epidermal-growth-factor and tyrphostin-25

Phospholipase Cgamma1 (PLCgamma1) catalyses hydrolysis of phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and inositol 1,4,5-trisphosphate (IP(3)), two second messengers which play important roles in cell proliferation and differentiation. The purpose of the current study was to identify PLCgamma1 in corneal epithelial cells and investigate whether epidermal growth factor (EGF) stimulates the activity of this enzyme. Addition of EGF to [(3)H]myo-inositol-labeled, cultured corneal epithelial cells stimulated production of IP(3), indicating activation of PLC. Western immunoblot analysis and an in vitro assay of PLC activity revealed that EGF activates gamma1 isoform of PLC, which is localized predominantly in the cytosolic fraction of the epithelial cells. EGF receptors were detected in the epithelial cells by EGF receptor antibody. Addition of EGF to the cells caused tyrosine phosphorylation of the receptors, translocation of PLCgamma1 from cytosol to plasma membrane, and phosphorylation of the enzyme at tyrosine residues. Addition of tyrphostin A-25, an inhibitor of receptor tyrosine kinase, attenuated the tyrosine phosphorylation of PLCgamma1 as well as its enzyme activity. These findings suggest that EGF stimulates PLCgamma1 in rabbit corneal epithelial cells, and that this effect is probably mediated by tyrosine phosphorylation of the enzyme.

Topics: Animals; Epidermal Growth Factor; Epithelial Cells; Epithelium, Corneal; ErbB Receptors; Hydrolysis; Isoenzymes; Phospholipase C gamma; Phosphorylation; Rabbits; Receptor Protein-Tyrosine Kinases; Recombinant Proteins; Type C Phospholipases; Tyrosine; Tyrphostins

To study whether the esophageal mucosa was able to elicit mucosal adaptation, we induced esophageal damage by perfusing acidified pepsin in rabbits. Mucosal adaptation was induced by preexposing the esophageal mucosa to a mild irritant (acidified saline) for 60 min prior to acidified pepsin (strong irritant). Macroscopic and microscopic esophageal injury, cell proliferation, and mucosal barrier function (H+, K+, hemoglobin flux rates) were studied. Preexposure of the esophageal mucosa to acidified saline significantly decreased both the mucosal damage and the mucosal barrier dysfunction induced by acidified pepsin. The development of this phenomenon was nondependent on cell proliferation. Concomitant treatment with either the nitric oxide synthase inhibitor, N(G)-nitro-L-arginine, or the perfusion of immunospecific EGF-receptor antibodies or tyrphostin-25, an inhibitor of the tyrosine kinase activities ligated to the intracytoplasmatic domain of the EGF receptor, during the preexposure period completely reversed the protection induced by acid. We conclude that the rabbit esophageal mucosa shows mucosal adaptation to acid and pepsin. The development of this phenomenon is fast, not dependent on cell proliferation, and dependent, at least in part, on nitric oxide and EGF-receptor-mediated mechanisms.

Topics: Adaptation, Physiological; Animals; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Esophagitis, Peptic; Esophagus; Mucous Membrane; Nitric Oxide; Nitric Oxide Synthase; Nitriles; Nitroarginine; Pepsin A; Protein-Tyrosine Kinases; Rabbits; Sodium Chloride; Tyrphostins

Exposing normal human breast epithelia (HBE) cells, which were growth arrested by a 3-day culture in EGF-deprived medium, to the microtubule disrupting agent, demecolcin (N-deacetyl-N-methyl-colchicine), for 2 hr significantly stimulated the initiation of DNA synthesis 22 hr later. The demecolcin-induced DNA synthesis was not accompanied by activation of the EGF receptor and it was inhibited by calphostin C, an inhibitor of protein kinase C (PKC), and U-73122, an inhibitor of phospholipase C (PLC). Contrary to this, the EGF-induced DNA synthesis was inhibited by tyrphostin A25, a specific inhibitor of the EGF receptor tyrosine kinase, and calphostin C. The results suggested that the involvement of PLC and PKC in the demecolcin-induced signal transduction pathway leads to the initiation of DNA synthesis.

Topics: Androstadienes; Breast; Cell Line; Cinnamates; Demecolcine; DNA; DNA Replication; Enzyme Inhibitors; Epidermal Growth Factor; Epithelium; ErbB Receptors; Estrenes; Female; Humans; Kinetics; Microtubules; Naphthalenes; Nitriles; Phosphorylation; Protein Kinase C; Pyrrolidinones; Type C Phospholipases; Tyrphostins; Wortmannin

FG human pancreatic carcinoma cells adhere to vitronectin using integrin alpha v beta 5 yet are unable to migrate on this ligand whereas they readily migrate on collagen in an alpha 2 beta 1-dependent manner. We report here that epidermal growth factor receptor (EGFR) activation leads to de novo alpha v beta 5-dependent FG cell migration on vitronectin. The EGFR specific tyrosine kinase inhibitor tyrphostin 25 selectively prevents EGFR autophosphorylation thereby preventing the EGF-induced FG cell migration response on vitronectin without affecting constitutive migration on collagen. Protein kinase C (PKC) activation also leads to alpha v beta 5-directed motility on vitronectin; however, this is not blocked by tyrosine kinase inhibitors. In this case, PKC activation appears to be associated with and downstream of EGFR signaling since calphostin C, an inhibitor of PKC, blocks FG cell migration on vitronectin induced by either PKC or EGF. These findings represent the first report implicating a receptor tyrosine kinase in a specific integrin mediated cell motility event independent of adhesion.

Topics: Cell Adhesion; Cell Line; Cell Movement; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Flow Cytometry; Genistein; Glycoproteins; Humans; Insulin-Like Growth Factor I; Integrins; Isoflavones; Kinetics; Naphthalenes; Nitriles; Pancreatic Neoplasms; Phosphotyrosine; Polycyclic Compounds; Protein Kinase C; Receptor Protein-Tyrosine Kinases; Receptor, IGF Type 1; Receptors, Vitronectin; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Tyrosine; Tyrphostins; Vitronectin