Gestational diabetes reduces the equilibrative nucleoside transporter 1 (hENT1) expression and activity in human umbilical vein endothelial cells (HUVEC), an effect associated with nitric oxide (NO)-dependent reduction of SLC29A1 (for hENT1) promoter activity (Farías et al., 2006). The human C/EBP homologous protein (hCHOP) is a transcription factor whose repressor effect on BCL2 depends on NO in murine fibroblasts (McCullough et al., 2001). We performed a serial deletion analysis of a SLC29A1 promoter region to study the involvement of hCHOP as transcriptional regulator of this gene in HUVEC. HUVEC from normal (n=20) or gestational diabetic (n=20) pregnancies were cultured under standard conditions (Farías et al.,2006). hENT1 mRNA and protein levels, and hCHOP nuclear abundance were determined. Six fragments of SLC29A1 upstream region (up to -3198 bp from ATG) were cloned into pGL3 vector (pGL3-hENT1-3198, pGL3-hENT1-2154, pGL3-hENT1-1810, pGL3-hENT1-1670, pGL3-hENT1-1114, pGL3-hENT1-795). These constructs were transfected into HUVEC by electroporation (320 V, 30 ms) and luciferase activity was compared with pRL-TK control vector. hCHOP specific binding to the consensus site for hCHOP-C/EBPα protein complex in the SLC29A1 promoter (-1853 to -1841 bp) was evaluated by chromatin immunoprecipitation. Reduced hENT1 protein (60 ± 5%)(P<0.05, unpaired Student t test, mean ± S.E.M.) and mRNA (25 ± 3%, P<0.05) levels, but increased hCHOP nuclear protein abundance (1.32-fold, P<0.05) were detected in HUVEC from gestational diabetic compared with normal pregnancies. Transcriptional activity of pGL3-hENT1-3198 or pGL3-hENT1-2154 constructs was lower (27 ± 3% or 31 ± 2%, respectively, P<0.05) in HUVEC from gestational diabetes compared with normal pregnancies. However, transcriptional activity was higher (P<0.05) when HUVEC from gestational diabetes were transfected with pGL3-hENT1-1810 (1.3-fold), pGL3-hENT1-1670 (1.4-fold) or pGL3-hENT1-1114 (1.42-fold), compared with cells from normal pregnancies. hCHOP binding to SLC29A1 promoter in HUVEC from gestational diabetes was higher (1.4-fold, P<0.05) than in cells from normal pregnancies. The reduced SLC29A1 promoter activity exhibited by HUVEC from gestational diabetes could be due to increased binding of the transcription factor hCHOP to its consensus sequence in this genomic region. Since NO synthesis is higher in HUVEC from gestational diabetes and reduces hENT1 expression, this vasodilator could be crucial for hCHOP repression of SLC29A1 promoter activity in this cell type.
Life Sciences 2007 (2007) Proc Life Sciences, PC513
Poster Communications: Transcriptional repression of equilibrative nucleoside transporter 1 in human fetal endothelium from gestational diabetes is associated with activation of C/EBP homologous protein
M. Farias1, C. Puebla1, A. Vecchiola1, M. Pastor-Anglada2, P. Casanello1, L. Sobrevia1
1. Cellular and Molecular Physiology Laboratory (CMPL) and Perinatology Research Laboratory (PRL), Department of Obstetrics & Gynaecology, Medical Research Centre (CIM), School of Medicine, Pontificia Universidad Catolica de Chile, Santiago, Chile. 2. Department of Biochemistry and Molecular Biology, Faculty of Biology, Universitat de Barcelona, Barcelona, Spain.
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Where applicable, experiments conform with Society ethical requirements.