Abboud Elizabeth R, Shelby Bryan D, Angelova Magdalena, Nelson Anne B, Ferris Marybeth, McFerrin Harris E, Morris Cindy A, Sullivan Deborah E
Department of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, LA.
Ochsner J. 2013 Spring;13(1):66-75.
Kaposi sarcoma-associated herpesvirus (KSHV) encoded G protein-coupled receptor (vGPCR) is a constitutively active lytic phase protein with significant homology to the human interleukin-8 receptor. vGPCR is necessary and sufficient to induce angiogenesis as well as the spindle cell proliferation characteristic of Kaposi sarcoma (KS) lesions. We previously demonstrated that Bcl-2, an antiapoptotic protein, is upregulated in KS lesions. The aim of this study was to determine if vGPCR enhances endothelial cell survival through upregulation of Bcl-2 expression and to elucidate the signaling pathways involved.
Primary human umbilical vein endothelial cells were transduced with a recombinant retrovirus expressing vGPCR and then subjected to serum starvation. Cell viability and apoptosis were analyzed by fluorescence-activated cell sorting. Bcl-2 expression was determined by real-time quantitative reverse transcription polymerase chain reaction and immunoblotting. Specific pharmacological inhibitors of phosphatidylinositol 3-kinase (PI3K)/Akt and the mammalian target of rapamycin (mTOR) were employed to elucidate the signaling pathways involved. Bcl-2 expression was knocked down using small interfering RNA (siRNA).
Endothelial cells expressing vGPCR showed increased survival after serum starvation and upregulation of Bcl-2 messenger RNA (mRNA) and protein. The vGPCR-induced increases in both Bcl-2 mRNA and protein levels were dependent on PI3K signaling but not on mTOR. Moreover, siRNA inhibition of Bcl-2 resulted in significant abrogation of the observed vGPCR-mediated cell survival advantage.
Taken together, the results demonstrate that Bcl-2 is a mediator of vGPCR-induced endothelial cell survival and is a downstream effector of Akt in this process.
卡波西肉瘤相关疱疹病毒（KSHV）编码的G蛋白偶联受体（vGPCR）是一种组成型激活的裂解期蛋白，与人白细胞介素-8受体具有显著同源性。vGPCR对于诱导血管生成以及卡波西肉瘤（KS）病变特征性的梭形细胞增殖是必需且充分的。我们之前证明，抗凋亡蛋白Bcl-2在KS病变中上调。本研究的目的是确定vGPCR是否通过上调Bcl-2表达来增强内皮细胞存活，并阐明其中涉及的信号通路。
用表达vGPCR的重组逆转录病毒转导原代人脐静脉内皮细胞，然后进行血清饥饿处理。通过荧光激活细胞分选分析细胞活力和凋亡情况。通过实时定量逆转录聚合酶链反应和免疫印迹法测定Bcl-2表达。使用磷脂酰肌醇3激酶（PI3K）/Akt和雷帕霉素哺乳动物靶蛋白（mTOR）的特异性药理抑制剂来阐明其中涉及的信号通路。使用小干扰RNA（siRNA）敲低Bcl-2表达。
表达vGPCR的内皮细胞在血清饥饿后显示出存活率增加，且Bcl-2信使核糖核酸（mRNA）和蛋白上调。vGPCR诱导的Bcl-2 mRNA和蛋白水平增加均依赖于PI3K信号传导，而不依赖于mTOR。此外，siRNA抑制Bcl-2导致观察到的vGPCR介导的细胞存活优势显著消除。
综上所述，结果表明Bcl-2是vGPCR诱导的内皮细胞存活的介质，并且在此过程中是Akt的下游效应物。
