Elsevier

Molecular Immunology

Volume 43, Issue 7, March 2006, Pages 980-986
Molecular Immunology

A functional polymorphism in the Eta-1 promoter is associated with allele specific binding to the transcription factor Sp1 and elevated gene expression

https://doi.org/10.1016/j.molimm.2005.05.012Get rights and content

Abstract

Early T lymphocyte activator 1 (Eta-1), also known as Osteopontin, is a cytokine produced by macrophages and T lymphocytes. It is involved in the regulation of IL-12 and IL-10 expression in macrophages and stimulates the polarization of T cells to the Th1 subset. Three promoter polymorphisms of the human Eta-1 gene, −443T/C, −156delG/G, −66T/G, were investigated for possible influence on gene expression. Electrophoretic mobility shift assays (EMSA) with nuclear extract from the human myeloid leukaemia premonocyte cell line, THP-1, revealed sequence specific binding of the transcription factor Sp1 to the −66T allele but not the −66G allele, and haplotype −443C/−156G/−66T showed a marked increase in promoter activity of a luciferase reporter gene. Thus, a substitution of the T-base with G at position −66 in the Eta-1 promoter modulates the promoter activity of the Eta-1 gene, which might influence the Th1 versus Th2 balance. These observations are discussed in relation to a recently reported related observation on the same gene, and it is argued that discrepancies between reporter gene assays in the two studies may be due to the use of different cell lines and may reflect requirements for different transcription factors in cells involved in immune responses compared with other cells.

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).

Section snippets

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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    Nucleotide sequence: accession number: D14813, SNPs: have been submitted to GenBank (NCBI).

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