Role of membrane-bound heparin-binding epidermal growth factor-like growth factor (HB-EGF) in renal epithelial cell branching

Background. The developing metanephros is characterized by growth and differentiation of the ureteric bud and the surrounding mesenchymal tissue. These processes can be influenced by several growth factors, including epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha). We examined whether another member of the EGF family of growth factors, heparin-binding epidermal growth factor (HB-EGF), might act as a morphogen in renal epithelial tubulogenesis. Methods. Expression of HB-EGF mRNA and immunoreactive protein were examined in fetal, neonatal and adult rat kidneys. For in vitro studies of tubulogenesis, a rat renal epithelial cell line (NRK52E) stably transfected with proHB-EGF (NRKproHB-EGF ) was treated with TPA for 30 minutes, washed with 2 mol/L NaCl to remove soluble HB-EGF trapped by cell surface heparan sulfate proteoglycan and replated onto plastic dishes in the absence of fetal calf serum. In further experiments, NRKproHB-EGF were suspended in a type I collagen gel in serum-free media. Results. Northern blot analysis indicated that HB-EGF was strongly expressed in embryonic rat kidney (embryonic days 18-20) and was still increased in the neonatal kidney (day 10), compared to the low basal levels in adult kidney. Immunohistochemical analysis confirmed that immunoreactive HB-EGF expression in the fetal rat kidney was localized predominantly to the ureteric bud. When NRKproHB-EGF were plated onto plastic substrata, they became progressively flattened and enlarged and exhibited filopoidia. By 10 hours after plating, NRKproHB-EGF began to migrate and subsequently developed cell-cell contact and fully established tubular-like structures. Immunoelectron microscopy revealed that the initial recovery of cellular proHB-EGF was localized predominantly to areas of cell-cell attachment. No tubule-like structures were observed in similarly treated NRK52E cells transfected with the vector alone. In collagen gels, NRKproHB-EGF developed short tubule-like structures in the absence of TPA treatment, but with simultaneous TPA treatment, longer and more arborized structures developed. MMP-1 mRNA and immunoreactive protein increased in the TPA-treated cells, suggesting that protein kinase C-mediated collagenase activity was important for the observed tubulogenesis. However, inhibition of EGF receptor tyrosine kinase with AG 1478 significantly blunted the TPA-induced tubulogenesis by NRKproHB-EGF grown in collagen gels. Conclusions. These results indicate that membrane-bound HB-EGF can mediate both epithelial cell branching and cell motility. Localization of proHB-EGF to the site of cell-cell contact and development of tubule-like structures in collagen gels suggests that proHB-EGF may be an important morphogen for renal epithelial cells.