Expression of human inducible nitric oxide synthase in response to cytokines is regulated by hypoxia-inducible factor-1

• Published in: Free radical biology & medicine Vol. 130; pp. 278 - 287
• Main Authors: Lee, Martin, Wang, Christine, Jin, Steven W., Labrecque, Mark P., Beischlag, Timothy V., Brockman, Mark A., Choy, Jonathan C.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.01.2019
• Subjects: A549 Cells; Astrocytes - metabolism; Cell Hypoxia - genetics; CRISPR-Cas Systems - genetics; Cytokine; Cytokines - metabolism; Cytokines - pharmacology; Gene Expression Regulation - drug effects; Gene regulation; Humans; Hypoxia-inducible factor (HIF); Hypoxia-Inducible Factor 1, alpha Subunit - genetics; Interferon-gamma - drug effects; Interferon-gamma - genetics; Interferon-gamma - pharmacology; Interleukin-1 - pharmacology; Nitric oxide; Nitric Oxide - biosynthesis; Nitric Oxide - metabolism; Nitric oxide synthase; Nitric Oxide Synthase Type II - genetics; Promoter Regions, Genetic - genetics; Response Elements - genetics; Tumor Necrosis Factor-alpha - pharmacology; NO; HIF; HK-1; Gene regulation; Cytokine; VEGF-A; Hypoxia-inducible factor (HIF); PHD; sgRNA; Nitric oxide; iNOS; Nitric oxide synthase; HRE; CoCl2
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/j.freeradbiomed.2018.10.441

The production of nitric oxide (NO) by inducible NO synthase (iNOS) and the regulation of gene expression by hypoxia-inducible factors (HIFs) are important for many aspects of human cell biology. However, little is known about whether iNOS expression is controlled by HIFs in human cells. Stimulation of A549 human lung epithelial cells with cytokines (TNF, IL-1 and IFNγ) increased the nuclear accumulation of HIF-1 in normoxic conditions. Activation of HIF-1 by hypoxia or CoCl2 was not sufficient to induce iNOS expression. However, pharmacological inhibition of HIF-1 reduced the induction of iNOS expression in A549 cells and primary human astrocytes. Moreover, elimination of HIF-1α expression and activity by CRISPR/Cas9 gene editing significantly reduced the induction of human iNOS gene promoter, mRNA and protein expression by cytokine stimulation. Three putative hypoxia response elements (HRE) are present within the human iNOS gene promoter and elimination of an HRE at −4981 bp reduced the induction of human iNOS promoter activity in response to cytokine stimulation. These findings establish an important role for HIF-1α in the induction of human iNOS gene expression in response to cytokine stimulation. [Display omitted] •Cytokines induce nuclear translocation of HIF-1α in normoxia.•HIF-1 is needed for maximal induction of human iNOS expression.•There is a functional hypoxia response element at −4981 bp of the human iNOS gene promoter.