Hypoxia and glucocorticoid signaling converge to regulate macrophage migration inhibitory factor gene expression.
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 1alpha. Further studies demonstrated that the cis elements are both required for glucocorticoid-dependent inhibition. ChIP demonstrated glucocorticoid-dependent recruitment of glucocorticoid receptor alpha to the MIF promoter in lymphocytes within 1 hour of treatment and a concomitant decrease in acetylated histone H3.


CONCLUSIONS
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.
Elsby
Laura M
LM
University of Manchester, Manchester, UK.
Donn
Rachelle
R
Alourfi
Zaynab
Z
Green
Laura M
LM
Beaulieu
Elaine
E
Ray
David W
DW
D001483:Base Sequence; D015687:Cell Hypoxia / Q000235:genetics*; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods; D004247:DNA / Q000737:chemistry; D003907:Dexamethasone / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D005938:Glucocorticoids / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D008263:Macrophage Migration-Inhibitory Factors / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D011485:Protein Binding / Q000235:genetics; D020545:Respiratory Mucosa / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics*; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects / Q000235:genetics
D005938:Glucocorticoids; D008263:Macrophage Migration-Inhibitory Factors; D009841:Oligonucleotides; D003907:Dexamethasone; D004247:DNA