Involvement of dihydroceramide desaturase in cell cycle progression in human neuroblastoma cells
Involvement of dihydroceramide desaturase in cell cycle progression in human neuroblastoma cells
复制标题
DOI：
10.1074/jbc.m700647200
复制
发表时间：
2007-06-08
影响因子：
4.8
通讯作者：
Bielawska, Alicja
中科院分区：
文献类型：
--
作者：
Kraveka, Jacqueline M.;Li, Li;Bielawska, Alicja
在人神经母细胞瘤细胞（SMS-KCNR）中研究了二氢神经酰胺去饱和酶作为神经酰胺从头生成途径中的关键酶的作用。使用二氢神经酰胺的水溶性类似物二氢神经酰胺类（D-dichloro-dhCCPS类似物）的新测定法用于原位测量去饱和酶活性。通过液相色谱/质谱分析测定D-溴代-2-N-[12 '-（1“-吡啶鎓）-十二烷酰基]-4，5-二氢鞘氨醇溴化物（C-12-dhCCPS）向其4，5-去饱和对应物D-溴代-2-N-[12 '-（1”-吡啶鎓）十二烷酰基]鞘氨醇溴化物（C-12-CCPS）的转化。使用C-8-环丙烯基神经酰胺（二氢神经酰胺去饱和酶的竞争性抑制剂）证实了测定的有效性。果蝇des-1基因的人类同源物（DEGS-1）最近被鉴定并报道具有去饱和酶活性。转染SMS-KCNR细胞的DEGS-1的小干扰RNA显著阻断C-12-dhCCPS向C-12-CCPS的转化。内源性二氢神经酰胺的相关积累证实DEGS-1是这些细胞中主要的活性二氢神经酰胺去饱和酶。DEGS-1的部分缺失抑制细胞生长，细胞周期停滞在G（0）/G（1）。这伴随着磷酸化视网膜母细胞瘤蛋白的量的显著减少。这种低磷酸化抑制互变霉素，而不是由冈田酸，这表明蛋白磷酸酶1的参与。此外，我们发现用芬维A胺处理SMS-KCNR细胞以剂量依赖性方式抑制去饱和酶活性。通过液相色谱/质谱法测量，二氢神经酰胺（但不是神经酰胺）的增加证实了这一过程。对DEGS-1的mRNA或蛋白水平没有影响，表明芬维A胺在翻译后水平作为该酶的抑制剂起作用。在芬维A胺治疗后观察到的视网膜母细胞瘤蛋白的低磷酸化，他莫霉素也能够阻断。这些发现表明二氢神经酰胺具有新的生物学功能。
The role of dihydroceramide desaturase as a key enzyme in the de novo pathway of ceramide generation was investigated in human neuroblastoma cells (SMS-KCNR). A novel assay using water-soluble analogs of dihydroceramide, dihydroceramidoids (D-erythro-dhCCPS analogs), was used to measure desaturase activity in situ. Conversion of D-erythro-2-N-[12 '-(1 ''-pyridinium)-dodecanoyl]- 4,5-dihydrosphingosine bromide (C-12-dhCCPS) to its 4,5-desaturated counterpart, D-erythro-2-N-[12 '-(1 ''-pyridinium) dodecanoyl]sphingosine bromide (C-12-CCPS), was determined by liquid chromatography/mass spectrometry analysis. The validity of the assay was confirmed using C-8-cyclopropenylceramide, a competitive inhibitor of dihydroceramide desaturase. A human homolog (DEGS-1) of the Drosophila melanogaster des-1 gene was recently identified and reported to have desaturase activity. Transfection of SMS-KCNR cells with small interfering RNA to DEGS-1 significantly blocked the conversion of C-12-dhCCPS to C-12-CCPS. The associated accumulation of endogenous dihydroceramides confirmed DEGS-1 as the main active dihydroceramide desaturase in these cells. The partial loss of DEGS-1 inhibited cell growth, with cell cycle arrest at G(0)/G(1). This was accompanied by a significant decrease in the amount of phosphorylated retinoblastoma protein. This hypophosphorylation was inhibited by tautomycin and not by okadaic acid, suggesting the involvement of protein phosphatase 1. Additionally, we found that treatment of SMS-KCNR cells with fenretinide inhibited desaturase activity in a dose-dependent manner. An increase in dihydroceramides (but not ceramides) paralleled this process as measured by liquid chromatography/mass spectrometry. There were no effects on the mRNA or protein levels of DEGS-1, suggesting that fenretinide acts at the post-translational level as an inhibitor of this enzyme. Tautomycin was also able to block the hypophosphorylation of the retinoblastoma protein observed upon fenretinide treatment. These findings suggest a novel biological function for dihydroceramides.