Promotion of survival and prevention of apoptosis in rat mesangial cells by a membrane-anchored form of heparin-binding EGF-like growth factor

Abstract

Background. We previously demonstrated that heparin-binding epidermal growth factor-like growth factor (HB-EGF) was expressed in rat mesangial cells and was involved in the progression of of glomerulonephritis in human and animal models. Soluble HB-EGF promotes cultured rat mesangial cell proliferation and the synthesis of type I and type III collagen mRNA. However, the precise role of proHB-EGF in mesangial cell function has not yet been clarified. In the present study, we address this question.

Methods. ProHB-EGF-transfected rat mesangial cells (MsC; MsC^proHB-EGF) and empty vector-transfected rat MsC (MsC^vector) were constructed. Cells were cultured in fetal calf serum (FCS)-containing (10%) or FCS-deficient medium, and the growth rates and numbers of surviving cells were determined. To elucidate the anti-apoptotic effect of proHB-EGF, cells were exposed to hydrogen peroxide (H₂O₂) or dexamethasone (DEX). Apoptosis was identified by qualitative and quantitative analysis. Expression of Bcl-2 protein in MsC^proHB-EGF after exposure to apoptosis inducers was also evaluated.

Results. When cells were plated in a medium containing 10% FCS, the growth rate of MsC^proHB-EGF was not different from that of MsC transfected with a plasmid alone (MsC^vector). When cultured in the absence of FCS, MsC^vector showed decreased cell numbers, indicating apoptotic cell death. In contrast, MsC^proHB-EGF formed small colonies, although they did not show increased cell numbers, while cell viability was 90%–100% of the initial cell number after subculture. When quiescent MsC were exposed to DEX or H₂O₂, MsC^vector exhibited significant DNA laddering, whereas MsC^proHB-EGF showed resistance to these stimuli. The release of caspase-3 from MsC^proHB-EGF after exposure to DEX or H₂O₂ was significantly lower than that from MsC^vector. Bcl-2 protein expression was weak in MsC^proHB-EGF at the baseline, but this expression was significantly upregulated after exposure to these stimuli. Confluent MsC^proHB-EGF spontaneously expressed high level of p21 protein. Northern blot analysis revealed that MsC^proHB-EGF expressed increased levels of type I and type III collagen mRNA and proteins compared with those of MsC^vector.

Conclusions. These results indicate that proHB-EGF contributes to mesangial cell survival by promoting cell viability and by inhibiting apoptosis. The anti-apoptotic effect of proHB-EGF could be closely related to the upregulation of Bcl-2 and p21 expression.