Intracellular signaling pathways involved in Gas6-Ax1-mediated survival of endothelial cells
Intracellular signaling pathways involved in Gas6-Ax1-mediated survival of endothelial cells
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DOI：
10.1152/ajpheart.00020.2004
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发表时间：
2004-09-01
影响因子：
4.8
通讯作者：
Blostein, MD
中科院分区：
文献类型：
--
作者：
Hasanbasic, I;Cuerquis, J;Blostein, MD
Gas6是受体酪氨酸激酶Axl的一种伽马羧化配体。Gas6-Axl的相互作用可以使内皮细胞免于凋亡，本研究探讨了导致这一现象的细胞内信号机制。应用流式细胞术首次证实Gas6能抑制原代培养的人脐静脉内皮细胞(HUVECs)血清饥饿诱导的细胞凋亡。这种作用是通过丝氨酸-苏氨酸激酶Akt的磷酸化来实现的，在100 ng/mlGas6处理4小时后达到最大的磷酸化。Wortmannin抑制Akt磷酸化并阻断Gas6介导的人脐静脉内皮细胞存活涉及磷脂酰肌醇3-激酶作为Akt磷酸化的中介。显性负性Akt结构在很大程度上消除了Gas6对HUVECs的保护作用，强调了Akt激活在Gas6介导的存活中的重要性。在HUVECs中发现了该存活通路的几个下游调控因子，即核因子-kappaB以及抗凋亡和促凋亡蛋白Bcl2和caspase3。我们发现，在Gas6处理后的早期，核因子-kappaB被磷酸化，这从Western blotting上的双联形成中得到了证明。同时，Bc l-2蛋白水平升高，支持Bc l-2抗细胞凋亡途径被刺激的观点。Gas6处理后，caspase3激活产物p12和p20的表达水平降低，与促凋亡caspase3活性降低一致。综上所述，这些实验为Gas6保护原代培养内皮细胞免受凋亡的机制提供了新的信息。
Gas6 is a gamma-carboxylated ligand for the receptor tyrosine kinase Axl. Gas6-Axl interactions can rescue endothelial cells from apoptosis, and this study examined the intracellular signaling mechanisms responsible for this phenomenon. Using flow cytometry, we first confirmed that Gas6 can abrogate apoptosis induced by serum starvation of primary cultures of human umbilical vein endothelial cells (HUVECs). This effect is mediated through phosphorylation of the serine-threonine kinase Akt, with maximal phosphorylation observed after 4 h of treatment with 100 ng/ml Gas6. Inhibition of Akt phosphorylation and abrogation of gas6-mediated survival of HUVECs by wortmannin implicated phosphatidylinositol 3-kinase as the mediator of Akt phosphorylation. Dominant negative Akt constructs largely abrogated the protective effect of Gas6 on HUVECs, underscoring the importance of Akt activation in Gas6-mediated survival. Several downstream regulators of this survival pathway were identified in HUVECs, namely, NF-kappaB as well as the antiapoptotic and proapoptotic proteins Bcl-2 and caspase 3, respectively. We showed that NF-kappaB is phosphorylated early after Gas6 treatment as evidenced by doublet formation on Western blotting. As well, the level of Bcl-2 protein increased, supporting the notion that the Bcl-2 antiapoptotic pathway is stimulated. The levels of expression of the caspase 3 activation products p12 and p20 decreased with Gas6 treatment, consistent with a reduction in proapoptotic caspase 3 activation. Taken together, these experiments provide new information about the mechanism underlying Gas6 protection from apoptosis in primary endothelial cell cultures.