Hypoxia and glucocorticoid signaling converge to regulate macrophage migration inhibitory factor gene expression

Abstract

Objective. Macrophage migration inhibitory factor (MIF) is a proinflammatory mediator involved in the pathogenesis of rheumatoid arthritis. This study was undertaken to identify the MIF promoter elements responsible for regulating gene expression.Methods. Luciferase reporter gene assays were used to identify the MIF promoter sequence responsible for basal activity. Bioinformatic analysis was used to predict transcription factor binding sites, and electrophoretic mobility shift assay (EMSA) was used to demonstrate transcription factor binding. Chromatin immunoprecipitation (ChIP) was used to demonstrate transcription factor loading on the MIF promoter.Results. We identified the minimal promoter sequence required for basal MIF promoter activity that was also capable of conferring glucocorticoid-dependent inhibition in a T lymphocyte model cell line. Deletion studies and EMSA revealed 2 elements in the MIF promoter that were responsible for basal promoter activity. The 5 element binds CREB/activating transcription factor 1, and the 3 element is a functional hypoxia-responsive element binding hypoxia-inducible factor 1␣. Further studies demonstrated that the cis elements are both required for glucocorticoiddependent inhibition. ChIP demonstrated glucocorticoid-dependent recruitment of glucocorticoid receptor ␣ to the MIF promoter in lymphocytes within 1 hour of treatment and a concomitant decrease in acetylated histone H3.Conclusion. Our findings indicate that hypoxia and glucocorticoid signaling converge on a single element regulating MIF; this regulatory unit is a potential interacting node for microenvironment sensing of oxygen tension and glucocorticoid action in foci of inflammation.

Hypoxia stimulates the expression of macrophage migration inhibitory factor in human vascular smooth muscle cells via HIF-1α dependent pathway

“…EMSA assay was performed using primers encompassing the hypoxia response element (HRE) and adjacent flanking regions in the promoter of human MIF gene [18]. Our data showed that HIF-1α binding activity could be increased by exposure to hypoxia (3% O 2 ) for 24 h (Figure 2C).…”

Cell Cycle Phase Regulates Glucocorticoid Receptor Function

“…24 hours later cells were treated as specified in results prior to lysis, then assayed for luciferase activity using a dual-luciferase reporter assay system following manufacturers instructions (Promega, Southampton, UK), and as previously described [45].…”

Hypoxia-dependent regulation of inflammatory pathways in immune cells

“…AMPK is a metabolic sensor that is activated in response to ATP depletion that occurs as a result of the metabolic crisis caused by hypoxia, and has recently emerged as a key regulator of inflammatory cell activity (90). The glucocorticoid receptor (GR), which plays a key role in the activation of antiinflammatory genes and the suppression of proinflammatory genes, is activated in response to hypoxia and likely contributes to the overall control of inflammation, although the mechanisms underpinning the activation of the GR by hypoxia remain unclear (91,92). Members of the cAMP response element-binding protein (CREB) family, including CREB and ATF4, have been shown to be regulated by hypoxia and can in turn regulate the expression of inflammatory genes in immune cells (93,94).…”