PRL-Induced ER{alpha} Gene Expression Is Mediated by Janus Kinase 2 (Jak2) While Signal Transducer and Activator of Transcription 5b (Stat5b) Phosphorylation Involves Jak2 and a Second Tyrosine Kinase

Jonna Frasor, Uriel Barkai, Liping Zhong, Asgerally T. Fazleabas and Geula Gibori

Departments of Physiology and Biophysics (J.F., U.B., L.Z., G.G.) and Obstetrics and Gynecology (A.T.F.), University of Illinois at Chicago, Chicago, Illinois 60612

Address all correspondence and requests for reprints to: Geula Gibori, Ph.D., 835 South Wolcott, M/C 901, University of Illinois at Chicago, Chicago, Illinois 60612. E-mail: ggibori{at}uic.edu


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the rat corpus luteum of pregnancy, PRL stimulation of ER expression is a prerequisite for E2 to have any luteotropic effect. Previous work from our laboratory has established that PRL stimulates ER{alpha} expression at the level of transcription and that the transcription factor Stat5 (signal transducer and activator of transcription 5) mediates this stimulation. Since it is well established that PRL activates Stat5 through the tyrosine kinase, Janus kinase 2 (Jak2), the role of Jak2 in PRL regulation of ER{alpha} expression was investigated. In primary luteinized granulosa cells, the general tyrosine kinase inhibitors, genistein and AG18, and the Jak2 inhibitor, AG490, prevented PRL stimulation of ER{alpha} mRNA levels, suggesting that PRL signaling to the ER{alpha} gene requires Jak2 activity. However, using an antibody that recognizes the tyrosine-phosphorylated forms of both Stat5a and Stat5b (Y694/Y699), it was found that AG490 could inhibit PRL-induced Stat5a phosphorylation only and had little or no effect on Stat5b phosphorylation. These effects of AG490 were confirmed in COS cells overexpressing Stat5b. Also in COS cells, a kinase-negative Jak2 prevented PRL stimulation of ER{alpha} promoter activity and Stat5b phosphorylation while a constitutively active Jak2 could stimulate both in the absence of PRL. Furthermore, kinase-negative-Jak2, but not AG490, could inhibit Stat5b nuclear translocation and DNA binding. Therefore, it seems that in the presence of AG490, Stat5b remains phosphorylated, is located in the nucleus and capable of binding DNA, but is apparently transcriptionally inactive. These findings suggest that PRL may activate a second tyrosine kinase, other than Jak2, that is capable of phosphorylating Stat5b without inducing transcriptional activity. To investigate whether another signaling pathway is involved, the src kinase inhibitor PP2 and the phosphoinositol-3 kinase inhibitor (PI3K), LY294002, were used. Neither inhibitor alone had any major effect on PRL regulation of ER{alpha} promoter activity or on PRL-induced Stat5b phosphorylation. However, the combination of AG490 and LY294002 largely prevented PRL-induced Stat5b phosphorylation. These findings indicate that PRL stimulation of ER{alpha} expression requires Jak2 and also that PRL can induce Stat5b phosphorylation through two tyrosine kinases, Jak2 and one downstream of PI3K. Furthermore, these results suggest that the role of Jak2 in activating Stat5b may be through a mechanism other than simply inducing Stat5b phosphorylation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
IN RODENTS, PRL plays a unique role in both the rescue and continued function of the corpus luteum during pregnancy. One well established function of PRL in regulating luteal function is its ability to stimulate ER expression, thereby maintaining luteal responsiveness to E2 (1, 2). Previous investigations in our laboratory have demonstrated that PRL can stimulate both ER{alpha} and ERß expression (3). Furthermore, this stimulation occurs at the level of transcription and is mediated by the transcription factor Stat5 (signal transducer and activator of transcription 5) (4).

The PRL receptor (PRL-R) is a member of the cytokine/hematopoietic receptor superfamily. These receptors are characterized by four conserved cysteines, a Trp-Ser-X-Trp-Ser (WSXWS) motif in their extracellular domain, and no intrinsic kinase activity (5, 6). It is well established that the PRL-R is associated with the tyrosine kinase Janus kinase 2 (Jak2) and activates Jak2 rapidly upon exposure to PRL (7, 8, 9, 10, 11). Signaling through Jak2 has been shown to be necessary for both PRL-induced proliferation of Nb2 cells and regulation of gene transcription through the transcription factor Stat5 (12, 13, 14). Jak2 has been shown to activate Stat5 through phosphorylation of both Stat5a and Stat5b on specific tyrosine residues in the C terminus (15, 16). The ability of PRL to utilize Jak2 appears to be specific since overexpression of Jak1 or Jak3 does not amplify PRL signaling to milk proteins (17, 18).

In addition to the Jak/Stat pathway, PRL has been shown to activate other signaling pathways including PKC (19, 20, 21, 22, 23, 24), phosphatidylinositol 3-kinase (PI3K) (25, 26, 27), MAPK (26, 27, 28, 29, 30, 31, 32, 33), and the src family of tyrosine kinases (36, 37, 38). Recently, a role for PKC{delta} in PRL regulation of relaxin expression in the rat corpus luteum has been identified (39, 40, 41). Also, PRL is known to stimulate PI3K activity in both Nb2 and CHO cells stably transfected with the PRL-R (25, 26). PRL stimulation of PI3K activity is necessary for the activation of PKB kinase and the prevention of apoptosis in rat decidual cells (42) and Nb2 cells (27). Although the function of src tyrosine kinase activity in PRL signaling has not been well studied, it may play a role in PRL activation of the PI3K pathway (43, 44).

Our previous studies have indicated that PRL utilizes the transcription factor Stat5 to mediate regulation of ER{alpha} gene transcription (4). It is well established that PRL activates Stat5 through phosphorylation on specific tyrosine residues in the C terminus of Stat5a and Stat5b (Y694 and Y699) by the tyrosine kinase Jak2 (15, 16). Although PRL has been shown to activate many different signaling pathways, Stat5 activation has been shown only to occur by PRL through Jak2. Therefore, it seems likely that the tyrosine kinase Jak2 is involved in PRL regulation of ER{alpha} expression. The purpose of this investigation was to examine the role of Jak2 in PRL-stimulated, Stat5- mediated regulation of ER{alpha} expression. We have found that while Jak2 activity is required for both PRL stimulation of ER{alpha} expression and Stat5a and Stat5b phosphorylation, a second tyrosine kinase, downstream of PI3K, can also mediate PRL-induced Stat5b phosphorylation specifically, but not Stat5a phosphorylation. This second tyrosine kinase, however, does not appear to be involved in either Stat5b transcriptional activity or ER{alpha} expression.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To investigate whether PRL stimulation of ER{alpha} mRNA expression is tyrosine kinase dependent, primary luteinized granulosa cells were treated with PRL in the presence of two different general tyrosine kinase inhibitors, genistein or AG18. Interestingly, both of these inhibitors have been used to prevent PRL stimulation of gene expression (45, 46, 47) but failed to prevent PRL inhibition of 20{alpha}-hydroxysteroid dehydrogenase expression (10). When cells were treated with genistein, both basal and PRL-stimulated levels of ER{alpha} mRNA were decreased, as determined by semiquantitative RT-PCR (Fig. 1Go, A and B). Similar results were observed when primary luteinized granulosa cells were treated with AG18, although the lowest dose used was less effective (Fig. 1Go, C and D). These findings indicate that PRL stimulation of ER{alpha} expression is dependent upon the activation of a tyrosine kinase.



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Figure 1. Effect of General Tyrosine Kinase Inhibitors on PRL Stimulation of ER{alpha} mRNA Expression in Primary Luteinized Granulosa Cells

Primary cells were cultured for 72 h followed by a 12-h treatment with PRL (1 µg/ml) either alone or in the presence of Genistein (A and B) or AG18 (C and D). ER{alpha} mRNA expression was examined by semiquantitative RT-PCR. In addition, PCR for the ribosomal protein L19 was performed in identical aliquots of cDNA as an internal control (A and C). After PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) analysis, the intensity of the ER{alpha} signal was normalized to that of L19 (B and D). Experiments were conducted twice in triplicate with PCR for each sample performed in triplicate. Within each experiment, the data were normalized to the control (untreated) values and expressed as a percentage of control.

 
To examine whether the tyrosine kinase required for PRL action is Jak2, the inhibitor, AG490, was used. AG490 was originally described as a specific Jak2 inhibitor (48). However, recent investigations indicate that AG490 can inhibit Jak3 as well as activation of the MAPK pathway, although MAPK appears to be downstream of Jak2 or Jak3 (49, 50, 51, 52). Since PRL has not been shown to activate Jak3 and since no other specific inhibitors of Jak2 have been identified, we used AG490 to block PRL activation of Jak2. Primary luteinized granulosa cells were treated with PRL for 12 h, either alone or in the presence of 25 µM AG490, and the effect on ER{alpha} mRNA expression was examined using real-time quantitative RT-PCR (Fig. 2AGo). It was found that AG490 completely prevented PRL stimulation of ER{alpha} mRNA expression in this model, suggesting that the tyrosine kinase required for PRL action on ER{alpha} expression is Jak2. Interestingly, AG490 alone (data not shown) as well as genistein or AG18 alone appeared to decrease ER{alpha} mRNA expression, suggesting that some level of signaling through Jak2 may be required to maintain the basal level of ER{alpha} expression in primary luteinized granulosa cells.



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Figure 2. Effect of AG490, a Jak2 Kinase Inhibitor, on PRL Stimulation of ER{alpha} Expression and Stat5 Phosphorylation

A, Primary luteinized granulosa cells were treated for 12 h with PRL (1 µg/ml) alone or in combination with AG490 (25 µM). RNA was isolated and quantitative, real-time RT-PCR was carried out for ER{alpha} as described in Materials and Methods. The data for each treatment group were normalized to the level of ER{alpha} in the untreated control group and presented here as the mean and SEM for the six different samples in each group. B, Primary luteinized granulosa cells were treated with PRL (1 µg/ml) for 5 min following a 30-min pretreatment with vehicle or AG490 (25 µM). Stat5a and Stat5b were immunoprecipitated with specific antibodies, and Western blotting was performed using an antibody, which recognizes the phosphorylated form (P-Stat5) of Stat5a and Stat5b (Y694/Y699), and then with the same antibodies used for immunoprecipitation. Data presented here are representative of three independent experiments. C, COS cells were cultured in six-well plates and transfected with 0.5 µg/well ER{alpha}-luc and expression vectors for ß-gal (0.5 µg/well), Stat5b (1 µg/well), and either PRL-RL (2 µg/well) or PRL-RCA (2 µg/well). Twenty-four hours after the start of transfection, cells were treated with AG490 (25 µM) for an additional 24 h. Luciferase activity was measured in each well and normalized to the ß-gal activity within that well. The experiment was repeated three times in triplicate. Within each experiment the data were normalized to control values. Data presented here represent the combined mean and SEM for three experiments. D, COS cells were transfected and treated as described. Western blotting was performed on WCE using the same antibodies as in panel B. Data presented here are representative of three independent experiments.

 
Since we have previously shown that PRL regulation of ER{alpha} expression is mediated by Stat5 (4), we next investigated whether the effect of AG490 was due to its ability to block activation of Stat5. Primary luteinized granulosa cells were treated with PRL for 5 min, either alone or after a 30-min pretreatment with AG490. Stat5a and Stat5b were then immunoprecipitated, and phosphorylation was examined by Western blotting. Both Stat5a and Stat5b become highly phosphorylated on Y694 and Y699, respectively, in response to PRL in these cells; however, only PRL-induced phosphorylation of Stat5a was prevented by AG490 whereas phosphorylation of Stat5b was unaffected (Fig. 2BGo). These results suggest that Jak2 appears to be sufficient for PRL activation of Stat5a, whereas a second tyrosine kinase may be involved in PRL activation of Stat5b.

It is possible that the inhibitory effect of AG490 on PRL-induced ER{alpha} mRNA expression may be due to the prevention of Stat5a phosphorylation. However, as we have previously shown, both Stat5a and Stat5b can mediate ER{alpha} expression to an equal extent (4). Furthermore, we have found that Stat5b is highly expressed in luteal cells whereas Stat5a is expressed at nearly undetectable levels (data not shown). This suggests then that Stat5b may, in fact, be the major regulator of ER{alpha} expression in vivo. Therefore, we decided to further investigate the role of Jak2 in Stat5b phosphorylation and transcriptional activity using COS cells, since these cells express undetectable levels of endogenous Stat5 by Western analysis. In addition, these experiments may eliminate the possibility that the effect of AG490 on ER{alpha} mRNA expression was the result of blocking Stat5a phosphorylation. COS cells were transfected with an approximately 700-bp fragment of the ER{alpha} promoter, which has been linked to the luciferase reporter gene and previously shown to be responsive to PRL (4). These cells were also transfected with an expression vector for Stat5b and for either the long form of the PRL-R (PRL-RL), which in the absence of PRL treatment served as the control, or with an expression vector for a constitutively active PRL-R (PRL-RCA). This active receptor has previously been shown to mediate gene transcription of PRL-regulated genes (4, 53). Cells were then treated with vehicle or 25 µM AG490 for 24 h. PRL-RCA stimulation of ER{alpha} promoter-driven luciferase activity was prevented by AG490 (Fig. 2CGo). The effect of AG490 was also examined on Stat5b phosphorylation in COS cells and, as was the case in primary cells, AG490 had no inhibitory effect on Stat5b phosphorylation (Fig. 2DGo). These findings suggest that AG490 can prevent PRL stimulation of ER{alpha} promoter activity and apparent Stat5b transcriptional activity without affecting Stat5b phosphorylation.

Because it is well accepted that PRL activates Stat5b through Jak2, we decided to confirm that Jak2 is involved in PRL stimulation of ER{alpha} promoter activity and Stat5b phosphorylation. To do this, we used expression vectors for a kinase-negative and a constitutively active Jak2 (KN-Jak2, CA-Jak2). The KN-Jak2 was created by mutation to the kinase domain, rendering it inactive, while the CA-Jak2 was made by deletion of a pseudokinase domain, which acts to negatively regulate Jak2 kinase activity (54, 55). When COS cells were transfected with the ER{alpha} promoter, Stat5b, and PRL-RCA, the KN-Jak2 could completely prevent PRL-RCA stimulation of ER{alpha} promoter activity (Fig. 3AGo). The effect of KN-Jak2 on Stat5b phosphorylation was also examined. In contrast to AG490, KN-Jak2 could, in large part, reverse the effect of PRL-RCA on Stat5b phosphorylation (Fig. 3BGo). When COS cells were transfected with only the ER{alpha} promoter and Stat5b, CA-Jak2 could stimulate promoter activity in the absence of any PRL signal transduction (Fig. 3CGo). In addition, CA-Jak2 induced a high degree of Stat5b phosphorylation in the absence of PRL (Fig. 3DGo). These findings indicate that Jak2 is involved in PRL stimulation of ER{alpha} expression and can mediate Stat5b phosphorylation.



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Figure 3. Effect of Kinase-Negative and Constitutively Active Jak2 on ER{alpha} Promoter Activity and Stat5b Phosphorylation

COS cells were cultured and transfected as described in Fig. 2Go. In addition, cells were transfected with 1 µg/well either empty vector or expression vector for KN-Jak2 (A and B). Alternatively, the PRL-R expression vectors were omitted and 1 µg/well empty vector or expression vector for CA-Jak2 was included (C and D). Forty-eight hours after the start of transfection, luciferase activity was measured in each well and normalized to the ß-gal activity within that well (A and C). The experiments were repeated three times in triplicate. Within each experiment the data were normalized to control values. Data presented here represent the combined mean and SEM for three experiments. Western blotting was also performed for phosphorylated Stat5 and for Stat5b as described in Fig. 2Go. Data presented here are representative of three independent experiments.

 
Our data suggest that Stat5b is not transcriptionally active although still phosphorylated in the presence of AG490. Therefore, we next examined the effect of AG490 on Stat5b nuclear translocation and DNA binding. COS cells were transfected with Stat5b and either PRL-RL or PRL-RCA and then treated with AG490 for 24 h. As a control, cells were also transfected with KN-Jak2 or CA-Jak2. To determine whether Stat5b was located in the nucleus, Western blotting was performed for Stat5b on nuclear extracts from these transfected cells (Fig. 4AGo). In the absence of PRL-RCA, Stat5b could not be detected in the nuclear fractions. PRL-RCA induced Stat5b nuclear translocation, and this was prevented by KN-Jak2, but not AG490, while the CA-Jak2 could induce Stat5b nuclear translocation in the absence of PRL. In parallel groups, Western blotting was performed on whole-cell extracts (WCE) to ensure that Stat5b was expressed at equivalent levels (data not shown). To examine whether AG490 could prevent Stat5b DNA binding, the same nuclear extracts were incubated with a labeled probe corresponding to a consensus Stat5 response element. PRL-RCA induced the formation of one DNA-protein complex, which has been shown to contain Stat5 (15) (Fig. 4BGo). AG490 treatment had no inhibitory effect on Stat5b DNA binding while KN-Jak2 could prevent the formation of this complex. In addition, the same complex was formed by CA-Jak2 in the absence of PRL-RCA. These results indicate that in the presence of AG490, Stat5b remains phosphorylated, located in the nucleus, capable of binding DNA, and yet unable to activate transcription of the ER{alpha} gene promoter.



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Figure 4. Role of Jak2 in Stat5b Nuclear Translocation and DNA Binding

COS cells were transfected and treated as described in Figs. 2Go and 3Go. Nuclear extracts were prepared and Western blotting was performed on 20 µg of nuclear protein using a specific antibody for Stat5b (A). Data presented here are representative of three independent experiments. Alternatively, 10 µg nuclear protein were incubated with 50K cpm of end-labeled oligonucleotide corresponding to the ß-casein Stat5 response element (B). The DNA-protein complexes were separated on a 4.5% polyacrylamide gel. The gel was then exposed to a PhosphoImager for analysis. These results are representative of three independent experiments.

 
As a control, the effects of Jak2 activity on Stat5a phosphorylation and nuclear translocation were also examined in COS cells. As was the case in primary luteinized granulosa cells, AG490 could prevent PRL-RCA-induced phosphorylation of Stat5a (Fig. 5AGo). In addition, KN-Jak2 also prevented Stat5a phosphorylation in COS cells (Fig. 5BGo). Nuclear extracts were also examined for the presence of Stat5a, and it was found that both AG490 and KN-Jak2 could prevent, while CA-Jak2 could stimulate, Stat5a translocation to the nucleus (Fig. 5CGo).



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Figure 5. Role of Jak2 in Stat5a Phosphorylation and Nuclear Translocation

COS cells were cultured, transfected, and treated as described for Figs. 2Go and 3Go except that Stat5a (1 µg/well) was transfected in place of Stat5b. Western blotting was performed on WCE for phosphorylated Stat5 and Stat5a (A and B). Western blotting was also performed on nuclear extracts for Stat5a (C). Data presented here are representative of two independent experiments.

 
One possible explanation for the lack of effect of AG490 on Stat5b could be that PRL can induce Stat5b phosphorylation through two different signaling pathways. Perhaps when Jak2 is active, Stat5b is transcriptionally active, but when Jak2 is inactivated by AG490, a second tyrosine kinase can induce Stat5b phosphorylation but not its ability to regulate gene transcription. This possibility is similar to that observed for Stat5b activation by the tyrosine kinase src. It has been shown that src can induce Stat5b phosphorylation, nuclear translocation, and DNA binding. However, when Stat5b is activated by src it is not capable of stimulating gene promoter activity (56). PRL is known to activate src family members in the T-cell line Nb2, rat liver, chicken embryo fibroblasts, and embryonic astrocytes (37, 38, 57, 58). It could be possible that PRL can activate src in the presence of AG490 and that src could be responsible for a phosphorylated but transcriptionally inactive Stat5b. To examine this, COS cells were transfected with ER{alpha}-luc, Stat5b, and either PRL-RL or PRL-RCA. Cells were then treated for 24 h with PP2, a specific inhibitor of src, and the effects on ER{alpha} promoter activity and Stat5b phosphorylation were examined. PP2 was found to have no effect on PRL-RCA-induced ER{alpha} promoter activity (Fig. 6AGo). In addition, Stat5b phosphorylation was unaffected by PP2, either alone or in combination with AG490 (Fig. 6BGo). Although we have not investigated whether PRL-RCA can activate src in COS cells or whether PP2 can prevent this activation, our findings suggest that this tyrosine kinase may not be involved in PRL-RCA-induced Stat5b phosphorylation or transcriptional activity.



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Figure 6. Effect of PP2, a c-Src Kinase Inhibitor, on PRL-RCA Stimulation of ER{alpha} Promoter Activity and Stat5b Phosphorylation in COS Cells

A, COS cells were cultured and transfected as described in Fig. 2Go. Twenty-four hours after the start of transfection, cells were treated with PP2 (25 µM) for an additional 24 h. Luciferase activity was measured in each well and normalized to the ß-gal activity within that well (A). The experiment was repeated three times in triplicate. Within each experiment the data were normalized to control values. Data presented here represent the combined mean and SEM for three experiments. B, COS cells were transfected and treated as described above. In addition, cells were treated with 25 µM AG490 or 25 µM PP2, either alone or in combination. WCE were prepared, and Western blotting was performed using an antibody that recognizes the phosphorylated form of Stat5 and then with a specific antibody for Stat5b. Data presented here are representative of three independent experiments.

 
In addition to src, PRL has also been shown to activate the PI3K signaling pathway in various cell types (25, 26, 42). Therefore, the PI3K inhibitor LY294002 was used to treat COS cells that had been transfected with ER{alpha}-luc, Stat5b, and PRL-RL or PRL-RCA. LY294002 had little effect on either ER{alpha} promoter activity or Stat5b phosphorylation in the presence of PRL-RCA (Fig. 7Go). Surprisingly, the combination of AG490 and LY294002 was capable of markedly reducing Stat5b phosphorylation. These findings suggest that Stat5b can be phosphorylated by two tyrosine kinases, Jak2 and a second tyrosine kinase, which is downstream of PI3K.



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Figure 7. Effect of LY294002, a PI3K Inhibitor, on PRL-RCA Stimulation of ER{alpha} Promoter Activity and Stat5b Phosphorylation in COS Cells

A, COS cells were cultured and transfected as described in Fig. 2Go. Twenty-four hours after the start of transfection, cells were treated with LY294002 (25 µM) for an additional 24 h. Luciferase activity was measured in each well and normalized to the ß-gal activity within that well (A). The experiment was repeated three times in triplicate. Within each experiment the data were normalized to control values. Data presented here represent the combined mean and SEM for three experiments. B, COS cells were transfected and treated as described above. In addition, cells were treated with 25 µM LY294002, either alone or in combination with 25 µM AG490. WCE were prepared and Western blotting was performed using an antibody that recognizes the phosphorylated form of Stat5 and then with a specific antibody for Stat5b. Data presented here are representative of three independent experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The results of these studies indicate that PRL stimulation of ER{alpha} transcription requires Jak2 tyrosine kinase activity. Furthermore, PRL activation of Stat5a appears to be dependent on Jak2 activity only since both AG490 and KN-Jak2 could prevent PRL-induced phosphorylation of Stat5a. PRL activation of Stat5b, however, appears to be multifaceted (summarized in Table 1Go). Both PRL-induced phosphorylation of Stat5b and Stat5b-mediated ER{alpha} transcription could be inhibited by KN-Jak2 and stimulated by CA-Jak2. In contrast, the Jak2 inhibitor, AG490, prevented Stat5b transcriptional activity without affecting its phosphorylation, nuclear translocation, or DNA binding activity. As a control, AG490 was found to prevent Stat5a phosphorylation and nuclear translocation. Only when the combination of AG490 and the PI3K inhibitor, LY294002, was used could PRL-induced Stat5b phosphorylation be prevented, suggesting that Jak2 and a tyrosine kinase downstream of PI3K may be involved in Stat5b phosphorylation. However, this second tyrosine kinase does not appear to be involved in PRL stimulation of ER{alpha} promoter activity.


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Table 1. Effect of Jak2 Expression Vectors and Kinase Inhibitors on PRL Induction of Stat5b Phosphorylation, Nuclear Translocation, DNA Binding and Transcriptional Activity

 
Several potential questions and implications arise from these findings. First, two tyrosine kinases appear to be capable of mediating PRL-induced Stat5b phosphorylation on the same tyrosine residue, Y699. The first kinase is Jak2, which is well established, whereas the second tyrosine kinase would be a novel kinase apparently downstream of PI3K. The Btk/Tec family of tyrosine kinases have been shown to be activated by PI3K, presumably by virtue of their pleckstrin homology domain, which interacts with phosphoinositide products of PI3K and induces membrane localization (59, 60, 61, 62). Furthermore, one member of this family, Bmx, can induce Stat1, Stat3, and Stat5 phosphorylation and DNA binding activity (63). However, Bmx could also activate Stat5-mediated transcription (63). Whether any members of the Tec family of kinases are involved in PRL signaling remains to be investigated.

Second, it is not clear whether PRL activation of PI3K is dependent on Jak2 activity. The KN-Jak2 could, in large part, prevent Stat5b phosphorylation, suggesting that the tyrosine kinase downstream of PI3K is also prevented by KN-Jak2. In contrast, AG490 did not prevent Stat5b phosphorylation, suggesting that activation of this second tyrosine kinase is not Jak2 dependent. Previous studies in this area suggest that PRL can activate PI3K through two pathways, one involving Jak2 and the insulin receptor substrate and the other involving the src kinase fyn and the adaptor protein Cbl (43, 44, 64, 65, 66). It is possible that the activation of PI3K depends on the presence of Jak2 protein and not its activity. If the mutation to KN-Jak2 significantly changes its conformation, then the formation of a signaling complex could be disrupted and prevent PI3K activation. AG490, on the other hand, is a tyrphostin tyrosine kinase inhibitor, which presumably would only block the substrate binding site and perhaps not affect the ability of Jak2 to interact with PI3K.

Third, when Jak2 is active, Stat5b can regulate gene transcription. In the absence of Jak2 activity, Stat5b can no longer stimulate ER{alpha} promoter activity even though it is phosphorylated, located in the nucleus, and capable of binding DNA. These data suggest that Jak2 activity is required for PRL regulation of gene transcription in some fashion beyond its ability to induce Stat5b phosphorylation on Y699. The ability of src kinase to activate Stat5b phosphorylation and DNA binding without transcriptional activity support this possibility (56). Whether Jak2 may be involved in the activation of signaling molecules in addition to Stat5, such as coactivators or other transcription factors, has not been investigated.

And finally, the ability of PRL to activate Stat5b phosphorylation through two kinases appears to be specific for Stat5b and not Stat5a. Very few instances of differential regulation of Stat5a and Stat5b have been reported; however, one in particular is of interest. Insulin can induce tyrosine phosphorylation of both Stat5a and Stat5b (67). AG490 prevented phosphorylation of Stat5a only and had no effect on Stat5b phosphorylation. When an insulin receptor kinase inhibitor was used, Stat5b phosphorylation induced by insulin was completely prevented. Although additional data suggested that Stat5b is a direct target of the insulin receptor (67, 68), one of the downstream pathways of insulin is PI3K. Perhaps a similar tyrosine kinase downstream of both PRL and insulin activation of PI3K is involved in Stat5b-specific phosphorylation.

Taken together, our results indicate that PRL regulation of ER{alpha} expression requires Jak2 activity. In addition, our data suggest the ability of PRL to induce Stat5b phosphorylation and transcriptional activity through Jak2 whereas Stat5b phosphorylation, but not transcriptional activity, may be mediated by novel tyrosine kinase downstream of PI3K.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
PMSG, human CG (hCG), DMEM/F12 (1:1), DMEM, horseradish peroxidase-conjugated secondary antibodies, LY294002, and all other reagent grade chemicals were obtained from Sigma (St. Louis, MO). Genistein and AG18 were obtained from ICN Biomedicals, Inc. (Aurora, OH). AG490 and PP2 were from Calbiochem (San Diego, CA). Protogel, a 30% acrylamide/bis-acrylamide mixture (37.5:1) was from National Diagnostics (Atlanta, GA). T4 polynucleotide kinase and TRIzol were purchased from Life Technologies, Inc. (Gaithersburg, MD). The Advantage RT-for-PCR kit and the chemiluminescence ß-galactosidase (ß-gal) substrate were from CLONTECH Laboratories, Inc. (Palo Alto, CA). FBS was from HyClone Laboratories, Inc. (Logan, UT). ExTaq DNA polymerase, ExTaq PCR buffer, and deoxynucleotide triphosphate (dNTP) were obtained from Panvera (Madison, WI). 32P-Labeled nucleotides were from Amersham Pharmacia Biotech (Arlington Heights, IL). DNA Master SYBR Green I was purchased from Roche Molecular Biochemicals (Indianapolis, IN). Trypsin-EDTA, antibiotics, and Amphotericin were from Mediatech (Herndon, VA). Antibodies to Stat5a, Stat5b, and phosphorylated Stat5a/5b were from Upstate Biotechnology, Inc. (Lake Placid, NY). Protein A/G agarose beads and the enhanced chemiluminescence detection reagents were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The luciferase assay substrates and reporter lysis buffer were purchased from Promega Corp. (Madison, WI). Ovine PRL (PRL-18, 30 IU/mg) was obtained from NIDDK, NIH (Bethesda, MD).

Primary Luteinized Granulosa Cell Culture and Transfection
Immature female Sprague Dawley rats were obtained from Sasco Animal Labs (Madison, WI) and housed under controlled conditions of light and temperature with free access to standard rat chow and water. All experiments were conducted in accordance with the principles and procedures of the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee. Follicular development was induced in immature rats (24–26 d of age) by injection of 15 IU PMSG ip. An ovulatory dose of hCG (10 IU, ip) was given 48 h later. Luteinized granulosa cells were harvested from large preovulatory follicles 7–8 h after hCG injection. The ovarian bursa and surrounding fat were removed, and the ovaries were incubated sequentially in DMEM/F12 (1:1) containing 6 mM EGTA and then 0.5 M sucrose. Granulosa cells were harvested by puncturing the follicles with 25-g needles, washed in DMEM/F12, and then cultured in six-well plates at a density of 0.25 x 106 cells per well for RNA extraction and at a density of 1 x 106 cells per 60-mm plate for protein extraction. The culture media was DMEM/F12 supplemented with 1% FBS, 100 IU/ml penicillin G, 100 µg/ml streptomycin, and 0.25 µg/ml Amphotericin. The cells were cultured for 72 h at 37 C in a 5% CO2, humidified atmosphere. After 72 h, media were changed and cells were cultured overnight before the start of treatment. After treatment, cells were washed twice with cold PBS and stored at -80 until processed.

RNA Isolation and Reverse Transcription
RNA from cell cultures was isolated using Trizol according to the manufacturer’s instructions. Reverse transcription was carried out using reagents from the Advantage RT-for-PCR kit. One microgram of total RNA was incubated with 1 µl oligo (dT)18 and 0.5 µl random hexamer at 70 C for 2 min. Four microliters of 5x reaction buffer, 1 µl dNTP (10 mM each), 0.5 µl RNase inhibitor, and 1 µl Moloney murine leukemia virus reverse transcriptase were added to each sample, and the total volume was brought to 20 µl with diethyl pyrocarbonate-treated H2O. The reaction was carried out for 1 h at 42 C followed by 5 min at 94 C. The reverse transcribed product was then diluted to a final volume of 100 µl by adding DEPC-treated H2O. Five-microliter aliquots of the diluted product were used for either semiquantitative or quantitative, real-time RT-PCR.

Semiquantitative RT-PCR
For each sample to be analyzed by semiquantitative RT-PCR, 5-µl identical aliquots of diluted reverse transcribed mRNA were used for the gene of interest and for the ribosomal protein L19, which served as an internal control. Diluted RT product (5 µl, representing 50 ng of total RNA) was combined with 20 pmol primers, 1x PCR buffer, 150 µM dNTP, 0.8 U ExTaq, and {alpha}-32P-dCTP (2.5 µCi of 3,000 µCi/mmol) in a final volume of 40 µl. The primers used have previously been described (3, 69). The samples were overlaid with light mineral oil, and PCR was carried out in two parts. First, five cycles were carried out with an annealing and extension temperature of 69 C for 5 min followed by denaturing at 95 C for 1 min. The second set consisted of a varying number of cycles with annealing at 65 C for 25 sec, extension at 72 C for 30 sec, and denaturing at 95 C for 25 sec. PCR reactions were carried out in a Perkin-Elmer/Cetus Thermal Cycler (Perkin-Elmer Corp., Norwalk, CT). For ER{alpha}, 25 step 2 cycles were used and 18 cycles for L19. The conditions were such that the amplification of the products was in the exponential phase and the assay was linear with respect to the amount of input RNA. Reaction products for ER{alpha} were combined with the corresponding L19 products and electrophoresed on an 8% polyacrylamide nondenaturing gel. After autoradiography, data were analyzed using a Molecular Dynamics, Inc. PhosphorImager and ImageQuant version 3 software (Molecular Dynamics, Inc., Sunnyvale, CA). The intensity of the ER{alpha} signal was normalized to that of the ribosomal protein L19 internal control.

Real-time, Quantitative RT-PCR
To generate standard curves for quantitative PCR, rat ER{alpha} cDNA, which was kindly provided by Dr. Maruyama, was diluted to concentrations ranging from 103 to 107 copies/µl. Five-microliter aliquots of standards or diluted RT products were combined with 2 µl 10x DNA Master SYBR Green I, 1.6 µl MgCl2 (3 mM final concentration), and specific primers for rat ER{alpha} (0.5 µM 77 final concentration). Reactions were carried out in glass capillary tubes in a total volume of 20 µl. The DNA Master SYBR Green I mix contains Taq DNA polymerase, reaction buffer, dNTP, 10 mM MgCl2, and SYBR Green I dye, which is a specific fluorescence dye for double-stranded DNA. PCR reactions were performed in the Roche Lightcycler instrument, and the accompanying software was used for data analysis (Roche Molecular Biochemicals). After a 2-min denaturation, PCR cycles were carried out as follows: 0 sec at 95 C, 10 sec at the annealing temperature, and 15 sec at 72 C. For ER{alpha}, 40 cycles at an annealing temperature of 63 C was used. At the end of each cycle, the amount of double-stranded DNA was monitored by measuring the level of SYBR Green I fluorescence. After the completion of all cycles, a level of fluorescence was selected at which all of the standards and samples were within the linear range of amplification. The crossing point, or the number of cycles necessary for each sample or standard to obtain the selected level of fluorescence, was calculated using the Roche Lightcycler software. Based on these crossing points, a standard curve was generated, and the number of ER{alpha} copies was calculated for each sample. The data presented represent the number of copies of ER{alpha} in 1 µl of diluted RT product, which corresponds to 10 ng of starting RNA.

Culture and Transfection of COS Cells
COS cells were routinely cultured in DMEM medium supplemented with 10% FBS, 100 IU/ml penicillin G, 100 µg/ml streptomycin, and 0.25 µg/ml Amphotericin. Cultures were carried out at 37 C in a 5% CO2, humidified atmosphere. For transient transfections, 100K cells were seeded per well in six-well plates and cultured as described above for 24 h. In general, cells were 50% confluent at the start of transfection. For transfections, DNA was combined with water (90 µl/well) and 2.5 M CaCl2 (10 µl/well) and mixed. In general, a total of 4–5 µg DNA were transfected per well, and the total amount of DNA was equalized with empty vector when necessary. An equal volume of 2x N,-N-bis[2-hydroxyethyl]-2-amino-ethanesulfonic acid-buffered saline was added, and DNA was allowed to precipitate at room temperature for 10 min. The DNA was then added dropwise to each well, and cells were cultured for 24 h at 3% CO2. Twenty-four hours after the start of transfection, media were changed to standard culture media supplemented with 1% FBS, and cells were cultured for an additional 24 h at 5% CO2 in the presence or absence of various inhibitors. After treatment, cells were washed twice with cold PBS and stored at -80 C for reporter assays.

Reporter Assays
Luciferase and ß-gal activities were measured by first preparing cell lysates in 1x reporter lysis buffer. Luciferase activity driven by the ER{alpha} promoter was measure by combining lysate with firefly luciferase assay substrate and measuring luminescence for 10 sec on a Lumat LB 9507 luminometer (EG&G Berthold, Oak Ridge, TN). As a control, cells were cotransfected with an expression vector for ß-gal. ß-gal Activity was measured in a separate aliquot of lysate by incubating with a luminescent ß-gal substrate for 1 h at room temperature and then measuring luminescence for 5 sec. The luciferase activity was normalized to the ß-gal activity within the same well.

Immunoprecipitation and Western Blotting
WCE from primary luteinized granulosa cells and COS cells were prepared by lysing cells in RIPA buffer (1x PBS, 1% Nonidet, 0.5% sodium deoxycholate, 0.1% SDS) containing 1 µM sodium orthovanadate, 10 µg/ml PMSF, and 30 µl/ml aprotinin. For immunoprecipitation, 500 µg of WCE were incubated with 4-µl anti-Stat5a or anti-Stat5b antibodies for 1 h at 4 C. Protein A/G agarose beads were added, and the mixture was incubated overnight at 4 C on a rocking platform. The beads were washed four times in PBS, resuspended in 2x electrophoresis buffer, and boiled for 5 min. For Western blots performed on WCE, protein was diluted in an equal volume of 2x electrophoresis buffer and boiled for 5 min. Twenty microliters of immunoprecipitated protein or 20 µg of WCE were separated on a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. Western blotting was performed by blocking nonspecific binding with 5% dry milk in Tris-buffered saline buffer containing 0.05% Tween 20 for 1 h. Blots were then incubated with the primary antibody overnight at 4 C on a rocking platform. After a series of washes, blots were incubated with a secondary antibody linked to horseradish peroxidase for 1 h. After extensive washing, blots were analyzed using an enhanced chemiluminescence detection system and exposed to x-ray film.

EMSA
Nuclear extracts were prepared from cell cultures by a published method (70). A probe corresponding to the bovine ß-casein Stat5 response element (15) was labeled by incubating 0.5 pmol annealed oligonucleotides with 11 U T4 kinase and 25 µCi {gamma}-32P ATP (3,000 µCi/mmol). The specific activity of the probes was greater than 8,000 cpm/fmol. Ten-microgram nuclear extracts were incubated with 50K cpm of labeled probes in 1x binding buffer (12 mM HEPES, pH 7.9, 40 mM KCl, 5 mM MgCl2, 0.12 mM EDTA, 0.06 mM EGTA, 0.5 mM dithiothreitol, 10% glycerol) at room temperature for 30 min. The samples were then run on a 4.5% nondenaturing polyacrylamide gel in 0.25x Tris-buffered EDTA buffer at 200 V for 2–3 h. The gels were dried and analyzed by autoradiography.


    ACKNOWLEDGMENTS
 
We are grateful to O. Silvennoinen for the Jak2 expression vectors, Jean Djiane and Paul Kelly for the PRL-R expression vectors, and Toshio Kitamura for the Stat5 expression vectors.


    FOOTNOTES
 
This work was supported by NIH Grants HD-11119 and HD-12356 (to GG).

Abbreviations: ß-gal, ß-Galactosidase; CA-Jak2, constitutively active Jak2; dNTP, deoxynucleotide triphosphate; Jak, Janus kinase; KN-Jak2; kinase-negative Jak2; PRL-R, PRL receptor; PRL-RCA, constitutively active PRL-R; PRL-RL, long form of PRL-R; STAT, signal transducer and activator of transcription; WCE, whole-cell extract.

Received for publication March 26, 2001. Accepted for publication July 24, 2001.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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