Phosphatidylinositol transfer proteins in cell survival and apoptosis

Abstract

Mouse fibroblast cells overexpressing phosphatidylinositol transfer protein alpha [PI-TPalpha; sense PI-TPalpha (SPIalpha) cells] show a significantly increased rate of proliferation and an extreme resistance toward ultraviolet- or tumor necrosis factor-alpha-induced apoptosis. The fact that the conditioned medium (CM) from SPIalpha cells demonstrates an increased mitogenic and anti-apoptotic activity compared with CM from wild-type NIH3T3 (wtNIH3T3) cells, shows that PI-TPalpha is involved in the production and secretion of bio-active eicosanoids, the production of which is cyclooxygenase 2 dependent. The PI-TPalpha-dependent anti-apoptotic factors act via activation of G protein-coupled receptor, most probably a cannabinoid 1-like receptor. Upon receptor activation, both the anti-apoptotic ERK/MAP kinase and the Akt/PKB pathway are activated. The ensuing activation of the transcription factor NF-kappaB is in line with the increased resistance towards UV-induced apoptosis. On the other hand, receptor activation by CM from SPIalpha cells was not linked to phospholipase C. Characterization of the CM showed that CM from SPIalpha cells contains more than one anti-apoptotic factor, with the bulk of the anti-apoptotic factors displaying very hydrophobic properties. The fact that a large number of COX-2-dependent differences between CM from SPIalpha cells and CM from wtNIH3T3 cells have hydrophobic properties and masses similar to arachidonic acid, 2-arachidonyl glycerol (2AG) and anandamide (AEA) and that the anti-apoptotic activity present in CM from SPI? cells can be inhibited by a specific antagonist for the CB1 receptor, strongly suggests that the some of the anti-apoptotic factors secreted by SPIalpha cells belong to the family of endocannabinoids. On the other hand, the involvement of endocannabinoids 2AG and AEA was excluded. In contrast to SPIalpha cells, mouse fibroblast cells overexpressing PI-TPbeta (SPIbeta cells) demonstrate a low rate of proliferation and a high sensitivity towards UV-induced apoptosis compared with wtNIH3T3 cells. SPIbetaS262A cells overexpressing a mutant PI-TPbeta that lacks the protein kinase C-dependent phosphorylation site Ser-262, demonstrate a phenotype comparable to wtNIH3T3 cells. This suggests that the sensitivity towards apoptosis is related to the phosphorylation of Ser-262 in PI-TPbeta. CM from SPIbeta cells lacks the anti-apoptotic activity demonstrated by CM from SPIalpha cells and to a lesser extent by CM from wtNIH3T3 cells. However, after heat denaturation CM from SPIbeta cells regains a protective activity comparable to that of CM from wtNIH3T3 cells. This indicates that CM from SPIbeta cells contains an antagonistic factor interfering with the anti-apoptotic activity present. SPIbetaS262A cells do not produce the antagonist suggesting that phosphorylation of Ser-262 is required. Since PI-TPalpha is involved in the production and secretion of mitogenic and anti-apoptotic arachidonic acid metabolites, we investigated the effect of prostaglandin (PG)E2 and PGF2alpha on cell survival. These prostaglandins, which are prominently present in CM from SPIalpha, were found to protect wtNIH3T3 and SPIbetaS262A cells against UV-induced apoptosis but failed to rescue SPIbeta cells. Concomitantly, upon incubation with PGE2 and PGF2alpha, an increased expression of COX-2 and activation of p42/p44 MAP kinase were observed in wtNIH3T3 and SPIbetaS262A cells but not in SPIbeta cells. Hence, it appears that specific mechanisms of cell survival are impaired in SPIbeta cells.