Transcription Factor SP2 Enhanced the Expression of Cd14 in Colitis-Susceptible C3H/HeJBir.

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

<i>Cd14</i> promoter activity after site-directed mutagenesis.

<p><b>A</b> After inactivation of OCT1, Nrf2 and proximal AP1 binding sites in the B6 allele reduced activities were detected while in promoter constructs lacking Stat1/Cdx2 and SP1 led to complete transcriptional silencing. <b>B</b> In the C3Bir allele deletion of AP1 and SP2 binding sites resulted in significant transcriptional decrease, while after the mutation of SP1 only a slight reduction of Luciferase expression was seen. ***: P < 0,05.</p

Transfection system establishment.

<p><b>A</b> Western blot analysis of CD14 expression in untreated and LPS stimulated murine RAW264.7 macrophages and NIH/3T3 fibroblasts, β-actin as endogeous control. Transfection efficiency test by determination of GFP-positive RAW264.7 cells by cyto fluorescence 24 hours after transfection with <b>B</b> Xtreme Gene HP, <b>C</b> Xtreme Gene 9 DNA, <b>D</b> Superfect, <b>E</b> Polyfect, and <b>F</b> by β-galactosidase assay, ***: p < 0,05.</p

Physical interaction of SP2 with the Cd14 promoter derived from C3Bir.

<p><b>A</b> SP2 electrophoretic mobility shift was performed running a biotin-labelled Sp2 consensus oligonucleotide alone (lane 1) or incubated with a nuclear extracts isolated from RAW264.7 cells (lane 2). In the latter two complex formations (I and II) from the Sp2 consensus oligo with nuclear extract components were detected. Competition of increasing amounts of unlabelled SP2 oligo for binding to the nuclear extract proteins led to a stepwise disappearance of biotin-Sp2-oligo protein complexes (lane 3–5). After pre-incubation of the nuclear extracts with an SP2 specific antibody new complexes (III-V) were established in this supershift assay demonstrating physical interaction of the SP2 protein with its consensus oligo (lane 6). Specific SP2 DNA complex formation was also demonstrated by the failure of SP1 and SP3 antibodies to bind to the protein DNA complexes (lane 7 and 8). <b>B</b> BMMs after four days of culture. The scale bar represents 10μm at a 10x magnification. <b>C</b> The MMULV-mediated overexpression of mouse SP2 in primary BMMs derived from C3Bir mice correlated with a higher Cd14 transcription rate compared to untreated and control virus infected macrophages. ***: p<0.01; ** p< 0.05</p

Patterns of transcription factor binding sites associated with inflammation and colitis.

<p>Bioinformational analysis of the <i>Cd14</i> alleles from the B6 and the C3Bir strain revealed striking changes in transcription factor binding sites caused by SNPs. AP1 and Sp1 sites were common in both alleles contributing to the control of <i>Cd14</i> expression in mice; +1: translation start site; grey boxes: exons 1 and 2.</p

Truncations of the <i>Cd14</i> promoter alleles.

<p>Constructs of the 5’ truncated <i>Cd14</i> promoter were cloned in the pGL4.17 reporter plasmid and transfected in RAW264.7 cells. <b>A</b> The strong luciferase activity increase after removal of the distal 100 bp and of the sequence between -474 and -385 in the B6 allele indicated inhibitory elements in these regions. Activating binding motifs were identified through the drop of luciferase activity after cutting of the -844 to -722 and -298 to -181 regions. <b>B</b> Stepwise truncation of the C3Bir allele revealed significant expression peaks after removal of the -474 to -385 region and the -181 to -52 sequence while the -298 to -181 and the -52 to +85 regions seemed to contain stimulating binding motifs. ***: P < 0,05.</p