The anti-apoptotic MAP kinase pathway is inhibited in NIH3T3 fibroblasts with increased expression of phosphatidylinositol transfer protein beta.
Mouse NIH3T3 fibroblast cells overexpressing phosphatidylinositol transfer protein beta (PI-TPbeta, SPIbeta cells) demonstrate a low rate of proliferation and a high sensitivity towards UV-induced apoptosis when compared with wtNIH3T3 cells. In contrast, SPIbetaS262A cells overexpressing a mutant PI-TPbeta 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-TPbeta 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 SPIbeta cells against UV-induced apoptosis. CM from SPIbeta cells lacks this protective activity. However, after heat denaturation CM from SPIbeta cells regains a protective activity comparable with that of 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. Moreover, in line with the apparent lack of anti-apoptotic activity, CM from SPIbeta cells does not induce the expression of COX-2 or the activation of p42/p44 MAP kinase in SPIbeta cells. In contrast, CM from wtNIH3T3 and SPIbetaS262A cells or heat-treated CM from SPIbeta 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) E(2) and PGF(2alpha), we investigated the effect of these PGs on cell survival. Although PGE(2) and PGF(2alpha) were found to protect wtNIH3T3 and SPIbetaS262A cells against UV-induced apoptosis, these PGs failed to rescue SPIbeta cells. The fact that the concentrations of PGE(2) and PGF(2alpha) in the CM from SPIbeta cells and wtNIH3T3 cells were found to be comparable suggests that the failure of these PGs to protect SPIbeta cells could render these cells more apoptosis sensitive. Concomitantly, upon incubation with PGE(2) and PGF(2alpha), 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.
Bijvoet Center, Department of Biochemistry of Lipids, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. MSchenning@dundee.ac.uk
Martijn
M
Schenning
Claudia M
CM
van Tiel
Karel W A
KW
Wirtz
Gerry T
GT
Snoek
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D017077:Culture Media, Conditioned; D051545:Cyclooxygenase 1 / Q000378:metabolism; D051546:Cyclooxygenase 2 / Q000378:metabolism; D015232:Dinoprostone / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects / Q000528:radiation effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology* / Q000528:radiation effects; D015870:Gene Expression / Q000187:drug effects; D020935:MAP Kinase Signaling System / Q000187:drug effects; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors*; D008954:Models, Biological; D050505:Mutant Proteins / Q000378:metabolism; D041681:NIH 3T3 Cells; D049294:Phospholipid Transfer Proteins / Q000235:genetics* / Q000378:metabolism*; D014466:Ultraviolet Rays
D017077:Culture Media, Conditioned; D004791:Enzyme Inhibitors; D050505:Mutant Proteins; D049294:Phospholipid Transfer Proteins; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D019950:Mitogen-Activated Protein Kinase 1; D020928:Mitogen-Activated Protein Kinases; D015232:Dinoprostone