Cancer Secretome May Influence BSP and DSP Expression in Human Salivary Gland Cells.

One of the biggest challenges in managing head and neck cancers, especially salivary gland cancers, is the identification of secreted biomarkers of the disease that can be evaluated noninvasively. A relevant source of enriched tumor markers could potentially be found in the tumor secretome. Although numerous studies have evaluated secretomes from various cancers, the influence of the cancer secretome derived from salivary gland cancers on the behavior of normal cells has not yet been elucidated. Our data indicate that secretome derived from salivary gland cancer cells can influence the expression of two potential biomarkers of oral cancer-namely, bone sialoprotein (BSP) and dentin sialoprotein (DSP)-in normal salivary gland cells. Using routine immunohistochemistry, immunofluorescence, and immunoblotting techniques, we demonstrate an enrichment of BSP and DSP in human salivary gland (HSG) cancer tissue, unique localizations of BSP and DSP in HSG cancer cells, and enriched expression of BSP and DSP in normal salivary gland cells exposed to a cancer secretome. The secretome domain of the cancer microenvironment could alter signaling cascades responsible for normal cell proliferation, migration, and invasion, thus enhancing cancer cell survival and the potential for cancer progression. The cancer secretome may be critical in maintaining and stimulating "cancer-ness," thus potentially promoting specific hallmarks of metastasis.

Dentin phosphophoryn in the matrix activates AKT and mTOR signaling pathway to promote preodontoblast survival and differentiation.

Dentin phosphophoryn (DPP) is an extracellular matrix protein synthesized by odontoblasts. It is highly acidic and the phosphorylated protein possesses a strong affinity for calcium ions. Therefore, DPP in the extracellular matrix can promote hydroxyapatite nucleation and can regulate the size of the growing crystal. Besides its calcium binding property, DPP can initiate signaling functions from the ECM (Extracellular matrix). The signals that promote the cytodifferentiation of preodontoblasts to fully functional odontoblasts are not known. In this study, we demonstrate that preodontoblasts on a DPP matrix, generates mechanical and biochemical signals. This is initiated by the ligation of the integrins with the RGD containing DPP. The downstream biochemical response observed is the activation of the AKT(protein kinase B) and mTOR (mammalian target of rapamycin) signaling pathways leading to the activation of the transcription factor NF-κB (Nuclear factor κB). Terminal differentiation of the preodontoblasts was assessed by identifying phosphate and calcium deposits in the matrix using von Kossa and Alizarin red staining respectively. Identifying the signaling pathways initiated by DPP in the dentin matrix would help in devising strategies for dentin tissue engineering.

Dentin phosphophoryn activates Smad protein signaling through Ca2+-calmodulin-dependent protein kinase II in undifferentiated mesenchymal cells.

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.

Stimulation of periodontal ligament stem cells by dentin matrix protein 1 activates mitogen-activated protein kinase and osteoblast differentiation.

BACKGROUND: Periodontitis can ultimately result in tooth loss. Many natural and synthetic materials have been tried to achieve periodontal regeneration, but the results remain variable and unpredictable. We hypothesized that exogenous treatment with dentin matrix protein 1 (DMP1) activates specific genes and results in phenotypic and functional changes in human periodontal ligament stem cells (hPDLSCs). METHODS: hPDLSCs were isolated from extracted teeth and cultured in the presence or absence of DMP1. Quantitative polymerase chain reactions were performed to analyze the expression of several genes involved in periodontal regeneration. hPDLSCs were also processed for immunocytochemical and Western blot analysis using phosphorylated extracellular signal-regulated kinase (pERK) and ERK antibodies. Alkaline phosphatase and von Kossa staining were performed to characterize the differentiation of hPDLSCs into osteoblasts. Field emission scanning electron microscopic analysis of the treated and control cell cultures were also performed. RESULTS: Treatment with DMP1 resulted in the upregulation of genes, such as matrix metalloproteinase-2, alkaline phosphatase, and transforming growth factor ß1. Activation of ERK mitogen-activated protein kinase signaling pathway and translocation of pERK from the cytoplasm to the nucleus was observed. Overall, DMP1-treated cells showed increased expression of alkaline phosphatase, increased matrix, and mineralized nodule formation when compared with untreated controls. CONCLUSION: DMP1 can orchestrate a coordinated expression of genes and phenotypic changes in hPDLSCs by activation of the ERK signaling pathway, which may provide a valuable strategy for tissue engineering approaches in periodontal regeneration.

DPP in the matrix mediates cell adhesion but is not restricted to stickiness: a tale of signaling.

Cell adhesion to DPP substrate is an integrin-mediated event and involves integrin binding, clustering, assembly of focal adhesion complexes and cytoskeletal organization. Cells perceive the DPP substrate through the integrin receptor αvß1 and bind the actin cytoskeleton to the membrane via focal adhesion sites. The cells respond to this proteinaceous rigid substrate by activating the mechano-chemical signaling events leading to cell spreading and formation of focal adhesions. Focal adhesions, which are sites of integrin binding to the extracellular matrix, form in the leading edge during cell migration. These sites are dynamic and the supramolecular assemblies contain structural and signaling components regulating cell functions. In our study, we present a scenario that integrins utilize the actin network to permit activation of the mitogen-activated kinase modules to transduce signals through the cytoplasm to the nucleus in the presence of DPP. We specifically demonstrate that ERK-mediated transcriptional events impinge on activation of transcription factors leading to cell differentiation.

Dentin phosphoprotein (DPP) activates integrin-mediated anchorage-dependent signals in undifferentiated mesenchymal cells.

Dentin phosphoprotein (DPP), a major noncollagenous protein of the dentin matrix, is a highly acidic protein that binds Ca(2+) avidly and is thus linked to matrix mineralization. Here, we demonstrate that the RGD domain in DPP can bind to integrins on the cell surface of undifferentiated mesenchymal stem cells and pulp cells. This coupling generates intracellular signals that are channeled along cytoskeletal filaments and activate the non-receptor tyrosine kinase focal adhesion kinase, which plays a key role in signaling at sites of cellular adhesion. The putative focal adhesion kinase autophosphorylation site Tyr(397) is phosphorylated during focal adhesion assembly induced by DPP on the substrate. We further demonstrate that these intracellular signals propagate through the cytoplasm and activate anchorage-dependent ERK signaling. Activated ERK translocates to the nucleus and phosphorylates the transcription factor ELK-1, which in turn coordinates the expression of downstream target genes such as DMP1 and dentin sialoprotein (DSP). These studies suggest a novel paradigm demonstrating that extracellular DPP can induce intracellular signaling that can be propagated to the nucleus and thus alter gene activities.

Activation of the ERK1/2 mitogen-activated protein kinase cascade by dentin matrix protein 1 promotes osteoblast 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.

Calcium-mediated stress kinase activation by DMP1 promotes osteoblast differentiation.

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.

MMP2-cleavage of DMP1 generates a bioactive peptide promoting differentiation of dental pulp stem/progenitor cell.

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.

Endoplasmic reticulum chaperone protein GRP-78 mediates endocytosis of dentin matrix protein 1.

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.