Department of Pediatrics, Xiangya Hospital of Central South University, No,87 Xiangya Road, Changsha, Hunan 410008, China.
J Neuroinflammation. 2011 Apr 8;8:28. doi: 10.1186/1742-2094-8-28.
Tumor necrosis factor-α (TNF-α), a proinflammatory cytokine, is capable of activating the small GTPase RhoA, which in turn contributes to endothelial barrier dysfunction. However, the underlying signaling mechanisms remained undefined. Therefore, we aimed to determine the role of protein kinase C (PKC) isozymes in the mechanism of RhoA activation and in signaling TNF-α-induced mouse brain microvascular endothelial cell (BMEC) barrier dysfunction.
Bend.3 cells, an immortalized mouse brain endothelial cell line, were exposed to TNF-α (10 ng/mL). RhoA activity was assessed by pull down assay. PKC-α activity was measured using enzyme assasy. BMEC barrier function was measured by transendothelial electrical resistance (TER). p115RhoGEF phosphorylation was detected by autoradiography followed by western blotting. F-actin organization was observed by rhodamine-phalloidin staining. Both pharmacological inhibitors and knockdown approaches were employed to investigate the role of PKC and p115RhoGEF in TNF-α-induced RhoA activation and BMEC permeability.
We observed that TNF-α induces a rapid phosphorylation of p115RhoGEF, activation of PKC and RhoA in BMECs. Inhibition of conventional PKC by Gö6976 mitigated the TNF-α-induced p115RhoGEF phosphorylation and RhoA activation. Subsequently, we found that these events are regulated by PKC-α rather than PKC-β by using shRNA. In addition, P115-shRNA and n19RhoA (dominant negative mutant of RhoA) transfections had no effect on mediating TNF-α-induced PKC-α activation. These data suggest that PKC-α but not PKC-β acts as an upstream regulator of p115RhoGEF phosphorylation and RhoA activation in response to TNF-α. Moreover, depletion of PKC-α, of p115RhoGEF, and inhibition of RhoA activation also prevented TNF-α-induced stress fiber formation and a decrease in TER.
Taken together, our results show that PKC-α phosphorylation of p115RhoGEF mediates TNF-α signaling to RhoA, and that this plays a critical role in signaling F-actin rearrangement and barrier dysfunction in BMECs.
肿瘤坏死因子-α（TNF-α）是一种促炎细胞因子，能够激活小 GTP 酶 RhoA，进而导致内皮屏障功能障碍。然而，其潜在的信号机制仍未得到明确。因此，我们旨在确定蛋白激酶 C（PKC）同工酶在 RhoA 激活机制以及在 TNF-α诱导的小鼠脑微血管内皮细胞（BMEC）屏障功能障碍信号中的作用。
Bend.3 细胞是一种永生化的小鼠脑内皮细胞系，用 TNF-α（10ng/mL）孵育。通过下拉测定法评估 RhoA 活性。使用酶测定法测量 PKC-α 活性。通过跨内皮电阻（TER）测量 BMEC 屏障功能。通过放射性自显影后进行 Western 印迹检测 p115RhoGEF 磷酸化。通过罗丹明鬼笔环肽染色观察 F-肌动蛋白的组织。使用药理学抑制剂和敲低方法研究 PKC 和 p115RhoGEF 在 TNF-α诱导的 RhoA 激活和 BMEC 通透性中的作用。
我们观察到 TNF-α在 BMECs 中诱导 p115RhoGEF 的快速磷酸化、PKC 的激活和 RhoA 的激活。Gö6976 抑制传统 PKC 减轻了 TNF-α诱导的 p115RhoGEF 磷酸化和 RhoA 激活。随后，我们通过 shRNA 发现这些事件是由 PKC-α而不是 PKC-β调节的。此外，P115-shRNA 和 n19RhoA（RhoA 的显性失活突变体）转染对介导 TNF-α诱导的 PKC-α 激活没有影响。这些数据表明，PKC-α而不是 PKC-β作为 TNF-α 反应中 p115RhoGEF 磷酸化和 RhoA 激活的上游调节剂。此外，PKC-α、p115RhoGEF 的耗竭以及 RhoA 激活的抑制也阻止了 TNF-α诱导的应力纤维形成和 TER 的降低。
综上所述，我们的结果表明 PKC-α 对 p115RhoGEF 的磷酸化介导了 TNF-α 信号向 RhoA 的传递，这在 BMECs 中 F-肌动蛋白重排和屏障功能障碍的信号转导中起着关键作用。
