Activation of MAPK cascades by GnRH: ERK and Jun N-terminal kinase are involved in basal and GnRH-stimulated activity of the glycoprotein hormone LHbeta-subunit promoter

The role of ERK and Jun N-terminal kinase (JNK) in basal- and GnRH-stimulated LHbeta-promoter activity was examined in the gonadotroph cell line LbetaT-2. GnRH agonist (GnRH-A) stimulates the MAPK cascades ERK, JNK, and p38MAPK, with a peak at 7 min for ERK and at 60 min for JNK and p38MAPK. The rat glycoprotein hormone LHbeta-subunit promoter, linked to the chloramphenicol acetyl transferase (CAT) reporter gene, was used to follow its activation. Addition of GnRH-A (10 nM) to LbetaT-2 cells resulted in a 6-fold increase in LHbeta-CAT activity at 8 h, which was markedly reduced by a GnRH antagonist. The PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA), but not the Ca(2+) ionophore ionomycin, stimulated LHbeta-CAT activity. Addition of GnRH-A and TPA together did not produce an additive response. Down-regulation of PKC, but not removal of Ca(2+), abolished the GnRH-A and the TPA response. Cotransfection of the LHbeta-promoter and the constitutively active form of Raf-1 stimulated basal and GnRH-A-induced LHbeta-CAT activity. The dominant negative forms of the ERK cascade members Ras, Raf-1, and MAPK/ERK kinase (MEK) markedly reduced basal and GnRH-A-induced LHbeta-CAT activity, Similar results were obtained with the MEK inhibitor PD 098059. Cotransfection of the LHbeta-promoter and the constitutively active CDC42 stimulated basal and GnRH-A-induced LHbeta-CAT activity. The dominant negative forms of the JNK cascade members Rac, CDC42, and SEK markedly diminished basal and GnRH-A-induced LHbeta-CAT activity. Interestingly, the constitutively active form of c-Src stimulated the basal and the GnRH-A response, whereas the dominant negative form of c-Src, or the c-Src inhibitor PP1 diminished basal and the GnRH-A response. We conclude that ERK and JNK are involved in basal and GnRH-A stimulation of LHbeta-CAT activity. c-Src participates also in LHbeta-promoter activation by a mechanism which might be linked to ERK and JNK activation.     

Extracellular signal-regulated kinase, Jun N-terminal kinase, p38, and c-Src are involved in gonadotropin-releasing hormone-stimulated activity of the glycoprotein hormone follicle-stimulating hormone beta-subunit promoter

The role of ERK, Jun N-terminal kinase (JNK), p38, and c-Src in GnRH-stimulated FSHbeta-subunit promoter activity was examined in the LbetaT-2 gonadotroph cell line. Incubation of the cells with a GnRH agonist resulted in activation of ERK, JNK, p38, and c-Src. The peak of ERK activation was observed at 5 min, whereas that of JNK, p38, and c-Src at 30 min, declining thereafter. ERK activation by GnRH is dependent on protein kinase C (PKC), as evident by activation, inhibition, and depletion of 12-O-tetradecanoylphorbol-13-acetate-sensitive PKC subspecies. Ca(2+) influx, but not Ca(2+) mobilization, is required for ERK activation. GnRH signaling to ERK is partially mediated by dynamin and a protein tyrosine kinase, apparently c-Src. ERK activation by GnRH in LbetaT-2 cells does not involve transactivation of epidermal growth factor receptor or mediation via Gbetagamma or beta-arrestin. Once activated by GnRH, ERK translocates to the nucleus. We examined the role of ERK, JNK, p38, and c-Src in GnRH-stimulated ovine FSHbeta promoter, linked to a luciferase reporter gene (-4741oFSHbeta-LUC). The PKC activator 12-O-tetradecanoylphorbol-13-acetate, but not the Ca(2+) ionophore ionomycin, stimulated FSHbeta-luciferase (LUC) activity. Furthermore, down-regulation of PKC, but not removal of Ca(2+), inhibited the GnRH response. Cotransfection of FSHbeta-LUC and the constitutively active forms of Raf-1 and MEK stimulated FSHbeta-LUC activity, whereas the dominant negatives of Ras, Raf-1, and MEK and the selective MEK inhibitor PD98059, abolished GnRH-induced FSHbeta-LUC activity. The dominant negatives of CDC42 and JNK reduced the GnRH response by 36 and 49%, respectively. Incubation of the cells with the p38 or the c-Src inhibitors SB203580 and PP1 also reduced the GnRH response. Surprisingly, two proximal activator protein-1 sites contribute very little to the GnRH response. Thus, PKC, ERK, JNK, p38, and c-Src, but not Ca(2+), are involved in GnRH induction of the ovine FSHbeta gene.     

Activation of mitogen-activated protein kinase phosphatase 2 by gonadotropin-releasing hormone

The aim of these studies was to identify the signaling mechanism(s) that contribute to GnRH-induced expression of MAPK phosphatase (MKP)-2, a dual specificity phosphatase that selectively inactivates MAPKs. GnRH receptor activation induced MKP-2 expression in both clonal (alphaT3-1) and primary gonadotropes. Activation of PKC isozymes was sufficient and required for MKP-2 induction. Inhibition of the extracellular signal-regulated kinase (ERK) or c-Jun N-terminal kinase (JNK) but not the p38 MAPK cascade was sufficient to block GnRH-induced MKP-2 expression. Induction of MKP-2 by GnRH was dependent on elevation in intracellular Ca(2+). Inhibition of Ca(2+) influx through L-type voltage-gated calcium channels blocked GnRH-induced MKP-2 expression. Depletion of intracellular Ca(2+) stores with thapsigargin blocked MKP-2 activation by GnRH independent of ERK and JNK activity. These results support the conclusion that MKP-2 induction by GnRH occurs via MAPK-dependent and -independent pathways. One mechanism requires GnRH-induced ERK and JNK activation, while a second MAPK-independent pathway requires a thapsigargin-sensitive calcium signal.     

Role of MAP kinase phosphatases in GnRH-dependent activation of MAP kinases

GnRH controls the synthesis and release of the pituitary gonadotropic hormones. MAP kinase (MAPK) cascades, including extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways, are crucial for GnRH-induced gene activation. In the present study, we investigated the function of GnRH-induced MAPK phosphatases (MKPs) using an in vivo mouse model as well as the alphaT3-1 cell line. Following GnRH agonist stimulation, in vivo gene profiling demonstrated that both MKP-1 and MKP-2 are induced with distinct temporal profiles, suggesting differential roles of these MKPs in the regulation of MAPK activation. Elevated activity of MKP-2 in alphaT3-1 cells, through either overexpression or activation of the endogenous MKP-2 gene, was correlated with inhibition of GnRH-induced activation of ERK and JNK, as well as the expression of ERK- and JNK-dependent proto-oncogenes. These data supported the conclusion that GnRH-induced MKPs likely serve as negative feedback regulators that modulate MAPK activity and function in the GnRH signaling pathway.     

Activation of mitogen-activated protein kinase (MAPK) by GnRH is cell-context dependent

The interaction of GnRH with its cognate receptor (GnRHR) in pituitary gonadotropes includes activation of Gq/G11 and phospholipase Cbeta (PLCbeta), which generates the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), which are required for Ca2+ mobilization and PKC isoforms activation. Activation of PKC in pituitary gonadotropes leads to the activation of the major members of the mitogen-activated protein kinase superfamily (MAPK), namely: extracellular signal-regulated kinase (ERK), jun-N-terminal Kinase (JNK) and p38MAPK. The above pathways mediate GnRH-induced gonadotropin release and synthesis. Here we summarise the diverse mechanisms utilized by GnRH to activate the MAPK members and show that they depend on "cell-context".     

Activation, differential localization, and regulation of the stress-activated protein kinases, extracellular signal-regulated kinase, c-JUN N-terminal kinase, and p38 mitogen-activated protein kinase, in synovial tissue and cells in rheumatoid arthritis

OBJECTIVE: To investigate whether stress- and mitogen-activated protein kinases (SAPK/MAPK), such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK, are significantly activated in rheumatoid arthritis (RA) synovial tissue compared with their activation in degenerative joint disease; to assess the localization of SAPK/MAPK activation in rheumatoid synovial tissue; and to search for the factors leading to stress kinase activation in human synovial cells. METHODS: Immunoblotting and immunohistology by antibodies specific for the activated forms of SAPK/MAPK were performed on synovial tissue samples from patients with RA and osteoarthritis (OA). In addition, untreated and cytokine-treated human synovial cells were assessed for SAPK/MAPK activation and downstream signaling by various techniques. RESULTS: ERK, JNK, and p38 MAPK activation were almost exclusively found in synovial tissue from RA, but not OA, patients. ERK activation was localized around synovial microvessels, JNK activation was localized around and within mononuclear cell infiltrates, and p38 MAPK activation was observed in the synovial lining layer and in synovial endothelial cells. Tumor necrosis factor alpha, interleukin-1 (IL-1), and IL-6 were the major inducers of ERK, JNK, and p38 MAPK activation in cultured human synovial cells. CONCLUSION: Signaling through SAPK/MAPK pathways is a typical feature of chronic synovitis in RA, but not in degenerative joint disease. SAPK/MAPK signaling is found at distinct sites in the synovial tissue, is induced by proinflammatory cytokines, and could lead to the design of highly targeted therapies.     

Extracellular signal-regulated protein kinase, but not c-Jun N-terminal kinase, is activated by type II gonadotropin-releasing hormone involved in the inhibition of ovarian cancer cell proliferation

Although a novel second form of GnRH (GnRH-II) has been reported to have an antiproliferative effect on gynecologic cancer cells, its biological mechanism remains to be elucidated. We have previously demonstrated that GnRH-II activates p38 MAPK. There is accumulating evidence that activation of MAPKs by GnRH-I and -II is important for cell proliferation, differentiation, and apoptosis. In the present study, we further investigated the involvement of GnRH-II in the inhibition of cell proliferation and activation of ERK1/2 and c-Jun N-terminal protein kinase/stress-activated protein kinase (JNK/SAPK) in ovarian cancer cells, OVCAR-3. The [(3)H]thymidine incorporation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays revealed that treatment with GnRH-II suppresses cell proliferation of ovarian cancer cells. Western blot analysis demonstrated that ERK1/2 was activated by GnRH-II (100 nm). Moreover, PD98059 (10 mum), an inhibitor of a MAPK/ERK kinase, reversed the activation of ERK1/2 induced by GnRH-II. The activation of ERK1/2 by GnRH-II subsequently phosphorylated Elk-1 as a downstream pathway, which was blocked by PD98059. On the other hand, it is not likely that GnRH-II activates the JNK/SAPK pathway. Taken together, these results indicate that the ERK1/2 pathway is involved in the effect of GnRH-II on antiproliferation and may be an important target for ovarian cancer therapy.     

Activation of the p38 mitogen-activated protein kinase pathway by gonadotropin-releasing hormone

Previous studies have shown that interaction of GnRH with its serpentine, G protein-coupled receptor results in activation of the extracellular signal regulated protein kinase (ERK) and the Jun N-terminal protein kinase (JNK) pathways in pituitary gonadotropes. In the present study, we examined GnRH-stimulated activation of an additional member of the mitogen-activated protein kinase (MAPK) superfamily, p38 MAPK GnRH treatment of alphaT3-1 cells resulted in tyrosine phosphorylation of several intracellular proteins. Separation of phosphorylated proteins by ion exchange chromatography suggested that GnRH receptor stimulation can activate the p38 MAPK pathway. Immunoprecipitation studies using a phospho-tyrosine antibody resulted in increased amounts of immunoprecipitable p38 MAPK from alphaT3-1 cells treated with GnRH. Immunoblot analysis of whole cell lysates using a phospho-specific antibody directed against dual phosphorylated p38 kinase revealed that GnRH-induced phosphorylation of p38 kinase was dose and time dependent and was correlated with increased p38 kinase activity in vitro. Activation of p38 kinase was blocked by chronic phorbol ester treatment, which depletes protein kinase C isozymes alpha and epsilon. Overexpression of p38 MAPK and an activated form of MAPK kinase 6 resulted in activation of c-jun and c-fos reporter genes, but did not alter the expression of the glycoprotein hormone alpha-subunit reporter. Inhibition of p38 activity with SB203580 resulted in attenuation of GnRH-induced c-fos reporter gene expression, but was not sufficient to reduce GnRH-induced c-jun or glycoprotein hormone alpha-subunit promoter activity. These studies provide evidence that the GnRH signaling pathway in alphaT3-1 cells includes protein kinase C-dependent activation of the p38 MAPK pathway. GnRH integration of c-fos promoter activity may include regulation by p38 MAPK.     

Gonadotropin-releasing hormone-desensitized LbetaT2 gonadotrope cells are refractory to acute protein kinase C,cyclic AMP,and calcium-dependent signaling

Sustained exposure of gonadotropes to GnRH causes a pronounced desensitization of gonadotropin release, but the mechanisms involved are poorly understood. It is known that desensitization is associated with decreased GnRH receptor and Gq/11 levels in alphaT3-1 cells, but it is not known whether downstream signaling is impaired. We have shown previously that chronic stimulation of signaling via expression of an active form of Galphaq causes GnRH resistance in LbetaT2 cells. In this study we investigated whether chronic GnRH treatment could down-regulate protein kinase C (PKC), cAMP, or Ca2+-dependent signaling in LbetaT2 cells. We found that chronic GnRH treatment desensitizes cells to acute GnRH stimulation not only by reducing GnRH receptor and Gq/11 expression but also by down-regulating PKC, cAMP, and calcium-dependent signaling. Desensitization was observed for activation of ERK and p38 MAPK and induction of c-fos and LHbeta protein expression. Activation of individual signaling pathways was able to partially mimic the desensitizing effect of GnRH on ERK, p38 MAPK, c-fos, and LHbeta but not on Gq/11. Chronic stimulation with phorbol esters reduced GnRH receptor expression to the same extent as chronic GnRH. Sustained GnRH also desensitized PKC signaling by down-regulating the delta, epsilon, and theta isoforms of PKC. We further show that chronic GnRH treatment causes heterologous desensitization of other Gq-coupled receptors.     

Implication of Gbetagamma proteins and c-SRC tyrosine kinase in parathyroid hormone-induced signal transduction in rat enterocytes

Parathyroid hormone (PTH) interacts in target tissues with a G protein-coupled receptor (GPCR) localized in the plasma membrane. Although activation of GPCR can elicit rapid stimulation of cellular protein tyrosine phosphorylation, the mechanism by which G proteins activate protein-tyrosine kinases is not completely understood. In the present work, we demonstrate that PTH rapidly increases the activity of non-receptor tyrosine kinase c-Src in rat intestinal cells (enterocytes). The response is biphasic, the early phase is fast and transient, peaking at 30 s (+120%), while the second phase progressively increases up to 5 min (+220%). The hormone activates c-Src in intestinal cells through fast changes in tyrosine phosphorylation of the enzyme. The first event in the activation of c-Src is the dephosphorylation of Tyr527 (which happens after a few seconds of PTH treatment), followed by a second event of activation with phosphorylation at Tyr416 (+twofold, 5 min). Removal of external Ca(2+) (EGTA, 0.5 mM) and chelation of intracellular Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetracetic acid acetoxymethyl ester (BAPTA) (5 microM) suppressed Tyr527 dephosphorylation and Tyr416 phosphorylation, indicating that Ca(2+) is an upstream activator of c-Src in enterocytes stimulated with PTH. The G protein subunits, Galphas and Gbeta, are associated with c-Src in basal conditions and this association increases two- to threefold in cells treated with PTH. Blocking of Gbeta subunits by preincubation of cells with a Gbeta antibody abolished hormone-dependent c-Src Tyr416 phosphorylation and ERK1/ERK2 activation. The results of this work indicate that PTH activates c-Src in intestinal cells through conformational changes via G proteins and calcium-dependent modulation of tyrosine phosphorylation of the enzyme, and that PTH receptor activation leads via Gbetagamma-c-Src to the phosphorylation of the MAP kinases, ERK1 and ERK2.     

c-Jun N-terminal kinase activation of activator protein-1 underlies homologous regulation of the gonadotropin-releasing hormone receptor gene in alpha T3-1 cells

Reproductive function is dependent on the interaction between GnRH and its cognate receptor found on gonadotrope cells of the anterior pituitary gland. GnRH activation of the GnRH receptor (GnRHR) is a potent stimulus for increased expression of multiple genes including the gene encoding the GnRHR itself. Thus, homologous regulation of the GnRHR is an important mechanism underlying gonadotrope sensitivity to GnRH. Previously, we have found that GnRH induction of GnRHR gene expression in alpha T3-1 cells is partially mediated by protein kinase C activation of a canonical activator protein-1 (AP-1) element. In contrast, protein kinase A and a cAMP response element-like element have been implicated in mediating the GnRH response of the GnRHR gene using a heterologous cell model (GGH(3)). Herein we find that selective removal of the canonical AP-1 site leads to a loss of GnRH regulation of the GnRHR promoter in transgenic mice. Thus, an intact AP-1 element is necessary for GnRH responsiveness of the GnRHR gene both in vitro and in vivo. Based on in vitro analyses, GnRH appeared to enhance the interaction of JunD, FosB, and c-Fos at the GnRHR AP-1 element. Although enhanced binding of cFos reflected an increase in gene expression, GnRH appeared to regulate both FosB and JunD at a posttranslational level. Neither overexpression of a constitutively active Raf-kinase nor pharmacological blockade of GnRH-induced ERK activation eliminated the GnRH response of the GnRHR promoter. GnRH responsiveness was, however, lost in alpha T3-1 cells that stably express a dominant-negative c-Jun N-terminal kinase (JNK) kinase, suggesting a critical role for JNK in mediating GnRH regulation of the GnRHR gene. Consistent with this possibility, we find that the ability of forskolin and membrane-permeable forms of cAMP to inhibit the GnRH response of the GnRHR promoter is associated with a loss of both JNK activation and GnRH-mediated recruitment of the primary AP-1-binding components.     

PTH regulation of c-Jun terminal kinase and p38 MAPK cascades in intestinal cells from young and aged rats

In the present study, we examined the role of Parathyroid hormone (PTH) on the c-Jun N-terminal kinase (JNK) 1/2 and p38 mitogen-activated protein kinase (MAPK) members of the MAPK family as it relates to ageing by measuring hormone-induced changes in their activity in enterocytes isolated from young (3 month old) and aged (24 month old) rats. Our results show that PTH induces a transient activation of JNK 1/2, peaking at 1 min (+threefold). The hormone also stimulates JNK 1/2 tyrosine phosphorylation, in a dose-dependent fashion, this effect being maximal at 10 nM. PTH-induced JNK 1/2 phosphorylation was suppressed by its selective inhibitor SP600125. Moreover, hormone-dependent activation of JNK 1/2 was dependent on calcium, since pretreatment of cells with BAPTA-AM or EGTA blocked PTH effects. With ageing, the response to PTH was significantly reduced. JNK basal protein expression was not different in the enterocytes from young and aged rats, however, basal protein phosphorylation increased with ageing. PTH did not stimulate, within 1–10 min, the basal activity and phosphorylation of p38 MAPK in rat intestinal cells. The hormone increased enterocyte DNA synthesis; the response was dose-dependent and decreased (-40%) with ageing. In agreement with the mitogenic role of the MAPK cascades, this effect was blocked by specific inhibitors of extracellular signal-regulated protein kinase (ERK) 1/2 and JNK 1/2. The results obtained in this work expand our knowledge on the mechanism of action of PTH in duodenal cells.     

Differential roles of extracellular signal-regulated kinase 1/2 and p38MAPK in mechanical load-induced procollagen alpha1(I) gene expression in cardiac fibroblasts

OBJECTIVE AND METHODS: We have previously demonstrated that mechanical loading of cardiac fibroblasts leads to increased synthesis and gene expression of the extracellular matrix protein collagen. We hypothesised that the upregulation of procollagen gene expression in cardiac fibroblasts, in response to cyclic mechanical load, is mediated by one or more members of the MAP kinase family. To test this hypothesis, the effect of mechanical load on the activation of extracellular signal-regulated kinase (ERK) 1/2, p46/54JNK, and p38MAPK was examined in rat cardiac fibroblasts. RESULTS: Peak phosphorylation of ERK 1/2, p38MAPK kinases, and p46/54JNK was observed following 10-20 min of continuous cyclic mechanical load. Mechanical load significantly increased procollagen alpha1(I) mRNA levels up to twofold above static controls after 24 h. This increase was completely abolished by the MEK 1/2 inhibitor U0126, with no effect on basal levels. In contrast, SB203580, a specific inhibitor of p38MAPK, enhanced both basal and stretch-stimulated levels of procollagen mRNA. Consistent with this finding, selective activation of the p38MAPK signalling pathway by expression of MKK6(Glu), a constitutive activator of p38MAPK, significantly reduced procollagen alpha1(I) promoter activity. SB203580-dependent increase in procollagen alpha1(I) was accompanied by ERK 1/2 activation, and inhibition of this pathway completely prevented SB203580-induced procollagen alpha1(I) expression. CONCLUSIONS: These results suggest that mechanical load-induced procollagen alpha1(I) gene expression requires ERK 1/2 activation and that the p38MAPK pathway negatively regulates gene expression in cardiac fibroblasts. These pathways are likely to be key in events leading to matrix deposition during heart growth and remodelling induced by mechanical load.     

Mitogen-activated protein kinase cascade in human normal and tumoral parathyroid cells

The calcium-sensing receptor (CaR) activation has recently been shown to modulate the ERK1 and ERK2 cascade in different cell lines. The present study investigated this pathway in human normal and tumoral parathyroid cells. In cells from normal parathyroids and almost all hyperplasia increasing extracellular calcium concentrations (Ca(o)(2+)) induced a significant activation of ERK1 and -2, the percent stimulation over basal activity (at 0.5 mM Ca(o)(2+)) being 545 +/- 140 and 800 +/- 205 in normal cells and 290 +/- 71 and 350 +/- 73 in hyperplasia at 1 and 2 mM Ca(o)(2+), respectively. This effect was mediated by CaR because it was mimicked by the receptor agonist gadolinium and neomycin. Basal and Ca(o)(2+)-stimulated ERK1 and -2 activity was nearly abolished by the PKC inhibitor calphostin C, and PKA changes did not affect ERK1 and -2 activity. PI3K blockade by wortmannin, known to prevent G protein betagamma subunit effect on ERK1 and -2, induced a 30% reduction of the Ca(o)(2+)-stimulated ERK1 and -2 activity. Adenomatous cells showed high PKC-dependent ERK1 and -2 activity in resting conditions that was unresponsive to high Ca(o)(2+). A role of MAPK on PTH secretion was suggested by the finding that PD98059, a specific MEK inhibitor, abolished the inhibitory effect of 1.5 mM Ca(o)(2+) on PTH release from normal parathyroid cells. In conclusion, these data first demonstrate that CaR activation, through the PKC pathway and, to a lesser extent, PI3K, increases ERK1 and -2 activity in normal parathyroid cells and this cascade seems to be involved in the modulation of PTH secretion by Ca(o)(2+). Interestingly, this signaling pathway is disrupted in parathyroid tumors.     

Angiotensin II-induced cardiac hypertrophy is associated with different mitogen-activated protein kinase activation in normotensive and hypertensive mice

OBJECTIVE: In addition to its haemodynamic effects, angiotensin II (AngII) is thought to contribute to the development of cardiac hypertrophy via its growth factor properties. The activation of mitogen-activated protein kinases (MAPK) is crucial for stimulating cardiac growth. Therefore, the present study aimed to determine whether the trophic effects of AngII and the AngII-induced haemodynamic load were associated with specific cardiac MAPK pathways during the development of hypertrophy. Methods The activation of the extracellular-signal-regulated kinase (ERK), the c-jun N-terminal kinase (JNK) and the p38 kinase was followed in the heart of normotensive and hypertensive transgenic mice with AngII-mediated cardiac hypertrophy. Secondly, we used physiological models of AngII-dependent and AngII-independent renovascular hypertension to study the activation of cardiac MAPK pathways during the development of hypertrophy. RESULTS: In normotensive transgenic animals with AngII-induced cardiac hypertrophy, p38 activation is associated with the development of hypertrophy while ERK and JNK are modestly stimulated. In hypertensive transgenic mice, further activation of ERK and JNK is observed. Moreover, in the AngII-independent model of renovascular hypertension and cardiac hypertrophy, p38 is not activated while ERK and JNK are strongly stimulated. In contrast, in the AngII-dependent model, all three kinases are stimulated. CONCLUSIONS: These data suggest that p38 activation is preferentially associated with the direct effects of AngII on cardiac cells, whereas stimulation of ERK and JNK occurs in association with AngII-induced mechanical stress.     

5-hydroxytryptamine 2B receptor regulates cell-cycle progression: cross-talk with tyrosine kinase pathways

In this paper, we present evidence that activation of 5-hydroxytryptamine 2B (5-HT2B) receptors by serotonin (5-HT) leads to cell-cycle progression through retinoblastoma protein hyperphosphorylation and through activation of both cyclin D1/cdk4 and cyclin E/cdk2 kinases by a mechanism that depends on induction of cyclin D1 and cyclin E protein levels. The induction of cyclin D1 expression, but not that of cyclin E, is under mitogen-activated protein kinase (MAPK) control, indicating an independent regulation of these two cyclins in the 5-HT2B receptor mitogenesis. Moreover, by using the specific platelet-derived growth factor receptor (PDGFR) inhibitor AG 1296 or by overexpressing a kinase-mutant PDGFR, we show that PDGFR kinase activity is essential for 5-HT2B-triggered MAPK/cyclin D1, but not cyclin E, signaling pathways. 5-HT2B receptor activation also increases activity of the Src family kinase, c-Src, Fyn, and c-Yes. Strikingly, c-Src, but not Fyn or c-Yes, is the crucial molecule between the G(q) protein-coupled 5-HT2B receptor and the cell-cycle regulators. Inhibition of c-Src activity by 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) or depletion of c-Src is sufficient to abolish the 5-HT-induced (i) PDGFR tyrosine kinase phosphorylation and MAPK activation, (ii) cyclin D1 and cyclin E expression levels, and (iii) thymidine incorporation. This paper elucidates a model of 5-HT2B receptor mitogenesis in which c-Src acts alone to control cyclin E induction and in concert with the receptor tyrosine kinase PDGFR to induce cyclin D1 expression via the MAPK/ERK pathway.