Dentin Matrix Protein 1 mRNA Research Articles

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.

The Effects of Inorganic Phosphorus Levels on Phosphorus Utilization, Local Bone-Derived Regulators, and BMP/MAPK Pathway in Primary Cultured Osteoblasts of Broiler Chicks.

Understanding the underlying mechanisms that regulate the bone phosphorus (P) utilization would be helpful for developing feasible strategies to improve utilization efficiency of P in poultry. We aimed to investigate the effects of inorganic P levels on P utilization, local bone-derived regulators and bone morphogenetic protein/mitogen-activated protein kinase (BMP/MAPK) pathway in primary cultured osteoblasts of broiler chicks in order to address whether local bone-derived regulators or BMP/MAPK pathway was involved in regulating the bone P utilization of broilers using an in vitro model. The primary cultured tibial osteoblasts of broiler chicks were randomly divided into one of five treatments with six replicates for each treatment. Then, cells were respectively incubated with 0.0, 0.5, 1.0, 1.5, or 2.0 mmol/L of added P as NaH2PO4 for 24 days. The results showed that as added P levels increased, tibial osteoblastic P retention rate, number and area of mineralized nodules, the mRNA expressions of endopeptidases on the X chromosome (PHEX), dentin matrix protein 1 (DMP1), bone morphogenetic protein 2 (BMP2), and the mRNA and protein expressions of matrix extracellular phosphoglycoprotein (MEPE) increased linearly (p < 0.001) or quadratically (p < 0.04), while extracellular signal-regulated kinase 1 (ERK1) mRNA expression and c-Jun N-terminal kinase 1 (JNK1) phosphorylated level decreased linearly (p < 0.02) or quadratically (p < 0.01). Correlation analyses showed that tibial osteoblastic P retention rate was positively correlated (r = 0.452–0.564, p < 0.03) with MEPE and BMP2 mRNA expressions. Furthermore, both number and area of mineralized nodules were positively correlated (r = 0.414–0.612, p < 0.03) with PHEX, DMP1, MEPE, and BMP2 mRNA expressions but negatively correlated (r = −0.566 to −0.414, p < 0.04) with the ERK1 mRNA expression and JNK1 phosphorylated level. These results suggested that P utilization in primary cultured tibial osteoblasts of broiler chicks might be partly regulated by PHEX, DMP1, MEPE, BMP2, ERK1, and JNK1.

Regulation of bone phosphorus retention and bone development possibly by related hormones and local bone-derived regulators in broiler chicks

BackgroundPhosphorus is essential for bone mineralization in broilers, however, the underlying mechanisms remain unclear. We aimed to investigate whether bone phosphorus retention and bone development might be regulated by related hormones and local bone-derived regulators in broilers.MethodsBroilers were fed diets containing different levels of non-phytate phosphorus (NPP) 0.15%, 0.25%, 0.35%, 0.45% and 0.55% or 0.15%, 0.22%, 0.29%, 0.36% and 0.43% from 1 to 21 or 22 to 42 days of age. Serum and tibia samples were collected for determinations of bone phosphorus retention and bone development parameters, related hormones and local bone-derived regulators of broiler chickens on d 14, 28 and 42, respectively.ResultsTibia ash phosphorus, total phosphorus accumulation in tibia ash (TPTA), bone mineral concentration (BMC), bone mineral density (BMD), bone breaking strength (BBS), and ash on d 14, 28 or 42, serum 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on d 28 and 42, mRNA expressions of tibia fibroblast growth factor 23 (FGF23) and dentin matrix protein 1 (DMP1) on d 14 and 28 increased linearly or quadratically (P < 0.05), while serum parathyroid hormone (PTH) on d 28, tibia alkaline phosphatase (ALP) on d 14, 28 and 42, bone gal protein (BGP) on d 14, and mRNA expression of tibia phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) on d 14 and 28 decreased linearly or quadratically (P < 0.04) as dietary NPP level increased. TPTA, BMC, BMD, and ash on d 28 and 42, BBS on d 28, and ash phosphorus on d 42 were positively correlated (r = 0.389 to 0.486, P < 0.03) with serum 1,25(OH)2D3. All of the above parameters were positively correlated (r = 0.380 to 0.689, P < 0.05) with tibia DMP1 mRNA expression on d 14, 28 and 42, but negatively correlated (r = − 0.609 to − 0.538, P < 0.02) with serum PTH on d 28, tibia ALP on d 14, 28 and 42, and BGP on d 14. TPTA, BMC and ash on d 14 and BMD on d 28 were negatively correlated (r = − 0.397 to − 0.362, P < 0.03) with tibia PHEX mRNA expression, and BMD on d 28 was positively correlated (r = 0.384, P = 0.04) with tibia FGF23 mRNA expression.ConclusionsThese results suggested that bone phosphorus retention and bone development parameters had moderate to strong correlations with serum PTH and 1,25(OH)2D3 and tibia DMP1, PHEX, FGF23, ALP and BGP in broilers during the whole growth period, and thus they might be partly regulated by these related hormones and local bone-derived regulators.

Plasma Membrane Ca2+-ATPase in Rat and Human Odontoblasts Mediates Dentin Mineralization.

Intracellular Ca2+ signaling engendered by Ca2+ influx and mobilization in odontoblasts is critical for dentinogenesis induced by multiple stimuli at the dentin surface. Increased Ca2+ is exported by the Na+–Ca2+ exchanger (NCX) and plasma membrane Ca2+–ATPase (PMCA) to maintain Ca2+ homeostasis. We previously demonstrated a functional coupling between Ca2+ extrusion by NCX and its influx through transient receptor potential channels in odontoblasts. Although the presence of PMCA in odontoblasts has been previously described, steady-state levels of mRNA-encoding PMCA subtypes, pharmacological properties, and other cellular functions remain unclear. Thus, we investigated PMCA mRNA levels and their contribution to mineralization under physiological conditions. We also examined the role of PMCA in the Ca2+ extrusion pathway during hypotonic and alkaline stimulation-induced increases in intracellular free Ca2+ concentration ([Ca2+]i). We performed RT-PCR and mineralization assays in human odontoblasts. [Ca2+]i was measured using fura-2 fluorescence measurements in odontoblasts isolated from newborn Wistar rat incisor teeth and human odontoblasts. We detected mRNA encoding PMCA1–4 in human odontoblasts. The application of hypotonic or alkaline solutions transiently increased [Ca2+]i in odontoblasts in both rat and human odontoblasts. The Ca2+ extrusion efficiency during the hypotonic or alkaline solution-induced [Ca2+]i increase was decreased by PMCA inhibitors in both cell types. Alizarin red and von Kossa staining showed that PMCA inhibition suppressed mineralization. In addition, alkaline stimulation (not hypotonic stimulation) to human odontoblasts upregulated the mRNA levels of dentin matrix protein-1 (DMP-1) and dentin sialophosphoprotein (DSPP). The PMCA inhibitor did not affect DMP-1 or DSPP mRNA levels at pH 7.4–8.8 and under isotonic and hypotonic conditions, respectively. We also observed PMCA1 immunoreactivity using immunofluorescence analysis. These findings indicate that PMCA participates in maintaining [Ca2+]i homeostasis in odontoblasts by Ca2+ extrusion following [Ca2+]i elevation. In addition, PMCA participates in dentinogenesis by transporting Ca2+ to the mineralizing front (which is independent of non-collagenous dentin matrix protein secretion) under physiological and pathological conditions following mechanical stimulation by hydrodynamic force inside dentinal tubules, or direct alkaline stimulation by the application of high-pH dental materials.

Effects of bergapten on damages of osteocytes MLO-Y4 induced by TCP wear particles and its mechanism

To study the effects of bergapten (BP) on damages of osteocytes MLO-Y4 induced by tricalcium phosphate (TCP) wear particles and its mechanism. ;Methods: MLO-Y4 cells were treated with TCP wear particles for 48 h to establish the model of osteocytes injuries in vitro. The MLO-Y4 cells were divided into the following five groups: control group, TCP wear particles treated (0.1 mg/ml) group, bergapten (1, 5 and 20 μmol/L) treated groups. MTT assay and Calcein-AM staining were used to determine the viability of MLO-Y4 cells; Hoechst 33342 staining and the flow cytometry were applied to detect the apoptosis of MLO-Y4; real-time PCR was performed to examine the mRNA levels of dentin matrix protein1 (DMP-1), sclerostin (SOST) and fibroblast growth factor23 (FGF23); Western blot was performed to examine protein expressions of glucose-regulated protein 78 (GRP78), protein kinase R-like ER kinase (PERK) phospho-PERK (p-PERK), eukaryotic initiation factor 2α (eIF2α), phospho-eIF2α (p-eIF2α), activating transcription factor 4 (AFT4), C/EBP homologous protein (CHOP) and caspase-3 in MLO-Y4 cells. ;Results: Compared with control group, the MLO-Y4 viability and DMP-1 mRNA level in TCP group were decreased significantly (P＜0.05), while the percentage of apoptosis and mRNA levels of SOST and FGF23 were obviously increased (P＜0.05), and protein expressions of GRP78, AFT4, CHOP, p-PERK/PERK and p-eIF2α/eIF2α were up-regulated significantly in MLO-Y4 cells (P＜0.05). Compared with TCP group, the damages of MLO-Y4 and cell apoptosis in bergapten treated groups were decrease obviously (P＜0.05), the expressions of GRP78, AFT4, CHOP, p-PERK/PERK and p-eIF2α/eIF2α were down-regulated remarkably (P＜0.05). ;Conclusion: Bergapten can inhibit osteocytes damages induced by TCP wear particles, which may be related to reducing ER stress and PERK pathway activation.

Genome-wide identification of chromatin-enriched RNA reveals that unspliced dentin matrix protein-1 mRNA regulates cell proliferation in squamous cell carcinoma

Chromatin-enriched noncoding RNAs (ncRNAs) have emerged as key molecules in epigenetic processes by interacting with chromatin-associated proteins. Recently, protein-coding mRNA genes have been reported to be chromatin-tethered, similar with ncRNA. However, very little is known about whether chromatin-enriched mRNA is involved in the chromatin modification process. Here, we comprehensively examined chromatin-enriched RNA in squamous cell carcinoma (SQCC) cells by RNA subcellular localization analysis, which was a combination of RNA fractionation and RNA-seq. We identified 11 mRNAs as highly chromatin-enriched RNAs. Among these, we focused on the dentin matrix protein-1 (DMP-1) gene because its expression in SQCC cells has not been reported. Furthermore, we clarified that DMP-1 mRNA was retained in chromatin in its unspliced form in SQCC in vitro and in vivo. As the inhibition of the unspliced DMP-1 mRNA (unspDMP-1) expression resulted in decreased cellular proliferation in SQCC cells, we performed ChIP-qPCR to identify cell cycle-related genes whose expression was epigenetically modified by unspDMP-1, and found that the CDKN1B promoter became active in SQCC cells by inhibiting unspDMP-1 expression. This result was further validated by the increased CDKN1B gene expression in the cells treated with siRNA for unspDMP-1 and by restoration of the decreased cellular proliferation rate by simultaneously inhibiting CDKN1B expression in SQCC cells. Further, to examine whether unspDMP-1 was able to associate with the CDKN1B promoter region, SQCC cells stably expressing PP7-mCherry fusion protein were transiently transfected with the unspDMP-1 fused to 24 repeats of the PP7 RNA stem loop (unspDMP-1-24xPP7) and we found that unspDMP-1-24xPP7 was efficiently precipitated with the antibody against mCherry and was significantly enriched in the CDKN1B promoter region. Thus, unspDMP-1 is a novel chromatin-enriched RNA that epigenetically regulates cellular proliferation of SQCC.

Effect of bone morphogenetic protein 2 and dexamethason on proliferation and differentiation of human dental pulp cells in vitro

To investigate the effect of bone morphogenetic protein 2 (BMP-2) and dexamethason (DXM) on proliferation and differentiation of human dental pulp cells in vitro. Primary human dental pulp cells were cultured in vitro by tissue culture method. The 3rd generation cells were used to identify cell phenotype for vimentin and cytokeratin by immunocytochemistry staining. The 3-5 generations of human dental pulp cells were randomly divided into 4 groups: 100 ng/mL BMP-2 (group A), 1×10 -8 mol/L DXM (group B), and both 100 ng/mL BMP-2 and 1×10 -8 mol/L DXM (group C) were added; neither BMP-2 nor DXM was added in group D as control group. The cell growth curve was drawn at 1, 3, 5, and 7 days after culture. The expressions of osteo/dentanogenic genes including alkaline phosphatase (ALP), dentin sialophoshoprotein (DSPP), and dentin matrix protein 1 (DMP-1) were detected by RT-PCR analysis at 5 and 7 days after culture, the ratio between the positive staining area and the total area by ALP staining at 14 days, and absorbance ( A) value at 562 nm by alizarin red staining at 21 days after culture. Human dental pulp cells were successfully isolated and cultured, which were long fusiform and showed a positive reaction for vimentin and a negative reaction for cytokeratin. The growth curve indicated that cells increased with the extending of incubation time, reached a peak at 5 days, then reduced at 7 days to the level at 3 days. At 5 days after culture, the cells were significantly more in groups A, B, and C than group D ( P<0.05), in group C than group A ( P<0.05), and in group A than group B ( P<0.05). RT-PCR analysis showed that the mRNA expressions of ALP, DSPP, and DMP-1 at 5 days were significantly higher in groups A, B, and C than group D ( P<0.05), and in group C than groups A and B ( P<0.05), but no significant difference was found between groups A and B ( P>0.05); the mRNA expression of DSPP in groups A, B, and C was significantly higher than that in group D ( P<0.05), but there was no significant difference in mRNA expressions between other groups at 7 days ( P>0.05). At 14 days, positive staining in varying degrees was observed in each group, especially in group C; the ratio between the positive staining area and the total area was significantly higher in group C than groups A, B, and D ( P<0.05), and in groups A and B than group D ( P<0.05), but there was no significant difference between groups A and B ( P>0.05). At 21 days, there were a variety of mineralized nodules in groups A, B, and C in nonuniformly scattered or clustered distribution, but no mineralized nodules were observed in group D. The A values of mineralized nodules showed significant difference between groups ( P<0.05). BMP-2 may be more effective in promoting proliferation of human dental pulp cells than DXM. Combined application of BMP-2 and DXM can remarkably promote the proliferation and differentiation of human dental pulp cells.

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.

Effects of CO2 Lasers on Dental Pulp Biology in Rats.

The purpose of this study was to investigate the effects of CO2 lasers on the proliferation and differentiation of dental pulp cells, and their latent self-recovery in connection with their stemness using reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. The first molars from male Sprague-Dawley rats, each weighing ∼150-200 g, were used for this study. The upper first molars were irradiated with a 10,600 nm wavelength CO2 laser under identical parameters (2 W CO2 laser, energy 4J, energy density 203.84 J/cm(2) for 8.8 sec) through the dentin of the occlusal surface. The molars were extracted immediately, or at 1, 3 or 5 days after the laser irradiation. RT-PCR analysis using primers specific for heat shock protein 70 (Hsp70), adenosine triphosphate (ATP)-binding cassette transporter G2 (ABCG2), dentin sialophosphoprotein (DSPP), and dentin matrix protein 1 (DMP1), and immunohistochemistry using antibodies specific for proliferating cell nuclear antigen (PCNA), ABCG2, CD34, and CD44 were performed. RT-PCR analysis revealed that Hsp70 mRNA expression in the immediate group and ABCG2 mRNA expression at day 1 were the highest. DSPP and DMP1 mRNA expression in the laser-irradiated groups increased gradually, reaching its peak on the 5th day of the experiment, although no significant difference found among groups with regard to DMP1 expression. Immunohistochemically, PCNA-positive cells were observed at all times after the laser irradiation; however, they were most evident on day 3. CD44-positive cells were observed strongly on day 1 and day 3, while ABCG2-positive cells were the most evident on day 3. These results demonstrate that CO2 laser irradiation induces degeneration in the pulp tissue, which is then repaired by newly formed odontoblast-like cells.

Vitamin D Represses Dentin Matrix Protein 1 in Cementoblasts and Osteocytes

Calcium and phosphorus homeostasis is achieved by interplay among hormones, including 1,25(OH)2D3 (1,25D), parathyroid hormone, and fibroblast growth factor 23 (FGF23), and their interactions with other proteins. For example, mutations in dentin matrix protein 1 (DMP-1) result in increased FGF23 and hypophosphatemic rickets. 1,25D is reported to modulate FGF23; thus, we hypothesized that 1,25D may be involved in modulating DMP-1 in an intermediary step. Murine cementoblasts (OCCM-30) and osteocyte-like cells (MLO-Y4 and MLO-A5), known to express DMP-1, were used to analyze effects of 1,25D on DMP-1 expression in vitro. DMP-1 mRNA levels decreased by 50% (p < .05) in the presence of 1,25D in all cell types, while use of a vitamin D receptor (VDR) agonist (EB1089) and antagonist (23S,25S)-DLAM-2P confirmed that VDR pathway activation was required for this response. Further analysis showed that histone deacetylase recruitment was necessary, but neither protein kinase A nor C pathways were required. In conclusion, our results support the hypothesis that 1,25D regulates DMP-1 expression through a VDR-dependent mechanism, possibly contributing to local changes in bone/tooth mineral homeostasis.

Identification, characterization, and expression of dentin matrix protein 1 gene inXenopus laevis

Dentin matrix protein 1 (DMP1) is an acidic extracellular matrix protein expressed mainly in bone and dentin, and is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family. The DMP1 gene, however, appears to evolve rapidly in comparison with other SIBLING genes, even though such functionally important molecules usually evolve more slowly than less important ones. The purpose of this study was to identify and characterize an ortholog of the DMP1 gene in an amphibian (Xenopus laevis; X. laevis) to clarify molecular evolutionary alterations in DMP1 associated with calcified tissues in tetrapods. Furthermore, we analyzed the mRNA expression of this gene to elucidate its functional change in bone and developing tooth germ in comparison with amniote DMP1s. The similarities of the deduced amino acid sequence of X. laevis DMP1 to that of the corresponding amniote proteins were low, although they did share several unique features specific to DMP1 and have similar properties. Expression of X. laevis DMP1 mRNA was predominant in osteocytes and odontoblasts, but only transiently observed in ameloblasts, as in amniotes. These results suggest that DMP1 has conserved several functions during tetrapod evolution. This indicates that continuity of biochemical properties has been more important in maintaining DMP1 functionality than that of the sequence of amino acid residues, which has undergone change over the course of molecular evolution.

Distinct differentiation properties of human dental pulp cells on collagen, gelatin, and chitosan scaffolds

The growth and differentiation properties of human dental pulp cells (HDPC) were investigated on a variety of natural scaffolds, including 2 types of collagen, gelatin, and chitosan. Cell attachment and growth rates of HDPC on collagen (type I and type III), gelatin, and chitosan were observed. Alkaline phosphatase (ALP) activity, mRNA expression of differentiation-related genes, and mineralization of the HDPC on each scaffold were assessed. Dental pulp cells attached and proliferated rapidly on collagen and gelatin, but chitosan did not properly support cell growth. The cells plated on gelatin exhibited high ALP activity, but not as high as cells plated on collagen. The expression peak of osteocalcin (OCN) mRNA from cells grown on collagen was found earlier and followed by dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP-1) mRNA expression. In cells grown on gelatin, however, OCN mRNA transcripts appeared at a later period of culture with no increase in DSPP or DMP-1 mRNA. Intensely mineralized extracellular matrix was seen in cells grown on collagen, but gelatin did not allow enough mineralization of cells in differentiation-inducing media. Collagen supported proliferation and differentiation of HDPC, and the expression of DSPP and DMP-1 mRNA was reduced on gelatin.

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.

Dexamethasone promotes DMP1 mRNA expression by inhibiting negative regulation of Runx2 in multipotential mesenchymal progenitor, ROB‐C26

Dentin matrix protein 1 (DMP1) is an acidic phosphorylated extracellular protein and essential for mineralization of dentin and bone; however, the precise mechanism regulating DMP1 expression is not fully understood. A synthetic glucocorticoid (GC), dexamethasone (Dex), promotes an early osteoblast differentiation of a mesenchymal progenitor, ROB-C26 (C26), in parallel with inductive expression of an osteoblast-specific transcription factor, Runx2, and other extracellular matrix proteins such as osteocalcin and bone sialoprotein (BSP). We have examined the effect of Dex on DMP1 expression via induction of Runx2 in C26 cells. Real time RT-PCR showed that Dex increases DMP1 mRNA expression levels at time- and dose-dependent manners and a GC antagonist, RU486, drastically inhibited DMP1 mRNA expression levels. Furthermore, Dex increased the luciferase activity of six-repeated osteoblast-specific cis-acting element 2 (6 x OSE2), which is the binding sequence of Runx2, suggesting that Dex stimulates DMP1 expression via activation of Runx2. However, unexpected results showed that overexpression of exogenous Runx2 depressed DMP1 mRNA expression level, even after cells had been treated with Dex, while downregulated expression of endogenous Runx2 enhanced Dex-induced DMP1 mRNA expression level. These results imply that large amounts of exogenous Runx2 inhibit DMP1 expression, whereas small amounts are more effective for Dex-induced DMP1 expression in C26 cells. Therefore, Dex may activate some factors that inhibit negative action of Runx2 on DMP1 expression. Since mitogen-activating protein kinase (MAPK) phosphatase-1 (MKP-1) has been reported to affect the Dex-induced osteoblast differentiation via decrease of Runx2-phosphorylation, we focus on the relationship between MKP-1 and DMP1 expression. Dex increases MKP-1 expression, and overexpression of exogenous MKP-1 showed significant increase of luciferase activity of 6 x OSE up to the level detected in Dex-treated C26 cells. However, no inductive DMP1 mRNA expression level was found in C26 cells unlike BSP and OPN. These results suggest that although MKP-1 increases DNA-binding activity of Runx2, DMP1 expression may require the collaboration of MKP-1 and additive factors to stimulate Runx2-mediated DMP1 expression in the post-transcriptional event of Dex-treated C26 cells.

Expression of dentin matrix protein 1 (DMP1) in nonmineralized tissues.

Dentin matrix protein 1 (DMP1) is an Arg-Gly-Asp-containing acidic phosphoprotein that was originally identified from a rat incisor cDNA library and was thought to be a dentin-specific protein. DMP1 was later shown to express in a number of hard tissue-forming cells, including osteoblasts, osteocytes, ameloblasts, and cementoblasts, and was considered to play important roles in mineralization. Further, DMP1 gene expression was also detected in fetal bovine brain and in newborn mouse brain. These findings indicate the possibility of DMP1 expression in other soft tissues. In the present study, to clarify the significance of DMP1 expression in nonmineralized tissues, we made a specific antibody to mouse DMP1 peptides and demonstrated that DMP1 protein was localized in mouse brain, pancreas, and kidney by immunohistochemistry. Further DMP1 mRNA was detected in nonmineralized mouse tissues including liver, muscle, brain, pancreas, and kidney by RT-PCR. Based on the evidence that the localization and the expression of DMP1 are not restricted to mineralized tissues, we assume that DMP1 may have functions other than the regulation of mineralization.

Expression of dentin matrix protein 1 (DMP1) during fracture healing

Dentin matrix protein 1 (DMP1) is one of the acidic phosphorylated extracellular matrix proteins called the SIBLING (small integrin-binding ligand, N-linked glycoproteins) family. Recent studies showed that DMP1 is expressed in the mineralized tissues and suggested that DMP1 is involved in the mineralization. We investigated the precise localization of DMP1 messenger RNA (mRNA) and protein during fracture healing. In situ hybridization demonstrated that DMP1 mRNA was strongly expressed in preosteocytes and osteocytes in the bony callus during intramembranous and endochondral ossification while DMP1 mRNA was not detected in osteoblasts and chondrocytes. During endochondral ossification, however, a low number of DMP1-expressing cells were identified in the cluster of hypertrophic chondrocytes. However, these DMP1-expressing cells were not hypertrophic and were likely to be osteoblast-lineage cells, which were embedded in the matrix of bone or cartilage, because type I collagen-expressing cells and invasion of capillary vessels were observed in the same area. Northern blot, in situ hybridization, and immunohistochemical analyses showed that DMP1 mRNA and protein expressions were increased until day 14 postfracture, when bony callus was formed, and then declined to a lower level during remodeling of the bony callus. Therefore, DMP1 is likely to play an important role in the mineralization of the bony callus.

MRNA expression and protein localization of dentin matrix protein 1 during dental root formation

Dentin matrix protein 1 (DMP1) is an acidic phosphoprotein. DMP1 was initially detected in dentin and later in other mineralized tissues including cementum and bone, but the DMP1 expression pattern in tooth is still controversial. To determine the precise localization of DMP1 messenger RNA (mRNA) and the protein in the tooth, we performed in situ hybridization and immunohistochemical analyses using rat molars and incisors during various stages of root formation. During root dentin formation of molars, DMP1 mRNA was detected in root odontoblasts in parallel with mineralization of the dentin. However, the level of DMP1 mRNA expression in root odontoblasts decreased near the coronal part and was absent in coronal odontoblasts. DMP1 protein was localized along dentinal tubules and their branches in mineralized root dentin, and the distribution of DMP1 shifted from the end of dentinal tubules to the base of the tubules as dentin formation progressed. During the formation of the acellular cementum, DMP1 mRNA was detected in cementoblasts lining the acellular cementum where its protein was localized. During the formation of the cellular cementum, DMP1 mRNA was detected in cementocytes embedded in the cellular cementum but not in cementoblasts, and its protein was localized in the pericellular cementum of cementocytes including their processes. During dentin formation of incisors, DMP1 mRNA was detected in odontoblasts on the cementum-related dentin, where its protein was localized along dentinal tubules near the mineralization front. The localization of DMP1 mRNA and protein in dentin and cementum was related to their mineralization, suggesting that one of the functions of DMP1 may be involved in the mineralization of dentin and cementum during root formation.

Dentin matrix protein 1 is expressed in human lung cancer.

We have previously shown that breast and prostate cancers express bone matrix proteins. DMP1 expression was evaluated in 59 human lung cancer samples at the protein and mRNA levels. It was detectable in 80% of the cases, suggesting a potential role for DMP1 in tumor progression and bone metastasis. Previously, we and others have shown that bone extracellular matrix proteins such as bone sialoprotein (BSP) and osteopontin (OPN) are expressed in various types of cancer that are characterized by a high affinity for bone including breast, prostate, and lung adenocarcinoma. Based on biochemical and genetic features, BSP, OPN, dentin matrix protein 1 (DMP1), and dentin sialophosphoprotein (DSPP) have been recently classified in a unique family named SIBLING (small integrin-binding ligand, N-linked glycoprotein). Therefore, we investigated whether DMP1 could also be detected in osteotropic cancers. We first used a cancer array for evaluating the relative abundance of DMP1 transcript in a broad spectrum of human cancer tissues. This screening showed that DMP1 was strongly detectable in lung tumors compared with normal corresponding tissue. In a second step, we used an immunophosphatase technique and a specific polyclonal antibody directed against DMP1 to examine the expression of DMP1 in 59 human non-small cell lung cancer samples, including 29 squamous carcinoma, 20 adenocarcinoma, and 10 bronchioloalveolar carcinoma. Student's t-test was used to determine the statistical significance of immunostaining scores between the lung cancer histological groups studied and between cancer and normal lung tissues. Our results show that DMP1 is detectable in 90% of the adenocarcinoma and squamous carcinoma analyzed while 8 of 10 bronchioloalveolar specimens were negative. DMP1 immunostaining intensity and extent scores were significantly higher in adenocarcinoma (p = 0.0004) and squamous carcinoma (p < 0.0001) samples compared with adjacent normal lung tissue. In situ hybridization experiments confirmed that DMP1 mRNA is localized in lung cancer cells. In this study, we show that a third SIBLING protein is ectopically expressed in lung cancer. The role of DMP1 in lung cancer is largely unknown. Further studies are required to determine the implication of this protein, next to its sisters SIBLING proteins, in tumor progression and bone metastasis development.

Mechanical loading stimulates dentin matrix protein 1 (DMP1) expression in osteocytes in vivo.

Dentin matrix protein 1 (DMP1) was originally postulated to be dentin specific. Further analysis showed that DMP1 is also expressed in mature cartilage and bone. In bone tissue, DMP1 is expressed predominantly in late osteoblasts and osteocytes. DMP1 belongs to the SIBLING (Small Integrin Binding Ligand N-linked Glycoprotein) family of cellular matrix proteins that also includes osteopontin, bone sialoprotein, dentin sialophosphoprotein, and others. In this study, we examined the effect of mechanical loading on expression of DMP1 mRNA and DMP1 protein in alveolar bone in the mouse tooth movement model by in situ hybridization and immunocytochemistry. The expression of DMP1 mRNA was determined quantitatively in mechanically loaded and control sites of dento-alveolar tissue at several time points from 6 h to 7 days after loading. The tooth movement model allows simultaneous evaluation of bone resorption and bone formation sites. Expression of DMP1 mRNA in osteocytes increased 2-fold as early as 6 h after treatment in both the bone formation and bone resorption sites. After 4 days, DMP1 expression in osteocytes increased to a maximum of 3.7-fold in the bone formation sites and 3.5-fold in the resorption sites. Osteoblasts responded in the opposite manner and showed a transient 45% decrease of DMP1 mRNA in bone formation sites and a constant decrease of DMP1 mRNA during the entire course of treatment in the bone resorption sites, with a peak inhibition of 67% at day 2. By immunocytochemistry using a C-terminal region peptide antibody to DMP1, we found that there was a transient decrease in immunoreactivity at 3 days after treatment on both the formation side and the resorption side compared with the matched contralateral control tissue. However by 7 days of loading, there was a dramatic increase in DMP1 protein immunoreactivity on both the formation side and the resorption side. These results represent changes in epitope availability using this antibody or true changes in protein levels. The observations imply that the DMP1 protein is undergoing dynamic changes in either synthesis or other protein/matrix interaction after mechanical loading of alveolar bone. The findings indicate that DMP1 is involved in the responses of osteocytes and osteoblasts to mechanical loading of bone. These results support the hypothesis that osteocytes alter their matrix microenvironment in response to mechanical loading.