Dentin Matrix Protein 1 (DMP1) plays a regulatory role in dentin mineralization and can also function as a signaling molecule. MMP-2 (matrix metalloproteinase-2) is a predominant protease in the dentin matrix that plays a prominent role in tooth formation and a potential role during the carious process. The possibility that MMP-2 can cleave DMP1 to release biologically active peptides was investigated in this study. DMP1, both in the recombinant form and in its native state within the dentin matrix, was shown to be a substrate for MMP-2. Proteolytic processing of DMP1 by MMP-2 produced two major peptides, one that contains the C-terminal region of the protein known to carry both the ASARM (aspartic acid and serine rich domain) domain involved in biomineralization and the DNA binding site of DMP1. In vitro experiments with recombinant N-and C-terminal polypeptides mimicking the MMP-2 cleavage products of DMP1 demonstrated an effect of the C-polypeptide on the differentiation of dental pulp stem/progenitor cells to a putative odontoblast phenotype. In vivo implantation of this peptide in a rat injured pulp model induced a rapid formation of a homogeneous dentin bridge covered by a palisade of orientated cells expressing dentin sialoprotein (DSP) and DMP1, attesting an efficient repair process. These data suggest that a peptide generated through the proteolytic processing of DMP1 by MMP-2 can regulate the differentiation of mesenchymal cells during dentinogenesis and thus sustain reparative dentin formation in pathological situations such as carious decay. In addition, these data open a new therapeutic possibility of using this peptide to regenerate dentin after an injury.

Dentin matrix protein 1 (DMP1), a phosphorylated protein present in the mineral phase of both vertebrates and invertebrates, is a key regulatory protein during biogenic formation of mineral deposits. Previously we showed that DMP1 is localized in the nuclear compartment of preosteoblasts and preodontoblasts. In the nucleus DMP1 might play an important role in the regulation of genes that control osteoblast or odontoblast differentiation. Here, we show that cellular uptake of DMP1 occurs through endocytosis. Interestingly, this process is initiated by DMP1 binding to the glucose-regulated protein-78 (GRP-78) localized on the plasma membrane of preodontoblast cells. Binding of DMP1 to GRP-78 receptor was determined to be specific and saturable with a binding dissociation constant K D ‫؍‬ 85 nM. We further depict a road map for the endocytosed DMP1 and demonstrate that the internalization is mediated primarily by caveolae and that the vesicles containing DMP1 are routed to the nucleus along microtubules. Immunohistochemical analysis and binding studies performed with biotin-labeled DMP1 confirm spatial co-localization of DMP1 and GRP-78 in the preodontoblasts of a developing mouse molar. Co-localization of DMP1 with GRP-78 was also observed in T4-4 preodontoblast cells, dental pulp stem cells, and primary preodontoblasts. By small interfering RNA techniques, we demonstrate that the receptor for DMP1 is GRP-78. Therefore, binding of DMP1 with GRP-78 receptor might be an important mechanism by which DMP1 is internalized and transported to the nucleus during bone and tooth development.Acidic noncollagenous proteins play a pivotal role during biomineral formation. Dentin matrix protein 1 (DMP1) 2 was the first of the acidic proteins cloned from the dentin matrix.Although initially isolated from the dentin matrix and thought to be unique to dentin and named accordingly, DMP1 has now been found to be present in all mineralized tissues of the vertebrate system (1-3).We previously showed that DMP1 may act as a transcriptional regulator as DMP1 is localized in the nucleus of preosteoblasts (2). In the nucleus DMP1 played a regulatory role in the regulation of specific genes that control osteoblast and odontoblast differentiation (2, 3). Specifically, we showed that DMP1 functions as a transcriptional regulator of the odontoblast specific gene dentin sialophosphoprotein (3). The export of DMP1 from the nucleus during maturation of osteoblasts was found to be in response to a stimulus from calcium ions. In the extracellular matrix DMP1 can nucleate the formation and growth of hydroxyapatite (4, 5). Initial observations from the DMP1-deficient mouse showed no apparent skeletal or tooth phenotype during early development, suggesting that DMP1 function may be redundant. However, postnatal DMP1-null mice developed tooth formation defects, characterized by a partial failure of maturation from predentin to dentin (6). Collectively, these data indicate that DMP1 may play a pivotal role in regulating mineralized matrix formation. In ...

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.Osteoblasts can react to a variety of biological signals. Among these, calcium signaling is essential for the proliferation and differentiation of osteoblasts. Earlier studies have shown that treating osteoblasts with parathyroid hormone or vitamin D 3 induces an increase in intracellular calcium ([Ca 2ϩ ] i ) by increasing the release of Ca 2ϩ from the intracellular stores (1-5). Store-operated Ca 2ϩ channels, which are activated in response to Ca 2ϩ store depletion, control homeostasis between the extracellular Ca 2ϩ reservoir and intracellular Ca 2ϩ storage and control a wide range of cellular functions.Dentin matrix protein 1 (DMP1) initially identified and localized in the mineralized dentin and bone matrix (6) is thought to play a regulatory role only in the calcification of the extracellular matrix. Apart from its role in mineralization, one of the putative functions of DMP1 is its involvement during differentiation of osteoblasts and odontoblasts (7-9). DMP1-null mice displayed severe defects in bone formation (10). We had shown earlier that DMP1 is specifically localized in the nucleus of differentiating osteoblasts and odontoblasts, and this translocation from the extracellular matrix is facilitated by the endocytic receptor GRP78 (11). The 78-kDa glucose-regulated protein (GRP78) is a calcium-binding molecular chaperone expressed in the endoplasmic reticulum of eukaryotic cells. Identification of GRP78 as a cell surface receptor for DMP1 is particularly interesting as its induction is a protective response against several kinds of stress, including ER 2 Ca 2ϩ depletion and accumulation of unglycosylated proteins (12, 13). However, the specific signaling pathways activated following DMP1 stimulus and osteoblast differentiation are not delineated yet.p38 MAPKs are widely e...

Background: DPP mediates activation of anchorage-dependent signals. Results: DPP activates focal adhesion complexes and MAPK signaling in undifferentiated mesenchymal cells and primary pulp cells, leading to their terminal differentiation into odontoblast-like cells. Conclusion: DPP on the substrate provides a tight association between the structural and signaling elements in undifferentiated mesenchymal cells. Significance: DPP promotes adhesion-based odontogenic cell differentiation.

DMP1 has been shown to play many roles in osteogenesis. We recently demonstrated that calcium-mediated stress kinase activation by DMP1 leads to osteoblast differentiation. In this study we demonstrate that DMP1 can also activate the extracellular signal-regulated kinase (ERK)-MAPK pathway. This activation was mediated through the RGD integrin-binding domain in DMP1. Further, we demonstrate that Runx2, an essential transcription factor, is stimulated by the ERK-MAPK pathway.