In vitro and in vivo effects of the overexpression of osteopontin on osteoblast differentiation using a recombinant adenoviral vector.
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 alphaVbeta3 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.
Kojima
Hiroko
H
Age Dimension Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central-6, Tsukuba 305-8566, Japan.
Uede
Toshimitsu
T
Uemura
Toshimasa
T
D000256:Adenoviridae / Q000235:genetics* / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000880:Anthraquinones / Q000494:pharmacology; D015152:Blotting, Northern; D015153:Blotting, Western; D001854:Bone Marrow Cells / Q000166:cytology; D016723:Bone Remodeling; D001842:Bone and Bones / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002448:Cell Adhesion; D002454:Cell Differentiation; D002516:Ceramics; D018360:Crystallography, X-Ray; D018076:DNA, Complementary / Q000378:metabolism; D005822:Genetic Vectors; D008297:Male; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism; D015675:Osteocalcin / Q000378:metabolism; D053495:Osteopontin; D010750:Phosphoproteins / Q000737:chemistry; D051381:Rats; D011916:Rats, Inbred F344; D012795:Sialoglycoproteins / Q000096:biosynthesis*; D013997:Time Factors
D000880:Anthraquinones; D018076:DNA, Complementary; D010750:Phosphoproteins; D012795:Sialoglycoproteins; C506897:Spp1 protein, rat; D015675:Osteocalcin; D053495:Osteopontin; C010078:alizarin; D000469:Alkaline Phosphatase; D002118:Calcium