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PubMed1989

Phorbol ester modulation of integrin-mediated cell adhesion: a postreceptor event.

Chinese hamster ovary (CHO) suspension culture cells adhere readily to substrata coated with extracellular matrix proteins such as fibronectin, vitronectin, or laminin. In the case of fibronectin, it is known that adhesion is mediated by an integrin-type, cell surface fibronectin receptor (FnR). We demonstrate here that treatment of CHO cells with submicromolar concentrations of phorbol ester produces a remarkable increase in the ability of these cells to adhere to fibronectin. Both the rate of adhesion and the efficiency of adhesion are enhanced about four- to fivefold. Further, phorbol ester treatment renders the fibronectin-mediated adhesion process less sensitive to inhibitors, including GRGDSP peptide and PB1, a monoclonal anti-FnR antibody. By contrast, nonspecific adhesion processes, for example cell attachment to substrata coated with polylysine or concanavalin A, are not affected by phorbol ester treatment. Thus integrin-mediated adhesion is modulated by phorbol esters, but nonspecific adhesion is not. Neither the number of cell surface FnRs nor the receptor affinity, as measured by 125I-fibronectin and 125I-anti-FnR antibody binding, is altered by phorbol ester treatment. Thus, the effect of phorbol ester on cell adhesion seems to occur at a step subsequent to initial ligand-receptor binding events. Since phorbol ester is a potent activator of protein kinase C, we examined phosphorylation patterns in control and phorbol-treated cells. In immunoprecipitates of lysates from suspension culture cells, there was no evidence of phorbol ester-stimulated phosphorylation of FnR or of talin, a protein thought to interact with FnR. These results suggest that phorbol ester effects on fibronectin-dependent adhesion are not due to phosphorylation of the FnR itself but rather may be due to postreceptor events, possibly the phosphorylation of cytoskeletal proteins involved in integrin-mediated adhesion.

Animals↗
PubMed1989

Alterations of phospholipid metabolism by phorbol esters and fatty acids occur by different intracellular mechanisms in cultured glioma, neuroblastoma, and hybrid cells.

Differences between the influences of phorbol esters (such as 4 beta-12-O-tetradecanoylphorbol 13-acetate) and of fatty acids (such as oleic acid) on the synthesis and turnover of phosphatidylcholine (PtdCho) and other phospholipids have been studied in glioma (C6), neuroblastoma (N1E-115), and hybrid (NG108-15) cells in culture using [methyl-3H]choline, [32P]Pi, [1,2-14C]ethanolamine, or 1-14C-labeled fatty acids as lipid precursors. 100-500 microM oleic acid stimulated PtdCho synthesis 3- to 5-fold in all three cell lines, but had little influence on chase of choline label following a 24-h pulse. Phorbol ester (50-200 nM) stimulated PtdCho synthesis 1.5- to 3-fold in C6 cells, was without effect in N1E-115 cells, and had intermediate effects on NG108-15 cells. Phorbol ester stimulated both uptake of extracellular choline and synthesis of PtdCho, whereas fatty acid stimulated only synthesis. Release of radioactivity from 24-h pulse-labeled PtdCho to the medium was enhanced by phorbol ester in C6 cells. Incorporation of [32P]Pi, primarily into PtdCho, was stimulated, whereas utilization of [1,2-14C]ethanolamine or 1-14C-fatty acid was little altered by phorbol ester. C6 cells "down-regulated" with phorbol ester lost the stimulatory response of subsequent treatment with phorbol esters on PtdCho synthesis, but the response to fatty acid was enhanced. Fatty acid had little influence on the relative binding of phorbol ester or "translocation" of phorbol ester binding sites. Accordingly, metabolism of phospholipids in these cultured cells of neural origin is markedly influenced by cell type, phospholipid class, condition of incubation medium, and nature of stimulator. Phorbol esters and fatty acids appear to enhance phospholipid synthesis and turnover by distinct intracellular mechanisms.

Animals↗
PubMed1986

Up regulation of the phorbol ester receptor-protein kinase C in HL-60 variant cells.

The human promyelocytic leukemia cell line HL-60 can be induced to differentiate into macrophage-like cells by nanomolar concentrations of phorbol esters. A phorbol ester-resistant variant R1B6 obtained by culturing HL-60 cells with increasing concentrations of 12-O-tetradecanoylphorbol-13-acetate, is reversibly resistant. These cells have been growing continuously in the presence of phorbol esters for more than 1 yr, but when the phorbol ester is removed, the cells gradually regain their sensitivity and express characteristics of macrophage-like cells upon readdition of phorbol ester. The concentration of phorbol ester receptors in R1B6 is about one-third that in the parental HL-60 cells. The reversion of the variants to sensitivity to phorbol esters is associated with the up regulation of the cytosol and membrane phorbol ester receptors. When partially purified, these receptor populations contain protein kinase C activity, in support of the identity of protein kinase C and the receptor. This study demonstrates that a phenotypic change in a clonal cell population correlates with the up regulation of the phorbol ester receptor-calcium-activated phospholipid-dependent protein kinase. This variant cell line is a useful model for analyzing the relationship between phorbol ester binding and protein kinase C during differentiation of HL-60 cells.

Caenorhabditis elegans Proteins↗
PubMed1987

A correlation between phorbol diester-induced protein phosphorylation and superoxide anion generation in HL-60 cells during granulocytic maturation.

As HL-60 cells matured along the granulocytic pathway, phorbol diester-induced superoxide anion production was compared to phorbol diester-induced protein phosphorylation using an in vitro phosphorylation technique. Maturation was induced by 0, 2, 4, or 6 days incubation with dimethyl sulfoxide (Me2SO). In 0 day Me2SO HL-60 cells, phorbol 12-myristate 13-acetate induced phosphorylation of protein pp29 (Mr = 28,600) and to a lesser extent protein pp76 (Mr = 76,300). With increased time of Me2SO incubation, phorbol 12-myristate 13-acetate induced phosphorylation of pp212 (Mr = 211,800), pp134 (Mr = 134,200), and pp76, whereas the phosphorylation of pp29 did not change appreciably. In close agreement with this increase in protein phosphorylation was the observed increase in phorbol diester-induced superoxide anion formation. Morphological characterization of cells during Me2SO-induced differentiation reveals that these increases in phorbol diester responses are probably attributable to the proportional rise in metamyelocytes, band, and segmented neutrophils. A variety of phorbol diesters increased superoxide anion generation in HL-60 cells differentiated into granulocyte-like cells by 6-day incubation with Me2SO. The structure-activity relationship of these phorbol diester derivatives for protein phosphorylation was strongly correlated to their ability to increase superoxide anion generation. Thus, we propose that phorbol diester-induced phosphorylation of pp212, pp134, and pp76, but not pp29 may play a role in mediating the functional response of phorbol diester-induced superoxide anion generation in HL-60 cells differentiated into mature granulocyte-like cells.

Cell Differentiation↗
PubMed1985

Characterization of phorbol diester binding to isolated cardiac myocytes.

There are specified and saturable binding sites for [20-3H]phorbol-12,13-dibutyrate on enzymatically dissociated rat cardiac myocytes. At 37 degrees C, maximal binding occurs within 20 min, with a KD of 3.9 nM and Bmax of 0.275 pmol/mg. [3H]Phorbol dibutyrate binding is blocked by 12-O-tetradecanoyl phorbol-13-acetate but not by 4 alpha-phorbol or 4 alpha-phorbol-12,13-dibutyrate. Dibucaine, tetracaine, chlorpromazine, and phospholipase C lowered phorbol binding through a competitive mechanism. Similarly, unsaturated (but not saturated) diacylglycerols competed with [3H]phorbol dibutyrate for the binding site. There was a progressive decline in specific binding of phorbol diesters to cardiac myocytes which occurred primarily during the first 3 weeks of postnatal life. Cardiac phorbol diester receptors may mediate protein kinase C-dependent effects on important cellular functions such as Ca2+ transport.

Aging↗
PubMed1988

Phorbol esters potentiate glucocorticoid-induced cytotoxicity in CEM-C7 human T-leukemia cell line.

Phorbol ester tumor promoter, such as 12-O-tetradecanoylphorbol-13-acetate (TPA), is synergistic with dexamethasone to cause growth inhibition of CEM-C7 human T-leukemia cell line. A specific saturable binding component which may mediate the phorbol ester effects has been identified by using [20-(3)H]phorbol 12,13-dibutyrate in a whole cell binding assay. Saturation of the specific binding occurs at a concentration (approx. 100 nM) consistent with causing maximal cytotoxicity. Scatchard analysis of the binding after 15 min at 37 degrees C demonstrates a single class of binding sites. The number is 194,000 sites per cell. Other phorbol esters are also cytotoxic to CEM-C7 cell in the presence of 30 nM dexamethasone in an approximate proportion to their activity in competing for [20-(3)H]phorbol 12,13-dibutyrate binding. Phorbol 12, 13-didecanoate, 4-O-methyl PMA, 4-beta-phorbol do not compete for specific binding. The synergism of phorbol esters and dexamethasone on CEM-C7 cells is reversible by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine(H-7). However, by treating CEM-C7 cells with TPA for 48 h there is not any increase in the affinity or levels of glucocorticoid receptor. It is tentatively concluded that phorbol esters may play an important role linked to the glucocorticoid-induced growth inhibition in CEM-C7 human T-leukemic cell.

Binding, Competitive↗
PubMed1983

Modification of glycogen synthase activity in isolated rat hepatocytes by tumor-promoting phorbol esters: evidence for differential regulation of glycogen synthase and phosphorylase.

Glycogen synthase (UDPglucose:glycogen 4-alpha-D-glucosyltransferase, EC 2.4.1.11), in isolated rat hepatocytes, has been identified as a novel intracellular target for tumor-promoting phorbol esters such as phorbol 12-tetradecanoate 13-acetate (TPA). Exposure of hepatocytes to TPA resulted in a 50% decrease in the activity ratio of glycogen synthase without/with glucose 6-phosphate. The inactivation was dose dependent and was half-maximal at a TPA concentration of approximately 16 nM (10 ng/ml). Phorbol and phorbol 13-monoacetate, ineffective tumor promoters, had little influence on glycogen synthase activity. Other biologically active diesters, phorbol 12,13-didecanoate, phorbol 12,13-dibutyrate, and phorbol 12,13-dibenzoate, caused significant inactivation of glycogen synthase. Glycogen phosphorylase (1,4-alpha-D-glucan:orthophosphate alpha-D-glucosyltransferase, EC 2.4.1.1) activity, however, was unaffected by TPA or any of the tumor-promoting phorbol esters mentioned above. It is concluded that phorbol diesters can interact in the regulatory pathway for glycogen synthase, but the lack of effect on phosphorylase argues that distinct mechanisms can operate for the control of glycogen synthase and phosphorylase.

Animals↗
PubMed2004

Activation of arylalkylamine N-acetyltransferase by phorbol esters in bovine pinealocytes suggests a novel regulatory pathway in melatonin synthesis.

In all mammalian species investigated, noradrenaline activates a beta-adrenoceptor/cAMP/protein kinase A-dependent mechanism to switch on arylalkylamine N-acetyltransferase and melatonin biosynthesis in the pineal gland. Other compounds which are known to influence the melatonin-generating system are phorbol esters. The effect of phorbol esters on regulation of melatonin synthesis has been mainly investigated in rat pinealocytes. In these cells, phorbol esters do not increase cAMP levels and arylalkylamine N-acetyltransferase on their own; however, phorbol esters potentiate the effects on cAMP and AANAT activity induced upon beta-adrenoceptor stimulation. In the present study, we investigated the effect of phorbol esters on the regulation of melatonin synthesis in bovine pinealocytes. We show that, in these cells, the phorbol esters 4beta-phorbol 12-myristate 13-acetate (PMA) or phorbol 12,13-dibutyrate have a direct stimulatory effect and induced 4-10-fold increases in AANAT protein levels, AANAT activity and melatonin production. The extent of these effects was similar to those induced by noradrenaline. Notably, responses to PMA were not accompanied by increases in cAMP levels. Northern blot analysis showed that Aanat mRNA levels did not change upon PMA treatment indicating that phorbol esters control AANAT at a post-transcriptional level. The effects on AANAT and melatonin production were reduced by use of protein kinase C inhibitors, but not by blockade of the cyclic AMP/protein kinase A pathway. Our results point towards a novel mechanism in the regulation of melatonin production that is cAMP-independent and involves protein kinase C. The study is of particular interest because regulation of melatonin biosynthesis in bovines may resemble that in primates more closely than that in rodents.

Animals↗
PubMed1987

Inhibition by bryostatin 1 of the phorbol ester-induced blockage of differentiation in hexamethylene bisacetamide-treated Friend erythroleukemia cells.

Phorbol esters inhibit chemically induced differentiation in Friend erythroleukemia cells. This study examines the effect of the macrocyclic lactone bryostatin 1 on phorbol ester responses in a Friend erythroleukemia cell clone, PS 7. In several biological systems, bryostatin 1 was reported to mimic phorbol ester action, including activation of protein kinase C, but in HL-60 cells it blocked phorbol ester-induced differentiation. We report here that bryostatin 1 blocks phorbol ester action in Friend cells (clone PS 7), a second differentiating system. In this system, in contrast to HL-60 cells, the phorbol esters inhibit rather than induce differentiation. Bryostatin 1 restores the differentiation response [50% effective dose, 15 +/- 3.5 nM (SEM)] as well as blocks a second phorbol ester effect, induction of cellular adherence. The inhibition of erythroid differentiation by dexamethasone, a nonphorbol compound whose action presumably is not protein kinase C mediated, is unaffected by bryostatin 1. Although bryostatin 1 inhibits [3H]phorbol 12,13-dibutyrate binding in intact Friend erythroleukemia cell clone PS 7, the mechanism for the antagonism of phorbol ester action by bryostatin 1 in Friend cells cannot be explained by simple competition at the binding site.

Acetamides↗
PubMed2000

Eyes wide shut: protein kinase C isozymes are not the only receptors for the phorbol ester tumor promoters.

In addition to the well-characterized interaction with classical and novel protein kinase C (PKC) isozymes, the phorbol ester tumor promoters bind to other receptors lacking kinase activity. Among these novel phorbol ester receptors, two families of proteins may play a role in the regulation of cell growth and malignant transformation: chimaerins and ras guanyl-releasing protein (ras-GRP). These proteins possess a single copy of the C1 domain that is involved in binding of phorbol esters and the lipid second messenger diacylglycerol. Four isoforms of chimaerins (alpha1-, alpha2-, beta1-, and beta2-chimaerins) have been isolated to-date, all of them possessing GTPase-activating protein activity for Rac, a small GTP-binding protein that controls actin cytoskeleton organization, cell-cycle progression, adhesion, and migration. Ras-GRP is a guanine nucleotide exchange factor for ras and promotes malignant transformation in fibroblasts in a phorbol ester-dependent manner. The C1 domain in Ras-GRP may, therefore, have a dominant role in Ras-GRP activation and is essential for phorbol ester-dependent activation of downstream effectors of ras, i.e., the mitogen-activated protein kinase cascade. Thus, a novel concept emerges in which phorbol esters may exert cellular responses through pathways not involving phorbol ester-responsive PKC isozymes. The discovery of "nonPKC" phorbol ester receptors adds an additional level of complexity to the understanding of phorbol ester effects and the molecular mechanisms of carcinogenesis.

Animals↗
PubMed1989

Activation of the Na+/H+ exchanger by phorbol ester and osmotic shock is dependent on the degree of neutrophilic maturation.

Activation of neutrophils leading to superoxide production is accompanied by cytoplasmic alkalinization, which results from stimulation of the Na+/H+ exchanger. Since the exchanger undergoes permanent alterations during neutrophilic maturation of HL-60 cells (Costa-Casnellie et al.: Journal of Biological Chemistry 263:11851-11855, 1988), we investigated whether its response to external stimuli such as phorbol esters or osmotic shock also was modified during cell maturation. Mature HL-60 cells produce superoxide in response to active phorbol esters, whereas immature HL-60 cells do not. Stimulation of the exchanger by active phorbol esters (phorbol 12-myristate 13-acetate or phorbol 12,13-dibutyrate) was observed in mature neutrophilic HL-60 cells but not in their immature counterparts. Inactive 4-alpha phorbol had no effect in either cell population. Compound H7 inhibited phorbol ester activation by 65%. In mature neutrophilic cells activation of the exchanger by phorbol esters caused two novel changes of its properties: 1) its apparent Km for Na+ transport increased 2-fold; 2) its Vmax increased 2.6-fold. Phorbol esters also caused a shift in pH dependence of activation similar to that induced in other cells. Osmotic shock, a different method known to activate the exchanger of other cells, induced activation in mature neutrophilic cells but not in immature cells. Thus, the response of the exchanger to external stimuli is affected by alterations occurring in association with cell maturation.

Carrier Proteins↗
PubMed1986

Phorbol diester treatment promotes enhanced adenylate cyclase activity in frog erythrocytes.

Incubation of intact frog erythrocytes with 12-O-tetradecanoyl phorbol-13-acetate (TPA), a tumor-promoting phorbol diester which activates protein kinase C, results in an approximate two- to threefold increase in subsequently tested beta-adrenergic agonist-stimulated adenylate cyclase activity. This increase is due to an elevation in the Vmax of the enzyme rather than to a change in affinity for the agonist. TPA treatment of frog erythrocytes does not alter the affinity (KD) or the binding capacity (Bmax) for the beta-adrenergic antagonist [125I]cyanopindolol. In addition, agonist/[125I]cyanopindolol competition curves are not affected by TPA pretreatment nor is their sensitivity to guanine nucleotides. Incubation of frog erythrocyte membranes alone with TPA does not promote sensitization or activation of adenylate cyclase activity. Pretreatment of intact frog erythrocytes with TPA also produces approximately two- to threefold increases in basal, guanine nucleotide-, prostaglandin E1-, forskolin-, NaF-, and MnCl2-stimulated adenylate cyclase activities in frog erythrocyte membranes. This enhancement of adenylate cyclase activity by TPA is induced rapidly (t1/2 approximately equal to 5 min) and with an EC50 of about 10(-7) to 10(-6) M. Other tumor-promoting phorbol diesters or phorbol diester-like compounds including 4 beta-phorbol 12,13-dibutyrate, 4 beta-phorbol 12,13-didecanoate, and mezerein are effective in promoting enhanced adenylate cyclase activity. In contrast, phorbols such as 4 beta-phorbol, 4 alpha-phorbol 12,13-didecanoate, and 4-O-methylphorbol 12-myristate 13-acetate, which are inactive in tumor promotion and which do not activate protein kinase C, do not affect frog erythrocyte adenylate cyclase activity. These data are suggestive of a protein kinase C-mediated phosphorylation of one of the adenylate cyclase components that is distal to the receptor, i.e., the nucleotide regulatory and/or catalytic components.

Adenylyl Cyclases↗
PubMed1992

Glucose transport by cultured human fibroblasts: regulation by phorbol esters and insulin.

The regulation of 3-O-methyl-D-glucose (OMG) uptake by insulin and phorbol esters was studied in cultured human skin fibroblasts. Insulin rapidly stimulated OMG uptake through a mechanism independent of new protein synthesis. Maximal insulin effect was reached in 30 min and remained constant up to 12 h. The protein kinase C activators 12-O-tetradecanoyl phorbol 13-acetate (TPA) and phorbol 12,13-dibutyrate (PdBU) promoted an initial rapid stimulation followed by a secondary long-term rise of OMG influx. This latter effect of phorbol esters on OMG influx began after 1 h, reached a maximum in 12-15 h, and was prevented by the simultaneous addition of protein synthesis inhibitors, suggesting that phorbol esters increased the synthesis of new glucose transporters. In accord with this interpretation, phorbol esters, but not insulin, increased mRNA levels for two distinct glucose transporters (GLUT1 and GLUT3) in human fibroblasts. Both the rapid and the long-term effects of phorbol esters on OMG influx were dose-dependent and half-maximal stimulations occurred at 15 nM for both PdBU and TPA. Kinetic analysis of OMG uptake indicated that both effects of phorbol esters were associated with an increase in the Vmax of the transport process, with no significant changes of the Km (4-6 mM). These results suggest that, in human fibroblasts, phorbol esters, unlike insulin, produce a long-term stimulation of OMG uptake, which is dependent upon protein synthesis and is associated with increased levels of GLUT1 and GLUT3 mRNA.

3-O-Methylglucose↗
PubMed1988

Divalent cation requirements for mounting a respiratory burst in response to phorbol diesters by macrophages from SENCAR and C57BL/6 mice.

Oxygen radicals are thought to play an important role in the promotion phase of carcinogenesis and the action of phorbol esters. Inflammatory cells are an abundant source of reactive oxygen intermediates (ROI) in the body and release large quantities of ROI when exposed to phorbol esters. Both protein kinase C (PKC), the receptor for phorbol esters, and the NADPH oxidase which generates ROI are Ca2+- and Mg2+-dependent. We investigated the requirements for Ca2+ and Mg2+ of macrophages from strains of mice sensitive and resistant to the promotion of tumors by phorbol esters. Macrophages from SENCAR mice, which are sensitive to phorbol ester promotion, required much lower levels of Ca2+ or Mg2+ to mount a full respiratory burst, as measured by the release of H2O2 in response to phorbol ester stimulation, than macrophages from C57BL/6 mice, which are resistant to promotion by phorbol esters. Conversely, when the particulate stimulus zymosan was used, there was little difference between macrophages from the two strains in requirements for Ca2+ and Mg2+ to release H2O2. Lowering the concentration of either cation in the absence of the other was more inhibitory than in the presence of the other cation. The studies demonstrate that differences in sensitivity to divalent cations by macrophages from these two strains is selective for phorbol ester stimulation and that lower requirements for Ca2+ and Mg2+ for ROI release correlates with sensitivity to the promotion of tumors by phorbol esters.

Animals↗
PubMed1991

Phorbol esters induce oscillatory contractions of intestinal smooth muscles.

The actions of tumor-promoting phorbol esters in smooth muscle excitation-contraction coupling were studied in isolated guinea pig ileum in the presence of various contractile agents. Muscarinic agonists, histamine and bradykinin elicited an initial transient phasic contraction and a subsequent sustained tonic contraction in guinea pig ileum. The Ca2+ channel antagonist nifedipine selectively inhibited the tonic contraction. Phorbol esters, protein kinase C activators, induced immediate muscle relaxation followed by oscillatory contractions when added during the tonic phase of contraction. Phorbol esters, when added in advance, slightly altered the ligand-induced phasic contraction but converted tonic contractions into oscillatory spikes. The amplitude, frequency and shape of the oscillation induced by phorbol esters were dependent upon the dose of phorbol ester: amplitude was increased and frequency was decreased by increasing the doses of phorbol ester. In contrast, the phorbol ester potentiated the tonic contraction induced by high potassium chloride with little effect on the phasic component. It also sensitized the muscles to Bay K 8644. Bay K 8644, which was ineffective in stimulating muscle contraction at 1 nM, became a very effective stimulator in the presence of the phorbol ester. All of these phorbol ester-induced potentiations and oscillations were sensitive to inhibition by staurosporine or nifedipine. These data suggest that in guinea pig ileum, protein kinase C plays a positive regulatory role in Ca2+ channel activation and promotes a complex regulatory effect on Ca(2+)-mobilizing ligand-stimulated Ca2+ channel activity, which results in oscillatory contractile responses to carbachol, methacholine, histamine and bradykinin.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed2000

Pharmacology of the receptors for the phorbol ester tumor promoters: multiple receptors with different biochemical properties.

The phorbol ester tumor promoters and related analogs are widely used as potent activators of protein kinase C (PKC). The phorbol esters mimic the action of the lipid second messenger diacylglycerol (DAG). The aim of this commentary is to highlight a series of important and controversial concepts in the pharmacology and regulation of phorbol ester receptors. First, phorbol ester analogs have marked differences in their biological properties. This may be related to a differential regulation of PKC isozymes by distinct analogs. Moreover, it seems that marked differences exist in the ligand recognition properties of the C1 domains, the phorbol ester/DAG binding sites in PKC isozymes. Second, an emerging theme that we discuss here is that phorbol esters also target receptors unrelated to PKC isozymes, a concept that has been largely ignored. These novel receptors lacking kinase activity include chimaerins (a family of Rac-GTPase-activating proteins), RasGRP (a Ras exchange factor), and Unc-13/Munc-13 (a family of proteins involved in exocytosis). Unlike the classical and novel PKCs, these "non-kinase" phorbol ester receptors possess a single copy of the C1 domain. Interestingly, each receptor class has unique pharmacological properties and biochemical regulation. Lastly, it is well established that phorbol esters and related analogs can translocate each receptor to different intracellular compartments. The differential pharmacological properties of the phorbol ester receptors can be exploited to generate specific agonists and antagonists that will be helpful tools to dissect their cellular function.

Animals↗
PubMed1993

Enhancement of phorbol ester induced cell aggregation after alterations in asparagine-linked oligosaccharides.

Human erythroleukemia (K-562) cells grown in the presence of phorbol 12,13-dibutyrate formed aggregates of cells not seen in untreated control cultures. Furthermore, the proportion of cells in aggregates and the size of the aggregates both increased dramatically in cultures treated with both phorbol ester and kifunensine, an inhibitor of asparagine-linked oligosaccharide processing. Relative to control cells, phorbol ester treated cells exhibited a greater proportion of N-linked oligosaccharides of the complex-type. Kifunensine prevented this change and caused an accumulation of Man9GlcNAc2. The enhanced aggregation of cells treated with phorbol ester plus kifunensine depended on phorbol ester concentration and was blocked by inhibitors of protein kinase C (H7, sphinganine and sangivamycin). In flow cytometry analysis, phorbol ester treated K-562 cells showed an increase in CD44, a glycoprotein involved in cell adhesion. Moreover, monoclonal antibody to CD44 augmented reaggregation of phorbol ester treated cells. The results implicate phorbol ester induction of CD44 in aggregation of K-562 cells and demonstrate that the presence of high mannose-type asparagine-linked oligosaccharides on cell glycoproteins correlates with increased aggregation of phorbol ester treated cells.

Alkaloids↗
PubMed1999

Prevention of neuronal apoptosis by phorbol ester-induced activation of protein kinase C: blockade of p38 mitogen-activated protein kinase.

Consistent with previous studies on cell lines and non-neuronal cells, specific inhibitors of protein kinase C induced mouse primary cultured neocortical neurons to undergo apoptosis. To examine the complementary hypothesis that activating protein kinase C would attenuate neuronal apoptosis, the cultures were exposed for 1 h to phorbol-12-myristate-13-acetate, which activated protein kinase C as evidenced by downstream enhancement of the mitogen-activated protein kinase pathway. Exposure to phorbol-12-myristate-13-acetate, or another active phorbol ester, phorbol-12,13-didecanoate, but not to the inactive ester, 4alpha-phorbol-12,13-didecanoate, markedly attenuated neuronal apoptosis induced by serum deprivation. Phorbol-12-myristate-13-acetate also attenuated neuronal apoptosis induced by exposure to beta-amyloid peptide 1-42, or oxygen-glucose deprivation in the presence of glutamate receptor antagonists. The neuroprotective effects of phorbol-12-myristate-13-acetate were blocked by brief (non-toxic) concurrent exposure to the specific protein kinase C inhibitors, but not by a specific mitogen-activated protein kinase 1 inhibitor. Phorbol-12-myristate-13-acetate blocked the induction of p38 mitogen-activated protein kinase activity and specific inhibition of this kinase by SB 203580 attenuated serum deprivation-induced apoptosis. c-Jun N-terminal kinase 1 activity was high at rest and not modified by phorbol-12-myristate-13-acetate treatment. These data strengthen the idea that protein kinase C is a key modulator of several forms of central neuronal apoptosis, in part acting through inhibition of p38 mitogen-activated protein kinase regulated pathways.

Amyloid beta-Peptides↗