Biotechnology and Bioengineering Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Microcirculation. 2010 May;17(4):259-70. doi: 10.1111/j.1549-8719.2010.00031.x.
Previous studies have shown that physiological levels of shear stress can protect endothelial cells (ECs) from apoptotic stimuli. Here, we differentiate between acute and chronic protection and demonstrate the use of proteomic technologies to uncover mechanisms associated with chronic protection of ECs. We hypothesized that changes in abundance of proteins associated with the TNF-alpha signaling cascade orchestrate shear stress-mediated protection from TNF-alpha when cells are preconditioned with shear prior to the exposure of apoptotic stimuli. Detection of cleaved caspase 3 through Western blot analysis confirmed chronic shear stress-mediated protection from TNF-alpha. In the presence of the nitric oxide synthase inhibitor, LNMA (N(omega)-monomethyl-l-arginine), chronic protection remained. Treatment with a de novo protein synthesis inhibitor, cycloheximide, eliminated this protective effect. Isotopic-labeling experiments, coupled with LC-MS/MS (liquid chromatography-tandem mass spectrometry) of isolated components of the TNF-alpha pathway revealed that CARD9, a known activator of the NF-kappaB pathway, was increased (60%) in sheared cells versus nonsheared cells. This result was confirmed through Western blot analysis. Our data suggest that de novo formation of proteins is required for protection from TNF-alpha in ECs chronically exposed to shear stress, and that CARD9 is a candidate protein in this response.
先前的研究表明，生理水平的切应力可保护内皮细胞（ECs）免受凋亡刺激。在这里，我们区分了急性和慢性保护，并展示了使用蛋白质组学技术来揭示与 ECs 慢性保护相关的机制。我们假设与 TNF-α信号级联相关的蛋白质丰度的变化，在细胞在暴露于凋亡刺激之前用切应力预处理时，可调节 TNF-α介导的切应力保护。通过 Western blot 分析检测到 cleaved caspase 3 证实了 TNF-α的慢性切应力介导的保护。在存在一氧化氮合酶抑制剂 LNMA（N（ω）-单甲基-l-精氨酸）的情况下，慢性保护仍然存在。用新的蛋白质合成抑制剂环己亚胺处理后，消除了这种保护作用。同位素标记实验，与 TNF-α途径的分离成分的 LC-MS/MS（液相色谱-串联质谱）相结合，表明 CARD9（NF-κB 途径的已知激活剂）在剪切细胞中比非剪切细胞增加了 60%。这一结果通过 Western blot 分析得到了证实。我们的数据表明，新形成的蛋白质是 ECs 慢性暴露于切应力时免受 TNF-α的保护所必需的，而 CARD9 是该反应中的候选蛋白。
