NFAT restricts osteochondroma formation from entheseal progenitors
Xianpeng Ge, Kelly Tsang, Lizhi He, Roberto A. Garcia, Joerg Ermann, Fumitaka Mizoguchi, Minjie Zhang, Bin Zhou, Bin Zhou, Antonios O. Aliprantis
Xianpeng Ge, Kelly Tsang, Lizhi He, Roberto A. Garcia, Joerg Ermann, Fumitaka Mizoguchi, Minjie Zhang, Bin Zhou, Bin Zhou, Antonios O. Aliprantis
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Research Article Bone biology

NFAT restricts osteochondroma formation from entheseal progenitors

Abstract

Osteochondromas are common benign osteocartilaginous tumors in children and adolescents characterized by cartilage-capped bony projections on the surface of bones. These tumors often cause pain, deformity, fracture, and musculoskeletal dysfunction, and they occasionally undergo malignant transformation. The pathogenesis of osteochondromas remains poorly understood. Here, we demonstrate that nuclear factor of activated T cells c1 and c2 (NFATc1 and NFATc2) suppress osteochondromagenesis through individual and combinatorial mechanisms. In mice, conditional deletion of NFATc1 in mesenchymal limb progenitors, Scleraxis-expressing (Scx-expressing) tendoligamentous cells, or postnatally in Aggrecan-expressing cells resulted in osteochondroma formation at entheses, the insertion sites of ligaments and tendons onto bone. Combinatorial deletion of NFATc1 and NFATc2 gave rise to larger and more numerous osteochondromas in inverse proportion to gene dosage. A population of entheseal NFATc1- and Aggrecan-expressing cells was identified as the osteochondroma precursor, previously believed to be growth plate derived or perichondrium derived. Mechanistically, we show that NFATc1 restricts the proliferation and chondrogenesis of osteochondroma precursors. In contrast, NFATc2 preferentially inhibits chondrocyte hypertrophy and osteogenesis. Together, our findings identify and characterize a mechanism of osteochondroma formation and suggest that regulating NFAT activity is a new therapeutic approach for skeletal diseases characterized by defective or exaggerated osteochondral growth.

Authors

Xianpeng Ge, Kelly Tsang, Lizhi He, Roberto A. Garcia, Joerg Ermann, Fumitaka Mizoguchi, Minjie Zhang, Bin Zhou, Bin Zhou, Antonios O. Aliprantis

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Figure 7

Ablation of nuclear factor of activated T cells c1 or c2 (NFATc1 or NFATc2) differentially affects proliferation of entheseal progenitors.

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Ablation of nuclear factor of activated T cells c1 or c2 (NFATc1 or NFAT...
(A) Schematic for isolating GFP+ cells from the tibial medial collateral ligament (MCL) entheses of mice with indicated genotypes 1 week after tamoxifen. GFP+ cells were flow-sorted after 7 days of ex vivo expansion. (B and C) Cells from Nfatc1-KO;mTmG mice displaying increased proliferation as measured by cell count (B, n = 3) and alamarBlue reduction (C, n = 6). (D) Representative anti–proliferating cell nuclear antigen (PCNA) IHC and quantification of PCNA+ cells at the tibial entheses of Nfatc1fl/flNfatc2+/– and Nfatc1AggCreERNfatc2+/– mice 2 weeks after tamoxifen (n = 3 animals). Scale bars: 100 μm. (E) Cell proliferation assay of ATDC5 lines lacking Nfatc1, Nfatc2, or both under nondifferentiating conditions (n = 3 per sample). (F and G) Cell proliferation analyses by alamarBlue assay (F, n = 8 per sample), micromass diameter (G, n = 10 per sample), and representative micromass images after 2 weeks of micromass culture in chondrogenic media. Original magnification for the image in G: ×1. (H) Expression of cell cycle genes in ATDC5 cells overexpressing constitutively active NFATc1 (caNFATc1) by real-time PCR (n = 3 independent infections per sample). (I) Expression of c-Myc and P21 genes in ATDC5 lines lacking Nfatc1, Nfatc2, or both (n = 6 per sample). Representative results of 3 independent experiments are presented. Two-way ANOVA followed by Tukey’s (in B and E) or Sidak’s (in H) tests were performed. P values in C, F, G, and I were determined by 1-way ANOVA followed by Tukey’s tests. Two-tailed Student’s t tests were performed for D.