Nkx2-3 Regulates Madcam1 And Vcam1 Expression In Human Intestinal Microvascular Endothelial Cells

NKX2-3 (NK2 transcription factor related, locus 3) has been shown to be associated with both Crohn's disease (CD) and ulcerative colitis (UC) by recent genome-wide association studies. NKX2-3 is a member of the Nkx family of homeodomain transcription factors that play critical roles in regulating tissue-specific gene expression essential for determining tissue differentiation. During development, NKX2-3 is primarily expressed in the midgut and hindgut mesoderm and spleen, as well as in pharyngeal endoderm. Analysis of NKX2-3-deficient mice has revealed a critical role for this homeobox transcription factor in spleen development and organization, and in establishing the correct environment for normal B cell development and T cell dependent immune response. NKX2-3 is also expressed in microvascular endothelial cells within the lamina propria and submucosa of the intestine, where it is required for expression of the lymphocyte adhesion molecule MAdCAM-1 in the mouse. Cell adhesion molecules (CAMs) are proteins located on the cell surface involved with the binding with other cells or with the extracellular matrix (ECM) in the process celled cell adhesion. Human intestinal microvascular endothelial cells (HIMEC) are recognized as a cell population actively involved in the pathogenesis of inflammatory bowel diseases (IBD) and IBDassociated microvascular dysfunction. Microvascular endothelial cells have a critical “gatekeeper” role in the inflammatory process through their ability to recruit circulating immune cells to foci of inflammation. Endothelial activation in response to cytokines and bacterial products results in cell adhesion molecule expression and chemokine production, which mediate increased binding and transmigration of leukocytes across the vascular wall. This process is medicated by leukocyte-endothelial adhesion molecules. To understand the cellular function of NKX2-3 and its potential role underlying IBD pathogenesis, we investigated a role of NKX2-3 in the regulation of MAd-CAM1 and VCAM1 expression in siRNA knockdown HIMECs. 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. The transcriptional profile of genes affected by NKX2-3 knockdown was highly consistent in the two HIMEC lines. The top 100 downregulated and top 100 up-regulated genes by NKX2-3 knockdown are mainly involved in immune and inflammatory response, cell proliferation and growth, metabolic process, and angiogenesis. G-Protein Coupled Receptor Signaling (p=1.71×10-14), Axonal Guidance Signaling (p=5.80×10-11), and ERK/MAPK Signaling (p=5.42×10-9) were the top three canonical pathways affected by NKX2-3 knockdown. 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. Expressions of MAdCAM1 and VCAM1 were decreased in two HIMEC lines detected by cDNA microarray. mRNA expression levels of MAdCAM1 and VCAM1 were decreased in two NKX2-3 knockdown HIMEC lines compared with control HIMEC lines by RT-PCR analysis. Leukocyteendothelial adhesion assays show that NKX2-3 can affect leukocyte-endothelial adhesion. Leukocyte-endothelial adhesion was decreased in two NKX2-3 knockdown HIMEC lines compared with control HIMEC lines. NKX2-3 may play an important role in IBD pathogenesis by regulating MAdCAM1 and VCAM1 expression and leukocyte-endothelial adhesion in HIMEC.