Dentin Sialoprotein Protein Research Articles

Reparative Dentin Formation Following Dental Pulp Capping is Mediated by TNFR1 In Vivo

IntroductionTumor necrosis factor (TNF)-α is a pro-inflammatory cytokine that promotes biomineralization in vitro in dental pulp cells. However, the role of TNF-α-TNF receptor 1 (TNFR1) signaling in reparative dentin formation and related inflammatory pathways is not known. Therefore, the aim of this study was to evaluate the role of the TNF-α-TNFR1 axis in dental pulp repair following pulp capping in vivo. MethodsDental pulp repair response of genetically deficient TNF-α receptor-1 mice (TNFR1−/−; n = 20) was compared with that of C57Bl6 mice (wild type [WT]; n = 20). Pulp capping was performed with mineral trioxide aggregate on the mandibular first molars of mice. After 7 and 70 days, tissues were collected and stained with hematoxylin and eosin for histopathological and histometric evaluation, and assessed by the Brown and Brenn methods for histomicrobiological analysis and by immunohistochemistry to localize TNF-α, Runt-related transcription factor 2, Dentin Sialoprotein (DSP) and Osteopontin (OPN) expression. ResultsCompared with WT mice, TNFR1−/− mice showed significantly decreased reparative dentin formation with a lower mineralized tissue area (P < .0001). Unlike WT mice, TNFR1−/− mice also exhibited significant dental pulp necrosis, neutrophil recruitment, and apical periodontitis formation (P < .0001) without bacterial tissue invasion. TNFR1−/− animals further exhibited decreased TNF-α, DSP, and OPN expression (P < .0001), whereas Runt-related transcription factor 2 expression was unchanged (P > .05). ConclusionThe TNF-α-TNFR1 axis is involved in reparative dentin formation following dental pulp capping in vivo. Genetic ablation of TNFR1 modified the inflammatory process and inhibited the expression of the DSP and OPN mineralization proteins, which culminated in dental pulp necrosis and development of apical periodontitis.

The functional significance of dentin sialoprotein-phosphophoryn and dentin sialoprotein

Phosphophoryn (PP) and dentin sialoprotein (DSP) are the most dominant non-collagenous proteins in dentin. PP is an extremely acidic protein that can function as a mineral nucleator for dentin mineralization. DSP was first identified in 1981, yet its functional significance is still controversial. Historically, these two proteins were considered to be independently synthesized and secreted by dental pulp cells into the developing dentin matrix. However, with the identification of the DSP coding sequence in 1994, followed 2 years later by the finding that the PP coding sequence was located immediately downstream from the DSP sequence, it became immediately clear that DSP and PP proteins were derived from a single DSP-PP (i.e., dentin sialophosphoprotein, DSPP) transcript. Since DSPP cDNA became available, tremendous progress has been made in studying DSP-PP mRNA distribution and DSP generation from the DSP-PP precursor protein at specific cleavage sites by protease tolloid-related-1 (TLR1) or bone morphogenetic protein 1 (BMP1). The functions of DSP-PP and DSP were investigated via DSP-PP knockout (KO) and DSP knockin in DSP-PP KO mice. In addition, a number of in vitro studies aimed to elucidate DSPP and DSP function in dental pulp cells.

Four and a Half LIM Domain Protein 2 Enhances Differentiation and Mineralization of Human Dental Pulp Cells

IntroductionFour and a half LIM domain protein 2 (FHL2) is 1 member of the LIM-only protein family, which is a transcriptional cofactor, playing important roles in osteoblast differentiation and bone formation. Our previous studies showed that the FHL2 protein was abundantly expressed in odontoblasts both during tooth development and in mature teeth, indicating that FHL2 might play a role in odontoblast differentiation and dentin formation. The aim of this study was to investigate the effects of FHL2 overexpression on odontoblast differentiation and mineralization of human dental pulp cells (hDPCs), exploring the function of FHL2 on dentin formation. MethodsFHL2 was steadily transfected into hDPCs. Then, alkaline phosphatase activity assay was shown; the formation of mineralized nodules was assessed by alizarin red staining; and the expression of odontoblast differentiation and mineralization-related molecules including alkaline phosphate (ALP), dentin sialoprotein (DSP), bone sialoprotein (BSP), and osteopontin (OPN) at messenger RNA and protein levels were determined by quantitative real-time polymerase chain reaction and Western blot analysis. ResultsIt was found that FHL2 overexpression could increase ALP activity and the formation of mineralized nodules of dental pulp cells. Moreover, FHL2 overexpression could up-regulate the expression of ALP, DSP, BSP, and OPN messenger RNA and protein levels significantly. ConclusionsFHL2 overexpression could enhance the differentiation and mineralization of hDPCs.

Immunohistochemical localization of the NH2-terminal and COOH-terminal fragments of dentin sialoprotein in mouse teeth

Dentin sialoprotein (DSP) is a major non-collagenous protein in dentin. Mutation studies in human, along with gene knockout and transgenic experiments in mice, have confirmed the critical role of DSP for dentin formation. Our previous study reported that DSP is processed into fragments in mouse odontoblast-like cells. In order to gain insights into the function of DSP fragments, we further evaluated the expression pattern of DSP in the mouse odontoblast-like cells using immunohistochemistry and western blot assay with antibodies against the NH(2)-terminal and COOH-terminal regions of DSP. Then, the distribution profiles of the DSP NH(2)-terminal and COOH-terminal fragments and osteopontin (OPN) were investigated in mouse teeth at different ages by immunohistochemistry. In the odontoblast-like cells, multiple low molecular weight DSP fragments were detected, suggesting that part of the DSP protein was processed in the odontoblast-like cells. In mouse first lower molars, immunoreactions for anti-DSP-NH(2) antibody were intense in the predentin matrix but weak in mineralized dentin; in contrast, for anti-DSP-COOH antibody, strong immunoreactions were found in mineralized dentin, in particular dentinal tubules but weak in predentin. Therefore, DSP NH(2)-terminal and COOH-terminal fragments from odontoblasts were secreted to different parts of teeth, suggesting that they may play distinct roles in dentinogenesis. Meanwhile, both DSP antibodies showed weak staining in reactionary dentin (RD), whereas osteopontin (OPN) was clearly positive in RD. Therefore, DSP may be less crucial for RD formation than OPN.

Transcriptional repression of the Dspp gene leads to dentinogenesis imperfecta phenotype in Col1a1-Trps1 transgenic mice

Dentinogenesis imperfecta (DGI) is a hereditary defect of dentin, a calcified tissue that is the most abundant component of teeth. Most commonly, DGI is manifested as a part of osteogenesis imperfecta (OI) or the phenotype is restricted to dental findings only. In the latter case, DGI is caused by mutations in the DSPP gene, which codes for dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). Although these two proteins together constitute the majority of noncollagenous proteins of the dentin, little is known about their transcriptional regulation. Here we demonstrate that mice overexpressing the Trps1 transcription factor (Col1a1-Trps1 mice) in dentin-producing cells, odontoblasts, present with severe defects of dentin formation that resemble DGI. Combined micro–computed tomography (µCT) and histological analyses revealed tooth fragility due to severe hypomineralization of dentin and a diminished dentin layer with irregular mineralization in Col1a1-Trps1 mice. Biochemical analyses of noncollagenous dentin matrix proteins demonstrated decreased levels of both DSP and DPP proteins in Col1a1-Trps1 mice. On the molecular level, we demonstrated that sustained high levels of Trps1 in odontoblasts lead to dramatic decrease of Dspp expression as a result of direct inhibition of the Dspp promoter by Trps1. During tooth development Trps1 is highly expressed in preodontoblasts, but in mature odontoblasts secreting matrix its expression significantly decreases, which suggests a Trps1 role in odontoblast development. In these studies we identified Trps1 as a potent inhibitor of Dspp expression and the subsequent mineralization of dentin. Thus, we provide novel insights into mechanisms of transcriptional dysregulation that leads to DGI. © 2012 American Society for Bone and Mineral Research.

Expression of mineralized tissue associated proteins: Dentin sialoprotein and phosphophoryn in rodent hair follicles

Mammalian hair development and tooth development are controlled by a series of reciprocal epithelial-mesenchymal interactions. Similar growth factors and transcription factors, such as fibroblast growth factor (FGF), sonic hedgehog homolog (SHH), bone morphogenetic proteins (BMPs) and Wnt10a, were reported to be involved in both of these interactions. Dentin sialoprotein (DSP) and phosphophoryn (PP) are the two major non-collagenous proteins secreted by odontoblasts that participate in dentin mineralization during tooth development. Because of striking similarities between tooth development and hair follicle development, we investigated whether DSP and/or PP proteins may also play a role in hair follicle development. In this study, we examined the presence and location of DSP/PP proteins during hair follicle development. Rat PP proteins were detected using immunohistochemical/immunofluorescent staining. DSP-PP mRNAs were detected by in situ hybridization with riboprobes. LacZ expression was detected in mouse tissues using a DSP-PP promoter-driven LUC in transgenic mice. We found that PP proteins and DSP-PP mRNAs are present in rat hair follicles. We also demonstrate that an 8 kb DSP-PP promoter is able to drive lacZ expression in hair follicles. We have firmly established the presence of DSP/PP in mouse and rat hair follicles by immunohistochemical/immunofluorescent staining, in situ hybridization with riboprobes and transgenic mice studies. The expression of DSP/PP in hair follicles is the first demonstration that major mineralization proteins likely may also contribute to soft tissue development. This finding opens a new avenue for future investigations into the molecular-genetic management of soft tissue development.

Effects of muramyl dipeptide on the differentiation of human dental pulp cells

To investigate the nucleotide-binding oligomerization domain-2 (NOD-2) gene expression in deep caries and the effects of NOD-2 agonist muramyl dipeptide (MDP) on the differentiation of human dental pulp cells (hDPC). NOD-2 gene level in deep caries and healthy pulp tissue was determined by real-time quantitative polymerase chain reaction (realtime-PCR). Realtime-PCR, Western blotting and immunofluorescence were performed to evaluate NOD-2 gene and protein expression. Dentin sialoprotein (DSP) protein level was assessed when hDPC were challenged by different concentrations of MDP for 24 hours, and sialophosphoprotein (DSPP), osteocalcin (OCN) mRNA and osteopontin (OPN) protein level were detected at different time points after incubation with 0.1 mg/L MDP. NOD-2 mRNA level was higher in pulp tissue of deep caries (0.2610 ± 0.0824) than that in healthy controls (0.0024 ± 0.0002), P < 0.05. The expression of NOD-2 gene and protein increased in a time denpendent manner upon stimulation with MDP. Immunofluorescence confirmed that NOD-2 protein was located in cytoplasm. Moreover, 0.1 mg/L MDP augmented DSP protein level. DSPP and OCN mRNA were elevated with time and reached the peak at 12 h and down-regulated. OPN protein level also increased with time. Dental pulp NOD-2 expression are up-regulated in pulp tissue of deep caries. MDP may be related to the differentiation of hDPC.

Bone Morphogenetic Protein 2 Mediates Dentin Sialophosphoprotein Expression and Odontoblast Differentiation via NF-Y Signaling

Dentin sialophosphoprotein (DSPP), an important odontoblast differentiation marker, is necessary for tooth development and mineralization. Bone morphogenetic protein 2 (BMP2) plays a vital role in odontoblast function via diverse signal transduction systems. We hypothesize that BMP2 regulates DSPP gene transcription and thus odontoblast differentiation. Here we report that expression of BMP2 and DSPP is detected during mouse odontogenesis by in situ hybridization assay, and BMP2 up-regulates DSPP mRNA and protein expression as well as DSPP-luciferase promoter activity in mouse preodontoblasts. By sequentially deleting fragments of the mouse DSPP promoter, we show that a BMP2-response element is located between nucleotides -97 and -72. By using antibody and oligonucleotide competition assays in electrophoretic mobility shift analysis and chromatin immunoprecipitation experiments, we show that the heterotrimeric transcription factor Y (NF-Y) complex physically interacts with the inverted CCAAT box within the BMP2-response element. BMP2 induces NF-Y accumulation into the nucleus increasing its recruitment to the mouse DSPP promoter in vivo. Furthermore, forced overexpression of NF-Y enhances promoter activity and increases endogenous DSPP protein levels. In contrast, mutations in the NF-Y-binding motif reduce BMP2-induced DSPP transcription. Moreover, inhibiting BMP2 signaling by Noggin, a BMP2 antagonist, results in significant inhibition of DSPP gene expression in preodontoblasts. Taken together, these results indicate that BMP2 mediates DSPP gene expression and odontoblast differentiation via NF-Y signaling during tooth development.

Dynamic Processing of Recombinant Dentin Sialoprotein-Phosphophoryn Protein

Dentin sialoprotein (DSP) and phosphophoryn (PP) are the two noncollagenous proteins classically linked to dentin but more recently found in bone, kidney, and salivary glands. These two proteins are derived from a single copy DSP-PP gene. Although this suggests that the DSP-PP gene is first transcribed into DSP-PP mRNAs, which later undergo processing to yield the DSP and PP proteins, this mechanism has not yet been demonstrated because of the inability to identify a DSP-PP precursor protein from any cell or tissue sample. To study this problem, we utilized a baculovirus expression system to produce recombinant DSP-PP precursor proteins from a DSP-PP(240) cDNA, which represents one of several endogenous DSP-PP transcripts that influence various tooth mineralization phases. Our in vitro results demonstrate that DSP-PP(240) precursor proteins are produced by this system and are capable of self-processing to yield both DSP and PP proteins. We further demonstrated that purified recombinant DSP-PP(240), purified recombinant PP(240), and the native highly phosphorylated protein (equivalent to the PP(523) isoform) have proteolytic activity. These newly identified tissue proteases may play key roles in tissue modeling during organogenesis.

Dentin-Pulp Complex Responses to Carious Lesions

To understand the molecular events underlying the dentin-pulp complex responses to carious progression, we systematically analyzed tissue morphology and dentin matrix protein distribution in non-carious teeth and in teeth with enamel and dentin caries. Dentin matrix proteins analyzed included collagen type I, phosphophoryn (PP) and dentin sialoprotein (DSP), all of which play decisive roles in the dentin mineralization process. Human non-carious and carious third molar teeth were freshly collected, demineralized, and processed for hematoxylin and eosin staining. The ABC-peroxidase method was used for immunohistochemical staining of collagen type I, PP and DSP proteins using specific antibodies. In situ hybridization was also performed. In contrast to elongated odontoblasts in non-carious teeth, odontoblasts subjacent to dentin caries were cuboidal and fewer in number. The predentin zone was also dramatically reduced in teeth with dentin caries. The staining intensity for collagen type I, PP and DSP in the dentin-pulp complex increased progressively from non-carious teeth, to teeth with enamel and dentin caries. In situ hybridization studies showed DSP-PP mRNA expression in odontoblasts and dental pulp that was consistent with our immunohistochemical results. These results suggest that carious lesions stimulate the dentin-pulp complex to actively synthesize collagen type I, PP and DSP proteins. This response to carious lesions is likely to provide a basis for reparative and/or reactionary dentin formation.

New cellular models for tracking the odontoblast phenotype

Odontoblasts and osteoblasts differ functionally and histologically. Because of their close relationship, mesenchymal cells derived from teeth and bone are difficult to distinguish ex vivo. Indeed, the main non-collagenous components of the odontoblastic extracellular matrix, dentin sialoprotein (DSP) or dentin matrix protein 1 (DMP1), have also been detected in osteoblasts. The need to develop cellular models of odontoblast differentiation and to identify markers specific for the odontoblast lineage, has led us to establish clonal cell lines from tooth germs of day 18 mouse embryos transgenic for an adenovirus-SV40 recombinant plasmid. In this study, we analyzed the phenotypes of three independent clones by RT-PCR and Western blot. These clones synthesised DSP, DMP1 and other extracellular matrix proteins typical of the odontoblast and are therefore likely to be derived from the pulp. Transcripts encoding a set of homeobox proteins involved in craniofacial development, such as Pax9, Msx1, Cbfa1, Dlx2 and 5 were also expressed albeit at a different level. These features of the pulpal clones are shared by the C1 mesodermal cells that are capable of differentiating along osteogenic, chondrogenic or adipogenic lineages In contrast, transcripts for two LIM-domain homeobox family genes (Lhx6 and Lhx7) were only detected in the dental clones. Since these genes are preferentially expressed in the mesenchyme of the developing tooth, this suggests that our transgenic-derived cell lines retain intrinsic properties of odontoblastic cells. They may help to characterise genes specifying the odontoblast phenotype and the signalling pathways underlying odontoblast differentiation.