MAPK Pathways Activate and Phosphorylate the Osteoblast-specific Transcription Factor, Cbfa1

Abstract

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 examines 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, while a dominant negative mutant, MEK(DN), was inhibitory. MEK(SP) also stimulated activity of a 147-base pair minimal OCN promoter, and this stimulation 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 purified His-tagged Cbfa1 fusion protein was directly phosphorylated by activated recombinant MAPK in vitro. Furthermore, (32)P metabolic labeling studies demonstrated that MEK(SP) clearly enhanced phosphorylation of Cbfa1 in intact cells, while MEK(DN) decreased phosphorylation. The specific MEK1/MEK2 inhibitor, 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 extracellular 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.

c-Jun N-terminal kinase 1 negatively regulates osteoblastic differentiation induced by BMP2 via phosphorylation of Runx2 at Ser104

“…The roles of ERK and p38 activation in osteoblastic differentiation have been well studied, (17)(18)(19)21) whereas the role of JNK1 activation in osteoblastic differentiation remains to be elucidated. To determine the role of JNK1 in osteoblastic differentiation, C2C12 and MC3T3-E1 cells were treated with BMP2 in the presence and absence of specific JNK inhibitor, SP600125, and two osteoblastic functions, ALP activity and bone mineralization, were compared.…”

“…(8,14,16) For example, both phosphorylation and transcriptional activity of Runx2 can be stimulated by the ERK and p38 pathways. (17)(18)(19)(20)(21)(22) Although the involvements of ERK and p38 pathways in osteoblastic differentiation have been well studied, the phosphorylation and activity of Runx2 modified by JNK during osteoblastic differentiation remains to be clarified.…”

Focal adhesion kinase signaling pathways regulate the osteogenic differentiation of human mesenchymal stem cells

“…Specifically, this interaction triggers osteoblast-specific expression of alkaline phosphatase and osteocalcin mRNAs, and ultimately, mineralization of bone tissue in a stage-specific sequence [12,13]. Matrix responsiveness during osteogenesis is caused by the aggregation of the α2β1 [9,[14][15][16] and αvβ3 [17] integrins that subsequently activate intracellular signaling cascades, resulting in the phosphorylation of the osteoblast-specific transcription factor Runx2/Cbfa-1 [15,18,19]. Understanding the molecular signaling mechanisms associated with these events and the osteogenic differentiation of hMSC as a whole, will accelerate the practical application of stem cell engineering.…”

“…However, the kinases and the specific amino acid residues responsible for the activation of Runx2/Cbfa-1 transcriptional activity are not fully known. Members of the mitogen-activated protein kinase (MAPK) family, including ERK, play a role in controlling Runx2/Cbfa-1 activity in differentiated osteoblasts [18]. Bonelike cells expressing constitutively active MEK, which controls ERK activity, increases expression of the bone protein osteocalcin.…”

The static magnetic field accelerates the osteogenic differentiation and mineralization of dental pulp cells

“…Cbfa1 is essential for osteoblastic differentiation, bone formation and tooth development (D'Souza et al 1999). It can be phosphorylated by the integrin-mediated activation of the extracellular signal-regulated kinase 1/2 (ERK1/2), the subgroups of mitogen-activated protein kinase (MAPK) (MEK-ERK1/2-Cbfa1 signaling), to control transcription of osteogenic gene (Xiao et al 2000;Kanno et al 2007). Moreover, mechanical force can increase the differentiation of osteoblasts, by altering the interaction of cell-extracellular matrices (ECM) and inducing the ECM integrindependent ERK1/2 to regulate the osteogenic genes (Kanno et al 2007;Hughes-Fulford 2004).…”