一氧化氮降低 SLC29A1 启动子活性和腺苷转运，涉及到妊娠期糖尿病患者人脐静脉内皮细胞中的转录因子复合物 hCHOP-C/EBPalpha。
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.
机构信息
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.
出版信息
Cardiovasc Res. 2010 Apr 1;86(1):45-54. doi: 10.1093/cvr/cvp410. Epub 2009 Dec 23.
AIMS
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.
METHODS AND 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.
CONCLUSION
The hCHOP-C/EBPalpha complex down-regulates SLC29A1 expression in an NO-dependent manner in HUVECs from gestational diabetes.
目的
一氧化氮（NO）依赖性降低人脐静脉内皮细胞（HUVEC）中 SLC29A1 转录活性导致人类核苷转运蛋白 1（hENT1）表达减少。由于 C/EBP 同源蛋白 10（hCHOP，与 C/EBPalpha 转录因子形成异二聚体）的转录因子的表达受 NO 激活，并在糖尿病中诱导，我们假设 hCHOP 在妊娠期糖尿病中 HUVEC 中 hENT1 表达减少中起作用。
方法和结果
使用来自 42 例正常妊娠和 42 例妊娠期糖尿病的 HUVEC 原代培养物进行腺苷摄取测定。实时 PCR（mRNA 定量），western blot（蛋白质丰度）和荧光素酶活性（SLC29A1 启动子活性）用于 hCHOP-C/EBPalpha 活性通过染色质免疫沉淀测定。重叠延伸诱变用于在 SLC29A1 启动子上生成突变的 hCHOP-C/EBPalpha 共有序列，并用内皮型一氧化氮合酶（eNOS）siRNA 重组腺病毒敲低 eNOS。在妊娠期糖尿病中，hCHOP 核蛋白含量和与 DNA 的结合更高，同时 SLC29A1 启动子活性，hENT1 表达和转运活性降低。这些变化通过 hCHOP 共有序列突变（-1845G> T 和-1844C> A），eNOS-siRNA 诱导的敲低和 N（G）-硝基-L-精氨酸甲酯（NOS 抑制剂）阻断，并在来自正常妊娠的细胞中被 S-亚硝基-N-乙酰-L，D-青霉胺（NO 供体）模拟。hCHOP 和 C/EBPalpha 的过表达模拟了来自正常妊娠的细胞中的妊娠期糖尿病效应，但不会改变来自妊娠期糖尿病的细胞中的 SLC29A1 启动子活性或 hENT1-腺苷转运。
结论
hCHOP-C/EBPalpha 复合物以依赖于 NO 的方式下调妊娠期糖尿病 HUVEC 中 SLC29A1 的表达。
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