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JAK inhibition increases bone mass in steady-state conditions and ameliorates pathological bone loss by stimulating osteoblast function

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Science Translational Medicine  12 Feb 2020:
Vol. 12, Issue 530, eaay4447
DOI: 10.1126/scitranslmed.aay4447
  • Fig. 1 JAKi increases trabecular bone mass under steady-state conditions.

    C57BL/6 mice (5 weeks old) received tofacitinib (50 mg/kg) or 0.5% methylcellulose and 0.025% Tween solution as vehicle (control) once daily by oral gavage for 6 weeks. Trabecular (trab) bone phenotype was quantified by μCT, and serum was analyzed by ELISA. (A and B) μCT quantification of trabecular bone mass in tibiae and vertebrae. Trabecular bone volume/total volume (BV/TV), trabecular thickness, and trabecular numbers are shown. (C) Representative μCT images of trabecular bone in tibiae (top) and vertebrae (bottom). Arrows indicate regions of decreased trabecular density. Scale bars, 500 μm. (D) RANKL and OPG serum concentrations and RANKL/OPG ratio. Data are presented as means ± SEM. Statistical significance was calculated by Mann-Whitney U test (*P < 0.05, n = 6).

  • Fig. 2 JAKi mitigates OVX-induced bone loss.

    C57BL/6 (8 weeks old) mice were bilaterally ovariectomized or underwent sham surgery. Bone loss was allowed to establish for 2 weeks. Mice then started to receive tofacitinib (50 mg/kg), baricitinib (10 mg/kg), or vehicle (control) twice daily by oral gavage for 6 weeks. Trabecular (trab) bone phenotype was quantified by μCT, and serum was analyzed via ELISA. (A and B) μCT quantification of trabecular bone mass in tibiae and vertebrae. Trabecular bone volume/total volume, trabecular thickness, and trabecular numbers are shown. (C) Representative μCT images of trabecular bone in the tibiae (top, each) and vertebrae (bottom, each) of mice receiving vehicle, tofacitinib, or baricitinib. Arrows indicate regions of decreased trabecular density. Scale bars, 500 μm. (D) RANKL and OPG serum concentrations and RANKL/OPG ratio. Data are presented as means ± SEM. Statistical significance was calculated by Mann-Whitney U test (*P < 0.05, **P ≤ 0.01, and ***P ≤ 0.001, n = 5 to 14).

  • Fig. 3 JAKi halts bone loss in the context of arthritis.

    C57BL/6 mice (8 weeks old) induced for serum-transfer arthritis (STA) received treatment with tofacitinib (50 mg/kg), baricitinib (10 mg/kg), or vehicle [control (ctrl)] twice daily by oral gavage for 2 weeks. An uninduced group, receiving vehicle twice daily by oral gavage, accompanied the experiment. Animals were scored daily for clinical disease parameters. Trabecular (trab) and cortical (cort) bone phenotype was quantified by μCT. (A to C) Clinical arthritis score, ankle swelling, and grip strength (top) and quantification of area under the curve (bottom). (D and E) μCT quantification of trabecular and cortical bone phenotype in tibiae. (D) Trabecular bone volume/total volume (BV/TV), trabecular thickness, and connectivity density. (E) Cortical bone area/total cross-sectional area inside the periosteal envelope (Ct.Ar/Tt.Ar) and cortical thickness. (F) Representative μCT images of tibial trabecular bone in uninduced mice and STA mice receiving vehicle, tofacitinib, or baricitinib. Scale bars, 500 μm. Data are expressed as means ± SEM. Statistical significance was calculated by one-way ANOVA with Dunnett post hoc test (*P < 0.05, **P ≤ 0.01, and ***P ≤ 0.001, n = 7 to 11).

  • Fig. 4 JAKi does not affect osteoclastogenesis or osteoclastic bone resorption.

    Osteoclasts were differentiated from human peripheral blood mononuclear cells or murine bone marrow cells in the presence or absence of JAK inhibitors. Osteoclasts were stained for TRAP and quantified. Osteoclastic resorption capability was evaluated by culturing osteoclasts in calcium phosphate matrix–coated wells and by quantifying the remaining calcium phosphate matrix. (A and B) Human osteoclastogenesis assays showing (A) representative microphotographs and (B) quantification of osteoclast numbers in untreated (control), baricitinib-, or tofacitinib-treated conditions. TRAP+ cells appear purple. Scale bars, 500 μm (n = 3). (C to F) Murine osteoclastogenesis assays showing (C and E) representative microphotographs of (C) baricitinib- and (E) tofacitinib-treated samples, respectively, compared with the untreated control. TRAP+ cells appear purple. Scale bars, 500 μm. (D and F) Quantification of murine osteoclast numbers of (D) baricitinib- and (F) tofacitinib-treated conditions, respectively, compared with untreated control sample (n = 3 to 4). (G and H) Von Kossa staining of calcium phosphate matrix–coated wells after murine osteoclastogenesis. The black area is the remaining calcium phosphate matrix, and the white area indicates resorbed spots. (G) Representative images of untreated (control), baricitinib-, and tofacitinib-treated conditions. (H) Quantification of von Kossa staining (n = 8). Scale bars, 500 μm. Data are expressed as means ± SEM. Statistical significance was calculated by one-way ANOVA with Dunnett post hoc test.

  • Fig. 5 JAKi induces an anabolic network in osteoblasts supporting osteoblastogenesis.

    Osteoblastogenesis was induced in MSC- and calvaria-derived osteoblasts challenged with dimethyl sulfoxide (DMSO) vehicle control, 300 nM baricitinib (B), 500 nM tofacitinib (T), or as indicated. (A) Representative photographs and quantification of calcium nodules deposited by MSC-derived osteoblasts and stained with Alizarin Red after 5 to 6 days of osteogenic induction. Scale bars, 5 mm (n = 6). (B) Representative photographs and quantification of calcium nodules deposited by calvaria-derived osteoblasts and stained with Alizarin Red after 21 days of osteogenic induction. Scale bars, 5 mm (n = 8). (C) RNAseq analysis of MSC-derived osteoblasts 1 day after osteogenic induction. Minus-Average (MA) plots for baricitinib- and tofacitinib-treated MSCs compared with vehicle control. Red and gray dots show genes with adjusted P value below or above 0.1, respectively (n = 4). (D and E) During osteoblastogenesis of MSCs, intracellular protein content was analyzed by Western blot (n = 5 to 6). (D) Representative Western blot for P-STAT3 and STAT3, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as loading control (top) and quantification of P-STAT3 and STAT3 (bottom panels). (E) Representative Western blot for P-STAT1 and STAT1, P-STAT1–positive control (Pos Ctrl), with GAPDH as loading control (top) and quantification of P-STAT1 and STAT1 (bottom panels). (F) Ingenuity pathway analysis for upstream mediators: Shades of blue and orange indicate decreased and increased activation z score, respectively. Data are expressed as means ± SEM. Statistical significance was calculated by (A and B) repeated-measures ANOVA and (D and E) one-way ANOVA with Dunnett post hoc test (*P < 0.05, **P ≤ 0.01, and ***P ≤ 0.001).

  • Fig. 6 JAKi increases Wnt1 and β-catenin protein expression in osteoblasts and induces repair of bone erosions.

    Osteoblastogenesis was induced in MSC- and calvaria-derived osteoblasts, with DMSO vehicle control, 300 nM baricitinib (B), 500 nM tofacitinib (T), or as indicated. (A to C) qPCR analysis of gene expression at day 1 in MSC-derived osteoblasts (n = 4). (D and E) qPCR analysis of gene expression at day 21 in calvaria-derived osteoblasts (n = 6). (F to H) During osteoblastogenesis of MSCs, intracellular protein content was analyzed by Western blot. Representative Western blots for (F) β-catenin, (G) Wnt1, and (H) SOCS2 with GAPDH as loading control and corresponding quantification of protein quantity (n = 4 to 6). Data are expressed as means ± SEM. Statistical significance was calculated by (A to E) one-way ANOVA and (F to H) repeated-measures ANOVA, each with Dunnett post hoc test (*P < 0.05, **P ≤ 0.01, and ***P ≤ 0.001).

  • Fig. 7 JAKi by tofacitinib induces repair of bone erosions in patients with RA.

    3D HR-pQCT images of metacarpophalangeal joints were generated before (left, each) and after treatment (right, each) from patients with RA (n = 2) who received 5 mg of tofacitinib twice daily for (top) 4 years and (bottom) 2 years. Scale bars, 5 mm. Arrows indicate exemplary regions of erosion where bone formation occurred.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/530/eaay4447/DC1

    Fig S1. JAKi does not affect cortical bone parameters or bone cell numbers under steady-state conditions.

    Fig S2. Confirmation of OVX and cortical bone effects of JAKi after OVX.

    Fig S3. Effects of JAKi on arthritis-mediated bone loss.

    Fig S4. Gene network changes in MSCs during JAKi.

    Fig. S5. Proposed model for the role of JAKi by baricitinib and tofacitinib in osteoblasts.

    Table S1. Genetic targets and primers used for qPCR.

    Table S2. Protein targets, antibodies, and control extracts used for Western blot analysis.

    Data file S1. Raw data.

  • The PDF file includes:

    • Fig S1. JAKi does not affect cortical bone parameters or bone cell numbers under steady-state conditions.
    • Fig S2. Confirmation of OVX and cortical bone effects of JAKi after OVX.
    • Fig S3. Effects of JAKi on arthritis-mediated bone loss.
    • Fig S4. Gene network changes in MSCs during JAKi.
    • Fig. S5. Proposed model for the role of JAKi by baricitinib and tofacitinib in osteoblasts.
    • Table S1. Genetic targets and primers used for qPCR.
    • Table S2. Protein targets, antibodies, and control extracts used for Western blot analysis.

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    Other Supplementary Material for this manuscript includes the following:

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