Systemic lupus erythematosus (SLE) is an autoimmune disease with a multifactorial etiology that is characterized by impaired T cell responses and dysregulation of B cell activation, leading to B cell hyperactivity and production of autoantibodies. Several lines of evidence suggest that early T cell activation gene 1, or osteopontin (OPN), may have a role in the pathogenesis of SLE as well as other autoimmune disorders.
OPN is a 60-kd secreted phospho-protein functioning as a free cytokine in body fluids or as an immobilized extracellular matrix molecule in mineralized tissues (1, 2). OPN may influence autoimmune diseases through its immunoregulatory effects, enhancing the proinflammatory Th1 cell response and inhibiting the Th2 responses (3, 4). Moreover, OPN stimulates T cell proliferation, interferon-γ production, and CD40 ligand expression, which in turn sustains the proliferation of B cells and production of antibodies (5). In mice, transgenic overexpression of OPN on a nonautoimmune background produces an autoimmune pattern with accumulation of B1 lymphocytes, hypergammaglobulinemia, and production of autoantibodies, including anti–double-stranded DNA antibodies (6). In humans, the serum level of OPN is increased in patients with SLE (7), and lupus nephritis is associated with increased OPN expression in renal tissue (8, 9). A synonymous sequence variation in exon 7 (Ala236Ala) of the OPN gene was significantly associated with SLE in a sample of 81 American patients with SLE (10).
A pathogenetic role of OPN has been strongly suggested in MRL-lpr/lpr mice (11–14) and in patients affected by the autoimmune lymphoproliferative syndrome (ALPS) (15); in both mice and patients with ALPS, an autoimmune pattern partially similar to SLE develops, associated with hypergammaglobulinemia, lymphadenopathy and/or splenomegaly, and peripheral expansion of CD4/CD8 double-negative T cells. In both mice and humans, the disease has been attributed to inherited mutations targeting the function of the Fas death receptor involved in switching of the immune response (16, 17). Several studies have suggested that high OPN levels contribute to the disease. Observations in mice revealed that 1) CD4/CD8 double-negative T cells constitutively expressed high levels of OPN, and this elevation of OPN coincided with the appearance of immunologic abnormalities (11–13), and 2) onset of polyclonal B cell activation was delayed by crossing MRL-lpr/lpr with opn−/− mice (13, 14), while observations in humans demonstrated that 3) high OPN levels directly correlated with hypergammaglobulinemia (15), and 4) 2 SNPs in the OPN 3′-untranslated region (3′-UTR) (+1083A/G and +1239A/C) were associated with high production of OPN and increased the risk of ALPS by 8-fold (15). Intriguingly, families of ALPS patients display an increased frequency of several common autoimmune diseases, including SLE, which suggests that they carry an autoimmune-prone genetic background.
The present study sought to test the involvement of OPN in SLE. To this purpose, the coding 5′ and 3′ flanking regions of the OPN gene were screened for sequence variations in SLE patients. The identified SNPs were tested for an association with SLE in a large panel of Italian patients and controls. The frequencies of the associated SNPs were then compared in patient subsets subdivided according to their clinical and immunologic features. Finally, the association of SLE-associated OPN genotypes with OPN serum production was investigated.
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- PATIENTS AND METHODS
The association of OPN gene polymorphisms with SLE susceptibility was tested in a large group of Italian patients and controls. Sequence variations were searched by a systematic screen of the coding regions as well as the 3′-UTR and 2,083 bp of the 5′ flanking OPN regions in 23 patients. This search was designed to ensure that potentially causative variants were considered among those tested, given that the SNPs available in the public database and in the literature may not include those specifically relevant to SLE. A total of 13 SNPs were detected, of which 5 are described for the first time herein. Six SNPs (none of which have been previously tested for association with SLE) were selected for further testing in a large panel of Italian patients.
Two sequence variations located in the 5′ flanking region (−156G/GG) and in the 3′-UTR (+1239A/C) were significantly associated with the disease. SNP at position +1239 was in perfect linkage disequilibrium with the synonymous variation in exon 7 (Ala236Ala), which was associated with SLE in a group of 81 American individuals (10). Thus, the previously reported result has been confirmed in a larger and different population.
A multivariate analysis showed that the effect on SLE susceptibility of the 2 SNPs in the 5′ and 3′ regions was independent of each other, i.e., not a consequence of linkage disequilibrium between them. Moreover, individuals carrying susceptibility alleles for both SNPs had a higher risk of developing SLE than did individuals carrying susceptibility alleles only at position −156 (Table 6): the risk associated with homozygosity for susceptibility alleles at both SNPs was 1.7-fold higher relative to homozygosity only at −156, and almost 4-fold higher relative to the complete absence of susceptibility alleles. These data suggest a synergism of the 2 sequence variations for SLE susceptibility, which is consistent with a growing number of other examples reported in the literature (31, 32), and indicates that both variations should be tested in any study investigating the role of OPN in disease etiology. The remaining haplotype background does not seem to influence the effect of these 2 SNPs.
The perfect linkage disequilibrium between alleles at positions −156, −1748, and intron 1 as well as between +1239, +282, and +750 does not allow us, in the absence of functional data, to determine which, if any, of these sequence variations is primarily involved in SLE susceptibility. The polymorphism in intron 1 does not seem to have a functional relevance, according to recently published observations (30). The SNPs in the 5′ flanking region may affect the transcription capability of the OPN promoter by a differential binding of transcription factors. Recent evidence suggests that position −156 falls in a putative binding site for a component of the RUNX family of transcription factors (33). Interestingly, SNPs in RUNX binding sites in 3 different genes were recently associated with 3 autoimmune diseases, namely SLE, psoriasis, and rheumatoid arthritis, suggesting an important role for this family of DNA binding proteins in autoimmunity (34).
As for the 3 associated SNPs in the 3′ part of the OPN gene, 2 of them, namely, +282T/C and +750C/T, are synonymous variations located in exons 6 and 7, respectively. Synonymous variations that modify exonic splicing enhancer or exonic splicing silencer sites have been reported to be causative mutations of genetic diseases (35). However, the involvement of OPN exon 6 and exon 7 synonymous SNPs in this mechanism seems unlikely, since the 2 known alternatively spliced isoforms of human OPN involve exons 4 and 5 (36).
The third SNP (+1239A/C) is located in the 3′-UTR region. This region is strongly implicated in the regulation of gene expression because it specifically controls stability, translational activity, and nuclear export of messenger RNA (mRNA) (37–39). Sequence variations in the 3′-UTR may affect some of these functions and can be causative mutations of genetic diseases (38, 39). Interestingly, even if this SNP is not part of sequences known to bind regulative factors, it falls in an 18-bp sequence conserved in the human, bovine, ovine, and porcine OPN 3′-UTR, suggesting a possible functional role of this region. Preliminary mRNA quantitative analysis performed in 3 individuals heterozygous for +1239A/C and homozygous for all of the promoter SNPs showed that mRNA carrying the SLE-associated allele (+1239C) was 4.4-fold more expressed than mRNA carrying +1239A. These data suggest a possible influence of this SNP in the control of mRNA stability. Accordingly, the +1239C allele was significantly associated with a higher serum protein level in healthy controls, directly relating OPN levels to this OPN gene variation and to SLE susceptibility. Conversely, no association with the baseline protein serum level was detected for position −156.
Thus, the mechanism by which this polymorphism contributes to SLE susceptibility is less clear. We can speculate that it might be relevant in the regulation of OPN production in response to the initial immunostimulating trigger. The fact that neither SNP was correlated with the protein level in SLE patients could be explained by an overwhelming effect of the immune activation in the patients following the initial trigger.
Intriguingly, the −156 susceptibility allele (−156G) was also associated, albeit with borderline significance (Pcorr = 0.046), with development of lymphadenopathy in SLE patients. This pattern recalls the characterization of ALPS as involving autoimmunities partly similar to SLE and lymphadenopathy, and as displaying high OPN levels and association with the +1239C allele (the −156 SNP was not evaluated in ALPS patients ). Since OPN favors proliferation and inhibits death of lymphocytes in vitro (15), it was suggested that OPN acts in synergy with the inherited Fas defect, which is considered the main cause of ALPS. This synergy may favor lymphocyte accumulation in the secondary lymphoid tissues and development of autoimmunity. A similar synergy might also work in SLE patients, who do not carry inherited defects of Fas function, but rather, overproduce a soluble form of Fas that may inhibit Fas function (40). Alternatively, OPN might act in synergy with other apoptosis defects not identified to date in SLE.
In summary, these data strongly suggest that OPN genetic variations have a key role in building up an autoimmune-prone background favoring lymphocyte accumulation in peripheral lymphoid tissues and leading to the development of autoimmunity. For at least one of these genetic variations, an association with increased OPN levels was demonstrated. OPN may exert its preferential effect through its capacity to stimulate proliferation and inhibit death of lymphocytes (15, 41) or through its capacity to modulate the immune response by inducing Th1 responses and potentiating polyclonal activation of B cells (3–5).