Expression of c-ret promotes morphogenesis and cell survival in mIMCD-3 cells

c-Ret, a protein tyrosine kinase receptor, and its ligand glial-derived neu rotropic factor (GDNF) are critical for early regulation of ureteric bud de velopment and nephrogenesis. To address whether c-ret directly initiates ep ithelial cell morphogenesis, the c-ret receptor was expressed in murine inn er medullary collecting duct cells (mIMCD-3, a cell line of ureteric bud or igin, which has no detectable endogenous c-ret expression). Stable expressi on of wild-type c-ret was found to yield a constitutively tyrosine-phosphor ylated receptor, with no change after the addition of GDNF. Examination of mRNA from these cells demonstrated the message for endogenous GDNF, suggest ing that c-ret was potentially being constitutively activated by an autocri ne mechanism. When mIMCD-3 cells stably expressing the phosphorylated c-ret receptor were cultured in a type I collagen matrix, they exhibited little GDNF-independent or -dependent branching process formation at early time po ints compared with the known morphogen hepatocyte growth factor (HGF) (48 h ; control, 0.33 +/- 0.33; GDNF, 1.0 +/- 0.58, P = nonsignificant; and HGF, 6.33 +/- 0.33 processes/20 cell clusters, P < 0.001), whereas extended cult ure (7 days) under serum-free conditions revealed a marked increase in cell survival and the spontaneous development of rudimentary branching process formation. Extended culture (7 days) of c-ret-expressing clones in type I c ollagen with the epithelial morphogens HGF and/or epidermal growth factor ( EGF) resulted in the development of complex three-dimensional spiny cysts, whereas parental mIMCD-3 cells died under these conditions. We conclude tha t activated c-ret appears to mediate epithelial morphogenesis by prolonging cell survival and, in conjunction with activation of the morphogenic recep tors c-met and the EGF receptor, initiates the events required for very ear ly branching morphogenesis.