BMP-4 induction of arrest and differentiation of osteoblast-like cells via p21^CIP1 and p27^KIP1 regulation

Cell cycle regulation by differentiation signals is critical for eukaryote development. We investigated the roles of bone morphogenetic protein (BMP)-4, an important stimulator of osteoblast differentiation and bone formation, in regulating cell cycle distribution in four osteoblast-like celllines and mouse primary osteoblasts, and the underlying mechanisms. In all cells used, BMP-4 induced G₀/G₁ arrest. The molecular basis of the BMP-4 effect was analyzed, and the presentation on molecular mechanism is focused on human MG63 cells. BMP-4 induced p21^CIP1 and p27^KIP1 expressions and hence cell differentiation but had no effects on the expressions of cyclins A, B1, D1, and E, cyclin-dependent protein kinase-2, -4, and -6. Using specific small interfering RNA (siRNA), we found that BMP-4-induced G ₀/G₁ arrest, and p21^CIP1 and p27^KIP1 expressions were mediated by BMP receptor type IA (BMPRIA)-specific Sma- and Mad-related protein (Smad)1/5. BMP-4 induced transient phosphorylations of ERK; transfection of MG63 cells with ERK2, but not ERK1, -specific siRNA inhibited the BMP-4-induced responses in MG63 cells. Pretreatment of MG63 cells with Arg-Gly-Asp-Ser, which blocks the cell-extracellular matrix interaction, or transfection with β₃ integrin-specific siRNA inhibited BMP-4-induced ERK and Smad1/5 phosphorylations. BMP-4 induced transient increases in associations of β₃-integrin with focal adhesion kinase and Shc, the dominant-negative mutants of which inhibited BMP-4-induced ERK and Smad1/5 phosphorylations. Our results indicate that BMP-4 induces G ₀/G₁ arrest and hence differentiation in osteoblast-like cells through increased expressions of p21^CIP1 and p27 ^KIP1, which are mediated by BMPRIA-specific Smad1/5. The extracellular matrix/β₃ integrin/ focal adhesion kinase/Shc/ERK2 signaling pathway is involved in these BMP-4-induced responses in osteoblast-like cells.