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Endocrinology Vol. 139, No. 4 1872-1882
Copyright © 1998 by The Endocrine Society

ARTICLES

Lactogenic Hormone-Inducible Phosphorylation and Gamma-Activated Site-Binding Activities of Stat5b in Primary Rat Leydig Cells and MA-10 Mouse Leydig Tumor Cells1

Masanori Kanzaki and Patricia L. Morris

Population Council (M.K., P.L.M.) and The Rockefeller University (P.L.M.), New York, New York 10021

Address all correspondence and requests for reprints to: Dr. Patricia L. Morris, Center for Biomedical Research, Population Council and The Rockefeller University, 1230 York Avenue, New York, New York 10021. E-mail: p-morris{at}popcbr.rockefeller.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The signal transducer and activator of transcription Stat5b has been implicated in signal transduction pathways for a number of cytokines and growth factors, including GH and PRL. Although these lactogenic hormones have the potential to enhance gonadotropin-induced steroidogenesis, the role of GH and PRL in the testis has long been and remains the subject of controversy. In this report we provide, to our knowledge, the first evidence of Stat5b protein expression in the testis and characterize the activation of Stat5b by these lactogenic hormones in primary rat progenitor, immature and adult Leydig cells, and mouse MA-10 Leydig tumor cells. In MA-10 cells, both GH and PRL mediate tyrosine phosphorylation of Janus kinase (JAK) 2 and Stat5b and induce DNA-binding activity of Stat5b. GH enhances both PIE (PRL-inducible element) and Fc{gamma}RI gamma-activated sites (GAS), but PRL modulates only PIE GAS. In primary Leydig cells isolated from 18-day-old rats, GH, but not PRL, activates cytoplasmic Stat5b and induces the binding of translocated nuclear Stat5b to GAS elements. Although Stat5b protein is expressed in both Percoll- and elutriator-purified adult rat Leydig cells, neither GH nor PRL treatment results in Stat5b-DNA binding. Our studies indicate that the MA-10 cell has the capacity to bind both GH and PRL and provides a useful model system with which to study the distinct testicular roles of these hormones. Moreover, our findings suggest that progenitor and immature Leydig cells are functional targets for GH in the immature rat, suggestive of a role for GH-Stat5b in testicular development. Our data indicate that lactogenic hormone-inducible transcriptional activation may target distinct gene expression in a signaling cascade(s) involving Stat5b but also imply coordinate control by multiple Leydig cell factors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GH receptor and PRL receptor (PRLR) are members of a class of plasma membrane receptors that were first characterized on the basis of several conserved features, including a single transmembrane domain and conserved amino acid sequences in the extracellular domain (1, 2, 3). GH receptor/GH binding protein immunoreactivity is localized mainly in the Leydig and Sertoli cell in adult rat testis (4). PRL binding sites and two forms of PRLR messenger RNAs (mRNAs) are demonstrated in testicular Leydig cells (5, 6, 7, 8). Due to conflicting evidence, the role of GH and PRL in male reproductive function has long been and remains the subject of controversy. GH treatment enhances testicular weight, LH receptor expression, and steroidogenic response to human CG (hCG) (9, 10, 11). PRL increases the number of LH receptors in Leydig cells and potentiates the effects of exogenous LH on testosterone production (12, 13, 14). Although PRL has no effect on the proliferation of MA-10 Leydig tumor cells, a dose-dependent biphasic effect of PRL on hCG-induced progesterone secretion is seen (15, 16). In the immature hypophysectomized rat model, LH, PRL, and GH treatment in vivo are all capable of increasing precursor mesenchymal cells, a finding that suggests hormonal and differentiation status affect responses to lactogenic stimulation (17).

The GH/PRL receptor superfamily shows neither a direct structural or functional relationship to the classical seven transmembrane-spanning receptors signaling via G proteins nor to those possessing inherent tyrosine kinase activity (1). Recent studies show that GH, PRL, and many cytokines use Stat proteins (signal transducers and activators of transcription) to regulate the transcription of specific genes through the Janus kinase (JAK)-Stat pathway (18, 19, 20). Ligand binding triggers the dimerization or oligomerization of receptors. Receptor-associated tyrosine-kinase (JAKs) cross-phosphorylate each other as well as the tyrosine residues on the cognate receptors. Subsequently, specific SH2-containing latent cytoplasmic Stat proteins are recruited, phosphorylated, and translocated into the nucleus where they activate gene transcription by binding to the promoters of target genes. The ability of individual receptors to activate overlapping but distinct sets of homo- and heterodimerized Stat proteins is thought to contribute to their signal specificity. In the search for the identification of PRL response elements, a gamma-activated site (GAS) sequence (TTCNNNGAA) in the promoter of the ß-casein gene and the putative transcription factor, named mammary gland factor, were identified (21). Mammary gland factor was cloned from sheep mammary tissue and identified as Stat5, a new member of the family of Stat proteins (22). In the mouse, Stat5 exists as two isoforms (5a and 5b) with a 96% similarity at the amino acid level; the major difference resides at the carboxyl termini, resulting in distinct transcriptional activation (23). Stat5a and Stat5b are expressed in most, if not all, tissues and can be activated by GH, PRL, and several cytokines (24, 25, 26, 27, 28). Both GH and PRL activate at least four different Stat family members (Stat1, 3, 5a, and 5b) but not Stat2, 4, and 6 in several cell types (25, 29, 30).

In the testis, we recently demonstrated that cell-specific testicular cytokine receptors are present and that Stat1 and Stat3 are differentially phosphorylated by interferon-{gamma} and interleukin-6, which mediate gene transcription (31, 32, 33, 34). In this report we provide, to our knowledge, the first evidence of testicular Stat5 protein expression and characterize the GH and PRL activation of Stat5b in primary testicular Leydig cells and MA-10 Leydig tumor cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies
Mouse anti-phosphotyrosine monoclonal antibody (mAb) (clone 4G10) and anti-JAK2 polyclonal antibody (pAb) were obtained from Upstate Biotechnology Inc. (Lake Placid, NY). Anti-Stat5b(SC) pAb, raised against a peptide corresponding to amino acids 706–722 of mouse Stat5b, was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Using transfection studies, this anti-Stat5b(SC) pAb has been shown not to cross-react with Stat5a (25). Anti-Stat5a pAb and anti-Stat5b(LH) pAb, raised against peptides corresponding to the C-terminal amino acids of cognate mouse Stat5 were kindly provided by L. Hennighausen (NIH). Specificity of these antibodies has been shown previously (35). Polyclonal antibodies raised against Stat1, Stat3, and pan-Stat5 (i.e. it recognizes both Stat5a and Stat5b) used in the supershift analysis in gel shift assay were kindly provided by J. E. Darnell, Jr. (Rockefeller University, New York, NY). Anti-3{alpha}-hydroxysteroid dehydrogenase (HSD) pAb and preimmune sera were generous gifts of T. Penning (University of Pennsylvania, Philadelphia, PA).

Cell culture
The MA-10 cell line (kindly provided by M. Ascoli, University of Iowa, Iowa City, IA) used in these experiments was maintained using standard protocols (36). For experiments, 1 x 107 cells were plated in 100-mm polystyrene plates and grown in Waymouth’s MB 752/1 medium with 15% horse serum (tested to maintain hCG binding in MA-10 cells). After 3 days, the medium was removed, cells were washed twice with PBS (Ca2+-, Mg2+-free), and serum-free Waymouth’s medium was added to the cells. MA-10 cells were stimulated with or without ovine GH (oGH, NIDDK-oGH-15), ovine PRL (oPRL, NIDDK-oPRL-15), or hCG (a generous gift of Y.Y. Tsong, Population Council). oGH, oPRL, and rat PRL (rPRL) were kindly provided by the National Hormone and Pituitary Distribution Program, NIDDK (Baltimore, MD).

Primary Leydig cells were prepared by the methods described previously (37). For the present studies, the progenitor and immature Leydig cells were prepared from 18-day-old Sprague-Dawley (SD) rats (Charles River, Kingston, NY); the freshly isolated primary Leydig cells were 45% 3{alpha}-HSD positive (Fig. 1Go, upper left) and 42% 3ß-HSD positive (Fig. 1Go, upper right) [these enzymes were used as indicators of the ratio of progenitor to immature Leydig cells as well as the purity and maturational status of the Leydig cells (38, 39)] and were cultured in serum-free medium supplemented with 2.5 µg/ml bovine insulin (Sigma, St. Louis, MO), 5 µg/ml transferrin (Calbiochem, La Jolla, CA), and 10 µg/ml bacitracin (Sigma). After 2 days in culture, the spindle-shaped Leydig cells were strongly positive for 3{alpha}-HSD using immunocytochemistry (Fig. 1Go, lower left) and clearly positive for 3ß-HSD using histochemistry (Fig. 1Go, lower right). Adult Leydig cells were obtained fromSD rats (55–65 days of age), purified by Percoll gradient separation (85% 3ß-HSD positive), and cultured in serum-free medium with the above factors. As required to confirm negative findings with the Percoll-purified Leydig cells, elutriator-purified adult Leydig cells (>97% 3ß-HSD positive) were prepared as described previously (37). Leydig cells were cultured for 2 days as above, washed, and then stimulated with oGH, oPRL, or rPRL (NIDDK-rPRL-B8SIAFP) for the indicated times.



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  		Figure 1. Characterization of Leydig cells isolated from 18-day-old rats. Freshly isolated Leydig cells from immature rats were cytocentrifuged on slides (upper panels) or the Leydig cells were cultured on chamber slides for 2 days (lower panels). For immunocytochemistry using anti-3{alpha}-HSD pAb, cells were fixed with methanol. Magnification, x 400. Freshly isolated Leydig cells from immature rats stained for 3{alpha}-HSD (upper left) or 3ß-HSD (upper right). Cultured Leydig cells from immature rats stained for 3{alpha}-HSD (lower left) or 3ß-HSD (lower right).

 
All experiments were repeated three to five times using different cell primary preparations or with the MA-10 cell line with comparable results obtained in the replicates of each. The results are presented as those typical for each experiment illustrated.

Total cellular lysate isolation and immunoprecipitation
Cells (2 x 107) were lysed in 1 ml RIPA buffer (50 mM Tris-HCl, pH 7.5, containing 1% NP-40, 0.1% SDS, 0.1% sodium deoxycholate, 150 mM NaCl, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml pepstein, 1 mM sodium orthovanadate; Sigma) for 30 min on ice. Cellular debris was pelleted by centrifugation at 12,000 x g for 15 min. Total cellular lysates were precleared for 1 h at 4 C with protein G-Sepharose beads (Santa Cruz) and were incubated overnight at 4 C with indicated antibody. Antibodies were captured by incubation for 1 h at 4 C with protein G-Sepharose beads and washed, and the samples were then dissolved in 2 x Laemmli’s buffer and boiled for 2 min.

Cytoplasmic protein and nuclear extract preparation
Cells (1 x 107) were lysed in 1 ml hypotonic buffer A (10 mM HEPES-KOH, pH 7.9; 10 mM KCl; 1.5 mM MgCl2; 0.5% NP-40; 2 mM dithiothreitol; 0.5 mM sodium orthovanadate; 0.5 mM phenylmethylsulfonyl fluoride; 1 µg/ml leupeptin; 2 µg/ml aprotinin) (Sigma). Cytoplasmic proteins were collected by centrifugation at 2,000 x g for 20 sec. Pellets were dissolved and washed once with 1 ml of buffer A containing 20% glycerol. Nuclei were collected by centrifugation at 2,000 x g for 20 sec, and nuclear proteins were extracted in 100 µl of high salt buffer B (20 mM HEPES-KOH, pH 7.9; 0.55 M KCl; 1.5 mM MgCl2; 2 mM dithiothreitol; 20% glycerol with the above protease and phosphatase inhibitors) for 30 min on ice with occasional vortexing. Supernatants (nuclear extracts) were collected by centrifugation at 12,000 x g for 15 min.

Immunoblotting
Cytoplasmic proteins (15 µg per lane), nuclear extracts (15 µg per lane), or immunoprecipitates were subjected to SDS-PAGE using 6.5 or 7.5% polyacrylamide gels under reducing conditions and electrophoretically transferred to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH). The membranes were probed with antibodies diluted at the following concentrations: anti-phosphotyrosine mAb (1:4,000), anti-Stat5a pAb (1:20,000), anti-Stat5b(LH) pAb (1:10,000), anti-Stat5b(SC) pAb (1:1,000), and anti-JAK2 pAb (1:2,000). Blots were developed with the enhanced chemiluminescence Western blotting system (Amersham, Arlington Heights, IL).

Electrophoretic gel mobility shift assay (EMSA)
The DNA probes used for gel mobility shift assay were as follows: 1) PIE (PRL-inducible element) probe (Santa Cruz, sc-2565) and 2) Fc{gamma}RI double-stranded probe made by annealing the following two synthetic complementary oligonucleotides: 5'-GATCGTATTTCCCAGAAAAGGAAC-3' (sense) and 5'-GGTTCCTTTTCTGGGAAATAC-3' (antisense) (Protein/DNA Technology Center, The Rockefeller University, New York, NY). PIE probe was end-labeled on one strand using T4 polynucleotide kinase (Promega, Madison, WI) and [{gamma}-32P]ATP (DuPont NEN, Boston, MA). Fc{gamma}RI GAS probe was labeled with a fill-in reaction using Klenow enzyme (Amersham) and [32P]deoxycytidine triphosphate (Amersham). Nuclear extracts (5 µg) were incubated at room temperature with the labeled oligonucleotide probe (0.5 ng, 2 x 105 cpm) in binding buffer with 1 µg poly (dI-dC) acid (Pharmacia Biotech, Piscataway, NJ) for 30 min. before separation by electrophoresis through a 4% polyacrylamide gel in 0.5 x Tris-borate-EDTA. Antibody supershifts were performed by adding 1:10 diluted preimmune rabbit serum or polyclonal antibodies raised against specific Stat proteins to binding reactions for a 30-min incubation before the addition of the probe.

Histo- and immunocytochemistry
Cells were attached to glass microscope slides using a StatSpin cytocentrifuge (Norwood, MA) or were cultured on glass chamber slides (Lab Tek, NUNC, Naperville, IL). The cells were fixed using 0.3% H2O2-methanol for 10 min at room temperature to quench the endogeneous peroxidase activity. The cells were then processed with the avidin-biotin complex (ABC) method using the Vectastain ABC kit (Vector, Burlingame, CA). The cells were incubated overnight at 4 C with anti-Stat5b(SC) pAb (1:1,000) or anti-3{alpha}-HSD pAb (1:1,000). After washing with PBS, the samples were treated with biotinylated anti-rabbit IgG antibody, followed by the avidin-biotin complex reagents and then developed with 3,3'-diaminobenzidine chromogen (DAKO, Carpinteria, CA). Preimmune serum and PBS were employed in place of primary antisera to determine nonspecific immunoreactivity.

Histochemical staining for {Delta}5-3ß-HSD enzyme activity was completed on air-dried attached or cultured Leydig cells with 0.4 mM etiocholanolone as the steroid substrate as previously described (39).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Nuclear translocation of Stat5b protein in MA-10 cells and primary rat Leydig cells
MA-10 cells were treated with either 100 ng/ml oGH or oPRL for various time periods up to 60 min; cytoplasmic protein and nuclear extracts were then isolated. The specificity of the testicular Stat5b activation response was investigated using Western blot analyses to determine the changes in the electrophoretic mobility of Stat5b protein, previously shown to be indicative of changes in the phosphorylation status of Stat5b (25, 40). In cytoplasmic proteins of unstimulated MA-10 cells, Stat5b migrated as a doublet that corresponds to the nonphosphorylated form (designated band 0) and a serine/threonine-phosphorylated form (designated band 1a) (Fig. 2AGo, lanes 1, 6, and 11). Stat5b immunoreactive band, designated band 2, induced by oGH corresponds to the tyrosine + serine/threonine-phosphorylated form (Fig. 2AGo, lanes 3, 4, 5, and 12). Band 1a migrates somewhat more slowly than band 1 (tyrosine-phosphorylated form) on Western blots of SDS gels. In most cases and in other published studies, however, these two bands cannot readily be distinguished electrophoretically and are therefore labeled as ‘band 1/1a’ (Fig. 2AGo, lower panel). In extracts from untreated cells, this band corresponds to band 1a. Since it represents a mixture of bands 1 and 1a in oGH- or oPRL-stimulated cytoplasmic proteins, we used nuclear extracts for Western analyses to identify the activated form of Stat5b, especially in oPRL-treated cells. Before the treatment, no nonactivated forms (neither nonphosphorylated nor serine/threonine-phosphorylated forms) were found in the nuclear extracts (Fig. 2BGo, lanes 14 and 19). In cells treated with either oGH (5 min) or oPRL (15 min), only activated forms (tyrosine-phosphorylated and/or tyrosine + serine/threonine-phosphorylated forms) of Stat5b proteins were detected in the nuclear extracts. In the nucleus, the activated Stat5b protein was detected up to 60 min. Bands designated NS were occasionally detected in the nuclear extracts; however, changes in the intensities of these bands did not consistently correlate with the presence of lactogenic hormones nor were these bands supershifted with specific Stat antisera. Using immunocytochemistry, MA-10 cells were examined for Stat5b localization at time zero and 30 min after the addition of the lactogenic hormones (100 ng/ml). Before treatment, Stat5b was detected in the cytoplasm (Fig. 4AGo). After oGH or oPRL treatment for 30 min, there was significant Stat5b immunostaining in the nucleus with very weak immunoreactivity seen remaining in the cytoplasm (Fig. 4Go, B and C).



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  		Figure 2. GH- or PRL-induced nuclear translocation of activated Stat5b in MA-10 cells. MA-10 cells were incubated with either 100 ng/ml oGH or oPRL for the times indicated. A, Cytoplasmic proteins were subjected to 6.5% SDS-PAGE and analyzed by immunoblot with anti-Stat5b(SC) pAb (1:1,000 dilution). The Stat5b immunoreactive band, designated band 2 (lane 12), was induced by oGH and corresponds to tyrosine + serine/threonine-phosphorylated forms. Band 1a (serine/threonine-phosphorylated form) and band 1 (tyrosine-phosphorylated form) are not readily distinguished electrophoretically and are, therefore, designated as band 1/1a on this figure. In extracts from untreated cells, this band corresponds to band 1a (lane 11), whereas in oGH- or oPRL-stimulated extracts, it corresponds to a mixture of band 1 and 1a. P indicates phosphorylated forms, and 0 indicates nonphosphorylated forms. B, Nuclear extracts were subjected to 7.5% SDS-PAGE and analyzed by immunoblot with anti-Stat5b(SC) pAb. * Indicates activated forms, and NS indicates nonspecific band.

 


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  		Figure 4. Immunocytochemical analyses showing the translocation of Stat5b into the nuclei of MA-10 cells or primary rat Leydig cells (magnification, x 400) using anti-Stat5b(SC) pAb. Cells were cultured within chamber slides and stimulated with 100 ng/ml oGH or oPRL for 30 min and then fixed with methanol. The specificity of the staining was confirmed using nonimmune rabbit serum (see insets of panels A, D, and G). MA-10 cells: Cytoplasmic localization of Stat5b in untreated MA-10 cells (panel A); nuclear localization of Stat5b after GH treatment (panel B); nuclear localization of Stat5b after PRL treatment (panel C). Primary Leydig cells from immature rats: cytoplasmic localization of Stat5b in untreated, cultured Leydig cells (panel D); nuclear localization of Stat5b in GH-treated Leydig cells (panel E); cytoplasmic localization of Stat5b following PRL treatment (panel F). Primary Leydig cells from adult rats: Cytoplasmic localization of Stat5b in untreated, cultured Leydig cells (panel G), GH-treated (panel H), and PRL treated (panel I) rat Leydig cells.

 
We next applied the same analytical methods for primary Leydig cells isolated from both immature and adult rats. In Leydig cells isolated from immature rats and cultured for 2 days in serum-free medium, oGH, but not PRL (ovine and rat origin), activated Stat5b within 60 min. In oGH-treated Leydig cells, the slower migrating band corresponding to activated Stat5b was observed as cytoplasmic protein (Fig. 3AGo, lane 6). In the nuclear extracts from these oGH-treated Leydig cells, activated Stat5b was detected up to 60 min (lanes 7, 9, and 12). Lanes 6 and 7 of Fig. 3Go illustrate that activated Stat5b from the nuclei of oGH-treated Leydig cells runs slower than nonactivated forms in the cytoplasm. Neither oPRL nor rPRL (1 and 100 ng/ml doses) had an effect on the nuclear translocation of Stat5b within 60 min. To determine whether Stat5b is activated by lactogenic hormone stimulation of adult Leydig cells, primary Leydig cells were purified, cultured for 2 days in serum-free medium, and then treated with 100 ng/ml oGH, oPRL, or rPRL for 30 min. Although nonactivated forms of Stat5b protein were detected in the cytoplasm, neither GH nor PRL translocated Stat5b into the nucleus of adult Leydig cells (Fig. 3BGo).



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  		Figure 3. GH- or PRL-regulated nuclear translocation of activated Stat5b in primary Leydig cells. Leydig cells were isolated from 18-day-old or adult (55–65 days of age) rats and were cultured in serum-free media for 2 days. Cells were then incubated with indicated doses of oGH, oPRL, rPRL, or with vehicle for 30 min (panel A, lanes 1–11, and panel B) or 60 min (panel A, lanes 12–14). Cytoplasmic proteins (panel A, lanes 1–6, and panel B) or nuclear extracts (panel A, lanes 7–14) from immature cells were subjected to 6.5% SDS-PAGE and analyzed by immunoblot with anti-Stat5b(SC) pAb (1:1,000 dilution). A, In Leydig cells from immature rats, oGH, but not ovine or rat PRL (1 or 100 ng/ml) activated Stat5b (designated *). NE (lane 7), Nuclear extract; NA, nonactivated Stat5b. B, Neither oGH (100 ng/ml) nor ovine or rat PRL (100 ng/ml) activates Stat5b within 30 min. NA, Nonactivated Stat5b.

 
Next, using immunocytochemistry, primary Leydig cells were examined for Stat5b localization at experimental time zero and 30 min after the addition of lactogenic hormones (100 ng/ml). Leydig cell-enriched primary preparations isolated from 18-day-old rats contain a combination of mesenchymal Leydig progenitors and immature Leydig cells. In vivo, the progenitor Leydig cell differs in morphology from the immature Leydig cell into which it will transform as new functional capabilities are acquired with maturation (41). In culture, both progenitor and immature Leydig cells assume a spindle-shaped morphology similar to that of elutriator-purified (>97% 3ß-HSD) and Percoll-purified Leydig cells isolated from adult rats and cultured under the conditions of the present study (see Fig. 1Go, lower panels, and Fig. 4Go, D–I). In cultured Leydig cells from immature rats, treatment with oGH for 30 min resulted in the nuclear translocation of Stat5b compared with the cytoplasmic localization of Stat5b seen in vehicle-treated matched controls (Fig. 4Go, D vs. E). In contrast, no nuclear Stat5b was observed in the same primary cells treated with either oPRL (Fig. 4FGo) or rPRL (data not shown). In Percoll-purified adult Leydig cells, lactogenic hormones (oGH, oPRL, Fig. 4Go, H and I, respectively) did not stimulate Stat5b translocation into the nucleus although there was apparently ample cytoplasmic Stat5b in untreated 3ß-HSD- positive Leydig cells (Fig. 4GGo). When these experiments were repeated with elutriator-purified, macrophage-negative, adult Leydig cells, similar results were found (data not shown).

Tyrosine phosphorylation of JAK2 and Stat5 proteins in MA-10 cells
Previous studies revealed that tyrosine phosphorylation of JAK2, but not JAK1, JAK3, or Tyk2, is required for Stat5-mediated cell signaling (42, 43). Therefore, we next examined the tyrosine phosphorylation of JAK2 protein after treatment with oGH or oPRL for 15 and 30 min using MA-10 cells. Our data (Fig. 5Go) demonstrate that JAK2 is tyrosine phosphorylated after the addition of oGH or oPRL as shown by immunoprecipitation with JAK2 antiserum and subsequent Western blotting with anti-phosphotyrosine antibody, 4G10. When blots were stripped and reprobed with JAK2 antibody, similar levels of protein were observed (Fig. 5Go, lower panel).



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  		Figure 5. GH- and PRL-induced tyrosine phosphorylation of JAK2 in MA-10 cells. MA-10 cells were incubated with 100 ng/ml oGH or oPRL for the times indicated. Whole-cell lysates were immunoprecipitated with anti-JAK2 pAb. Immunoprecipitated proteins were immunoblotted first with anti-phosphotyrosine 4G10 mAb (1:4,000 dilution), stripped, and reprobed with anti-JAK2 pAb (1:2,000 dilution). The molecular weight of protein standards are indicated.

 
We next studied the tyrosine phosphorylation of Stat5b proteins in MA-10 cells treated with oGH or oPRL. Cells were treated for 30 min and lysed. Total cellular proteins were immunoprecipitated with 4G10 mAb, and tyrosine- phosphorylated Stat5b was detected by Western blotting using anti-Stat5b(SC) pAb. Although Stat5b was not phosphorylated in nontreated MA-10 cells or in cells treated with hCG, oGH and oPRL treatment resulted in phosphorylation of Stat5b (Fig. 6AGo). To investigate the kinetic effect of oGH or oPRL on tyrosine phosphorylation of Stat5b, proteins were immunoprecipitated with Stat5b(SC) pAb and Western blotted with 4G10 mAb. Tyrosine phosphorylation of the Stat5b band was seen in both oGH- and oPRL-treated MA-10 cells by 15 min. The amount of phosphorylated Stat5b decreased after 30 min, but was still apparent at 60 min despite similar levels of total Stat5b proteins, ascertained by reblotting with anti-Stat5b(SC) pAb (Fig. 6BGo, lower panels).



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  		Figure 6. GH- and PRL-promoted tyrosine phosphorylation of Stat5b in MA-10 cells. A, MA-10 cells were incubated with 100 ng/ml oGH, oPRL, hCG, or with vehicle for 30 min. Whole-cell lysates were immunoprecipitated with anti-phosphotyrosine 4G10 mAb. Immunoprecipitated proteins were immunoblotted with anti-Stat5b(SC) pAb (1:1,000 dilution). The molecular weight of protein standards are indicated. B, MA-10 cells were incubated with 100 ng/ml oGH or oPRL for the times indicated. Whole cell lysates were immunoprecipitated with anti-Stat5b(SC) pAb. Immunoprecipitated proteins were immunoblotted first with anti-phosphotyrosine 4G10 mAb (1:4,000 dilution), stripped, and reprobed with anti-Stat5b(SC) pAb (1:1,000 dilution).

 
To address the issue of heterodimerization of Stat5a with Stat5b, MA-10 cells were treated with oGH or oPRL for 30 min and then lysed in RIPA buffer. Immunoprecipitations were performed using anti-Stat5a pAb or anti-Stat5b(LH) pAb. Western analyses using anti-phosphotyrosine mAb demonstrated that Stat5a protein (94 kDa), which appears as a slower migrating band than Stat5b (92 kDa) (Fig. 7Go, upper panel), is tyrosine phosphorylated as well as Stat5b in lactogenic hormone-treated MA-10 cells. In immunoprecipitates using the anti-Stat5a pAb, no phosphorylated Stat5b protein was detected. Very faint bands corresponding to Stat5b were detected in immunoprecipitates using the anti-Stat5b(LH) pAb when membranes were blotted with anti-Stat5a pAb (middle panel). However, there was no evidence for the formation of Stat5a-Stat5b heterodimers in other studies when the membrane was blotted with either anti-Stat5b(LH) pAb (lower panel) or anti-Stat5b(SC) pAb (data not shown).



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  		Figure 7. GH- and PRL-stimulated tyrosine phosphorylation of Stat5a and Stat5b in MA-10 cells. MA-10 cells were incubated with 100 ng/ml oGH, oPRL, or with vehicle for 30 min. Whole-cell lysates were immunoprecipitated with anti-Stat5a pAb or anti-Stat5b(LH) pAb. Immunoprecipitated proteins were subsequently immunoblotted with anti-phosphotyrosine mAb (4G10, 1:4,000 dilution), anti-Stat5a pAb (1:20,000 dilution), or anti-Stat5b(LH) pAb (1:10,000 dilution).

 
Binding of Stat5 proteins to distinct GAS elements in GH- or PRL-treated MA-10 cells
We next studied whether the effects of these lactogenic hormones on Stat5b protein tyrosine phosphorylation were associated with enhanced binding of Stat5b to GAS elements. The DNA binding of nuclear protein from MA-10 cells to synthetic, double-stranded oligonucleotides containing a DNA response element derived from either PIE or Fc{gamma}RI was examined using a gel mobility shift assay. The oligonucleotide representing the PIE of the rat ß-casein promoter predominantly binds to nuclear extracts of oGH- or oPRL-treated cells and forms a single mobility shift complex X (Fig. 8AGo). The intensity of complex X increased in a dose-dependent manner in both oGH- and oPRL-treated cells. The presence of Stat5 proteins in the oGH- or oPRL-activated PIE complex was verified by the ability of polyclonal antibodies to form a gel "supershift" of complex X (Fig. 8CGo). The complex was supershifted by both anti-pan Stat5 (lane 4) and anti-Stat5b(SC) antiserum (lane 5), but not by preimmune serum, anti-Stat1, or -Stat3 antiserum (Fig. 8CGo, upper panel). The lower panel in Fig. 8CGo shows that complex X is composed of two different bands Xa and Xb. Band Xa was supershifted when anti-Stat5a pAb was added (lanes 7 and 10), and band Xb was supershifted by anti-Stat5b(LH) (lanes 8 and 11) in both oGH- and oPRL-treated MA-10 cells. The kinetics of the formation of PIE-Stat5 complex were consistent with the time-dependent nuclear translocation of activated Stat5b proteins shown in Fig. 2Go. Interestingly, when the Fc{gamma}RI probe was used, only nuclear extracts from oGH-treated, but not oPRL-treated, MA-10 cells bound this probe (Fig. 9AGo). This complex I was also shown by supershift analyses to contain both Stat5a and Stat5b proteins, but not Stat1 and Stat3 (Fig. 9BGo).



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  		Figure 8. GH- and PRL-modulated Stat5b protein binding to PIE oligonucleotide in MA-10 cells. Nuclear extracts were subjected to EMSA with the PIE probe. A, Cells were incubated with 100 ng/ml oGH or oPRL for the times indicated. Extracts from cells and PIE probe formed a specific protein-DNA complex, designated complex X. B, Cells were incubated with increasing doses of oGH or oPRL for 30 min. The intensity of complex X showed the dose-dependent increase up to 500 ng/ml. C, PIE gel shift complex X was identified as containing Stat5b protein by supershifting with both anti-pan-Stat5 pAb (S5) and anti-Stat5b(SC) pAb (S5b(SC))(upper panel). In the lower panel, the complex X consists of two complexes (Xa and Xb). Xa was supershifted by anti-Stat5a pAb (S5a) and Xb by anti-Stat5b(LH) (S5b(LH)) pAb. s.s., Supershifted band; NI, preimmune serum; S1, anti-Stat1 pAb; S3, anti-Stat3 pAb.

 


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  		Figure 9. GH- and PRL-mediated Stat5 protein binding to Fc{gamma}RI probe in MA-10 cells. Nuclear extracts were subjected to EMSA with the Fc{gamma}RI probe. A, Cells were incubated with 100 ng/ml oGH or oPRL for the times indicated. Extracts from oGH-treated MA-10 cells and Fc{gamma}RI probe formed a specific protein-DNA complex, designated complex I. B, Fc{gamma}RI gel shift complex I was identified as containing Stat5a and Stat5b by supershifting with anti-pan-Stat5 pAb (S5), anti-Stat5b(SC) pAb (S5b(SC)), anti-Stat5b(LH) pAb (S5b(LH)), and anti-Stat5a pAb (S5a). s.s., Supershifted band; NI, preimmune serum; S1, anti-Stat1 pAb; S3, anti-Stat3 pAb.

 
In primary Leydig cells isolated from immature rats, nuclear extracts from oGH-treated cells bound both PIE and Fc{gamma}RI DNA (Fig. 10AGo, lane 4, and 9B, lane 4), but when cells were treated with oPRL or rat PRL, we could not detect specific DNA-protein complex in EMSA using either PIE and Fc{gamma}RI oligonucleotides. In lane 3 of Fig. 10Go, A and B, 1 µg of nuclear extract instead of 5 µg was used to check the specificity. In oGH- or PRL (ovine and rat)-treated adult Leydig cells, no specific DNA-protein complex was detectable using GAS probes (data not shown).



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  		Figure 10. Stat5 protein binding to GAS DNA elements in Leydig cells from immature rats. Leydig cells were incubated with oGH or ovine or rat PRL (1 or 100 ng/ml) for the times indicated. EMSA was performed using PIE (A) or Fc{gamma}RI (B) probe. EMSA using nuclear extracts of oGH-treated Leydig cells and each of these probes formed specific protein-DNA complexes, designated either complex X or I. In lane 3 of both panels A and B, 1 µg nuclear extract instead of 5 µg was added to the reaction.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Herein we describe the identification and characterization of Stat5b protein in Leydig cells using MA-10 mouse Leydig tumor cells as well as primary rat Leydig cells. GH and PRL mediate tyrosine phosphorylation of Stat5a and Stat5b protein and DNA binding to the GAS elements in MA-10 cells. In Leydig cells from 18-day-old rats, GH, but not PRL, activates Stat5b and modulates the binding of translocated nuclear Stat5b to GAS elements. Although Stat5b protein is expressed in cultured adult Leydig cells and despite the presence of their respective receptors, neither GH nor PRL activates Stat5b within 30 min. In contrast to freshly isolated nonproliferating, differentiated primary rat Leydig cells, MA-10 cells are immortalized mouse Leydig cells with tumorigenic ability. An additional difference between primary Leydig cells and the cell line is a lesion in the 17{alpha}-hydroxylase activity in MA-10 cells that results in the production of progesterone, rather than androgen, as the major steroid secreted (36). Alternatively, recent studies indicate that inhibitors of Stat proteins may be involved at a cytoplasmic level in the regulation of ligand-specific activation of distinct cell types (44). Such underlying factors, as well as the species of their origin, may be relevant to the ability to differentially activate the Leydig cell Stat5b-signaling cascade in these two systems. However, LH/hCG receptor affinity, receptor number, and many of the steroidogenic processes are very similar in MA-10 and normal rat Leydig cells (15, 36). Thus, the MA-10 mouse testicular Leydig tumor cell line has provided a useful model for the study of steroidogenesis in Leydig cells. Whether the regulation of Leydig cell steroidogenesis involves the JAK2/Stat5 system is not clear at this time. hCG increases cAMP and progesterone production in MA-10 cells but did not phosphorylate Stat5b in our studies. A recent study showed that although PRL activates the JAK2/Stat5 pathway in the ovary, the down-regulation of 20{alpha}-HSD mRNA by PRL does not appear to involve tyrosine kinase activity but rather depends on de novo synthesis of protein(s) (45). Thus, similar events may occur in Leydig cells.

Studies of the rat Leydig cell led to the recognition of three distinct developmental stages: Leydig cell progenitors, immature, and adult Leydig cells. By day 14, mesenchymal-like stem cells become Leydig cell progenitors, intermediates in the Leydig cell lineage. In situ between days 14 and 28, progenitors transform to morphologically recognizable immature Leydig cells that are rounder due to more abundant smooth endoplasmic reticulum, have a more intense 3ß-HSD staining, and express higher levels of functional LH receptors (41). Our findings indicate that >90% of the Leydig cells in our primary preparation from immature rats contain cytoplasmic Stat5b. Therefore, it appears that both progenitors and immature Leydig cells contain the latent cytoplasmic transcription factor. Also, our immunocytochemical data demonstrate the nuclear translocation of Stat5b after GH but not PRL treatment in almost all the primary Leydig cells from the immature rat. Taken together, our findings suggest that both the progenitors and immature Leydig cells in the testis of 18-day-old rats can respond to GH stimulation with Stat5b-DNA binding. In contrast, neither GH nor PRL induce Stat5b DNA binding in the adult Leydig cell that has been isolated and then cultured as indicated, findings consistent with a role for Stat5b during Leydig cell differentiation.

The PRLR is a single chain transmembrane protein that belongs to the cytokine receptor superfamily and is expressed in a wide variety of tissues. Both forms of the PRLR can dimerize upon ligand binding and activate JAK2, Fyn, and mitogen-activated protein (MAP) kinases to promote cell growth (19, 46). Only the long form activates transcription of the ß-casein gene in transfected cell lines (47), and the short form acts as a dominant negative inhibitor through the formation of inactive heterodimers resulting in an inhibition of JAK2 activation (48). Dual activation of the glucocorticoid receptor by glucocorticoids and Stat5 by PRL is required for efficient induction of the ß-casein gene (49). The tyrosine-docking sites of the rat PRLR required for maximal Stat5 activation were recently identified, but these tyrosine residues were not implicated in the activation of JAK2 (50).

In the testis, both short and long forms of PRLR mRNAs are observed, although the long form mRNA is preferentially expressed in Leydig cells (6, 8). While PRL has been shown to bind to both immature and adult rat Leydig cells in vitro (5, 14), PRL did not activate Stat5b in primary rat Leydig cells. Although chronic (24 h) exposure to oPRL has been shown to exert biphasic dose-dependent effects on gonadotropin-induced steroidogenesis in primary rat Leydig cells or mouse MA-10 cells (14, 16), our results reveal no biphasic effect on Stat5-PIE binding in MA-10 cells. Moreover, in the primary Leydig cells, neither 1 nor 100 ng/ml of PRL activated Stat5. It is interesting to note that although both granulosa and luteal ovarian cells respond to PRL in PMSG-primed PRL-treated rats, PIE-binding activity was detected only after treatment with hCG (51). Thus, the kinetics of PRL regulation of Leydig cell function may require a multiplicity of events modulated by cross-talk among other signaling pathways such as Fyn and MAP kinases and cAMP.

A potential involvement of GH in testicular function is suggested by both experimental and clinical data. In humans and animals, either isolated GH deficiency or GH resistance is associated with delayed puberty and poor responsiveness to hCG stimulation, indications of a Leydig cell defect(s) secondary to the GH deficit (52, 53). These data imply that GH is involved in the functional development of Leydig cells in vivo. However, one of the major problems associated with defining the actions of GH on the Leydig cell is the ability of GHs to bind to both somatogenic (GH) and lactogenic (PRL) receptors, as well as binding proteins in nonprimate tissues. Additionally, the use of [125I]hGH in binding studies has led to some confusion between the roles of GH and PRL receptors in Leydig cells. Our present study is the first, to our knowledge, to report a direct effect of GH on Leydig cell-signaling components. GH, but not PRL, activates Stat5b in Leydig cells cultured from immature rats only. In other tissues, the local production of insulin-like growth factor-1 (IGF-1) mediates the effects of GH (54). Using immunohistochemistry, IGF-1 was shown in Sertoli and Leydig cells in immature but not adult rats; in adult rats the staining was localized to the germ cells (55). IGF-1 enhanced LH-stimulated testosterone production and exerted a modest effect on DNA synthesis in immature rat Leydig cells in vitro (56, 57). Activation of Stat5b after GH treatment in Leydig cells from immature but not adult rats is compatible with these maturational profiles of IGF and the Leydig cell. Therefore, one potential mediator of the action of GH on immature Leydig cell function is IGF-1. Taken together, our present studies suggest that the GH-Stat5b-signaling system may regulate gene expression by transcriptional activation during the functional development of Leydig cells.

Cell type-restricted Stat activation has been described by others studying GH and PRL signaling. Stat1 and/or Stat3, as well as Stat5, are activated by GH and PRL in several cell types and lines (25, 29, 30). Since PIE and Fc{gamma}RI probes bind activated Stat1 and Stat5, we also detected the binding of Stat1 in interferon-{gamma}-treated MA-10 cells using these probes in EMSA (data not shown)(58). Thus, it appears that selectivity involving specific Stat proteins and/or specific DNA elements is a general feature of lactogenic hormone-signal transduction and raises the possibility that selective activation can contribute to the regulation of defined subsets of genes in a cell type-specific manner. When we used Fc{gamma}RI (TTCCCAGAA) probe in EMSA, tyrosine-phosphorylated Stat5b from PRL-treated MA-10 cells did not bind to this GAS element, whereas the same DNA-binding protein sample did bind to PIE (TTCTAGGAA). In GH-treated MA-10 cells, activated Stat5b binds to the Fc{gamma}RI GAS element. Similarly, a previous study showed that activated Stat5b bound preferentially to the PIE oligonucleotide, but not to the IRF-1 GAS probe in PRL-treated Nb2 lymphocytes (59). In our results, the amount of activated Stat5b in PRL-treated MA-10 cells was equal to or greater than in GH-treated cells, and there is no difference in the form of dimerization: only Stat5a-Stat5a or Stat5b-Stat5b homodimers exist in PRL- or GH-treated cells. The only difference in the forms of Stat5b seen in GH- and PRL-treated MA-10 cells was the appearance of band 2 using Western analysis. Consequently, in GH-treated MA-10 cells, the tyrosine- + serine/threonine-phosphorylated form of Stat5b may result in a differential binding to Fc{gamma}RI GAS elements. Lactogenic hormone-inducible Stat5b transcription factor activation may target distinct DNA-binding sites with subsequently hormone-specific gene expression in the Leydig cell. Further experimentation, including the modulation of other Stat proteins by lactogenic hormones in MA-10 and/or primary Leydig cells, is required to address this issue. Our present studies indicate that the MA-10 cell with its capacity to bind both PRL and GH is a useful model system in which to study the distinct roles of these lactogenic hormones.

Stat5b-deficient male mice are characterized by a decrease in body growth profile relative to that of the slower rate of wild-type females (60). This growth defect first emerges at puberty and is due to a loss of sexual dimorphism of liver gene expression induced by pulsatile plasma GH. Our present studies show that testicular Leydig cells are also targets of GH in the immature male rat, data suggestive of a role for GH-Stat5b in testis development. Recent data demonstrated that, in addition to their important roles in coupling many receptors to the JAK/Stat cascade, JAKs may also function to couple certain receptors to other signaling pathways (61). Interestingly, PRL- and GH-induced phosphorylation of MAP kinase, ras, and raf-1 has been observed in several cell types (62, 63), suggestive of specific roles for these kinases in PRL- and GH-signaling pathways in the Leydig cell. Taken together, our data indicate that testicular lactogenic hormone-inducible transcriptional activation may target distinct, male-specific gene expression in a signaling cascade(s) involving Stat5b but also imply coordinate control by multiple Leydig cell factors.


   Acknowledgments
 
We are grateful for the skilled technical expertise of L. Mitchell and D. Policarpio and editorial assistance of J. Schweis.


   Footnotes
 
1 This study was funded by NIH Grant R01-HD-16149 (to P.L.M.). Fellowship support (to M.K.) was provided by The Andrew W. Mellon Foundation. Back

Received September 3, 1997.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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