Lu Changming, Pelech Steven, Zhang Hong, Bond Jeffrey, Spach Karen, Noubade Rajkumar, Blankenhorn Elizabeth P, Teuscher Cory
Department of Medicine, University of Vermont, Burlington, Vermont 05405, USA.
J Neurosci Res. 2008 Sep;86(12):2624-40. doi: 10.1002/jnr.21716.
Pertussis toxin (PTX) is an ancillary adjuvant used to elicit experimental allergic encephalomyelitis (EAE), the principal autoimmune model of multiple sclerosis. One mechanism whereby PTX potentiates EAE is to increase blood-brain barrier (BBB) permeability. To elucidate further the mechanism of action of PTX on the BBB, we investigated the genomic and proteomic responses of isolated mouse brain endothelial cells (MBEC) following intoxication. Among approximately 14,000 mouse genes tracked by cDNA microarray, 34 showed altered expression in response to PTX. More than one-third of these genes have roles in angiogenesis. Accordingly, we show that intoxication of MBEC induces tube formation in vitro and angiogenesis in vivo. The global effect of PTX on signaling protein levels and phosphorylation in MBEC was investigated by using Kinex antibody microarrays. In total, 113 of 372 pan-specific and 58 of 258 phospho-site-specific antibodies revealed changes >or=25% following intoxication. Increased STAT1 Tyr-701 and Ser-727 phosphorylation; reduced phosphorylation of the activating phospho-sites in Erk1, Erk2, and MAPKAPK2; and decreased phosphorylation of arrestin beta1 Ser-412 and Hsp27 Ser-82 were confirmed by Kinetworks multi-immunoblotting. The importance of signal transduction pathways on PTX-induced MBEC tube formation was evaluated pharmacologically. Inhibition of phospholipase C, MEK1, and p38 MAP kinase had little effect, whereas inhibition of cAMP-dependent protein kinase, protein kinase C, and phosphatidylinositol 3-kinase partially blocked tube formation. Taken together, these findings are consistent with the concept that PTX may lead to increased BBB permeability by altering endothelial plasticity and angiogenesis.
百日咳毒素（PTX）是一种辅助佐剂，用于引发实验性自身免疫性脑脊髓炎（EAE），这是多发性硬化症的主要自身免疫模型。PTX增强EAE的一种机制是增加血脑屏障（BBB）的通透性。为了进一步阐明PTX对血脑屏障的作用机制，我们研究了中毒后分离的小鼠脑内皮细胞（MBEC）的基因组和蛋白质组反应。在通过cDNA微阵列追踪的约14000个小鼠基因中，有34个基因的表达因PTX而发生改变。这些基因中超过三分之一在血管生成中发挥作用。因此，我们表明MBEC中毒可在体外诱导管形成，并在体内诱导血管生成。使用Kinex抗体微阵列研究了PTX对MBEC中信号蛋白水平和磷酸化的整体影响。在中毒后，372种泛特异性抗体中的113种和258种磷酸化位点特异性抗体中的58种显示变化≥25%。通过Kinetworks多重免疫印迹证实了STAT1 Tyr-701和Ser-727磷酸化增加；Erk1、Erk2和MAPKAPK2中激活磷酸化位点的磷酸化减少；以及抑制蛋白β1 Ser-412和Hsp27 Ser-82的磷酸化减少。通过药理学评估了信号转导通路对PTX诱导的MBEC管形成的重要性。抑制磷脂酶C、MEK1和p38丝裂原活化蛋白激酶几乎没有作用，而抑制cAMP依赖性蛋白激酶、蛋白激酶C和磷脂酰肌醇3激酶可部分阻断管形成。综上所述，这些发现与PTX可能通过改变内皮可塑性和血管生成导致血脑屏障通透性增加的概念一致。
