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Endocrinology Vol. 143, No. 1 228-238
Copyright © 2002 by The Endocrine Society

GROWTH FACTORS-CYTOKINES-ONCOGENES

PRL Signal Transduction in the Epithelial Compartment of Rat Prostate Maintained as Long-Term Organ Cultures in Vitro

Tommi J. Ahonen, Pirkko L. Härkönen, Hallgeir Rui and Marja T. Nevalainen

United States Military Cancer Institute (H.R., M.T.N.) and Department of Pathology (H.R., M.T.N.), Uniformed Services University of Health Sciences, Bethesda, Maryland 20814; and Department of Anatomy and Cell Biology (T.J.A., P.L.H.), and the Medicity Research Laboratory (T.J.A., P.L.H.) University of Turku, Institute of Biomedicine, 20520 Turku, Finland

Address all correspondence and requests for reprints to: Dr. Marja T. Nevalainen, Department of Pathology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, Maryland 20814. E-mail: mnevalainen{at}usuhs.mil


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Using long-term organ cultures of rat prostate tissue explants, we previously demonstrated that PRL both stimulates proliferation and acts as an androgen-independent suppressor of apoptosis in prostate epithelial cells, leading to epithelial hyperplasia. In this work we delineate intracellular signaling molecules activated by PRL in prostate tissue to identify candidate signaling proteins that are responsible for maintaining survival and proliferation of prostate epithelium in androgen-deprived growth environment. We now show that signal transducer and activator of transcription-5a (Stat5a) and Stat5b become tyrosine phosphorylated in response to PRL stimulation in rat prostate using prostate organ culture as an experimental model. Stat5 was translocated to the nuclei of epithelial cells of prostate tissue as demonstrated by immunohistochemistry. Furthermore, EMSA showed PRL-inducible binding of Stat5a homodimers and Stat5a/5b heterodimers to the PRL response element of the ß-casein gene promoter. Signaling molecules Stat3, Stat1, MAPK, or protein kinase B, which can be activated by PRL in other target cells, were not activated by PRL in prostate tissue. Furthermore, we show that Stat5a and Stat5b are continuously phosphorylated in rat prostate in vivo, although they are expressed to varying degree in separate lobes of rat prostate.

Collectively, our results suggest that PRL signaling in rat prostate tissue is primarily transduced via Stat5a and Stat5b. The Stat5 pathway represents one candidate signaling mechanism, used by PRL and possibly other growth factors and cytokines, that supports the viability of prostate epithelial cells during long-term androgen deprivation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ABLATION OF ANDROGENS inhibits androgen-dependent proliferation of prostate cancer cells and induces tumor cell apoptosis. As most prostate cancers overcome the growth arrest induced by withdrawal of androgens, one of the central issues in understanding the progression of prostate cancer is to define the growth factors and signaling pathways that provide prostate cells with the ability to survive in growth environment lacking androgens.

PRL, a member of the helix bundle peptide hormone/cytokine superfamily (1), regulates the growth and differentiation of prostate (2, 3, 4). PRL might also be involved in the development of prostate tumors (5), as hyperplastic enlargement of the prostate was induced by hyperprolactinemia in transgenic mice overexpressing PRL (6). In organ cultures of rat dorsolateral prostate and human prostate we have shown previously that PRL induced hyperplastic changes in the prostate epithelium (7, 8). In addition, PRL directly stimulated the proliferation of prostate epithelium in culture (7, 8) and antagonized apoptotic epithelial cell death induced by androgen deprivation (9). The observation of local production of PRL in rat and human prostate tissue (8, 10) and the presence of receptors for PRL in prostate epithelium (11, 12) imply an autocrine loop of PRL action in prostate tissue.

In this work we specifically investigated the signaling molecules that mediate the effects of PRL in prostate tissue to identify candidate transcription factors that are responsible for maintaining the survival and proliferation of prostate epithelium in an androgen-deprived growth environment. In mammary gland, signal transducer and activator of transcription-5a (Stat5a) is known to be the principal mediator of PRL responses (13, 14, 15, 16). Stat5 belongs to the Stat family of transcription factors of seven members, of which Stat5 comprises two distinct, but highly homologous, isoforms, 94-kDa Stat5a and 92-kDa Stat5b (15). Upon PRL binding to its receptor, Janus tyrosine kinase-2 preassociated with the cytoplasmic domain of PRL receptor is activated (17). This leads to recruitment of Stat5 proteins to the activated receptor complex and their activation by phosphorylation on a specific tyrosine residue (13, 14). Phosphorylated Stat proteins dimerize and translocate to the nucleus, where they bind to specific response elements of target gene promoters to regulate transcription (14, 18). In addition to Stat5a (15, 16, 19), Stat5b (19, 20) as well as Stat3 (21, 22, 23) and Stat1 (21, 23) have been shown to be activated by PRL in cells of myeloid, lymphoid, and mammary origin. Parallel to Janus tyrosine kinase-Stat pathways, signaling routes of predominantly serine/threonine kinases (24, 25), including ERK (extracellular signal regulated protein kinase) (22, 23, 26, 27) and protein kinase B (PKB) (28, 29), transduce PRL signals in a variety of cell types.

To identify signaling molecules that mediate the effects of PRL in prostate tissue, we used organ culture of prostate as an experimental model. The androgen responsiveness and tissue-specific functions of prostate epithelium are successfully maintained in organ culture, probably due to the maintenance of the tissue architecture with intact epithelial-stromal interactions in this in vitro model. We now show that Stat5a and -5b are the principal signaling proteins activated by PRL in rat prostate epithelium. Other known PRL signaling molecules, such as Stat3, Stat1, MAPK, or PKB, were not activated by PRL in rat prostate tissue. Stat5 became phosphorylated on tyrosine in response to PRL also in the absence of androgens. Furthermore, we show that Stat5 proteins are continuously activated in rat prostate in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Prostate samples and organ culture
Adult Sprague Dawley rats, outbred strain (10–12 wk old, 250–280 g) were killed by cervical dislocation under carbon dioxide anesthesia, and dorsal, lateral, and ventral prostate lobes were excised and frozen for in vivo studies. For studies of PRL-activated signaling proteins in prostate tissues the organ culture method of Trowell (30) was used with some modifications (31) as described previously (7, 8, 9, 10, 11, 32). Separate lobes of rat prostate were cut into approximately 1-mm3 pieces in culture medium, and the tissue pieces were transferred to lens papers lying on stainless steel grids in petri dishes. The medium was phenol-free culture medium 199 with Earle’s salts (Sigma, St. Louis, MO), supplemented with penicillin G (100 IU/ml), streptomycin sulfate (100 µl/ml), and glutamine (100 µg/ml; Sigma). In addition, the basal medium contained the combination of insulin (0.08 IU/ml; Insulin Lente, Eli Lilly & Co., Indianapolis, IN) and corticosterone (0.1 µM; Sigma). No serum was added to any culture medium. The gas atmosphere was a mixture of O2, CO2, and N2 (40:5:55), and the temperature was 37 C. The explants were cultured for 7 d with or without T (0.1 µM; Sigma), which was dissolved in ethanol. The final concentration of ethanol in the culture medium was 0.01%. The medium was changed every other day.

After 7 d of organ culture, half of the prostate explants of each prostate lobe cultured with or without T were stimulated with 100 nM ovine PRL (oPRL; a gift from Dr. A. F. Parlow, National Pituitary Hormone Program, NIDDK, Bethesda, MD) for 1 h. Also, a group of the explants was cultured in the presence of 100 nM oPRL in the culture medium for 7 d to study the effects of PRL on the morphology of separate rat prostate lobes. A T concentration of 100 nM, which is approximately 6–10 times higher than circulating levels in adult male rats (33), was used in these studies. As shown previously, 100 nM T is required for maintenance of normal epithelial morphology in rat prostate organ culture (34). As diffusion of peptide hormones in the tissue compartments of prostate explants was expected to be less efficient than that of steroid hormones, the concentration of PRL used in organ culture was approximately 10–100 times higher than the levels of PRL in male rat circulation (33, 35).

Solubilization of proteins, immunoprecipitation, and immunoblotting
Uncultured (n = 6 rats) and cultured (n = 6 cultures) prostate tissues (n = 60 rats) were homogenized with an Ultraturrax homogenizer (Janke & Kunkel, IKA Labortechnik, Staufen, Germany) in lysis buffer (1 g/5 ml) containing 10 mM Tris-HCl (pH 7.6), 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 1% Triton X-100, 1 mM phenylmethylsulfonylfluoride, 5 µg/ml aprotinin, 1 µg/ml pepstatin A, and 2 µg/ml leupeptin. Tissue homogenates were rotated end over end at 4 C for 60 min, and insoluble material was pelleted at 12,000 x g for 30 min at 4 C. The protein concentrations of clarified tissue lysates were determined by a simplified Bradford method (Bio-Rad Laboratories, Inc., Hercules, CA). Depending on the experiment, 1 ml lysate containing 6.0 mg total protein was used for immunoprecipitation for 3 h at 4 C with polyclonal rabbit antiserum specific to Stat5a, Stat5b, Stat3, or Stat1 (2 µl/ml; Advantex Bioreagents, Conroe, TX). Antibodies were captured by incubation for 60 min with protein A-Sepharose beads (Pharmacia Biotech, Piscataway, NJ) and washed three times in 1 ml lysis buffer, and samples were subjected to 7.5% SDS-PAGE under reducing conditions. For some analyses, 30 µg total protein of prostate tissue homogenates were directly separated by 7.5% SDS-PAGE without preceding immunoprecipitation (immunoblotting of anti-active MAPK, anti-pan-ERK, anti-Actin, anti-Stat5a, an anti-Stat5b). The proteins were transferred to a polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA), using a semidry transfer unit (Multiphor Novablot, Amersham Pharmacia Biotech, Piscataway, NJ).

After transfer, the blots were incubated for at least 1 h at room temperature in blocking buffer [0.02 M Tris-HCl (pH 7.6), 0.137 M NaCl, 1% BSA, and 0.01% sodium azide] before immunoblotting. Blots were incubated overnight with primary antibodies diluted in blocking buffer at the following concentrations: antiphosphotyrosine-Stat5a/b (Y694/Y699) monoclonal antibody AX1 (1 µg/ml; Advantex Bioreagents), anti-Stat5a polyclonal antibody (1:3000; Advantex Bioreagents), anti-Stat5b polyclonal antibody (1:3000; Advantex Bioreagents), antiphosphotyrosine-Stat3 (Tyr705) polyclonal antibody (1:1000; New England Biolabs, Inc., Beverly, MA), antiphosphotyrosine-Stat1 (Tyr701) polyclonal antibody (1:1000; New England Biolabs, Inc., Beverley, MA), anti-Stat3 monoclonal antibody (0.1 µg/ml; Transduction Laboratories, Inc., Lexington, KY), anti-Stat1 monoclonal antibody (0.1 µg/ml; Transduction Laboratories, Inc.), anti-active MAPK polyclonal antibody (0.05 µg/ml; Promega Corp., Madison, WI), anti-pan-ERK mAb (0.05 µg/ml; Transduction Laboratories), and anti-Actin polyclonal antibody (1:100; Sigma). For reblotting, the filters were incubated in stripping buffer [100 mM NaCl, 62.5 mM Tris-HCl (pH 7.6), 100 mM mercaptoethanol, and 2% SDS] at 60 C for 30 min. Coimmunoprecipitation of Stat5a/b with AR in rat prostate was studied by blotting the immunoprecipitated Stat5 proteins from uncultured and cultured rat dorsal, lateral, and ventral lobes with polyclonal antibodies against rat AR (36) (1:5000; a gift from Prof. Olli Jänne, University of Helsinki, Helsinki, Finland) and with a monoclonal antibody recognizing both Stat5a and Stat5b ({alpha}-panStat5) (1:1000; Transduction Laboratories, Inc.). Control immunoprecipitations were performed with normal rabbit serum (NRS). The ability of anti-AR antibody to recognize ARs was shown by blotting of immunoprecipitated AR proteins and whole tissue lysates from rat ventral prostate with anti-AR antibody (1:5000). The blots were washed [50 mM Tris-HCl (pH 7.6), 200 mM NaCl, and 0.25% Tween 20] and incubated with horseradish peroxidase-conjugated goat antibodies to mouse or rabbit IgG (5 µg/ml; Transduction Laboratories, Inc.), followed by incubation with enhanced chemiluminescence substrate mixture (Amersham Pharmacia Biotech) and exposure to x-ray films (Eastman Kodak Co., Rochester, NY). Densitometric normalization and comparison of phosphorylation of Stat5a and Stat5b in cultured rat dorsal, lateral, and ventral prostate from three organ culture experiments were performed using an Eagle Eye system (Stratagene, La Jolla, CA).

Immunohistochemistry
Formalin-fixed paraffin sections of cultured rat prostate tissues were deparaffinized, followed by rehydration in graded alcohol. Immunohistochemistry of activated Stat5 was performed as described.1 Briefly, parallel tissue sections for detection of activated Stat5 were microwave-treated with antigen retrieval solution AXAR1 (Advantex Bioreagents) and for phosphorylated PKB with citrate solution (BioGenex Laboratories, Inc., San Ramon, CA). Endogenous peroxidase activity was blocked by incubating the slides in 0.3% hydrogen peroxide for 10 min at room temperature. Nonspecific binding of IgGs was minimized by preincubation in normal goat serum for 2 h at room temperature. The primary monoclonal antibody recognizing phosphorylated tyrosine 694/699 of activated Stat5 and the polyclonal antibody against phosphorylated PKB were both diluted in 1% BSA in PBS at concentrations of 0.6 µg/ml and 1:50, respectively. Antigen-antibody complexes were detected using appropriate biotinylated goat secondary antibodies, followed by streptavidin-horseradish peroxidase complex (BioGenex Laboratories, Inc.). As a chromogen, 3,3'-diaminobenzidine was used, and Mayer hematoxylin was used as a counterstain. For controls subtype-specific mouse IgG or NRS was used as appropriate.

EMSA
Rat prostate explants were cultured for 7 d in organ culture and stimulated with 100 nM oPRL for 1 h. Prostate tissues were homogenized (1 g/10 ml) in 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 phenylmethylsulfonylfluoride, 5 µg/ml aprotinin, 1 µg/ml pepstatin A, and 2 µg/ml leupeptin]. Tissue homogenates were pelleted by centrifugation at 800 x g for 10 min at 4 C, and the pellets were solubilized in EMSA lysis buffer containing 300 mM NaCl. Lysates were incubated on ice for 10 min, then clarified by centrifugation at 20,000 x g for 10 min at 4 C. For the EMSA (22), 1 ng 32P-labeled oligonucleotide (5'-agatttctaggaattcaaatc-3') corresponding to the PRL response element of the rat ß-casein gene was incubated with 10 µg protein from tissue lysates in 30 µl binding mixture [50 mM Tris-HCl (pH 7.4), 25 mM MgCl2, 5 mM dithiothreitol, and 50% glycerol] at room temperature for 20 min. The samples were preincubated with either NRS or polyclonal antibodies specific to Stat5a or Stat5b proteins (Advantex Bioreagents) as indicated. Polyacrylamide gels (5%) containing 5% glycerol and 0.25x Tris borate/EDTA were prerun in 0.25x Tris borate/EDTA buffer at 4-10 C for 1.5 h at 300 V. After loading of samples, the gels were run at room temperature for about 3 h at 250 V, dried by heating under vacuum, and exposed to x-ray films (X-Omat, Eastman Kodak Co.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Stat5a and Stat5b, but not Stat3 or Stat1, are activated by PRL in rat prostate in organ culture
Signaling molecules that are activated by PRL in prostate tissue were studied using organ culture of rat prostate as an experimental model (7, 8, 9, 10, 11, 30, 31). We have previously shown that hormone responsiveness and tissue-specific functions of prostate epithelium are well maintained in this in vitro model of prostate (7, 8, 9, 10, 11, 32). Tissue explants from dorsal, lateral, and ventral lobes of rat prostate were cultured with or without T for 7 d, after which half of the explants were stimulated with PRL.

First, Stat5a, Stat5b, Stat3, and Stat1 were immunoprecipitated from cultured rat dorsal, lateral, and ventral prostate tissues and blotted with anti-phosphoTyr-Stat5, anti-phosphoTyr-Stat3, and anti-phosphoTyr-Stat1 antibodies, respectively. Western blot analysis showed that in all rat prostate lobes both Stat5a and Stat5b were phosphorylated on tyrosine in response to PRL (Fig. 1Go, A and B). In contrast to Stat5a and Stat5b, neither Stat3 nor Stat1 was activated by PRL in any of the rat prostate lobes (Fig. 1Go, C and D). Reblotting of the samples with corresponding Stat antibodies verified equal levels of Stat proteins loaded per lane (Fig. 1Go). The kinetics of tyrosine phosphorylation of Stat5 in rat prostate were tested using different PRL stimulation times. Tyrosine phosphorylation of Stat5 peaked between 30–60 min of PRL stimulation and was barely detectable 4 h after the start of the PRL stimulation (data not shown). In all rat prostate lobes the PRL-induced activation of both Stat5a and Stat5b was enhanced by the presence of T in the culture medium (Fig. 1Go, A, B, and E). To more directly assess the enhancement by T of PRL-induced activation of Stat5, phosphoprotein immunoblots were analyzed by densitometry and normalized for Stat5 protein levels (Fig. 1EGo). Overall, after normalization there was a moderate, but consistent, enhancement of PRL-induced tyrosine phosphorylation of both Stat5a and Stat5b by T in all rat prostate lobes in organ culture. However, the T enhancement of PRL-induced phosphorylation of Stat5a and Stat5b was generally less than 2-fold, except in rat lateral prostate, where the phosphorylation of Stat5a was increased 2.5-fold by the presence of T in the culture medium (Fig. 1EGo). A low level of constitutive phosphorylation of serine residue S725 or S730 of Stat5a and Stat5b, respectively, was observed in all rat prostate lobes maintained as organ cultures (data not shown). However, stimulation of the prostate explants with PRL did not increase phosphorylation of Stat5 on serine residues (data not shown), which, when phosphorylated, may have an inhibitory effect on transcriptional regulation (38).



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  		Figure 1. Stat5a and Stat5b, but not Stat3 or Stat1, are phosphorylated on tyrosine in response to PRL stimulation in all lobes of rat prostate in organ culture. Explants from dorsal, lateral, and ventral lobes of rat prostate (n = 60 rats) were cultured with or without 100 nM T for 7 d in organ culture (n = 6 organ cultures). At the end of each culture half the explants were stimulated with 100 nM oPRL for 1 h. Stat5a, Stat5b, Stat3, and Stat1 were immunoprecipitated (IP) from tissue homogenates and blotted with a monoclonal anti-phosphoTyr-Stat5 ({alpha}pY-Stat5a/b) antibody (A and B, upper panels), a polyclonal anti-phosphoTyr-Stat3 ({alpha}pY-Stat3) antibody (C, upper panel), or a polyclonal anti-phosphoTyr-Stat1 ({alpha}pY-Stat3) antibody (D, upper panel). Filters from three experiments were stripped and reblotted with polyclonal antisera against Stat5a (A, lower panel) or Stat5b (B, lower panel) or with a monoclonal antibody against Stat3 (C, lower panel) or Stat1 (D, lower panel). E, Densitometric normalization and comparison of PRL-induced tyrosine phosphorylation of Stat5a and Stat5b in rat dorsal, lateral, and ventral prostate in organ culture.

 
Activation of Stat5 by PRL in rat prostate was also shown by a novel immunohistochemical detection of tyrosine-phosphorylated Stat5 in situ1 in paraffin-embedded tissue sections. Epithelial cells of dorsal, lateral, and ventral prostates displayed intense nuclear immunostaining for activated Stat5 in explants stimulated with PRL (Fig. 2Go, B and D, F, H, J, and L), indicating nuclear translocation of phosphorylated Stat5. In contrast, no immunoreaction for activated Stat5 was present in the epithelium of prostate explants that had not been stimulated with PRL (Fig. 2Go, A and C, E, G, I, and K). Consistent with results obtained by immunoblotting (Fig. 1Go, A and B), the presence of T in the culture medium enhanced immunostaining for activated Stat5 in all rat prostate lobes (Fig. 2Go, D and H, L). Lactating mouse mammary gland was used as a positive tissue control1 (Fig. 2MGo) (16), and sections of lactating mammary gland incubated with subtype-specific IgG showed no positive reaction (Fig. 2NGo).



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  		Figure 2. Activated Stat5 translocates to the nuclei of the epithelial cells of PRL-stimulated explants of dorsal, lateral, and ventral rat prostate in organ culture. Explants from dorsal (A–D), lateral (E–H), and ventral (I–L) prostate were cultured without (A, B, E, F, I, and J) or with 100 nM T (C, D, G, H, K, and L) for 7 d in organ culture. At the end of the culture (n = 3 cultures), half of the explants in each group were stimulated with 100 nM oPRL for 1 h (B, D, F, H, J, and L). Paraffin-embedded tissue sections were immunostained with a monoclonal antiphosphoTyr-Stat5 antibody and counterstained with hematoxylin. Biotin-streptavidin-amplified peroxidase-antiperoxidase immunodetection shows intensive positive nuclear immunostaining of epithelial cells of PRL-stimulated explants from rat dorsal (B and D), lateral (F and H) and ventral (J and L) prostate (arrows). In the presence of T in the culture media more cells showed nuclear immunostaining of tyrosine-phosphorylated Stat5 (D, H, and L; arrows). In contrast, epithelial cells from unstimulated prostate explants (A, C, E, G, I, and K) show no specific immunoreaction. Lactating mouse mammary gland was used as a positive control tissue (m), and incubation with subtype-specific IgG showed no positive reaction (N). Bar, 33 µm.

 
The binding of phosphorylated Stat5 to DNA in rat prostate epithelium was studied by EMSA (Fig. 3Go).The PRL response element of the ß-casein gene promoter was used as a probe, and the ability of anti-Stat5a and anti-Stat5b antibodies to properly supershift Stat5a and Stat5b, respectively, was verified by parallel EMSA analysis of COS-7 cells that were transfected with expression vectors encoding PRL receptor and either Stat5a or Stat5b (data not shown). Nuclear extracts of rat dorsal (Fig. 3Go), lateral, or ventral prostate (data not shown) that had been cultured for 7 d in organ culture and stimulated with PRL were incubated with NRS or antisera against Stat5a and/or Stat5b before incubation with the oligonucleotide probe. The supershift analysis of nuclear extracts of cultured rat prostate showed that antiserum against Stat5a almost completely supershifted the Stat5 complex bound to the ß-casein gene promoter, whereas antiserum against Stat5b shifted it to only a small extent (Fig. 3Go). These results demonstrated that PRL-activated Stat5 in rat prostate epithelial cells binds to DNA and suggested that Stat5 binds to the PRL response element of the ß-casein gene promoter as both Stat5a homodimers and Stat5a/5b heterodimers, but only to lesser extent as Stat5b homodimers. Collectively, our results indicate that both Stat5a and Stat5b transduce PRL signals in the epithelial cells of rat prostate tissue.



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  		Figure 3. Activated Stat5a and Stat5b of cultured rat prostate bind to the PRL response element of the ß-casein gene promoter shown by EMSA. Nuclear extracts of cultured rat dorsal prostate explants were incubated with normal rabbit serum (NRS) or with antisera against Stat5a ({alpha}Stat5a) and/or Stat5b ({alpha}Stat5b) before incubation with the oligonucleotide probe corresponding to the PRL response element of ß-casein gene. An arrow indicates the complex that binds to ß-casein promoter in prostate explants that were stimulated with 100 nM oPRL (+). The complex was supershifted by antiserum against Stat5a almost completely, whereas anti-Stat5b antiserum supershifted the complex less efficiently. A combination of anti-Stat5a and anti-Stat5b ({alpha}Stat5a/b) antisera supershifted the DNA binding complex most efficiently.

 
MAPKs ERK1 and ERK2 are stimulated by PRL in a variety of tissues (22, 23, 24, 26, 27). These kinases may also be indirectly involved in the regulation of Stat5a serine phosphorylation (39). In rat dorsal, lateral, and ventral prostate, ERK1/2 were in an activated state, as assessed by anti-active MAPK immunoblotting of whole tissue lysates of cultured prostate explants (data presented to reviewers). Stimulation of prostate explants with PRL did not, however, further increase phosphorylation of ERK1/2 in any rat prostate lobe (data presented to reviewers). In addition to signaling routes including ERK, another serine/threonine kinase pathway involving PI3K and PKB has been shown to transduce PRL signals in both Nb2 rat lymphoma cells (28, 29) and human breast cancer cells (40). PKB (also referred to as Akt) is a downstream enzyme of PI3K (41) and is involved in the phosphorylation and inactivation of substrates involved in the regulation of apoptosis (42). Immunohistochemistry of activated PKB in tissue sections of cultured explants of rat dorsal, lateral, and ventral prostate showed that PRL did not increase the phosphorylation of PKB in any rat prostate lobe (data presented to reviewers).

Stat5 does not directly interact with AR in rat prostate tissue
Stat5 interacts functionally with GR, MR, and PR of the steroid receptor superfamily on transcription from the ß-casein gene (43). Furthermore, Stat5 has been shown to directly associate with GR via protein-protein interactions in mammary cells (44, 45). We have previously shown that androgens and PRL have a synergistic effect on the expression of probasin gene in rat dorsolateral prostate in organ culture (7). In addition, our present results show increased phosphorylation of Stat5 in rat prostate in the presence of T (Figs. 1Go and 2Go). These observations prompted us to investigate whether AR and Stat5 proteins also directly interact in rat prostate tissue. Stat5a and Stat5b were immunoprecipitated from cultured (Fig. 4AGo) and uncultured (data not shown) rat dorsal, lateral, and ventral prostates, resolved on SDS-PAGE, and immunoblotted with a polyclonal anti-AR antibody (Fig. 4AGo, upper panel). The filters were stripped and reblotted with a monoclonal antibody that recognizes both Stat5a and Stat5b (anti-panStat5). No coimmunoprecipitation of AR and Stat5 was detected in any of the samples (Fig. 4AGo). The ability of anti-AR antibody to properly immunoprecipitate AR was verified by immunoprecipitation and subsequent immunoblotting of ARs from rat ventral prostate (Fig. 4BGo). To reveal potential unspecific binding of anti-AR antibody to IgGs, control immunoprecipitations were performed with NRS. Furthermore, T had no effect on the expression of Stat5 proteins in any of the lobes of rat prostate (Fig. 1Go, A and B, and Fig. 4AGo).



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  		Figure 4. Stat5 does not directly interact with AR in rat prostate tissue. A, Explants of rat dorsal, lateral, and ventral prostate were cultured without (-) or with (+) T for 7 d and stimulated with 100 nM oPRL for 1 h. Stat5a and Stat5b proteins were collectively immunoprecipitated (IP), resolved on SDS-PAGE and immunoblotted with a polyclonal antibody against androgen receptor ({alpha}AR; upper panel). The filters were stripped and reblotted with a monoclonal antibody recognizing both Stat5a and Stat5b ({alpha}panStat5). Control immunoprecipitations were performed with normal rabbit serum (NRS) and immunoblotted with anti-AR ({alpha}AR) or anti-Stat5 ({alpha}panStat5) antibodies. B, The ability of anti-AR antibody to recognize ARs was shown by blotting immunoprecipitated androgen proteins and whole tissue lysates from rat ventral prostate with anti-AR ({alpha}AR) antibody (1:5000).

 
PRL induces hyperplastic epithelium in rat dorsolateral prostate, but has no effect on the epithelial morphology of ventral lobe of rat prostate in organ culture
Dorsal, lateral, and ventral lobes of rodent prostate arise from different parts of urogenital sinus, and separate rat prostate lobes differ in their patterns of ductal branching and secretory activity (46). Developmentally the dorsal and lateral lobes are considered the parts of rat prostate corresponding to human prostate, whereas no equivalent for the rat ventral prostate lobe has been defined in adult human prostate (47). Based on in vivo experiments, biological responses of dorsolateral and ventral parts of rat prostate to PRL have also been suggested to differ (3, 4, 48). As our results indicated that Stat5 proteins transmit PRL signals similarly in all rat prostate lobes, we wanted to compare the direct effects of PRL on the morphology of ventral prostate to those on dorsal and lateral prostate using organ culture.

Explants of dorsal, lateral, and ventral prostate were cultured for 7 d with or without PRL. PRL induced hyperplastic changes in both rat dorsal and lateral prostate epithelium, whereas in ventral lobe PRL had no effect on epithelial morphology (Fig. 5Go). In the presence of PRL, epithelial cells of rat dorsal and lateral prostate were large and cuboidal, and the epithelial cells were arranged in multiple layers in acini with small glandular lumina (Fig. 5Go, B and E). In contrast, in rat ventral prostate the epithelium showed involutive changes, with large glandular lumina and flat and squamous epithelial cells when cultured with PRL (Fig. 5HGo), a morphology closely resembling that of explants cultured in basal medium (Fig. 5GGo). To confirm the maintenance of hormone responsiveness of prostate tissue in organ culture conditions in each experiment, a group of explants of each prostate lobe was cultured with T (Fig. 5Go, C and F, I). In the presence of T in the culture medium the epithelium of all three prostate lobes was columnar (Fig. 5Go, C and F, I), as expected (7, 31), whereas in explants cultured in basal medium the glandular lumina were large, and the epithelial cells were mostly flat and varied in size (Fig. 5Go, A and D, G). In summary, PRL induced epithelial hyperplasia in rat dorsolateral prostate, but had no effect on the epithelial morphology of the ventral lobe of rat prostate.



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  		Figure 5. PRL induces hyperplastic epithelium in rat dorsolateral prostate, but has no effect on epithelial morphology of ventral lobe of rat prostate in organ culture. Explants of dorsal (A–C), lateral (D–F), and ventral (G–I) lobes of rat prostate were cultured for 7 d in organ culture in the basal medium (A, D, and G), in the presence of 100 nM oPRL (B, E, and H) or in the presence of 100 nM T (C, F, and I). Note (arrows) large and cuboidal epithelial cells arranged in layers in rat dorsal (B) and lateral (E) prostate cultured with PRL, whereas in rat ventral prostate (H) PRL had no effect on the epithelial morphology (arrow). T induced columnar epithelium in all rat prostate lobes (C, F, and I). Bar, 55 µm.

 
Stat5a and Stat5b are activated, but differentially expressed, in rat dorsal, lateral, and ventral prostate in vivo
In addition to pituitary PRL, PRL or PRL-like proteins produced locally by prostate epithelium (8, 11) and other cytokines and growth factors (49) may activate Stat5, Stat3, Stat1, MAPK kinase, and PKB in prostate epithelium. Therefore, we examined the activation state of different PRL signaling proteins in separate lobes of normal adult rat prostate in vivo. Stat5a, Stat5b, Stat3, and Stat1 were immunoprecipitated from dorsal, lateral, and ventral lobes of rat prostate. Immunoblotting of Stat5a immunoprecipitations with anti-phosphoTyr-Stat5 showed that Stat5a was tyrosine phosphorylated in both rat dorsal and lateral prostate, but not in rat ventral prostate (Fig. 6AGo). When filters were reblotted with Stat5a antibody, however, Stat5a protein levels differed in the immunoprecipitations from dorsal, lateral, and ventral rat prostate lobes (Fig. 6AGo, second panel from the top). Specifically, the level of Stat5a proteins in rat ventral prostate was noticeably lower compared with those in dorsal and lateral lobes. Furthermore, immunoblotting of whole tissue lysates from separate rat prostate lobes with anti-Stat5a antibody verified that Stat5a was expressed at a lower level in rat ventral prostate compared with dorsal and lateral lobes (Fig. 6AGo, third panel from the top). Reblotting of the filters with anti-Actin antibody demonstrated that equal amounts of protein from whole tissue lysates were loaded per lane (Fig. 6AGo, bottom panel).



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  		Figure 6. Stat5a and Stat5b are activated, but differentially expressed in rat dorsal, lateral, and ventral prostate in vivo. Stat5a (A), Stat5b (B), Stat3 (C), and Stat1 (D) were immunoprecipitated (IP) from dorsal, lateral, and ventral lobes of rat prostate; resolved on SDS-PAGE; and immunoblotted with antiphosphoTyr-Stat5 ({alpha}pYStat5a/b; A and B, panel 1), antiphosphoTyr-Stat3 ({alpha}pYStat3; C, panel 1), or anti-phosphoTyr-Stat1 ({alpha}pYStat1; D, panel 1) antibodies. The filters were reblotted with anti-Stat5a ({alpha}Stat5a; A, panel 2), anti-Stat5b ({alpha}Stat5b; B, panel 2), a polyclonal anti-Stat3 antibody recognizing the C-terminal part of Stat3 ({alpha}Stat3; C, panel 2), a monoclonal anti-Stat3 antibody recognizing the N-terminal part of Stat3 ({alpha}Stat3; C, panel 3), or anti-Stat1 ({alpha}Stat1; D, panel 2) antibody. Whole tissue lysates from dorsal, lateral, and ventral rat prostate were immunoblotted with anti-Stat5a ({alpha}Stat5a; A, panel 3) antibody or with anti-Stat5b ({alpha}Stat5b; B, panel 3) antibody. The filters containing whole tissue lysates were stripped and reblotted with anti-actin ({alpha}Actin; A and B, panel 4) antibody.

 
In contrast, Stat5b (Fig. 6BGo) was activated to an equal extent in dorsal, lateral, and ventral lobes of rat prostate. Both reblotting the filters containing immunoprecipitated Stat5b from rat dorsal, lateral, and ventral prostate and immunoblotting of whole tissue lysates with anti-Stat5b antibody showed equal expression of Stat5b in all rat prostate lobes (Fig. 6BGo). In conclusion, our results indicate that in vivo Stat5a is expressed at a lower level in rat ventral prostate than in dorsal and lateral prostate, and that both Stat5a and Statb are in a phosphorylated state in rat prostate.

As opposed to Stat5a and Stat5b, neither Stat3 (Fig. 6CGo, upper panel) nor Stat1 (Fig. 6DGo, upper panel) was phosphorylated on tyrosine or serine (data not shown) in any of the rat prostate lobes in vivo. Reblotting of the filters containing the immunoprecipitations of Stat3 showed expression of Stat3 in rat dorsal and lateral prostate, but not in ventral prostate, when immunoblotted with a polyclonal antibody against the carboxyl-terminal end of Stat3 (Fig. 6CGo, middle panel), in contrast to the results obtained by organ culture (Fig. 1CGo, lower panel). However, immunoblotting of the same filters with a monoclonal anti-Stat3 antibody that recognizes the amino-terminal region of Stat3 indicated expression of a shorter Stat3 form of 80–90 kDa (50) in rat ventral prostate and a 70- to 80-kDa form of Stat3 (51) in rat lateral prostate (Fig. 6CGo, bottom panel). Carboxyl-terminally truncated Stat5 and Stat3 isoforms lack the trans-activation domain and thus might act as dominant negative regulators of transcription (52, 53). It is possible that in vivo the splicing of Stat3 mRNA (50) in rat ventral prostate is different from that in vitro. Another possibility is that a protease (51, 52), which cleaves Stat3 into a shorter protein form in rat prostate in vivo, is deactivated in in vitro conditions. Reblotting of the filters containing immunoprecipitations of Stat1 with anti-Stat1 antibody showed equal expression of Stat1 in rat dorsal, lateral, and ventral lobes and equal loading of Stat1 proteins per lane (Fig. 6DGo, bottom panel).

In addition to Stat5a, -5b, -3, and -1, whole tissue lysates of separate rat prostate lobes were immunoblotted with an antibody recognizing phosphorylated MAPKs ERK1 and ERK2. Immunoblot analysis indicated that ERK1/2 proteins were in an activated state in all rat prostate lobes in vivo (data presented to reviewers). Furthermore, activation of PKB of the PI3K pathway in separate rat prostate lobes in vivo was studied by immunohistochemistry. Scattered epithelial cells in dorsal, lateral, and ventral lobes displayed intense nuclear immunostaining for activated PKB, suggesting that PKB was continuously activated in vivo in all rat prostate lobes (data presented to reviewers).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present work we demonstrate that transcription factors Stat5a and Stat5b transduce PRL signals in rat prostate epithelium. The present study also shows that prostate organ culture provides an important in vitro model for mapping signaling pathways triggered by a specific ligand in prostate tissue. Furthermore, in this work Stat5 proteins were shown to be continuously activated, but differentially expressed, in vivo in separate lobes of rat prostate.

In search of new effective treatment modalities for prostate cancer, elucidating mechanisms of androgen-independent proliferation and survival of prostate cancer cells is crucial. We have demonstrated previously that PRL, independent of androgens, both induces proliferation (7, 8) and inhibits apoptosis (9) of prostate epithelial cells. These effects were associated with hyperplastic changes in epithelial morphology in rat dorsolateral prostate and human prostate (7, 8). Consistent with these results, mice overexpressing PRL develop hyperplastic enlargement of prostate (6). PRL has also been shown to stimulate in vitro growth of both primary prostate epithelial cells (54) and androgen refractory prostate cancer cell lines (55, 56). The detection of locally produced PRL in prostate epithelium (8, 10) indicates an autocrine action of PRL in prostate tissue. PRL could thus be one of the factors that promote proliferation and survival of prostate cancer cells in a growth environment deprived of androgens. Local production of PRL in prostate epithelium might also explain the limited success of the suppression of pituitary PRL production in prostate cancer patients (57). Therefore, characterizing the signaling pathways of PRL in prostate epithelium is of direct relevance for identifying new therapeutic target molecules.

In this work we demonstrate for the first time the use of prostate organ culture as an experimental model for studies of activation of specific intracellular signaling pathways regulating the biology and function of prostate tissue. The advantage of prostate organ culture as an in vitro model for these studies is that all tissue components are present, which therefore allows the interactions of prostate epithelium and stroma (7, 8, 9, 32). This preserved function of prostate epithelium in organ culture is distinct from primary cultures of normal prostate epithelial cells, which frequently dedifferentiate and lose androgen sensitivity (58). By using this model we have previously shown biological effects of PRL on prostate epithelium (7, 8). Here we apply organ culture to identify the signaling cascades regulated by PRL in rat prostate epithelium. In general, this study provides a basis for future studies of signaling routes triggered by any peptide factor in normal prostate epithelial cells, and interactions of kinase pathways with steroid receptor-mediated signaling systems.

We show in this work that PRL selectively induced epithelial hyperplasia in explant organ cultures of dorsal and lateral lobes of the rat prostate, but not in the ventral prostate. However, the dorsolateral prostate was not associated with a pattern of PRL-induced signal transduction different from that induced in ventral prostate. In organ cultures of dorsal, lateral, and ventral lobes of the rat prostate, PRL stimulation led to activation of transcription factors Stat5a and Stat5b, but not Stat3, Stat1, or protein kinases MAPK and PKB, other mediators of PRL actions described in breast cancer cells and lymphocytes (21, 22, 23, 24, 25, 26, 27, 28, 29, 40).

The mechanism of selective PRL-induced hyperplasia in dorsolateral prostate therefore remains to be explained. First, different biological effects of PRL in ventral compared with dorsolateral lobes could be due to yet to be identified signals that are differentially activated by PRL. Second, interaction and cross-talk between PRL-induced Stat5 activation and signaling pathways activated by other factors may differ between the prostate lobes. Third, the repertoire of transcriptionally responsive, Stat5-regulated genes may differ between the various lobes due to differences in transcriptional coactivators and chromatin structure (59, 60). Dorsal, lateral, and ventral lobes of rodent prostate arise from different parts of urogenital sinus, and both cytodifferentiation and androgen responsiveness of dorsolateral compared with ventral lobes differ (46).

Based on the present work, Stat5a and Stat5b are to date the only identified candidate mediators of the selective PRL-induced hyperplasia of rat dorsolateral, and not ventral, prostate. Yet PRL also activates Stat5 in rat ventral prostate, and in Stat5a null mice we previously observed a prostate phenotype in ventral prostate that was characterized by cystic changes in morphology (61). These cystic changes were not apparent in dorsolateral prostates from Stat5a-null mice, where the gross morphology appeared to be normal. The phenotype observed in ventral prostates of Stat5a-null mice may further underscore the distinct biological effects of PRL in ventral vs. dorsolateral prostates. Increased cystic changes may also be related to the generally more convoluted and larger glandular structures of ventral prostates compared with shorter and simpler ductal structures in dorsolateral prostates. Furthermore, thorough analysis of dorsolateral prostates of mice was hampered due to the limited size of these anatomical structures in the mouse (61). We therefore have not excluded the existence of more subtle changes, for instance a reduction in total epithelial cell numbers, in the dorsolateral lobes of the prostates of Stat5a-deficient mice. Although it is evident that PRL has distinct biological effects in the various lobes of rodent prostate, further studies are needed to determine the mechanisms underlying the distinct effects. These studies include examination of prostates of mice that lack both Stat5a and Stat5b as well as identification of Stat5-regulated gene repertoires of the various lobes of rodent prostate. Prostate organ culture would provide an excellent model for such studies, because the concomitant changes in other hormone levels in serum associated with in vivo PRL injections would be bypassed in organ culture.

The PRL-induced phosphorylation of Stat5 in rat prostate epithelium was increased by T. The synergistic biological effects of T and PRL on prostate growth and differentiation have been shown both in vivo (62) and in vitro in prostate organ culture (7). The underlying mechanisms of synergistic effects of T and PRL have been suggested to be related to induction of the expression of AR by PRL in prostate epithelium and to increased binding of androgens to their receptors in the presence of PRL (63). Our study provides the first evidence that androgens enhance intracellular signal transduction of PRL in prostate tissue at least to some extent. Although androgens did not up-regulate levels of Stat5 proteins, the moderately increased tyrosine phosphorylation of Stat5a and Stat5b may be due to increased levels of PRL receptors, because mRNAs encoding PRL receptor are up-regulated by androgens in rat prostate (11). Moreover, several Stat5-specific tyrosine phosphatases have recently been described, such as protein tyrosine phosphatase B1 and SH2 domain-containing tyrosine phosphatase (64, 65), which directly dephosphorylate Stat5 and thereby negatively regulate Stat5 signaling. It is possible that androgens down-regulate the expression or activity of Stat5-specific tyrosine phosphatases in prostate epithelium, as shown for a prostate-specific acid phosphatase (66), which has tyrosine phosphatase activity (67). The finding of enhancement of PRL-stimulated Stat5 activation by androgens in prostate tissue requires further study. Stat5 and AR did not directly interact with each other in rat prostate epithelium as shown for GR and Stat5 in mammary epithelial cells (44, 45). However, the lack of detection of AR associated with the Stat5a/5b complex could also be due to the limited sensitivity of the detection method.

Both Stat5a and Stat5b were in an activated state in vivo in separate rat prostate lobes. Stat5a was expressed at a considerably lower level in rat ventral prostate compared with dorsal and lateral lobes, whereas Stat5b was equally expressed in all three lobes of rat prostate. This is interesting, because 1) our EMSA results suggested a major role for Stat5a as a PRL signaling molecule in rat prostate, and 2) in a majority of experimental models of PRL effects on rodent prostate, the biological effects of PRL are manifested specifically in dorsolateral parts of rat prostate (3, 6, 62). Thus, the responsiveness of rat dorsolateral prostate to PRL could be due to the higher expression level of Stat5a in dorsolateral parts compared with ventral parts of the rat prostate. The activation of Stat5 in rat prostate epithelium might be caused by pituitary PRL and/or PRL that is locally produced in prostate epithelium (8, 10). In addition, a number of cytokines of both class I and II cytokine families, such as GH, erythropoietin, thrombopoietin, granulocyte colony-stimulating factor, IL-2 group ILs, IL-3 group ILs, and IL-6 group ILs, have been shown to activate Stat5 in a variety of tissues (49). Their contributions to basal activation of Stat5 in rat prostate epithelium remain to be studied.

In summary, our results suggest that the transcription factors Stat5a and Stat5b primarily transduce PRL signals in rat prostate epithelium. Our results further demonstrate that Stat5a proteins are continuously phosphorylated in rat prostate in vivo, although they are expressed to varying degree in separate lobes of rat prostate. It is possible that the Stat5 pathway represents a specific signaling mechanism used by PRL and other growth factors and cytokines to support prostate epithelial cell growth during long-term androgen deprivation. Therapy-based killing of prostate cancer cells may require combined blockade of distinct signaling pathways of several growth factors and cytokines, among which Stat5 proteins may provide a good candidate target. Prostate organ culture provides an excellent model for mapping the signal transduction routes regulating the proliferation and apoptosis of prostate epithelium.


   Acknowledgments
 
We thank Prof. Olli Jänne and Dr. Jorma Palvimo (University of Helsinki, Helsinki, Finland) for providing the anti-AR antibody. The NIDDK National Pituitary Hormone Program and Dr. A. F. Parlow (Pituitary Hormone and Antisera Center, Torrance, CA) are gratefully acknowledged for providing oPRL. Also, we thank Ms. Sue Pletcher for technical assistance with histology.


   Footnotes
 
This work was supported by NIH Grants RO1-DK-52013 and RO1-CA-83813, DOD Department of Defense Prostate Cancer Grant DAMD-17-00-1-0022, Turku University Graduate School of Biomedical Sciences (University of Turku, Turku, Finland), and the Foundation for the Finnish Cancer Institute.

Abbreviations: NRS, Normal rabbit serum; oPRL, ovine PRL; PKB, protein kinase B; Stat, signal transducer and activator of transcription.

Received May 22, 2001.

Accepted for publication September 11, 2001.


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