IL-10 production induced by HIV-1 Tat stimulation of human monocytes is dependent on the activation of PKC beta(II) and delta isozymes

The effect of HIV-1 Tat protein on the production of IL-10, an immunosuppressive cytokine, was examined in human primary monocytes obtained from healthy HIV-1-negative blood donors. As expected and in agreement with our previous data, a dose-dependent induction of IL-10 was observed. In addition, we showed that this induction is mediated by the PKC pathway: in the presence of Ro 31-8220, an inhibitor of all PKC isozymes, or after 48 h of PMA treatment, Tat protein becomes unable to stimulate IL-10 production. Among the 11 PKC isozymes, eight (PKC alpha, beta(I), beta(II), delta, epsilon, eta, zeta, mu) are expressed in monocytes. In this study, by analyzing the translocation to the membrane after Tat stimulation, we showed that PKC alpha, beta(I), beta(II), delta and epsilon isozymes are activated by Tat. Moreover, by combining different approaches including selective PKC inhibitors (G?6983, G?6976, hispidin and rottlerin), we showed that PKC beta(II) and delta isozymes are essential for the activation of IL-10 production in human monocytes following stimulation by HIV-1 Tat protein.     

Increased AP-1 and NF-kappaB activation and recruitment with the combination of the proinflammatory cytokines IL-1beta, tumor necrosis factor alpha and IL-17 in rheumatoid synoviocytes

To determine the contribution of IL-1beta, tumor necrosis factor alpha (TNF-alpha) and IL-17 to AP-1, NF-kappaB and Egr-1 activation in rheumatoid arthritis, the effect of the cytokines used alone or in combination was measured on TF expression in rheumatoid synoviocytes. Effects on mRNA expression were measured by RT-PCR and effects on nuclear translocation were measured by immunocytochemistry. To assess the functional consequences of cytokine induction, osteoprotegerin levels were measured in synoviocyte supernatants.IL-1beta and TNF-alpha alone at optimal concentration (100 pg/ml) induced the nuclear translocation of NF-kappaB and almost all AP-1 members, except JunB and Egr-1 for IL-1beta and except Fra-2 and Egr-1 for TNF-alpha. IL-17 was clearly less potent since no nuclear translocation was observed, except for a weak activation of Fra-1 and NF-kappaB. More importantly, when these cytokines were used at low concentrations, their combination showed a synergistic effect on almost all the TFs, except for Egr-1, with a particular effect on Fra-1 and NF-kappaB. Increased recruitment of additional factors was induced when the three cytokines were combined. IL-1 and TNF-alpha induced mRNA expression of c-jun while IL-17 had no effect. A synergistic effect was seen with their combination. A similar synergistic effect was observed for osteoprotegerin production when these three cytokines were combined at low concentrations.AP-1 and NF-kappaB pathways were highly sensitive to the combination through synergistic mechanisms. These effects observed in rheumatoid arthritis synoviocytes may reflect the conditions found in the rheumatoid arthritis joint and may contribute to the mode of action of cytokine inhibitors.     

Activation of protein kinase C beta II by the stereo-specific phosphatidylserine receptor is required for phagocytosis of apoptotic thymocytes by resident murine tissue macrophages

We showed previously that protein kinase C (PKC) is required for phagocytosis of apoptotic leukocytes by murine alveolar (AM?) and peritoneal macrophages (PM?) and that such phagocytosis is markedly lower in AM? compared with PM?. In this study, we examined the roles of individual PKC isoforms in phagocytosis of apoptotic thymocytes by these two M? populations. By immunoblotting, AM? expressed equivalent PKC eta but lower amounts of other isoforms (alpha, betaI, betaII, delta, epsilon, mu, and zeta), with the greatest difference in betaII expression. A requirement for PKC betaII for phagocytosis was demonstrated collectively by phorbol 12-myristate 13-acetate-induced depletion of PKC betaII, by dose-response to PKC inhibitor Ro-32-0432, and by use of PKC betaII myristoylated peptide as a blocker. Exposure of PM? to phosphatidylserine (PS) liposomes specifically induced translocation of PKC betaII and other isoforms to membranes and cytoskeleton. Both AM? and PM? expressed functional PS receptor, blockade of which inhibited PKC betaII translocation. Our results indicate that murine tissue M? require PKC betaII for phagocytosis of apoptotic cells, which differs from the PKC isoform requirement previously described in M? phagocytosis of other particles, and imply that a crucial action of the PS receptor in this process is PKC betaII activation.     

Microgravity modifies protein kinase C isoform translocation in the human monocytic cell line U937 and human peripheral blood T-cells

Individual protein kinase C (PKC) isoforms fulfill distinct roles in the regulation of the commitment to differentiation, cell cycle arrest, and apoptosis in both monocytes and T-cells. The human monocyte like cell line U937 and T-cells were exposed to microgravity, during spaceflight and the translocation (a critical step in PKC signaling) of individual isoforms to cell particulate fraction examined. PKC activating phorbol esters induced a rapid translocation of several PKC isoforms to the particulate fraction of U937 monocytes under terrestrial gravity (1 g) conditions in the laboratory. In microgravity, the translocation of PKC beta II, delta, and epsilon in response to phorbol esters was reduced in microgravity compared to 1 g, but was enhanced in weak hypergravity (1.4 g). All isoforms showed a net increase in particulate PKC following phorbol ester stimulation, except PKC delta which showed a net decrease in microgravity. In T-cells, phorbol ester induced translocation of PKC delta was reduced in microgravity, compared to 1 g, while PKC beta II translocation was not significantly different at the two g-levels. These data show that microgravity differentially alters the translocation of individual PKC isoforms in monocytes and T-cells, thus providing a partial explanation for the modifications previously observed in the activation of these cell types under microgravity.     

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.     

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.     

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.     

The role of p38 mitogen-activated protein kinase in IL-6 and IL-8 production from the TNF-alpha- or IL-1beta-stimulated rheumatoid synovial fibroblasts

We examined the role of p38 mitogen-activated protein (MAP) kinase in the tumor necrosis factor alpha (TNF-alpha)- or interleukin-1beta (IL-1beta)-induced production of interleukin-6 (IL-6) and interleukin-8 (IL-8) in fresh rheumatoid synovial fibroblast (RSF) cultures concomitantly with the induction of p38 MAP kinase activity. Pretreatment of RSF with a specific p38 MAP kinase inhibitor, SB203580, blocked the induction of IL-6 and IL-8 without affecting nuclear translocation of nuclear factor kappaB (NF-kappaB) or IL-6 and IL-8 mRNA levels. These findings suggest that p38 MAP kinase inhibitor may have synergistic, rather than additive, effect for the treatment of rheumatoid arthritis.     

Mechanism of inhibition of sequestration of protein kinase C alpha/betaII by ceramide. Roles of ceramide-activated protein phosphatases and phosphorylation/dephosphorylation of protein kinase C alpha/betaII on threonine 638/641

Sustained activation of protein kinase C (PKC) isoenzymes alpha and betaII leads to their translocation to a perinuclear region and to the formation of the pericentrion, a PKC-dependent subset of recycling endosomes. In MCF-7 human breast cancer cells, the action of the PKC activator 4beta-phorbol-12-myristate-13-acetate (PMA) evokes ceramide formation, which in turn prevents PKCalpha/betaII translocation to the pericentrion. In this study we investigated the mechanisms by which ceramide negatively regulates this translocation of PKCalpha/betaII. Upon PMA treatment, HEK-293 cells displayed dual phosphorylation of PKCalpha/betaII at carboxyl-terminal sites (Thr-638/641 and Ser-657/660), whereas in MCF-7 cells PKCalpha/betaII were phosphorylated at Ser-657/660 but not Thr-638/641. Inhibition of ceramide synthesis by fumonisin B1 overcame the defect in PKC phosphorylation and restored translocation of PKCalpha/betaII to the pericentrion. To determine the involvement of ceramide-activated protein phosphatases in PKC regulation, we employed small interference RNA to silence individual Ser/Thr protein phosphatases. Knockdown of isoforms alpha or beta of the catalytic subunits of protein phosphatase 1 not only increased phosphorylation of PKCalpha/betaII at Thr-638/641 but also restored PKCbetaII translocation to the pericentrion. Mutagenesis approaches in HEK-293 cells revealed that mutation of either Thr-641 or Ser-660 to Ala in PKCbetaII abolished sequestration of PKC, implying the indispensable roles of phosphorylation of PKCalpha/betaII at those sites for their translocation to the pericentrion. Reciprocally, a point mutation of Thr-641 to Glu, which mimics phosphorylation, in PKCbetaII overcame the inhibitory effects of ceramide on PKC translocation in PMA-stimulated MCF-7 cells. Therefore, the results demonstrate a novel role for carboxyl-terminal phosphorylation of PKCalpha/betaII in the translocation of PKC to the pericentrion, and they disclose specific regulation of PKC autophosphorylation by ceramide through the activation of specific isoforms of protein phosphatase 1.     

Protein kinase C alpha, betaI, and betaII isozymes regulate cytokine production in mast cells through MEKK2/ERK5-dependent and -independent pathways

Regulation of MAPK pathways by PKC isoforms was examined in murine bone marrow-derived mast cells (BMMCs). The PKCalpha, betaI, and betaII isoforms showed the most robust activation after FcepsilonR1-mediated stimulation by anti-ovalbumin specific IgE and ovalbumin (IgE-ova). PKCalpha, betaI, and betaII were all involved in activation of JNK, MEKK2, and ERK5, with differential relative contributions of each isoform to specific MAPK pathway components. BMMCs from mice lacking MEKK2 showed reduced production (50-60%) of IL-6, IL-13, and TNF-alpha after stimulation, demonstrating MEKK2-dependent and -independent pathways for cytokine production. Cytokine production was stimulated by over-expression of PKC in cells from MEKK2-deficient and wild-type mice. Activation of ERK5 did not occur in BMMCs lacking MEKK2, indicating that MEKK2-independent cytokine production was also ERK5-independent. Since MAPK modules differentially regulate mast cell functions, including degranulation and cytokine production, it is suggested that specific functions could be targeted by inhibiting specific PKC isoforms.