Abnormal PTPN11 enhancer methylation promotes rheumatoid arthritis fibroblast-like synoviocyte aggressiveness and joint inflammation
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
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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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 2

The putative PTPN11 enhancer region drives gene expression and is regulated by the glucocorticoid receptor.

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The putative PTPN11 enhancer region drives gene expression and is regula...
(A) PTPN11 enhancer luciferase reporter vector or empty control vector was cotransfected into rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) with renilla plasmid. Firefly luciferase activity was measured and normalized to renilla luciferase activity 24 hours after transfection. 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. (B) Scheme of location and sequences of the PTPN11 enhancer glucocorticoid receptor–binding (GR-binding) motif and primers used in C. (C) After serum starvation for 24 hours, RA FLS were treated with ethanol (EtOH) or 100 nM dexamethasone (DEX) for 90 minutes. ChIP-PCR was performed using anti-GR antibody or control IgG. Values were normalized to corresponding input DNA and are expressed as fold enrichment relative to control IgG. Scatter plot shows the mean ± SEM of 3 independent experiments with different RA FLS lines. Data were analyzed using the 2-tailed paired t test. (D) PTPN11 enhancer luciferase reporter vector or empty control vector was cotransfected into RA FLS with renilla plasmid. After serum starvation for 24 hours, RA FLS were treated with medium or 100 nM DEX for 24 hours. Firefly luciferase activity was measured and normalized to renilla luciferase activity. Box-and-whisker plots depict the ratio of relative luciferase activity of DEX-treated cells versus medium-treated cells from 14 independent experiments with different RA FLS lines. Data were analyzed using the 2-tailed paired t test.