Expression and distribution of SIBLING proteins in the predentin/dentin and mandible of hyp mice

Oral Diseases (2010) 16 , 453–464 Objectives: Human X‐linked hypophosphatemia (XLH) and its murine homologue, Hyp are caused by inactivating mutations in PHEX gene. The protein encoded by PHEX gene is an endopeptidase whose physiological substrate(s) has not been identified. Dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP), two members of the Small Integrin‐Binding LIgand, N‐linked Glycoprotein (SIBLING) family are proteolytically processed. It has been speculated that PHEX endopeptidase may be responsible for the proteolytic cleavage of DMP1 and DSPP. To test this hypothesis and to analyse the distribution of SIBLING proteins in the predentin/dentin complex and mandible of Hyp mice, we compared the expression of four SIBLING proteins, DMP1, DSPP, bone sialoprotein (BSP) and osteopontin (OPN) between Hyp and wild‐type mice. Methods: These SIBLING proteins were analysed by protein chemistry and immunohistochemistry. Results: (1) Dentin matrix protein 1 and DSPP fragments are present in the extracts of Hyp predentin/dentin and bone; (2) the level of DMP1 proteoglycan form, BSP and OPN is elevated in the Hyp bone. Conclusions: The PHEX protein is not the enzyme responsible for the proteolytic processing of DMP1 and DSPP. The altered distribution of SIBLING proteins may be involved in the pathogenesis of bone and dentin defects in Hyp and XLH.     

Expression of SIBLINGs and their partner MMPs in salivary glands

Three members of the SIBLING family of integrin-binding phosphoglycoproteins (bone sialoprotein, BSP; osteopontin, OPN; and dentin matrix protein-1, DMP1) were recently shown to bind with high affinity (nM) and to activate 3 different matrix metalloproteinases (MMP-2, MMP-3, and MMP-9, respectively) in vitro. The current study was designed to document the possible biological relevance of the SIBLING-MMP activation pathway in vivo by showing that these 3 SIBLINGs and their known MMP partners are co-expressed in normal adult tissue. BSP, OPN, and DMP1 were invariably co-expressed with their partner MMPs in salivary glands of humans and mice. The 2 SIBLING proteins without known MMP partners, dentin sialophosphoprotein (DSPP) and matrix extracellular phosphoglycoprotein (MEPE), were also expressed in salivary glands. Expression of all SIBLINGs in this normal, non-mineralizing epithelial tissue suggests that they serve at least one function in vivo other than directly promoting matrix mineralization--a function we hypothesize involves local activation of MMPs.     

Distribution of Small Integrin-Binding LIgand,N-linked Glycoproteins (SIBLING) in the condylar cartilage of rat mandible

The Small Integrin-Binding LIgand, N-linked Glycoprotein (SIBLING) family is one category of non-collagenous proteins closely related to osteogenesis. In this study, the authors systematically evaluated the presence and distribution of four SIBLING family members, dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), bone sialoprotein (BSP) and osteopontin (OPN), in rat mandibular condylar cartilage using protein chemistry and immunohistochemistry. For protein chemistry, SIBLING proteins in the dissected condylar cartilage were extracted with 4 M guanidium–HCl, separated by ion-exchange chromatography, and analyzed by Western immunoblotting. Immunohistochemistry was employed to assess the distribution of these four SIBLING proteins in the condylar cartilage of 2-, 5- and 8-week-old rats. Results from both approaches showed that all four members are expressed in the condylar cartilage. DSPP, unlike that observed in dentin and bone, exists as a full-length form (uncleaved) in the condylar cartilage. The NH₂-terminal fragment of DMP1 is mainly detected in the matrix of the cartilage while the COOH-terminal fragment is primarily localized in the nuclei of cells in the chondroblastic and hypertrophic layers. The data obtained in this investigation provide clues about the potential roles of these SIBLING proteins in chondrogenesis.     

Distribution of SIBLING proteins in the organic and inorganic phases of rat dentin and bone

The SIBLING protein family is a group of non-collagenous proteins (NCPs) that includes dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), bone sialoprotein (BSP), and osteopontin (OPN). In the present study, we compared these four proteins in different phases of rat dentin and bone. First, we extracted NCPs in the unmineralized matrices and cellular compartments using guanidium-HCl (G1). Second, we extracted NCPs closely associated with hydroxyapatite using an EDTA solution (E). Last, we extracted the remaining NCPs again with guanidium-HCl (G2). Each fraction of Q-Sepharose ion-exchange chromatography was analyzed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), Stains-All stain, and with western immunoblotting. In dentin, the NH₂-terminal fragment of DSPP and its proteoglycan form were primarily present in the G1 extract, whereas the COOH-terminal fragment of DSPP was present exclusively in the E extract. The processed NH₂-terminal fragment of DMP1 was present in G1 and E extracts, whereas the COOH-terminal fragment of DMP1 existed mainly in the E extract. Bone sialoprotein was present in all three extracts of dentin and bone, whereas OPN was present only in the G1 and E extracts of bone. The difference in the distribution of the SIBLING proteins between organic and inorganic phases supports the belief that these molecular species play different roles in dentinogenesis and osteogenesis.     

Immunohistochemical study of small integrin-binding ligand, N-linked glycoproteins in reactionary dentin of rat molars at different ages

Small integrin-binding ligand, N-linked glycoproteins (SIBLING) are believed to play key roles in the process of biomineralization. Reactionary dentin (RD), formed by odontoblasts in response to external stimuli, differs morphologically from primary dentin (PD). To test our hypothesis that the microscopic changes reflect variations in molecular mechanisms involved in formation of the two forms of dentin, and to characterize RD further, we compared the distributions of four SIBLING proteins [bone sialoprotein (BSP), osteopontin (OPN), dentin matrix protein 1 (DMP-1) and dentin sialophosphoprotein (DSPP)] in naturally occurring RD with those in PD. Molars of rats aged 12, 18, 24 and 36 wk were analyzed using immunohistochemistry with antibodies against BSP, OPN, DMP-1, and dentin sialoprotein (a fragment of DSPP). Differences in the distribution of the four SIBLING proteins were evident. Bone sialoprotein, not seen in PD, was consistently observed in RD. Osteopontin, almost absent from PD, was clearly observed in RD. The expression levels of DMP-1 and DSP in RD were lower than in PD. Elevated expression of BSP and OPN, along with a marked decrease of dentin sialoprotein and DMP-1 in RD, suggests a difference in the mechanism of formation of the two forms of dentin.     

Establishment of porcine pulp-derived cell lines and expression of recombinant dentin sialoprotein and recombinant dentin matrix protein-1

The major non-collagenous proteins in dentin have extensive post-translational modifications (PTMs) that appear to be odontoblast-specific, so expression of recombinant dentin proteins in other cell types does not achieve the in vivo pattern of PTMs. We established cell lines from developing porcine dental papillae and used them to express recombinant dentin sialoprotein (DSP) and dentin matrix protein-1 (DMP1). Pulp cells were immortalized with pSV3-neo and clonally selected. Cell lines were characterized by reverse transcruption-polymerase chain reaction (RT-PCR) and assayed for alkaline phosphatase activity and mineralized nodule formation. One of the five cell lines (P4-2) exhibited an odontoblastic phenotype, as determined by expression of tooth-specific markers, response to cytokines, and ability to form mineralized nodules. DSP and DMP1 expression constructs were transiently transfected into various cell lines. DSP, expressed by P4-2 cells, contained chondroitin 6-sulfate, which is a defining modification of the DSP proteoglycan. DMP1 was secreted and cleaved by proteases, even in human kidney 293 cells, which normally do not express DMP1, demonstrating susceptibility to non-specific proteolysis. Both recombinant proteins enhanced P4-2 cell attachment in a dose-dependent manner. We conclude that we have immortalized porcine odontoblast-like cells which express recombinant dentin extracellular matrix components with post-translational modifications that closely resemble those produced in vivo.     

Nuclear factor I‐C expression pattern in developing teeth and its important role in odontogenic differentiation of human molar stem cells from the apical papilla

Nuclear factor I‐C (NFIC) has an important role in the development of murine dental roots, but its role in human root formation is unreported. We thus elucidated the regulatory role of NFIC in the differentiation of human stem cells from the apical papilla (hSCAPs). The first step for this was to determine the expression of NFIC in human teeth, and it was found that NFIC expression was restricted to the odontoblasts and preodontoblasts of the developing molars of humans and mice. NFIC was found to be expressed in odontoblast‐like cells after the subcutaneous transplantation of hSCAPs. NFIC expression was concomitant with dentin sialophosphoprotein (DSPP) in the mineralization of hSCAPs. NFIC knockdown in hSCAPs significantly inhibited expression of DSPP and promoted that of dentin matrix protein 1 (DMP1), meanwhile upregulated the expression of TGF‐β1 and downregulated SMAD3 and SMAD4. NFIC expression was significantly upregulated after TGF‐β1 treatment in hSCAPs. NFIC knockdown prolonged G1 phase of the cell cycle, but had no effect on cell proliferation and migration. These results suggest that NFIC is involved in the development of human root dentin and the regulation of odontoblastic differentiation of hSCAPs. NFIC may participate in the DMP1‐DSPP signaling pathway and comprises a complex signaling cycle with TGF‐β1.     

Effects of lipopolysaccharide on newly established rat dental pulp-derived cell line with odontoblastic properties

To clarify mechanisms of pulp wound healing and regeneration, it is important to establish continuous odontoblast-lineage cell lines. In this study, we established the proliferating pulp progenitor cell lines from dental papilla cells of rat incisor. These cell lines showed high levels of alkaline phosphatase (ALP) activity, expression of Runx2 and dentin sialophosphoprotein (DSPP), and extracellular formation of mineralized nodules. By using the cell line with high expression level of DSPP and the prominent mineral deposition, we examined whether bacterial lipopolysaccharide (LPS) had effects on its odontoblastic properties and found that ALP activity, expression of DSPP and Runx2, and the formation of mineralized nodules were suppressed in LPS dose-dependent manner. These results indicate that our established pulp progenitor cell line exhibits odontoblastic properties, which were suppressed by LPS, suggesting that gram-negative bacterial infection might downregulate the odontoblast function.     

MEPE expression in osteocytes during orthodontic tooth movement

MEPE and DMP1 may play a role in mineralisation and demineralisation within the osteocyte microenvironment. Our earlier studies showed that DMP1 is mechanically responsive [Gluhak-Heinrich J, Ye L, Bonewald LF, Feng JQ, MacDougall M, Harris SE, et al. Mechanical loading stimulates dentin matrix protein 1 (DMP1) in osteocytes in vivo. J Bone Min Res 2003;18(5):807-17]. OBJECTIVES: To examine the effect of mechanical loading on the expression of MEPE using mouse tooth movement model, and compare this effect to that on DMP1. METHODS: In situ hybridisation and immunohistochemistry was performed on 38 treated and 38 control bone sites loaded 6-72 h. ImageJ was used for quantification of mRNA expression in osteocytes. RESULTS: Alveolar osteocytes showed high basal level of MEPE that decreased during the first day of loading, followed by 2.8-fold stimulation at day 3, and returning to a control level by day 7. CONCLUSION: The osteocyte specific mechanical stimulation of MEPE was delayed and different, compared to that of DMP1. This suggests a distinct role of MEPE and DMP1 in the response of osteocytes to mechanical loading in vivo.     

The role of bone morphogenetic proteins 2 and 4 in mouse dentinogenesis

ObjectiveThe bone morphogenetic proteins (BMPs) play crucial roles in tooth development. However, several BMPs retain expression in the dentin of the fully patterned and differentiated tooth. We hypothesized that BMP signaling therefore plays a role in the function of the differentiated odontoblast, the job of which is to lay down and mineralize the dentin matrix.DesignWe generated mice deficient in Bmp2 and 4 using a dentin matrix protein 1 (Dmp1) promoter-driven cre recombinase that was expressed in differentiated odontoblasts.ResultsThe first and second molars of these Bmp2 and Bmp4 double conditional knockout (DcKO) mice displayed reduced dentin and enlarged pulp chambers compared to cre-negative littermate controls. DcKO mouse dentin in first molars was characterized by small, disorganized dentinal fibers, a wider predentin layer, and reduced expression of dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), and bone sialoprotein (BSP). DcKO mouse odontoblasts demonstrated increased type I collagen mRNA production, indicating that the loss of BMP signaling altered the rate of collagen gene expression in these cells. Bmp2 and Bmp4 single Dmp1-cre knockout mice displayed no discernable dentin phenotype.ConclusionsThese data demonstrate that BMP signaling in differentiated odontoblasts is necessary for proper dentin production in mature teeth.     

永生化成牙本质细胞表达牙本质基质蛋白的实验研究

目的　探讨永生化人成牙本质细胞样细胞系hTERT-hOd-l表达牙本质基质蛋白的情况。方法　矿化液培养hTERT-hOd-l细胞5周,检测骨钙素(OC)分泌量和碱性磷酸酶(ALP)活性。采用免疫组织化学、RT-PCR和原位杂交方法检测Ⅰ型胶原、骨涎蛋白(BSP)、牙本质基质蛋白1 (DMP1)以及成牙本质细胞标志物牙本质涎磷蛋白(DSPP) 和牙本质涎蛋白(DSP)在细胞中的表达。结果　在矿化液诱导下,hTERT-hOd-l细胞ALP活性和OC分泌量升高。 hTERT-hOd-l细胞在mRNA水平上表达BSP、DMP1和DSPP,在蛋白质水平上表达DSP和Ⅰ型胶原。结论　hTERT- hOd-l细胞在体外表达牙本质基质蛋白,具有矿化的潜能。     

Phenotypic variation in dentinogenesis imperfecta/dentin dysplasia linked to 4q21

Dentinogenesis imperfecta (DGI) and dentin dysplasia (DD) are allelic disorders that primarily affect the formation of tooth dentin. Both conditions are autosomal-dominant and can be caused by mutations in the dentin sialophosphoprotein gene (DSPP, 4q21.3). We recruited 23 members of a four-generation kindred, including ten persons with dentin defects, and tested the hypothesis that these defects are linked to DSPP. The primary dentition showed amber discoloration, pulp obliteration, and severe attrition. The secondary dentition showed either pulp obliteration with bulbous crowns and gray discoloration or thistle-tube pulp configurations, normal crowns, and mild gray discoloration. Haplotype analyses showed no recombination between three 4q21-q24 markers and the disease locus. Mutational analyses identified no coding or intron junction sequence variations associated with affection status in DMP1, MEPE, or the DSP portion of DSPP. The defects in the permanent dentition were typically mild and consistent with a diagnosis of DD-II, but some dental features associated with DGI-II were also present. We conclude that DD-II and DGI-II are milder and more severe forms, respectively, of the same disease.     

骨髓间质干细胞转染牙本质涎磷蛋白基因的实验研究

目的 评价牙本质涎磷蛋白（DSPP）转基因修饰骨髓问质干细胞（BM-MSC）后，对BM-MSC生物学特性及DSPP表达的影响。方法构建含小鼠DSPP基因的真核表达载体pcDNA3．1（＋）／DSPP，用脂质体介导转染大鼠BM-MSC；RT-PCR检测转染后细胞的Pax-9和DMP-1基因表达情况；检测转染细胞矿化诱导后Von Kossa钙盐染色计算单位面积钙结节形成率。结果成功构建DSPP真核表达载体pcDNA3．1（＋）／DSPP，酶切后得到3．0kb和5．4kb的片段，与回收的目的基因和载体基因片段大小相符；经转染BM-MSC后24h可见DSPP基因表达，48h后可见有Pax-9基因表达，无DMP-1基因表达；转基因后的BM-MSC免疫组化染色显示DSPP阳性；转染细胞矿化诱导后钙结节形成率高于未转染细胞。结论BM-MSC转基因表达DSPP能够增强其矿化能力，并诱导牙齿发育相关基因的表达，提示DSPP可能在牙齿发育早期具有一定作用。     

Dentinogenesis and Dentin Sialophosphoprotein (DSPP)

Dentin sialophosphoprotein (DSPP) is critical for proper mineralization of tooth dentin, and understanding its structure and function should yield important insights into how dentin biomineralization is controlled. During the recent six years, DSPP-derived proteins isolated from developing porcine teeth have been characterized. Porcine DSPP is expressed and secreted by odontoblasts and is processed by BMP-1, MMP-20 and MMP-2 into three main parts: dentin sialoprotein (DSP), dentin glycoprotein (DGP), and dentin phosphoprotein (DPP). We have learned that DSP is a proteoglycan that forms covalent dimers, DGP is a phosphorylated glycoprotein, and DPP is a highly phosphorylated intrinsically disordered protein that shows extensive length polymorphisms due to the genetic heterogeneity of its coding region.     

高迁移率族蛋白B1在人牙髓细胞成牙本质分化中的表达

目的:检测人牙髓细胞(human dental pulp cells,hDPCs)诱导矿化过程中高迁移率族蛋白B1(high-mobility group box 1,HMGB1)的表达变化,探讨HMGB1在人牙髓细胞损伤修复中的可能作用。方法:组织块法分离培养hDPCs,收集矿化诱导0、3、7、11、14d后hDPCs的mRNA、蛋白质及细胞爬片,实时荧光定量反转录聚合酶链反应(RT-PCR)和蛋白质印迹法(western blot)分别检测HMGB1、牙本质涎磷蛋白(dentin sialophosphoprotein, DSPP)、牙本质基质蛋白1(dentin matrix protein 1, DMP1)及碱性磷酸酶(alkaline phosphatase, ALP)的mRNA及蛋白表达水平;并检测ALP活性,免疫荧光检测HMGB1在hDPCs矿化过程中的表达。结果:hDPCs矿化诱导后,DMP1、DSPP、ALP及HMGB1的mRNA表达显著性上调,DMP1、DSPP和ALP的mRNA以及碱性磷酸酶活性在诱导7 d、11 d和14 d后与对照组间差异有统计学意义(P<0.05),HMGB1mRNA在矿化诱导11d和14d后与对照组的差异具有统计学意义(P<0.05)。蛋白印迹法检测示细胞内各矿化标记的蛋白表达均较对照组上调,而细胞内HMGB1的蛋白表达较对照组下调。免疫荧光结果显示hDPCs矿化过程中,HMGB1逐渐从胞核转移至胞浆。结论:在hDPCs诱导成牙本质细胞分化过程中,HMGB1在mRNA水平上与DMP1、DSPP和ALP的表达变化趋势相似,而细胞内HMGB1蛋白水平表达下调,且HMGB1在hDPCs细胞内出现转位,提示HMGB1与hDPCs的成牙本质分化有关,可能在牙髓细胞损伤修复中发挥作用。     

Expression of mineralization markers in dental pulp cells

There is an increasing interest in the utility of dental pulp stem cells (DPSCs) for dentin regeneration. The mechanisms involved in DPSC differentiation remain poorly understood. The purpose of the study was to investigate the mineralization capacity of human dental pulp cells (DPCs) and identify potential markers for odontoblast differentiation. The isolated DPCs expressed mesenchymal stem-cell markers as shown by flow cytometry and could differentiate in vitro into odontogenic, adipogenic, and chondrogenic lineages. Alkaline phosphatase activity of DPCs elevated over time, with significant upregulation on day 21 in odontogenic induction. Quantitative RT-PCR revealed that osteocalcin, dentin sialophosphoprotein (DSPP), and matrix extracellular phosphoglycoprotein (MEPE) expression also increased time dependently in the induction cultures. In conclusion, we isolated DPCs with stem cell characteristics. MEPE and DSPP showed a similar regulatory pattern of DPCs mineralization. MEPE along with DSPP may be potential odontogenetic differentiation markers.     

The Effect of SIRT6 on the Odontoblastic Potential of Human Dental Pulp Cells

Introduction

The aim of this study was to investigate whether SIRT6 is expressed in human dental pulp as well as the effect of SIRT6 on proliferation and odontoblastic differentiation of human dental pulp cells (HDPCs).

Methods

Immunohistochemical and immunocytochemical assays were used to detect the expression of SIRT6 in human dental pulp tissue and HDPCs. To determine the effect of SIRT6 on odontoblast differentiation, HDPCs with loss (HDPCs SIRT6 knockdown) and gain (HDPCs SIRT6 overexpression) of SIRT6 function were developed, and their proliferation ability was examined. Odontogenic differentiation of HDPCs was determined by alkaline phosphatase (ALP) activity, ALP-positive cell staining, alizarin red staining, and von Kossa staining. Mineralization-related genes, including ALP, dentin sialophosphoprotein (DSPP), and dentin matrix acidic phosphoprotein 1, were determined by real-time quantitative polymerase chain reaction. Western blot analysis was performed to detect the expression of DSPP protein.

Results

SIRT6 was found in the dental pulp tissue and HDPCs. SIRT6 knockdown decreased ALP activity in HDPCs; calcium nodule formation ability; and the expression of mineralization-related genes such as ALP, DSPP, and DMP1, whereas these were increased with the overexpression of SIRT6.

Conclusions

SIRT6 is expressed in human dental pulp and participates in the odontoblast differentiation of HDPCs.     

KLF4 promotes the odontoblastic differentiation of human dental pulp cells

Introduction

Krüppel-like factor 4 (KLF4) plays an important role in cytodifferentiation and proliferation. Our previous study showed that KLF4 was specifically expressed in polarizing and elongating odontoblasts. However, the role of KLF4 in odontoblast differentiation was still unknown. The purpose of this study was to investigate the role of KLF4 in odontoblastic differentiation of human dental pulp cells (hDPCs).

Methods

hDPCs were treated with odontoblastic induction medium. Odontoblastic differentiation was determined by the detection of alkaline phosphatase (ALPase) activity and the expression of mineralization-related genes including ALP, dentin sialophosphoprotein (DSPP), and dentin matrix protein-1 (DMP-1). Also, cell proliferation ability was examined by the 5-ethynyl-2’-deoxyuridine (EdU) incorporation assay. Simultaneously, messenger RNA and protein levels of KLF4 were detected. pKLF4-IRES2-EGFP plasmid encoding full-length KLF4 was constructed to overexpress KLF4, and biologic effects of KLF4 on hDPCs were investigated by the evaluation of ALPase activity and the detection of ALP, DSPP, and DMP-1 expression and analysis of cell proliferation ability.

Results

ALPase activity and the expression of odontoblastic differentiation markers progressively increased in hDPCs cultured with odontoblastic induction medium. Meanwhile, the proliferation ability decreased in this procedure; messenger RNA and protein levels of KLF4 increased significantly on day 5 after the odontoblastic induction of hDPCs and kept increasing until day 14. hDPCs showed up-regulated activity of ALPase and the expression of mineralization-related genes, including ALP, DMP-1, and dentin sialoprotein (DSP), after KLF4 overexpression. Besides, the proliferation ability of hDPCs decreased significantly in the KLF4 overexpression group by EdU incorporation assay.

Conclusions

Our findings suggest that KLF4 is able to promote odontoblastic differentiation of hDPCs and inhibit proliferation of hDPCs.