Much attention has recently focused upon hepatocyte growth factor (HGF) as a potential regulator of epithelial branching morphogenesis. However, since neither the HGF nor c-met "knockout" mice show abnormal kidney branching morphogenesis, we sought to analyze the relative importance of HGF in in vitro branching morphogenesis compared with other factors secreted by the embryonic kidney. Exploiting an assay that employs kidney epithelial cells (murine inner medullary collecting duct, mIMCD3) seeded in collagen cocultured with the embryonic kidney, we found that a tyrosine kinase inhibitor that is highly specific for the epidermal growth factor (EGF) receptor (EGFR), tyrphostin AG1478, inhibited mIMCD3 cell process formation (an early step in branching tubulogenesis) by 40%, whereas high concentrations of neutralizing anti-HGF antibodies had a lesser effect (20% inhibition), suggesting that EGFR ligands account for a larger fraction of branching morphogens secreted by the embryonic kidney than HGF. In addition, when an embryonic epithelial cell line derived from c-met (-/-) mice was cocultured with the embryonic kidney, these c-met (-/-) cells underwent process formation. EGFR ligands but not HGF were able to induce branching tubulogenesis in these cells. All EGFR ligands tested, including EGF, transforming growth factor-alpha, heparin-binding EGF, betacellulin, and amphiregulin, induced mIMCD3 cell tubulogenesis. EGFR ligands caused upregulation of urokinase, urokinase receptor, and matrix metalloprotease-1, and tubulogenesis could be inhibited by the metalloprotease inhibitor 1,10-phenanthroline. Our results support the notion that multiple parallel and potentially redundant growth factor-dependent pathways regulate branching tubulogenesis.

Grb2-associated binder 1 (GAB1) is a docking protein found to associate with the activated c-MET receptor via the MET-binding domain (MBD) and appears to be critical for the tubulogenic actions of this receptor. Pulldown experiments with bacterially expressed MBD and full-length GAB1 revealed the presence of c-MET as well as phosphorylated ERK2 (pERK2). By using purified pERK2 and non-pERK2, we found that GAB1 associates exclusively with the phosphorylated form of the enzyme and that this association does not require mediation by a third protein. When epitope-tagged GAB1 was cotransfected with constitutively active MEK1 into A293 cells, co-immunoprecipitation of GAB1 and pERK2 was observed, demonstrating that this interaction can occur in intact cells. In vitro, both the MBD and full-length GAB1 were found to be substrates for activated ERK2. In intact cells, epitope-tagged GAB1 was found to be basally phosphorylated on serine with an increase following co-transfection with constitutively active MEK1 and the appearance of novel phosphorylation sites detected by phosphopeptide mapping. Thus, it appears that GAB1 can associate directly with phosphorylated ERK2 via the MET-binding domain and that GAB1 then acts as a substrate for the enzyme.In cultured epithelial cells, the tyrosine kinase receptor c-MET and its ligand hepatocyte growth factor (HGF) 1 are capable of inducing mitogenesis, motogenesis (scattering/chemotaxis), and morphogenesis (formation of branching tubules) (1). In the kidney, these distinct actions may comprise the components of more complex events such as ureteric bud branching and repair of renal tubules after injury. Whereas mitogenesis and motogenesis have been described following activation of many tyrosine kinase receptors, epithelial tubulogenesis appears to be relatively specific for ligand activation of members of the MET receptor family and the epidermal growth factor receptor (EGFR) family (2). Recently a docking protein, GAB1 (Grb-2-associated binding protein 1), was found to associate with both c-MET and the EGFR in a ligand-dependent fashion and to be capable of inducing the tubulogenic phenotype (3-5).GAB1 was cloned by as a protein that associated with Grb-2 in an expression library assay. The amino acid sequence of GAB1 suggested that it was a docking protein similar to the insulin receptor substrate 1 family with an amino-terminal pleckstrin homology (PH) domain and a proline-rich carboxyl terminus, as well as tyrosine phosphorylation sites for potential SH2 binding by the phosphoinositide 3-kinase, Grb2, phospholipase C-␥, and SHPTP2. By using a yeast two-hybrid screen with the carboxyl terminus of c-MET as the bait, Weidner et al. (4), and Grb-2-mediated interaction between the proline-rich region of GAB1 and Y 1356 VNV of c-MET (6). The finding that the MBD of GAB1 associates directly with c-MET in a tyrosine-dependent fashion suggests that this domain might associate with other tyrosine-phosphorylated proteins and thus act in a novel fashion to mediate epithelial signaling ...

The growth factor/receptor combination of hepatocyte growth factor (HGF)/c-met has been postulated to be critical for mesenchymal-to-epithelial conversion and tubule formation in the developing kidney. We therefore isolated and immortalized cells from embryonic kidneys of met -/- transgenic mice to determine whether these cells were epithelial and able to chemotax and form tubules in vitro. The cells were immortalized with retrovirus expressing human papillomavirus 16 (HPV 16) E6/E7 genes. Two rapidly dividing clones were isolated and found to express the epithelial cell markers cytokeratin, zonula occludens-1, and E-cadherin but not to express the fibroblast marker vimentin. The met -/- cells were able to chemotax in response to epidermal growth factor and transforming growth factor-alpha (TGF-alpha) and form tubules in vitro in response to TGF-alpha but not HGF. These experiments suggest that the HGF/c-met axis is not essential for epithelial cell development in the embryonic kidney and demonstrate that other growth factors are capable of supporting early tubulogenesis.

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.