Evans Justin, Ko YooSeung, Mata Wilmer, Saquib Muhammad, Eldridge Joel, Cohen-Gadol Aaron, Leaver H Anne, Wang Shukun, Rizzo Maria Teresa
Signal Transduction Laboratory, Methodist Research Institute, Indiana University Health, Indiana University School of Medicine, Indianapolis, IN, USA.
Goodman Campbell Brain and Spine, Department of Neurological Surgery, Indiana University School of Medicine, Indianapolis, IN, USA.
Microvasc Res. 2015 Mar;98:145-58. doi: 10.1016/j.mvr.2014.04.011. Epub 2014 May 5.
Arachidonic acid (AA), a bioactive fatty acid whose levels increase during neuroinflammation, contributes to cerebral vascular damage and dysfunction. However, the mode of injury and underlying signaling mechanisms remain unknown. Challenge of primary human brain endothelial cells (HBECs) with AA activated a stress response resulting in caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and disruption of monolayer integrity. AA also induced loss of mitochondrial membrane potential and cytochrome c release consistent with activation of intrinsic apoptosis. HBEC stimulation with AA resulted in sustained p38-MAPK activation and subsequent phosphorylation of mitogen-activated protein kinase activated protein-2 (MAPKAP-2) kinase and heat shock protein-27 (Hsp27). Conversely, other unsaturated and saturated fatty acids had no effect. Pharmacological and RNA interference-mediated p38α or p38β suppression abrogated AA signaling to caspase-3 and Hsp27, suggesting involvement of both p38 isoforms in AA-induced HBEC apoptosis. Hsp27 silencing also blocked caspase-3 activation. AA stimulated intracellular calcium release, which was attenuated by inositol 1,4,5-trisphosphate (IP3) receptor antagonists. Blockade of intracellular calcium release decreased caspase-3 activation, but had no effect on AA-induced p38-MAPK activation. However, inhibition of p38-MAPK or blockade of intracellular calcium mobilization abrogated AA-induced cytochrome c release. AA-induced caspase-3 activation was abrogated by pharmacological inhibition of lipooxygenases. These findings support a previously unrecognized signaling cooperation between p38-MAPK/MAPKAP-2/Hsp27 and intracellular calcium release in AA-induced HBEC apoptosis and suggest its relevance to neurological disorders associated with vascular inflammation.
花生四烯酸（AA）是一种生物活性脂肪酸，其水平在神经炎症期间会升高，它会导致脑血管损伤和功能障碍。然而，损伤模式和潜在的信号传导机制仍不清楚。用AA刺激原代人脑内皮细胞（HBECs）会激活应激反应，导致半胱天冬酶-3激活、聚（ADP-核糖）聚合酶裂解以及单层完整性破坏。AA还会导致线粒体膜电位丧失和细胞色素c释放，这与内源性凋亡的激活一致。用AA刺激HBECs会导致p38丝裂原活化蛋白激酶（p38-MAPK）持续激活，随后丝裂原活化蛋白激酶激活蛋白-2（MAPKAP-2）激酶和热休克蛋白-27（Hsp27）发生磷酸化。相反，其他不饱和脂肪酸和饱和脂肪酸则没有影响。药理学和RNA干扰介导的p38α或p38β抑制消除了AA向半胱天冬酶-3和Hsp27的信号传导，表明这两种p38亚型都参与了AA诱导的HBEC凋亡。Hsp27沉默也会阻止半胱天冬酶-3激活。AA刺激细胞内钙释放，这被肌醇1,4,5-三磷酸（IP3）受体拮抗剂所减弱。细胞内钙释放的阻断会降低半胱天冬酶-3激活，但对AA诱导的p38-MAPK激活没有影响。然而，p38-MAPK的抑制或细胞内钙动员的阻断消除了AA诱导的细胞色素c释放。AA诱导的半胱天冬酶-3激活通过脂氧合酶的药理学抑制而被消除。这些发现支持了p38-MAPK/MAPKAP-2/Hsp27与细胞内钙释放在AA诱导的HBEC凋亡中之前未被认识到的信号合作，并表明其与血管炎症相关的神经系统疾病的相关性。
