Sensitization of regulated exocytosis by protein kinase C

Activation of protein kinase C (PKC) increases vesicular secretion in many cell types. We determined the calcium dependence of secretion and the size of the readily releasable pool of secretory granules in pituitary gonadotropes by photorelease of caged-calcium. The calcium affinity for exocytosis was roughly doubled by activation of PKC by a phorbol ester, whereas the size of the readily releasable pool was not greatly increased. The effect was due to activation of PKC, because it was blocked by a PKC inhibitor and was not mimicked by an inactive phorbol ester analogue. A similar increase in calcium sensitivity was induced by preincubation with gonadotropin-releasing hormone, the physiological releasing hormone. These findings provide direct evidence for physiological regulation of secretion by enhancement of Ca2+-sensing steps. Because exocytosis depends on the third- to fourth-power of intracellular free Ca2+ concentration, this mechanism ensures a powerful up-regulation of hormone release and may explain how PKC can stimulate exocytosis without an increase of Ca2+ above the resting level.     

Altered expression of protein kinase C in adult T-cell leukemia cells.

Protein kinase C (PKC) has been shown to be involved in the mitogenic response and in oncogenic cell transformation in many experimental models. We analyzed the expression of PKC in both highly purified leukemic T cells freshly isolated from adult T-cell leukemia (ATL) patients and control T lymphocytes obtained from healthy volunteers. PKC activity was decreased in the ATL cells as compared with the control T cells. Cytosolic PKC activity in the ATL cells was remarkably decreased, whereas particulate membrane PKC activity was similar to the control level. The percentage of PKC activity in the particulate fraction was 34% in the ATL cells and 19% in the control cells. Regarding the altered subcellular localization of PKC activity, phorbol ester-induced translocation of cytosolic PKC was inhibited in some ATL cases. Similarly to the decrease in PKC activity, there was a decrease in the expression of the major PKC isozymes II(beta) and III(alpha) in ATL cells. These results suggest impaired regulation of PKC expression in ATL as well as in many experimental cancers.     

Induction of exocytosis in permeabilized pituitary cells by alpha- and beta-type protein kinase C.

Protein kinase C is now recognized to comprise a family of closely related subspecies (PKCs). When cultured rat pituitary cells were permeabilized by digitonin for 5 min in the absence of Ca2+, endogenous PKC activity was decreased by 72%. PKC depletion was also achieved by prior treatment (24 hr) with high concentrations of phorbol 12-myristate 13-acetate (PMA). When purified activated brain PKCs were added for 30 min to PMA-pretreated, digitonin-permeabilized cells, only alpha- and beta- but not gamma-type PKC stimulated luteinizing hormone release. Since PKC was implicated as a mediator of gonadotropin secretion, gonadotropin-releasing hormone might utilize alpha- and beta-type PKCs for stimulation of gonadotropin secretion; alpha- and beta-type PKCs might participate also in other exocytotic responses in diverse biological systems in which PKC was implicated.     

Antigen-induced increase in protein kinase C activity in plasma membrane of mast cells.

Bridging of cell-bound IgE antibody molecules on colony-stimulating factor-dependent mouse mast cell line (PT-18) cells by multivalent antigen induces phospholipid methylation, a transient rise in intracellular cAMP, intracellular mobilization and uptake of Ca2+, and formation of diacylglycerol followed by histamine release. Exposure of the sensitized cells to antigen also induces a substantial increase in protein kinase C activity in the plasma membrane, which is accompanied by a slight decrease in the enzyme in cytosol. Protein kinase C activity in the membrane fraction reached maximum within 30 sec after antigen challenge and then gradually declined. The increase of the enzyme activity in the membrane could not be explained by a shift of the enzyme from cytosol, and it suggested that bridging of IgE-receptor may induce a modulation of existing enzyme to a state of higher catalytic activity. Phorbol 12-myristate 13-acetate also induced a rapid but persistent increase in protein kinase C activity in the membrane fraction of mast cells. However, the increase in the enzyme activity in the membrane was accompanied by a marked decrease in the enzyme in cytosol.     

Glucocorticoid-dependent complementation of a hepatoma cell variant defective in viral glycoprotein sorting.

We have utilized the rat hepatoma (HTC) cell sorting variant CR4 to examine the glucocorticoid-regulated pathways that localize mouse mammary tumor virus glycoproteins to the cell surface. The defective sorting of cell surface mouse mammary tumor virus glycoproteins in CR4 cells was complemented after fusion with either normal rat hepatocytes or uninfected HTC cells. Indirect immunofluorescence of transient heterokaryons revealed that the regulated localization of mouse mammary tumor virus glycoproteins was dependent upon glucocorticoid treatment and required de novo RNA and protein synthesis. Thus, a glucocorticoid-regulated trafficking activity, unrelated to mouse mammary tumor virus sequences, which is induced in both adult rat liver and cultured hepatoma cells, can act in trans to mediate an intracellular sorting pathway for membrane glycoproteins.     

Altered cardiac endothelin receptors and protein kinase C in deoxycorticosterone-salt hypertensive rats

The aim of the present study was to assess the status of ET-1 receptor subtypes (ET(A)and ET(B)) in ventricular myocytes and fibroblasts and to determine the role of PKC-dependent pathways in ET-1-stimulated cardiac cells in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Systolic blood pressure and relative heart to body weight were significantly increased in DOCA-salt rats. In unilaterally nephrectomized (Uni-Nx) control rats, more than 90% of cardiomyocyte ET receptors were of the ET(A)subtype, whereas in fibroblasts ET(A)and ET(B)receptors were present in a 1:3 ratio. In DOCA-salt rats, the density of the ET(A)receptor subtype was reduced by 31% in cardiomyocytes and in cardiac fibroblasts only ET(B)receptor density was decreased by 29%. Affinity was unchanged. The relative expression of immunoreactive PKC alpha, gamma and epsilon was significantly increased, whereas PKC delta was not altered in cardiac extracts of DOCA-salt rats. In cardiac fibroblasts from DOCA-salt rats PKC delta was significantly increased and PKC epsilon was not translocated after ET-1 stimulation. The hearts of DOCA-salt hypertensive rats are thus characterized by: (1) decreased density of cardiomyocyte ET(A)receptors and fibroblast ET(B)receptors; (2) cell-specific enhanced expression of some PKC isoenzymes (alpha, gamma, delta and epsilon); and (3) unresponsiveness of PKC epsilon to translocate in the presence of ET-1. Together with alterations of ET-1-induced Ca(2+)handling in cardiac myocytes and fibroblasts, which we previously reported, results from the present study indicate a marked modification of the cardiac ET-1 system of DOCA-salt hypertensive rats.     

Protein kinase C as a signal for exocytosis

We have studied signaling mechanisms that stimulate exocytosis and luteinizing hormone secretion in isolated male rat pituitary gonadotropes. As judged by reverse hemolytic plaque assays, phorbol-12-myristate-13-acetate (PMA) stimulates as many gonadotropes to secrete as does gonadotropin-releasing hormone (GnRH). However, PMA and GnRH use different signaling pathways. The secretagogue action of GnRH is not very sensitive to bisindolylmaleimide I, an inhibitor of protein kinase C, but is blocked by loading cells with a calcium chelator, 1,2-bis-(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid. The secretagogue action of PMA is blocked by bisindolylmaleimide I and is not very sensitive to the intracellular calcium chelator. GnRH induces intracellular calcium elevations, whereas PMA does not. As judged by amperometric measurements of quantal catecholamine secretion from dopamine- or serotonin-loaded gonadotropes, the secretagogue action of PMA develops more slowly (in several minutes) than that of GnRH. We conclude that exocytosis of secretory vesicles can be stimulated independently either by calcium elevations or by activation of protein kinase C.     

Targeting protein kinase C in mantle cell lymphoma

Although targeting the Bruton tyrosine kinase (BTK) with ibrutinib has changed lymphoma treatment, patients with mantle cell lymphoma (MCL) remain incurable. In this study, we characterized a broad range of MCL cell lines and primary MCL cells with respect to the response to the BTK inhibitor, ibrutinib, and compared it with the response to the protein kinase C (PKC) inhibitor, sotrastaurin. At clinically relevant concentrations, each drug induced potent cell death only in the REC‐1 cell line, which was accompanied by robust inhibition of AKT and ERK1/ERK2 (ERK1/2, also termed MAPK3/MAPK1) phosphorylation. In sensitive REC‐1 cells, the drug‐mediated impaired phosphorylation was obvious on the levels of B‐cell receptor‐induced and basal phosphorylation. Similar results were obtained in primary MCL cells with ibrutinib and in a subset with sotrastaurin. The various drug‐resistant MCL cell lines showed very distinct responses in terms of basal AKT and ERK1/2 phosphorylation. Interestingly, targeting PKC and BTK at the same time led to ibrutinib‐mediated rescue of a weak sotrastaurin‐induced apoptosis in MINO cells. Additional targeting of AKT sensitized MINO cells to inhibitor‐mediated cytotoxicity. In summary, MCL cells are heterogeneous in their response to BTK or PKC inhibition, indicating the need for even more individualized targeted treatment approaches in subsets of MCL patients.     

The role of protein kinase C in alpha-thrombin-mediated endothelial cell activation.

Thrombin, the key regulatory protein of hemostasis, has been implicated in a variety of important endothelial cell processes closely linked to endothelial signal transduction mechanisms. An initial event, following receptor binding by catalytically active alpha-thrombin, appears to be the activation of a G-protein-coupled, PI-specific PLC, with resultant generation of IP3 and DAG, with increases in [Ca2+]i, and activation and translocation of PKC (Fig. 9). PKC activation results in down-regulation of PLC, as demonstrated by inhibition of agonist-induced increases in [Ca2+]i, whereas PLA2 activity is up-regulated, with a resultant increase in endothelial PGI2 synthesis. Recently, we have demonstrated that activity of membrane-bound, endothelial PLD, is also up-regulated by PKC activation. In addition to its modulatory role in endothelial cell phospholipase activities, PKC activation appears to play a critical role in thrombin-mediated endothelial barrier dysfunction, likely via specific cytoskeletal protein phosphorylation. A temporal relationship between alpha-thrombin-mediated signal transduction and specific cellular responses, such as PGI2 synthesis and barrier dysfunction, can be established (Fig. 2). Further investigations are ongoing to identify more clearly the precise biochemical intermediates involved in the endothelial cell response to thrombin, as well as the role of differential phosphorylation by various protein kinase systems in thrombin-mediated signal transduction in vascular endothelium.     

Mammalian mitogen-activated protein kinase kinase kinase (MEKK) can function in a yeast mitogen-activated protein kinase pathway downstream of protein kinase C.

Mitogen-activated protein kinase cascades are conserved in fungal, plant, and metazoan species. We expressed murine MAP kinase kinase kinase (MEKK) in the yeast Saccharomyces cerevisiae to determine whether this kinase functions as a general or specific activator of genetically and physiologically distinct MAP-kinase-dependent signaling pathways and to investigate how MEKK is regulated. Expression of MEKK failed to correct the mating deficiency of a ste11 delta mutant that lacks an MEKK homolog required for mating. MEKK expression also failed to induce expression of a reporter gene controlled by the HOG1 gene product (Hog1p), a yeast MAP kinase homolog involved in response to osmotic stress. Expression of MEKK did correct the cell lysis defect of a bck1 delta mutant that lacks an MEKK homolog required for cell-wall assembly. MEKK required the downstream MAP kinase homolog in the BCK1-dependent pathway, demonstrating that it functionally replaces the BCK1 gene product (Bck1p) rather than bypassing the pathway. MEKK therefore selectively activates one of three distinct MAP-kinase-dependent pathways. Possible explanations for this selectivity are discussed. Expression of the MEKK catalytic domain, but not the full-length molecule, corrected the cell-lysis defect of a pkc1 delta mutant that lacks a protein kinase C homolog that functions upstream of Bck1p. MEKK therefore functions downstream of the PKC1 gene product (Pkc1p). The N-terminal noncatalytic domain of MEKK, which contains several consensus protein kinase C phosphorylation sites, may, therefore, function as a negative regulatory domain. Protein kinase C phosphorylation may provide one mechanism for activating MEKK.     

Inhibition of gonadotropin-induced granulosa cell differentiation by activation of protein kinase C.

The induction of granulosa cell differentiation by follicle-stimulating hormone (FSH) is characterized by cellular aggregation, expression of luteinizing hormone (LH) receptors, and biosynthesis of steroidogenic enzymes. These actions of FSH are mediated by activation of adenylate cyclase and cAMP-dependent protein kinase and can be mimicked by choleragen, forskolin, and cAMP analogs. Gonadotropin releasing hormone (GnRH) agonists inhibit these maturation responses in a calcium-dependent manner and promote phosphoinositide turnover. The phorbol ester phorbol 12-myristate 13-acetate (PMA) also prevented FSH-induced cell aggregation and suppressed cAMP formation, LH receptor expression, and progesterone production, with an ID50 of 0.2 nM. In FSH-treated cells, PMA did not reduce the initial increase in cAMP formation during the first 24 hr of culture but prevented its secondary increase from 24 to 48 hr. PMA also inhibited LH receptor induction by cholera toxin, forskolin, and 8-bromo-cAMP, but it did not impair cAMP responses to the former two agents, indicating that the site of action of the phorbol ester is distal to adenylate cyclase. The early stimulation of cAMP-dependent protein kinase activity by FSH was also unaffected by PMA, consistent with its lack of effect on the initial cAMP response to FSH. However, PMA caused a marked decrease in cytosolic protein kinase C activity within 1 min of its addition to the cells. The permeant diacylglycerols, 1-oleoyl-2-acetoyl-sn-glycerol and sn-1,2-dioctanoyl glycerol, also inhibited LH receptor formation, while the nonpermeant diacylglycerol, diolein, was inactive. These results indicate that in situ activation of protein kinase C by PMA or permeant diacylglycerols inhibits cAMP-dependent granulosa cell differentiation, and suggest that the inhibitory actions of GnRH agonists on granulosa cell maturation are also mediated by protein kinase C.     

Calcium antagonists ameliorate ischemia-induced endothelial cell permeability by inhibiting protein kinase C.

BACKGROUND: Dihydropyridines block calcium channels; however, they also influence endothelial cells, which do not express calcium channels. We tested the hypothesis that nifedipine can prevent ischemia-induced endothelial permeability increases by inhibiting protein kinase C (PKC) in cultured porcine endothelial cells. METHODS AND RESULTS: Ischemia was induced by potassium cyanide/deoxyglucose, and permeability was measured by albumin flux. Ion channels were characterized by patch clamp. [Ca2+]i was measured by fura 2. PKC activity was measured by substrate phosphorylation after cell fractionation. PKC isoforms were assessed by Western blot and confocal microscopy. Nifedipine prevented the ischemia-induced increase in permeability in a dose-dependent manner. Ischemia increased [Ca2+]i, which was not affected by nifedipine. Instead, ischemia-induced PKC translocation was prevented by nifedipine. Phorbol ester also increased endothelial cell permeability, which was dose dependently inhibited by nifedipine. The effects of non-calcium-channel-binding dihydropyridine derivatives were similar. Analysis of the PKC isoforms showed that nifedipine prevented ischemia-induced translocation of PKC-alpha and PKC-zeta. Specific inhibition of PKC isoforms with antisense oligodeoxynucleotides demonstrated a major role for PKC-alpha. CONCLUSIONS: Nifedipine exerts a direct effect on endothelial cell permeability that is independent of calcium channels. The inhibition of ischemia-induced permeability by nifedipine seems to be mediated primarily by PKC-alpha inhibition. Anti-ischemic effects of dihydropyridine calcium antagonists could be due in part to their effects on endothelial cell permeability.     

A self-assembling protein kinase C inhibitor.

Previous studies have described a dicationic anticarcinoma agent that can chemically assemble in situ from monocationic phosphonium salts. The chemical combination of these monocationic precursors in the micromolar concentration range, occurring after their uptake by cells, was probably responsible for their synergistic inhibition of cell growth and for their selective cytotoxicity to Ehrlich ascites murine carcinoma cells relative to untransformed epithelial cells. Here, we report that the dicationic product that forms in this assembly reaction is an in vitro inhibitor of protein kinase C (PKC) alpha and beta 1 isoforms, exhibiting IC50 values of 20.4 microM and 35 microM, respectively. The monocationic precursors proved to be much weaker inhibitors of PKC (IC50 values greater than 200 microM). When PKC is exposed to combinations of the two precursors, the enzymatic activity decreases steadily as a function of time. Using dose-response data and HPLC kinetic studies, we show that when the two precursor compounds are added as a combination to PKC under these conditions, the rate of formation of the inhibitory product follows the observed time course of decline in PKC activity under identical conditions. We discuss the possibility that antiproliferative effects against carcinoma cells of the preformed dication and of the combined monocationic precursors involve inhibition of PKC.     

Expression of protein kinase C isoforms in various steroidogenic cell types

Different endocrine, steroidogenic cell types were examined for their content in protein kinase C (PKC) subtypes I, II and III. The expression of the PKC isoforms was assayed following high-performance liquid chromatography separation and characterization using a set of antipeptide antibodies specific for each enzyme isotype. Bovine and rat adrenocortical cells, as well as porcine Sertoli cells expressed only the type III PKC. By contrast, Leydig cell expressed both the isotypes I, II and II at similar levels. Taking into account the biological effect observed in these various cell types upon PKC activation, it may be suggested that the type III iso-PKC is involved in the steroidogenic activation pathways, whereas the expression of the types I, II, and III, only in Leydig cells, may contribute to a different array of cross-talk regulation pathways in these cells.     

Production of matrix metalloproteinases in human cultured mast cells: involvement of protein kinase C-mitogen activated protein kinase kinase-extracellular signal-regulated kinase pathway.

BACKGROUND: Matrix metalloproteinases (MMPs) have been reported to play crucial roles in the migration of inflammatory cells through basement membrane components. To confirm the role of mast cells as a source of MMPs, we investigated the production of MMP and its pathway in human cultured mast cells (HCMC). We also investigated the production of tissue inhibitors of metalloproteinase (TIMPs). METHODS: HCMC was stimulated with phorbor 12-miristate 13-acetate (PMA) and/or calcium ionophore A23187 (A23187), and the resulting MMP production was evaluated by gelatin zymography and western blotting. Expression of MMP and TIMP mRNA was also examined. Granulocyte macrophage-colony stimulating factor (GM-CSF) was measured by ELISA and activation of extracellular signal-regulated kinase (ERK) was evaluated by western blotting. RESULTS: We detected the de novo synthesis of MMP-9 in HCMC after stimulation with PMA and found that the synthesis was mediated through protein kinase C-mitogen activated protein kinase kinase (MEK)-ERK pathway. The MMP-9 production induced by PMA was suppressed by simultaneous treatment with A23187, whereas GM-CSF production was potentiated. We also detected the expression of mRNA for membrane-type 1 (MT1)-MMP, TIMP-1 and TIMP-2 after stimulation with PMA. Glucocorticoids and flavonoids inhibited MMP-9 production, and TIMPs and MMP inhibitors inhibited the gelatinolytic activity of mast cell-derived MMP-9. Furthermore, phenylmethylsulfonyl fluoride, a protease inhibitor, inhibited the conversion from proMMP-9 to active MMP-9. CONCLUSIONS: These results suggest that the human mast cell is a leading member of MMP production, and the production, activation and activity are controllable by pharmacological agents.     

Reconstitution of a variant macrophage cell line defective in oxygen metabolism with a H2O2-generating system.

A variant clone, C3C, derived from the cloned macrophage cell line J774.16 lacks the capacity to produce O2- or H2O2 after appropriate stimulation. When the parental and variant cell lines were infected with epimastigotes of Trypanosoma cruzi, the parasites were killed or their growth was inhibited by the parental line, but they grew readily in the variant clone C3C. It was possible to reconstitute the variant cell line with an enzyme system targeted to the lysosomal compartment capable of generating a single oxygen metabolite, H2O2. This was accomplished by allowing the cells to phagocytize zymosan particles covalently coupled with glucose oxidase (GO-Zy particles). Approximately one-third of the H2O2 theoretically expected to be produced by the ingested GO-Zy particles could be detected outside the cells by the cytochrome c peroxidase assay; this fraction may represent the efficiency of extracellular assays for H2O2 production. When T. cruzi-infected clone C3C cells were reconstituted with GO-Zy particles, upon addition of glucose, intracellular killing of the parasites occurred. It was possible to estimate the level of H2O2 production required to kill a single parasite (8.7 x 10(-7) nmol/min) by GO-Zy particles in suspension and to formulate a first approximation of the killing potency of the reconstituted cells--i.e., number of parasites expected to be killed--that correlated well with the observed growth of the parasites intracellularly.     

Role of protein kinase C in eosinophil function.

Protein kinase C (PKC) isoforms are being elucidated as an increasingly diverse family of enzymes involved in the downstream signal transduction and cell function in various types of cells. To date, 11 PKC isoforms have been identified; they are grouped according to their molecular structure and mode of activation: conventional PKCs (alpha, beta I, beta II, and gamma), novel PKCs (delta, epsilon, mu, theta, and eta), and atypical PKCs (zeta, and iota/lambda). Eosinophils are involved in the pathogenesis of allergic diseases such as bronchial asthma, pollinosis, and atopic dermatitis as well as in the inflammatory response to parasitic infections. Recent studies using selective activators and inhibitors of individual PKC isoforms have revealed that this enzyme is involved in eosinophil dynamics such as cell motility and other functions. However, the role of PKCs in eosinophil functions has been not wholly understood. In this review, we have focused upon and summarized the current knowledge regarding the role of PKC isoforms in eosinophil functions.