Using differential display PCR, we have identified two novel transcripts that are induced as a prechondroblastic cell line, MLB 13 MYC clone 17, acquires markers of the osteoblast phenotype in response to BMP-2 treatment. The first gene encodes a novel kinase that, when stably expressed in MC3T3E1 cells, results in a dramatic attenuation of the program of osteoblast differentiation. The second gene encodes a new member of the WD40 repeat family of proteins that markedly accelerates the program of osteoblast differentiation when stably expressed in MC3T3E1 cells. The presence of 7 WD repeats and of a short amino terminal non-beta-propeller region, coupled with the absence of a carboxy terminal extension and of a putative transactivation domain, link this protein most closely to the beta-transducin subfamily. However, the lack of a coiled-coil structure in the N- terminal extension of BIG-3 (BMP-2 Induced Gene-3Kb), and its cytoplasmic location, support the hypothesis that this protein is not a beta-transducin homolog. The studies proposed will address the hypothesis that the effects of BIG-3 on osteoblast differentiation require other actions of BMPs, by examining the program of osteoblast differentiation in noggin-treated MC3T3-E1 cells stably transfected with BIG-3 or empty vector. To address whether BIG-3 is essential for osteoblast differentiation, BIG-3 protein synthesis, in untransfected MC3T3-E 1 cells, will be blocked using antisense RNA strategies. Inhibition of BIG-3 protein expression will be evaluated and the impact of this reduction in BIG-3 protein levels on the differentiation of MC3T3-E1 cells will be examined. Deletion analyses will be performed to address the hypothesis that the amino terminal, non-WD repeat sequences contribute both to subcellular location and to the function of BIG-3. Yeast two-hybrid analyses will be performed to isolate proteins that interact with BIG-3 and contribute to its effects on osteoblast differentiation. Transgenic mice overexpressing BIG-3 in early osteoblasts (Col1-BIG-3) will be generated to address the hypothesis that BIG-3 accelerates the program of osteoblast differentiation in vivo. Analyses will be performed to assess whether this is associated with a parallel increase in osteoclast activity or whether uncoupling of bone formation and bone resorption is observed in the Col1-BIG-3 mice. Studies in primary osteoblasts isolated from these mice will determine if the overexpression of BIG-3 accelerates the differentiation of primary osteoblasts and whether it has effects on the differentiation and number of """"""""osteoprogenitors"""""""" that give rise to endochondral and intramembranous bone.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK036597-17
Application #
6692970
Study Section
Special Emphasis Panel (ZRG1-OBM-2 (01))
Program Officer
Malozowski, Saul N
Project Start
1986-07-01
Project End
2008-01-31
Budget Start
2004-02-01
Budget End
2005-01-31
Support Year
17
Fiscal Year
2004
Total Cost
$392,826
Indirect Cost
Name
Massachusetts General Hospital
Department
Type
DUNS #
073130411
City
Boston
State
MA
Country
United States
Zip Code
02199
Gori, Francesca; Zhu, Eric D; Demay, Marie B (2009) Perichondrial expression of Wdr5 regulates chondrocyte proliferation and differentiation. Dev Biol 329:36-43
Zhu, Eric D; Demay, Marie B; Gori, Francesca (2008) Wdr5 is essential for osteoblast differentiation. J Biol Chem 283:7361-7
Gori, Francesca; Friedman, Lauren G; Demay, Marie B (2006) Wdr5, a WD-40 protein, regulates osteoblast differentiation during embryonic bone development. Dev Biol 295:498-506
Yu, Xijie; Sabbagh, Yves; Davis, Siobhan I et al. (2005) Genetic dissection of phosphate- and vitamin D-mediated regulation of circulating Fgf23 concentrations. Bone 36:971-7
Gori, Francesca; Demay, Marie B (2005) The effects of BIG-3 on osteoblast differentiation are not dependent upon endogenously produced BMPs. Exp Cell Res 304:287-92
Gori, Francesca; Demay, Marie B (2004) BIG-3, a novel WD-40 repeat protein, is expressed in the developing growth plate and accelerates chondrocyte differentiation in vitro. Endocrinology 145:1050-4
Sooy, Karen; Demay, Marie B (2002) Transcriptional repression of the rat osteocalcin gene by deltaEF1. Endocrinology 143:3370-5
Kearns, A E; Donohue, M M; Sanyal, B et al. (2001) Cloning and characterization of a novel protein kinase that impairs osteoblast differentiation in vitro. J Biol Chem 276:42213-8
Gori, F; Schipani, E; Demay, M B (2001) Fibromodulin is expressed by both chondrocytes and osteoblasts during fetal bone development. J Cell Biochem 82:46-57
Gori, F; Divieti, P; Demay, M B (2001) Cloning and characterization of a novel WD-40 repeat protein that dramatically accelerates osteoblastic differentiation. J Biol Chem 276:46515-22

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