Calreticulin Destabilizes Glucose Transporter-1 mRNA in Vascular Endothelial and Smooth Muscle Cells Under High-Glucose Conditions

Abstract

Abstract-Substrate autoregulation of glucose transporter-1 (GLUT-1) mRNA and protein expression provides vascular endothelial and smooth muscle cells a sensitive mechanism to adapt their rate of glucose transport in response to changing glycemic conditions. Hyperglycemia-induced downregulation of glucose transport is particularly important in protecting these cells against an excessive influx of glucose and consequently increased intracellular protein glycation and generation of free radicals; both are detrimental in the development of vascular disease in diabetes. We aimed to investigate the molecular mechanism of high glucose-induced downregulation of GLUT-1 mRNA expression in primary bovine aortic vascular endothelial (VEC) and smooth muscle (VSMC) cell cultures. Using RNA mobility shift, UV cross-linking, and in vitro degradation assays, followed by mass-spectrometric analysis, we identified calreticulin as a specific destabilizing trans-acting factor that binds to a 10-nucleotide cis-acting element (CAE 2181(CAE -2190 ) in the 3Ј-untranslated region of GLUT-1 mRNA. Pure calreticulin accelerated the rate of GLUT-1 mRNA-probe degradation in vitro, whereas overexpression of calreticulin in vascular cells decreased significantly the total cell content of GLUT-1 mRNA and protein. The expression of calreticulin was augmented in vascular cells exposed to high glucose in comparison with low-glucose conditions. Similarly, increased expression of calreticulin was observed in aortae of diabetic Psammomys obesus in comparison with normoglycemic controls. These data suggest that CAE 2181-2190 -calreticulin complex, which is formed in VSMC and VEC exposed to hyperglycemic conditions, renders GLUT-1 mRNA susceptible to degradation. This interaction underlies the process of downregulation of glucose transport in vascular cells under high-glucose conditions. (Circ Res. 2005;97:1001-1008.)Key Words: calreticulin Ⅲ glucose transporter-1 Ⅲ hyperglycemia Ⅲ mRNA turnover Ⅲ vascular smooth muscle cells Ⅲ vascular endothelial cells H yperglycemia is a major risk factor in the development of cardiovascular complications associated with the metabolic syndrome and diabetes. 1,2 Chronic hyperglycemia alters the normal function of endothelial and smooth muscle cells in blood vessels, which undergo morphological and functional modifications, because of an excessive extra-and intracellular protein glycation and an uncontrolled production of free radicals. Collectively, these processes contribute to basement-membrane thickening, vascular occlusion, increased permeability, and initiation and progression of vascular disease and atherosclerosis. [3][4][5][6] The transport of glucose across the plasma membrane of cells is the rate-limiting step for subsequent glucose metabolism. A family of glucose transporters (GLUTs) mediates the entry of glucose into cells by facilitated diffusion. 7 Vascular endothelial (VEC) and vascular smooth muscle (VSMC) cells express predominantly the ubiquitous GLUT-1 and to a very small extent the insulin-sensi...

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