Role of protein kinase C and intracellular calcium mobilization in the induction of macrophage tumoricidal activity by interferon-gamma

These studies were designed to test the hypothesis that changes in intracellular Ca2+ levels and activation of the calcium ion- and phospholipid-dependent protein kinase C were required for the induction of macrophage tumoricidal activity by interferon-gamma (IFN-gamma). Phenothiazines and R24571, known antagonists of calcium-binding proteins and therefore nonspecific inhibitors of protein kinase C, blocked in a dose-dependent manner the induction of macrophage cytocidal activity by either natural or recombinant IFN-gamma. Macrophages depleted of intracellular Ca2+ by chelation with Quin 2, were also unresponsive to IFN-gamma. These treatments effected neither the binding of IFN-gamma to its cell surface receptor nor the normal intracellular processing of IFN-gamma. Activators of protein kinase C (such as phorbol esters) and Ca2+ ionophores when added alone did not effect the activation state of the macrophage population. However, macrophages exposed to both drugs in combination were elevated into the primed activation state such that in the presence of a second signal (lipopolysaccharide or heat killed Listeria monocytogenes), the cells were triggered to express full levels of tumoricidal activity. The capacity of phorbol esters to induce cellular activation correlated with their ability to bind and to activate protein kinase C. No synergistic effect was observed between IFN-gamma and protein kinase C activators and/or Ca2+ ionophores, indicating that the drugs could only prime and could not trigger macrophages for tumor cell killing. These results thus support the concept that protein kinase C activation and mobilization of intracellular Ca2+ are essential steps in the pathway of IFN-gamma-dependent induction of non-specific tumoricidal activity in macrophages.     

Calcium transients in human platelets monitored by aequorin, fura-2 and quin-2: effects of protein kinase C activation and inhibition

Tumour-promoting phorbol esters and 1,2-dioctanoyl-sn-glycerol both induce calcium transients in platelets. However, these can only be detected in platelets loaded with aequorin, but not in those loaded with the fluorescent probes quin-2 and fura-2 presumably because of intracellular calcium buffering. Several effects induced by phorbol esters and diacylglycerols, including the rise in (Ca2+)i, the stimulation of Na+/H+ transporter and the inhibition of the effects of thrombin alone on (Ca2+)i are potently antagonised by staurosporine, a compound known to inhibit protein kinase C. Higher concentrations of staurosporine themselves inhibit the thrombin-induced calcium transient. Staurosporine inhibits the effects of phorbol esters and dioctanoyl glycerol with equal potency although the latter does not cause enzyme translocation of cytosolic protein kinase C to membranes. These results therefore suggest that some, if not all, the effects of protein kinase C activation can occur without translocation of the enzyme.     

Antagonistic effects of phorbol esters on lymphocyte activation. Evidence that protein kinase C provides an early signal associated with lytic function

There is increasing evidence that protein kinase C plays a role in the transduction of an activation signal in lymphocytes. The bulk of this evidence is based on pharmacological experiments involving the tumor promoter phorbol myristate acetate (PMA) as a protein kinase C agonist. However, in cytotoxic T lymphocytes, PMA has been shown to both stimulate and inhibit lytic function. By examining the effects of a series of phorbol esters on protein kinase C activity in lymphocytes, we will demonstrate that these antagonistic effects of PMA on cytotoxic T lymphocyte function are related to multiple effects of PMA on protein kinase C activity.     

Activation of the C3b receptor: effect of diacylglycerols and calcium mobilization

The plasma membrane expression and the phagocytic function of the C3b receptor (CR1) on human neutrophils (PMN) are under the control of cellular regulatory mechanisms, and phorbol esters are one class of agents that modulate both membrane expression and function. Phorbol esters also activate protein kinase C; however, the physiologic activation of protein kinase C is thought to be mediated by diacylglycerol. Diacylglycerols are generated during phosphatidyl inositol turnover, which is associated with a rise in intracellular calcium due to another product of polyphosphoinositide metabolism, inositol trisphosphate. We therefore studied the effects of synthetic diacylglycerols and calcium mobilization on CR1 function. In our experiments, treatment of neutrophils with two synthetic diacylglycerols, 1-oleoyl-2-acetoyl-sn-3-glycerol (OAG) and sn-1,2-dioctanoylglycerol, like phorbol esters, induced ligand-independent internalization of CR1. In contrast, the addition of exogenous phospholipase C had no effect on receptor internalization over the time course studied. OAG treatment also enabled neutrophils to specifically phagocytose via CR1. Calcium mobilization with the calcium ionophore A23187 (1 microM) had a synergistic effect on phorbol ester-induced internalization of CR1, but abrogated the phorbol ester enhancement of CR1-dependent phagocytosis. Both trimethoxybenzoate, the intracellular calcium antagonist, and chlorpromazine inhibited phorbol ester-induced internalization of CR1, whereas chelation of extracellular calcium did not. We conclude that activation of protein kinase C modulates the expression and function of CR1, and that calcium mobilization also influences these processes. We speculate that polyphosphoinositide turnover may be involved in the physiologic regulation of CR1.     

Activation of phospholipase D: a signaling system set in motion by perturbation of the T lymphocyte antigen receptor/CD3 complex.

A number of cellular signaling systems are called into play by interaction of the T lymphocyte antigen receptor/CD3 complex with its cognate antigen. Well-described signaling systems include phosphoinositide turnover, tyrosine phosphorylation, protein kinase C activation, and increased cytosolic calcium. We have explored the possibility that another recently described signaling system, activation of phospholipase D, may be operative. Data presented here demonstrate that stimulation of Jurkat T cells with anti-CD3 antibodies or phorbol esters resulted in activation of phospholipase D, as measured by production of phosphatidylethanol and phosphatidic acid. The combination of anti-CD3 antibody plus phorbol ester led to a greater than additive production of phosphatidylethanol and to the additive production of phosphatidic acid (in the absence of ethanol). Phorbol esters as a second stimulus with anti-CD3 antibody led to a additive increase in cellular diacylglycerol content but provided no increased production of inositol phosphates, suggesting that diacylglycerol production in these cells results from hydrolysis of noninositol containing lipids as well as from phosphinositides. Exogenous addition of phosphatidic acid led to increases in cytosolic calcium that, depending on the concentration used, resulted from release of an intracellular store of calcium and influx of extracellular calcium. Changes in cytosolic calcium occurred in the absence of inositol phosphates production. These studies establish a role for increased phospholipase D activity in T lymphocyte activation.     

Phorbol Ester Enhancement of Neurotransmitter Release from Rat Brain Synaptosomes

Neurotransmitter release from rat brain synaptosomes was measured following pretreatment with various phorbol esters. Ca2+-dependent, evoked neurotransmitter release was increased by phorbol esters that were active in stimulating protein kinase C. Protein kinase C activation was demonstrated by increased incorporation of 32P into 87-kilodalton phosphoprotein, a specific substrate for that kinase. Inactive phorbol esters had no effect on neurotransmitter release or on the phosphorylation of 87-kilodalton phosphoprotein. The increased release was observed in either crude cortical synaptosomal fractions (P2) or purified cortical synaptosomal fractions. The enhancement was found for all neurotransmitters (norepinephrine, acetylcholine, gamma-aminobutyric acid, serotonin, dopamine, and aspartate), all brain regions (cerebral cortex, hippocampus, and corpus striatum), and all secretagogues (elevated extracellular K+ level, veratridine, or A23187) examined. It was also observed at all calcium concentrations present during stimulation of release. The phorbol ester enhancement of Ca2+-dependent release occurred whether or not calcium was present during pretreatment. These results indicate that stimulation of protein kinase C leads to an enhanced sensitivity of the stimulus-secretion coupling processes to calcium within the nerve terminal. The results support the possibility that presynaptic activation of protein kinase C modulates nerve terminal neurotransmitter release in the CNS.     

Rapid priming of calcium mobilization and superoxide anion production in human neutrophils by substimulatory concentrations of phorbol esters: a novel role for protein kinase C and tyrosine phosphorylation in the up-modulation of signal transduction.

The modulatory influences of phorbol esters on the functional responsiveness of human peripheral blood neutrophils have been investigated. These studies focused on measurements of the levels of cytoplasmic free calcium and of tyrosine phosphorylation as well as on their ability to mount an oxidative response. Short incubation times (< 1 min) with low concentrations of phorbol esters (5-50 nM) were shown to enhance the above indices of neutrophil responsiveness to chemotactic factors such as fMet-Leu-Phe and leukotriene B4. On the other hand, a time- and concentration-dependent inhibition of calcium mobilization and superoxide production was also observed. The effects of the phorbol esters were stereo-specific and were antagonized by a novel protein kinase C inhibitor (RO 318220) but were not affected by the oxidative burst inhibitor diphenyleneiodonium. Pre-incubation of the cells with phorbol 12,13-dibutyrate (PDBu) altered in a concentration-dependent manner the tyrosine phosphorylation pattern stimulated by fMet-Leu-Phe. In addition, the tyrosine kinase inhibitor erbstatin inhibited the priming of the mobilization of calcium induced by PDBu. These data demonstrate the rapidity of the effects of the activation of protein kinase C, their potential to modulate positively the early events of the excitation-response coupling sequence and the complexity of the functional interrelationships among the various cellular activation pathways available to human neutrophils and other non-muscle cells.     

Involvement of three intracellular messenger systems, protein kinase C, calcium ion and cyclic AMP, in the regulation of c-fos gene expression in Swiss 3T3 cells

In quiescent cultures of Swiss 3T3 cells, platelet-derived growth factor or fibroblast growth factor known to induce both protein kinase C activation and Ca2+ mobilization raised c-fos mRNA. This action of the growth factors was mimicked by the specific activators for protein kinase C, such as phorbol esters and a membrane-permeable synthetic diacylglycerol, and also by the Ca2+ ionophores, such as A23187 and ionomycin. Prostaglandin E1 known to elevate cyclic AMP also raised c-fos mRNA, and this action was mimicked by 8-bromo-cyclic AMP, dibutyryl cyclic AMP and forskolin. These results suggest that expression of the c-fos gene is regulated by three different intracellular messenger systems, protein kinase C, Ca2+ and cyclic AMP, in Swiss 3T3 cells.     

Regulation of platelet phospholipase C

We have investigated factors affecting the activation of phospholipase C in human platelets. Prior exposure of platelets to phorbol esters that stimulated protein kinase C inhibits the activation of phospholipase C in response to a variety of receptor-directed agonists, including alpha- and gamma-thrombin and thromboxane A2 analogues. Such activation has been assayed by measurements of accumulated InsP3 (including Ins(1,4,5)P3 and Ins(1,3,4)P3) and PtdOH. Inhibition is not overcome by Ca2+ ionophores, and substances that block or mimic Na+-H+ exchange neither block nor mimic these inhibitory effects. Cyclic AMP and cyclic GMP, other agents known to inhibit phospholipase C activation, do not accumulate in platelets exposed to phorbol esters. Although a portion of the effects of phorbol ester on InsP3 accumulation may be explained by 5-phosphomonoesterase activity, it is likely that more direct effects on phospholipase C are being exerted as well, and contribute the major inhibitory route. We have examined the susceptibility of adenylyl cyclase-associated Gi and 'Gp'-activated phospholipase C to inhibitory ADP-ribosylation by pertussis toxin-derived enzyme (S1 protomer) administered to saponin-permeabilized platelets. The effects of alpha-thrombin on adenylyl cyclase can be inhibited by up to 50% by S1, at which point inhibition of phospholipase C is barely detectable. Thromboxane A2 analogues, which do not affect adenylyl cyclase (Gi), stimulate phospholipase C; this effect is not impaired by S1. We therefore propose that the inhibitory effects of phorbol esters on the activation of phospholipase C are not mediated primarily by effects on Gi.     

Regulation of chromaffin cell secretion and protein kinase C activity by chronic phorbol ester treatment

Bovine adrenal chromaffin cells were exposed to phorbol esters to determine the effects of reduced levels of protein kinase C on secretion of hormones. Treatment with active phorbol esters such as 4 beta-phorbol 12, 13-didecanoate (PDD) reduced levels of protein kinase C activity with a maximal 80-90% reduction in activity after 16-24 h treatment (greater than or equal to 500 nM PDD). Treatment with PDD also inhibited catecholamine secretion from chromaffin cells evoked by nicotine, barium, and scorpion venom (50-70%, t1/2 approximately 6 h) and by veratridine (80%, t1/2 less than 15 min). Secretion induced by these agents in phorbol ester-treated cells returned to that of untreated cells by 3-4 days despite no recovery of protein kinase C activity. Potassium-evoked secretion was not inhibited by phorbol ester treatment. Catecholamine secretion from digitonin-permeabilized cells was more sensitive to calcium between 1 and 24 h, but not greater than or equal to 48 h, after addition of phorbol ester. The results suggest that phorbol esters inhibit secretion by activation of protein kinase C resulting in inhibition of ion channels or receptors but not of the secretory machinery itself; hence, protein kinase C may usually machinery itself; hence, protein kinase C may usually attenuate secretory responses in the adrenal chromaffin cell.     

Osmotic activation of the Na+/H+ antiport in protein kinase C-depleted lymphocytes

The Na+/H+ antiport of rat thymic lymphocytes is activated when protein kinase C is stimulated by phorbol esters. A similar activation of the antiport is obtained when the cells are treated with hypertonic solutions. We tested the possibility that protein kinase C also mediates the osmotic activation of Na+/H+ exchange. Protein kinase C was depleted by preincubation of thymocytes for 24 hr in the presence of high concentrations of phorbol ester. Disappearance of the enzyme was assessed by direct measurement of phosphotransferase activity, and by the loss of biological responses to phorbol esters. The Na+/H+ antiport in protein kinase C-depleted cells was not stimulated by addition of phorbol ester, but responded normally to hypertonic treatment. The results indicate that the osmotic activation of countertransport does not require stimulation of protein kinase C.     

Specific binding of phorbol esters to nuclei of human promyelocytic leukemia cells

In this report, we demonstrate that HL-60 nuclei isolated in calcium but not EGTA containing buffers specifically bind PE and express approximately 37,000 receptor sites/nucleus. Nuclear phorbol ester binding is lost by isolation in the absence of calcium, but can be repleted by the addition of partially purified protein kinase C and calcium. When HL-60 cells are treated with bryostatin 1, a compound which activates protein kinase C in a similar fashion to phorbol esters but does not induce differentiation of HL-60 cells, and nuclei are isolated in the presence of EGTA, these nuclei continue to bind phorbol esters. These experiments suggest that HL-60 nuclei bind PE in vitro, and that compounds that activate protein kinase C may increase nuclear binding of PE in situ.     

The activation of protein kinase G by daphnane, ingenane and tigliane diterpenoid esters

The activation of protein kinase C by daphnane, ingenane and tigliane diterpenoid eaters. In this review, the mechanism of action of phorbol esters and related diterpenes is described. These compounds have been shown to stimulate a Ca^{2 +} and phospholipid dependent protein kinase, termed kinase C. Phorbol esters activate protein kinase C by substituting for the natural effector, the second messenger, diacylglycerol. The various known protein substrates of this enzyme are described. Many of these substrates are involved in regulation of protein synthesis, DNA expression, cell transformation etc. This provides the explanation for the tumour promotion effects of some phorbol esters. Evidence for the biochemical mechanisms of action of phorbol esters that have other biological effects are also described. Recent evidence from our laboratories indicates that phorbol esters with limited biological effects, e.g. inflammatory but not tumour promoting, also act through this protein kinase. These phorbol esters appear to stimulate the phosphorylation of a different range of substrate proteins in vivo.     

Antiviral activity of phorbol myristate acetate and possible relationships with interferon action

Signal-induced turnover of membrane phospholipids represents a fundamental transducing mechanism that induces a signal cascade resulting in mobilization of calcium, activation of protein kinase C by diacylglycerol, release of arachidonic acid and stimulation of cyclic GMP production. In this pathway tumor-promoting phorbol esters such as phorbol myristate acetate (PMA) may substitute for diacylglycerol. The interferonlike antiviral effect of PMA described here suggests that the inositol phospholipid-diacylglycerol-protein kinase C signal-transducing mechanism may be involved in interferon action.     

Phorbol esters inhibit the synthesis of acetylcholine receptors in cultured muscle cells

The acetylcholine receptor (AChR) synthesis, insertion and degradation rates are regulated by numerous intracellular and extracellular agents. Recent studies have shown that Ca2+ and Ca2+ ionophores have a profound regulatory effect on the appearance of AChR clusters and AChR synthesis. These regulatory effects may be mediated through the activation of calcium and phospholipid-dependent protein kinases by agents such as phorbol esters. In this study, we have utilized 4-beta-phorbol-12-myristate-13-acetate (PMA) in order to determine whether the activation of protein kinase C exerts a regulatory effect on the expression of AChRs in cultured chick myotubes. Our results show that 4-beta-phorbol-12-myristate-13-acetate decreased intracellular AChRs and suppressed AChR synthesis without affecting the turnover rate. Control and PMA treated cells labeled with [35S] methionine and immunoprecipitated with a monoclonal antibody to the alpha subunit of AChRs (mAb35) revealed a significant decrease in radioactivity precipitated after exposure to PMA. Polyacrylamide gel electrophoresis revealed no major changes in protein patterns, or in newly synthesized proteins as determined by [35S] methionine incorporation and autoradiography. Other enzymes important in muscle metabolism were not affected by PMA treatment. Our results indicate that activation of protein kinase C results in the suppression of AChRs synthesis and dispersal of AChR clusters.     

Protein kinase C activation in a 3T3 cell variant morphologically unresponsive to tumor-promoting phorbol esters

To investigate the mechanism of the morphological changes induced in cells by tumor-promoting phorbol esters, we isolated a 3T3 cell variant which was morphologically unresponsive to phorbol esters and analyzed the activation of protein kinase C induced by the phorbol esters in it. The variant resembled the parent cells in its activation and appeared to have been altered at some step distal to the early events of protein kinase C activation.     

Possible involvement of protein kinase C and calcium ion in growth factor-induced expression of c-myc oncogene in Swiss 3T3 fibroblasts

The addition of platelet-derived growth factor and fibroblast growth factor to quiescent cultures of Swiss 3T3 fibroblasts rapidly induced protein kinase C activation and Ca2+ mobilization and afterwards markedly increased c-myc mRNA levels. 1-Oleoyl-2-acetylglycerol, a membrane-permeable synthetic diacylglycerol, and 12-O-tetradecanoylphorbol 13-acetate, a tumor-promoting phorbol ester, stimulated protein kinase C activation without Ca2+ mobilization. Inversely, Ca2+ ionophores, A23187 and ionomycin, elicited Ca2+ mobilization without protein kinase C activation. Both protein kinase C-activating and Ca2+-mobilizing agents were able to increase c-myc mRNA levels in an additive manner. Prolonged treatment of the cells with phorbol 12,13-dibutyrate, another protein kinase C-activating phorbol ester, led to the down-regulation and complete disappearance of protein kinase C. In these cells, 1-oleoyl-2-acetylglycerol and 12-O-tetradecanoylphorbol 13-acetate did not increase c-myc mRNA levels, but platelet-derived growth factor, fibroblast growth factor, and the Ca2+ ionophores, all of which still induced Ca2+ mobilization, stimulated the increase of c-myc mRNA levels. These results strongly suggest that both protein kinase C and Ca2+ may be involved in platelet-derived growth factor- as well as fibroblast growth factor-induced expression of the c-myc oncogene in Swiss 3T3 cells.     

Regulation of S6 kinase activity in Madin-Darby canine kidney renal epithelial cells

Mitogenic stimulation of mammalian cells results in increased serine phosphorylation of ribosomal protein S6. Phorbol esters, which stimulate protein kinase C activity, can also increase S6 phosphorylation. In order to further investigate the role of protein kinase C in the activation S6 kinase, we studied the stimulation of an S6 kinase activity in response to phorbol ester and epinephrine in a renal epithelial cell line, Madin-Darby canine kidney cells (MDCK). In these cells, S6 phosphorylating activity in cytosolic extracts was increased following the addition of phorbol ester to the intact cells. S6 kinase and protein kinase C activities were measured in separate fractions prepared by DEAE-Sephacel fractionation of cytosolic extracts prepared from the same cells. The time course and dose-response curves for the effects of phorbol 12-myristate 13-acetate (PMA) on S6 kinase activity were similar to those for its effects on protein kinase C binding to the membrane fraction, indicating that S6 kinase activation was correlated with protein kinase C activation. Epinephrine, acting via alpha1-adrenergic receptors, also stimulated S6 kinase activity in MDCK cells; the magnitude of this effect was similar to that of PMA. However, epinephrine causes only a slight and transient association of protein kinase C with the membrane. The effect of epinephrine on S6 kinase activity, unlike that of PMA, was dependent on the presence of extracellular calcium. A23187, a calcium ionophore, could also stimulate S6 kinase activity. These results suggest that S6 kinase can be activated through more than one signaling pathway in MDCK cells. The properties of the PMA-stimulated S6 kinase were further investigated following partial purification of the enzyme. The S6 kinase was distinct from protein kinase C by several criteria. Noteably, the S6 kinase was highly specific for S6 as substrate. These results show that phorbol esters, acting through protein kinase C, stimulate the activity of a unique S6 kinase. This S6 kinase can also be activated through a signaling pathway that appears to be dependent on increased intracellular calcium.     

Lymphoma models for B-cell activation and tolerance. VII. Pathways in anti-Ig-mediated growth inhibition and its reversal

WEHI-231, CH33, and CH31 are B-cell lymphomas that are inhibited in their growth by crosslinking of surface Ig receptors during early G1. This "negative signaling" process can be prevented or reversed under certain conditions. In the present paper, we use a cell synchronization procedure to demonstrate that activation of protein kinase C (PKC) is not involved in the negative signal for growth, but rather that PKC activation prevents growth inhibition when present early in the cell cycle. Indeed, the prevention of negative signaling is only accomplished by active phorbol esters. Moreover, phorbol esters and a calcium ionophore fail to deliver a negative signal under conditions in which anti-Ig can significantly prevent cell cycle progression into S phase, thereby ruling out synergy between PKC and calcium in growth inhibition. Whether phorbol esters reverse negative signaling by preventing internalization of the immune complex or phosphorylation of a critical intracellular protein is discussed.     

Effects of phorbol esters on contraction and protein kinase C activation in rabbit aorta

This study investigates the relationship between the contractile efficacy of phorbol esters and their ability to activate protein kinase C in intact rabbit aorta. Phorbol dibutyrate (PDB) induced a maximal contraction approximately 3.5-fold greater than that to phorbol myristate acetate (PMA). The magnitude of maximal PDB- and PMA-induced contraction correlated with the magnitude of protein kinase C activation, as assessed by the decrease in cytosolic protein kinase C activity. KCl (60mM) did not potentiate the PMA-induced decrease in cytosolic protein kinase C activity. These results suggest that the lack of efficacy of PMA is due to its inability to activate protein kinase C in the intact rabbit aorta. It is speculated that the different contractile efficacies of phorbol esters result from selective activation of protein kinase C isoforms, and that the amounts of these isoforms varies amongst vascular tissues.