Properties of the (DSS) n triplet repeat domain of rat dentin phosphophoryn

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Mineralized tissues such as dentin and bone assemble extracellular matrices uniquely rich in a variety of acidic phosphoproteins. Although these proteins are presumed to play a role in the process of biomineralization, key questions regarding the nature of their contributions remain unanswered. First, it is not known whether highly phosphorylated proteins alone can induce matrix mineralization, or whether this activity requires the involvement of other bone/dentin non-collagenous proteins. Second, it remains to be established whether the protein kinases that phosphorylate these acidic proteins are unique to cells responsible for producing mineralized tissues. To begin to address these questions, we consider the case of phosphophoryn (PP), due to its high content of phosphate, high affinity for Ca 2؉ , and its potential role in hydroxyapatite nucleation. We have created a model system of biomineralization in a cellular environment by expressing PP in NIH3T3 fibroblasts (which do not produce a mineralized matrix); as a positive control, PP was expressed in MC3T3-E1 osteoblastic cells, which normally mineralize their matrices. We show that expression of PP in NIH3T3 cells is sufficient for the induction of matrix mineralization. In addition, assessment of the phosphorylation status of PP in these cells reveals that the transfected NIH3T3 cells are able to phosphorylate PP. We suggest that the phosphorylation of PP is essential for mineral formation. The principle goal of this study is to enrich the current knowledge of mineralized tissue phosphorylation events by analyzing them in the context of a complete cellular environment.In biologically induced mineralization, crystals are generally produced by a heterogeneous nucleation mechanism. The deposition of mineral crystals in bone, dentin, and cartilage is orchestrated by cells, and the growth of these crystals is facilitated through mineral-matrix interactions. Current data indicate an important role for non-collagenous extracellular matrix (ECM) 2 proteins associated with collagen fibrils in the process of biomineralization. The high affinity of these non-collagenous proteins for divalent cations may facilitate control of the nucleation and growth of the initial mineral deposits. In addition, these proteins may subsequently regulate the size, composition, microstructure, morphology, and the orientation of the resulting crystals formed (crystal growth) (1). Mineralized tissues are unique in their production of extracellular acidic phosphoproteins. These phosphoproteins may be of structural and/or regulatory significance to the formation of mineralized tissues, potentially acting as a key interface between the nucleating mineral crystal and surrounding collagen fibrils (2, 3). These phosphoproteins include: osteopontin (OPN) (4), bone sialoprotein (5, 6), dentin matrix protein 1 (DMP1) (7), phosphophoryn (PP) (8), osteonectin (9), bone acidic glycoprotein 75 kDa (BAG 75) (10), and MEPE (11), each accumulating in the extracellular matrices of bone and dentin to diffe...

Section: Resultssupporting

confidence: 60%

Section: Transfection Of Pshooter-er-pp Construct Into Mc3t3-e1 and Nmentioning

confidence: 99%

Expression of Phosphophoryn Is Sufficient for the Induction of Matrix Mineralization by Mammalian Cells

Sfeir

Lee

et al. 2011

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“…In situ hybridization experiments have shown that DMP-1 is expressed by hypertrophic chondrocytes, osteoblasts, and odontoblasts (28). Another dentin ECM protein, phosphophoryn (PP), a cleavage product of DSPP (16), has been implicated as a regulator of mineral crystal formation (16,18,19,29). DSPP is localized to chromosome 4, linking mutations in the gene to dentinogenesis imperfecta type II (15,16).…”

mentioning

confidence: 99%

“…Odontoblasts secrete PP along the mineralization front (21,37,38) and, typical of other NCPs, the physiochemical properties of PP dictate high affinity for Ca 2ϩ , which implicates a role in the nucleation or modulation of HA crystal formation (13,22,24). An RGD domain is present at the N-terminal domain of PP (13,19), suggesting an auxiliary function in ECM-cell communication and in the initiation of intracellular signaling pathways. PP, like DMP-1, is a SIB-LING protein family member and could have an important regulatory role in osteogenic signaling and growth factor activity.…”

mentioning

confidence: 99%

Phosphophoryn Regulates the Gene Expression and Differentiation of NIH3T3, MC3T3-E1, and Human Mesenchymal Stem Cells via the Integrin/MAPK Signaling Pathway

Jadlowiec

Koch

Zhang

et al. 2004

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148

142

Extracellular matrix proteins (ECMs) serve as both a structural support for cells and a dynamic biochemical network that directs cellular activities. ECM proteins such as those of the SIBLING family (small integrinbinding ligand glycoprotein) could possess inherent growth factor activity. In this study, we demonstrate that exon 5 of dentin matrix protein 3 (phosphophoryn (PP)), a non-collagenous dentin ECM protein and SIB-LING protein family member, up-regulates osteoblast marker genes in primary human adult mesenchymal stem cells (hMSCs), a mouse osteoblastic cell line (MC3T3-E1), and a mouse fibroblastic cell line (NIH3T3). Quantitative real-time PCR technology was used to quantify gene expression levels of bone markers such as Runx2, Osx (Osterix), bone/liver/kidney Alp (alkaline phosphatase), Ocn (osteocalcin), and Bsp (bone sialoprotein) in response to recombinant PP and stably transfected PP. PP up-regulated Runx2, Osx, and Ocn gene expression. PP increased OCN protein production in hMSCs and MC3T3-E1. ALP activity and calcium deposition was increased by PP in hMSC. Furthermore, an ␣ v ␤ 3 integrin-blocking antibody significantly inhibited recombinant PP-induced expression of Runx2 in hMSCs, suggesting that signaling by PP is mediated through the integrin pathway. PP was also shown to activate p38, ERK1/2, and JNK, three components of the MAPK pathway. These data demonstrate a novel signaling function for PP in cell differentiation beyond the hypothesized role of PP in biomineralization.

“…PP is localized in dentin and bone and is a regulator of matrix mineralization (27). PP is highly negatively charged and consists of many repeats of Asp-Ser-Ser (DSS) n , where n could be as high as 28 (28). Our recent work has demonstrated that PP activates many of the same target genes as BMP-2, including Runx2, Osx, alkaline phosphatase (Alp), and osteocalcin (Ocn) (1).…”

mentioning

confidence: 99%

Extracellular Matrix-mediated Signaling by Dentin Phosphophoryn Involves Activation of the Smad Pathway Independent of Bone Morphogenetic Protein

Jadlowiec

Zhang

et al. 2006

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Cells have ingenious mechanisms for interpreting complex signals from their external microenvironment. Previously, we have shown that phosphophoryn (PP) regulates the expression of bone/ dentin marker genes via the integrin/MAPK signaling pathway (Jadlowiec, J., Koch, H., Zhang, X., Campbell, P. G., Seyedain, M., and Sfeir, C. (2004) J. Biol. Chem. 279, 53323-53330). We hypothesize that other signaling pathways important for mineralized tissue morphogenesis such as the Smad pathway could be involved in PP signaling. We determined activation of the Smad pathway in human adult mesenchymal stem cells following treatment with recombinant PP (rPP). We observed that PP enhanced phosphorylation of Smad1 within 30 min and Smad1 translocation to the nucleus within 1 h. PP up-regulated the expression of Smad1 target genes, Smad6, Dlx5, and Runx2. The timing of PP activation of Smad1 implies this is a direct effect; however, we also investigated the possible involvement of bone morphogenetic proteins in PP stimulation of the Smad pathway. PP was shown to up-regulate Bmp-2 gene expression 12 h post-treatment with PP, which is much later than initial detection of Smad1 phosphorylation at 30 min. Furthermore, addition of Noggin did not block Smad1 phosphorylation by PP. We propose that PP could signal via the Smad pathway by either directly stimulating the phosphorylation of Smad1 via integrins or other mechanisms. These might include integrin/bone morphogenetic protein receptor interactions or involvement of PP with other growth factors leading to the modulation of intracellular signaling. It is noteworthy that a non-transforming growth factor-␤ family member activates the Smad pathway. The role of PP in regulating the Smad pathway raises very interesting questions regarding the role of PP during bone and tooth development.The ECM relays complex signals from the microenvironment to cells. This directs proliferation, differentiation, and apoptosis for proper tissue development and remodeling. During formation of mineralized tissues such as bone and dentin, the cell utilizes several intracellular signaling pathways representing an interconnected network of proteins with the goal to decipher cues from the ECM. We focused our current work on the role of the non-collagenous proteins of dentin such as phosphophoryn (PP) 2 in cell signaling and differentiation. We have determined the involvement of the MAPK pathway in PP's signaling (1). In the present study, our hypothesis is that the Smad pathway might be involved since many of the genes up-regulated by PP are critical to bone differentiation.The Smad pathway is one such pathway important in signal interpretation from the ECM during osteogenesis/dentinogenesis. Smad proteins are intracellular signaling molecules that are activated by members of the transforming growth factor (TGF-␤) family of growth factors which includes the bone morphogenetic proteins (BMPs). Smads are the initial responders to receptor activation of the TGF-␤ family and have been studied as transcriptional a...