Coexpression of CD9 augments the ability of membrane-bound heparin- binding epidermal growth factor-like growth factor (proHB-EGF) to preserve renal epithelial cell viability

Abstract

Background. Transfection of renal epithelial cells (NRK 52E) with membrane-associated heparin-binding epidermal growth factor-like growth factor (proHB-EGF) increased renal epithelial cell survival by promoting cell-cell and cell-extracellular matrix interactions. ProHBEGF has been shown to form a complex in the plasma membrane with the tetraspanin CD9, an interaction that significantly increases the effectiveness of proHB-EGF as a juxtacrine mitogenic agent. Methods. We examined whether the coexpression of proHB-EGF and CD9 would increase renal epithelial cell survival. CD9 was stably transfected into NRK 52E cells, either alone (NRK(CD9)) or together with proHB-EGF (NRK(both)). Results. Juxtacrine mitogenic activity of NRK(CD9) was no different than in cells transfected with vector alone (NRK(vector)), but was increased by NRK(both); juxtacrine mitogenic activity by NRK(both) was twofold greater than when proHB-EGF was transfected alone (NRK(proHB-EGF)). When grown in 10% fetal calf serum, growth rates were similar among all transfectants. However, in 1% fetal calf serum, NRK(proHB- EGF) grew 50% faster than NRK(vector) or NRK(CD9), and NRK(both) grew 20% to 50% faster than NRK(proHB-EGF) at one, two, and three days of culture. NRK(proHB-EGF) attachment to plastic substratum at one, two, and three hours was 250% greater than that of NRK(vector), and NRK(both) was 20% to 30% greater than that of NRK(proHB-EGF). Coating plates with either poly 2- hydroxyethyl methacrylate or the GRGDTP peptide prevented normal cell- extracellular matrix attachment, and NRK(vector) or NRK(CD9) failed to attach or form cell-cell attachments. NRK(proHB-EGF) exhibited 300% and NRK(both) exhibited 600% greater cell viability under these conditions. Expression of type I and type III collagen mRNA was enhanced similarly in NRK(proHB-EGF) and NRK(both), but the expression of β1 integrin was up-regulated only in NRK(both) Conclusions. Coexpression of proHB-EGF and CD9 may render the renal epithelial cells more resistant to disruption of cell-cell and cell-matrix interactions and could accelerate the re-establishment of these attachments.