Transcriptional Activation of the Macrophage Migration-Inhibitory Factor Gene by the Corticotropin-Releasing Factor is Mediated by the Cyclic Adenosine 3′,5′-Monophosphate Responsive Element-Binding Protein CREB in Pituitary Cells

Abstract

Macrophage migration-inhibitory factor (MIF) has recently been identified as a pituitary hormone that functions as a counterregulatory modulator of glucocorticoid action within the immune system. In the anterior pituitary gland, MIF is expressed in TSH- and ACTH-producing cells, and its secretion is induced by CRF. To investigate MIF function and regulation within pituitary cells, we initiated the characterization of the MIF 5'-regulatory region of the gene. The -1033 to +63 bp of the murine MIF promoter was cloned 5' to a luciferase reporter gene and transiently transfected into freshly isolated rat anterior pituitary cells. This construct drove high basal transcriptional activity that was further enhanced after stimulation with CRF or with an activator of adenylate cyclase. These transcriptional effects were associated with a concomitant rise in ACTH secretion in the transfected cells and by an increase in MIF gene expression as assessed by Northern blot analysis. A cAMP-responsive element (CRE) was identified within the MIF promoter region which, once mutated, abolished the cAMP responsiveness of the gene. Using this newly identified CRE, DNA-binding activity was detected by gel retardation assay in nuclear extracts prepared from isolated anterior pituitary cells and AtT-20 corticotrope tumor cells. Supershift experiments using antibodies against the CRE-binding protein CREB, together with competition assays and the use of recombinant CREB, allowed the detection of CREB-binding activity with the identified MIF CRE. These data demonstrate that CREB is the mediator of the CRF-induced MIF gene transcription in pituitary cells through an identified CRE in the proximal region of the MIF promoter.

Up-Regulation of Macrophage Migration Inhibitory Factor Gene and Protein Expression in Glial Tumor Cells during Hypoxic and Hypoglycemic Stress Indicates a Critical Role for Angiogenesis in Glioblastoma Multiforme

“…This construct has been previously shown to be sufficient to drive reporter gene expression in pituitary cells. 22 A prominent basal transcriptional activity was detected in unstimulated rat glioma cells (29.3 Ϯ 4.5 ϫ 10 3 relative light units (RLU), ϳ80-fold more than the promoterless parent vector). Under hypoxic conditions, the transcriptional activity was found to be 3.9-fold induced in the MIFluc-transfected cells (118.9 Ϯ 11.1 ϫ 10 3 RLU) (Figure 4).…”

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

“…Limited analysis of regulatory elements present in the MIF promoter has been performed previously; a CRE-binding protein CREB, which was present at positions Ϫ48 to Ϫ41 in the murine MIF promoter, was shown to be essential for corticotropin-releasing factorinduced MIF transcription (35). This corresponds to position Ϫ12 in the human MIF gene promoter (36).…”

“…In this study, we identified the minimal MIF promoter sequence that is required for basal gene expression and discovered within this sequence the 2 regulatory elements that control MIF expression both in the basal state and in activated/repressed states. The first, MIF-1, encompasses the previously described CREB binding element (27,35), although bioinformatic analysis suggested greater homology with the AP-1 consensus sequence. The second, MIF-3, overlaps with the putative HIF-1␣ binding site (27).…”

Polymorphisms of the macrophage migration inhibitory factor gene in a UK population with Type 1 diabetes mellitus

“…Variation in reporter gene luciferase activity for the different CATT alleles has been described, with the CATT 5 allele having the lowest level of basal and stimulated MIF promoter activity in human lung epithelial (A549) and fibroblast cell lines [18]. In contrast, higher reporter gene luciferase activity is found for the MIF-173*C allele in human T lymphoblast cell line (CEMC7A) and for MIF-173*G in human A549 lung epithelial cells [14].…”

“…This work suggests that MIF promoter activity is altered in different cell types by binding of several transcription factors to the CATT and MIF-173G>C polymorphic sites. There are sequences within the proximal promoter of MIF which resemble cAMP responsive elements; however, no tissue-specific regulatory elements have been identified [18]. These important gene regulatory elements can map to distant sites both 5¢ and 3¢ of the gene coding region [19], therefore it is necessary to comprehensively assess the genomic region surrounding the MIF gene for such sequences.…”