Interaction of Ceramides, Sphingosine, and Sphingosine 1-Phosphate in Regulating DNA Synthesis and Phospholipase D Activity

doi:10.1074/jbc.270.44.26318

Interaction of Ceramides, Sphingosine, and Sphingosine 1-Phosphate in Regulating DNA Synthesis and Phospholipase D Activity (*)

From the Department of Biochemistry, Signal Transduction Laboratories and the Lipid and Lipoprotein Research Group, 328 Heritage Medical Research Centre, University of Alberta, Edmonton, Alberta T6G 2S2, Canada

^¶
Medical Scientist of the Alberta Heritage Foundation for Medical Research.
To whom correspondence and reprint requests should be addressed. Tel.: 403-492-2078; Fax: 403-492-3383.

↵^§ Present address: Instituto de Investigaciones Biomedicas, Consejo Superior de Investigaciones Científicas, C/Arturo Duperier, 4, 28029 Madrid, Spain.

Abstract

C- and C-ceramides (N-acetylsphingosine and N-hexanoylsphingosine, respectively) abolished the stimulation of DNA synthesis by sphingosine 1-phosphate in rat fibroblasts. This inhibition by ceramide was partially prevented by insulin. C-ceramide did not alter the stimulation of DNA synthesis by insulin and decreased the sphingosine-induced stimulation by only 16%. The ceramides did not significantly modify the actions of sphingosine or sphingosine 1-phosphate in decreasing cAMP concentrations. C- and C-ceramides blocked the activation of phospholipase D by sphingosine 1-phosphate, and this inhibition was not affected by insulin. Okadaic acid decreased the activation of phospholipase D by sphingosine 1-phosphate and did not reverse the inhibitory effect of C-ceramide on this activation. Therefore, this effect of C-ceramide is unlikely to involve the stimulation of phosphoprotein phosphatase activity. Sphingosine did not activate phospholipase D activity significantly after 10 min. C-ceramide stimulated the conversion of exogenous [H]sphingosine 1-phosphate to sphingosine and ceramide in fibroblasts. Ceramides can inhibit some effects of sphingosine 1-phosphate by stimulating its degradation via a phosphohydrolase that also hydrolyzes phosphatidate. Furthermore, C- and C-ceramides stimulated ceramide production from endogenous lipids, and this could propagate the intracellular signal. This work demonstrates that controlling the production of ceramide versus sphingosine and sphingosine 1-phosphate after sphingomyelinase activation could have profound effects on signal transduction.

Footnotes

↵* This work was supported by grants from the Medical Research Council of Canada, the Canadian Neuroscience Network Centres of Excellence, and the Cancer Research Fund of the University of Alberta. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

SM

sphingomyelin

TNF-α

tumor necrosis factor-α

PA

phosphatidate

PLD

phospholipase D

SPP

sphingosine 1-phosphate

DMEM

Dulbecco's modified Eagle's medium

C²

acetyl

C⁶

hexanoyl

C⁸

octanoyl.
↵² D. W. Waggoner, A. Gómez-Muñoz, J. Dewald, and D. N. Brindley, unpublished data.
- Received June 1, 1995.
- Revision received August 25, 1995.