Inflammation Research Unit, Pfizer Global Research and Development, Chesterfield, Missouri 63017, USA.
J Biol Chem. 2010 Apr 23;285(17):12536-42. doi: 10.1074/jbc.M109.099630. Epub 2010 Feb 17.
Endothelial cells play an important role in the recruitment of immune cells to a disease locus through the induced expression of chemokines and cell adhesion molecules (CAMs). The proinflammatory lysophospholipid, lysophosphatidic acid (LPA), which is elevated in multiple inflammatory diseases, is a potent activator of the RhoA/Rho kinase signaling pathway and has been shown to induce the expression of CAMs in endothelial cells. The present study was undertaken to map signal transduction downstream of LPA and to investigate the contributions of the Rho kinase isoforms ROCK1 and ROCK2 to adhesion molecule expression in human umbilical vein endothelial cells. LPA activated Rho kinase within minutes and subsequently the NF-kappaB pathway through phosphorylation of the p65 subunit. The lipid also induced the late expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Pharmacologic inhibition of Rho kinase signaling blocked LPA-induced p65 phosphorylation and suppressed ICAM-1 and VCAM-1 expression. Inhibition of the NF-kappaB pathway had no impact on LPA-induced Rho kinase activation, but inhibited adhesion molecule expression. Small interfering RNA-facilitated knockdown of each isoform identified ROCK2 as the mediator of LPA-driven phosphorylation of NF-kappaB p65 and of ICAM-1 and VCAM-1 mRNA and protein induction. Taken collectively, our data are consistent with Rho kinase being upstream of NF-kappaB in driving LPA-mediated adhesion molecule expression. This study also provides the first evidence of the critical involvement of ROCK2 in LPA-induced CAM expression through activation of the NF-kappaB pathway in human endothelial cells.
内皮细胞通过诱导趋化因子和细胞黏附分子（CAM）的表达，在募集免疫细胞到疾病部位方面发挥着重要作用。在多种炎症性疾病中升高的促炎溶血磷脂酸（LPA）是 RhoA/Rho 激酶信号通路的有效激活剂，已被证明可诱导内皮细胞中 CAM 的表达。本研究旨在绘制 LPA 下游的信号转导图谱，并研究 Rho 激酶同工型 ROCK1 和 ROCK2 对人脐静脉内皮细胞黏附分子表达的贡献。LPA 在数分钟内激活 Rho 激酶，随后通过磷酸化 p65 亚基激活 NF-κB 途径。该脂质还诱导细胞间黏附分子-1（ICAM-1）和血管细胞黏附分子-1（VCAM-1）的晚期表达。Rho 激酶信号的药理学抑制阻断了 LPA 诱导的 p65 磷酸化，并抑制了 ICAM-1 和 VCAM-1 的表达。NF-κB 途径的抑制对 LPA 诱导的 Rho 激酶激活没有影响，但抑制了黏附分子的表达。每种同工型的小干扰 RNA 介导的敲低鉴定 ROCK2 是 LPA 驱动的 NF-κB p65 磷酸化以及 ICAM-1 和 VCAM-1 mRNA 和蛋白诱导的介体。总的来说，我们的数据与 Rho 激酶在驱动 LPA 介导的黏附分子表达中作为 NF-κB 的上游一致。这项研究还首次提供了证据，证明 ROCK2 通过激活人内皮细胞中的 NF-κB 途径，在 LPA 诱导的 CAM 表达中起关键作用。
