Induction of Relaxin Messenger RNA Expression in Response to Prolactin Receptor Activation Requires Protein Kinase C {delta} Signaling

Carl A. Peters, Evelyn T. Maizels, May C. Robertson, Robert P.C. Shiu, Melvin S. Soloff and Mary Hunzicker-Dunn

Departments of Cell and Molecular Biology (C.A.P., E.T.M., M.H.-D.) Northwestern University Medical School Chicago, Illinois 60611
Department of Physiology (M.C.R., R.P.C.S.) University of Manitoba Winnipeg, Manitoba, Canada R3E0W3
Department of Obstetrics and Gynecology (M.S.S.) University of Texas Medical Branch Galveston, Texas 77555


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The ability of PRL or rat placental lactogen (rPL)-1 to induce relaxin mRNA expression was analyzed in a luteinized rat granulosa cell culture model. PRL receptor activation induced relaxin mRNA expression in a concentration- and time-dependent manner. High concentrations of PRL receptor agonist, equivalent to those of the second half of pregnancy in rats, were required to elicit relaxin mRNA expression. A 40-fold induction of relaxin mRNA was observed in cells treated 24 h with 1 µg/ml of rPL-1. Estrogen enhanced relaxin expression induced by PRL but did not affect relaxin expression on its own. PRL/rPL-1 induction of relaxin expression was independent of the extracellular regulated kinase (ERK) members of the mitogen-activated protein kinase (MAPK) pathway, based on the inability of the ERK kinase inhibitor PD98059 to block induction of relaxin expression. PRL/rPL-1 induction of relaxin expression required protein kinase C (PKC) {delta}, based on the ability of the preferential PKC {delta} inhibitor rottlerin to abolish induction of relaxin expression. Direct activation of PKC by phorbol myristate acetate, however, was not sufficient to promote induction of relaxin mRNA expression. Stats (signal transducers and activators of transcription) 3 and 5 DNA binding activities were induced by PRL/rPL-1 treatment of luteinized granulosa cells but only Stat 3 DNA binding was reduced by rottlerin. PRL/rPL-1 treatment of luteinized granulosa cells resulted in increased phosphorylation on tyrosine-705 and serine-727 of Stat 3, and these responses were reduced and blocked, respectively, by rottlerin. Tyrosine and serine phosphorylations of Stat 3 in the corpus luteum were also increased in the second half of pregnancy when PL levels are highest. Stat 3, but not Stat 1 or 5, coimmunoprecipitated with luteal PKC {delta} during pregnancy; Stat 3 transiently coimmunoprecipitated with PKC {delta} from luteinized granulosa cells in response to PRL receptor activation; and Stat 3/PKC {delta} complex formation required PKC {delta} kinase activity. Taken together, these results show that PKC {delta} is obligatory for PRL/rPL-1-dependent relaxin expression, that PKC {delta} complexes with Stat 3 in response to PRL receptor activation, and that PKC {delta} is involved in the regulation of Stat 3 phosphorylation downstream of the PRL receptor. These results demonstrate that PRL/rPL-1 promotes relaxin expression in luteal cells and that this event is mediated, at least in part, via PKC {delta}.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Relaxin is a 6000-Da polypeptide hormone that has been implicated in the successful delivery and survival of pups in the rat (1, 2). The corpus luteum is the primary site of relaxin expression (3). Relaxin mRNA is initially detected in luteal samples on day 11 of pregnancy; expression increases until day 19 or 20 of pregnancy and declines before parturition (3, 4). Translated relaxin is stored in secretory granules in the corpus luteum before secretion (5, 6). Secreted relaxin in the serum of pregnant rats is increasingly detected during the second half of pregnancy and peaks on the day before parturition (7). Both relaxin mRNA expression in corpora lutea and serum levels of relaxin during pregnancy in rats have been shown to depend upon a placental factor and to be enhanced by estrogen (E2) (8, 9, 10, 11, 12). Coincident with the increase in relaxin mRNA expression, the corpus luteum switches from reliance on pituitary PRL or decidual luteotropin to reliance on rat placental lactogen (rPL)-1 for maintenance of luteal function, as assessed by progesterone production (13, 14). In contrast to the pulsatile release of PRL in the first half of pregnancy, in which serum PRL levels never exceed 100 ng/ml, rPL-1 and rPL-2 release, although of limited duration, is continuous, and serum levels reach 3 µg/ml in the second half of pregnancy (13, 15). PRL, rPL-1, and rPL-2 all bind to the two forms of the PRL receptor, designated as long and short forms, detected in the ovary of the rat (16, 17).

PRL receptors do not have intrinsic kinase activity. They instead rely on janus kinase (JAK)-2 which is constitutively bound to the membrane proximal domain of the PRL receptor (18, 19, 20). Upon agonist binding, PRL receptors dimerize and JAK-2 tyrosine kinase activity is induced causing the phosphorylation of JAK-2 and the PRL receptor (19, 21). Tyrosine-phosphorylated receptor and JAK-2 likely serve as docking sites for additional signal transduction proteins (22). Signal transducers and activators of transcription (Stat)-family members 1, 3, 5a, and 5b have been shown in various cellular models to be tyrosine phosphorylated after PRL receptor activation, presumably by JAK-2 (23, 24). Tyrosine phosphorylation of the Stat proteins is required for their dimerization and subsequent translocation to the nucleus where they bind to response elements and induce gene transcription. Stats 5a and 5b are well characterized for their role in induction of PRL-responsive genes such as ß-casein in breast tissue (22, 25, 26). In the ovary, Stat 5 expression is induced concomitant with luteal formation (27) and Stat 5 has recently been shown to mediate the PRL-dependent expression of the steroidogenic enzyme responsible for conversion of pregnenolone to progesterone (28). Stat 5b has been shown to be vital for PRL-dependent induction of {alpha}2-macroglobulin (M) expression in rat corpora lutea (29, 30). Consistent with the evidence that Stat 5 plays a major role in transducing PRL actions in luteal cells, mice deficient in Stat 5b abort in the second half of pregnancy (31).

In addition to PRL-stimulated tyrosine phosphorylation of the Stats, Stats 1, 3, 5a, and 5b are each phosphorylated on at least one serine residue. However, the functional significance of the serine phosphorylation of the Stats is still controversial. Serine-725 of Stat 5a is constitutively phosphorylated as is a second unidentified serine residue of Stat 5a, while phosphorylation of serine-730 of Stat 5b is induced in response to PRL in COS-7 and Nb2 cells (32). However, phosphorylation of serine-725 or -730 on Stats 5a or 5b, respectively, is not essential for DNA binding or transcriptional activation of a ß-casein reporter gene in COS-7 cells (29). In contrast to Stats 5a and 5b, ligand-dependent phosphorylation of serine-727 of Stat 1 is reported to enhance its transcriptional activity but not its DNA binding activity (33, 34, 35). Like Stat 1, ligand-dependent phosphorylation of serine-727 of Stat 3 does not affect Stat 3 DNA binding but does enhance transcriptional activity (34, 36, 37). Although the serine kinases responsible for the phosphorylation of Stats 1 and 5 have yet to be identified, experimental evidence in some cellular models supports a role for the extracellular regulated kinase (ERK) members of the mitogen-activated protein kinase (MAPK) family (36, 38, 39) and, in other cells, for protein kinase C (PKC) {delta} as Stat 3 kinases (40).

Consistent with these reports, PRL is reported in mammary and lymphoma cells to activate the ERKs (25, 41, 42). Signaling through the PRL receptor has also been shown to activate the serine/threonine kinases of the PKC family in Nb2 cells, astrocytes, and vascular smooth muscle cells, based on the ability of PRL to induce translocation of PKC to the Triton-soluble fraction in these cells (43, 44, 45).

The PKC family is a group of 11 related but separate isoforms. These isoforms exhibit distinct responsiveness to physiological stimuli and have unique tissue distributions, subcellular localizations, and substrate specificities, indicating isoform-specific functions (46). Ovarian tissues in the rat express the {alpha}, ßI, ßII, {delta}, {epsilon}, and {zeta} isoforms of PKC (47, 48). PKC {delta} is distinguished from the other isoforms expressed in the rat ovary by the striking increase in PKC {delta} mRNA and protein that is observed beginning on day 11 of pregnancy in the corpus luteum (49). PKC {delta} protein and mRNA expression in corpora lutea continue to increase up to 25-fold by day 18 of pregnancy, remain high until day 21, and then decrease just before parturition (49). In addition, based on its translocation to the Triton-soluble cellular fraction, immunecomplex kinase activity, and phosphorylation, we have shown that PKC {delta} is activated in the corpus luteum during pregnancy and after PRL treatment in cultured luteinized granulosa cells (50).

These results taken together led us to hypothesize that relaxin expression in the second half of pregnancy was stimulated by PRL receptor activation and mediated, in part, by PKC {delta}. Current studies were therefore undertaken to begin to address the possible role of signaling through the PRL receptor by the high concentrations of rPL-1 present in the second half of pregnancy in the induction of relaxin mRNA expression. We employed a luteinized granulosa cell culture system and found that treatment with high concentrations of PRL or rPL-1 strongly induced relaxin mRNA expression in a concentration- and time-dependent manner. In addition, our results showed that induction of relaxin mRNA expression required PKC {delta} kinase activity and implicated Stat 3 as a PKC {delta} target leading to relaxin mRNA expression. Our results position PKC {delta} as a vital regulator of relaxin expression in response to PRL signaling in rat luteal cells.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PRL Induces Relaxin Expression
Although relaxin expression is known to require a placental-derived factor (8, 9, 10, 11), this factor has not been identified. We analyzed whether this factor might be rPL-1, which is detected at high levels in the serum of pregnant rats immediately before the increase in relaxin mRNA expression (3, 4, 14). Since rPL-1 employs the same receptor as PRL (16), we initially analyzed the ability of PRL to induce relaxin mRNA expression. Luteinized granulosa cells were cultured in the presence of increasing concentrations of PRL for 9 days. Little or no relaxin mRNA could be detected when cells were cultured in the absence of PRL or in the presence of 20 ng/ml of PRL (Fig. 1Go). However, treatment with 500 or 3000 ng/ml PRL induced relaxin mRNA expression 20-fold and greater than 30-fold, respectively.



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Figure 1. PRL Induces Relaxin Expression in a Concentration-Dependent Manner

Luteinized granulosa cells were cultured for 9 days in the presence of the indicated concentrations of PRL and mRNA isolated. A, Relaxin mRNA expression was assessed by Northern blot analysis followed by reprobing of the same membrane to detect L19 to assess loading. B, Fold induction of relaxin mRNA expression ([relaxin probe auto-radiographic density/L 19 autoradiographic density for indicated PRL concentrations]/[relaxin probe auto-radiographic density/L 19 autoradiographic density for control]) is shown; 0 PRL = 1. For rest of details see Materials and Methods. Result is representative of two experiments.

 
Treatment of luteinized granulosa cells with 1 µg/ml of PRL induced detectable relaxin mRNA expression in as little as 4 h (Fig. 2AGo). However, relaxin mRNA expression was more evident after 24 and 48 h of PRL treatment. PRL-dependent relaxin mRNA expression was still readily detected after 5 and 9 days of PRL treatment (Fig. 2BGo). Cells cultured in the absence of PRL exhibited no relaxin mRNA expression regardless of the length of the culture period.



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Figure 2. PRL Induces Relaxin Expression in a Time-Dependent Manner

Luteinized granulosa cells were cultured 4–48 h (A) or 5 or 9 days (B) as indicated in the absence (-) or presence (+) of 1 µg/ml PRL, and mRNA was isolated. Relaxin mRNA expression was assessed by Northern blot analysis followed by reprobing of the same membrane to detect L19 for assessment of loading. For remainder of details see Materials and Methods. Each result is representative of two experiments.

 
PRL-Dependent Induction of Relaxin mRNA Expression is Enhanced by E2
E2 has been shown to enhance both the expression of relaxin mRNA in the corpus luteum and the production of relaxin protein, as detected in the serum of pregnant rats (9, 12). We therefore analyzed whether E2 could induce relaxin mRNA expression either in the absence or presence of rPL-1. Results presented in Fig. 3Go show that E2 by itself did not induce relaxin mRNA expression (lanes 1 and 2). However, E2 enhanced relaxin mRNA expression induced by either 0.5 or 3 µg/ml rPL-1.



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Figure 3. PRL-Dependent Induction of Relaxin mRNA Expression Is Enhanced by E2

Luteinized granulosa cells were cultured for 12 days in the absence (-) or presence (+) of 10 nM E2. For the final 4 days of culture, cells were also treated with the indicated concentration of rPL-1. After mRNA isolation, relaxin mRNA expression was assessed by Northern blot analysis followed by reprobing of the same membrane to detect L19 for assessment of loading. Results are representative of five experiments.

 
PKC Signaling Is Necessary but Not Sufficient for PRL-Dependent Induction of Relaxin mRNA Expression
We have previously shown that PKC {delta} is the sole PKC isoform whose expression is increased in the second half of pregnancy, beginning between days 10 and 12 of pregnancy (49). Days 10–12 of pregnancy target a time of elevated serum rPL-1 and rising serum androgen titers and of increasing relaxin expression by the corpus luteum (3, 7, 15, 51). PKC {delta} protein expression is increased by E2 in rat corpora lutea of pseudopregnancy (49) and in luteinized granulosa cells (48). We have also shown that PKC {delta} is acutely activated by PRL in luteinized granulosa cells cultured in the presence of E2, based on the translocation of PKC {delta} to a Triton-soluble membrane fraction, its increased immune complex kinase activity, and its phosphorylation on serine-662 in response to PRL (50). Therefore, we assessed whether PKC signaling was required for PRL-mediated relaxin mRNA expression in luteinized granulosa cells cultured in the presence of E2. Cells were pretreated either overnight with 10 nM phorbol myristate acetate (PMA) to down-regulate PKC isoforms or for 30 min with the PKC {delta} preferential inhibitor rottlerin (40, 52) before overnight treatment with rPL-1. As shown in Fig. 4Go, A and B, and depicted graphically in Fig. 4CGo, rPL-1 strongly induced relaxin mRNA expression. Pretreatment with the PKC {delta} preferential inhibitor rottlerin blocked the ability of rPL-1 to induce relaxin mRNA expression (Fig. 4Go, A and C). Similarly, overnight pretreatment with PMA before treatment with rPL-1 down-regulated PKC isoform expression (data not shown) and blocked rPL-1-dependent induction of relaxin mRNA expression (Fig. 4Go, B and C). Pretreatment of cells for 30 min with the general PKC inhibitor GF109203X (53) also abated rPL-1-dependent relaxin induction (not shown). However, PKC signaling was not sufficient to induce relaxin mRNA expression since direct activation of PKC with PMA did not induce relaxin mRNA expression (Fig. 4Go, A and C). These results suggest that the induction of relaxin expression by rPL-1 requires PKC {delta} kinase activity.



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Figure 4. PKC Signaling Is Necessary but Not Sufficient for PRL-Dependent Induction of Relaxin mRNA Expression

Luteinized granulosa cells were cultured for 8 days in the presence of E2. On day 8 cells were pretreated 30 min with 10 µM rottlerin or 50 µM PD98059 as indicated (A) or overnight with 10 nM PMA (B). Cells were subsequently treated overnight with the rPL-1 vehicle (control) or with 3 µg/ml rPL-1 or 10 nM PMA, as indicated, and RNA collected. Relaxin mRNA expression was assessed by Northern blot analysis followed by reprobing of the same membrane to detect L19 for assessment of loading. Results are representative of four experiments. C, Average (± SEM) fold induction from four experiments of relaxin mRNA expression ([relaxin probe autoradiographic density/L19 autoradiographic density for the indicated treatment]/[relaxin probe autoradiographic density/L19 autoradiographic density for control), control = 1. D, Cells were pretreated for 30 min with or without 50 µM PD98059 (PD) and then treated for 10 min with rPL-1 vehicle (control), 3 µg/ml rPL-1, or 10 nM PMA. Protein from the cells was then collected in a membrane extracting buffer and 50 µg of total protein/lane were analyzed for ERK/MAPK activation using an antibody that detects dually phosphorylated p44 MAPK (upper panel). Blots were subsequently reprobed with a MAPK antibody (lower panel). For remainder of details see Materials and Methods. Results are representative of three experiments.

 
ERK/MAPK Signaling is Not Necessary for PRL-Dependent Induction of Relaxin mRNA Expression
The ERK members of the MAPK family have also been reported to be activated downstream of the PRL receptor (25, 41, 42); therefore, we analyzed whether or not the ERK/MAPK kinase (MEK) inhibitor PD98059 (54) affected the ability of rPL-1 to induce relaxin mRNA expression. As shown in Fig. 4Go, A and C, pretreatment of luteinized granulosa cells with the MEK inhibitor had no significant effect on the rPL-1-dependent induction of relaxin mRNA expression. Figure 4DGo shows that the inability of the MEK inhibitor to block rPL-1-induced relaxin mRNA expression was not due to a lack of activation of ERK/MAPK by rPL-1. rPL-1 promoted the activation of p44 MAPK, as detected with an antibody that detects dually phosphorylated MAPK, and this activation was completely blocked when cells were pretreated with PD98059. Consistent with reports that several PKC isoforms are capable of activating ERK/MAPK (55), PMA treatment of luteinized granulosa cells also induced activation of p44 MAPK (Fig. 4DGo), yet PMA did not promote relaxin mRNA expression (Fig. 4AGo). These results suggest that rPL-1-dependent relaxin expression is independent of the ERK/MAPK signaling pathway.

Effect of Rottlerin on PRL-Induced Stat 3 DNA Binding
Stat 5b has been shown to be activated in corpora lutea during the second half of pregnancy, based on the ability of Stat 5b to bind to DNA response elements of the {alpha}2-M promoter (29, 30). Minimal Stat 5a and Stat 3 DNA binding and no Stat 1 DNA binding to the {alpha}2-M promoter could be identified (29). However, since PRL receptor signaling can employ Stats 1, 3, and 5 (24) after JAK-2 activation (19), any of these Stats could play a role in regulation of relaxin mRNA expression downstream of the PRL receptor. As the relaxin promoter sequence has not been published, we sought to determine whether or not Stats 1, 3, or 5 DNA binding activity was induced by PRL and whether or not pretreatment with rottlerin altered this DNA binding. E2-primed luteinized granulosa cells were treated with PRL for 10 min and nuclear extracts were prepared. Electrophoretic mobility shift assays (EMSAs) were performed using Stat 1, 3, and 5 consensus DNA binding elements. Figure 5AGo shows that Stat 1 DNA binding is not induced by PRL in luteinized granulosa cells. In contrast, oligonucleotide probes containing the consensus Stat 3 (panel B) and Stat 5 (panel D) DNA binding sites were shifted in response to PRL. Bands observed with Stat 3- (panel B) and Stat 5- (panel D) specific oligonucleotide probes were competed with their respective unlabeled oligonucleotides (data not shown). The specificity of the Stat 3 complex was confirmed by the ability of a Stat 3 antibody to supershift the complex (panel C). Since the PKC {delta} inhibitor rottlerin blocks relaxin expression, the effect of rottlerin on PRL-stimulated DNA binding was also determined. Stat 3 DNA binding was reduced in cells pretreated with rottlerin (panel B) while Stat 5 DNA binding was unaffected (panel D). Furthermore, we confirmed that the complex formed using the Stat 5-specific oligonucleotide probe indeed contained Stat 5. Incubation with antibodies to Stat 5a or 5b both supershifted the complexes (panels E and F), confirming that PRL activates the DNA binding of both Stat 5a and Stat 5b in luteinized granulosa cells.



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Figure 5. Effect of Rottlerin on Stat 3 DNA Binding after rPL-1 Treatment

Luteinized granulosa cells were cultured 8 days in the presence of E2. On day 8, cells were pretreated with 10 µM rottlerin (30 min) as indicated, and cells were subsequently treated with rPL-1 vehicle (control) or 3 µg/ml rPL-1 for 10 min. Nuclear extracts were prepared and employed in EMSAs. For details see Materials and Methods. Oligonucleotides containing Stat 1(A), Stat 3 (B and C), or Stat 5 (D–F) consensus DNA-binding elements were end-labeled with [32P] and mixed with nuclear extracts either alone (A, B, and D) or in the presence of Stat 3 (C), Stat 5a (E), or 5b (F) polyclonal antibodies (Ab). Results are representative of three separate experiments.

 
Effect of Rottlerin on Stat 3 Phosphorylation and Coimmunoprecipitation with PKC {delta} after rPL-1 Treatment
We next sought to identify the PKC {delta} target downstream of the PRL receptor. Activation of transcription by Stat 3 has been shown to be regulated by both tyrosine and serine phosphorylation (34, 36, 37), and PKC {delta} has recently been identified as a Stat 3 serine kinase (40). In contrast, neither Stat 5a nor 5b requires serine phosphorylation for DNA binding or activation of transcription (32). Therefore, we considered Stat 3 the most likely PKC {delta} target. Using the E2-primed luteinized granulosa cell model, we assessed whether PRL receptor activation increased the tyrosine and serine phosphorylation of Stat 3. As shown in Fig. 6AGo, a basal level of tyrosine-705 and serine-727 Stat 3 phosphorylation was detected, consistent with reports in other cells (36). Treatment of cells with rPL-1 for 10 min increased the phosphorylation of Stat 3 both on tyrosine-705 (top panel) and serine-727 (middle panel). We then determined whether rPL-1-stimulated serine phosphorylation of Stat 3 was dependent on PKC {delta} activation by pretreating cells with rottlerin. Pretreatment of cells with rottlerin nearly completely blocked phosphorylation of Stat 3 on serine-727 and reduced the phosphorylation of Stat 3 on tyrosine-705 to the level detected before rPL-1 treatment. Consistent with evidence that Stat 3 DNA binding is regulated by tyrosine but not serine phosphorylation (34, 36, 37), our results show a basal level of Stat 3 DNA binding and tyrosine phosphorylation (compare Figs. 5BGo and 6AGo, top panel). Stat 3 DNA binding and tyrosine phosphorylation exhibit a similar increase with PRL receptor activation and reduction by rottlerin. In contrast to Stat 3, Stat 1 phosphorylation of tyrosine-701 was not affected by treatment of luteinized granulosa cells with rPL-1 (Fig. 6BGo, top panel) even though Stat 1 protein is clearly present in these cells (Fig. 6BGo, bottom panel) consistent with our DNA binding results (Fig. 5AGo). Hela cells treated with (positive) and without (negative) interferon {alpha} served as controls for Stat 1 tyrosine phosphorylation. Despite the ability of PRL to enhance Stat 5 DNA binding activity (Fig. 5DGo), the tyrosine phosphorylation of Stat 5 in PRL-treated cells was not detected (not shown).



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Figure 6. Effect of Rottlerin on Stat 3 Phosphorylation after rPL-1 Treatment

Luteinized granulosa cells were cultured for 8 days in the presence of E2. On day 8, cells were pretreated with 10 µM rottlerin (30 min) as indicated, and cells were subsequently treated with rPL-1 vehicle (control) or 3 µg/ml rPL-1 for 10 min. Cell lysates were prepared in a membrane extracting buffer and analyzed for Stat 3 and Stat 1 phosphorylation by Western blotting. For details see Materials and Methods. A, Stat 3 phosphorylation on tyrosine-705 (upper panel) and serine-727 (second panel) was detected by Western blotting with phospho-epitope-specific antibodies, and the presence of Stat 3 (bottom panel) was detected by Western blotting with a Stat 3 monoclonal antibody. B, Phosphorylation of Stat 1 on tyrosine-701 (upper panel) was detected by Western blotting with phospho-epitope-specific antibody, and the presence of Stat 1 (lower panel) was detected by Western blotting with a Stat 1 monoclonal antibody. Results are representative of two experiments.

 
To assess whether a Stat 3/PKC {delta} complex is formed in response to activation of the PRL receptor, we determined whether Stats 1, 3, or 5 coimmunoprecipitated with PKC {delta} after rPL-1 treatment of E2-primed luteinized granulosa cells. Treatment of cells with rPL-1 for 5 min caused both Stats 3 and 1 to coimmunoprecipitate with PKC {delta} (Fig. 7Go). This effect was blocked by rottlerin treatment, suggesting that PKC {delta} activation is obligatory for complex formation between PKC {delta} and Stats 1 and 3. Coimmunoprecipitation of Stats 1 and 3 with PKC {delta} in response to rPL-1 was transient since neither Stat 1 nor Stat 3 coimmunoprecipitated with PKC {delta} in samples from cells treated with rPL-1 for 30 min. Stat 5 did not coimmunoprecipitate with PKC {delta}. As blots were first probed for PKC{delta} (bottom panel) and subsequently probed for Stats 1, 3, and 5 (first, second, and third panels, respectively), residual PKC {delta} immunoreactivity is detected in the Stat Western blots.



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Figure 7. Stat 3 Coimmunoprecipitation with PKC {delta} After rPL-1 Treatment: Effect of Rottlerin

Luteinized granulosa cells were cultured for 8 days in the presence of E2. On day 8 cells were pretreated with 10 µM rottlerin (30 min), as indicated, and cells were subsequently treated with 3 µg/ml rPL-1 for the indicated times, and cell lysates were prepared in membrane extracting buffer. PKC {delta} immunoprecipitations were performed as described in Materials and Methods. The PKC {delta} (lower panel) followed sequentially by Stat 1 (upper panel), Stat 3 (second panel), Stat 5 (third panel). Western blots were performed on PKC {delta} immunoprecipitates. Migration positions of Stat 1 at 92 kDa (upper panel), Stat 3 at 91 kDA (second panel), and PKC {delta} (panels 1–3) are indicated on the right. Results are representative of three experiments.

 
Phosphorylation and Coimmunoprecipitation of Stat 3 with PKC {delta} during Pregnancy
Finally, we wanted to determine whether the association between PKC {delta} and Stat 3 was reproduced in an in vivo setting in which luteal cells are exposed to sustained and high levels of PLs. We thus assessed Stat 3 phosphorylation on tyrosine-705 and serine-727 in luteal extracts obtained on days 11 and 18 of pregnancy. Stat 3 tyrosine (Fig. 8AGo) and serine (Fig. 8BGo) phosphorylations both increased as pregnancy progressed, as shown by Western blots of luteal lysates from the indicated days of pregnancy. In contrast to Stat 3, Stat 1 was not active during pregnancy, as assessed by Western blot analysis to detect Stat 1 phosphorylated on tyrosine-701 (Fig. 8CGo). Neither total luteal Stat 1 nor, as previously shown (29), Stat 3 protein expression was regulated in the second half of pregnancy (Fig. 8Go, lower portion of each panel).



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Figure 8. Phosphorylation of Luteal Stat 3 on Serine-727 and Tyrosine-705 during Pregnancy

Lysates were prepared from rat corpora lutea, obtained on indicated days of pregnancy, in a membrane extracting buffer, as described in Materials and Methods. Fifty micrograms of total protein/lane were analyzed for phosphorylation of Stat 3 on tyrosine-705 (A) or serine-727 (B) by Western blotting with phospho-epitope specific antibodies (upper panels), and the presence of Stat 3 was detected by Western blotting with a Stat 3 monoclonal antibody (lower panels). Phosphorylation of Stat 1 on tyrosine-701 (C) was detected by Western blotting with phospho-epitope-specific antibody (upper panel), and the presence of Stat 1 was detected by Western blotting with a Stat 1 monoclonal antibody (lower panel). Results are representative of three experiments.

 
We also determined whether Stat 3 coimmunoprecipitated with PKC {delta} from luteal extracts obtained on days 11 and 18 of pregnancy. Results (Fig. 9AGo, top right panel) showed that Stat 3 indeed coimmunoprecipitated with PKC {delta} on days 11 and 18 of pregnancy and that higher levels of Stat 3 were detected in PKC {delta} immunoprecipitates on day 18 compared with day 11, consistent with results of Fig. 8AGo. Neither Stats 1 nor 5 coimmunoprecipitated with PKC {delta}, although both were detected in luteal membrane extracts on these two representative days of pregnancy (Fig. 9AGo, left panels). Coimmunoprecipitation of PKC {delta} with JAK-2 from luteal membrane extracts was also detected on day 18 of pregnancy (Fig. 9BGo). The absence of detectable PKC {delta} in the Jak-2 immunoprecipitate from corpora lutea obtained on day 11 of pregnancy could reflect the lower luteal levels of both PKC {delta} and Jak-2 on this day (Fig. 9Go, A and B, bottom panels).



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Figure 9. Coimmunoprecipitation of Luteal Stat 3 and JAK-2 with PKC {delta} during Pregnancy

A, Lysates were prepared in membrane extracting buffer from corpora lutea obtained on the indicated days of pregnancy, PKC {delta} immunoprecipitations from 500 µg of total protein were performed as described in Materials and Methods. Right panels display Stat 3, Stat 5, Stat 1, and PKC {delta} Western blots performed on PKC {delta} immunoprecipitates from the indicated days of pregnancy. For comparison, left panels display Stat 3, Stat 5, Stat 1, and PKC {delta} Western blots performed on 50 µg of total protein extract from the indicated days of pregnancy. B, JAK-2 immunoprecipitation from 500 µg of total luteal protein extract. Blot was probed for PKC {delta} and reprobed with a JAK-2 antibody. Results are representative of two experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The results presented herein demonstrate that rPL-1/PRL, at concentrations equivalent to those of rPL-1 present in rat serum in the second half of pregnancy (14), strongly induces relaxin mRNA expression in luteinized rat granulosa cells. Concentrations of PRL receptor agonist equivalent to those present in the first half of pregnancy do not elicit relaxin expression in luteinized granulosa cells, consistent with the absence of relaxin expression during the first half of pregnancy. E2 synergized with PRL receptor agonist in the luteinized granulosa cell model to promote increased relaxin expression, consistent with the presence in the second half of pregnancy not only of rPL-1 and, subsequently rPL-2, but also of increasing concentrations of serum testosterone, aromatized to E2 in the corpus luteum (51). Although relaxin expression could be induced rapidly (in as little as 4 h), PRL receptor agonist continued over time (up to 9 days) to promote relaxin expression in the luteinized granulosa cell model, also consistent with the sustained expression of relaxin by the corpus luteum between days 12 and 20 of pregnancy (3, 7). Thus, the temporal pattern of relaxin expression during pregnancy appears to be mimicked in the luteinized granulosa cell model by the luteotrophic complex of hormones known to be required to sustain progesterone production by the corpus luteum in the second half of pregnancy (56). This is the first demonstration, to our knowledge, of direct evidence for the regulation of relaxin expression by PRL receptor agonists in concert with E2. These results are consistent with earlier reports showing that relaxin expression depended on a signal or signals derived from the placental unit, which was lost by removal of conceptuses (fetus and placenta) or hysterectomy of pregnant rats and temporarily rescued by E2 (11, 12, 57). Our results thus support the hypothesis that the placental derived signal consists of a combination of rPL-1 (and later, rPL-2) and androgen, which is aromatized to estrogen by the corpus luteum (14, 51, 58).

Our results demonstrate that the induction of relaxin mRNA expression in response to PRL receptor activation in luteinized granulosa cells requires signaling through PKC {delta}. PKC {delta} is strongly induced in the second half of pregnancy, and its pattern of expression temporally coincides with the pattern of relaxin expression (3, 7, 49). Although we do not know how PKC {delta} expression is regulated in corpora lutea during the second half of pregnancy, we have observed that E2 increases PKC {delta} protein expression >= 2-fold in every rat ovarian system that we have analyzed to date (48, 49). It is therefore possible that the enhancing effect of E2 on PRL/rPL-1-stimulated relaxin expression by luteinized granulosa cells, as seen in Fig. 3Go, results from the E2-stimulated increase in PKC {delta} expression. Since rPL-1/PRL, in concentrations present in the second half of pregnancy, also synergizes with E2 to further increase PKC {delta} protein and mRNA expression in luteinized rat granulosa cells (48), the effect of PRL/rPL-1 on relaxin expression in E2-primed cells might also be due in part to increased expression of PKC {delta}.

Although signaling through PKC {delta} is required for PRL-dependent relaxin expression in luteinized granulosa cells, PKC signaling is not sufficient to induce relaxin mRNA expression since treatment of cells with PMA, a direct activator of PKC isoforms such as PKC {delta}, does not induce relaxin mRNA expression. Therefore, relaxin expression requires not only activation of PKC {delta} but also of additional pathways downstream of the PRL receptor. This is the first report, to our knowledge, that links a specific PKC isoform to PRL receptor action. However, our results implicating PKC as a necessary mediator of PRL induction of relaxin mRNA expression are not the first instance in which PKC has been implicated in the actions of PRL. PRL-dependent proliferation in Nb2 lymphoma and liver cells is either potentiated or mimicked by the PKC activator phorbol ester and is inhibited by inhibition of PKC activity (43, 44). PRL-dependent activation of tyrosine hydroxylase in neurons is blocked by a PKC inhibitor but not by protein kinase A or calmodulin inhibitors (59).

PRL could activate PKC {delta} through various routes. PRL has been reported to induce production of diacylglycerol by the liver both in vivo and in vitro (60), as well as by rat granulosa cells (61), apparently independent of PI 4,5-bisphosphate hydrolysis. Alternatively, PKC {delta} may be activated downstream of the PRL receptor by an increase in the level of phosphatidyl inositol (3, 4, 5)triphosphate [PI(3, 4, 5)P3] in a PI3-kinase-dependent fashion (33, 62, 63). PI(3, 4, 5)P3 has been shown to activate several PKC isoforms both in vitro and after activation of PI3-kinase in vivo (64). Furthermore, a recent report by Le Good et al. (65) showed that PKC {delta} is phosphorylated on its activation loop by 3-phosphoinositide-dependent protein kinase-1 (65). This activation loop phosphorylation of PKC {delta} is enhanced by PI(3, 4, 5)P3 in vitro and is dependent on PI3-kinase in vivo, leading to increased catalytic activity of PKC {delta} (65). Clearly, several pathways could be employed downstream of the PRL receptor to affect the activation state of PKC {delta}.

Our results indicate that Stat 3 is a likely target for PKC {delta} downstream of the PRL receptor to affect relaxin expression. Tyrosine phosphorylation of the Stats is required for their dimerization, translocation to the nucleus, and transcriptional activity (66). Transcriptional activation by Stat 3 additionally appears to be optimized, at least in some cells, by phosphorylation on serine-727 (34, 36, 37). PKC {delta} is a Stat 3 serine kinase (40). We show that rPL-1 treatment of luteinized granulosa cells promotes increased phosphorylation of Stat 3 both on tyrosine-705 and serine-727. Pretreatment of cells with the PKC {delta} inhibitor before rPL-1 treatment nearly ablated serine-727 phosphorylation of Stat 3 and reduced tyrosine-705 phosphorylation to a level similar to that seen on Stat 3 before rPL-1/PRL treatment. The rottlerin-dependent reduction of Stat 3 tyrosine-705 phosphorylation coincides with a reduction in Stat 3 DNA binding activity. These results show that the PRL/rPL-1-dependent phosphorylation of Stat 3 on both serine and tyrosine is dependent on the activity of PKC{delta}. Consistent with our evidence supporting Stat 3 as a PKC {delta} target in luteinized granulosa cells, the extent of Stat 3 tyrosine-705 and serine-727 phosphorylations is higher in corpora lutea obtained on day 18 compared with day 11 of pregnancy, consistent with higher expression of both PKC {delta} and relaxin mRNA at this time of pregnancy (3, 49). Moreover, Stat 3 transiently coimmunoprecipitates with PKC {delta} after rPL-1 treatment of luteinized granulosa cells, and this coprecipitation is blocked when cells are pretreated with rottlerin. This result suggests that a Stat 3/PKC {delta} complex is formed in response to PRL/rPL-1-dependent PKC {delta} activation. In support of this hypothesis, we have observed that during pregnancy, luteal Stat 3 and JAK-2, but not Stats 1 or 5, coimmunoprecipitate with PKC {delta}, especially on day 18 of pregnancy when PKC {delta} levels are elevated. Perhaps JAK-2 serves as a docking site for Stat 3 and PKC {delta} after PRL receptor activation (24, 67). Consistent with our results, Stat 3 has recently been reported to transiently associate with PKC {delta} in an interleukin-6-dependent manner (40). While PKC {delta}-dependent Stat 3 phosphorylations on serine-727 and tyrosine-705 closely correlate with PRL-dependent relaxin expression, it is not clear whether Stat 3 exerts a positive or negative effect on relaxin expression. Stat 3 serine-727 phosphorylation has been shown both to enhance transcription (33, 34) and to inhibit Stat 3 transcriptional activity (38, 40). Additional studies will be necessary to determine how Stat 3 modulates PRL- dependent relaxin expression.

There are a number of possible mechanisms by which PKC {delta} could regulate the serine and tyrosine phosphorylations of Stat 3. These include direct phosphorylation of Stat 3 on serine-727 by PKC {delta}, as recently demonstrated in a PKC {delta} immune complex kinase assay using tagged Stat 3 as substrate (40), and/or modulation via PKC {delta}-catalyzed phosphorylation of other Stat 3 serine kinases or Stat 3 tyrosine kinases, including JAK-2 and Src, or of associated phosphatases (19, 60, 68, 69, 70, 71). Future studies are needed to elucidate the precise mechanism of the PKC {delta}-dependent regulation of Stat 3 phosphorylations in luteinized granulosa cells.

Our results suggest that Stat 1 but not Stat 5 may also be a PKC {delta} target in luteinized rat granulosa cells. Stat 1 did not exhibit either DNA binding activity or tyrosine-701 phosphorylation in response to PRL in luteinized granulosa cells, and Stat 1 tyrosine-701 phosphorylation was undetectable in luteal extracts of pregnancy. These results are consistent with the previously reported lack of Stat 1 DNA binding during pregnancy (29). However, while Stat 1 was not readily detected in PKC {delta} immunoprecipitates from corpora lutea obtained during pregnancy, Stat 1 did coimmunoprecipitate with PKC {delta} in response to acute rPL-1 treatment of luteinized granulosa cells. The coimmunoprecipitation of Stat 1 with PKC {delta} in luteinized granulosa cells was transient, an observation that perhaps explains why Stat 1 did not readily coimmunoprecipitate with PKC {delta} from corpora lutea of pregnancy that are chronically exposed to rPLs. However, scrutiny of the Stat 1 blot in Fig. 9Go shows that a very small amount of Stat 1 is detectable on day 18 in the PKC {delta} immunoprecipitate, consistent with a very transient association of Stat 1 with PKC {delta}. In luteinized granulosa cells, the coimmunoprecipitation of Stat 1 with PKC {delta} appeared to be dependent on PKC {delta} kinase activity, as it was inhibited by pretreatment of cells with rottlerin, but to be independent of Stat 1 tyrosine phosphorylation since rPL-1 did not stimulate detectable tyrosine phosphorylation of Stat 1. PKC {delta} has been shown to inhibit the tyrosine phosphorylation of Stat 1 catalyzed by Bmx kinase (72). Perhaps a similar mechanism might explain the absence of the tyrosine phosphorylation of Stat 1 and therefore the apparent absence of signaling through Stat 1 by PRL in luteal cells. Consistent with this hypothesis, the transient association of PKC {delta} and Stat 1 in luteinized granulosa cells might reflect an inhibitory serine phosphorylation of Stat 1. In contrast to Stat 1, Stat 5 has been shown to be activated during the second half of pregnancy (29, 30) at the appropriate time to play a role in regulating relaxin expression (3). Corpora lutea of Stat 5b null mice cease to produce progesterone on day 12 of pregnancy, and these mice abort their fetuses (31), consistent with recent evidence that Stat 5 regulates expression of 3ß-hydroxysteroid dehydrogenase expression in a PRL-dependent manner (28). In luteinized granulosa cells, Stat 5 DNA binding is clearly induced by PRL (see Fig. 5Go). However, unlike Stat 3, Stat 5 DNA binding and transcriptional activities are solely regulated by tyrosine phosphorylation (29) and, in luteinized granulosa cells, Stat 5 DNA binding is unchanged by PKC {delta} inhibition. Moreover, Stat 5 did not coimmunoprecipitate with PKC {delta} from either luteinized granulosa cells or corpora lutea of pregnant rats. Therefore, although a role for Stat 5 in regulation of relaxin expression cannot be ruled out, it is apparent that Stat 5 is not a direct target for PKC {delta}-dependent regulation of relaxin expression.

In contrast to PKC {delta}, ERK/MAPK signaling is not necessary for relaxin expression although PRL/rPL-1 does induce ERK/MAPK activation, as detected by the tyrosine and threonine phosphorylations of p44 MAPK in luteinized granulosa cells. ERK/MAPK activation is blocked by pretreatment with the MEK inhibitor while this inhibitor does not alter the PRL/rPL-1-dependent induction relaxin expression. Furthermore, PMA treatment of luteinized granulosa cells induces p44 MAPK tyrosine and threonine phosphorylations but does not induce relaxin mRNA expression. While the lack of a direct effect of PMA on relaxin expression indicates that typical PMA-regulated transcription involving activating protein-1 (AP-1) and non-AP-1 transacting factors through PMA and serum response elements (73) are not sufficient to induce relaxin expression, synergy between these transcription factors and Stat transcription factors has not been ruled out.

In conclusion, we have found that signaling through the PRL receptor is capable of strongly inducing relaxin mRNA expression in luteinized granulosa cells. PKC {delta} is necessary but not sufficient for induction of relaxin mRNA expression. PKC {delta} associates with Stat 3 during pregnancy and after PRL/rPL-1 treatment in luteinized granulosa cells. PKC {delta}/Stat 3 association in luteinized granulosa cells after PRL/rPL-1 treatment is transient and blocked by the PKC {delta} inhibitor rottlerin. Similarly, Stat 3 serine phosphorylation is induced after PRL treatment and blocked by pretreatment with rottlerin. These results support a role for PKC {delta} in the induction of relaxin mRNA expression during pregnancy in response to activation of the PRL receptor by saturating agonist concentrations at a time when PKC {delta} is highly expressed and active.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
The following materials were purchased: [{alpha}-32P] deoxy-CTP (specific activity, 3000 Ci/mmol), [{gamma}-32P] ATP (specific activity, 3000 Ci/mmol) from NEN Life Science Products (Boston, MA); SDS/PAGE reagents from Bio-Rad Laboratories, Inc. (Hercules, CA); protein standards from Diversified Biotech; Nytran nylon membranes from Schleicher & Schuell, Inc. (Keene, NH); Hybond C-extra nitrocellulose and ECL reagents from Amersham Pharmacia Biotech (Arlington Heights, IL); TRIzol reagent from Life Technologies, Inc. (Gaithersburg, MD); PKC {delta} specific monoclonal antibody (directed to the N terminus) and Stats 1, 3, and 5 monoclonal antibodies from Transduction Laboratories, Inc. (Lexington, KY); 4G-10 (antiphosphotyrosine) monoclonal antibody and antibodies for JAK-2 and PI3 kinase from Upstate Biotechnology, Inc. (Lake Placid, NY); active-ERK/MAPK antibody and T4 polynucleotide kinase from Promega Corp. (Madison, WI); ERK/MAPK antibody from Zymed Laboratories, Inc. (South San Francisco, CA); phospho-epitope antibodies that detect Stat 3 phosphorylated on serine-727 or tyrosine-705 or Stat 1 phosphorylated on tyrosine-710, and HeLa cell extracts prepared with and without interferon {alpha} treatment from New England Biolabs, Inc. (Beverly, MA); Stats 1 p84/91, 3, and 5 gel shift oligonucleotides, and Stats 3, 5a, and 5b polyclonal antibodies from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); rottlerin from Alexis; PD98059 from Calbiochem (La Jolla, CA). All other biochemical reagents were purchased from Sigma (St. Louis, MO). Final concentrations are indicated throughout.

Granulosa Cell Culture
Rats (Sasco strains) were obtained at 21 days of age from Charles River Laboratories, Inc. (Wilmington, MA) and maintained in accordance with "Guidelines for the Care and Use of Experimental Animals" by protocols approved by the Northwestern University Animal Care and Use Committee. Follicles were collected from 30-day-old rats that had been administered a low dose of hCG (0.15 IU) given subcutaneously twice daily for 2 days. On the following day a high dose of hCG (10 IU) was given to rats via tail vein injection, and ovaries were isolated 7 h later (74). Cells were harvested by mechanical dispersion and put into culture (75). The medium used for all procedures was DMEM/Ham’s F-12 (DMEM/F-12, 1:1) without phenol red and with 15 mM HEPES, 3.15 g/liter glucose, 1% charcoal-stripped FBS, 100 IU penicillin G, and 100 µg/ml streptomycin. After sequential incubations at 37 C in 6 mM EGTA in DMEM/F-12 and 0.5 M sucrose in DMEM/F-12, ovaries were returned to DMEM/F-12. Granulosa cells were released into the medium from all follicles using 30-gauge needles and gentle pressure. Cells were pelleted at 100 x g for 15 min, counted using trypan blue, and plated at a density of approximately 1 x 106 cells/ml on plastic dishes (Falcon, Becton Dickinson and Co, Lincoln Park, NJ). Cells were cultured in humidified atmosphere at 37 C, 5% CO2 with or without 10 nM E2 (in ethanol, final concentration, 0.5%) for up to 12 days as indicated. Alternatively cells were cultured in the presence of the indicated concentration of PRL (ovine PRL-20, NIDDK) for up to 9 days. Media was changed every 3 days.

Pregnant Rats
Pregnant rats were Sasco strains obtained from Charles River Laboratories, Inc. and maintained as described above. On the appropriate day of pregnancy rats were killed, ovaries removed, corpora lutea dissected, and luteal lysates prepared as described below. Day 1 of pregnancy is sperm positive day.

RNA Preparation and Northern Blot Analysis
Equivalent results were obtained when total RNA was isolated by a one-step isolation procedure according to Life Technologies, Inc. specifications for use of TRIzol reagent or isolated in a buffer containing 3 M LiCl and 6 M urea (76). Ten micrograms of RNA were separated by electrophoresis in a 1% agarose-formaldehyde gel. RNA was transferred to nylon membrane, covalently attached using UV cross-linking, and the membrane hybridized with relaxin cDNA that had been labeled with [{alpha}-32P] deoxy-CTP using random hexamer primers and the Klenow fragment of Escherichia coli DNA polymerase. Northern blots were reprobed with L19 (77), a probe that detects the LLrep3 gene family (78), to assess the amount of mRNA present in each lane. Hybridizations were carried out in 50% formamide, 5 x SSPE, 2 x Denhardt’s reagent, 10% dextran sulfate, 0.1% SDS, and 100 µg/ml salmon sperm DNA at 42 C. Membranes were washed in 2 x SSC at room temperature for 15 min, at 50 C for 30 min, and in 1 x SSC at 50 C for 30 min and then exposed to X-AR film Eastman Kodak Co. (Rochester, NY) at -80 C.

Electrophoretic Mobility Shift Assay
Cells were solubilized in 100 µl/60-mm culture dish of EMSA lysis buffer [20 mM HEPES, pH 7.0, 10 mM KCl, 1 mM MgCl2, 20% glycerol, 0.2% Nonidet P-40, 1 mM orthovanadate, 25 mM NaF, 200 µM phenylmethylsulfonyl fluoride (PMSF), 5 µg/ml aprotinin, 1 µg/ml pepstatin A, and 2 µg/ml leupeptin]. Lysates were incubated on ice for 20 min and then clarified by centrifugation at 20,000 x g for 20 min at 4 C. Oligonucleotides were labeled with [{gamma}-32P] ATP, using T4 polynucleotide kinase according to manufacturers specifications. Binding reactions (20 µl) were incubated at room temperature for 20 min and contained 0.5 ng DNA probe, ± 10 µg extract in 10 mM Tris (pH 7.5), 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 5% glycerol, and 1 µg poly (dI-dC). Polyacrylamide gels (5%) containing 2.5% glycerol and 0.5 x TBE were prerun in 0.5 x TBE for 30 min at 350 V and run at room temperature at 350 V after samples were loaded. Gels were wrapped in plastic wrap and exposed to film with intensifying screen at -70 C. The oligonucleotide probes used for gel mobility shift studies were as follows: Stat 1, 5'-CAT-GTT-ATG-CAT-ATT-CCT-GTA-AGT-G-3'; Stat 3, 5'-GAT-CCT-TCT-GGG-AAT-TCC-TAG-ATC-3'; Stat 5, 5'-AGA-TTT-CTA- GGA-ATT-CAA-TCC.

Lysate Preparation and Western Immunoblot Analysis
Clarified lysates were prepared by homogenization of cells in 100 µl/60 mm culture dish of a lysis buffer (10 mM potassium phosphate, pH 7.0, 1 mM EDTA, 5 mM EGTA, 10 mM MgCl2, 2 mM dithiothreitol, 1 mM sodium vanadate, 50 mM ß-glycerophosphate, 1 mM PMSF, 0.5% NP-40, 0.1% deoxycholic acid) followed by centrifugation of homogenates at 15,000 x g for 10 min. Samples were denatured by addition of 3 x stop (3% SDS, 150 mM Tris-HCl, 2.4 mM EDTA, 3% ß-mercaptoethanol, 30% glycerol, and 0.5% bromphenol blue). A similar procedure was employed for pregnant rat corpora lutea tissue samples. Protein concentrations were determined (79) using BSA as a standard. Protein samples were separated by SDS-PAGE and transferred to membranes for Western blot analysis. Western blot analysis was performed using the Amersham Pharmacia Biotech ECL detection system following the provided protocol. Where appropriate, membranes were stripped of antibodies according to the protocol provided with the ECL detection system. Densitometric quantitation was performed by image analysis using Molecular Analyst software from Bio-Rad Laboratories (Hercules, CA).

Immunoprecipitation
Immunoprecipitations were performed on lysates containing 500 µg of total protein using the indicated antibodies. Antibody-antigen complexes were precipitated by further incubation with an antimouse Ig antibody, where applicable, and protein A-conjugated Sepharose or with protein A/G-conjugated agarose alone. After washing the pelleted proteins with low-salt (10 mM Tris-HCl, pH 7.2, 150 mM NaCl, 1% deoxycholate, 1% Triton X-100, 0.1% SDS, 1 mM sodium vanadate, and 40 µg/ml PMSF) and high-salt (10 mM Tris-HCl, pH 7.2, 1 M NaCl, 0.1% NP-40, 1 mM sodium vanadate, and 40 µg/ml PMSF) radioimmunoprecipitation assay (RIPA) buffer, precipitated proteins were stopped in a 1x stop solution and denatured in a boiling water bath.


    FOOTNOTES
 
Address requests for reprints to: Mary Hunzicker-Dunn, Department of Cell and Molecular Biology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago Illinois 60611.

Supported by NIH Grant P01 HD-21921 (M.H.D.) and the P30 Center for Research on Fertility and Infertility, Northwestern University (NIH Grant P30 HD-28048).

Received for publication February 8, 1999. Revision received January 11, 2000. Accepted for publication January 18, 2000.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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