Mouse fibroblast cells overexpressing phosphatidylinositol transfer protein ␣ [PI-TP ␣ ; sense PI-TP ␣ (SPI ␣ ) cells]show a significantly increased rate of proliferation and an extreme resistance toward ultraviolet-or tumor necrosis factor-␣ -induced apoptosis. The conditioned medium (CM) from SPI ␣ cells or the neutral lipid extract from CM stimulated the proliferation of quiescent wild-type NIH3T3 cells. CM was also highly effective in increasing resistance toward induced apoptosis in both wild-type cells and the highly apoptosis-sensitive SPI ␤ cells (i.e., wild-type cells overexpressing PI-TP ␤ ). CM from SPI ␣ cells grown in the presence of NS398, a specific cyclooxygenase-2 (COX-2) inhibitor, expressed a diminished mitogenic and antiapoptotic activity. This strongly suggests that at least one of the bioactive factor(s) is an eicosanoid. In accordance, SPI ␣ cells express enhanced levels of COX-1 and COX-2. The antiapoptotic activity of CM from SPI ␣ cells tested on SPI ␤ cells was inhibited by ‫ف‬ 50% by pertussis toxin and suramin as well as by SR141716A, a specific antagonist of the cannabinoid 1 receptor. These inhibitors had virtually no effect on the COX-2-independent antiapoptotic activity of CM from SPI ␣ cells.The latter results imply that PI-TP ␣ mediates the production of a COX-2-dependent eicosanoid that activates a G-protein-coupled receptor, most probably a cannabinoid 1-like receptor.

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Background: Phosphoinositide 3-kinase lipid signals exert important biological effects through proteins with specific recognition domains.Results: We identify a novel such protein domain in IQGAP proteins and define its crystal structure and phosphoinositide binding preferences.Conclusion: This domain is a distinct cellular phosphatidylinositol 3,4,5-trisphosphate sensor, characteristic of select IQGAP proteins.Significance: These observations open a new and unexpected window on phosphoinositide 3-kinase signaling networks.

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The conditioned medium (CM) from mouse NIH3T3 fibroblast cells overexpressing phosphatidylinositol transfer protein alpha (PI-TPalpha; SPIalpha cells) demonstrates an increased anti-apoptotic activity compared with CM from wild type NIH3T3 (wtNIH3T3) cells. As previously shown, the anti-apoptotic activity acts by activating a G protein-coupled receptor, most probably a cannabinoid 1 (CB1)-like receptor as the activity was blocked by both pertussis toxin and rimonabant [M. Schenning, C.M. van Tiel, D. Van Manen, J.C. Stam, B.M. Gadella, K.W. Wirtz and G.T. Snoek, Phosphatidylinositol transfer protein alpha regulates growth and apoptosis of NIH3T3 cells: involvement of a cannabinoid 1-like receptor, J. Lipid Res. 45 (2004) 1555-1564]. The CB1 receptor appears to be expressed in mouse fibroblast cells, at levels in the order SPIalpha>wtNIH3T3>SPIbeta cells (i.e. wild type cells overexpressing PI-TPbeta). Upon incubation of SPIbeta cells with the PI-TPalpha-dependent anti-apoptotic factors, both the ERK/MAP kinase and the Akt/PKB pathway are activated in a CB1 receptor dependent manner as shown by Western blotting. In addition, activation of ERK2 was also shown by EYFP-ERK2 translocation to the nucleus, as visualized by confocal laser scanning microscopy. The subsequent activation of the anti-apoptotic 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 activation as the YFP-labelled C2-domain of protein kinase C was not translocated to the plasma membrane of SPIbeta cells as visualized by confocal laser scanning microscopy.