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 3

PTPN11 expression in rheumatoid arthritis fibroblast-like synoviocytes is glucocorticoid responsive.

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PTPN11 expression in rheumatoid arthritis fibroblast-like synoviocytes ...
(A) After serum starvation for 24 hours, RA FLS were treated with increasing concentrations of dexamethasone (DEX) for 24 hours. PTPN11 mRNA expression was analyzed by qPCR, and results were normalized to GAPDH mRNA expression. 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 5 independent experiments with different rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) lines. Data were analyzed using Dunnett’s multiple comparisons test. (B) Female C57BL/6 mice (6–8 weeks of age) were administered 1 μg DEX or vehicle (n = 4 per group) by i.p. injection. Ptpn11 mRNA expression in ankle homogenates was measured after 24 hours by qPCR performed in triplicate. Box-and-whisker plots depict Ptpn11 expression normalized to Gapdh expression. Data were analyzed using the 2-tailed unpaired t test. (C) RA FLS were transfected with control siRNA or siRNA against glucocorticoid receptor (GR) mRNA. After 24 hours, RA FLS were serum starved for 24 hours and then cultured in the absence (left) or presence (right) of 100 nM DEX for 24 hours. qPCR was performed to assess transcript levels of GR and PTPN11. Results were normalized to GAPDH expression. Box-and-whisker plots depict 4 independent experiments with different RA FLS lines. Data were analyzed using the 2-tailed paired t test. (D) After serum starvation for 24 hours, RA FLS were treated with 100 nM β-estradiol (E2), 100 nM Cl-4AS-1, or 100 nM DEX for 24 hours. PTPN11 mRNA expression was analyzed by qPCR. Results were normalized to GAPDH mRNA. 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.