Triplett Jason W, Pavalko Fredrick M
Dept. of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
Am J Physiol Cell Physiol. 2006 Nov;291(5):C909-21. doi: 10.1152/ajpcell.00113.2006. Epub 2006 Jun 28.
Maintenance of bone structural integrity depends in part on the rate of apoptosis of bone-forming osteoblasts. Because substrate adhesion is an important regulator of apoptosis, we have investigated the role of focal adhesions in regulating bone cell apoptosis. To test this, we expressed a truncated form of alpha-actinin (ROD-GFP) that competitively displaces endogenous alpha-actinin from focal adhesions, thus disrupting focal adhesions. Immunofluorescence and morphometric analysis of vinculin and tyrosine phosphorylation revealed that ROD-GFP expression dramatically disrupted focal adhesion organization and reduced tyrosine phosphorylation at focal adhesions. In addition, Bcl-2 protein levels were reduced in ROD-GFP-expressing cells, but caspase 3 cleavage, poly(ADP-ribose) polymerase cleavage, histone H2A.X phosphorylation, and cytotoxicity were not increased due to ROD-GFP expression alone. Increases in both ERK and Akt phosphorylation were also observed in ROD-GFP-expressing cells, although inhibition of either ERK or Akt individually or together failed to induce apoptosis. However, we did find that ROD-GFP expression sensitized, whereas alpha-actinin-GFP expression protected, cells from TNF-alpha-induced apoptosis. Further investigation revealed that activation of TNF-alpha-induced survival signals, specifically Akt phosphorylation and NF-kappaB activation, was inhibited in ROD-GFP-expressing cells. The reduced expression of antiapoptotic Bcl-2 and inhibited survival signaling rendered ROD-GFP-expressing cells more susceptible to TNF-alpha-induced apoptosis. Thus we conclude that alpha-actinin plays a role in regulating cell survival through stabilization of focal adhesions and regulation of TNF-alpha-induced survival signaling.
骨结构完整性的维持部分取决于成骨细胞的凋亡速率。由于底物黏附是凋亡的重要调节因子，我们研究了黏着斑在调节骨细胞凋亡中的作用。为了验证这一点，我们表达了一种截短形式的α-辅肌动蛋白（ROD-GFP），它能竞争性地将内源性α-辅肌动蛋白从黏着斑中置换出来，从而破坏黏着斑。对纽蛋白和酪氨酸磷酸化的免疫荧光及形态计量分析表明，ROD-GFP的表达显著破坏了黏着斑的组织，并降低了黏着斑处的酪氨酸磷酸化。此外，在表达ROD-GFP的细胞中Bcl-2蛋白水平降低，但仅因ROD-GFP的表达，半胱天冬酶3的切割、聚（ADP-核糖）聚合酶的切割、组蛋白H2A.X的磷酸化及细胞毒性并未增加。在表达ROD-GFP的细胞中还观察到ERK和Akt磷酸化均增加，尽管单独或共同抑制ERK或Akt均未能诱导细胞凋亡。然而，我们确实发现ROD-GFP的表达使细胞对肿瘤坏死因子-α诱导的凋亡敏感，而α-辅肌动蛋白-GFP的表达则保护细胞免受这种凋亡。进一步研究表明，在表达ROD-GFP的细胞中，肿瘤坏死因子-α诱导的生存信号激活（特别是Akt磷酸化和核因子-κB激活）受到抑制。抗凋亡Bcl-2表达的降低及生存信号的抑制使表达ROD-GFP的细胞更易受到肿瘤坏死因子-α诱导的凋亡。因此我们得出结论，α-辅肌动蛋白通过稳定黏着斑和调节肿瘤坏死因子-α诱导的生存信号在调节细胞存活中发挥作用。
