Abstract 257: Sphingosine-1-Phosphate Inhibits Erk1/2 Expression in Type 1 Non-Obese Diabetic (NOD) Mouse Aortic Endothelium Through Induction of MAP Kinase Phosphatase-3
Endothelial activation is a key early event in vascular complications of Type 1 diabetes. The non-obese diabetic (NOD) mouse is a well-characterized mouse model of Type 1 diabetes that develops autoimmune destruction of the pancreatic beta cells resulting in spontaneous hyperglycemia within 18 weeks of age. We found that diabetic NOD mice have increased endothelial activation, with increased production of MCP-1 and IL-6, and significant induction (30%) of surface VCAM-1 expression. Using freshly isolated aorta in an ex vivo adhesion assay, we found that diabetic NOD mice have a 4-fold increase in monocyte adhesion to aortic endothelium (15±3 monocytes bound in control NOD mouse aorta versus 71±9 monocytes bound in diabetic NOD mouse aorta, p<0.0001). Further, the sphingolipid sphingosine-1-phosphate (S1P) prevents endothelial activation and monocyte adhesion in these diabetic NOD mice. In the current study, we found that 100nM S1P induced extracellular regulated kinase (Erk1/2) activation in diabetic NOD aortic endothelial cells (EC) within 10 minutes of S1P addition. After 4h, S1P significantly reduced Erk1/2 phosphorylation in diabetic NOD mice; levels of Erk1/2 phosphorylation were reduced by 90% after 4h of S1P incubation compared to PBS-treated diabetic NOD EC. Total levels of Erk1/2 protein were not significantly changed. To determine the mechanism causing the dramatic downregulation of erk1/2 phosphorylation by S1P, we examined expression of mitogen-activated kinase phosphatases (MKP), a family of dual specificity phosphatases. MKP-3 is a cytoplasmic phosphatase expressed in EC that dephosphorylates Erk1/2 to prevent its mobilization to the nucleus for gene transcription. S1P induced MKP-3 mRNA approximately 3-fold in EC. Further, S1P significantly increased expression of MKP-3 protein by 2-fold in diabetic NOD EC. Interestingly, we observed similar upregulation of MKP-3 expression and dephosphorylation of Erk1/2 when EC were treated with 100nM SEW2871, a S1P1 receptor-specific agonist. Thus, S1P acts through the S1P1 receptor to regulate Erk1/2 phosphorylation via MKP-3 activation in Type 1 diabetic NOD EC. Understanding the mechanisms of S1P in diabetic endothelium may be important for preventing complications of Type 1 diabetes.