ABSTRACT
Dentin phosphophoryn (DPP) is a major noncollagenous protein in the dentin matrix. In this study, we demonstrate that pluripotent stem cells such as C3H10T1/2 and human bone marrow cells can be committed to the osteogenic lineage by DPP. Treatment with DPP can stimulate the release of intracellular Ca(2+). This calcium flux triggered the activation of Ca(2+)-calmodulin-dependent protein kinase II (CaMKII). Activated CaMKII induced the phosphorylation of Smad1 and promoted nuclear translocation of p-Smad1. Inhibition of store Ca(2+) depletion by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) or down-regulation of CaMKII by KN-62, a selective cell-permeable pharmacological inhibitor or a dominant negative plasmid of CaMKII, blocked DPP-mediated Smad1 phosphorylation. Activation of Smad1 resulted in the expression of osteogenic markers such as Runx2, Osterix, DMP1, Bone sialoprotein, Osteocalcin, NFATc1, and Schnurri-2, which have been implicated in osteoblast differentiation. These findings suggest that DPP is capable of triggering commitment of pluripotent stem cells to the osteogenic lineage.
Subject(s)
Calcium Signaling , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Cell Differentiation , Extracellular Matrix Proteins/physiology , Mesenchymal Stem Cells/enzymology , Phosphoproteins/physiology , Sialoglycoproteins/physiology , Smad1 Protein/metabolism , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/analogs & derivatives , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/pharmacology , Animals , Antigens, Differentiation/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/antagonists & inhibitors , Calcium-Calmodulin-Dependent Protein Kinase Type 2/physiology , Cattle , Cell Nucleus/metabolism , Cells, Cultured , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Enzyme Activation , Humans , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/physiology , Mice , NFATC Transcription Factors/genetics , NFATC Transcription Factors/metabolism , Osteoblasts/metabolism , Osteogenesis , Phosphorylation , Protein Processing, Post-Translational , Protein Transport , Smad Proteins, Receptor-Regulated/metabolism , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Calcium signaling and calcium transport play a key role during osteoblast differentiation and bone formation. Here, we demonstrate that DMP1 mediated calcium signaling, and its downstream effectors play an essential role in the differentiation of preosteoblasts to fully functional osteoblasts. DMP1, a key regulatory bone matrix protein, can be endocytosed by preosteoblasts, triggering a rise in cytosolic levels of calcium that initiates a series of downstream events leading to cellular stress. These events include release of store-operated calcium that facilitates the activation of stress-induced p38 MAPK leading to osteoblast differentiation. However, chelation of intracellular calcium and inhibition of the p38 signaling pathway by specific pharmacological inhibitors and dominant negative plasmid suppressed this activation. Interestingly, activated p38 MAPK can translocate to the nucleus to phosphorylate transcription factors that coordinate the expression of downstream target genes such as Runx 2, a key modulator of osteoblast differentiation. These studies suggest a novel paradigm by which DMP1-mediated release of intracellular calcium activates p38 MAPK signaling cascade to regulate gene expression and osteoblast differentiation.
