Figure 5
Involvement of heparin-binding epidermal growth-like growth factor (HB-EGF) shedding in sPLA 2 -IIA-induced a proliferative response. BV-2 cells were treated with the indicated inhibitors for 30 minutes at 37°C and then incubated with 1 μg/ml of sPLA2-IIA for 5 minutes. (A) HB-EGF expression was analyzed by flow cytometry: untreated cells (open black curves) were compared with sPLA2-IIA-treated cells in the absence (open dark grey curves) or presence of the indicated inhibitors (open blue curves). Solid light grey curves show negative controls in which the primary antibody was omitted. (B, C) BV-2 cells were incubated for 30 minutes at 37°C in the presence of the indicated dose of an anti-HB-EGF neutralizing antibody, and then stimulated with 1 μg/ml of sPLA2-IIA. (B) After 15 minutes incubation, phosphorylation of EGFR was analyzed by flow cytometry: untreated cells are represented by open black curves, whereas sPLA2-IIA-treated cells both in the absence and in the presence of the neutralizing Ab are represented by open dark grey curves. Solid grey curves represent isotype controls. (C) After 15 minutes, whole cell lysates were extracted and protein phosphorylation was assessed by western blotting using p-ERK, p-P70S6, p-rS6 and actin antibodies. Membranes also were stained with Ponceau S as a loading control. Results shown are representative of three independent experiments. (D) Primary and immortalized BV-2 microglial cells were stimulated as indicated in the presence or absence of an anti-HB-EGF neutralizing antibody. After 24 h, cell proliferation was investigated and expressed as optical density values ± SD. Values are the average of three separate experiments in quadruplicate (*P < 0.001 compared to control cells, **P < 0.001 compared to sPLA2-IIA-stimulated cells).