Fig. 5.

MG63 cell differentiation and Runx2 binding activity in the nucleus were inhibited by shear stress acting through α_vβ₃ and β₁ integrins and Smad5. (A–C) MG63 cells were kept as controls (C) or subjected to shear stress (12 dynes/cm²) for 30 min to 24 h, as indicated. (B, D–F) The cells were transfected with empty vector control PSRα or OCN-Luc (1 μg/ml each) (B), transfected with siRNA (siCL for control, or specific siRNA of Smad1 or Smad5) (40 nM each) (D and E) for 48 h, or pretreated with control IgG or a specific antibody against α_vβ₃ or β₁ (10 μg/ml each) for 2 h (F) and then were kept under static conditions or subjected to shear stress for 6 and 24 h (B) or 24 h (D–F). The OCN and ALP mRNA expressions (A, D, and E), OCN promoter and ALP activities (B), and Runx2-DNA binding activity (C and E) were determined by real-time PCR, luciferase and ALP activity assays, and EMSA, respectively. In C, total nuclear extracts of cells and ³²P-labeled oligonucleotides containing human OCN Runx2-binding sites were used. Nuclear extracts were preincubated with 20-fold excess unlabeled oligonucleotides (+20×). As positive controls, MG63 cells were treated with BMP-4 (100 ng/ml) for 6 and 24 h (C). Nuclear extracts preincubated with the Runx2 antibody (+Ab) show a super shift band (SH). Results in A, B, D, and F are means ± SEM from three to four independent experiments. Results in C and E are representative of two or three independent experiments with similar results. *, P < 0.05 vs. static control cells (A and B); #, P < 0.05 vs. control treatments (D and F).