Abnormal PTPN11 enhancer methylation promotes rheumatoid arthritis fibroblast-like synoviocyte aggressiveness and joint inflammation
Keisuke Maeshima, … , Nunzio Bottini, Gary S. Firestein
Keisuke Maeshima, … , Nunzio Bottini, Gary S. Firestein
Published May 19, 2016
Citation Information: JCI Insight. 2016;1(7):e86580. https://doi.org/10.1172/jci.insight.86580.
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Research Article Inflammation

Abnormal PTPN11 enhancer methylation promotes rheumatoid arthritis fibroblast-like synoviocyte aggressiveness and joint inflammation

Abstract

The PTPN11 gene, encoding the tyrosine phosphatase SHP-2, is overexpressed in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) compared with osteoarthritis (OA) FLS and promotes RA FLS invasiveness. Here, we explored the molecular basis for PTPN11 overexpression in RA FLS and the role of SHP-2 in RA pathogenesis. Using computational methods, we identified a putative enhancer in PTPN11 intron 1, which contained a glucocorticoid receptor–binding (GR-binding) motif. This region displayed enhancer function in RA FLS and contained 2 hypermethylation sites in RA compared with OA FLS. RA FLS stimulation with the glucocorticoid dexamethasone induced GR binding to the enhancer and PTPN11 expression. Glucocorticoid responsiveness of PTPN11 was significantly higher in RA FLS than OA FLS and required the differentially methylated CpGs for full enhancer function. SHP-2 expression was enriched in the RA synovial lining, and heterozygous Ptpn11 deletion in radioresistant or innate immune cells attenuated K/BxN serum transfer arthritis in mice. Treatment with SHP-2 inhibitor 11a-1 reduced RA FLS migration and responsiveness to TNF and IL-1β stimulation and reduced arthritis severity in mice. Our findings demonstrate how abnormal epigenetic regulation of a pathogenic gene determines FLS behavior and demonstrate that targeting SHP-2 or the SHP-2 pathway could be a therapeutic strategy for RA.

Authors

Keisuke Maeshima, Stephanie M. Stanford, Deepa Hammaker, Cristiano Sacchetti, Li-fan Zeng, Rizi Ai, Vida Zhang, David L. Boyle, German R. Aleman Muench, Gen-Sheng Feng, John W. Whitaker, Zhong-Yin Zhang, Wei Wang, Nunzio Bottini, Gary S. Firestein

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Figure 1

Identification of a putative intragenic PTPN11 enhancer displaying epigenetic alterations in rheumatoid arthritis fibroblast-like synoviocytes versus osteoarthritis fibroblast-like synoviocytes.

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Identification of a putative intragenic PTPN11 enhancer displaying epige...
(A) Scheme of the putative intragenic enhancer in chromosomal region 12q24.13, depicting an ENCODE-derived DNase I hypersensitivity cluster, chromatin state annotation tracks produced from ChromHMM, H3K4me1 marker, transcription factor ChIP sequencing, and human/mouse/rat conserved transcription factor–binding sites. Data were downloaded from the UCSC Genome Browser (http://genome.ucsc.edu/; assembly GRCh37/hg19). (B) After serum starving rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) for 24 hours, ChIP-PCR was performed using anti-H3K4me1 antibody on the PTPN11 enhancer or MYT1 exon 1 as a control or control (Ctrl) IgG on the PTPN11 enhancer. The occupancy of H3K4me1 or Ctrl IgG is shown as the percentage of input. Box-and-whisker plots depict median (line within box), 25th percentile and 75th percentile (bottom and top borders), and range of minimum to maximum values (whiskers) from 4 independent experiments with different RA FLS lines. Data were analyzed using the 2-tailed paired t test. (C) Sequence of DNase I hypersensitivity region in the PTPN11 enhancer (chr12:112860281-112860755) with 8 CpG dinucleotides. (D) Analysis of differentially methylated loci between RA (n = 8) and osteoarthritis (OA) (n = 6) FLS in the PTPN11 DNase I hypersensitivity region through pyrosequencing. Box-and-whisker plots depict the percentage of methylation of each CpG site. Data were analyzed using the 2-tailed unpaired t test with Welch’s correction.