Thrombin-induced rapid geranylgeranylation of RhoA as an essential process for RhoA activation in endothelial cells
Thrombin-induced rapid geranylgeranylation of RhoA as an essential process for RhoA activation in endothelial cells
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DOI：
10.1074/jbc.m409547200
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发表时间：
2005-03-18
影响因子：
4.8
通讯作者：
Maruyama, Y
中科院分区：
文献类型：
--
作者：
Ohkawara, H;Ishibashi, T;Maruyama, Y
RhoA在凝血酶诱导的内皮功能障碍中起着关键的信号传导作用。可能的凝血酶调节的香叶基香叶基化，脂质修饰，未加工的RhoA和RhoA激活中的内皮细胞（EC）的香叶基香叶基化的意义还没有很好地理解。在未刺激的培养的人主动脉EC中，RhoA的未加工形式和香叶基香叶基化形式的量分别为31 +/-8和69 +/-8%总细胞RhoA（n = 6，p < 0.0001），如通过Triton X-114分配方法测定的。凝血酶通过刺激香叶基香叶基转移酶I（GGT酶I）活性，在1分钟内诱导大部分未加工的RhoA快速转化为香叶基香叶基化形式。凝血酶诱导的快速geranylgeranylation抑制急性短期（3分钟）预处理阿托伐他汀以及抑制剂的GGT酶I（GGTI-286）。凝血酶也迅速刺激GTP加载RhoA，这是阻断急性预处理阿托伐他汀或GGTI-286。这些观察结果表明凝血酶刺激RhoA对未加工RhoA的快速geranylgeranylation的依赖性。重要的是，添加香叶基香叶基焦磷酸到用阿托伐他汀预处理的EC中迅速逆转了阿托伐他汀对凝血酶刺激RhoA的抑制。这些结果表明，未加工的RhoA的香叶基香叶基化可能会限制凝血酶诱导的内皮细胞中RhoA的完全激活。细胞骨架分析表明，阿托伐他汀和GGTI-286抑制凝血酶诱导的应力纤维形成。我们提供的证据表明，在凝血酶刺激的内皮细胞，RhoA的未加工形式迅速geranylgeranylated成为成熟的形式，然后转化为GTP结合的活性RhoA。
RhoA plays a critical signaling role in thrombin-induced endothelial dysfunction. The possible thrombin regulation of geranylgeranylation, a lipid modification, of unprocessed RhoA and the significance of the geranylgeranylation in RhoA activation in endothelial cells (ECs) are not well understood. The amounts of the unprocessed and geranylgeranylated forms of RhoA in non-stimulated cultured human aortic ECs were 31 +/- 8 and 69 +/- 8% total cellular RhoA, respectively (n = 6, p < 0.0001), as determined by the Triton X-114 partition method. Thrombin-induced rapid conversion of most of the unprocessed RhoA into the geranylgeranylated form within 1 min through stimulating geranylgeranyltransferase I ( GGTase I) activity. Thrombin-induced rapid geranylgeranylation was inhibited by acute short term ( 3 min) pretreatment with atorvastatin as well as by an inhibitor of GGTase I (GGTI-286). Thrombin also rapidly stimulated GTP loading of RhoA, which was blocked by acute pretreatment with either atorvastatin or GGTI-286. These observations indicate the dependence of thrombin stimulation of RhoA on the rapid geranylgeranylation of unprocessed RhoA. Importantly, the addition of geranylgeranylpyrophosphate to ECs pretreated with atorvastatin quickly reversed the atorvastatin inhibition of thrombin stimulation of RhoA. These results suggest that geranylgeranylation of unprocessed RhoA may limit thrombin-induced full activation of RhoA in ECs. Cytoskeleton analysis demonstrated that atorvastatin and GGTI-286 inhibited thrombin-induced stress fiber formation. We provide the evidence that, in thrombin-stimulated ECs, the unprocessed form of RhoA is rapidly geranylgeranylated to become the mature form, which then is converted into GTP-bound active RhoA.