Rafiq Khadija, Kolpakov Mikhail A, Abdelfettah Malika, Streblow Daniel N, Hassid Aviv, Dell'Italia Louis J, Sabri Abdelkarim
Cardiovascular Research Center, Department of Anatomy and Cell Biology, Temple University, Philadelphia, Pennsylvania 19140, USA.
J Biol Chem. 2006 Jul 14;281(28):19781-92. doi: 10.1074/jbc.M513040200. Epub 2006 May 11.
Inflammatory cells and their proteases contribute to tissue reparation at site of inflammation. Although beneficial at early stages, excessive inflammatory reaction leads to cell death and tissue damage. Cathepsin G (Cat.G), a neutrophil-derived serine protease, has been shown to induce neonatal rat cardiomyocyte detachment and apoptosis by anoikis through caspase-3 dependent pathway. However the early mechanisms that trigger Cat.G-induced caspase-3 activation are not known. This study identifies focal adhesion kinase (FAK) tyrosine dephosphorylation as an early mechanism that regulates Cat.G-induced anoikis in cardiomyocytes. Both FAK tyrosine phosphorylation at Tyr-397 and kinase activity decrease rapidly upon Cat.G treatment and was associated with a decrease of FAK association with adapter and cytoskeletal proteins, p130(Cas) and paxillin, respectively. FAK-decreased tyrosine phosphorylation is required for Cat.G-induced myocyte anoikis as concurrent expression of phosphorylation-deficient FAK mutated at Tyr-397 or pretreatment with a protein-tyrosine phosphatase (PTP) inhibitor, pervanadate, blocks Cat.G-induced FAK tyrosine dephosphorylation, caspase-3 activation and DNA fragmentation. Analysis of PTPs activation shows that Cat.G treatment induces an increase of SHP2 and PTEN phosphorylation; however, only SHP2 forms a complex with FAK in response to Cat.G. Expression of dominant negative SHP2 mutant markedly attenuates FAK tyrosine dephosphorylation induced by Cat.G and protects myocytes to undergo apoptosis. In contrast, increased SHP2 expression exacerbates Cat.G-induced FAK tyrosine dephosphorylation and myocyte apoptosis. Taken together, these results show that Cat.G induces SHP2 activation that leads to FAK tyrosine dephosphorylation and promotes cardiomyocyte anoikis.
炎症细胞及其蛋白酶有助于炎症部位的组织修复。虽然在早期阶段有益，但过度的炎症反应会导致细胞死亡和组织损伤。组织蛋白酶G（Cat.G）是一种源自中性粒细胞的丝氨酸蛋白酶，已被证明可通过依赖半胱天冬酶-3的途径，通过失巢凋亡诱导新生大鼠心肌细胞脱离和凋亡。然而，触发Cat.G诱导的半胱天冬酶-3激活的早期机制尚不清楚。本研究确定粘着斑激酶（FAK）酪氨酸去磷酸化是调节Cat.G诱导的心肌细胞失巢凋亡的早期机制。Cat.G处理后，Tyr-397处的FAK酪氨酸磷酸化和激酶活性均迅速降低，分别与FAK与衔接蛋白和细胞骨架蛋白p130（Cas）和桩蛋白的结合减少有关。Cat.G诱导的心肌细胞失巢凋亡需要FAK酪氨酸磷酸化降低，因为同时表达在Tyr-397处突变的磷酸化缺陷型FAK或用蛋白酪氨酸磷酸酶（PTP）抑制剂过氧钒酸盐预处理可阻断Cat.G诱导的FAK酪氨酸去磷酸化、半胱天冬酶-3激活和DNA片段化。对PTP激活的分析表明，Cat.G处理可诱导SHP2和PTEN磷酸化增加；然而，只有SHP2在响应Cat.G时与FAK形成复合物。显性负性SHP2突变体的表达显著减弱了Cat.G诱导的FAK酪氨酸去磷酸化，并保护心肌细胞免于凋亡。相反，增加SHP2表达会加剧Cat.G诱导的FAK酪氨酸去磷酸化和心肌细胞凋亡。综上所述，这些结果表明，Cat.G诱导SHP2激活，导致FAK酪氨酸去磷酸化，并促进心肌细胞失巢凋亡。
