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. 2016 Feb;68(2):359-69.
doi: 10.1002/art.39442.

Receptor Protein Tyrosine Phosphatase α-Mediated Enhancement of Rheumatoid Synovial Fibroblast Signaling and Promotion of Arthritis in Mice

Stephanie M Stanford  1 , Mattias N D Svensson  1 , Cristiano Sacchetti  1 , Caila A Pilo  1 , Dennis J Wu  1 , William B Kiosses  2 , Annelie Hellvard  3 , Brith Bergum  3 , German R Aleman Muench  1 , Christian Elly  1 , Yun-Cai Liu  1 , Jeroen den Hertog  4 , Ari Elson  5 , Jan Sap  6 , Piotr Mydel  3 , David L Boyle  7 , Maripat Corr  7 , Gary S Firestein  7 , Nunzio Bottini  1
Affiliations

Receptor Protein Tyrosine Phosphatase α-Mediated Enhancement of Rheumatoid Synovial Fibroblast Signaling and Promotion of Arthritis in Mice

Stephanie M Stanford et al. Arthritis Rheumatol. 2016 Feb.

Abstract

Objective: During rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) critically promote disease pathogenesis by aggressively invading the extracellular matrix of the joint. The focal adhesion kinase (FAK) signaling pathway is emerging as a contributor to the anomalous behavior of RA FLS. The receptor protein tyrosine phosphatase α (RPTPα), which is encoded by the PTPRA gene, is a key promoter of FAK signaling. The aim of this study was to investigate whether RPTPα mediates FLS aggressiveness and RA pathogenesis.

Methods: Through RPTPα knockdown, we assessed FLS gene expression by quantitative polymerase chain reaction analysis and enzyme-linked immunosorbent assay, invasion and migration by Transwell assays, survival by annexin V and propidium iodide staining, adhesion and spreading by immunofluorescence microscopy, and activation of signaling pathways by Western blotting of FLS lysates. Arthritis development was examined in RPTPα-knockout (KO) mice using the K/BxN serum-transfer model. The contribution of radiosensitive and radioresistant cells to disease was evaluated by reciprocal bone marrow transplantation.

Results: RPTPα was enriched in the RA synovial lining. RPTPα knockdown impaired RA FLS survival, spreading, migration, invasiveness, and responsiveness to platelet-derived growth factor, tumor necrosis factor, and interleukin-1 stimulation. These phenotypes correlated with increased phosphorylation of Src on inhibitory Y(527) and decreased phosphorylation of FAK on stimulatory Y(397) . Treatment of RA FLS with an inhibitor of FAK phenocopied the knockdown of RPTPα. RPTPα-KO mice were protected from arthritis development, which was due to radioresistant cells.

Conclusion: By regulating the phosphorylation of Src and FAK, RPTPα mediates proinflammatory and proinvasive signaling in RA FLS, correlating with the promotion of disease in an FLS-dependent model of RA.

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Figures

Figure 1
Figure 1. RPTPα is enriched in the RA synovial lining and promotes TNF and IL-1β signaling in RA FLS
(A) Immunohistochemical staining of RA synovial sections using anti-RPTPα or control IgG antibodies. (B–C) RA FLS (n=4) were treated with 2.5 µM control non-targeting (Ctl) or PTPRA PMO for 7 d. (B) PTPRA mRNA expression levels were measured by qPCR. Median and interquartile range (IQR) is shown. *, p<0.05, Mann-Whitney test. (C) RPTPα protein levels were measured by Western blotting. (D) Following treatment with PMO, RA FLS (n=4) were stimulated with 50 ng/ml TNF or 2 ng/ml IL-1 for 24 hr. mRNA expression was analyzed by qPCR. Median and IQR is shown. Protein expression in cell supernatants was measured by ELISA. Mean ± standard error of the mean (SEM) is shown. *, p<0.05, Mann-Whitney test.
Figure 2
Figure 2. RPTPα promotes RA FLS invasiveness
(A) Following treatment with PMO for 7 d, RA FLS (n=4) invaded through Matrigel-coated transwell chambers in response to 50 ng/ml PDGF-BB for 48 hr. (B) PMO-treated RA FLS (n=4) migrated through uncoated transwell chambers in response to 5% FBS for 24 hr. (A–B) Median and IQR % maximum number of cells per field is shown. *, p<0.05, Mann-Whitney test. (C) PMO-treated RA FLS were washed and stimulated with 50 ng/ml PDGF for 24 hr. Cells were collected and stained with Annexin V and PI, and cell fluorescence was assessed by FACS. Graphs show gating strategy to detect early apoptotic (Annexin V+PI−) and necrotic/late apoptotic (Annexin V+PI+) cells. Significance was calculated using the Chi square test (p<0.0001, Chi-square=2294, df=2). Data is representative of 4 independent experiments. (D) PMO-treated RA FLS (n=4) were plated on fibronectin (FN)-coated coverslips in the presence of 5% FBS. Graphs show median and IQR cells per field after 15 min (left) or cell area after 15, 30 and 60 min (right). *, p<0.05, Wilcoxon matched-pairs signed rank test.
Figure 3
Figure 3. RPTPα promotes RA FLS signaling downstream SRC
(A) anti-pSRC-Y527 levels in PMO-treated RA FLS lysates were measured by Western blotting. Data is representative of 4 independent experiments. (B–C) Western blotting of lysates of PMO-treated RA FLS stimulated with 50 ng/ml TNF or left unstimulated. (B) Signal intensities of Western blots of TNF-activated proteins from lysates were quantified by densitometric scanning. Mean ± SEM of signal relative to GAPDH from 6 RA FLS lines is shown. (C) Representative image is shown. (D) Signal intensities of Western blots of lysates from unstimulated PMO-treated RA FLS. Mean ± SEM of signal relative to p65 from 6 RA FLS lines is shown. *, p<0.05; NS, non-significant, Wilcoxon matched-pairs signed rank test.
Figure 4
Figure 4. FAK inhibition impairs activation of JNK and TNF and IL-1β-induced gene expression in RA FLS
(A) RA FLS (n=4) were stimulated with 50 ng/ml TNFα or 2 ng/ml IL-1β in the presence of DMSO, the FAK inhibitor PF573228, or the AKT inhibitor MK2206 for 24 hr. mRNA expression was analyzed by qPCR. Mean ± SEM is shown. *, p<0.05, Mann-Whitney test. (B) RA FLS were stimulated with 50 ng/ml TNF or 2 ng/ml IL-1β for 30 min or left unstimulated, in the presence of DMSO or 10 µM FAK inhibitor PF573228. Data is representative of 4 independent experiments.
Figure 5
Figure 5. Ptpra KO mice are resistant to K/BxN serum transfer arthritis
WT and Ptpra KO littermate mice were administered 200 µl K/BxN sera at 8 weeks of age. (A) Ankle thickness was measured every 2 days (WT, n=16; KO, n=17). Mean ± SEM ankle swelling is shown. *, p<0.05, 2way ANOVA. (B) 7 days post-sera transfer, mice (n=3) were injected with intravital inflammation probe and luminescence of wrist and ankle joints was measured. Mean ± SEM luminescent counts per joint are shown. *, p<0.05, Wilcoxon matched-pairs signed rank test. (C) Histological analysis of ankles stained with H&E or Safranin-O at the end of the disease course. Left, histological scores of bone and cartilage erosions (WT, n=16; KO, n=17). Mean ± SEM is shown. *, p<0.05, Wilcoxon matched-pairs signed rank test. Right, representative images of H&E-stained (upper panels; yellow arrows indicate regions of inflammatory infiltrate) or Safranin-O-stained (lower panels; black arrows indicate regions of cartilage erosion) joints.
Fig. 6
Fig. 6. Arthritis protection in Ptpra KO mice is dependent upon radioresistant cells
(A–B) Mice were lethally irradiated and administered bone-marrow from donor mice. 10–11 weeks post-irradiation, arthritis was induced in recipients by administration of K/BxN sera. (A) Male WT congenic CD45.1 mice were administered bone-marrow cells from WT or Ptpra KO CD45.2 donor mice (WT donors, n=19; KO donors, n=18). (B) Male WT (n=11) or Ptpra KO (n=11) mice were administered bone-marrow cells from WT congenic CD45.1 mice. Mean ± SEM is shown. *, p<0.05, 2way ANOVA. (C) WT (n=5) and Ptpra KO (n=3) littermate mice were administered Angiosense 680 dye, followed by administration of K/BxN serum. Ankle fluorescence was monitored after 60 min. Median and IQR is shown. NS, non-significant, Mann-Whitney test. (D) WT (n=7) or Ptpra KO (n=7) mice were administered K/BxN sera. 8 days post-sera transfer, ankle joints were homogenized and mRNA expression was analyzed by qPCR. Median and IQR is shown. *, p<0.05, NS, non-significant, Mann-Whitney test.
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