Rho kinase-2 activation in human endothelial cells drives lysophosphatidic acid-mediated expression of cell adhesion molecules via NF-kappaB p65

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.

FIGURE 1.

LPA activates Rho kinase and NF-κB pathways and induces expression of ICAM-1 and VCAM-1 protein in HUVECs. HUVECs were cultured in EGM-2 growth medium and serum-starved overnight in 0.5% serum prior to addition of 50 μm LPA. At the indicated times, cells were harvested and lysates were prepared for either Western blotting or ELISA as described under “Experimental Procedures.” Western blot analysis of Rho kinase substrates ppMLC and pMYPT-1 (A) and adhesion molecules ICAM-1 and VCAM-1 (B) at the indicated times after LPA addition. ELISAs of phospho NF-κB P65 and total NF-κB P65 in HUVEC lysates were performed at the indicated times after LPA addition (C). NF-κB activity is expressed as a ratio of these two measurements. Values are means (n = 3) ± S.E.

FIGURE 2.

LPA-induced expression of ICAM-1 and VCAM-1 protein in HUVECs is mediated by Rho kinase activation upstream of NF-κB. Serum-starved HUVECs were preincubated for 1 h with the specified concentrations of either H1152P, a Rho kinase inhibitor, or Bay 11-7082, an NF-κB inhibitor (NFkBi), prior to treatment with LPA for the indicated durations. Cell lysates were analyzed by Western blot analysis of pMYPT-1 and ppMLC using phosphospecific antibodies (A) and ICAM-1, VCAM-1, ROCK1, and ROCK2 protein (B). Total MYPT-1, total MLC, and GAPDH were used as normalization controls (C). NF-κB activities were assessed for the different treatments, as described in the legend to Fig. 1. Values are means (n = 3) ± S.E. *, p < 0.05 and ***, p < 0.0001 compared with no compound treatment. ROCKi, ROCK inhibitor.

FIGURE 3.

Both Rho kinase isoforms ROCK1 and ROCK2 phosphorylate MYPT-1 and MLC and can compensate for the loss of the other. HUVECs were incubated with 50 nm οf either a nonspecific control siRNA (NC) or a ROCK1-specific, ROCK2-specific, or dual ROCK1/ROCK2 siRNAs for 24 h in a transfection medium. The medium was then replaced with standard growth medium (EGM-2), and cells were incubated for an additional 24 h. Cells were then serum-starved overnight prior to LPA exposure for 5 min. A, cell viability was assessed for each treatment with CellTiter-Glo luminescent cell viability assay (Promega) following the manufacturer's instructions. Values are means (n = 4) ± S.E. B, efficiency of gene knockdown was assessed by Western blotting with ROCK1- and ROCK2-specific antibodies (top two panels). Phosphorylation of MYPT-1 and MLC was assessed by Western blotting with phosphospecific antibodies. Total MYPT-1, total MLC, and GAPDH were used as normalization controls.

FIGURE 4.

ROCK2 activates NF-κB by phosphorylating the p65 subunit. siRNA-mediated gene knockdowns were performed as described in the legend to Fig. 3, and cells were then exposed to LPA for 30 min. Effectiveness of gene knockdown was assessed by Western blotting with ROCK1- and ROCK2-specific antibodies (top two panels). Phospho-NF-κB p65 was measured with a phosphospecific antibody and total NF-κB p65 and GAPDH were used as normalization controls.

FIGURE 5.

ROCK2 is the dominant mediator of LPA-driven ICAM-1 and VCAM-1 mRNA and protein expression in HUVECs. siRNA-mediated gene knockdowns were performed as described in the legend to Fig. 3, and cells were then incubated with LPA for an additional 4 h. Knockdown efficacy was evaluated by Western blotting (A). ICAM-1 (B) and VCAM-1 (C) mRNA levels were measured by quantitative real-time PCR using gene-specific primers. Values are means (n = 4) ± S.E. ***, p < 0.0001 compared with nonspecific control (NC) siRNA treatment. D, following siRNA treatment and exposure to LPA for 4 h, ICAM-1 and VCAM-1 proteins were visualized by Western blotting with gene-specific antibodies. GAPDH was used as a normalization control.