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Endocrinology Vol. 145, No. 11 4846-4852
Copyright © 2004 by The Endocrine Society

BRIEF COMMUNICATION

Role of Calcium-Calmodulin-Dependent Protein Kinase Cascade in Thyrotropin (TSH)-Releasing Hormone Induction of TSH and Prolactin Gene Expression

Koji Murao, Hitomi Imachi, Wen M. Cao, Xiao Yu, Hiroshi Tokumitsu, Hiroyuki Inuzuka, Norman C. W. Wong, Margaret A. Shupnik, Ryoji Kobayashi and Toshihiko Ishida

First Department of Internal Medicine (K.M., H.I., W.M.C., X.Y., T.I.) and Department of Signal Transduction Sciences (H.T., H.I., R.K.), Faculty of Medicine, Kagawa University, Kagawa 761-0793, Japan; Departments of Medicine and Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary (N.C.W.W.), Calgary, Canada T2N 4N1; and Department of Internal Medicine, Division of Endocrinology and Metabolism, University of Virginia (M.A.S.), Charlottesville, Virginia 22903

Address all correspondence and requests for reprints to: Dr. Koji Murao, First Department of Internal Medicine, Faculty of Medicine, Kagawa University, 1750-1 Miki-cho, Kita-gun, Kagawa 761-0793, Japan. E-mail: mkoji{at}kms.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
TRH binds to a membrane receptor that activates several intracellular signaling pathways and increases transcription of the TSH and prolactin (PRL) genes. Although TRH induces TSH and PRL gene expression, the underlying mechanism is not clear. In this report we examined the role of the Ca2+/calmodulin-dependent protein (CaM) kinase cascade in mediating TRH-stimulated transcription of TSH and PRL. RT-PCR and Western blot analysis were used to show that CaM kinase kinase (CaM-KK) and CaM IV (CaM-KIV) were present in rat anterior pituitary and its cell line GH3. Next, the effects of constitutively active CaM-KIV (CaM-KIVc) or its dominant negative mutant (CaM-KIVdn) on TSH and PRL promoter activity were tested in GH3 cells. The results showed that either CaM-KIVc alone or an upstream kinase, CaM-KK, induced the activity of both TSH and PRL promoters. Exposure of GH3 cells to 100 µM TRH induced CaM-KIV activity within 5 min and, as expected, also increased both TSH and PRL promoter activity. In contrast, cells carrying the CaM-KIVdn isoform had suppressed TRH induction of both TSH and PRL promoter activity. These results indicate that the CaM-KK-CaM-KIV cascade probably plays an important role in TRH induction of TSH and PRL transcriptional activity in pituitary cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE ANTERIOR pituitary, TRH binds to its receptor to activate phospholipase C. This interaction leads to calcium mobilization and protein kinase C (PKC) activation (1, 2). The combination of these intracellular signaling pathways ultimately results in increased transcription of the TSH-ß, {alpha}-subunit, and PRL genes (3, 4). Although many pituitary proteins are phosphorylated in response to TRH, protein kinase C, and Ca2+, most have not been identified (1).

TSH is a member of a pituitary glycoprotein hormone family that includes FSH, LH, and chorionic gonadotropin. These hormones consist of two subunits, termed {alpha} and ß. The {alpha}-subunit ({alpha}-glycoprotein hormone subunit) is common to all members of this family, and the ß-subunit is unique and confers specific biological activity to each dimeric hormone (1, 2). The TSH subunit genes are coordinately regulated at a transcriptional level by thyroid hormone (4), dopamine (5), and TRH (1, 2). Human PRL (hPRL) gene expression and secretion are regulated by various hormones and growth factors, including dopamine, epidermal growth factor, and TRH (6). These factors modulate different signaling pathways (cAMP, Ca2+, PKC, and MAPKs) that regulate hPRL gene transcription. These second messengers regulate hPRL gene activity via the proximal promoter region (4, 6, 7).

Ca2+/calmodulin-dependent protein (CaM) kinase type IV (CaM-KIV) is of particular interest in neuronal Ca2+ signaling because of its expression profile and its function as a transcriptional activator. CaM-KIV is expressed in both nuclei and cytosol of neurons of several brain regions, including the cortex, cerebellum, hippocampus, and amygdala (8). These kinases belong to a diverse group of enzymes that participate in many cellular responses and are activated by increasing concentrations of intracellular Ca2+. There are two multifunctional CaM kinases, CaM-KI and CaM-IV, that are activated by an upstream CaM kinase kinase (CaM-KK) through phosphorylation of a threonine residue within the active loop of the protein. This phosphorylation strongly up-regulates the catalytic activity of both enzymes (8, 9, 10, 11, 12).

In the present study we examined the role of the CaM-KK-CaM-KIV cascade on both TSH and PRL gene expression in pituitary cells. The results of our studies suggest a mechanism requiring the CaM-KK-CaM-KIV cascade in TRH induction of TSH and PRL gene transcription.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and animals
GH3 cells (Japan Health Sciences Foundation, Osaka, Japan) were cultured in Ham’s F-10 medium (ICN Biomedicals, Inc., Aurora, OH) supplemented with 15% fetal bovine serum and 2.5% horse serum in a humidified atmosphere containing 5% CO2. Male Wistar rats were killed by decapitation. The isolated rat pituitary cells were suspended in DMEM containing 5% horse serum, 2.5% fetal calf serum, and 1% nonessential amino acids, and the cells were seeded at a concentration of 1 x 105 cells/dish. All experimental procedures were approved by the animal care and experimentation committee of Kagawa University.

PCR
Total RNA was isolated from GH3 cells and pituitary of Wistar rats using a single-step acid guanidinium thiocyanate-phenol-chloroform extraction method (13). The expression of CaM-KIV and CaM-KK was examined using RT-PCR as described previously (14). A primer pair matching the published sequence (15, 16) of CaM-KIV and CaM-KK (sense, 5'-GCCCTATGCTCTCAAAGTGT-3' and 5'-GGAGGTGAAGAACTCAGTC-3'; antisense, 5'-CACACCGTCTTCATGAGCAC-3' and 5'-GGATGCAGCCTCATCTTCCT-3', respectively) was synthesized and used in separate RT-PCRs. Thirty cycles of PCR for CaM-KIV and CaM-KK were performed using a thermal cycler (Sanko Junyaku, Tokyo, Japan) according to a step program of 60 sec at 94 C, 60 sec at 51 C, and 60 sec at 72 C, followed by a 15-min extension at 72 C.

CaM-KIV activity in GH3 cells
GH3 cells were treated with 10 nM TRH for varying times as indicated, then the cells were lysed, and endogenous CaM-KIV was immunoprecipitated with anti-CaM-KIV antibody (an affinity-purified goat polyclonal antibody, sc-1546, Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Confluent GH3 cells in 100-mm culture dishes were rinsed with PBS and then lysed with 1.0 ml cold immunoprecipitation buffer [1% Triton X-100, 150 mM NaCl, 10 mM Tris (pH 7.4), 1 mM EDTA, 1 mM EGTA, 0.2 mM sodium vanadate, 0.2 mM phenylmethylsulfonylfluoride, and 0.5% Nonidet P-40] by constant agitation for 30 min at 4 C. The cells were scraped from the dish and passed several times through a 26-gauge needle to disperse large aggregates. Insoluble material was removed by centrifuging the lysate for 15 min at 4 C in a microcentrifuge, and the supernatant was called the total cell lysate. Next, we added to the lysate 5 µl CaM-KIV polyclonal antibody (Santa Cruz Biotechnology, Inc.), and the mixture was incubated at 4 C for 1 h. To this solution were added 20 µl protein G-Sepharose 4 Fast Flow, then it was vortexed and incubated with agitation at 4 C for 30 min, followed by three washes with immunoprecipitation buffer. CaM-KIV activity in the immunocomplex was measured at 30 C for 10 min in 30 µl 50 mM HEPES (pH7.5), 10 mM Mg(Ac)2, 1 mM dithiothreitol, 400 µM [{gamma}-32P]ATP (1000–2000 cpm/pmol), and 40 µM syntide-2 containing 2 mM CaCl2/8 µM CaM.

Transfection of GH3 cells and luciferase reporter gene assay
The PRL luciferase reporter gene (pPRL-LUC) was constructed using 940 bp from the hPRL promoter (–888/+52; +1 is defined as the start site of exon 1) (17) linked to the luciferase reporter gene (PGBV2, ToyoInk, Tokyo, Japan) as described previously (17). The reporter plasmid containing the rat TSH-ß promoter (pTSH-LUC) was constructed as described previously (2). Purified reporter plasmid was transfected into GH3 cells (at 60% confluence) using a conventional cationic liposome transfection methods (Lipofectamine, Invitrogen Life Technologies, Gaithersburg, MD). One microgram of Rous sarcoma virus-ß-galactosidase was added to all transfections as a monitor of the efficiency of DNA uptake by the cells (18). All assays were corrected for ß-galactosidase activity, and the total amount of protein per reaction was identical. Both cDNA of Ca2+/CaM-independent mutant of CaM-KIV (CaM-KIVc, 305 HMDT to DEDD), CaM-KIV kinase-negative mutant (CaM-KIVdn, 305 HMDT to DEDD, K71E), and CaM-KK mutant (CaM-KKc, residues 1–434) were constructed as described previously (9, 11). Transfected cells were maintained in control medium with or without cotransfection of a vector expressing the constitutively active form of CaM-KIV or an expression plasmid encoding a dominant negative mutant of CaM-KIV for 24 h as described previously (11). Transfected cells were harvested, and ß-galactosidase activity was measured in an aliquot of the cytoplasmic fraction (14). Twenty-microliter aliquots were taken for the luciferase assay and performed according to the manufacturer’s instructions (ToyoInk).

Western blot analysis
The proteins extracts were resuspended under reducing conditions, and 15 µg were fractionated by size on 7.5% sodium dodecyl sulfate-polyacrylamide gel, then transferred to polyvinylidene difluoride membrane for immunoblotting as described previously. The membranes were incubated for 1 h at 4 C with 0.2% Tween 20 in PBS containing anti-CaM-KK or anti-CaM-KIV antiserum (diluted 1:250) as described previously (19). Antibody binding was visualized using a chemiluminescence detection kit (ECL, Amersham Biosciences, Little Chalfont, UK).

Other assay
The levels of TSH and PRL were measured using a commercially available RIA kit (Amersham Biosciences, Piscataway, NJ).

Statistical analysis
Statistical comparisons were made by one-way ANOVA and t test, with P < 0.05 considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of CaM-KK-CaM-KIV in GH3 cells and rat pituitary
The participation of the CaM-KK/CaM-KIV-cascade in Ca2+-dependent gene expression has been demonstrated in various cell types, including hippocampal neuron and T cells (10). However, whether it is present in the rat pituitary and the GH3 cell line is not known. Therefore, we examined the mRNA expression of CaM-KK{alpha} and CaM-KIV using RT-PCR. The results (Fig. 1AGo) showed that CaM-KIV and an upstream protein kinase, CaM-KK{alpha}, was present in rat pituitary and GH3 cells. Consistent with the RT-PCR studies, both CaM-KK{alpha} and CaM-KIV proteins were detected using Western blot analysis (Fig. 1BGo) in lysate from rat pituitary and GH3 cells. Together these findings show that the mRNAs encoding and the corresponding proteins of CaM-KK{alpha} and CaM-KIV were present in rat pituitary and GH3 cells.



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  		FIG. 1. Expression of CaM-KK/CaM-KIV in rat anterior pituitary. A, RT-PCR of CaM-KK (left panel)/CaM-KIV (right panel) in anterior pituitary. ß-Actin was amplified as an internal control. Lane M, Markers; lane 1, GH3 cells; lane 2, rat anterior pituitary; lane 3, GH3 cells, negative control without reverse transcriptase; lane 4, rat anterior pituitary, negative control without reverse transcriptase. B, Total cell protein extracted from GH3 and rat anterior pituitary was blotted with anti-CaM-KK or CaM-KIV antibody, respectively. Lane 1, GH3 cells; lane 2, rat anterior pituitary.

 
Effect of CaM kinase cascade on both TSH and PRL gene transcription
Previous studies showed that CaM-KIV stimulated only PRL promoter activity (20), but whether other components of the CaM kinase cascade are a part of this process is not known. Reported data suggest a role for CaM-KIV in transcriptional regulation, and because the kinase is activated by CaM-KK, we postulated that CaM-KK is a likely participant (12). To answer this question, we examined the potential role of the CaM-K cascade by cotransfecting GH3 cells with pPRL-LUC or pTSH-LUC plus CaM-KIV and/or CaM-KK to determine whether these latter proteins affected PRL or TSH promoter activity. Results (Fig. 2Go) showed that GH3 cells expressing constitutively active CaM-KIVc alone induced 3- and 4-fold increases in TSH and PRL promoter activity, respectively. That CaM-KK participated in this process was demonstrated by coexpression of nonactivated wild-type CaM-KIV plus constitutively active CaM-KKc. This combination augmented TSH and PRL promoter activity by 4- and almost 7-fold, respectively. In contrast, the cotransfection of nonactivated wild-type CaM-KIV and CaM-KK did not activate the reporter, pTSH-LUC or pPRL-LUC. Together these findings suggest that both CaM-KIV and an upstream kinase, CaM-KK, activated TSH and PRL promoter activity in GH3 cells.



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  		FIG. 2. Effects of the CaM-KK/CaM-KIV cascade on TSH and PRL promoter activity. The CaM-KK/CaM-KIV cascade activated TSH (A) and PRL (B) promoter activity in GH3 cells. These cells were transfected with pTSH-LUC (A) or pPRL-LUC (B) and empty vector, CaM-KKc, CaM-KIVc/CaM-KIV [wild-type (w)] expression vectors, and cells were harvested 24 h later. All assays were corrected for ß-galactosidase activity, and the total amount of protein per reaction was identical. The results are expressed as relative luciferase activity compared with control cells, which were arbitrarily set at 100. Each data point shows the mean and SEM of three separate transfections. The asterisk denotes a significant difference (P < 0.05).

 
These findings prompted us to determine the physiological role of CaM-KIV in TSH or PRL secretion. Therefore, rat pituitary cells were transfected with the constitutively active CaM-KIVc, then we measured the TSH or PRL concentration in the medium by RIA. The results confirm that the transfection of CaM-KIVc stimulated both TSH (control, 3.65 ± 0.24 ng/ml; CaM-KIVc, 5.01 ± 0.48 ng/ml; P < 0.01; n = 5) and PRL (control, 1982 ± 154 ng/ml; CaM-KIVc, 2861 ± 224 ng/ml; P < 0.01; n = 5).

TRH induction of CaM-KIV activity
TRH is a well known physiological stimulant of both TSH and PRL gene expression (1). To determine whether the CaM kinase cascade is part of this induction process, we examined the ability of TRH to activate CaM-KIV in GH3 cells. To examine this possibility, GH3 cells were treated with 100 nM TRH for at least 1 min before harvest at 5, 10, 15, and 30 min after treatment (Fig. 3Go). Cell extracts were subjected to immunoprecipitation using an anti-CaM-KIV antibody, and the precipitated material was subjected to measurement of kinase activities using syntide-2. In comparison with nonstimulated cells, TRH treatment caused roughly a 2.5-fold increase in CaM-KIV activity. The induction kinetics of CaM-KIV activity reached a peak after 5 min of exposure to the TRH and returned to a basal level after 30 min. These data show that TRH can activate CaM-KIV, and the observation is consistent with the idea that TRH induction of both TRH and PRL activity may involve the CaM kinase cascade.



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  		FIG. 3. TRH stimulates the activity of CaM-KIV in GH3 cells. GH3 cells were exposed to 100 nM TRH for the indicated time, and CaM-KIV activity was determined as described in Materials and Methods. Each data point shows the mean and SEM of four separate determinations. The asterisk denotes a significant difference (P < 0.05).

 
Participation of CaM-KIV in TRH induction of TSH and PRL gene expression
The preceding studies have identified the components of the CaM kinase cascade in TRH induction of TSH and PRL promoter activity. Whether this signal transduction cascade mediates the actions of TRH in GH3 cells is not known. This lack of clarity in the mechanism of TRH action led us to examine whether CaM-KIV participates in TRH-stimulated transcription of both TSH and PRL genes.

To determine whether CaM-KIV plays a role in TRH-induced signaling to enhance TSH and PRL promoter activity, we cotransfected GH3 cells with pTSH-LUC or pPRL-LUC together with either an expression plasmid encoding a dominant negative mutant of CaM-KIVdn or control, an empty expression plasmid. The results (Fig. 4Go) showed that the activity of the reporter, pTSH-LUC, plus either empty vector and that of CaM-KIVdn in transfected cells exposed to medium without TRH was the same. In contrast, when transfected cells were exposed to medium containing 100 nM TRH pTSH-LUC activity increased 3.5-fold (Fig. 4AGo, column 2). The TRH-stimulated increase was attenuated only 2-fold in the presence of overexpression of CaM-KIVdn (Fig. 4AGo, column 3). When the PRL promoter was analyzed in a similar fashion, TRH caused a 5-fold increase in activity, and in the presence of CaM-KIVdn, the stimulatory effect of TRH was attenuated only 2.2-fold. Together these findings support the idea that CaM-KIV participates in the TRH induction of TSH and PRL promoter activity in GH3 cells, but it dose not seem to be the sole mediator.



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  		FIG. 4. CaM-KIVdn inhibits the up-regulation of TSH and PRL promoter activity by TRH stimulation in GH3 cells. A, Activity of TSH promoter; B, activity of PRL promoter. GH3 cells were transfected with pTSH-LUC (A) or pPRL-LUC (B) and CaM-KIVdn or empty vector. The cells were grown in control medium for 48 h after transfection. The cells were then exposed to medium containing 100 nM TRH for 24 h before measuring luciferase activity. All assays were corrected for ß-galactosidase activity, and the total amount of protein per reaction was identical. Control (no treatment with TRH); pTSH-LUC (A)/pPRL-LUC (B) and pcDNA3, CaM-KIVdn; pTSH-LUC (A)/pPRL-LUC (B) and CaM-KIVdn expression vector. The results are expressed as relative luciferase activities compared with control cells, which were arbitrarily set at 100. Each data point shows the mean and SEM of four separate transfections.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we have examined the role of the CaM-KK/CaM-KIV cascade in TRH induction of TSH and PRL promoter activity. Our interest in this potential role of CaM kinases stems from their participation in a wide variety of biological events that require intracellular Ca2+. For example, CaM kinases are involved in muscle contraction, neurotransmitter release, and the regulation of gene expression (8, 10). Recent studies have indicated that so-called multifunctional CaM kinases, including CaM-KI, -II, and -IV, are regulated by phosphorylation, either alone or with an upstream protein kinase (12, 13), CaM-KK.

A previous report showed that a specific inhibitor of CaM kinases, KN-62, reduced the ability of TRH to increase the activity of the PRL promoter and concluded that CaM-KII was involved in this process (20). Although KN-62 is thought to be highly specific for CaM-KII, KN-62 may also inhibit CaM-KIV. This finding prompted us to probe the role of CaM-KIV in TRH-induced signaling in the pituitary cell line GH3. The results presented here show that the mRNAs encoding the CaM-KIV and CaM-KK gene products and the corresponding proteins were present in both anterior pituitary and the rat pituitary cell line GH3. That the components of the CaM kinase cascade could induce TSH and PRL promoter activity was demonstrated in GH3 cells using cotransfection of reporter constructs plus CaM-KIV alone or with CaM-KK. The ability of constitutively active CaM-KIV to induce both TSH and PRL promoter suggested that this component activates gene expression. Data showing that constitutively active CaM-KK in the presence of wild-type CaM-KIV mimicked the effects of CaM-KIVc pointed to a role for the upstream kinase, CaM-KK. The rapid induction of intracellular CaM-KIV phosphorylation in GH3 cells after exposure to TRH was consistent with the idea that the CaM kinase cascade participated in TRH induction of TSH and PRL promoter activity. Finally the ability of a dominant negative CaM-KIV to attenuate the TSH- and PRL-inducing effects of TRH provided strong evidence that this cascade played an important role in mediating the actions of TRH. However, the observation that CaM-KIVdn does not completely block the stimulatory actions of TRH on TSH and PRL promoters suggests other pathways may also participate in the TRH induction of these genes. Another possibility for the results shown in Fig. 4Go is a technical explanation that may arise from prior induction of TSH and PRL promoter activity that precedes the expression of CaM-KIVdn.

Pituitary TSH production is stimulated physiologically by the actions of the hypothalamic peptide TRH (2). This effect leads to an increase in the intracellular free Ca2+ concentration and subsequent activation of PKC. Studies have revealed a positive regulation of the synthesis and secretion of TSH by agents acting through the cAMP protein kinase A system (21). However, there is growing evidence that the promoter of TSH-ß gene is also regulated by both protein kinase A/cAMP- and PKC/Ca2+-mediated pathways (1, 2). The mechanism by which TRH activates gene transcription is not completely defined; however, the POU domain protein Pit-1 plays a crucial role in the basal expression of TSH-ß (22, 23), but Pit-1 alone cannot restore TSH-ß promoter activity in thyrotrope cells that have lost the ability to express this gene (24). Our current report adds to the list the CaM-KK/CaM-KIV cascade and its ability to induce both TSH and PRL promoter activity in response to TRH stimulation.

One of the most common mechanisms by which elevated intracellular Ca2+ regulates cellular events is through its association with calmodulin. The Ca2+/CaM complex binds to and modulates the functions of multiple key regulatory proteins, including a family of CaM kinases (25). A role for transcriptional regulation by CaM kinase is suggested by the observation that the Ca2+-dependent transcription of three immediate early genes [c-fos, nerve growth factor-induced gene (NGFI)-A, and NGFI-B] is blocked 80% by the CaM kinase inhibitor KN-62 (12). CaM-KK has been identified and cloned as an activator for two multifunctional CaM-KKs, CaM-KI and CaM–IV. Phosphorylation by CaM-KK of Thr residues located in the activation loop of the catalytic domain of CaM-KIV results in a large increase in the catalytic efficiency of CaM kinase (9, 26).

CaM-KIV, which has significant nuclear localization, phosphorylates transcription factors, such as cAMP-responsive element-binding protein (CREB) and serum response factor (8). Several studies have shown that CaM-KIV can mediate transcriptional stimulation through CREB phosphorylation (27, 28). More recently, we have demonstrated that cotransfection of CaM-KIV with CaM-KK stimulates CREB-mediated transcription 14-fold relative to either kinase alone (26). This is consistent with the in vitro observation that the maximum velocity/Km ratio of CREB phosphorylation was increased 30-fold by activation of CaM-KIV by CaM-KK (29). Moreover, previous reports have shown that CREB-binding protein (CBP) enhances the expression of pituitary genes, including PRL and TSH, and suggested that CBP may play an important role in TRH stimulation of the anterior pituitary (21). Chawla et al. (30) suggested that CBP action is also controlled by CaM-KIV via the C terminus. Together with our results these findings indicate that activation of CBP by CaM-KIV may be involved in TRH-mediated pituitary hormone gene expression.

In summary, we have examined the role of the CaM-KK/CaM-KIV cascade in TRH induction of TSH and PRL promoter activity using the pituitary cell line, GH3. The results indicate that the cascade stimulates the transcription of pituitary hormones genes TSH and PRL, suggesting that activation of the CaM-KK-CaM-KIV cascade may play an important role in TRH signaling in the anterior pituitary.


   Acknowledgments
 
We thank Miss Kazuko Yamaji and Kiyo Ueeda for their technical assistance.


   Footnotes
 
This work was supported in part by Grant-in-Aid for Scientific Research 14770601 (to K.M.).

Abbreviations: CaM, Ca2+/calmodulin-dependent protein; CaM-KIV, Ca2+/calmodulin-dependent protein kinase IV; CaM-KIVc, constitutively active Ca2+/calmodulin-dependent protein kinase IV; CaM-KIVdn, dominant negative mutant of Ca2+/calmodulin-dependent protein kinase IV; CaM-KK, Ca2+/calmodulin-dependent protein kinase kinase; CBP, cAMP-responsive element-binding protein-binding protein; CREB, cAMP-responsive element-binding protein; h, human; LUC, luciferase; PKC, protein kinase C; PRL, prolactin.

Received April 28, 2004.

Accepted for publication July 28, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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