Effect of ageing on the expression of protein kinase C and its activation by 1,25(OH)2-vitamin D3 in rat skeletal muscle

To characterize age-induced effects on muscle protein kinase C (PKC) and its regulation by the steroid hormone 1,25(OH)2-vitamin D3 [1,25(OH)2D3], changes in PKC activity and the expression and translocation of the specific PKC conventional isoforms alpha and beta, novel isoforms delta, epsilon, and theta and atypical isoform zeta were studied in homogenates and subcellular fractions from skeletal muscle of young (3 months) and aged (24 months) rats treated in vitro with 1,25(OH)2D3. The hormone (10(-9) M) increased total and membrane PKC activity, within 1 min, and these effects were completely blunted in muscle from aged rats. The presence of PKC isoenzymes was shown by Western blot analysis with the use of specific antibodies. The expression of PKC alpha, beta and delta was greatly diminished in old rats, whereas age-related changes were less pronounced in the isoforms epsilon, theta and zeta. After a short exposure (1 min) of muscle to 1,25(OH)2D3, increased amounts of PKC alpha and beta in muscle membranes and reverse translocation (from membrane to cytosol) of PKC epsilon were observed only in young animals. The data indicate that, in rat muscle, ageing impairs calcium-dependent PKC (alpha and beta) and calcium-independent PKC (delta, epsilon, theta and zeta) signal transduction pathways under selective regulation by 1,25(OH)2D3.     

Respiratory syncytial virus infection results in activation of multiple protein kinase C isoforms leading to activation of mitogen-activated protein kinase

Respiratory syncytial virus (RSV) is an important respiratory pathogen that preferentially infects epithelial cells in the airway and causes a local inflammatory response. Very little is known about the second messenger pathways involved in this response. To characterize some of the acute response pathways involved in RSV infection, we used cultured human epithelial cells (A549) and optimal tissue culture-infective doses (TCID(50)) of RSV. We have previously shown that RSV-induced IL-8 release is linked to activation of the extracellular signal-related kinase (ERK) mitogen-activated protein kinase pathway. In this study, we evaluated the upstream events involved in ERK activation by RSV. RSV activated ERK at two time points, an early time point consistent with viral binding and a later sustained activation consistent with viral replication. We next evaluated the role of protein kinase C (PKC) isoforms in RSV-induced ERK kinase activity. We found that A549 cells contain the Ca(2+)-dependent isoforms alpha and beta1, and the Ca(2+)-independent isoforms delta, epsilon, eta, mu, theta, and zeta. Western analysis showed that RSV caused no change in the amounts of these isoforms. However, kinase activity assays demonstrated activation of isoform zeta within 10 min of infection, followed by a sustained activation of isoforms beta1, delta, epsilon, and mu 24-48 h postinfection. A cell-permeable peptide inhibitor specific for the zeta isoform decreased early ERK kinase activation by RSV. Down-regulation of the other PKC isoforms with PMA blocked the late sustained activation of ERK by RSV. These studies suggest that RSV activates multiple PKC isoforms with subsequent downstream activation of ERK kinase.     

Differential action of nerve growth factor and phorbol ester TPA on rat synaptosomal PKC isoenzymes.

The subcellular redistribution of protein kinase C family members (alpha, beta, gamma, delta, epsilon and zeta isoforms) was examined in response to treatment with 12-O-tetradecanoyl-phorbol-13 acetate (TPA) or nerve growth factor (NGF) in a synaptosomal-enriched P2 fraction from rat brain. Treatment with TPA affected members of the classical-PKC family (alpha, beta and gamma), resulting in a final loss of total protein of each isoenzyme. The kinetics of changes of members of the novel-PKC family are different, the delta isoform being translocated, but not down-regulated, while the epsilon isoform showing only a slight diminishing of immunoreactivity in the soluble and particulate fractions. The atypical-PKC zeta isoform was not translocated in response to TPA. Incubation with NGF induced a loss of immunoreactivity of the cytosolic alpha, beta and epsilon isoforms, but the membrane fractions of these isoforms were not appreciably affected. In contrast, a marked translocation from cytosol to membrane was observed in the case of the gamma and delta isoforms. The zeta isoform presented a slight translocation from the particulate fraction to the soluble fraction. Thus, the results show that the effects of TPA and NGF on PKC isoforms are not coincident in synaptosomes, the 6 isoform being activated and not down-regulated by both treatments, whereas the gamma isoform is only down-regulated in the case of TPA, but presents sustained translocation with NGF, indicating that PKC isoform-specific degradation pathways exist in synaptic terminals. The effects of NGF on PKC isoforms coexist with an increase in NGF-induced polyphosphoinositide hydrolysis, suggesting the participation of phospholipases.     

Chronic exposure to ammonia induces isoform-selective alterations in the intracellular distribution and NMDA receptor-mediated translocation of protein kinase C in cerebellar neurons in culture

Hyperammonemia is responsible for most neurological alterations in patients with hepatic encephalopathy by mechanisms that remain unclear. Hyperammonemia alters phosphorylation of neuronal protein kinase C (PKC) substrates and impairs NMDA receptor-associated signal transduction. The aim of this work was to analyse the effects of hyperammonemia on the amount and intracellular distribution of PKC isoforms and on translocation of each isoform induced by NMDA receptor activation in cerebellar neurons. Chronic hyperammonemia alters differentially the intracellular distribution of PKC isoforms. The amount of all isoforms (except PKC zeta) was reduced (17-50%) in the particulate fraction. The contents of alpha, beta1, and epsilon isoforms decreased similarly in cytosol (65-78%) and membranes (66-83%), whereas gamma, delta, and theta; isoforms increased in cytosol but decreased in membranes, and zeta isoform increased in membranes and decreased in cytosol. Chronic hyperammonemia also affects differentially NMDA-induced translocation of PKC isoforms. NMDA-induced translocation of PKC alpha and beta is prevented by ammonia, whereas PKC gamma, delta, epsilon, or theta; translocation is not affected. Inhibition of phospholipase C did not affect PKC alpha translocation but reduced significantly PKC gamma translocation, indicating that NMDA-induced translocation of PKC alpha is mediated by Ca2+, whereas PKC gamma translocation is mediated by diacylglycerol. Chronic hyperammonemia reduces Ca+2-mediated but not diacylglycerol-mediated translocation of PKC isoforms induced by NMDA.     

The epsilon isoform of protein kinase C is involved in regulation of the LTD(4)-induced calcium signal in human intestinal epithelial cells

We investigated the potential roles of specific isoforms of protein kinase C (PKC) in the regulation of leukotriene D(4)-induced Ca(2+) signaling in the intestinal epithelial cell line Int 407. RT-PCR and Western blot analysis revealed that these cells express the PKC isoforms alpha, betaII, delta, epsilon, zeta, and mu, but not betaI, gamma, eta, or theta;. The inflammatory mediator leukotriene D(4) (LTD(4)) caused the TPA-sensitive PKC isoforms alpha, delta, and epsilon, but not betaII, to rapidly translocate to a membrane-enriched fraction. The PKC inhibitor GF109203X at 30 microM but not 2 microM significantly impaired the LTD(4)-induced Ca(2+) signal, indicating that the response involves a novel PKC isoform, such as delta or epsilon, but not alpha. LTD(4)-induced Ca(2+) signaling was significantly suppressed in cells pretreated with TPA for 15 min and was abolished when the pretreatment was prolonged to 2 h. Immunoblot analysis revealed that the reduction in the LTD(4)-induced calcium signal coincided with a reduction in the cellular content of PKCepsilon and, to a limited extent, PKCdelta. LTD(4)-induced Ca(2+) signaling was also markedly suppressed by microinjection of antibodies against PKCepsilon but not PKCdelta. These data suggest that PKCepsilon plays a unique role in regulation of the LTD(4)-dependent Ca(2+) signal in intestinal epithelial cells.     

Expression and localization of protein kinase C theta isoform in mouse testis.

Protein kinase C (PKC) is encoded by a complex of a gene family, and its multiple isoforms are expressed in various mammalian tissues. The objective of this study was to investigate the expression and localization of a PKC theta isoform in mouse testis. PKC theta displays the highest homology to PKC delta, lacks the Ca2+-binding C2 domain and, thus, belongs to the subfamily of Ca2+-independent PKC enzymes which also includes the delta, epsilon, zeta and eta isoforms. We analyzed the PKC theta mRNA and protein by Northern blotting, in situ hybridization, and immunohistochemistry. In testes of normal mice, signals of PKC theta isoform expression were detected specifically in the interstitial cells of testes. The expression of PKC theta isoform was also detected in testes of germ cell-deficient W/W(v) mice. These results suggest that PKC theta isoform has the specific biological functions in the interstitial cells of testis.     

Analysis of protein kinase C isoforms involved in the activation of laminin receptor in Raw264.7 macrophages

Adherence of hematopoietic macrophages to a laminin (LM) substratum requires protein kinase C (PKC)-dependent activation of LM receptor. This study was performed to analyze PKC isoform(s) leading to the activation of LM receptor during Raw264.7 macrophage-like cell adhesion to a LM substratum. Raw264.7 cells expressed multiple PKC isoforms, including alpha, beta I, delta, epsilon, zeta, lambda/iota, and mu. Among the PKC isoforms expressed, selective activation of PKC delta and epsilon was sufficient to induce cell adhesion to LM. PKC-dependent cell adherence was blocked by the selective inhibition of PKC delta, suggesting that PKC delta was the responsible PKC isoform leading to activation of LM receptor. PKC delta appeared to activate LM receptor in an intact microfilament-dependent pathway, because disruption of microfilament inhibited cell adhesion to LM without affecting PKC delta activation.     

Multiple isoforms of protein kinase C in lymphocytes and airway smooth muscle of guinea pig

The isoenzyme pattern of protein kinase C (PKC) in lymphocytes and airway smooth muscles (ASM) was examined by Western blot using commercially available monoclonal antibodies. The results showed the presence of PKC alpha, beta, gamma, epsilon, eta, mu and zeta in lymphocytes and PKC alpha, gamma, epsilon, eta and zeta in ASM. The unexpected feature was the presence of PKCgamma in both lymphocytes and ASM of guinea pigs. Expression of this PKC isoform is usually restricted to tissues in the central nervous system or spinal cord. Expression of PKC delta, theta, lambda and tau was not detected in either lymphocytes or ASM.     

Different translocation of three distinct PKC isoforms with tumor-promoting phorbol ester in human platelets

Protein kinase C (PKC), a family of related but distinct enzymes whose cellular functions are poorly understood, acts in synergy with Ca2+ mobilization for the activation of platelets. Using specific antibodies for the different isoforms, immunoblot analysis revealed the presence in human platelets of three different PKC subtypes which specifically react with alpha, beta and zeta-PKC antibodies. Whereas the subcellular distribution of the alpha PKC remained unaffected, incubation of platelets with 1 microM PMA for 2 min resulted in a significant subcellular distribution from cytosol to membrane of beta-PKC (25%) and zeta (15%). The beta-PKC isoform is more sensitive than alpha and zeta-PKC to PMA, since 100 nM PMA resulted in a translocation of 85%, 64% and 66% respectively of a maximum translocation observed with 1 microM PMA.     

Evidence for clonal heterogeneity of the expression of six protein kinase C isoforms in murine B and T lymphocytes.

Protein kinase C (PKC), which plays a pivotal role in lymphocyte activation, represents a homologous family of at least nine proteins. Seven genes that encode PKC proteins have been identified. Since the regulatory properties and substrate specificities of the isoforms are not identical in vitro, it is possible that each isoform plays a unique role in cell activation. Toward an understanding of the role of PKC isoforms in lymphocyte activation we have studied the expression of mRNA encoding six of the isoforms (alpha, beta, gamma, delta, epsilon, and zeta) in T cell clones and B cell lines. PKC isoform phenotyping was done by MAPPing using isoform-specific primers and slot-blot analyses of mRNA were performed using specific probes. T cell clones and B cell lines were determined to express levels of the delta, epsilon, and zeta isoforms of PKC that were detectable by MAPPing. Plasmacytomas did not express PKC-beta message detectable by MAPPing. Slot blot analyses and Western blot analyses with peptide-specific antibody confirmed that B cell plasmacytomas did not express PKC-beta mRNA or protein. T cell clones and B cell lines were similar in that none expressed PKC-gamma. In cells that expressed PKC isoforms that were detectable by the MAPPing protocol, there was heterogeneity in the relative abundance of isoform mRNA (PKC-delta and -beta) and protein (PKC-beta and -epsilon). Such diversity of isoform expression could be responsible for the differential responsiveness of lymphocyte clones to activating stimuli.     

Selective protein kinase C isoforms are involved in endothelin-1-induced human uterine contraction at the end of pregnancy

The role of protein kinase C (PKC) in contraction of the human myometrium induced by endothelin-1 (ET-1) was investigated at the end of pregnancy. The expression and subcellular distribution of PKC isoforms were examined by Western blot analysis using isoform-specific antibodies. At least three conventional PKC isoforms (cPKC; alpha, beta1, and beta2), two novel PKC isoforms (epsilon and delta), and an atypical PKC isoform (zeta) were detected in pregnant myometrium. Quantitative immunoblotting revealed that all these isoforms were mainly distributed in the particulate fraction. The lack of a calcium chelator to modify the particulate sequestration of cPKC suggests an interaction with an anchoring protein such as receptor-activated C kinase-1, which is evidenced in the particulate fraction of the pregnant myometrium. Of the six isoforms, only PKCbeta1, PKCbeta2, PKCdelta, and PKCzeta were translocated to the particulate fraction, and PKCepsilon to the cytoskeletal fraction, after stimulation with ET-1. Involvement of PKC in the ET-1-induced contractile response is supported by the inhibition caused by the PKC inhibitor calphostin C. However, we demonstrated that the selective cPKC isoform inhibitor, G? 6976, as well as the substantial depletion of PKCbeta1 and PKCepsilon and the partial depletion of PKCalpha and PKCdelta by a long-term treatment with phorbol 12,13-dibutyrate did not prevent ET-1-induced contraction. Accordingly, our results suggest that PKCdelta and PKCzeta activation mediated ET-1-induced contraction, whereas cPKC isoforms were not implicated in the human pregnant myometrium.     

Tamoxifen inhibits phorbol ester stimulated osteoclastic bone resorption: an effect mediated by calmodulin.

Tamoxifen inhibits bone resorption by disrupting calmodulin-dependent processes. Since tamoxifen inhibits protein kinase C in other cells, we compared the effects of tamoxifen and the phorbol ester, phorbol myristate acetate, on osteoclast activity. Phorbol esters stimulate bone resorption and calmodulin levels four-fold (k0.5 = 0.1-0.3 microM). In contrast, tamoxifen inhibited osteoclast activity approximately 60% with an IC50 of 1.5 microM, had no apparent effect on protein kinase C activity in whole-cell lysates, and reduced protein kinase C alpha recovered by immunoprecipitation 75%. Phorbol esters stimulated resorption in a time-dependent manner that was closely correlated with a similar-fold increase in calmodulin. Protein kinase C alpha, beta, delta, epsilon, and zeta were all down-regulated in response to phorbol ester treatment. Tamoxifen and trifluoperazine inhibited PMA-dependent increases in bone resorption and calmodulin by 85 +/- 10%. Down-regulation of protein kinase C isoforms by phorbol esters suggests that the observed increases in bone resorption and calmodulin levels are most likely due to a mechanism independent of protein kinase C and dependent on calmodulin. In conclusion, the data suggest that protein kinase C negatively regulates calmodulin expression and support the hypothesis that the effects of both phorbol esters and tamoxifen on osteoclast activity is mediated by calmodulin.     

Protein kinase CbetaI interacts with the beta1-adrenergic signaling pathway to attenuate lipolysis in rat adipocytes

We have shown previously that insulin attenuates beta1-adrenergic receptor (beta1-AR)-mediated lipolysis via activation of protein kinase C (PKC) in rat adipocytes. This antilipolysis persists after removal of insulin and is independent of the phosphodiesterase 3B activity, and phorbol 12-myristate 13-acetate (PMA) could substitute for insulin to produce the same effect. Here, we attempted to identify the PKC isoform responsible for antilipolysis. Isolated adipocytes were treated with high and low concentrations of PMA for up to 6 h to degrade specific PKC isoforms. In the PMA-treated cells, the downregulation profiles of PKC isoforms alpha and betaI, but not betaII, delta, epsilon, or zeta, correlated well with a decrease of lipolysis-attenuating effect of PMA. After rats fasted for 24 h, adipocyte expression of PKC isoform alpha increased, while expression of PKCdelta decreased. Fasting did not change the potency of PMA to attenuate lipolysis, however. The lipolysis-attenuating effect of PMA was blocked by the PKCbetaI/betaII inhibitor LY 333531, but not by the PKCbetaII inhibitor CGP 53353 or the PKCdelta inhibitor rottlerin. These data suggest that PKCbetaI interacts with beta1-AR signaling and attenuates lipolysis in rat adipocytes.     

Expression of four protein kinase C isoforms in rat fibroblasts. Distinct subcellular distribution and regulation by calcium and phorbol esters.

Protein kinase C (PKC), the major receptor for tumor-promoting phorbol esters, consists of a family of at least eight distinct lipid-regulated enzymes. How the various PKC isozymes are regulated in vivo and how they couple to particular cellular responses is largely unknown. We have examined the expression and regulation of PKC isoforms in R6 rat embryo fibroblasts. Northern and Western blot analyses indicate that these cells express four PKC isoforms, cPKC alpha, nPKC epsilon, nPKC delta, and nPKC zeta; of which nPKC epsilon and nPKC delta are the most abundant. In agreement with the simultaneous presence of cPKC and nPKC isozymes, both Ca(2+)-dependent and -independent PKC activities were detected in extracts of these cells. cPKC alpha and nPKC zeta were predominantly localized in the cytosol when subcellular fractionation was carried out in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. When cell lysis was carried out in the presence of Ca2+, greater than 50% of cPKC alpha redistributed to the particulate fraction, whereas nPKC zeta remained in the cytosol. In contrast to cPKC alpha and nPKC zeta, 60-80% of nPKC epsilon and nPKC delta were located in a Ca(2+)-insensitive, membrane-bound form. Treatment of R6 cells with 12-O-tetradecanoyl phorbol 13-acetate (TPA), resulted in the translocation of all four PKC isozymes to the membrane fraction, and the subsequent down-regulation of cPKC alpha, nPKC zeta, and nPKC delta, nPKC epsilon, however, was only partially down-regulated in response to long-term TPA exposure. Overproduction of exogenous cPKC beta I in R6 cells conferred partial resistance of nPKC delta to TPA-induced down-regulation and potentiated the resistance of nPKC epsilon to down-regulation. These results demonstrate that the multiple isoforms of PKC which coexist within a single cell type are differentially regulated by extra- and intracellular stimuli and may thereby influence growth control and transformation via distinct mechanisms.     

Study of synthetic peptides derived from the PKI55 protein, a protein kinase C modulator, in human neutrophils stimulated by the methyl ester derivative of the hydrophobic N-formyl tripeptide for-Met-Leu-Phe-OH

Elucidation of the involvement of protein kinase C subtypes in several diseases is an important challenge for the future development of new drug targets. We previously identified the PKI55 protein, which acts as a protein kinase C modulator, establishing a feedback loop of inhibition. The PKI55 protein is able to penetrate the cell membrane of activated human T-lymphocytes and to inhibit the activity of alpha, beta(1) and beta(2) protein kinase C isoforms. The present study aimed to identify the minimal amino acid sequence of PKI55 that is able to inhibit the enzyme activity of protein kinase C. Peptides derived from both C- and N-terminal sequences were synthesized and initially assayed in rat brain protein kinase C to identify which part of the entire protein maintained the in vitro effects described for PKI55, and then the active peptides were tested on the isoforms alpha, beta(1), beta(2), gamma, delta, epsilon and zeta to identify their specific inhibition properties. Specific protein kinase C isoforms have been associated with the activation of specific signal transduction pathways involved in inflammatory responses. Thus, the potential therapeutic role of the selected peptides has been studied in polymorphonuclear leukocytes activated by the methyl ester derivative of the hydrophobic N-formyl tripeptide for-Met-Leu-Phe-OH to evaluate their ability to modulate chemotaxis, superoxide anion production and lysozyme release. These studies have shown that only chemotactic function is significantly inhibited by these peptides, whereas superoxide anion production and lysozyme release remain unaffected. Western blotting experiments also demonstrated a selective reduction in the levels of the protein kinase C beta(1) isoform, which was previously demonstrated to be associated with the polymorphonuclear leukocyte chemotactic response.     

Nicergoline stimulates protein kinase C mediated alpha-secretase processing of the amyloid precursor protein in cultured human neuroblastoma SH-SY5Y cells.

We investigated the ability of the antidementia agents, nicergoline, aniracetam and hydergine to stimulate PKC mediated alpha-secretase amyloid precursor protein (APP) processing in cultured human neuroblastoma SH-SY5Y cells. Western immunoblotting of cell conditioned media using the Mabs 22C11 and 6E10 revealed the presence of 2 bands with molecular mass of 90 and 120 kDa, corresponding to possible alternatively glycosylated forms of secreted APP (APPs). Short-term (30 min and 2 h) treatment of cells with nicergoline gave an increased intensity of both bands, compared to non-treated cells. Maximal nicergoline effects, of the order of 150-200% over basal APPs release, were seen at concentrations between 1 and 10 microM. Under the same condition, 1 microM PdBu, used as a positive control, gave 500-1000% increases of basal APPs release. In contrast, aniracetam and hydergine, did not show any effect on APPs secretion. 2 h treatment with nicergoline had no effect on cellular full-length APP levels, as determined by immunoblotting of cell extracts with 22C11 and CT15 antibodies. Immunoblotting with PKC isoform specific antibodies of soluble and membrane fractions prepared from 2 h treated cells, showed that nicergoline (50 microM) and PdBu (1 microM) both induced translocation of PKC alpha, gamma and epsilon, but not PKC beta. The involvement of PKC in mediating nicergoline stimulated APPs release was also studied using specific inhibitors. 1 microM calphostin C, a broad range PKC inhibitor, significantly reduced both PdBu (1 microM) and nicergoline (10 microM) induced APPs release. In contrast, Go6976 (1 microM), a selective PKC alpha and beta1 inhibitor, as well as the cAMP-dependent protein kinase inhibitor, H89 (1 microM) were without effect. These results indicate that nicergoline can modulate alpha-secretase APP processing by a PKC dependent mechanism that is likely to involve the gamma and epsilon isoforms of this enzyme.