Rapid replenishment of sphingomyelin in the plasma membrane upon degradation by sphingomyelinase in NIH3T3 cells overexpressing the phosphatidylinositol transfer protein β

Abstract

In order to study the in vivo function of the phosphatidylinositol transfer protein beta (PI-TPbeta), mouse NIH3T3 fibroblasts were transfected with cDNA encoding mouse PI-TPbeta. Two stable cell lines were isolated (SPIbeta2 and SPIbeta8) in which the levels of PI-TPbeta were increased 16- and 11-fold respectively. The doubling time of the SPIbeta cells was about 1.7 times that of the wild-type (wt) cells. Because PI-TPbeta expresses transfer activity towards sphingomyelin (SM) in vitro, the SM metabolism of the overexpressors was investigated. By measuring the incorporation of [methyl-(3)H]choline chloride in SM and phosphatidylcholine (PtdCho), it was shown that the rate of de novo SM and PtdCho synthesis was similar in transfected and wt cells. We also determined the ability of the cells to resynthesize SM from ceramide produced in the plasma membrane by the action of bacterial sphingomyelinase (bSMase). In these experiments the cells were labelled to equilibrium (60 h) with [(3)H]choline. At relatively low bSMase concentrations (50 munits/ml), 50% of [(3)H]SM in wt NIH3T3 cells was degraded, whereas the levels of [(3)H]SM in SPIbeta cells appeared to be unaffected. Since the release of [(3)H]choline phosphate into the medium was comparable for both wt NIH3T3 and SPIbeta cells, these results strongly suggest that breakdown of SM in SPIbeta cells was masked by rapid resynthesis of SM from the ceramide formed. By increasing the bSMase concentrations to 200 munits/ml, a 50% decrease in the level of [(3)H]SM in SPIbeta cells was attained. During a recovery period of 6 h (in the absence of bSMase) the resynthesis of SM was found to be much more pronounced in these SPIbeta cells than in 50% [(3)H]SM-depleted wt NIH3T3 cells. After 6 h of recovery about 50% of the resynthesized SM in the SPIbeta cells was available for a second hydrolysis by bSMase. When monensin was present during the recovery period, the resynthesis of SM in bSMase-treated SPIbeta cells was not affected. However, under these conditions 100% of the resynthesized SM was available for hydrolysis. On the basis of these results we propose that, under conditions where ceramide is formed in the plasma membrane, PI-TPbeta plays an important role in restoring the steady-state levels of SM.

Phosphatidylinositol Transfer Proteins: Emerging Roles in Cell Proliferation, Cell Death and Survival

“…The intracellular concentration of PI-TPb is about a 100-fold lower than the concentration of PI-TPa in most mammalian tissues (36). It was shown that PI-TPb is involved in the traffic of SM from the Golgi membranes to the plasma membrane (40). In contrast to the effect of PI-TPa, increased expression of PI-TPb leads to an increase in cell cycle duration from 21 h to 34 h. The saturation density of confluent cultures decreased and the cells were much more sensitive to UV-and TNFa-induced apoptosis when compared to wtNIH3T3 cells (36).…”

“…Involvement of PI-TPa and PI-TPb in the regulation of PC synthesis and/or SM transfer (8,33,40), was not confirmed in COS-7 cells that were transiently transfected with either PI-TPa or PI-TPb (13). An explanation for this discrepancy might be that the effects are strongly dependent on the cell type used for transfection or that transiently transfected cell cultures demonstrate different properties when compared to stable cell lines in particular when autocrine as well as paracrine stimulation processes are involved.…”

“…PI-TPb (Fig. 3) is localized at the Golgi (8,28), stimulates SM transport to the plasma membrane (40) , is able to stimulate the formation of secretory vesicles from the Golgi membranes (24,25) and could be involved in various secretory events (22). NIH3T3 cells with increased expression of PI-TPb grow very slowly and are extremely sensitive to induction of apoptosis (36).…”

Current thoughts on the phosphatidylinositol transfer protein family

“…Two separate studies have examined the e¡ects of over-expression of PITPL (which localises at the Golgi) on sphingolipid metabolism. In a study using NIH 3T3 ¢broblasts, hydrolysis of SM at the external lea£et of the plasma membrane by exogenous SMase, led to enhanced replenishment of SM at the plasma membrane [48]. In a second study using COS-7 cells, no changes in replenishment was observed [33].…”

The anti-apoptotic MAP kinase pathway is inhibited in NIH3T3 fibroblasts with increased expression of phosphatidylinositol transfer protein β

“…In previous studies on wtNIH3T3 cells that have a 10-fold increase in PI-TPβ levels (SPIβ cells) we observed that these cells display a decreased growth rate relative to the wtNIH3T3 cells [7]. In addition, under conditions where sphingomyelin (SM) in the plasma membrane was hydrolyzed to ceramide by exogenous sphingomyelinase, SPIβ cells in contrast to wtNIH3T3 cells maintained the steady-state levels of SM in the plasma membrane suggesting that PI-TPβ was involved in this process [7]. This is in agreement with the finding that in vitro PI-TPβ binds and transfers SM in addition to PI and PC while PI-TPα lacks the ability to transfer SM [8].…”