Abstract
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.
Highlights
Lysophosphatidic acid (LPA), a pleiotropic proinflammatory lipid mediator, is elevated in multiple disease states
LPA Activates the Rho Kinase and NF-B Pathways and Induces cell adhesion molecules (CAMs) Expression in HUVECs—HUVECs were cultured to confluence in growth medium (EGM-2) containing 2% fetal bovine serum prior to being serum-starved overnight in 0.5% fetal bovine serum
Due to the propensity of LPA to stick to plastic and bind serum protein, the mediator is usually added at micromolar concentrations
Summary
Lysophosphatidic acid (LPA), a pleiotropic proinflammatory lipid mediator, is elevated in multiple disease states. Two isoforms of Rho kinase have been described and are widely referred to in the published literature as ROCK1 and ROCK2 ( known as ROK and ROK␣, respectively) [23] They are both important regulators of the cytoskeleton, mediating RhoA effects on stress fiber formation, membrane ruffling, cell contraction, adhesion, and motility [24]. We have shown for the first time that LPA-induced ICAM-1 and VCAM-1 expression is mediated by activation of the Rho kinase-NF-B pathway in human endothelial cells. The targeted knockdown of ROCK2 with siRNA, but not ROCK1, blocked LPA-induced NF-kB activation, thereby attenuating adhesion molecule expression These findings suggest that ROCK2 is essential for LPA-mediated CAM expression in human endothelial cells and could be a key regulator of the trans-endothelial migration of leukocytes in inflammatory disease
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