Extracellular Signal-Regulated Kinase and c-Src But Not Jun N-Terminal Kinase Are Involved in Basal and Gonadotropin-Releasing Hormone-Stimulated Act
Abstract2q, http://www.100md.com
Addition of a GnRH agonist (GnRH-A) to {alpha} T3-1 cells stimulates different MAPK cascades: ERK, Jun N-terminal kinase (JNK), and p38. Activation of JNK, ERK, and p38 shows a unique fold activation ratio of 25:12:2, which might encode signal specificity. ERK is translocated to the nucleus within 20 min with a peak at 120 min of GnRH-A stimulation. We used the human -subunit promoter linked to chloramphenicol acetyl transferase (CAT) to examine the role of ERK, JNK, and c-Src, which is implicated in MAPK activation, in basal and GnRH-stimulated CAT. Addition of GnRH-A resulted in a 3-fold increase in CAT, whereas the Ca2+ ionophore ionomycin and the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol-13-acetate (TPA) had no effect. Addition of GnRH-A and TPA, but not GnRH-A and ionomycin, produced a synergistic response, whereas removal of Ca2+, but not down-regulation of TPA-sensitive PKCs, abolished GnRH-A-stimulated {alpha} CAT. Thus, regulation of -promoter activity by GnRH is Ca2+ dependent and is further augmented by PKC. Cotransfection of CAT and constitutively active or dominant negative plasmids of ERK and JNK cascade members, or the use of the ERK inhibitor PD98059, revealed that ERK, but not JNK, is involved in basal and GnRH-A-stimulated {alpha} CAT. Because c-Src participates in MAPK activation by GnRH, we also studied its role. Cotransfection of {alpha} CAT and the dominant negative form of c-Src or incubation with the c-Src inhibitor PP1 reduced GnRH-A-stimulated {alpha} CAT. The 5'-deletion analysis revealed that the -846/-420 region participated in basal {alpha} -transcription. In addition, the -346/-156 region containing the pituitary glycoprotein hormone basal element, {alpha} -basal elements, glycoprotein-specific element, and upstream response element is involved in basal and GnRH-A-stimulated {alpha} CAT. ERK contribution to GnRH maps to -346/-280 containing the pituitary glycoprotein hormone basal element and {alpha} -basal elements 1/2. Surprisingly, although c-Src is involved in GnRH-A-stimulated ERK, its involvement is mapped to another region (-280/-180) containing the glycoprotein-specific element. Thus, ERK and c-Src but not JNK are involved in basal and GnRH-A-stimulated-{alpha} CAT, whereas c-Src contribution is independent of ERK activation.
Introductionwdc, 百拇医药
GnRH STIMULATES THE release and synthesis of the heterodimeric glycoprotein hormones LH and FSH from pituitary gonadotrophs. Common to pituitary glycoprotein hormones LH, FSH, and TSH is the {alpha} -subunit, which binds noncovalently to a specific ß-subunit (1, 2). Signaling by the GnRH receptor (GnRHR) involves rapid and sequential stimulation of the phospholipases C and D, Ca2+ mobilization and influx, activation of protein kinase C (PKC) subspecies, and the MAPK cascades ERK, Jun N-terminal kinase (JNK), and p38 and release of arachidonic acid and formation of bioactive lipoxygenase products (3, 4, 5, 6, 7, 8). Although GnRHR activates a large array of signaling molecules, the mechanisms involved in gonadotropin synthesis have not yet been clarified (2, 9, 10).wdc, 百拇医药
Each of the MAPK signaling cascades consists of up to five tiers of protein kinases, which sequentially activate each other by phosphorylation (6, 11). At present, four distinct MAPK cascades have been identified in mammals. They have been named according to their respective MAPK components: the p42 and p44 MAPKs, also known as ERK1/2; JNK1/3; p38, ß, and ; and ERK5 (6, 11).
A small G protein of the Ras family often initiates the cascade. The signal is then transmitted to the MAPK kinase (MAPK/ERK kinase, MEK) kinase (MAP3K, MEK kinase, MEKK), followed by activation of MEK and MAPK. Raf kinases often serve as the MEKK in the ERK cascade, and on activation the signal is transmitted to MEK and ERK. In the JNK cascade, MEKK1 is the most common kinase at the MAP3K level followed by MKK4/7 and JNK1–3 (6, 11). The unique property of MAPKs to translocate to the nucleus, phosphorylate, and activate transcription factors implies a potential role for MAPK in transcription.$?, http://www.100md.com
GnRH stimulates different MAPK cascades: ERK, JNK, and p38 in pituitary and GnRHR-transfected cells (Refs. 6 , 8 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 and present results). The role of MAPKs in GnRHstimulation of common {alpha} -subunit gene transcription is not clear. Although some reports claim that ERK is involved in basal as well as GnRH-stimulated {alpha} -transcription (13, 24), others reported that ERK is involved mainly in the regulation of the basal but not the GnRH response (14). Controversy also exists concerning the role of ERK in GnRH regulation of LHß gene expression. We and others reported a positive role for ERK (25, 26), whereas others have found no role for ERK in the transcriptional regulation of LHß by GnRH (21, 24).
Activation of JNK by GnRH (16, 20, 21) was expected to be important in {alpha} -transcription, because Jun and activating transcription factor-2, known substrates of JNK, have been shown to contribute to basal -transcription via binding to the cAMP response element (CRE; Ref. 27). We therefore decided to investigate the role of MAPKs in the transcriptional control of gonadotropin subunit genes by GnRH. First, we demonstrated that both ERK and JNK mediate the GnRH response on LHß (26). Here we show that Ca2+, ERK, and c-Src, but not JNK, are involved in transcriptional regulation of the human -subunit gene by GnRH. 5'-Deletion analysis has identified four regions involved in basal activation, out of which three are also involved in the GnRH response. Of these, one is mediated by ERK and, surprisingly, a different one by c-Src. Although c-Src is involved in GnRH-stimulated MAPK activation (16, 22), this first protooncogene and nonreceptor tyrosine kinase mediates the {alpha} -gene transcriptional response by GnRH in ERK- and JNK-independent mechanism.
Materials and Methodsa3, 百拇医药
Materialsa3, 百拇医药
A stable GnRH agonist, [D-Trp (6)]-GnRH (GnRH-A), ionomycin, and 12-O-tetradecanoylphorbol-13-acetate (TPA) were purchased from Sigma (St. Louis, MO). Selective inhibitors for ERK (PD 98059) and c-Src (PP1) were from Calbiochem (La Jolla, CA). Mouse monoclonal antiactive and rabbit polyclonal anti-general MAPKs (ERK, JNK, and p38), protein A-Sepharose, and rhodamine-conjugated goat-antirabbit IgG were purchased from Sigma (Rehovot, Israel). Polyclonal anti-hemagglutinin (HA) antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The plasmids for the various ERK and JNK cascade members were recently described (16, 22). The human {alpha} -subunit promoter linked to chloramphenicol acetyl transferase (CAT) was kindly provided by Dr. P. Mellon (University of California, San Diego, CA), and 5' deletion mutants of h-846{alpha} LUC were kindly provided by Dr. J. L. Jameson (Northwestern University Medical School, Chicago, IL).
Activation of MAPK cascadesd^+*{0, http://www.100md.com
The {alpha} T3-1 cells were grown in 6-well plates and serum starved [0.1% fetal calf serum (FCS)] for 16 h. After stimulation, the cells were washed twice with ice-cold PBS and once with ice-cold buffer A: 50 mM ß-glycerophosphate, 1.5 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, and 1 mM sodium orthovanadate. Cells were harvested in 0.3 ml buffer H: buffer A containing 1 mM benzamidine, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride, followed by sonication (2 x 7 sec, 40 W) and centrifugation (15,000 x g, 15 min, 4 C). The supernatants, which contained cytosolic proteins, were collected, and aliquots from each sample (20 µg) were separated on 10% SDS-PAGE followed by Western blotting with mouse monoclonal anti-active MAPKs (ERK, JNK, and p38; Ref. 22). Total MAPKs were detected with polyclonal antibodies for the various MAPKs as a control. The blots were developed with alkaline phosphatase or horseradish peroxidase-conjugated antimouse or antirabbit Fab antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). The blots were autoradiographed on X-100 films (Kodak, Rochester, NY), and the phosphorylation was quantitated by densitometry (690 densitometer, Bio-Rad Laboratories, Inc., Hercules, CA).
Cell culture and transfection.5]y;e, 百拇医药
Subconfluent {alpha} T3-1 cells were cotransfected for 8 h with 5 µg each {alpha} CAT or 5' deletion mutants linked to luciferase (LUC), with or without the CA or the DN mutants of MAPK cascade members, using the calcium phosphate method. The total amount of plasmid was adjusted to 20 µg with vector DNA in control experiments. The transfection efficiency was 10–30%, as determined by transfection with a plasmid that contained Rous sarcoma virus-LUC. Then 24–36 h after transfection, the cells were serum starved for 16 h and incubated with GnRH-A or other stimulant as described in the figure legends. Cells were then harvested and the cell extracts were analyzed for CAT or LUC activities (26), which were normalized to the level of Rous sarcoma virus-LUC activity where appropriate. Results were then calculated as fold increase relative to expression in control wells, and the data are shown as the mean ± SEM. ANOVA was used to determine statistically different groups; significance was set at P < 0.05: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.
Immunoprecipitation{, 百拇医药
Subconfluent T3-1 cells were cotransfected with 10 µg HA-tagged wild-type ERK2 (HA-ERK) and 10 µg of the mutants to be analyzed. Subsequently, the cells were serum starved (0.1% FCS, 16 h) and stimulated with GnRH-A (10 nM) for 7 min. Following stimulation the cells were washed, lysed as above, and ERK immunoprecipitated with polyclonal anti-HA antibodies using protein A-Sepharose (30 µl, Sigma). Cell lysates (500 µg protein) were incubated with the conjugated beads and rotated end to end for 2 h at 4 C. Following incubation the beads were washed once with 1 ml RIPA assay buffer [137 mM NaCl, 20 mM Tris at pH 7.4, 10% (vol/vol) glycerol, 1% Triton X-100, 0.5% (wt/vol) deoxycholate, 0.1% (wt/vol) SDS, 2.0 mM EDTA, 1.0 mM phenylmethylsulfonyl fluoride, and 20 µM leupeptin]; twice with 1 ml LiCl, 0.5 mM in 100 mM Tris at pH 8.0; and twice with 1 ml buffer A. The immunoprecipitates were subjected to 10% SDS-PAGE and Western blotting using the monoclonal anti-active ERK antibody. Total HA-ERK was detected using a polyclonal anti-HA antibody. The blots were developed as described above.
Fluorescent cell staining&+umv5, http://www.100md.com
Subconfluent T3-1 cells were serum starved (16 h, 0.1% FCS) and treated with GnRH-A for various time points. Subsequently, the cells were washed with PBS, fixed in 3% paraformaldehyde in PBS, and permeabilized with 0.2% Triton X-100 in PBS for 5 min. Staining was performed by 45-min incubation with polyclonal antigeneral ERK antibody (diluted 1:100), followed by 45-min incubation with rhodamine-conjugated goat-antirabbit IgG (diluted 1:100). Fluorescent staining was viewed with a fluorescent microscope (EFD-3, Nikon, Melville, NY).&+umv5, http://www.100md.com
Results&+umv5, http://www.100md.com
Effect of GnRH-A on MAPK activity&+umv5, http://www.100md.com
To study the activation of MAPKs in response to GnRH, we used antigeneral MAPK antibodies and also monoclonal antibodies, which specifically recognize the doubly phosphorylated, active forms of the various MAPKs (Ref. 22 ; Fig. 1A). The T3-1 cells were treated with GnRH-A for different time periods, and cell lysates were subsequently prepared. A Western blot analysis was carried out using total cell lysates and the antibodies as above. Each band from the antiphospho MAPK (ERK, JNK, and p38) was normalized to the corresponding band from the antigeneral MAPK antibodies blot for even loading. The results indicated that ERK, JNK, and p38 were activated on GnRH-A stimulation. JNK1 was activated (~ 25-fold) similar to previous results using glutathione-S-transferase-Jun as a substrate (16), and a more recent report (19), peaking at 30–45 min and declining thereafter. ERKs were activated (~ 12-fold), similar to previous observations using myelin basic protein as a substrate (15), and more recent reports (18, 19, 20). ERK activation, which peaked at 15 min, returned to basal levels at 60–90 min. The p38 was phosphorylated only to a small extent (2- to 3-fold), in agreement with others (17). Activation was transient and peaked at 30 min after stimulation with GnRH. Under the same conditions, the selective inhibitor for MEK (PD98059, 50 µM) abolished GnRH-A-stimulated ERK but not JNK or p38 activation (Ref. 22 and Fig. 1B). The protein tyrosine kinase inhibitor genistein produced a 40% and 80% inhibition on ERK and JNK activation, respectively (Ref. 22 and Fig. 1B). The PKC selective inhibitor GF109203X inhibited GnRH-A-stimulated ERK, JNK, and p38 activation by 70%, 50%, and 50%, respectively (Ref. 22 and Fig. 1B). The c-Src selective inhibitor PP1 (2 µM) reduced ERK activation by GnRH-A by 50% at 15 min of incubation (22).
fig.ommitteedfig.ommitteed&-!4n, http://www.100md.com
Figure 1. Effect of GnRH-A on MAPK activity in T3-1 cells. Subconfluent T3-1 cells were treated with GnRH-A (100 nM) in the absence (A) or presence (B) of PD98059 (50 µM), GF109203X (5 µM), or genistein (200 µM) for the indicated times, and cell lysates were subsequently prepared. MAPK (ERK, JNK, and p38) activity was determined by Western blotting with type-specific antibodies for phospho-MAPK (active form) and anti-MAPK antibodies. Each band form the antiphospho MAPK was normalized to the corresponding band from the anti-MAPK blot for even loading. Fold stimulation was determined by densitometry as described in Materials and Methods. Inhibition of activated ERK by the drugs was shown elsewhere (22 ), and representative experiments are shown here, whereas similar results were observed in two other experiments (n = 3, SEM 10%).&-!4n, http://www.100md.com
Effect of GnRH-A on ERK translocation&-!4n, http://www.100md.com
To study the effect of GnRH-A on the subcellular localization of ERK, {alpha} T3-1 cells were treated with or without GnRH-A and later stained with the general polyclonal anti-MAPK antibody (Fig. 2). The staining of nonstimulated cells with this antibody was detected primarily in the cytosol. GnRH-A treatment of {alpha} T3-1 cells induced significant translocation of ERK to the nucleus. This nuclear staining was detected first after 20 min of GnRH-A stimulation and continued progressively to reach a peak at 120 min, in which all cells examined were stained. Hence, ERK can mediate nuclear transcriptional responses. The nuclear import and export signal is under investigation. Here we focused on the contribution of ERK, JNK, and c-Src to transcription from the common {alpha} -subunit promoter, whereas others (17) have shown that p38 is not involved in this process.
fig.ommitteedfig.ommitteed@1y$', http://www.100md.com
Figure 2. Effect of GnRH-A on ERK translocation in T3-1 cells. Subconfluent T3-1 cells were serum starved (16 h, 0.1% FCS) and treated in the absence (A) or presence of GnRH-A (100 nM) for 60 min (B). Following stimulation, the cells were washed, fixed, permeabilized, and stained with polyclonal antigeneral ERK antibody (1:100) followed by rhodamine-conjugated secondary antibody (1:100) as in Materials and Methods. Control staining was detected primarily in the cytosol, whereas nuclear staining was detected progressively within 20–120 min of GnRH stimulation. A representative experiment is shown.@1y$', http://www.100md.com
Effect of GnRH, TPA, and ionomycin on -promoter activity@1y$', http://www.100md.com
We first characterized the effect of GnRH-A and its known second messenger modulators, TPA and ionomycin (3, 4, 5, 6, 7, 8), on {alpha} CAT. The {alpha} T3-1 cells were transiently transfected with {alpha} CAT and treated with GnRH-A, TPA, and ionomycin. Maximal activation of {alpha} CAT was observed at 1 nM GnRH-A (3-fold) at 6 h of incubation (Fig. 3, A and B). Both ionomycin (0.1–5 µM) and TPA (1–100 ng/ml) had no significant effect on {alpha} CAT activity at 1–8 h of incubation (Fig. 3, C and D, and data not shown). Down-regulation of TPA-responsive PKCs (28) had no significant effect on stimulation by GnRH-A, whereas removal of Ca2+, with or without EGTA, abolished the response (Fig. 4, A and B). Although TPA had no effect on its own, when added together with GnRH-A, TPA but not ionomycin enhanced the response on {alpha} CAT activity (Fig. 4C). Thus, the signaling pathway used by GnRH for regulating -promoter activity is Ca2+ dependent and is further augmented by a TPA-sensitive PKC subspecies such as a member of a conventional PKC or more likely a novel PKC (28), most likely PKC{delta} and/or PKC{epsilon} , which is activated by GnRH (29) and is now thought to mediate ERK activation by G protein-coupled receptors (GPCRs; Refs. 6 , 8 , 11) that should be confirmed.
fig.ommitteedfig.ommitteedwh|ed(, 百拇医药
Figure 3. Effect of GnRH-A, TPA, and ionomycin on CAT activity. Subconfluent T3-1 cells were transfected with CAT for 8 h, washed several times, and incubated for 24 h. Stimulants were then added for 8 h (A, C, D) or the indicated times (B, GnRH-A, 10 nM). The CAT activity was determined as described in Materials and Methods. Results are expressed as fold increase relative to untreated controls and represent the average of three experiments. **, P < 0.01; ***, P < 0.001.wh|ed(, 百拇医药
fig.ommitteedfig.ommitteedwh|ed(, 百拇医药
Figure 4. Role of Ca2+ and PKC in GnRH-A stimulation of CAT. Subconfluent T3-1 cells were transfected with CAT for 8 h, washed several times, and incubated for 24 h. A, To remove endogenous TPA-sensitive PKCs, cells were down-regulated by including TPA (100 ng/ml) during the 24-h incubation (D-reg). GnRH-A (10 nM) and TPA (100 ng/ml) were then added for 8 h. B, The transfected cells were transferred to normal DMEM, Ca2+-free DMEM, or Ca2+-free DMEM also containing EGTA (0.25 M) and incubated with or without GnRH-A (10 nM) for 8 h. C, The transfected cells were incubated with GnRH-A (10 nM), TPA (100 ng/ml), or ionomycin (1 µM) or with the indicated combinations for 8 h. The CAT activity was determined as described in Materials and Methods. Results are expressed as fold increase relative to untreated controls and represent the average of three experiments. *, P < 0.05; **, P < 0.01.
ERK is partially involved in -subunit promoter activationw1w), 百拇医药
We next examined the role of ERK in basal and GnRH-A-stimulated {alpha} CAT. Cotransfection of the cells with {alpha} CAT and a constitutively active c-Raf, the downstream effector of Ras in ERK signaling, resulted in a 2-fold activation of basal {alpha} CAT activity and a larger response to GnRH-A (Fig. 5A), which seems to result from enhanced ERK activation (not shown). The role of ERK was further examined by the use of the dominant negative mutants of the ERK cascade members Ras, Raf-1, and MEK, with or without GnRH-A. As seen in Fig. 5B, the mutants reduced basal and GnRH-A-stimulated {alpha} CAT activity. Nevertheless, the fold stimulation of the GnRH-A response (14) was reduced by the mutants of Raf-1 and MEK but not by Ras, which was more active on basal expression. The MEK inhibitor PD98059 was more potent and reduced significantly basal and GnRH-A-stimulated {alpha} CAT activity, and the fold stimulation of the GnRH-A response (14) was also markedly inhibited (Fig. 5C). Thus, ERK is partially involved in basal and GnRH-A-stimulated {alpha} CAT activity.
fig.ommitteedfig.ommitteedm+g62gz, 百拇医药
Figure 5. Role of ERK in GnRH-A stimulation of CAT. Subconfluent T3-1 cells were transfected with CAT with or without the constitutively active (CA) form of Raf1 (A) or dominant negative (DN) plasmids of Ras, Raf-1, or MEK (B) for 8 h. Cells were washed several times and incubated for 36 h. GnRH-A (10 nM) was then added for 8 h alone (B) or in the in the presence of the MEK inhibitor PD98059 (50 µM, C), and {alpha} CAT activity was determined as described in Materials and Methods. Results are expressed as fold increase relative to untreated controls and represent the average of three experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.m+g62gz, 百拇医药
The partial inhibition of the GnRH-A response by the mutants suggest the involvement of other signaling pathways or lack of full inhibition of ERK activation by GnRH-A. To that end, we investigated the effect of PD98059 and the mutants used above upon GnRH-stimulated ERK activation. We found that PD98059, at the concentration used for inhibition of GnRH-stimulated {alpha} CAT (50 µM), abolished GnRH-induced ERK activity (22), confirming the relative contribution of ERK to {alpha} CAT. To examine the effect of the mutants on ERK activity, we contransfected the cells with HA-ERK and the mutants, immunoprecipitated HA-ERK, and performed Western blotting with antiphospho-ERK (Fig. 6). Basal and GnRH-A-induced HA-ERK was markedly reduced by the dominant negative mutants of Raf-1, Ras, and MEK; the latter abolished the response. Indeed, PD98059 was more effective than the mutants in inhibition of GnRH-stimulated {alpha} CAT activity. Also the rank order for inhibition of GnRH-A-stimulated {alpha} CAT activity by the mutants (MEK > Raf-1 > Ras) is in agreement with that of HA-ERK. Nevertheless, the data imply that additional signaling pathways are involved in GnRH-regulated {alpha} CAT activity.
fig.ommitteedfig.ommitteede, 百拇医药
Figure 6. Effect of dominant negatives (DN) of Ras, Raf-1, and MEK on GnRH-A-induced HA-ERK activation. Subconfluent {alpha} T3-1 cells were cotransfected with HA-ERK and the DN plasmids of Ras, Raf-1, and MEK. GnRH-A (10 nM) was then added for 7 min. Unstimulated cells served as control. HA-ERK was immunoprecipitated from cell lysates with anti-HA antibodies, and samples from each lysate were resolved by 10% SDS-PAGE and immunoblotted with either antiphospho ERK or anti-HA. Blots were quantified by densitometry (arbitrary units) and are shown in the bar graphs. A representative experiment is shown.e, 百拇医药
JNK is not involved in -subunit promoter activatione, 百拇医药
We next examined the role of JNK in GnRH-A-stimulated {alpha} CAT. Cotransfection of the cells with CAT and constitutively active CDC42 or with the dominant negative mutants of the JNK cascade members Rac, CDC42, and SEK (MKK4) had no significant effect on basal or GnRH-A-stimulated CAT (Fig. 7). Under similar conditions we have recently found that the constitutively active CDC42 stimulated LHßCAT and the dominant negative mutants of Rac, CDC42, and SEK markedly reduced GnRH-A-stimulated LHßCAT activity in LßT-2 cells (26), confirming the effectiveness of the plasmids used. Hence, JNK is not involved in basal or GnRH-A-stimulated -subunit promoter activation.
fig.ommitteedfig.ommitteeda|, http://www.100md.com
Figure 7. Role of JNK in GnRH-A stimulation ofCAT. Subconfluent T3-1 cells were transfected with CAT with or without the constitutively active (CA) form of CDC42 (A) or the dominant negative (DN) plasmids of Rac, CDC42, and SEK (B) for 8 h. Cells were washed several times and incubated for 36 h. GnRH-A (10 nM) was then added for 8 h, and CAT activity was determined as described in Materials and Methods. Results are expressed as fold increase relative to untreated controls and represent the average of three experiments. *, P < 0.05; **, P < 0.01.a|, http://www.100md.com
c-Src is involved in -subunit promoter activationa|, http://www.100md.com
We also examined the role of c-Src in basal and GnRH-A-stimulated {alpha} CAT because the protooncogene is involved in GnRH-stimulated MAPK (16, 22). Cotransfection of the cells with {alpha} CAT and constitutively active c-Src stimulated basal and GnRH-A-stimulated CAT (Fig. 8A). Incubation of the cells with the c-Src selective inhibitor, PP1, or cotransfection of the cells with CAT and dominant negative c-Src has only a slight or no effect on basal {alpha} CAT, unlike the marked inhibition seen with the mutant of Ras (Fig. 5) but markedly reduced the GnRH response as well as the fold stimulation, which went down by 50% (Fig. 8B).
fig.ommitteedfig.ommitteedg:22, http://www.100md.com
Figure 8. Role of c-Src in GnRH-A stimulation of CAT. Subconfluent T3-1 cells were transfected with CAT with or without the constitutively active (CA) form of c-Src (A) or the dominant negative plasmid of c-Src (CSK) for 8 h. Cells were washed several times and incubated for 36 h. GnRH-A (10 nM) was then added for 8 h alone or in cells treated with the c-Src inhibitor PP1 (2 µM). TheCAT activity was determined as described in Materials and Methods. Results are expressed as fold increase relative to untreated controls and represent the average of three experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.g:22, http://www.100md.com
5'-deletion analysis of the -subunit promoterg:22, http://www.100md.com
We then used 5'-deletion analysis (30) to map the basal and GnRH-responsive regions as well as those involved in ERK and c-Src activation (Fig. 9). The cells were transfected with the various {alpha} -promoter deletion mutants linked to LUC (Fig. 9A and Ref. 30) and later incubated with or without GnRH-A in the presence and absence of the selective ERK and c-Src inhibitors as above. Basal h-846{alpha} LUC expression showed a decrease with the first deletion to -420 with no decrease to the second deletion to -346 (Fig. 9B). Basal and GnRH-A-stimulated h-846LUC expression showed a gradual decrease with further deletions to -280, -180, and -156. Thus, basal h-846LUC activity in T3-1 cells is dependent on the -846/-420 region, which is not characterized yet. The basal and GnRH-A response map to the -346/-280 region including the pituitary glycoprotein hormone basal element (PGBE) and basal elements (BE1/2; Refs. 30, 31, 32), the -280/-180 region including the glycoprotein-specific element (GSE; Refs. 33), and the -180/-156 region, which includes the upstream response element (URE; Refs. 31 , 34 , 35). Others have also shown the importance of the CRE for basal {alpha} -subunit expression (31, 36, 37).
fig.ommitteedfig.ommitteed^/p$, 百拇医药
Figure 9. Basal, GnRH, ERK, and c-Src-responsive regions in-promoter deletion mutants. Subconfluent T3-1 cells were transfected with 5'-deletion mutants of h-846LUC (A, Ref. 30 ) for 8 h. Cells were washed several times and incubated for 36 h. GnRH-A (10 nM) was then added for 8 h with (C and D) or without (B) PD98059 (50 µM) or with PP1 (2 µM), and LUC activity was determined as described in Materials and Methods. Results represent the average of three experiments. *, P < 0.05.^/p$, 百拇医药
To explore the regions involved in ERK involvement in h-846LUC activity we looked for the loss of PD98069 inhibition of the response (Fig. 9C). Loss of PD98059 inhibition was mapped to the -346/-280 region including the PGBE and BE1/2, indicating that this region mediates the ERK contribution to the GnRH-A response. Similarly, to explore the c-Src-responsive regions, we followed the loss of PP1 inhibition of the GnRH-A response (Fig. 9D). Surprisingly, the loss of PP1 inhibition did not match the ERK-responsive region as anticipated and mapped to the -280/-180 region, which includes the GSE. We anticipate that sequences, other than the PGBE, BE1/2, and GSE, which are not known as ERK and c-Src response elements, are involved in mediating the contribution of ERK and c-Src to the GnRH response. Further studies using site-specific and block mutants of the human -promoter (31) and gel retardation assays are underway to identify the ERK and c-Src-responsive sequences and the transcription factors involved. Thus, although c-Src is involved in GnRH-stimulated ERK and JNK (16, 22), its contribution to the GnRH effect on the -subunit gene is independent of ERK and JNK activation.
Discussion4, 百拇医药
GnRH is not a major player in pituitary gonadotroph differentiation and proliferation and is not a stress signal; nevertheless, GnRH stimulates a robust activation of JNK (25-fold), ERK (~ 12-fold), and a minor effect on p38 (2- to 3-fold) in T3-1 cells. This robust activation of MAPK by GnRH, which is unique in comparison with other GPCRs (6), suggests that MAPK plays an important role in GnRH actions. We suggest the existence of differentiated functions, which are elicited by GnRH and mediated by the MAPK gene family that should be identified.4, 百拇医药
Activation of ERK by GnRH in {alpha} T3-1 cells involves direct activation of Raf-1 by PKC and Ras activation in a dynamine-dependent manner downstream to c-Src (15, 22). A prevailing dogma in the field of signaling implicates transactivation of epithelial growth factor receptor (EGFR) during MAPK activation by GPCRs (38). Transactivation of EGFR by GPCRs involves activation of a metalloproteinase that cleaves pro-HB-EGF resulting in an autocrine action of EGF on MAPK (38). Indeed, it was reported that activation of ERK by GnRH in {alpha} T3-1 cells is mediated by the EGFR (39). However, we found no evidence for transactivation of EGFR by GnRH in {alpha} T3-1 cells during MAPK activation (22). The reason for the discrepancy is not clear, although it is possible that under different growing conditions, certain cell types are modified and express different repertoires of signaling molecules.
We demonstrate here that ERK is translocated to the nucleus within 20–30 min of GnRH stimulation of {alpha} T3-1 cells. ERK accumulation reaches a peak in 120 min and declines thereafter. The nuclear import and export signal is under investigation. Indeed, the MEK inhibitor PD98059, which blocks ERK activation by GnRH (22), was shown to inhibit GnRH-stimulated mRNA accumulation of {alpha} -subunit and FSHß, but not LHß, in adult male rat pituitary cells (40). Our results that ERK is involved in the transcriptional regulation of an {alpha} -gene, those of Haisenleder et al. (40) at the mRNA level and further studies at the protein level (41) will confirm that ERK is involved in GnRH-induced {alpha} -gene expression.xf?{k, 百拇医药
The role of MAPK in GnRH action is controversial. Some reported that ERK is involved in GnRH-stimulated {alpha} promoter activity (13), whereas others found that ERK is mainly involved in basal -transcription (14). Some reported that PKC and ERK are involved in GnRH regulation of -, but not LHß, which is mediated by Ca2+ (24), whereas others reported that Ca2+ mediates the GnRH effect on and PKC mediates the effect on LHß (42). Similarly, it was reported that PKC and ERK, but not Ca2+, are involved in GnRH regulation of LHß (25). Others found a role for JNK, but not for PKC and ERK, in regulation of LHß by GnRH (21). We and others have recently reported a role for ERK (26, 43) and JNK (26) in the transcriptional regulation of LHß by GnRH. The opposing results can be explained by different cell types, promoters, and species used by the various authors.
We examined here the role of MAPK in GnRH-induced CAT. The results that TPA and ionomycin had no significant effect on CAT presented here are surprising because others have found stimulation by TPA (24, 32). The known effect of TPA on -mRNA accumulation (44, 45, 46) was found mainly to be due to mRNA stabilization (44). Also, others have found a diminished role for PKC in GnRH regulation of {alpha} vs. its role in LHß and FSHß (42). Although we found no direct effect of TPA on CAT activity, the PKC activator, but not the Ca2+ ionophore, enhanced the GnRH response in addition to its effect on -subunit mRNA stability (44). Thus, the signaling pathway used by GnRH for regulating is Ca2+ dependent and further augmented by a TPA-sensitive PKC isoform. Potential candidates are conventional PKCs or more likely members of the novel PKC (28), such as PKC{delta} and/or PKC{epsilon} , which are known to be activated by GnRH (29), and are linked to ERK activation by GPCRs (6, 8, 11).
The role of ERK observed here was demonstrated by activation of Raf-1, inhibition with the selective MEK inhibitor PD98059, and the use of mutants of Raf-1 and MEK. Because PD98059 abolished GnRH-activated ERK (22), its inhibition of GnRH-induced CAT (50%) confirms the role of ERK. Similarly, the rank order for inhibition of GnRH-stimulated CAT by the mutants (MEK > Raf-1 > Ras) is in good agreement with that of HA-ERK inhibition. Nevertheless, because the mutants of Raf-1 and Ras did not abolish GnRH-induced HA-ERK, the results suggest that the contribution of ERK to regulate {alpha} CAT activity should be corrected to the level of inhibition obtained by PD989059. The data suggest that other signaling pathways are also involved in the GnRH response.6hv3q, http://www.100md.com
JNK and p38 were discovered as stress-induced MAPKs, which are activated by UV radiation and cytokines (11). As mentioned above, we and others have found activation of JNK and p38 by GnRH (16, 17, 20, 21, 43). Because c-Jun and activating transcription factor-2 have been implicated in {alpha} -transcription (27) and both are JNK substrates, it was expected that JNK would participate in GnRH action. Therefore, our findings that JNK has no role in the GnRH response are surprising. Nevertheless, JNK is involved in the transcription control of LHß by GnRH (21, 26). It was also shown that p38 is not involved in GnRH stimulation of -subunit gene expression (17).
To identify elements of the -subunit involved in ERK and c-Src activation, we first characterized the basal and GnRH-responsive regions. Using 5'-deletion analysis (30), we found that basal -transcription maps to the -846/-420 region, which has not been characterized yet. The basal and GnRH response map to the -346/-280 region also includes the PGBE (-329/-320), BE1 (-316/-302), BE2 (-296/-285; Ref. 30, 31, 32), the (-280/-180) region including the GSE (33), and the -180/-156 region, which includes the URE (31, 34, 35). Others (31, 36, 37, 47) have also shown the importance of the CRE for basal -transcription. Hence, transcription of the human {alpha} -gene in {alpha} T3-1 cells differs from that in rat pituitary cells in which more proximal sequences (-180/-132) including the TSE, URE-2, -gene activator, and a CRE are also involved (30). The GnRH-responsive regions observed here are in good agreement with those identified in primary cultures of rat pituitary cells (30).l:4}i[, 百拇医药
We then used the 5' deletion analysis and selective inhibitors of ERK and c-Src and reasoned that loss of inhibition of the GnRH response will indicate the removal of an enhancer region. We found that the -346/-280 region, known to include the PGBE and BE1/2, is involved in ERK mediation of the GnRH response. Because this region is involved in basal and GnRH activation of the -gene in rat pituitary cells (30), it is a proper site for ERK mediation. Because two additional regions, which include the GSE and URE, are also responsive to GnRH, it is evident that additional mediators are also involved.
The pLim-Lhx3 homeobox gene is involved in the differentiation of several pituitary cell lineages (48). In addition, pLim-LH2 transactivates gonadotroph basal -subunit by binding to the PGBE (49). Similarly, the {alpha} BE1/2 is also associated with basal -subunit expression (31). Although we cannot rule out the possibility that the PGBE and BE1/2 mediate the ERK effect, it is more likely that other responsive sequences in the -346/-280 region, which should be identified, are involved in ERK activation of -gene transcription. Indeed, Roberson et al. (13) have implicated ERK in the regulation of mouse -gene promoter during GnRH activation and presented preliminary evidence for participation of Elk-1 via a potential Ets factor-binding site. The possibility that ERK acts via the activation of c-jun and c-fos acting on activator protein-1 (AP-1) sites (11), which are important for FSHß expression (50), is also examined, although there are no classical AP-1 sites in the human-promoter. Another potential way for ERK activation of the -gene is via the CRE. The cAMP response element-binding protein can bind the CRE of the human -promoter in T3-1 cells (9, 10) and can be activated by ERK via RSK 1, 2, and 3 (11). However, our 5'-deletion analysis did not identify the CRE as a GnRH- or ERK-responsive region, in agreement with others (30).
We found here, for the first time, a role for c-Src in GnRH-stimulated {alpha} -subunit transcription. Because c-Src is involved in ERK and JNK activation by GnRH (16, 22) and c-Src and ERK can transactivate similar response elements, we anticipated that c-Src contribution will coincide with that of ERK. Surprisingly, we found here that c-Src exerts its effect via a different region, namely the -280/-180 region, which is known to include the GSE. Studies are in progress to identify the specific sequences mediating the ERK and the c-Src responses and identify the transcription factors involved. Moreover, a series of weak elements, which are transactivated by Ca2+ (9, 24, 51), ERK, c-Src, and additional upstream signaling cascades, might act as a composite response element as observed for basal {alpha} -subunit expression (31). The Ca2+-dependent mechanism observed here and previously by others (51) might be involved in ERK (19) and c-Src activation, or act independently, via up-regulation of an unknown factor, which transactivates -promoter (9, 24, 51), and add to the contribution of ERK and c-Src to the GnRH response.
Alterations in the frequency of GnRH pulses and/or the density of GnRHRs are thought to be responsible for differential expression of gonadotropin subunit genes (40, 52). We suggest that differential signaling might also mediate signal specificity during GnRH action (20, 24, 26, 42, 45, 46). We therefore propose that Ca2+, ERK, and c-Src are involved in regulating common , whereas PKC, ERK, JNK, and c-Src are involved in regulating the LHß gene (21, 26, 43 and the present results). PKC and two AP-1 sites were shown to be involved in basal and GnRH regulation of the ovine FSHß promoter (50). The role of MAPK in gonadotropin subunit gene regulation is evolutionarily conserved. The PKC-ERK pathway is now implicated also in GnRH elevation of {alpha} and LHß gene expression, but not FSHß, which is mediated by protein kinase A in primary cultures of tilapia pituitary cells (53).7f@-, 百拇医药
Acknowledgments7f@-, 百拇医药
We thank Dr. O. Benard for his interest and help during the study. We also thank Drs. P. Mellon for the {alpha} T3-1cells and for the human {alpha} CAT promoter and J. L. Jameson for the 5'-deletion mutants.
Received October 25, 2002.5, http://www.100md.com
Accepted for publication October 30, 2002.5, http://www.100md.com
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Gur G, Bonfil D, Safarian H, Naor Z, Yaron Z 2002 GnRH signaling pathways regulate differentially the tilapia gonadotropin subunit genes Mol Cell Endocrinol 189:125–134(Dagan Harris Dana Chuderland David Bonfil Sarah Kraus Rony Seger and Zvi Naor)
Addition of a GnRH agonist (GnRH-A) to {alpha} T3-1 cells stimulates different MAPK cascades: ERK, Jun N-terminal kinase (JNK), and p38. Activation of JNK, ERK, and p38 shows a unique fold activation ratio of 25:12:2, which might encode signal specificity. ERK is translocated to the nucleus within 20 min with a peak at 120 min of GnRH-A stimulation. We used the human -subunit promoter linked to chloramphenicol acetyl transferase (CAT) to examine the role of ERK, JNK, and c-Src, which is implicated in MAPK activation, in basal and GnRH-stimulated CAT. Addition of GnRH-A resulted in a 3-fold increase in CAT, whereas the Ca2+ ionophore ionomycin and the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol-13-acetate (TPA) had no effect. Addition of GnRH-A and TPA, but not GnRH-A and ionomycin, produced a synergistic response, whereas removal of Ca2+, but not down-regulation of TPA-sensitive PKCs, abolished GnRH-A-stimulated {alpha} CAT. Thus, regulation of -promoter activity by GnRH is Ca2+ dependent and is further augmented by PKC. Cotransfection of CAT and constitutively active or dominant negative plasmids of ERK and JNK cascade members, or the use of the ERK inhibitor PD98059, revealed that ERK, but not JNK, is involved in basal and GnRH-A-stimulated {alpha} CAT. Because c-Src participates in MAPK activation by GnRH, we also studied its role. Cotransfection of {alpha} CAT and the dominant negative form of c-Src or incubation with the c-Src inhibitor PP1 reduced GnRH-A-stimulated {alpha} CAT. The 5'-deletion analysis revealed that the -846/-420 region participated in basal {alpha} -transcription. In addition, the -346/-156 region containing the pituitary glycoprotein hormone basal element, {alpha} -basal elements, glycoprotein-specific element, and upstream response element is involved in basal and GnRH-A-stimulated {alpha} CAT. ERK contribution to GnRH maps to -346/-280 containing the pituitary glycoprotein hormone basal element and {alpha} -basal elements 1/2. Surprisingly, although c-Src is involved in GnRH-A-stimulated ERK, its involvement is mapped to another region (-280/-180) containing the glycoprotein-specific element. Thus, ERK and c-Src but not JNK are involved in basal and GnRH-A-stimulated-{alpha} CAT, whereas c-Src contribution is independent of ERK activation.
Introductionwdc, 百拇医药
GnRH STIMULATES THE release and synthesis of the heterodimeric glycoprotein hormones LH and FSH from pituitary gonadotrophs. Common to pituitary glycoprotein hormones LH, FSH, and TSH is the {alpha} -subunit, which binds noncovalently to a specific ß-subunit (1, 2). Signaling by the GnRH receptor (GnRHR) involves rapid and sequential stimulation of the phospholipases C and D, Ca2+ mobilization and influx, activation of protein kinase C (PKC) subspecies, and the MAPK cascades ERK, Jun N-terminal kinase (JNK), and p38 and release of arachidonic acid and formation of bioactive lipoxygenase products (3, 4, 5, 6, 7, 8). Although GnRHR activates a large array of signaling molecules, the mechanisms involved in gonadotropin synthesis have not yet been clarified (2, 9, 10).wdc, 百拇医药
Each of the MAPK signaling cascades consists of up to five tiers of protein kinases, which sequentially activate each other by phosphorylation (6, 11). At present, four distinct MAPK cascades have been identified in mammals. They have been named according to their respective MAPK components: the p42 and p44 MAPKs, also known as ERK1/2; JNK1/3; p38, ß, and ; and ERK5 (6, 11).
A small G protein of the Ras family often initiates the cascade. The signal is then transmitted to the MAPK kinase (MAPK/ERK kinase, MEK) kinase (MAP3K, MEK kinase, MEKK), followed by activation of MEK and MAPK. Raf kinases often serve as the MEKK in the ERK cascade, and on activation the signal is transmitted to MEK and ERK. In the JNK cascade, MEKK1 is the most common kinase at the MAP3K level followed by MKK4/7 and JNK1–3 (6, 11). The unique property of MAPKs to translocate to the nucleus, phosphorylate, and activate transcription factors implies a potential role for MAPK in transcription.$?, http://www.100md.com
GnRH stimulates different MAPK cascades: ERK, JNK, and p38 in pituitary and GnRHR-transfected cells (Refs. 6 , 8 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 and present results). The role of MAPKs in GnRHstimulation of common {alpha} -subunit gene transcription is not clear. Although some reports claim that ERK is involved in basal as well as GnRH-stimulated {alpha} -transcription (13, 24), others reported that ERK is involved mainly in the regulation of the basal but not the GnRH response (14). Controversy also exists concerning the role of ERK in GnRH regulation of LHß gene expression. We and others reported a positive role for ERK (25, 26), whereas others have found no role for ERK in the transcriptional regulation of LHß by GnRH (21, 24).
Activation of JNK by GnRH (16, 20, 21) was expected to be important in {alpha} -transcription, because Jun and activating transcription factor-2, known substrates of JNK, have been shown to contribute to basal -transcription via binding to the cAMP response element (CRE; Ref. 27). We therefore decided to investigate the role of MAPKs in the transcriptional control of gonadotropin subunit genes by GnRH. First, we demonstrated that both ERK and JNK mediate the GnRH response on LHß (26). Here we show that Ca2+, ERK, and c-Src, but not JNK, are involved in transcriptional regulation of the human -subunit gene by GnRH. 5'-Deletion analysis has identified four regions involved in basal activation, out of which three are also involved in the GnRH response. Of these, one is mediated by ERK and, surprisingly, a different one by c-Src. Although c-Src is involved in GnRH-stimulated MAPK activation (16, 22), this first protooncogene and nonreceptor tyrosine kinase mediates the {alpha} -gene transcriptional response by GnRH in ERK- and JNK-independent mechanism.
Materials and Methodsa3, 百拇医药
Materialsa3, 百拇医药
A stable GnRH agonist, [D-Trp (6)]-GnRH (GnRH-A), ionomycin, and 12-O-tetradecanoylphorbol-13-acetate (TPA) were purchased from Sigma (St. Louis, MO). Selective inhibitors for ERK (PD 98059) and c-Src (PP1) were from Calbiochem (La Jolla, CA). Mouse monoclonal antiactive and rabbit polyclonal anti-general MAPKs (ERK, JNK, and p38), protein A-Sepharose, and rhodamine-conjugated goat-antirabbit IgG were purchased from Sigma (Rehovot, Israel). Polyclonal anti-hemagglutinin (HA) antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The plasmids for the various ERK and JNK cascade members were recently described (16, 22). The human {alpha} -subunit promoter linked to chloramphenicol acetyl transferase (CAT) was kindly provided by Dr. P. Mellon (University of California, San Diego, CA), and 5' deletion mutants of h-846{alpha} LUC were kindly provided by Dr. J. L. Jameson (Northwestern University Medical School, Chicago, IL).
Activation of MAPK cascadesd^+*{0, http://www.100md.com
The {alpha} T3-1 cells were grown in 6-well plates and serum starved [0.1% fetal calf serum (FCS)] for 16 h. After stimulation, the cells were washed twice with ice-cold PBS and once with ice-cold buffer A: 50 mM ß-glycerophosphate, 1.5 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, and 1 mM sodium orthovanadate. Cells were harvested in 0.3 ml buffer H: buffer A containing 1 mM benzamidine, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride, followed by sonication (2 x 7 sec, 40 W) and centrifugation (15,000 x g, 15 min, 4 C). The supernatants, which contained cytosolic proteins, were collected, and aliquots from each sample (20 µg) were separated on 10% SDS-PAGE followed by Western blotting with mouse monoclonal anti-active MAPKs (ERK, JNK, and p38; Ref. 22). Total MAPKs were detected with polyclonal antibodies for the various MAPKs as a control. The blots were developed with alkaline phosphatase or horseradish peroxidase-conjugated antimouse or antirabbit Fab antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). The blots were autoradiographed on X-100 films (Kodak, Rochester, NY), and the phosphorylation was quantitated by densitometry (690 densitometer, Bio-Rad Laboratories, Inc., Hercules, CA).
Cell culture and transfection.5]y;e, 百拇医药
Subconfluent {alpha} T3-1 cells were cotransfected for 8 h with 5 µg each {alpha} CAT or 5' deletion mutants linked to luciferase (LUC), with or without the CA or the DN mutants of MAPK cascade members, using the calcium phosphate method. The total amount of plasmid was adjusted to 20 µg with vector DNA in control experiments. The transfection efficiency was 10–30%, as determined by transfection with a plasmid that contained Rous sarcoma virus-LUC. Then 24–36 h after transfection, the cells were serum starved for 16 h and incubated with GnRH-A or other stimulant as described in the figure legends. Cells were then harvested and the cell extracts were analyzed for CAT or LUC activities (26), which were normalized to the level of Rous sarcoma virus-LUC activity where appropriate. Results were then calculated as fold increase relative to expression in control wells, and the data are shown as the mean ± SEM. ANOVA was used to determine statistically different groups; significance was set at P < 0.05: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.
Immunoprecipitation{, 百拇医药
Subconfluent T3-1 cells were cotransfected with 10 µg HA-tagged wild-type ERK2 (HA-ERK) and 10 µg of the mutants to be analyzed. Subsequently, the cells were serum starved (0.1% FCS, 16 h) and stimulated with GnRH-A (10 nM) for 7 min. Following stimulation the cells were washed, lysed as above, and ERK immunoprecipitated with polyclonal anti-HA antibodies using protein A-Sepharose (30 µl, Sigma). Cell lysates (500 µg protein) were incubated with the conjugated beads and rotated end to end for 2 h at 4 C. Following incubation the beads were washed once with 1 ml RIPA assay buffer [137 mM NaCl, 20 mM Tris at pH 7.4, 10% (vol/vol) glycerol, 1% Triton X-100, 0.5% (wt/vol) deoxycholate, 0.1% (wt/vol) SDS, 2.0 mM EDTA, 1.0 mM phenylmethylsulfonyl fluoride, and 20 µM leupeptin]; twice with 1 ml LiCl, 0.5 mM in 100 mM Tris at pH 8.0; and twice with 1 ml buffer A. The immunoprecipitates were subjected to 10% SDS-PAGE and Western blotting using the monoclonal anti-active ERK antibody. Total HA-ERK was detected using a polyclonal anti-HA antibody. The blots were developed as described above.
Fluorescent cell staining&+umv5, http://www.100md.com
Subconfluent T3-1 cells were serum starved (16 h, 0.1% FCS) and treated with GnRH-A for various time points. Subsequently, the cells were washed with PBS, fixed in 3% paraformaldehyde in PBS, and permeabilized with 0.2% Triton X-100 in PBS for 5 min. Staining was performed by 45-min incubation with polyclonal antigeneral ERK antibody (diluted 1:100), followed by 45-min incubation with rhodamine-conjugated goat-antirabbit IgG (diluted 1:100). Fluorescent staining was viewed with a fluorescent microscope (EFD-3, Nikon, Melville, NY).&+umv5, http://www.100md.com
Results&+umv5, http://www.100md.com
Effect of GnRH-A on MAPK activity&+umv5, http://www.100md.com
To study the activation of MAPKs in response to GnRH, we used antigeneral MAPK antibodies and also monoclonal antibodies, which specifically recognize the doubly phosphorylated, active forms of the various MAPKs (Ref. 22 ; Fig. 1A). The T3-1 cells were treated with GnRH-A for different time periods, and cell lysates were subsequently prepared. A Western blot analysis was carried out using total cell lysates and the antibodies as above. Each band from the antiphospho MAPK (ERK, JNK, and p38) was normalized to the corresponding band from the antigeneral MAPK antibodies blot for even loading. The results indicated that ERK, JNK, and p38 were activated on GnRH-A stimulation. JNK1 was activated (~ 25-fold) similar to previous results using glutathione-S-transferase-Jun as a substrate (16), and a more recent report (19), peaking at 30–45 min and declining thereafter. ERKs were activated (~ 12-fold), similar to previous observations using myelin basic protein as a substrate (15), and more recent reports (18, 19, 20). ERK activation, which peaked at 15 min, returned to basal levels at 60–90 min. The p38 was phosphorylated only to a small extent (2- to 3-fold), in agreement with others (17). Activation was transient and peaked at 30 min after stimulation with GnRH. Under the same conditions, the selective inhibitor for MEK (PD98059, 50 µM) abolished GnRH-A-stimulated ERK but not JNK or p38 activation (Ref. 22 and Fig. 1B). The protein tyrosine kinase inhibitor genistein produced a 40% and 80% inhibition on ERK and JNK activation, respectively (Ref. 22 and Fig. 1B). The PKC selective inhibitor GF109203X inhibited GnRH-A-stimulated ERK, JNK, and p38 activation by 70%, 50%, and 50%, respectively (Ref. 22 and Fig. 1B). The c-Src selective inhibitor PP1 (2 µM) reduced ERK activation by GnRH-A by 50% at 15 min of incubation (22).
fig.ommitteedfig.ommitteed&-!4n, http://www.100md.com
Figure 1. Effect of GnRH-A on MAPK activity in T3-1 cells. Subconfluent T3-1 cells were treated with GnRH-A (100 nM) in the absence (A) or presence (B) of PD98059 (50 µM), GF109203X (5 µM), or genistein (200 µM) for the indicated times, and cell lysates were subsequently prepared. MAPK (ERK, JNK, and p38) activity was determined by Western blotting with type-specific antibodies for phospho-MAPK (active form) and anti-MAPK antibodies. Each band form the antiphospho MAPK was normalized to the corresponding band from the anti-MAPK blot for even loading. Fold stimulation was determined by densitometry as described in Materials and Methods. Inhibition of activated ERK by the drugs was shown elsewhere (22 ), and representative experiments are shown here, whereas similar results were observed in two other experiments (n = 3, SEM 10%).&-!4n, http://www.100md.com
Effect of GnRH-A on ERK translocation&-!4n, http://www.100md.com
To study the effect of GnRH-A on the subcellular localization of ERK, {alpha} T3-1 cells were treated with or without GnRH-A and later stained with the general polyclonal anti-MAPK antibody (Fig. 2). The staining of nonstimulated cells with this antibody was detected primarily in the cytosol. GnRH-A treatment of {alpha} T3-1 cells induced significant translocation of ERK to the nucleus. This nuclear staining was detected first after 20 min of GnRH-A stimulation and continued progressively to reach a peak at 120 min, in which all cells examined were stained. Hence, ERK can mediate nuclear transcriptional responses. The nuclear import and export signal is under investigation. Here we focused on the contribution of ERK, JNK, and c-Src to transcription from the common {alpha} -subunit promoter, whereas others (17) have shown that p38 is not involved in this process.
fig.ommitteedfig.ommitteed@1y$', http://www.100md.com
Figure 2. Effect of GnRH-A on ERK translocation in T3-1 cells. Subconfluent T3-1 cells were serum starved (16 h, 0.1% FCS) and treated in the absence (A) or presence of GnRH-A (100 nM) for 60 min (B). Following stimulation, the cells were washed, fixed, permeabilized, and stained with polyclonal antigeneral ERK antibody (1:100) followed by rhodamine-conjugated secondary antibody (1:100) as in Materials and Methods. Control staining was detected primarily in the cytosol, whereas nuclear staining was detected progressively within 20–120 min of GnRH stimulation. A representative experiment is shown.@1y$', http://www.100md.com
Effect of GnRH, TPA, and ionomycin on -promoter activity@1y$', http://www.100md.com
We first characterized the effect of GnRH-A and its known second messenger modulators, TPA and ionomycin (3, 4, 5, 6, 7, 8), on {alpha} CAT. The {alpha} T3-1 cells were transiently transfected with {alpha} CAT and treated with GnRH-A, TPA, and ionomycin. Maximal activation of {alpha} CAT was observed at 1 nM GnRH-A (3-fold) at 6 h of incubation (Fig. 3, A and B). Both ionomycin (0.1–5 µM) and TPA (1–100 ng/ml) had no significant effect on {alpha} CAT activity at 1–8 h of incubation (Fig. 3, C and D, and data not shown). Down-regulation of TPA-responsive PKCs (28) had no significant effect on stimulation by GnRH-A, whereas removal of Ca2+, with or without EGTA, abolished the response (Fig. 4, A and B). Although TPA had no effect on its own, when added together with GnRH-A, TPA but not ionomycin enhanced the response on {alpha} CAT activity (Fig. 4C). Thus, the signaling pathway used by GnRH for regulating -promoter activity is Ca2+ dependent and is further augmented by a TPA-sensitive PKC subspecies such as a member of a conventional PKC or more likely a novel PKC (28), most likely PKC{delta} and/or PKC{epsilon} , which is activated by GnRH (29) and is now thought to mediate ERK activation by G protein-coupled receptors (GPCRs; Refs. 6 , 8 , 11) that should be confirmed.
fig.ommitteedfig.ommitteedwh|ed(, 百拇医药
Figure 3. Effect of GnRH-A, TPA, and ionomycin on CAT activity. Subconfluent T3-1 cells were transfected with CAT for 8 h, washed several times, and incubated for 24 h. Stimulants were then added for 8 h (A, C, D) or the indicated times (B, GnRH-A, 10 nM). The CAT activity was determined as described in Materials and Methods. Results are expressed as fold increase relative to untreated controls and represent the average of three experiments. **, P < 0.01; ***, P < 0.001.wh|ed(, 百拇医药
fig.ommitteedfig.ommitteedwh|ed(, 百拇医药
Figure 4. Role of Ca2+ and PKC in GnRH-A stimulation of CAT. Subconfluent T3-1 cells were transfected with CAT for 8 h, washed several times, and incubated for 24 h. A, To remove endogenous TPA-sensitive PKCs, cells were down-regulated by including TPA (100 ng/ml) during the 24-h incubation (D-reg). GnRH-A (10 nM) and TPA (100 ng/ml) were then added for 8 h. B, The transfected cells were transferred to normal DMEM, Ca2+-free DMEM, or Ca2+-free DMEM also containing EGTA (0.25 M) and incubated with or without GnRH-A (10 nM) for 8 h. C, The transfected cells were incubated with GnRH-A (10 nM), TPA (100 ng/ml), or ionomycin (1 µM) or with the indicated combinations for 8 h. The CAT activity was determined as described in Materials and Methods. Results are expressed as fold increase relative to untreated controls and represent the average of three experiments. *, P < 0.05; **, P < 0.01.
ERK is partially involved in -subunit promoter activationw1w), 百拇医药
We next examined the role of ERK in basal and GnRH-A-stimulated {alpha} CAT. Cotransfection of the cells with {alpha} CAT and a constitutively active c-Raf, the downstream effector of Ras in ERK signaling, resulted in a 2-fold activation of basal {alpha} CAT activity and a larger response to GnRH-A (Fig. 5A), which seems to result from enhanced ERK activation (not shown). The role of ERK was further examined by the use of the dominant negative mutants of the ERK cascade members Ras, Raf-1, and MEK, with or without GnRH-A. As seen in Fig. 5B, the mutants reduced basal and GnRH-A-stimulated {alpha} CAT activity. Nevertheless, the fold stimulation of the GnRH-A response (14) was reduced by the mutants of Raf-1 and MEK but not by Ras, which was more active on basal expression. The MEK inhibitor PD98059 was more potent and reduced significantly basal and GnRH-A-stimulated {alpha} CAT activity, and the fold stimulation of the GnRH-A response (14) was also markedly inhibited (Fig. 5C). Thus, ERK is partially involved in basal and GnRH-A-stimulated {alpha} CAT activity.
fig.ommitteedfig.ommitteedm+g62gz, 百拇医药
Figure 5. Role of ERK in GnRH-A stimulation of CAT. Subconfluent T3-1 cells were transfected with CAT with or without the constitutively active (CA) form of Raf1 (A) or dominant negative (DN) plasmids of Ras, Raf-1, or MEK (B) for 8 h. Cells were washed several times and incubated for 36 h. GnRH-A (10 nM) was then added for 8 h alone (B) or in the in the presence of the MEK inhibitor PD98059 (50 µM, C), and {alpha} CAT activity was determined as described in Materials and Methods. Results are expressed as fold increase relative to untreated controls and represent the average of three experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.m+g62gz, 百拇医药
The partial inhibition of the GnRH-A response by the mutants suggest the involvement of other signaling pathways or lack of full inhibition of ERK activation by GnRH-A. To that end, we investigated the effect of PD98059 and the mutants used above upon GnRH-stimulated ERK activation. We found that PD98059, at the concentration used for inhibition of GnRH-stimulated {alpha} CAT (50 µM), abolished GnRH-induced ERK activity (22), confirming the relative contribution of ERK to {alpha} CAT. To examine the effect of the mutants on ERK activity, we contransfected the cells with HA-ERK and the mutants, immunoprecipitated HA-ERK, and performed Western blotting with antiphospho-ERK (Fig. 6). Basal and GnRH-A-induced HA-ERK was markedly reduced by the dominant negative mutants of Raf-1, Ras, and MEK; the latter abolished the response. Indeed, PD98059 was more effective than the mutants in inhibition of GnRH-stimulated {alpha} CAT activity. Also the rank order for inhibition of GnRH-A-stimulated {alpha} CAT activity by the mutants (MEK > Raf-1 > Ras) is in agreement with that of HA-ERK. Nevertheless, the data imply that additional signaling pathways are involved in GnRH-regulated {alpha} CAT activity.
fig.ommitteedfig.ommitteede, 百拇医药
Figure 6. Effect of dominant negatives (DN) of Ras, Raf-1, and MEK on GnRH-A-induced HA-ERK activation. Subconfluent {alpha} T3-1 cells were cotransfected with HA-ERK and the DN plasmids of Ras, Raf-1, and MEK. GnRH-A (10 nM) was then added for 7 min. Unstimulated cells served as control. HA-ERK was immunoprecipitated from cell lysates with anti-HA antibodies, and samples from each lysate were resolved by 10% SDS-PAGE and immunoblotted with either antiphospho ERK or anti-HA. Blots were quantified by densitometry (arbitrary units) and are shown in the bar graphs. A representative experiment is shown.e, 百拇医药
JNK is not involved in -subunit promoter activatione, 百拇医药
We next examined the role of JNK in GnRH-A-stimulated {alpha} CAT. Cotransfection of the cells with CAT and constitutively active CDC42 or with the dominant negative mutants of the JNK cascade members Rac, CDC42, and SEK (MKK4) had no significant effect on basal or GnRH-A-stimulated CAT (Fig. 7). Under similar conditions we have recently found that the constitutively active CDC42 stimulated LHßCAT and the dominant negative mutants of Rac, CDC42, and SEK markedly reduced GnRH-A-stimulated LHßCAT activity in LßT-2 cells (26), confirming the effectiveness of the plasmids used. Hence, JNK is not involved in basal or GnRH-A-stimulated -subunit promoter activation.
fig.ommitteedfig.ommitteeda|, http://www.100md.com
Figure 7. Role of JNK in GnRH-A stimulation ofCAT. Subconfluent T3-1 cells were transfected with CAT with or without the constitutively active (CA) form of CDC42 (A) or the dominant negative (DN) plasmids of Rac, CDC42, and SEK (B) for 8 h. Cells were washed several times and incubated for 36 h. GnRH-A (10 nM) was then added for 8 h, and CAT activity was determined as described in Materials and Methods. Results are expressed as fold increase relative to untreated controls and represent the average of three experiments. *, P < 0.05; **, P < 0.01.a|, http://www.100md.com
c-Src is involved in -subunit promoter activationa|, http://www.100md.com
We also examined the role of c-Src in basal and GnRH-A-stimulated {alpha} CAT because the protooncogene is involved in GnRH-stimulated MAPK (16, 22). Cotransfection of the cells with {alpha} CAT and constitutively active c-Src stimulated basal and GnRH-A-stimulated CAT (Fig. 8A). Incubation of the cells with the c-Src selective inhibitor, PP1, or cotransfection of the cells with CAT and dominant negative c-Src has only a slight or no effect on basal {alpha} CAT, unlike the marked inhibition seen with the mutant of Ras (Fig. 5) but markedly reduced the GnRH response as well as the fold stimulation, which went down by 50% (Fig. 8B).
fig.ommitteedfig.ommitteedg:22, http://www.100md.com
Figure 8. Role of c-Src in GnRH-A stimulation of CAT. Subconfluent T3-1 cells were transfected with CAT with or without the constitutively active (CA) form of c-Src (A) or the dominant negative plasmid of c-Src (CSK) for 8 h. Cells were washed several times and incubated for 36 h. GnRH-A (10 nM) was then added for 8 h alone or in cells treated with the c-Src inhibitor PP1 (2 µM). TheCAT activity was determined as described in Materials and Methods. Results are expressed as fold increase relative to untreated controls and represent the average of three experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.g:22, http://www.100md.com
5'-deletion analysis of the -subunit promoterg:22, http://www.100md.com
We then used 5'-deletion analysis (30) to map the basal and GnRH-responsive regions as well as those involved in ERK and c-Src activation (Fig. 9). The cells were transfected with the various {alpha} -promoter deletion mutants linked to LUC (Fig. 9A and Ref. 30) and later incubated with or without GnRH-A in the presence and absence of the selective ERK and c-Src inhibitors as above. Basal h-846{alpha} LUC expression showed a decrease with the first deletion to -420 with no decrease to the second deletion to -346 (Fig. 9B). Basal and GnRH-A-stimulated h-846LUC expression showed a gradual decrease with further deletions to -280, -180, and -156. Thus, basal h-846LUC activity in T3-1 cells is dependent on the -846/-420 region, which is not characterized yet. The basal and GnRH-A response map to the -346/-280 region including the pituitary glycoprotein hormone basal element (PGBE) and basal elements (BE1/2; Refs. 30, 31, 32), the -280/-180 region including the glycoprotein-specific element (GSE; Refs. 33), and the -180/-156 region, which includes the upstream response element (URE; Refs. 31 , 34 , 35). Others have also shown the importance of the CRE for basal {alpha} -subunit expression (31, 36, 37).
fig.ommitteedfig.ommitteed^/p$, 百拇医药
Figure 9. Basal, GnRH, ERK, and c-Src-responsive regions in-promoter deletion mutants. Subconfluent T3-1 cells were transfected with 5'-deletion mutants of h-846LUC (A, Ref. 30 ) for 8 h. Cells were washed several times and incubated for 36 h. GnRH-A (10 nM) was then added for 8 h with (C and D) or without (B) PD98059 (50 µM) or with PP1 (2 µM), and LUC activity was determined as described in Materials and Methods. Results represent the average of three experiments. *, P < 0.05.^/p$, 百拇医药
To explore the regions involved in ERK involvement in h-846LUC activity we looked for the loss of PD98069 inhibition of the response (Fig. 9C). Loss of PD98059 inhibition was mapped to the -346/-280 region including the PGBE and BE1/2, indicating that this region mediates the ERK contribution to the GnRH-A response. Similarly, to explore the c-Src-responsive regions, we followed the loss of PP1 inhibition of the GnRH-A response (Fig. 9D). Surprisingly, the loss of PP1 inhibition did not match the ERK-responsive region as anticipated and mapped to the -280/-180 region, which includes the GSE. We anticipate that sequences, other than the PGBE, BE1/2, and GSE, which are not known as ERK and c-Src response elements, are involved in mediating the contribution of ERK and c-Src to the GnRH response. Further studies using site-specific and block mutants of the human -promoter (31) and gel retardation assays are underway to identify the ERK and c-Src-responsive sequences and the transcription factors involved. Thus, although c-Src is involved in GnRH-stimulated ERK and JNK (16, 22), its contribution to the GnRH effect on the -subunit gene is independent of ERK and JNK activation.
Discussion4, 百拇医药
GnRH is not a major player in pituitary gonadotroph differentiation and proliferation and is not a stress signal; nevertheless, GnRH stimulates a robust activation of JNK (25-fold), ERK (~ 12-fold), and a minor effect on p38 (2- to 3-fold) in T3-1 cells. This robust activation of MAPK by GnRH, which is unique in comparison with other GPCRs (6), suggests that MAPK plays an important role in GnRH actions. We suggest the existence of differentiated functions, which are elicited by GnRH and mediated by the MAPK gene family that should be identified.4, 百拇医药
Activation of ERK by GnRH in {alpha} T3-1 cells involves direct activation of Raf-1 by PKC and Ras activation in a dynamine-dependent manner downstream to c-Src (15, 22). A prevailing dogma in the field of signaling implicates transactivation of epithelial growth factor receptor (EGFR) during MAPK activation by GPCRs (38). Transactivation of EGFR by GPCRs involves activation of a metalloproteinase that cleaves pro-HB-EGF resulting in an autocrine action of EGF on MAPK (38). Indeed, it was reported that activation of ERK by GnRH in {alpha} T3-1 cells is mediated by the EGFR (39). However, we found no evidence for transactivation of EGFR by GnRH in {alpha} T3-1 cells during MAPK activation (22). The reason for the discrepancy is not clear, although it is possible that under different growing conditions, certain cell types are modified and express different repertoires of signaling molecules.
We demonstrate here that ERK is translocated to the nucleus within 20–30 min of GnRH stimulation of {alpha} T3-1 cells. ERK accumulation reaches a peak in 120 min and declines thereafter. The nuclear import and export signal is under investigation. Indeed, the MEK inhibitor PD98059, which blocks ERK activation by GnRH (22), was shown to inhibit GnRH-stimulated mRNA accumulation of {alpha} -subunit and FSHß, but not LHß, in adult male rat pituitary cells (40). Our results that ERK is involved in the transcriptional regulation of an {alpha} -gene, those of Haisenleder et al. (40) at the mRNA level and further studies at the protein level (41) will confirm that ERK is involved in GnRH-induced {alpha} -gene expression.xf?{k, 百拇医药
The role of MAPK in GnRH action is controversial. Some reported that ERK is involved in GnRH-stimulated {alpha} promoter activity (13), whereas others found that ERK is mainly involved in basal -transcription (14). Some reported that PKC and ERK are involved in GnRH regulation of -, but not LHß, which is mediated by Ca2+ (24), whereas others reported that Ca2+ mediates the GnRH effect on and PKC mediates the effect on LHß (42). Similarly, it was reported that PKC and ERK, but not Ca2+, are involved in GnRH regulation of LHß (25). Others found a role for JNK, but not for PKC and ERK, in regulation of LHß by GnRH (21). We and others have recently reported a role for ERK (26, 43) and JNK (26) in the transcriptional regulation of LHß by GnRH. The opposing results can be explained by different cell types, promoters, and species used by the various authors.
We examined here the role of MAPK in GnRH-induced CAT. The results that TPA and ionomycin had no significant effect on CAT presented here are surprising because others have found stimulation by TPA (24, 32). The known effect of TPA on -mRNA accumulation (44, 45, 46) was found mainly to be due to mRNA stabilization (44). Also, others have found a diminished role for PKC in GnRH regulation of {alpha} vs. its role in LHß and FSHß (42). Although we found no direct effect of TPA on CAT activity, the PKC activator, but not the Ca2+ ionophore, enhanced the GnRH response in addition to its effect on -subunit mRNA stability (44). Thus, the signaling pathway used by GnRH for regulating is Ca2+ dependent and further augmented by a TPA-sensitive PKC isoform. Potential candidates are conventional PKCs or more likely members of the novel PKC (28), such as PKC{delta} and/or PKC{epsilon} , which are known to be activated by GnRH (29), and are linked to ERK activation by GPCRs (6, 8, 11).
The role of ERK observed here was demonstrated by activation of Raf-1, inhibition with the selective MEK inhibitor PD98059, and the use of mutants of Raf-1 and MEK. Because PD98059 abolished GnRH-activated ERK (22), its inhibition of GnRH-induced CAT (50%) confirms the role of ERK. Similarly, the rank order for inhibition of GnRH-stimulated CAT by the mutants (MEK > Raf-1 > Ras) is in good agreement with that of HA-ERK inhibition. Nevertheless, because the mutants of Raf-1 and Ras did not abolish GnRH-induced HA-ERK, the results suggest that the contribution of ERK to regulate {alpha} CAT activity should be corrected to the level of inhibition obtained by PD989059. The data suggest that other signaling pathways are also involved in the GnRH response.6hv3q, http://www.100md.com
JNK and p38 were discovered as stress-induced MAPKs, which are activated by UV radiation and cytokines (11). As mentioned above, we and others have found activation of JNK and p38 by GnRH (16, 17, 20, 21, 43). Because c-Jun and activating transcription factor-2 have been implicated in {alpha} -transcription (27) and both are JNK substrates, it was expected that JNK would participate in GnRH action. Therefore, our findings that JNK has no role in the GnRH response are surprising. Nevertheless, JNK is involved in the transcription control of LHß by GnRH (21, 26). It was also shown that p38 is not involved in GnRH stimulation of -subunit gene expression (17).
To identify elements of the -subunit involved in ERK and c-Src activation, we first characterized the basal and GnRH-responsive regions. Using 5'-deletion analysis (30), we found that basal -transcription maps to the -846/-420 region, which has not been characterized yet. The basal and GnRH response map to the -346/-280 region also includes the PGBE (-329/-320), BE1 (-316/-302), BE2 (-296/-285; Ref. 30, 31, 32), the (-280/-180) region including the GSE (33), and the -180/-156 region, which includes the URE (31, 34, 35). Others (31, 36, 37, 47) have also shown the importance of the CRE for basal -transcription. Hence, transcription of the human {alpha} -gene in {alpha} T3-1 cells differs from that in rat pituitary cells in which more proximal sequences (-180/-132) including the TSE, URE-2, -gene activator, and a CRE are also involved (30). The GnRH-responsive regions observed here are in good agreement with those identified in primary cultures of rat pituitary cells (30).l:4}i[, 百拇医药
We then used the 5' deletion analysis and selective inhibitors of ERK and c-Src and reasoned that loss of inhibition of the GnRH response will indicate the removal of an enhancer region. We found that the -346/-280 region, known to include the PGBE and BE1/2, is involved in ERK mediation of the GnRH response. Because this region is involved in basal and GnRH activation of the -gene in rat pituitary cells (30), it is a proper site for ERK mediation. Because two additional regions, which include the GSE and URE, are also responsive to GnRH, it is evident that additional mediators are also involved.
The pLim-Lhx3 homeobox gene is involved in the differentiation of several pituitary cell lineages (48). In addition, pLim-LH2 transactivates gonadotroph basal -subunit by binding to the PGBE (49). Similarly, the {alpha} BE1/2 is also associated with basal -subunit expression (31). Although we cannot rule out the possibility that the PGBE and BE1/2 mediate the ERK effect, it is more likely that other responsive sequences in the -346/-280 region, which should be identified, are involved in ERK activation of -gene transcription. Indeed, Roberson et al. (13) have implicated ERK in the regulation of mouse -gene promoter during GnRH activation and presented preliminary evidence for participation of Elk-1 via a potential Ets factor-binding site. The possibility that ERK acts via the activation of c-jun and c-fos acting on activator protein-1 (AP-1) sites (11), which are important for FSHß expression (50), is also examined, although there are no classical AP-1 sites in the human-promoter. Another potential way for ERK activation of the -gene is via the CRE. The cAMP response element-binding protein can bind the CRE of the human -promoter in T3-1 cells (9, 10) and can be activated by ERK via RSK 1, 2, and 3 (11). However, our 5'-deletion analysis did not identify the CRE as a GnRH- or ERK-responsive region, in agreement with others (30).
We found here, for the first time, a role for c-Src in GnRH-stimulated {alpha} -subunit transcription. Because c-Src is involved in ERK and JNK activation by GnRH (16, 22) and c-Src and ERK can transactivate similar response elements, we anticipated that c-Src contribution will coincide with that of ERK. Surprisingly, we found here that c-Src exerts its effect via a different region, namely the -280/-180 region, which is known to include the GSE. Studies are in progress to identify the specific sequences mediating the ERK and the c-Src responses and identify the transcription factors involved. Moreover, a series of weak elements, which are transactivated by Ca2+ (9, 24, 51), ERK, c-Src, and additional upstream signaling cascades, might act as a composite response element as observed for basal {alpha} -subunit expression (31). The Ca2+-dependent mechanism observed here and previously by others (51) might be involved in ERK (19) and c-Src activation, or act independently, via up-regulation of an unknown factor, which transactivates -promoter (9, 24, 51), and add to the contribution of ERK and c-Src to the GnRH response.
Alterations in the frequency of GnRH pulses and/or the density of GnRHRs are thought to be responsible for differential expression of gonadotropin subunit genes (40, 52). We suggest that differential signaling might also mediate signal specificity during GnRH action (20, 24, 26, 42, 45, 46). We therefore propose that Ca2+, ERK, and c-Src are involved in regulating common , whereas PKC, ERK, JNK, and c-Src are involved in regulating the LHß gene (21, 26, 43 and the present results). PKC and two AP-1 sites were shown to be involved in basal and GnRH regulation of the ovine FSHß promoter (50). The role of MAPK in gonadotropin subunit gene regulation is evolutionarily conserved. The PKC-ERK pathway is now implicated also in GnRH elevation of {alpha} and LHß gene expression, but not FSHß, which is mediated by protein kinase A in primary cultures of tilapia pituitary cells (53).7f@-, 百拇医药
Acknowledgments7f@-, 百拇医药
We thank Dr. O. Benard for his interest and help during the study. We also thank Drs. P. Mellon for the {alpha} T3-1cells and for the human {alpha} CAT promoter and J. L. Jameson for the 5'-deletion mutants.
Received October 25, 2002.5, http://www.100md.com
Accepted for publication October 30, 2002.5, http://www.100md.com
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