Bone morphogenetic proteins, extracellular matrix, and mitogen-activated protein kinase signaling pathways are required for osteoblast-specific gene expression and differentiation in MC3T3-E1 cells

Osteoblasts secrete a complex extracellular matrix (ECM) containing collagenous and noncollagenous proteins, bone morphogenetic proteins (BMPs), and growth factors. Osteoblast-specific gene expression requires ascorbic acid (AA)-dependent assembly of a collagenous ECM. Matrix responsiveness requires an α₂β₁ integrin-collagen interaction and mitogen-activated protein kinase (MAPK) activity, which phosphorylates and activates the osteoblast-specific transcription factor Cbfa1. This study examines interactions between this integrin/MAPK-mediated pathway and signals initiated by BMPs contained in the osteoblast matrix. MC3T3-E1 cells were shown to constitutively express BMP-2, BMP-4, and BMP-7. Noggin, a specific BMP inhibitor, reversibly blocked AA-induced gene expression, indicating that BMP production by MC3T3-E1 cells was necessary for differentiation. The ability of exogenously added BMP-2, BMP-4, or BMP-7 to stimulate osteocalcin (OCN) and bone sialoprotein (BSP) mRNAs or OCN promoter activity was synergistically increased in cells that were actively synthesizing an ECM (i.e., were grown in the presence of AA). A minimum of 4 days of ECM accumulation was required for this synergistic response to be observed. Neither BMP-7, AA, nor a combination of these two treatments had major effects on Cbfa1 messenger RNA (mRNA) or protein levels, as would be expected if regulation was mainly at the post-transcriptional level. U0126, a specific inhibitor of MAPK/extracellular signal-regulated kinase (MEK), blocked AA- or BMP-7/AA-dependent gene expression in a time- and dose-dependent manner that was closely correlated with inhibition of extracellular signalregulated kinase (ERK) phosphorylation. This work establishes that autocrine BMP production as well as integrin-mediated cell-collagen interactions are both required for osteoblast differentiation, and both these pathways require MAP kinase activity.