTACG-3') were used. For supershift assays, 1 µg of antibody was incubated with nuclear extracts for 45 min at 25 C before addition of radiolabeled probe.
Immunoprecipitation and Western Analysis
Nuclear extracts (
200 µg) were precleared by adding 0.25 µg control rabbit IgG and 20 µl Protein A-agarose and incubating at 4 C for 30 min. Agarose beads were pelleted by centrifugation at 1,000 x g for 5 min at 4 C, and supernatants were removed to fresh tubes. For immunoprecipitation, pre-cleared lysate (70100 µg) was mixed with 2 µg of Stat antibody and incubated for 1 h at 4 C. Protein A-agarose (20 µl) was then added and the reactions were incubated overnight at 4 C. Agarose beads were pelleted by centrifugation at 1,000 x g for 5 min at 4 C, and supernatants were removed and discarded. Pellets were washed 4 times with 1 ml PBS, and the final pellet was resuspended in 40 µl 1x electrophoresis sample buffer. For Western analyses, nuclear extracts or immunoprecipitated protein was fractionated by SDS-PAGE, and Stat5 was detected as described (30).
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ACKNOWLEDGMENTS
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The authors wish to thank Dr. Paul Bertics and Dr. Jon Houtman for the Stat5a dominant negative construct, and Tom Hnasko for technical assistance with mutagenesis of promoter constructs.
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FOOTNOTES
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This work was supported in part by NIH R01-CA-78312 and T32-HD-07259, the University of Wisconsin Comprehensive Cancer Center, and the University of Wisconsin Center for Womens Health and Womens Health Research.
Abbreviations: AP-1, Activator protein-1; ATF, activating transcription factor; CHO, Chinese hamster ovary; CREB, cAMP response element binding protein; GAS,
-interferon activation sequence; MEK, MAPK kinase; PRE, PRL response element; PRLR, PRL receptor; Stat, signal transducers and activators of transcription.
Received for publication October 8, 2001.
Accepted for publication December 21, 2001.
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