Abstract 11455: Mechanism Underlying Increased eNOS Expression and Neuroprotection in Mice With Targeted Deletion of ROCK2
Background: The Rho kinases (ROCK1 and ROCK2) are important regulators of the actin cytoskeleton. We have recently shown that ROCK activity is increased during the onset of ischemic stroke and that the ROCK inhibitor, fasudil, decreased the severity of focal cerebral ischemia, in part, through the upregulation of endothelial nitric oxide synthase (eNOS). However, the precise mechanism by which ROCK regulates eNOS expression is unknown.
Methods and Results: Using RNA affinity chromatography with 3’UTR of eNOS mRNA followed by MALDI-TOF-MS analysis, we have identified eukaryotic elongation factor 1 alpha 1 (eEF1A1) as an actin-binding protein, which directly binds to eNOS mRNA and regulates eNOS mRNA stability. Overexpression of eEF1A1 decreased, and knock-down of eEF1A1 by siRNA increased, eNOS expression. Protein kinase assays using eEF1A1 with purified ROCK1 and ROCK2 showed that eEF1A1 is strongly phosphorylated by ROCK2, but not ROCK1, and that eEF1A1 phosphorylation by ROCK2 is required for binding to and destabilization of eNOS mRNA. Indeed, treatment of human endothelial cells (EC) with fasudil decreased eEF1A1 phosphorylation, but not expression. This was associated with increased eNOS mRNA stability and expression, and NO production. Furthermore, compared to that of WT and ROCK1+/- mice, eNOS mRNA stability and expression were increased in ECs from ROCK2+/- mice and endothelium-dependent relaxation was enhanced in both ROCK2+/- and EC-specific ROCK2 KO mice (EC-ROCK2-/-). These results correlated with decreased cerebral infarct size and neurological deficits in both ROCK2+/- and EC-ROCK2-/- mice subjected to transient focal cerebral ischemia. When ROCK2+/- mice were place on eNOS-/- background (double mutant ROCK2+/-/eNOS-/- mice), the neuroprotective effects observed in ROCK2+/- mice were abolished, indicating that eNOS mediates neuroprotective effects in ROCK2-deficient mice.
Conclusion: ROCK2 negatively regulates eNOS expression by phosphorylating eEF1A1, which is required for eEF1A1 to bind to eNOS mRNA. The binding of eEF1A1 decreases eNOS mRNA stability and expression. These findings suggest a novel mechanism by which ROCK2 could impair endothelial function and worsen cerebral ischemia.
Author Disclosures: Y. Hiroi: None. K. Noma: None. H. Kim: None. Y. Li: None. R. Okamoto: None. J. Sun: None. M.A. Michael A. Moskowitz: None. J.K. Liao: None.
- © 2014 by American Heart Association, Inc.