Vav1 鸟嘌呤核苷酸交换因子在 Jurkat 白血病 T 细胞中 Bcl-2 转录和凋亡抑制中的独特作用。
The distinct role of guanine nucleotide exchange factor Vav1 in Bcl-2 transcription and apoptosis inhibition in Jurkat leukemia T cells.
机构信息
Tianjin Key Laboratory of Protein Sciences, Department of Biochemistry and Molecular Biology, College of Life Sciences, Nankai University, Tianjin 300071, China.
出版信息
Acta Pharmacol Sin. 2011 Jan;32(1):99-107. doi: 10.1038/aps.2010.185. Epub 2010 Dec 13.
AIM
To investigate a novel function of proto-oncogene Vav1 in the apoptosis of human leukemia Jurkat cells.
METHODS
Jurkat cells, Jurkat-derived vav1-null cells (J.Vav1) and Vav1-reconstituted J.WT cells were treated with a Fas agonist antibody, IgM clone CH11. Apoptosis was determined using propidium iodide (PI) staining, Annexin-V staining, DNA fragmentation, cleavage of caspase 3/caspase 8, and poly (ADP-ribose) polymerase (PARP). Mitochondria transmembrane potential (ΔΨ(m)) was measured using DiOC(6)(3) staining. Transcription and expression of the Bcl-2 family of proteins were evaluated using semi-quantitative RT-PCR and Western blot, respectively. Bcl-2 promoter activity was analyzed using luciferase reporter assays.
RESULTS
Cells lacking Vav1 were more sensitive to Fas-mediated apoptosis than Jurkat and J.WT cells. J.Vav1 cells lost mitochondria transmembrane potential (ΔΨ(m)) more rapidly upon Fas induction. These phenotypes could be rescued by re-expression of Vav1 in J.Vav1 cells. The expression of Vav1 increased the transcription of pro-survival Bcl-2. The guanine nucleotide exchange activity of Vav1 was required for enhancing Bcl-2 promoter activity, and the Vav1 downstream substrate, small GTPase Rac2, was likely involved in the control of Bcl-2 expression.
CONCLUSION
Vav1 protects Jurkat cells from Fas-mediated apoptosis by promoting Bcl-2 transcription through its GEF activity.
目的
研究原癌基因 Vav1 在人白血病 Jurkat 细胞凋亡中的新功能。
方法
用 Fas 激动型抗体 IgM 克隆 CH11 处理 Jurkat 细胞、Jurkat 衍生的 vav1 缺失细胞（J.Vav1）和 Vav1 重建的 J.WT 细胞。用碘化丙啶（PI）染色、Annexin-V 染色、DNA 片段化、caspase 3/caspase 8 的切割和多聚（ADP-核糖）聚合酶（PARP）来确定细胞凋亡。使用 DiOC(6)(3)染色测量线粒体跨膜电位（ΔΨ(m)）。使用半定量 RT-PCR 和 Western blot 分别评估 Bcl-2 家族蛋白的转录和表达。通过荧光素酶报告基因分析来分析 Bcl-2 启动子活性。
结果
缺乏 Vav1 的细胞比 Jurkat 和 J.WT 细胞对 Fas 介导的凋亡更敏感。J.Vav1 细胞在 Fas 诱导时更快地失去线粒体跨膜电位（ΔΨ(m)）。这些表型可以通过在 J.Vav1 细胞中重新表达 Vav1 来挽救。Vav1 的表达增加了抗凋亡 Bcl-2 的转录。Vav1 的鸟嘌呤核苷酸交换活性对于增强 Bcl-2 启动子活性是必需的，并且 Vav1 的下游底物，小 GTPase Rac2，可能参与了 Bcl-2 表达的控制。
结论
Vav1 通过其 GEF 活性促进 Bcl-2 转录，从而保护 Jurkat 细胞免受 Fas 介导的凋亡。
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