Autoimmunity
ISSN: 0891-6934 (Print) 1607-842X (Online) Journal homepage: http://www.tandfonline.com/loi/iaut20
Association of OSMR Gene Polymorphisms with Rheumatoid Arthritis and Systemic Lupus Erythematosus Patients Yuan-Zhao Lin, Ruei-Nian Li, Chia-Hui Lin, Tsan-Teng Ou, Cheng-Chin Wu, Wen-Chan Tsai, Hong-Wen Liu & Jeng-Hsien Yen To cite this article: Yuan-Zhao Lin, Ruei-Nian Li, Chia-Hui Lin, Tsan-Teng Ou, Cheng-Chin Wu, Wen-Chan Tsai, Hong-Wen Liu & Jeng-Hsien Yen (2014) Association of OSMR Gene Polymorphisms with Rheumatoid Arthritis and Systemic Lupus Erythematosus Patients, Autoimmunity, 47:1, 23-26, DOI: 10.3109/08916934.2013.849701 To link to this article: https://doi.org/10.3109/08916934.2013.849701
Published online: 12 Nov 2013.
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http://informahealthcare.com/aut ISSN: 0891-6934 (print), 1607-842X (electronic) Autoimmunity, 2014; 47(1): 23–26 ! 2014 Informa UK Ltd. DOI: 10.3109/08916934.2013.849701
RESEARCH ARTICLE
Association of OSMR Gene Polymorphisms with Rheumatoid Arthritis and Systemic Lupus Erythematosus Patients Yuan-Zhao Lin1*, Ruei-Nian Li2*, Chia-Hui Lin1, Tsan-Teng Ou3, Cheng-Chin Wu3, Wen-Chan Tsai1,3, Hong-Wen Liu3, and Jeng-Hsien Yen1,3 1
Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan, 2Department of Biomedical Science and Environmental Biology, and 3Division of Rheumatology, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan Abstract
Keywords
Cytokines are involved in the pathogenesis of autoimmune diseases. Oncostatin M receptor (OSMR) activates JAK/STAT and MAPK pathways leading to the stimulation of a variety of cytokines and inflammatory substances. Many pro-inflammatory cytokines are involved in the inflammatory process of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). In this study, we carried out experiments to examine the relationship of OSMR promoter polymorphisms with RA and SLE patients. 241 patients of RA, 143 patients of SLE and 203 healthy controls were enrolled in their recruitment from the Kaohsiung Medical University Hospital. Genomic DNA was extracted from peripheral blood mononuclear cells and gene polymorphism was genotyped by TaqMan real-time polymerase chain reaction. The OMSR promoter region 100 G/T (rs22922016) genotype was in no relation to the susceptibility of RA, but 100 T/T (rs22922016) genotype could prevent the patients with sicca syndrome and the existence of anti-Ro antibodies. In contrast, the 100 G/TþT/T (rs22922016) genotypes were significantly associated with an increased risk of SLE (odds ratio, OR¼1.62, 95% confidence interval (CI), 1.01–2.62). 94.38% of SLE patients with arthritis were belonged to the 1687C/C (rs540558) genotype. The T allele of promoter region 100 T/T (rs22922016) has protective effect and could ameliorate the disease condition in RA patients, whereas the same T allele was a risk allele in the susceptibility of SLE. The disease severity of rheumatoid arthritis and systemic lupus erythematosus can be partially affected by the OSMR promoter polymorphisms.
Oncostatin M receptor, rheumatoid arthritis, systemic lupus erythematosus, gene polymorphism, SNP
Oncostatin M (OSM) is a multifunctional cytokine and is mainly produced by T cells and monocytes [1]. OSM is a member of interleukin-6 family which includes IL-6, IL-11, leukemia inhibitory factor (LIF), cardiotropin-1 (CT-1) and ciliary neurotrophic factor (CNTF) [2]. These cytokines are well known for their roles in controlling immune responses, inflammatory mediators, cell proliferation and they are involved in the development of aberrant inflammation. These cytokines share common receptor subunits. OSM has two types of receptors, gp130 with LIF binding protein (gp130/LIFR) and gp130 with OSM-specific receptor (gp130/ OSMR) [2,3]. When OSM binds to gp130 subunit, it triggers the formation of heterodimer of receptors and activates the Janus kinases family JAK1 and JAK2 [4]. The JAKs enzymes
*These authors contributed equally to this work. Correspondence: Jeng-Hsien Yen, Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan. E-mail: [email protected]
Received 16 July 2013 Revised 29 August 2013 Accepted 25 September 2013 Published online 12 November 2013
then phosphorylate the STATs, which are subsequently transported to the nucleus and bind to their cognate DNA binding sites. Activated-STATs bind to target DNA and regulate a variety of genes which are often associated with inflammatory reactions [5,6]. OSM is highly expressed in synovial fluid of rheumatoid arthritis (RA) patients, stimulates synovial fibroblast proliferation and production of cytokines to recruit inflammatory cell during disease progression [1,7,8]. High concentration of OSM may elicit the inflammation responses in joints and leading to bone erosion. OSM has been reported that it is detected in the serum of 9.4% of SLE patients [9]. These results indicated that OSM and OSMR are in relation to autoimmune diseases. SLE is a complex systemic autoimmune disease and the detailed etiology is still unclear. It is involved in multiple organs and systems. RA is a chronic inflammation disease that may involve extra-articular organs in addition to joints [10]. Many pro-inflammatory cytokines, like IL-6, IL-8, IL-18 and TNF are involved in the inflammatory process of autoimmune diseases [11–13]. Therefore, in this study, we have carried out experiments to examine the relationship of OSMR promoter polymorphisms with RA and SLE patients.
20 13
Introduction
History
24
Y.-Z. Lin et al.
Autoimmunity, 2014; 47(1): 23–26
Methods
analysis was conducted by the IBM SPSS Statistics version 19.
Clinical subjects Two hundred and forty-one patients of RA, 143 patients of SLE and 203 healthy controls were enrolled in their recruitment from the Kaohsiung Medical University Hospital. The diagnosis of RA was according to the American Rheumatism Association 1987 revised criteria for the classification of RA. The diagnosis of SLE and all patients were fulfilled the revised criteria of American College Rheumatology 1997 for the classification of SLE. This study was approved by the Institutional Review Board of Kaohsiung Medical University Hospital (KMUH). Genomic DNA and total RNA extraction Peripheral blood mononuclear cells (PBMC) were collected by Ficoll-paque (GE Healthcare, Little Chalont, UK) purification and genomic DNA was extracted from PBMC of collected patients from KMUH. Total RNA are extracted by the method of QIAmp RNA Blood Mini Kit (Qiagen, Hilden, Germany). RNA is reversely transcribed to cDNA with random primers and high-capacity cDNA Archive kit (Applied Biosystems, Life Technologies, Carlsbad, CA). SNP genotyping and data analysis Two different polymorphic sites on the OSMR promoter region 1687C/G (rs540558) and 100 G/T (rs22922016) were genotyped. The OSMR SNP genotyping was performed by TaqMan SNP Genotyping Assays (Applied Biosystems, Life Technologies, Carlsbad, CA) by real-time polymerase chain reaction (PCR) with ABI 7500 Real-Time PCR systems. The odds ratio (OR) and its 95% confidence interval (CI) were calculated to evaluate the risk of disease. The statistical
OMSR RNA expression A total of 30 RA patients, 30 SLE patients and 30 control individuals were assayed for the OMSR expression. The OMSR RNA expression was performed by the TaqMan Gene Expression Assay (Applied Biosystems, cat. no. 4369016) and real-time PCR reaction mix (Applied Biosystems, cat. no. 4369016). The reverse transcription was at 50 C for 2 min, PCR condition was at 95 C for 10 min, 95 C for 10 s, 60 C for 1 min (repeat 40 cycles) on ABI 7500 Real-Time PCR systems. H1299 is a lung cancer cell line which serves as a positive control, and RNA polymerase II is the input control [14].
Results 100 T/T genotype ameliorates the rheumatoid arthritis syndrome Previous study has demonstrated that the OSM and OSMR were associated with the susceptibility to autoimmune diseases [4,9,15]. Therefore, in this study, we have carried out experiments to determine two different polymorphic sites on the OSMR promoter region in RA and SLE patients. The genotypes of OMSR promoter sequence 1687C/G and 100 G/T were not found in relation to the susceptibility of patients with RA (Table 1). Moreover, in clinical manifestation, the genotype 100 G/T was associated with sicca syndrome and anti-Ro/SSA antibodies but not 1687C/G (Table 2). The odds ratio of 100 T/T genotype with sicca syndrome was 0.28 (95%CI, 0.12–0.69), and 9.1% of RA patients exhibited sicca syndrome with 100 T/T genotype
Table 1. Genotypes frequencies of OSMR promoter in RA and SLE patients. RA (N ¼ 241)
Control (N ¼ 203)
N(%)
N(%)
OSMR Genotype
SLE (N ¼ 143)
Control (N ¼ 203)
N(%)
N(%)
p Value
OR (95% CI)
p Value
1687C/G (rs540558) C/C C/G G/G C/GþG/G C/CþC/G
221 19 1 20 240
(91.7%) (7.88%) (0.41%) (8.3%) (99.59%)
182 21 0 21 203
(89.66%) (10.34%) (0%) (10.34%) (100%)
NS NS NS NS NS
127 15 1 16 142
(88.81%) (10.49%) (0.70%) (11.19%) (99.3%)
182 21 0 21 203
(89.66%) (10.34%) (0%) (10.34%) (100%)
– – – – –
NS NS NS NS NS
100 G/T (rs22922016) G/G G/T T/T C/TþT/T G/GþG/T
76 117 48 168 193
(31.54%) (48.55%) (19.92%) (68.46%) (80.08%)
70 98 35 133 168
(34.48%) (48.28%) (17.24%) (65.52%) (82.76%)
NS NS NS NS NS
35 77 31 108 112
(24.48%) (53.85%) (21.68%) (75.52%) (78.32%)
70 98 35 133 168
(34.48%) (48.28%) (17.24%) (65.52%) (82.76%)
– 0.89 (0.50–1.58) 1.77 (0.94–3.33) 1.62 (1.01–2.62) –
NS 0.69 0.08 0.046 NS
OR: odds ratio. CI: confidence interval. NS: no significant.
Table 2. Genotypes frequencies of OSMR promoter in RA patients and controls and stratified by sicca syndrome or anti-Ro antibodies. Sicca syndrome OSMR Genotype 100 G/T (rs22922016) G/GþG/T T/T
Anti-Ro antibodies
þ N(%)
N(%)
OR (95% CI)
p Value
þ N(%)
N(%)
OR (95% CI)
p Value
70 (90.9%) 7 (9.1%)
82 (73.88%) 29 (26.12%)
1 0.28 (0.12–0.69)
0.0004
47 (92.16%) 4 (7.84%)
91 (75.2%) 30 (24.8%)
1 0.26 (0.09–0.78)
0.01
OMSR gene polymorphisms with autoimmune diseases
DOI: 10.3109/08916934.2013.849701
(Table 2). Whereas, 70 out of 152 RA patients of genotypes 100 G/G and 100 G/T presented sicca syndrome. The odds ratio of 100 T/T genotype with anti-Ro antibodies was 0.26 (95% CI, 0.09–0.78) (Table 2). The presence of anti-Ro antibodies existed only in 4 (7.84%) of 100 T/T genotype RA patients (Table 2). We observed that the OMSR promoter region 100 T/T genotype could prevent rheumatoid arthritis patients with sicca syndrome and the existence of anti-Ro antibodies. T allele of 100 G/T is associated with SLE The genotype 1687C/G was not found in relation to SLE patients (Table 1). However, the odds ratio of 100 T/ T genotype was notably associated with the susceptibility of SLE (OR ¼ 1.77, 95% CI, 0.94–3.33), but without statistical significance. On the contrary, the 100 G/TþT/T genotypes were significantly associated with an increased risk of SLE (OR ¼ 1.62, 95% CI, 1.01–2.62) (Table 1). This result indicated that T allele was a risk allele in the susceptibility of SLE. 1687C/C genotype is associated with arthritis syndrome in SLE In clinical manifestation, the genotype 1687C/C was found in association with arthritis syndrome in SLE patients but not 100 C/GþG/G (Table 3). The percentage of 1687C/C genotype was 94.38% in SLE patients with arthritis. The odds ratio of 1687C/G and 1687G/G was 0.18 (95% CI, 0.04–1.58) (Table 3). We have performed the TaqMan realtime polymerase chain reaction to evaluate the RNA expression of OSMR gene in RA and SLE patients. However, OSMR gene expression was not detected in RA and SLE patients (Table 4).
Discussion We have investigated two different polymorphic sites on OSMR promoter region in RA and SLE patients. The results indicated that the OSMR promoter genotypes 1687C/G and 100 G/T were found in no relation to the susceptibility with rheumatoid arthritis patients. In RA clinical outcome, there was no correlation with genotypes 1687C/G as well. Table 3. Genotypes frequencies of OSMR promoter in SLE patients and controls and stratified by arthritis. Arthritis OSMR Genotype
þ N(%)
N(%)
OR (95% CI)
1687C/G (rs540558) C/C 84 (94.38%) 12 (75%) 1 C/GþG/C 5 (5.62%) 4 (25%) 0.18 (0.04–0.76)
p Value
0.01
Table 4. The gene expression of OSMR in RA and SLE patients.
OSMR expression (2DCT) NS: no significant.
RA
SLE
Control
H1299 (Positive control)
NS
NS
NS
0.2984
25
However, we noticed that sicca syndrome was associated with 100 G/T allele in RA patients. Genotype 100 T/T of RA patients were also found in decrease concentration of anti-Ro antibodies. Anti-Ro antibodies are associated with different autoimmune diseases, such as RA, SLE and Sjo¨gren syndrome (SS) [16,17]. One of the main pathologies of SLE and SS is anti-Ro antibodies [18,19]. These results indicate that the ameliorative effect of T allele in RA patients disease condition. In contrast, the presence of G allele would cause an increased risk of sicca syndrome and the rise of anti-Ro antibodies in RA patients. Cytokines are involved in the pathogenesis of autoimmune diseases [13,15]. Oncosatin M receptor (OSMR) is the main receptor for oncostatin M (OSM), one of the IL-6 cytokine families [1]. IL-6 and OSM activates JAK1 and JAK2, which subsequently activate the activity of transcriptional factors STAT1, STAT3 and STAT5. Therefore, OMSR activates JAK/ STAT and MAPK pathways and stimulates the production of a variety of cytokines and inflammatory substances [4,6,20]. Recent study has reported that CD4 T cells secrete IL-31 which is also a ligand to OSMR [21]. 100 G/T was found in no relation with RA patients. However, T allele of 100 G/T was a risk allele to the susceptibility of SLE. Besides, OSMR promoter 1687C/G was associated with arthritis (þ) in SLE patients. The increased risk of arthritis in SLE patients is associated to the presence of C allele of the polymorphic site 1687C/G. On the other side, the haplotypes of OSMR promoter 1687C 100 G and 1687C 100 T were related to SLE (data not shown). We tried to determine the expression level of OSMR mRNA by real-time polymerase chain reaction. The result indicated that OSMR mRNA was undetected and the protein was supposed to be no expression both in RA and SLE patients. However, the promoter polymorphism shows evidence of association to the susceptibility of SLE. Alternatively, SNP can perform synergic pair effect in SNPSNP interaction [22,23]. The complicate network may interfere the expression or the activity of a second gene. The promoter site 100 of OSMR contains transcriptional factor binding sites of STAT1 and STAT3, whereas 1687 site contains STAT1 binding sequence [24]. Both STAT1 and STAT3 are the transcriptional activators of many inflammatory mediators and cytokines. The pathway of STAT1 is activated in RA as previously reported [20]. In addition, 100 site contains binding sequence for interferon regulatory factors (IRF) [25], and IRFs are important mediators in the pathogenesis of SLE [26]. This suggests that 100 T allele has a critical role in the severity of disease condition in SLE. The change of OSMR promoter sequence 1687C4G or 100 G4T in a single nucleotide would affect the transcriptional factor binding affinity, and subsequently would downregulate the promoter activity. Pre-B cell colony-enhancing factor (PBEF) is a secretory cytokine which regulates cell apoptosis and B cell development and maturation [27]. Recent report has demonstrated that the mRNA and protein of PBEF are expressed in a high concentration in synovial fluid of RA patients [28,29]. The regulation of PBEF is involved in the STAT3 dependent IL6 signal pathway [30]. Promoter polymorphism was associated with different inflammatory diseases, such as RA, SLE,
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Sjo¨gren’s syndrome [31–33]. In conclusion, we have suggested that the possible role of the OSMR promoter polymorphism affect the expression level of different cytokines and inflammatory mediators. Therefore, the disease severity of RA and SLE can be partially affected by the OSMR promoter polymorphism.
Declaration of interest No potential conflicts of interest were disclosed.
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17. Menendez, A., J. Gomez, E. Escanlar, et al. 2013. Clinical associations of anti-SSA/Ro60 and anti-Ro52/TRIM21 antibodies: diagnostic utility of their separate detection. Autoimmunity 46: 32–39. 18. Gal, I., G. Lakos, and M. Zeher. 2000. Comparison of the anti-Ro/ SSA autoantibody profile between patients with primary and secondary Sjogren’s syndrome. Autoimmunity 32: 89–92. 19. Xue, D., H. Shi, J. D. Smith, et al. 2003. A lupus-like syndrome develops in mice lacking the Ro 60-kDa protein, a major lupus autoantigen. Proc. Natl. Acad. Sci. USA 100: 7503–7508. 20. Hintzen, C., S. Quaiser, T. Pap, et al. 2009. Induction of CCL13 expression in synovial fibroblasts highlights a significant role of oncostatin M in rheumatoid arthritis. Arthritis Rheumat. 60: 1932–1943. 21. Zhang, Q., P. Putheti, Q. Zhou, et al. 2008. Structures and biological functions of IL-31 and IL-31 receptors. Cytokine Growth Factor Rev. 19: 347–356. 22. Julia, A., J. Ballina, J. D. Canete, et al. 2008. Genome-wide association study of rheumatoid arthritis in the Spanish population: KLF12 as a risk locus for rheumatoid arthritis susceptibility. Arthritis Rheumat. 58: 2275–2286. 23. Curk, T., G. Rot, and B. Zupan. 2011. SNPsyn: detection and exploration of SNP-SNP interactions. Nucleic Acids Res. 39: W444–W449. 24. van der Pouw Kraan, T. C., F. A. van Gaalen, P. V. Kasperkovitz, et al. 2003. Rheumatoid arthritis is a heterogeneous disease: evidence for differences in the activation of the STAT-1 pathway between rheumatoid tissues. Arthritis Rheumat. 48: 2132–2145. 25. Marinescu, V. D., I. S. Kohane, and A. Riva. 2005. MAPPER: a search engine for the computational identification of putative transcription factor binding sites in multiple genomes. BMC Bioinform. 6: 79. 26. Patel, D. R. 2011. Linking interferon regulatory factors to the pathogenesis of human lupus. Transl. Res.: J. Lab. Clin. Med. 157: 323–325. 27. Luk, T., Z. Malam, and J. C. Marshall. 2008. Pre-B cell colonyenhancing factor (PBEF)/visfatin: a novel mediator of innate immunity. J. Leukoc. Biol. 83: 804–816. 28. Otero, M., R. Lago, R. Gomez, et al. 2006. Changes in plasma levels of fat-derived hormones adiponectin, leptin, resistin and visfatin in patients with rheumatoid arthritis. Ann. Rheum. Dis. 65: 1198–1201. 29. Brentano, F., O. Schorr, C. Ospelt, et al. 2007. Pre-B cell colonyenhancing factor/visfatin, a new marker of inflammation in rheumatoid arthritis with proinflammatory and matrix-degrading activities. Arthritis Rheumat. 56: 2829–2839. 30. Nowell, M. A., P. J. Richards, C. A. Fielding, et al. 2006. Regulation of pre-B cell colony-enhancing factor by STAT3-dependent interleukin-6 trans-signaling: implications in the pathogenesis of rheumatoid arthritis. Arthritis Rheumat. 54: 2084–2095. 31. Harrison, P., J. J. Pointon, K. Chapman, et al. 2008. Interleukin-1 promoter region polymorphism role in rheumatoid arthritis: a metaanalysis of IL-1B-511A/G variant reveals association with rheumatoid arthritis. Rheumatology (Oxford). 47: 1768–1770. 32. Li, R. N., Y. H. Hung, C. H. Lin, et al. 2010. Inhibitor IkappaBalpha promoter functional polymorphisms in patients with rheumatoid arthritis. J. Clin. Immunol. 30: 676–680. 33. Lin, C. H., S. C. Wang, T. T. Ou, et al. 2008. I kappa B alpha promoter polymorphisms in patients with systemic lupus erythematosus. J. Clin. Immunol. 28: 207–213.
ISSN: 0891-6934 (Print) 1607-842X (Online) Journal homepage: http://www.tandfonline.com/loi/iaut20
Association of OSMR Gene Polymorphisms with Rheumatoid Arthritis and Systemic Lupus Erythematosus Patients Yuan-Zhao Lin, Ruei-Nian Li, Chia-Hui Lin, Tsan-Teng Ou, Cheng-Chin Wu, Wen-Chan Tsai, Hong-Wen Liu & Jeng-Hsien Yen To cite this article: Yuan-Zhao Lin, Ruei-Nian Li, Chia-Hui Lin, Tsan-Teng Ou, Cheng-Chin Wu, Wen-Chan Tsai, Hong-Wen Liu & Jeng-Hsien Yen (2014) Association of OSMR Gene Polymorphisms with Rheumatoid Arthritis and Systemic Lupus Erythematosus Patients, Autoimmunity, 47:1, 23-26, DOI: 10.3109/08916934.2013.849701 To link to this article: https://doi.org/10.3109/08916934.2013.849701
Published online: 12 Nov 2013.
Submit your article to this journal
Article views: 137
View Crossmark data
Citing articles: 3 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=iaut20
http://informahealthcare.com/aut ISSN: 0891-6934 (print), 1607-842X (electronic) Autoimmunity, 2014; 47(1): 23–26 ! 2014 Informa UK Ltd. DOI: 10.3109/08916934.2013.849701
RESEARCH ARTICLE
Association of OSMR Gene Polymorphisms with Rheumatoid Arthritis and Systemic Lupus Erythematosus Patients Yuan-Zhao Lin1*, Ruei-Nian Li2*, Chia-Hui Lin1, Tsan-Teng Ou3, Cheng-Chin Wu3, Wen-Chan Tsai1,3, Hong-Wen Liu3, and Jeng-Hsien Yen1,3 1
Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan, 2Department of Biomedical Science and Environmental Biology, and 3Division of Rheumatology, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan Abstract
Keywords
Cytokines are involved in the pathogenesis of autoimmune diseases. Oncostatin M receptor (OSMR) activates JAK/STAT and MAPK pathways leading to the stimulation of a variety of cytokines and inflammatory substances. Many pro-inflammatory cytokines are involved in the inflammatory process of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). In this study, we carried out experiments to examine the relationship of OSMR promoter polymorphisms with RA and SLE patients. 241 patients of RA, 143 patients of SLE and 203 healthy controls were enrolled in their recruitment from the Kaohsiung Medical University Hospital. Genomic DNA was extracted from peripheral blood mononuclear cells and gene polymorphism was genotyped by TaqMan real-time polymerase chain reaction. The OMSR promoter region 100 G/T (rs22922016) genotype was in no relation to the susceptibility of RA, but 100 T/T (rs22922016) genotype could prevent the patients with sicca syndrome and the existence of anti-Ro antibodies. In contrast, the 100 G/TþT/T (rs22922016) genotypes were significantly associated with an increased risk of SLE (odds ratio, OR¼1.62, 95% confidence interval (CI), 1.01–2.62). 94.38% of SLE patients with arthritis were belonged to the 1687C/C (rs540558) genotype. The T allele of promoter region 100 T/T (rs22922016) has protective effect and could ameliorate the disease condition in RA patients, whereas the same T allele was a risk allele in the susceptibility of SLE. The disease severity of rheumatoid arthritis and systemic lupus erythematosus can be partially affected by the OSMR promoter polymorphisms.
Oncostatin M receptor, rheumatoid arthritis, systemic lupus erythematosus, gene polymorphism, SNP
Oncostatin M (OSM) is a multifunctional cytokine and is mainly produced by T cells and monocytes [1]. OSM is a member of interleukin-6 family which includes IL-6, IL-11, leukemia inhibitory factor (LIF), cardiotropin-1 (CT-1) and ciliary neurotrophic factor (CNTF) [2]. These cytokines are well known for their roles in controlling immune responses, inflammatory mediators, cell proliferation and they are involved in the development of aberrant inflammation. These cytokines share common receptor subunits. OSM has two types of receptors, gp130 with LIF binding protein (gp130/LIFR) and gp130 with OSM-specific receptor (gp130/ OSMR) [2,3]. When OSM binds to gp130 subunit, it triggers the formation of heterodimer of receptors and activates the Janus kinases family JAK1 and JAK2 [4]. The JAKs enzymes
*These authors contributed equally to this work. Correspondence: Jeng-Hsien Yen, Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan. E-mail: [email protected]
Received 16 July 2013 Revised 29 August 2013 Accepted 25 September 2013 Published online 12 November 2013
then phosphorylate the STATs, which are subsequently transported to the nucleus and bind to their cognate DNA binding sites. Activated-STATs bind to target DNA and regulate a variety of genes which are often associated with inflammatory reactions [5,6]. OSM is highly expressed in synovial fluid of rheumatoid arthritis (RA) patients, stimulates synovial fibroblast proliferation and production of cytokines to recruit inflammatory cell during disease progression [1,7,8]. High concentration of OSM may elicit the inflammation responses in joints and leading to bone erosion. OSM has been reported that it is detected in the serum of 9.4% of SLE patients [9]. These results indicated that OSM and OSMR are in relation to autoimmune diseases. SLE is a complex systemic autoimmune disease and the detailed etiology is still unclear. It is involved in multiple organs and systems. RA is a chronic inflammation disease that may involve extra-articular organs in addition to joints [10]. Many pro-inflammatory cytokines, like IL-6, IL-8, IL-18 and TNF are involved in the inflammatory process of autoimmune diseases [11–13]. Therefore, in this study, we have carried out experiments to examine the relationship of OSMR promoter polymorphisms with RA and SLE patients.
20 13
Introduction
History
24
Y.-Z. Lin et al.
Autoimmunity, 2014; 47(1): 23–26
Methods
analysis was conducted by the IBM SPSS Statistics version 19.
Clinical subjects Two hundred and forty-one patients of RA, 143 patients of SLE and 203 healthy controls were enrolled in their recruitment from the Kaohsiung Medical University Hospital. The diagnosis of RA was according to the American Rheumatism Association 1987 revised criteria for the classification of RA. The diagnosis of SLE and all patients were fulfilled the revised criteria of American College Rheumatology 1997 for the classification of SLE. This study was approved by the Institutional Review Board of Kaohsiung Medical University Hospital (KMUH). Genomic DNA and total RNA extraction Peripheral blood mononuclear cells (PBMC) were collected by Ficoll-paque (GE Healthcare, Little Chalont, UK) purification and genomic DNA was extracted from PBMC of collected patients from KMUH. Total RNA are extracted by the method of QIAmp RNA Blood Mini Kit (Qiagen, Hilden, Germany). RNA is reversely transcribed to cDNA with random primers and high-capacity cDNA Archive kit (Applied Biosystems, Life Technologies, Carlsbad, CA). SNP genotyping and data analysis Two different polymorphic sites on the OSMR promoter region 1687C/G (rs540558) and 100 G/T (rs22922016) were genotyped. The OSMR SNP genotyping was performed by TaqMan SNP Genotyping Assays (Applied Biosystems, Life Technologies, Carlsbad, CA) by real-time polymerase chain reaction (PCR) with ABI 7500 Real-Time PCR systems. The odds ratio (OR) and its 95% confidence interval (CI) were calculated to evaluate the risk of disease. The statistical
OMSR RNA expression A total of 30 RA patients, 30 SLE patients and 30 control individuals were assayed for the OMSR expression. The OMSR RNA expression was performed by the TaqMan Gene Expression Assay (Applied Biosystems, cat. no. 4369016) and real-time PCR reaction mix (Applied Biosystems, cat. no. 4369016). The reverse transcription was at 50 C for 2 min, PCR condition was at 95 C for 10 min, 95 C for 10 s, 60 C for 1 min (repeat 40 cycles) on ABI 7500 Real-Time PCR systems. H1299 is a lung cancer cell line which serves as a positive control, and RNA polymerase II is the input control [14].
Results 100 T/T genotype ameliorates the rheumatoid arthritis syndrome Previous study has demonstrated that the OSM and OSMR were associated with the susceptibility to autoimmune diseases [4,9,15]. Therefore, in this study, we have carried out experiments to determine two different polymorphic sites on the OSMR promoter region in RA and SLE patients. The genotypes of OMSR promoter sequence 1687C/G and 100 G/T were not found in relation to the susceptibility of patients with RA (Table 1). Moreover, in clinical manifestation, the genotype 100 G/T was associated with sicca syndrome and anti-Ro/SSA antibodies but not 1687C/G (Table 2). The odds ratio of 100 T/T genotype with sicca syndrome was 0.28 (95%CI, 0.12–0.69), and 9.1% of RA patients exhibited sicca syndrome with 100 T/T genotype
Table 1. Genotypes frequencies of OSMR promoter in RA and SLE patients. RA (N ¼ 241)
Control (N ¼ 203)
N(%)
N(%)
OSMR Genotype
SLE (N ¼ 143)
Control (N ¼ 203)
N(%)
N(%)
p Value
OR (95% CI)
p Value
1687C/G (rs540558) C/C C/G G/G C/GþG/G C/CþC/G
221 19 1 20 240
(91.7%) (7.88%) (0.41%) (8.3%) (99.59%)
182 21 0 21 203
(89.66%) (10.34%) (0%) (10.34%) (100%)
NS NS NS NS NS
127 15 1 16 142
(88.81%) (10.49%) (0.70%) (11.19%) (99.3%)
182 21 0 21 203
(89.66%) (10.34%) (0%) (10.34%) (100%)
– – – – –
NS NS NS NS NS
100 G/T (rs22922016) G/G G/T T/T C/TþT/T G/GþG/T
76 117 48 168 193
(31.54%) (48.55%) (19.92%) (68.46%) (80.08%)
70 98 35 133 168
(34.48%) (48.28%) (17.24%) (65.52%) (82.76%)
NS NS NS NS NS
35 77 31 108 112
(24.48%) (53.85%) (21.68%) (75.52%) (78.32%)
70 98 35 133 168
(34.48%) (48.28%) (17.24%) (65.52%) (82.76%)
– 0.89 (0.50–1.58) 1.77 (0.94–3.33) 1.62 (1.01–2.62) –
NS 0.69 0.08 0.046 NS
OR: odds ratio. CI: confidence interval. NS: no significant.
Table 2. Genotypes frequencies of OSMR promoter in RA patients and controls and stratified by sicca syndrome or anti-Ro antibodies. Sicca syndrome OSMR Genotype 100 G/T (rs22922016) G/GþG/T T/T
Anti-Ro antibodies
þ N(%)
N(%)
OR (95% CI)
p Value
þ N(%)
N(%)
OR (95% CI)
p Value
70 (90.9%) 7 (9.1%)
82 (73.88%) 29 (26.12%)
1 0.28 (0.12–0.69)
0.0004
47 (92.16%) 4 (7.84%)
91 (75.2%) 30 (24.8%)
1 0.26 (0.09–0.78)
0.01
OMSR gene polymorphisms with autoimmune diseases
DOI: 10.3109/08916934.2013.849701
(Table 2). Whereas, 70 out of 152 RA patients of genotypes 100 G/G and 100 G/T presented sicca syndrome. The odds ratio of 100 T/T genotype with anti-Ro antibodies was 0.26 (95% CI, 0.09–0.78) (Table 2). The presence of anti-Ro antibodies existed only in 4 (7.84%) of 100 T/T genotype RA patients (Table 2). We observed that the OMSR promoter region 100 T/T genotype could prevent rheumatoid arthritis patients with sicca syndrome and the existence of anti-Ro antibodies. T allele of 100 G/T is associated with SLE The genotype 1687C/G was not found in relation to SLE patients (Table 1). However, the odds ratio of 100 T/ T genotype was notably associated with the susceptibility of SLE (OR ¼ 1.77, 95% CI, 0.94–3.33), but without statistical significance. On the contrary, the 100 G/TþT/T genotypes were significantly associated with an increased risk of SLE (OR ¼ 1.62, 95% CI, 1.01–2.62) (Table 1). This result indicated that T allele was a risk allele in the susceptibility of SLE. 1687C/C genotype is associated with arthritis syndrome in SLE In clinical manifestation, the genotype 1687C/C was found in association with arthritis syndrome in SLE patients but not 100 C/GþG/G (Table 3). The percentage of 1687C/C genotype was 94.38% in SLE patients with arthritis. The odds ratio of 1687C/G and 1687G/G was 0.18 (95% CI, 0.04–1.58) (Table 3). We have performed the TaqMan realtime polymerase chain reaction to evaluate the RNA expression of OSMR gene in RA and SLE patients. However, OSMR gene expression was not detected in RA and SLE patients (Table 4).
Discussion We have investigated two different polymorphic sites on OSMR promoter region in RA and SLE patients. The results indicated that the OSMR promoter genotypes 1687C/G and 100 G/T were found in no relation to the susceptibility with rheumatoid arthritis patients. In RA clinical outcome, there was no correlation with genotypes 1687C/G as well. Table 3. Genotypes frequencies of OSMR promoter in SLE patients and controls and stratified by arthritis. Arthritis OSMR Genotype
þ N(%)
N(%)
OR (95% CI)
1687C/G (rs540558) C/C 84 (94.38%) 12 (75%) 1 C/GþG/C 5 (5.62%) 4 (25%) 0.18 (0.04–0.76)
p Value
0.01
Table 4. The gene expression of OSMR in RA and SLE patients.
OSMR expression (2DCT) NS: no significant.
RA
SLE
Control
H1299 (Positive control)
NS
NS
NS
0.2984
25
However, we noticed that sicca syndrome was associated with 100 G/T allele in RA patients. Genotype 100 T/T of RA patients were also found in decrease concentration of anti-Ro antibodies. Anti-Ro antibodies are associated with different autoimmune diseases, such as RA, SLE and Sjo¨gren syndrome (SS) [16,17]. One of the main pathologies of SLE and SS is anti-Ro antibodies [18,19]. These results indicate that the ameliorative effect of T allele in RA patients disease condition. In contrast, the presence of G allele would cause an increased risk of sicca syndrome and the rise of anti-Ro antibodies in RA patients. Cytokines are involved in the pathogenesis of autoimmune diseases [13,15]. Oncosatin M receptor (OSMR) is the main receptor for oncostatin M (OSM), one of the IL-6 cytokine families [1]. IL-6 and OSM activates JAK1 and JAK2, which subsequently activate the activity of transcriptional factors STAT1, STAT3 and STAT5. Therefore, OMSR activates JAK/ STAT and MAPK pathways and stimulates the production of a variety of cytokines and inflammatory substances [4,6,20]. Recent study has reported that CD4 T cells secrete IL-31 which is also a ligand to OSMR [21]. 100 G/T was found in no relation with RA patients. However, T allele of 100 G/T was a risk allele to the susceptibility of SLE. Besides, OSMR promoter 1687C/G was associated with arthritis (þ) in SLE patients. The increased risk of arthritis in SLE patients is associated to the presence of C allele of the polymorphic site 1687C/G. On the other side, the haplotypes of OSMR promoter 1687C 100 G and 1687C 100 T were related to SLE (data not shown). We tried to determine the expression level of OSMR mRNA by real-time polymerase chain reaction. The result indicated that OSMR mRNA was undetected and the protein was supposed to be no expression both in RA and SLE patients. However, the promoter polymorphism shows evidence of association to the susceptibility of SLE. Alternatively, SNP can perform synergic pair effect in SNPSNP interaction [22,23]. The complicate network may interfere the expression or the activity of a second gene. The promoter site 100 of OSMR contains transcriptional factor binding sites of STAT1 and STAT3, whereas 1687 site contains STAT1 binding sequence [24]. Both STAT1 and STAT3 are the transcriptional activators of many inflammatory mediators and cytokines. The pathway of STAT1 is activated in RA as previously reported [20]. In addition, 100 site contains binding sequence for interferon regulatory factors (IRF) [25], and IRFs are important mediators in the pathogenesis of SLE [26]. This suggests that 100 T allele has a critical role in the severity of disease condition in SLE. The change of OSMR promoter sequence 1687C4G or 100 G4T in a single nucleotide would affect the transcriptional factor binding affinity, and subsequently would downregulate the promoter activity. Pre-B cell colony-enhancing factor (PBEF) is a secretory cytokine which regulates cell apoptosis and B cell development and maturation [27]. Recent report has demonstrated that the mRNA and protein of PBEF are expressed in a high concentration in synovial fluid of RA patients [28,29]. The regulation of PBEF is involved in the STAT3 dependent IL6 signal pathway [30]. Promoter polymorphism was associated with different inflammatory diseases, such as RA, SLE,
26
Y.-Z. Lin et al.
Sjo¨gren’s syndrome [31–33]. In conclusion, we have suggested that the possible role of the OSMR promoter polymorphism affect the expression level of different cytokines and inflammatory mediators. Therefore, the disease severity of RA and SLE can be partially affected by the OSMR promoter polymorphism.
Declaration of interest No potential conflicts of interest were disclosed.
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