J Biol Chem, Vol. 274, Issue 50, 35393-35399, December 10, 1999

Overexpression of Phosphatidylinositol Transfer Protein  in NIH3T3 Cells Activates a Phospholipase A^*

Gerry T. Snoek^§, Christopher P. Berrie^¶, Teunis B. H. Geijtenbeek, Hester A. van der Helm, Jenny A. Cadeé, Cristiano Iurisci^¶, Daniela Corda^¶, and Karel W. A. Wirtz

From the  Centre for Biomembranes and Lipid Enzymology, Department of Lipid Biochemistry, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands and ^¶ Istituto de Richerche Farmacologiche "Mario Negri," Consorzio Mario Negri Sud, Department of Cell Biology and Oncology, 66030 Santa Maria Imbaro, Chieti, Italy

In order to investigate the cellular function of the mammalian phosphatidylinositol transfer protein  (PI-TP), NIH3T3 fibroblast cells were transfected with the cDNA encoding mouse PI-TP. Two stable cell lines, i.e. SPI6 and SPI8, were isolated, which showed a 2- and 3-fold increase, respectively, in the level of PI-TP. Overexpression of PI-TP 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 cells to 13-14 h for SPI6 and SPI8 cells. Analysis of exponentially growing cultures by fluorescence-activated cell sorting showed that a shorter G₁ phase is mainly responsible for this decrease. The saturation density of the cells increased from 0.20 × 10⁵ cells/cm² for wtNIH3T3 cells to 0.53 × 10⁵ cells/cm² for SPI6 and SPI8 cells. However, anchorage-dependent growth was maintained as shown by the inability of the cells to grow in soft agar.

Upon equilibrium labeling of the cells with myo-[³H] inositol, the relative incorporation of radioactivity in the total inositol phosphate fraction was 2-3-fold increased in SPI6 and SPI8 cells when compared with wtNIH3T3 cells. A detailed analysis of the inositol metabolites showed increased levels of glycerophosphoinositol, Ins(1)P, Ins(2)P, and lysophosphatidylinositol (lyso-PtdIns) in SPI8 cells, whereas the levels of phosphatidylinositol (PtdIns) and phosphatidylinositol 4,5-bisphosphate were the same as those in control cells. The addition of PI-TP to a total lysate of myo-[³H]inositol-labeled wtNIH3T3 cells stimulated the formation of lyso-PtdIns. The addition of Ca²⁺ further increased this formation. Based on these observations, we propose that PI-TP is involved in the production of lyso-PtdIns 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 and SPI6 cells. The addition of growth factors (platelet-derived growth factor, bombesin) to these overexpressers did not activate the phospholipase C-dependent degradation of phosphatidylinositol 4,5-bisphosphate.