Genome-wide expression profiling implicates a MAST3-regulated gene set in colonic mucosal inflammation of ulcerative colitis patients

Abstract

Background: Crohn's disease (CD) and ulcerative colitis (UC) are inflammatory bowel diseases (IBDs) presumably caused by dysregulated immune responses to the gut microbiota. Genetic association studies have implicated dozens of chromosomal regions or loci in IBD susceptibility. The next challenge is to explain the individual role of each of these modest effect loci in the disease state. We have previously identified MAST3 as an IBD susceptibility gene through genetic fine-mapping of the 19p linkage region. Testing MAST3 in a reporter assay provided preliminary evidence that MAST3 modulates the activity of inflammation-related transcription factor nuclear factor kappa B.

Methods: Here we characterized the function of MAST3 through an examination of the influence of the modulation of MAST3 expression on endogenous genome-wide expression patterns. More specifically, we looked at differential gene expression resulting from overexpression and knockdown of the MAST3 gene in epithelial and macrophage cell lines. From we highlight a group of genes whose expression is modulated by MAST3 and correlate their expression with NF-jB activity. Their expression was found to be enriched in inflamed mucosal tissue of UC patients, confirming the importance of these genes in IBD.

Results: We highlight a group of genes whose expression is modulated by MAST3 and correlate their expression with NF-κB activity. Their expression was found to be enriched in inflamed mucosal tissue of UC patients, confirming the importance of these genes in IBD. These MAST3-regulated genes are central to mucosal immune responses. Among them are proinflammatory cytokines (e.g., CCL20, IL8), regulators of NF-κB (e.g., TNFAIP3, LY96, NFKBIA), genes involved in interferon-induced defense against pathogen invasion (e.g., IFIT1, ISG15), and genes involved in cell adhesion and/or migration (e.g., CD44, TMOD1).

Conclusions: Taken together, these results confirm MAST3 as a modulator of the inflammatory response through regulation of immune gene expression in the gut of IBD patients.

Figure 1. MAST3-regulated genes are involved in critical immune functions

Genes in the MAST3-regulated gene set (genes upregulated by 2-fold or more in MAST3 overexpressed cells) were classified according to their function recovered from literature searches. The functions of the genes mainly cluster around the activity of NF-κB. Sixty-one percent of the 28 genes have experimentally confirmed NF-κB binding site in their promoter and are regulated by NF-κB. Thirty-two percent regulates the activity of NF-κB. Twenty-one percent are pro-inflammatory cytokines. Other functions include cell adhesion and migration and interferon mediated defense against pathogen invasion and tumor progression. Two genes out of the 28, CALCB and SLC25A24 do not seem to be directly involved in immune functions and were not included in the diagram.

Figure 2. MAST3 regulates gene expression through NF-κB

A. The overexpression of MAST3 increases the activity of NF-κB. The graph shows the activity of NF-κB tested in HEK293 cells that overexpress the MAST3 gene at different times post-transfection (MAST3 over). The increase in activity is significantly detected after 12h (*P=0.01). B. The knockdown of MAST3 decreases the activity of NF-κB. The graph shows the activity of NF-κB tested in THP1 cells at different stages of differentiation and stimulation following different treatment. The decrease in activity is significantly detected in phorbol 12-myristate 13-acetate (PMA) differentiated cells after 6h of lipopolysaccharide (LPS) stimulation (*P=0.02). C. and D. Expression of MAST3-regulated genes IL8 (C) and CCL20 (D) post-transfection in cells overexpressing the MAST3 gene. Expression is normalized to HPRT1 expression. Error bar represent standard deviation of 3 independent biological replicates. KD MAST3: knockdown MAST3, KD NT: non target knockdown control.

Figure 2. MAST3 regulates gene expression through NF-κB

A. The overexpression of MAST3 increases the activity of NF-κB. The graph shows the activity of NF-κB tested in HEK293 cells that overexpress the MAST3 gene at different times post-transfection (MAST3 over). The increase in activity is significantly detected after 12h (*P=0.01). B. The knockdown of MAST3 decreases the activity of NF-κB. The graph shows the activity of NF-κB tested in THP1 cells at different stages of differentiation and stimulation following different treatment. The decrease in activity is significantly detected in phorbol 12-myristate 13-acetate (PMA) differentiated cells after 6h of lipopolysaccharide (LPS) stimulation (*P=0.02). C. and D. Expression of MAST3-regulated genes IL8 (C) and CCL20 (D) post-transfection in cells overexpressing the MAST3 gene. Expression is normalized to HPRT1 expression. Error bar represent standard deviation of 3 independent biological replicates. KD MAST3: knockdown MAST3, KD NT: non target knockdown control.