Cardiovascular Research Advance Access [Accepted Manuscript] published online on February 4, 2009
Cardiovascular Research, doi:10.1093/cvr/cvp045
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TGF-β1 Inhibits Expression and Activity of hENT1 in a Nitric Oxide-dependent Manner in Human Umbilical Vein Endothelium
1 Cellular and Molecular Physiology Laboratory (CMPL) and Perinatology Research Laboratory (PRL), Department of Obstetrics and Gynaecology, Medical Research Centre (CIM), School of Medicine, Faculty of Medicine, Pontificia Universidad Católica de Chile, P.O. Box 114-D, Santiago, Chile.
2 Department of Basic Sciences, Faculty of Sciences, Universidad del Bío-Bío, Chillán, Chile.
3 Department of Biochemistry and Molecular Biology, Institute of Biomedicine at the University of Barcelona (IBUB), Universitat de Barcelona and CIBER EHD, Barcelona, Spain.
* Correspondence: Dr Luis Sobrevia Cellular and Molecular Physiology Laboratory (CMPL) Department of Obstetrics and Gynaecology School of Medicine Pontificia Universidad Católica de Chile P.O. Box 114-D, Santiago, Chile. Telephone: +562-3548118 Fax: +562-6321924 E-mail: sobrevia{at}med.puc.cl
Aims: We studied whether transforming growth factor β1 (TGF-β1) modulates human equilibrative nucleoside transporters 1 (hENT1) expression and activity in human umbilical vein endothelial cells (HUVEC). 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-β1 in this cell type. TGF-β1 increases NO synthesis via activation of TGF-β receptor type II (TβRII), and NO inhibits hENT1 expression and activity in HUVEC.
Methods: HUVEC (passage 2) were used for experiments. Total and hENT1-mediated adenosine transport was measured in the absence or presence of TGF-β1, NG-nitro-L-arginine methyl ester (L-NAME, NO synthase inhibitor), S-nitroso-L,D-acetyl penicillamine (SNAP, NO donor) and/or KT-5823 (protein kinase G inhibitor) in control cells and cells expressing a truncated form of TGF-β1 receptor type II (TTβRII). Western blot and real-time PCR were used to determine hENT1 protein abundance and mRNA expression. SLC29A1 gene promoter and Sp1 transcription factor activity was assayed. Vascular reactivity was assayed in endothelium-intact or -denuded umbilical vein rings.
Results: TGF-β1 reduced hENT1-mediated adenosine transport, hENT1 protein abundance, hENT1 mRNA expression and SLC29A1 gene promoter activity, but increased Sp1 binding to DNA. TGF-β1 effect was blocked by L-NAME and KT-5823 and mimicked by SNAP in control cells. However, TGF-β1 was ineffective in cells expressing TTβRII or a mutated Sp1 consensus sequence. Vasodilatation in response to TGF-β1 and nitrobenzylthioinosine (an ENTs inhibitor) was endothelium dependent and blocked by KT-5823 and ZM-241385.
Conclusion: hENT1 is down-regulated by activation of TGF-β receptor type II by TGF-β1 in HUVEC, a phenomenon where NO and Sp1 play key roles. These findings comprise physiological mechanisms that could be important in diseases where TGF-β1 plasma level is increased as in gestational diabetic mothers or patients with diabetes mellitus.
Time for primary review: 42 Days