Abstract 10548: The Novel Calmodulin-Related Proteins, Death Associated Protein Kinase 3 and Eukaryotic Elongation Factor 2 Kinase Mediate Inflammation and Apoptosis in Vascular Smooth Muscle Cells
Calmodulin (CaM) is associated with a variety of cell functions including muscular contraction. It is recently clarified that CaM-related proteins are responsible for cardiovascular diseases. Moreover our recent study demonstrated that protein expression of death associated protein kinase (DAPK)3 (a CaM kinase (CaMK) family protein) and eukaryotic elongation factor 2 kinase (eEF2K) (also known as CaMKIII) increases in aorta and mesenteric artery from spontaneous hypertensive rats compared with Wistar Kyoto rats. Vascular inflammation and apoptosis are essential for the pathogenesis of hypertension. We therefore examined whether DAPK and eEF2K affects on vascular inflammation and apoptosis. In rat mesenteric arterial smooth muscle cells (SMCs), pro-inflammatory stimulations (for 24 h) such as TNF-α (10 ng/ml), interleukin-1β (4 ng/ml), H2O2 (300 μM), angiotensinII (0.1 μM) and endothelin-1 (0.1 μM) increased DAPK3 mRNA (n=5-7, P<0.01). Small interfering RNA (siRNA) against DAPK3 significantly inhibited TNF-induced vascular cell adhesion molecule (VCAM)-1 expression (n=3-4, P<0.05). A DAPK3 siRNA inhibited TNF-induced phosphorylation of JNK (n=5, P<0.05), p38 (n=5, P<0.05) and Akt (n=5, P<0.01) but not ERK and NF-κB. Furthermore, a DAPK3 siRNA inhibited H2O2-induced cell apoptosis (n=5-9, P<0.01) as determined by TUNEL staining. A DAPK3 siRNA also inhibited H2O2-induced expression of cleaved caspase3 (n=4, P<0.05) and phosphorylation of JNK (n=4, P<0.05) and p38 (n=4, P<0.01). Protein expression (n=5, P<0.01) of eEF2K in SMCs was increased by TNF-α (10 ng/ml, 20 min). An eEF2K inhibitor, NH125 significantly inhibited TNF-induced VCAM-1 expression (n=4, P<0.01). An eEF2K siRNA also inhibited TNF-induced expression of VCAM-1 (n=4, P<0.05) as well as phosphorylation of JNK (n=4-5, P<0.01) and Akt (n=6, P<0.05) but not p38 and ERK. In NF-κB signals, an eEF2K siRNA inhibited phosphorylation of NF-κB (n=5-6, P<0.05) but not activation of IκB-α. Furthermore, an eEF2K siRNA inhibited H2O2-induced cell apoptosis (n=4, P<0.01). We for the first time demonstrated that the novel CaM-related proteins (DAPK3 and eEF2K) may be related to the pathogenesis of hypertensive vascular diseases via stimulating inflammation and apoptosis.
- © 2011 by American Heart Association, Inc.