ABSTRACT
TRH has been found to stimulate tyrosine phosphorylation of the epidermal growth factor (EGF) receptor. A specific EGF receptor kinase inhibitor, tyrphostin AG1478, substantially reduced TRH-stimulated tyrosine phosphorylation of the EGF receptor. TRH-induced EGF receptor phosphorylation was found to lead to the recruitment of the adapter proteins Grb2 and Shc. TRH treatment also led to phosphorylation of the related receptor tyrosine kinase, HER2. HER2 activation likely contributes to downstream signaling events and enhances EGF receptor action. TRH-induced tyrosine phosphorylation of the EGF receptor was reduced by incubation with a protein kinase C (PKC) kinase inhibitor, GF109203X. EGF receptor phosphorylation was required for full TRH-induced activation of mitogen-activated protein kinase (MAPK) and stimulation of specific transcriptional responses.
Subject(s)
ErbB Receptors/metabolism , Thyrotropin-Releasing Hormone/pharmacology , Animals , Enzyme Inhibitors/pharmacology , Genes, Reporter , Indoles/pharmacology , Luciferases/genetics , Maleimides/pharmacology , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation , Phosphotyrosine/metabolism , Pituitary Neoplasms , Protein Kinase C/metabolism , Quinazolines , Rats , Recombinant Proteins/biosynthesis , Transcriptional Activation , Transfection , Tumor Cells, Cultured , Tyrphostins/pharmacologyABSTRACT
The mouse Wnt-1 gene plays an essential role in fetal brain development and can contribute to tumorigenesis when activated aberrantly in the mammary gland. The gene encodes secretory glycoproteins associated with the extracellular or pericellular matrix, and it has been proposed that Wnt-1, as well as its Drosophila homolog wingless, may function in intercellular signalling. We show here that fibroblasts expressing Wnt-1 protein, although not transformed themselves, are able to elicit morphological transformation of neighboring C57MG mammary epithelial cells in coculture experiments. Heparin inhibits this effect, possibly by displacing Wnt-1 protein from its normal site of action. Our results indicate that the Wnt-1 gene can act via a paracrine mechanism in cell culture and strongly support the notion that in vivo the gene may function in cell-to-cell communication.
Subject(s)
Cell Transformation, Neoplastic/genetics , Mammary Glands, Animal , Proto-Oncogene Proteins/genetics , Zebrafish Proteins , Animals , Cell Line , Cells, Cultured , Epithelium , Fibroblasts , Heparin/pharmacology , Immunoblotting , Mice , Wnt Proteins , Wnt1 ProteinABSTRACT
The mouse genome contains a retrovirus-like sequence, designated VL30, which is expressed at high levels in transformed cells and which can be induced by exogenously supplied epidermal growth factor (EGF). Binding of EGF to the EGF receptor produces changes in intracellular calcium levels and phospholipase activity which indirectly lead to activation of protein kinase C. We treated AKR-2B cells, Swiss 3T3 cells, and the 3T3 variants NR6 (EGF receptorless) and TNR9 (phorbol ester nonresponsive) with various phorbol ester tumor promoters and with the synthetic diacylglycerol sn-1,2-dioctanoylglycerol. Tumor-promoting phorbol esters (e.g. 12-O-tetradecanoyl phorbol acetate (TPA] increased the level of VL30 expression. Stimulation with either TPA or EGF produced a similar time course of VL30 expression. TPA induced VL30 expression in the EGF-receptorless NR6 cell line, indicating that neither EGF ligand-receptor binding nor phosphorylation of the EGF receptor was required for induction of VL30 expression. Protein synthesis was not required for the TPA-mediated increase in VL30 expression, as pretreatment with cycloheximide did not block or reduce the TPA effect. VL30 expression was also stimulated by treatment with sn-1,2-dioctanoylglycerol, an analog of a probable endogenous activator of protein kinase C. These results suggest that activation of protein kinase C plays a direct role in regulating VL30 expression.
