Effects of Cytokines on Gonadotropin-Releasing Hormone (GnRH) Gene Expression in Primary Hypothalamic Neurons and in GnRH Neurons
http://www.100md.com
《内分泌学杂志》
Service of Endocrinology (P.I., R.S., J.-P.R., M.G., F.P.P., R.C.G.), Diabetology and Metabolism, Department of Medicine, University Hospital, CH-1011 Lausanne, Switzerland
Service of Endocrinology (F.P.P.), Diabetology and Metabolism, University Hospital, 1211 Geneva, Switzerland
Abstract
Various cytokines produced during the immune reaction can modulate the neuroendocrine reproductive axis, probably by inducing changes in the activity of hypothalamic GnRH neurons. However, the precise cellular and molecular effects of cytokines on these neurons have not been reported yet. To gain a better insight into these regulations, we first examined the pattern of expression of cytokine receptors in a novel neuronal cell line expressing GnRH (Gnv-4 cells). Among others, gp130 is expressed in Gnv-4 cells, together with the ligand receptor subunits specific for IL-6 as well as oncostatin M (OSM). Consistent with the latter observation, we show that OSM stimulates the expression of the immediate early genes c-fos and early growth response-1 in Gnv-4 cells, an effect dependent upon the activation of the MAPK Erk1/2 intracellular signaling pathway. Functional studies performed in parallel in Gnv-4 cells and in primary hypothalamic neuronal cell cultures show that OSM, although devoid of any effect of its own on GnRH gene expression, can inhibit dose-dependently the stimulation of GnRH expression by N-methyl-D-aspartic acid. In conclusion, these data demonstrate that a GnRH-expressing neuronal cell line can be modulated in vitro by cytokines implicated in the regulation of the reproductive axis. Moreover, they provide the first evidence of an involvement of OSM in these regulations.
Introduction
THE SOPHISTICATED interconnections existing between the immune and the neuroendocrine systems are based on the mutual sharing of receptors and mediators: neuroendocrine cells express various cytokines and their corresponding receptors, whereas immune cells synthesize neurohormones and also express their receptors (1). In this context, the release of some potent proinflammatory cytokines such as TNF-, IL-1, and IL-6 is likely to play a central pathophysiological role in the inhibition of the neuroendocrine reproductive axis observed during severe illnesses (2, 3). Hypothalamic neurons expressing GnRH represent the key activators of that axis, and it has been suggested that this inhibition of the reproductive axis is mediated centrally (4).
However, very little is known about the precise mechanisms involved. In addition, available data regarding the effects of individual cytokines on the neuroendocrine reproductive axis remain somewhat controversial. For example, the intracerebroventricular administration of IL-6 has no effect on the circulating levels of LH (5), whereas this cytokine was found to stimulate GnRH release from primary rat hypothalamic cultures in a dose- and time-dependent manner (6). Similarly, the central administration of IL-1 was found to decrease circulating LH levels, and therefore to disrupt the estrous cycle of female rats (7, 8), but the same cytokine was shown to stimulate GnRH release in vitro in some experimental settings (7, 9).
Some of the uncertainties regarding the effects of cytokines on hypothalamic GnRH neurons might be related to the difficulties inherent in their study: GnRH neurons are present in low number, and scattered throughout the medio-basal hypothalamus. Here we used primary hypothalamic neuronal cell cultures in parallel with Gnv-4 cells, one of the novel GnRH neuronal cell lines recently generated in our laboratory by conditional immortalization of adult rat hypothalamic neurons (10) to better define the potential effects of various cytokines on GnRH neurons. We report that several cytokine receptors are expressed by Gnv-4 cells, including the IL-1 receptor 1 and 2, the -subunit of the IL-2 receptor, and members of the gp130 family: the IL-6 receptor and the receptor for oncostatin M (OSMR). After the latter observation, we found that OSM stimulates the expression of several immediate early genes via the MAPK Erk1/2 signaling pathway, but that it has no effect on GnRH gene expression when administered alone. However, it was found to inhibit in a dose-dependent manner the expression of GnRH stimulated by the glutamate agonist N-methyl-D-aspartic acid (NMDA), an effect that we observed both in Gnv-4 cells and in primary hypothalamic neurons. In conclusion, we have demonstrated that cytokines implicated in the regulation of the reproductive axis can modulate the activity of GnRH-expressing immortalized neuronal cells. This suggests that these effects are acting at the level of hypothalamic GnRH neurons, a hypothesis that should be confirmed in vivo.
Materials and Methods
Cell cultures
Gnv-4 cells are one of the eleven clones of GnRH-expressing cells obtained by conditional immortalized of adult rat hypothalamic cell cultures (10). Two clones expressed the highest levels of GnRH mRNA: Gnv-3 cells described in the companion paper of the present manuscript (10), and Gnv-4 cells. Like Gnv-3 cells, Gnv-4 cells express markers of well differentiated neurons, do not express markers of glial cells, and respond to NMDA stimulation with increases in both the secretion and the expression of GnRH (data not shown). Thus, they are essentially indistinguishable from Gnv-3 cells.
Cells are grown in Neurobasal A medium with fetal bovine serum, B27 supplement (all from Invitrogen AG, Basel Switzerland) and doxycycline 5 μg/ml (Sigma, Fluka Chemie GmbH, Buchs, Switzerland). Doxycycline is added to the medium to promote proliferation of these cells because they were immortalized conditionally using a modified Tet-On system (11). Before the experiments, cells from an early passage (3 or 4) are grown in medium without doxycycline for 3 d, and then starved for 12 h in DMEM containing 4.5 g/liter glucose (Invitrogen AG).
Primary rat hypothalamic cell cultures are performed as described (12, 13). Briefly, hypothalami are obtained from 10- to 12-wk-old female Wistar rats. After rats were killed by decapitation, the entire hypothalamus is quickly dissected from the rest of the brain, and cells dispersed mechanically in PBS buffer (PBS without calcium or magnesium (GIBCO) supplemented with 0.06% glucose (Fluka Chemie GmbH) and 100 U/100 μg/ml Penicillin/Streptomycin (Seromed, Fakola, Basel, Switzerland). Freshly excised whole hypothalami are gently passed several times through Pasteur pipettes flamed in the middle of the procedure to decrease the diameter of their opening. Nondispersed tissue is allowed to settle for 5 min and supernatant transferred to a clean tube. The remaining pellet is resuspended in 4 ml of PBS buffer and mechanical dispersion repeated. Supernatants from first and second dispersion are mixed, centrifuged for 5 min at 100 x g and the cellular pellet gently resuspended in 5 ml of Neurobasal Medium (GIBCO)-0.04% B27 supplement (GIBCO) containing 500 μM glutamine and 25 μM glutamate (Sigma). After dispersion, cells are plated at a density of 400,000 live cells/well in six-well plates (Corning Costar Corp., Cambridge, MA) coated with 5 μg/ml poly-D-lysine (Sigma, Fluka Chemie GmbH), and grown in Neurobasal-A Medium (Invitrogen AG) with B27 supplement (Invitrogen AG) containing 500 μM glutamine and 25 μM glutamate (Sigma, Fluka Chemie GmbH). Half of the medium is changed every fourth day, and the cells are used between 3 and 4 wk after dispersion.
Experimental design
For the study of the effects of OSM on the immediate early genes c-fos, c-jun, Egr-1 (early growth response-1), MKP-1 (MAPK phosphatase 1), Myd118 and GADD45, GnV-4 cells were treated for 30 min with recombinant murine OSM (R&D Systems, Abingdon, UK) at concentrations varying from 10–250 ng/ml. Expression of these genes, as well as expression of cytokines and of cytokine receptors, was assessed by RT-PCR of total RNA extracts (Tripure reagent; Roche Diagnostics, Rotkreuz, Switzerland), using the primer pairs listed in Table 1. The RT reaction was performed using Superscript reverse transcriptase (Invitrogen AG) with oligo deoxythymidine (Promega, Madison, WI), and PCR amplification was achieved with a Gene Amp PCR System 9700 thermocycler (PE Applied Biosystems, Rotkreuz, Switzerland). mRNA levels were quantified by densitometric analysis of the gels, normalized to the mRNA levels of the ribosomal protein S12. All PCR products were confirmed by restriction enzyme digestion.
For the analysis of signal transduction events, OSM or IL-6 (Peprotech, London, UK) were applied at a concentration of 100 ng/ml for short time periods between 2 and 60 min. The induction of Erk1/2 by OSM was assessed by Western blot, using monoclonal antibodies against phosphospecific and total Erk1/2 (Cell Signaling Technology, Inc., Beverly, MA). To allow quantification, protein concentration in each extract was measured precisely using the bicinchoninic acid protein assay reagent (Pierce, Rockford, IL), and equal amounts of proteins were loaded on the gels. These extracts were resolved by 10% SDS-PAGE electrophoresis, and then transferred to a polyvinylidene difluoride membrane (Hybond-P; Amersham Biosciences, Otelfingen, Switzerland). Proteins-antibody complexes were visualized by the ECL system (Amersham, Arlington Heights, IL). Quantification of phosphorylated Erk1/2 was achieved by densitometric analysis of the blots, using a Typhoon System (Amersham Biosciences, Otelfingen, Switzerland), after verifying that total Erk1/2 content was affected by neither OSM or IL-6 treatment (data not shown). Next, the role of the MAPK Erk1/2 pathway in mediating the effects of OSM (250 ng/ml) on the expression of c-fos, Egr-1, MKP-1, and GADD45 was evaluated by pretreating the cells for 1 h with the enzyme inhibitor PD98059 (50 μM, Sigma, Fluka Chemie GmbH) before stimulation. Gene expression was measured at 30 min after stimulation with OSM.
Finally, the effects of OSM were studied either on basal or NMDA (Sigma, Fluka Chemie GmbH)-stimulated GnRH gene expression, both in Gnv-4 cells and in primary hypothalamic neuronal cell cultures. Cells (Gnv-4 or primary neurons) were incubated for 6 h with OSM at concentrations between 2 and 100 ng/ml, either alone or together with NMDA (50 μM). Protein fragments exhibiting a terminal glutamic acid act as NMDA antagonists at its receptor (14, 15). Because OSM was found to inhibit NMDA-stimulated expression of GnRH, experiments were repeated in the presence of a protease inhibitor (aprotinin, Trasylol) to exclude a role of potential degradation products of OSM in this inhibition.
GnRH mRNA levels were measured in total RNA extracts by real-time, semiquantitative RT-PCR using LightCycler technology (Roche Diagnostics) as described (13). All measurements were performed in triplicates. The interassay coefficient of variation was between 6 and 15%, and a fourth run was performed for samples with an interassay coefficient of variation above 10%. The results are expressed in relation to the control sample run in the same reaction, and all experiments were repeated at least three times. Statistical significance of all results was assessed by ANOVA, using the JMP4 statistical package (SAS Institute, Cary, NC). These experiments were approved by both the institutional and the state ethical committees on animal research.
Results
Figure 1A demonstrates that Gnv-4 cells express gp130, the common signal transducing chain of the gp130 family of cytokine receptors, together with the ligand-specific chains IL-6 receptor and OSM receptor . Other receptors expressed by these cells include the IL-1 receptor 1 and 2, and the -subunit of the IL-2 receptor (data not shown). The IL-1 receptor accessory protein, which is mandatory for proper IL-1 signal transduction (16), is also expressed. In contrast, we failed to amplify the receptors for IL-10, IL-11, leukemia inhibitory factor (LIF) and ciliary neurotrophic factor. In addition to these receptors, Gnv-4 cells also express IL-6 at the mRNA level (data not shown), whereas IL-1, IL-1, IL-1 receptor antagonist, IL-6, and OSM were not found.
Because Gnv-4 cells express the OSM receptor, the effects of OSM on cellular activity were first examined by studying the expression of different immediate early genes. Figure 1, B–D, demonstrate the rapid, dose-dependent stimulation of c-fos, Egr-1, and GADD45 by OSM. Similar results were observed with MKP-1 (data not shown). Among these four genes, the inductions of GADD45 and c-fos were the most remarkable.
Activation of either c-fos, Egr-1, or MKP-1 has been associated with the MAPK signal-transduction cascade (17, 18, 19). Therefore, we next examined the involvement of this signaling pathway in the actions of OSM. OSM induced a rapid and strong activation of the MAPK Erk1/2 pathway, as demonstrated by the increase in Erk1/2 phosphorylation displayed on Fig. 2A. The activation of this pathway after OSM treatment occurs earlier and lasts longer than what is observed after the same concentration of IL-6. Figure 2, B and C, illustrates the effects of blocking the MAPK Erk1/2 pathway before stimulating the cells with OSM: preincubation of Gnv-4 cells with the Erk1/2 inhibitor PD98059 completely abolished the OSM-induced stimulation of c-fos and Egr-1.
Figure 3 illustrates the effects of OSM on GnRH gene expression. Figure 3A demonstrates that OSM alone has no effect, but that it can inhibit the NMDA-induced increase in GnRH mRNA levels observed in Gnv-4 cells. This inhibition is dose dependent, reaching a maximum (70% inhibition) at the concentration of 50 ng/ml. Addition of an inhibitor of proteases (aprotinin) did not modify the inhibition exerted by OSM on the effect of NMDA (data not shown). Very similar results were obtained in primary hypothalamic neuronal cell cultures (Fig. 3B). However, the maximal inhibition was observed at a slightly higher concentration of OSM of 100 ng/ml.
Discussion
Cytokines produced during the immune reaction can alter the activity of the neuroendocrine reproductive axis (2, 3). The capacity reported for various cytokines to modulate GnRH gene expression and secretion (6, 9, 20, 21) suggests that these effects are essentially mediated at the hypothalamic level (4), but the precise mechanisms involved remain largely unknown. To gain a better insight into these regulations, we first examined the profile of cytokine receptors expressed by Gnv-4 cells. Gnv-4 is a novel GnRH-expressing cell line obtained by conditional immortalization of primary hypothalamic neuronal cell cultures as described in the companion paper of the present article (10).
Gnv-4 cells express both the IL-1 receptor and the IL-1 receptor accessory protein, a mandatory component of the IL-1 signal-transduction pathway (16). These data demonstrate that the required components of the IL-1 receptor complex are found in these GnRH-expressing neurons, a finding consistent with the modulation of the expression of GnRH by IL-1 reported in vivo (20). Gnv-4 cells also express both IL-6 and the IL-6 receptor. The simultaneous expression of IL-6 and its receptor in these cells raises the possibility of the existence of an autocrine regulatory loop, as previously suggested (6). In contrast, the receptors for LIF and ciliary neurotrophic factor, which exhibit a relatively widespread expression among several neuronal cell types (22), were not found in these cells. Our present finding that Gnv-4 cells express several different cytokine receptors suggests that GnRH neurons may possess the cellular machinery to respond directly to these cytokines, a hypothesis that should be further tested in vivo.
Gnv-4 cells also express the OSM-specific receptor subunit, OSMR (23). In humans, two forms of OSM receptors have been identified. The type I OSM receptor is also a LIF receptor, and is composed of gp130 and of the ligand-specific LIFR. In murine cells, OSM binds exclusively to the type II OSM receptor, which is a heterodimer composed of gp130 and OSMR (24). To the best of our knowledge, no data are available in the rat, but because the LIFR is not expressed in Gnv-4 cells, the effects of OSM reported here are probably transduced via the type II OSM receptor.
OSM itself is a cytokine structurally and functionally related to LIF that has pleiotropic actions in a wide variety of tissues. In the periphery, OSM has been involved in the process of hematopoiesis, in the development and regeneration of the liver, in local inflammation or in cell migration (25, 26, 27, 28, 29, 30, 31). In contrast, very limited information is available regarding its potential actions as an endocrine modulator: it can stimulate the secretion of pituitary ACTH (32) or placental human chorionic gonadotropin (33), and it has been proposed to function as an autocrine growth factor for Sertoli cells (34). In the central nervous system, previous work has shown that the OSMR is expressed predominantly in the olfactory bulb (35). This could be relevant to our present observation because neurons of the olfactory bulb and hypothalamic GnRH neurons share a common embryonic origin in the olfactory placode (36).
Stimulation of Gnv-4 cells with OSM activates the MAPK Erk1/2 pathway, followed by a stimulation of the expression of a number of immediate early genes. These data demonstrate that the OSM receptor expressed by Gnv-4 cells is functional. Among the four immediate early genes induced by OSM, c-fos is generally regarded as a marker of neuronal activation (37, 38), whereas Egr-1 is related to the regulation of cell proliferation and programmed cell death (19). Their stimulation by OSM has already been described in liver and smooth muscle cells (39, 40, 41). The activation of the dual-specificity MKP-1 by OSM in Gnv-4 cells may be regarded as a negative feedback mechanism to control the MAPK cascade (42) and is consistent with our present finding that the OSM receptor is linked to the MAPK Erk1/2 pathway. Finally, the GADD45 family of immediate early genes are protein products involved in maintaining genomic stability and regulating growth arrest (43) that can also be induced by OSM in NIH3T3 cells (44). Therefore, our finding that GADD45 is stimulated by OSM in Gnv-4 cells suggests that OSM may participate in these regulation in GnRH neurons, as already demonstrated for other neuronal cell types (45).
Because Gnv-4 cells express GnRH, we then investigated the effects of OSM on GnRH gene expression in these cells. We also performed parallel experiments in primary hypothalamic neurons in culture (12, 13) to evaluate the potential effects of OSM in nontransformed cells. OSM alone does not influence basal GnRH gene expression, but it can inhibit in both models the increase induced by the glutamate agonist NMDA, a potent GnRH secretagogue (46) that also stimulates GnRH expression both in Gnv-4 cells and in primary neuronal cultures. Overall, our observations in Gnv-4 cells and in primary neurons are in line with reports demonstrating that different cytokines can modulate GnRH expression and/or secretion (5, 6, 7, 8, 9, 20, 22). Because Gnv-4 cells exhibit many features of adult GnRH neurons, the present results suggest that these neurons are directly modulated by cytokines, a hypothesis that should be further tested in vivo. In addition, our data allow to add OSM to the list of potential actors in this neuro-endocrine-immunologic interplay. OSM is produced mainly by activated T cells and mononuclear cells in the periphery (31), but the microglia seems also to be an important source of OSM in the CNS (47, 48). In addition, OSM could be expressed in inflammatory brain lesions (48). It can therefore be hypothesized that local brain OSM production might influence the functioning of GnRH neurons during inflammatory or immune diseases, acting locally as a paracrine factor.
Acknowledgments
The authors thank Jean-Pierre Bourguignon for his helpful comments on the data during the preparation of the manuscript.
Footnotes
Present address for P.I.: 2nd Department of Medicine, Faculty of Medicine, Semmelweis University, H-1088 Budapest, Hungary.
This work was supported by Grant Nos. 3200B0-105 657/1 (to R.C.G.) and 32-00B0-100 858/1 (to F.P.P.) from the Swiss National Science Foundation, a grant by the Novartis Foundation (to F.P.P.), and a scholarship from the Swiss Federal Commission for Scholarships for Foreign Students (to P.I.).
First Published Online November 10, 2005
Abbreviations: Egr-1, Early growth response-1; LIF, leukemia inhibitory factor; MKP-1, MAPK phosphatase 1; NMDA, N-methyl-D-aspartic acid; OSM, oncostatin M; OSMR, receptor for OSM.
Accepted for publication November 1, 2005.
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Service of Endocrinology (F.P.P.), Diabetology and Metabolism, University Hospital, 1211 Geneva, Switzerland
Abstract
Various cytokines produced during the immune reaction can modulate the neuroendocrine reproductive axis, probably by inducing changes in the activity of hypothalamic GnRH neurons. However, the precise cellular and molecular effects of cytokines on these neurons have not been reported yet. To gain a better insight into these regulations, we first examined the pattern of expression of cytokine receptors in a novel neuronal cell line expressing GnRH (Gnv-4 cells). Among others, gp130 is expressed in Gnv-4 cells, together with the ligand receptor subunits specific for IL-6 as well as oncostatin M (OSM). Consistent with the latter observation, we show that OSM stimulates the expression of the immediate early genes c-fos and early growth response-1 in Gnv-4 cells, an effect dependent upon the activation of the MAPK Erk1/2 intracellular signaling pathway. Functional studies performed in parallel in Gnv-4 cells and in primary hypothalamic neuronal cell cultures show that OSM, although devoid of any effect of its own on GnRH gene expression, can inhibit dose-dependently the stimulation of GnRH expression by N-methyl-D-aspartic acid. In conclusion, these data demonstrate that a GnRH-expressing neuronal cell line can be modulated in vitro by cytokines implicated in the regulation of the reproductive axis. Moreover, they provide the first evidence of an involvement of OSM in these regulations.
Introduction
THE SOPHISTICATED interconnections existing between the immune and the neuroendocrine systems are based on the mutual sharing of receptors and mediators: neuroendocrine cells express various cytokines and their corresponding receptors, whereas immune cells synthesize neurohormones and also express their receptors (1). In this context, the release of some potent proinflammatory cytokines such as TNF-, IL-1, and IL-6 is likely to play a central pathophysiological role in the inhibition of the neuroendocrine reproductive axis observed during severe illnesses (2, 3). Hypothalamic neurons expressing GnRH represent the key activators of that axis, and it has been suggested that this inhibition of the reproductive axis is mediated centrally (4).
However, very little is known about the precise mechanisms involved. In addition, available data regarding the effects of individual cytokines on the neuroendocrine reproductive axis remain somewhat controversial. For example, the intracerebroventricular administration of IL-6 has no effect on the circulating levels of LH (5), whereas this cytokine was found to stimulate GnRH release from primary rat hypothalamic cultures in a dose- and time-dependent manner (6). Similarly, the central administration of IL-1 was found to decrease circulating LH levels, and therefore to disrupt the estrous cycle of female rats (7, 8), but the same cytokine was shown to stimulate GnRH release in vitro in some experimental settings (7, 9).
Some of the uncertainties regarding the effects of cytokines on hypothalamic GnRH neurons might be related to the difficulties inherent in their study: GnRH neurons are present in low number, and scattered throughout the medio-basal hypothalamus. Here we used primary hypothalamic neuronal cell cultures in parallel with Gnv-4 cells, one of the novel GnRH neuronal cell lines recently generated in our laboratory by conditional immortalization of adult rat hypothalamic neurons (10) to better define the potential effects of various cytokines on GnRH neurons. We report that several cytokine receptors are expressed by Gnv-4 cells, including the IL-1 receptor 1 and 2, the -subunit of the IL-2 receptor, and members of the gp130 family: the IL-6 receptor and the receptor for oncostatin M (OSMR). After the latter observation, we found that OSM stimulates the expression of several immediate early genes via the MAPK Erk1/2 signaling pathway, but that it has no effect on GnRH gene expression when administered alone. However, it was found to inhibit in a dose-dependent manner the expression of GnRH stimulated by the glutamate agonist N-methyl-D-aspartic acid (NMDA), an effect that we observed both in Gnv-4 cells and in primary hypothalamic neurons. In conclusion, we have demonstrated that cytokines implicated in the regulation of the reproductive axis can modulate the activity of GnRH-expressing immortalized neuronal cells. This suggests that these effects are acting at the level of hypothalamic GnRH neurons, a hypothesis that should be confirmed in vivo.
Materials and Methods
Cell cultures
Gnv-4 cells are one of the eleven clones of GnRH-expressing cells obtained by conditional immortalized of adult rat hypothalamic cell cultures (10). Two clones expressed the highest levels of GnRH mRNA: Gnv-3 cells described in the companion paper of the present manuscript (10), and Gnv-4 cells. Like Gnv-3 cells, Gnv-4 cells express markers of well differentiated neurons, do not express markers of glial cells, and respond to NMDA stimulation with increases in both the secretion and the expression of GnRH (data not shown). Thus, they are essentially indistinguishable from Gnv-3 cells.
Cells are grown in Neurobasal A medium with fetal bovine serum, B27 supplement (all from Invitrogen AG, Basel Switzerland) and doxycycline 5 μg/ml (Sigma, Fluka Chemie GmbH, Buchs, Switzerland). Doxycycline is added to the medium to promote proliferation of these cells because they were immortalized conditionally using a modified Tet-On system (11). Before the experiments, cells from an early passage (3 or 4) are grown in medium without doxycycline for 3 d, and then starved for 12 h in DMEM containing 4.5 g/liter glucose (Invitrogen AG).
Primary rat hypothalamic cell cultures are performed as described (12, 13). Briefly, hypothalami are obtained from 10- to 12-wk-old female Wistar rats. After rats were killed by decapitation, the entire hypothalamus is quickly dissected from the rest of the brain, and cells dispersed mechanically in PBS buffer (PBS without calcium or magnesium (GIBCO) supplemented with 0.06% glucose (Fluka Chemie GmbH) and 100 U/100 μg/ml Penicillin/Streptomycin (Seromed, Fakola, Basel, Switzerland). Freshly excised whole hypothalami are gently passed several times through Pasteur pipettes flamed in the middle of the procedure to decrease the diameter of their opening. Nondispersed tissue is allowed to settle for 5 min and supernatant transferred to a clean tube. The remaining pellet is resuspended in 4 ml of PBS buffer and mechanical dispersion repeated. Supernatants from first and second dispersion are mixed, centrifuged for 5 min at 100 x g and the cellular pellet gently resuspended in 5 ml of Neurobasal Medium (GIBCO)-0.04% B27 supplement (GIBCO) containing 500 μM glutamine and 25 μM glutamate (Sigma). After dispersion, cells are plated at a density of 400,000 live cells/well in six-well plates (Corning Costar Corp., Cambridge, MA) coated with 5 μg/ml poly-D-lysine (Sigma, Fluka Chemie GmbH), and grown in Neurobasal-A Medium (Invitrogen AG) with B27 supplement (Invitrogen AG) containing 500 μM glutamine and 25 μM glutamate (Sigma, Fluka Chemie GmbH). Half of the medium is changed every fourth day, and the cells are used between 3 and 4 wk after dispersion.
Experimental design
For the study of the effects of OSM on the immediate early genes c-fos, c-jun, Egr-1 (early growth response-1), MKP-1 (MAPK phosphatase 1), Myd118 and GADD45, GnV-4 cells were treated for 30 min with recombinant murine OSM (R&D Systems, Abingdon, UK) at concentrations varying from 10–250 ng/ml. Expression of these genes, as well as expression of cytokines and of cytokine receptors, was assessed by RT-PCR of total RNA extracts (Tripure reagent; Roche Diagnostics, Rotkreuz, Switzerland), using the primer pairs listed in Table 1. The RT reaction was performed using Superscript reverse transcriptase (Invitrogen AG) with oligo deoxythymidine (Promega, Madison, WI), and PCR amplification was achieved with a Gene Amp PCR System 9700 thermocycler (PE Applied Biosystems, Rotkreuz, Switzerland). mRNA levels were quantified by densitometric analysis of the gels, normalized to the mRNA levels of the ribosomal protein S12. All PCR products were confirmed by restriction enzyme digestion.
For the analysis of signal transduction events, OSM or IL-6 (Peprotech, London, UK) were applied at a concentration of 100 ng/ml for short time periods between 2 and 60 min. The induction of Erk1/2 by OSM was assessed by Western blot, using monoclonal antibodies against phosphospecific and total Erk1/2 (Cell Signaling Technology, Inc., Beverly, MA). To allow quantification, protein concentration in each extract was measured precisely using the bicinchoninic acid protein assay reagent (Pierce, Rockford, IL), and equal amounts of proteins were loaded on the gels. These extracts were resolved by 10% SDS-PAGE electrophoresis, and then transferred to a polyvinylidene difluoride membrane (Hybond-P; Amersham Biosciences, Otelfingen, Switzerland). Proteins-antibody complexes were visualized by the ECL system (Amersham, Arlington Heights, IL). Quantification of phosphorylated Erk1/2 was achieved by densitometric analysis of the blots, using a Typhoon System (Amersham Biosciences, Otelfingen, Switzerland), after verifying that total Erk1/2 content was affected by neither OSM or IL-6 treatment (data not shown). Next, the role of the MAPK Erk1/2 pathway in mediating the effects of OSM (250 ng/ml) on the expression of c-fos, Egr-1, MKP-1, and GADD45 was evaluated by pretreating the cells for 1 h with the enzyme inhibitor PD98059 (50 μM, Sigma, Fluka Chemie GmbH) before stimulation. Gene expression was measured at 30 min after stimulation with OSM.
Finally, the effects of OSM were studied either on basal or NMDA (Sigma, Fluka Chemie GmbH)-stimulated GnRH gene expression, both in Gnv-4 cells and in primary hypothalamic neuronal cell cultures. Cells (Gnv-4 or primary neurons) were incubated for 6 h with OSM at concentrations between 2 and 100 ng/ml, either alone or together with NMDA (50 μM). Protein fragments exhibiting a terminal glutamic acid act as NMDA antagonists at its receptor (14, 15). Because OSM was found to inhibit NMDA-stimulated expression of GnRH, experiments were repeated in the presence of a protease inhibitor (aprotinin, Trasylol) to exclude a role of potential degradation products of OSM in this inhibition.
GnRH mRNA levels were measured in total RNA extracts by real-time, semiquantitative RT-PCR using LightCycler technology (Roche Diagnostics) as described (13). All measurements were performed in triplicates. The interassay coefficient of variation was between 6 and 15%, and a fourth run was performed for samples with an interassay coefficient of variation above 10%. The results are expressed in relation to the control sample run in the same reaction, and all experiments were repeated at least three times. Statistical significance of all results was assessed by ANOVA, using the JMP4 statistical package (SAS Institute, Cary, NC). These experiments were approved by both the institutional and the state ethical committees on animal research.
Results
Figure 1A demonstrates that Gnv-4 cells express gp130, the common signal transducing chain of the gp130 family of cytokine receptors, together with the ligand-specific chains IL-6 receptor and OSM receptor . Other receptors expressed by these cells include the IL-1 receptor 1 and 2, and the -subunit of the IL-2 receptor (data not shown). The IL-1 receptor accessory protein, which is mandatory for proper IL-1 signal transduction (16), is also expressed. In contrast, we failed to amplify the receptors for IL-10, IL-11, leukemia inhibitory factor (LIF) and ciliary neurotrophic factor. In addition to these receptors, Gnv-4 cells also express IL-6 at the mRNA level (data not shown), whereas IL-1, IL-1, IL-1 receptor antagonist, IL-6, and OSM were not found.
Because Gnv-4 cells express the OSM receptor, the effects of OSM on cellular activity were first examined by studying the expression of different immediate early genes. Figure 1, B–D, demonstrate the rapid, dose-dependent stimulation of c-fos, Egr-1, and GADD45 by OSM. Similar results were observed with MKP-1 (data not shown). Among these four genes, the inductions of GADD45 and c-fos were the most remarkable.
Activation of either c-fos, Egr-1, or MKP-1 has been associated with the MAPK signal-transduction cascade (17, 18, 19). Therefore, we next examined the involvement of this signaling pathway in the actions of OSM. OSM induced a rapid and strong activation of the MAPK Erk1/2 pathway, as demonstrated by the increase in Erk1/2 phosphorylation displayed on Fig. 2A. The activation of this pathway after OSM treatment occurs earlier and lasts longer than what is observed after the same concentration of IL-6. Figure 2, B and C, illustrates the effects of blocking the MAPK Erk1/2 pathway before stimulating the cells with OSM: preincubation of Gnv-4 cells with the Erk1/2 inhibitor PD98059 completely abolished the OSM-induced stimulation of c-fos and Egr-1.
Figure 3 illustrates the effects of OSM on GnRH gene expression. Figure 3A demonstrates that OSM alone has no effect, but that it can inhibit the NMDA-induced increase in GnRH mRNA levels observed in Gnv-4 cells. This inhibition is dose dependent, reaching a maximum (70% inhibition) at the concentration of 50 ng/ml. Addition of an inhibitor of proteases (aprotinin) did not modify the inhibition exerted by OSM on the effect of NMDA (data not shown). Very similar results were obtained in primary hypothalamic neuronal cell cultures (Fig. 3B). However, the maximal inhibition was observed at a slightly higher concentration of OSM of 100 ng/ml.
Discussion
Cytokines produced during the immune reaction can alter the activity of the neuroendocrine reproductive axis (2, 3). The capacity reported for various cytokines to modulate GnRH gene expression and secretion (6, 9, 20, 21) suggests that these effects are essentially mediated at the hypothalamic level (4), but the precise mechanisms involved remain largely unknown. To gain a better insight into these regulations, we first examined the profile of cytokine receptors expressed by Gnv-4 cells. Gnv-4 is a novel GnRH-expressing cell line obtained by conditional immortalization of primary hypothalamic neuronal cell cultures as described in the companion paper of the present article (10).
Gnv-4 cells express both the IL-1 receptor and the IL-1 receptor accessory protein, a mandatory component of the IL-1 signal-transduction pathway (16). These data demonstrate that the required components of the IL-1 receptor complex are found in these GnRH-expressing neurons, a finding consistent with the modulation of the expression of GnRH by IL-1 reported in vivo (20). Gnv-4 cells also express both IL-6 and the IL-6 receptor. The simultaneous expression of IL-6 and its receptor in these cells raises the possibility of the existence of an autocrine regulatory loop, as previously suggested (6). In contrast, the receptors for LIF and ciliary neurotrophic factor, which exhibit a relatively widespread expression among several neuronal cell types (22), were not found in these cells. Our present finding that Gnv-4 cells express several different cytokine receptors suggests that GnRH neurons may possess the cellular machinery to respond directly to these cytokines, a hypothesis that should be further tested in vivo.
Gnv-4 cells also express the OSM-specific receptor subunit, OSMR (23). In humans, two forms of OSM receptors have been identified. The type I OSM receptor is also a LIF receptor, and is composed of gp130 and of the ligand-specific LIFR. In murine cells, OSM binds exclusively to the type II OSM receptor, which is a heterodimer composed of gp130 and OSMR (24). To the best of our knowledge, no data are available in the rat, but because the LIFR is not expressed in Gnv-4 cells, the effects of OSM reported here are probably transduced via the type II OSM receptor.
OSM itself is a cytokine structurally and functionally related to LIF that has pleiotropic actions in a wide variety of tissues. In the periphery, OSM has been involved in the process of hematopoiesis, in the development and regeneration of the liver, in local inflammation or in cell migration (25, 26, 27, 28, 29, 30, 31). In contrast, very limited information is available regarding its potential actions as an endocrine modulator: it can stimulate the secretion of pituitary ACTH (32) or placental human chorionic gonadotropin (33), and it has been proposed to function as an autocrine growth factor for Sertoli cells (34). In the central nervous system, previous work has shown that the OSMR is expressed predominantly in the olfactory bulb (35). This could be relevant to our present observation because neurons of the olfactory bulb and hypothalamic GnRH neurons share a common embryonic origin in the olfactory placode (36).
Stimulation of Gnv-4 cells with OSM activates the MAPK Erk1/2 pathway, followed by a stimulation of the expression of a number of immediate early genes. These data demonstrate that the OSM receptor expressed by Gnv-4 cells is functional. Among the four immediate early genes induced by OSM, c-fos is generally regarded as a marker of neuronal activation (37, 38), whereas Egr-1 is related to the regulation of cell proliferation and programmed cell death (19). Their stimulation by OSM has already been described in liver and smooth muscle cells (39, 40, 41). The activation of the dual-specificity MKP-1 by OSM in Gnv-4 cells may be regarded as a negative feedback mechanism to control the MAPK cascade (42) and is consistent with our present finding that the OSM receptor is linked to the MAPK Erk1/2 pathway. Finally, the GADD45 family of immediate early genes are protein products involved in maintaining genomic stability and regulating growth arrest (43) that can also be induced by OSM in NIH3T3 cells (44). Therefore, our finding that GADD45 is stimulated by OSM in Gnv-4 cells suggests that OSM may participate in these regulation in GnRH neurons, as already demonstrated for other neuronal cell types (45).
Because Gnv-4 cells express GnRH, we then investigated the effects of OSM on GnRH gene expression in these cells. We also performed parallel experiments in primary hypothalamic neurons in culture (12, 13) to evaluate the potential effects of OSM in nontransformed cells. OSM alone does not influence basal GnRH gene expression, but it can inhibit in both models the increase induced by the glutamate agonist NMDA, a potent GnRH secretagogue (46) that also stimulates GnRH expression both in Gnv-4 cells and in primary neuronal cultures. Overall, our observations in Gnv-4 cells and in primary neurons are in line with reports demonstrating that different cytokines can modulate GnRH expression and/or secretion (5, 6, 7, 8, 9, 20, 22). Because Gnv-4 cells exhibit many features of adult GnRH neurons, the present results suggest that these neurons are directly modulated by cytokines, a hypothesis that should be further tested in vivo. In addition, our data allow to add OSM to the list of potential actors in this neuro-endocrine-immunologic interplay. OSM is produced mainly by activated T cells and mononuclear cells in the periphery (31), but the microglia seems also to be an important source of OSM in the CNS (47, 48). In addition, OSM could be expressed in inflammatory brain lesions (48). It can therefore be hypothesized that local brain OSM production might influence the functioning of GnRH neurons during inflammatory or immune diseases, acting locally as a paracrine factor.
Acknowledgments
The authors thank Jean-Pierre Bourguignon for his helpful comments on the data during the preparation of the manuscript.
Footnotes
Present address for P.I.: 2nd Department of Medicine, Faculty of Medicine, Semmelweis University, H-1088 Budapest, Hungary.
This work was supported by Grant Nos. 3200B0-105 657/1 (to R.C.G.) and 32-00B0-100 858/1 (to F.P.P.) from the Swiss National Science Foundation, a grant by the Novartis Foundation (to F.P.P.), and a scholarship from the Swiss Federal Commission for Scholarships for Foreign Students (to P.I.).
First Published Online November 10, 2005
Abbreviations: Egr-1, Early growth response-1; LIF, leukemia inhibitory factor; MKP-1, MAPK phosphatase 1; NMDA, N-methyl-D-aspartic acid; OSM, oncostatin M; OSMR, receptor for OSM.
Accepted for publication November 1, 2005.
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