Research ArticleRheumatoid Arthritis

Targeting phosphatase-dependent proteoglycan switch for rheumatoid arthritis therapy

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Science Translational Medicine  20 May 2015:
Vol. 7, Issue 288, pp. 288ra76
DOI: 10.1126/scitranslmed.aaa4616
  • Fig. 1. RPTPσ-dependent PG switch controls FLS invasiveness and migration.

    (A) Ptprs expression in freshly sorted FLS from joints of nonarthritic mice or mice with K/BxN serum–induced arthritis. (B) Ptprs and Ptprc expression in freshly sorted FLS and synovial macrophages from mice with chronic K/BxN arthritis. (A and B) Mean ± SEM of expression relative to Polr2a; ****P < 0.0001, unpaired t test and Welch’s correction. (C) Pathology of safranin O–stained ankles from mice on day 14 of acute, or day 24 of chronic, K/BxN arthritis. Representative of three ankles per group; S, synovial lining; Ta, talus; Ti, tibia. (D) Ptprs expression in mouse FLS cultured at 0.2 × 106 (subconfluent) to 0.5 × 106 (maximum density) cells per well of a six-well plate. Mean ± SEM of expression relative to Polr2a (n = 3; *P = 0.0418, two-tailed unpaired t test). (E) (Left) Invasion of wild-type (WT) or Ptprs KO FLS through Matrigel in response to 5% fetal bovine serum (FBS) in the presence or absence of CS (100 μg/ml). (Right) Western blot of RPTPσ expression in Ptprs WT and KO FLS. (F) WT or Ptprs KO FLS migrated through Transwells in response to 5% FBS in the presence or absence of CS with or without chondroitinase (C’ase) ABC treatment. (G and H) Transwell migration of WT or Ptprs KO FLS in response to 5% FBS (G) in the presence or absence of HS (100 μg/ml) or (H) with or without pretreatment with 10 mU of heparinase III. (I) RA FLS were treated for 7 days with cell-permeable antisense oligonucleotides (ASO) to knock down PTPRS or control ASO and allowed to migrate through Transwells in response to 5% FBS in the presence of CS (100 μg/ml) or vehicle. (E to I) Mean ± SEM fold change of invasion or migration relative to the vehicle-treated cells from the same experiment is shown. Data are from three independent experiments (n = 45 fields; ****P < 0.0001, Mann-Whitney).

  • Fig. 2. RPTPσ Ig1&2 inhibits FLS migration and invasiveness in an RPTPσ-dependent manner.

    (A) FLS migrated through Transwells in response to 5% FBS in the presence of the indicated concentrations of Ig1&2. (B and C) Migration of WT (B and C) or Ptprs KO FLS (B) through Transwells in response to (B) 5% FBS or (C) PDGF-BB (50 ng/ml) in the presence of 20 nM Ig1&2 or vehicle. (D) Invasion of WT or Ptprs KO FLS through Matrigel in response to 5% FBS in the presence of 20 nM Ig1&2 or vehicle. (E) Migration of FLS through Transwells in response to 5% FBS in the presence of vehicle or 20 nM Ig1&2 or non-GAG binding Ig1&2 ΔK mutant. n.s., not significant. (F and G) FLS migrated through Transwells in response to 5% FBS in the presence of (F) vehicle or 20 nM Ig1&2, with or without 10 mU of heparinase III pretreatment, or (G) vehicle, 20 nM Ig1&2, CS (100 μg/ml), or both Ig1&2 and CS. (H) RA FLS migrated through Transwells in response to 5% FBS in the presence of vehicle or 20 nM Ig1&2. (I) RA FLS monolayers were serum-starved before scratch-wounding and stimulated with 10% FBS in the presence of 20 nM Ig1&2 or vehicle. Wound width was measured at three points at the indicated times. (A to H) Mean ± SEM fold change of invasion or migration relative to vehicle-treated cells. Data are from three independent experiments (n = 45 fields; ****P < 0.0001, Mann-Whitney). (I) Mean ± SEM wound width from two independent experiments (n = 6 fields; 12 hours, **P = 0.0087; 24 hours, *P = 0.0411; 48 hours, **P = 0.0043; Mann-Whitney).

  • Fig. 3. RPTPσ Ig1&2 decreases FLS attachment to cartilage in vitro in an RPTPσ-dependent manner.

    (A) RA FLS were allowed to attach to cartilage explants in the presence of 20 nM bovine serum albumin (BSA) or Ig1&2. Representative images (left) and quantification of RA FLS (right) attached to cartilage explants in vitro in the presence of 20 nM Ig1&2 or BSA (n = 20 fields; ****P < 0.0001, Mann-Whitney). (B) Quantification of Ptprs WT or KO FLS attached to cartilage explants in vitro in the presence of 20 nM Ig1&2 or BSA (n = 15 fields; ****P < 0.0001, Mann-Whitney). (C) Quantification of Ptprs WT FLS attached to cartilage explants in vitro in the presence of 20 nM Ig1&2 or BSA with or without pretreatment with heparinase III (10 mU/ml) (n = 15 fields; **P = 0.0033, ****P < 0.0001, Mann-Whitney). (A to C) Mean ± SEM of fold change of number of cells attached to cartilage per 100× field relative to the BSA-treated cells from the same experiment is shown. Data are from three independent experiments.

  • Fig. 4. Regulation of FLS migration by RPTPσ Ig1&2 is dependent on syndecan-4.

    (A) mRNA expression of syndecan and glypican family members in RA FLS. Mean ± SEM of expression normalized to POL2RA housekeeping gene and relative to SDC2; n = 5 RA lines. (B) RA FLS were treated with cell-permeable ASO to knock down SDC2/GPC1 or SDC4 or control ASO and allowed to migrate through Transwells in response to 5% FBS in the presence of 20 nM Ig1&2 or vehicle. Mean ± SEM of the fold change of migration relative to that of the same cells treated with vehicle. Data are representative of three independent experiments (n = 45 fields; ***P = 0.0002, ****P < 0.0001, Mann-Whitney). (C and D) Sdc3 (C) or Sdc4 (D) KO FLS were allowed to migrate through Transwells for 4 hours in response to 5% FBS in the presence of vehicle or 20 nM Ig&2. Mean ± SEM fold change of migration relative to that of the same cells treated with vehicle. Data are representative of two independent experiments in triplicate (n = 30 fields; ****P < 0.0001, Mann-Whitney). (E) Syndecan-4 was coexpressed with empty vector or RPTPσ in HEK293T cells. Representative Western blot of input and immunoprecipitation of syndecan-4 probed with anti-hemagglutinin (HA) and anti-FLAG antibodies (n = 3). (F) Syndecan-4 was expressed in HEK293T cells, immunoprecipitated, and then incubated with Ig1&2. Representative Western blot of input and pull-down of Ig1&2 probed with anti-6×His (Ig1&2) and anti-FLAG antibodies (representative of n = 2 independent experiments). (G) Ankle sections of mice with chronic arthritis were incubated with purified Ig1&2 for 2 hours and then stained for syndecan-4 and 6×His (Ig1&2) and imaged by confocal microscopy. Representative image of three ankles from three individual mice; S, synovial infiltrate; B, bone; C, cartilage.

  • Fig. 5. Ezrin is regulated by the PG switch through RPTPσ-mediated dephosphorylation.

    (A) Transwell migration in response to 5% FBS of Ptprs KO FLS expressing WT or C/S RPTPσ or empty vector (EV) and treated with vehicle or Ig1&2. Mean ± SEM fold change of migration relative to vehicle-treated EV FLS from the same experiment (n = 45 fields; ****P < 0.0001, Mann-Whitney). Data are from three independent experiments. (B) FLS were unstimulated or stimulated with PDGF (50 ng/ml) for 15 min in the presence of 20 nM Ig1&2 or vehicle, and ezrin was immunoprecipitated from cell lysates. Representative Western blot (left) and densitometric analysis (right) of PDGF-induced ezrin phosphorylation from three independent experiments. Mean ± SEM (n = 3; *P = 0.0138, one-tailed unpaired t test). (C) Western blot of lysates from unstimulated or pervanadate-stimulated RA FLS incubated with glutathione S-transferase (GST) or GST-RPTPσ D1516A substrate-trapping mutant. (D) Transwell migration in response to 5% FBS of mouse FLS expressing WT or phosphomimetic Y146E or Y353E ezrin mutants or EV and treated with vehicle or Ig1&2. (Left) Mean ± SEM fold change of migration relative to the vehicle-treated EV sample from the same experiment (n = 30 fields; ****P < 0.0001, Mann-Whitney). (Right) Representative Western blot of lysates from FLS transfected with EV or WT or phosphomimetic mutant ezrin and immunoblotted for Xpress tag or glyceraldehyde phosphate dehydrogenase (GAPDH) (loading control). Data are from three independent experiments. (E and F) Monolayers of (E) WT FLS or (F) Ptprs KO FLS expressing WT or C/S RPTPσ were scratch-wounded and stimulated with PDGF for 8 hours in the presence of vehicle or 20 nM Ig1&2. Pseudocolored map of intensity of ezrin staining in cells migrating toward the wound edge (blue, low; red, high). Representative fields are from three independent experiments.

  • Fig. 6. RPTPσ Ig1&2 administration decreases arthritis severity in an RPTPσ-dependent manner.

    (A to D) BALB/c mice were induced with acute K/BxN arthritis and treated with vehicle or 0.5 mg of Ig1&2 intravenously daily (days 0 to 6). (A) Clinical score (left) and change in ankle thickness (right) (n = 6; **P = 0.0022, Mann-Whitney). (B) Ankle luminescence (left) and luminescence counts (right) of mice treated with inflammation probe on day 4 of arthritis (n = 6; **P = 0.0043, Mann-Whitney). (C) Three-dimensional microCT of ankles on day 14. White arrows indicate bone erosion or reactive bone deposition. (D) Histopathological score of bone erosion and cartilage damage of ankle sections from mice in (A) on day 14 (n = 6; bone erosion *P = 0.0299, cartilage damage *P = 0.0056, two-tailed unpaired t test). (E and F) Ptprs KO mice were induced with arthritis as in (A). (E) Clinical score (left) and change in ankle thickness (right); n = 3 per group. (F) Mice from (E) were injected with RediJect Inflammation Probe on day 4 of arthritis induction. Mean ± SEM of luminescence in each ankle joint measured. n = 3 mice per treatment. (G and H) BALB/c mice were induced with chronic K/BxN arthritis (serum administration shown by arrows) and treated daily with vehicle or 0.5 mg of Ig1&2 intravenously (days 12 to 24). (G) Clinical score and change in ankle thickness (n = 3; *P = 0.0135, **P = 0.0058, two-tailed unpaired t test). (H) Mice were injected with RediJect Inflammation Probe on day 20 of arthritis, and luminescence in each ankle joint was measured. Graphs represent mean ± SEM (n = 6; *P = 0.0260, Mann-Whitney).

  • Fig. 7. RPTPσ Ig1&2 decreases FLS attachment to cartilage in vivo.

    (A) BALB/c mice were induced with acute K/BxN arthritis and treated with vehicle or 0.5 mg of Ig1&2 intravenously daily (days 0 to 6). Pathology of safranin O–stained ankle sections from mice on day 14 of arthritis. Arrow indicates inflammation; arrowhead indicates bone erosion. FLS crawling over cartilage (arrow) (yellow insets) and reduced chondrocyte layer (red insets) in (left) vehicle-treated versus (right) Ig1&2-treated mice. Cartilage PG content (red insets) assessed by safranin O staining. (B) SCID mice were implanted subcutaneously with cartilage explants and RA FLS in surgical sponge and treated with BSA or Ig1&2 intravenously for 35 days. (Left) Representative images showing cartilage (C) and sponge (Sp). Arrows indicate FLS attaching to and invading cartilage. (Right) Mean ± SEM invasion scores; n = 3 mice per group (n = 30 fields; ****P < 0.0001, Mann-Whitney).

  • Fig. 8. Working model for the RPTPσ-dependent PG switch in FLS.

    RPTPσ interacts with the HS PG syndecan-4 on the surface of FLS and is maintained in an inactive oligomeric state. Tyrosine phosphorylation of ezrin downstream of the PDGFR promotes its localization to the actin cytoskeleton, enabling cell migration and invasion. Disruption of the RPTPσ-HS interaction by the HS-binding decoy RPTPσ Ig1&2 fragment displaces RPTPσ from HS. This leads to dephosphorylation of ezrin and disassociation of ezrin from the actin cytoskeleton, decreasing FLS migration, invasion, and attachment to cartilage.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/288/288ra76/DC1

    Materials and Methods

    Fig. S1. Expression of PTPRS in OA and RA FLS.

    Fig. S2. Reduction of surface HS inhibits migration of FLS in an RPTPσ-dependent manner.

    Fig. S3. Knockdown of PTPRS in RA FLS.

    Fig. S4. RPTPσ Ig1&2 does not affect FLS survival.

    Fig. S5. RPTPσ Ig1&2 treatment does not affect FLS response to cytokine stimulation.

    Fig. S6. RPTPσ Ig1&2 decreases cell attachment to fibronectin.

    Fig. S7. Knockdown of SDC2/GPC1 and SDC4 in RA FLS.

    Fig. S8. Reconstitution of Ptprs KO FLS with RPTPσ mutants.

    Fig. S9. RPTPσ Ig1&2 does not affect paxillin, FAK, or p130Cas phosphorylation in FLS.

    Fig. S10. RPTPσ Ig1&2 decreases colocalization of ezrin with the actin cytoskeleton during migration.

    Fig. S11. RPTPσ Ig1&2 does not alter β-catenin or cadherin-11 phosphorylation and does not affect cadherin-11 functions in FLS.

    Fig. S12. RPTPσ Ig1&2 does not affect PDGFR expression or downstream Akt/MAPK signaling in FLS.

    Fig. S13. Ptprs deficiency does not affect severity of K/BxN acute serum transfer arthritis.

    Fig. S14. Expression of R2A subclass of PTPs in FLS.

    Fig. S15. RPTPσ Ig1&2 does not affect macrophage response to stimulation.

    Fig. S16. Blockade of peripheral nervous system arms does not affect arthritis in the K/BxN model.

    Fig. S17. RPTPσ Ig1&2 does not affect K/BxN serum–induced vascular permeabilization of peripheral joints.

    Table S1. Source data for Fig. 1 (A, B, and D).

    Table S2. Source data for fig. S1.

    Table S3. Source data for fig. S4.

    Table S4. Source data for fig. S5.

    Table S5. Source data for Fig. 2H.

    Table S6. Source data for fig. S6.

    Table S7. Source data for Fig. 3 (B and C).

    Table S8. Source data for Fig. 4A.

    Table S9. Source data for fig. S7 (A and B).

    Table S10. Source data for Fig. 5B.

    Table S11. Source data for Fig. 6 (A to H).

    Table S12. Source data for fig. S13A.

    Table S13. Source data for fig. S14.

    Table S14. Source data for fig. S15.

    Table S15. Source data for fig. S16 (A to C).

    Table S16. Source data for fig. S17.

    Source Data 1. Full Western blots (Fig. 1E).

    Source Data 2. Full Western blots (Fig. 4, E and F).

    Source Data 3. Full Western blots (Fig. 5, B to D).

    Source Data 4. Flow cytometry data (fig. S2A).

    Source Data 5. Flow cytometry gating (fig. S4).

    Source Data 6. Flow cytometry gating (fig. S8).

    Source Data 7. Full Western blots (fig. S9).

    Source Data 8. Full Western blots (fig. S11).

    Source Data 9. Full Western blots (fig. S12).

    References (3644)

  • Supplementary Material for:

    Targeting phosphatase-dependent proteoglycan switch for rheumatoid arthritis therapy

    Karen M. Doody, Stephanie M. Stanford, Cristiano Sacchetti, Mattias N. D. Svensson, Charlotte H. Coles, Nikolaos Mitakidis, William B. Kiosses, Beatrix Bartok, Camille Fos, Esther Cory, Robert L. Sah, Ru Liu-Bryan, David L. Boyle, Heather A. Arnett, Tomas Mustelin, Maripat Corr, Jeffrey D. Esko, Michel L. Tremblay, Gary S. Firestein, A. Radu Aricescu, Nunzio Bottini*

    *Corresponding author. E-mail: nunzio{at}lji.org

    Published 20 May 2015, Sci. Transl. Med. 7, 288ra76 (2015)
    DOI: 10.1126/scitranslmed.aaa4616

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Expression of PTPRS in OA and RA FLS.
    • Fig. S2. Reduction of surface HS inhibits migration of FLS in an RPTPσ-dependent manner.
    • Fig. S3. Knockdown of PTPRS in RA FLS.
    • Fig. S4. RPTPσ Ig1&2 does not affect FLS survival.
    • Fig. S5. RPTPσ Ig1&2 treatment does not affect FLS response to cytokine stimulation.
    • Fig. S6. RPTPσ Ig1&2 decreases cell attachment to fibronectin.
    • Fig. S7. Knockdown of SDC2/GPC1 and SDC4 in RA FLS.
    • Fig. S8. Reconstitution of Ptprs KO FLS with RPTPσ mutants.
    • Fig. S9. RPTPσ Ig1&2 does not affect paxillin, FAK, or p130Cas phosphorylation in FLS.
    • Fig. S10. RPTPσ Ig1&2 decreases colocalization of ezrin with the actin cytoskeleton during migration.
    • Fig. S11. RPTPσ Ig1&2 does not alter β-catenin or cadherin-11 phosphorylation and does not affect cadherin-11 functions in FLS.
    • Fig. S12. RPTPσ Ig1&2 does not affect PDGFR expression or downstream Akt/MAPK signaling in FLS.
    • Fig. S13. Ptprs deficiency does not affect severity of K/BxN acute serum transfer arthritis.
    • Fig. S14. Expression of R2A subclass of PTPs in FLS.
    • Fig. S15. RPTPσ Ig1&2 does not affect macrophage response to stimulation.
    • Fig. S16. Blockade of peripheral nervous system arms does not affect arthritis in the K/BxN model.
    • Fig. S17. RPTPσ Ig1&2 does not affect K/BxN serum–induced vascular permeabilization of peripheral joints.
    • References (3644)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Source data for Fig. 1 (A, B, and D).
    • Table S2 (Microsoft Excel format). Source data for fig. S1.
    • Table S3 (Microsoft Excel format). Source data for fig. S4.
    • Table S4 (Microsoft Excel format). Source data for fig. S5.
    • Table S5 (Microsoft Excel format). Source data for Fig. 2H.
    • Table S6 (Microsoft Excel format). Source data for fig. S6.
    • Table S7 (Microsoft Excel format). Source data for Fig. 3 (B and C).
    • Table S8 (Microsoft Excel format). Source data for Fig. 4A.
    • Table S9 (Microsoft Excel format). Source data for fig. S7 (A and B).
    • Table S10 (Microsoft Excel format). Source data for Fig. 5B.
    • Table S11 (Microsoft Excel format). Source data for Fig. 6 (A to H).
    • Table S12 (Microsoft Excel format). Source data for fig. S13A.
    • Table S13 (Microsoft Excel format). Source data for fig. S14.
    • Table S14 (Microsoft Excel format). Source data for fig. S15.
    • Table S15 (Microsoft Excel format). Source data for fig. S16 (A to C).
    • Table S16 (Microsoft Excel format). Source data for fig. S17.
    • Source Data 1 (.pdf format). Full Western blots (Fig. 1E).
    • Source Data 2 (.pdf format). Full Western blots (Fig. 4, E and F).
    • Source Data 3 (.pdf format). Full Western blots (Fig. 5, B to D).
    • Source Data 4 (.pdf format). Flow cytometry data (fig. S2A).
    • Source Data 5 (.pdf format). Flow cytometry gating (fig. S4).
    • Source Data 6 (.pdf format). Flow cytometry gating (fig. S8).
    • Source Data 7 (.pdf format). Full Western blots (fig. S9).
    • Source Data 8 (.pdf format). Full Western blots (fig. S11).
    • Source Data 9 (.pdf format). Full Western blots (fig. S12).

    [Download Data Files S1 to S9]

    [Download Tables S1 to S16]

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