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Ulcerative colitis (UC) and Crohn's disease (CD) are two related yet different forms of chronic intestinal inflammation. We investigated the genes regulated by NKX2-3 in B cells from a UC patient by cDNA microarray and compared the results to those genes regulated by NKX2-3 in B cells from a CD patient. Genes regulated by NKX2-3 in B cells from UC were mainly involved in cell growth, inflammation, and immune response. Among the genes regulated by NKX2-3 in both UC and CD, expression of 145 genes was similarly altered and 34 genes was differentially affected by NKX2-3 knockdown. EGR1 was up-regulated in NKX2-3 knockdown B cells from UC while down-regulated in NKX2-3 knockdown B cells from CD. mRNA expressions of NKX2-3 and EGR1 were increased in diseased intestinal tissues from 19 CD patients. NKX2-3 may play different roles in UC and CD pathogenesis by differential regulation of EGR1.

PTPN2 is a risk gene for Crohn's disease (CD). We investigated whether PTPN2 genetic variants (rs2542151 and rs2542152) were associated with CD in a familial IBD registry. Both rs2542151 and rs2542152 are associated with CD, but not ulcerative colitis (UC). mRNA expression levels of PTPN2 were significantly increased in intestinal tissues (p=0.0493), and nearly significantly increased in B cells (p=0.0889) from CD patients, but not significantly altered in UC. cDNA microarray results found that PTPN2 was down-regulated by NKX2-3 knockdown in human cells. We confirmed this observation by RT-PCR analyses in NKX2-3 knockdown in B cells from IBD patients and human intestinal microvascular endothelial cells (HIMEC). In addition, we found that mRNA expression of another IBD-associated gene, NKX2-3, was increased in intestinal tissues and B cells from CD patients, but not significantly increased in UC patients. A positive correlation was observed between mRNA expression of PTPN2 and NKX2-3 in B cells and in intestinal tissues from both CD and UC patients. These results suggest that PTPN2 may have an important role in CD pathogenesis and may represent a potential diagnostic and therapeutic target for IBD.

NKX2-3 SNP rs11190140 is associated with inflammatory bowel disease (IBD). The T allele is over-transmitted in IBD and the C allele represents a potential CpG methylation site. We hypothesize that genetic variation and/or methylation of SNP rs11190140 may play a role in NKX2-3 gene expression by affecting transcription factor binding. We studied 233 IBD cases and 250 unrelated healthy individuals from an IBD population from central Pennsylvania and performed genotype analyses of the genetic variation and methylation status analysis using PCR-based RFLP. For transcription factor binding, nuclear extracts from human B cells were incubated with biotin-labeled oligonucleotide sequences of the NKX2-3 promoter region containing the genetic variation of T, non-methylated C or methylated C at rs11190140, followed by biotin pull-down and Western blot analysis for transcription factors SP1, NFAT1, NF-κB, and ETS-1. In case-control analysis, the genetic variation was significantly associated with IBD (OR=0.503, 95% CI=0.330-0.764, p<0.001). Methylation status analyses revealed that the C allele is subject to modification by DNA methylation. transcription factor binding assay indicated distinct differential binding of NFAT1 to the NKX2-3 promoter sequence, with higher binding to those with non-methylated and methylated C than to T. The binding of NFAT1 to the NKX2-3 promoter region with rs1190140 was confirmed by ChIP assay. We speculate that the rs11190140 may regulate NKX2-3 expression and have a role in IBD pathogenesis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: NKX2-3 is associated with inflammatory bowel disease (IBD). NKX2-3 is expressed in microvascular endothelial cells and the muscularis mucosa of the gastrointestinal tract. Human intestinal microvascular endothelial cells (HIMECs) are actively involved in the pathogenesis of IBD and IBD-associated microvascular dysfunction. To understand the cellular function of NKX2-3 and its potential role underlying IBD pathogenesis, we investigated the genes regulated by NKX2-3 in HIMEC using cDNA microarray., Methodology/principal Findings: NKX2-3 expression was suppressed by shRNA in two HIMEC lines and gene expression was profiled by cDNA microarray. Pathway Analysis was used to identify gene networks according to biological functions and associated pathways. Validation of microarray and genes expression in intestinal tissues was assessed by RT-PCR. NKX2-3 regulated genes are involved in immune and inflammatory response, cell proliferation and growth, metabolic process, and angiogenesis. Several inflammation and angiogenesis related signaling pathways that play important roles in IBD were regulated by NKX2-3, including endothelin-1 and VEGF-PI3K/AKT-eNOS. Expression levels of NKX2-3, VEGFA, PI3K, AKT, and eNOS are increased in intestinal tissues from IBD patients and expression levels of EDN1 are decreased in intestinal tissues from IBD patients. These results demonstrated the important roles of NKX2-3, VEGF, PI3K, AKT, eNOS, and EDN1 in IBD pathogenesis. Correlation analysis showed a positive correlation between mRNA expression of NKX2-3 and VEGFA and a negative correlation between mRNA expression of NKX2-3 and EDN1 in intestinal tissues from IBD patients., Conclusion/relevance: NKX2-3 may play an important role in IBD pathogenesis by regulating endothelin-1 and VEGF signaling in HIMECs.

Background: Nkx2-3 has been reported to be up-regulated in B cell lines and intestinal tissues from Crohn's disease patients and down-regulated in colorectal cancer., Aims: The purpose of the current study is to determine genes regulated by Nkx2-3 in sporadic (CRS61) and inflammatory bowel disease-associated (CRS4) colorectal cancer cell lines., Methods: Small interfering RNA-mediated knockdown of Nkx2-3 in both cell lines was generated and high-density cDNA microarrays representing over 25,000 genes were performed. Microarray results were validated by RT-PCR and immunofluorescence. Pathway analysis was used to identify gene networks associated with Nkx2-3 knockdown in these cell lines., Results: A total of 1,677 genes were regulated by Nkx2-3 in CRS4 cells; 1,375 genes were regulated by Nkx2-3 in CRS61 cells. Among those genes regulated by Nkx2-3, 254 genes were similarly regulated by Nkx2-3 knockdown in both cell lines; 159 genes were differentially regulated by Nkx2-3 knockdown between the two lines. Genes regulated by Nkx2-3 were grouped primarily within the following two functional categories: (1) immune and inflammatory response; and (2) cell proliferation, growth, and oncogenesis. Among the genes with similarly changed expression in the two cell lines, the top affected pathways included antigen presentation and cell-cell signaling. Among the genes with differentially changed expression between the two cell lines, ingenuity pathway analysis indicated that the top affected pathway included genes directly involved in Wnt signaling., Conclusions: Nkx2-3 may contribute to the pathogenesis of IBD-associated CRC and sporadic CRC by regulating the Wnt signaling pathway.

Nkx2-3 gene variants are strongly associated with inflammatory bowel disease (IBD) and its expression is up-regulated in Crohn's disease (CD). However, the nature of its role underlying IBD pathogenesis is unknown. We investigated the genes regulated by Nkx2-3 using cDNA microarray. A small interfering RNA (siRNA)-mediated knockdown of Nkx2-3 in a B cell line from a CD patient was generated. Gene expression was profiled on high-density cDNA microarrays representing over 25,000 genes. Ingenuity pathway analysis (IPA) was used to identify gene networks according to biological functions and associated pathways. Expression profiling analysis by cDNA microarray showed that 125 genes were regulated by Nkx2-3 knockdown (fold change >or=3.0, p<0.01), among which 51 genes were immune and inflammatory response genes. Microarray results were validated by RT-PCR and further confirmed in a B cell line expressing siRNA of Nkx2-3 from an additional CD patient. The results showed that Nkx2-3 was up-regulated (p<0.05) and EDN1 was down-regulated (p<0.05) in B cell lines from CD patients. mRNA expression levels of Nkx2-3 were negatively correlated with those of EDN1 (r=-0.6044, p<0.05). EDN1 was also down-regulated in intestinal tissues from UC patients (p<0.05). Our present results demonstrate that a decrease in Nkx2-3 gene expression level can profoundly alter the expression of genes and cellular functions relevant to the pathogenesis and progression of IBD, such as EDN1., ((c) 2010 Elsevier Inc. All rights reserved.)