Inhibition of the protein kinase C pathway promotes anti-CD95-induced apoptosis in Jurkat T cells

The role of the basal activity of the serine/threonine protein kinase, protein kinase C (PKC) in the regulation of anti-CD95-induced apoptosis in Jurkat T cells was investigated. The PKC-specific inhibitor GF 109203X and the proposed cPKC-specific inhibitor Go 6976, in a concentration-dependent manner, increased the percentage of cells undergoing apoptosis induced by anti-CD95 mAb as demonstrated by propidium iodide (PI) staining, TUNEL assay and DNA fragmentation by gel electrophoresis. Furthermore, Go 6976 and GF 109203X abrogated phorbol myristate acetate-induced inhibition of anti-CD95-induced apoptosis. To examine the molecular mechanism by which PKC modulates anti-CD95-induced apoptosis, the effects of Go 6976 on known effector and regulatory molecules of cell death were studied. Increased recruitment of cells undergoing apoptosis was associated with enhanced anti-CD95-induced proteolytic cleavage of the most receptor-proximal cysteine protease caspase-8, subsequent cleavage and activation of the machinery protease caspase-3, and cleavage of the caspase substrates DNA-dependent protein kinase catalytic subunit, poly-(ADP-ribose) polymerase and lamin B1. CD95 and FADD protein levels in Jurkat T cells were not altered by Go 6976 treatment. In addition, Go 6976 did not alter protein levels and subcellular distribution of the anti-apoptotic molecules Bcl-2 and Bcl-xL. These data suggest indirectly that basal PKC activity acts at an early stage in the anti-CD95-induced caspase pathway to attenuate subsequent activation of downstream effector molecules and associated apoptosis in Jurkat T cells.      

Cell desensitization by sublytic C5b-9 complexes and calcium ionophores depends on activation of protein kinase C

Basal cell resistance to lysis by complement C5b-9 complexes depends on extracellular and intracellular protection. Cell membrane regulatory proteins and enzymes interfere with complement activation and intracellular processes of protein phosphorylation and synthesis support cell resistance and damage repair. K562 human erythroleukemic cells treated with sublytic complement doses become protected from lytic doses of complement within 50 min. The early signaling processes leading to cell desensitization to complement-mediated lysis were studied. Treatment with calcium ionophores or phorbol 12-myristate 13-acetate rapidly induced in K562 cells protection from complement as well as synthesis of a large protein complex similar to the large complement-induced protein complex L-CIP induced by sublytic complement. Both ionophore- and complement-induced protection were blocked by treatment with protein kinase C (PKC) inhibitors. Calphostin C, sphingosine and GF109203X abrogated complement-induced protection almost completely, whereas Go6976 inhibited it only partially. Since Go6976 is a selective inhibitor of the conventional PKC type, it is proposed that sublytic complement doses activate both conventional and non-conventional PKC types. Immunofluorescence analysis of K562 cells demonstrated sublytic complement-induced translocation of the conventional PKCalpha and PKCbetaII from the cytoplasm to the plasma membrane. These results indicate that PKC activation is an early obligatory signal in cell desensitization by sublytic C5b-9 or calcium ionophore.     

Role of protein kinase C isoforms in rat epididymal microvascular endothelial barrier function

Endothelial barrier dysfunction is involved in a variety of diseased states. We investigated the role of protein kinase C (PKC) in monolayer permeability using endothelial cells (EC) overexpressing PKC alpha (PKC alpha EC), PKC delta (PKC delta EC) or vector (vector control EC) cDNAs. Thrombin induced permeability changes in all EC, and induced significantly elevated rates of monolayer permeability in PKC alpha EC. Conversely, the basal level of permeability was significantly blunted in PKC delta EC, resulting in diminished thrombin-induced changes in permeability. PKC inhibitors, G?6976 and rottlerin, reversed the effects of PKC alpha and PKC delta overexpression on permeability, respectively. Immunoblot analyses demonstrated significantly less beta-catenin associated with the cytoskeletal subcellular fraction in thrombin-treated PKC alpha EC, an effect blocked by pretreatment with G?6976. PKC delta EC contained significantly greater numbers of focal contacts per cell. Thrombin enhanced RhoA GTPase activity in all EC; with a 3-fold greater level of activity in PKC delta EC. Rottlerin significantly blunted RhoA GTPase activity in all EC. Overexpression of RhoA dominant-negative cDNA diminished the size and number of focal contacts in EC, and significantly enhanced the basal rate of PKC delta EC monolayer permeability. These findings demonstrate that monolayer permeability changes are differentially regulated by PKC isoenzymes, suggesting that PKC alpha promotes endothelial barrier dysfunction and PKC delta enhances basal endothelial barrier function.     

Lysophosphatidic acid-induced changes in cAMP profiles in young and senescent human fibroblasts as a clue to the ageing process

This study attempts to elucidate the molecular mechanisms underlying the ageing-dependent cAMP profiles in human diploid fibroblasts stimulated by lysophosphatidic acid (LPA). In senescent cells, LPA-dependent Gialpha activation was reduced, with a consequent reduction in Gi-suppressed cAMP levels, without alterations in the levels of Gialpha proteins. In young cells, when Gialpha activity was inhibited by pertussis toxin pretreatment, or when its expression was blocked by siRNA, the pattern of changes in cAMP levels in response to LPA was similar to that seen in senescent cells. An increase in protein kinase C (PKC)-dependent isoforms of adenylyl cyclase (AC) types II, IV, and VI was also observed in these senescent fibroblasts. In senescent cells treated with PKC-specific inhibitors, bis-indolylmaleimide, G?6976, rottlerin, and PKCvarepsilonV1, LPA-induced cAMP accumulation was inhibited, indicating that increased ACs in response to LPA occur via the activation of protein kinase Cs. When the expression of AC II, IV, and VI was blocked by siRNA in senescent fibroblasts, LPA-induced cAMP accumulation was also blocked. These results suggest that the senescence-associated increase of cAMP levels after LPA treatment is associated with reduced Gialpha, increased AC II, IV, and VI proteins, and PKC-dependent stimulation of their activities and provide an explanation for the age-dependent differences in cAMP-related physiological responses.     

Linkage of protein kinase C-beta activation and intracellular interleukin-2 accumulation in human naive CD4 T cells.

A critical role for protein kinase C (PKC) in signal transduction events has been well established. Moreover, studies of regulation in PKC levels suggest participation in mediating long-term cellular functions. Protein kinase C-beta (PKC-beta) has been reported to be involved in interleukin-2 (IL-2) synthesis in T lymphocytes. In this study, the role of PKC-beta in intracellular accumulation of IL-2 was investigated using specific inhibitors. Preincubation with two different PKC inhibitors, one specific for classical isotypes (alpha and beta I) Go6976, and one which inhibits both classical and non-classical isotypes, GF109203X, caused a complete block in cytoplasmic IL-2 accumulation when naive CD4 T cells were stimulated in the presence of CD2+CD28+phorbol myristate acetate (PMA). In contrast, preincubation with up to 1000 ng/ml of cyclosporin A (CsA) resulted in a reduction in the intracellular IL-2 detected, as observed by a decrease in the proportion of positive cells as well as a fall in the mean fluorescence intensity (MFI). CsA did not influence PKC-beta translocation. Flow cytometric assessments of PKC-beta and its isoforms beta I and beta II correlated with Western blotting analysis and these results were further supported by the use of PKC-beta-positive (HUT 78) and -negative (BW5147) T-cell lines. Using the specific inhibitors, Go6976 and GF109203X, the findings in this study suggest that activation and translocation of PKC-beta is critical for accumulation of intracellular IL-2. The influence of CsA in reducing but not blocking IL-2 synthesis is discussed. PMA-induced down-regulation of the CD4 antigen was observed in the presence of Go6976 and but not GF109203X, suggesting regulation by non-classical PKC isoforms.     

Pasteurella (Mannheimia) haemolytica leukotoxin-induced cytolysis of bovine leukocytes: role of arachidonic acid and its regulation

Pasteurella (Mannheimia) haemolytica leukotoxin (Lkt) is the major factor that contributes to lung injury in bovine pneumonic pasteurellosis. Lkt is a pore-forming exotoxin that has the unique property of inducing cytolysis only in ruminant leukocytes and platelets. Cytolysis of many cell types is mediated by arachidonic acid (AA) and its generation by phospholipases is regulated by G-protein-coupled receptors. However, the contribution of Lkt-induced AA generation to cytolysis and the signalling cascade underlying AA generation in bovine leukocytes have not been determined. We have determined whether AA mediates Lkt-induced cytolysis and delineated the signalling mechanisms underlying AA generation in bovine leukocytes. Bovine lymphoma cells were used as an experimental system to investigate the Lkt-induced [(3)H] AA release, an index of AA generation and lactate dehydrogenase release, an index of cytolysis. The results indicate that Lkt induces AA release and cytolysis in a concentration- and time-dependent fashion. The AA analog, 5,8,11,14-eicosatetraynoic acid inhibited Lkt-induced cytolysis, but not AA release. Lkt-induced AA release and cytolysis were inhibited by pertussis toxin, inhibitors of cytosolic phospholipase A(2)(cPLA(2)), phospholipase C and protein kinase C (PKC), and by chelation of intracellular calcium. Furthermore, Western blot analysis revealed the presence of G(i), G(s)and G(q)type G-proteins. These results demonstrate that AA metabolites from cPLA(2)activation contribute to Lkt-induced cytolysis and G(i)type G-proteins, Ca(2+)and PKC, regulate the cPLA(2)activity.     

Short-term delay of Fas-stimulated apoptosis by GM-CSF as a result of temporary suppression of FADD recruitment in neutrophils: evidence implicating phosphatidylinositol 3-kinase and MEK1-ERK1/2 pathways downstream of classical protein kinase C

Granulocyte/macrophage colony-stimulating factor (GM-CSF) inhibits Fas-induced apoptosis of neutrophils. However, the exact step in the apoptotic pathway blocked by GM-CSF remained unclear. Here, we found that pretreatment of neutrophils with GM-CSF inhibits the recruitment of Fas-associated protein with death domain (FADD) to Fas, abolishing the formation of the death-inducing signaling complex required for Fas-induced apoptosis. Two-dimensional electrophoresis revealed that GM-CSF modifies the ratio of FADD subspecies. These GM-CSF-triggered changes were abrogated, and Fas-induced apoptosis was restored by an inhibitor of classical protein kinase C (PKC), Go6976, and by the combination of a phosphatidylinositol 3-kinase (PI-3K) inhibitor, LY294002, and an inhibitor of mitogen-activated protein kinase kinase (MEK)1, PD98059. Go6976 blocked GM-CSF-elicited phosphorylation of Akt/PKB and extracellular signal-regulated kinase (ERK)1/2. These results indicated that GM-CSF suppresses Fas-induced neutrophil apoptosis by inhibiting FADD binding to Fas, through redundant actions of PI-3K and MEK1-ERK1/2 pathways downstream of classical PKC.     

Translocation of protein kinase-C with IP3-induced calcium mobilization by heat-stable enterotoxin of Vibrio cholerae non-O1 in isolated rat enterocytes

The activity of the calcium- and phospholipid-dependent enzyme protein kinase C (PKC) in response to heat-stable enterotoxin (NAG-ST) of Vibrio cholerae non-O1 was examined in isolated rat enterocytes. Optimal stimulation of the membrane-bound PKC activity (about 4.3-fold) was observed after 1 min of incubation of cells with 10 ng/ml toxin; and the effects were dose dependent. Following NAG-ST treatment an increase in PKC activity in the membrane fraction was found with a concomitant decrease in the cytosolic fraction suggesting the redistribution of the enzyme. The pronounced enzyme activity in presence of a classical pseudosubstrate and its complete inhibition by G? 6976 suggested the involvement of a calcium-dependent isoform of PKC (PKC-alpha). A time course study employing an immunoblot assay provided evidence that NAG-ST led to almost complete translocation of PKC-alpha to the membrane. A 65% inhibition of enzyme activity in the membrane fraction and inhibition of its translocation to some extent by dantrolene treatment further suggested that the enzyme was translocated with the rise of intracellular calcium ([Ca2+]i). The phosphorylation of three membrane proteins by toxin-induced PKC in vitro and abolition of this phosphorylation by G? 6976 demonstrated that phosphorylation of these membrane proteins was PKC-alpha mediated and might be involved in the alteration of membrane functions.     

Contractile potentiation by endothelin-1 involves protein kinase C-delta activity in the porcine coronary artery.

The relationship between potentiation of serotonin (5-hydroxytryptamine, 5-HT)-induced contraction by endothelin-1 (ET-1) and the activity of protein kinase C (PKC) was examined in the porcine coronary artery. Low concentrations (30-100 pM) of ET-1 selectively potentiated the 5-HT-induced contraction 1.5 to 2 times over the control without any additional increase in myosin light-chain phosphorylation. The potentiation was attenuated by PKC down-regulation caused by phorbol 12-myristate 13-acetate. By Western blot analysis with isoform-specific antibodies to PKC, at least four isoforms (PKCalpha, PKCbeta1, PKCdelta and PKCzeta) were identified in the porcine coronary artery. PKCalpha and PKCdelta were mostly in the cytosol fraction, whereas PKCbeta1 and PKCzeta were almost equally distributed in the cytosol and membrane fractions in the resting and contractile states. Of the four isoforms, only PKCdelta was translocated from the cytosol to the membrane fraction during the contractile potentiation by ET-1. These results suggest that the activity of PKCdelta, a Ca2+-independent PKC isoform, is involved in the potentiation of 5-HT-induced contraction by ET-1 in the porcine coronary artery.     

PKCalpha mediates CCL18-stimulated collagen production in pulmonary fibroblasts

A CC chemokine, CCL18, has been previously reported to stimulate collagen production in pulmonary fibroblasts. This study focused on the role of protein kinase C (PKC) in the profibrotic signaling activated by CCL18 in pulmonary fibroblasts. Of the three PKC isoforms that are predominantly expressed in fibroblasts (PKCalpha, PKCdelta, and PKCepsilon), two isoforms (PKCdelta and PKCepsilon) have been implicated in profibrotic intracellular signaling. The role of PKCalpha-mediated signaling in the regulation of collagen production remains unclear. In this study, PKCalpha was found mostly in the cytoplasm, whereas PKCdelta and PKCepsilon were found mostly in the nucleus of cultured primary pulmonary fibroblasts. In response to stimulation with CCL18, PKCalpha but not PKCdelta or PKCepsilon underwent rapid (within 5-10 min) transient phosphorylation and nuclear translocation. Inhibition with dominant-negative mutants of PKCalpha and ERK2, but not PKCdelta or PKCepsilon, abrogated CCL18-stimulated ERK2 phosphorylation and collagen production. The effect of CCL18 on collagen production and the activity of collagen promoter reporter constructs were also abrogated by a selective pharmacologic inhibitor of PKCalpha G?6976. Stimulation of fibroblasts with CCL18 caused an increase in intracellular calcium concentration. Consistent with the known calcium dependence of PKCalpha signaling, blocking of the calcium signaling with the intracellular calcium-chelating agent BAPTA led to abrogation of PKCalpha nuclear translocation, ERK2 phosphorylation, and collagen production. These observations suggest that in primary pulmonary fibroblasts, PKCalpha but not PKCdelta or PKCepsilon mediate the profibrotic effect of CCL18. PKCalpha may therefore become a viable target for future antifibrotic therapies.     

Pertussis toxin-induced lung edema. Role of manganese superoxide dismutase and protein kinase C

The mechanism by which pertussis toxin (Ptx) causes lung edema is not clear. We investigated the role of pulmonary manganese superoxide dismutase (MnSOD) and protein kinase C (PKC) in Ptx-induced lung edema. We demonstrated that intraperitoneal injection of Ptx at a concentration of 5 microg/100 g body weight caused a similar degree of lung edema in 2 d, as measured by lung wet weight/dry weight ratio, in heterozygous MnSOD gene (Sod2)-knockout mice (Sod2(+/-)) and in their wild-type littermates (Sod2(+/+)). The level of lung MnSOD activity in Sod2(+/-) mice was approximately half that of Sod2(+/-) mice. Ptx had no effect on levels of lung MnSOD messenger RNA, immunoreactive protein, or enzyme activity in either Sod2(+/+) or Sod2(+/-) mice. Ptx also had no effect on lung copper-zinc SOD, catalase, and glutathione peroxidase activities in these mice. On the other hand, Ptx caused the activation of lung PKC, for example, by translocation of a 72-kD PKC isoform from the cytosolic fraction to the membrane fraction. Pretreatment of mice with bisindolylmaleimide, a PKC inhibitor, prevented both the Ptx-induced activation of PKC and lung edema. These data suggest that Ptx-induced lung edema in mice is, at least in part, due to the activation of lung PKC.     

Protein kinase Cdelta regulates p67phox phosphorylation in human monocytes

Phosphorylation of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase components p67phox and p47phox accompanies the assembly and activation of this enzyme complex. We have previously reported that activation of human monocytes with opsonized zymosan (ZOP), a potent stimulator of NADPH oxidase activity, results in the phosphorylation of p67phox and p47phox. In this study, we investigated the regulation of p67phox phosphorylation. Although protein kinase C (PKC)alpha has previously been shown to regulate NADPH oxidase activity, we found that inhibition of PKCalpha had no effect on p67phox phosphorylation. Our studies demonstrate that pretreatment of monocytes with antisense oligodeoxyribonucleotides specific for PKCdelta or rottlerin, a selective inhibitor for PKCdelta, inhibited the phosphorylation of p67phox in monocytes, and Go6976, a specific inhibitor for conventional PKCs, PKCalpha and PKCbeta, had no such inhibitory effect. Additional studies indicate that ZOP stimulation of monocytes induces PKCdelta and p67phox to form a complex. We also demonstrate that lysates from activated monocytes as well as PKCdelta immunoprecipitates from activated monocytes can phosphorylate p67phox in vitro and that pretreatment of monocytes with rottlerin blocked the phosphorylation in each case. We further show that recombinant PKCdelta can phosphorylate p67phox in vitro. Finally, we show that PKCdelta-deficient monocytes produce significantly less superoxide anion in response to ZOP stimulation, thus emphasizing the functional significance of the PKCdelta regulation of p67phox phosphorylation. Taken together, this is the first report to describe the requirement of PKCdelta in regulating the phosphorylation of p67phox and the related NADPH oxidase activity in primary human monocytes.     

Steroid hormone action in musculoskeletal cells involves membrane receptor and nuclear receptor mechanisms

Steroid hormones regulate target cells through traditional nuclear mechanisms as well as by membrane mechanisms. 1alpha,25(OH)2D3 and 24R,25(OH)2D3 bind membrane receptors (mVDR) and mediate their effects on the physiological responses of musculoskeletal cells via protein kinase C (PKC). In cultures of costochondral growth plate chondrocytes, 1alpha,25(OH)2D3 binds the 1,25-mVDR in growth zone cells, activating phospholipase C (PLC), leading to diacylglycerol (DAG) production and PKC translocation to the plasma membrane. It also activates PLA2, increasing arachidonic acid release and prostaglandin synthesis. 24R,25(OH)2D3 binds its membrane receptor in resting zone chondrocytes, activating phospholipase D (PLD), and increasing DAG and PKC activity, but translocation does not occur. PLA2 activity is decreased, reducing arachidonic acid and prostaglandin production. 17Beta-estradiol (E2) activates PKC in both cartilage cells, but DAG is not involved. 1alpha,25(OH)2D3 and 24R,25(OH)2D3 also increase PKC in osteoblasts in a cell-specific manner. Antibodies to the 1,25-mVDR block PKC activation. Membrane-mediated events influence gene expression via signaling cascades, including the ERK1/2 MAP kinases. The ability of steroid hormones to initiate events nongenomically is important for regulation of matrix vesicle (MV) function in the extracellular matrix. MVs have mVDRs, but ligand binding inhibits PKC-zeta (PKCzeta) via a mechanism that differs from PKCalpha activation in the plasma membranes. Treatment of MVs from growth zone chondrocyte cultures with 1alpha,25(OH)2D3 releases stromelysin-1 (MMP-3) and increases TGF-beta activation. MMP-3 is also involved in proteoglycan degradation, facilitating calcification. 24R,25(OH)2D3 inhibits PKCzeta in MV from resting zone cell cultures and inhibits MMP-3 release. Chondrocytes and osteoblasts produce 1,25(OH)2D3, 24,25(OH)2D3, and E2; thus, locally produced steroids may function as autocrine regulators of matrix events, including matrix vesicle enzyme activity and matrix protein remodelling during longitudinal growth, calcification, and growth factor activation.     

NK-active cytokines IL-2, IL-12, and IL-15 selectively modulate specific protein kinase C (PKC) isoforms in primary human NK cells.

Natural killer (NK) cell function is largely modulated by growth factors and cytokines. In particular, interleukin (IL)-2, IL-12, and IL-15 have major effects on the proliferative and cytotoxic activities of NK cells against tumor and virus-infected cells. It is thought that the members of the protein kinase C (PKC) family of serine/threonine kinases play an important role in mediating the pleiotropic effects of cytokines on their target cells. We have investigated the downstream effects generated in purified human NK cells by IL-2, IL-12, and IL-15 on PKCalpha and PKCepsilon--a canonical and a novel isoform of PKC, respectively. By means of Western blotting, PKC activity assays, and immunofluorescence performed on highly purified preparations of primary human NK cells, we demonstrate that: 1) the three cytokines have similar effects on PKCalpha and PKCepsilon activities; 2) whereas PKCepsilon activity is induced by cytokine stimulation, PKCalpha activity is inhibited; and 3) both the induction of PKCepsilon and the inhibition of PKCalpha functional activity are relatively early events in NK cells, while longer cytokine stimulations do not generate significant variations in enzyme activity, suggesting that the activation of both the canonical and novel isoforms of PKC are events required in the early phases of cytokine-induced NK cell stimulation.     

Reciprocal inhibitory interactions between interferon γ and histamine in melanoma

BACKGROUND: Although protein kinase C (PKC) isoenzymes have been implicated as mediators for multiple physiological processes, PKC also mediates cellular and intestinal mucosal injury. We have investigated the expression of the isoenzymes, PKCalpha, PKCdelta, PKCepsilon and PKCzeta in colonic mucosal tissue from TNBS-treated and HLA-B27 spontaneous colitis animals. METHODS: Colonic mucosal samples were taken at various times (2 h-14 d) after instillation of TNBS (75 mg/kg in 50% ethanol) or from HLA-B27 rats at 16-18 weeks of age. Tissues were homogenized and separated into membrane and cytosolic fractions by centrifugation. PKC activity was measured radioenzymatically. PKC protein for isoforms alpha, delta, epsilon and delta was assessed by Western blot while corresponding mRNA was analyzed by RT-PCR. RESULTS: PKC activity increased in cytosolic and membrane fractions by 1d after TNBS and returned to normal by d3. PKCalpha protein was translocated from cytosol to membrane by 2 h after TNBS followed by down-regulation until d3. Increases in PKCdelta, PKCepsilon and PKCzeta protein occurred initially in membrane fractions as early as 2 h after TNBS. Increases in cytosolic protein occurred at later times after induction of colitis. Protein levels for all isoenzymes remained increased up to 7d after TNBS. RT-PCR revealed that mRNA for PKCalpha decreased while PKC mRNA increased correspondingly with their respective protein levels. In HLA-B27 rats, protein levels for all isoforms were less than was detected in normal colonic tissue. Conclusions: The early increase in gene expression and protein levels for PKCalpha and zeta suggest that these isozymes may play roles in an acute model of colitis induced by TNBS. In contrast, the increase in PKCdelta and epsilon protein was not associated with mRNA changes suggesting that these isozymes are not similarly regulated in the inflamed colonic mucosa. In a chronic model of experimental colonic inflammation (HLA-B27), all of these isoforms appeared to be down-regulated.     

Activation of protein kinase C by 4-aminopyridine dependent on Na+ channel activity in rat hippocampal slices

The convulsant drug 4-aminopyridine (4-AP) stimulates the phosphorylation of the neuron-specific presynaptic protein B-50 in hippocampal slices. This effect could be attenuated by the protein kinase C (PKC) inhibitor staurosporine. Moreover, the endogenous phosphorylation of B-50 was found to be restricted to the 15 kDa Staphylococcus aureus protease fragment of B-50, known to contain the PKC acceptor site. The effect of 4-AP on B-50 phosphorylation was sensitive to the Na+ channel blocker tetrodotoxin. These results indicate that 4-AP stimulates PKC activity in hippocampal slices by a mechanism dependent on Na+ channel activity.     

Conventional protein kinase C and atypical protein kinase Czeta differentially regulate macrophage production of tumour necrosis factor-alpha and interleukin-10

In chronic inflammatory diseases such as rheumatoid arthritis, joint macrophages/monocytes are the major source of pro- and anti-inflammatory cytokines. Little is understood regarding the signalling pathways which determine the production of the pro-inflammatory cytokine, tumour necrosis factor-alpha (TNF-alpha) and the anti-inflammatory cytokine, interleukin-10 (IL-10). Two pathways integral to macrophage function are the protein kinase C (PKC)- and the cAMP-dependent pathways. In this report, we have investigated the involvement of PKC and cAMP in the production of TNF-alpha and IL-10 by peripheral blood monocyte-derived macrophages. The utilization of the PKC inhibitors Go6983, Go6976 and RO-32-0432 demonstrated a role for conventional PKCs (alpha and beta) in the production of TNF-alpha in response to stimulation by lipopolysaccharide and phorbol 12-myristate 13-acetate (PMA)/ionomycin. PKC stimulation resulted in the downstream activation of the p42/44 mitogen-activated protein kinase (MAPK) pathway which differentially regulates TNF-alpha and IL-10. The addition of cAMP however, suppressed activation of this MAPK and TNF-alpha production. Cyclic-AMP augmented IL-10 production and cAMP response element binding protein activation upon stimulation by PMA/ionomycin. In addition, cAMP activated PKCzeta; inhibition of which, by a dominant negative adenovirus construct, selectively suppressed IL-10 production. These observations suggest that pro-inflammatory and anti-inflammatory cytokines are differentially regulated by PKC isoforms; TNF-alpha being dependent on conventional PKCs (alpha and beta) whereas IL-10 is regulated by the cAMP-regulated atypical PKCzeta.     

The synthetic chemoattractant peptide, Trp-Lys-Tyr-Met-Val-D-Met, enhances monocyte survival via PKC-dependent Akt activation

Previously, we showed that Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm) stimulates superoxide generation and chemotactic migration in monocytes and neutrophils. In this study, we examined the effect of WKYMVm on monocyte survival. Serum starvation-induced monocyte death was attenuated in the presence of WKYMVm, which was abated when the cells were preincubated with LY294002, suggesting the involvement of phosphoinositide-3-kinase (PI 3-kinase) in the peptide-induced monocyte survival. WKYMVm stimulated ERK and Akt activity via PI 3-kinase activation in monocytes. We also investigated the signaling pathway of WKYMVm-induced ERK and Akt activation. The WKYMVm-induced ERK activation was PI 3-kinase-dependent but PKC-independent. However, Akt activation by WKYMVm was dependent not only on PI 3-kinase but also on the PKC pathway. When monocytes were incubated with WKYMVm, caspase-3 activity, which is important for cell death, was inhibited. Pretreatment of the cells with LY294002, GF109203X, and Go 6976 but not PD98059 blocked WKYMVm-induced monocyte survival and caspase-3 inhibition. In summary, the novel chemoattractant WKYMVm enhances monocyte survival via Akt-mediated pathways, and in this process, PKC and PI 3-kinase act upstream of Akt.     

Receptor-stimulated phospholipase A(2) liberates arachidonic acid and regulates neuronal excitability through protein kinase C

Type B photoreceptors in Hermissenda exhibit increased excitability (e.g., elevated membrane resistance and lowered spike thresholds) consequent to the temporal coincidence of a light-induced intracellular Ca(2+) increase and the release of GABA from presynaptic vestibular hair cells. Convergence of these pre- and postsynaptically stimulated biochemical cascades culminates in the activation of protein kinase C (PKC). Paradoxically, exposure of the B cell to light alone generates an inositol triphosphate-regulated rise in diacylglycerol and intracellular Ca(2+), co-factors sufficient to stimulate conventional PKC isoforms, raising questions as to the unique role of synaptic stimulation in the activation of PKC. GABA receptors on the B cell are coupled to G proteins that stimulate phospholipase A(2) (PLA(2)), which is thought to regulate the liberation of arachidonic acid (AA), an "atypical" activator of PKC. Here, we directly assess whether GABA binding or PLA(2) stimulation liberates AA in these cells and whether free AA potentiates the stimulation of PKC. Free fatty-acid was estimated in isolated photoreceptors with the fluorescent indicator acrylodan-derivatized intestinal fatty acid-binding protein (ADIFAB). In response to 5 microM GABA, a fast and persistent increase in ADIFAB emission was observed, and this increase was blocked by the PLA(2) inhibitor arachidonyltrifluoromethyl ketone (50 microM). Furthermore, direct stimulation of PLA(2) by melittin (10 microM) increased ADIFAB emission in a manner that was kinetically analogous to GABA. In response to simultaneous exposure to the stable AA analogue oleic acid (OA, 20 microM) and light (to elevate intracellular Ca(2+)), B photoreceptors exhibited a sustained (>45 min) increase in excitability (membrane resistance and evoked spike rate). The excitability increase was blocked by the PKC inhibitor chelerythrine (20 microM) and was not induced by exposure of the cells to light alone. The increase in excitability in the B cell that followed exposure to light and OA persisted for > or =90 min when the pairing was conducted in the presence of the protein synthesis inhibitor anisomycin (1 microm), suggesting that the synergistic influence of these signaling agents on neuronal excitability did not require new protein synthesis. These results indicate that GABA binding to G-protein-coupled receptors on Hermissenda B cells stimulates a PLA(2) signaling cascade that liberates AA, and that this free AA interacts with postsynaptic Ca(2+) to synergistically stimulate PKC and enhance neuronal excitability. In this manner, the interaction of postsynaptic metabotropic receptors and intracellular Ca(2+) may serve as the catalyst for some forms of associative neuronal/synaptic plasticity.