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 neurotropic factor (GDNF) are critical for early regulation of ureteric bud development and nephrogenesis. To address whether c-ret directly initiates epithelial cell morphogenesis, the c-ret receptor was expressed in murine inner medullary collecting duct cells (mIMCD-3, a cell line of ureteric bud origin, which has no detectable endogenous c-ret expression). Stable expression of wild-type c-ret was found to yield a constitutively tyrosine-phosphorylated receptor, with no change after the addition of GDNF. Examination of mRNA from these cells demonstrated the message for endogenous GDNF, suggesting that c-ret was potentially being constitutively activated by an autocrine 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 points 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 culture (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 collagen 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 that activated c-ret appears to mediate epithelial morphogenesis by prolonging cell survival and, in conjunction with activation of the morphogenic receptors c-met and the EGF receptor, initiates the events required for very early branching morphogenesis.
O'Rourke
D A
DA
Division of Nephrology, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215, USA.
Sakurai
H
H
Spokes
K
K
Kjelsberg
C
C
Takahashi
M
M
Nigam
S
S
Cantley
L
L
D000818:Animals; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology; D002470:Cell Survival / Q000502:physiology; D003094:Collagen; D004285:Dogs; D029721:Drosophila Proteins; D004847:Epithelial Cells / Q000378:metabolism; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; D017228:Hepatocyte Growth Factor / Q000494:pharmacology; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D007679:Kidney Medulla; D007685:Kidney Tubules, Collecting / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000378:metabolism / Q000502:physiology*; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism / Q000502:physiology*
D029721:Drosophila Proteins; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; D011518:Proto-Oncogene Proteins; D017228:Hepatocyte Growth Factor; D003094:Collagen; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases; C416649:Ret protein, Drosophila; C494999:Ret protein, mouse