Tendon-derived cathepsin K-expressing progenitor cells activate Hedgehog signaling to drive heterotopic ossification

Abstract

Heterotopic ossification (HO) is pathological bone formation characterized by ossification within muscle, tendons, or other soft tissues. However, the cells of origin and mechanisms involved in the pathogenesis of HO remain elusive. Here we show that deletion of suppressor of fused (Sufu) in cathepsin K-Cre-expressing (Ctsk-Cre-expressing) cells resulted in spontaneous and progressive ligament, tendon, and periarticular ossification. Lineage tracing studies and cell functional analysis demonstrated that Ctsk-Cre could label a subpopulation of tendon-derived progenitor cells (TDPCs) marked by the tendon marker Scleraxis (Scx). Ctsk+Scx+ TDPCs are enriched for tendon stem cell markers and show the highest self-renewal capacity and differentiation potential. Sufu deficiency caused enhanced chondrogenic and osteogenic differentiation of Ctsk-Cre-expressing tendon-derived cells via upregulation of Hedgehog (Hh) signaling. Furthermore, pharmacological intervention in Hh signaling using JQ1 suppressed the development of HO. Thus, our results show that Ctsk-Cre labels a subpopulation of TDPCs contributing to HO and that their cell-fate changes are driven by activation of Hh signaling.

Keywords: Bone disease; Mouse models; Stem cells; Therapeutics.

Figure 1. Sufu deletion in Ctsk-Cre–expressing cells induces spontaneous HO in the ligament and tendon.

(A) X-ray images and μCT scans reveal that spontaneous ossification (red arrows) of the tibiotalar ligament and Achilles tendon progresses gradually in Ctsk-CKO mice at 4, 9, and 20 weeks. Images are representative of 3 different sets of experiments. n = 3 per group. (B) H&E staining, Safranin O/fast green (SOFG) staining, and immunofluorescence staining of the chondrogenic marker type II collagen (COLII) and the osteogenic marker osteopontin (OPN) of the Achilles tendon from 20-week-old Ctsk-Ctrl and Ctsk-CKO mice. Images are representative of 3 different sets of experiments. Scale bars: 100 μm (2 left panels); 20 μm (8 right panels).

Figure 2. Ctsk-Cre–expressing cells in tendons and ligaments contribute to HO.

(A) Schematic of the Ctsk-Cre Rosa26-Ai9 reporter mice. (B) Confocal images of hind limbs from Ctsk-Cre Rosa26-Ai9 mice showing Ctsk expression in the cells of the Achilles tendon, patellar tendon, and quadriceps tendon. Scale bars: 50 μm. (C) Immunofluorescence showing Ctsk⁺ (Ai9⁺) cells in the Achilles tendon of 6-week-old Ctsk-CKO Rosa26-Ai9 mice displays the chondrogenic marker COLII (green) and the osteogenic marker OPN (green). Scale bars: 50 μm (left panels); 25 μm (right panels). (D) Quantitative analysis of the percentage of Ctsk⁺ (Ai9⁺) cells that express the chondrogenic marker COLII in Achilles tendons. n = 4 per group. (E) Quantitative analysis of the percentage of Ctsk⁺ (Ai9⁺) cells that express the osteogenic marker OPN in Achilles tendons. n = 4 per group. (F–I) qRT-PCR analysis of Hh target gene (F), chondrogenic gene (G), osteogenic gene (H), and tendon-related gene (I) expression in sorted Ctsk⁺ Achilles tendon cells from 6-week-old Ctsk-CKO Rosa26-Ai9 mice compared with Ctsk-Ctrl Rosa26-Ai9 mice. n = 3 per group. (J and K) Immunofluorescence showing Ctsk⁺Scx⁺ (orange) cells in the Achilles tendon of 6-week-old Ctsk-CKO Rosa26-Ai9 ScxGFP mice displays the chondrogenic marker COLII and aggrecan (gray). Scale bars: 20 μm. Results are presented as the mean ± SEM; unpaired t test, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3. Ctsk-Cre labels a unique subpopulation of TDPCs.

(A) Fluorescence imaging of the Achilles tendon of 2-week-old Ctsk-Cre Rosa26-Ai9 ScxGFP mice. Scale bars: 50 μm. (B) FACS analysis of percentages of Ctsk⁺Scx⁺, Ctsk^–Scx⁺, and Ctsk⁺Scx^– tendon-derived cells from the Achilles tendon of 4-week-old Ctsk-Cre Rosa26-Ai9 ScxGFP mice. Images are representative of 3 independent experiments. (C) FACS analysis of stem cell surface markers of Ctsk⁺Scx⁺, Ctsk^–Scx⁺, and Ctsk⁺Scx^– tendon-derived cells from the Achilles tendon of 4-week-old Ctsk-Cre Rosa26-Ai9 ScxGFP mice. Images are representative of 3 independent experiments. (D) Colony formation assay using Ctsk^–Scx^–, Ctsk⁺Scx^–, Ctsk^–Scx⁺, and Ctsk⁺Scx⁺ tendon-derived cells from the Achilles tendon of 4-week-old Ctsk-Cre Rosa26-Ai9 ScxGFP mice. Colonies were stained with crystal violet after 7 days of culture (n= 5). (E) Colony numbers were counted, and colony-formation efficiencies in each group are shown (n= 5). Data are presented as the mean ± SEM; 1-way ANOVA with Tukey’s test was used for comparison of multiple groups; ***P < 0.001. (F) Fluorescence imaging of the cultured Ctsk⁺Scx⁺ TDPCs after 96 hours with or without TGF-β3. (G) In vitro differentiation of sorted Ctsk⁺Scx⁺ TDPCs into osteogenic lineage (3 weeks) by alizarin red S staining, adipogenic lineage (1 week) by Oil Red O staining, and chondrogenic lineage (2 weeks) by Alcian blue and Safranin O staining.

Figure 4. Sufu-deficient tendon-derived cells exhibit enhanced chondrogenic and osteogenic differentiation.

(A) Chondrogenic differentiation of high-density sorted Achilles tendon cells from 4-week-old Ctsk-CKO Rosa26-Ai9 mice and Ctsk-Ctrl Rosa26-Ai9 mice cultured in chondrogenic medium was assessed by Alcian blue staining at day 10 (n = 4). (B) qRT-PCR analysis of gene markers of chondrogenesis. (C) Osteogenic differentiation analysis of Sufu-deficient tendon-derived cells and control cells by ALP staining (1 week) and alizarin red S staining (2 weeks) (n = 5). Insets represent the fluorescence signals of cells after differentiation. (D) Quantification of ALP activity relative to cell numbers. n = 5 per group. (E and F) Osteogenic differentiation of sorted Achilles tendon cells from 4-week-old Ctsk-CKO Rosa26-Ai9 mice and Ctsk-Ctrl Rosa26-Ai9 mice. Gene expression analysis of markers of Hh signaling and osteogenesis by qRT-PCR. (G) TUNEL assays revealed apoptosis of the Achilles tendon from 4-week-old Ctsk-CKO Rosa26-Ai9 mice and control mice. n = 3 per group. DNase I was used as positive control. Scale bars: 50 μm. Results are presented as the mean ± SEM; unpaired t test, **P < 0.01, ***P < 0.001.

Figure 5. Gli1/Gli2 deficiency prevents HO progression in Ctsk-CKO mice.

(A) X-ray and μCT radiographs show HO formation of the Achilles tendon of Ctsk-Ctrl, Gli1^lacZ/lacZ, Ctsk-Cre Gli2^fl/fl, Ctsk-CKO, Ctsk-CKO Gli1^lacZ/lacZ, and Ctsk-CKO Gli2^fl/fl mice. (B) Quantitative analysis of the HO volume around the ankles of Ctsk-Ctrl (n = 4), Gli1^lacZ/lacZ (n = 4), Ctsk-Cre Gli2^fl/fl (n = 4), Ctsk-CKO (n = 6), Ctsk-CKO Gli1^lacZ/lacZ (n = 6), and Ctsk-CKO Gli2^fl/fl (n = 5) mice. (C) SOFG staining shows HO of the Achilles tendon of Ctsk-Ctrl, Gli1^lacZ/lacZ, Ctsk-Cre Gli2^fl/fl, Ctsk-CKO, Ctsk-CKO Gli1^lacZ/lacZ, and Ctsk-CKO Gli2^fl/fl mice. Scale bars: 100 μm. (D) Immunofluorescence staining of the osteogenic marker OCN in the Achilles tendon of Ctsk-Ctrl, Gli1^lacZ/lacZ, Ctsk-Cre Gli2^fl/fl, Ctsk-CKO, Ctsk-CKO Gli1^lacZ/lacZ, and Ctsk-CKO Gli2^fl/fl mice. Scale bars: 50 μm. Data are presented as the mean ± SEM; 1-way ANOVA with Tukey’s test was used for comparison of multiple groups; **P < 0.01, ***P < 0.001.

Figure 6. Pharmacological inhibition of Hh signaling by JQ1 ameliorates HO progression in Ctsk-CKO mice.

(A) Alcian blue staining of high-density sorted Achilles tendon cells from 4-week-old Ctsk-CKO Rosa26-Ai9 mice and Ctsk-Ctrl Rosa26-Ai9 mice cultured in chondrogenic medium treated with Gant61 and JQ1 at day 10 (n = 4). (B) qRT-PCR analysis of gene markers of chondrogenesis. (C) ALP staining (day 7) and alizarin red S staining (day 14) of sorted Ctsk⁺ cells from the Achilles tendon of Ctsk-CKO Rosa26-Ai9 mice and Ctsk-Ctrl Rosa26-Ai9 mice treated with Gant61 and JQ1 in osteogenic medium for 14 days (n = 4). Insets represent the fluorescence signals of the cells after differentiation. (D) Gene expression of markers of Hh signaling and osteogenic differentiation after Gant61 and JQ1 treatment. (E) μCT radiographs showed the HO of Ctsk-Ctrl mice with vehicle treatment (n = 3), Ctsk-Ctrl mice with JQ1 treatment (n = 4), Ctsk-CKO mice with vehicle treatment (n = 6), and Ctsk-CKO mice with JQ1 treatment (n = 6). (F) Quantitative analysis of the whole HO volume of Ctsk-Ctrl mice treated with vehicle (n = 3), Ctsk-Ctrl mice treated with JQ1 (n = 4), Ctsk-CKO mice treated with vehicle (n = 6), and Ctsk-CKO mice treated with JQ1 (n = 6). (G) SOFG staining of the Achilles tendon of Ctsk-Ctrl and Ctsk-CKO mice with JQ1 or vehicle treatment. Images are representative of 4 different sets of experiments. Scale bars: 100 μm. Data are presented as the mean ± SEM; 1-way ANOVA with Tukey’s test was used for comparison of multiple groups; *P < 0.05, **P < 0.01, ***P < 0.001.