Endothelin 1 transcription is controlled by nuclear factor-kappa B in AGE-stimulated cultured endothelial cells

Incubation of bovine aortic endothelial cells (BAECs) with erythrocytes fro m 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 g lycemic control. ET-1 levels after incubation with erythrocytes derived fro m patients with HbA(1c) levels <6% were just half the levels observed after incubation with erythrocytes from patients with HbA(1c) levels >8%. N-r-(c arboxymethyl)lysine (CML)-containing protein isolated from patients' erythr ocytes induced ET-1, and CML-containing protein-dependent ET-1 induction wa s blocked by the recombinant decoy peptide soluble receptor for advanced gl ycation end products (AGEs), which comprises the NH2-terminal Ig domain of the receptor for AGEs, In vitro-generated AGEs induced ET-1 mRNA transcript ion (nuclear run-on assay and Northern blot;) in a time- and dose-dependent manner. Transient; transfection of BAECs with a chimeric construct contain ing the 5' promoter region of the ET-1 gene linked to a reporter gene confi rmed that AGE induced ET-1 promoter activity. Electrophoretic mobility shif t assay confirmed AGE-inducible binding of members of the nuclear factor-ka ppa b (NF-kappa B) family to a potential binding site at -2,090 bp, Binding was functionally significant because overexpression of the cytoplasmic inh ibitor of NF-kappa B or deletion of the NF-kappa B binding site reduced ET- 1 induction, whereas overexpression of NF-kappa B p65 induced ET-1 even in the absence of AGEs, Thus, ET-1 transcription is controlled by the AGE-indu cible redox-sensitive transcription factor NF-kappa B.