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Intestinal DMBT1 expression is modulated by Crohn's disease-associated IL23R variants and by a DMBT1 variant which influences binding of the transcription factors CREB1 and ATF-2.

Diegelmann J, Czamara D, Le Bras E, Zimmermann E, Olszak T, Bedynek A, Göke B, Franke A, Glas J, Brand S - PLoS ONE (2013)

Bottom Line: Several DMBT1 SNPs were associated with CD susceptibility.All haplotype groups tested showed highly significant associations with CD (including omnibus P-values as low as 6.1 × 10(-18)).Intestinal DMBT1 expression is decreased in CD patients carrying the rs2981804 CD risk allele.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine II - Grosshadern, Ludwig-Maximilians-University (LMU), Munich, Germany ; Department of Preventive Dentistry and Periodontology, Ludwig-Maximilians-University (LMU), Munich, Germany.

ABSTRACT

Objectives: DMBT is an antibacterial pattern recognition and scavenger receptor. In this study, we analyzed the role of DMBT1 single nucleotide polymorphisms (SNPs) regarding inflammatory bowel disease (IBD) susceptibility and examined their functional impact on transcription factor binding and downstream gene expression.

Methods: Seven SNPs in the DMBT1 gene region were analyzed in 2073 individuals including 818 Crohn's disease (CD) patients and 972 healthy controls in two independent case-control panels. Comprehensive epistasis analyses for the known CD susceptibility genes NOD2, IL23R and IL27 were performed. The influence of IL23R variants on DMBT1 expression was analyzed. Functional analysis included siRNA transfection, quantitative PCR, western blot, electrophoretic mobility shift and luciferase assays.

Results: IL-22 induces DMBT1 protein expression in intestinal epithelial cells dependent on STAT3, ATF-2 and CREB1. IL-22 expression-modulating, CD risk-associated IL23R variants influence DMBT1 expression in CD patients and DMBT1 levels are increased in the inflamed intestinal mucosa of CD patients. Several DMBT1 SNPs were associated with CD susceptibility. SNP rs2981804 was most strongly associated with CD in the combined panel (p = 3.0 × 10(-7), OR 1.42; 95% CI 1.24-1.63). All haplotype groups tested showed highly significant associations with CD (including omnibus P-values as low as 6.1 × 10(-18)). The most strongly CD risk-associated, non-coding DMBT1 SNP rs2981804 modifies the DNA binding sites for the transcription factors CREB1 and ATF-2 and the respective genomic region comprising rs2981804 is able to act as a transcriptional regulator in vitro. Intestinal DMBT1 expression is decreased in CD patients carrying the rs2981804 CD risk allele.

Conclusion: We identified novel associations of DMBT1 variants with CD susceptibility and discovered a novel functional role of rs2981804 in regulating DMBT1 expression. Our data suggest an important role of DMBT1 in CD pathogenesis.

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The Th17 cytokine IL-22 induces DMBT1 expression in intestinal epithelial cells dependent on STAT3.(A) DMBT1 expression is induced by IL-22 in intestinal epithelial cells as determined by quantitative PCR in HT-29 and DLD-1 cells stimulated with 100 ng/ml IL-22 for 6 and 24 hours, respectively. Data are from five independent experiments. DMBT1 expression in unstimulated cells was arbitrarily set to 1.0 and expression in IL-22-stimulated cells was calculated accordingly. *p<0.01 vs. unstimulated cells. (B) Silencing of STAT3 expression by siRNA transfection reduces IL-22-induced DMBT1 expression in DLD-1 cells. *p<0.01 vs. unstimulated; #p<0.05 vs. control+IL-22.
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pone-0077773-g002: The Th17 cytokine IL-22 induces DMBT1 expression in intestinal epithelial cells dependent on STAT3.(A) DMBT1 expression is induced by IL-22 in intestinal epithelial cells as determined by quantitative PCR in HT-29 and DLD-1 cells stimulated with 100 ng/ml IL-22 for 6 and 24 hours, respectively. Data are from five independent experiments. DMBT1 expression in unstimulated cells was arbitrarily set to 1.0 and expression in IL-22-stimulated cells was calculated accordingly. *p<0.01 vs. unstimulated cells. (B) Silencing of STAT3 expression by siRNA transfection reduces IL-22-induced DMBT1 expression in DLD-1 cells. *p<0.01 vs. unstimulated; #p<0.05 vs. control+IL-22.

Mentions: We and others have shown that IL-22 is an inducer of antimicrobial peptides like defensins in IEC and other tissues [35], [36], [37]. DMBT1 is an antibacterial scavenger receptor (9) and we recently demonstrated that the STAT3-activating cytokine IL-27 induces DMBT1 expression in IEC [23]. Given that IL-22 is a known STAT3 activator [35], we hypothesized that IL-22 might induce DMBT1 expression in IEC. To test this hypothesis, we stimulated HT-29 and DLD-1 cells with 100 ng/ml IL-22 for 6 and 24 hours, respectively. Quantitative PCR revealed a significant up-regulation of DMBT1 expression in both cell lines (Fig. 2A). To determine the signaling pathways involved, we transfected DLD-1 cells with siRNA against STAT3 or an unspecific control siRNA prior to IL-22 stimulation. The IL-22-induced DMBT1 expression was significantly reduced in cells with knocked-down STAT3 expression (Fig. 2B).


Intestinal DMBT1 expression is modulated by Crohn's disease-associated IL23R variants and by a DMBT1 variant which influences binding of the transcription factors CREB1 and ATF-2.

Diegelmann J, Czamara D, Le Bras E, Zimmermann E, Olszak T, Bedynek A, Göke B, Franke A, Glas J, Brand S - PLoS ONE (2013)

The Th17 cytokine IL-22 induces DMBT1 expression in intestinal epithelial cells dependent on STAT3.(A) DMBT1 expression is induced by IL-22 in intestinal epithelial cells as determined by quantitative PCR in HT-29 and DLD-1 cells stimulated with 100 ng/ml IL-22 for 6 and 24 hours, respectively. Data are from five independent experiments. DMBT1 expression in unstimulated cells was arbitrarily set to 1.0 and expression in IL-22-stimulated cells was calculated accordingly. *p<0.01 vs. unstimulated cells. (B) Silencing of STAT3 expression by siRNA transfection reduces IL-22-induced DMBT1 expression in DLD-1 cells. *p<0.01 vs. unstimulated; #p<0.05 vs. control+IL-22.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3818382&req=5

pone-0077773-g002: The Th17 cytokine IL-22 induces DMBT1 expression in intestinal epithelial cells dependent on STAT3.(A) DMBT1 expression is induced by IL-22 in intestinal epithelial cells as determined by quantitative PCR in HT-29 and DLD-1 cells stimulated with 100 ng/ml IL-22 for 6 and 24 hours, respectively. Data are from five independent experiments. DMBT1 expression in unstimulated cells was arbitrarily set to 1.0 and expression in IL-22-stimulated cells was calculated accordingly. *p<0.01 vs. unstimulated cells. (B) Silencing of STAT3 expression by siRNA transfection reduces IL-22-induced DMBT1 expression in DLD-1 cells. *p<0.01 vs. unstimulated; #p<0.05 vs. control+IL-22.
Mentions: We and others have shown that IL-22 is an inducer of antimicrobial peptides like defensins in IEC and other tissues [35], [36], [37]. DMBT1 is an antibacterial scavenger receptor (9) and we recently demonstrated that the STAT3-activating cytokine IL-27 induces DMBT1 expression in IEC [23]. Given that IL-22 is a known STAT3 activator [35], we hypothesized that IL-22 might induce DMBT1 expression in IEC. To test this hypothesis, we stimulated HT-29 and DLD-1 cells with 100 ng/ml IL-22 for 6 and 24 hours, respectively. Quantitative PCR revealed a significant up-regulation of DMBT1 expression in both cell lines (Fig. 2A). To determine the signaling pathways involved, we transfected DLD-1 cells with siRNA against STAT3 or an unspecific control siRNA prior to IL-22 stimulation. The IL-22-induced DMBT1 expression was significantly reduced in cells with knocked-down STAT3 expression (Fig. 2B).

Bottom Line: Several DMBT1 SNPs were associated with CD susceptibility.All haplotype groups tested showed highly significant associations with CD (including omnibus P-values as low as 6.1 × 10(-18)).Intestinal DMBT1 expression is decreased in CD patients carrying the rs2981804 CD risk allele.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine II - Grosshadern, Ludwig-Maximilians-University (LMU), Munich, Germany ; Department of Preventive Dentistry and Periodontology, Ludwig-Maximilians-University (LMU), Munich, Germany.

ABSTRACT

Objectives: DMBT is an antibacterial pattern recognition and scavenger receptor. In this study, we analyzed the role of DMBT1 single nucleotide polymorphisms (SNPs) regarding inflammatory bowel disease (IBD) susceptibility and examined their functional impact on transcription factor binding and downstream gene expression.

Methods: Seven SNPs in the DMBT1 gene region were analyzed in 2073 individuals including 818 Crohn's disease (CD) patients and 972 healthy controls in two independent case-control panels. Comprehensive epistasis analyses for the known CD susceptibility genes NOD2, IL23R and IL27 were performed. The influence of IL23R variants on DMBT1 expression was analyzed. Functional analysis included siRNA transfection, quantitative PCR, western blot, electrophoretic mobility shift and luciferase assays.

Results: IL-22 induces DMBT1 protein expression in intestinal epithelial cells dependent on STAT3, ATF-2 and CREB1. IL-22 expression-modulating, CD risk-associated IL23R variants influence DMBT1 expression in CD patients and DMBT1 levels are increased in the inflamed intestinal mucosa of CD patients. Several DMBT1 SNPs were associated with CD susceptibility. SNP rs2981804 was most strongly associated with CD in the combined panel (p = 3.0 × 10(-7), OR 1.42; 95% CI 1.24-1.63). All haplotype groups tested showed highly significant associations with CD (including omnibus P-values as low as 6.1 × 10(-18)). The most strongly CD risk-associated, non-coding DMBT1 SNP rs2981804 modifies the DNA binding sites for the transcription factors CREB1 and ATF-2 and the respective genomic region comprising rs2981804 is able to act as a transcriptional regulator in vitro. Intestinal DMBT1 expression is decreased in CD patients carrying the rs2981804 CD risk allele.

Conclusion: We identified novel associations of DMBT1 variants with CD susceptibility and discovered a novel functional role of rs2981804 in regulating DMBT1 expression. Our data suggest an important role of DMBT1 in CD pathogenesis.

Show MeSH
Related in: MedlinePlus