Abstract
We previously showed that degradation of cellular sphingomyelin (SM) by SMase C results in a greater stimulation of cholesterol translocation to endoplasmic reticulum, compared to its degradation by SMase D. Here we investigated the hypothesis that the effect of SMase C is partly due to the generation of ceramide, rather than due to depletion of SM alone. Inhibition of hydroxymethylglutaryl CoA reductase (HMGCR) activity was used as a measure of cholesterol translocation. Treatment of fibroblasts with SMase C resulted in a 90% inhibition of HMGCR, whereas SMase D treatment inhibited it by 29%. Treatment with exogenous ceramides, or increasing the endogenous ceramide levels also inhibited HMGCR by 60–80%. Phosphorylation of HMGCR was stimulated by SMase C or exogenous ceramide. The effects of ceramide and SMase D were additive, indicating the independent effects of SM depletion and ceramide generation. These results show that ceramide regulates sterol trafficking independent of cellular SM levels.
Highlights
The distribution of sphingomyelin among the various cellular membranes is remarkably similar to that of cholesterol, and the two membrane lipids are known to interact with each other [1,2]
Our previous studies showed that treatment of fibroblasts with SMase C increased the translocation of Plasma membrane SM (PM) cholesterol to endoplasmic reticulum (ER) by 4-fold, as measured by the stimulation of acyl CoA: cholesterol acyltransferase (ACAT) activity, compared to only a 70% stimulation by SMase D [6]
Since the percent of SM hydrolyzed by the two enzymes was comparable, we proposed that the greater response to SMase C treatment may be partly due to the generation of the cytoactive ceramide, which affected either cholesterol trafficking or the enzyme activity
Summary
The distribution of sphingomyelin among the various cellular membranes is remarkably similar to that of cholesterol, and the two membrane lipids are known to interact with each other [1,2]. Our previous studies in human foreskin fibroblasts showed that unlike the effect of SMase C, treatment of the cells with SMase D (which degrades SM to ceramide phosphate) results in relatively modest stimulation of ACAT, the extent of SM hydrolysis by the two enzymes was comparable [6]. This raised the possibility that the cellular response to SMase C treatment with respect to cholesterol trafficking is at least partly due to the generation of ceramide rather than the loss of SM alone from the PM. In the present study we tested the hypothesis that endogenous or exogenous ceramides modulate intracellular cholesterol trafficking (and) or the activity of enzymes of cholesterol homeostasis, independent of membrane SM concentration
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