title: TGF-beta1 inhibits expression and activity of hENT1 in a nitric oxide-dependent manner in human umbilical vein endothelium.
abstract: OBJECTIVE
  We studied whether transforming growth factor beta1 (TGF-beta1) modulates human equilibrative nucleoside transporters 1 (hENT1) expression and activity in human umbilical vein endothelial cells (HUVECs). hENT1-mediated adenosine transport and expression are reduced in gestational diabetes and hyperglycaemia, conditions associated with increased synthesis and release of nitric oxide (NO) and TGF-beta1 in this cell type. TGF-beta1 increases NO synthesis via activation of TGF-beta receptor type II (TbetaRII), and NO inhibits hENT1 expression and activity in HUVECs.
  
  
  RESULTS
  HUVECs (passage 2) were used for experiments. Total and hENT1-mediated adenosine transport was measured in the absence or presence of TGF-beta1, NG-nitro-L-arginine methyl ester (L-NAME, NO synthase inhibitor), S-nitroso-N-acetyl-L,D-penicillamine (SNAP, NO donor), and/or KT-5823 (protein kinase G inhibitor) in control cells and cells expressing a truncated form of TGF-beta1 receptor type II (TTbetaRII). Western blot and real-time PCR were used to determine hENT1 protein abundance and mRNA expression. SLC29A1 gene promoter and specific protein 1 (Sp1) transcription factor activity was assayed. Vascular reactivity was assayed in endothelium-intact or -denuded umbilical vein rings. TGF-beta1 reduced hENT1-mediated adenosine transport, hENT1 protein abundance, hENT1 mRNA expression, and SLC29A1 gene promoter activity, but increased Sp1 binding to DNA. TGF-beta1 effect was blocked by L-NAME and KT-5823 and mimicked by SNAP in control cells. However, TGF-beta1 was ineffective in cells expressing TTbetaRII or a mutated Sp1 consensus sequence. Vasodilatation in response to TGF-beta1 and S-(4-nitrobenzyl)-6-thio-inosine (an ENT inhibitor) was endothelium-dependent and blocked by KT-5823 and ZM-241385.
  
  
  CONCLUSIONS
  hENT1 is down-regulated by activation of TbetaRII by TGF-beta1 in HUVECs, a phenomenon where NO and Sp1 play key roles. These findings comprise physiological mechanisms that could be important in diseases where TGF-beta1 plasma level is increased as in gestational diabetic mothers or patients with diabetes mellitus.
authors.affiliation: 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, PO Box 114-D, Santiago, Chile.
authors.forename: José L
authors.initials: JL
authors.lastname: Vega
authors.forename: Carlos
authors.initials: C
authors.lastname: Puebla
authors.forename: Rodrigo
authors.initials: R
authors.lastname: Vásquez
authors.forename: Marcelo
authors.initials: M
authors.lastname: Farías
authors.forename: Julio
authors.initials: J
authors.lastname: Alarcón
authors.forename: Marçal
authors.initials: M
authors.lastname: Pastor-Anglada
authors.forename: Bernardo
authors.initials: B
authors.lastname: Krause
authors.forename: Paola
authors.initials: P
authors.lastname: Casanello
authors.forename: Luis
authors.initials: L
authors.lastname: Sobrevia
mesh_terms: D000241:Adenosine / Q000378:metabolism*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism*; D033721:Equilibrative Nucleoside Transporter 1 / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006801:Humans; D009569:Nitric Oxide / Q000378:metabolism*; D011247:Pregnancy; D011248:Pregnancy Complications / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism*; D014158:Transcription, Genetic; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*; D014664:Vasodilation
chemical_list: D033721:Equilibrative Nucleoside Transporter 1; D018125:Receptors, Transforming Growth Factor beta; C438649:SLC29A1 protein, human; C508960:TGFB1 protein, human; D053773:Transforming Growth Factor beta1; D009569:Nitric Oxide; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II; D000241:Adenosine