Regulation of protein kinase C by nerve growth factor, epidermal growth factor, and phorbol esters in PC12 pheochromocytoma cells

We have used a permeabilized cell assay and a synthetic peptide substrate (KRTLRR) to specifically monitor the activity of protein kinase C in PC12 cells preincubated with nerve growth factor (NGF), epidermal growth factor (EGF), or phorbol esters. Pretreatment of PC12 cells with 1 microM 12-O-tetradecanoylphorbol 13-acetate or 1 microM phorbol dibutyrate stimulated the rate of KRTLRR peptide phosphorylation 4.8- and 2.6-fold, respectively. Furthermore, pretreatment of cells with NGF or EGF transiently increased the KRTLRR peptide kinase activity. Peak stimulations of KRTLRR peptide kinase (1.3-2-fold) were observed after 1-5 min of growth factor treatment and returned to control levels within 15-20 min. The KRTLRR peptide kinase activity fulfilled two criteria of protein kinase C. A synthetic peptide inhibitor of protein kinase C inhibited both growth factor- and phorbol ester-stimulated KRTLRR peptide kinase activity. In addition, growth factors and phorbol esters failed to stimulate KRTLRR peptide kinase activity in cells rendered protein kinase C-deficient by long-term treatment with 1 microM 12-O-tetradecanoylphorbol 13-acetate. In contrast to the transient activation of protein kinase C, ribosomal S6 kinase, assayed with the synthetic peptide RRLSSLRA, was persistently activated by NGF and EGF. The findings indicate that protein kinase C serves an early and transient role in the molecular actions of NGF and EGF in PC12 cells.     

Regulation of platelet-derived growth factor gene expression by transforming growth factor beta and phorbol ester in human leukemia cell lines.

We studied the expression of the genes encoding the A and B chains of platelet-derived growth factor (PDGF) in a number of human leukemia cell lines. Steady-state expression of the A-chain RNA was seen only in the promonocytic leukemia cell line U937 and in the T-cell leukemia cell line MOLT-4. It has previously been reported that both PDGF A and PDGF B genes are induced during megakaryoblastic differentiation of the K562 erythroleukemia cells and transiently during monocytic differentiation of the promyelocytic leukemia cell line HL-60 and U937 cells. In this study we show that PDGF A RNA expression was induced in HL-60 and Jurkat T-cell leukemia cells and increased in U937 and MOLT-4 cells after a 1- to 2-h stimulation with an 8 pM concentration of transforming growth factor beta (TGF-beta). PDGF A RNA remained at a constant, elevated level for at least 24 h in U937 cells, but returned to undetectable levels within 12 h in HL-60 cells. No PDGF A expression was induced by TGF-beta in K562 cells or in lung carcinoma cells (A549). Interestingly, essentially no PDGF B-chain (c-sis proto-oncogene) RNA was expressed simultaneously with PDGF A. In the presence of TGF-beta and protein synthesis inhibitors, PDGF A RNA was superinduced at least 20-fold in the U937 and HL-60 cells. PDGF A expression was accompanied by secretion of immunoprecipitable PDGF to the culture medium of HL-60 and U937 cells. The phorbol ester tumor promoter tetradecanoyl phorbol acetate also increased PDGF A expression with similar kinetics, but with a mechanism distinct from that of TGF-beta. These results suggest a role for TGF-beta in the differential regulation of expression of the PDGF genes.     

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.     

Regulation by the protein kinase C activator phorbol ester of calcium channels in polymorphonuclear leukocytes

The Quin fluorescence in gamma-hexachlorocyclohexane-stimulated polymorphonuclear leukocytes is rapidly increased, which points to the increase in Ca2+in concentration during leukotriene B4 synthesis in leukocytes. An addition of EGTA and calcium antagonists (nifedipine, verapamil, diltiazem) to cell suspensions does not affect the basal level of internal Ca2+ but results in the inhibition of the gamma-hexachlorocyclohexane-induced Ca2+ increase. Two mechanisms of calcium homeostasis regulation in neutrophils are proposed. One of them, cAMP regulation, is coupled with a potent inhibiting effect of prostacyclin, an adenylate cyclase activator, on Ca2+in increase in stimulated neutrophils. The other one is the activation of protein kinase C catalyzed by 4 beta-phorbol-12 beta-myristate-13 alpha-acetate. The experimental results suggest that such an activation blocks Ca2+ influx into the cells via the closure of Ca2+ channels. The synergism of action of the above mechanisms in the regulation of calcium homeostasis in neutrophils is demonstrated.     

Retention of specific protein kinase C isozymes following chronic phorbol ester treatment in BC3H-1 myocytes

Since insulin effects on glucose transport persist in phorbol ester "desensitized" or "down-regulated" BC3H-1 myocytes, we reexamined the evidence for protein kinase C (PKC) depletion. After 24 hrs of 5 microM 12-0-tetradecanoyl phorbol-13-acetate (TPA) treatment, PKC-directed histone phosphorylation and acute TPA effects on glucose transport were lost, but PKC-dependent vinculin phosphorylation was still evident. Hydroxylapatite (HAP) chromatography revealed loss of a type III, but not a type II, PKC-dependent vinculin phosphorylation. Immunoblots of cytosolic preparations of PKC-"depleted" myocytes confirmed the retention of PKC. Our findings indicate that TPA "down-regulated" BC3H-1 myocytes contain immunoreactive and functionally active PKC. The latter may explain the continued effectiveness of both insulin and diacylglycerol (DiC8) for stimulating glucose transport in "down-regulated" cells.     

Transforming growth factor beta-1 attenuates endothelin-1-induced functions in neonatal cardiac myocytes

In the present study we characterized a "crosstalk" mechanism between transforming growth factor beta-1 (TGF beta-1) and endothelin-1 (ET1) signaling pathways in neonatal cardiac myocytes. A 5 minute pretreatment with 1 ng/ml concentrations of TGF beta-1 attenuated ET1-induced negative chronotropic effects and translocation of the alpha, delta and varepsilonPKC isozymes to the particulate cell fraction. We found no effect of TGF beta-1 on responses induced by the P(2) purinergic agonist ATP or phorbol ester. Treatment of cardiac myocytes with acidic fibroblast growth factor (aFGF) did not alter ET1- or ATP-mediated effects on contraction rate or translocation of PKC isozymes to the particulate fraction. Our studies suggest that TGF beta-1 may act as a negative modulator of ET1- but not ATP- or phorbol ester-induced PKC isozyme signaling events in neonatal cardiac myocytes. A better understanding of the complex ET1 and TGF beta-1 signaling mechanisms in neonatal heart cells should enhance our knowledge regarding the interplay between these pathways.     

Growth-promoting effect of platelet-derived growth factor on rat cardiac myocytes

Platelet-derived growth factor (PDGF) is a dimeric molecule consisting of disulfide-bonded A- and B-polypeptide chains. Homodimeric (PDGF-AA, PDGF-BB) as well as heterodimeric (PDGF-AB) isoforms exert their effects on target cells by binding with different specificities to two structurally related protein tyrosine kinase receptors, denoted alpha- and beta-receptors. PDGF stimulates growth in various cell types, but little is known about its effect on mammalian cardiomyocytes. Therefore, growth-promoting effect of PDGF on rat cardiomyocytes was investigated. Primary culture of neonatal rat ventricular myocytes was prepared and cellular growth was estimated by [3H]-leucine incorporation assay. Tyrosine-phosphorylated PDGF-beta receptor of cardiomyocytes was determined by immunoblotting analysis after immunoprecipitation. PDGF-beta receptor, extracellular signal-regulated kinase (ERK) 1/2 and phosphorylated ERK1/2 of cardiomyocytes were measured by immunoblotting analysis. [3H]-leucine incorporation into the cultured myocytes was increased in a time- and dose-dependent manner after PDGF-BB stimulation. Phosphorylation of PDGF-beta receptor and ERK1/2 in cardiomyocytes was increased after short-term stimulation of PDGF-BB. Protein expression of PDGF-beta receptor and ERK1/2 was increased after long-term stimulation of PDGF-BB. [(3)H]-leucine incorporation into the cultured myocytes induced by PDGF-BB was partly blocked by mitogen-activated ERK-activating kinase (MEK) inhibitor PD98059, phospholipase C (PLC) inhibitor U73122, and protein kinase C (PKC) inhibitor staurosporin aglycone, respectively. Therefore, PDGF beta receptor, ERK1/2, PLC and PKC are involved in the signal transduction of PDGF-induced growth response of rat cardiac myocytes.     

Activation of protein kinase C by a tumor-promoting phorbol ester in pancreatic islets

Rat pancreatic islet homogenates display protein kinase C activity. This phospholipid-dependent and calcium-sensitive enzyme is activated by diacylglycerol or the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). In the presence of TPA, the Ka for Ca2+ is close to 5 microM. TPA does not affect phosphoinositide turnover but stimulates [32P]- and [3H]choline-labelling of phosphatidylcholine in intact islets. Exogenous phospholipase C stimulates insulin release, in a sustained and glucose-independent fashion. The secretory response to phospholipase C persists in media deprived of CaCl2. It is proposed that protein kinase C participates in the coupling of stimulus recognition to insulin release evoked by TPA, phospholipase C and, possibly, those secretatogues causing phosphoinositide breakdown in pancreatic islets.     

Inhibition of phorbol ester binding and protein kinase C activity by heavy metals

Other laboratories have reported biphasic effects of heavy metals on protein kinase C activity: stimulation followed by inhibition at higher concentrations. We demonstrate that these earlier findings most likely resulted from a combination of the effect of the heavy metals to liberate Ca2+ from Ca2+-EGTA buffer systems and the direct inhibitory effects of the metals on protein kinase C. Simulations of such interactions substantiate this conclusion. When soluble protein kinase C is prepared without the addition of Ca2+ or chelator, heavy metals (Cd2+, Cu2+, Hg2+, Zn2+, in the 10 microM range) inhibit the activity of, and the binding of regulatory ligands to, protein kinase C. Heavy metals inhibit the extent of [3H]phorbol dibutyrate binding without affecting the affinity of the interaction, an inhibition that is not surmounted by excess phospholipid. Heavy metals also inhibit the phospholipid-dependent catalytic activity of protein kinase C in a manner that excess phosphatidylserine can overcome. The inhibition of enzyme activity by heavy metals cannot be surmounted by excess Ca2+ or Mg2+. The inhibitory effects of heavy metals are not confined to protein kinase C. Heavy metals also inhibit cyclic AMP binding to cyclic AMP-dependent protein kinase and the catalytic activity of that kinase, but in a distinctly different pattern.     

Protein kinase C negatively modulated by phorbol ester

Pretreatment of protein kinase C with 12-O-tetradecanoylphorbol-13-acetate (TPA) and phospholipid resulted in complete inhibition of ATP/phosphotransferase activity, irreversibly. The inactivation by TPA required the phospholipid, and TPA alone did not cause inactivation. Ca2+ and diacylglycerol mimicked TPA. This action of TPA was not general for all protein kinases as it did not accelerate the inactivation of the catalytic subunit of cAMP-dependent protein kinase by phospholipid. The addition of MgATP to the reaction mixture completely protected protein kinase C from being inactivated by TPA, in the presence of phospholipid. The nucleotide-binding site of the enzyme was probably influenced by the binding of TPA and phospholipid.     

Conventional protein kinase C isoforms mediate neuroprotection induced by phorbol ester and estrogen

Rapid signal transduction pathways play a prominent role in mediating neuroprotective actions of estrogen in the CNS. We have previously shown that estrogen-induced neuroprotection of primary cerebrocortical neurons from beta-amyloid peptide (Abeta) toxicity depends on activation of protein kinase C (PKC). PKC activation with phorbol-12-myristate-13-acetate (PMA) also provides neuroprotection in this paradigm. Because the PKC family includes several isoforms that have opposing roles in regulating cell survival, we sought to identify which PKC isoforms contribute to neuroprotection induced by PMA and estrogen. We detected protein expression of multiple PKC isoforms in primary neuron cultures, including conventional (alpha, betaI, betaII), novel (delta, epsilon, theta) and atypical (zeta, iota/lambda) PKC. Using a panel of isoform-specific peptide inhibitors and activators, we find that novel and atypical PKC isoforms do not participate in the mechanism of either PMA or estrogen neuroprotection. In contrast, a selective peptide activator of conventional PKC isoforms provides dose-dependent neuroprotection against Abeta toxicity. In addition, peptide inhibitors of conventional, betaI, or betaII PKC isoforms significantly reduce protection afforded by PMA or 17beta-estradiol. Taken together, these data provide evidence that conventional PKC isoforms mediate phorbol ester and estrogen neuroprotection of cultured neurons challenged by Abeta toxicity.     

Subcellular distribution and immunocytochemical localization of protein kinase C in myocardium, and phosphorylation of troponin in isolated myocytes stimulated by isoproterenol or phorbol ester

Protein kinase C (PKC) catalytic activity was found in the cytosol, sarcolemma and sarcoplasmic reticulum, and PKC immunoreactivity was found in the striated regions and sarcolemma of rat hearts. Enhanced phosphorylation of troponin T and, to a lesser extent, troponin I was noted in isolated rat cardiac myocytes incubated with PKC activator phorbol ester, but only the phosphorylation of troponin I was stimulated by isoproterenol. It is suggested that PKC-mediated phosphorylation of troponin might be involved in regulation of myocardial function or in pathophysiology of the heart.     

Participation of protein kinase C in the activation of human neutrophils by phorbol ester

The calmodulin antagonist N(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7) has been examined as an inhibitor of superoxide anion production and granule exocytosis in phorbol ester (PMA)-activated neutrophils. Inhibition of the respiratory burst was observed at a concentration of W-7 identical to that required for inhibition of native protein kinase C (PKC), whereas the concentration required to inhibit the secretory response was found to correspond to that required for inhibition of the proteolytically converted fully active PKC. The IC50 of W-7 was in both cases 5 and 12 fold higher than that required for inhibition of calmodulin dependent kinases. The results confirm the essential role for the membrane-bound PKC in the production of O2- radicals and provide a clear evidence of the direct participation of the proteolytically activated cytosolic PKC to the secretory response of PMA activated neutrophils.     

Protection of cardiac myocytes via delta(1)-opioid receptors, protein kinase C, and mitochondrial K(ATP) channels

The objective of the present study was to investigate the role of delta(1)-opioid receptors in mediating cardioprotection in isolated chick cardiac myocytes and to investigate whether protein kinase C and mitochondrial ATP-sensitive K(+) (K(ATP)) channels act downstream of the delta(1)-opioid receptor in mediating this beneficial effect. A 5-min preexposure to the selective delta(1)-opioid receptor agonist (-)-TAN-67 (1 microM) resulted in less myocyte injury during the subsequent prolonged ischemia compared with untreated myocytes. 7-Benzylidenenaltrexone, a selective delta(1)-opioid receptor antagonist, completely blocked the cardioprotective effect of (-)-TAN-67. Naltriben methanesulfonate, a selective delta(2)-opioid receptor antagonist, had only a slight inhibitory effect on (-)-TAN-67-mediated cardioprotection. Nor-binaltorphimine dihydrochloride, a kappa-opioid receptor antagonist, did not affect (-)-TAN-67-mediated cardioprotection. The protein kinase C inhibitor chelerythrine and the K(ATP) channel inhibitors glibenclamide, a nonselective K(ATP) antagonist, and 5-hydroxydecanoic acid, a mitochondrial selective K(ATP) antagonist, reversed the cardioprotective effect of (-)-TAN-67. These results suggest that the delta(1)-opioid receptor is present on cardiac myocytes and mediates a potent cardioprotective effect via protein kinase C and the mitochondrial K(ATP) channel.     

Modulation of the insulin growth factor II/mannose 6-phosphate receptor in microvascular endothelial cells by phorbol ester via protein kinase C

Phosphorylation of hormone receptors by protein kinase C (PKC) may be involved in the regulation of receptor recycling. We have studied the recycling and the phosphorylation state of the insulin growth factor (IGF) II/mannose 6-phosphate (Man-6-P) receptor in microvascular endothelial cells from rat adipose tissue. Scatchard analysis showed these cells have over 2 x 10(6) receptors/cell with an affinity constant of 1 x 10(9) M-1. In the presence of phorbol myristate acetate (PMA), an activator of PKC and analog of diacylglycerol, IGF-II receptor number increased in the plasma membrane by 60% without changes in the binding affinity. This increase in cell surface receptor number was confirmed by affinity cross-linking and 125I-surface labeling studies, occurred with a half-time of 20 min, and was reversible upon withdrawal of PMA. The redistribution of IGF-II/Man-6-P receptors was not due to an inhibition of internalization which was in fact stimulated by PMA. The effect of PMA on IGF-II receptor recycling correlated with its stimulation of PKC activity. Furthermore, after down-regulation of cellular PKC levels by preincubation with PMA, PMA was unable to activate residual PKC activity in the membranous pool or increase IGF-II receptor number at the cell surface. The phosphorylation state of the IGF-II/Man-6-P receptor was determined by 32P labeling of intact cells and immunoprecipitation with anti-receptor antibodies. In the basal state, the receptor was phosphorylated only on serine residues which was increased by 75% after treatment with PMA. In contrast, IGF-II decreased receptor phosphorylation and plasma membrane binding in a parallel and dose-dependent manner. Thus, PKC-stimulated serine phosphorylation of IGF-II/Man-6-P receptor may promote the translocation of the receptor to the cell surface, whereas IGF-II-stimulated dephosphorylation of the receptor may lead to a decrease in the number of cell surface receptors. These data suggest a role for PKC-mediated serine phosphorylation in the regulation of intracellular trafficking of receptors in endothelial cells.     

Altered catalytic properties of protein kinase C in phorbol ester treated cells

Exposure of various cell types (rat-1 fibroblasts, bovine adrenocortical cells, human lymphoid cells) to nanomolar concentrations of TPA, resulted in a rapid, apparent loss of cellular protein kinase C content, when the enzyme was assayed by its phospholipid and Ca2+-dependent histone (H1)-kinase activity, following solubilization and DEAE-cellulose chromatography isolation. By contrast, no loss of protein kinase C was detected when the enzyme was probed by its high affinity PDBu binding capacity nor when the kinase activity was assayed with protein substrates other than histones, such as vinculin and a cytochrome P-450. It is concluded that, in addition to the previously reported enzyme subcellular redistribution, following TPA treatment, the phorbol ester induces striking alterations of the cellular protein kinase C catalytic activities. The molecular mechanisms of these changes and their implication in the tumor promotion process remain to be clarified.     

Molecular biology of protein kinase C signaling in cardiac myocytes

The PKC family of serine/threonine kinases have been implicated in a diverse array of cellular responses. Adult cardiac myocytes express multiple PKC isozymes, which participate in the response of muscle cells to extracellular stimuli, modulate contractile properties, and promote cell growth and survival. Recently, the classification of this ubiquitous family of signaling molecules has been expanded from three to four subfamilies. This review will focus on the application of pharmacologic and molecular approaches to explore the biology of cardiac PKC isozymes. The availability of transgenic mice and peptide PKC modulators have been instrumental in identifying target substrates for activated cardiac PKC isozymes, as well as the identification of specific isozymes linked to distinct growth characteristics and cell phenotype. The rapid growth of knowledge in the area of PKC signaling and PKC substrate interactions, may result in the development of therapeutic modalities with the potential to arrest or reverse the progression of cardiovascular diseases.     

A phorbol ester and phospholipid-activated, calcium-unresponsive protein kinase in mouse epidermis: characterization and separation from protein kinase C

The phosphorylation of an Mr 82,000 protein (p82) in the Triton X-100 extract of the particulate fraction of mouse epidermis is dependent on the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) or diacylglycerol and phospholipid and, contrary to protein kinase C (PKC)-catalyzed phosphorylation, cannot be activated by calcium plus phospholipid. The novel p82 kinase differs also from PKC in many other respects, such as substrate specificity, turnover rate, and sensitivity to inhibitors. The p82 kinase can be separated from PKC by chromatography on phenyl sepharose and does not react with a polyclonal PKC antiserum. Like PKC, the novel kinase phosphorylates its substrate on threonine and serine, but not on tyrosine. Similar to PKC, the epidermal p82-kinase system is down-modulated after TPA treatment of mouse skin, with a half-life of around 5 h. Down-modulation is also accomplished by the phorbol ester RPA, but not by the Ca2+ ionophore A23187, and it is inhibited by the immunosuppressive agent cyclosporin A. In addition to down-modulation, TPA treatment of the animals activates a phosphatase that dephosphorylates phosphorylated p82 in the extract of the particulate fraction.     

Extracellular regulated kinase, but not protein kinase C, is an antiapoptotic signal of insulin-like growth factor-1 on cultured cardiac myocytes

This study aims to elucidate the signaling pathway for insulin-like growth factor-1 (IGF-1) in cultured neonatal rat cardiomyocytes and particularly the role of IGF-1 in cardiac apoptosis. IGF-1 stimulated polyphosphoinositide turnover, translocation of protein kinase C (PKC) isoforms (alpha, epsilon, and delta) from the soluble to the particulate fraction, activation of phospholipid-dependent and Ca(2+)-, phospholipid-dependent PKC, and activation of the extracellular-regulated kinase (ERK). IGF-1 attenuated sorbitol-induced cardiomyocyte viability and nuclear DNA fragmentation. These antiapoptotic effects of IGF-1 were blocked by PD-098059 (an MEK inhibitor) but not by bisindolylmaleimide I (BIM, a specific PKC inhibitor). The ERK pathway may therefore be an important component in the mechanism whereby IGF-1 exerts its antiapoptotic effect on the cardiomyocyte.