OBJECTIVES: We have previously reported that interleukin (IL)-6 is induced by hypoxic stimulation in cardiac myocytes. In the present study, we examined the induction of potent transcription factors of IL-6, nuclear factor (NF)-kappa B and NF-IL6, in cardiac myocytes subjected to hypoxia. METHODS: Five different lengths of IL-6 promoter-luciferase reporter plasmids and three mutant plasmids, in which the binding sites of NF-kappa B and/or NF-IL6 were disrupted, were transfected into neonatal rat cardiac myocytes. Luciferase activities after hypoxic stimulation were measured. Electrophoretic mobility shift assays were performed using oligonucleotides containing the binding site for NF-kappa B or NF-IL6 as a probe. RESULTS: Hypoxic stimulation for 4 h increased luciferase activity by 5.7 fold in -179/+12-luciferase reporter plasmid, whereas no significant increase was observed in -60/+12-luciferase plasmid. Decrease in luciferase activity was more prominent when the NF-kappa B binding site was disrupted rather than when the NF-IL6 binding site was disrupted. Moreover, when both sites were disrupted, luciferase activity increased only by 1.5 fold. Electrophoretic mobility shift assays demonstrated enhanced binding activity to oligonucleotides containing the NF-kappa B binding site in hypoxic cardiac myocytes, which displayed a supershift with antibody to its subunit, p50 or p65. The binding activity to the NF-IL6 probe also enhanced and displayed a supershift with antibody to NF-IL6. CONCLUSIONS: Although hypoxic stimulation induced NF-kappa B and NF-IL6 in cardiac myocytes, NF-kappa B may be the primary positive regulator of transcriptional activation of the IL-6 gene in the context of hypoxia.
OBJECTIVES: We have previously reported that interleukin (IL)-6 is induced by hypoxic stimulation in cardiac myocytes. In the present study, we examined the induction of potent transcription factors of IL-6, nuclear factor (NF)-kappa B and NF-IL6, in cardiac myocytes subjected to hypoxia. METHODS: Five different lengths of IL-6 promoter-luciferase reporter plasmids and three mutant plasmids, in which the binding sites of NF-kappa B and/or NF-IL6 were disrupted, were transfected into neonatal rat cardiac myocytes. Luciferase activities after hypoxic stimulation were measured. Electrophoretic mobility shift assays were performed using oligonucleotides containing the binding site for NF-kappa B or NF-IL6 as a probe. RESULTS:Hypoxic stimulation for 4 h increased luciferase activity by 5.7 fold in -179/+12-luciferase reporter plasmid, whereas no significant increase was observed in -60/+12-luciferase plasmid. Decrease in luciferase activity was more prominent when the NF-kappa B binding site was disrupted rather than when the NF-IL6 binding site was disrupted. Moreover, when both sites were disrupted, luciferase activity increased only by 1.5 fold. Electrophoretic mobility shift assays demonstrated enhanced binding activity to oligonucleotides containing the NF-kappa B binding site in hypoxic cardiac myocytes, which displayed a supershift with antibody to its subunit, p50 or p65. The binding activity to the NF-IL6 probe also enhanced and displayed a supershift with antibody to NF-IL6. CONCLUSIONS: Although hypoxic stimulation induced NF-kappa B and NF-IL6 in cardiac myocytes, NF-kappa B may be the primary positive regulator of transcriptional activation of the IL-6 gene in the context of hypoxia.
Authors: Arkadi Beloiartsev; Maria da Glória Rodrigues-Machado; Guo Ling Zhou; Timothy C Tan; Luca Zazzeron; Robert E Tainsh; Patricio Leyton; Rosemary C Jones; Marielle Scherrer-Crosbie; Warren M Zapol Journal: Am J Respir Cell Mol Biol Date: 2015-05 Impact factor: 6.914