Endocrine Abstracts

MIF is a potent pro-inflammatory mediator that opposes the anti-inflammatory actions of glucocorticoids (Gc) and is involved in the pathogenesis of multiple diseases. The MIF promoter is inhibited by Gc in lymphocytes (CEMC7A) but not in epithelial cells (A549), despite expression of functional glucocorticoid receptors (GR) in both. Deletion studies localized the cis element responsible for Gc inhibition to between MIF −71 and +85. This sequence contained no GR binding sites; however, three potential AP-1 binding sites were found, one upstream (MIF1) and two downstream (MIF2 and MIF3) of the transcription start site. Electrophoretic mobility shift assays identified a complex composed of ATF-1/CREB bound to MIF1, whereas MIF2 did not bind any sequence-specific protein, and MIF3 bound a lymphocyte-specific complex. When MIF1 and MIF3 were deleted from the minimal (−71 to +85) MIF reporter construct the individual deletions did not prevent Gc repression of promoter activity, but loss of both abolished Gc repression. Further analysis of the MIF3 sequence revealed a hypoxic responsive element (HRE), and western blotting indicated constitutive expression of hypoxia inducible factor 1 alpha (HIF1 alpha) in CEMC7A cells. MIF promoter activity was shown to be enhanced under hypoxic conditions in CEM cells, however this effect was abrogated when MIF3 was deleted, indicating that MIF3 is indeed a functional hypoxia responsive element. Chromatin immunoprecipitation demonstrated Gc dependent recruitment of GR to the proximal MIF promoter in lymphocytes, within 1 h of treatment, accompanied by a time-dependent reduction in acetylated histone H3. In contrast, in the epithelial cells there was no GR recruitment, and no histone modification.

In summary, we identify convergence of hypoxia and gc signalling on a single element regulating the angiogenic and pro inflammatory cytokine MIF; an integrating node for micro environment sensing and glucocorticoid action in inflammatory foci.