Dentin Matrix Protein 1 Protein Research Articles

Dentin Matrix Protein 1 Regulates Mineralization of MC3T3-E1 Cells via the TNAP-ANK-ENPP1 Axis.

Dentin matrix protein 1 (DMP1) is central to matrix mineralization. Clarification of the function of DMP1 is crucial to understanding normal bone formation and pathological calcification. The tissue-nonspecific alkaline phosphatase (TNAP) -progressive ankylosing enzyme (ANK) -extracellular nucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1) axis induces deposition of hydroxyapatite (HA) and pyrophosphate dehydrate (CPPD) by regulating pyrophosphate (PPi). Here, we investigated the mechanism by which DMP1 and the TNAP-ANK-ENPP1 axis participate in mineralization. Expression of DMP1, TNAP, NPP1, and ANK genes in MC3T3-E1 cells was detected by RT-qPCR before and after treatment with DMP1 siRNA. An enzyme-linked immunosorbent assay was used to determine expression of DMP1 protein, TNAP activity was detected by SIGMAFAST p-nitrophenyl phosphate tablets, and mineralization of osteoblasts was determined by alizarin red staining. PPi levels were determined radiometrically and equalized for cell DNA. Levels of calcium, inorganic phosphate, zinc, and magnesium were assessed by standard laboratory techniques. After DMP1 gene silencing, expressions of TNAP, ENPP1, and ANK were correspondingly reduced. DMP1 altered extravesicular and intravesicular ion levels through the TNAP-ENPP1-ANK axis in MC3T3-E1 cells. DMP1 regulated mineralization of MC3T3-E1 cells via the TNAP-ANK-ENPP1 axis and affected TNAP activity by two processes-rapid regulation of the Zn2+ transporter (ZnT) and transcriptional regulation of hysteresis. However, DMP1 may affect expression of ENPP1 and ANK only via hysteresis transcriptional regulation. DMP1, as a calcium trap or catalytic enzyme, appears to have a role in collagen mineralization.

Dentin Matrix Protein 1 Silencing Inhibits Phosphorus Utilization in Primary Cultured Tibial Osteoblasts of Broiler Chicks.

Three experiments were carried out in the present study to investigate whether dentin matrix protein 1 (DMP1) was involved in regulating phosphorus (P) metabolic utilization in primary cultured tibial osteoblasts of broiler chicks. Experiment 1 was conducted to select the optimal osteogenic inductive culture medium and the optimal induction time in primary cultured tibial osteoblasts of broiler chicks. In experiment 2, the siRNAs against DMP1 were designed, synthesized and transfected into primary cultured tibial osteoblasts of broiler chicks, and then the inhibitory efficiencies of siRNAs against DMP1 were determined, and the most efficacious siRNA was selected to be used for the DMP1 silencing. In experiment 3, with or without siRNA against DMP1, primary cultured tibial osteoblasts of broiler chicks were treated with the medium supplemented with 0.0, 1.0 or 2.0 mmol/L of P as NaH2PO4 for 12 days. The P metabolic utilization-related parameters were measured. The results showed that the osteogenic induced medium 2 and 12 days of the optimal induction time were selected; Among the designed siRNAs, the si340 was the most effective (P < 0.05) in inhibiting the DMP1 expression; DMP1 silencing decreased (P < 0.05) the expressions of DMP1 mRNA and protein, P retention rate, mineralization formation, alkaline phosphatase activity and bone gla-protein content in tibial osteoblasts at all of added P levels. It is concluded that DMP1 silencing inhibited P utilization, and thus DMP1 was involved in regulating P metabolic utilization in primary cultured tibial osteoblasts of broiler chicks, which provides a novel insight into the regulation of the P utilization in the bone of broilers, and will contribute to develop feasible strategies to improve the bone P utilization efficiency of broilers so as to decrease its excretion.

Recombinant human dentin matrix protein 1 (DMP1) induces the osteogenic differentiation of human periodontal ligament cells

The study aimed to produce recombinant human dentin matrix protein 1 (DMP1) and to test, whether the recombinant DMP1 produced in Escherichia coli possesses functional activity. A gene construction comprising a gene encoding for DMP1 protein with polyhistidine sequence at its C-terminus was created using the pET22b plasmid and expressed in E. coli. The optimization of cultivation conditions has enabled the induction of the gene expression with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and DMP1 recombinant protein production at 37 °C for 6 h. The recombinant protein was purified using Ni affinity chromatography. DMP1 influence on the viability, osteogenic differentiation and calcification of human periodontal ligament (PDL) cells was examined. The purified DMP1 could induce the expression of osteogenesis related genes and calcium deposition in PDL cells. These findings indicate that DMP1 produced in E. coli can induce the osteogenic differentiation of human PDL cells, leading to improved tooth repair and regeneration.

MicroRNA let-7c-5p Suppressed Lipopolysaccharide-Induced Dental Pulp Inflammation by Inhibiting Dentin Matrix Protein-1-Mediated Nuclear Factor kappa B (NF-κB) Pathway In Vitro and In Vivo.

BackgroundLet-7c-5p is down-regulated in dental pulp tissues in inflammatory disorders. The microRNA (miR) molecule shows an anti-inflammation potential due to its direct regulation of dentin matrix protein-1 (DMP1), which promotes inflammation changes in dental pulp tissues. In the present study, the effect of let-7c-5p on lipopolysaccharide (LPS)-induced pulpitis was detected and the associated mechanism was explored.Material/MethodsDental pulp stem cells (DPSCs) were isolated from rat dental tissues, infected with let-7c-5p lentivirus particles, and subjected to LPS administration to induce inflammation. Then, the effect of let-7c-5p overexpression on LPS-induced impairments on DPSCs were detected and the mechanism was explained by focusing on the DMP1 expression and NF-κB pathway. The role of DMP1 in the anti-inflammation effect of let-7c-5p was assessed by incubating let-7c-5p-expressed DPSCs with DMP1 protein. The results of in vitro assays were verified in LPS-induced rat pulpitis models.ResultsLPS administration increased the production of IL-1β and TNF-α and decreased DPSCs viability by increasing the expression of DMP1 and activating NF-κB pathway. However, the induced expression of let-7c-5p relieved DPSCs from LPS-induced inflammation and suppressed DMP1 as well as NF-κB pathway. The incubation of let-7c-5p-expressed DPSCs with DMP1 protein blocked the effect of let-7c-5p. In in vivo experiments, the injection of let-7c-5p attenuated LPS-induced pulpitis by inhibiting DMP1-mediated NF-κB pathway.ConclusionsFindings outlined in the current study demonstrated the dental pulp protecting function of let-7c-5p during LPS-induced inflammation, which was exerted by inhibiting the DMP1-mediated NF-κB pathway.

Asiaticoside induces osteogenic differentiation of human periodontal ligament cells through the Wnt pathway.

Asiaticoside is a compound isolated from Herb Centella asiatica, which has been shown to promote osteogenic differentiation of human periodontal ligament (hPDL) cells. This study investigated the molecular mechanism underlying the asiaticoside-induced osteogenic differentiation of hPDL cells. hPDL cells were incubated with various concentrations of asiaticoside to test cell viability by MTT assay. The mRNA expression levels were analyzed by using quantitative real-time polymerase chain reaction (PCR). Osteogenic differentiation was determined by alkaline phosphatase activity assay and alizarin red staining. The subcellular localization of β-catenin was analyzed by both immunofluorescence and western blot. The results showed that asiaticoside had no effect on the cell viability at any of the tested concentrations. Real-time PCR revealed that osterix (OSX) and dentin matrix protein1 (DMP1) mRNA were significantly enhanced by asiaticoside treatment. Alkaline phosphatase activity and in vitro mineralization were also significantly induced. Interestingly, asiaticoside dose-dependently increased WNT3A mRNA expression, but not WNT5A and WNT10B. The activation of Wnt signaling was shown to result in nuclear accumulation of β-catenin as evaluated by immunofluorescence staining and western blot analysis. Pre-treatment with recombinant human Dickkopf1 (rhDKK1) inhibited asiaticoside-induced β-catenin nuclear translocation and osteoblast marker gene expression. Moreover, rhDKK1 attenuated asiaticoside-induced DMP1 protein expression. The data demonstrate that asiaticoside induces osteogenic differentiation of hPDL cells by activating the Wnt/β-catenin signaling pathway. The findings suggest that asiaticoside could be used as a novel therapeutic drug for periodontal tissue regeneration.

TCP wear particles causes injury of periprosthetic osteocytes in the mouse calvaria

To study whether tricalcium phosphate(TCP) wear particles cause injuries of periprosthetic osteocytes in the mouse calvaria, and to explain its molecular mechanism. Thirty six-week(ICR)male mice were randomly divided into sham group, model (TCP) group and 3-methyladenine (3-MA) group. A murine calvarial model of osteolysis was established by 30 mg of TCP wear particles implantation over the periosteum around the middle suture of calvaria in mice. On the second postoperative day, the autophagy specific inhibitor 3-MA (1.0 mg/kg) was subcutaneously injected to the calvaria in the 3-MA-treated mice every other day. After 2 weeks, blood and the calvaria were obtained. Micro-CT was used to detect bone mineral density(BMD), bone volume fraction (BVF) and porosity number. HE staining and flow cytometry were performed to analyze the viability and apoptosis of periprosthetic osteocytes. The serum levels of dentin matrix protein 1(DMP-1) and sclerostin (SOST) were determined by ELISA. The proteins expressions of DMP-1, SOST, Beclin-1 and microtuble-associated protein 1 light chain 3 (LC-3) were detected by Western blot in the calvaria osteocytes. Compared with the sham group, the mice in the TCP group showed that a significant decrease in the viability of periprosthetic osteocytes, but obvious increases in number of osteocytes death and osteocytes apoptosis (P<0.05), and in serum level and protein expression of SOST; significant decreases in serum level and protein expression of DMP-1 (P<0.05), and remarkable up-regulation of autophagy-related factors beclin-1 and the conversion of LC3-Ⅱ from LC3-I in the calvaria osteocytes. Compared with TCP group, the mice in the 3-MA group showed that injuries of calvaria osteocytes were obviously aggravated, and osteocytes apoptosis was significantly increased (P<0.05). TCP wear particles can cause injuries of periprosthetic osteocytes via activation of apoptosis and autophagy, which promotes osteolysis around the prosthesis osteolysis and joint aseptic loosening.

GaAlAs laser-induced pulp mineralization involves dentin matrix protein 1 and osteopontin expression.

GaAlAs lasers induce pulp mineralization by promoting reparative dentinogenesis. This study analyzed the expression of dentin matrix protein 1 (DMP1) and osteopontin in GaAlAs laser-irradiated rat molars, to examine the hypothesis that these proteins play a role in the laser-induced reparative dentinogenic process. The mesial surfaces of the upper first molars of 8-week-old Wistar rats were irradiated with a pulsed GaAlAs laser. After 1-14days, mRNA expression of DMP1 and osteopontin in the coronal pulp was analyzed using real-time PCR. DMP1, osteopontin, and heat shock protein 25 (HSP25) were immunolocalized at 1-21days. The pulp exhibited a degenerative zone in its mesial portion on days 1-3, and progressive formation of reparative dentin lined with HSP25-immunoreactive odontoblast-like cells, from day 7 onwards. DMP1 and osteopontin mRNA expression were significantly upregulated on days 1-7 and 3-7, respectively. From day 7 onwards, DMP1 and osteopontin immunoreactivity colocalized along the boundary between the primary and reparative dentin. GaAlAs laser irradiation of rat molars induced upregulated DMP1 and osteopontin mRNA expression in the coronal pulp, followed by the formation of reparative dentin and the colocalization of DMP1 and osteopontin immunoreactivity at the site at which this tissue first appeared.

Glycosylation of Dentin Matrix Protein 1 is critical for osteogenesis.

Proteoglycans play important roles in regulating osteogenesis. Dentin matrix protein 1 (DMP1) is a highly expressed bone extracellular matrix protein that regulates both bone development and phosphate metabolism. After glycosylation, an N-terminal fragment of DMP1 protein was identified as a new proteoglycan (DMP1-PG) in bone matrix. In vitro investigations showed that Ser89 is the key glycosylation site in mouse DMP1. However, the specific role of DMP1 glycosylation is still not understood. In this study, a mutant DMP1 mouse model was developed in which the glycosylation site S89 was substituted with G89 (S89G-DMP1). The glycosylation level of DMP1 was down-regulated in the bone matrix of S89G-DMP1 mice. Compared with wild type mice, the long bones of S89G-DMP1 mice showed developmental changes, including the speed of bone remodeling and mineralization, the morphology and activities of osteocytes, and activities of both osteoblasts and osteoclasts. These findings indicate that glycosylation of DMP1 is a key posttranslational modification process during development and that DMP1-PG functions as an indispensable proteoglycan in osteogenesis.

PTH and Vitamin D Repress DMP1 in Cementoblasts

A complex feedback mechanism between parathyroid hormone (PTH), 1,25(OH)2D3 (1,25D), and fibroblast growth factor 23 (FGF-23) maintains mineral homeostasis, in part by regulating calcium and phosphate absorption/reabsorption. Previously, we showed that 1,25D regulates mineral homeostasis by repressing dentin matrix protein 1 (DMP1) via the vitamin D receptor pathway. Similar to 1,25D, PTH may modulate DMP1, but the underlying mechanism remains unknown. Immortalized murine cementoblasts (OCCM.30), similar to osteoblasts and known to express DMP1, were treated with PTH (1–34). Real-time quantitative polymerase chain reaction (PCR) and Western blot revealed that PTH decreased DMP1 gene transcription (85%) and protein expression (30%), respectively. PTH mediated the downregulation of DMP1 via the cAMP/protein kinase A (PKA) pathway. Immunohistochemistry confirmed the decreased localization of DMP1 in vivo in cellular cementum and alveolar bone of mice treated with a single dose (50 µg/kg) of PTH (1–34). RNA-seq was employed to further identify patterns of gene expression shared by PTH and 1,25D in regulating DMP1, as well as other factors involved in mineral homeostasis. PTH and 1,25D mutually upregulated 36 genes and mutually downregulated 27 genes by ≥2-fold expression (P ≤ 0.05). Many identified genes were linked with the regulation of bone/tooth homeostasis, cell growth and differentiation, calcium signaling, and DMP1 transcription. Validation of RNA-seq results via PCR array confirmed a similar gene expression pattern in response to PTH and 1,25D treatment. Collectively, these results suggest that PTH and 1,25D share complementary effects in maintaining mineral homeostasis by mutual regulation of genes/proteins associated with calcium and phosphate metabolism while also exerting distinct roles on factors modulating mineral metabolism. Furthermore, PTH may modulate phosphate homeostasis by downregulating DMP1 expression via the cAMP/PKA pathway. Targeting genes/proteins mutually governed by PTH and 1,25D may be a viable approach for designing new therapies for preserving mineralized tissue health.

Osteocytic protein expression response to doxercalciferol therapy in pediatric dialysis patients.

BackgroundOsteocytic protein expression is dysregulated in CKD and is affected by changes in mineral metabolism; however the effects of active vitamin D sterol therapy on osteocyte protein expression in advanced CKD is unknown.MethodsEleven pediatric patients with end stage kidney disease underwent bone biopsy, were treated for 8 months with doxercalciferol, and then underwent a second bone biopsy. Bone expression of fibroblast growth factor 23 (FGF23), dentin matrix protein 1 (DMP1), and sclerostin were determined by immunohistochemistry and quantified by Ariol Scanning. Western blot analysis and qRT-PCR was performed on bone abstracts of a subset of study subjects to determine the nature (i.e. size) of FGF23 and DMP1 in bone before and after therapy.ResultsAs assessed by immunohistochemistry, bone FGF23, DMP1 and sclerostin protein all increased with therapy. In the case of FGF23, this increase was due to an increase in the full-length molecule without the appearance of FGF23 fragments. DMP1 was present primarily in its full-length form in healthy controls while 57kDa and 37kDa fragments of DMP1 were apparent in bone of dialysis patients at baseline and the 57 kDa appeared to decrease with therapy.ConclusionMarked changes in osteocytic protein expression accompany doxercalciferol therapy, potentially impacting bone mineralization and the skeletal response to PTH. The effects of these bone changes on long-term outcomes remain to be determined.

Products of dentin matrix protein-1 degradation by interleukin-1β-induced matrix metalloproteinase-3 promote proliferation of odontoblastic cells.

We have previously reported that interleukin (IL)-1β induces matrix metalloproteinase (MMP)-3-regulated cell proliferation in mouse embryonic stem cell (ESC)-derived odontoblast-like cells, suggesting that MMP-3 plays a potentially unique physiological role in regeneration by odontoblast-like cells. MMPs are able to process virtually any component of the extracellular matrix, including collagen, laminin and bioactive molecules. Because odontoblasts produce dentin matrix protein-1 (DMP-1), we examined whether the degraded products of DMP-1 by MMP-3 contribute to enhanced proliferation in odontoblast-like cells. IL-1β increased mRNA and protein levels of odontoblastic marker proteins, including DMP-1, but not osteoblastic marker proteins, such as osteocalcin and osteopontin. The recombinant active form of MMP-3 could degrade DMP-1 protein but not osteocalcin and osteopontin in vitro. The exogenous degraded products of DMP-1 by MMP-3 resulted in increased proliferation of odontoblast-like cells in a dose-dependent manner. Treatment with a polyclonal antibody against DMP-1 suppressed IL-1β-induced cell proliferation to a basal level, but identical treatment had no effect on the IL-1β-induced increase in MMP-3 expression and activity. Treatment with siRNA against MMP-3 potently suppressed the IL-1β-induced increase in DMP-1 expression and suppressed cell proliferation (p < 0.05). Similarly, treatment with siRNAs against Wnt5a and Wnt5b suppressed the IL-1β-induced increase in DMP-1 expression and suppressed cell proliferation (p < 0.05). Rat KN-3 cells, representative of authentic odontoblasts, showed similar responses to the odontoblast-like cells. Taken together, our current study demonstrates the sequential involvement of Wnt5, MMP-3, DMP-1 expression, and DMP-1 degradation products by MMP-3, in effecting IL-1β-induced proliferation of ESC-derived odontoblast-like cells.

Role of dentin matrix protein 1 in cartilage redifferentiation and osteoarthritis

The aim of this study was to test the possible involvement, relevance and significance of dentin matrix protein 1 (DMP1) in chondrocyte redifferentiation and OA. To examine the function of DMP1 in vitro, bone marrow stromal cells (BMSCs) and articular chondrocytes (ACs) were isolated and differentiated in micromasses in the presence or absence of DMP1 small interfering RNA and analysed for chondrogenic phenotype. The association of DMP1 expression with OA progression was analysed time dependently in the OA menisectomy rat model and in grade-specific OA human samples. It was found that DMP1 was strongly related to chondrogenesis, which was evidenced by the strong expression of DMP1 in the 14.5-day mouse embryonic cartilage development stage and in femoral heads of post-natal days 0 and 4. In vitro chondrogenesis in BMSCs and ACs was accompanied by a gradual increase in DMP1 expression at both the gene and protein levels. In addition, knockdown of DMP1 expression led to decreased chondrocyte marker genes, such as COL2A1, ACAN and SOX9, and an increase in the expression of COL10A and MMP13 in ACs. Moreover, treatment with IL-1β, a well-known catabolic culprit of proteoglycan matrix loss, significantly reduced the expression of DMP1. Furthermore, we also observed the suppression of DMP1 protein in a grade-specific manner in knee joint samples from patients with OA. In the menisectomy-induced OA model, an increase in the Mankin score was accompanied by the gradual loss of DMP1 expression. Observations from this study suggest that DMP1 may play an important role in maintaining the chondrogenic phenotype and its possible involvement in altered cartilage matrix remodelling and degradation in disease conditions like OA.

Nuclear localization of DMP1 proteins suggests a role in intracellular signaling

Dentin matrix protein 1 (DMP1) is highly expressed in odontoblasts and osteoblasts/osteocytes and plays an essential role in tooth and bone mineralization and phosphate homeostasis. It is debatable whether DMP1, in addition to its function in the extracellular matrix, can enter the nucleus and function as a transcription factor. To better understand its function, we examined the nuclear localization of endogenous and exogenous DMP1 in C3H10T1/2 mesenchymal cells, MC3T3-E1 preosteoblast cells and 17IIA11 odontoblast-like cells. RT-PCR analyses showed the expression of endogenous Dmp1 in all three cell lines, while Western-blot analysis detected a major DMP1 protein band corresponding to the 57kDa C-terminal fragment generated by proteolytic processing of the secreted full-length DMP1. Immunofluorescent staining demonstrated that non-synchronized cells presented two subpopulations with either nuclear or cytoplasmic localization of endogenous DMP1. In addition, cells transfected with a construct expressing HA-tagged full-length DMP1 also showed either nuclear or cytoplasmic localization of the exogenous DMP1 when examined with an antibody against the HA tag. Furthermore, nuclear DMP1 was restricted to the nucleoplasm but was absent in the nucleolus. In conclusion, these findings suggest that, apart from its role as a constituent of dentin and bone matrix, DMP1 might play a regulatory role in the nucleus.

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.

Molecular analysis of DMP1 mutants causing autosomal recessive hypophosphatemic rickets

We previously demonstrated that the mutations Met1Val (M1V) and the deletion of nucleotides 1484–1490 (1484–1490del) in Dentin matrix protein-1 (DMP1) cause the novel disorder autosomal recessive hypophosphatemic rickets (ARHR), which is associated with elevated fibroblast growth factor-23 (FGF23). To further understand the role of DMP1 in ARHR, we undertook molecular genetic and in vitro expression studies. First, we examined a kindred with a severe hypophosphatemic rickets phenotype and recessive inheritance. Analyses of this family demonstrated that the affected members had elevated serum FGF23 and carried a large, biallelic deletion that removed the majority of DMP1. At a minimum, this deletion encompassed 49 kb between DMP1 exon 3 and an intergenic region 5′ to the next telomeric gene, integrin-binding sialoprotein (IBSP). We next performed immunofluorescent studies in cells to understand the effects of the known ARHR mutations on DMP1 cellular processing. These analyses showed that the M1V DMP1 mutant was not sorted to the trans-Golgi network (TGN) and secretory pathway, but filled the entire cytoplasm. In contrast, the 1484–1490del mutant localized to the TGN and was secreted, similar to wild type DMP1. The 1484–1490del mutation replaces the DMP1 18 C-terminal amino acids with 33 non-native residues. Truncation of wild type DMP1 by these native 18 residues followed by Western blot and confocal microscopic analyses demonstrated a wild type expression pattern when compared with the 1484–1490del mutant, indicating that the last 18 residues are not critical for cellular trafficking, but that the 33 additional residues arising from the 1484–1490del mutation likely compromise DMP1 processing. The relationship between DMP1 and FGF23 is unclear. To test endogenous DMP1 response to serum metabolites that also regulate FGF23, UMR-106 cells were treated with 1,25(OH) 2 vitamin D (1 × 10 − 7 M) and showed a 12-fold increase in DMP1 mRNA and protein at 24 h. In summary, we have identified a novel DMP1 deletion as the cause of ARHR, as well as demonstrated that the ARHR mutations alter DMP1 cellular processing, and that DMP1 can be regulated by vitamin D. Taken together, this work expands our understanding of the genetic and molecular mechanisms associated with DMP1 alterations causing ARHR.

Generation of a conditional null allele for Dmp1 in mouse

Dentin matrix protein1 (DMP1), highly conserved in humans and mice, is highly expressed in teeth, the skeleton, and to a lesser extent in nonskeletal tissues such as brain, kidney, and salivary gland. Pathologically, DMP1 is associated with several forms of cancers and with tumor-induced osteomalacia. Conventional disruption of the murine Dmp1 gene results in defects in dentin in teeth and in the skeleton, including hypophosphatemic rickets, and abnormalities in phosphate homeostasis. Human DMP1 mutations are responsible for the condition known as autosomal recessive hypophosphatemic rickets. For better understanding of the roles of DMP1 in different tissues at different stages of development and in pathological conditions, we generated Dmp1 floxed mice in which loxP sites flank exon 6 that encodes for over 80% of DMP1 protein. We demonstrate that Cre-mediated recombination using Sox2-Cre, a Cre line expressed in epiblast during early embryogenesis, results in early deletion of the gene and protein. These homozygous Cre-recombined null mice display an identical phenotype to conventional null mice. This animal model will be useful to reveal distinct roles of DMP1 in different tissues at different ages.

Degradation of MEPE, DMP1, and Release of SIBLING ASARM-Peptides (Minhibins): ASARM-Peptide(s) Are Directly Responsible for Defective Mineralization in HYP

Mutations in PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) and DMP1 (dentin matrix protein 1) result in X-linked hypophosphatemic rickets (HYP) and autosomal-recessive hypophosphatemic-rickets (ARHR), respectively. Specific binding of PHEX to matrix extracellular phosphoglycoprotein (MEPE) regulates the release of small protease-resistant MEPE peptides [acidic serine- and aspartate-rich MEPE-associated motif (ASARM) peptides]. ASARM peptides are potent inhibitors of mineralization (minhibins) that also occur in DMP1 [MEPE-related small integrin-binding ligand, N-linked glycoprotein (SIBLING) protein]. It is not known whether these peptides are directly responsible for the mineralization defect. We therefore used a bone marrow stromal cell (BMSC) coculture model, ASARM peptides, anti-ASARM antibodies, and a small synthetic PHEX peptide (SPR4; 4.2 kDa) to examine this. Surface plasmon resonance (SPR) and two-dimensional (1)H/(15)N nuclear magnetic resonance demonstrated specific binding of SPR4 peptide to ASARM peptide. When cultured individually for 21 d, HYP BMSCs displayed reduced mineralization compared with wild type (WT) (-87%, P < 0.05). When cocultured, both HYP and WT cells failed to mineralize. However, cocultures (HYP and WT) or monocultures of HYP BMSCs treated with SPR4 peptide or anti-ASARM neutralizing antibodies mineralized normally. WT BMSCs treated with ASARM peptide also failed to mineralize properly without SPR4 peptide or anti-ASARM neutralizing antibodies. ASARM peptide treatment decreased PHEX mRNA and protein (-80%, P < 0.05) and SPR4 peptide cotreatment reversed this by binding ASARM peptide. SPR4 peptide also reversed ASARM peptide-mediated changes in expression of key osteoclast and osteoblast differentiation genes. Western blots of HYP calvariae and BMSCs revealed massive degradation of both MEPE and DMP1 protein compared with the WT. We conclude that degradation of MEPE and DMP-1 and release of ASARM peptides are chiefly responsible for the HYP mineralization defect and changes in osteoblast-osteoclast differentiation.

Porcine dentin matrix protein 1: gene structure, cDNA sequence, and expression in teeth

Dentin matrix protein 1 (DMP1) is an acidic non-collagenous protein that is necessary for the proper biomineralization of bone, cartilage, cementum, dentin, and enamel. Dentin matrix protein 1 is highly phosphorylated and potentially glycosylated, but there is no experimental data identifying which specific amino acids are modified. For the purpose of facilitating the characterization of DMP1 from pig, which has the advantage of large developing teeth for obtaining protein in quantity and extensive structural information concerning other tooth matrix proteins, we characterized the porcine DMP1 cDNA and gene structure, raised anti-peptide immunoglobulins that are specific for porcine DMP1, and detected DMP1 protein in porcine tooth extracts and histological sections. Porcine DMP1 has 510 amino acids, including a 16-amino acid signal peptide. The deduced molecular weight of the secreted, unmodified protein is 53.5 kDa. The protein has 93 serines and 12 threonines in the appropriate context for phosphorylation, and four asparagines in a context suitable for glycosylation. Dentin matrix protein 1 protein bands with apparent molecular weights between 30 and 45 kDa were observed in partially purified dentin extracts. In developing teeth, immunohistochemistry localized DMP1 in odontoblasts and the dentinal tubules of mineralized dentin and in ameloblasts, but not in the enamel matrix.

DMP1 depletion decreases bone mineralization in vivo: an FTIR imaging analysis.

The role of DMP1 in mineralization was analyzed by comparing bone mineral and matrix properties in dmp1-null female mice to heterozygous and wildtype controls by FTIR imaging spectroscopy. The observed decreased mineral content in dmp1 null mice indicates a key role for dmp1 in bone mineralization. Indirect effects of DMP1 on other systems also determine the KO phenotype. Dentin matrix protein 1 (DMP1), an acidic phosphorylated extracellular matrix protein, is highly expressed in mineralized tissues. In vitro, DMP1 peptides can promote or inhibit mineralization depending on the extent of phosphorylation, the peptide size, and concentration. To clarify the biological function of DMP1 protein on in vivo mineralization, this study analyzed bone properties of dmp1 knockout (KO) mice compared with heterozygous (HET) and wildtype (WT) controls. Tibias from dmp1 KO and age-, sex-, and background-matched HET and WT mice at 4 and 16 weeks (N(total) = 60) were examined by Fourier transform infrared imaging (FTIRI), histology (n = 6 per genotype and age; N = 36), and geometry by muCT (n = 4 per genotype and age; N = 24). Serum ionic calcium and phosphate concentrations were also determined. The mineral-to-matrix ratios (spectroscopic parameter of relative mineral content) were significantly lower in dmp1 KO mice tibias compared with WT and HET at 4 and 16 weeks. The mineral crystallinity (crystal size/perfection) was significantly increased in dmp1 KO and HET mice relative to WT. Collagen cross-link ratios (a spectroscopic parameter related to the relative amounts of nonreducible/reducible collagen cross-links) in dmp1 KO were not significantly different from WT and HET. Based on muCT, cortical bone cross-sectional areas at 16 but not 4 weeks were significantly reduced in the KO compared with controls. Maximum, minimum, and polar cross-sectional moments of inertia were significantly lower in dmp1 KO than in HET at 16 weeks but not at 4 weeks. Histological analysis and muCT 3-D images suggested that dmp1 KO mice had osteomalacia. Dmp1 KO mice had significantly lower ionic calcium and phosphate concentrations relative to WT, whereas in the HET, values for phosphate were equivalent, and calcium values were decreased relative to WT values. The findings of decreased mineral-to-matrix ratio and increased crystal size in bones of dmp1 KO mice suggest that DMP1 has multiple roles (both direct and indirect) in the regulation of postnatal mineralization. We suggest that direct effects on mineral formation, crystal growth, and indirect effects on regulation of Ca x P concentrations and matrix turnover all contribute to the dominant phenotype in the dmp1 KO mouse.

Different patterns of selection on the nuclear genes IRBP and DMP-1 affect the efficiency but not the outcome of phylogeny estimation for didelphid marsupials

Selection at the protein-level can influence nucleotide substitution patterns for protein-coding genes, which in turn can affect their performance as phylogenetic characters. In this study, we compare two protein-coding nuclear genes that appear to have evolved under markedly different selective constraints and evaluate how selection has shaped their phylogenetic signal. We sequenced 1100+ bp of exon 6 of the gene encoding dentin matrix protein 1 (DMP1) from most of the currently recognized genera of New World opossums (family: Didelphidae) and compared these data to an existing matrix of sequences from the interphotoreceptor retinoid-binding protein gene (IRBP) and morphological characters. In comparison to IRBP, DMP1 has far fewer sites under strong purifying selection and exhibits a number of sites under positive directional selection. Furthermore, selection on the DMP1 protein appears to conserve short, acidic, serine-rich domains rather than primary amino acid sequence; as a result, DMP1 has significantly different nucleotide substitution patterns from IRBP. Using Bayesian methods, we determined that DMP1 evolves almost 30% faster than IRBP, has 2.5 times more variable sites, has less among-site rate heterogeneity, is skewed toward A and away from CT (IRBP has relatively even base frequencies), and has a significantly lower rate of change between adenine and any other nucleotide. Despite these different nucleotide substitution patterns, estimates of didelphid relationships based on separate phylogenetic analyses of these genes are remarkably congruent whether patterns of nucleotide substitution are explicitly modeled or not. Nonetheless, DMP1 contains more phylogenetically informative characters per unit sequence and resolves more nodes with higher support than does IRBP. Thus, for these two genes, relaxed functional constraints and positive selection appear to improve the efficiency of phylogenetic estimation without compromising its accuracy.