MAPK pathways activate and phosphorylate the osteoblast-specific transcription factor, Cbfa1

The bone-specific transcription factor, Cbfa1, regulates expression of the osteocalcin (OCN) gene and is essential for bone formation. However, little is known about the mechanisms regulating Cbfa1 activity. This work examine s the role of the MAPK pathway in regulating Cbfa1-dependent transcription. Stimulation of MAPK by transfecting a constitutively active form of MEK1, MEK(SP), into MC3T3-E1 preosteoblast cells increased endogenous OCN mRNA, w hile a dominant negative mutant, MEK(DN), was inhibitory. MEK(SP) also stim ulated activity of a 147-base pair minimal OCN promoter, and this stimulati on required an intact copy of OSE2, the DNA binding site for Cbfa1. Effects of MEK(SP) were specific to Cbfa1-positive osteoblast-like cells. A purifi ed His-tagged Cbfa1 fusion protein was directly, phosphorylated by activate d recombinant MAPK in vitro, Furthermore, P-32 metabolic labeling studies d emonstrated that MEK(SP) clearly enhanced phosphorylation of Cbfa1 in intac t cells, while MEK(DN) decreased phosphorylation. The specific MEK1/MER2 in hibitor, PD98059, inhibited extracellular matrix-dependent up-regulation of the OCN promoter, indicating that the MAPK pathway and, presumably, Cbfa1 phosphorylation are also required for responsiveness of osteoblasts to extr acellular matrix signals. This study is the first demonstration that Cbfa1 is controlled by MAPKs and suggests that this pathway has an important role in the control of osteoblast-specific gene expression.