Quehenberger P, Bierhaus A, Fasching P, Muellner C, Klevesath M, Hong M, Stier G, Sattler M, Schleicher E, Speiser W, Nawroth P P
Department of Medical and Chemical Laboratory Diagnostics, University of Vienna, Austria.
Diabetes. 2000 Sep;49(9):1561-70. doi: 10.2337/diabetes.49.9.1561.
Incubation of bovine aortic endothelial cells (BAECs) with erythrocytes from patients with type 2 diabetes induced an increase in endothelin 1 (ET-1) production. The effect of erythrocytes on ET-1 synthesis was dependent on glycemic control. ET-1 levels after incubation with erythrocytes derived from patients with HbA(1c) levels <6% were just half the levels observed after incubation with erythrocytes from patients with HbA(1c) levels >8%. Nepsilon-(carboxymethyl)lysine (CML)-containing protein isolated from patients' erythrocytes induced ET-1, and CML-containing protein-dependent ET-1 induction was blocked by the recombinant decoy peptide soluble receptor for advanced glycation end products (AGEs), which comprises the NH2-terminal Ig domain of the receptor for AGEs. In vitro-generated AGEs induced ET-1 mRNA transcription (nuclear run-on assay and Northern blot) in a time- and dose-dependent manner. Transient transfection of BAECs with a chimeric construct containing the 5' promoter region of the ET-1 gene linked to a reporter gene confirmed that AGE induced ET-1 promoter activity. Electrophoretic mobility shift assay confirmed AGE-inducible binding of members of the nuclear factor-kappab (NF-kappaB) family to a potential binding site at -2,090 bp. Binding was functionally significant because overexpression of the cytoplasmic inhibitor of NF-kappaB or deletion of the NF-kappaB binding site reduced ET-1 induction, whereas overexpression of NF-kappaB p65 induced ET-1 even in the absence of AGEs. Thus, ET-1 transcription is controlled by the AGE-inducible redox-sensitive transcription factor NF-kappaB.
用2型糖尿病患者的红细胞孵育牛主动脉内皮细胞（BAECs）会导致内皮素1（ET-1）生成增加。红细胞对ET-1合成的影响取决于血糖控制情况。与糖化血红蛋白（HbA）(1c)水平<6%患者的红细胞孵育后的ET-1水平仅为与HbA(1c)水平>8%患者的红细胞孵育后观察到水平的一半。从患者红细胞中分离出的含Nε-（羧甲基）赖氨酸（CML）的蛋白质可诱导ET-1生成，且含CML蛋白质依赖性ET-1诱导被晚期糖基化终产物（AGEs）的重组诱饵肽可溶性受体所阻断，该受体由AGEs受体的NH2末端免疫球蛋白结构域组成。体外生成的AGEs以时间和剂量依赖性方式诱导ET-1 mRNA转录（核转录分析和Northern印迹法）。用含有与报告基因相连的ET-1基因5'启动子区域的嵌合构建体对BAECs进行瞬时转染，证实AGE可诱导ET-1启动子活性。电泳迁移率变动分析证实AGE可诱导核因子-κB（NF-κB）家族成员与位于-2,090 bp处的潜在结合位点结合。这种结合在功能上具有重要意义，因为NF-κB的细胞质抑制剂过表达或NF-κB结合位点缺失会降低ET-1诱导，而NF-κB p65过表达即使在没有AGEs的情况下也能诱导ET-1生成。因此，ET-1转录受AGE诱导的氧化还原敏感转录因子NF-κB调控。
