epidermal-growth-factor and rottlerin

Protein tyrosine phosphatase alpha (PTPα) functions as an activator of Src by dephosphorylating Tyr527/530, a critical negative regulatory site. The increase of PTPα catalytic activity requires its phosphorylation at Ser180 and/or Ser204 and its dissociation from PTPα/Grb2 complex. Here, we show that epidermal growth factor (EGF) stimulation increases the ability of PTPα to activate Src by dephosphorylating Tyr530 in BT-20 and SKBR3 breast cancer cell lines. Treatment of these cells with EGF transiently decreased the association of PTPα with Grb2 and enhanced PTPα catalytic activity via Ser180 and Ser204 phosphorylation that was blocked by the protein kinase C delta (PKCδ) inhibitor rottlerin or knockdown of PKCδ by siRNA or by the overexpression of PTPαS180A/S204A mutant. PTPα siRNA blocked EGF-mediated Src activation in cancer cells and inhibited on colony formation, whereas control siRNA did not. These results suggested that PTPα links activation of epidermal growth factor receptor (EGFR) signaling with Src activation and may provide a novel therapeutic target for treatment of breast cancer.

Topics: Acetophenones; Benzopyrans; Biocatalysis; Breast Neoplasms; Cell Line, Tumor; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; GRB2 Adaptor Protein; Humans; Immunoblotting; Mutation; Phosphorylation; Protein Binding; Protein Kinase C-delta; Proto-Oncogene Proteins pp60(c-src); Receptor-Like Protein Tyrosine Phosphatases, Class 4; RNA Interference; Serine; Signal Transduction; Tyrosine

Protein kinase C (PKC) is a member of serine/threonine protein kinase family that plays important roles in the control of vast variety of cellular functions. Nevertheless, the regulatory effect of PKC on adipogenesis remained not well understood. In this study, we investigated the effect of several PKC isoforms on the adipogenic conversion of 3T3-L1 preadipocytes induced by dexamethasone, isobutylmethylxanthine and insulin. Treatment of cells with broad-spectrum PKC inhibitor Rö318220 suppressed the adipogenesis. Gö6976, a selective inhibitor for PKC isoforms-alpha, -betaI and -mu, also inhibited the adipogenesis of cells. Pretreatment of cells with peroxisomal proliferator activated receptor-gamma (PPARgamma) agonist troglitazone abolished the inhibitory effect of Gö6976 on adipogenesis. The plasmic membrane translocation of PKC-betaI was observed at the first 2 days of differentiation. Whereas no translocation of PKC-alpha and -mu was observed. Overexpression of dominant negative PKC-betaI, but not wild-type PKC-betaI, blocked adipogenesis. This effect of dominant negative PKC-betaI can be reversed by troglitazone, suggesting that PKC-betaI is required for the initiation of adipogenesis. In addition, rottlerin, a specific inhibitor of PKC-delta, can reverse the suppression of adipogenesis mediated by 12-O-tetradecanoyl-phorbol-13-acetate, transforming growth factor-beta1, and epidermal growth factor. These data suggest that PKC-betaI is important in the induction of adipogenesis, while the PKC-delta has an inhibitory role for adipogenesis.

Topics: 3T3-L1 Cells; Acetophenones; Adipocytes; Adipogenesis; Animals; Benzopyrans; Carbazoles; Cell Differentiation; Cell Membrane; Cell Proliferation; Chromans; Cytosol; Epidermal Growth Factor; Gene Expression; Genes, Dominant; Indoles; Mice; Mutant Proteins; Protein Kinase C; Protein Kinase C beta; Protein Kinase C-delta; Protein Transport; Tetradecanoylphorbol Acetate; Thiazolidinediones; Transforming Growth Factor beta; Troglitazone

Myosin-based cell contractile force is considered to be a critical process in cell motility. However, for epidermal growth factor (EGF)-induced fibroblast migration, molecular links between EGF receptor (EGFR) activation and force generation have not been clarified. Herein, we demonstrate that EGF stimulation increases myosin light chain (MLC) phosphorylation, a marker for contractile force, concomitant with protein kinase C (PKC) activity in mouse fibroblasts expressing human EGFR constructs. Interestingly, PKCdelta is the most strongly phosphorylated isoform, and the preferential PKCdelta inhibitor rottlerin largely prevented EGF-induced phosphorylation of PKC substrates and MARCKS. The pathway through which EGFR activates PKCdelta is suggested by the fact that the MEK-1 inhibitor U0126 and the phosphatidylinositol 3-kinase inhibitor LY294002 had no effect on PKCdelta activation, whereas lack of PLCgamma signaling resulted in delayed PKCdelta activation. EGF-enhanced MLC phosphorylation was prevented by a specific MLC kinase inhibitor ML-7 and the PKC inhibitors chelerythrine chloride and rottlerin. Further indicating that PKCdelta is required, a dominant-negative PKCdelta construct or RNAi-mediated PKCdelta depletion also prevented MLC phosphorylation. In the absence of PLC signaling, MLC phosphorylation and cell force generation were delayed similarly to PKCdelta activation. All of the interventions that blocked PKCdelta activation or MLC phosphorylation abrogated EGF-induced cell contractile force generation and motility. Our results suggest that PKCdelta activation is responsible for a major part of EGF-induced fibroblast contractile force generation. Hence, we identify here a new pathway helping to govern cell motility, with PLC signaling playing a role in activation of PKCdelta to promote the acute phase of EGF-induced MLC activation.

Topics: Acetophenones; Alkaloids; Animals; Benzophenanthridines; Benzopyrans; Butadienes; Cell Line; Cell Movement; Chromones; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Fibroblasts; Genes, Dominant; Genetic Vectors; Immunoblotting; Isometric Contraction; Mice; Morpholines; Myosin Light Chains; Nitriles; Phenanthridines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Plasmids; Precipitin Tests; Protein Isoforms; Protein Kinase C; Protein Kinase C-delta; RNA Interference; RNA, Small Interfering; Signal Transduction; Time Factors; Transfection

Epidermal growth factor (EGF) and fibronectin are known to play an important role in wound healing. In this study, we demonstrated that EGF upregulates the expression of fibronectin mRNA and protein in human dermal fibroblasts. Actinomycin D, an RNA synthesis inhibitor, significantly blocked basal mRNA expression, but the addition of EGF compensated the blockage. Cycloheximide, a protein synthesis inhibitor, did not block this upregulation by EGF. In addition, the treatment with EGF significantly reduced the degradation rate of fibronectin mRNA. But EGF did not increase fibronectin promoter activity. EGF-mediated induction of fibronectin expression was inhibited by the treatment of fibroblasts with protein kinase C (PKC) inhibitor, Calphostin C and Rottlerin. The transfection of a dominant-negative mutant of PKCdelta into fibroblasts significantly reduced the induction of fibronectin protein expression by EGF. EGF enhanced PKCdelta protein expression and also translocated PKCdelta to the membrane. Rottlerin blocked the EGF-mediated reduction of mRNA degradation rate. These results indicate that EGF-mediated induction of fibronectin expression occurs at the post-transcriptional level and involves PKCdelta signaling pathway.

Topics: Acetophenones; Benzopyrans; Cells, Cultured; Dermis; Enzyme Inhibitors; Epidermal Growth Factor; Fibroblasts; Fibronectins; Gene Expression Regulation; Humans; Protein Kinase C; Protein Kinase C-alpha; Protein Kinase C-delta; Protein Kinase C-epsilon; RNA, Messenger; Signal Transduction; Up-Regulation

Reactive oxygen species (ROS) are implicated in cardiovascular diseases. ROS, such as H2O2, act as second messengers to activate diverse signaling pathways. Although H2O2 activates several tyrosine kinases, including the epidermal growth factor (EGF) receptor, JAK2, and PYK2, in vascular smooth muscle cells (VSMCs), the intracellular mechanism by which ROS activate these tyrosine kinases remains unclear. Here, we identified two distinct signaling pathways required for receptor and nonreceptor tyrosine kinase activation by H2O2 involving a metalloprotease-dependent generation of heparin-binding EGF-like growth factor (HB-EGF) and protein kinase C (PKC)-delta activation, respectively. H2O2-induced EGF receptor tyrosine phosphorylation was inhibited by a metalloprotease inhibitor, whereas the inhibitor had no effect on H2O2-induced JAK2 tyrosine phosphorylation. HB-EGF neutralizing antibody inhibited H2O2-induced EGF receptor phosphorylation. In COS-7 cells expressing an HB-EGF construct tagged with alkaline phosphatase, H2O2 stimulates HB-EGF production through metalloprotease activation. By contrast, dominant negative PKC-delta transfection inhibited H2O2-induced JAK2 phosphorylation but not EGF receptor phosphorylation. Dominant negative PYK2 inhibited H2O2-induced JAK2 activation but not EGF receptor activation, whereas dominant negative PKC-delta inhibited PYK2 activation by H2O2. These data demonstrate the presence of distinct tyrosine kinase activation pathways (PKC-delta/PYK2/JAK2 and metalloprotease/HB-EGF/EGF receptor) utilized by H2O2 in VSMCs, thus providing unique therapeutic targets for cardiovascular diseases.

Topics: Acetophenones; Adenoviridae; Animals; Benzopyrans; Cells, Cultured; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; Genes, Dominant; Humans; Hydrogen Peroxide; Immunoblotting; Janus Kinase 2; Metalloendopeptidases; Models, Biological; Muscle, Smooth, Vascular; Oxidation-Reduction; Precipitin Tests; Protein Kinase C; Protein Kinase C-delta; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Rats; Rats, Sprague-Dawley; Receptor Protein-Tyrosine Kinases; Signal Transduction; Time Factors; Transfection

Epidermal growth factor (EGF) and TRH both produce enhanced prolactin (PRL) gene transcription and PRL secretion in GH4 rat pituitary tumor cell lines. These agents also activate protein kinase C (PKC) in these cells. Previous studies have implicated the PKCepsilon isozyme in mediating TRH-induced PRL secretion. However, indirect studies using phorbol ester down-regulation to investigate the role of PKC in EGF- and TRH-induced PRL gene transcription have been inconclusive. In the present study, we examined the role of multiple PKC isozymes on EGF- and TRH-induced activation of the PRL promoter by utilizing general and selective PKC inhibitors and by expression of genes for wild-type and kinase-negative forms of the PKC isozymes. Multiple nonselective PKC inhibitors, including staurosporine, bisindolylmaleimide I, and Calphostin C, inhibited both EGF and TRH induced rat PRL promoter activity. TRH effects were more sensitive to Calphostin C, a competitive inhibitor of diacylglycerol, whereas Go 6976, a selective inhibitor of Ca(2+)-dependent PKCs, produced a modest inhibition of EGF but no inhibition of TRH effects. Rottlerin, a specific inhibitor of the novel nPKCdelta isozyme, significantly blocked both EGF and TRH effects. Overexpression of genes encoding PKCs alpha, betaI, betaII, delta, gamma, and lambda failed to enhance either EGF or TRH responses, whereas overexpression of nPKCeta enhanced the EGF response. Neither stable nor transient overexpression of nPKCepsilon produced enhancement of EGF- or TRH-induced PRL promoter activity, suggesting that different processes regulate PRL transcription and hormone secretion. Expression of a kinase inactive nPKCdelta construct produced modest inhibition of EGF-mediated rPRL promoter activity. Taken together, these data provide evidence for a role of multiple PKC isozymes in mediating both EGF and TRH stimulated PRL gene transcription. Both EGF and TRH responses appear to require the novel isozyme, nPKCdelta, whereas nPKCeta may also be able to transmit the EGF response. Inhibitor data suggest that the EGF response may also involve Ca(2+)-dependent isozymes, whereas the TRH response appears to be more dependent on diacylglycerol.

Topics: Acetophenones; Animals; Benzopyrans; Calcium; Carbazoles; Cell Line; Enzyme Inhibitors; Epidermal Growth Factor; Gene Expression Regulation; Indoles; Isoenzymes; Maleimides; Naphthalenes; Phorbol Esters; Pituitary Gland; Prolactin; Promoter Regions, Genetic; Protein Kinase C; Protein Kinase C-delta; Protein Kinase C-epsilon; Rats; Staurosporine; Thyrotropin-Releasing Hormone; Transcription, Genetic

The protein kinase C involvement in bradykinin (BK)-induced phospholipase D (PLD) activation in A-431 cells was examined. Treatment of cells with BK induced the rapid activation of intracellular PLD activity. The PLD activation induced by BK was blocked by pretreatment of A-431 cells with staurosporine, or by prolonged treatment with phorbol-12-myristate-13-acetate (PMA). PKC inhibitors Ro-31-8220 and bisindolylmaleimide I, showed the same inhibitory effects on the BK-stimulated increase of PLD activity, indicating a role of PKC in this activation process. Similar results were observed in PMA-induced PLD activation. In contrast, PKC down-regulation or PKC inhibitors had no obvious effect on the PLD activation stimulated by epidermal growth factor (EGF). Furthermore, rottlerin and Go 6976, the PKC inhibitors specific for PKC-delta, -alpha and -betaI, respectively, markedly inhibited the PLD activity stimulated by BK. These results indicated that PKC, at least PKC-delta and Ca2+-dependent PKC-alpha or -betaI, plays an important role in BK-induced but not EGF-induced PLD activation in A-431 cells.

Topics: Acetophenones; Benzopyrans; Bradykinin; Carbazoles; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; Humans; Immunoblotting; Indoles; Isoenzymes; Kinetics; Maleimides; Phospholipase D; Phospholipids; Protein Kinase C; Staurosporine; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Epidermal growth factor (EGF) receptor-overexpressing p53-deficient A431 cells response to toxic dose of EGF by G1 arrest and apoptosis was studied. We previously reported an increased expression of growth arrest and DNA-damage-inducible gene, Gadd45, in EGF-overexposed A431 cells. The mechanism for this induction was increased half-lives of mRNA and protein. In this study, using phorbol ester (a PKC activator) and specific inhibitors of PKC isoforms, we showed that protein kinase C-delta (PKCdelta) was involved in the increase of Gadd45 protein stability. We further demonstrated that Gadd45 is ubiquitinated and is regulated by proteolysis. While EGF induced ubiquitination of total cellular proteins, there was a decrease in Gadd45 ubiquitination, which could be inhibited by Rottlerin, a PKCdelta-specific inhibitor. These results suggest that an increase in Gadd45 stability may involve PKCdelta-dependent ubiquitin-proteasome pathway.

Topics: Acetophenones; Benzopyrans; Cell Division; Cycloheximide; DNA Damage; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Flavonoids; GADD45 Proteins; Humans; Imidazoles; Intracellular Signaling Peptides and Proteins; Isoenzymes; Kinetics; Leupeptins; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Kinase C; Protein Kinase C-delta; Proteins; Pyridines; Recombinant Proteins; Staurosporine; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Ubiquitins

Downregulation of protein kinase C delta (PKC delta) by treatment with the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) transforms cells that overexpress the non-receptor class tyrosine kinase c-Src (Z. Lu et al., Mol. Cell. Biol. 17:3418-3428, 1997). We extended these studies to cells overexpressing a receptor class tyrosine kinase, the epidermal growth factor (EGF) receptor (EGFR cells); like c-Src, the EGF receptor is overexpressed in several human tumors. In contrast with expectations, downregulation of PKC isoforms with TPA did not transform the EGFR cells; however, treatment with EGF did transform these cells. Since TPA downregulates all phorbol ester-responsive PKC isoforms, we examined the effects of PKC delta- and PKC alpha-specific inhibitors and the expression of dominant negative mutants for both PKC delta and alpha. Consistent with a tumor-suppressing function for PKC delta, the PKC delta-specific inhibitor rottlerin and a dominant negative PKC delta mutant transformed the EGFR cells in the absence of EGF. In contrast, the PKC alpha-specific inhibitor Go6976 and expression of a dominant negative PKC alpha mutant blocked the transformed phenotype induced by both EGF and PKC delta inhibition. Interestingly, both rottlerin and EGF induced substantial increases in phospholipase D (PLD) activity, which is commonly elevated in response to mitogenic stimuli. The elevation of PLD activity in response to inhibiting PKC delta, like transformation, was dependent upon PKC alpha and restricted to the EGFR cells. These data demonstrate that PKC isoforms alpha and delta have antagonistic effects on both transformation and PLD activity and further support a tumor suppressor role for PKC delta that may be mediated by suppression of tyrosine kinase-dependent increases in PLD activity.

Topics: Acetophenones; Animals; Benzopyrans; Carbazoles; Cell Transformation, Neoplastic; Cells, Cultured; Crosses, Genetic; Epidermal Growth Factor; ErbB Receptors; Indoles; Isoenzymes; Models, Genetic; Mutagenesis, Insertional; Phospholipase D; Protein Kinase C; Protein Kinase C-alpha; Protein Kinase C-delta; Rats; Recombinant Proteins; Tetradecanoylphorbol Acetate