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. 2013 Jul 25;499(7459):491-5.
doi: 10.1038/nature12396. Epub 2013 Jul 17.

Ptpn11 deletion in a novel progenitor causes metachondromatosis by inducing hedgehog signalling

Wentian Yang  1 , Jianguo Wang, Douglas C Moore, Haipei Liang, Mark Dooner, Qian Wu, Richard Terek, Qian Chen, Michael G Ehrlich, Peter J Quesenberry, Benjamin G Neel
Affiliations

Ptpn11 deletion in a novel progenitor causes metachondromatosis by inducing hedgehog signalling

Wentian Yang et al. Nature. .

Abstract

The tyrosine phosphatase SHP2, encoded by PTPN11, is required for the survival, proliferation and differentiation of various cell types. Germline activating mutations in PTPN11 cause Noonan syndrome, whereas somatic PTPN11 mutations cause childhood myeloproliferative disease and contribute to some solid tumours. Recently, heterozygous inactivating mutations in PTPN11 were found in metachondromatosis, a rare inherited disorder featuring multiple exostoses, enchondromas, joint destruction and bony deformities. The detailed pathogenesis of this disorder has remained unclear. Here we use a conditional knockout (floxed) Ptpn11 allele (Ptpn11(fl)) and Cre recombinase transgenic mice to delete Ptpn11 specifically in monocytes, macrophages and osteoclasts (lysozyme M-Cre; LysMCre) or in cathepsin K (Ctsk)-expressing cells, previously thought to be osteoclasts. LysMCre;Ptpn11(fl/fl) mice had mild osteopetrosis. Notably, however, CtskCre;Ptpn11(fl/fl) mice developed features very similar to metachondromatosis. Lineage tracing revealed a novel population of CtskCre-expressing cells in the perichondrial groove of Ranvier that display markers and functional properties consistent with mesenchymal progenitors. Chondroid neoplasms arise from these cells and show decreased extracellular signal-regulated kinase (ERK) pathway activation, increased Indian hedgehog (Ihh) and parathyroid hormone-related protein (Pthrp, also known as Pthlh) expression and excessive proliferation. Shp2-deficient chondroprogenitors had decreased fibroblast growth factor-evoked ERK activation and enhanced Ihh and Pthrp expression, whereas fibroblast growth factor receptor (FGFR) or mitogen-activated protein kinase kinase (MEK) inhibitor treatment of chondroid cells increased Ihh and Pthrp expression. Importantly, smoothened inhibitor treatment ameliorated metachondromatosis features in CtskCre;Ptpn11(fl/fl) mice. Thus, in contrast to its pro-oncogenic role in haematopoietic and epithelial cells, Ptpn11 is a tumour suppressor in cartilage, acting through a FGFR/MEK/ERK-dependent pathway in a novel progenitor cell population to prevent excessive Ihh production.

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Conflict of interest statement

Competing interests statement

W.Y. and B.G. N. have filed a provisional patent application on the use of Smoothened inhibitors for the treatment of metachondromatosis (Title: Hedgehog Pathway Inhibition for Cartilage Tumor and Metachondromatosis Treatment; UPA#61/614,449).

Figures

Figure 1
Figure 1
Ptpn11 deletion in Cathepsin K-expressing cells causes metachondromatosis. a, Schemes for generating Ctsk-KO, LysM-KO, and Control mice. Gross images (b) and Faxitron/μ-CT radiographs (c) of 12 week-old Ctsk-KO mice showing dwarfism and scoliosis (b.ii, white arrow; c.ii, black arrow), increased bone mineral density (c.ii,iv) and multiple exostoses of knees, ankles, and metatarsals (b.iv; c.ii,iv,vi,viii; arrowheads) with joint destruction. d, Sagittal sections of metatarsal joints stained with H&E (i–iii), Safranin O (iv–vi) and Alcian blue (vii–ix) showing cartilaginous exostoses and enchondromas (arrows) in Ctsk-KO mice. Images in iii, vi and ix are magnified (10X) views of boxed areas in ii, iv and viii, respectively. Data shown are representative images; each analysis was performed on at least 5 mice/genotype.
Figure 2
Figure 2
Skeletal tumors in Ctsk-KO mice originate from Perichondrial Groove of Ranvier cells. a, X-gal staining of knee joint sections from 1-week-old R26LSLlacZ;Ctsk-Cre and R26LSLlacZ;LysM-Cre reporter mice showing that the Ctsk (but not the LysM) promoter is active not only in osteoclasts, but also in a subset of cells from the Perichondrial Groove of Ranvier (arrows). b, H&E and Safranin O staining of knee joint sections from P10 Ctsk-Control (i,iv) and Ctsk-KO (ii,iii,v,vi) mice showing expansion of cells within the Perichondrial Groove of Ranvier region in Ctsk-KO mice. Images in iii & vi are magnified (10x) views of boxed areas in ii & v respectively; c, H&E-and Safranin O-stained sections showing expanding YFP+ population within the Perichondrial Groove of Ranvier (boxed region in top panels, magnified below) that also stains with Safranin O, indicative of cartilage. Dashed line marks boundary between marrow/growth plate and perichondrial groove. d, frozen section of an exostosis from the metatarsal joint of Ctsk-KO/YFP mice showing co-localization of YFP reporter with cartilaginous tumor cells (boxed area). Note that the lesion is enriched in proliferating and pre-hypertrophic chondrocytes, as shown by overlapping Col2α1 and Col10α1 immunostaining. Each panel is a representative image from one mouse; each analysis was performed on at least 3 mice/genotype.
Figure 3
Figure 3
Ptpn11 deletion in Ctsk-expressing cells causes expansion of novel chondroprogenitor cell population within the Perichondrial Groove of Ranvier. a, Flow cytometric analysis showing YFP+ cells from pooled epiphyseal cartilage from 5–7 Ctsk-Control/YFP mice; note increased percentage of such cells in 2-week-old Ctsk-KO/YFP mice. CC: Chondroid cells. b, Flow cytometric analysis of YFP+ perichondrial cells showing staining for CD31, CD44, CD90, and CD166. Data in panels a and b are from a single experiment; similar results were obtained in 2 additional experiments. c, Immunofluorescence micrograph showing Stro1 and Jagged1 expression in YFP+ perichondrial cells. Nuclei are stained with DAPI. Note enhanced intensity of Stro1 and Jagged staining in Ctsk-KO cells. Data shown are from single mice of each genotype; two additional mice were analyzed for each genotype with similar results. d, CCPs give rise to cartilage, fat and bone. FACS-purified YFP+ cells from 5–7 mice were subjected to differentiation assays in triplicate. After 2–3 weeks of culture (see Methods), cells were fixed and stained with Alcian blue, Oil red, and Alizarin red to visualize the formation of cartilage, fat, and bone tissue, respectively.
Figure 4
Figure 4
Shp2 deficiency impairs Erk activation but promotes Ihh and Pthrp expression. a, (Left panel) qRT-PCR showing increased Col2α1, Col10α1, Ihh, and Pthrp expression in laser-captured cartilaginous cells from exostoses in 4 mice/genotype, compared with normal articular cartilage cells (mean±S.D; *p<0.05, 2-tailed Student’s t test). (Right panel) Immunostaining of representative paraffin sections from Perichondrial Groove of Ranvier region of Ctsk-KO and Control mice. Note the decreased number of p-Erk+ cells (75.4% in Ctsk-Control vs 32.2% in Ctsk-KO; n=3 mice), but increased Ihh expression in Ctsk-KO, compared with Control, mice. b, (Left Panel) Immunoblot showing Shp2 in ATDC5 cells stably expressing shRNAs against mouse Ptpn11 (ATDC5-KD1, ATDC5-KD2, respectively) or scrambled control hairpin. (Right panels). Representative blot showing that Shp2 deficiency decreases Erk activation in response to Fgf18 (top); data from multiple experiments (n=3) showing pErk levels (compared with control at 5 minutes, mean±S.D.; p<0.05, 2-tailed Student’s t test) are quantified below. qRT-PCR (bottom left) shows increased Ihh and Pthrp expression in Shp2-deficient ATDC5 cells (mean ± S.D.; n=3, *p<0.05, 2-tailed Student’s t test). c, FGFR (PD173074, 10nM) or MEK (UO126, 1μM) inhibitor treatment of parental ATDC5 cells enhances Ihh and Pthrp expression, as shown by qRT-PCR (mean±S.D; n=3, *p<0.05, 2-tailed Student’s t test). d, Faxitron radiographs showing that Hedgehog pathway blockade following administration of the Smoothened inhibitor PF-04449913 (100μg/g body weight) to Ctsk-KO mice ameliorates tumor formation, compared with vehicle control (0.5% methylcellulose)-treated mice. Images of representative posterior paws (i–iv) and knees (v–vii) taken pre- (i,iii,v,vii) and post-treatment with Vehicle (ii,vi) or Smoothened inhibitor (SMOi) (iv,viii) for 4 weeks. Note continued development of exostoses and endochromas in Vehicle-treated mice, and their amelioration in SMOi-treated group (arrows). Also, see Figs. S4–S7 and Supplemental video clips 1,2.
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Comment in

  • Cell biology: tumour stem cells in bone.
    Zaidi M, Méndez-Ferrer S. Zaidi M, et al. Nature. 2013 Jul 25;499(7459):414-6. doi: 10.1038/nature12412. Epub 2013 Jul 17. Nature. 2013. PMID: 23863941 Free PMC article. No abstract available.

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