Polymorphisms in the hypoxia inducible factor binding site of the macrophage migration inhibitory factor gene promoter in schizophrenia

Abstract

Background: Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine that promotes neurogenesis and neuroprotection. MIF is predominantly expressed in astrocytes in the brain. The serum MIF level and microsatellites/single nucleotide polymorphisms (SNPs) in the MIF gene promoter region are known to be associated with schizophrenia (SCZ). Interestingly, previous studies reported that hypoxia, an environmental risk factor for SCZ, induced MIF expression through binding of the hypoxia inducible factor (HIF)-1 to the hypoxia response element (HRE) in the MIF promoter.

Methods: We investigated the involvement of MIF in SCZ while focusing on the HIF pathway. First, we conducted an association study of the SNP rs17004038 (C>A) in the HRE of the MIF promoter between 1758 patients with SCZ and 1507 controls. Next, we investigated the effect of hypoxia on MIF expression in primary cultured astrocytes derived from neonatal mice forebrain.

Results: SNP rs17004038 was significantly associated with SCZ (p = 0.0424, odds ratio = 1.445), indicating that this SNP in the HRE of the MIF promoter was a genetic risk factor for SCZ. Hypoxia induced MIF mRNA expression and MIF protein production and increased HIF-1 binding to the MIF promoter, while the activity of the MIF promoter was suppressed by mutations in the HRE and by deletion of the HRE in astrocytes.

Conclusion: These results suggest that SNP rs17004038 in the HRE of the MIF promoter was significantly associated with SCZ and may be involved in the pathophysiology of SCZ via suppression of hypoxia and HIF pathway-induced MIF expression.

Fig 1. Human and mouse macrophage migration inhibitory factor (MIF) gene promoter region.

Fig 2. Hypoxia induced macrophage migration inhibitory factor (MIF) mRNA expression and protein production via the hypoxia inducible factor (HIF) pathway.

A. The effects of ML228, a HIF activator, on MIF mRNA expression in primary cultured astrocytes (PCAs). PCAs were incubated for 48 h in 0, 0.25, 0.5, or 1.0 μM ML228. MIF mRNA expression was analyzed by qRT-PCR. The values are shown as the ratio of MIF mRNA to beta-actin (ACTB) mRNA (Dunnett’s test vs. vehicle; n = 3). B. The time-dependent effects of hypoxia on MIF mRNA expression in PCAs. PCAs were incubated for 3, 6, 12, 24, or 48 h in 21.0% or 0.1% O₂. MIF mRNA expression was analyzed by qRT-PCR. The values are shown as the ratio of MIF mRNA to ACTB mRNA (Student’s t-test; n = 5–6). C. The effects of the HIF inhibitor YC-1 on hypoxia-induced MIF mRNA expression in PCAs. PCAs were incubated for 6 h in 21.0% or 0.1% O₂ and treated with 0 or 10 μM of YC-1. MIF mRNA expression was analyzed by qRT-PCR. The values are shown as the ratio of MIF mRNA to ACTB mRNA (Tukey’s test; n = 6). D. The effects of hypoxia on MIF protein production in cell lysate of PCAs. PCAs were incubated for 24 or 48 h in 21.0% or 0.1% O₂. The MIF protein level per 1 μg of total protein was analyzed using a MIF enzyme-linked immunosorbent assay (ELISA) (Student’s t-test; n = 6). The data are expressed as the mean ± SEM. *p < 0.05, **p < 0.01, or ***p < 0.001.

Fig 3. Hypoxia-inducible factor (HIF) binding to the hypoxia response element (HRE) induced activation of the macrophage migration inhibitory factor (MIF) gene promoter under hypoxia, that was abrogated by the allelic variant of the HRE.

A. The effects of hypoxia on HIF binding at the MIF gene promoter in primary cultured astrocytes (PCAs). PCAs were incubated for 6, 24, or 48 h in 21.0% or 0.1% O₂. HIF-1α binding to the MIF gene promoter was analyzed by chromatin immunoprecipitation (ChIP) assay. Real-time PCR was performed on DNA purified from each of the ChIP reactions by using a primer set specific for the MIF gene promoter. The % input values indicate the ratio of immunoprecipitated DNA fragments to the input DNA fragments (Student’s t-test after logarithmic transformation; n = 6). B. The allelic variant of the HRE abrogates MIF induction by hypoxia in PCAs. PCAs were transfected with a reporter plasmid containing the MIF genomic region (−335 to −1) upstream of the Nanoluc luciferase reporter gene and a control plasmid containing the firefly luciferase reporter gene. Where indicated, the wild type (WT) HRE sequence (CACGT) was mutated to AACGT (SNP), CTAGC (mutHRE), or deletion (delHRE). At 24 h after transfection, PCAs were incubated for 24 h in 21.0% or 0.1% O₂. The graph represents the corrected luciferase activity values of each construct in cells exposed to hypoxia over the luciferase activity obtained in normoxic cells (Dunnett’s test; n = 9–14). The data are expressed as the mean ± SEM. *p < 0.05, **p < 0.01, or ***p < 0.001.

Fig 4. Putative roles of single nucleotide polymorphism (SNP) rs17004038 in the hypoxia response element (HRE) of the macrophage migration inhibitory factor (MIF) gene promoter in perinatal hypoxia-associated schizophrenia (SCZ) risk.

Abbreviation: HIF, Hypoxia-inducible factor.