Inhibition of a signaling pathway in cardiac muscle cells by active mitogen-activated protein kinase kinase.

Signaling via the Ras pathway involves sequential activation of Ras, Raf-1, mitogen-activated protein kinase kinase (MKK), and the extracellular signal-regulated (ERK) group of mitogen-activated protein (MAP) kinases. Expression from the c-Fos, atrial natriuretic factor (ANF), and myosin light chain-2 (MLC-2) promoters during phenylephrine-induced cardiac muscle cell hypertrophy requires activation of this pathway. Furthermore, constitutively active Ras or Raf-1 can mimic the action of phenylephrine in inducing expression from these promoters. In this study, we tested whether constitutively active MKK, the molecule immediately downstream of Raf, was sufficient to induce expression. Expression of constitutively active MKK induce ERK2 kinase activity and caused expression from the c-Fos promoter, but did not significantly activate expression of reporter genes under the control of either the ANF or MLC-2 promoters. Expression of CL100, a phosphatase that inactivates ERKs, prevented expression from all of the promoters. Taken together, these data suggest that ERK activation is required for expression from the Fos, ANF, and MLC-2 promoters but MKK and ERK activation is sufficient for expression only from the Fos promoter. Constitutively active MKK synergized with phenylephrine to increase expression from a c-Fos- or an AP1-driven reporter. However, active MKK inhibited phenylephrine- and Raf-1-induced expression from the ANF and MLC-2 promoters. A DNA sequence in the MLC-2 promoter that is a target for inhibition by active MKK, but not CL100, was mapped to a previously characterized DNA element (HF1) that is responsible for cardiac specificity. Thus, activation of cardiac gene expression during phenylephrine-induced hypertrophy requires ERK activation but constitutive activation by MKK can inhibit expression by targeting a DNA element that controls the cardiac specificity of gene expression.     

Opposing Effects of Jun Kinase and p38 Mitogen-Activated Protein Kinases on Cardiomyocyte Hypertrophy

c-Jun N-terminal protein kinase (JNK) and p38, two distinct members of the mitogen-activated protein (MAP) kinase family, regulate gene expression in response to various extracellular stimuli, yet their physiological functions are not completely understood. In this report we show that JNK and p38 exerted opposing effects on the development of myocyte hypertrophy, which is an adaptive physiological process characterized by expression of embryonic genes and unique morphological changes. In rat neonatal ventricular myocytes, both JNK and p38 were stimulated by hypertrophic agonists like endothelin-1, phenylephrine, and leukemia inhibitory factor. Expression of MAP kinase kinase 6b (EE), a constitutive activator of p38, stimulated the expression of atrial natriuretic factor (ANF), which is a genetic marker of in vivo cardiac hypertrophy. Activation of p38 was required for ANF expression induced by the hypertrophic agonists. Furthermore, a specific p38 inhibitor, SB202190, significantly changed hypertrophic morphology induced by the agonists. Surprisingly, activation of JNK led to inhibition of ANF expression induced by MEK kinase 1 (MEKK1) and the hypertrophic agonists. MEKK1-induced ANF expression was also negatively regulated by expression of c-Jun. Our results demonstrate that p38 mediates, but JNK suppresses, the development of myocyte hypertrophy.     

Inhibition of phenylephrine-induced cardiac hypertrophy by docosahexaenoic acid

Many of the cardiovascular benefits of fish oil result from the antiarrhythmic actions of the n-3 polyunsaturated lipids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). The beneficial effects of DHA/EPA in patients with coronary artery disease and myocardial infarction may also result from modulation of the myocardial hypertrophic response. Hypertrophy was assessed in neonatal cardiomyocytes exposed to phenylephrine (PE) by measuring cell surface area, total protein synthesis ((14)C leucine incorporation), and the organization of sarcomeric alpha-actinin and by monitoring expression of atrial natriuretic factor (ANF). We report that PE induced a twofold increase in cell surface area and protein synthesis in cardiomyocytes. The hypertrophied cardiomyocytes also exhibited increased expression of ANF in perinuclear regions and organization of sarcomeric alpha-actinin into classical z-bands. Treatment of cardiomyocytes with 5 microM DHA effectively prevented PE-induced hypertrophy as shown by inhibition of surface area expansion and protein synthesis, inhibition of ANF expression, and prevention of alpha-actinin organization into z-bands. DHA treatment prevented PE-induced activation of Ras and Raf-1 kinase. The upstream inhibition of Ras --> Raf-1 effectively prevented translocation and nuclear localization of phosphorylated extracellularly regulated kinase 1 and 2 (Erk1/2). These effects consequently led to inhibition of nuclear translocation, and hence, activation of the downstream signaling enzyme p90 ribosomal S6 kinase (p90(rsk)). These results indicate that PE-induced cardiac hypertrophy can be minimized by DHA. Our results suggest that inhibition of Ras --> Raf-1 --> Erk1/2 --> p90(rsk) --> hypertrophy is one possible pathway by which DHA can inhibit cardiac hypertrophy. In vivo studies are needed to confirm these in vitro effects of DHA.     

MAP kinase- and Rho-dependent signals interact to regulate gene expression but not actin morphology in cardiac muscle cells.

Post-natal growth of cardiac muscle cells occurs by hypertrophy rather than division and is associated with changes in gene expression and muscle fiber morphology. We show here that the protein kinase MEKK1 can induce reporter gene expression from the atrial natriuretic factor (ANF) promoter, a genetic marker that is activated during in vivo hypertrophy. MEKK1 induced both stress-activated protein kinase (SAPK) and extracellular signal-regulated protein kinase (ERK) activity; however, while the SAPK cascade stimulated ANF expression, activation of the ERK cascade inhibited expression. C3 transferase, a specific inhibitor of the small GTPase Rho, also inhibited both MEKK- and phenylephrine-induced ANF expression, indicating an additional requirement for Rho-dependent signals. Microinjection or transfection of C3 transferase into the same cells did not disrupt actin muscle fiber morphology, indicating that Rho-dependent pathways do not regulate actin morphology in cardiac muscle cells. While active MEKK1 was a potent activator of hypertrophic gene expression, this kinase did not induce actin organization and prevented phenylephrine-induced organization. These data suggest that multiple signals control hypertrophic phenotypes. Positive and negative signals mediated by parallel MAP kinase cascades interact with Rho-dependent pathways to regulate hypertrophic gene expression while other signals induce muscle fiber morphology in cardiac muscle cells.     

Differential effects of Pyk2 and FAK on the hypertrophic response of cardiac myocytes

The related cytoplasmic non-receptor tyrosine kinases Pyk2 (proline-rich tyrosine kinase 2) and FAK (focal adhesion kinase) have been implicated in phenylephrine-induced G-protein-coupled receptor-mediated signaling mechanisms leading to cardiomyocyte hypertrophy. We report that, in phenylephrine-stimulated neonatal rat ventricular myocytes (NRVM), Pyk2 augments expression of the hypertrophic marker atrial natriuretic factor (ANF) but reduces cytoskeletal organization and cell spreading. In contrast, FAK attenuates ANF production but does not alter cytoskeletal organization and cell spreading. Pyk2 and FAK exhibit differential localization in both unstimulated and phenylephrine-stimulated myocytes. Pyk2 catalytic activity is required for Pyk2 to augment ANF secretion but is not necessary to reduce cell spreading. Pyk2 autophosphorylation is required but not sufficient for Pyk2 to augment ANF secretion. Expression of the Pyk2 FERM domain as an autonomous fragment inhibits phenylephrine-mediated ANF secretion and reduces cell spreading. In addition, expression of the Pyk2 FERM domain inhibits the ability of Pyk2 to augment ANF secretion; this is correlated with reduced Pyk2 autophosphorylation. These data indicate that Pyk2 and FAK have different roles and occupy different positions in signaling pathways leading to the development of cardiomyocyte hypertrophy. This work was supported by grant HL67938 from the National Institutes of Health (J.C.L.)     

RapV12 antagonizes Ras-dependent activation of ERK1 and ERK2 by LPA and EGF in Rat-1 fibroblasts.

Rap1 is a small Ras-related GTPase which when over-expressed is able to revert transformation by Ki-Ras. We have investigated the role of Rap1 in regulating 'normal' Ras function by studying the activation of the mitogen-activated protein (MAP) kinases ERK1 and ERK2 by two fundamentally different growth factors, epidermal growth factor (EGF) and 1-oleoyl-lyso-phosphatidic acid (LPA). Conditional expression of RasN17 (a dominant-negative mutant) in Rat-1 cells inhibited activation of MAP kinases by EGF and also LPA, the first time a defined G-protein-coupled receptor mitogen has been shown to require Ras to exert its effects. Conditional or constitutive expression of even low levels of RapV12 (a mutant insensitive to Rap-GAP) attenuated activation of MAP kinases by EGF and LPA, but did not interfere with growth factor-stimulated increases in Ras-GTP, indicating that signalling from receptors to Ras was not impaired. Inhibition of Ras-mediated signalling with either RasN17 or RapV12 attenuated DNA synthesis by EGF and LPA. We conclude that receptor tyrosine kinases and G-protein-coupled receptors use Ras as a common step in signalling to MAP kinases and that Rap-GTP (RapV12) at physiological levels interferes with downstream signalling from Ras to MAP kinases in vivo.     

A diacylglycerol-protein kinase C-RasGRP1 pathway directs Ras activation upon antigen receptor stimulation of T cells

Ras GTPases are on/off switches regulating numerous cellular responses by signaling to various effector molecules. In T lymphocytes, Ras can be activated by two Ras exchange factors, SOS and RasGRP1, which are recruited through the adapters Grb2 and LAT and via the second-messenger diacylglycerol (DAG), respectively. Mitogen-activated protein (MAP) kinase phosphorylation patterns induced by active Ras can vary and contribute to distinct cellular responses. The different consequences of Ras activation by either guanine exchange factor are unknown. DAG also recruits and activates the kinase protein kinase Ctheta (PKCtheta) turning on the Erk MAP kinase pathway, but the biochemical mechanism responsible is unclear. We generated T-cell clones deficient in phorbol myristate acetate (a surrogate for DAG)-induced Ras activation. Analysis of a RasGRP1-deficient Jurkat T-cell clone and RasGRP1 RNA interference in wild-type cells revealed that RasGRP1 is required for optimal, antigen receptor-triggered Ras-Erk activation. RasGRP1 relies on its DAG-binding domain to selectively activate Erk kinases. Activation of Erk correlates with the phosphorylation of threonine residue 184 in RasGRP1. This phosphorylation event requires the activities of novel PKC kinases. Conversely, active PKCtheta depends on RasGRP1 sufficiency to effectively trigger downstream events. Last, DAG-PKC-RasGRP1-driven Ras-Erk activation in T cells is a unique signaling event, not simply compensated for by SOS activity.     

Critical contribution of linker proteins to Raf kinase activation.

Genetic analysis of Ras signaling has unveiled the participation of non-enzymatic accessory proteins in signal transmission. These proteins, KSR, CNK, and Sur-8, can interact with multiple core components of the Ras/MAP kinase cascade and may contribute to the structural organization of this cascade. However, the precise biochemical nature of the contribution of these proteins to Ras signaling is currently unknown. Here we show directly that CNK and KSR are required for stimulus dependent Raf kinase activation. CNK is required for membrane recruitment of Raf, while KSR is likely required to couple Raf to upstream kinases. These results demonstrate that CNK and KSR are integral components of the cellular machinery mediating Raf activation.     

The alpha-adrenergic stimulation of atrial natriuretic factor expression in cardiac myocytes requires calcium influx, protein kinase C, and calmodulin-regulated pathways.

It has been shown recently that alpha-adrenergic agonists can stimulate atrial natriuretic factor (ANF) expression in ventricular cardiac myocytes; however, little is known about the intracellular signals mediating this activation. The present study focused on the potential roles of calcium-regulated kinases and calcium influx in the alpha-adrenergic stimulation of ANF gene expression in ventricular myocardial cell cultures. Myocardial cells maintained for 48 h in serum-free medium supplemented with phenylephrine (PE) possessed up to 15-fold higher levels of ANF peptide and ANF mRNA than control cells. The removal of PE, or the addition of nifedipine, resulted in a rapid decline in ANF expression, suggesting that the sustained elevation of some intracellular messenger (e.g. calcium and/or phospholipid hydrolysis products) was required for the adrenergic response. The calcium channel agonist BAY K 8644 was capable of increasing ANF expression in a nifedipine-sensitive manner; however, unlike PE, it did not stimulate phosphoinositide hydrolysis. The protein kinase C inhibitor, H7, caused an approximate 75% reduction in PE-stimulated ANF expression, but had no effect on BAY K-stimulated expression. W7, a calcium/calmodulin inhibitor, completely blocked the effects of both PE and BAY K 8644. The addition of either H7 or W7 24 h after the PE addition resulted in a decline of ANF expression. These results indicate that alpha-adrenergic agonists augment ANF gene expression through at least two pathways, one that is H7-sensitive, perhaps involving the sustained activation of protein kinase C, and the other that is W7-sensitive, perhaps involving the sustained activation of calmodulin-regulated kinases. Further, it appears that BAY K 8644-mediated increases in ANF expression are independent of protein kinase C activation and dependent on calmodulin-regulated events.     

Regulation of Ras.GTP loading and Ras-Raf association in neonatal rat ventricular myocytes by G protein-coupled receptor agonists and phorbol ester. Activation of the extracellular signal-regulated kinase cascade by phorbol ester is mediated by Ras.

The small G protein Ras has been implicated in hypertrophy of cardiac myocytes. We therefore examined the activation (GTP loading) of Ras by the following hypertrophic agonists: phorbol 12-myristate 13-acetate (PMA), endothelin-1 (ET-1), and phenylephrine (PE). All three increased Ras.GTP loading by 10-15-fold (maximal in 1-2 min), as did bradykinin. Other G protein-coupled receptor agonists (e.g. angiotensin II, carbachol, isoproterenol) were less effective. Activation of Ras by PMA, ET-1, or PE was reduced by inhibition of protein kinase C (PKC), and that induced by ET-1 or PE was partly sensitive to pertussis toxin. 8-(4-Chlorophenylthio)-cAMP (CPT-cAMP) did not inhibit Ras.GTP loading by PMA, ET-1, or PE. The association of Ras with c-Raf protein was increased by PMA, ET-1, or PE, and this was inhibited by CPT-cAMP. However, only PMA and ET-1 increased Ras-associated mitogen-activated protein kinase kinase 1-activating activity, and this was decreased by PKC inhibition, pertussis toxin, and CPT-cAMP. PMA caused the rapid appearance of phosphorylated (activated) extracellular signal-regulated kinase in the nucleus, which was inhibited by a microinjected neutralizing anti-Ras antibody. We conclude that PKC- and Gi-dependent mechanisms mediate the activation of Ras in myocytes and that Ras activation is required for stimulation of extracellular signal-regulated kinase by PMA.     

Activation of Ras in vitro and in intact fibroblasts by the Vav guanine nucleotide exchange protein.

We recently identified Vav, the product of the vav proto-oncogene, as a guanine nucleotide exchange factor (GEF) for Ras. Vav is enzymatically activated by lymphocyte antigen receptor-coupled protein tyrosine kinases or independently by diglycerides. To further evaluate the physiological role of Vav, we assessed its GDP-GTP exchange activity against several Ras-related proteins in vitro and determined whether Vav activation in transfected NIH 3T3 fibroblasts correlates with the activity status of Ras and mitogen-activated protein (MAP) kinases. In vitro translated purified Vav activated by phorbol myristate acetate (PMA) or phosphorylation with recombinant p56lck displayed GEF activity against Ras but not against recombinant RacI, RacII, Ral, or RhoA proteins. Expression of vav or proto-vav in stably transfected NIH 3T3 cells led to a approximately 10-fold increase in basal or PMA-stimulated Ras exchange activity, respectively, in total-cell lysates and Vav immunoprecipitates. Elevated GEF activity was paralleled in each case by a significant increase in the proportion of active, GTP-bound Ras. PMA had a minimal effect on the low Ras. GTP level in untransfected control fibroblasts but increased it from 20 to 37% in proto-vav-transfected cells. vav-transfected cells displayed a constitutively elevated Ras. GTP level (35%), which was not increased further by PMA treatment. MAP kinases, known downstream intermediates in Ras-dependent signaling pathways, similarly exhibited increased basal or PMA-stimulated activity in Vav-expressing cells by comparison with normal NIH 3T3 cells. These results demonstrate a physiologic interaction between Vav and its target, Ras, leading to MAP kinase activation.     

Thyrotropin-induced mitogenesis is Ras dependent but appears to bypass the Raf-dependent cytoplasmic kinase cascade.

Cellular growth control requires the coordination and integration of multiple signaling pathways which are likely to be activated concomitantly. Mitogenic signaling initiated by thyrotropin (TSH) in thyroid cells seems to require two distinct signaling pathways, a cyclic AMP (cAMP)-dependent signaling pathway and a Ras-dependent pathway. This is a paradox, since activated cAMP-dependent protein kinase disrupts Ras-dependent signaling induced by growth factors such as epidermal growth factor and platelet-derived growth factor. This inhibition may occur by preventing Raf-1 protein kinase from binding to Ras, an event thought to be necessary for the activation of Raf-1 and the subsequent activation of the mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinases (MEKs) and MAP kinase (MAPK)/ERKs. Here we report that serum-stimulated hyperphosphorylation of Raf-1 was inhibited by TSH treatment of Wistar rat thyroid cells, indicating that in this cell line, as in other cell types, increases in intracellular cAMP levels inhibit activation of downstream kinases targeted by Ras. Ras-stimulated expression of genes containing AP-1 promoter elements was similarly inhibited by TSH. On the other hand, stimulation of thyroid cells with TSH resulted in stimulation of DNA synthesis which was Ras dependent but both Raf-1 and MEK independent. We also show that Ras-stimulated DNA synthesis required the use of this kinase cascade in untreated quiescent cells but not in TSH-treated cells. These data suggest that in TSH-treated thyroid cells, Ras might be able to signal through effectors other than the well-studied cytoplasmic kinase cascade.     

Platelet-derived growth factor activates p38 mitogen-activated protein kinase through a Ras-dependent pathway that is important for actin reorganization and cell migration.

Members of the mitogen activated protein (MAP) kinase family, extracellular signal-regulated kinase, stress-activated protein kinase-1/c-Jun NH2-terminal kinase, and p38, are central elements that transduce the signal generated by growth factors, cytokines, and stressing agents. It is well known that the platelet-derived growth factor (PDGF) activates extracellular signal-regulated kinase, which leads to cellular mitogenic response. On the other hand, the role of the other MAP kinases in mediating the cellular function of PDGF remains unclear. In the present study, we have investigated the functional role of the other MAP kinases in PDGF-mediated cellular responses. We show that ligand stimulation of PDGF receptors leads to the activation of p38 but not stress-activated protein kinase-1/c-Jun NH2-terminal kinase. Experiments using a specific inhibitor of p38, SB203580, show that the activation of p38 is required for PDGF-induced cell motility responses such as cell migration and actin reorganization but not required for PDGF-stimulated DNA synthesis. Analyses of tyrosine residue-mutated PDGF receptors show that Src homology 2 domain-containing proteins including Src family kinases, phosphatidylinositol 3-kinase, the GTPase-activating protein of Ras, the Src homology 2 domain-containing phosphatase SHP-2, phospholipase C-gamma, and Crk do not play a major role in mediating the PDGF-induced activation of p38. Finally, the expression of dominant-negative Ras but not dominant-negative Rac inhibited p38 activation by PDGF, suggesting that Ras is a potent mediator in the p38 activation pathway downstream of PDGF receptors. Taken together, our present study proposes the existence of a Ras-dependent pathway for the activation of p38, which is important for cell motility responses elicited by PDGF stimulation.     

Regulation of mitogen-activated protein kinases in cardiac myocytes through the small G protein Rac1

Small guanine nucleotide-binding proteins of the Ras and Rho (Rac, Cdc42, and Rho) families have been implicated in cardiac myocyte hypertrophy, and this may involve the extracellular signal-related kinase (ERK), c-Jun N-terminal kinase (JNK), and/or p38 mitogen-activated protein kinase (MAPK) cascades. In other systems, Rac and Cdc42 have been particularly implicated in the activation of JNKs and p38-MAPKs. We examined the activation of Rho family small G proteins and the regulation of MAPKs through Rac1 in cardiac myocytes. Endothelin 1 and phenylephrine (both hypertrophic agonists) induced rapid activation of endogenous Rac1, and endothelin 1 also promoted significant activation of RhoA. Toxin B (which inactivates Rho family proteins) attenuated the activation of JNKs by hyperosmotic shock or endothelin 1 but had no effect on p38-MAPK activation. Toxin B also inhibited the activation of the ERK cascade by these stimuli. In transfection experiments, dominant-negative N17Rac1 inhibited activation of ERK by endothelin 1, whereas activated V12Rac1 cooperated with c-Raf to activate ERK. Rac1 may stimulate the ERK cascade either by promoting the phosphorylation of c-Raf or by increasing MEK1 and/or -2 association with c-Raf to facilitate MEK1 and/or -2 activation. In cardiac myocytes, toxin B attenuated c-Raf(Ser-338) phosphorylation (50 to 70% inhibition), but this had no effect on c-Raf activity. However, toxin B decreased both the association of MEK1 and/or -2 with c-Raf and c-Raf-associated ERK-activating activity. V12Rac1 cooperated with c-Raf to increase expression of atrial natriuretic factor (ANF), whereas N17Rac1 inhibited endothelin 1-stimulated ANF expression, indicating that the synergy between Rac1 and c-Raf is potentially physiologically important. We conclude that activation of Rac1 by hypertrophic stimuli contributes to the hypertrophic response by modulating the ERK and/or possibly the JNK (but not the p38-MAPK) cascades.     

Mitogen-activated protein kinase kinase kinase kinase 4 as a putative effector of Rap2 to activate the c-Jun N-terminal kinase

Little is known about the specific signaling roles of Rap2, a Ras family small GTP-binding protein. In a search for novel Rap2-interacting proteins by the yeast two-hybrid system, we isolated isoform 3 of the human mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), a previously described but uncharacterized isoform. Other isoforms of MAP4K4 in humans and mice are known as hematopoietic progenitor kinase (HPK)/germinal center kinase (GCK)-like kinase and Nck-interacting kinase, respectively. MAP4K4 belongs to the STE20 group of protein kinases and regulates c-Jun N-terminal kinase (JNK). MAP4K4 interacted with Rap2 through its C-terminal citron homology domain but did not interact with Rap1 or Ras. Interaction with Rap2 required the intact effector region of Rap2. MAP4K4 interacted preferentially with GTP-bound Rap2 over GDP-bound Rap2 in vitro. In cultured cells, MAP4K4 colocalized with Rap2, while a mutant MAP4K4 lacking the citron homology domain failed to do so. Furthermore, Rap2 enhanced MAP4K4-induced activation of JNK. These results suggest that MAP4K4 is a putative effector of Rap2 mediating the activation of JNK by Rap2.     

A role for focal adhesion kinase in phenylephrine-induced hypertrophy of rat ventricular cardiomyocytes

A variety of agonists including phenylephrine (PE) induce hypertrophy in neonatal ventricular cardiomyocytes. Here we report that signals provided by extracellular matrix proteins (ECM) augment the PE-induced hypertrophic response of cardiomyocytes and provide evidence that ECM-dependent signaling is mediated in part by the protein tyrosine kinase, focal adhesion kinase (FAK). Addition of PE to cultured neonatal cardiomyocytes stimulated sarcomeric organization, increased cell size, and induced atrial natriuretic factor in cardiomyocytes plated on the ECM protein laminin or fibronectin. In contrast, cardiomyocytes plated on the non-adhesive substrate gelatin exhibited a reduced capacity to undergo these PE-stimulated hypertrophic changes. In cardiomyocytes cultured on ECM, PE stimulated a rapid increase in tyrosine phosphorylation of focal adhesion proteins including FAK, paxillin, and p130 Crk-associated substrate and subsequent formation of peripheral focal complexes. Inhibition of the PE-induced hypertrophic response by genistein and herbimycin-A indicated a requirement for protein tyrosine kinases in PE signaling. To determine whether activation of FAK is required for PE-induced hypertrophy, a dominant-interfering mutant form of FAK, termed FRNK (FAK-related non-kinase), was ectopically expressed in cardiomyocytes using a replication-defective adenovirus expression system. FRNK expression attenuated PE-stimulated hypertrophy as assessed by cell size, sarcomeric organization, and induction of atrial natriuretic factor. These data indicate that the signal transduction pathways leading to cardiomyocyte hypertrophy are strongly influenced by and/or dependent upon an integrin-mediated signaling process requiring FAK.     

beta 2-adrenergic receptor activates extracellular signal-regulated kinases (ERKs) via the small G protein rap1 and the serine/threonine kinase B-Raf

G protein-coupled receptors can induce cellular proliferation by stimulating the mitogen-activated protein (MAP) kinase cascade. Heterotrimeric G proteins are composed of both alpha and betagamma subunits that can signal independently to diverse intracellular signaling pathways including those that activate MAP kinases. In this study, we examined the ability of isoproterenol, an agonist of the beta(2)-adrenergic receptor (beta(2)AR), to stimulate extracellular signal-regulated kinases (ERKs). Using HEK293 cells, which express endogenous beta(2)AR, we show that isoproterenol stimulates ERKs via beta(2)AR. This action of isoproterenol requires cAMP-dependent protein kinase and is insensitive to pertussis toxin, suggesting that Galpha(s) activation of cAMP-dependent protein kinase is required. Interestingly, beta(2)AR activates both the small G proteins Rap1 and Ras, but only Rap1 is capable of coupling to Raf isoforms. beta(2)AR inhibits the Ras-dependent activation of both Raf isoforms Raf-1 and B-Raf, whereas Rap1 activation by isoproterenol recruits and activates B-Raf. beta(2)AR activation of ERKs is not blocked by expression of RasN17, an interfering mutant of Ras, but is blocked by expression of either RapN17 or Rap1GAP1, both of which interfere with Rap1 signaling. We propose that isoproterenol can activate ERKs via Rap1 and B-Raf in these cells.     

Expression of cyclin D1 and CDK4 causes hypertrophic growth of cardiomyocytes in culture: a possible implication for cardiac hypertrophy

Differentiated cardiomyocytes have little capacity to proliferate and show the hypertrophic growth in response to alpha1-adrenergic stimuli via the Ras/MEK pathway. In this study, we investigated a role of cyclin D1 and CDK4, a positive regulator of cell cycle, in cultured neonatal rat cardiomyocyte hypertrophy. D-type cyclins including cyclin D1 were induced in cells stimulated by phenylephrine. This induction was inhibited by MEK inhibitor PD98059 and the dominant negative RasN17, but mimicked by expression of the constitutive active Ras61L. Over-expression of cyclin D1 and CDK4 using adenovirus gene transfer caused the hypertrophic growth of cardiomyocytes, as evidenced by an increase of the cell size as well as the amount of cellular protein and its rate of synthesis. However, the cyclin D1/CDK4 kinase activity was not up-regulated in cells treated by hypertrophic stimuli or in cells over-expressing the cyclin D1 and CDK4. Furthermore, a CDK inhibitor, p16, did not inhibit the hypertrophic growth of cardiomyocytes. These results clearly indicated that cyclin D1 and CDK4 have a role in hypertrophic growth of cardiomyocytes through a novel mechanism(s) which appears not to be related to its activity required for cell cycle progression.