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Vol. 19, Issue 3, 1083-1092, March 2008

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StARD13(Dlc-2) RhoGap Mediates Ceramide Activation of Phosphatidylglycerolphosphate Synthase and Drug Response in Chinese Hamster Ovary Cells

Grant M. Hatch^*,, Yuan Gu, Fred Y. Xu^*, Jeannick Cizeau, Shannon Neumann, Ji-Seon Park, Shauna Loewen^,, and Michael R.A. Mowat^,

Manitoba Institute of Cell Biology, CancerCare Manitoba, Winnipeg, Manitoba, Canada R3E 0V9; and Departments of Biochemistry and Medical Genetics and *Pharmacology and Therapeutics, University of Manitoba, Winnipeg, Manitoba, Canada R3E 0V9

Submitted August 22, 2006; Revised November 15, 2007; Accepted December 14, 2007
Monitoring Editor: Howard Riezman

To identify genes involved in etoposide drug response, we used promoter trap mutagenesis to isolate an etoposide-resistant Chinese hamster ovary (CHO) cell line. This resistant CHO-K1 line, named E91, showed cross-resistance to C₂-ceramide (N-acetylsphingosine). The promoter trap retrovirus was found integrated into intron 1–2 of the Dlc-2 (Stard13) RhoGap gene. The E91 cells showed elevated guanosine triphosphate (GTP)-bound RhoA levels compared with the parental line, suggesting that retrovirus integration had inactivated one of the Dlc-2 RhoGap alleles. To test whether E91 cells were impaired in an intracellular ceramide-regulated process not directly related to cell killing, we measured mitochondrial phosphatidylglycerolphosphate (PGP) synthase and phospholipase A2 enzyme activities in cells after C₂-ceramide addition. Parental cells showed elevated enzyme activities after treatment with C₂-ceramide or tumor necrosis factor , but not the E91 cells. These results suggested that intracellular ceramide signaling was defective in E91 cells due to increased levels of active GTP-bound RhoA. RNA knockdown experiments of the Dlc2 RhoGap resulted in increased GTP-bound RhoA and reduced induction of PGP synthase after C₂-ceramide addition compared with controls. Expression of a dominant-negative RhoA in the E91 cell line allowed induction of PGP synthase by ceramide. The RNA interference knockdown cell line also showed increased etoposide resistance. This study is the first report for the regulation of a phospholipid biosynthetic enzyme through RhoGap expression.

Address correspondence to: Michael R.A. Mowat (mmowat{at}cc.umanitoba.ca)

Abbreviations used: C₂-Cer, N-acetylsphingosine; CHO, Chinese hamster ovary; CL, cardiolipin; CDP-DG, cytidine-5'-diphosphate-1,2-diacyl-sn-glycerol; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LPA, lysophosphatic acid; LTR, long terminal repeat; PG, phosphatidylglycerol; PLA2, phospholipase A2; SM, sphingomyelin; siRNA, short interfering RNA; shRNAi, short hairpin interfering RNA.