Ablation of Dihydroceramide Desaturase Confers Resistance to Etoposide-Induced Apoptosis In Vitro

Monowarul M Siddique,Benjamin T Bikman,Scott A Summers,Li Ying,Erin Reinhardt,Guanghou Shui,Markus R Wenk,Liping Wang,Rafael Linden

doi:10.1371/journal.pone.0044042

Abstract

Sphingolipid biosynthesis is potently upregulated by factors associated with cellular stress, including numerous chemotherapeutics, inflammatory cytokines, and glucocorticoids. Dihydroceramide desaturase 1 (Des1), the third enzyme in the highly conserved pathway driving sphingolipid biosynthesis, introduces the 4,5-trans-double bond that typifies most higher-order sphingolipids. Surprisingly, recent studies have shown that certain chemotherapeutics and other drugs inhibit Des1, giving rise to a number of sphingolipids that lack the characteristic double bond. In order to assess the effect of an altered sphingolipid profile (via Des1 inhibition) on cell function, we generated isogenic mouse embryonic fibroblasts lacking both Des1 alleles. Lipidomic profiling revealed that these cells contained higher levels of dihydroceramide than wild-type fibroblasts and that complex sphingolipids were comprised predominantly of the saturated backbone (e.g. sphinganine vs. sphingosine, dihydrosphingomyelin vs. sphingomyelin, etc.). Des1 ablation activated pro-survival and anabolic signaling intermediates (e.g. Akt/PKB, mTOR, MAPK, etc.) and provided protection from apoptosis caused by etoposide, a chemotherapeutic that induces sphingolipid synthesis by upregulating several sphingolipid biosynthesizing enzymes. These data reveal that the double bond present in most sphingolipids has a profound impact on cell survival pathways, and that the manipulation of Des1 could have important effects on apoptosis.

Highlights

Sphingolipids are comprised of a sphingosine backbone coupled to a fatty acyl-chain
The third reaction in the ceramide biosynthesis pathway is catalyzed by dihydroceramide desaturase, an enzyme that introduces a 4,5-trans-double bond into the sphinganine backbone of dihydroceramide
Ceramide was replaced with dihydroceramide (Fig. 1C), sphingomyelin with dihydrosphingomyelin (Fig. 1D), and sphingosine with sphinganine (Fig. 1E)

Summary

Introduction

Sphingolipids are comprised of a sphingosine backbone coupled to a fatty acyl-chain. De novo sphingolipid biosynthesis involves a highly conserved pathway initiated by the condensation of palmitate and serine. The third reaction in the ceramide biosynthesis pathway is catalyzed by dihydroceramide desaturase, an enzyme that introduces a 4,5-trans-double bond into the sphinganine backbone of dihydroceramide. Lipidomic analysis has recently revealed that a number of factors including various chemotherapeutics [9,10], reactive oxygen species [11], and resveratrol [12] inhibit dihydroceramide desaturation, promoting accumulation of the dihydro form of high-order sphingolipids. These observations have prompted studies on this enzyme and this unique class of lipids [12,13]

Methods

Results

Conclusion