Mitogen-activated protein (MAP) kinases are a multigene family activated by many extracellular stimuli. There are three groups of MAP kinases based on their dual phosphorylation motifs, TEY, TPY, and TGY, which are termed extracellular signal-regulated protein kinases (ERK1/2), c-Jun N-terminal kinases, and p38, respectively. A new MAP kinase family member termed Big MAP kinase 1 (BMK1) or ERK5 was recently cloned. BMK1 has a TEY sequence similar to ERK1/2 but has unique COOH-terminal and loop-12 domains. To define BMK1 regulation, its activation in cultured rat vascular smooth muscle cells was characterized. Angiotensin II, phorbol ester, platelet-derived growth factor, and tumor necrosis factor-␣ were the strongest stimuli for ERK1/2 but were weak activators of BMK1. In contrast, H 2 O 2 caused concentration-dependent activation of BMK1 but not ERK1/2. Sorbitol activated both BMK1 and ERK1/2. BMK1 activation by H 2 O 2 was calcium-dependent and appeared ubiquitous as shown by stimulation in human skin fibroblasts, human vascular smooth muscle cells, and human umbilical vein endothelial cells. These findings demonstrate that activation of BMK1 is different from ERK1/2 and suggest an important role for BMK1 as a redox-sensitive kinase.
Abstract-Osteopontin (OPN) is a soluble secreted phosphoprotein that binds with high affinity to several integrins and it has been found at the site of atherosclerotic lesions. However, the role of OPN expression in vivo is still poorly understood. To investigate the physiological role of OPN in detail, we generated transgenic mice (Tg) overexpressing the OPN gene under control of the cytomegalovirus enhancer/chicken -actin promoter. Key Words: aorta Ⅲ atherosclerosis Ⅲ genes Ⅲ inflammation Ⅲ smooth muscle O steopontin (OPN) is a phosphoprotein that was originally isolated from the bone. It possesses the tripeptide sequence Arg-Gly-Asp like other extracellular matrix proteins and serum proteins such as fibronectin, vitronectin, and collagen. 1,2 OPN functions as a cell adhesion and migration molecule that can bind to several ligands including ␣ v  3 integrin, CD44, and fibronectin. 3-5 A wide range of cell types including some epithelia, macrophages, T cells, and vascular smooth muscle cells (SMCs) express OPN in a constitutive or inducible fashion. 6 -9 OPN has recently emerged as a key factor in both vascular remodeling and in the development of atherosclerosis. 10 -12 In vitro studies have shown that OPN also promotes the proliferation of cultured rat vascular SMCs 13 and human coronary artery SMCs. 14 Furthermore, a previous study has shown OPN to stimulate Arg-Gly-Aspdependent endothelial migration in vitro. 15 However, the function of OPN in the vessel walls is still not yet well understood. The present study uses OPN transgenic mice (Tg) to determine the effect of OPN on SMCs in vivo and to investigate the effect of cuff-induced injury to the femoral artery in vivo. Our results show that OPN overexpression is associated with a significant increase in medial thickening with aging in vivo and in intimal thickening after arterial injury. Materials and Methods Construction of TransgeneWe prepared a transgene construct containing the cytomegalovirus enhancer/chicken -actin (CAG) promoter with cDNA to murine OPN. The CAG-OPN transgene was constructed by inserting the 1.35-kb EcoRI fragment containing mouse OPN cDNA sequence into the EcoRI site of the third exon of the rabbit -globin gene in pBsCAG-2. 16,17 OPN cDNA contains 34 bp of the 5Ј noncoding region, 885 bp of the coding region, and 429 bp of the 3Ј untranslated region. Sequencing revealed that the OPN cDNA was identical to the described mouse OPN cDNA. 18 The resulting plasmid was termed pCAG/OPN-9. The CAG-OPN transgene was isolated from the pCAG/OPN-9 backbone by SalI and BamHI digestion.
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