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A Mouse Model of Chondrocyte-Specific Somatic

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A Mouse Model of Chondrocyte-Specific Somatic A mouse model of chondrocyte-specific somatic mutation reveals a role for Ext1 loss of heterozygosity in multiple hereditary exostoses Kazu Matsumotoa, Fumitoshi Iriea, Susan Mackemb, and Yu Yamaguchia,1 aSanford Children’s Health Research Center, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037; and bCancer and Developmental Biology Laboratory, National Cancer Institute, Frederick, MD 21702 Edited* by Erkki Ruoslahti, University of California, Santa Barbara, CA, and approved May 12, 2010 (received for review December 23, 2009) Multiple hereditary exostoses (MHE) is one of the most common The current consensus is that the copolymerase activity resides skeletal dysplasias, exhibiting the formation of multiple cartilage- in the EXT1 protein, and that the association of the EXT2 capped bony protrusions (osteochondroma) and characteristic protein with the EXT1 protein is necessary for the proper lo- bone deformities. Individuals with MHE carry heterozygous loss- calization of EXT1 in the Golgi apparatus (14, 15). Genetic of-function mutations in Ext1 or Ext2, genes which together encode ablation of either gene results in essentially complete abrogation an enzyme essential for heparan sulfate synthesis. Despite the of HS production in cells and tissues (16, 17). The majority of identification of causative genes, the pathogenesis of MHE remains cases of MHE carry frameshift or missense mutations in EXT1 or unclear, especially with regard to whether osteochondroma results EXT2 (18, 19). from loss of heterozygosity of the Ext genes. Hampering elucida- Despite the unambiguous identification of causative genes and +/− +/− tion of the pathogenic mechanism of MHE, both Ext1 and Ext2 their function, the pathogenic mechanism of MHE remains heterozygous mutant mice, which mimic the genetic status of hu- elusive. It was originally hypothesized that EXT1 and EXT2 are man MHE, are highly resistant to osteochondroma formation, es- tumor suppressor genes, and the loss of heterozygosity (LOH) at pecially in long bones. To address these issues, we created a mouse these loci plays a key role in osteochondroma formation (8, 9). model in which Ext1 is stochastically inactivated in a chondrocyte- However, the data from genetic analysis of osteochondromas are specific manner. We show that these mice develop multiple osteo- equivocal (20–23). It is also uncertain whether osteochondroma chondromas and characteristic bone deformities in a pattern and is a true neoplasm, which by definition is derived from clonal a frequency that are almost identical to those of human MHE, sug- expansion of progenitor cells, or whether it represents a focal gesting a role for Ext1 LOH in MHE. Surprisingly, however, geno- developmental malformation (20). Further confounding the is- +/− +/− typing and fate mapping analyses reveal that chondrocytes sue is the fact that both Ext1 and Ext2 heterozygous mu- constituting osteochondromas are mixtures of mutant and wild- tant mice, which mimic the genetic conditions of human MHE type cells. Moreover, osteochondromas do not possess many typi- faithfully, develop osteochondromas only in rib bones with a low cal neoplastic properties. Together, our results suggest that inacti- penetrance (17, 24). Ext1 fi vation of in a small fraction of chondrocytes is suf cient for To address these unsolved issues, we created a mouse model the development of osteochondromas and other skeletal defects based on stochastic, tissue-specific inactivation of Ext1. We show associated with MHE. Because the observed osteochondromas in that mice with inactivation of Ext1 in a minor fraction of chon- our mouse model do not arise from clonal growth of chondrocytes, drocytes develop multiple osteochondromas and bone deform- they cannot be considered true neoplasms. ities in a pattern almost identical to human MHE, whereas mice carrying monoallelic inactivation do not, suggesting a role for bone development | heparan sulfate | osteochondroma LOH in the process. Surprisingly, we found that osteochondromas do not develop in these mice by clonal growth of mutant chon- ultiple hereditary exostoses (MHE) is an autosomal drocytes. Our results provide answers to long-standing questions Mdominant disorder characterized by the formation of concerning the pathogenic mechanism of this disorder, and fur- multiple cartilage-capped bony protrusions (osteochondroma). thermore, indicate that, despite the likely contribution of LOH in It is one of the most common skeletal dysplasias in humans, its initiation, osteochondroma in MHE is not a true neoplasm in with a prevalence of 1 in 18,000 (1, 2). In MHE, osteochon- its strictest sense. dromas occur in almost all types of bones, including flat bones, rib bones, and vertebrae, although they most typically form at Results the distal end of long bones. MHE patients also exhibit various To test the hypothesis that inactivation of Ext1 occurring in deformities of the skeletal system, such as short stature, limb a small fraction of chondrocytes is the pathogenic mechanism of length inequalities, bowing of the limb bones, and scoliosis (3– MHE, we used a method of stochastic inactivation of loxP- F 5). Malignant transformation into metastatic chondrosarcoma flanked Ext1 alleles (Ext1 ) using a tamoxifen-dependent Cre ERT is the most serious complication of MHE, but its frequency is transgene driven by the Col2a1 promoter (Col2-Cre ) (25). not particularly high (6). Genetic linkage analysis has identified We originally intended to control the level of recombination ERT two genes as being associated with the vast majority of MHE: using different doses of tamoxifen. Unexpectedly, Col2-Cre ; EXT1, located on chromosome 8q24.1, and EXT2, located on chromosome 11p11 (7–11). It has been established that EXT1 EXT2 and jointly encode a glycosyltransferase essential for Author contributions: Y.Y. designed research; K.M. and F.I. performed research; S.M. heparan sulfate (HS) synthesis (12, 13). contributed new reagents/analytic tools; K.M. and F.I. analyzed data; and Y.Y. wrote Heparan sulfate (HS) is a highly sulfated linear polysaccharide the paper. with a backbone of alternating N-acetylglucosamine (GlcNAc) The authors declare no conflict of interest. and glucuronic acid (GlcA) residues. The EXT1 and EXT2 pro- *This Direct Submission article had a prearranged editor. teins form an oligomeric complex that catalyzes the copoly- 1To whom correspondence should be addressed. E-mail: [email protected]. merization of GlcNAc and GlcA residues, thereby elongating the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. HS backbone. 1073/pnas.0914642107/-/DCSupplemental. 10932–10937 | PNAS | June 15, 2010 | vol. 107 | no. 24 www.pnas.org/cgi/doi/10.1073/pnas.0914642107 Downloaded by guest on September 24, 2021 F/F Ext1 mice developed multiple osteochondromas and other the radial head were observed in 91.7% of Ext1-SKO mice, where- MHE-like skeletal defects without tamoxifen treatment (Fig. 1). as scoliosis was found in 58.3% (Fig. 1 D and E, and Table S1). As described below, this turned out to be due to a low-level Together with the pattern of osteochondroma formation men- leakiness of the transgene, which causes random deletion of tioned above, these results show that Ext1-SKO mice phenocopy Ext1 in a small fraction of chondrocytes (see below). For con- the skeletal defects of MHE quite faithfully. In comparison, het- ERT F/F ERT F/+ venience, Col2-Cre ;Ext1 mice that are raised without ta- erozygous SKO mice (Col2-Cre ;Ext1 ) never developed moxifen treatment are called Ext1-SKO mice (“SKO” repre- osteochondromas or other skeletal defects (Table S1). The same is senting “stochastic/sporadic knockout”) in this article. true for another conditional heterozygous Ext1 mutant model in In Ext1-SKO mice, multiple bony protrusions involving the which Ext1 is monoallelically inactivated in the entire limb bud F/+ wrist, fibula, shoulder, and rib were identified by radiographic and mesenchyme (Prx1-Cre;Ext1 )(Table S1). These results strongly macroscopic observations (Fig. 1 A and B). Histological exami- suggest a requirement for biallelic inactivation of Ext1 in the de- nation of these lesions revealed bony tuberosities with a cartilage velopment of MHE-like phenotype. In addition to the develop- cap (Fig. 1C), which is consistent with the histological features of ment of osteochondromas, Ext1-SKO mice display mild abnormal- osteochondromas. Table S1 summarizes the occurrence of osteo- ities in the growth plate and joint cartilage (Fig. S1). ERT chondromas at different locations in Ext1-SKO mice at P28. The The Col2-Cre transgene used in this study has been repor- penetrance of the long bone exostosis phenotype (defined as the ted to exhibit a low-level leakiness (25). Thus, we hypothesized presence of at least one conspicuous osteochondroma in long that the phenotypes observed in Ext1-SKO mice are caused by Ext1 leaky Cre activity. To test this hypothesis, we analyzed the spa- bones) in -SKO mice was 100%. This is in stark contrast to the ERT observation that mice carrying the same genotype as human MHE tiotemporal recombination pattern of the Col2-Cre transgene +/− +/− patients (i.e., Ext1 and Ext2 ) never develop osteochon- using the Rosa26-lacZ reporter (26) in whole-mount skeleton dromas in long bones (17, 24) (Table S1). (Fig. S2) and sections through the growth plate (Fig. 2A). In the Individuals with MHE frequently exhibit skeletal defects other whole-mount forelimb at E14.5, a small number of lacZ+ clusters than osteochondromas, such as short stature, bowing deformity in developing limb skeleton were observed (Fig. S2, E14.5), in- of the forearm, subluxation/dislocation of the radius, and scoli- dicating that stochastic recombination occurs as early as E14.5. + osis. Ext1-SKO mice had short stature [wild type, 8.04 ± 0.13 cm Postnatally, a small number of lacZ clusters were found in the + (n = 16); Ext1-SKO, 5.57 ± 0.15 cm (n = 12); at P28]. The femoral head cartilage at P0, and the number of lacZ clusters bowing deformity of the radius and the subluxation/dislocation of showed no significant increase during the period from P0 to P28 (Fig.
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