Gonadotropin-Releasing Hormone Induction of Extracellular-Signal Regulated Kinase Is Blocked by Inhibition of Calmodulin
Mark S. Roberson,
Stuart P. Bliss,
Jianjun Xie,
Amy M. Navratil,
Todd A. Farmerie,
Michael W. Wolfe and
Colin M. Clay
Department of Biomedical Sciences (M.S.R., S.P.B., J.X.), Cornell University, Ithaca, New York 14853; Department of Biomedical Sciences (A.M.N., T.A.F., C.M.C.), Colorado State University, Fort Collins, Colorado 80523; and Department of Molecular and Integrative Physiology (M.W.W.), University of Kansas Medical Center, Kansas City, Kansas 66160
Address all correspondence and requests for reprints to: Mark S. Roberson Ph.D., T3-004d Veterinary Research Tower, Department of Biomedical Sciences, Cornell University, Ithaca, New York 14853. E-mail: msr14{at}cornell.edu.
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ABSTRACT
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Our previous studies demonstrate that GnRH-induced ERK activation required influx of extracellular Ca2+ in
T3-1 and rat pituitary cells. In the present studies, we examined the hypothesis that calmodulin (Cam) plays a fundamental role in mediating the effects of Ca2+ on ERK activation. Cam inhibition using W7 was sufficient to block GnRH-induced reporter gene activity for the c-Fos, murine glycoprotein hormone
-subunit, and MAPK phosphatase (MKP)-2 promoters, all shown to require ERK activation. Inhibition of Cam (using a dominant negative) was sufficient to block GnRH-induced ERK but not c-Jun N-terminal kinase activity activation. The Cam-dependent protein kinase (CamK) II inhibitor KN62 did not recapitulate these findings. GnRH-induced phosphorylation of MAPK/ERK kinase 1 and c-Raf kinase was blocked by Cam inhibition, whereas activity of phospholipase C was unaffected, suggesting that Ca2+/Cam modulation of the ERK cascade potentially at the level of c-Raf kinase. Enrichment of Cam-interacting proteins using a Cam agarose column revealed that c-Raf kinase forms a complex with Cam. Reconstitution studies reveal that recombinant c-Raf kinase can associate directly with Cam in a Ca2+-dependent manner and this interaction is reduced in vitro by addition of W7. Cam was localized in lipid rafts consistent with the formation of a Ca2+-sensitive signaling platform including the GnRH receptor and c-Raf kinase. These data support the conclusion that Cam may have a critical role as a Ca2+ sensor in specifically linking Ca2+ flux with ERK activation within the GnRH signaling pathway.
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INTRODUCTION
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GnRH IS A HYPOTHALAMIC decapeptide that is required for normal production and secretion of pituitary gonadotropins. As such, GnRH is required for normal reproduction and fertility in mammals. The GnRH receptor is a plasma membrane-associated heptahelical receptor capable of orchestrating the activation of numerous interconnecting signaling pathways via activation of G
q, phospholipase C, and the elaboration of inositol 1,4,5 trisphosphate (IP3) and diacylglycerol (1, 2). The second messenger IP3 is critical for release of Ca2+ from internal stores (such as the endoplasmic reticulum), whereas diacylglycerol is required for activation of protein kinase C (PKC) isozymes. In addition to changes in IP3-dependent Ca2+, GnRH receptor activation also induces Ca2+ influx from the extracellular space via L-type voltage-gated Ca2+ channels (VGCC; Refs.3, 4, 5, 6, 7). Increased PKC isozyme activity along with changes in Ca2+ concentrations within pituitary cells is central to the activation of several of the MAPK family members. Our studies supported the conclusion that GnRH-induced activation of the ERK cascade requires PKC activation along with specific influx of extracellular Ca2+ through VGCCs in both the gonadotrope cell model
T3-1 and in primary cultures of rat pituitary cells (5). GnRH-responsive, ERK-dependent genes such as c-Fos and the MAPK dual-specificity phosphatase MAPK phosphatase (MKP)-2 are also affected by inhibition of VGCC Ca2+ and consequently the inhibition of the ERK pathway (5, 6, 7). In contrast to inhibition of VGCC Ca2+, chelation of intracellular Ca2+ using compounds such as Bapta-AM were not sufficient to block GnRH-induced ERK phosphorylation but did reduce GnRH-induced c-Jun N-terminal kinase activity (6). These studies supported speculation that influx of VGCC Ca2+ to create local or compartmentalized increases in cell Ca2+ may be requisite for the effects of VGCC inhibition on the ERK pathway in pituitary cells. Consistent with this hypothesis, we have recently demonstrated that the GnRH receptor, G
q and c-Raf kinase are all compartmentalized to specific membrane microdomains (lipid rafts) in a constitutive manner (8). Regulation of GnRH receptor localization to these flotillin 1-positive rafts was abolished by cholesterol perturbation of the plasma membrane and repletion of cholesterol to membranes resulted in a restoration of the GnRH receptor to lipid rafts. Moreover, GnRH signaling to the ERK pathway was blocked by cholesterol depletion and rescued by cholesterol repletion. Our hypothesis is that membrane-associated Ca2+ influx through VGCCs may contribute regionally to a putative signaling platform(s) containing (minimally) the GnRH receptor, G
q, and c-Raf kinase in the plasma membrane associated with raft compartments. Ca2+-dependent initiation of ERK signaling by GnRH may be associated with this putative membrane compartmentalization of key signaling molecules within rafts. What remains unclear in the context of this mechanism is how Ca2+ is recognized and at what level within the signaling pathway Ca2+ mediates activation of the ERK pathway.
Calmodulin (Cam) is the prototypical Ca2+-binding protein and serves important roles as a Ca2+ sensor in a number of different intracellular signaling scenarios. The molecular structure of Cam includes the presence of four Ca2+ binding sites within two globular domains tethered via an
helix (reviewed in Ref.9). Upon Ca2+ binding, structural conformation of Cam is altered exposing domains for association with Cam-binding proteins. Cam has been linked to activation of signaling through Ca2+-dependent molecules such as Cam-dependent protein kinases (CamK) and cAMP response element binding protein (CREB) phosphorylation, myosin light chain kinase, and the regulation of Ca2+-sensitive adrenergic stimulation of smooth muscle contractility, Cam-dependent adenylyl cyclase, and phosphodiesterase activities and regulation of calcineurin (Cam-dependent protein phosphatase 2B) activity (9). Cam has also been associated with regulation of Ca2+ flux through VGCCs where Cam plays a role in the detection of Ca2+ influx and facilitates the inactivation of the L-type channel (10, 11). Early studies by Conn et al. (12) suggested that Cam may serve as a Ca2+ sensor in gonadotropes whereby GnRH action induced changes in the subcellular localization of Cam to the plasma membrane. Collectively, these studies articulate an important role for Cam as a Ca2+ sensor, modulating Ca2+-dependent signaling as a consequence. The present studies demonstrate that pharmacological disruption of Cam resulted in alteration in GnRH-mediated gene regulation of primary gene targets such as c-Fos, glycoprotein hormone
-subunit, and MKP-2. A common mechanism among the gene promoters sensitive to Cam inhibition was a shared requirement for GnRH-induced ERK activation. Cam disruption via several methodologies resulted in inhibition of GnRH-induced ERK signaling. A key signaling intermediate within the ERK cascade, c-Raf kinase, was capable of forming a complex with Cam in a Ca2+-dependent manner suggesting that Ca2+ may impact ERK signaling at the level of c-Raf kinase. Importantly, Cam appears to partition into lipid rafts along with c-Raf kinase and the GnRH receptor consistent with a Ca2+-sensitive signaling platform within this membrane compartment.
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RESULTS
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We have shown previously that influx of extracellular Ca2+ through VGCCs was essential for stimulation of the ERK cascade by GnRH in the gonadotrope cell model,
T3-1, and in rat pituitary cells in primary culture (5). These studies supported the prediction that Cam may be an important Ca2+ sensor and inhibition of Cam may impact expression of ERK-dependent genes such as c-Fos, the murine glycoprotein hormone
-subunit, and the promoter regulating the expression of MKP-2, a dual-specificity phosphatase putatively involved in regulating the duration of ERK signaling in gonadotropes (7). To test this hypothesis,
T3-1 cells were transfected with reporter genes for c-Fos,
-subunit, and the GnRH receptor promoter coupled to luciferase. Transfected cells were pretreated with either control solution or the Cam inhibitor W7, 30 min before treatment with the GnRH analog, buserelin (GnRHa). Cells were harvested 6 h after GnRHa treatment and assayed for luciferase activity. Pretreatment with W7 inhibited GnRH-induced luciferase activity from the c-Fos and
-subunit promoters but not from the GnRH-receptor promoter (Fig. 1
). The latter has been shown to be mediated by GnRH-induced c-Jun N-terminal kinase (JNK) activity and not ERK activity in
T3-1 cells (13). To examine the role of Cam inhibition on gene transcription in more detail, the MKP-2 promoter and regulation of early growth response protein 1 (Egr-1) was used as a transcriptional model (Fig. 2
). Consistent with observations on the c-Fos and
-subunit promoters, pretreatment of transfected cells with W7 reduced responsiveness of the MKP-2 promoter to GnRHa (Fig. 2A
). MKP-2 promoter activity is enhanced by GnRH-induced Egr-1 up-regulation and transcription-stimulating activity (14). Pretreatment with W7 caused a marked dose-dependent reduction in Egr-1 protein up-regulation induced by GnRHa (2249% compared with GnRH treatment alone; Fig. 2B
). Furthermore, using a Gal-4-Egr-1 fusion protein, basal and GnRH-induced Egr-1 transcription-stimulating activity were essentially abolished with W7 treatment (Fig. 2C
).

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Fig. 2. Inhibition of Cam with W7 Attenuates MKP-2 Promoter Activity, Egr-1 Protein Up-Regulation, and Transcription-Stimulating Activity
Transfection studies were carried out as described in Fig. 1 . A, T3-1 cells were transfected with the MKP-2 promoter-luciferase reporter and pretreated with control solution or W7 (15 µM) for 30 min. Response to 6 h of GnRHa is shown ± SEM. B, T3-1 cells were serum starved for 2 h, pretreated with increasing doses of W7 for 30 min, and then administered GnRHa for 1 h. Nuclear extracts were prepared and subjected to Western blot analysis using an antibody against Egr-1. Equal amounts of protein (20 µg) were added to each lane. C, T3-1 cells were cotransfected with 5xGal4-E1b-luciferase reporter and either an expression vector for Gal4 alone or the Gal4-Egr-1 fusion protein. Twenty-four hours later, cells were pretreated with control solution or W7 (15 µM) followed by GnRHa for 6 h, then assayed for luciferase activity. Relative luciferase activity was standardized by constant level of protein. Experiments were conducted in triplicate within an experiment on at least three separate occasions with equivalent results. Data are presented as means of a representative experiment ± SEM. Bars with different letters (a, b, and c) indicate differences (P < 0.05). The legend reflects treatment designations for all transfection-luciferase experiments in panels A and C.
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A common mechanistic theme with all of these affected reporter systems was the requirement for GnRH-induced ERK activation. We therefore assessed the effect of W7 on global signaling through tyrosine-phosphorylation of intracellular proteins. This included an analysis of the phosphorylation status of ERK family members. GnRHa treatment of
T3-1 cells induced marked changes in several tyrosine phosphorylated proteins (Fig. 3
) as we have demonstrated previously, including p42 and p44 ERKs (15). Pretreatment of cells with W7 reduced phosphorylation of a number of these intracellular proteins including bands at p42/44. Using phosphorylation-specific antibodies, ERK phosphorylation was shown to be reduced in a dose-dependent manner by pretreatment with W7 (Fig. 4A
). Dual phosphorylation of ERKs detected by this antibody was highly correlated with increased catalytic activity as determined by in vitro kinase assays using recombinant Elk-1 as a substrate (Zhang, T., and M. S. Roberson, unpublished observations). Interestingly, the Cam inhibitor did not affect GnRH-induced phosphorylation of JNK (Fig. 4B
) providing evidence for the specificity of the action of W7 in these studies. Consistent with the effects of W7, two additional inhibitors of Cam (W13 and trifluoperazine dimaleate or TFD) both blocked GnRH-induced ERK activation (Fig. 5
, A and B). Using a genetic approach to Cam inhibition, wild type and mutant forms of Cam were overexpressed in
T3-1 cells to determine the effect of Cam on ERK phosphorylation. The mutant form of Cam used here was deficient in all four Ca2+ binding sites (10, 11). Overexpression of wild-type Cam did not alter GnRH-induced ERK or JNK phosphorylation, whereas overexpression of the Ca2+-binding-deficient Cam mutant was effective at reducing GnRH-induced ERK but not JNK phosphorylation (Fig. 6
). Thus, these overexpression studies serve to confirm observations using the pharmacological approach to Cam inhibition.

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Fig. 3. Cam Inhibition with W7 Attenuates GnRH-Induced Tyrosine Phosphorylation in T3-1 Cells
T3-1 cells were serum starved for 2 h, pretreated with control solution [dimethylsulfoxide (DMSO)] or W7 (15 µM) for 30 min then GnRHa for 0, 15, or 30 min. The cells were lysed, lysates clarified and equal amounts of total protein (10 µg) were resolved by SDS-PAGE. Western blot analysis was then conducted using antiphosphotyrosine monoclonal antiserum. Arrows indicate W7-induced changes in tyrosine phosphorylation including changes in ERK phosphorylation. Molecular size standards are depicted at the left of the blot (MW).
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Fig. 6. Overexpression of a Ca2+-Binding Deficient Form of Cam Disrupts Signaling to ERK But Not JNK within the GnRH Pathway
T3-1 cells were cotransfected by lipofection with a control vector or increasing doses of wild-type (WT) or Ca2+-binding-deficient Cam (Mut; 2.5 or 5 µg). The lower doses of Cam expression vector were supplemented with control plasmid such that all transfections received similar amounts of DNA. Forty-eight hours later, all cells were serum-starved for 2 h then administered GnRHa for 0, 15 or 30 min. Whole cell lysates were then prepared and resolved by SDS-PAGE for Western blot analysis for phospho-ERK, total ERK, phospho-JNK and total JNK. Assessment of total ERK or JNK was used to standardize lane loading.
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W7, W13, and TFD all putatively have broad relative specificity for interference between Cam and association with interacting proteins. The possibility exists that the actions of these broad specificity Cam inhibitors may reflect inhibition of CamKs, such as CamK II. To examine this possibility, KN62 was used in dose response studies examining its effects on GnRH-induced ERK phosphorylation (Fig. 7
). Unlike W7, W13, and TFD, KN62 has greater relative specificity for inhibition of CamK II. Pretreatment of
T3-1 cells with KN62 did not affect GnRH-induced ERK activation (Fig. 7A
) but was sufficient to reduce basal CREB phosphorylation (Fig. 7B
), demonstrating the effectiveness of this compound in
T3-1 cells at the doses used. These studies suggest that either Cam alone or Cam association with activities other than CamKII are likely involved in mediating the effect of W7, W13, and TFD on GnRH-induced ERK phosphorylation.

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Fig. 7. The CamKII-Specific Inhibitor KN62 Does Not Mimic the Effects of W7 on ERK Phosphorylation
T3-1 cells were prepared as described in Fig. 4 except that some cells were pretreated with increasing doses of KN62 (5 or 10 µM) or W7 (15 µM; A). ERK phosphorylation was assayed using phospho-specific ERK antiserum, whereas total ERKs were measured to standardize lane loading. In panel B, control and KN62 (10 µM)-treated cells were examined for changes in phosphorylation of CREB using a phospho-specific CREB antibody. Similar levels of CREB protein (Pan CREB) were present regardless of phosphorylation state.
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To determine the level within the ERK cascade where W7 may be exerting inhibition, studies focused on examining the effects of W7 on upstream catalytic activities that are known to affect GnRH-induced ERK activation. Pretreatment of
T3-1 cells with W7 resulted in inhibition of MAPK/ERK kinase (MEK) 1 phosphorylation and c-Raf kinase phosphorylation at serine 338 (Fig. 8
, A and B). A possible target of Cam interaction that may additionally affect ERK phosphorylation in this model is phospholipase C (PLC) ß. PLCß has recently been shown to physically interact with Cam where Cam association was correlated with catalytic activity and signaling via muscarinic receptors (16). In
T3-1 cells, IP3 accumulation after GnRHa treatment was used as a readout for PLCß activity (Fig. 8C
). Cells pretreated with W7 at doses shown to block ERK activation did not affect IP3 accumulation. Collectively, these studies suggest that a possible target of Cam inhibition is upstream of MEK1 but downstream of PLC, potentially at the level of c-Raf kinase.
c-Raf kinase was shown to form a complex with Cam in the context of signaling via integrins (17). Studies therefore focused on determining whether c-Raf kinase could form a complex with Cam using Cam-agarose affinity chromatography.
T3-1 cell lysates were bound to a Cam-agarose column, washed extensively, and then Cam-interacting complexes were eluted by chelating Ca2+ within the column. As a positive control, IQGAP (a known Cam-interacting protein; Refs.18 and 19) was shown to bind this column (Fig. 9A
). Interestingly, c-Raf kinase also bound this column and could be eluted with chelation of Ca2+, suggesting the binding was Ca2+ dependent. The interaction between c-Raf kinase and Cam-agarose was specific because ERK binding to this column was not detected. 14-3-3 proteins have been shown to be a Cam-interacting partner (20, 21) and also associated with c-Raf kinase binding (22, 23). Consistent with these observations, 14-3-3 proteins were retained on the Cam column as suggested using a pan-specific 14-3-3 antibody. A 14-3-3ß-specific antibody provided evidence for the specific association between 14-3-3ß and Cam (Fig. 9A
). These studies supported speculation that interactions between Cam and 14-3-3ß may provide a scaffold for retention of c-Raf kinase on the Cam agarose column. To test this hypothesis, we used reconstitution assays where c-Raf kinase and 14-3-3ß were prepared as recombinant 35S-labeled proteins and subjected to binding reactions with Cam agarose in the form of a pull-down assay (Fig. 9B
). These studies revealed that both 14-3-3ß and c-Raf kinase can bind Cam agarose independently and that when bound together, the combination of proteins did not enhance Cam binding. These studies suggested that a direct interaction between c-Raf kinase and Cam may be important and supported the prediction that binding of c-Raf kinase to Cam agarose would be disrupted by addition of EGTA to chelate Ca2+ or W7 to disrupt association between Cam and c-Raf kinase. W7 is thought to bind to the amino and carboxyl-terminal hydrophobic binding pockets of Cam and interfere with Ca2+-dependent binding of target enzymes. As depicted in Fig. 9C
, addition of EGTA completed disrupted c-Raf kinase association with Cam and W7 reduced the Cam-c-Raf kinase association markedly.

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Fig. 9. c-Raf Kinase Forms a Specific Complex with Cam on a Cam-Agarose Column
T3-1 whole cell extracts were prepared and allowed to batch bind with a Cam agarose matrix. After binding, the Cam agarose was washed and then eluted using EGTA to chelate Ca2+. Fractions were collected and resolved on SDS-PAGE along with an aliquot of the starting material (20% input). A, Blots were probed with antibodies for IQGAP (a known Cam-interacting protein) as a positive control, c-Raf kinase ERKs, a pan-specific 14-3-3 and 14-3-3ß. B, A reconstitution assay was performed using recombinant 14-3-3ß and c-Raf kinase (35S methionine labeled) within the context of a Cam agarose pull-down assay. Input reflects 10% of total recombinant protein used in binding reactions. The Cam agarose pull down was carried out with 14-3-3ß or c-Raf kinase alone or in combination. Products of the binding reactions were resolved by SDS-PAGE and visualized by autoradiography. C, Cam agarose pull down was used in binding reactions containing c-Raf kinase (35S methionine labeled) in the presence of control solution, EGTA (10 mM) or W7 (15 µM). The protein bands were visualized as described above.
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We have previously shown that the GnRH receptor and c-Raf kinase colocalize to low-buoyant density compartments within the plasma membrane referred to as lipid rafts (8). Disruption of these raft compartments by cholesterol depletion blocked GnRH signaling to the ERK cascade, whereas cholesterol depletion followed by repletion rescued ERK signaling by GnRH. The current studies thus far support the prediction that Cam may also partition into these discrete plasma membrane compartments in conjunction with c-Raf kinase establishing a Ca2+-sensitive signaling platform facilitating the activation of the ERK cascade initially from lipid rafts. To address this possibility, we sought to determine whether 14-3-3ß and Cam were present in lipid rafts in
T3-1 cells (Fig. 10
). Western blot analyses of fractions obtained from sucrose density centrifugation of membrane preparations from
T3-1 cells revealed that 14-3-3ß and Cam partitioned into lipid rafts. Flotillin-1 was used as a putative marker for noncaveolar lipid rafts in these studies (data not shown). 14-3-3ß and Cam, along with the GnRH receptor, G
q, and c-Raf kinase (shown previously; Ref.8), all comigrate with flotillin-1 within the sucrose gradient suggesting all of these signaling molecules may be present in a similar low-density membrane compartment. The compartmentalization of a putative Ca2+-sensitive signaling complex (c-Raf kinase and Cam) into lipid rafts with the GnRH receptor supports the possibility that this complex is poised to respond to GnRH and integrate Ca2+ and ERK signaling in gonadotrope-derived cells.

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Fig. 10. Cam Partitions to Low-Buoyant Density Lipid Rafts in T3-1 Cells
T3-1 cell lysates were prepared using 0.1% Triton X-100 and subjected to sucrose density gradient centrifugation. Fractions were collected representing low to high density with regard to sucrose concentration and assayed by Western blot for 14-3-3ß and Cam.
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DISCUSSION
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The original premise of the current studies was that influx of Ca2+ through VGCCs was required for GnRH-induced ERK activation (5), and this regulation was likely mediated regionally within the gonadotrope by virtue of a putative signaling platform(s) localizing the GnRH receptor with G
q and c-Raf kinase into lipid rafts (8). The central hypothesis was that Ca2+ influx was sensed within the GnRH pathway by a Ca2+-binding protein such as Cam and disruption of Ca2+ sensing would attenuate GnRH-induced ERK signaling. The current studies provide evidence in support of this hypothesis. Cam disruption by W7 (and other pharmacological Cam inhibitors) and overexpression of a Ca2+-binding-deficient Cam mutant resulted in blockade of GnRH-induced ERK (but not JNK) phosphorylation and ERK-dependent genes such as c-Fos, the murine glycoprotein hormone
-subunit, and Egr-1-dependent MKP-2 activation. The c-Fos promoter has been shown to be regulated by a ternary complex involving serum-response factor, the Ets factor Elk-1, and the serum response element. Activation of the ERK pathway results in activation of this ternary complex through Elk-1 phosphorylation (24). The murine glycoprotein hormone
-subunit promoter has also been reported to be regulated via a putative Ets factor binding to the GnRH-responsive element within this promoter; however, a definitive Ets family member has not been reported (25, 26). In the case of the
-subunit promoter, the GnRH-responsive element appears to be the target of GnRH regulation through the ERK cascade. We have described an Egr-1-dependent mechanism for the up-regulation of the dual-specificity phosphatase, MKP-2 by GnRH (14). In the context of this GnRH- and ERK-dependent gene, Cam inhibition partially reduced up-regulation of Egr-1 protein levels and abolished Egr-1 transcription activating capabilities suggesting that Gal4-Egr-1 activity was Cam/ERK dependent. The differential effect(s) of Cam inhibition on Egr-1 protein vs. Gal4-Egr-1 activity likely reflects differences in the molecular basis of protein up-regulation when compared with potential posttranslational modification of Egr-1 protein (such as phosphorylation and recruitment of coregulators) that lead to transcriptional activation by this factor. In contrast to these ERK-dependent gene promoters, the GnRH receptor promoter has been shown to be sensitive to overexpression of a dominant-negative acting mutant of JNK and not to inhibition of the ERK cascade. These studies supported the conclusion that JNK but not ERK was central to GnRH induction of the GnRH receptor gene (13). The current studies are consistent with this conclusion because W7 inhibited ERK but not JNK phosphorylation and regulation of the GnRH receptor promoter by GnRH was unaffected by Cam inhibition. Collectively, these studies provide evidence for the central role for Cam as a putative Ca2+ sensor in the regulation of ERK-dependent genes within the GnRH pathway.
The importance of Ca2+ influx through VGCCs on ERK activity in gonadotrope cell models is currently controversial. In the
T3-1 model, our studies of the inhibition of VGCC Ca2+ was accomplished with the use of nifedipine, a dihydropyridine receptor antagonist (5, 6). In these studies, intracellular Ca2+ changes in the absence or presence of this inhibitor were examined using Indo-1 fluorescence to visualize and determine the relative effectiveness of the VGCC inhibitor. In
T3-1 cells, nifedipine specifically blocked GnRH-induced ERK (but not JNK) phosphorylation at levels of nifedipine that quantitatively blocked VGCC Ca2+ as measured by Indo-1 fluorescence. Acute replacement of Ca2+ with Mg2+ in the
T3-1 cell culture medium also was effective at blocking GnRH-induced ERK phosphorylation providing additional evidence for the importance of extracellular Ca2+. Importantly, the effect(s) of nifedipine on ERK phosphorylation was recapitulated in rat pituitary cells dispersed into primary culture suggesting fidelity between the
T3-1 cell model and differentiated pituitary cells. The effects of VGCC blockade with nifedipine on ERK phosphorylation were also consistent with reports by Yokoi and colleagues (27) using LßT2 cells, although Ca2+ measurements using fluorescent reporter dyes were not reported. In a different series of studies, effects of dihydropyridine receptor antagonists or extracellular Ca2+ chelation with EGTA on GnRH-induced ERK activation again in the LßT2 cell model were not observed (28). In these studies (28), nimodipine did not affect GnRH-induced ERK phosphorylation and EGTA had minimal effects. Thus, in contrast to the
T3-1 cell model, these studies in LßT2 cells (28) were not consistent with responses in rat pituitary cells in primary culture. It is plausible that elucidation of the disparity between the studies conducted in LßT2 cells may require quantification of Ca2+ signals in this cell model using indicator dyes to fully appreciate the efficacy and pharmacological activities of the VGCC antagonists and their effects on ERK phosphorylation by GnRH.
Initially, we considered the possible impact of Cam inhibition on L-type VGCC activity in the gonadotrope and the role this might play on modulation of the ERK cascade by GnRH. The role of Cam in the context of the L-type VGCC channel subunits is to modulate the inactivation of channel conductance as local Ca2+ concentrations increase (10, 11). Thus, Cam inhibition in this case would likely lead to potentially greater Ca2+ influx through L-type VGCCs rather than reduced Ca2+ signaling. As such, this potential mechanism was discounted because it was unlikely that inhibition of Cam at the level of the L-type channel would lead to reduced Ca2+ influx and inhibition of the ERK pathway.
Cam is a ubiquitously expressed molecule shown to associate with a large number of interacting partners (directly and indirectly), and Cam activity is subject to regulation dependent upon phosphorylation state (for review see Ref.9). In the current studies, we have made use of IQGAP as a positive control (for review see Ref.19) and investigated the possibility that PLCß (16) may be affected by Cam inhibition within the GnRH signaling pathway. In the latter situation, we find no evidence supporting the possibility that Cam inhibition alters IP3 accumulation, suggesting that if a Cam-PLCß interaction exists in
T3-1 cells, the impact of this interaction is likely minimal on GnRH signaling. Our studies demonstrating c-Raf kinase as a Cam-binding protein were based upon the specific retention of c-Raf kinase on a Cam-agarose column in a Ca2+-dependent manner. Others have demonstrated c-Raf kinase as a Cam-interacting protein in different cell systems. In a series of studies by Agell and colleagues (31, 32) using NIH 3T3 cells, both c-Raf kinase and specific isoforms of Ras were observed to bind to a Cam agarose column. Neither MEK nor ERKs were found to bind to the Cam affinity column, consistent with our studies. Cam association with Ras appeared to be specific to K-Ras and association favored the GTP bound state. Cam association with K-Ras served as a negative modulator because inhibition of Cam in these studies enhanced signaling through K-Ras and increased c-Raf kinase activity (31, 32). These studies likely define a mechanism different from that supported by the present studies because Cam inhibition effectively blocked c-Raf kinase phosphorylation at S338, presumably leading to inhibition of ERK phosphorylation by GnRH. There is evidence for Ras involvement in GnRH signaling in
T3-1 and Cos cell models; GTP binding of Ras appeared to be mediated by GnRH-induced cleavage of membrane-associated EGF and activation of the EGF receptor (33, 34, 35). Based upon responses to Cam inhibition in the present studies, it appears unlikely that Ras isoforms such as K-Ras may be playing a role in Ca2+-modulated ERK activation by GnRH.
Other studies have shown c-Raf kinase association with Cam via participation in a complex that includes CamKII (17). This interesting series of studies supports the possibility that c-Raf kinase activation was mediated by CamKII binding and subsequent direct phosphorylation. This mechanism has helped define a role for Cam in integrin-mediated ERK activation. However, this scenario is again less likely in the context of the current studies because we can rule out a putative role for CamKII based upon our studies using the specific CamKII inhibitor KN62. CamKII has been reported as a signaling intermediate within the GnRH pathway by Haisenleder and colleagues (36, 37). These studies implicate CamKII in the regulation of the gonadotropin subunit genes; however, consistent with our studies, activation of ERKs by GnRH was not associated with CamKII activity in gonadotrope cell models and in rat pituitary cells in primary culture.
Our studies have investigated the possibility that 14-3-3 proteins may serve as a bridge or scaffold between Cam and c-Raf kinase. Interestingly, 14-3-3ß did bind to the Cam agarose column in a Ca2+-sensitive manner consistent with previous reports of Cam association with 14-3-3
(21, 23). However, c-Raf kinase also bound the Cam column, presumably independent of 14-3-3ß. Importantly, association between Cam and c-Raf kinase were reduced in vitro by chelation of Ca2+ or the addition of W7, providing a potential mechanism for W7 action in blocking ERK activation by GnRH in
T3-1 cells. The remaining question is how Ca2+/Cam engages c-Raf kinase to potentially alter its catalytic activity. Several possibilities may exist. First, Ca2+/Cam may affect c-Raf kinase structurally to promote catalytic activity consistent with Cam association with myosin light chain kinase (9). Alternatively, Ca2+/Cam may provide a scaffold for other molecules to participate in binding and ultimately facilitate changes in catalytic activity of c-Raf kinase. Future studies are focused on elucidation of these possibilities.
Collectively, the results of these studies support speculation that the GnRH receptor, c-Raf kinase, and Cam occupy discrete membrane compartments associated with lipid rafts. GnRH receptor activation leads to changes in intracellular Ca2+. ERK activation by GnRH requires influx of Ca2+ through VGCCs. It is plausible that a raft-associated signaling platform containing Cam serves to sense local Ca2+ influx and leads to alteration of ERK signaling through c-Raf kinase activity. Disruption of Cam activity by W7 leads to reduced c-Raf kinase activation correlated with a reduction in Cam/c-Raf kinase association in vitro and ultimately a loss of ERK activation by GnRH in this system. The more global effect(s) of Cam inhibition on tyrosine phosphorylation induced by GnRH in this gonadotrope cell model makes it tempting to speculate a key role for Ca2+/Cam in GnRH signaling potentially beyond the role of Cam in the activation of the ERK pathway.
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MATERIALS AND METHODS
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Reagents
Antibodies for phospho-tyrosine (titer 1:1000), c-Raf kinase (titer 1:1000), MEK1 (titer 1:1000), ERK2 (titer 1:1000), IQGAP (titer 1:1000), 14-3-3ß (titer 1:1000) and all horseradish peroxidase-coupled secondary antibodies (titer 1:5000) were purchased from Santa Cruz Biotechnology (Santa Cruz CA) and were used according to the manufacturers instructions. The phospho-ERK, -JNK, and -MEK1 antibodies (titer 1:1000) were purchased from Cell Signaling Technologies (Beverly, MA) and were used according to the manufacturers instructions. The phospho-c-Raf kinase S338 (titer 1:500), phospho-CREB (titer 1:1000), CREB (titer 1:1000), pan-14-3-3 (titer 1:500) and Cam (titer 1:100) antibodies were obtained from Upstate Biotechnology (Lake Placid, NY) and used according to the manufacturers instructions. The GnRH analog buserelin (referred to as GnRHa) was a gift from Dr. Richard Maurer (Oregon Health Sciences University, Portland, OR) and was used at 10 nM for all studies. W7, W13, trifluoperazine dimaleate (TFD), and KN62 were obtained from Calbiochem (San Diego, CA) and were used at doses reported in individual figures. Calmodulin agarose was purchased from Stratagene (La Jolla, CA) and the matrix was prepared according to the manufacturers instructions. Expression vectors for wild-type and Ca2+-binding-deficient Cam were a gift from Dr. David Yue (Johns Hopkins University, Baltimore, MD). Expression vectors for ERK2 and JNK1 were gifts from Drs. Melanie Cobb (University of Texas, Southwestern, Dallas, TX) and Roger Davis (University of Massachusetts, Worchester, MA), respectively. Expression vector for 14-3-3ß was a gift from Dr. Jun-Lin Guan (Cornell University, Ithaca, NY).
Cell Culture
T3-1 cells were generously provided by Dr. Pamela Mellon (University of California, San Diego, CA; Ref.38). Cells were cultured in monolayers in the presence of DMEM containing 10% fetal bovine serum and supplemented with penicillin and streptomycin. Cells were maintained at 37 C in a 5% CO2, humidified atmosphere. For all studies,
T3-1 cells were split within 2 d of experimentation and used as subconfluent (approximately 50%) cultures.
Transient Transfection Studies and Luciferase Assays
T3-1 cells were transfected using Ca2+ phosphate precipitation as previously described (14). The c-Fos-luciferase (15), mouse
-subunit-luciferase (25), GnRH receptor-luciferase (39, 40), and the MKP-2-luciferase (14) have been previously described. For these reporter genes, cells were transfected for 4 h, serum-containing medium was replaced and transfected cells were immediately administered GnRHa (10 nM) for a 6-h period. Cell lysates were prepared by three freeze-thaw cycles and luciferase activity was determined in samples standardized by protein levels. The Gal4-Egr-1 system has been previously described (14). Briefly, expression vectors for Gal4 or Gal4-Egr-1 were cotransfected with the 5xGal4-E1B-luciferase reporter. The following morning, transfected cells were pretreated with W7 (15 µM) for 30 min followed by control solution or GnRHa and cells were harvested 6 h later and assayed for luciferase activity as described above. Transient transfection studies were conducted in triplicate on at least three separate occasions with similar results.
Preparation of Whole Cell Lysates and Western Blot Analysis
For all blotting studies, cells were serum-starved for a 2-h period followed by pretreatment with inhibitors and subsequently treated with GnRHa for the designated time courses. After treatments within individual experiments, cells were lysed in a standard RIA immunoprecipitation buffer as described (7). Lysates were clarified by centrifugation and denatured by boiling in an equal volume of buffer containing 100 mM Tris (pH 6.8), 4% sodium dodecyl sulfate, 20% glycerol, and 200 mM dithiothreitol (2x SDS-loading buffer). Samples were resolved on 10% polyacrylamide gels, transferred to polyvinylidene difluoride membranes, blocked in either 5% BSA (phospho-tyrosine antibody) or nonfat dried milk in Tris-buffered saline (pH 7.5)/0.1% Tween 20 (TBST). Primary antibodies were added at the appropriate dilution/titer and incubated at 4 C overnight with constant agitation. Blots were washed in TBST, then exposed to secondary antibody for 12 h at room temperature, washed again, and protein bands were visualized using enhanced chemiluminescence reagents (PerkinElmer, Boston MA). With the use of phospho-specific antibodies, lane loading was determined using the pan-specific antibodies to the corresponding phospho-specific antisera used in individual studies. For studies examining nuclear Egr-1 levels, equal amount of nuclear protein (20 µg) was used in each lane. Lane loading was verified using Ponceau S staining of the membrane before Western blot. All Western blotting studies were conducted on at least three separate occasions with similar results. Representative blots are shown.
Overexpression Studies with Wild-Type and Ca2+ Binding-Deficient Cam
T3-1 cells were cotransfected by lipofection (Invitrogen, Carlsbad, CA) with expression vectors for control vector (pcDNA3; 5 µg) or increasing doses (2.5 or 5.0 µg) of expression vector for wild type Cam or a mutant form of Cam with all four Ca2+ binding sites mutated. All transfections were carried out using equivalent amounts of total DNA where pcDNA3 was used to bring the total DNA level up to 5 µg. Forty-eight hours after transfection, cells were serum starved for 2 h and administered control solution or GnRHa for 0, 15, or 30 min. RIA immunoprecipitation whole cell lysates were obtained as outlined above and Western blot analyses for phospho-ERK and -JNK were conducted.
[3H]Inositol Assays
Accumulation of phosphorylated inositol using a previously described (8) method was used to quantify phospholipase C activity in
T3-1 cells. Briefly,
T3-1 cells were plated at approximately 50% confluence overnight in 24-well culture plates. The serum-containing culture medium was washed from the cells with serum-free M199 culture medium (Mediatech, Herndon, VA). After washing, cells were incubated at 37 C for 5 h in 0.3 ml of serum-free M199 containing 2 µCi of myo-[2-3H]inositol. The labeled cells were then washed with serum-free DMEM containing 5 mM LiCl. Medium was removed and cells remained untreated or were administered GnRHa in 1 ml serum-free DMEM containing 5 mM LiCl. These treatment conditions were maintained at 37 C for the indicated times, after which the medium was removed, and 1 ml of water heated to 95 C. The cells were then frozen overnight and thawed at room temperature. Cell lysates were collected and loaded separately onto Dowex 1-X8, 200400 mesh, formate-form columns with an approximate bed volume of 0.4 ml. Free, unphosphorylated, and monophosphorylated inositol was eluted from the lysate by the addition of 10-column vol of water. After collection of the eluent containing the free inositol, total remaining inositol phosphates (di- and greater) were collected by the addition of 10 bed volumes of 1 M ammonium formate in 0.1 M formic acid. Radioactivity in the free and phosphorylated inositol eluents were quantitated using a Beckman LS-5000CE liquid scintillation counter. Data are presented as phosphorylated inositol expressed as a percentage of the total [3H]inositol.
Cam Agarose Affinity Chromatography
T3-1 cell lysates were prepared in 50 mM Tris (pH 7.5), 1.0% Triton X-100, 5 mM EDTA, 250 mM NaCl, 1 mM sodium vanadate, 25 mM ß-glycerophosphate, 5 mM benzamidine, and 0.2 mM phenylmethylsulfonyl fluoride (Buffer A). This buffer was supplemented with a protease inhibitor cocktail (Sigma, catalog no. P-8340; St. Louis, MO). Lysates were subjected to two freeze-thaw cycles (80 C, then thawed on ice) and clarified by centrifugation. To prepare the calmodulin agarose, beads were sedimented by low-speed centrifugation, the storage buffer removed, and beads were washed four times in a buffer containing 20 mM Tris (pH 7.5), 4 mM MgCl2, 2 mM CaCl2, 10 mM KCl, and 2 mM phenylmethylsulfonyl fluoride (Buffer B). Cell lysates in Buffer A were diluted 10-fold in Buffer B. Diluted lysates were then mixed with washed Cam agarose beads for 34 h at 4 C with constant mixing. The lysate/agarose solutions were then loaded into a disposable column and the retained calmodulin agarose matrix was washed with 20-column vol of Buffer B. The column was eluted with a buffer containing 20 mM Tris (pH 7.5), 4 mM MgCl2, 10 mM EGTA, 10 mM KCl, and 1 mM phenylmethylsulfonyl fluoride. Fractions were collected in 250 µl volumes. Equal volumes of fractions were used for Western blotting analysis. In some studies, Cam agarose beads were used to pull-down Cam-interacting proteins in reconstitution assays. In these studies, recombinant c-Raf kinase and 14-3-3ß were prepared using a coupled transcription/translation wheat germ lysate system in the presence of 35S-methionine according to the manufacturers instructions (Promega, Madison WI). Recombinant labeled proteins were then added to Cam agarose binding reactions (400 µl) in Buffer B as described for individual experiments in the absence or presence of EGTA (10 mM) or W7 (15 µM). Binding reactions were carried out for 2 h at 4 C. Complexes were then washed in Buffer B (in the absence or presence of EGTA or W7). Samples were then boiled and resolved by SDS-PAGE. The gels were fixed in 15% methanol/acetic acid, washed in 20% isopropanol and dried. Bands were visualized by autoradiography. These pull-down studies were completed twice with equivalent results.
Isolation of Low-Buoyant Density Membrane Compartments or Lipid Rafts
Isolation of lipid rafts was carried out as previously described (8). Briefly,
T3-1 cells were lysed in a buffer containing a low concentration (0.1%) of Triton X-100 and subjected to density gradient centrifugation through a step gradient of sucrose. Fractions were collected whereby low fraction numbers reflect low relative buoyant density. Equal volumes of fractions were then resolved on SDS-PAGE gels and Western blot analysis was used to examine expression of 14-3-3ß and calmodulin. Three independent sets of raft fractions from control and GnRHa-treated cells were used in these studies.
Statistical Analysis
For transfection studies, data were subjected to ANOVA, and treatment differences were determined by Tukeys Studentized range test. Differences were considered statistically significant at P < 0.05.
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ACKNOWLEDGMENTS
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Appreciation is extended to Drs. Richard Maurer, David Yue, Melanie Cobb, Roger Davis, Pamela Mellon, and Jun-Lin Guan for providing valuable reagents. Special thanks to Drs. Mike Kotlikoff and George Ignotz for helpful discussion during the preparation of this manuscript.
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FOOTNOTES
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These studies were supported by grants from the National Institute of Child Health and Human Development (R01 HD34722 to M.S.R. and F3244379 to S.P.B.).
First Published Online May 12, 2005
Abbreviations: Cam, Calmodulin; CamK, Cam-dependent protein kinase; CREB, cAMP response element binding protein; Egr-1, early growth response protein 1; GnRHa, buserelin; IP3, inositol 1,4,5 trisphosphate; JNK, c-Jun N-terminal kinase activity; MEK, MAPK/ERK kinase; MKP, MAPK phosphatase; PKC, protein kinase C; PLC, phospholipase C; TFD, trifluoperazine dimaleate;VGCC, voltage-gated Ca2+ channels.
Received for publication February 22, 2005.
Accepted for publication May 4, 2005.
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