Downregulation Of PKCα Research Articles

Selective Relocalization and Proteasomal Downregulation of PKCα Induced by Platelet-Activating Factor in Retinal Pigment Epithelium

Protein kinases C (PKCs) are key cell-signaling mediators in retinal physiology and pathophysiology. The cellular localization of PKC isoforms is important in defining their activity and specificity; the present study investigated the modulatory potential of the proinflammatory mediator platelet-activating factor (PAF) on the subcellular distribution of PKCalpha, beta, and delta isotypes. This study used real-time visualization of green fluorescent protein fused to PKCalpha, beta, or delta in the human retinal pigment epithelial (RPE) cell line ARPE-19. In PAF-stimulated ARPE-19 cells, PKCalpha translocated to the plasma membrane and then colocalized with Golgi markers p230 and GM130; PKCbeta translocated to the plasma membrane but not to the Golgi; and PKCdelta translocated to the Golgi. Pretreatment with PKC inhibitor calphostin C abolished the PAF-induced translocation of PKCalpha to the plasma membrane or to the Golgi, but the Golgi inhibitor Brefeldin A only prevented the accumulation of PKCalpha in Golgi, without affecting its membrane relocalization. PAF promoted depletion of PKCalpha and delta isoforms but not that of PKCbeta. Proteasome inhibitors lactacystin and MG-132 prevented the PAF-induced depletion of PKCalpha, but the inhibitor of lysosomal proteolysis E-64d was ineffective in rescuing PKCalpha. These results suggest that the PAF-induced downregulation of PKCalpha occurs principally through the proteasomal pathway. This remarkable PAF-mediated diversity in PKC translocation and downregulation highlights the significance of isotype-specific PKC activation in signaling pathways in ARPE-19 cells. These signaling events may be critical during RPE responses to oxidative stress, inflammation, and retinal degenerations, when PAF production is enhanced.

Role of PKC isoforms in the FcγR-mediated inhibition of LPS-stimulated IL-12 secretion by macrophages

Ligation of Fc receptors for immunoglobulin G (FcgammaRs) inhibits lipopolysaccharide (LPS)-stimulated secretion of interleukin (IL)-12 by macrophages. FcgammaR activation of protein kinase C (PKC) contributes to several functions of this receptor including phagocytosis, activation of the reduced nicotinamide adenine dinucleotide phosphate oxidase, and secretion of certain cytokines. Therefore, we tested the hypothesis that PKC mediates the FcgammaR inhibition of IL-12 secretion by macrophages. In murine macrophages, FcgammaR ligation augmented LPS-stimulated activation of PKC-alpha and PKC-delta but reduced IL-12p40 secretion. Similarly, activation of PKC with phorbol 12-myristate 13-acetate (PMA) depressed LPS-stimulated IL-12p40 secretion, and depletion of PKC augmented LPS-stimulated IL-12p40 secretion. Antisense down-regulation of PKC-delta increased LPS-stimulated IL-12p40 secretion and fully prevented the effects of FcgammaR ligation or PMA on IL-12p40 secretion. In contrast, down-regulation of PKC-epsilon blocked LPS-stimulated secretion of IL-12p40. Down-regulation of PKC-alpha had no effect on LPS-stimulated IL-12p40 secretion. The results suggest a negative role for PKC-delta and a positive role for PKC-epsilon in the regulation of LPS-stimulated IL-12p40 secretion.

Enhancement of Cisplatin Sensitivity of Cisplatin-Resistant Human Cervical Carcinoma Cells by Bryostatin 1

Bryostatin 1, a unique protein kinase C (PKC) activator, is already in the clinical trials. An understanding of complex regulation of PKC by bryostatin 1 is essential for effective use of bryostatin 1 in the clinic. We have previously shown that the ability of bryostatin 1 to enhance cisplatin sensitivity correlated with its ability to down-regulate PKCdelta in HeLa cells. We have investigated how bryostatin 1 influences PKCdelta regulation in cisplatin-resistant HeLa (HeLa/CP) cells, and if bryostatin 1 could be used to reverse cisplatin resistance. Phorbol 12,13-dibutyrate (PDBu), bryostatin 1, and small interfering RNA were used to manipulate PKC level/activation status. Cell death was monitored by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Annexin V dye-binding assay, and analysis of hypodiploid peak in a flow cytometer. Bryostatin 1 elicited a biphasic concentration response on PKCdelta down-regulation and cisplatin-induced cell death in HeLa/CP cells; the maximum effect was achieved with 1 nmol/L bryostatin 1. Down-regulation of PKCalpha increased with increasing concentrations of bryostatin 1. PDBu induced down-regulation of PKCalpha in HeLa and HeLa/CP cells but it had little effect on PKCdelta down-regulation in HeLa/CP cells. However, both PDBu and bryostatin 1 enhanced the sensitivity of HeLa/CP cells to cisplatin. Knockdown of PKCdelta by small interfering RNA inhibited cisplatin-induced apoptosis but knockdown of PKCalpha enhanced cisplatin-induced cell death. These results suggest that although PKCdelta acts as a proapoptotic protein, full-length PKCdelta may inhibit cisplatin-induced cell death. Thus, persistent activation/down-regulation of PKCdelta by bryostatin 1 was associated with cisplatin sensitization. Furthermore, PKCalpha acts as an antiapoptotic protein and down-regulation of PKCalpha by PDBu was associated with cellular sensitization to cisplatin.

Activation-Induced Depletion of Protein Kinase Cα Provokes Desensitization of Monocytes/Macrophages in Sepsis

Sepsis accounts for the majority of fatal casualties in critically ill patients, because extensive research failed to significantly improve appropriate therapy strategies. Thus, understanding molecular mechanisms initiating the septic phenotype is important. Symptoms of septic disease are often associated with monocyte/macrophage desensitization. In this study, we provide evidence that a desensitized cellular phenotype is characterized by an attenuated oxidative burst. Inhibition of the oxidative burst and depletion of protein kinase C alpha (PKC alpha) were correlated in septic patients. To prove that PKC alpha down-regulation indeed attenuated the oxidative burst, we set up a cell culture model to mimic desensitized monocytes/macrophages. We show that LPS/IFN-gamma-treatment of RAW264.7 and U937 cells lowered PKC alpha expression and went on to confirm these data in primary human monocyte-derived macrophages. To establish a role of PKC alpha in cellular desensitization, we overexpressed PKC alpha in RAW264.7 and U937 cells and tested for phorbolester-elicited superoxide formation following LPS/IFN-gamma-pretreatment. Inhibition of the oxidative burst, i.e., cellular desensitization, was clearly reversed in cells overexpressing PKC alpha, pointing to PKC alpha as the major transmitter in eliciting the oxidative burst in monocytes/macrophages. However, PKC alpha inactivation by transfecting a catalytically inactive PKC alpha mutant attenuated superoxide formation. We suggest that depletion of PKC alpha in monocytes from septic patients contributes to cellular desensitization, giving rise to clinical symptoms of sepsis.

Syndecan-4 regulates localization, activity and stability of protein kinase C-alpha.

During cell-matrix adhesion, syndecan-4 transmembrane heparan sulphate proteoglycan plays a critical role in the formation of focal adhesions and stress fibres. We have shown previously that the syndecan-4 cytoplasmic domain directly binds to and activates PKC-alpha (protein kinase C-alpha) in vitro [Oh, Woods and Couchman (1997) J. Biol. Chem. 272, 8133-8136]. However, whether syndecan-4 has the same activity in vivo needs to be addressed. Using mammalian two-hybrid assays, we showed that syndecan-4 interacted with PKC-alpha in vivo and that this interaction was mediated through syndecan-4 cytoplasmic domain. Furthermore, the activation of PKC increased the extent of interaction between syndecan-4 and PKC-alpha. Overexpression of syndecan-4, but not a mutant lacking its cytoplasmic domain, specifically increased the level of endogenous PKC-alpha and enhanced the translocation of PKC-alpha into both detergent-insoluble and membrane fractions. In addition, rat embryo fibroblasts overexpressing syndecan-4 exhibited a slowed down-regulation of PKC-alpha in response either to a prolonged treatment with PMA or to maintaining cells in suspension culture. PKC-alpha immunocomplex kinase assays also showed that syndecan-4 overexpression increased the activity of membrane PKC-alpha. Taken together, these results suggest that syndecan-4 interacts with PKC-alpha in vivo and regulates its localization, activity and stability.

Cisplatin resistance is associated with deregulation in protein kinase C-δ

Proteolytic activation of protein kinase C (PKC)-δ has been associated with cell death induced by the DNA damaging agent cisplatin. In the present study, we have examined if PKCδ is affected when cells acquire resistance to cisplatin. The level of PKCδ was elevated in cisplatin-resistant HeLa (HeLa/CP) cells compared to parental HeLa cells. Prolonged cellular exposure to the PKC activator phorbol-12,13-dibutyrate (PDBu), caused downregulation of PKCδ in HeLa cells but not in HeLa/CP cells. Treatment of HeLa cells with PDBu resulted in the translocation of PKCδ from the cytosol to the membrane but it failed to induce PKCδ translocation in HeLa/CP cells. PDBu, however, induced translocation and downregulation of PKCα in both HeLa and HeLa/CP cells. The ability of PDBu to enhance cisplatin-induced cell death was attenuated in cisplatin-resistant HeLa cells. Thus, a deregulation in PKCδ was associated with reduced cellular sensitivity to cisplatin.

Protein kinase Cα negatively regulates cell spreading and motility in MDA-MB-231 human breast cancer cells downstream of epidermal growth factor receptor

Previous work has shown that phorbol esters modulate chemotaxis. Here, we demonstrate that PKC activation via phorbol 12-myristate 13-acetate (PMA) treatment of MDA-MB-231 cells inhibits EGF-induced cell spreading, the initial event of motility and chemotaxis. Of five PKC isoforms ( α, ι, λ, δ, and ε ) identified in this cell line, PMA treatment only induced PKCα translocation from the cytosol to the membrane, an event that correlated with the development of the rounded morphology. Cell recovery was linked to PKCα downregulation in part via the proteasome pathway since treatment with MG101 in the presence of PMA did not lead to PKCα degradation and cell recovery. Co-immunoprecipitation and immunolocalization demonstrated that EGF co-localized with PKCα and EGFR, however, PMA did not abrogate EGFR transactivation. This work suggests that PKCα is the primary target of PMA acting as a transient negative regulator of cell spreading and motility in MDA-MB-231 breast cancer cells.

Suppression of Synaptotagmin II restrains phorbolester-induced downregulation of protein kinase Calpha by diverting the kinase from a degradative pathway to the recycling endocytic compartment.

Downregulation of protein kinase Calpha (PKCalpha) following long-term exposure to phorbol esters such as TPA is traffic dependent and involves delivery of the active, membrane-associated PKCalpha to endosomes. In this study, we show that synaptotagmin II (Syt II), a member of the Syt family of proteins, is required for TPA-induced degradation of PKCalpha. Thus, whereas the kinase half-life in TPA-treated cultured mast cells (the mast cell line rat basophilic leukemia RBL-2H3) is 2 hours, it is doubled in RBL-Syt II(-) cells, in which the cellular level of Syt II is reduced by >95% by transfection with Syt II antisense cDNA. We demonstrate that in TPA-treated RBL cells, PKCalpha travels from the cytosol to the plasma membrane, where it is delivered to early endosomes on its route to degradation. By contrast, in TPA-treated RBL-Syt II(-) cells, PKCalpha is diverted to recycling endosomes and remains distributed between the plasma membrane and the perinuclear recycling endocytic compartment. Notably, in both RBL and RBL-Syt II(-) cells, a fraction of PKCalpha is delivered and maintained in the secretory granules (SG). These results implicate Syt II as a critical factor for the delivery of internalized cargo for degradation. As shown here, one consequence of Syt II suppression is a delay in PKCalpha downregulation, resulting in its prolonged signaling.

Gene therapy for the management of pain: part II: molecular targets.

TREATMENT of chronic pain, particularly of neuropathic etiology, is extremely difficult and resistant to many available pharmacologic therapies. Current analgesic agents may be limited with regard to analgesic efficacy or side effects. Newer and experimental pharmacologic agents may also have significant limitations. By targeting a specific receptor or other specific protein targets, a gene therapy approach to the treatment of pain may provide greater analgesic efficacy without the limitations associated with current pharmacotherapy. Advances in the field of gene therapy, along with significant increases in our understanding of the neurobiology of nociception and knowledge of the fundamental genetic structure of many nociceptive targets, have made gene therapy for the management of pain a conceivable reality. In part I of this review, we introduced the basic concepts of gene therapy with an emphasis on the available tools (e.g., viral vectors and antisense oligonucleotides) and strategies for upregulating antinociceptive or downregulating pronociceptive targets. In part II, we summarize current knowledge regarding the nociceptive role, molecular biology, and antisense and knockout data of several novel nociceptive targets for gene therapy. We base our selection of the targets included in this review on the three aforementioned criteria. The targets selected are the best characterized and, in our opinion, most likely amenable to the gene therapeutic approach. A simple but feasible strategy and potential gene therapy targets for the management of pain are summarized in figure 1. However, the list is admittedly incomplete, and the readers are referred to other recent reviews cited in part I of this review for a broader perspective on potential targets for the management of pain.

The Transient Increase of Tight Junction Permeability Induced by Bryostatin 1 Correlates with Rapid Downregulation of Protein Kinase C-α

The role of PKC-α in altered epithelial barrier permeability following the activation of PKC by TPA (12-O-tetradecanoyl phorbol 13-acetate) and bryostatin 1 in LLC-PK1 cells was investigated in this study. Like TPA, bryostatin 1 binds to and activates PKC but unlike TPA, it is not a tumor promoter. TPA at 10−7 M induced a sustained 95% decrease in transepithelial electrical resistance (Rt) across LLC-PK1 epithelial cell sheets, while 10−7 M bryostatin 1 caused only a 30% decrease in Rt, which spontaneously reversed after 5 h. Simultaneous exposure of cell sheets to 10−7 M TPA and 10−7 M bryostatin 1 blunted the increase in epithelial permeability observed with 10−7 M TPA alone. Co-incubation of cell sheets with bryostatin 1 and MG-132, a proteasomal inhibitor, caused a further decrease in Rt at the 6-h time point and inhibited the recovery in Rt seen with bryostatin 1 alone at this time point. TPA caused a rapid translocation of PKC-α from the cytosol to the membrane of the cell where it remained elevated. Bryostatin 1 treatment resulted in a slower translocation of PKC-α from the cytosol to the membrane and a much more rapid downregulation of PKC-α, with disappearance from this compartment after only 6 h. The classical PKC inhibitor Go6976 prevented the decrease in Rt seen with TPA. Treatment of cells with TPA and bryostatin 1 resulted in a PKC-α translocation and downregulation profile which more closely resembled that seen with bryostatin 1 alone. Co-incubation of cells with MG-132 and bryostatin 1 caused a slower downregulation of PKC-α from the membrane fraction. Bryostatin 1 treatment of cells expressing a dominant/negative form of PKC-α resulted in a slower and less extensive decrease in Rt compared to the corresponding control cells. For both TPA and bryostatin 1, the level of PKC-α in the membrane-associated fraction of the treated cells correlated closely with increased transepithelial permeability. Due to its transient effect on tight junction permeability, bryostatin 1 offers a novel pharmacological tool to investigate junctional physiology.

Disruption of Actin Cytoskeleton Induces Chondrogenesis of Mesenchymal Cells by Activating Protein Kinase C-α Signaling

Disruption of actin cytoskeleton with cytochalasin D has been known to induce chondrogenic differentiation of chick embryo limb bud mesenchymal cells. However, the mechanism(s) for the induction of chondrogenesis by cytochalasin D is not yet clearly known. In the present study, we examined possible involvement of protein kinase C (PKC) and extracellular signal-regulated protein kinase (Erk-1) in chondrogenesis of mesenchymal cells induced by disruption of actin cytoskeleton. Disruption of actin cytoskeleton with cytochalasin D or latrunculin B induced chondrogenesis of mesenchymal cells cultured at subconfluent cell density, as determined by type II collagen expression. Among the expressed PKC isoforms, cytochalasin D dramatically increased expression and activation of PKCα in a dose-dependent manner, and inhibition or downregulation of PKCα blocked cytochalasin D-induced chondrogenesis. Cytochalasin D also downregulated Erk-1 phosphorylation that is associated with chondrogenesis. Our results, therefore, suggest that disruption of actin cytoskeleton induces chondrogenesis of mesenchymal cells by activating PKCα and by inhibiting Erk-1 signaling.

Mitogenic Signal of Epidermal Growth Factor in Chick Embryo Hepatocytes: Role of PKCα

In the present study we investigated the role of two isoforms of protein kinase C in the mitogenic signal of epidermal growth factor (EGF) in primary culture of chick embryo hepatocytes. The down-regulation of PKCα by long-term exposure to phorbol myristate acetate (PMA) provoked a reduced mitogenic response to EGF while the down-regulation of PKCε with oligonucleotide antisense had no effect on the stimulation of DNA synthesis, assayed as thymidine incorporation. EGF enhanced H3 diacylglycerol (DAG) production by cells preincubated with H3myristic acid, but did not increase the production of inositol 1-4-5-trisphosphate (IP3). EGF produced an increase in the release of arachidonic acid and prostaglandin E2 (PGE2) in the extracellular medium. The increase was blocked by specific inhibitors (quinacrine and AACOCF3) of phospholipase A2 (PLA2) and was inhibited by down-regulation of PKCα, demostrating that this isoform is involved in arachidonic acid production. DAG and arachidonic acid produced an additional effect on thymidine incorporation. The treatment with PLA2 inhibitors, which block the increase in arachidonic acid, decreased the effect of EGF on DNA synthesis. These results suggest that in chick embryo hepatocytes PKCα is the main isoform involved in EGF-induced DNA synthesis. Its rapid activation is dependent on DAG production and induces an increased production of arachidonic acid and prostaglandin which are involved in the mitogenic activity.

The proliferative effect of vascular endothelial growth factor requires protein kinase C-alpha and protein kinase C-zeta.

The heparin-binding protein vascular endothelial growth factor (VEGF) is a highly specific growth factor for endothelial cells. VEGF binds to specific tyrosine kinase receptors, which mediate intracellular signaling. We investigated 2 hypotheses: (1) VEGF affects intracellular calcium [Ca2+]i regulation and [Ca2+]i-dependent messenger systems; and (2) these mechanisms are important for VEGF's proliferative effects. [Ca2+]i was measured in human umbilical vein endothelial cells using fura-2 and fluo-3. Protein kinase C (PKC) activity was measured by histone-like pseudosubstrate phosphorylation. PKC isoform distribution was observed with confocal microscopy and Western blot. Inhibition of PKC isoforms was assessed by specific antisense oligonucleotides (ODN) for the PKC isoforms. VEGF (10 ng/mL) induced a transient increase in [Ca2+]i followed by a sustained elevation. The sustained [Ca2+]i plateau was abolished by EGTA. Pertussis toxin also abolished the plateau phase, whereas the initial peak was not affected. The PKC isoforms alpha, delta, epsilon, and zeta were identified in endothelial cells. VEGF induced a translocation of PKC-alpha and PKC-zeta toward the nucleus and the perinuclear area, whereas cellular distribution of PKC-delta and PKC-epsilon was not influenced. Cell exposure to TPA led to a down-regulation of PKC-alpha and reduced the proliferative effect of VEGF. VEGF-induced endothelial cell proliferation also was reduced by the PKC inhibitors staurosporine and calphostin C. Specific down-regulation of PKC-alpha and PKC-zeta with antisense ODN reduced the proliferative effect of VEGF significantly. Our data show that VEGF induces initial and sustained Ca2+ influx. VEGF leads to the translocation of the [Ca2+]i-sensitive PKC isoform alpha and the atypical PKC isoform zeta. Antisense ODN for these PKC isoforms block VEGF-induced proliferation. These findings suggest that PKC isoforms alpha and zeta are important for VEGF's angiogenic effects.

Antisense Oligonucleotides Targeting Protein Kinase C-α, -βI, or -δ But Not -η Inhibit Lipopolysaccharide-Induced Nitric Oxide Synthase Expression in RAW 264.7 Macrophages: Involvement of a Nuclear Factor κB-Dependent Mechanism

AbstractThe signaling pathway for protein kinase C (PKC) activation and the role of PKC isoforms in LPS-induced nitric oxide (NO) release were studied in RAW 264.7 macrophages. The tyrosine kinase inhibitor genestein attenuated LPS-induced NO release and inducible nitric oxide synthase (iNOS) expression, as did the phosphoinositide-specific phospholipase C (PI-PLC) inhibitor U73122 and the phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor D609. LPS stimulated phosphatidylinositol (PI) hydrolysis and PKC activity in RAW cells; both were inhibited by genestein. The PKC inhibitors (staurosporine, calphostin C, Ro 31-8220, or Go 6976) or long-term 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment also resulted in inhibition of LPS-induced NO release and iNOS expression. Western blot analysis showed expression of PKC-α, -βI, -δ, -η, and -ζ in RAW cells; down-regulation of PKC-α, -βI, and -δ, but not -η, was seen after long-term TPA treatment, indicating the possible involvement of one or all of PKC-α, -βI, and -δ, but not -η, in LPS-mediated effects. Treatment with antisense oligonucleotides for these isoforms further demonstrated the involvement of PKC-α, -βI, and δ, but not -η, in LPS responses. Stimulation of cells with LPS for 1 h caused activation of NF-κB in the nuclei by detection of NF-κB-specific DNA-protein binding; this was inhibited by genestein, U73122, D609, calphostin C, or antisense oligonucleotides for PKC-α, -βI, and -δ, but not -η. These data suggest that LPS activates PI-PLC and PC-PLC via an upstream tyrosine kinase to induce PKC activation, resulting in the stimulation of NF-κB DNA-protein binding, then initiated the expression of iNOS and NO release. PKC isoforms α, βI, and δ were shown to be involved in the regulation of these LPS-induced events.

Divergent influences of protein kinase C isoforms on intracellular levels of phosphorylated and non-phosphorylated tau immunoreactivity

Stable transfectant subclones of SH-SY-5Y cells expressing antisense-oriented mRNA directed against PKCα (“α-transfectant”) and PKCϵ (“ϵ-transfectant”) expressed reduced levels of target isoforms, yet maintenance of normal levels of heterologous PKC isoforms as compared to those transfected with the plasmid vector only or the parental SH-SY-5Y cell line. Downregulation of PKCα and ϵ had differential effects on phospho-dependent and -independent tau immunoreactivity. Total tau immunoreactivity was reduced in both transfectant subclones as compared to that in the parental line. By contrast, only the ϵ-transfectant contained significantly lower levels of PHF-1 immunoreactivity, while the α-transfectant contained similar levels to that of the parental line. Our use of the complementary antibodies AT-8 and Tau-1, which recognize the same site when phosphorylated or nonphosphorylated, respectively, allowed us to monitor more precisely the relationship between tau phosphorylation and proteolytic degradation. Although significantly reduced in both transfectants, approximately twice as much AT-8 immunoreactivity was present in the α-transfectant as compared to the ϵ-transfectant. Tau-1 immunoreactivity was also drastically reduced in both transfectants. These data provide suggest that site-specific phosphorylation of tau renders it resistant to degradation, and, given the pivotal role of PKC in signal transduction pathways, suggest that a complex interplay of signal transduction events may underscore the onset and/or progression of AD.

Phosphorylation events mediated by protein kinase Cα and ε participate in regulation of tau steady-state levels and generation of certain “Alzheimer-like” phospho-epitopes

Hyperactivation of protein kinase C (PKC) in intact neuroblastoma cells by several methods increases site-specific tau phosphorylation as shown by increases in paired helical filament-1 (PHF-1) and ALZ-50 but not AT-8 immunoreactivity. In the present study, the influence of PKC on tau metabolism was further examined by isoform-specific antisense oligonucleotide-mediated PKC downregulation in human SH-SY-5Y neuroblastoma cells and by generation of stably-transfected subclones expressing isoform-specific anti-PKC mRNA sequences. Downregulation of PKCε by both of these methods reduced PHF-1 and ALZ-50 immunoreactivity, suggesting that this PKC isoform, perhaps via downstream kinase cascades, regulated tau phosphorylation events that normally generate these epitopes. By contrast, downregulation of either PKCε or PKCα reduced immunoreactivity towards the phosphate-independent anti-tau antibodies 5E2 and JM, suggesting that both of these isoforms participated in regulation of tau steady-state levels. Downregulation of PKCβ did not affect any of the above changes. The above roles were apparently unique for PKCε and PKCα, since activation of multiple PKC isoforms by phorbol ester treatment andlor other calcium-dependent kinase(s) by ionophore-mediated calcium influx could not compensate for downregulation of PKCα or PKCε in maintaining tau steady-state levels or PHF-1/ALZ-50 immunoreactivity, respectively. These findings suggest that hyperactivation of signal transduction pathways, including those regulated by PKC, could evoke changes in neuronal cells reminiscent of those seen in affected neurons in Alzheimer's disease.

Differential desensitization of thromboxane A2 receptor subtypes.

Two subtypes of the thromboxane A2 (TxA2) receptor (TxA2R-E and TxA2R-P), which differ in their alternatively spliced cytoplasmic tails, have been identified. The initial concentration of the TxA2 mimetic IBOP required to reduce peak intracellular Ca2+ concentration ([Ca2+]i) induced by a second addition of IBOP (100 nmol/L) was similar (IC50 for TxA2R-E and TxA2R-P, 0.46 +/- 0.16 and 0.40 +/- 0.07 nmol/L) in fibroblasts overexpressing either the TxA2R-E or -P subtype. Although the number of TxA2 binding sites decreased in TxA2R-P cells after prolonged stimulation with a TxA2 mimetic, those in the TxA2R-E cells increased markedly. To determine whether the mechanism for desensitization differs between subtypes, the effect of activation of protein kinase C (PKC) or cAMP-dependent kinase on TxA2-induced [Ca2+]i mobilization was measured. Forskolin reduced the IBOP-induced peak [Ca2+]i in neither TxA2R-E nor TxA2R-P cells; however, treatment with phorbol esters (IC50, 0.57 +/- 0.70 nmol/L) strongly prevented IBOP-mediated [Ca2+]i rise in TxA2R-E but not in TxA2R-P cells. Desensitization of TxA2R-E by phorbol esters was prevented by the PKC inhibitor calphostin C or by downregulation of PKC-alpha. Thus, the response of TxA2R-E to prolonged stimulation differs from that of TxA2R-P in both the regulation of the number of binding sites and the mechanism for desensitization; agonists that activate PKC-alpha might interfere with TxA2R-E-mediated signaling.

Protein kinase C in beta-cells: expression of multiple isoforms and involvement in cholinergic stimulation of insulin secretion

The mammalian protein kinase C (PKC) family consists of at least 11 distinct isotypes with marked differences in tissue distribution, localization, cofactor depdence and substrate specificity. Evidence exists for the expression of some of the PKC isoforms in pancreatic beta-cells but no comprehensive analysis of all the known PKC types has been accomplished. To assess the functional relevance of phosphorylation by PKC in the mechanism of insulin secretion we firstly investigated the expression of PKC isoforms in pancreatic beta-cells. The combination of reverse transcription-polymerase chain reaction (RT-PCR), Northern analysis and immunoblotting demonstrated the expression of PKC-α, βII, ϵ, ζ, λ and μ in MIN6 beta-cells. PKC-μ has not previously been detected in beta-cells. Expression of PKC-δ was also observed at the mRNA level; however, the protein could not be detected by Western blotting in MIN6 cells but res readily observed in RINm5F beta-cells. In short-term incubations, insulin release from MIN6 cells was augmented by 12- O-tetradecanoyl-phorbol-13-acetate (TPA), by carbachol, and by 40 mM K + Culture of MIN6 cells overnight with TPA resulted in down-regulation of PKC-α (totally) and ϵ (partially), without significant change in the other isoforms. In such TPA-treated cells, the secretory response to TPA and to carbachol was abolished but not that elicited by high K +. It is suggested that PKC-α and/or ϵ may play a role in cholinergic potentiation of insulin secretion.

Downregulation of Protein Kinase C Suppresses Induction of Apoptosis in Human Prostatic Carcinoma Cells

Protein kinase C (PKC) has been implicated in propagating signals for apoptosis. We have investigated the effect of pharmacological modulation of PKC activity in DU-145 human androgen-independent prostatic carcinoma cells. The apoptotic death of these cells is characterized by the acquisition of classical apoptotic morphology and generation of ≥1-mbp and 450- to 600-kbp DNA fragments in attached preapoptotic cell populations prior to cellular detachment and accrual of 30- to 50-kbp DNA fragments. We found that induction of apoptosis was arrested by downregulation of PKC activity and not by transient activation or inhibition of the enzyme. Concentrations and durations of exposure to phorbol esters that downregulated PKC activity correlated with inhibition of VP-16 or melphalan-induced morphological apoptosis and generation of the 30- to 50-kbp DNA fragments. Chronic exposure to phorbol-12,13-dibutyrate (PDBu) did not, however, suppress production of the ≥1-mbp and 450- to 600-kbp DNA fragments found in preapoptotic cell populations, suggesting that PKC downregulation may interfere with the transition between a preapoptotic cell and an apoptotic cell. PKC isozyme analysis revealed that chronic PDBu treatment caused downregulation of PKC-α and -ϵ in DU-145 cells. Using concentrations of the PKC inhibitor UCN-01 that were consistent with PKC-α inhibition (but not PKC-ϵ inhibition), however, did not mimic the effects of chronic PDBu treatment, implying that downregulation of PKC-ϵ may be of particular importance. Together, these findings suggest that phorbol esters may act as tumor promoters by suppressing apoptosis.

The role of protein kinase C in activation and termination of mitogen-activated protein kinase activity in angiotensin II-stimulated rat aortic smooth-muscle cells

Mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases activated by both tyrosine kinase and G-protein-linked receptor agonists. In rat aorta vascular smooth-muscle cells (VSMC), vasoconstrictors, angiotension II (AII), and α-thrombin (α-thr), as well as platelet-derived growth factor ββ (PDGF) stimulated the tyrosine phosphorylation and activation of MAP kinase in a time- and concentration-dependent manner. Pre-treatment of cells with the protein kinase C (PKC) inhibitor Ro-318220, inhibited the initial increase in tyrosine phosphorylation of MAP kinase in response to vasoconstrictors, suggesting the involvement of PKC. Four isoforms of PKC were identified in VSMC by western blotting: α, β, ϵ, and ζ. Downregulation of PKCα and PKCε isoforms following chronic phorbol myristate 12, 13-acetate (PMA) pre-treatment resulted in the abolition of AII-stimulated MAP kinase activation. Selective downregulation of PKCα following pre-treatment with bryostatin 1 did not affect AII-stimulated MAP kinase. Preincubation of cells with Ro-318220 enhanced the activation of MAP kinase at later time points. In addition, Ro-318220 pre-treatment inhibited the induction by AII of a novel transcriptionally regulated phosphatase, MAP kinase phosphatase-1 (MKP-1). However, AII-mediated activation of MAP kinase was not prolonged by cycloheximide pre-treatment and was not maintained indefinitely by Ro-318220. These results demonstrate a specific role for the Ca 2+-independent PKC isoform, PKCε, in the activation of MAP kinase in response to vasoconstrictors, and suggest that PKC-mediated induction of MKP-1 plays no role in the termination of transiently activated MAP kinase.