Yi Fan, Chen Qi-Zheng, Jin Si, Li Pin-Lan
Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298, USA.
Cell Physiol Biochem. 2007;20(6):909-18. doi: 10.1159/000110451.
We have demonstrated that homocysteine (Hcys) stimulates de novo ceramide synthesis and thereby induces NADPH oxidase activation by increase of Rac GTPase activity in rat mesangial cells (RMCs). However, which isofrom of Rac GTPases is involved in Hcys-induced NADPH oxidase activity and what mechanism mediates Hcys-induced Rac GTPase activation remain unknown. The present study first addressed the role of Rac1 and then determined the contribution of a subfamily of Guanine Nucleotide Exchange Factors (GEFs), Vav, to the action of Hcys on Rac and NADPH oxidase activities in RMCs. By small interfering RNA (siRNA), it was found that Rac1-siRNA attenuated Hcys-induced superoxide (O(2)(-)) production. To explore the mechanism activating Rac by Hcys, GEF-Vav was examined. Vav2 was found to be a predominant isoform among Vav family in RMCs. In Vav2-siRNA transfected RMCs, Hcys-induced Rac activity was blocked, which was accompanied by significant reduction of Hcys-induced O(2)(-). production. This Vav2-siRNA also blocked Rac activation induced by C16-Ceramide (C16-Cer), an intermediate lipid product stimulated by Hcys. Furthermore, we found that Hcys induced Vav2 phosphorylation in a time-dependent manner, which could be induced by C16-Cer and blocked by inhibition of de novo ceramide synthesis. These results suggest that Vav2 importantly contributes to Hcys-induced increase in Rac1 activity and consequent activation of NADPH oxidase in RMCs via ceramide-associated tyrosine phosphorylation.
我们已经证明，同型半胱氨酸（Hcys）刺激神经酰胺的从头合成，从而通过增加大鼠系膜细胞（RMCs）中Rac GTP酶活性来诱导NADPH氧化酶激活。然而，哪种Rac GTP酶同工型参与Hcys诱导的NADPH氧化酶活性以及什么机制介导Hcys诱导的Rac GTP酶激活仍然未知。本研究首先探讨了Rac1的作用，然后确定了鸟嘌呤核苷酸交换因子（GEFs）亚家族Vav对Hcys在RMCs中对Rac和NADPH氧化酶活性作用的贡献。通过小干扰RNA（siRNA）发现，Rac1-siRNA减弱了Hcys诱导的超氧化物（O(2)(-)）产生。为了探索Hcys激活Rac的机制，对GEF-Vav进行了研究。发现Vav2是RMCs中Vav家族中的主要同工型。在转染了Vav2-siRNA的RMCs中，Hcys诱导的Rac活性被阻断，同时Hcys诱导的O(2)(-)产生显著减少。这种Vav2-siRNA也阻断了由Hcys刺激的中间脂质产物C16-神经酰胺（C16-Cer）诱导的Rac激活。此外，我们发现Hcys以时间依赖性方式诱导Vav2磷酸化，这可由C16-Cer诱导并被从头神经酰胺合成的抑制所阻断。这些结果表明，Vav2通过神经酰胺相关的酪氨酸磷酸化对Hcys诱导的RMCs中Rac1活性增加及随后的NADPH氧化酶激活起重要作用。
