High D-glucose reduces SLC29A1 promoter activity and adenosine transport involving specific protein 1 in human umbilical vein endothelium.
High D-glucose reduces human equilibrative nucleoside transporter 1 (hENT1)-mediated adenosine uptake involving endothelial nitric oxide synthase (eNOS), mitogen-activated protein (MAP) kinase kinases 1 and 2/MAP kinases p42/44 (MEK/ERKs), and protein kinase C (PKC) activation in human umbilical vein endothelium (HUVEC). Since NO represses SLC29A1 gene (hENT1) promoter activity we studied whether D-glucose-reduced hENT1-adenosine transport results from lower SLC29A1 expression in HUVEC primary cultures. HUVEC incubation (24 h) with high D-glucose (25 mM) reduced hENT1-adenosine transport and pGL3-hENT1(-1114) construct SLC29A1 reporter activity compared with normal D-glucose (5 mM). High D-glucose also reduced pGL3-hENT1(-1114) reporter activity compared with cells transfected with pGL3-hENT1(-795) construct. N(G)-nitro-L-arginine methyl ester (L-NAME, NOS inhibitor), PD-98059 (MEK1/2 inhibitor), and/or calphostin C (PKC inhibitor) blocked D-glucose effects. Insulin (1 nM) and phorbol 12-myristate 13-acetate (PMA, 100 nM, PKC activator), but not 4alpha-phorbol 12,13-didecanoate (4alphaPDD, 100 nM, PMA less active analogue) reduced hENT1-adenosine transport. L-NAME and PD-98059 blocked insulin effects. L-NAME, PD-98059, and calphostin C increased hENT1 expression without altering protein or mRNA stability. High D-glucose increased Sp1 transcription factor protein abundance and binding to SLC29A1 promoter, phenomena blocked by L-NAME, PD-98059, and calphostin C. Sp1 overexpression reduced SLC29A1 promoter activity in normal D-glucose, an effect reversed by L-NAME and further reduced by S-nitroso-N-acetyl-L,D-penicillamine (SNAP, NO donor) in high D-glucose. Thus, reduced hENT1-mediated adenosine transport in high D-glucose may result from increased Sp1 binding to SLC29A1 promoter down-regulating hENT1 expression. This phenomenon depends on eNOS, MEK/ERKs, and PKC activity, suggesting potential roles for these molecules in hyperglycemia-associated endothelial dysfunction.
Puebla
Carlos
C
Cellular and Molecular Physiology Laboratory, Department of Obstetrics and Gynaecology, Medical Research Centre (CIM), School of Medicine, Faculty of Medicine, Pontificia Universidad Católica de Chile, Santiago, Chile.
Farías
Marcelo
M
González
Marcelo
M
Vecchiola
Andrea
A
Aguayo
Claudio
C
Krause
Bernardo
B
Pastor-Anglada
Marçal
M
Casanello
Paola
P
Sobrevia
Luis
L
D020121:5' Untranslated Regions / Q000235:genetics; D000241:Adenosine / Q000378:metabolism*; D001483:Base Sequence; D001692:Biological Transport / Q000187:drug effects; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D033721:Equilibrative Nucleoside Transporter 1 / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D005799:Genes, Dominant; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D013816:Thermodynamics; D014158:Transcription, Genetic / Q000187:drug effects; D014471:Umbilical Veins / Q000187:drug effects / Q000378:metabolism*
D020121:5' Untranslated Regions; D033721:Equilibrative Nucleoside Transporter 1; D012333:RNA, Messenger; C438649:SLC29A1 protein, human; D016329:Sp1 Transcription Factor; D004247:DNA; D005947:Glucose; D000241:Adenosine