Transcriptional repression of equilibrative nucleoside transporter 1 by D-glucose involves nitric oxide and Sp1 in human foetal endothelium
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2007
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Abstract
Adenosine uptake via the human equilibrative nucleoside transporters 1 (hENT1) is reduced in human umbilical vein endothelial cells (HUVEC) exposed to high D-glucose. Since specific mechanisms by which D-glucose reduces hENT1 expression and activity in these cells are unknown, we examined the role of nitric oxide (NO) and Sp1 as modulators of SLC29A1 (for hENT1) promoter activity in response to D-glucose. HUVEC from normal pregnancies were isolated and exposed (24 h) to 5 mM (normal) or 25 mM (high) D-glucose in absence or presence of NG-nitro-L-arginine methyl ester (LNAME, 100 mM). Sp1 protein levels were evaluated by western blot in nuclear fractions. Reporter activity of plasmid constructs containing a promoter region of SLC29A1 (-1114 bp to ATG, pGL3-hENT1-1114), Sp1 overexpression experiments (co-transfection with the expression vector pCGN-Sp1) and Sp1 binding to this region (chromatin immunoprecipitation) was assayed. pGL3-hENT1-1114 transcriptional activity was reduced by high Dglucose and in cells overexpressing Sp1. Nuclear Sp1 abundance and specific binding to its consensus sequence in SLC29A1 promoter (-815 to -801 bp) were higher in high D-glucose (1.9- and 1.7-fold, respectively) compared with normal D-glucose. All these effects were blocked by L-NAME. Thus, a NO-dependent Sp1 repression on SLC29A1 promoter could explain the reduced hENT1 expression and activity exhibited by HUVEC in high extracellular D-glucose. FONDECYT 1070865, VRAID2007-PUC (Chile). AECI A/5484/06 (Spain). C. Puebla holds a PUC-VRAID PhD fellowship. M. Farías hold CONICYT and PUC-School of Medicine PhD fellowships