Rho kinase mediates Porphyromonas gingivalis outer membrane vesicle-induced suppression of endothelial nitric oxide synthase through ERK1/2 and p38 MAPK

• P. gingivalis OMVs suppressed eNOS expression both at RNA and protein levels.
• ROCK is effectively activated by the stimulation of OMVs.
• ROCK regulates OMVs-induced suppression of eNOS activity.
• ERK1/2 and p38 MAPK are downstream regulators of ROCK.
• OMVs-induced reduction of eNOS is via ERK1/2 and p38 MAPK-dependent ways.

ObjectiveTo investigate the effect of Rho kinase (ROCK) on Porphyromonas gingivalis outer membrane vesicles (OMVs)-induced suppression of endothelial nitric oxide synthase (eNOS) and explore the potential mechanism.DesignFirstly, we investigated the effect of OMVs on total eNOS expression and eNOS activity in Human Umbilical Vein Endothelial Cells (HUVECs) and if ROCK activation is involved. Furthermore, we estimated the effect of ROCK in regulating eNOS expression and the possible underlying mechanism in vitro. At last we confirmed the results by immunohisochemisty for eNOS expression in mouse aorta endothelium exposed to OMVs and inhibitors.ResultsWe found that OMVs suppressed eNOS expression both at RNA and protein levels in a time- and dose-dependent manner. ROCK activity was observed in this process by detecting phosphorylation of myosin light chain (MLC) and myosin-associated phosphatase type 1 (MYPT-1), which lead to reduced eNOS expression. The suppression of eNOS was significantly reversed by ROCK inhibitor Y-27632. Moreover, Y-27632 pretreatment obviously inhibited the activation of ERK1/2 and p38 MAPKs induced by OMVs, whereas that of JNK was not affected. In addition, blocking ERK1/2 or p38 MAPK by PD98059 and SB203580, respectively attenuated the OMVs-induced eNOS phosphorylation. Ex vivo study shows that OMVs reduced eNOS expression in mouse aorta endothelium. Co-treatment with OMVs and inhibitors could significantly reverse the eNOS suppression. Taken together, these results demonstrate that ROCK mediated OMVs-induced eNOS suppression through ERK1/2 and p38 MAPK.ConclusionsThese data suggest that ROCK may mediate OMVs-induced eNOS expression through ERK1/2 and p38 MAPK.