Session Title: Rheumatoid Arthritis - Human Etiology and Pathogenesis III
Session Type: ACR Concurrent Abstract Session
Session Time: 2:30PM-4:00PM
Background/Purpose: Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) mediate disease pathogenesis by invading the joint extracellular matrix. The PTPN11 gene, encoding the tyrosine phosphatase SHP-2, is overexpressed in RA FLS compared to osteoarthritis (OA) FLS and promotes RA FLS invasiveness. Here we sought to elucidate the molecular basis for PTPN11 overexpression in RA and explore whether pharmacologic inhibition or genetic deficiency of its expressed protein, SHP-2, reduces RA FLS aggressiveness and arthritis severity.
Methods: FLS were obtained from arthroplasty RA and OA synovium. Dexamethasone (DEX)-induced DNA binding of the glucocorticoid receptor (GR) in RA FLS was assessed by chromatin immunoprecipitation (ChIP)-PCR. Arthritis severity in the K/BxN serum transfer model was assessed by measuring ankle swelling. Contribution of PTPN11 in FLS to arthritis severity was assessed by reconstitution of irradiated wild type (WT) or Ptpn11-/+mice with bone-marrow from WT mice.
Results: Potential regulatory regions of PTPN11 were identified in the UCSC genome browser and the ENCODE database. Based on the co-localization of histone marks and transcription factor binding motifs, we identified a possible 1.4 kb enhancer in intron 1 of PTPN11 containing a glucocorticoid receptor (GR)-binding motif. To confirm function of the enhancer, the region was subcloned into a luciferase reporter construct and transfected into FLS. The FLS were stimulated with DEX (100 nM) and ChIP-PCR showed that GR binding to the enhancer increased 5.6±0.4-fold (p<0.01) and increased luciferase expression by 13±5-fold (p<0.05). We then showed that DEX increased PTPN11 expression 1.4-fold higher in RA than OA FLS (p<0.05). To determine why PTPN11 expression is higher in RA, we pyrosequenced the 8 enhancer CpG sites near the GR-binding site. We found 2 hypermethylation sites in RA FLS compared to OA FLS: chr12 112860522 (28±4.1% in RA and 8.2±0.7% in OA; p<0.01) and chr12 112860601 (30%±3.6 in RA and 17%±2.4 in OA; p<0.05). Deletion of the two CpGs reduced enhancer activity by 40±9% (p<0.05), showing they are required for enhancer function. Having discovered that PTPN11 dysregulation is associated with abnormal enhancer DNA methylation in two CpGs, we examined functional consequences of high PTPN11. Using a transwell assay, a small molecule SHP-2 inhibitor reduced RA FLS migration by 83±8.3% (p<0.0001). Furthermore, heterozygous deletion of Ptpn11 in radioresistant cells (e.g., fibroblasts) attenuated passive K/BxN arthritis severity in mice (54±3.4% reduction, p<0.05). Treatment of mice daily with 7.5 mg/kg SHP-2 inhibitor reduced arthritis severity by 20±5% (AUC, p<0.05).
Conclusion: We identified a new intronic GR-responsive enhancer that is abnormally methylated in RA FLS, resulting in increased expression of the pathogenic gene PTPN11. Blocking function of its encoded protein, SHP-2, reduces RA FLS aggressiveness and arthritis severity. Our findings show how the interplay of abnormal epigenetics with a pathogenic gene determines FLS behavior and joint inflammation. SHP-2 inhibition could be an important new strategy that targets FLS invasiveness in RA.
To cite this abstract in AMA style:Maeshima K, Stanford SM, Hammaker D, Sacchetti C, Ai R, Zhang V, Boyle DL, Zeng L, Muench G, Feng GS, Whitaker J, Zhang ZY, Wang W, Bottini N, Firestein GS. Abnormal DNA Methylation in a Novel PTPN11 Enhancer Increases Destructive Potential of Rheumatoid Arthritis (RA) Fibroblast-like Synoviocytes (FLS) and Joint Inflammation [abstract]. Arthritis Rheumatol. 2015; 67 (suppl 10). https://acrabstracts.org/abstract/abnormal-dna-methylation-in-a-novel-ptpn11-enhancer-increases-destructive-potential-of-rheumatoid-arthritis-ra-fibroblast-like-synoviocytes-fls-and-joint-inflammation/. Accessed December 3, 2021.
« Back to 2015 ACR/ARHP Annual Meeting
ACR Meeting Abstracts - https://acrabstracts.org/abstract/abnormal-dna-methylation-in-a-novel-ptpn11-enhancer-increases-destructive-potential-of-rheumatoid-arthritis-ra-fibroblast-like-synoviocytes-fls-and-joint-inflammation/