Transcription factor ICBP90 regulates the MIF promoter and immune susceptibility locus
Jie Yao, … , Patty Lee, Richard Bucala
Jie Yao, … , Patty Lee, Richard Bucala
Published January 11, 2016
Citation Information: J Clin Invest. 2016;126(2):732-744. https://doi.org/10.1172/JCI81937.
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Research Article Immunology

Transcription factor ICBP90 regulates the MIF promoter and immune susceptibility locus

Abstract

The immunoregulatory cytokine macrophage migration inhibitory factor (MIF) is encoded in a functionally polymorphic locus that is linked to the susceptibility of autoimmune and infectious diseases. The MIF promoter contains a 4-nucleotide microsatellite polymorphism (–794 CATT) that repeats 5 to 8 times in the locus, with greater numbers of repeats associated with higher mRNA levels. Because there is no information about the transcriptional regulation of these common alleles, we used oligonucleotide affinity chromatography and liquid chromatography–mass spectrometry to identify nuclear proteins that interact with the –794 CATT5–8 site. An analysis of monocyte nuclear lysates revealed that the transcription factor ICBP90 (also known as UHRF1) is the major protein interacting with the MIF microsatellite. We found that ICBP90 is essential for MIF transcription from monocytes/macrophages, B and T lymphocytes, and synovial fibroblasts, and TLR-induced MIF transcription is regulated in an ICBP90- and –794 CATT5–8 length–dependent manner. Whole-genome transcription analysis of ICBP90 shRNA–treated rheumatoid synoviocytes uncovered a subset of proinflammatory and immune response genes that overlapped with those regulated by MIF shRNA. In addition, the expression levels of ICBP90 and MIF were correlated in joint synovia from patients with rheumatoid arthritis. These findings identify ICBP90 as a key regulator of MIF transcription and provide functional insight into the regulation of the polymorphic MIF locus.

Authors

Jie Yao, Lin Leng, Maor Sauler, Weiling Fu, Junsong Zheng, Yi Zhang, Xin Du, Xiaoqing Yu, Patty Lee, Richard Bucala

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Figure 6

ICBP90 regulates MIF expression in human lymphocytes and rheumatoid synovial fibroblasts.

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ICBP90 regulates MIF expression in human lymphocytes and rheumatoid syno...
Cultured Raji B cells were transfected with control or ICBP90 shRNA. Eight hours later (A) ICBP90 mRNA and (B) intracellular ICBP90 protein content were measured by qPCR and Western blotting, respectively. (C) Cellular MIF mRNA was determined by qPCR, and (D) secreted MIF was determined by ELISA. Human Jurkat T cells were transfected with control or ICBP90 shRNA and analyzed for (E) ICBP90 mRNA, (F) intracellular ICBP90 protein content, (G) MIF mRNA, and (H) secreted MIF as above. (I) Jurkat T cells were transfected with MIF promoter-luciferase reporter plasmids, treated with an ICBP90 or control shRNA, cultured for 6 hours, and stimulated or not with PMA/ionomycin (100 ng/ml). Cultured human synovial fibroblasts were transfected with control or ICBP90 shRNA. Eight hours later (J) ICBP90 mRNA and (K) intracellular ICBP90 protein content were measured by qPCR and Western blotting, respectively. (L) Cellular MIF mRNA was determined by qPCR, and (M) cytosolic and (N) secreted MIF were quantified by ELISA. Cytosolic MIF is expressed as ng immunoreactive MIF/total cellular protein. Data are presented as the mean+SD of 3 measurements, with all experiments replicated twice (n = 3 measurements per experiment). *P < 0.05, **P < 0.01 by 2-tailed Student’s t test for control shRNA vs. ICBP90 shRNA (A, C–E, G–J, and L–N) and for PMA/ionomycin stimulated vs. unstimulated conditions (I).