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.     

Phorbol esters down-regulate protein kinase C in rat brain cerebral cortical slices

The effect of phorbol esters on cyclic AMP production in rat cerebral cortical slices was studied using a prelabelling technique to measure cyclic nucleotide accumulation. Cholera toxin-stimulated cyclic AMP accumulation was enhanced approximately 2-fold by phorbol 12-myristate, 13-acetate (PMA) which alone had no effect on cyclic AMP production. The augmentation by PMA was maximal within the first hour of incubation, decreasing progressively thereafter. Protein kinase C activity was decreased 80-90% during a 3 hr exposure to PMA, as was 3H-phorbol 12,13-dibutyrate binding. Both phosphatidyl serine and arachidonic acid were found to enhance protein kinase C activity in a concentration-dependent manner, an effect that was attenuated by prolonged incubation of the brain tissue with PMA. The results indicate that exposure of brain slices to phorbol esters causes a down-regulation of rat brain protein kinase C, and that this modification corresponds with a decrease in the ability of PMA to augment cyclic AMP production, suggesting a functional relationship between the two systems in rat brain.     

Phorbol esters and calcium-mobilizing hormones increase membrane-associated protein kinase C activity in rat hepatocytes

Vasopressin, angiotensin II, epinephrine (alpha 1-adrenergic action) and phorbol 12-myristate 13-acetate (PMA) induce increases in membrane-associated protein kinase C activity concomitant with decreases in the cytosolic activity. The data indicate that the calcium-mobilizing hormones and the active phorbol ester induce translocation from the cytosol to the plasma membrane of this protein kinase. The protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, blocked the translocation to the membrane of this protein kinase induced by PMA and vasopressin.     

Changes in membrane potential and postsynaptic potentials evoked by strychnine in neocortical neurons

Intracellular responses of neurons of the suprasylvian fissure to intracortical stimulation before and during topical cortical strychnine application was studied in experiments on immobilized, unanesthetized cats (a local anesthetic was used). Untreated cortical neurons responded to intracortical stimulation with a monosynaptic excitatory postsynaptic potential (EPSP) followed by an inhibitory postsynaptic potential (IPSP). Application of strychnine evoked epileptiform population activity and paroxysmal depolarizations of neuronal membrane potentials (MPs), followed by hyperpolarization. Increased hyperpolarizations, and the prolonged duration of their summation were responsible for an increased MP and reduced or abolished tonic spike activity. Intracellular application (as a result of diffusion from the microelectrode) of ethyleneglycoltetraacetate (EGTA) that blocked the calcium-dependent potassium membrane conductance (gK(Ca)) abolished the hyperpolarization. The development of epileptiform activity was accompanied by reduction of the IPSP, and an increase in the monosynaptic EPSP. The role of gK(Ca) and postsynaptic inhibition in epileptogenesis is discussed.I. I. Mechnikov State University, Odessa. Translated from Neirofiziologiya, Vol. 24, No. 6, pp. 684–691, November–December, 1992.     

Agents that activate protein kinase C reduce acetylcholine sensitivity in cultured myotubes

We have examined acetylcholine (ACh)-elicited potentials or currents in current- or voltage-clamped cultured myotubes exposed to 12-O- tetradecanoyl-phorbol-13-acetate (TPA), a potent tumor promoter that activates protein kinase C. Although this agent had little action on either membrane resting potential or electrical resistance, a reversible decrease in ACh sensitivity was induced on 3-4-d-old chick myotubes. Depression of transmitter action by TPA was extended to 7-8-d mouse myotubes only when they were treated with phosphatidylserine. Glyceryl dioleate had effects on myotubes similar to those of TPA but with a reduced efficacy. We conclude that the activation of protein kinase C might be involved with the capacity of ACh receptors to respond to transmitter stimulation.     

Phorbol esters induce translocation of the nPKC p105 to membrane in mussel hemocytes

Previous works revealed the presence of the nPKC enzyme p105 in hemocytes of M. galloprovincialis Lmk. Specific mussel antibodies were obtained from mouse and used in confocal microscopy and Western blotting. These techniques allowed the observation of p105 cytosol-to-membrane translocation induced by TPA for the first time in hemocytes of molluscs. The incubation of mussel immune cells with TPA for longer than 30 min also triggered a down-regulation process. Mussel hemocytes are an excellent model to study the molecular processes of innate immunity.     

Generalized posttetanic changes in the excitatory postsynaptic and acetylcholine-evoked currents in snail neurons

Heteroreceptor posttetanic changes in excitatory postsynaptic currents (EPSC) and inward currents evoked by the local iontophoretic application of acetylcholine (ACh) on the dorsal surface of PLa3 and PRa3 Helix lucorum neurons were studied. The following changes in the currents were revealed over the course of 1-1.5 h after tetanization. The rhythmical ACh application (0.5-1.0 cps, 10-40 s) evokes potentiation of the orthodromic EPSC. The tetanic orthodromic stimulation of one of the nerves (n. intestinalis, n. pallialis dexter, or n. pallialis sinister; 1-5 cps, 1-2 min) causes the potentiation of the ACh current and also heterosynaptic depression of the EPSC. It is concluded that activation of subsynaptic and nonsynaptic neurotransmitter chemoreceptors evokes the development of generalized posttetanic changes in neuronal responses.     

Phorbol esters, but not the hormonal form of vitamin D, induce changes in protein kinase C during differentiation of human histiocytic lymphoma cell line (U-937)

Human histiocytic lymphoma cells (U-937) undergo similar differentiation when incubated with the phorbol ester 12-0-tetradecanoyl phorbol-13-acetate (TPA) and 1,25-dihydroxycholecalciferol. In this action, TPA somehow implicates calcium-sensitive and phospholipid-dependent protein kinase (protein kinase C), which is rapidly and significantly affected by this inducer. On the contrary, 1,25-dihydroxycholecalciferol in its differentiating action does not involve protein kinase C thus suggesting that the secosteroid induces monocytic differentiation possible through a different mechanism of that of phorbol ester.     

Phorbol esters induce two distinct changes in GH3 pituitary cell adenylate cyclase activity

Phorbol esters alter cyclic AMP levels in a number of tissues, including the anterior pituitary. We report that membrane preparations from GH3 cells exposed to phorbol esters exhibit decreased vasoactive intestinal peptide (VIP)-stimulated and enhanced forskolin-stimulated adenylate cyclase activity. The responsiveness of adenylate cyclase activity to NaF, guanylyl-imidodiphosphate, and Mn2+ was also reduced by phorbol ester treatment. The ability of somatostatin to inhibit forskolin-stimulated adenylate cyclase activity was reduced while phorbol ester exposure had no apparent effect on somatostatin inhibition of VIP-stimulated adenylate cyclase activity. We suggest that protein kinase C alters at least two distinct components of the adenylate cyclase system. One modification disrupts hormone receptor-Gs interaction (lowering VIP efficacy) and the second perturbation augments the activity of the adenylate cyclase catalytic subunit.     

Phorbol esters, but not insulin, promote depletion of cytosolic protein kinase C in rat adipocytes

The tumor-promoting phorbol esters have insulinomimetic effects in several tissues. Employing two different assay systems, we have compared the effects of phorbol ester and insulin on the activity and intracellular distribution of the Ca++ and phospholipid dependent protein kinase (protein kinase C) in isolated rat adipocytes. Phorbol ester leads to a prompt depletion of kinase activity from the cytosolic fraction and appearance of activity in membrane extracts; neither of these effects is mimicked by insulin. These results, taken together with other data, emphasize important divergences between the actions of these agonists and suggest that changes in protein kinase C activity or intracellular distribution are not a necessary concomitant of the cascade of insulin action.     

Depolarizing stimuli and neurotransmitters utilize separate pathways to activate protein kinase C in sympathetic neurons.

Several types of extracellular signals affect the function of peripheral neurons. Depolarizing stimuli cause sudden increases in permeability to various ions leading to propagation of nerve impulses and release of transmitter substances. Neurons also receive external signals via neurotransmitter receptors located on the membrane. Different types of receptors present on sympathetic neurons are believed to modulate stimulation-evoked release of norepinephrine. We have investigated the effects of depolarizing stimuli and neurotransmitters on different signaling pathways in homogeneous cultures of chick sympathetic neurons. Depolarizing stimuli (35 mM KCl; electrical stimulation, 1 Hz for 5 min) and neurotransmitters (acetylcholine and 5-hydroxytrypatmine) enhanced membrane binding of protein kinase C by 2-5-fold. 35 mM KCl increased formation of 1,2-diacylglycerol and hydrolysis of [3H]phosphatidycholine without affecting [3H] phosphoinositide hydrolysis. Neurotransmitters increased [3H]inositol phosphates and 1,2-diacylglycerol without affecting the hydrolysis of [3H]phosphatidylcholine. 5-Hydroxytryptamine and acetylcholine (muscarinic component) did not increase Ca2+ concentration in the Indo-1-loaded neuronal cell body or the growth cone, but 35 mM KCl and electrical stimulation caused a marked increase in Ca2+ concentration in both regions of sympathetic neurons. We believe this to be the first demonstration of these two types of signalling mechanisms co-existing in sympathetic neurons; depolarization activate the phosphatidylcholine pathway and neurotransmitters activate the phosphatidylinositol pathway. The importance of two pathways in controlling neuronal Ca2+ concentration and the release of transmitter is discussed.     

Evidence that bacteria induce translocation of protein kinase C activity in human polymorphonuclear leukocytes

Particulate fraction associated protein kinase activity was studied in human polymorphonuclear leukocytes stimulated by bacteria. Staphylococcus aureus was found to increase particulate fraction associated protein kinase C activity in a time and concentration dependent manner. The increase comprised both the phospholipid dependent and independent kinase activity and was augmented by addition of serum. Similar observations were done using Staphylococcus epidermidis and Klebsiellae pneumoniae. However, Escherichia coli only increased the phospholipid independent kinase activity in the particulate fraction, which suggests the presence of protease activity.     

Phorbol esters induce synthesis of thromboplastin activity in human monocytes.

12-O-Tetradecanoylphorbol 13-acetate (TPA), phorbol 12,13-diacetate and phorbol 12,13-didecanoate were all potent inducers of thromboplastin activity in human monocytes in vitro, whereas 4 alpha-phorbol 12,13-didecanoate and 4 alpha-phorbol had no such effect. A concomitant increase in titrable apoprotein III antigen was found (apoprotein III is the protein component of thromboplastin). The increase was inhibited by cycloheximide and actinomycin D and partly by alpha-amanitin. The increase of thromboplastin activity was therefore most likely due to synthesis de novo of apoprotein III. The response was approximately halved in the absence of serum or Ca2+. Retinol had a weak inhibitory effect, and retinoic acid was inhibitory only at concentrations that also induced signs of cytotoxicity. TPA caused an initial rise in monocyte cyclic AMP concentration of about 90-120 min duration. No increase in 45Ca2+ influx was induced over 2 h. Good correlation exists between induction of apoprotein III synthesis in monocytes in vitro and mouse skin-tumour promotion in vivo by the various phorbol derivatives. Substances inactive in tumour promotion do not induce the synthesis of apoprotein III. General activating and cytotoxic effects of TPA were monitored by determining release of lysozyme, beta-glucuronidase and lactate dehydrogenase.     

Phorbol esters induce transient changes in the accessibility of the carboxy-terminal domain of nuclear lamin A.

Treatment of human epithelial cells in culture with phorbol esters (TPA) gives rise to a transient and reversible loss of accessibility to antibodies of the nonhelical carboxy-terminal domain of nuclear lamin A that distinguishes it from lamin C. No change in the accessibility of epitopes present in the common domain of lamins A and C was observed. Loss of accessibility of lamin A was not due to proteolytic degradation nor to modification of the isoelectric point of lamin A and did not depend upon protein kinase C activation nor protein synthesis. Perturbation of desmosome organization by growth in low calcium blocked the effect of TPA on lamin A. Prolonged exposure to nocodazole, one of the effects of which is a perinuclear collapse of intermediate filaments, also blocked the effect of TPA on lamin A. These results suggest that the initial target of TPA may be at the level of cell-cell contacts and that the perturbation induced by TPA may be propagated via the structural link formed by intermediate filaments between the cell surface and the nucleus, giving rise to a change in conformation of the carboxy-terminal domain of lamin A or to an interaction of this domain with another nuclear component. These results form the basis for the hypothesis that the interphase nuclear lamina may play an active role in the process of mechanochemical signal transduction.     

Nonphorbol tumor promoters okadaic acid and calyculin-A induce membrane translocation of protein kinase C.

The cell-permeable inhibitors of type 1 and 2A protein phosphatases, okadaic acid and calyculin-A, induced a redistribution of protein kinase C (PKC) activity and immunoreactivity (40 to 60%) from cytosol to membrane in some cell types. Calyculin-A was 100-fold more potent than okadaic acid and required only 5 to 10 nM concentrations to induce this PKC translocation. The concentration of these agents required to induce the redistribution of PKC correlated with the potency of these agents to inhibit both type 1 and 2A protein phosphatases. There was a lag period of 15 to 30 min before the onset of PKC translocation, as this process might have been induced by indirect cellular events triggered by inhibitions of protein phosphatases (1 and 2A). Taken together these results suggest that although the okadaic acid class of tumor promoters and phorbol ester-related agents bind to two different cellular receptors having counteracting enzymic activities, they share a common mechanism of action, namely the induction of cytosol to membrane translocation of PKC.     

Phorbol ester-induced actin assembly in neutrophils: role of protein kinase C

The shape changes and membrane ruffling that accompany neutrophil activation are dependent on the assembly and reorganization of the actin cytoskeleton, the molecular basis of which remains to be clarified. A role of protein kinase C (PKC) has been postulated because neutrophil activation, with the attendant shape and membrane ruffling changes, can be initiated by phorbol esters, known activators of PKC. It has become apparent, however, that multiple isoforms of PKC with differing substrate specificities exist. To reassess the role of PKC in cytoskeletal reorganization, we compared the effects of diacylglycerol analogs and of PKC antagonists on kinase activity and on actin assembly in human neutrophils. Ruffling of the plasma membrane was assessed by scanning EM, and spatial redistribution of filamentous (F)-actin was assessed by scanning confocal microscopy. Staining with NBD-phallacidin and incorporation of actin into the Triton X-100-insoluble ("cytoskeletal") fraction were used to quantify the formation of (F)-actin. [32P]ATP was used to detect protein phosphorylation in electroporated cells. Exposure of neutrophils to 4 beta-PMA (an activator of PKC) induced protein phosphorylation, membrane ruffling, and assembly and reorganization of the actin cytoskeleton, whereas the 4a-isomer, which is inactive towards PKC, failed to produce any of these changes. Moreover, 1,2-dioctanoylglycerol, mezerein, and 3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol, which are nonphorbol activators of PKC, also promoted actin assembly. Although these effects were consistent with a role of PKC, the following observations suggested that stimulation of conventional isoforms of the kinase were not directly responsible for actin assembly: (a) Okadaic acid, an inhibitor of phosphatases 1 and 2A, potentiated PMA-induced protein phosphorylation, but not actin assembly; and (b) PMA-induced actin assembly and membrane ruffling were not prevented by the conventional PKC inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, staurosporine, calphostin C, or sphingosine at concentrations that precluded PMA-induced protein phosphorylation and superoxide production. On the other hand, PMA-induced actin assembly was inhibited by long-chain fatty acid coenzyme A esters, known inhibitors of nuclear PKC (nPKC). We conclude that PMA-induced actin assembly is unlikely to be mediated by the conventional isoforms of PKC, but may be mediated by novel isoforms of the kinase such as nPKC.     

Phorbol esters and diacylglycerols amplify bradykinin-stimulated prostaglandin synthesis in Swiss 3T3 fibroblasts. Possible independence from protein kinase C

When Swiss 3T3 fibroblasts were incubated with bradykinin, prostaglandin E2 (PGE2) synthesis was stimulated. Phorbol esters or the diacylglycerol analog 1-oleoyl-2-acetylglycerol (OAG), by themselves, did not acutely stimulate PGE2 synthesis. However, when cells were preincubated with phorbol esters or OAG, bradykinin-stimulated PGE2 synthesis was potentiated markedly. When phorbol esters and OAG were added together, bradykinin-stimulated PGE2 synthesis was potentiated in an additive manner. When cells were preincubated for 48 h with phorbol esters, then bradykinin added, amplification of bradykinin-stimulated PGE2 synthesis by phorbol ester or OAG was still apparent, even though prolonged pretreatment with phorbol esters abolished protein kinase C (Ca2+/phospholipid-dependent enzyme) activity in cell-free preparations. Further, the protein kinase C antagonist, H-7, only slightly inhibited phorbol ester or OAG amplification of bradykinin-stimulated PGE2 synthesis. The possibility is raised that diacylglycerol, formed in response to many receptors, may serve as a transducer of receptor-receptor interactions. Since desensitization or inhibition of protein kinase C only partially reduced the amplification of bradykinin-stimulated PGE2 synthesis by phorbol esters or OAG, the possibility is raised that diacylglycerol mimetics may have actions in addition to activation of protein kinase C.