Abstract
Desmosterolosis is an autosomal recessive disease caused by mutations in the 3β-hydroxysterol-Delta24 reductase (DHCR24) gene, with severe developmental anomalies including short limbs. We utilized DHCR24 knockout (KO) mice to study the underlying bone pathology. Because the KO mice died within a few hours after birth, we cultured metatarsal bones from newborn mice. The growth of bones from KO mice was significantly retarded after 1 week of culture. Absence of proliferating chondrocytes in the growth plate and abnormal hypertrophy of prehypertrophic chondrocytes were observed in the bones from KO mice. Hypertrophic differentiation was evidenced by higher expression of Indian hedgehog, alkaline phosphatase, and matrix metalloproteinase 13. Since elevated levels of reactive oxygen species (ROS) during chondrogenesis are known to inhibit proliferation and to initiate chondrocyte hypertrophy in the growth plate, and since DHCR24 acts as a potent ROS scavenger, we hypothesized that the abnormal chondrocyte proliferation and differentiation in KO mice were due to decreased ROS scavenging activity. Treatment with an antioxidant, N-acetyl cysteine, could correct the abnormalities observed in the bones from KO mice. Treatment of bones from wild-type mice with U18666A, a chemical inhibitor of DHCR24, resulted in short broad bones with a disrupted proliferating zone. Treatment of ATDC cells with hydrogen peroxide (H2O2) induced hypertrophic changes as evidenced by the expression of the marker genes specific for hypertrophic chondrocyte differentiation. H2O2-induced hypertrophic change was prevented by adenoviral delivery of DHCR24. Induction of chondrocyte differentiation in ATDC cells by insulin was associated with increased ROS production that was markedly enhanced by treatment of ATDC5 cells with DHCR24 siRNA. This is the first demonstration that DHCR24 plays an important role in long bone growth by protecting chondrocytes from ROS
Similar content being viewed by others
References
Andersson HC, Kratz L, Kelley R (2002) Desmosterolosis presenting with multiple congenital anomalies and profound developmental delay. Am J Med Genet 113:315–319
Waterham HR, Koster J, Romeijn GJ, Hennekam RC, Vreken P, Andersson HC, FitzPatrick DR, Kelley RI, Wanders RJ (2001) Mutations in the 3beta-hydroxysterol Delta24-reductase gene cause desmosterolosis, an autosomal recessive disorder of cholesterol biosynthesis. Am J Hum Genet 69:685–694
FitzPatrick DR, Keeling JW, Evans MJ, Kan AE, Bell JE, Porteous ME, Mills K, Winter RM, Clayton PT (1998) Clinical phenotype of desmosterolosis. Am J Med Genet 75:145–152
Mirza R, Hayasaka S, Takagishi Y, Kambe F, Ohmori S, Maki K, Yamamoto M, Murakami K, Kaji T, Zadworny D, Murata Y, Seo H (2006) DHCR24 gene knockout mice demonstrate lethal dermopathy with differentiation and maturation defects in the epidermis. J Invest Dermatol 126:638–647
Chagin AS, Karimian E, Zaman F, Takigawa M, Chrysis D, Savendahl L (2007) Tamoxifen induces permanent growth arrest through selective induction of apoptosis in growth plate chondrocytes in cultured rat metatarsal bones. Bone 40:1415–1424
Chagin AS, Chrysis D, Takigawa M, Ritzen EM, Savendahl L (2006) Locally produced estrogen promotes fetal rat metatarsal bone growth; an effect mediated through increased chondrocyte proliferation and decreased apoptosis. J Endocrinol 188:193–203
Mukherjee A, Rotwein P (2009) Akt promotes BMP2-mediated osteoblast differentiation and bone development. J Cell Sci 122:716–726
Serrat MA, King D, Lovejoy CO (2008) Temperature regulates limb length in homeotherms by directly modulating cartilage growth. Proc Natl Acad Sci USA 105:19348–19353
Wagner EF, Karsenty G (2001) Genetic control of skeletal development. Curr Opin Genet Dev 11:527–532
Olsen BR, Reginato AM, Wang W (2000) Bone development. Annu Rev Cell Dev Biol 16:191–220
Kronenberg HM (2003) Developmental regulation of the growth plate. Nature 423:332–336
Kielty CM, Kwan AP, Holmes DF, Schor SL, Grant ME (1985) Type X collagen, a product of hypertrophic chondrocytes. Biochem J 227:545–554
Reichenberger E, Aigner T, von der Mark K, Stoss H, Bertling W (1991) In situ hybridization studies on the expression of type X collagen in fetal human cartilage. Dev Biol 148:562–572
Tuckermann JP, Pittois K, Partridge NC, Merregaert J, Angel P (2000) Collagenase-3 (MMP-13) and integral membrane protein 2a (Itm2a) are marker genes of chondrogenic/osteoblastic cells in bone formation: sequential temporal, and spatial expression of Itm2a, alkaline phosphatase, MMP-13, and osteocalcin in the mouse. J Bone Miner Res 15:1257–1265
Lai LP, Mitchell J (2005) Indian hedgehog: its roles and regulation in endochondral bone development. J Cell Biochem 96:1163–1173
Morita K, Miyamoto T, Fujita N, Kubota Y, Ito K, Takubo K, Miyamoto K, Ninomiya K, Suzuki T, Iwasaki R, Yagi M, Takaishi H, Toyama Y, Suda T (2007) Reactive oxygen species induce chondrocyte hypertrophy in endochondral ossification. J Exp Med 204:1613–1623
Kuehnle K, Crameri A, Kalin RE, Luciani P, Benvenuti S, Peri A, Ratti F, Rodolfo M, Kulic L, Heppner FL, Nitsch RM, Mohajeri MH (2008) Prosurvival effect of DHCR24/Seladin-1 in acute and chronic responses to oxidative stress. Mol Cell Biol 28:539–550
Lu X, Kambe F, Cao X, Kozaki Y, Kaji T, Ishii T, Seo H (2008) 3beta-Hydroxysteroid-delta24 reductase is a hydrogen peroxide scavenger, protecting cells from oxidative stress-induced apoptosis. Endocrinology 149:3267–3273
Di Stasi D, Vallacchi V, Campi V, Ranzani T, Daniotti M, Chiodini E, Fiorentini S, Greeve I, Prinetti A, Rivoltini L, Pierotti MA, Rodolfo M (2005) DHCR24 gene expression is upregulated in melanoma metastases and associated to resistance to oxidative stress-induced apoptosis. Int J Cancer 115:224–230
Greeve I, Hermans-Borgmeyer I, Brellinger C, Kasper D, Gomez-Isla T, Behl C, Levkau B, Nitsch RM (2000) The human DIMINUTO/DWARF1 homolog seladin-1 confers resistance to Alzheimer’s disease-associated neurodegeneration and oxidative stress. J Neurosci 20:7345–7352
Shukunami C, Ishizeki K, Atsumi T, Ohta Y, Suzuki F, Hiraki Y (1997) Cellular hypertrophy and calcification of embryonal carcinoma-derived chondrogenic cell line ATDC5 in vitro. J Bone Miner Res 12:1174–1188
Watanabe H, de Caestecker MP, Yamada Y (2001) Transcriptional cross-talk between Smad, ERK1/2, and p38 mitogen-activated protein kinase pathways regulates transforming growth factor-beta-induced aggrecan gene expression in chondrogenic ATDC5 cells. J Biol Chem 276:14466–14473
Mushtaq T, Farquharson C, Seawright E, Ahmed SF (2002) Glucocorticoid effects on chondrogenesis, differentiation and apoptosis in the murine ATDC5 chondrocyte cell line. J Endocrinol 175:705–713
Wechsler A, Brafman A, Shafir M, Heverin M, Gottlieb H, Damari G, Gozlan-Kelner S, Spivak I, Moshkin O, Fridman E, Becker Y, Skaliter R, Einat P, Faerman A, Bjorkhem I, Feinstein E (2003) Generation of viable cholesterol-free mice. Science 302:2087
Cenedella RJ (1980) Concentration-dependent effects of AY-9944 and U18666A on sterol synthesis in brain. Variable sensitivities of metabolic steps. Biochem Pharmacol 29:2751–2754
Shukunami C, Shigeno C, Atsumi T, Ishizeki K, Suzuki F, Hiraki Y (1996) Chondrogenic differentiation of clonal mouse embryonic cell line ATDC5 in vitro: differentiation-dependent gene expression of parathyroid hormone (PTH)/PTH-related peptide receptor. J Cell Biol 133:457–468
Trayner ID, Rayner AP, Freeman GE, Farzaneh F (1995) Quantitative multiwell myeloid differentiation assay using dichlorodihydrofluorescein diacetate (H2DCF-DA) or dihydrorhodamine 123 (H2R123). J Immunol Methods 186:275–284
Naski MC, Ornitz DM (1998) FGF signaling in skeletal development. Front Biosci 3:d781–d794
Cho SY, Kim JH, Paik YK (1998) Cholesterol biosynthesis from lanosterol: differential inhibition of sterol delta 8-isomerase and other lanosterol-converting enzymes by tamoxifen. Mol Cells 8:233–239
Goldring MB, Tsuchimochi K, Ijiri K (2006) The control of chondrogenesis. J Cell Biochem 97:33–44
Koh CH, Whiteman M, Li QX, Halliwell B, Jenner AM, Wong BS, Laughton KM, Wenk M, Masters CL, Beart PM, Bernard O, Cheung NS (2006) Chronic exposure to U18666A is associated with oxidative stress in cultured murine cortical neurons. J Neurochem 98:1278–1289
Mahadev K, Zilbering A, Zhu L, Goldstein BJ (2001) Insulin-stimulated hydrogen peroxide reversibly inhibits protein-tyrosine phosphatase 1b in vivo and enhances the early insulin action cascade. J Biol Chem 276:21938–21942
Mahadev K, Motoshima H, Wu X, Ruddy JM, Arnold RS, Cheng G, Lambeth JD, Goldstein BJ (2004) The NAD(P)H oxidase homolog Nox4 modulates insulin-stimulated generation of H2O2 and plays an integral role in insulin signal transduction. Mol Cell Biol 24:1844–1854
Acknowledgments
This work was supported in part by Japan Society for Promotion of Science (P06463) to R.M. and H.S. and grants from Chubu University to H.S. and S.Q. We are indebted to the Quark Biotech Inc. for the provision of DHCR24−/− mice. DHCR24−/− mice were produced by Lexicon Genetics Incorporated for Quark Biotech Inc.
Author information
Authors and Affiliations
Corresponding author
Additional information
R. Mirza and S. Qiao contributed equally to this work.
About this article
Cite this article
Mirza, R., Qiao, S., Tateyama, K. et al. 3β-Hydroxysterol-Delta24 reductase plays an important role in long bone growth by protecting chondrocytes from reactive oxygen species. J Bone Miner Metab 30, 144–153 (2012). https://doi.org/10.1007/s00774-011-0303-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00774-011-0303-7