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 5

Aggrecan- and nuclear factor of activated T cells c1–expressing (NFATc1-expressing) cells at the enthesis are precursors of osteochondromas.

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Aggrecan- and nuclear factor of activated T cells c1–expressing (NFATc1-...
(A) Fluorescent images of frozen sections of the knee joints from Nfatc1AggCreERNfatc2–/–;Rosa26-mTmGfl/+ (DKO;mTmG) mice 1 month after tamoxifen showing the majority of cells in osteochondromas are GFP+ (left panel). The image of higher magnification (right panel) reveals that cells without Cre recombination (white arrowheads) are also present. (B) Fluorescent images of knee joints from Nfatc1Cre;mTmG mice displaying Nfatc1 not expressed in growth plate (GP) cells. (C) Illustration showing tamoxifen-induced Cre-mediated recombination of the red fluorescence protein (RFP) reporter allele in Nfatc1+ cells from Nfatc1-CreERT2;Rosa26-RFPfl/+ (Nfatc1CreER;RFP) mice leading to RFP expression (left panel). RFP fluorescence not detectable in GP cells 48 hours after tamoxifen pulse in 8-week-old Nfatc1CreER;RFP mice (right panel). (D) Fluorescent images of knee joints showing Nfatc1 expression in cells of the tibial medial collateral ligament (MCL) entheses and the perichondrium (PC)/periosteum (PO). (E) Fluorescent images of knee joints from AggCreER;mTmG and DKO;mTmG mice demonstrating cells in the periosteum adjacent to the MCL enthesis do not express Aggrecan (right panel, white arrowheads). GFP+ cells in left panel are in continuity with those at the MCL enthesis. OCM, osteochondroma. Images are representative of 4 or 5 mice per genotype except C, in which 2 mice are used. (F) Schematics showing a subpopulation of cells at the tibial MCL enthesis expressing both Nfatc1 and Aggrecan. (G–I) Oil Red O, Alizarin Red S, and Safranin O staining, as well as quantitative PCR (qPCR) analyses, demonstrating adipogenic, osteogenic, and chondrogenic capacities of Aggrecan+ cells cultured ex vivo under lineage-specific differentiating conditions. (J) Flow cytometric analysis of stem cell (Sca1, CD90, and CD44), hematopoietic cell (CD45), and endothelial cell (CD31) markers in Aggrecan+ cells from AggCreER;mTmG mice. n = 3 animals per group. Scale bars: 100 μm (except in A, right panel, 25 μm).