BRANCHING TUBULOGENESIS INDUCED BY HEPARIN-BINDING EGF-LIKE GROWTH FACTOR

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Soluble form of HB-EGF (s-HB-EGF) induce tubulogenesis in ureteric bud cells (UBC) cultured in type I collagen gel. s-HB-EGF induced tubule-like structures with abundant arborized branches. UBC were isolated from the embryonic ureteric buds of mice transgenic for SV-40 antigen. UBC that were stably transfected with mouse proHB-EGF (UBC^<proHB-EGF>) were used in this study. proHB-EGF contains the sequence PVENPLYTYDHT in the juxtamembrane extracellular domain and s-HB-EGF is cleaved at P (proline). Then, we constructed a truncated mutant of proHB-EGF that has a 9 amino acid deletion (PVENPLYTY) at the site where cleavage occur (UBC^<del9aa>). There was almost no cleavage of s-HB-EGF in UBC^<del9aa> compared to wild type proHB-EGF transfected UBC (UBC^<proHB-EGF>) after treatment with TPA.Both UBC^<proHB-EGF> and UBC^<del9aa> induced tyrosine phosphorylation of EGF-receptors in A431 cells that express high levels of EGF-receptor. Both UBC^<proHB-EGF> and UBC^<del9aa> showed long straight tubule-like structures with few branches mimicking renal tubules in vivo. Immunoprecipitates from the UBC^<proHB-EGF> and UBC^<del9aa> showed upregulation of phosphoinositide-3 kinase (PI-3 kinase) activity, concomitantly with morphological changes in elongation of the branching cords. UBC^<proHB-EGF> and UBC^<del9aa> showed apical-basolateral polarization and expression of the water channel aquaporin-2. In conclusion, proHB-EGF can induce collecting duct formation in UBC, which was mediated by activating PI-3 kinase via the EGF-receptor. Juxtacrine activation with proHB-EGF could be important for well-balanced morphogenesis of the collecting duct system.