Interleukin 2 modulation of adenylate cyclase. Potential role of protein kinase C

Interleukin 2 (IL 2) stimulated DNA synthesis of murine T lymphocytes (CT6) in a concentration-dependent manner, over a range of 1-1000 units/ml. This proliferative effect of IL 2 was attenuated by simultaneous exposure to prostaglandin E2 (PGE)2. In intact cells, IL 2 inhibited both basal and PGE2-stimulated cAMP production; the amount of cAMP generated was dependent upon the relative concentrations of IL 2 and PGE2. The effect of IL 2 on CT6 cell proliferation and cAMP production was mimicked by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), which, like IL 2, causes a translocation and activation of protein kinase C. While PGE2 stimulated adenylate cyclase activity in membrane preparations, neither IL 2 nor TPA inhibited either basal or stimulated membrane adenylate cyclase activity. However, when CT6 cells were pretreated with IL 2 or TPA and membranes incubated with calcium and ATP, both basal and PGE2-and NaF-stimulated membrane adenylate cyclase activity was inhibited. This inhibition of adenylate cyclase activity was also observed if membranes from untreated cells were incubated with protein kinase C purified from CT6 lymphocytes in the presence of calcium and ATP. The data suggest that the decreased cAMP production which accompanies CT6 cell proliferation results from an inhibition of adenylate cyclase activity mediated by protein kinase C and that these two distinct protein phosphorylating systems interact to modulate the physiological response to IL 2.     

Possible involvement of protein kinase C in parathyroid hormone degradation by osteoblast-like rat osteosarcoma cell line UMR106

The effects of 12-O-tetraadecanoyl phorbol-13-acetate (TPA), 1-oleoyl-2-acetyl-glycerol (OAG), and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) on the parathyroid hormone (PTH) degrading activity in a PTH-responsive osteoblast-like rat osteosarcoma cell line UMR106 were investigated to assess the role of Ca2+-activated. Phospholipid dependent protein kinase (protein kinase C) on the degradation of hormones. TPA and OAG, activators of protein kinase C, enhanced the PTH degrading activity dose-dependently, whereas H-7, an inhibitor of protein kinase C, exhibited a dose-dependent inhibition on this activity. These data suggest that protein kinase C activation may enhance PTH degrading activity by UMR106 cells as a possible regulator of PTH degradation.     

Activation by phorbol esters of protein kinase C in MCF-7 human breast cancer cells

Exposure of MCF-7 human breast cancer cells to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) leads to the inhibition of cell proliferation. We investigate here the short-term effects of TPA on subcellular distribution of protein kinase C, and on protein phosphorylation in cultured MCF-7 cells. We report a rapid and dramatic decrease in cytosolic protein kinase C activity after TPA treatment. Only 30% of the enzymatic activity lost in the cytosol was recovered in the particulate fraction. These data suggest that subcellular translocation of protein kinase C is accompanied by a rapid down-regulation of the enzyme (70%). Furthermore, TPA and other protein kinase C activators rapidly induce the phosphorylation of a 28 kDa protein in intact MCF-7 cells. Phorbol esters devoid of tumor-promoting activity are ineffective both for inducing these early biochemical events and for inhibiting cell proliferation.     

Biphasic effect of protein kinase C activators on spontaneous and glucocorticoid-induced apoptosis in primary mouse thymocytes

Spontaneous and glucocorticoid (fluocinolone acetonide, FA)-induced apoptosis of primary mouse thymocytes was inhibited by protein kinase C (PKC) activators such as bryostatin-1 and phorbol ester 12-O-tetradecanoyl-phorbol-13 acetate (TPA) within the first 2-4 h of incubation but was enhanced upon prolonged treatment. Only the anti-apoptotic but not the pro-apoptotic effect of TPA was completely suppressed by the PKC inhibitor Goe 6983 and moderately inhibited by Goe 6976. Immunoblot analysis revealed distinct PKC alpha, beta, delta, eta, theta, mu and zeta signals, a very faint PKCepsilon and no PKCgamma signal. Upon prolonged TPA treatment all PKC isoenzymes became downregulated, albeit at different rates (PKCdelta>alpha>mu>beta,theta>eta,zeta). No significant generation of caspase-derived catalytic PKC fragments, as found to be produced upon induction of apoptosis and to be pro-apoptotic in other systems, was observed in FA- or TPA-treated thymocytes. It is concluded that the early anti-apoptotic effect of TPA depends on the activation of n-type PKC isoenzymes, whereas stimulation of spontaneous and FA-induced apoptosis by TPA ensues, at least partially, from a downregulation (or inactivation) of anti-apoptotic PKC species, i.e. in primary thymocytes PKC activation is primarily involved in a negative regulation of apoptosis.     

Downregulation of tumor necrosis factor receptors of macrophages by interferons and interleukin-1. Role of protein kinase C activation

Freshly harvested murine peritoneal macrophages and a line of transformed murine macrophages (RAW) were used in experiments designed to investigate the effect of different interferons (IFN) and interleukin-1 (IL-1) on tumor necrosis factor (TNF) receptors. Low concentrations of IFN-gamma or somewhat higher concentrations of IFN-alpha drastically downregulated the TNF receptors of RAW cells. A similar, but less pronounced, downregulation of TNF receptors was observed in peritoneal macrophages treated with these IFNs. This downregulation could not be accounted for by an induction of TNF secretion. Furthermore, IFN-alpha and gamma interacted synergistically in downregulating TNF receptors of RAW cells. IL-1 also downregulated TNF receptors. When RAW cells were treated with inhibitors of protein kinase C, the downregulation of TNF receptors by IFNs or IL-1 was reversed, and TNF binding increased up to 2-fold over that of untreated cells. Such increase was also observed in RAW cells treated only with the inhibitor of protein kinase C, staurosporine. However, TNF receptors decreased in peritoneal macrophages treated with staurosporine. This finding was explained by activation of macrophages by staurosporine, which induced secretion of TNF. These findings indicate that protein kinase C activity regulates TNF receptors in macrophages.     

Effects of protein kinase C activators on germinal vesicle breakdown and polar body emission of mouse oocytes

Protein phosphorylation mediated by cAMP-dependent protein kinase is instrumental in maintaining meiotic arrest of mouse oocytes. To assess whether protein phosphorylation mediated by calcium/phospholipid-dependent protein kinase (protein kinase C) might also inhibit the resumption of meiosis, we treated oocytes with activators of this enzyme. The active phorbol esters 12-O-tetra-decanoyl phorbol-13-acetate (TPA) and 4 beta-phorbol 12,13-didecanoate (4 beta-PDD) inhibited germinal vesicle breakdown (GVBD), as did a more natural activator of protein kinase, C, sn-1,2-dioctanoylglycerol (diC8). An inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), did not inhibit GVBD. We then examined whether protein kinase C activators inhibit a step in the cAMP-modulated pathway that regulates resumption of meiosis. TPA did not inhibit the maturation-associated decrease in oocyte cAMP. Microinjected heat-stable protein inhibitor of cAMP-dependent protein kinase failed to induce GVBD in the presence of TPA. Both TPA and diC8 partially inhibited specific changes in oocyte phosphoprotein metabolism that are tightly correlated with resumption of meiosis; these agents also induced the apparent phosphorylation of specific oocyte proteins. These results suggest that protein kinase C activators may inhibit resumption of meiosis by acting distal to a decrease in cAMP-dependent protein kinase activity, but prior to changes in oocyte phosphoprotein metabolism that are presumably required for resumption of meiosis. Finally, we compared the effects of db-cAMP and protein kinase C activators on polar body emission following GVBD. TPA, 4 beta-PDD or diC8, but not 4 alpha-PDD or db-cAMP, inhibited polar body emission in a dose-dependent manner. The morphology and cytology of oocytes in which polar body emission was inhibited by TPA or 4 beta-PDD differed from that of oocytes treated with diC8. Thirty to 60% of the former were round in shape and exhibited a clump of chromosomes but no spindle; the remainder were distended in shape and exhibited a metaphase I spindle. All oocytes treated with diC8, however, were round, had dispersed chromosomes, and no spindle. These results suggest that, in contrast to resumption of meiosis, polar body emission is inhibited by activation of protein kinase C but not cAMP-dependent protein kinase.     

Evidence of protein kinase C involvement in phorbol diester-stimulated arachidonic acid release and prostaglandin synthesis

Many stimulators of prostaglandin production are thought to activate the Ca2+- and phospholipid-dependent protein kinase first described by Nishizuka and his colleagues (Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T., and Nishizuka, Y. (1979) J. Biol. Chem. 254, 3692-3695. In this paper we report evidence that the activation of protein kinase C caused by 12-O-tetradecanoylphorbol-13-acetate (TPA) is involved in the increased prostaglandin production induced by 12-O-tetradecanoylphorbol-13-acetate in Madin-Darby canine kidney (MDCK) cells. We have shown that TPA activates protein kinase C in MDCK cells with similar dose response curve as observed for TPA induction of arachidonic acid release in MDCK cells. Activation of protein kinase C was associated with increased phosphorylation of proteins of 40,000 and 48,000 daltons. We used two compounds (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OMe) and 1-(5-isoquinolinesulfonyl)piperazine) known to inhibit protein kinase C by different mechanisms to further examine if activation of protein kinase C was involved in the increased synthesis of prostaglandins in TPA-treated MDCK cells. We found that both compounds inhibited protein kinase C partially purified from MDCK cells and that ET-18-OMe inhibited the phosphorylation of proteins by protein kinase C in the intact cells. Addition of either compound during or after TPA treatment decreased both release of arachidonic acid from phospholipids and prostaglandin synthesis. Release of [3H]arachidonic acid from phosphatidylethanolamine in TPA-treated cells was blocked by ET-18-OMe or 1-(5-isoquinolinesulfonyl)piperazine addition. However, arachidonic acid release stimulated by A23187 is not blocked by Et-18-OMe. When assayed in vitro, treatment of cells with Et-18-OMe did not prevent the enhanced conversion of arachidonic acid into prostaglandins induced by pretreatment of cells with TPA. Our results suggest that the stimulation of phospholipase A2 activity by TPA occurs via activation of protein kinase C by TPA.     

1-Oleoyl-2-acetylglycerol promotes immunoglobulin production independent of cell proliferation in human peripheral blood mononuclear cells

1-Oleoyl-2-acetylglycerol (OAG) stimulated IgG and IgM production in a dose-dependent manner in human peripheral blood mononuclear cells (PBM) but not PBM proliferation. 12-O-Tetradecanoyl phorbol-13-acetate (TPA) did not stimulate Ig production. OAG did not stimulate an increase in IL-2 generation or IL-2 receptor expression. H-7, a protein kinase C blocker completely inhibited OAG-stimulated Ig production. The results suggest that OAG stimulation of Ig production is independent of cell proliferation; a generalized increase in T-cell activation does not appear to be necessary in the OAG stimulation of Ig production. Finally, PBMs respond differently to OAG and TPA although both are protein kinase C activators.     

Inhibition of IL-2 receptor induction and IL-2 production in the human leukemic cell line Jurkat by a novel peptide inhibitor of protein kinase C

We recently reported that the myristoylated peptide N-myristoyl-Lys-Arg-Thr-Leu-Arg (N-m-KRTLR) is a novel protein kinase C inhibitor. In this study, we investigated the biological effects of N-m-KRTLR using as an in vitro model the induction of the IL-2 receptor and IL-2 secretion by Jurkat cells in response to stimulation with 12-O tetradecanoylphorbol-13-acetate (TPA) plus phytohemagglutinin (PHA) and TPA plus OKT3 mAb. N-m-KRTLR significantly suppressed induction of the IL-2 receptor on the surface of the Jurkat cells by TPA plus either PHA or OKT3 mAb. Furthermore, N-m-KRTLR inhibited the production and release of IL-2 from cultured Jurkat cells stimulated with TPA plus either PHA or OKT3 mAb. Similarly, this peptide significantly inhibited the IL-2 production in normal human peripheral blood mononuclear cells in response to stimulation by TPA and PHA. In contrast, this peptide did not affect expression of the CD3 complex on the surface of the Jurkat cells either alone or in the presence of TPA or PHA. Furthermore, N-m-KRTLR did not interfere with the spontaneous proliferation of the Jurkat cells, and its effects on IL-2 secretion and IL-2 receptor expression in the Jurkat cells were evident without loss of cell viability. These results suggest that the novel protein kinase C inhibitor N-m-KRTLR may selectively inhibit certain activation pathways of Jurkat cells and indicate the usefulness of N-m-KRTLR in the analysis of discrete events in T cell activation.     

Okadaic acid induction of the urokinase-type plasminogen activator gene occurs independently of cAMP-dependent protein kinase and protein kinase C and is sensitive to protein synthesis inhibition.

Urokinase-type plasminogen activator (uPA) gene expression in LLC-PK1 cells is induced by activation of cAMP-dependent protein kinase (cAMP-PK) or protein kinase C (PK-C). To determine whether protein phosphatases can also modulate uPA gene expression, we tested okadaic acid, a potent specific inhibitor of protein phosphatases 1 and 2A, in the presence and absence of cAMP-PK and PK-C activators. Okadaic acid by itself induced uPA mRNA accumulation. This induction was strongly attenuated by the inhibition of protein synthesis. In contrast, the inhibition of protein synthesis enhanced induction by 8-bromo-cAMP and only delayed induction by 12-O-tetradecanoylphorbol-13-acetate (TPA). In addition, down-regulation of PK-C by chronic treatment with TPA did not abrogate the okadaic acid-dependent induction. These results provide evidence for a novel signal transduction pathway leading to gene regulation that involves protein phosphorylation but is independent of both cAMP-PK and PK-C.     

Inhibition of T-cell antigen receptor-mediated transmembrane signaling by protein kinase C activation.

The murine T-lymphoma cell line LBRM-33 is known to require synergistic signals delivered through the antigen receptor (Ti-CD3) complex, together with interleukin 1 (IL-1), for activation of IL-2 gene expression and IL-2 production. Although 12-O-tetradecanoylphorbol-13-acetate (TPA) was capable of replacing IL-1 as an activating stimulus under certain conditions, biologic studies indicated that TPA failed to synergize with Ti-CD3-dependent stimuli under conditions in which IL-1 was clearly active. Acute exposure to TPA and other active phorbol esters resulted in a concentration-dependent inhibition of the increases in phosphoinositide hydrolysis and intracellular free Ca2+ concentration stimulated by phytohemagglutinin or anti-Ti antibodies. TPA treatment induced no direct alteration of phospholipase C enzymatic activities in LBRM-33 cells. In contrast, both Ti-CD3 cross-linkage and TPA rapidly stimulated the phosphorylation of identical CD3 complex polypeptides, presumably via activation of protein kinase C. Exposure of LBRM-33 cells to TPA resulted in a time-dependent, partial down-regulation of surface Ti-CD3 expression. Thus, TPA treatment inhibited the responsiveness of LBRM-33 cells to Ti-CD3-dependent stimuli by inducing an early desensitization of Ti-CD3 receptors, followed by a decrease in membrane receptor expression. These studies indicate that phorbol esters deliver bidirectional signals that both inhibit Ti-CD3-dependent phosphoinositide hydrolysis and augment IL-2 production in LBRM-33 cells.     

Effect of macrophages on the translocation of protein kinase C in concanavalin A-stimulated lymphocytes

The concanavalin A (Con A)-induced proliferation of lymph node lymphocytes is dependent on the presence of macrophages. When lymphocytes are depleted of macrophages, Con A is no longer mitogenic. Either 12-0-tetradecanoylphorbol-13-acetate (TPA), interleukin 1 (IL1), or macrophages in combination with Con A can restore proliferation. To establish where the proliferation process is blocked in the absence of macrophages, an early step in the signalling pathway, the activation of protein kinase C, was examined. It was found that although Con A caused translocation of protein kinase C from the cytosol to the membrane of lymph node cells, when the lymph node cells were depleted of macrophages and exposed to Con A, this translocation of protein kinase C did not occur. Instead, protein kinase C activity decreased in the membrane fraction and increased in the cytosol. On the other hand, TPA caused translocation of protein kinase C (PKC) from the cytosol to the membrane regardless of the presence of macrophages. However, the macrophage product, IL1, alone or in combination with Con A did not cause translocation of protein kinase C. In a reconstitution experiment, in which lymph node cells were depleted of macrophages and then macrophages were added back, the addition of Con A again lead to translocation of protein kinase C from the cytosol to the membrane. This combination also restored cell proliferation. Therefore, the Con A induced PKC translocation in T lymphocytes is macrophage mediated. TPA overcomes the macrophage requirement by directly activating PKC, while IL1 appears to act at a different step in proliferation.     

Regulation fo protein kinase C activity by various lipids

Protein kinase C has recently attracted considerable attention because of its importance in the control of cell division, cell differentiation, and signal transduction across the cell membrane. The activity of this enzyme is altered by several lipids such as diacylglycerol, free fatty acids, lipoxins, gangliosides, and sulfatides. These lipids may interact with protein kinase C either directly or through calcium ions and produce their regulatory effect (activation or inhibition) on the activities of the enzymes phosphorylated by this kinase. These processes widen our perspective of the regulation of intercellular and intracelluular communication.Abbreviations used (PK-C) Protein kinase C - (cAMP-PK) cAMP dependent protein kinase - (DAG) diacylglycerol - (PtdSer) phosphatidylserine - (InsP ₃) inositol 1,4,5-trisphosphate - (PtdIns 4,5-P₂) inositol 4,5 bisphosphate - (FFA) free fatty acid - (MBP) myelin basic protein - (ATP) adenosine triphosphate - (GTP) guanine triphosphate - (TPA) 12-tetradecanoylphorbol-13-acetate - (EGF) epidermal growth factor - (PDGF) platelet derived growth factor - (NeuNAc) and N-acetylneuraminic acid     

Control of parathyroid hormone-degrading activity in the opossum kidney cell: possible involvement of protein kinase C

To clarify the possible role of protein kinase C in the control of parathyroid hormone (PTH)-degrading activity (PTHDA) in a PTH-responsive opossum kidney (OK) cell line, we investigated the effects of protein kinase C activators, 12-O-tetradecanoyl phorbol 13-acetate (TPA), 1-oleoyl-2-acetyl-glycerol (OAG), and 4 beta-phorbol 12, 13-didecanoate (4 beta-PDD). TPA, OAG, and 4 beta-PDD enhanced PTHDA in a dose-dependent fashion (10-50 ng/ml, 10-100 microgram/ml, and 10-50 nM, respectively), whereas 4 alpha-PDD, a non-activator of protein kinase C, did not affect it. HPLC analysis of TPA-treated samples revealed increase of all immunoreactive PTH fragments produced by OK cells. These findings suggested that activation of protein kinase C in OK cells would augment PTHDA in the cells.     

The role of protein kinase C in the inactivation of hepatic glycogen synthase by calcium-mobilizing agonists.

The regulation of glycogen synthase by Ca2+-mobilizing hormones was studied by using rat liver parenchymal cells in primary culture. Long-term exposure of hepatocytes to 4 beta-phorbol 12-myristate 13-acetate (TPA) resulted in a decrease in vasopressin or ATP inhibition of glycogen synthesis and glycogen synthase activity, without any change in the activation of glycogen phosphorylase. In contrast, treatment with TPA did not diminish the effects of glucagon, isoprenaline or A23187 on glycogen synthase or phosphorylase. TPA treatment for 18 h did not change specific [3H]vasopressin binding, but abolished protein kinase C activity in a concentration-dependent manner. The effects of TPA to decrease protein kinase C activity and to reverse the inactivation of glycogen synthase by vasopressin were well correlated and were mimicked by mezerein, but not by 4 alpha-phorbol. However, 1 microM-TPA totally inhibited protein kinase C activity, but reversed only 60% of the vasopressin effect on glycogen synthase. It is therefore concluded that Ca2+-mobilizing hormones inhibit glycogen synthase partly, but not wholly, through a mechanism involving protein kinase C.     

Activation of protein kinase C is not required for exocytosis from bovine adrenal chromaffin cells. The effects of protein kinase C(19-31), Ca/CaM kinase II(291-317), and staurosporine

We examined whether protein kinase C activation plays a modulatory or an obligatory role in exocytosis of catecholamines from chromaffin cells by using PKC(19-31) (a protein kinase C pseudosubstrate inhibitory peptide), Ca/CaM kinase II(291-317) (a calmodulin-binding peptide), and staurosporine. In permeabilized cells, PKC (19-31) inhibited the phorbol ester-mediated enhancement of Ca2(+)-dependent secretion as much as 90% but had no effect on Ca2(+)-dependent secretion in the absence of phorbol ester. The inhibition of the phorbol ester-induced enhancement of secretion by PKC (19-31) was correlated closely with the ability of the peptide to inhibit in situ phorbol ester-stimulated protein kinase C activity. PKC(19-31) also blocked 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced phosphorylation of numerous endogenous proteins in permeabilized cells but had no effect on Ca2(+)-stimulated phosphorylation of tyrosine hydroxylase. Ca/CaM kinase II(291-317), derived from the calmodulin binding region of Ca/calmodulin kinase II, had no effect on Ca2(+)-dependent secretion in the presence or absence of phorbol ester. The peptide completely blocked the Ca2(+)-dependent increase in tyrosine hydroxylase phosphorylation but had no effect on TPA-induced phosphorylation of endogenous proteins in permeabilized cells. To determine whether a long-lived protein kinase C substrate might be required for secretion, the lipophilic protein kinase inhibitor, staurosporine, was added to intact cells for 30 min before permeabilizing and measuring secretion. Staurosporine strongly inhibited the phorbol ester-mediated enhancement of Ca2(+)-dependent secretion. It caused a small inhibition of Ca2(+)-dependent secretion in the absence of phorbol ester which could not be readily attributed to inhibition of protein kinase C. Staurosporine also inhibited the phorbol ester-mediated enhancement of elevated K(+)-induced secretion from intact cells while it enhanced 45Ca2+ uptake. Staurosporine inhibited to a small extent secretion stimulated by elevated K+ in the absence of TPA. The data indicate that activation of protein kinase C is modulatory but not obligatory in the exocytotoxic pathway.     

Phosphatidylethanol biosynthesis in ethanol-exposed NG108-15 neuroblastoma X glioma hybrid cells. Evidence for activation of a phospholipase D phosphatidyl transferase activity by protein kinase C

12-O-Tetradecanoylphorbol-13-acetate (TPA) stimulates the release of free choline from intact NG108-15 cells into the medium, without affecting the release of phosphocholine (Liscovitch, M., Blusztajn, J.K., Freese, A., and Wurtman, R.J. (1987) Biochem. J. 241, 81-86). To test the hypothesis that this response reflects activation of cellular phospholipase D, via protein kinase C (Ca2+/phospholipid-dependent enzyme), I examined in NG108-15 cells the biosynthesis of the abnormal phospholipid phosphatidylethanol, produced by phospholipase D in the presence of ethanol by transphosphatidylation. Phosphatidylethanol production was quantitated by measuring the incorporation of phosphatidyl moieties (prelabeled metabolically with [3H]oleic acid) into phosphatidylethanol. The production of phosphatidylethanol in NG108-15 cells was virtually dependent on stimulation by TPA, in a time- and concentration-dependent manner (EC50 = 18 nM). The rate of 3H-phosphatidylethanol formation reached a peak after 10 min of incubation with TPA and declined gradually thereafter. The levels of 3H-phosphatidylethanol in TPA-treated cells were directly related to ethanol concentration in the physiologically attainable range (20-80 mM). Phosphatidylethanol production was activated only by phorbol derivatives that are activators of protein kinase C (i.e. TPA, 4 beta-phorbol-12,13-dibutyrate, and 4 beta-phorbol-12,13-didecanoate) and could be mimicked by a cell-permeant diacylglycerol, 1,2-dioctanoyl-sn-glycerol, in a nonadditive manner. The effect of TPA was inhibited by the protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (0.1 mM) by 70% but not by N-(2-guanidinoethyl)-5-isoquinolinesulfonamide. Phosphatidylethanol formation was completely abolished in cells in which protein kinase C was down-regulated by pretreatment of the cells with TPA. These results indicate that phosphatidylethanol biosynthesis in NG108-15 cells depends largely on activation of protein kinase C. In contrast to its effects on the release of free choline and on the accumulation of phosphatidylethanol, TPA did not affect the levels of phosphatidic acid in NG108-15 cells. It is therefore proposed that protein kinase C selectively activates the phosphatidyl transferase activity of phospholipase D, reflecting a signal termination mechanism which may be operative in phospholipase D-mediated signal transduction cascades.     

Phorbol esters induce both intracellular translocation and down-regulation of protein kinase C in MCF-7 cells

Exposure of MCF-7 human breast cancer cells to phorbol ester 12-O-tetradecanoyl-13-acetate (TPA) results in a complete inhibition of cell proliferation. We investigated the effects of TPA on protein kinase C activity when cells were exposed to phorbol ester for various lengths of time. TPA induces within 5 min a drastic dose-dependent decrease of the cytosolic protein kinase C activity. The enzyme apparently lost at the cytosolic level was only partially recovered in the particulate fraction. The apparent down-regulation of the translocated enzyme which was only 34% after 1 min reached 72% and 84% after respectively 10 min and 15 min. Moreover, when cells are treated with TPA for longer periods of time, the particulate protein kinase C activity continues to decrease, dropping below control after 1 hour. This progressive decline leads to an almost complete disappearance of protein kinase C activity in MCF-7 cells after 45 hours of TPA treatment. The apparent loss of protein kinase C activity upon short- as well as long-exposure of cells to TPA was not accompanied by a concomitant increase of Ca, PL-independent protein kinase activity. We discuss the implication of these biochemical events in the inhibition of cell proliferation with regard to the respective short- and long-term effects of TPA on protein kinase C activity.     

Inhibition of human skin fibroblast proliferation by histamine and phorbol esters is mediated by protein kinase C

The proliferation of human skin fibroblasts in culture was examined using a [3H]thymidine incorporation assay. Histamine inhibited thymidine incorporation with an IC50 of about 0.2 microM. This effect was blocked by the H1 receptor antagonist mepyramine but not by the H2 receptor antagonist cimetidine. Protein kinase C activators, including several phorbol esters and mezerine, also inhibited thymidine incorporation. The IC50 for beta-phorbol 12,13-didecanoate was less than 0.1 nM. The alpha-isomer of this compound was inactive. Long-term treatment of cells with the beta-isomer eliminated the ability of both histamine and phorbol ester to inhibit thymidine incorporation, presumably due to downregulation of protein kinase C. Our results suggest that histamine H1 receptors are linked to activation of protein kinase C and that activation of this enzyme leads to an inhibition of cell proliferation.     

Estrogen-induced genes,WISP-2 and pS2, respond divergently to protein kinase pathway

Recently, we identified WISP-2 (Wnt-1 inducible signaling pathway protein 2) as a novel estrogen-inducible gene in the MCF-7 human breast cancer cell line. In this study, we examined whether WISP-2 expression is modulated by PK activators. Treatment with protein kinase A (PKA) activators [cholera toxin plus 3-isobutyl-1-methylxanthine (CT/IBMX)] induced WISP-2 expression. CT/IBMX induced expression of the other estrogen-responsive gene, pS2, more dramatically than maximum stimulation by 17beta-estradiol (E2). Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), which directly stimulates protein kinase C (PKC) activity, completely prevented WISP-2 mRNA induction by E2, whereas it increased pS2 mRNA expression more dramatically than maximum stimulation by E2. Results of treatments with the protein synthesis inhibitor cycloheximide and the pure antiestrogen ICI182,780 suggest that these PK pathways modulate WISP-2 gene expression via different molecular mechanisms than those for pS2. Because TPA inhibits cell proliferation, we investigated whether WISP-2 induction was dependent on cell growth. Cells were treated with insulin-like growth factor-1 (IGF-1) or interleukin-1alpha (IL-1alpha) to stimulate or inhibit cell growth, respectively. These treatments had no effect on WISP-2 mRNA expression either alone or in combination with E2, suggesting that WISP-2 induction is independent of cell growth.