Division of Cardiology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan.
Cardiovasc Res. 2012 Jan 1;93(1):181-9. doi: 10.1093/cvr/cvr280. Epub 2011 Oct 21.
The expression of PUMA (p53-up-regulated modulator of apoptosis), an apoptosis-regulating gene, increases during endoplasmic reticulum stress. The mechanisms by which cyclic stretch influences the regulation of PUMA in vascular smooth muscle cells (VSMCs) during apoptosis remain unclear. We hypothesized that cyclic stretch enhances PUMA expression in VSMCs undergoing apoptosis.
Human VSMCs grown on a Flexcell I flexible membrane base were stretched via vacuum to 20% of elongation at a frequency of 1 Hz. An in vivo model of volume overload with aorta-caval shunt and pressure overload with aortic banding in adult rats was used to study PUMA expression. Cyclic stretch markedly enhanced PUMA protein and gene expression after stretch. Addition of c-jun N-terminal kinase (JNK) inhibitor SP600125 and interferon-γ (IFN-γ) antibody 30 min before stretch inhibited PUMA expression. Gel shift assay demonstrated that stretch increased the DNA binding activity of interferon regulatory factor-1 (IRF-1). SP600125, JNK small interfering RNA, and IFN-γ antibody attenuated the DNA binding activity induced by stretch. PUMA-Mut plasmid, SP600125, and IRF-1 antibody attenuated the promoter activity. Stretch increased secretion of IFN-γ from VSMCs, and conditioned media from stretched VSMCs increased PUMA protein expression. The in vivo model of aorta-caval shunt and aortic banding also showed increased PUMA protein expression in the aorta.
Cyclic mechanical stretch increases PUMA expression in cultured human VSMCs. The PUMA expression induced by stretch is mediated by IFN-γ, JNK, and IRF-1 pathways. These findings suggest that PUMA is an important mediator in VSMC apoptosis induced by stretch.
凋亡调节基因 PUMA（p53 上调凋亡调节因子）的表达在内质网应激时增加。细胞周期拉伸通过何种机制影响血管平滑肌细胞（VSMCs）凋亡过程中 PUMA 的调节尚不清楚。我们假设细胞周期拉伸增强凋亡中的 VSMCs 中 PUMA 的表达。
在 Flexcell I 柔性膜基底上生长的人 VSMCs 通过真空拉伸至 20%的伸长率，频率为 1 Hz。采用主动脉-腔静脉分流的体内容量超负荷模型和主动脉缩窄的体内压力超负荷模型，研究 PUMA 表达。细胞周期拉伸后明显增强了 PUMA 蛋白和基因表达。在拉伸前 30 分钟加入 c-jun N 末端激酶（JNK）抑制剂 SP600125 和干扰素-γ（IFN-γ）抗体可抑制 PUMA 表达。凝胶迁移实验表明，拉伸增加了干扰素调节因子-1（IRF-1）的 DNA 结合活性。SP600125、JNK 小干扰 RNA 和 IFN-γ 抗体减弱了拉伸诱导的 DNA 结合活性。PUMA-Mut 质粒、SP600125 和 IRF-1 抗体减弱了启动子活性。拉伸增加了 VSMCs 中 IFN-γ 的分泌，并且来自拉伸 VSMCs 的条件培养基增加了 PUMA 蛋白表达。主动脉-腔静脉分流和主动脉缩窄的体内模型也显示主动脉中 PUMA 蛋白表达增加。
细胞周期拉伸增加了培养的人 VSMCs 中 PUMA 的表达。拉伸诱导的 PUMA 表达是通过 IFN-γ、JNK 和 IRF-1 途径介导的。这些发现表明 PUMA 是拉伸诱导的 VSMC 凋亡的重要介质。
