Figure 6

Extracellular domain of obif can promote osteoblast differentiation. (A) Schematic representation of partial obif proteins overexpressed in MC3T3-E1 cells. Upper: the full-length obif protein, middle: the partial obif fragment containing the N-terminal extracellular domain (ECD), lower: the partial obif fragment composed of the ECD and the transmembrane domain (TM). Numbers represent amino acid residues of mouse (human) obif proteins. (B-F) MC3T3-E1 cells infected with retroviruses were cultured in the differentiation medium and used for assays. Both of bands of partial obif poteins detected by Western blot were larger than predicted sizes (B). Immunocytochemistry analyses using anti-FLAG antibody (C-D). MC3T3-ECD+TM protein localizes to the plasma membranes whereas a large amount of obif-ECD protein localizes to the cytoplasm. Scale bars = 50 μm. MC3T3-E1 cells infected with retroviruses expressing a full-length obif or its partial proteins showed elevated ALP activities at all time points examined (E). Infection with retroviruses expressing full-length and partial proteins significantly promoted mineral deposition both at day 28 and 42 (F). (G) AP fusion proteins (obif-ECD-AP, AP-obif-ECD) and AP protein were detected by Western blotting analysis using anti-AP antibody. Fusion proteins in supernatants are larger than those in lysates from transfected cells. (H-I) AP-obif (ECD) bound to bone tissues whereas control AP protein did not bind at detectable levels. (J, K) Higher magnifications of the squares in H &I, respectively. Scale bars = 100 μm.