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.     

The phorbol ester TPA increases the affinity of exocytosis for calcium in ‘leaky’ adrenal medullary cells

Exposure of ‘leaky’ bovine adrenal medullary cells to the phorbol ester TPA causes a shift in the calcium-activation curve to lower calcium concentrations without altering the levels of secretion at the extremes of the activation curve. These results are consistent with a role for protein kinase C in exocytosis.     

Effect of RNA and protein synthesis inhibitors on the release of inflammatory mediators by macrophages responding to phorbol myristate acetate

The interaction of phorbol myristate acetate with resident populations of mouse peritoneal macrophages causes an increased release of arachidonic acid followed by increased synthesis and secretion of prostaglandin E₂ and 6-keto-prostaglandin F_1α. In addition, phorbol myristate acetate causes the selective release of lysosomal acid hydrolases from resident and elicited macrophages. These effects of phorbol myristate acetate on macrophages do not cause lactate dehydrogenase to leak into the culture media. The phorbol myristate acetate-induced release of arachidonic acid and increased synthesis and secretion of prostaglandins by macrophages can be inhibited by RNA and protein synthesis inhibitors, whereas the release of lysosomal hydrolases is unaffected. 0.1 μg/ml actinomycin D blocked the increased prostaglandin production due to this inflammatory agent by more than 80%, and 3 μg/ml cycloheximide blocked prostaglandin production by 78%. Similar results with these metabolic inhibitors were found with another stimulator of prostaglandin production, zymosan. However, these inhibitors do not interfere with lysosomal hydrolase releases caused by zymosan or phorbol myristate acetate. It appears that one of the results of the interaction of macrophages with inflammatory stimuli is the synthesis of a rapidly turning-over protein which regulates the production of prostaglandins. It is also clear that the secretion of prostaglandins and lysosomal hydrolyses are independently regulated.     

Phorbol ester (TPA) potentiates noradrenaline and acetylcholine-evoked amylase secretion in the rat pancreas

The effect of the phorbol ester 12-O-tetradeconyl-phorbol-13-acetate (TPA) on noradrenaline (NA) and acetylcholine (ACh)-evoked amylase secretion in isolated segments of rat pancreas was investigated. TPA alone evoked a relatively small increase in amylase output. However, when combined with either noradrenaline or ACh, the phorbol ester markedly enhanced the secretagogue-induced amylase secretion. These effects were dose related. TPA also enhanced the amylase secretion evoked by either the Ca²⁺ ionophore A23187 or dibutyryl cyclic AMP. This potentiation by TPA of noradrenaline, ACh, Ca²⁺ ionophore A23187 and dibutyryl cyclic AMP-evoked amylase output may suggest the existence of a third pathway controlling enzyme secretion in the pancreas.     

Phorbol ester induces intracellular translocation of phospholipid/Ca2+-dependent protein kinase and stimulates amylase secretion in isolated pancreatic acini

Treatment of intact rat pancreatic acini with phorbol ester (12-O-tetradecanoyl-phorbol-13-acetate, TPA) resulted in a time- and concentration-dependent translocation of phospholipid/Ca2+-dependent protein kinase (PL/Ca-PK) from the soluble fraction. Redistribution of PL/Ca-PK was concurrent with stimulation of amylase secretion by TPA-treated acini. Polymyxin B, a potent and selective inhibitor of PL/Ca-PK completely inhibited TPA-induced amylase secretion. These findings are consistent with a role for PL/Ca-PK in the regulation of pancreatic exocrine secretion.     

Bimodal effect of phorbol ester on B cell activation. Implication for the role of protein kinase C.

The role of protein kinase C PKC in B cell activation is controversial. These studies were undertaken to determine whether protein kinase C has a stimulatory or inhibitory role in B cell activation. We found that treatment of B cells for a short period of time (30 min) with the PKC activator phorbol 12,13-dibutyrate (PDBU) primed the cells for enhanced proliferative responses to anti-immunoglobulin (anti-Ig) antibody whereas treatment for a longer period of time (3 h or more) resulted in suppression of proliferation. The enhanced proliferative response to treatment of B cells with PDBU for short periods of time was associated with inhibition of anti-Ig-stimulated increases in phosphatidyl 4,5-bisphosphate (PIP2) hydrolysis and inhibition of increases in [Ca2+]i, indicating that activation of PKC per se might be sufficient for enhancing B cell activation. The time-dependent effect of phorbol esters on the inhibition of B cell proliferation was found to be closely correlated with the kinetics of disappearance of PKC as measured by Western blot and by enzymatic activity but not with inhibition of [Ca2+]i and PIP2. These data demonstrate a bimodal time-dependent effect of PDBU on B cell activation and suggest that (a) the inhibitory effect of phorbol ester on anti-Ig-induced proliferation may be due to the disappearance of PKC rather than to the inhibition of PIP2 and Ca2+; and (b) the early activation of PKC is a stimulatory rather than an inhibitory signal in the induction of B lymphocyte proliferation by anti-Ig.     

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.     

Effect of calmidazolium and phorbol ester on catecholamine secretion from adrenal chromaffin cells

Carbamylcholine-stimulated catecholamine release from adrenal chromaffin cells was completely inhibited by pretreatment of the cells for 10 min with 1 μM calmidazolium. Catecholamine release due to 55 mM K⁺ and ionophore A23187 was also inhibited by calmidazolium but less effectively than release due to carbamylcholine. Inhibition of release appeared to be due to an effect of calmidazolium on a step distal to Ca²⁺ entry, since the carbamylcholine-stimulated rise in the concentration of intracellular free calcium, monitored using quin-2, was unaffected by calmidazolium. The possibility was considered that calmidazolium inhibited secretion through an effect on protein kinase C rather than calmodulin. However, the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), had no demonstrable effect on catecholamine release, arguing against a significant role for protein kinase C in secretion from adrenal chromaffin cells. These results give further support to the notion that calmodulin plays a role in the secretory process in chromaffin cells.     

The effects of phorbol ester on mouse blastomeres: a role for protein kinase C in compaction?

The effects of phorbol myristate acetate (PMA) and other activators of protein kinase C on the cytoskeletal organization of mouse oocytes and early embryos have been examined. The effects observed depended on the developmental stage on exposure to PMA. PMA had little effect on the cytoskeletal or microvillous organization of unfertilized oocytes. Interphase cells from embryos prior to compaction showed limited disruption and loss of microvilli when exposed to PMA and foci of polymerized actin remained visible in the cytocortex of embryos up to the early 8-cell stage. When compacted late 8-cell embryos were exposed to PMA, most microvilli were lost and little polymerized actin remained in the cytocortex. PMA also caused loss of microtubules from compact 8-cell embryos under some experimental conditions. Intercellular flattening was both prevented and reversed. The relevance of these observations to the rearrangement of cell-cell contacts and cytoskeletal organization seen during compaction at the 8-cell stage is discussed and a possible role for protein kinase C in the generation of cell polarity proposed.     

Long-term phorbol ester treatment down-regulates protein kinase C and sensitizes the phosphoinositide signaling pathway to hormone and growth factor stimulation. Evidence for a role of protein kinase C in agonist-induced desensitization

Exposure of a nontransformed, continuous line of epithelial cells derived from rat liver (WB cells) to epidermal growth factor, angiotensin II, [Arg8]vasopressin, and epinephrine resulted in rapid accumulation of the inositol phosphates (InsP) InsP1, InsP2, and InsP3. Although short-term (5-60 min) pretreatment of WB cells with the phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) markedly attenuated InsP accumulation in response to all agonists, the inhibitory effects on the InsP response were lost after 2 h incubation with PMA; and, with extended (6-24 h) preincubation, a time-dependent potentiation of the InsP response to angiotensin II, epidermal growth factor and [Arg8]vasopressin was observed. The InsP response during a 15-min challenge with angiotensin II in cells pretreated for 18 h with 600 nM and 10 microM PMA was increased by 2-3-fold and 4-6-fold, respectively. Long-term (18 h) treatment with 600 nM and 10 microM PMA caused a similar 90-100% loss of measurable Ca2+/phospholipid-dependent enzyme (protein kinase C) activity in cytosolic and soluble particulate fractions. The effects of long-term PMA pretreatment do not represent a general enhancement of hormone responsiveness since the InsP response to epinephrine was not affected. In control cells, the InsP response to angiotensin II and epinephrine desensitized very rapidly. Long-term pretreatment with PMA greatly reduced the contribution of agonist-induced desensitization to the angiotensin II response; in contrast, the extent of desensitization occurring during incubation of WB cells with epinephrine was unaltered by long-term treatment with PMA suggesting that an additional mechanism may be involved in alpha 1-adrenergic receptor desensitization. No PMA-induced change in resting levels of [3H]phosphoinositides or the metabolism of exogenous [3H]inositol 1,4,5-trisphosphate by WB homogenates occurred. Stimulation of InsP formation in intact cells by NaF and activation of phospholipase C by GTP gamma S in membranes both were unaltered by short-term or long-term PMA pretreatment. These data are consistent with the idea that following long-term treatment of WB cells with PMA, the occurrence of agonist-induced desensitization of receptors linked to the phosphoinositide/Ca2+ signaling system is reduced, apparently at least in part due to the loss of contribution of a negative feedback regulatory role of protein kinase C.     

Bombesin and phorbol ester stimulate phosphatidylcholine hydrolysis by phospholipase C: evidence for a role of protein kinase C

Bombesin caused a marked stimulation of 32Pi into phosphatidylinositol (PI), with no apparent lag, and into phosphatidylcholine (PC), after a lag of about 20 min. Stimulation was blocked by the bombesin receptor antagonist, [D-Arg1, D-Pro2, D-Trp7,9, Leu11] substance P, indicating that the effects on both PI and PC were mediated through the same receptor. The tumor-promoting phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) and dioctanoylglycerol (diC8) both directly activate protein kinase C and in this report were shown to stimulate 32Pi incorporation into PC but not into Pl. In addition, TPA stimulated the release of [3H]choline and [3H]phosphocholine and the accumulation of [3H]diacyglycerol from prelabelled cells. These results strongly suggest that TPA activates a phospholipase C specific for PC. Pretreatment of cells with phorbol-12, 13-dibutyrate (PDBu) for 24 h depleted cellular protein kinase C activity and inhibited the ability of TPA to induce these effects suggesting a direct involvement of protein kinase C. Similarly the bombesin stimulation of 32Pi into PC and of [3H]choline and [3H]phosphocholine release was inhibited by PDBu pretreatment. DiC8 and, to a lesser extent, TPA stimulated the translocation of CTP:phosphocholine cytidylytransferase from the cytosolic to the particulate fraction. DiC8 also stimulated this translocation in cells depleted of protein kinase C. It was concluded that both bombesin and TPA activated protein kinase C leading to activation of a phospholipase C specific for PC.     

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.     

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.     

Protein kinase C isoforms in pituitary cells displaying differential sensitivity to phorbol ester

Investigations with protein kinase C (PKC) isoform-specific antisera, revealed distinct profiles of PKC isoform content amongst pituitary tissues. Western analysis revealed the and isoforms of PKC are present in rat anterior and posterior pituitary tissue as well as in the GH₃ somatomammotrophic cell line. AtT-20/D16-V corticotrophic and T3-1 gonadotrophic murine cell lines contained no PKC-. The or isoforms were undetected in any pituitary tissue. PKC activity measurements revealed Ca²⁺-independent PKCs in T3-1 and GH₃ cells which were more sensitive to activation by phorbol-dibutyrate (PDBu) than the corresponding PKC activity found in COS cells. However, Ca²⁺-dependent PKC activities were of similar sensitivity to PDBu in GH₃, T3-1 and COS cells, indicating that functional differences observed in PDBu-sensitivity in these cells may be due to differential activation of Ca²⁺-independent PKC isoforms. Moreover, substrate-specificity of these PKCs were also compared indicating that the amount of Ca²⁺-dependency of the observed PKC activity from the same pituitary tissue is dependent upon the substrate utilized by the PKC isotypes present. These findings explain differential sensitivities of PKC-mediated actions that have previously been observed in a range of pituitary cells. (Mol Cell Biochem 000-000, 1999)     

Effects of phorbol ester on immunoreactive protein kinase C,insulin binding,and glucose uptake in astrocytic glial and neuronal cells from the brain

Phorbol esters, potent stimulators of protein kinase C (PKC), stimulate [³H]2-deoxy-d-glucose (dGlc) uptake and [¹²⁵I] insulin binding in cultured glial cells but not neuronal cells from neonatal rat brains. Using an antibody to the and forms of PKC we have demonstrated that both neuronal and glial cells contain an immunoactive PKC of Mr 80 kD, although the PKC level in neurons is greater than 4-fold that in glia. The majority of immunoactive PKC (63%) is cytosolic in glial cells although the reverse is true in neuronal cells, in which 88% of the PKC is membrane-bound in the basal state. The most potent phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulates a redistribution of this enzyme in neuronal and glial cells. The TPA-stimulated translocation of PKC from cytosol to membrane precedes TPA's effecs of [³H]dGlc uptake and insulin binding in glial cells.     

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.     

Insulin-dependent alterations of phorbol ester binding to adipocyte subcellular constituents. Evidence for the involvement of protein kinase C in insulin action

The binding of tritiated phorbol-12,13-dibutyrate (3H-PBu2) was employed to estimate the mass of protein kinase C associated with plasma membranes and cytosol isolated from untreated and insulin-treated adipocytes. Binding of 3H-PBu2 to both plasma membranes and cytosol was rapid, achieving a steady state within minutes. Treatment of cells with physiological concentration of insulin (0.67 nM) caused a 42% increase (from 0.92 +/- 0.08 to 1.30 +/- 0.12 pmol 3H-PBu2/mg protein, p less than 0.0001) and a 27% decrease (from 0.41 +/- 0.07 to 0.30 +/- 0.05 pmol 3H-PBu2/mg protein, p less than 0.020) in phorbol ester bound to cytosol and plasma membranes, respectively. The half-maximal concentrations of unlabelled PBu2 needed to displace 3H-PBu2 bound to cytosol from control and insulin-treated cells were 54 and 13 pM, respectively. These data indicate that insulin modifies protein kinase C in adipocytes.     

Dual effect of the phorbol ester TPA on arachidonic acid release from HeLa cells

The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) induces release of arachidonic acid (AA) from HeLa cells with a maximum at 2-3 h. Subsequently the extracellular level of AA decreases. Cycloheximide (CH, 10(-5) M does not influence the release of AA, however, it causes the AA level to remain elevated. In the presence of TPA and CH (i) re-uptake of AA is not altered, (ii) re-incorporation of AA into phosphatidylinositol (and phosphatidylethanolamine) is largely increased, and (iii) the level of lysophosphatidylinositol is elevated. The latter two phenomena can be prevented by fluocinolone acetonide (10(-8) M), i.e. by inhibition of phospholipase A2 (PLA2). These data point to a continuously elevated PLA2 activity in the presence of TPA and CH. The phorbol ester appears to induce a proteinaceous principle which diminishes PLA2 activity.     

Structural insights into the interactions of phorbol ester and bryostatin complexed with protein kinase C: a comparative molecular dynamics simulation study

Protein kinase C (PKC) isozymes are important regulatory enzymes that have been implicated in many diseases, including cancer, Alzheimer’s disease, and in the eradication of HIV/AIDS. Given their potential clinical ramifications, PKC modulators, e.g. phorbol esters and bryostatin, are also of great interest in the drug development. However, structural details on the binding between PKC and its modulators, especially bryostatin – the highly potent and non-tumor promoting activator for PKCs, are still lacking. Here, we report the first comparative molecular dynamics study aimed at gaining structural insight into the mechanisms by which the PKC delta cys2 activator domain is used in its binding to phorbol ester and bryostatin-1. As anticipated in the phorbol ester binding, hydrogen bonds are formed through the backbone atoms of Thr242, Leu251, and Gly253 of PKC. However, the opposition of H-bond formation between Thr242 and Gly253 may cause the phorbol ester complex to become less stable when compared with the bryostatin binding. For the PKC delta-bryostatin complex, hydrogen bonds are formed between the Gly253 backbone carbonyl and the C30 carbomethoxy substituent of the ligand. Additionally, the indole Nε1 of the highly homologous Trp252 also forms an H-bond to the C20 ester group on bryostatin. Backbone fluctuations also suggest that this latter H-bond formation may abrogate the transient interaction between Trp252 and His269, thus dampening the fluctuations observed on the nearby Zn²⁺-coordinating residues. This new dynamic fluctuation dampening model can potentially benefit future design of new PKC modulators.