Role of FoxO3a in Osteoblast Differentiation and Matrix Calcification

Forkhead Box O transcription factors play important roles in bone metabolism via defending against oxidative stress and apoptosis (Almeida, 2011; Ambrogini et al., 2010). FoxO3a is of special interest as it is the predominant isoform in bone cells (Ambrogini et al., 2010). In osteoblasts, the administration of 1,25 dihydroxyvitamin D₃ (1,25D₃) increases FoxO3a expression, and alters calcium handling. We therefore queried whether FoxO3a participates in vitamin D-mediated calcium regulation or osteoblast differentiation and matrix calcification, independent of reactive oxygen species (ROS) formation. This dissertation investigates the regulating role of Forkhead Box O3 transcription factors (FoxO3) in osteoblast differentiation and matrix mineralization. FoxO3a expression increased throughout differentiation. At day 3 and 7, FoxO3a mRNA and protein expression levels were significantly higher than predifferentiation. 1,25D₃ further enhanced both FoxO3a mRNA and protein expression levels in the 7-day differentiated osteoblast cells. Immunocytofluorescence localization of FoxO3a demonstrated the addition of 1,25D₃ led to nuclear localization. 7-day differentiated osteoblast cells incubated with H₂O₂ showed no obvious difference from control, however, increased FoxO3a expression was attenuated when H₂O₂ was added with 1,25D₃. In order to study the effect of 1,25D₃ on calcium regulation in osteoblasts, expression of calcium homeostasis mediators were assessed. Increased expression of the calcium channel Ca_v3.1 and plasma membrane Ca²⁺ ATPase (PMCA-1b) was observed throughout osteoblast differentiation. Addition of 1,25D₃ enhanced expression of Calbindin-D_9K and the sodium-calcium exchanger (NCX). 1,25D₃ increased calcium uptake and calcium deposition in differentiated osteoblasts as demonstrated by ratiometric live cell calcium imaging and alizarin red staining, respectively. FoxO3a overexpression led to a reduction in calcium uptake and calcium deposition, resulting in uncalcified matrix. When FoxO3a was over-expressed in MC3T3-E1 cells, pre-osteoblasts failed to differentiate into osteoblasts and unmineralized osteoid failed to calcify its matrix, as assessed by the biomarkers Runx2 and osteocalcin, respectively. Based on expression and functional data, the results suggest that high levels of FoxO3a inversely regulate osteoblast differentiation and matrix calcification, with a role in the repression of both processes.

Year	Entry
1995	Riggs BL, Melton LJ III. The worldwide problem of osteoporosis: insights afforded by epidemiology. Bone. November 1995;17(5)(suppl 1):S505-S511.
1992	Riggs BL, Melton LJ III. The prevention and treatment of osteoporosis. NEJM. August 27, 1992;327(9):620-627.
1998	Riggs BL, Khosla S, Melton LJ III. A unitary model for involutional osteoporosis: estrogen deficiency causes both type I and type II osteoporosis in postmenopausal women and contributes to bone loss in aging men. J Bone Miner Res. May 1998;13(5):763-773.
2005	Day TF, Guo X, Garrett-Beal L, Yang Y. Wnt/β-catenin signaling in mesenchymal progenitors controls osteoblast and chondrocyte differentiation during vertebrate skeletogenesis. Dev Cell. May 2005;8(5):739-750.
1996	Duey P, Desbois C, Boyce B, Pinerot G, Story B, Dunstan C, Smith E, Bonadioll J, Goldstein S, Gundberg C, Bradley A, Karsenty G. Increased bone formation in osteocalcin-deficient mice. Nature. August 1, 1996;382(6590):448-452.
2010	Dallas SL, Bonewald LF. Dynamics of the transition from osteoblast to osteocyte. Annals NY Acad Sci. March 2010;1192(1):437-443.
2008	Clarke B. Normal bone anatomy and physiology. Clin J Am Soc Nephrol. November 2008;3(suppl 3):S131-S139.
1997	Ducy P, Zhang R, Geoffroy V, Ridall AL, Karsent G. Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell. May 30, 1997;89(5):747-754.
2002	Everts V, Delaissé JM, Korper W, Jansen DC, Tigchelaar-Gutter W, Saftig P, Beertsen W. The bone lining cell: its role in cleaning Howship's lacunae and initiating bone formation. J Bone Miner Res. January 2002;17(1):77-90.
1997	Otto F, Thornell AP, Crompton T, Denzel A, Gilmour KC, Rosewell IR, Stamp GW, Beddington RS, Mundlos S, Olsen BR, Selby PB, Owen MJ. Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell. May 30, 1997;89(5):765-771.
2012	Kanis JA, Odén A, McCloskey EV, Johansson H, Wahl DA, Cooper C; IOF Working Group on Epidemiology and Quality of Life. A systematic review of hip fracture incidence and probability of fracture worldwide. Osteoporos Int. September 2012;23(9):2239-2256.
1999	Suda T, Takahashi N, Udagawa N, Jimi E, Gillespie MT, Martin TJ. Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr Rev. June 1999;20(3):345-357.
1997	Kato Y, Windle JJ, Koop BA, Mundy GR, Bonewald LF. Establishment of an osteocyte‐like cell line, MLO‐Y4. J Bone Miner Res. December 1997;12(12):2014-2023.
1997	Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, Deguchi K, Shimizu Y, Bronson RT, Gao Y-H, Inada M, Sato M, Okamoto R, Kitamura Y, Yoshiki S, Kishimoto T. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell. May 30, 1997;89(5):755-764.
1999	Wang D, Christensen K, Chawla K, Xiao G, Krebsbach PH, Franceschi RT. Isolation and characterization of MC3T3-E1 preosteoblast subclones with distinct in vitro and in vivo differentiation/mineralization potential. J Bone Miner Res. June 1999;14(6):893-903.
1983	Sudo H, Kodama H-A, Amagai Y, Yamamoto S, Kasai S. In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria. J Cell Biol. January 1983;96(1):191-198.
1991	Fratzl P, Fratzl-Zelman N, Klaushofer K, Vogl G, Koller K. Nucleation and growth of mineral crystals in bone studied by small-angle X-ray scattering. Calcif Tiss Int. June 1991;48(6):407-413.
2011	Bonewald LF. The amazing osteocyte. J Bone Miner Res. 2011;26(2):229-238.
1984	Glimcher MJ, Muir H. Recent studies of the mineral phase in bone and its possible linkage to the organic matrix by protein-bound phosphate bonds. Philos Trans R Soc Lond B Biol Sci. February 13, 1984;304(1121):479-508.
2010	Papaioannou A, Morin S, Cheung AM, Atkinson S, Brown JP, Feldman S, Hanley DA, Hodsman A, Jamal SA, Kaiser SM, Kvern B, Siminoski K, Leslie WD; Scientific Advisory Council of Osteoporosis Canada. 2010 clinical practice guidelines for the diagnosis and management of osteoporosis in Canada: summary. Can Med Assoc J. November 23, 2010;182(17):1864-1873.

Year

Entry

1995

Riggs BL, Melton LJ III. The worldwide problem of osteoporosis: insights afforded by epidemiology. Bone. November 1995;17(5)(suppl 1):S505-S511.

1992

Riggs BL, Melton LJ III. The prevention and treatment of osteoporosis. NEJM. August 27, 1992;327(9):620-627.

1998

Riggs BL, Khosla S, Melton LJ III. A unitary model for involutional osteoporosis: estrogen deficiency causes both type I and type II osteoporosis in postmenopausal women and contributes to bone loss in aging men. J Bone Miner Res. May 1998;13(5):763-773.

2005

Day TF, Guo X, Garrett-Beal L, Yang Y. Wnt/β-catenin signaling in mesenchymal progenitors controls osteoblast and chondrocyte differentiation during vertebrate skeletogenesis. Dev Cell. May 2005;8(5):739-750.

1996

Duey P, Desbois C, Boyce B, Pinerot G, Story B, Dunstan C, Smith E, Bonadioll J, Goldstein S, Gundberg C, Bradley A, Karsenty G. Increased bone formation in osteocalcin-deficient mice. Nature. August 1, 1996;382(6590):448-452.