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J. Biochem, 2004, Vol. 136, No. 3 377-386
© 2004 The Japanese Biochemical Society

MOLECULAR BIOLOGY

In Vitro and In Vivo Effects of the Overexpression of Osteopontin on Osteoblast Differentiation Using a Recombinant Adenoviral Vector

Hiroko Kojima1,2, Toshimitsu Uede3 and Toshimasa Uemura1,2,4,*

1 Age Dimension Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central-6, Tsukuba 305-8566; 2 JST(Japan Science and Technology Agency); 3 Division of Molecular Immunology, Institute for Genetic Medicine, Hokkaido University, Sapporo; and 4 Department of Orthopedic Surgery, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519

Osteopontin (OPN) is a highly acidic secreted phosphoprotein that binds to cells via an RGD (arginine-glycine-aspartic acid) cell adhesion sequence that recognizes the {alpha}Vß3 integrin. OPN may regulate the formation and remodeling of bone. To elucidate the function of OPN in bone tissue, we examined the overexpression of OPN in osteoblasts in vitro and in vivo using an adenoviral vector carrying an OPN cDNA (Adv-OPN). Rat bone marrow–derived osteoblasts infected with Adv-OPN were examined by Western blotting, immunofluorescence, nodule formation measurements, assay of alkaline phosphatase (ALP) activity, and Northern blotting. The results suggested that not only osteoblast differentiation markers such as osteocalcin and ALP, but nodule formation and ALP activity are markedly enhanced by OPN overexpression in the case of viral infection. On the contrary, when Adv-OPN and uninfected osteoblasts were implanted into subcutaneous sites with a porous ceramic scaffold, the ALP activity and calcium content of the OPN-infected composite were higher than in uninfected composites, however, the differences were smaller than expected from the in vitro experiments. We speculate that the difference in the result of in vitro and in vivo experiments originates from the inhibitory effect of secreted OPN on the crystal growth of apatite in vivo, which competes with the induced activity of osteoblasts.

* To whom correspondence should be addressed. Tel: +81-29-861-2724, Fax: +81-29-861-2789, E-mail: t.uemura{at}aist.go.jp


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