Tendon-derived cathepsin K–expressing progenitor cells activate Hedgehog signaling to drive heterotopic ossification
Heng Feng, … , Qing Bi, Weiguo Zou
Heng Feng, … , Qing Bi, Weiguo Zou
Published August 27, 2020
Citation Information: J Clin Invest. 2020;130(12):6354-6365. https://doi.org/10.1172/JCI132518.
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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.

Authors

Heng Feng, Wenhui Xing, Yujiao Han, Jun Sun, Mingxiang Kong, Bo Gao, Yang Yang, Zi Yin, Xiao Chen, Yun Zhao, Qing Bi, Weiguo Zou

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Figure 3

Ctsk-Cre labels a unique subpopulation of TDPCs.

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Ctsk-Cre labels a unique subpopulation of TDPCs. (A) Fluorescence imagi...
(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.