Transcriptional repression of the equilibrative nucleoside transporter I in human umbilical vein endothelium from gestational diabetes
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Date
2006
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Abstract
Human umbilical vein endothelium (HUVEC) from gestational diabetes (GD) show increases nitric oxide (NO) synthesis by an adenosine receptor A2a dependent mechanism. A requisite for A2a signaling in GD is an increase in extracellular levels of adenosine by down regulation of the expression and activity of the equilibrative nucleoside transporter 1 (hENT1). We studied the transcriptional activity of the hENT1 promoter in HUVEC from GD. Methods: Primary culture of HUVEC from normal and GD pregnancies were maintain up to passage 2 in medium 199 (3.2 mM L-glutamine, Ethic committee approval and informed patient consent obtained). hENT1 gene promoter activity was measured in HUVEC exposed to adenosine (10 µM, 4 h) following transfection by electroporation with pGL3 (luciferase reporter gene) plasmids carrying -3100, -2056 and -1016 bp of the promoter sequence (320 Volt, 30 ms, 8-10% efficiency). hENT1 protein content in HUVEC was determined by Western blot. Results: Transcriptional activity of hENT1 promoter sequence from -2050 up to -3100 bp is decreased in HUVEC from GD compared to normal cells. This effect was also observed in normal cells exposed to adenosine. hENT1 protein content is reduced by ~50% in HUVEC exposed to adenosine. Conclusions: Down regulation of hENT1 in GD is mediated by a decreased transcriptional activity in the hENT1 promoter. In addition, hENT1 expression is repressed at transcriptional and post-translational level by adenosine in HUVEC. Supported by FONDECYT 1030781/1030607/7050030 (Chile) and Fundación Andes (C-14060/50).