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

Publisher: Published by Portland Press on behalf of the Biochemical Society Country of Publication: England NLM ID: 2984726R Publication Model: Print Cited Medium: Print ISSN: 0264-6021 (Print) Linking ISSN: 02646021 NLM ISO Abbreviation: Biochem J Subsets: MEDLINE

Original Publication: London, UK : Published by Portland Press on behalf of the Biochemical Society

Membrane Proteins*; Carrier Proteins/*metabolism ; Cell Membrane/*metabolism ; Sphingomyelin Phosphodiesterase/*metabolism ; Sphingomyelins/*metabolism; 3T3 Cells ; Animals ; Carrier Proteins/genetics ; Gene Expression Regulation ; Mice ; Phospholipid Transfer Proteins ; Sphingomyelin Phosphodiesterase/genetics ; Sphingomyelins/genetics

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.

J Biol Chem. 1999 Dec 10;274(50):35393-9. (PMID: 10585408)
J Biol Chem. 1960 Mar;235:769-75. (PMID: 13793161)
Anal Biochem. 1976 May 7;72:248-54. (PMID: 942051)
Biochim Biophys Acta. 1981 Oct 23;666(1):99-109. (PMID: 6271237)
Biochim Biophys Acta. 1982 Mar 12;710(3):314-23. (PMID: 6280771)
J Cell Biol. 1985 Jan;100(1):27-34. (PMID: 3965473)
Mol Cell Biol. 1987 Aug;7(8):2745-52. (PMID: 3670292)
J Biol Chem. 1989 Oct 5;264(28):16557-64. (PMID: 2777797)
FEBS Lett. 1990 Feb 12;261(1):155-7. (PMID: 2155131)
Biochim Biophys Acta. 1993 Feb 24;1166(2-3):305-8. (PMID: 8443249)
Oncogene. 1993 Apr;8(4):1075-81. (PMID: 8455935)
Biochem Soc Trans. 1993 May;21(2):240-4. (PMID: 8359473)
Biochim Biophys Acta. 1994 Apr 20;1191(1):52-8. (PMID: 8155684)
J Biochem. 1994 May;115(5):981-4. (PMID: 7961615)
Biochem J. 1994 Oct 15;303 ( Pt 2):341-5. (PMID: 7980390)
J Biol Chem. 1995 Feb 3;270(5):2047-52. (PMID: 7836432)
J Biol Chem. 1995 Jun 16;270(24):14263-6. (PMID: 7782280)
FEBS Lett. 1995 Jul 17;368(2):393-6. (PMID: 7628646)
Biochem J. 1995 Sep 1;310 ( Pt 2):643-9. (PMID: 7654206)
J Cell Biochem. 1995 Jun;58(2):191-8. (PMID: 7673327)
Nature. 1995 Oct 12;377(6549):544-7. (PMID: 7566155)
Curr Biol. 1995 Jul 1;5(7):775-83. (PMID: 7583124)
Curr Biol. 1996 Jun 1;6(6):730-8. (PMID: 8793299)
Exp Cell Res. 1996 Aug 25;227(1):33-9. (PMID: 8806448)
J Biol Chem. 1997 Apr 11;272(15):9609-12. (PMID: 9092485)
J Cell Sci. 1997 Mar;110 ( Pt 6):781-8. (PMID: 9099952)
Biochem J. 1997 Jun 1;324 ( Pt 2):353-60. (PMID: 9182690)
Biochem J. 1997 Oct 15;327 ( Pt 2):545-52. (PMID: 9359428)
Curr Opin Hematol. 1996 Jan;3(1):48-54. (PMID: 9372051)
Biochim Biophys Acta. 1997 Oct 30;1359(1):1-12. (PMID: 9398080)
FASEB J. 1998 Jan;12(1):101-9. (PMID: 9438415)
Biochem Soc Trans. 1997 Nov;25(4):1125-31. (PMID: 9449961)
Exp Cell Res. 1998 Jan 10;238(1):13-22. (PMID: 9457052)
Biochem J. 1998 Apr 1;331 ( Pt 1):251-6. (PMID: 9512487)
J Biol Chem. 1998 Jun 5;273(23):14550-9. (PMID: 9603970)
Biochem J. 1998 Jul 1;333 ( Pt 1):91-7. (PMID: 9639567)
Biochem J. 1998 Oct 1;335 ( Pt 1):175-9. (PMID: 9742227)
J Biol Chem. 1957 May;226(1):497-509. (PMID: 13428781)
Can J Biochem Physiol. 1959 Aug;37(8):911-7. (PMID: 13671378)
J Biol Chem. 1974 Mar 10;249(5):1506-12. (PMID: 4817756)

GM 43825 United States GM NIGMS NIH HHS

0 (Carrier Proteins)
0 (Membrane Proteins)
0 (Phospholipid Transfer Proteins)
0 (Sphingomyelins)
EC 3.1.4.12 (Sphingomyelin Phosphodiesterase)

Date Created: 20000324 Date Completed: 20000425 Latest Revision: 20190501

20221213

PMC1220883

10.1042/0264-6021:3460537

10677376