Signaling pathways involved in IL-8-dependent activation of adhesion through Mac-1

In human neutrophils, IL-8 induces chemotaxis, the respiratory burst, and granule release, and enhances cellular adhesion, a beta(2) integrin-dependent event. IL-8 stimulates neutrophil adhesion to purified fibrinogen in a Mac-1-dependent manner. Mitogen-activated protein kinase (MAPK) activation was detected in human neutrophil lysates after treatment with IL-8 and PMA, but not the activating mAb CBR LFA 1/2. IL-8-stimulated neutrophil adhesion to fibrinogen was blocked 50% by the MAPK/extracellular signal-related kinase-activating enzyme inhibitor PD098059. Adhesion was blocked approximately 75% by inhibition of the phosphatidylinositol-3 kinase (PI3K) pathway with LY294002, supporting that activation of both MAPK and PI3K may play a role in IL-8-dependent inside-out signals that activate Mac-1. Activation of MAPK was inhibited in IL-8-stimulated cells in the presence of PI3K inhibitors LY294002 or wortmannin, supporting a model in which PI3K is upstream of MAPK. IL-8-stimulated neutrophil adhesion was inhibited 50% by bisindolylmaleimide-I, implicating protein kinase C (PKC) in the intracellular signaling from the IL-8R to Mac-1. A 74-kDa molecular mass species was detected by an activation-specific Ab to PKC when cells were stimulated with PMA or IL-8, but not a beta(2)-activating Ab. Inhibition of either MAPK or PKC resulted in partial inhibition of IL-8-stimulated polymorphonuclear neutrophil adhesion, and treatment with both inhibitors simultaneously completely abolished IL-8-stimulated adhesion to ligand. Inhibition of PI3K blocked MAPK activation, but not PKC activation, suggesting a branch point that precedes PI3K activation. These data suggest that both MAPK and PKC are activated in response to IL-8 stimulation, and that these may represent independent pathways for beta(2) integrin activation in neutrophils.     

The role of atypical protein kinase C in CSF-1-dependent Erk activation and proliferation in myeloid progenitors and macrophages

Colony stimulating factor-1 (CSF-1 or M-CSF) is the major physiological regulator of the proliferation, differentiation and survival of cells of the mononuclear phagocyte lineage. CSF-1 binds to a receptor tyrosine kinase, the CSF-1 receptor (CSF-1R). Multiple pathways are activated downstream of the CSF-1R; however, it is not clear which pathways regulate proliferation and survival. Here, we investigated the role of atypical protein kinase Cs (PKCζ) in a myeloid progenitor cell line that expressed CSF-1R (32D.R) and in primary murine bone marrow derived macrophages (BMMs). In 32D.R cells, CSF-1 induced the phosphorylation of PKCζ and increased its kinase activity. PKC inhibitors and transfections with mutant PKCs showed that optimal CSF-1-dependent Erk activation and proliferation depended on the activity of PKCζ. We previously reported that CSF-1 activated the Erk pathway through an A-Raf-dependent and an A-Raf independent pathway (Lee and States, Mol. Cell. Biol.18, 6779). PKC inhibitors did not affect CSF-1 induced Ras and A-Raf activity but markedly reduced MEK and Erk activity, implying that PKCζ regulated the CSF-1-Erk pathway at the level of MEK. PKCζ has been implicated in activating the NF-κB pathway. However, CSF-1 promoted proliferation in an NF-κB independent manner. We established stable 32D.R cell lines that overexpressed PKCζ. Overexpression of PKCζ increased the intensity and duration of CSF-1 induced Erk activity and rendered cells more responsive to CSF-1 mediated proliferation. In contrast to 32D.R cells, PKCζ inhibition in BMMs had only a modest effect on proliferation. Moreover, PKCζ -specific and pan-PKC inhibitors induced a paradoxical increase in MEK-Erk phosphorylation suggesting that PKCs targeted a common negative regulatory step upstream of MEK. Our results demonstrated that CSF-1 dependent Erk activation and proliferation are regulated differentially in progenitors and differentiated cells.     

The effect of 24R,25-(OH)(2)D(3) on protein kinase C activity in chondrocytes is mediated by phospholipase D whereas the effect of 1alpha,25-(OH)(2)D(3) is mediated by phospholipase C

1alpha,25-(OH)(2)D(3) regulates protein kinase C (PKC) activity in growth zone chondrocytes by stimulating increased phosphatidylinositol-specific phospholipase C (PI-PLC) activity and subsequent production of diacylglycerol (DAG). In contrast, 24R,25-(OH)(2)D(3) regulates PKC activity in resting zone (RC) cells, but PLC does not appear to be involved, suggesting that phospholipase D (PLD) may play a role in DAG production. In the present study, we examined the role of PLD in the physiological response of RC cells to 24R,25-(OH)(2)D(3) and determined the role of phospholipases D, C, and A(2) as well as G-proteins in mediating the effects of vitamin D(3) metabolites on PKC activity in RC and GC cells. Inhibition of PLD with wortmannin or EDS caused a dose-dependent inhibition of basal [3H]-thymidine incorporation by RC cells and further increased the inhibitory effect of 24R,25-(OH)(2)D(3). Wortmannin also inhibited basal alkaline phosphatase activity and [35]-sulfate incorporation and decreased the stimulatory effect of 24R,25-(OH)(2)D(3). This inhibitory effect of wortmannin was not seen in cultures treated with the PI-3-kinase inhibitor LY294002, verifying that wortmannin affected PLD. Wortmannin also inhibited basal PKC activity and partially blocked the stimulatory effect of 24R,25-(OH)(2)D(3) on this enzyme activity. Neither inhibition of PI-PLC with U73122, nor PC-PLC with D609, modulated PKC activity. Wortmannin had no effect on basal PLD in GC cells, nor on 1alpha,25-(OH)(2)D(3)-dependent PKC. Inhibition of PI-PLC blocked the 1alpha,25-(OH)(2)D(3)-dependent increase in PKC activity but inhibition of PC-PLC had no effect. Activation of PLA(2) with melittin inhibited basal and 24R,25-(OH)(2)D(3)-stimulated PKC in RC cells and stimulated basal and 1alpha,25-(OH)(2)D(3)-stimulated PKC in GC cells, but wortmannin had no effect on the melittin-induced changes in either cell type. Pertussis toxin modestly increased the effect of 24R,25-(OH)(2)D(3) on PKC, whereas GDPbetaS had no effect, suggesting that PLD2 is the isoform responsible. This indicates that 1alpha,25-(OH)(2)D(3) regulates PKC in GC cells via PI-PLC and PLA(2), but not PC-PLC or PLD, whereas 24R,25-(OH)(2)D(3) regulates PKC in RC cells via PLD2.     

Involvement of ERK and AKT signaling in the growth effect of arginine vasopressin on adult rat cardiac fibroblast and the modulation by simvastatin

Arginine vasopressin (AVP) has been shown to directly induce neonatal rat cardiac fibroblasts (CFs) proliferation, a major component involved in cardiac hypertrophy. Herein, we explored whether AVP is also a growth factor for adult rat CFs and, if so, whether the growth effect could be inhibited by simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor. AVP significantly increased DNA synthesis in adult rat CFs by 73.5 +/- 5.1% (P < or = 0.05), an effect inhibited by V1 receptor antagonist, d(CH(2))(5)[Tyr(2)(Me), Arg(8)]-vasopressin. AVP also activated extracellular signal-regulated kinase 1/2 (ERK1/2) as assessed by MBP phosphotransferase activity (5.1 +/- 0.6 fold over basal level, P < or = 0.05) and Western blot analysis, and effects were mimicked by protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), but abolished by inhibiting cellular PKC through chronic PMA incubation. In addition, AVP induced PKC activation (27.2 +/- 3.8% from a basal value of 9.3 +/- 0.7%, P < or = 0.05). AVP-induced increase in DNA synthesis could be attenuated by the specific inhibitors of ERK1/2 (PD98059), PI3K (LY294002), and AKT (1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate, HIMO). Simvastatin inhibited the effects of AVP on DNA synthesis, ERK1/2, and PKC activation in a dose-dependent manner. Phosphatidylinositol-3-kinase (PI3K)-dependent AKT activation induced by AVP was also inhibited by simvastatin. The effects of simvastatin on ERK1/2, PKC, and AKT activation and DNA synthesis could be reversed by mevalonate. These results support a growth-inducing effect of AVP on adult rat CFs through ERK and AKT signalings and the growth effect could be attenuated by simvastatin via inhibiting these two pathways.     

Signaling pathways mediating gastrointestinal smooth muscle contraction and MLC20 phosphorylation by motilin receptors

The signaling cascades initiated by motilin receptors in gastric and intestinal smooth muscle cells were characterized. Motilin bound with high affinity (IC(50) 0.7 +/- 0.2 nM) to receptors on smooth muscle cells; the receptors were rapidly internalized via G protein-coupled receptor kinase 2 (GRK2). Motilin selectively activated G(q) and G(13), stimulated G alpha(q)-dependent phosphoinositide (PI) hydrolysis and 1,4,5-trisphosphate (IP(3))-dependent Ca(2+) release, and increased cytosolic free Ca(2+). PI hydrolysis was blocked by expression of G alpha(q) minigene and augmented by overexpression of dominant negative RGS4(N88S) or GRK2(K220R). Motilin induced a biphasic, concentration-dependent contraction (EC(50) = 1.0 +/- 0.2 nM), consisting of an initial peak followed by a sustained contraction. The initial Ca(2+)-dependent contraction and myosin light-chain (MLC)(20) phosphorylation were inhibited by the PLC inhibitor U-73122 and the MLC kinase inhibitor ML-9 but were not affected by the Rho kinase inhibitor Y27632 or the PKC inhibitor bisindolylmaleimide. Sustained contraction and MLC(20) phosphorylation were RhoA dependent and mediated by two downstream messengers: PKC and Rho kinase. The latter was partly inhibited by expression of G alpha(q) or G alpha(13) minigene and abolished by coexpression of both minigenes. Sustained contraction and MLC(20) phosphorylation were partly inhibited by Y27632 and bisindolylmaleimide and abolished by a combination of both inhibitors. The inhibition reflected phosphorylation of two MLC phosphatase inhibitors: CPI-17 via PKC and MYPT1 via Rho kinase. We conclude that motilin initiates a G alpha(q)-mediated cascade involving Ca(2+)/calmodulin activation of MLC kinase and transient MLC(20) phosphorylation and contraction as well as a sustained G alpha(q)- and G alpha(13)-mediated, RhoA-dependent cascade involving phosphorylation of CPI-17 by PKC and MYPT1 by Rho kinase, leading to inhibition of MLC phosphatase and sustained MLC(20) phosphorylation and contraction.     

Regulation of interleukin-1alpha expression by integrins and epidermal growth factor receptor in keratinocytes from a mouse model of inflammatory skin disease

Transgenic mice expressing beta1 integrins in the suprabasal epidermal layers have sporadic skin hyperproliferation and inflammation correlated with activation of extracellular signal-regulated kinase (Erk) mitogen-activated protein kinase and increased interleukin (IL)-1alpha production. We investigated the link between aberrant integrin expression, Erk activation, and expression of IL-1alpha. Transgenic keratinocytes had higher basal Erk activity and IL-1alpha levels than nontransgenic controls and were more sensitive to stimulation of Erk activity and IL-1alpha production by IL-1alpha, 12-O-tetradecanoylphorbol-13-acetate (TPA), epidermal growth factor (EGF), and serum. Inhibition of Erk in transgenic keratinocytes reduced basal IL-1alpha levels and the stimulation of IL-1alpha production by serum or phorbol ester, demonstrating that Erk could regulate IL-1alpha expression. TPA or IL-1alpha treatment resulted in rapid down-regulation of the EGF receptor in transgenic cells, indicative of transactivation. Inhibition of transactivation blocked basal and TPA or IL-1alpha induced Erk activation, but not IkappaBalpha degradation, and abolished increased IL-1alpha production in transgenic cells. In transgene-negative cells, constitutive activation of IL-1-dependent signaling by wild type or kinase-dead IRAK1 stimulated IL-1alpha production independent of Erk. We conclude that suprabasal integrin expression leads to Erk activation and increased IL-1alpha expression by potentiating activation of the EGF receptor. These results provide a mechanism by which aberrant integrin expression triggers epidermal hyperproliferation and inflammation.     

Eucapnic intermittent hypoxia augments endothelin-1 vasoconstriction in rats: role of PKCdelta

We reported previously that simulating sleep apnea by exposing rats to eucapnic intermittent hypoxia (E-IH) causes endothelin-dependent hypertension and increases constrictor sensitivity to endothelin-1 (ET-1). In addition, augmented ET-1-induced constriction in small mesenteric arteries (sMA) is mediated by increased Ca(2+) sensitization independent of Rho-associated kinase. We hypothesized that exposing rats to E-IH augments ET-1-mediated vasoconstriction by increasing protein kinase C (PKC)-dependent Ca(2+) sensitization. In sMA, the nonselective PKC inhibitor GF-109203x (3 microM) significantly inhibited ET-1-stimulated constriction in E-IH arteries but did not affect ET-1-stimulated constriction in sham arteries. Phospholipase C inhibitor U-73122 (1 microM) also inhibited constriction by ET-1 in E-IH but not sham sMA. In contrast, the classical PKC (cPKC) inhibitor G?-6976 (1 microM) had no effect on ET-1-mediated vasoconstriction in either group, but a PKCdelta-selective inhibitor (rottlerin, 3 microM) significantly decreased ET-1-mediated constriction in E-IH but not in sham sMA. ET-1 increased PKCdelta phosphorylation in E-IH but not sham sMA. In contrast, ET-1 constriction in thoracic aorta from both sham and E-IH rats was inhibited by G?-6976 but not by rottlerin. These observations support our hypothesis that E-IH exposure significantly increases ET-1-mediated constriction of sMA through PKCdelta activation and modestly augments ET-1 contraction in thoracic aorta through activation of one or more cPKC isoforms. Therefore, upregulation of a PKC pathway may contribute to elevated ET-1-dependent vascular resistance in this model of hypertension.     

Role of protein kinase C in arginine vasopressin-stimulated ERK and p70S6 kinase phosphorylation

We previously showed in rat renal glomerular mesangial cells, that arginine vasopressin (AVP)-stimulated cell proliferation was mediated by epidermal growth factor receptor (EGF-R) transactivation, and activation (phosphorylation) of ERK1/2 and p70S6 kinase (Ghosh et al. [2001]: Am J Physiol Renal Physiol 280:F972-F979]. In this paper, we extend these observations and show that different protein kinase C (PKC) isoforms play different roles in mediating AVP-stimulated ERK1/2 and p70S6 kinase phosphorylation and cell proliferation. AVP treatment for 0-60 min stimulated the serine/threonine phosphorylation of PKC isoforms alpha, delta, epsilon, and zeta. The activation of PKC was dependent on EGF-R and phosphatidylinositol 3-kinase (PI3K) activation. In addition, inhibition of conventional and novel PKC isoforms by chronic (24 h) exposure to phorbol 12-myristate 13-acetate (PMA) inhibited AVP-induced activation of ERK and p70S6 kinase as well as EGF-R phosphorylation. Rottlerin, a specific inhibitor of PKCdelta, inhibited both ERK and p70S6 kinase phosphorylation and cell proliferation. In contrast, a PKCepsilon translocation inhibitor decreased ERK1/2 activation without affecting p70S6 kinase or cell proliferation, while a dominant negative PKCzeta (K281W) cDNA delayed p70S6 kinase activation without affecting ERK1/2. On the other hand, G?6976, an inhibitor of conventional PKC isoforms, did not affect p70S6 kinase, but stimulated ERK1/2 phosphorylation without affecting cell proliferation. Our results indicate that PKCdelta plays an important role in AVP-stimulated ERK and p70S6 kinase activation and cell proliferation.     

Knockout of PKC alpha enhances insulin signaling through PI3K

Insulin stimulates glucose transport and certain other metabolic processes by activating atypical PKC isoforms (lambda, zeta, iota) and protein kinase B (PKB) through increases in D3-polyphosphoinositides derived from the action of PI3K. The role of diacylglycerol-sensitive PKC isoforms is less clear as they have been suggested to be both activated by insulin and yet inhibit insulin signaling to PI3K. Presently, we found that insulin signaling to insulin receptor substrate 1-dependent PI3K, PKB, and PKC lambda, and downstream processes, glucose transport and activation of ERK, were enhanced in skeletal muscles and adipocytes of mice in which the ubiquitous conventional diacylglycerol-sensitive PKC isoform, PKC alpha, was knocked out by homologous recombination. On the other hand, insulin provoked wortmannin-insensitive increases in immunoprecipitable PKC alpha activity in adipocytes and skeletal muscles of wild-type mice and rats. We conclude that 1) PKC alpha is not required for insulin-stimulated glucose transport, and 2) PKC alpha is activated by insulin at least partly independently of PI3K, and largely serves as a physiological feedback inhibitor of insulin signaling to the insulin receptor substrate 1/PI3K/PKB/PKC lambda/zeta/iota complex and dependent metabolic processes.     

BMP-2 regulates cardiomyocyte contractility in a phosphatidylinositol 3 kinase-dependent manner

Bone morphogenetic protein-2 (BMP-2) regulates development of heart during vertebrate embryogenesis. In vitro BMP-2 induces differentiation of precardiac cells into mature cardiomyocytes by inducing the expression of cardiac-specific genes. However, the role of BMP-2 and its signaling in other cardiac functions have not been studied. We examined the action of phosphatidylinositol (PI) 3 kinase in isolated adult rat cardiomyocytes. Incubation of rat ventricular cardiomyocytes with BMP-2 increased the PI 3 kinase activity. Ly294002, a pharmacological inhibitor of PI 3 kinase, blocked BMP-2-induced PI 3 kinase activity completely. To investigate the contractility of isolated cardiomyocytes, fractional shortening was examined. BMP-2 significantly increased the percent fractional shortening of the cardiomyocytes. Inhibition of PI 3 kinase activity completely abolished this action of BMP-2. These data indicate that PI 3 kinase regulates BMP-2-induced myocyte contractility. To further confirm this observation, we used adenovirus-mediated gene transfer to express a constitutively active myristoylated catalytic subunit of PI 3 kinase in rat cardiomyocytes. Infection of cardiomyocytes with the adenovirus vector increased the expression of constitutively active PI 3 kinase within 24 h. Expression of constitutively active PI 3 kinase significantly increased cardiomyocyte contractility. Together, these data show for the first time that the growth and differentiation factor, BMP-2, stimulates cardiomyocyte contractility. Also we provide the first evidence that BMP-2-induced PI 3 kinase activity regulates this cardiomyocyte function.     

Activation of P2Y2 receptor induces c-FOS protein through a pathway involving mitogen-activated protein kinases and phosphoinositide 3-kinases in HeLa cells

The effects of P2Y2 purinoceptor activation on c-Fos expression and the signaling pathways evoked by extracellular ATP/UTP in HeLa cells were investigated. We found that P2Y2 activation induced c-Fos protein and phosphorylated the extracellular signal-regulated kinases 1 and 2 (ERK1/2). The P2Y2-stimulated c-Fos induction was partly blocked (a) by U73122, a phospholipase C inhibitor, (b) by G?6976, a conventional PKC inhibitor, (c) by PD098059, a mitogen-activated protein kinase kinase inhibitor, and, moreover, (d) by the inhibitors of phosphoinositide 3-kinases (PI3K), LY294002 and wortmannin. When G?6976 and PD098059, or G?6976 and wortmannin, were combined there was a totally inhibition of P2Y2-induced c-Fos increase. Either U73122 or G?6976 did not inhibit ERK1/2 phosphorylation induced by ATP/UTP, while it was inhibited by LY294002 (or wortmannin) and by staurosporine. Additionally, wortmannin inhibited the cytosol-to-membrane translocation of PKC- epsilon induced by ATP/UTP. These data indicated that agonist-induced PI3K and downstream PKC- epsilon activation mediated the effect of ATP/UTP on ERK1/2 activation. To test the biological consequences of ERK1/2 activation, the effect of P2Y2 on cell functions were examined. P2Y2 stimulation increased cell proliferation and this effect was attenuated by PD098059 in a dose-dependent manner, thereby indicating that the ERK pathway mediates mitogenic signaling by P2Y2. In conclusion, the activation of conventional PKCs through P2Y2 receptor acts in concert with ERK and PI3K/PKC- epsilon pathways to induce c-Fos protein and HeLa cell proliferation.     

Zap-70-independent Ca(2+) mobilization and Erk activation in Jurkat T cells in response to T-cell antigen receptor ligation

The tyrosine kinase ZAP-70 has been implicated as a critical intermediary between T-cell antigen receptor (TCR) stimulation and Erk activation on the basis of the ability of dominant negative ZAP-70 to inhibit TCR-stimulated Erk activation, and the reported inability of anti-CD3 antibodies to activate Erk in ZAP-70-negative Jurkat cells. However, Erk is activated in T cells receiving a partial agonist signal, despite failing to activate ZAP-70. This discrepancy led us to reanalyze the ZAP-70-negative Jurkat T-cell line P116 for its ability to support Erk activation in response to TCR/CD3 stimulation. Erk was activated by CD3 cross-linking in P116 cells. However, this response required a higher concentration of anti-CD3 antibody and was delayed and transient compared to that in Jurkat T cells. Activation of Raf-1 and MEK-1 was coincident with Erk activation. Remarkably, the time course of Ras activation was comparable in the two cell lines, despite proceeding in the absence of LAT tyrosine phosphorylation in the P116 cells. CD3 stimulation of P116 cells also induced tyrosine phosphorylation of phospholipase C-gamma1 (PLCgamma1) and increased the intracellular Ca(2+) concentration. Protein kinase C (PKC) inhibitors blocked CD3-stimulated Erk activation in P116 cells, while parental Jurkat cells were refractory to PKC inhibition. The physiologic relevance of these signaling events is further supported by the finding of PLCgamma1 tyrosine phosphorylation, Erk activation, and CD69 upregulation in P116 cells on stimulation with superantigen and antigen-presenting cells. These results demonstrate the existence of two pathways leading to TCR-stimulated Erk activation in Jurkat T cells: a ZAP-70-independent pathway requiring PKC and a ZAP-70-dependent pathway that is PKC independent.     

Regulation of amphetamine-stimulated dopamine efflux by protein kinase C beta

Evidence suggests that protein kinase C (PKC) and intracellular calcium are important for amphetamine-stimulated outward transport of dopamine in rat striatum. In this study, we examined the effect of select PKC isoforms on amphetamine-stimulated dopamine efflux, focusing on Ca(2+)-dependent forms of PKC. Efflux of endogenous dopamine was measured in superfused rat striatal slices; dopamine was measured by high performance liquid chromatography. The non-selective classical PKC inhibitor G?6976 inhibited amphetamine-stimulated dopamine efflux, whereas rottlerin, a specific inhibitor of PKC delta, had no effect. A highly specific PKC beta inhibitor, LY379196, blocked dopamine efflux that was stimulated by either amphetamine or the PKC activator, 12-O-tetradecanoylphorbol-13-acetate. None of the PKC inhibitors significantly altered [3H]dopamine uptake. PKC beta(I) and PKC beta(II), but not PKC alpha or PKC gamma, were co-immunoprecipitated from rat striatal membranes with the dopamine transporter (DAT). Conversely, antisera to PKC beta(I) and PKC beta(II) but not PKC alpha or PKCg amma were able to co-immunoprecipitate DAT. Amphetamine-stimulated dopamine efflux was significantly enhanced in hDAT-HEK 293 cells transfected with PKC beta(II) as compared with hDAT-HEK 293 cells alone, or hDAT-HEK 293 cells transfected with PKCa lpha or PKC beta(I). These results suggest that classical PKC beta(II) is physically associated with DAT and is important in maintaining the amphetamine-stimulated outward transport of dopamine in rat striatum.     

PKC alpha regulates the hypertrophic growth of cardiomyocytes through extracellular signal-regulated kinase1/2 (ERK1/2)

Members of the protein kinase C (PKC) isozyme family are important signal transducers in virtually every mammalian cell type. Within the heart, PKC isozymes are thought to participate in a signaling network that programs developmental and pathological cardiomyocyte hypertrophic growth. To investigate the function of PKC signaling in regulating cardiomyocyte growth, adenoviral-mediated gene transfer of wild-type and dominant negative mutants of PKC alpha, beta II, delta, and epsilon (only wild-type zeta) was performed in cultured neonatal rat cardiomyocytes. Overexpression of wild-type PKC alpha, beta II, delta, and epsilon revealed distinct subcellular localizations upon activation suggesting unique functions of each isozyme in cardiomyocytes. Indeed, overexpression of wild-type PKC alpha, but not betaI I, delta, epsilon, or zeta induced hypertrophic growth of cardiomyocytes characterized by increased cell surface area, increased [(3)H]-leucine incorporation, and increased expression of the hypertrophic marker gene atrial natriuretic factor. In contrast, expression of dominant negative PKC alpha, beta II, delta, and epsilon revealed a necessary role for PKC alpha as a mediator of agonist-induced cardiomyocyte hypertrophy, whereas dominant negative PKC epsilon reduced cellular viability. A mechanism whereby PKC alpha might regulate hypertrophy was suggested by the observations that wild-type PKC alpha induced extracellular signal-regulated kinase1/2 (ERK1/2), that dominant negative PKC alpha inhibited PMA-induced ERK1/2 activation, and that dominant negative MEK1 (up-stream of ERK1/2) inhibited wild-type PKC alpha-induced hypertrophic growth. These results implicate PKC alpha as a necessary mediator of cardiomyocyte hypertrophic growth, in part, through a ERK1/2-dependent signaling pathway.