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Departments of Medicine (F.L., J.M.O., N.J.G.W.) and Reproductive Medicine (P.L.M.) and the University of California San Diego Cancer Center (P.L.M., N.J.G.W.), University of California, San Diego, California 92093; and the Medical Research Service (D.A.A., N.J.G.W.), San Diego Veterans Healthcare System, San Diego, California 92161
Address all correspondence and requests for reprints to: Nicholas Webster, Department of Medicine 0673, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0673. E-mail: nwebster{at}ucsd.edu.
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-subunit and a unique, hormone-defining, ß-subunit (1). Analysis of the regulation of gonadotropin subunit gene expression by GnRH in the pituitary has been hampered by the dearth of cell lines that show appropriate regulation. Much of the original work was performed on dispersed primary cultures of pituitary cells or on artificial systems created by the transfection of the GnRH receptor into heterologous cells (2). Although the primary cultures contain about 5–10% mature gonadotropes, they represent a heterogeneous population of cells and are difficult to manipulate in vitro. The heterologous cell systems do show some responses to GnRH, due to the transfected receptor, but do not express the gonadotropin genes. The development of immortalized pituitary cell lines by targeted expression of SV40 large T antigen driven by the common glycoprotein hormone -subunit promoter has greatly furthered our understanding of GnRH signaling (3). In particular, the T3–1 cells are committed to the gonadotrope lineage as they express both the -subunit gene and the GnRH receptor gene, but are considered immature, because they do not express the LHß and FSHß genes (4, 5). These cells have made possible the study of -subunit gene expression as well as the regulation of endogenous GnRH receptors both at the genomic and posttranslational level (6). They have also been useful for analyzing the complex intracellular signaling events that follow GnRH stimulation. However, it has not been possible to study LHß and FSHß regulation in these cells. More recently, other immortalized pituitary cell lines have been developed by utilizing the LHß promoter for targeted expression of SV40 T-antigen in transgenic mice. These cells, the LßT2 and LßT4 cells, express both LHß and FSHß genes (7, 8). Moreover, both genes are regulated by GnRH, as is secretion of LH protein, and the cells are, thus, representative of mature pituitary gonadotropes. The GnRH receptor is a member of the seven-transmembrane, or serpentine, class of receptors that couple to heterotrimeric G proteins (GPCRs). Binding of GnRH to its receptor initiates many signaling cascades similar to other GPCRs. One pathway leads to the activation of the MAPK family (9, 10, 11, 12, 13). MAPK cascades comprise one of the major signaling systems through which cells transduce and integrate diverse intracellular signals. The three subfamilies of the MAPKs are the ERKs, the c-Jun N-terminal kinases (JNK)/stress- activated protein kinases, and the p38 MAPKs (14, 15, 16, 17). The ERKs are strongly activated by polypeptide growth factors and phorbol esters but are weakly activated by environmental stresses such as osmotic or heat shock, UV light, and inhibitors of protein synthesis. In contrast, JNK and p38 MAPKs are strongly activated by cytokines and adverse stimuli but are poorly activated by growth factors.
All MAPKs are activated by phosphorylation on both threonine and tyrosine residues within the motif Thr-Xaa-Tyr (18). The Xaa represents Glu in the ERK subfamily, Pro in the JNK subfamily, and Gly in the p38 MAPK subfamily. Both the threonine and tyrosine residues are phosphorylated by a dual-specificity kinase or MKK. The central residue in the Thr-Xaa-Tyr motif allows for selective activation by different MKKs, such that MEK1 and MEK2 selectively phosphorylate and activate the ERKs; MKK4/SEK1 and MKK7 phosphorylate and activate JNK; and MKK3 and MKK6 phosphorylate and activate p38 MAPK (19, 20, 21, 22, 23). Many studies have shown that the phosphorylation of MAPK on both Thr and Tyr correlates very closely with enzyme activity. Recently, it has been shown that GnRH is capable of activating the MAPKs in whole-cell lysates from primary pituitary cultures (24, 25) and gonadotrope cell lines (26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36).
Upon activation, MAPKs dimerize and translocate to the nucleus (37). Once in the nucleus, the MAPKs phosphorylate and activate nuclear transcription factors involved in DNA synthesis and cell division leading to the induction of immediate-early response genes including c-fos, c-jun, and Egr-1. The fos/jun heterodimer binds to AP-1 sites, and the Egr-1 factor binds to GC-rich elements on various promoters to induce the transcription of downstream genes. Recent evidence indicates that GnRH can stimulate c-fos and Egr-1 expression in primary pituitary cells (38), and T3–1 cells (39, 40, 41), leading to the suggestion that some GnRH transcriptional effects could be mediated by these immediate-early genes. In T3–1 gonadotrope cells, MAPK may be involved in the GnRH regulation of the -subunit gene (29, 30). GnRH also regulates the transcriptional activities of LHß and FSHß chimeric reporter genes in the same cell line, possibly through differential use of PKC/MAPK and Ca2+ pathways (42, 43, 44, 45). However, the T3–1 gonadotrope cell line does not express the LHß gene; therefore, it is not possible to study the regulation of the native promoter. The LßT2 gonadotrope cell expresses both LHß and FSHß genes, so can be used to study the regulation of transfected native promoters (7, 8). Both promoters are induced by GnRH, but the exact signaling pathways involved are not clearly understood. Consequently, we investigated whether GnRH stimulates the activity of the MAPK subfamilies, causes translocation of activated ERK, and/or induces expression of c-fos and LHß proteins in LßT2 cells.
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T3–1, GGH3, and Cos-7/GnRHR cell lines. We tested whether these kinases are activated by GnRH in the LßT2 pituitary gonadotrope cell line. Serum-starved cells were treated with 100 nM GnRH for increasing periods. Whole-cell lysates were immunoblotted for the dually phosphorylated forms of ERK, JNK, and p38. The blots were then stripped and reprobed with antibodies for ERK1/2 protein, JNK2 protein, and p38 protein, respectively, to confirm equivalent sample loading. GnRH stimulated a rapid and very strong increase in ERK phosphorylation that was visible by 1 min (50-fold), reached its peak at 10 min (60-fold), and then declined over 90 min (Fig. 1A
). GnRH also stimulated p38 phosphorylation very strongly with a similar time course reaching 17-fold at 1 min with a maximum 30-fold at 10 min (Fig. 1C). In contrast, GnRH-induced JNK phosphorylation was much weaker, reaching only 10-fold at 30 min (Fig. 1B). Similar amounts of ERK, p38, and JNK2 were loaded in all lanes (bottom panels). A similar time course of ERK, p38, and JNK activation was observed in the related LßT4 gonadotrope cell line (data not shown).
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). GnRH and KCl activated ERK to similar extents, but PMA activation was noticeably stronger than GnRH (2-fold). This indicated that GnRH could potentially signal through either pathway in LßT2 cells.
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). GnRH and PMA caused a similar 2.5-fold increase, but KCl did not cause a significant activation of ras. The lack of a response to calcium elevation may be related to the observation that T3–1 and LßT2 cells express low levels of the calcium-dependent tyrosine kinase Pyk2 that mediates ras activation in other cells (Ref. 49 and our unpublished data).
A panel of pharmacological inhibitors was used to identify the signaling pathways involved in the activation of ERK by GnRH. Serum-starved cells were pretreated for 30 min with the PKC inhibitor bisindolylmaleimide I [BIM-1 (1 µM)], the MEK inhibitor PD98059 (20 µM), the cell-permeable calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester [BAPTA-AM (50 µM)], or the PI3K inhibitor LY294002 (20 µM) and then treated with 100 nM GnRH for 5 min. Whole-cell lysates were immunoblotted for active ERK as before. The MEK inhibitor PD98059 blocked activation of ERK almost completely (>95%) as expected. The PKC inhibitor BIM-1 caused a 40% reduction, indicating that ERK activation is partially mediated by conventional PKC isoforms (Fig. 2C). Chelation of intracellular calcium with BAPTA-AM had no effect, nor did inhibition of PI3K with LY294002. We were particularly intrigued by the lack of effect of calcium chelation, as calcium has been shown to be important in T3–1 cells. To examine the calcium requirement more closely, cells were pretreated with EGTA (3 mM) to chelate extracellular calcium, nimodipine (10 µM) to block L-type calcium channels, or BAPTA-AM (50 µM) to chelate intracellular calcium. Cells were then stimulated with either GnRH, PMA to activate PKC, KCl to depolarize the cell, or ionomycin (10 µM) to elevate calcium. Whole-cell lysates were blotted for dually phosphorylated ERK (Fig. 2D). EGTA caused a 30% reduction in GnRH stimulation, but nimodipine and BAPTA were without effect. PMA stimulation was not altered by any of the agents. The depolarization-induced increase in phospho-ERK with KCl was blocked more than 80% by chelation of extracellular calcium as expected, but was blocked only 50% by nimodipine or BAPTA. Ionomycin- induced ERK phosphorylation was blocked more than 80% by EGTA but not at all by nimodipine or BAPTA, consistent with the passive entry of calcium into the cells with ionomycin. These data suggest that GnRH-induced activation of ERK in LßT2 cells is mediated partially by PKC and may require calcium influx, but not via L-type calcium channels. Depolarization- induced ERK activation was more effectively blocked by EGTA than nimodipine, suggesting that other non- L-type calcium channels may be involved. This is consistent with our finding that the L-type channel agonist Bay-K 8644 is a much weaker activator of ERK than KCl (data not shown).
Inhibition of ERK activation by PD98059 implicated MEK in GnRH signaling. To confirm that GnRH activates ERK via the Raf-MEK pathway, cells were treated with the structurally unrelated MEK inhibitor U0126 that effectively inhibits both MEK1 and MEK2. Pretreatment of LßT2 cells with 1 µM U0126 prevented the activation of ERK by GnRH, PMA, and KCl, showing that all three agonists signal via MEK (Fig. 2E).
GnRH Stimulates Accumulation of Active ERK in the Nuclei of LßT2 Cells
ERKs are a family of protein serine/threonine kinases that transduce signals from the cell surface to the nucleus in response to a variety of extracellular stimuli. ERK1 and 2 have been shown to translocate to the nucleus when phosphorylated, where they activate nuclear transcription factors. We used an immunofluorescent staining technique using antibodies to the dually phosphorylated ERKs to assess the translocation of activated phospho-ERK to the nucleus after GnRH stimulation. LßT2 cells were plated on coverslips, rendered quiescent by serum starvation, and then stimulated with 100 nM GnRH, 100 nM PMA, or 50 mM KCl for 5 min. Cells were stained with the antibody to active ERK followed by a rhodamine-conjugated secondary antibody, counterstained with Hoechst 33258 to highlight the nuclei, and visualized by fluorescence microscopy. In the basal state, very little staining for active phospho-ERK was detected (Fig. 3A, left panels). GnRH stimulation caused the appearance of activated phospho-ERK in the nucleus of many LßT2 cells; similar results were obtained with PMA treatment (Fig. 3A, middle panels). Elevation of calcium with KCl failed to cause nuclear phospho-ERK staining (Fig. 3A, right panels) despite activation of ERK in whole-cell lysates by Western blot (Fig. 2), indicating that an additional signal is required for nuclear localization.
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). GnRH and PMA cause nuclear phospho-ERK staining in approximately 60% of cells. Inhibition of conventional PKC isoforms with BIM-1, or MEK with PD98059, completely blocked both the GnRH- and PMA-stimulated nuclear localization of phospho-ERK. As for the whole-cell immunoblotting, chelation of intracellular calcium with BAPTA-AM, chelation of extracellular calcium with EGTA, blockade of L-type calcium channels with nimodipine, inhibition of PI3K with LY294002, or inhibition of p38MAPK with SB203580 had no effect on nuclear staining for phospho-ERK.
To confirm a role for conventional PKCs in the nuclear localization of activated ERK, LßT2 cells were pretreated with vehicle or the PKC inhibitor BIM-1 (1 µM) for 30 min, and then stimulated with 100 nM GnRH for 5 min. The cells were fractionated into nuclear and cytoplasmic extracts, and equal amounts of protein were immunoblotted with the antibody against the active dually phosphorylated form of ERK (Fig. 3C). GnRH treatment causes the appearance of phospho-ERK in both the cytoplasmic and nuclear fractions. Inhibition of PKC signaling blocks the appearance of phospho-ERK in the nuclear fraction by 90% but has no effect on the cytoplasmic fraction (Fig. 3C). The nuclear accumulation of ERK was also monitored by immunofluorescence using antibodies to ERK2 protein (Fig. 3D). In basal, unstimulated cells most ERK2 staining is observed in the cytoplasm with very little in the nucleus. Upon GnRH stimulation, ERK2 redistributes so that staining is uniform over both the cytoplasm and nucleus. Pretreatment with the PKC inhibitor BIM-1 prevents the redistribution of ERK2 so that the staining is identical with that of basal cells (Fig. 3D).
The complete inhibition of nuclear phospho-ERK staining by inhibition of PKC with BIM-1 was very striking. To confirm this finding, diacylglycerol (DAG)-dependent PKC isoforms were down-regulated by chronic treatment with the phorbol ester PMA. LßT2 cells were pretreated with 1 µM PMA or vehicle control for 16 h. Whole-cell extracts were immunoblotted for PKC isoforms. Blots were stripped and reblotted for ERK2 to control for protein loading. The -, ß-, 
-, 
-, 
-, and 
-isoforms of PKC are expressed in LßT2 cells, and chronic PMA treatment reduced expression of the -, ß-, -, and -isoforms by more than 90% and the -isoform completely, but had no effect on the atypical DAG-independent -isoform (Fig. 4A). PMA-down-regulated cells were then stimulated with 100 nM GnRH for 5 min, and clarified cell lysates were immunoblotted for activation of ERK. PKC down-regulation reduces GnRH-stimulated ERK activation by more than 75% (Fig. 4B). The inhibition of ERK is somewhat greater than that observed with BIM-1, presumably because PMA down-regulates both the conventional PKCs and the novel PKC isoforms and that are less sensitive to BIM-1 inhibition. To confirm that PKC down-regulation blocks nuclear phospho-ERK staining, PMA-down-regulated cells were stimulated with 100 nM GnRH or 100 nM PMA for 5 min, and then fixed and stained for phospho-ERK as above. Unlike acute PMA treatment, chronic treatment with 1 µM PMA does not lead to nuclear localization of active ERK. Chronic PMA treatment, however, completely abolishes the subsequent acute GnRH and PMA-stimulated nuclear staining of phospho-ERK (Fig. 4C). These data strongly support the conclusion that GnRH activation of ERK is dependent on conventional DAG-sensitive PKCs in LßT2 cells.
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T3–1 cells, and Egr-1 in LßT2 cells. Therefore, we examined the effect of GnRH, PMA, or KCl on c-fos protein expression. Serum-starved LßT2 cells were stimulated with 100 nM GnRH, 100 nM PMA, or 50 mM KCl for 60 min. Whole-cell lysates were then immunoblotted for c-fos (Fig. 5A). All three agonists were able to stimulate c-fos protein expression (13-fold for GnRH, 9-fold for PMA, and 6-fold for KCl).
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). Inhibition of MEK with PD98059 causes a 70% reduction in c-fos levels. Inhibition of PKC with BIM-1, which completely blocks the nuclear staining of phospho-ERK, surprisingly does not block the induction of c-fos by GnRH. Conversely, chelation of intracellular calcium with BAPTA-AM, or extracellular calcium with EGTA, neither of which had any effect on ERK activation, completely blocked GnRH-induced c-fos expression (>90% for BAPTA, >80% for EGTA). As for MAPK, inhibition of PI3K with LY294002, or p38 MAPK with SB203580, had no effect on induction of c-fos.
To confirm the immunoblotting results, we used single-cell immunofluorescent staining to examine the nuclear expression of c-fos. LßT2 cells were plated on coverslips, rendered quiescent by serum starvation, and then stimulated with 100 nM GnRH, 100 nM PMA, or 50 mM KCl for 60 min. Cells were fixed and stained for c-fos, then counterstained for DNA, and visualized by fluorescence microscopy. Cells that were positive for nuclear c-fos staining were counted. Very few cells (<5%) stained for c-fos in the basal unstimulated state (Fig. 6A). GnRH and PMA were both able to cause c-fos staining in the majority (>70%) of cell nuclei. KCl was also able to induce c-fos staining but to a lesser extent (
50%).
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). Inhibition of PKC with BIM-1 again did not inhibit the GnRH stimulation of c-fos although the PMA effect was abrogated completely. BIM-1 also had no effect on the ability of KCl to stimulate c-fos, showing that this calcium effect is not mediated by PKC. Inhibition of MEK with PD98059 or U0126 blocked the effects of either PKC or calcium on c-fos expression, but only caused a partial (65%) inhibition of the GnRH effect. This suggests that GnRH has effects on c-fos expression that, unlike effects on MAPK, are independent of MEK. Chelation of intracellular calcium with BAPTA-AM completely inhibited c-fos staining with all three agonists. Inhibition of L-type calcium channels with nimodipine or chelation of extracellular calcium with EGTA prevented the KCl-induced increase in c-fos as expected, had no effect on the PMA induction, and reduced the GnRH effect by 50%. Inhibition of PI3K or p38MAPK had no effect on c-fos induction by GnRH.
To confirm the effect of the PKC inhibitor BIM-1 in the induction of c-fos, LßT2 cells were pretreated with 1 µM PMA for 16 h to down-regulate the DAG-sensitive isoforms of PKC, and then exposed to 100 nM GnRH or 100 nM PMA for 60 min. As expected, PMA-induced c-fos expression was completely abolished by PKC depletion, whereas GnRH still induced c-fos expression in 40% of the PKC-depleted cells (Fig. 6C). Because the number of positive cells after GnRH stimulation is reduced by 30%, signaling by DAG-dependent PKC isoforms that are not BIM-1 sensitive may be required for full induction. The concentration of BIM used in these experiments should efficiently inhibit the conventional -, ß-, and 
-isoforms of PKC, but not the novel isoforms , , and that are present in these cells. This observation suggests that novel, but not conventional PKC isoforms may play an important role in regulating GnRH-induced c-fos expression in LßT2 cells.
GnRH Induces LHß Protein via MEK and Calcium in LßT2 Cells
The LßT2 cells express both LHß and FSHß mRNAs and secrete mature LH in response to GnRH. The LHß mRNA is also induced by both GnRH and steroid hormones (8). We used immunofluorescence to examine LHß protein expression in these cells. LßT2 cells were plated on coverslips, rendered quiescent by serum starvation, and stimulated overnight with 100 nM GnRH, 100 nM PMA, or 50 mM KCl. Cells were fixed and stained for LHß, and then counterstained for DNA and visualized by fluorescence microscopy (Fig. 7A). LHß staining is observed in the perinuclear region consistent with the localization of the protein in secretory vesicles. Basal cells show staining in 18% of cells. Stimulation with GnRH, PMA, or KCl doubles the number of LHß-positive cells. A time course for GnRH induction of LHß protein was performed (Fig. 7B). A single dose of 100 nM GnRH was administered for 1, 2, 4, 8, or 24 h, and the number of LHß positive cells was counted. An increase in the number of LHß-positive cells is apparent by 2 h and reaches a maximum at 8 h, which is maintained for up to 24 h. Thus, protein expression is induced very rapidly after GnRH stimulation. A GnRH dose-response curve was then determined. Cells were treated with increasing doses of GnRH for 16 h and then fixed and stained for LHß (Fig. 7C). An increase in the number of LHß-positive cells is first apparent at 1 nM GnRH; the ED50 for the induction of LHß is approximately 3 nM.
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). Inhibition of MEK with PD98059 or U0126 blocked the increase in LHß- positive cells with all three agonists. Inhibition of PKC with BIM-1 blocked only the increase with PMA, but not GnRH or KCl. Blockade of L-type calcium channels with nimodipine prevented the GnRH or KCl-stimulated increase in LHß-positive cells but had no effect on the PMA stimulation. Chelation of intracellular calcium with BAPTA-AM or extracellular calcium with EGTA, as used in the earlier experiments, was toxic to the cells over the extended period required for induction of LHß protein and caused the cells to detach from the coverslip (data not shown). We were also unable to test down-regulation of PKC, as overnight treatment with PMA alone induced LHß expression. Inhibition of PI3K or p38MAPK did not prevent the induction of LHß protein by GnRH. |
DISCUSSION |
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T3–1 and LßT2 cells, and p38MAPK in T3–1 cells, have been published previously (24, 25, 26, 27, 28, 29, 30, 31). We show here that activation of ERK by GnRH in LßT2 cells is partially blocked by inhibition of conventional PKC signaling, either using BIM-I or by down-regulation of PKC with chronic PMA treatment, in agreement with previous reports (30, 31, 34, 49, 50). PKC signaling may activate the ERK pathway at two potential points. We demonstrate here that phorbol esters can activate ras in LßT2 cells. Phorbol esters can also activate Raf in the absence of ras activation by direct phosphorylation (46, 47, 48). Phosphorylation of Ser338 or Raf-1 may be mediated by the novel isoforms PKC and - that cause prolonged activation of ERK (51, 52, 53). The ras protein may still be required to target Raf to the membrane, however, to allow phosphorylation by PKC (54). Ser338 is also the target of the kinases Pak-1 and Pak-3, which are activated downstream of PI3K and Rac and cause activation of ERK (55, 56). Phosphorylation of Ser338 by novel PKCs may explain why inhibition of PKC with BIM-1 reduces ERK activation by 40% but PMA down-regulation reduces ERK activation by 75%. The novel PKCs are less sensitive to BIM-1 than conventional PKCs but are efficiently down-regulated by chronic PMA.
Calcium elevations have been shown to activate ERK in a number of cells, but the mechanism is unclear. In PC12 cells, calcium elevation induced by depolarization can activate both the classical ras pathway by activating either the epidermal growth factor receptor, Pyk2, or RasGRF, and a novel calmodulin-dependent pathway activating MEK in a Raf-independent manner (57). We did not find evidence for ras activation with depolarization of LßT2 cells but ERK is activated strongly. GnRH has been shown to cause both release of intracellular calcium and influx of extracellular calcium in LßT2 cells. Is either of these calcium signals important for ERK activation? Preventing calcium influx with EGTA causes only a very modest (30%) reduction in ERK activation by GnRH, and chelation of intracellular calcium with BAPTA-AM or blockade of L-type calcium channels with nimodipine has no effect on ERK activation. This is in contradiction to recent reports from two groups. Mulvaney and co-workers (32, 33) reported that inhibition of the initial calcium transient in T3–1 cells using BAPTA-AM or thapsigargin does not inhibit GnRH-stimulated ERK activation, in agreement with our findings in LßT2 cells. However, they found that sustained calcium elevation via influx through voltage-gated calcium channels is necessary for ERK activation in both T3–1 and primary pituitary cells, as it is almost completely blocked by EGTA or the L-type channel blocker nifedipine (33). Our results in LßT2 cells differ in that EGTA has only a small effect, and the channel blocker nimodipine has no effect on ERK activation. This may indicate an important difference between the immortalized LßT2 cell line and primary gonadotrope cells. Interestingly, Mulvaney and associates (33) observed that depolarization of the cell with potassium did not activate ERK despite sustained calcium elevation, but activation of L-type calcium channels with Bay-K 8644 activated ERK very strongly in the presence of a nearly identical elevation of calcium. They suggested that either depolarization does not activate the same channels or that depolarization and Bay-K 8644 differ in the mechanism of activation. In contrast, we observed that depolarization is a strong activator of ERK in LßT2 cells and that Bay-K 8644 is a very weak activator. This would be consistent with L-type channels playing a minor role in signaling to ERK in this cell type. More importantly, Yokoi et al. (36) reported that GnRH activation of ERK in LßT2 cells, the same cells used for our study, is dependent on calcium as it is blocked completely by EGTA, BAPTA-AM, or nifedipine. Why do our results in the same cells differ? The previous study measured ERK activation by immunoprecipitation with an antibody to ERK1 followed by an in vitro kinase assay using myelin basic protein as substrate. ERK2 is the major isoform in the LßT2 cells and it’s possible that the antibody used didn’t recognize this isoform, particularly as no controls were shown to demonstrate efficient immunoprecipitation. The ERKs have also been found in large signaling complexes assembled on scaffold proteins, which may also interfere with antibody recognition. In the present study, activation of ERK is measured by antibodies to the dually phosphorylated form of the enzyme on whole-cell lysates and thus would not be affected by the presence of large signaling complexes.
Activated ERK translocates to the nucleus in many cells, where it phosphorylates transcription factors of the ternary complex factor (Elk/SAP/ets) and nuclear receptor families. The mechanism of nuclear localization is poorly understood. Unphosphorylated inactive ERK is monomeric and is small enough to enter the nucleus by passive diffusion (37). Monomeric ERK is not retained in the nucleus, however, but is exported rapidly due to the presence of a nuclear export signal. Activation of ERK by phosphorylation causes dimerization, kinase activation, and accumulation in the nucleus. The ERK dimer is too large to enter the nucleus passively but is actively transported and retained in the nucleus. ERK kinase activity is not required for transport, as kinase-defective mutants are retained in the nucleus to the same extent as wild type (37). Import is via a Ran-dependent process, and ERK can interact with components of the nuclear pore complex (58). Retention in the nucleus requires dimerization, as mutant ERKs that fail to dimerize when activated do not accumulate in the nucleus. ERKs do contain a putative nuclear export sequence in the dimer interface that is hidden in the dimeric ERK. This is not the only mechanism, however, as mutation of this sequence does not result in nuclear retention of inactive ERK. The mechanism of transport of the dimerized ERK is not known. It may be that the nuclear localization sequence is intrinsic to dimeric ERK, or ERK may piggy-back into the nucleus in association with other proteins, or that ERK is retained in the cytoplasm by a scaffold protein.
We show here that GnRH or PMA stimulation of LßT2 cells causes an increase in phospho-ERK immunofluorescence that is localized exclusively to the nucleus. This nuclear staining is dependent on MEK and PKC. Depolarization of the cells with KCl does not lead to nuclear fluorescence despite strong activation of ERK by immunoblot, suggesting that calcium activates ERK by a different pathway. Inhibition of PKC only causes a partial reduction in ERK activation by immunoblot, but entirely eliminates nuclear staining and nuclear accumulation by subcellular fractionation. Thus, it appears that the nuclear accumulation of phospho-ERK requires a PKC signal that is distinct from that leading to activation. The sensitivity of the nuclear localization to the inhibitor BIM-1 might suggest that this signal is mediated by conventional PKC isoforms. Even in the presence of normal PKC signaling, the majority of phosphorylated ERK is present in the cytoplasm by fractionation after GnRH stimulation, but no cytoplasmic phospho-ERK immunofluorescence is observed under any conditions tested. We believe that most of the ERK is sequestered in the cytoplasm in large signaling complexes. Many components of the MAPK cascades are coordinated by scaffold proteins such as JIP1, MP-1, MEKK1, and ß-arrestin (59, 60, 61). The most likely explanation is that even though ERK may be phosphorylated in the cell, it is still bound in a signaling complex preventing the recognition of the epitope. Phosphorylation of a component of the complex by PKC may cause release of active ERK, allowing dimerization and nuclear translocation. We are currently investigating whether ERK is found associated with such a signaling complex and whether translocation of ERK to the nucleus requires a PKC-dependent signal.
We also examined the regulation of four known GnRH target genes, the immediate-early gene c-fos and the LHß, FSHß, and -subunit genes. The laboratory of Marshall and associates (62) has demonstrated that pre-mRNA transcripts of LHß and FSHß in primary pituitary cells correlate very well with serum gonadotropin levels. If a causal relationship exists between transcription and secretion, which may or may not be the case, it would imply that changes in the expression of the ß-subunit proteins also correlate with circulating hormone levels. Expression of both c-fos and LHß proteins is induced by treatment with GnRH, or with PMA to activate PKC, or by depolarization with KCl. The -subunit protein is detected in more than 50% of LßT2 cells in the basal state and does not increase with GnRH treatment (data not shown). This is consistent with the lack of induction of the -subunit mRNA by Northern blot in this cell line (8). We were unable to detect expression of the FSHß protein even after GnRH treatment (data not shown), as the mRNA is expressed at extremely low levels (63). Inhibition of MEK reduced the induction of both c-fos and LHß proteins by GnRH, showing that both genes are induced partially via the Raf-MEK-ERK cascade. Inhibition of PKC with BIM-1 had no effect on the induction of either gene by GnRH, yet blocked the nuclear localization of active ERK. This is in contrast to T3–1 cells where activation of MAPK correlated with induction of c-fos. Down-regulation of PKC, on the other hand, reduced the GnRH induction of c-fos, which may indicate the involvement of novel PKC isoforms, which are less sensitive to BIM-1 but have been shown to activate Raf directly and cause prolonged activation of ERK (53). Chelation of intracellular calcium had no effect on ERK activation but surprisingly eliminated the induction of c-fos. On this point the induction of c-fos by GPCRs or tyrosine kinase receptors differs, as chelation of intracellular calcium with BAPTA-AM prolongs the activation of ERK by epidermal growth factor (EGF) and enhances c-fos induction in mouse embryonic fibroblasts (64). Blockade of L-type calcium channels or chelation of extracellular calcium impaired the induction of c-fos by GnRH and prevented the induction of LHß, suggesting that influx of calcium as well as intracellular calcium is important. The pattern of inhibition was similar with the c-fos and LHß genes, which may indicate that GnRH signals via common calcium-dependent and MEK-dependent mechanisms to induce both genes.
The MEK dependence is noteworthy for a number of reasons. First, the MEK inhibitor implicates ERK in the induction of both genes. However, we have defined conditions in which activated ERK is excluded from the nucleus, arguing against the direct activation of nuclear transcription factors by ERK. One possible explanation is that activation of both c-fos and LHß genes is ERK dependent but via an intermediary kinase. A number of kinases including Rsk1, Rsk2, and Mnk1 can be activated by ERK. These kinases have also been shown to translocate to the nucleus upon activation. It is possible that one of these kinases is present in the signaling complex with MEK and ERK, is released upon activation by ERK, and translocates to the nucleus. Alternatively, the target transcription factor may be resident in the cytoplasm in quiescent cells and may translocate to the nucleus after phosphorylation by ERK, analogous to the nuclear translocation of the Smad (similar to mothers against decapentaplegic) and signal transducer and activator of transcription (Stat) transcription factors. Second, inhibition of MEK by either PD98059 or U0126 did not eliminate c-fos induction completely, indicating a parallel pathway signaling to the c-fos promoter. This may be via direct cdc42/Rac/Pak1 activation of the serum-response factor in the absence of ERK signaling that has been reported by Hill and Treisman (65), although additional studies will be necessary to investigate this non-MEK pathway.
In conclusion, we have shown that GnRH activates all three subfamilies of MAPKs to differing degrees and have identified a novel PKC-dependent translocation of activated ERK to the nucleus in LßT2 cells. We also found that expression of the c-fos and LHß proteins are coordinately regulated by GnRH via calcium and MEK-dependent pathways. This may have important implications for GnRH-regulated gene expression in gonadotropes, and studies are underway to determine whether other genes are under coordinate regulation in the LßT2 cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cell Culture
LßT2 cells were maintained in monolayer cultures in DMEM supplemented with 10% FBS and antibiotics in humidified 10% CO2 at 37 C. Cells were starved overnight in serum-free DMEM and then pretreated with inhibitors PD98059 (20 µM), BIM-1 (1 µM), BAPTA-AM (50 µM), LY 294002 (20 µM), U0126 (1 µM), and SB203580 (10 µM) for 30 min at 37 C. Agonists GnRH (100 nM), PMA (100 nM), KCl (50 mM), or PDGF (20 ng/ml) were added for the appropriate period.
Immunostaining
For c-fos and LHß staining, LßT2 cells were plated on 10-mm acid-washed glass coverslips and stimulated with agonists and inhibitors as above. Cells were washed with PBS and fixed with 3.7% formaldehyde in PBS for 20 min at room temperature. After two washes in PBS, the cells were permeabilized and blocked in PBS containing 5% BSA and 0.5% Nonidet P-40 for 10 min. Coverslips were incubated with the rabbit anti-c-fos antibody (1:400 dilution) or rabbit anti-LHß antibody (1:1,200 dilution) for 60 min at room temperature, washed once in PBS, and then incubated with TRITC-conjugated antirabbit IgG antibody (1:100 dilution) in PBS with 5% BSA and 0.5% Nonidet P-40 for 30 min at room temperature. After a wash with PBS, coverslips were incubated with a DNA intercalating dye (Hoechst 33258, Sigma) diluted 1:250 for 60 min to stain nuclei. Finally, the coverslips were extensively washed with PBS, rinsed with water, and mounted in PBS containing 15% gelvatol (polyvinyl alcohol), 33% glycerol, and 0.1% sodium azide.
For phospho-ERK staining, cells were washed with PBS, fixed in 3.7% formaldehyde in PBS as above, and then washed with Tris-buffered saline (TBS)-Triton (50 mM Tris-HCL, pH 7.4, 150 mM NaCl, and 0.1% Triton X-100). The cells were permeabilized in 100% methanol at -20 C for 10 min, washed with TBS-Triton, and then blocked with 5% normal horse serum in TBS-Triton for 60 min at room temperature to reduce nonspecific staining. Coverslips were incubated with the anti-ACTIVE MAPK antibody at a 1:400 dilution in 5% BSA in TBS-Triton overnight at 4 C. The cells were washed with 0.1% BSA in TBS-Triton and then incubated with a TRITC-conjugated antirabbit IgG antibody at a 1:100 dilution in 3% BSA in TBS-Triton for 60 min at room temperature. Coverslips were washed with TBS-Triton and incubated with Hoechst 33258 dye (1:250 dilution) in TBS-Triton for 60 min at room temperature. The coverslips were washed and mounted as described above. Staining was visualized with an Axiophot fluorescence microscope (Carl Zeiss, Thornwood, NY) and photographed using the ISEE imaging system (Inovision, Raleigh, NC).
Western Blotting
LßT2 cells were grown to confluence in six-well plates, washed once with PBS, and incubated in serum-free DMEM overnight. For inhibition experiments, the cells were pretreated with PD98059 (20 µM), BIM-1 (1 µM), BAPTA (50 µM), LY294002 (20 µM), SB203580 (10 µM), or nimodipine (10 µM) for 30 min, or EGTA (3 mM) for 15 min at 37 C. Cells were stimulated with 100 nM GnRH, 100 nM PMA, or 50 mM KCl for various periods of time at 37 C. Thereafter, cells were washed with ice-cold PBS, and then lysed on ice in SDS sample buffer (50 mM Tris, 5% glycerol, 2% SDS, 0.005% bromophenol blue, 84 mM dithiothreitol, 100 mM sodium fluoride, 10 mM sodium pyrophosphate, and 2 mM sodium orthovanadate, pH 6.8), boiled for 5 min to denature proteins, and sonicated for 5 min to shear the chromosomal DNA. Equal volumes (30–40 µl) of these lysates were separated by SDS-PAGE on 10% gels, electrotransferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P, Millipore Corp., Bedford, MA). The membranes were blocked with 5% BSA (for phospho-MAPK, -JNK, and -p38) or 5% nonfat dried milk (for c-fos and ras) in TBS-Tween (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween-20). Blots were incubated with primary antibodies in blocking buffer for 60 min at room temperature, and then incubated with horseradish peroxidase-linked secondary antibodies followed by chemiluminescent detection. Anti-ACTIVE MAPK or anti-ACTIVE JNK antibodies were used at a 1:2,500 dilution; anti-phospho-p38, anti-ERK, anti-JNK2, anti-p38, anti-ras, or anti-c-fos antibodies were used at a dilution of 1:1,000. Antibodies to individual isoforms of PKC were used at a dilution of 1:1,000. For the phospho-specific antibodies, the PVDF membranes were immediately stripped by placing the membrane in stripping buffer (0.5 M NaCl and 0.5 M acetic acid) for 10 min at room temperature. The membrane was then washed once for 10 min in TBS-Tween, reblocked, and blotted with antibodies to the unphosphorylated form of the enzyme to control for equal protein loading.
Ras-GTP Pull-Down Assay
LßT2 cells were plated in 10-cm dishes and grown until confluence. Cells were serum starved overnight and then stimulated with GnRH (100 nM), PMA (100 nM), PDGF (20 ng/ml), or KCl (50 mM) for 5 min at 37 C. Cells were washed with ice-cold PBS, lysed in RIPA buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 10% glycerol, 25 mM NaF, 10 mM MgCl2, 1 mM EDTA, 1 mM sodium orthovanadate, 10 µg/ml leupeptin, 10 µg/ml aprotinin) and clarified by centrifugation. A GST-fusion protein (10 µg) containing the ras-binding domain of Raf was added and incubated on ice for 60 min. This fusion protein binds the active GTP-bound form of ras. The fusion protein was precipitated on glutathione Sepharose beads and washed three times in binding buffer. The pellets were boiled in SDS-sample buffer, and the proteins were separated on 12% SDS polyacrylamide gels, transferred to PVDF membranes, and immunoblotted with a pan-ras antibody as above.
Isolation of Nuclear and Cytoplasmic Fractions
LßT2 cells were plated in 10-cm dishes and grown until confluence. Cells were serum starved overnight, then pretreated with vehicle or 1 µM BIM-1 for 30 min, and then stimulated with 100 nM GnRH for 5 min at 37 C. Cells were washed with cold PBS and harvested, and nuclei were prepared according to a published procedure (65). Briefly, cells were resuspended in nuclear isolation buffer [10 mM Tris-HCl (pH 7.8), 1% NP-40, 10 mM mercaptoethanol, 0.5 mM phenylmethylsulfonylfluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor, 15 µg/ml calpain inhibitor 1 and 2, 2 mM Na3VO4, and 5 mM NaF] for 3 min on ice. An equal volume of water was added, and the cells were allowed to swell for 3 min. The cells were sheared by eight passages through a 22-gauge needle. Nuclei were recovered by centrifugation at 400 x g at 4 C for 6 min and then washed once in 10 mM Tris-HCl (pH 7.8), 2 mM MgCl2 plus protease and phosphatase inhibitors as before. The supernatant was used as the cytoplasmic fraction. The nuclei were solubilized in 25 mM HEPES (pH 7.5), 10 mM MgCl2, 5 mM EDTA, 5 mM EGTA, 50 mM NaCl, 50 mM NaF, 30 mM sodium pyrophosphate, 10% glycerol, 1% Triton X-100 plus protease inhibitors as above for 20 min at 4 C with shaking. Cell debris was removed by centrifugation at 12,000 x g at 4 C for 5 min, and the supernatant was used as the nuclear fraction. Aliquots of nuclear and cytoplasmic fractions containing equal amounts of protein were resolved by SDS-PAGE, transferred to PVDF membranes, and immunoblotted with the Anti-ACTIVE ERK antibodies as before.
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FOOTNOTES |
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Abbreviations: BAPTA-AM, 1,2-bis(2-Aminophenoxy)ethane- N,N,N',N'-tetraacetic acid acetoxymethyl ester; BIM-1, bisindolylmaleimide; DAG, diacylglycerol; GPCR, G protein-coupled receptor; GST, glutathione-S-transferase; JNK, c-Jun N-terminal kinase; MEK, MAPK kinase; MKK, dual-specificity kinase; PDGF, platelet-derived growth factor; PMA, phorbol-12-myristate-13-acetate; PVDF, polyvinylidene difluoride; TBS, Tris-buffered saline; TRITC, tetramethylrhodamine isothiocyanate.
Received for publication July 13, 2001. Accepted for publication November 27, 2001.
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D. Harris, D. Chuderland, D. Bonfil, S. Kraus, R. Seger, and Z. Naor Extracellular Signal-Regulated Kinase and c-Src, But Not Jun N-Terminal Kinase, Are Involved in Basal and Gonadotropin-Releasing Hormone-Stimulated Activity of the Glycoprotein Hormone {alpha}-Subunit Promoter Endocrinology, February 1, 2003; 144(2): 612 - 622. [Abstract] [Full Text] [PDF]
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B. H. Shah, J.-W. Soh, and K. J. Catt Dependence of Gonadotropin-releasing Hormone-induced Neuronal MAPK Signaling on Epidermal Growth Factor Receptor Transactivation J. Biol. Chem., January 24, 2003; 278(5): 2866 - 2875. [Abstract] [Full Text] [PDF]
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J. G. Ellis IV, M. Davila, and R. Chakrabarti Potential Involvement of Extracellular Signal-regulated Kinase 1 and 2 in Encystation of a Primitive Eukaryote, Giardia lamblia. STAGE-SPECIFIC ACTIVATION AND INTRACELLULAR LOCALIZATION J. Biol. Chem., January 10, 2003; 278(3): 1936 - 1945. [Abstract] [Full Text] [PDF]
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F. Liu, I. Usui, L. G. Evans, D. A. Austin, P. L. Mellon, J. M. Olefsky, and N. J. G. Webster Involvement of Both Gq/11 and Gs Proteins in Gonadotropin-releasing Hormone Receptor-mediated Signaling in Lbeta T2 Cells J. Biol. Chem., August 23, 2002; 277(35): 32099 - 32108. [Abstract] [Full Text] [PDF]
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