StARD13(Dlc-2) RhoGap mediates ceramide activation of phosphatidylglycerolphosphate synthase and drug response in Chinese hamster ovary cells.
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 C(2)-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 C(2)-ceramide addition. Parental cells showed elevated enzyme activities after treatment with C(2)-ceramide or tumor necrosis factor alpha, 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 C(2)-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.
Hatch
Grant M
GM
Manitoba Institute of Cell Biology, CancerCare Manitoba, Winnipeg, Manitoba, Canada R3E 0V9.
Gu
Yuan
Y
Xu
Fred Y
FY
Cizeau
Jeannick
J
Neumann
Shannon
S
Park
Ji-Seon
JS
Loewen
Shauna
S
Mowat
Michael R A
MR
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001483:Base Sequence; D016466:CHO Cells; D002470:Cell Survival / Q000187:drug effects; D002518:Ceramides / Q000494:pharmacology*; D003114:Colony-Forming Units Assay; D006224:Cricetinae; D003412:Cricetulus; D004351:Drug Resistance / Q000187:drug effects*; D004789:Enzyme Activation / Q000187:drug effects; D005047:Etoposide / Q000494:pharmacology*; D005091:Exons / Q000235:genetics; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005799:Genes, Dominant; D008969:Molecular Sequence Data; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D017855:Transferases (Other Substituted Phosphate Groups) / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D025521:Tumor Suppressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D002518:Ceramides; D020690:GTPase-Activating Proteins; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; D025521:Tumor Suppressor Proteins; C085379:rho GTPase-activating protein; D005047:Etoposide; D017855:Transferases (Other Substituted Phosphate Groups); C020676:CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase