The anti-apoptotic MAP kinase pathway is inhibited in NIH3T3 fibroblasts with increased expression of phosphatidylinositol transfer protein β

Mouse NIH3T3 fibroblast cells overexpressing phosphatidylinositol transfer protein ß (PI-TPß, SPIß cells) demonstrate a low rate of proliferation and a high sensitivity towards UV-induced apoptosis when compared with wtNIH3T3 cells. In contrast, SPIßS262A cells overexpressing a mutant PI-TPß that lacks the protein kinase C-dependent phosphorylation site Ser-262, demonstrate a phenotype comparable with wtNIH3T3 cells. This suggests that the phosphorylation of Ser-262 in PI-TPß is involved in the regulation of apoptosis. Conditioned medium (CM) from wtNIH3T3 cells contains bioactive factors, presumably arachidonic acid metabolites [H. Bunte, et al., 2006; M. Schenning, et al., 2004] that are able to protect SPIß cells against UV-induced apoptosis.CMfrom SPIß cells lacks this protective activity. However, after heat denaturation CM from SPIß cells regains a protective activity comparable with that of wtNIH3T3 cells. This indicates that CM from SPIß cells contains an antagonistic factor interfering with the anti-apoptotic activity present. SPIßS262A cells do not produce the antagonist suggesting that phosphorylation of Ser-262 is required. Moreover, in line with the apparent lack of anti-apoptotic activity, CM from SPIß cells does not induce the expression of COX-2 or the activation of p42/p44 MAP kinase in SPIß cells. In contrast, CM from wtNIH3T3 and SPIßS262A cells or heat-treated CM from SPIß cells does induce these anti-apoptotic markers. Since we have previously shown that some of the arachidonic acid metabolites present in CM from wtNIH3T3 cells are prostaglandin (PG) E2 and PGF2a, we investigated the effect of these PGs on cell survival. Although PGE2 and PGF2a were found to protect wtNIH3T3 and SPIßS262A cells against UV-induced apoptosis, these PGs failed to rescue SPIß cells. The fact that the concentrations of PGE2 and PGF2a in the CM from SPIß cells and wtNIH3T3 cells were found to be comparable suggests that the failure of these PGs to protect SPIß cells could render these cells more apoptosis sensitive. Concomitantly, upon incubation with PGE2 and PGF2a, an increased expression of COX-2 and activation of p42/p44 MAP kinase were observed in wtNIH3T3 and SPIßS262A cells but not in SPIß cells. Hence, it appears that specific mechanisms of cell survival are impaired in SPIß cells.