BACKGROUND & AIMS: Incubation of purified (>95%) canine parietal cells in primary culture with epidermal growth factor for 7-16 hours stimulates H(+)K(+)-adenosine triphosphatase gene expression. In this study, we examined the effect of prolonged stimulation (72 hours) of the parietal cells with epidermal growth factor. METHODS: H(+)K(+)-adenosine triphosphatase protein and gene expression were assessed by immunohistochemistry and Northern blots. Mitogen-activated protein kinase and Akt activation were quantitated by kinase assays and Western blots with specific antiphospho antibodies. Akt overexpression was achieved by adenovirus-mediated gene transfer of a constitutively active Akt gene. RESULTS: Epidermal growth factor changed the morphology of the cultured cells, which acquired the appearance of fusiform cells, and it inhibited H(+)K(+)-adenosine triphosphatase gene expression. Staining of the cells both with anti-H(+)K(+)-adenosine triphosphatase antibodies and with Texas Red-labeled Dolichos biflorus lectin confirmed that the fusiform cells expressed markers of parietal cell differentiation. Epidermal growth factor stimulated mitogen-activated protein kinase with 2 peaks of activation, observed after 5 minutes and 72 hours, whereas it activated Akt after 5 minutes but not 72 hours of incubation. Overexpression of Akt blocked both epidermal growth factor-induced morphological transformation and inhibition of H + K + -adenosine triphosphatase gene expression. Identical results were observed in the presence of the mitogen-activated protein kinase inhibitor PD98059. CONCLUSIONS: Activation of the Akt signal-transduction pathway seems to be a crucial event for the induction of parietal cell maturation and differentiation.
BACKGROUND & AIMS: Incubation of purified (>95%) canine parietal cells in primary culture with epidermal growth factor for 7-16 hours stimulates H(+)K(+)-adenosine triphosphatase gene expression. In this study, we examined the effect of prolonged stimulation (72 hours) of the parietal cells with epidermal growth factor. METHODS: H(+)K(+)-adenosine triphosphatase protein and gene expression were assessed by immunohistochemistry and Northern blots. Mitogen-activated protein kinase and Akt activation were quantitated by kinase assays and Western blots with specific antiphospho antibodies. Akt overexpression was achieved by adenovirus-mediated gene transfer of a constitutively active Akt gene. RESULTS: Epidermal growth factor changed the morphology of the cultured cells, which acquired the appearance of fusiform cells, and it inhibited H(+)K(+)-adenosine triphosphatase gene expression. Staining of the cells both with anti-H(+)K(+)-adenosine triphosphatase antibodies and with Texas Red-labeled Dolichos biflorus lectin confirmed that the fusiform cells expressed markers of parietal cell differentiation. Epidermal growth factor stimulated mitogen-activated protein kinase with 2 peaks of activation, observed after 5 minutes and 72 hours, whereas it activated Akt after 5 minutes but not 72 hours of incubation. Overexpression of Akt blocked both epidermal growth factor-induced morphological transformation and inhibition of H + K + -adenosine triphosphatase gene expression. Identical results were observed in the presence of the mitogen-activated protein kinase inhibitor PD98059. CONCLUSIONS: Activation of the Akt signal-transduction pathway seems to be a crucial event for the induction of parietal cell maturation and differentiation.
Authors: Andrew J Bredemeyer; Jessica H Geahlen; Victoria G Weis; Won Jae Huh; Bernd H Zinselmeyer; Subhashini Srivatsan; Mark J Miller; Andrey S Shaw; Jason C Mills Journal: Dev Biol Date: 2008-10-31 Impact factor: 3.582