Conditional disruption of the osterix gene in chondrocytes during early postnatal growth impairs secondary ossification in the mouse tibial epiphysis

Abstract

In our previous studies, we have found that the prepubertal increase in thyroid hormone levels induces osterix (Osx) signaling in hypertrophic chondrocytes to transdifferentiate them into osteoblasts. To test if Osx expressed in chondrocytes directly contributes to transdifferentiation and secondary ossification, we generated Osx ^flox/flox; Col2-Cre-ERT2 mice and knocked out Osx with a single injection of tamoxifen at postnatal day (P) 3 prior to evaluation of the epiphyseal bone phenotype by µCT, histology, and immunohistochemistry (IHC) at P21. Vehicle (oil)-treated Osx ^flox/flox; Col2-Cre-ERT2 and tamoxifen-treated, Cre-negative Osx ^flox/flox mice were used as controls. µCT analysis of tibial epiphyses revealed that trabecular bone mass was reduced by 23% in the Osx conditional knockout (cKO) compared with control mice. Trabecular number and thickness were reduced by 28% and 8%, respectively, while trabecular separation was increased by 24% in the cKO mice. Trichrome staining of longitudinal sections of tibial epiphyses showed that bone area and bone area adjusted for total area were decreased by 22% and 18%, respectively. IHC studies revealed the presence of abundant Osx-expressing prehypertrophic chondrocytes in the epiphyses of control mice at P10, but not in the cKO mice. Furthermore, expression levels of MMP13, COL10, ALP, and BSP were considerably reduced in the epiphyses of cKO mice. We also found that Osx overexpression in ATDC5 chondrocytes increased expression of Col10, Mmp13, Alp, and Bsp. Our data indicate that Osx expressed in chondrocytes plays a significant role in secondary ossification by regulating expression of genes involved in chondrocyte hypertrophy and osteoblast transdifferentiation.

References

[1] Harada S, Rodan GA. Control of osteoblast function and regulation of bone mass. Nature, 2003, 423:349-355

[2] Ralston SH, de Crombrugghe B. Genetic regulation of bone mass and susceptibility to osteoporosis. Genes Dev., 2006, 20:2492-2506

[3] Dao DY et al. Cartilage-specific beta-catenin signaling regulates chondrocyte maturation, generation of ossification centers, and perichondrial bone formation during skeletal development. J. Bone Min. Res, 2012, 27:1680-1694

[4] Adams SL, Cohen AJ, Lassova L. Integration of signaling pathways regulating chondrocyte differentiation during endochondral bone formation. J. Cell Physiol., 2007, 213:635-641

[5] Chung UI, Schipani E, McMahon AP, Kronenberg HM. Indian hedgehog couples chondrogenesis to osteogenesis in endochondral bone development. J. Clin. Invest, 2001, 107:295-304

[6] Lefebvre V, Smits P. Transcriptional control of chondrocyte fate and differentiation. Birth Defects Res. Part C. Embryo Today Rev., 2005, 75:200-212

[7] Mackie EJ, Tatarczuch L, Mirams M. The skeleton: a multi-functional complex organ: the growth plate chondrocyte and endochondral ossification. J. Endocrinol., 2011, 211:109-121

[8] Maeda Y et al. Indian Hedgehog produced by postnatal chondrocytes is essential for maintaining a growth plate and trabecular bone. Proc. Natl Acad. Sci. USA, 2007, 104:6382-6387

[9] Nilsson O, Marino R, De Luca F, Phillip M, Baron J. Endocrine regulation of the growth plate. Horm. Res., 2005, 64:157-165

[10] van der Eerden BC, Karperien M, Wit JM. Systemic and local regulation of the growth plate. Endocr. Rev., 2003, 24:782-801

[11] Zuscik MJ, Hilton MJ, Zhang X, Chen D, O’Keefe RJ. Regulation of chondrogenesis and chondrocyte differentiation by stress. J. Clin. Invest., 2008, 118:429-438

[12] Staines KA, Pollard AS, McGonnell IM, Farquharson C, Pitsillides AA. Cartilage to bone transitions in health and disease. J. Endocrinol., 2013, 219:R1-R12

[13] Brown RS. Minireview: developmental regulation of thyrotropin receptor gene expression in the fetal and newborn thyroid. Endocrinology, 2004, 145:4058-4061

[14] Hume R et al. Thyroid G. Human fetal and cord serum thyroid hormones: developmental trends and interrelationships. J. Clin. Endocrinol. Metab., 2004, 89:4097-4103

[15] Mahony BS, Bowie JD, Killam AP, Kay HH, Cooper C. Epiphyseal ossification centers in the assessment of fetal maturity: sonographic correlation with the amniocentesis lung profile. Radiology, 1986, 159:521-524

[16] Xing W, Cheng S, Wergedal J, Mohan S. Epiphyseal chondrocyte secondary ossification centers require thyroid hormone activation of Indian hedgehog and osterix signaling. J. Bone Min. Res., 2014, 29:2262-2275

[17] Williams GR. Thyroid hormone actions in cartilage and bone. Eur. Thyroid J., 2013, 2:3-13

[18] Aghajanian P, Xing W, Cheng S, Mohan S. Epiphyseal bone formation occurs via thyroid hormone regulation of chondrocyte to osteoblast transdifferentiation. Sci. Rep., 2017, 7

[19] Xing W et al. Thyroid hormone receptor-beta1 signaling is critically involved in regulating secondary ossification via promoting transcription of the Ihh gene in the epiphysis. Am. J. Physiol. Endocrinol. Metab., 2016, 310:E846-E854

[20] Nakashima K et al. The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell, 2002, 108:17-29

[21] Nishimura R et al. Osterix regulates calcification and degradation of chondrogenic matrices through matrix metalloproteinase 13 (MMP13) expression in association with transcription factor Runx2 during endochondral ossification. J. Biol. Chem., 2012, 287:33179-33190

[22] Oh JH, Park SY, de Crombrugghe B, Kim JE. Chondrocyte-specific ablation of Osterix leads to impaired endochondral ossification. Biochem. Biophys. Res Commun., 2012, 418:634-640

[23] Hilton MJ, Tu X, Long F. Tamoxifen-inducible gene deletion reveals a distinct cell type associated with trabecular bone, and direct regulation of PTHrP expression and chondrocyte morphology by Ihh in growth region cartilage. Dev. Biol., 2007, 308:93-105

[24] Akiyama H et al. Osteo-chondroprogenitor cells are derived from Sox9 expressing precursors. Proc. Natl Acad. Sci. USA, 2005, 102:14665-14670

[25] Xing W, Kim J, Wergedal J, Chen ST, Mohan S. Ephrin B1 regulates bone marrow stromal cell differentiation and bone formation by influencing TAZ transactivation via complex formation with NHERF1. Mol. Cell Biol., 2010, 30:711-721

[26] Xing W, Pourteymoor S, Mohan S. Ascorbic acid regulates osterix expression in osteoblasts by activation of prolyl hydroxylase and ubiquitination-mediated proteosomal degradation pathway. Physiol. Genom., 2011, 43:749-757

[27] Bouxsein ML et al. Guidelines for assessment of bone microstructure in rodents using micro-computed tomography. J. Bone Min. Res, 2010, 25:1468-1486

[28] Iida A et al. Identification of biallelic LRRK1 mutations in osteosclerotic metaphyseal dysplasia and evidence for locus heterogeneity. J. Med. Genet., 2016, 53:568-574

[29] Xing W et al. Targeted disruption of leucine-rich repeat kinase 1 but not leucine-rich repeat kinase 2 in mice causes severe osteopetrosis. J. Bone Min. Res, 2013, 28:1962-1974

[30] Xing, W. et al. Genetic evidence that thyroid hormone is indispensable for prepubertal IGF-I expression and bone acquisition in mice. J. Bone Miner. Res. 27, 1067–1079 (2012).

[31] Cheng S, Xing W, Zhou X, Mohan S. Haploinsufficiency of osterix in chondrocytes impairs skeletal growth in mice. Physiol. Genom., 2013, 45:917-923

[32] Hu K, Olsen BR. Osteoblast-derived VEGF regulates osteoblast differentiation and bone formation during bone repair. J. Clin. Invest, 2016, 126:509-526

[33] Hu K, Olsen BR. Vascular endothelial growth factor control mechanisms in skeletal growth and repair. Dev. Dyn., 2017, 246:227-234

[34] Omoteyama K, Takagi M. The effects of Sp7/Osterix gene silencing in the chondroprogenitor cell line, ATDC5. Biochem. Biophys. Res Commun., 2010, 403:242-246

[35] Newton Phillip T., Li Lei, Zhou Baoyi, Schweingruber Christoph, Hovorakova Maria, Xie Meng, Sun Xiaoyan, Sandhow Lakshmi, Artemov Artem V., Ivashkin Evgeny, Suter Simon, Dyachuk Vyacheslav, El Shahawy Maha, Gritli-Linde Amel, Bouderlique Thibault, Petersen Julian, Mollbrink Annelie, Lundeberg Joakim, Enikolopov Grigori, Qian Hong, Fried Kaj, Kasper Maria, Hedlund Eva, Adameyko Igor, Sävendahl Lars, Chagin Andrei S.. A radical switch in clonality reveals a stem cell niche in the epiphyseal growth plate. Nature, 2019, 567 7747 234-238

[36] Zidan J, Keidar Z, Basher W, Israel O. Effects of tamoxifen on bone mineral density and metabolism in postmenopausal women with early-stage breast cancer. Med. Oncol., 2004, 21:117-121

[37] Wilson CH et al. The kinetics of ER fusion protein activation in vivo. Oncogene, 2014, 33:4877-4880

[38] Zhong ZA et al. Optimizing tamoxifen-inducible Cre/loxp system to reduce tamoxifen effect on bone turnover in long bones of young mice. Bone, 2015, 81:614-619

[39] Stickens D et al. Altered endochondral bone development in matrix metalloproteinase 13-deficient mice. Development, 2004, 131:5883-5895

[40] Ewing RM et al. Large-scale mapping of human protein-protein interactions by mass spectrometry. Mol. Syst. Biol., 2007, 3:89

Funding

U.S. Department of Health & Human Services | National Institutes of Health (NIH)(AR 048139)