Garin Gwenaele, Abe Jun-ichi, Mohan Amy, Lu Weimin, Yan Chen, Newby Andrew C, Rhaman Arshad, Berk Bradford C
University of Rochester, Cardiovascular Research Institute, Box 679, 601 Elmwood Avenue, Rochester, NY 14642, USA.
Circ Res. 2007 Jul 6;101(1):97-105. doi: 10.1161/CIRCRESAHA.107.148270. Epub 2007 May 24.
Unidirectional laminar flow is atheroprotective, in part by inhibiting cytokine-mediated endothelial cell (EC) inflammation and apoptosis. Previously, we showed that flow inhibited TNF-alpha signaling by preventing activation of JNK. Recently, PKCzeta was identified as the PKC isoform most strongly regulated by flow pattern, with increased PKCzeta activity in regions of disturbed flow versus unidirectional flow. Interestingly, PKCzeta is cleaved by caspases after TNF-alpha stimulation to generate a 50-kDa truncated form (CATzeta, catalytic domain of PKCzeta) with a higher kinase activity than the full-length protein. We hypothesized that flow would inhibit TNF-alpha-mediated PKCzeta cleavage and thereby CATzeta formation. We found that PKCzeta activity was required for TNF-alpha-mediated JNK and caspase-3 activation in ECs. PKCzeta was rapidly cleaved to generate CATzeta in cultured bovine and human aortic ECs and in intact rabbit vessels stimulated with TNF-alpha. This truncated form of PKCzeta enhanced JNK and caspase-3 activation. Interestingly, PKCzeta cleavage was prevented by inhibitors of PKCzeta, JNK, and caspase activities, suggesting that these enzymes, via regulating CATzeta formation, modulate caspase-3 activity in ECs. Finally, we found that flow reduced caspase-dependent processing of PKCzeta and caspase-3 activation. These results define a novel role for PKCzeta as a shared signaling mediator for flow and TNF-alpha, and important for flow-mediated inhibition of proinflammatory and apoptotic events in ECs.
单向层流具有抗动脉粥样硬化作用，部分原因是它能抑制细胞因子介导的内皮细胞（EC）炎症和凋亡。此前，我们发现血流通过阻止JNK的激活来抑制TNF-α信号传导。最近，PKCζ被确定为受血流模式调控最强的PKC同工型，在紊乱血流区域与单向血流区域相比，PKCζ活性增加。有趣的是，TNF-α刺激后，PKCζ会被半胱天冬酶切割，产生一种50 kDa的截短形式（CATζ，PKCζ的催化结构域），其激酶活性高于全长蛋白。我们推测血流会抑制TNF-α介导的PKCζ切割，从而抑制CATζ的形成。我们发现PKCζ活性是TNF-α介导的ECs中JNK和半胱天冬酶-3激活所必需的。在用TNF-α刺激的培养牛和人主动脉ECs以及完整兔血管中，PKCζ迅速被切割生成CATζ。这种截短形式的PKCζ增强了JNK和半胱天冬酶-3的激活。有趣的是，PKCζ的切割被PKCζ、JNK和半胱天冬酶活性抑制剂所阻止，这表明这些酶通过调节CATζ的形成来调节ECs中的半胱天冬酶-3活性。最后，我们发现血流减少了PKCζ的半胱天冬酶依赖性加工和半胱天冬酶-3的激活。这些结果确定了PKCζ作为血流和TNF-α共同信号介质的新作用，对血流介导的ECs中促炎和凋亡事件的抑制很重要。
