Gt. Snoek et al., Overexpression of phosphatidylinositol transfer protein alpha in NIH3T3 cells activates a phospholipase A, J BIOL CHEM, 274(50), 1999, pp. 35393-35399
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.