epidermal-growth-factor and erbstatin

Epidermal growth factor (EGF) decreases Na(+) reabsorption across distal nephron epithelia. Activity of the epithelial Na(+) channel (ENaC) is limiting for Na(+) transport in this portion of the nephron. Abnormal ENaC activity and EGF signaling are both associated with polycystic kidney disease localized to the distal nephron. We tested here whether EGF and other ligands for receptor tyrosine kinases (RTK) decrease ENaC activity. EGF markedly and quickly decreased ENaC activity. The RTK inhibitor erbstatin blocked EGF actions on ENaC and when added alone increased channel activity, uncovering basal suppression by endogenous RTK. The protein tyrosine phosphatase inhibitor vanadate, similar to EGF, decreased ENaC activity. Growth factors and vanadate decreased ENaC activity by decreasing open probability. ENaC was not phosphorylated in response to EGF, indicating that intermediary proteins transduce the inhibitory signal from the EGF receptor (EGFR) to ENaC. We find that neither MAPK 1/2 nor c-Src is signaling intermediaries between EGFR and ENaC. Inhibition of ENaC paralleled decreases in plasma membrane phosphatidylinositol 4,5-bisphosphate levels [PtdIns(4,5)P(2)] and was abolished by clamping PtdIns(4,5)P(2). We conclude that EGF and other ligands for RTK decrease ENaC open probability by decreasing membrane PtdIns(4,5)P(2) levels.

Topics: Animals; CHO Cells; Cricetinae; Cricetulus; Epidermal Growth Factor; Epithelial Cells; Hydroquinones; Membrane Potentials; Nephrons; Phosphatidylinositol 4,5-Diphosphate; Receptor Protein-Tyrosine Kinases; Signal Transduction; Sodium Channels

Epidermal growth factor (EGF) has been shown to influence FSH-stimulated estradiol (E2) and progesterone (P4) production from granulosa cells. RG 50810, a tyrosine kinase inhibitor (TKI), has previously been shown to inhibit the EGF-receptor tyrosine kinase. RG 50810 has also been shown to inhibit FSH-stimulated increases in mRNA for steroidogenic enzymes, implying a functional role of tyrosine kinases in FSH action in granulosa cells. However, inhibition of FSH-stimulated steroidogenesis by TKIs has not been evaluated in connection with the effects of EGF in granulosa cells. In the present studies, FSH-stimulated E2 production was inhibited similarly by inhibitors of protein kinase A (H-89) and protein kinase C (calphostin C) and by TKIs, and none of the inhibitors were capable of reversing the EGF-induced inhibition of FSH-stimulated E2 production. FSH-stimulated P4 production was enhanced dramatically in serum-containing medium with concentrations of TKI that were near previously reported IC50s. The enhancing effect of TKIs was less evident in serum-free medium. Addition of EGF to serum-free medium enhanced FSH-stimulated P4 production, and the TKIs reversed EGF-enhanced P4 production, but in a manner similar to that of protein kinase A inhibitor H-89. Compared to results in serum-free medium, the potency of RG 50810 and genistein to inhibit the effects of EGF on P4 production was 3- to 8-fold greater relative to H-89. These studies have demonstrated that TKIs RG 50810 and genistein selectively inhibit the effects of EGF on FSH-stimulated P4 production in granulosa cell cultures. In contrast, these studies have demonstrated nonselective inhibition of FSH-stimulated E2 and P4 production by TKIs in serum-free medium, in which it is not clear which enzyme system is affected by the compounds tested.

Topics: Animals; Cells, Cultured; Culture Media, Serum-Free; Enzyme Inhibitors; Epidermal Growth Factor; Estradiol; Female; Follicle Stimulating Hormone; Genistein; Granulosa Cells; Hydroquinones; Isoquinolines; Naphthalenes; Progesterone; Protein-Tyrosine Kinases; Rats; Rats, Wistar; Steroids; Sulfonamides; Tyrphostins

Epidermal growth factor (EGF) is a potent mitogen for normal mouse mammary epithelial cells grown in primary culture. EGF activation of the EGF-receptor (EGF-R) induces intrinsic tyrosine kinase activity which results in EGF-R autophosphorylation and tyrosine phosphorylation of other intracellular substrates involved in EGF-R signal transduction. Genistein and erbstatin are anticancer agents which have been shown to be potent tyrosine kinase inhibitors. However, the effects of these compounds in modulating EGF-dependent normal mammary epithelial cell proliferation is presently unknown. Therefore, studies were conducted to determine the effects of genistein and erbstatin on EGF-dependent proliferation, and EGF-R levels and autophosphorylation in normal mouse mammary epithelial cells grown in primary culture and maintained in serum-free media. Chronic treatment with 6.25-100 microM genistein or 1-16 microM erbstatin significantly decreased EGF-dependent mammary epithelial cell proliferation in a dose-responsive manner. However, the highest doses of genistein (100 microM) and erbstatin (16 microM) were found to be cytotoxic. Additional studies showed that acute treatment with 6.25-400 microM genistein did not affect EGF-R levels or EGF-induced EGF-R autophosphorylation, while acute treatment with 1-64 microM erbstatin caused a slight reduction in EGF-R levels, but had no effect on EGF-dependent EGF-R autophosphorylation in these cells. In contrast, chronic treatment with similar doses of genistein or erbstatin resulted in a large dose-responsive decrease in EGF-R levels, and a corresponding decrease in total cellular EGF-R autophosphorylation intensity. These results demonstrate that the inhibitory effects of chronic genistein and erbstatin treatment on EGF-dependent mammary epithelial cell proliferation is not due to a direct inhibition of EGF-R tyrosine kinase activity, but results primarily from a down-regulation in EGF-R levels and subsequent decrease in mammary epithelial cell mitogenic-responsiveness to EGF stimulation.

Topics: Animals; Cell Division; Cells, Cultured; Down-Regulation; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Female; Genistein; Growth Inhibitors; Hydroquinones; Mammary Glands, Animal; Mice; Mice, Inbred BALB C

Since the majority of endometrial carcinomas do not contain any detectable ras mutations, the precise contribution of aberrant Ras function, if any, to endometrial carcinoma development remains to be determined. Since there is considerable evidence that Ras transformation is associated with a decreased requirement for growth factors, we compared the growth response of endometrial carcinoma cells harbouring wild-type (Ishikawa cells) or mutated (HHUA cells) K-ras to epidermal growth factor (EGF). K-ras mutation did not significantly affect the level of the EGF receptor (EGFR) expressed in these carcinoma cells. EGF could stimulate the growth of Ishikawa, but not HHUA cells. Furthermore, EGF caused elevation of Ras-GTP levels in Ishikawa, but not HHUA cells. However, the introduction of mutated, but not normal, K-ras into Ishikawa cells rendered them non-responsive to EGF growth stimulation. Thus, the presence of mutated K-ras alone modulated the growth response of endometrial carcinoma cells to EGF. An inhibitor of the EGFR tyrosine kinase activity could prevent soft agar colony formation of Ishikawa cells, but not HHUA or mutant K-ras(12V)-transfected Ishikawa cells. Taken together, these results suggest that mutated K-ras causes a loss of responsiveness to EGF stimulation and that EGFR function is dispensable for the growth of mutant Ras-positive endometrial carcinoma cells.

Topics: Apoptosis; Cell Division; Endometrial Neoplasms; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Female; Genes, ras; Humans; Hydroquinones; Mutation; Signal Transduction; Transforming Growth Factor alpha; Tumor Cells, Cultured

Topics: Arachidonate 12-Lipoxygenase; Arachidonic Acid; Carcinoma, Squamous Cell; Enzyme Induction; Enzyme Inhibitors; Epidermal Growth Factor; Humans; Hydroquinones; Isoenzymes; Microsomes; Protein Kinase C; Protein-Tyrosine Kinases; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The modulation by epidermal growth factor (EGF) of the Na+/H+ antiport in fetal and adult rat hepatocytes was studied in nominally HCO3- free solution. EGF (10 nM) activated the antiport in adult rat hepatocytes by 0.22 +/- 0.03 (mean +/- SD;n=10) pH units over basal value, measured with the fluorescent pH-sensitive intracellular probe, 2',7'-bis(carboxyethyl)-5(6)- carboxyfluorescein (BCECF). The effect of EGF was inhibited by amiloride analogue 5-(N-ethyl-N-isopropyl) amiloride (EIPA), by ouabain, inhibitor of the Na+ pump, and by erbstatin analogue, an inhibitor of the tyrosine kinase activity of the EGF receptor. The effect of EGF on Na+/H+ antiport in adult rat hepatocytes appeared to be mediated by both protein kinase C (PKC) and G protein system. No effect of EGF and phorbol 12-myristate 13-acetate, an activator of PKC, on the Na+/H+ antiport was observed in fetal hepatocytes of 20 and 22 days. A different sensitivity of the antiport to high concentrations of amiloride and EIPA suggests that altered amount of the Na+/H+ antiport units or different isoforms could be expressed in fetal compared with adult cells.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Animals; Cells, Cultured; Enzyme Inhibitors; Epidermal Growth Factor; Fetus; Fluoresceins; Fluorescent Dyes; GTP-Binding Proteins; Hydroquinones; Kinetics; Liver; Ouabain; Protein Kinase C; Protein-Tyrosine Kinases; Rats; Rats, Wistar; Sodium-Hydrogen Exchangers; Tetradecanoylphorbol Acetate

1. The induction of cyclo-oxygenase-2 (COX-2) afforded by bacterial lipopolysaccharide (LPS, endotoxin) in bovine aortic endothelial cells (BAEC) is mediated by tyrosine kinase. LPS also causes the generation of several cytokines including interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). This study investigates whether endogenous IL-1 beta, TNF-alpha, EGF or PDGF contribute to the induction of COX-2 elicited by LPS in BAEC and if their action is due to activation of tyrosine kinase. Furthermore, we have studied the induction of COX-2 by exogenous cytokines. 2. Accumulation of 6-oxo-prostaglandin (PG) F1 alpha in cultures of BAEC was measured by radioimmunoassay at 24 h after addition of either LPS (1 microgram ml-1) alone or LPS together with a polyclonal antibody to one of the various cytokines. In experiments designed to measure 'COX activity', 6-oxo-PGF1 alpha generated by BAEC activated with recombinant human IL-1 beta, TNF-alpha, EGF or PDGF for 12 h was measured after incubation of washed cells with exogenous arachidonic acid (30 microM for 15 min). Western blot analysis determined the expression of COX-2 protein in BAEC. 3. The accumulation of 6-oxo-PGF1 alpha caused by LPS in BAEC was attenuated by co-incubation with one of the polyclonal antibodies, anti-IL-1 beta, anti-TNF-alpha, anti-EGF, anti-PDGF or with the IL-1 receptor antagonist, in a dose-dependent manner. Exogenous IL-1 beta, TNF-alpha or EGF also caused an increase in COX activity, while PDGF was ineffective. The increase in COX activity elicited by IL-1,beta(10 ng ml-1), TNF-alpha (100 ng ml-1) or EGF (1000 ng ml-1) in BAEC was attenuated by erbstatin (0.005 to 5 microg ml-1), as was the expression of COX-2 protein measured by Western blot analysis.4. PDGF (10 ng ml-1) significantly augmented the rise in COX activity and COX-2 protein caused by shorter incubation of BAEC with LPS (1 microg ml-1 for 3 h). Combination of PDGF (10 ng ml-1) with a low concentration of IL-l beta (1 ng ml-1) for 12 h, also increased 'COX activity', but combination of PDGF and TNF-alpha (10 ng ml-1) did not show any increased activity.5. These results suggest that (i) the induction of COX activity and COX-2 protein elicited by LPS in BAEC is mediated by TNF-alpha with lesser contributions from PDGF, EGF or IL-1 beta; (ii) exogenous IL-1 beta,TNF-alpha or EGF alone induce COX-2 activity and protein in BAEC; (iii)

Topics: 6-Ketoprostaglandin F1 alpha; Analysis of Variance; Animals; Aorta; Arachidonic Acid; Blotting, Western; Cattle; Cells, Cultured; Cytokines; Dose-Response Relationship, Drug; Endothelium, Vascular; Enzyme Activation; Enzyme Induction; Enzyme Inhibitors; Epidermal Growth Factor; Humans; Hydroquinones; Interleukin-1; Lipopolysaccharides; Platelet-Derived Growth Factor; Prostaglandin-Endoperoxide Synthases; Protein-Tyrosine Kinases; Radioimmunoassay; Recombinant Proteins; Tumor Necrosis Factor-alpha

Expression of epidermal growth factor (EGF) receptor is often increased in various human carcinomas. Therefore, inhibition of the EGF/EGF receptor-induced signaling pathway may help to suppress these carcinomas. In the presence of Ca2+, EGF induces elongation of A431 cells in approximately 30 min. The cell elongation was shown to be accompanied by a reorganization of actin filaments. These phenotypical changes were specifically inhibited by a tyrosine kinase inhibitor, erbstatin, and inhibitors of phosphatidylinositol (PI) turnover such as psi-tectorigenin and inostamycin. The amount of filamentous actin was increased by EGF, which was also inhibited by these compounds. Long-term treatment of A431 cells with EGF induced the disappearance of cytoskeleton and aggregation of the cells, which was again inhibited by the PI turnover inhibitors. Thus tyrosine kinase and phosphatidylinositol turnover inhibitors were shown to inhibit the signaling pathways of EGF-induced cytoskeletal organization of A431 cells.

Topics: Actins; Calcium; Carcinoma; Cell Aggregation; Cytoskeleton; Epidermal Growth Factor; Furans; Humans; Hydroquinones; Isoflavones; Microscopy, Phase-Contrast; Phosphatidylinositols; Protein-Tyrosine Kinases; Tumor Cells, Cultured

Topics: Animals; Cell Division; Cell Line; DNA Replication; Epidermal Growth Factor; ErbB Receptors; Humans; Hydroquinones; Phosphorylation; Protein-Tyrosine Kinases; Rats; Streptomyces

The effect of tyrosine kinase inhibitor, erbstatin, on cell growth and mRNA expression of growth-factor/receptor system was examined in 6 human gastric-carcinoma cell lines. Erbstatin inhibited both EGF-induced and serum-stimulated cell growth of all 6 cell lines (TMK-1, MKN-1, -7, -28, -45, -74) in a dose-dependent manner. 3H-thymidine incorporation by TMK-1 cells was also suppressed by erbstatin. Erbstatin inhibited protein kinase activity of EGF receptor, p185ERBB2 and pp60c-src in TMK-1 cells. The expression of mRNA of EGF receptor gene and ERBB-2 by TMK-1 cells was not changed by erbstatin treatment, whereas that of c-src was slightly decreased. Interestingly, erbstatin decreased membrane-bound TGF-alpha precursor as measured by anti-TGF-alpha antibody-binding assay, although mRNA expression for TGF-alpha was not altered by erbstatin. Our findings suggest that erbstatin may act as a growth inhibitor for human gastric-carcinoma cells and may not only inhibit tyrosine kinase activities but also negatively modulate the post-transcriptional step of TGF-alpha expression.

Topics: Cell Division; Cell Line; Culture Media; DNA Replication; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Humans; Hydroquinones; Kinetics; Protein-Tyrosine Kinases; Proto-Oncogenes; RNA, Messenger; Stomach Neoplasms; Transcription, Genetic; Transforming Growth Factor alpha

Erbstatin, a tyrosine kinase inhibitor, inhibited epidermal growth factor (EGF)-induced inositol phosphate production in cultured A431 cells. However, it did not inhibit ATP-induced inositol phosphate production. Cytosolic but not membrane-associated phospholipase C was activated by EGF, and erbstatin inhibited enhancement of the phospholipase C activity in EGF-treated cells. Thus, tyrosine kinase of A431 cells is suggested to be functionally involved in phospholipase C activation.

Topics: Cell Line; Cell Membrane; Cytosol; Enzyme Activation; Epidermal Growth Factor; Humans; Hydroquinones; Inositol; Inositol Phosphates; Kinetics; Protein-Tyrosine Kinases; Type C Phospholipases

We prepared methyl 2,5-dihydroxycinnamate as a stable analogue of erbstatin, a tyrosine kinase inhibitor. This analogue was about 4 times more stable than erbstatin in calf serum. It inhibited epidermal growth factor receptor-associated tyrosine kinase in vitro with an IC50 of 0.15 micrograms/ml. It also inhibited in situ autophosphorylation of epidermal growth factor receptor in A431 cells. Methyl 2,5-dihydroxycinnamate was shown to delay the S-phase induction by epidermal growth factor in quiescent normal rat kidney cells, without affecting the total amount of DNA synthesis. The effect of erbstatin on S-phase induction was smaller, possibly because of its shorter life time.

Topics: Animals; Cell Cycle; Cells, Cultured; DNA; Epidermal Growth Factor; ErbB Receptors; Hydroquinones; Kidney; Phosphorylation; Protein-Tyrosine Kinases; Rats; Thymidine

Erbstatin inhibited the kinase activity of the receptor for epidermal growth factor in cultured A431 cells, while it did not alter turnover of the receptor protein. It also inhibited autophosphorylation of the src gene product p60src in Rous sarcoma virus-infected normal rat kidney cells. Erbstatin did not inhibit the binding of epidermal growth factor to its receptor but did inhibit internalization of epidermal growth factor-receptor complexes. It did not inhibit the epidermal growth factor-stimulated phosphatidylinositol turnover in A431 cells. Thus, erbstatin inhibited two oncogene product-related tyrosine protein kinases in situ and thus is a useful tool to study the role of tyrosine protein kinase.

Topics: Animals; Cells, Cultured; Epidermal Growth Factor; ErbB Receptors; Hydroquinones; Phosphatidylinositols; Phospholipids; Phosphorylation; Protein-Tyrosine Kinases; Rats