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Center for Neuroendocrinology and Department of Anatomy and Structural Biology, University of Otago School of Medical Sciences (F.Y.M., G.M.A., T.D.G., D.R.G., S.J.B.), Dunedin 9001, New Zealand; and Institut National de la Santé et de la Recherche Médicale, Unité 584, Molecular Endocrinology, Faculté de Médecine Necker (V.G.), 75730 Paris, France
Address all correspondence and requests for reprints to: Dr. Stephen Bunn, Center for Neuroendocrinology and Department of Anatomy and Structural Biology, University of Otago School of Medical Sciences, P.O. Box 913, Dunedin 9001, New Zealand. E-mail: stephen.bunn{at}stonebow.otago.ac.nz.
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1–9-G129R-human prolactin and the JAK inhibitor AG490, but was unaffected by selected serine/threonine kinase inhibitors (H89, KN-93, bisindolymaleimide, or PD98059). Antibodies selective for STAT5a or STAT5b indicated that the response was restricted to STAT5b, with the number of TH cells displaying STAT5b nuclear immunoreactivity rising from less than 10% under basal conditions to approximately 70% after prolactin stimulation. STAT5a nuclear labeling remained unchanged at 6–10% of TH-positive cells. STAT5b selectivity was confirmed in vivo, where the injection of prolactin into bromocriptine-treated rats stimulated a time-dependent increase in STAT5b, but not STAT5a, nuclear staining in the TH-expressing neurons in the arcuate nucleus. These results extend our previous findings with STAT5b-deficient mice and strongly suggest that in NEDA neurons, prolactin signaling via the JAK/STAT pathway is mediated exclusively by STAT5b. |
Introduction |
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The NEDA neurons, comprised of tuberoinfundibular (TIDA), tuberohypophyseal, and periventricular hypothalamic dopaminergic neurons, are subject to regulatory inputs, of which prolactin itself is probably the most significant (2). NEDA neurons increase dopamine turnover in response to exogenous prolactin in vivo (4), and their basal activity is dependent on endogenous prolactin levels (5, 6).
The cellular mechanisms by which prolactin regulates NEDA neuron activity remain unresolved. Prolactin’s action is probably direct, because NEDA neurons express prolactin receptors (7, 8, 9). Similarly, we and others have shown that prolactin increases the expression and activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, in NEDA neurons maintained in culture (7, 10). As a member of the class I cytokine receptor family, prolactin receptors couple to a Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway (1). Activation of prolactin receptors in a variety of tissues activates specific STAT proteins, most notably STAT1, STAT3, STAT5a, and the closely related STAT5b (11, 12, 13). Prolactin-induced activation of STAT5 occurs in NEDA neurons (14), although this study did not distinguish between STAT5a and STAT5b. Our previous work indicates that STAT5b may be particularly important in this response, in that mice deficient in STAT5b have low levels of the dopamine metabolite dihydroxyphenylacetic acid in the median eminence and low TH mRNA expression in their arcuate nuclei compared with wild-type controls (15). As a presumed consequence of this breakdown in the NEDA-mediated prolactin signaling, these STAT5b-deficient mice are hyperprolactinemic (15). Although these observations suggest an obligatory role for STAT5b, they do not preclude additional pathways. We have, for example, reported that prolactin-mediated stimulation of TH activity in these neurons is dependent on a range of specific serine/threonine protein kinases (10). Furthermore, as noted above, prolactin activates other members of the STAT family in a number of cell types. In particular, recent data suggest that in addition to STAT5b, the closely related STAT5a is also expressed in NEDA neurons in the arcuate nucleus (16). To determine whether prolactin activates these additional STAT proteins in NEDA neurons, we have combined the use of a cell culture preparation with in vivo studies to characterize the influence of prolactin on the phosphorylation and nuclear translocation of specific STAT proteins in TH-expressing neurons in the rat mediobasal hypothalamus.
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Measurement of STAT expression in hypothalamic cell cultures
Cultures were removed from the incubator and washed briefly (twice for 5 min each time) with 400 µl carbogen-gassed HEPES-buffered saline of the following composition: 150 mM NaCl, 15 mM HEPES, 5.5 mM glucose, 3.8 mM K2HPO4, 1 mM MgSO4, 1.5 mM CaCl2, and 0.5 mM sodium ascorbate, adjusted to pH 7.4 at 37 C. After the last wash, cells were incubated with 200 µl ovine prolactin (routinely 1 µg/ml; obtained from Sigma-Aldrich Corp.) or buffer alone for the required time (5–240 min). The cells were then lysed in ice-cold 10 mM Tris buffer (three wells collected in a total of 200 µl) containing 5 mM EDTA, 50 mM sodium fluoride, 50 mM NaCl, 1% Triton X-100, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonylfluoride, and 10 µl/ml protease inhibitor mixture (obtained from Sigma-Aldrich Corp.). The lysate was sonicated and centrifuged at 12,000 x g for 10 min, and the resultant supernatant was fractionated by SDS-PAGE (35–75 µg/track) and transferred onto nitrocellulose membranes. Blots were then probed with antibodies (supplied by Zymed Laboratories, San Francisco, CA) against STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6 (1 µg/ml in Blotto) or phospho-STAT5 (2 µg/ml in 3% BSA) overnight at 4 C, then washed with 10 mM Tris-buffered saline containing 0.05% Tween 20, reincubated with the appropriate horseradish peroxidase-coupled secondary antibody (1:5000 dilution) for 60 min at room temperature, and washed again, and the image was developed using enhanced chemiluminescence according to the manufacturer’s instructions (Amersham Biosciences, Auckland, New Zealand). Relative levels of phospho-STAT5 were determined using densitometric image analysis (Image, National Institutes of Health, Bethesda, MD) and expressed as a percentage of that obtained from cells incubated with buffer alone and examined on the same immunoblot.
Hypothalamic cell culture immunocytochemistry
Cells cultured on poly-D-lysine-coated glass coverslips were removed from the incubator and washed with buffer as outlined above. When appropriate, cultures were then preincubated for 15 min with selected protein kinase inhibitors, H89 (10 µM), KN93 (3 µM), or PD 98059 (50 µM; all from Sigma-Aldrich Corp.); bisindolymaleimide (3 µM) or AG490 (25–400 µM; from Calbiochem, San Diego, CA); or the prolactin antagonist 1–9-G129R-hPRL (2–250 µg/ml) (18) before stimulation with or without prolactin (1 µg/ml) in the continued presence of the appropriate inhibitor. After the required incubation time (routinely 30 min), the buffer was removed, and the cells were immediately fixed for 20 min with 4% formaldehyde in PBS and then permeabilized with 10% acetic acid in ethanol at –20 C (5 min). Fixed cells were washed three times for 5 min each time in 1 ml PBS, followed by 1 h in PBS containing 5% goat serum and then overnight at 4 C with rabbit polyclonal anti-TH (AB151 diluted 1:200; from Chemicon International, Temecula, CA) in combination with monoclonal antibodies against a selected STAT (STAT1, STAT3, STAT5a, STAT5b, phospho-STAT1, phospho-STAT3, or phospho-STAT5, all at 1:2000 dilution; from Zymed Laboratories). After antibody incubation, cells were washed with PBS (six times, 5 min each time) and incubated with a combination of secondary antibodies (antimouse Alexa 488 and antirabbit Alexa 568; Molecular Probes, Eugene, OR; both diluted 1:1000 in PBS) for 30 min at room temperature. They were then mounted in Vectashield (Vector Laboratories, Inc., Burlingame, CA) and examined under fluorescence microscopy using an Olympus AX70 microscope (New Hyde Park, NY) fitted with a Spot-RT Color digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI). Analysis of immunocytochemical experiments was performed by counting the number of TH-immunoreactive cells present on a coverslip and then determining the percentage of these cells also displaying nuclear STAT immunoreactivity. A minimum of 100 TH-immunoreactive cells were assessed in this fashion for each experimental condition. Statistical analysis was performed using a Mann-Whitney U test or, in the case of multiple groups, ANOVA, followed by Dunnett’s multiple comparison test.
In vivo prolactin stimulation and STAT5 immunohistochemistry
Female rats (10–12 wk old; 200–250 g) were ovariectomized 7 d before experimentation. Endogenous prolactin secretion was then blocked by bromocriptine methanesulfonate (Research Biochemicals, Inc., Natick, MA) administration (200 µg, sc, in 200 µl 10% ethanol) 5 h before injection of ovine prolactin (250 µg sc in 200 µl saline; Sigma-Aldrich Corp.). After the required stimulation time the animals (n = 5/group) were deeply anesthetized with 60 mg sodium pentobarbital (containing 1000 IU heparin) and perfused via the ascending aorta with 30 ml PBS (containing 120 IU heparin) and then 250 ml 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Control rats were perfused 30 min after sc injection of vehicle only. Brains were removed, postfixed in the same fixative overnight, and then infiltrated until sinking in 30% sucrose. Cryostat sections (40 µm) were prepared at the level of the arcuate nucleus from each brain to provide several similar series of consecutive sections (240 µm apart), which were stored in cryoprotectant at –20 C until required for dual-label fluorescence immunohistochemistry. Free-floating sections were washed three times in 0.1 M phosphate buffer and incubated for 48 h with rabbit polyclonal anti-TH (AB151; 1:6000; Chemicon International) and mouse monoclonal anti-STAT5a or anti-STAT5b, (1:3000; Zymed Laboratories) in 5% normal goat serum. Sections were then washed three times for 5 min each time with PBS and incubated for an additional 3 h with antimouse Alexa 488 and antirabbit Alexa 568, both diluted 1:1000 in PBS containing 1% normal goat serum (Molecular Probes), washed again in PBS, mounted on 3-aminopropylthriethoxy-silane-coated slides, and coverslipped with Vectashield (Vector Laboratories, Inc.). Sections were then viewed and photographed using an LSM 510 Axioplan confocal microscope (Carl Zeiss, New York, NY). Image analysis of STAT nuclear translocation was performed using ImageJ software (National Institutes of Health). On each section, all TH-immunoreactive cells within the dorsomedial and ventrolateral regions of the arcuate nucleus (i.e. TIDA neurons) were identified, and the cell bodies and nuclear boundaries were traced. The average intensity of STAT immunoreactivity within these defined areas (cytoplasm and nucleus) was measured and expressed as a ratio of nuclear to cytoplasmic intensities, which was used as an index of STAT translocation (14). Cells with STAT immunoreactivity at or below background levels were excluded from this analysis. For each animal, all TH-immunoreactive cells on one side of the arcuate nucleus (
15 neurons, on the average) from two or three evenly spaced sections were analyzed in this way, and the average ratio was calculated to provide a single data point. Statistical analysis was performed using ANOVA, followed by Dunnett’s multiple comparison test.
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). In each case only a single immunoreactive band was detected running at the expected molecular weight. STAT4 and STAT6 were not expressed at detectable levels in these cultures. To identify the signaling pathways activated by prolactin in the dopaminergic cells present in these cultures, the cells were incubated with or without prolactin (1 µg/ml for 30 min) and then dual-labeled for TH and phospho-STAT1, -3, or -5. After incubation with buffer alone, no immunoreactivity was seen for any of the phospho-STAT proteins in TH-immunoreactive cells (data not shown). As shown in Fig. 2, incubation with prolactin resulted in nuclear phospho-STAT5 labeling in both TH-positive and TH-negative cells (Fig. 2C). Of the approximately 100 TH cells examined in each of three cultures, 54 ± 4% exhibited phospho-STAT5 immunoreactivity in their nuclei after 30-min incubation with prolactin. Nuclear phospho-STAT1 and phospho-STAT3 labeling was occasionally seen after incubation with prolactin, but this was only in TH-negative cells (Fig. 2, A and B). The identity of these prolactin-responsive cells has not been established, but it is noteworthy that they were not immunoreactive for the glial cell maker, glial fibrillary acidic protein (data not shown). Although phospho-STAT1 and phospho-STAT3 were not detected in TH-positive cells after incubation with prolactin, their nonphosphorylated forms were present, with 16 ± 3% and 19 ± 3% of TH neurons expressing STAT1 and STAT3, respectively (50 cells examined for each condition on two separate cell cultures).
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). A maximum response was observed after 15 min, with approximately 50% of the TH-positive cells exhibiting nuclear phospho-STAT5 labeling (Fig. 3). This level of response was maintained to 30 min and then declined to essentially basal levels by 240 min. It should be noted that in addition to there being fewer phospho-STAT5 cells labeled at longer times, the intensity of labeling in individual cells also declined (Fig. 3A).
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1–9-G129R-hPRL caused concentration-dependent inhibition of prolactin-stimulated phospho-STAT5 nuclear labeling in TH-positive cells (Fig. 4). With a prolactin concentration of 1 µg/ml (for 30 min), the antagonist exhibited an IC50 of approximately 2 µg/ml. At the highest concentration of antagonist used (250 µg/ml), there were still nuclear phospho-STAT5-labeled, TH-immunoreactive neurons present; although this number was small and variable (14 ± 12% of TH cells), it contrasted with the complete absence of phospho-STAT5 labeling under basal conditions. No phospho-STAT5 labeling was seen in cells incubated with antagonist alone (10 µg/ml for 30 min).
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). This inhibition was significant (P < 0.05) at 25 µM and appeared to reach a plateau of approximately 50% at 100 µM, with an additional sharp decline to only about 10% of responding cells by 400 µM. In contrast, concentrations of the serine/threonine kinase inhibitors H89 (10 µM), bisindolymaleimide I (3 µM), and KN-93 (3 µM) that are effective in inhibiting prolactin-induced TH activation (10) failed to have any significant effect on prolactin-induced phospho-STAT5 nuclear labeling in TH-positive cells (Fig. 5B). Similarly, inhibition of the MAP/ERK kinase MAPK kinase, with PD98059 (50 µM) also failed to reduce this response (Fig. 5B).
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). Prolactin stimulation resulted in the appearance of a second STAT5b immunoreactivity band. The higher molecular weight band probably corresponds to phospho-STAT5b, because it comigrates with the immunoreactive band that appears in response to prolactin stimulation (Fig. 6B). It should be noted that the time course of this phospho-STAT5 band correlates well with the time course of phospho-STAT5 immunoreactivity reported in Fig. 3
. In contrast, the immunoblot for STAT5a showed no indication of a second immunoreactive band in response to prolactin (Fig. 6A). The suggestion that STAT5b is primarily responsible for prolactin signaling in these cells was supported by immunocytochemical data. Under basal conditions (30-min incubation with buffer alone), a small proportion (6 ± 2%) of TH-positive cells displayed STAT5a-immunoreactive nuclei. Incubation with prolactin (1 µg/ml for 30 min) did not significantly increase the extent of this labeling (10 ± 3% of TH-positive cells). In contrast, although the level of STAT5b-labeled nuclei was similar to that of STAT5a-labeled nuclei under basal conditions (8 ± 9% of TH-positive cells), there was a marked increase after incubation with prolactin, with 70 ± 8% of TH-positive cells now exhibiting STAT5b immunoreactivity in their nuclei (Fig. 7).
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, lower panels), suggesting a prolactin-induced translocation of STAT5b from the cytoplasm into the nucleus. This response was apparent after 30 min, but reached a statistically significant maximum after 60 min before declining again to basal levels after 240 min (Fig. 8B). In contrast, the nuclear/cytoplasmic ratio of STAT5a remained unchanged after prolactin administration (Fig. 8B), although there did appear to be an increase in STAT5a staining in the nuclei of some non-TH-immunoreactive cells (Fig. 8A, upper panels).
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In the current context it was important to identify which STAT proteins are activated by prolactin in TH-positive cells. Our immunocytochemical investigations demonstrated that although STAT1, STAT3, and STAT5a/b were expressed in these cells, prolactin stimulation resulted exclusively in phospho-STAT5 nuclear staining. Although prolactin stimulation did not result in phospho-STAT1 or phospho-STAT3 nuclear labeling in TH-positive cells, a small population of non-TH cells displayed these responses. This observation is of interest because previous reports of prolactin-mediated STAT3 responses have been limited to cells of mammary, myeloid, or lymphoid origin (13, 20, 21). To our knowledge, this is the first suggestion that STAT3 may be involved in prolactin signaling in cells of neuronal origin. Prolactin has been reported to activate STAT1 in cultured astrocytes (12). In the current studies, however, we found no evidence that cells exhibiting phospho-STAT1 were immunoreactive for glial fibrillary acidic protein, suggesting possible regional differences in astrocytic sensitivity to prolactin.
Prolactin stimulation of phospho-STAT5 nuclear labeling was profound, with the number of TH-positive cells responding rising from zero under basal conditions to approximately 70% after a 30-min incubation with prolactin (1 µg/ml). Although this prolactin concentration was relatively high, similar levels have been used to elevate TH activity in these cultures (7, 10, 22), and this concentration is only slightly higher than that in the circulation during the preovulatory surge and lower than that reported in portal blood (23).
The specificity of the prolactin-induced phospho-STAT5 response was supported by the demonstration of inhibition by the prolactin receptor antagonist -9-G129R-hPRL at concentrations similar to that used to inhibit prolactin-stimulated TH activity in these cultures (10). Similar concentrations of this or related analogs inhibit prolactin responses in other cell types (24, 25, 26). Although 10 µg/ml -9-G129R-hPRL (a 10-fold molar excess of the prolactin concentration) reduced the number of TH-immunoreactive cells exhibiting nuclear phospho-STAT5 by 80%, a small population of TH-positive cells remained resistant even at much higher concentrations of antagonist. The reason for this apparent resistance is unknown, but it should be noted that no phospho-STAT5 immunoreactivity was seen with antagonist alone, supporting evidence that it is free of agonist action (26).
The prolactin-stimulated increase in nuclear phospho-STAT5 labeling was suppressed by the JAK inhibitor AG490. At 100 µM, the inhibitor suppressed the response by about 60%. Similar concentrations of AG490 fully inhibit prolactin-stimulated responses in other cell types (27, 28, 29). Although it is probable that at the concentrations required to abolish the prolactin response in these hypothalamic cultures (400 µM) the drug is having nonspecific effects, AG490 resistance may suggest that prolactin signaling in these cells involves additional mechanisms. A similar proposal has been made for insulin-induced STAT5 activation in pancreatic cells on the basis that AG490 only partially inhibited the response (30). The nature of alternative pathways is unknown, but prolactin has been shown to activate Src family kinases and phosphatidylinositol 3-kinase (1), both of which may have a role in activating STAT5b in response to other stimuli (31, 32, 33)
We have previously demonstrated that protein kinase A, protein kinase C, MAPK, and, to a lesser extent, Ca2+/calmodulin-dependent protein kinase II participated in prolactin stimulation of TH activity in cultured hypothalamic cells (10). Given that STAT5 can be serine phosphorylated, and that this phosphorylation may influence its nuclear translocation (34), we examined the effect of a range of protein kinase inhibitors (used in our previous study on prolactin-induced TH activation) on prolactin-induced phospho-STAT5 translocation. In contrast to their documented inhibition of TH activity at the concentrations employed, these inhibitors had no effect on phospho-STAT5 translocation. We have previously shown that these kinases are similarly not involved in prolactin stimulation of TH mRNA expression (10), an observation consistent with the proposal that this response is mediated by a STAT5-dependent, serine/threonine kinase-independent pathway, presumably involving tyrosine kinase phosphorylation of the STAT protein. In contrast, the acute prolactin activation of TH is dependent on specific serine/threonine kinases (10).
STAT5 exists as two highly homologous isomers, STAT5a and STAT5b, sharing approximately 96% amino acid identity (35). Studies using mice deficient in either STAT5a or STAT5b suggest that there may be a level of functional redundancy between the two isomers in some responses (36). Our previous studies indicate that this is not the case with prolactin regulation of TH activity and TH expression in NEDA neurons (15), because prolactin-mediated feedback is severely impaired in STAT5b-deficient animals. Hence, although STAT5a is expressed in a significant number of hypothalamic NEDA neurons (16), it apparently cannot compensate for the absence of STAT5b. In the present in vivo experiments, STAT5a immunoreactivity was found in approximately 40% of TH-positive neurons, but prolactin administration did not alter its nuclear to cytoplasmic ratio, indicating that it was not responsive to prolactin receptor activation. This conclusion is supported by the cell culture studies, which indicated that the number of TH-positive cells exhibiting nuclear STAT5a immunoreactivity was not increased after incubation with prolactin. These data suggest that although present, STAT5a does not participate in prolactin signaling in these cells. This is a particularly surprising observation, because STAT5a is well-established mediator of prolactin action in peripheral tissues (36). These data indicate a high degree of functional specialization of the JAK/STAT pathway in neurons.
In contrast to STAT5a, STAT5b underwent nuclear translocation in response to prolactin both in vivo and in vitro. After prolactin administration in vivo, TH-positive neurons exhibited an increase in STAT5b nuclear staining in the arcuate nucleus. This agrees well with the response to prolactin in these neurons measuring total STAT5 immunoreactivity (14). Selective nuclear translocation of STAT5b was also seen in culture, with approximately 70% of TH-positive neurons exhibiting STAT5b immunoreactivity in their nuclei after a 60-min incubation with prolactin. The proposal that STAT5b is phosphorylated in response to prolactin was supported by the observed decrease in the electrophoretic mobility of a proportion of the STAT5b protein. Given that STAT5b can be both serine and tyrosine phosphorylated, it is unclear which of these phosphorylation events was responsible for this change in mobility.
The reason why approximately 30% of the TH-positive neurons did not undergo STAT5b translocation in response to prolactin is unclear. The in vivo data suggest that approximately 90% of the TIDA neurons expressed STAT5b, suggesting that nonresponding cells may lack appropriate prolactin receptors rather than the transduction mechanism. Although tuberohypophyseal and periventricular hypothalamic dopaminergic neuronal populations were not analyzed in our in vivo study, we observed a similar degree of STAT5b staining as was seen in TIDA neurons. It is possible that in addition to NEDA neurons, these culture preparations may contain dopaminergic neurons from neighboring brain regions, such as the zona incerta, which do not express prolactin receptors (8), and were not immunoreactive for STAT5 proteins in our in vivo study (data not shown).
In conclusion, these current studies on rat hypothalamic neurons extend previous findings using STAT5b-deficient mice, supporting the proposal that prolactin-mediated transcriptional regulation of hypothalamic dopaminergic neurons is mediated exclusively by STAT5b. Although these neurons also express STAT1, STAT3, and STAT5a, these transcription factors do not participate in the prolactin response. STAT5a, in particular, is well established as a mediator of prolactin signal transduction in some tissues (36). Thus, the absence of a STAT5a-mediated response to prolactin in the present studies and the inability of STAT5a to compensate for the absence of STAT5b in mice (15) demonstrate that these closely related transcription factors have specific and nonredundant functions in these hypothalamic dopaminergic neurons. Although the significance, in terms of the cellular response resulting from the activation of STAT5b rather than STAT5a, remains to be resolved, it is important to note that the two transcription factors show different DNA-binding characteristics (37, 38). It will be of interest to determine whether prolactin signal transduction in other central nervous system and peripheral neurons is also selectively mediated by STAT5b.
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Footnotes |
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First Published Online August 25, 2005
Abbreviations: JAK, Janus kinase; NEDA, neuroendocrine dopaminergic; STAT, signal transducer and activator of transcription; TH, tyrosine hydroxylase; TIDA, tuberoinfundibular dopaminergic neuron.
Received June 24, 2005.
Accepted for publication August 17, 2005.
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2-macroglobulin, and decidual PRL involving the Jak2 and Stat5 pathway. Endocrinology 142:1242–1250 gene expression is mediated by Janus kinase 2 (Jak2) while signal transducer and activator of transcription 5b (Stat5b) phosphorylation involves Jak2 and a second tyrosine kinase. Mol Endocrinol 15:1941–1952This article has been cited by other articles:
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, Basal conditions; 
, prolactin stimulation. *, P < 0.05 compared with basal (by Mann-Whitney U test).