Figure 4.

Effects on expression levels of osteoblastic and osteocytic markers and involvement of the FAK-mediated MAPK pathway in the regulation of FGF23 by exogenous DMP1. (a) Expression levels of Fgf23, Runx2, Osterix, Col1a1, bone-type alikaline phosphatases (Alp), Osteocalcin (Osc), Dmp1 and SOST in the presence (100 ng ml⁻¹) and absence of DMP1 were compared. (b) UMR-106 cells were treated with FAK or MEK inhibitor (inh.) in the presence of DMP1 (100 ng ml⁻¹). After 24 h, total RNA was collected from the cells, and the expression levels of fgf23 were analyzed using quantitative real-time PCR. Protein levels of FGF23 in the cell culture supernatant and cell lysate were determined using ELISA. *Control vs DMP1; ^#DMP1 vs inh., P<0.05. (c) Cells were cultured with or without the PI3K or ROCK inh. in the presence of DMP1 (100 ng ml⁻¹). After 24 h, total RNA was extracted from the cells, and the expression levels of fgf23 were analyzed using quantitative real-time PCR. *Control vs DMP1, P<0.05. (d) After 24 h of treatment under the same conditions, cells were incubated in Alexa Fluor 568-conjugated phalloidin (red) and 4′-6-diamidino-2-phenylindole (blue) for actin and nuclei, respectively. Merged images are shown in the lowest panels. The data are representative of at least three independent experiments. MEK, MAPK/extracellular signal related kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.