title: Nitric oxide reduces SLC29A1 promoter activity and adenosine transport involving transcription factor complex hCHOP-C/EBPalpha in human umbilical vein endothelial cells from gestational diabetes.
abstract: OBJECTIVE
  Reduced expression of human equilibrative nucleoside transporter 1 (hENT1) results from nitric oxide (NO)-dependent reduced SLC29A1 transcriptional activity in human umbilical vein endothelial cells (HUVECs) from gestational diabetes. As expression of the transcription factor C/EBP homologous protein 10 (hCHOP, which forms heterodimers with C/EBPalpha transcription factor) is activated by NO and induced in diabetes mellitus, we hypothesize that hCHOP plays a role in the gestational diabetes-reduced hENT1 expression in HUVECs.
  
  
  RESULTS
  HUVEC primary cultures from 42 normal and 42 gestational diabetic pregnancies were used for adenosine uptake assays. Real-time PCR (mRNA quantification), western blotting (protein abundance), and luciferase activity (SLC29A1 promoter activity) were used. hCHOP-C/EBPalpha activity was assayed by chromatin immunoprecipitation. Overlap extension mutagenesis was used to generate a mutated hCHOP-C/EBPalpha consensus site at the SLC29A1 promoter, and endothelial NO synthase (eNOS) siRNA recombinant adenovirus was used to knock down eNOS. hCHOP nuclear protein abundance and binding to DNA were higher in gestational diabetes, paralleled by reduced SLC29A1 promoter activity, hENT1 expression, and transport activity. These changes were blocked by hCHOP consensus sequence mutation (-1845G > T and -1844C > A), eNOS-siRNA-induced knockdown, and N(G)-nitro-L-arginine methyl ester (NOS inhibitor), and were mimicked by S-nitroso-N-acetyl-L, D-penicillamine (NO donor) in cells from normal pregnancies. hCHOP and C/EBPalpha overexpression mimicked gestational diabetes effects in cells from normal pregnancies, but did not alter SLC29A1 promoter activity or hENT1-adenosine transport in cells from gestational diabetes.
  
  
  CONCLUSIONS
  The hCHOP-C/EBPalpha complex down-regulates SLC29A1 expression in an NO-dependent manner in HUVECs from gestational diabetes.
authors.affiliation: Cellular and Molecular Physiology Laboratory, Department of Obstetrics and Gynaecology, Faculty of Medicine, School of Medicine, Pontificia Universidad Católica de Chile, PO Box 114-D, Santiago, Chile.
authors.forename: Marcelo
authors.initials: M
authors.lastname: Farías
authors.forename: Carlos
authors.initials: C
authors.lastname: Puebla
authors.forename: Francisco
authors.initials: F
authors.lastname: Westermeier
authors.forename: Miguel J
authors.initials: MJ
authors.lastname: Jo
authors.forename: Marçal
authors.initials: M
authors.lastname: Pastor-Anglada
authors.forename: Paola
authors.initials: P
authors.lastname: Casanello
authors.forename: Luis
authors.initials: L
authors.lastname: Sobrevia
mesh_terms: D000241:Adenosine / Q000378:metabolism; D001120:Arginine / Q000378:metabolism; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D016640:Diabetes, Gestational / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D015536:Down-Regulation / Q000502:physiology; D042783:Endothelial Cells / Q000166:cytology / Q000502:physiology*; D033721:Equilibrative Nucleoside Transporter 1 / Q000235:genetics* / Q000378:metabolism; D005260:Female; D006801:Humans; D016296:Mutagenesis / Q000502:physiology; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism; D011247:Pregnancy; D011401:Promoter Regions, Genetic / Q000502:physiology; D034741:RNA, Small Interfering; D015398:Signal Transduction / Q000502:physiology; D051742:Transcription Factor CHOP / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation / Q000502:physiology; D014471:Umbilical Veins / Q000166:cytology
chemical_list: D022763:CCAAT-Enhancer-Binding Protein-alpha; C497249:DDIT3 protein, human; D033721:Equilibrative Nucleoside Transporter 1; D034741:RNA, Small Interfering; C438649:SLC29A1 protein, human; D051742:Transcription Factor CHOP; D009569:Nitric Oxide; D001120:Arginine; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D000241:Adenosine