Phenotypic Analysis of Dlx 5 Overexpression in Post-natal Bone

Abstract

Dlx5 plays an important role in the embryonic development of mineralized tissues. We hypothesized that Dlx5 also functions in regulating post-natal bone formation in mice. To prove this hypothesis, we infected 5-day-old bone sialoprotein (BSP)/avian retroviral receptor gene (TVA) transgenic mice with replication-competent retroviral vectors expressing wild-type Dlx5 (RCAS-Dlx5WT) and mutated Dlx5 at arginine (R) 31 of its homeodomain (RCAS-Dlx5RH). Immunohistochemistry indicated that RCAS-Dlx5WT increased BSP and osteopontin (OPN) expression, whereas it decreased that of osteocalcin (OC). RCAS-Dlx5RH mediated opposite effects. Semi-quantitative RT-PCR confirmed these results. Ex vivo overexpression of RCAS-Dlx5WT in BSP/TVA calvarial cells promoted, whereas that of RCAS-Dlx5RH inhibited, mineralized nodule formation as compared with that in control cells. Our results suggest that Dlx5 promotes expression of early markers of osteogenic differentiation and increases mineralization post-natally.

Get full access to this article

View all access options for this article.

References

Abe T, Nomura S, Nakagawa R, Fujimoto M, Kawase I, Naka T -2006-. Osteoblast differentiation is impaired in SOCS-1-deficient mice. J Bone Miner Metab 24:283–290.

Acampora D, Merlo GR, Paleari L, Zerega B, Postiglione MP, Mantero S, et al. -1999-. Craniofacial, vestibular and bone defects in mice lacking the Distal-less-related gene Dlx5. Development 126:3795–3809.

Chen J, Zhang Q, McCulloch CA, Sodek J -1991-. Immunohistochemical localization of bone sialoprotein in foetal porcine bone tissues: comparisons with secreted phosphoprotein 1 -SPP-1, osteopontin- and SPARC -osteonectin-. Histochem J 23:281–289.

Chen J, Shapiro HS, Sodek J -1992-. Development expression of bone sialoprotein mRNA in rat mineralized connective tissues. J Bone Miner Res 7:987–997.

D’Elia AV, Tell G, Paron I, Pellizzari L, Lonigro R, Damante G -2001-. Missense mutations of human homeoboxes: a review. Hum Mutat 18:361–374.

Depew MJ, Lufkin T, Rubenstein JL -2002-. Specification of jaw subdivisions by Dlx genes. Science 298:381–385.

Ducy P, Desbois C, Boyce B, Pinero G, Story B, Dunstan C, et al. -1996-. Increased bone formation in osteocalcin-deficient mice. Nature 382:448–452.

Ecarot-Charrier B, Bouchard F, Delloye C -1989-. Bone sialoprotein II synthesized by cultured osteoblasts contains tyrosine sulfate. J Biol Chem 264:20049–20053.

Federspiel MJ, Hughes SH -1997-. Retroviral gene delivery. Methods Cell Biol 52:179–214.

10.

Ferrari D, Kosher RA -2002-. Dlx5 is a positive regulator of chondrocyte differentiation during endochondral ossification. Dev Biol 252:257–270.

11.

Fisher LW, McBride OW, Termine JD, Young MF -1990-. Human bone sialoprotein. Deduced protein sequence and chromosomal localization. J Biol Chem 265:2347–2351.

12.

Ganss B, Kim RH, Sodek J -1999-. Bone sialoprotein. Crit Rev Oral Biol Med 10:79–98.

13.

Gehring WJ, Affolter M, Burglin T -1994-. Homeodomain proteins. Annu Rev Biochem 63:487–526.

14.

Holland EC, Varmus HE -1998-. Basic fibroblast growth factor induces cell migration and proliferation after glia-specific gene transfer in mice. Proc Natl Acad Sci USA 95:1218–1223.

15.

Lekic P, Sodek J, McCulloch CA -1996-. Osteopontin and bone sialoprotein expression in regenerating rat periodontal ligament and alveolar bone. Anat Rec 244:50–58.

16.

Li L, Zhu J, Tu Q, Yamauchi M, Sodek J, Karsenty G, et al. -2005-. An in vivo model to study osteogenic gene regulation: targeting an avian retroviral receptor -TVA- to bone with the bone sialoprotein -BSP- promoter. J Bone Miner Res 20:1403–1413.

17.

McIntosh I, Dreyer SD, Clough MV, Dunston JA, Eyaid W, Roig CM, et al. -1998-. Mutation analysis of LMX1B gene in nail-patella syndrome patients. Am J Hum Genet 63:1651–1658.

18.

Meinel L, Fajardo R, Hofmann S, Langer R, Chen J, Snyder B, et al. -2005-. Silk implants for the healing of critical size bone defects. Bone 37:688–698.

19.

Merlo GR, Zerega B, Paleari L, Trombino S, Mantero S, Levi G -2000-. Multiple functions of Dlx genes. Int J Dev Biol 44:619–626.

20.

Murshed M, Harmey D, Millan JL, McKee MD, Karsenty G -2005-. Unique coexpression in osteoblasts of broadly expressed genes accounts for the spatial restriction of ECM mineralization to bone. Genes Dev 19:1093–1104.

21.

Paz J, Wade K, Kiyoshima T, Sodek J, Tang J, Yamauchi M, et al. -2005-. Tissue- and bone cell-specific expression of bone sialoprotein is directed by a 9.0 kb promoter in transgenic mice. Matrix Biol 24:341–352.

22.

Ryoo HM, Hoffmann HM, Beumer T, Frenkel B, Towler DA, Stein GS, et al. -1997-. Stage-specific expression of Dlx-5 during osteoblast differentiation: involvement in regulation of osteocalcin gene expression. Mol Endocrinol 11:1681–1694.

23.

Semina EV, Reiter R, Leysens NJ, Alward WL, Small KW, Datson NA, et al. -1996-. Cloning and characterization of a novel bicoid-related homeobox transcription factor gene, RIEG, involved in Rieger syndrome. Nat Genet 14:392–399.

24.

Simeone A, Acampora D, Pannese M, D’Esposito M, Stornaiuolo A, Gulisano M, et al. -1994-. Cloning and characterization of two members of the vertebrate Dlx gene family. Proc Natl Acad Sci USA 91:2250–2254.

25.

Sodek J, Ganss B, McKee MD -2000-. Osteopontin. Crit Rev Oral Biol Med 11:279–303.

26.

Tadic T, Dodig M, Erceg I, Marijanovic I, Mina M, Kalajzic Z, et al. -2002-. Overexpression of Dlx5 in chicken calvarial cells accelerates osteoblastic differentiation. J Bone Miner Res 17:1008–1014.

27.

Tu Q, Valverde P, Chen J -2006-. Osterix enhances proliferation and osteogenic potential of bone marrow stromal cells. Biochem Biophys Res Commun 341:1257–1265.

28.

Valverde P, Tu Q, Chen J -2005-. BSP and RANKL induce osteoclastogenesis and bone resorption synergistically. J Bone Miner Res 20:1669–1679.

29.

Vastardis H, Karimbux N, Guthua SW, Seidman JG, Seidman CE -1996-. A human MSX1 homeodomain missense mutation causes selective tooth agenesis. Nat Genet 13:417–421.

30.

Volk SW, Diefenderfer DL, Christopher SA, Haskins ME, Leboy PS -2005-. Effects of osteogenic inducers on cultures of canine mesenchymal stem cells. Am J Vet Res 66:1729–1737.

31.

Wang J, Zhou HY, Salih E, Xu L, Wunderlich L, Gu X, et al. -2006-. Site-specific in vivo calcification and osteogenesis stimulated by bone sialoprotein. Calcif Tissue Int 79:179–189.

32.

Wilkie AO, Tang Z, Elanko N, Walsh S, Twigg SR, Hurst JA, et al. -2000-. Functional haploinsufficiency of the human homeobox gene MSX2 causes defects in skull ossification. Nat Genet 24:387–390.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.47 MB