Activation of PKC-epsilon and ERK1/2 participates in shear-induced endothelial MCP-1 expression that is repressed by nitric oxide

Vascular endothelial cells (ECs) continuously experience hemodynamic shear stress generated from blood flow. Previous studies have demonstrated that shear stress modulates monocyte chemotactic protein-1 (MCP-1) expression in ECs. This study explored the roles of protein kinase C (PKC), extracellular signal-regulated protein kinase (ERK1/2), and nitric oxide (NO) in sheared-induced MCP-1 expression in ECs. The activation of PKC-alpha and PKC-epsilon isoforms was observed in ECs exposed to shear stress. The use of an inhibitor (calphostin C) to PKC-alpha and PKC-epsilon decreased ERK1/2 activation and MCP-1 induction by shear, whereas an inhibitor (Go6976) to PKC-alpha did not affect ERK1/2 activation or MCP-1 induction. Inhibition of ERK1/2 activation by PD98059 blocked MCP-1 induction. Transfection of ECs with an antisense to PKC-epsilon abolished the shear inducibility of MCP-1 promoter. These results demonstrate that PKC-epsilon and ERK1/2 participate in shear-induced MCP-1 expression. We also examined the regulatory role of NO in MCP-1 expression. An NO donor (NOC18) suppressed shear-induced activation of PKC-epsilon and ERK1/2, and also repressed MCP-1 induction. Consistently, overexpression of endothelial nitric oxide synthase (eNOS) to enhance the endogenous generation of NO in ECs decreased the activation of PKC-epsilon and ERK1/2, and also inhibited MCP-1 expression. Taken together, these findings suggest that PKC-epsilon and ERK1/2 are critical in the signaling pathway(s) leading to the MCP-1 expression induced by shear stress. Additionally, this study indicates that NO, by repressing PKC-epsilon activity and ERK pathway activation, attenuates shear-induced MCP-1 expression.     

Cooperation between PKC-alpha and PKC-epsilon in the regulation of JNK activation in human lung cancer cells

Phorbol esters can induce activation of two mitogen-activated protein kinase (MAPK) pathways, the extracellular signal-regulated kinase (ERK) pathway and the c-Jun N-terminal kinase (JNK) pathway. Unlike ERK activation, JNK activation by phorbol esters is somehow cell-specific. However, the mechanism(s) that contribute to the cell-specific JNK activation remain elusive. In this study, we found that phorbol 12-myristate 13-acetate (PMA) induced JNK activation only in non-small cell lung cancer (NSCLC) cells, but not in small cell lung cancer (SCLC) cells, whereas ERK activation was detected in both cell types. In NSCLC cells, PMA induced JNK activation in a time- and dose-dependent manner. JNK activation was attenuated by protein kinase C (PKC) down-regulation through prolonged pre-treatment with PMA and significantly inhibited by PKC inhibitors G?6976 and GF109203X. Subcellular localization studies demonstrated that PMA induced translocation of PKC-alpha, -betaII, and -epsilon isoforms, but not PKC-delta, from the cytosol to the membrane. Analysis of various PKC isoforms revealed that PKC-epsilon was exclusively absent in the SCLC cell lines tested. Ectopic expression of PKC-epsilon in SCLC cells restored PMA activation of JNK signaling only in the presence of PKC-alpha, suggesting that PKC-alpha and PKC-epsilon act cooperatively in regulating JNK activation in response to PMA. Furthermore, using dominant negative mutants and pharmacological inhibitors, we define that a putative Rac1/Cdc42/PKC-alpha pathway is convergent with the PKC-epsilon/MEK1/2 pathway in terms of the activation of JNK by PMA.     

Cyclic strain activates redox-sensitive proline-rich tyrosine kinase 2 (PYK2) in endothelial cells

Proline-rich tyrosine kinase 2 (PYK2), structurally related to focal adhesion kinase, has been shown to play a role in signaling cascades. Endothelial cells (ECs) under hemodynamic forces increase reactive oxygen species (ROS) that modulate signaling pathways and gene expression. In the present study, we found that bovine ECs subjected to cyclic strain rapidly induced phosphorylation of PYK2 and Src kinase. This strain-induced PYK2 and Src phosphorylation was inhibited by pretreating ECs with an antioxidant N-acetylcysteine. Similarly, ECs exposed to H(2)O(2) increased both PYK2 and Src phosphorylation. An increased association of Src to PYK2 was observed in ECs after cyclic strain or H(2)O(2) exposure. ECs treated with an inhibitor to Src (PPI) greatly reduced Src and PYK2 phosphorylation, indicating that Src mediated PYK2 activation. Whereas the protein kinase C (PKC) inhibitor (calphostin C) pretreatment was shown to inhibit strain-induced NADPH oxidase activity, ECs treated with either calphostin C or the inhibitor to NADPH oxidase (DPI) reduced strain-induced ROS levels and then greatly inhibited the Src and PYK2 activation. In contrast to the activation of PYK2 and Src with calcium ionophore (ionomycin), ECs treated with a Ca(2+) chelator inhibited both phosphorylation, indicating that PYK2 and Src activation requires Ca(2+). ECs transfected with antisense to PKCalpha, but not antisense to PKCepsilon(,) reduced cyclic strain-induced PYK2 activation. These data suggest that cyclic strain-induced PYK2 activity is mediated via Ca(2+)-dependent PKCalpha that increases NADPH oxidase activity to produce ROS crucial for Src and PYK2 activation. ECs under cyclic strain thus activate redox-sensitive PYK2 via Src and PKC, and this PYK2 activation may play a key role in the signaling responses in ECs under hemodynamic influence.     

Sustained muscle contraction induced by agonists, growth factors, and Ca(2+) mediated by distinct PKC isozymes

The role of protein kinase C (PKC) in sustained contraction was examined in intestinal circular and longitudinal muscle cells. Initial contraction induced by agonists (CCK-8 and neuromedin C) was abolished by 1) inhibitors of Ca(2+) mobilization (neomycin and dimethyleicosadienoic acid), 2) calmidazolium, and 3) myosin light chain (MLC) kinase (MLCK) inhibitor KT-5926. In contrast, sustained contraction was not affected by these inhibitors but was abolished by 1) the PKC inhibitors chelerythrine and calphostin C, 2) PKC-epsilon antibody, and 3) a pseudosubstrate PKC-epsilon inhibitor. GDPbetaS abolished both initial and sustained contraction, whereas a Galpha(q/11) antibody inhibited only initial contraction, implying that sustained contraction was dependent on activation of a distinct G protein. Sustained contraction induced by epidermal growth factor was inhibited by calphostin C, PKC-alpha,beta,gamma antibody, and a pseudosubstrate PKC-alpha inhibitor. Ca(2+) (0.4 microM) induced an initial contraction in permeabilized muscle cells that was blocked by calmodulin and MLCK inhibitors and a sustained contraction that was blocked by calphostin C and a PKC-alpha,beta,gamma antibody. Thus initial contraction induced by Ca(2+), agonists, and growth factors is mediated by MLCK, whereas sustained contraction is mediated by specific Ca(2+)-dependent and -independent PKC isozymes. G protein-coupled receptors are linked to PKC activation via distinct G proteins.     

Evidence that insulin-like growth factor-1 requires protein kinase C-epsilon, PI3-kinase and mitogen-activated protein kinase pathways to protect human vascular smooth muscle cells from apoptosis

Insulin-like growth factor (IGF)-1 has been implicated in the development of occlusive vascular lesions. Although its role in vascular smooth muscle cell (VSMC) growth and migration are fairly well characterized, anti-apoptotic signals of IGF-1 in human VSMC remain largely unknown. In this study, we examined IGF-1 signals that protect human and rat VSMC from staurosporine (STAU)- and c-myc- induced apoptosis, respectively. Treatment with STAU resulted in apoptotic DNA fragmentation, phosphatidylserine externalization and cell shrinkage, but only occasional VSMC 'blebbing'. STAU-induced death and IGF-1-mediated survival were concentration dependent, while time-lapse video microscopy showed that IGF-1 inhibited c-myc-induced apoptosis by 90%. Pretreatment with mitogen-activated protein kinase/extracellular signal regulated kinase kinase (MEK) inhibitors UO126 and PD098059, or with the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin, reversed IGF-1-mediated human VSMC survival by 25-27% and 66%, respectively. Translocation studies showed that IGF-1 activated protein kinase C (PKC)-epsilon, but not PKC-alpha or PKC-delta, even in the presence of STAU, while pharmacological PKC inhibition (Ro-318220 or Go6976) implicated PKC-zeta or a novel PKC isozyme in IGF-1-mediated survival. Transient expression of activated PKC-epsilon but not activated PKC-zeta decreased myc-induced apoptosis in rat VSMC. In human VSMC, antisense oligodeoxynucleotides to PKC-epsilon partially reversed IGF-1-induced survival. In addition, IGF-1 elicited a mild but sustained activation of extracellular signal regulated kinase (ERK)1/2 in human VSMC that was abolished after 1 h in the presence of STAU. PKC downregulation reversed both IGF-1- and PMA-induced ERK activity, but platelet-derived growth factor (PDGF)-induced activity was unchanged. These results indicate for the first time that IGF-1 can protect human VSMC via multiple signals, including PKC-epsilon, PI3-K and mitogen-activated protein kinase pathways.     

Differential recovery of protein kinase C-alpha and -epsilon isozymes after long-term phorbol ester treatment in rat renal mesangial cells

Glomerular mesangial cells have been shown to express two protein kinase C (PKC) isozymes, PKC-alpha and PKC-epsilon. Upon long-term treatment with phorbol ester PKC-alpha is depleted faster than PKC-epsilon. Here we demonstrate that removal of phorbol ester results in a differential recovery of PKC-alpha and -epsilon isozymes. Whereas PKC-epsilon starts to recover within 1h, PKC-alpha does not begin to recover before 4 h after removal of phorbol ester. These data suggest a differential rate of protein synthesis of PKC-alpha and -epsilon. In parallel to the recovery of PKC isozymes mesangial cells also regained their functional responsiveness, i.e., stimulation of prostaglandin synthesis and feedback inhibition of angiotensin II-stimulated InsP3 formation.     

Protein kinase C-epsilon plays a role in neurite outgrowth in response to epidermal growth factor and nerve growth factor in PC12 cells.

In this study, we examined the role of specific protein kinase C (PKC) isoforms in the differentiation of PC12 cells in response to nerve growth factor (NGF) and epidermal growth factor (EGF). PC12 cells express PKC-alpha, -beta, -gamma, -delta, -epsilon, -mu, and -zeta. For PKC-delta, -epsilon, and -zeta, NGF and EGF exerted differential effects on translocation. Unlike overexpression of PKC-alpha and -delta, overexpression of PKC-epsilon caused enhanced neurite outgrowth in response to NGF. In the PKC-epsilon-overexpressing cells, EGF also dramatically induced neurite outgrowth, arrested cell proliferation, and induced a sustained phosphorylation of mitogen-activated protein kinase (MAPK), in contrast to its mitogenic effects on control cells or cells overexpressing PKC-alpha and -delta. The induction of neurite outgrowth by EGF was inhibited by the MAPK kinase inhibitor PD95098. In cells overexpressing a PKC-epsilon dominant negative mutant, NGF induced reduced neurite outgrowth and a more transient phosphorylation of MAPK than in controls. Our results suggest an important role for PKC-epsilon in neurite outgrowth in PC12 cells, probably via activation of the MAPK pathway.     

Cyclic strain stimulates isoform-specific PKC activation and translocation in cultured human keratinocytes

Previous studies have demonstrated that cyclic strain induces keratinocyte proliferative and morphological changes. Since protein kinase C (PKC) is known to play an important role in the regulation of keratinocyte growth and differentiation, the objective of this study was to determine the role of the PKC signaling pathway as a mediator of strain modulation of the keratinocyte phenotype. In particular, we tested the following specific hypotheses: (1) cyclic strain stimulates PKC activity and translocation, (2) cyclic strain activates PKC in an isoform-specific manner, and (3) PKC mediates the strain activated proliferative and morphological response in cultured human keratinocytes. To test these hypotheses, keratinocytes were subjected to vacuum-generated cyclic strain (10% average strain), followed by measurement of PKC activity, PKC isoform distribution by Western blot analysis and confocal microscopy, and examination of the effect of PKC inhibitors (calphostin C and staurosporine) on strain induced proliferative and morphological changes. We observed stimulation of PKC activity (62.3 ± 5.1% increase) coupled with translocation of PKC from the cytosolic to the membrane fraction in keratinocytes subjected to acute cyclic strain. Cyclic strain also caused translocation of PKC α and δ, but not ζ isoforms, from the cytosolic to the membrane fraction as demonstrated by both Western blot analysis and confocal microscopy. PKC β was not detected in these cells. PKC inhibitors, calphostin C (10 nM), and staurosporine (5 nM), inhibited strain-induced PKC activation and keratinocyte proliferation, but did not block the effects of strain on cellular morphology or alignment. We conclude that these data support our hypothesis that cyclic strain stimulates PKC activity and translocation in an isoform-specific manner in cultured human keratinocytes. Moreover, our studies with PKC inhibitors support the hypothesis that strain-induced changes in the keratinocyte phenotype may be selectively modulated by PKC. J. Cell. Biochem. 67:327–337, 1997. © 1997 Wiley-Liss, Inc.     

PKC-epsilon regulation of extracellular signal-regulated kinase: a potential role in phenylephrine-induced cardiocyte growth

Hypertrophic growth of cardiac muscle is dependent on activation of the PKC-epsilon isoform. To define the effectors of PKC-epsilon involved in growth regulation, recombinant adenoviruses were used to overexpress either wild-type PKC-epsilon (PKC-epsilon/WT) or dominant negative PKC-epsilon (PKC-epsilon/DN) in neonatal rat cardiocytes. PKC-epsilon/DN inhibited acute activation of PKC-epsilon produced in response to phorbol ester and reduced ERK1/2 activity as measured by the phosphorylation of p42 and p44 isoforms. The inhibitory effects were specific to PKC-epsilon because PKC-epsilon/DN did not prevent translocation of either PKC-alpha or PKC-delta. Overexpression of PKC-epsilon/DN blunted the acute increase in ERK1/2 phorphorylation induced by the alpha(1)-adrenergic agonist phenylephrine (PE ). Inhibition of PKC-delta with rottlerin potentiated the effects of PE on ERK1/2 phosphorylation. PKC-epsilon/DN adenovirus also blocked cardiocyte growth as measured after 48 h of PE treatment, although the multiplicity of infection was lower than that required to block acute ERK1/2 activation. PE activated p38 mitogen-activated protein kinase as measured by its phosphorylation, but the response was not blocked by PKC inhibitors or by overexpression of PKC-epsilon/DN. Taken together, these studies show that the hypertrophic agonist PE regulates ERK1/2 activity in cardiocytes by a pathway dependent on PKC-epsilon and that PE-induced growth is mediated by PKC-epsilon.     

Distinct PKC isoforms mediate cell survival and DNA synthesis in thrombin-induced myofibroblasts

Thrombin activates protease-activated receptor (PAR)-1 and induces a myofibroblast phenotype in normal lung fibroblasts that resembles the phenotype of scleroderma lung fibroblasts. We now demonstrate that PAR-1 expression is dramatically increased in lung tissue from scleroderma patients, where it is associated with inflammatory and fibroproliferative foci. We also observe that thrombin induces resistance to apoptosis in normal lung fibroblasts, and this process is regulated by protein kinase C (PKC)-epsilon but not by PKC-alpha. Overexpression of a constitutively active (c-a) form of PAR-1 or PKC-epsilon significantly inhibits Fas ligand-induced apoptosis in lung fibroblasts, whereas scleroderma lung fibroblasts are resistant to apoptosis de novo. Thrombin translocates p21Cip1/WAF1, a signaling molecule downstream of PKC, from the nucleus to cytoplasm in normal lung fibroblasts mimicking the localization of p21Cip1/WAF1 in scleroderma lung fibroblasts. Overexpression of c-a PKC-alpha or PKC-epsilon results in accumulation of p21Cip1/WAF1 in the cytoplasm. Depletion of PKC-alpha or inhibition of mitogen-activated protein kinase (MAPK) blocks thrombin-induced DNA synthesis in lung fibroblasts. Inhibition of PKC by calphostin or PKC-alpha, but not PKC-epsilon, by antisense oligonucleotides prevents thrombin-induced MAPK phosphorylation and accumulation of G(1) phase regulatory protein cyclin D1, suggesting that PKC-alpha, MAPK, and cyclin D1 mediate lung fibroblast proliferation. These data demonstrate that two distinct PKC isoforms mediate thrombin-induced resistance to apoptosis and proliferation and suggest that p21Cip1/WAF1 promotes both phenomena.     

Activation of Ras, Raf-1 and protein kinase C in differentiating human neuroblastoma cells after treatment with phorbolester and NGF

The human neuroblastoma cell line SH-SY5Y/TrkA differentiates in vitro and acquires a sympathetic phenotype in response to phorbolester (activator of protein kinase C, PKC) in the presence of serum or growth factors, or nerve growth factor (NGF). We have now investigated to what extent phorbolester and NGF cause activation of Ras and Raf-1 and the involvement of PKC in this response in differentiating SH-SY5Y/TrkA cells. NGF stimulated increased accumulation of Ras-GTP and a threefold activation of Raf-1. In contrast, 12-O-tetradecanoylphorbol-13-acetate (TPA) had no effect on the amount of Ras-GTP but led to a smaller activation of Raf-1. NGF caused a limited increase in phosphorylation of Raf-1 compared with TPA, and NGF-induced Raf activity was independent of PKC. Analysis of phosphorylation of the endogenous PKC substrate myristoylated alanine-rich C-kinase substrate (MARCKS), and of subcellular distribution of PKC-alpha, -delta, and -epsilon revealed that NGF only caused a very small activation of PKC in SH-SY5Y/TrkA cells. The results identify Raf-1 as a target for both TPA- and NGF-induced signals in differentiating SH-SY5Y/TrkA cells and demonstrate that signalling to Raf-1 was mediated via distinct mechanisms.     

Phosphatidylinositol-3 kinase dependent MAP kinase activation via p21ras in endothelial cells exposed to cyclic strain

Hemodynamic forces play a key role in the modulation of the morphology and function of the endothelium by activating several kinases. We have previously shown that cyclic strain, a repetitive mechanical stretch, induces activation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), members of the mitogen activated protein (MAP) kinase family. In order to investigate the upstream pathway of strain-induced ERK1/2 activation, we examined p21ras activation by cyclic strain and the effect of wortmannin and LY294002, phosphatidylinositol-3 kinase (PI 3-kinase) inhibitors on ERK1/2 phosphorylation. Cyclic strain induced a transient and rapid activation of p21ras at 1 min after strain. Wortmannin inhibited strain-induced ERK1/2 activation by 56.3 and 86.3 %, respectively. LY294002 inhibited ERK1 activation completely and ERK2 activation by 42.9%. These results suggest a possible involvement of p21ras and PI 3-kinase in the signal transduction pathway leading to the strain-induced ERK1/2 activation.     

Protein kinase C modulates agonist-sensitive release of Ca2+ from internal stores in HEK293 cells overexpressing the calcium sensing receptor

This study examined the mechanism of Ca2+ entry and the role of protein kinase C (PKC) in Ca2+ signaling induced by activation of the calcium sensing receptor (CaR) in HEK293 cells stably expressing the CaR. We demonstrate that influx of Ca2+ following CaR activation exhibits store-operated characteristics in being associated with Ca2+ store depletion and inhibited by 2-aminoethoxydiphenyl borate. Inhibition of PKC with GF109203X, Go6983, or Go6976 and down-regulation of PKC activity enhanced the release of Ca2+ from internal stores in response to the polyvalent cationic CaR agonist neomycin, whereas activation of PKC with acute 12-O-tetradecanoylphorbol-13-acetate treatment decreased the release. In contrast, overexpression of wild type PKC-alpha or -epsilon augmented the neomycin-induced release of Ca2+ from internal stores, whereas dominant negative PKC-epsilon strongly decreased the release, but dominant negative PKC-alpha had little effect. Prolonged treatment of cells with 12-O-tetradecanoylphorbol-13-acetate effectively down-regulated immunoreactive PKC-alpha but had little effect on the expression of PKC-epsilon. Together these results indicate that diacylglycerol-responsive PKC isoforms differentially influence CaR agonist-induced release of Ca2+ from internal stores. The fundamentally different results obtained when overexpressing or functionally down-regulating specific PKC isoforms as compared with pharmacological manipulation of PKC activity indicate the need for caution when interpreting data obtained with the latter approach.     

The synergistic activation of Raf-1 kinase by phorbol myristate acetate and hydrogen peroxide in NIH3T3 cells

We have previously demonstrated that a 33kDa C-terminal fragment of c-Raf-1 underwent a mobility shift in response to hydrogen peroxide (H(2)O(2)) and phorbol myristate acetate (PMA), respectively. In this study, we have demonstrated that H(2)O(2) induced the activation of N-terminal deletion mutant as well as full length Raf-1 kinase. The pharmacological PKC activator PMA also induced a weak increase in Raf-1 kinase activity through PKC-epsilon activation as determined by the transient expression of dominant negative mutants of PKC-epsilon-K436R. Interestingly, H(2)O(2) produced synergistic increase of PMA-stimulated Raf-1 kinase activation after simultaneous treatment of PMA and H(2)O(2). This synergistic activation of Raf-1 kinase was further enhanced by cypermethrin (an inhibitor of protein phosphatase 2B) and dephostatin (tyrosine kinase inhibitor) implying an inhibitory role for these phosphatases in the Raf-1 signaling pathway. Taken together, our data suggest that the synergistic activation of Raf-1 kinase in response to PMA and H(2)O(2) occurs via mechanisms that involve an interaction of Raf-1 kinase and PKC-epsilon, along with a transient phosphorylation of both Raf-1 kinase and PKC.     

Differential regulation of matrix metalloproteinase-2 and -9 expression and activity in adult rat cardiac fibroblasts in response to interleukin-1beta

Matrix metalloproteinases (MMPs), a family of endoproteinases, are implicated in cardiac remodeling. Interleukin-1beta (IL-1beta), which is increased in the heart following myocardial infarction, increases expression and activity of MMP-2 (gelatinase A) and -9 (gelatinase B) in cardiac fibroblasts. Previously, we have shown that IL-1beta activates ERK1/2, JNKs, and protein kinase C (PKC). However, signaling pathways involved in the regulation of MMP-2 and -9 expression and activity are not yet well understood. Using adult rat cardiac fibroblasts, we show that inhibition of ERK1/2 and JNKs inhibits IL-1beta-stimulated increases in MMP-9, not MMP-2, expression and activity. Chelerythrine, an inhibitor of PKC, inhibited activation of ERK1/2 and JNKs and expression and activity of both MMPs. Selective inhibition of PKC-alpha/beta1 using G?6976 inhibited JNKs activation and the expression and activity of MMP-9, not MMP-2. Inhibition of PKC-theta and PKC-zeta using pseudosubstrates inhibited IL-1beta-stimulated activation of ERK1/2 and JNKs and the expression and activity of MMP-2 and -9. Inhibition of PKC-epsilon had no effect. IL-1beta activated NF-kappaB pathway as measured by increased phosphorylation of IKKalpha/beta and IkappaB-alpha. Inhibition of ERK1/2, JNKs, and PKC-alpha/beta1 had no effect on NF-kappaB activation, whereas inhibition of PKC-theta and PKC-zeta inhibited IL-1beta-stimulated activation of NF-kappaB. SN50, NF-kappaB inhibitor peptide, inhibited IL-1beta-stimulated increases in MMP-2 and -9 expression and activity. These observations suggest that 1) activation of ERK1/2 and JNKs plays a critical role in the regulation of MMP-9, not MMP-2, expression and activity; 2) PKC-alpha/beta1 act upstream of JNKs, not ERK1/2; 3) PKC-zeta and -theta, not PKC-epsilon, act upstream of JNKs, ERK1/2, and NF-kappaB; and 4) activation of NF-kappaB stimulates expression and activity of MMP-2 and -9.     

The novel protein kinase C isoforms -delta and -epsilon modulate caerulein-induced zymogen activation in pancreatic acinar cells

Isoforms of protein kinase C (PKC) have been shown to modulate some cellular responses such as pathological secretion and generation of inflammatory mediators during acute pancreatitis (AP). We propose that PKC also participates in premature zymogen activation within the pancreatic acinar cell, a key event in the initiation of AP. This hypothesis was examined in in vivo and cellular models of caerulein-induced AP using PKC activators and inhibitors. Phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA, 200 nM), a known activator of PKC, enhanced zymogen activation at both 0.1 nM and 100 nM caerulein, concentrations which mimic physiological and supraphysiological effects of the hormone cholecystokinin, respectively, in preparations of pancreatic acinar cells. Isoform-specific PKC inhibitors for PKC-delta and PKC-epsilon reduced supraphysiological caerulein-induced zymogen activation. Using a cell-free reconstitution system, we showed that inhibition of PKC-delta and -epsilon, reduced zymogen activation in both zymogen granule-enriched and microsomal fractions. In dispersed acinar cells, 100 nM caerulein stimulation caused PKC-delta and -epsilon isoform translocation to microsomal membranes using cell fractionation and immunoblot analysis. PKC translocation was confirmed with in vivo studies and immunofluorescence microscopy in pancreatic tissues from rats treated with or without 100 nM caerulein. PKC-epsilon redistributed from an apical to a supranuclear region following caerulein administration. The signal for PKC-epsilon overlapped with granule membrane protein, GRAMP-92, an endosomal/lysosomal marker, in a supranuclear region where zymogen activation takes place. These results indicate that PKC-delta and -epsilon isoforms translocate to specific acinar cell compartments and modulate zymogen activation.     

Role of ras-dependent ERK activation in phorbol ester-induced endothelial cell barrier dysfunction

The treatment of endothelial cell monolayers with phorbol 12-myristate 13-acetate (PMA), a direct protein kinase C (PKC) activator, leads to disruption of endothelial cell monolayer integrity and intercellular gap formation. Selective inhibition of PKC (with bisindolylmaleimide) and extracellular signal-regulated kinases (ERKs; with PD-98059, olomoucine, or ERK antisense oligonucleotides) significantly attenuated PMA-induced reductions in transmonolayer electrical resistance consistent with PKC- and ERK-mediated endothelial cell barrier regulation. An inhibitor of the dual-specificity ERK kinase (MEK), PD-98059, completely abolished PMA-induced ERK activation. PMA also produced significant time-dependent increases in the activity of Raf-1, a Ser/Thr kinase known to activate MEK ( approximately 6-fold increase over basal level). Similarly, PMA increased the activity of Ras, which binds and activates Raf-1 ( approximately 80% increase over basal level). The Ras inhibitor farnesyltransferase inhibitor III (100 microM for 3 h) completely abolished PMA-induced Raf-1 activation. Taken together, these data suggest that the sequential activation of Ras, Raf-1, and MEK are involved in PKC-dependent endothelial cell barrier regulation.     

Protein kinase Cdelta overexpression enhances radiation sensitivity via extracellular regulated protein kinase 1/2 activation, abolishing the radiation-induced G(2)-M arrest.

Protein kinase C (PKC) has been widely implicated in regulation ofcell growth/cell cycle progression and apoptosis. However,the role of PKCdelta in radiosensitivity and cell cycle regulation remains unclear. Overexpression of PKCdelta increased Ca2+-independent PKC activity without altering other PKC isoforms (PKCalpha, -beta1, -epsilon, and -zeta), and extracellular regulated protein kinase (ERK) 1/2 activity was also increased in PKCdelta-specific manner. A clonogenic survival assay showed that PKCdelta-overexpressed cells had more radiosensitivity and pronounced induction of apoptosis than control cells. Flow cytometric analysis revealed that PKCdelta made the cells escape from radiation-induced G(2)-M arrest. Moreover, p53 and p21(Waf) induction by radiation were higher in PKCdelta-overexpressed cells than control cells, and PKCdelta-mediated apoptosis was reduced, when radiation-induced ERK1/2 activity was inhibited by PD98059. Furthermore, PKCdelta antisense and rottlerin, PKC inhibitor-abrogated PKCdelta-mediated radiosensitivity and reduced ERK1/2 activity to the control vector level. These results demonstrated that PKCdelta overexpression enhanced radiation-induced apoptosis and radiosensitivity via ERK1/2 activation, thereby abolishing the radiation-induced G(2)-M arrest and finally apoptosis.     

Protein kinase C-epsilon protects PC12 cells against methamphetamine-induced death: possible involvement of suppression of glutamate receptor

The involvement of PKC isoform in the methamphetamine (MA)-induced death of neuron-like PC12 cell was studied. The death and the enhanced terminal dUTP nick end labeling (TUNEL) staining were inhibited by a caspase inhibitor, z-Val-Ala-Asp- (OMe)-CH(2)F (z-VAD-fmk). However, the cell death shows neither morphological nor biochemical features of apoptosis or necrosis. The cell death was suppressed by a protein kinase C (PKC) activator, 12,13-phorbol myristate acetate, but was enhanced by PKC specific inhibitor calphostin C or bisindolylmaleimide, not by PKC inhibitor relatively specific for PKC-alpha (safingol) or PKC-delta (rottlerin). Western blotting demonstrated the expression of PKC-alpha, gamma, delta, epsilon and zeta, of which PKC-epsilon translocated from the soluble to the particulate fraction after MA-treatment. Antisense to PKC-epsilon enhanced MA-induced death. A glutamate receptor antagonist MK801 abrogated the cell death, which is reversed by PKC inhibition. These data suggest that PKC-epsilon promotes PC12 cell survival through glutamate receptor suppression.