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Abstract
Genetic analysis in the IL10-deficient mouse model revealed a modifier locus of experimental inflammatory bowel disease (IBD) on chromosome 18, with the allele of the strain C3H/HeJBir (C3Bir) conferring resistance and the allele of C57BL/6J (B6) conferring susceptibility. Differential Cd14 expression was associated with this background specific susceptibility to intestinal inflammation. Polymorphisms of the Cd14 promoter were found to be likely causative for strain specific expression, and Cd14-knockout mice revealed a protective role of this gene-product in experimental IBD. In this study, luciferase reporter assays confirmed an increased activity of the C3Bir derived Cd14 promoter compared to the one of B6. Promoter truncation experiments and site-directed mutagenesis in both strains resulted in reduced Cd14 promoter activity and confirmed that a central AP1 and the proximal SP1 transcription factor binding sites mediated the basal activity of the Cd14 promoter in the mouse. Moreover, a T to C exchange at position -259 replaced putative STAT1 and CDX1 sites in the Cd14 promoter from B6 by a SP2 site in C3Bir. Ablation of the Sp2 site through truncation was associated with a decreased promoter activity. Site-directed mutagenesis also demonstrated that the inactivation of SP2 led to a substantial loss of promoter activity in C3Bir. Performing electrophoretic mobility shift and supershift assays demonstrated interaction of SP2 with its potential binding site. In addition, retroviral—mediated overexpression of the SP2 transcription factor in primary bone marrow macrophages derived from C3Bir mice caused a significant increase in Cd14 transcription. These data characterized SP2 as important factor responsible for higher Cd14 expression and reduced IBD susceptibility mediated by the C3Bir allele.
Highlights
CD14 was identified as a marker and modifier gene in human inflammatory bowel disease (IBD) [1,2,3] and found to be associated to experimental IBD in the IL10-deficient mouse model [4,5,6,7]
Three AP1 and a single SP1 recognition site in close proximity to the transcription start site were identified. These results confirmed the work of Matsuura and colleagues who localized an AP1 binding elements in the Cd14 promoter derived from BALB/c mice identical to AP1 site at -367 to -356 in the B6 and C3H/ HeJBir (C3Bir) alleles [16,19]
AP1 binding sites were found in rat, cattle and human CD14 promoter controlling the basic promoter activity like in the mouse [17,18]
Summary
CD14 was identified as a marker and modifier gene in human IBD [1,2,3] and found to be associated to experimental IBD in the IL10-deficient mouse model [4,5,6,7]. CD14 is synthesized in a soluble (sCD14) and a membrane bound (mCD14) form. Together with lymphocyte antigen 96 and toll like receptor 4 (TLR4) mCD14 forms a receptor for lipopolysaccharides (LPS); sCD14 circulates as a plasma protein due to a missing GPI anchor which is either not synthesized or it is post-translational detached by matrix metalloproteinase [9,10]. Less is known about the molecular mechanisms controlling expression and post-translational editing of soluble or membrane bound CD14. Several AP1 binding site were identified in the CD14 promoter in cow, mouse, rat and man mediating basal transcription activity [16,17,18,19]. The tantamount polymorphism C-260T/C-159T was found in patients with Crohn’s disease and ulcerative colitis [32,33,35,36,37]
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