Down-regulation Of Protein Kinase C Isoforms Research Articles

Role of protein kinase C isoforms in locomotion of Walker 256 carcinosarcoma cells.

Treatment with low (nanomolar) concentrations of phorbol-12-myristate-13-acetate (PMA) for 5 to 30 min suppresses locomotion of Walker 256 carcinosarcoma cells, suggesting that activation of protein kinase C (PKC) is a stop signal for tumor cell locomotion. We have compared the effects of PMA on cell shape and motility with down-regulation of specific PKC isoforms. Using specific antibodies, we show that Walker carcinosarcoma cells express PKC isoforms alpha, betaI, betaII, gamma, lambda, mu, eta and zeta. Short-term incubation with PMA induced a marked shift of isoforms alpha, betaI, betaII, gamma and eta to the particulate fraction. Long-term incubation with PMA (0.1 microM, 6 hr) resulted in significant reduction of expression of conventional PKCs alpha, betaI, betaII and gamma and of the novel PKC eta to 10% to 26% of controls. Down-regulation of PKC alpha, betaI and betaII by long-term incubation with PMA was reversible after removal of PMA, whereas that of isoforms gamma and eta was not. The motile properties of cells after down-regulation of PKC isoforms were investigated. Concomitant with down-regulation of PKC isoforms, long-term incubation of cells with PMA resulted in recovery of the polar shape and the ability to migrate. Motility and polarized shape of the down-regulated cells were no longer susceptible to short-term treatment with PMA, showing that active PKC is indeed responsible for the inhibitory effects of PMA. Effects of long-term incubation with PMA on cell shape and motility were reversible. Our findings strongly suggest that PKCs alpha, betaI and betaII activated by PMA are involved in stopping Walker carcinosarcoma cell locomotion.

High glucose alters the response of mesangial cell protein kinase C isoforms to endothelin-1

High glucose causes glomerular mesangial growth and increased matrix synthesis contributing to diabetic glomerulopathy. Our purpose was to determine if high glucose alters endothelin-1 (ET-1) or platelet-derived growth factor-B activation of mesangial cell diacylglycerol-sensitive protein kinase C (PKC) isoforms and subsequent stimulation of mitogen-activated protein kinase (MAPK; p42, p44). Rat mesangial cells in primary culture were growth arrested for 48 hours in glucose 5.6 mM (NG) or 30 mM (HG). PKC-alpha, PKC-delta, and PKC-epsilon translocation from the cytosol-to-membrane and cytosol-to-particulate (cytoskeleton, nucleus) cellular fractions were measured by immunoblot using isoform-specific monoclonal antibodies. PKC isoforms were visualized also by confocal immunofluorescence microscopy. MAPK activation was measured by immunoblot using phospho-MAPK antibody and by detection of Elk-1 fusion protein phosphorylation following phospho-MAPK immunoprecipitation. In NG, ET-1 stimulated cytosol-to-membrane translocation of PKC-delta and PKC-epsilon but not PKC-alpha. In HG, the pattern of ET-1-stimulated PKC-delta and PKC-epsilon changed to a cytosol-to-particulate distribution, which was confirmed by confocal immunofluorescence imaging. Platelet-derived growth factor-B did not cause translocation of PKC-alpha, PKC-delta, or PKC-epsilon in either NG or HG. In HG, both basal and ET-1-stimulated MAPK activities were increased significantly. In HG, down-regulation of PKC isoforms with phorbol ester prevented the increased stimulation of MAPK by ET-1. In HG, the enhanced activation of mesangial cell MAPK by ET-1 is PKC dependent and associated with altered translocation of PKC-delta and PKC-epsilon. Enhanced mesangial cell signaling responsiveness to vasoactive peptides in HG may constitute an important mechanism contributing to diabetic nephropathy.

TPA-enhanced motility and invasion in a highly metastatic variant (L-10) of human rectal adenocarcinoma cell line RCM-1: selective role of PKC-alpha and its inhibition by a combination of PDBu-induced PKC downregulation and antisense oligonucleotides treatment.

We previously found that 12-O-tetradecanoylphorbol-13-acetate (TPA)-enhanced invasiveness was associated with augmentation of cell motility but not that of metalloproteinase activity in a highly metastatic variant (L-10) of the human colon adenocarcinoma cell line RCM-1 and that this enhancement was possibly mediated by protein kinase C (PKC). In this study, we first intended to determine the specific isoforms of PKC involved in this TPA-enhanced L-10 cell motility that leads to invasion, and then investigated the way to inhibit the enhanced motility and invasion by using antisense oligodeoxynucleotides (ODN) targeting the isoform. An activator of conventional PKC isoforms (cPKC), thymeleatoxin, enhanced L-10 cell motility and invasion like TPA, and an inhibitor of cPKC, Go-6976, efficiently inhibited TPA-enhanced motility and invasion. TPA treatment induced a shift of PKC-alpha, but not other isoforms, from the cytosol to the membrane fraction, indicating the activation of the isoform. During the assay period, only activation but not downregulation of PKC-alpha occurred with the low concentration of TPA used in our assays. Antisense ODNs specific for PKC-alpha efficiently reduced its expression at the protein levels and inhibited L-10 cell motility in the absence of TPA. With TPA treatment, however, the remaining PKC-alpha was sufficient for activation leading to enhanced invasion. Only a combination of depletion of PKC by prolonged stimulation with a high concentration of phorbol 12,13 dibutyrate (PDBu) and treatment with antisense ODNs effectively inhibited L-10 cell invasion even in the presence of TPA. These results suggested that downregulation of PKC isoforms by treatment with antisense ODNs alone is insufficient to suppress the isoform-mediated cellular events in the presence of PKC activators, and thus that some additional treatments are necessary for the successful downregulation of them.

Protein Kinase C Mediates the Mitogenic Action of Thrombopoietin in c-Mpl–Expressing UT-7 Cells

Protein kinase C (PKC) has been implicated in signal transduction events elicited by several hematopoietic growth factors. Thrombopoietin (TPO) is the major regulator of megakaryocytic lineage development, and its receptor, c-Mpl, transduces signals for the proliferation and differentiation of hematopoietic progenitors. In this study we have examined the effect of TPO on the subcellular distribution of PKC (a measure of enzyme activation) in a growth factor-dependent pluripotent hematopoietic cell line that was engineered to express the c-Mpl receptor (UT-7/mpl). In addition, we have assessed the significance of this activation for the induction of both mitogenesis and differentiation. Using a PKC translocation assay, TPO was found to stimulate a time- and dose-dependent increase in the total content of PKC activity present in the membrane fraction of UT-7/mpl cells (maximum increase = 2.3-fold above basal level after 15 minutes with 40 ng/mL TPO, EC50 = 7 ng/mL). Accordingly, a decrease of PKC content in the cytosolic fraction was observed. Immunoblot analysis using PKC isotype-specific antibodies showed that TPO treatment led to a marked increase of the Ca2+/diacylglycerol-sensitive PKC isoforms α and β found in the membrane fraction. In contrast, the subcellular distribution of these isoforms did not change after treatment with granulocyte-macrophage colony-stimulating factor (GM-CSF). Exposure of UT-7/mpl cells to the selective PKC inhibitor GF109203X completely inhibited the PKC activity associated to the membrane fraction after TPO treatment, and blocked the mitogenic effect of TPO. In contrast, GF109203X had no effect on the TPO-induced expression of GpIIb, a megakaryocytic differentiation antigen. Downregulation of PKC isoforms α and β to less than 25% of their initial level by treatment with phorbol 12,13-dibutyrate also abolished the TPO-induced mitogenic response, but had no significant effect when this response was induced by GM-CSF. Taken together, these findings suggest that (1) TPO stimulates the activation of PKC, (2) PKC activation mediates the mitogenic action of TPO, and (3) PKC activation is not required for TPO-induced expression of megakaryocytic surface markers.

Differential expression of protein kinase C isoforms in glial and neuronal cells. Translocation and down-regulation of PKC isoforms in C 6 glioma and ng 108-15 hybrid cells: Effects of extracellular Ca 2+-depletion

Protein kinase C (PKC), the major receptor for tumor-promoting phorbol esters, consists of a family of at least 12 distinct lipid-regulated enzymes. We examined the expression and regulation of PKC isoforms in C 6-glioma and NG 108-15 hybrid cells. Western blot analysis indicated that both cell lines express four PKC isoforms, PKCα, PKCδ, PKCε and PKCζ. The expression of PKCα and PKCδ in C 6-glioma cells was more abundant than NG 108-15 cells, however, PKCε in NG 108-15 was more abundant than C 6-glioma cells in which PKCε was almost undetectable. Treatment of both cells with TPA for 10 min resulted in the translocation of PKCα, PKCδ and PKCε to the membrane fraction. When the intact cells were treated with Ca 2+-free, EGTA containing physiological saline solution, the membrane bound conventional PKCα (cPKCα) was greatly reduced and cytosolic cPKCα was only slightly increased. However, neither membrane bound nor cytosolic new PKCδ (nPKCδ), nPKCε and atypical PKCζ (aPKCζ) was affected by extracellular Ca 2+ depletion. In this condition, the translocation of cPKCα, nPKCδ and nPKCε induced by TPA still occurred, however, that of cPKCα was reduced more than in the normal condition. After long-term treatment (17 h) with TPA, cPKCα, nPKCδ and nPKCε were down-regulated both in the cytosol and membrane. The phenomena of cPKCα were confirmed by measuring the PKC activity with histone as the substrate. From in vitro endogenous phosphorylation studies, a 31 kDa substrate protein phosphorylation in C 6 glioma cell membrane and 31 and 26 kDa proteins in NG 108-15 cell membrane were increased in the translocation but disappeared in the down-regulation of PKC.

Protein kinase C isoforms in multidrug resistant P388/ADR cells: a possible role in daunorubicin transport

To identify the role of protein kinase C (PKC) isoforms in multidrug resistance in tumor cells, we examined the PKC isoform pattern in the multidrug resistant P388/ADR cell line and studied the effect of down regulation of PKC isoforms on intracellular daunorubicin accumulation and P-glycoprotein expression. Using monoclonal antibodies to PKCα, β and γ and flow cytometry technique we showed that P388/ADR cells overexpressed PKCα and β as compared to drug sensitive P388 cells. Prolonged treatment of P388/ADR cells with phorbol myristate acetate (PMA), a procedure that is known to down regulate PKC, resulted in the down regulation of total PKC activity and the PKCβ isoform (at the protein level) that was accompanied by the correction of daunorubicin accumulation in P388/ADR cells. The level of expression of P-glycoprotein in PMA treated cells was similar to that of untreated cells. These results suggest that PKCβ regulates the drug efflux function of P-glycoprotein.