Genes regulated by Nkx2-3 in siRNA-mediated knockdown B cells: Implication of endothelin-1 in inflammatory bowel disease

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Abstract

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 ⩾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.

Introduction

IBD consists of two major forms, Crohn’s disease (CD) and ulcerative colitis (UC). Genetics plays an important role in the development of inflammatory bowel disease (IBD) [1]. Nkx2-3 has recently been strongly associated with IBD by genome-wide association studies (GWAs) [2], [3]. The functional role of Nkx2-3 in IBD pathogenesis is currently unknown. Recently, we observed that the expression of Nkx2-3 is up-regulated in intestinal tissues and B cells from CD patients [4].

Nkx2-3 is a member of the Nkx family of homeodomain transcription factors. Homeobox transcription factors regulate cell-specific expression of genes involved in tissue differentiation and development [5], [6]. Nkx2-3 is principally expressed in subsets of cells of mesodermal origin in the gastrointestinal tract and spleen [7], [8]. It also has an important role in spleen and Peyer patch development, affecting B cell maturation and T cell-dependent immune responses [9], [10]. Within the gastrointestinal tract, Nkx2-3 is expressed in microvascular endothelial and smooth muscle cells of the lamina propria and submucosa.

Evidence indicates that one of the Nkx family members, Nkx2-5, regulates heart development by regulating over 20 genes, such as Cx40, Cx43, Dio2, and Rae28 [11], [12], [13]. Nkx2-3, similarly to Nkx2-5 in the heart, may significantly influence gene expression within the intestine, and thereby play an important role in IBD pathogenesis, Nkx2-3-deficient mice have shown that Nkx2-3 is required for expression of MAdCAM-1, an endothelial cell adhesion molecule, which is critical for lymphocyte homing to the gut [9], [10]. In this context, Nkx2-3 likely influences migration of antigen-responsive lymphocytes to the gut and thus affects the intestinal inflammatory response. Expression of MAdCAM-1 has been shown to be dramatically up-regulated within focal sites of intestinal inflammation in both animal models of IBD [14] and in human tissue samples from patients with CD and UC [15].

Since transcription factors can regulate downstream genes, we performed a gene expression microarray analysis on an Nkx2-3 siRNA knockdown B cell line from a CD patient. Using this technique, we have identified genes differentially expressed in Nkx2-3 knockdown cells as compared to control cells, many of which are inflammation-related. Our further study on EDN1 (endothelin-1) regulation in IBD patients suggests possible pathways by which Nkx2-3 may contribute to the pathogenesis of human IBD.

Section snippets

Cell lines

B cell lines were established by infection with Epstein–Barr virus (EBV) [4]. CD-1 is a B cell line from a CD patient. This patient is a 50 year old woman with terminal ileum Crohn’s disease first diagnosed at age 18. She underwent an ileocecetomy for fibrous stricture of the ileum after recent repetitive bouts of disease flares (Montreal Classification: A2, L1, B2). CD-2 is a B cell line from a 27 years old female CD patient. All B cell lines used in this study were obtained from an established

Generation of Nkx2-3 knockdown cell lines

The genotype of the CD-1 cell line is homozygous GG at the Nkx2-3 SNP rs10883365. It has been reported that the G allele of SNP rs10883365 is strongly associated with risk for IBD [2]. First we examined the expression of Nkx2-3 in CD-1 cell line and two other non-IBD B cell lines: one from a non-IBD CD-1 family member (mother; AG at the Nkx2-3 SNP rs10883365), and another from a non-familial non-IBD healthy control (GG at the Nkx2-3 SNP rs10883365). mRNA and protein expression levels of Nkx2-3

Discussion

Genetic, infectious, and immunologic factors have all been implicated in influencing the development of IBD. B cells play an important role in human gastrointestinal health by producing IgA and IgM, which helps to protect the intestinal epithelial barrier [19]. IBD is associated with a reduction in J-chain expression, which leads to reduced secretory dimeric IgA, suggesting a malfunctioning of B cells [20]. Furthermore, B cells may promote pathology in UC by producing epithelial cell-specific

Acknowledgments

This work was supported by a Grant from the Philadelphia Health Care Trust (WAK), the Carlino fund for IBD research at the Milton S. Hershey Medical Center (WAK), Penn State College of Medicine, and the Milton S. Hershey Penn State College of Medicine Surgery Research Feasibility Grant (Z.L.).

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