A functional polymorphism in the Eta-1 promoter is associated with allele specific binding to the transcription factor Sp1 and elevated gene expression
Introduction
In 1989 investigations of gene expression in activated CD4+ cells identified a cytokine termed early T lymphocyte activator 1 (Eta-1) as the predominant transcript after bacterial infection (Forton et al., 2002, Patarca et al., 1989). The sequence was identical to the bone protein, Osteopontin. Eta-1 is a secreted phosphorylated glycoprotein produced by activated macrophages and may constitute the most abundant molecule secreted by activated T cells. It enhances Th1 and inhibits Th2 development by inducing macrophages to produce IL-12 and INF-γ, while inhibiting their IL-10 expression (Ashkar et al., 2000). In addition, Eta-1 increases CD3-mediated T cell production of INF-γ and CD40L, which augments the IL-12 production by human monocytes (O’Regan et al., 2000). Eta-1 contains a Gly-Arg-Gly-Asp-Ser (GRGDS) encoding motif, which promote cell attachment via the CD44 and αvβ1,3 integrins (Hu et al., 1995, Weber et al., 1997).
The Eta-1 gene is located on the human chromosome 4q13 (Young et al., 1990) and contains seven exons. In mice, the gene is located on chromosome 5 and maps to the locus of genetic resistance to the intracellular infection caused by Rickettsia tsutsugamushi (Ricr locus), an obligate intracellular bacterium that is the etiological agent for human scrub typhus (Patarca et al., 1993). Resistance to infections strongly depend on the presence of a particular Eta-1 allele differing from the other in at least ten amino acids (Ono et al., 1995). Inbred mouse strains expressing the Eta-1a allele are resistant to infection, whereas inbred mouse stains that express the Eta-1b allele are susceptible (Patarca et al., 1989). Furthermore, Eta-1 knock-out mice fail to respond against infections characterized by Th1 response (Ashkar et al., 2000) and show a five-fold reduction in macrophage infiltration after renal injury compared with wild-type controls (Ophascharoensuk et al., 1999).
Recently, Eta-1 was found to be expressed 19-fold higher in Th1 cells compared with Th2 cells (Nagai et al., 2001). This strongly suggests that Eta-1 could be involved in the polarization of an early Th1 cytokine response and thereby the T cell-dependent response to bacterial infection.
Several polymorphisms in the Eta-1 gene have been described, and some of those have been reported to be associated with systemic lupus erythematosus, multiple sclerosis, urolithiasis, primary biliary cirrhosis, and autoimmune lymphoproliferative syndrome (ALPS) (Forton et al., 2002, Yamate et al., 2000, Kikuchi et al., 2003, Niino et al., 2003, Chiocchetti et al., 2004, D’Alfonso et al., 2005).
In the present study, we screened for promoter polymorphisms of the Eta-1 gene and luciferase- and EMSA-assays were performed to elucidate the potential transcriptional effect of the detected polymorphisms. During the preparation of our manuscript, Giacopelli et al. (2004) reported observations on related studies of the same gene, but their results are at variance with ours concerning the influence of promoter polymorphisms in positions −66, −156, and −443 on gene expression. In addition, we have crucial information concerning a haplotype not studied by Giacopelli et al. (2004).
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Subjects
Peripheral venous blood samples were obtained from 104 healthy individuals and genomic DNA was prepared using the QIAamp® DNA Blood Maxi Kit (QIAGEN). All individuals included were unrelated Danish Caucasians. The Local Ethics Committee of Copenhagen, Denmark, approved the study protocol.
Screening the Eta-1 promoter for SNPs
The promoter region of the Eta-1 gene of 21 individuals, spanning from position −2267 to +52 related to the first base in exon 1, was amplified by PCR in six overlapping fragments (Table 1). PCR were carried
SNPs in the Eta-1 gene
A total of six nucleotide differences were detected in the human Eta-1 promoter spanning from position −2267 to +52, five substitutions in position −1776, −1748, −616, −443, and −66, and one deletion in position −156. SSP-PCR genotyping was used to genotype the −1748G/A, −616T/G, −443T/C, −156delG/G, and −66T/G SNPs in 104 normal healthy Caucasian. The genotype distributions of the promoter polymorphisms are all frequent in normal individuals (Table 3). None of the genotype distributions
Discussion
Eta-1 has been defined as a Th1 cytokine, which is expressed 19-fold higher in Th1-cells compared with Th2-cells (Nagai et al., 2001). It enhances a Th1 response by inducing macrophages to produce IL-12 and INF-γ, and by inhibiting their IL-10 expression (Ashkar et al., 2000). This strongly suggests that Eta-1 could be involved in the polarization of an early Th1 cytokine response. Genetic polymorphisms of Eta-1 might influence the level of expression and thus the balance between
Acknowledgements
The Danish Medical Research Council supported these studies as part of the Danish Allergy Research Centre (DARC).
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2015, GeneCitation Excerpt :For example, several studies demonstrated that SPP1 mRNA expression was enhanced in osteoarthritic chondrocytes as compared with normal cells (Pullig et al., 2000), and that SPP1 protein levels in plasma and synovial fluid were also positively correlated with disease severity of knee osteoarthritis (Honsawek et al., 2009). Recently, it has been reported that three functional polymorphisms rs17524488, rs11730582 and rs28357094 (also known as − 156delG>insG, − 443T>C and − 66T>G respectively) in the promoter region of SPP1 gene can influence transcriptional activity of this gene (Giacopelli et al., 2004; Hummelshoj et al., 2006), and that these genetic variants result in an increased risk for several kinds of diseases such as systemic lupus erythematosus (D'Alfonso et al., 2005), systemic sclerosis (Barizzone et al., 2011) and cancers (Schultz et al., 2009; Chiu et al., 2010). Based on these evidences, we proposed that functional polymorphisms of rs17524488, rs11730582 and rs28357094 in SPP1 gene promoter might be involved in the development of hip OA as well.
The implication of osteopontin (OPN) expression and genetic polymorphisms of OPN promoter in oral carcinogenesis
2010, Oral OncologyCitation Excerpt :These two polymorphic sites at −156 and −443 were reconstructed and showed linkage disequilibrium. The haplotype of −443C/−156GG/−66T is the least proportion in whole population which is similar to previous study on healthy Italian individuals.7 Our data reveals that haplotype frequency of −443T/−156GG/−66T and −443C/−156G/−66T in OSCC was 1.86 folds higher than controls.
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Nucleotide sequence: accession number: D14813, SNPs: have been submitted to GenBank (NCBI).