Biochemical Properties of Mammalian Neutral Sphingomyelinase2 and Its Role in Sphingolipid Metabolism

Abstract

Neutral sphingomyelinase (N-SMase) is one of the key enzymes involved in the generation of ceramide; however, the gene(s) encoding for the mammalian N-SMase is still not well defined. Previous studies on the cloned nSMase1 had shown that the protein acts primarily as lyso-platelet-activating factor-phospholipase C. Recently the cloning of another putative N-SMase, nSMase2, was reported. In this study, biochemical characterization of the mouse nSMase2 was carried out using the overexpressed protein in yeast cells in which the inositol phosphosphingolipid phospholipase C (Isc1p) was deleted. N-SMase activity was dependent on Mg 2؉ and was activated by phosphatidylserine and inhibited by GW4869. The ability of nSMase2 to recognize endogenous sphingomyelin (SM) as substrate was investigated by overexpressing nSMase2 in MCF7 cells. Mass measurements showed a 40% decrease in the SM levels in the overexpressor cells, and labeling studies demonstrated that nSMase2 accelerated SM catabolism. Accordingly, ceramide measurement showed a 60 ؎ 15% increase in nSMase2-overexpressing cells compared with the vector-transfected MCF7. The role of nSMase2 in cell growth was next investigated. Stable overexpression of nSMase2 resulted in a 30 -40% decrease in the rate of growth at the late exponential phase. Moreover, tumor necrosis factor induced ϳ50% activation of nSMase2 in MCF7 cells overexpressing the enzyme, demonstrating that nSMase2 is a tumor necrosis factor-responsive enzyme. In conclusion, these results 1) show that nSMase2 is a structural gene for nSMase, 2) suggest that nSMase2 acts as a bona fide N-SMase in cells, and 3) implicate nSMase2 in the regulation of cell growth and cell signaling.

Ceramide triggers an NF-κB-dependent survival pathway through calpain

“…1 A very recent report demonstrated that endogenous nSMase2 levels are induced upon confluence and are involved in induction of cell cycle arrest in MCF7 cells. 40,41 Furthermore, nSMase overexpression and treatment with cell-permeable ceramide analogues can induce growth arrest in a number of cells types including SK-Hep1, 6 U937 7 and HT-29. 8 Over 1000 research papers have described the induction of programmed cell death (apoptosis) by interventions that elevate the cell content of ceramide.…”

The sphingolipid salvage pathway in ceramide metabolism and signaling

“…Genetic defects in several of these hydrolytic enzymes cause various disorders with lysosomal accumulation of the substrate lipids, a group of disorders termed the sphingolipidoses [41,[90][91][92][93]. Particularly, some of the known sphingolipidoses might closely associate with aberrant metabolisms of ceramide because of defective activities of ceramide generating/degrading enzymes: Farber's disease, Gaucher disease, and Niemann-Pick type A and B diseases are caused by a deficiency of acid ceramidase [94,95], glucocerebrosidase (acid-β-glucosidase) [96][97][98], acid SMase [99,100], respectively. The salvage pathway is one of the routes for controlling cellular levels of ceramide.…”

Bone Morphogenic Protein (BMP) Signaling Up-regulates Neutral Sphingomyelinase 2 to Suppress Chondrocyte Maturation via the Akt Protein Signaling Pathway as a Negative Feedback Mechanism

“…This effect of Smpd3 knockdown against expression of Col2a1 was mimicked by the addition of 1 M GW4869, a specific inhibitor compound for nSMase (Fig. 3B) (49), suggesting that the data from the siSmpd3 experiment resulted from down-regulation of nSMase2. GW4869 also enhanced the production of the cartilage-specific extracellular component, glycosaminoglycan, by BMP-2 stimulation for 17 days, as assessed by Alcian blue staining, both in ATDC5 cells and in primary chondrocytes (Fig.…”