Overexpression of phosphatidylinositol transfer protein alpha in NIH3T3 cells activates a phospholipase A

In order to investigate the cellular function of the mammalian phosphatidyl inositol transfer protein alpha (PI-TP alpha), NIH3T3 fibroblast cells were transfected with the cDNA encoding mouse PI-TP alpha. Two stable cell line s, i.e. SPI6 and SPI8, were isolated, which showed a 2- and 3-fold increase , respectively, in the level of PI-TP alpha. Overexpression of PI-TP alpha resulted in a decrease in the duration of the cell cycle from 21 h for the wild type (nontransfected) NIH3T3 (wtNIH3T3) cells and mock-transfected cel ls to 13-14 h for SP16 and SP18 cells. Analysis of exponentially growing cu ltures by fluorescence-activated cell sorting showed that a shorter G(1) ph ase is mainly responsible for this decrease. The saturation density of the cells increased from 0.20 x 10(5) cells/cm(2) for wtNIH3T3 cells to 0.53 x 10(5) cells/cm(2) for SPI6 and SPI8 cells. However, anchorage-dependent gro wth was maintained as shown by the inability of the cells to grow in soft a gar. Upon equilibrium labeling of the cells with myo-[H-3] inositol, the relativ e incorporation of radioactivity in the total inositol phosphate fraction w as 2-3-fold increased in SPI6 and SPI8 cells when compared with wtNIH3T3 ce lls. A detailed analysis of the inositol metabolites showed increased level s of glycerophosphoinositol, Ins(1)P, Ins(2)P, and lysophosphatidylinositol (lyso-PtdIns) in SPI8 cells, whereas the levels of phosphatidylinositol (P tdIns) and phosphatidylinositol 4,5-bisphosphate were the same as those in control cells, The addition of PI-TP alpha to a total lysate of myo-[H-3]in ositol-labeled wtNIH3T3 cells stimulated the formation of lyso-PtdIns. The addition of Ca2+ further increased this formation. Based on these observati ons, we propose that PI-TP alpha is involved in the production of lyso-PtdI ns by activating a phospholipase A acting on PtdIns, The increased level of lyso-PtdIns that is produced in this reaction could be responsible for the increased growth rate and the partial loss of contact inhibition in SPI8 a nd SPI6 cells, The addition of growth factors (platelet-derived growth fact or, bombesin) to these overexpressers did not activate the phospholipase C- dependent degradation of phosphatidylinositol 4,5-bisphosphate.