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.     

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.     

Expression and processing of small integrin-binding ligand N-linked glycoproteins in mouse odontoblastic cells

OBJECTIVE: Small integrin-binding ligand N-linked glycoproteins (SIBLINGs) are expressed in dentin and believed to control dentinogenesis. Five members of SIBLING family include bone sialoprotein (BSP), osteopontin (OPN), matrix extracellular phosphoglycoprotein (MEPE), dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP). These genes are clustered on chromosome 4q in humans and share similar biological features. DSPP and DMP1 are processed into given structural/functional fragments in rat and porcine. It still remains unclear whether these evidences occur in mouse and other SIBLING members are also processed into given fragments from their parent precursors. The aim of this study was to identify expression and processing of the five proteins in two mouse odontoblastic cell lines. DESIGN: Two mouse odontoblastic cells were used to study expression and processing of the five SIBLING proteins by immunohistochemistry and Western blot analyses. RESULTS: Immunohistochemistry study showed that all of the five SIBLING members were expressed within the cytoplasm and cellular processes in the mouse odontoblastic cell lines. Expression levels of DMP1 and DSPP were higher in differentiated mouse odontoblasts than undifferentiated mouse odontoblasts. Immunolabelling signal of DSP and MEPE was also detected within the nucleus in the two cell lines. Western blot assay indicated that all five members were processed into at least two fragments in these cells. CONCLUSIONS: These results suggest that different processed products and expression levels of the SIBLING proteins may play distinct biological functions in tooth development and mineralisation.     

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.     

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。     

Refined mapping of the human dentin sialophosphoprotein (DSPP) gene within the critical dentinogenesis imperfecta type II and dentin dysplasia type II loci

Dentinogenesis imperfecta type II and dentin dysplasia type II are diseases resulting in abnormal dentin formation, which have been mapped to overlapping regions of human chromosome 4q defined by markers D4S2691 and D4S2692 (6.6 cM) and D4S3291 and SPP1 (14.1 cM), respectively. Recently, two of the major non-collagenous proteins of dentin, dentin sialoprotein (DSP) and dentin phosphoprotein (DPP, phosphophoryn) have been shown to be encoded by a single gene, termed dentin sialophosphoprotein (DSPP), which has been mapped to human chromosome 4. The purpose of this study was to perform refined mapping of DSPP related to these disease loci by gene content mapping, as well as to place the DSPP gene on the physical map of human chromosome 4 by sequence tagged site (STS) content mapping. Human genomic DSPP clones were isolated, and gene content mapping performed with specific primers for dentin matrix protein 1 (DMP1), bone sialoprotein (BSP) and osteopontin (secreted phosphoprotein 1, SPP1). STS content mapping was then performed with flanking STS markers to these dentin/bone gene loci. Our results demonstrate that the DSPP and DMP1 genes are within a maximum distance of 110 kb. Both DSPP and DMP-1 have been placed on the physical map of human chromosome 4 within the interval defined by markers D4S564 and D4S1292. DSPP is thereby strengthened as a candidate gene for both DGI-II and DD-II.     

Transglutaminase crosslinking of SIBLING proteins in teeth

Transglutaminase 2 (TG2), a protein-crosslinking enzyme, participates in extracellular matrix maturation and cell adhesion in cartilage and bone. We hypothesized that TG2 has similar roles in teeth. A TG activity assay and immunoblotting of rat tooth extracts showed TG activity and the presence of high-molecular-weight forms of the SIBLING (Small Integrin-Binding LIgand N-linked Glycoprotein) proteins: dentin matrix protein 1 (DMP1), dentin phosphoprotein (DPP), and bone sialoprotein (BSP). DMP1 and BSP, each containing both glutamine and lysine residues critical for crosslink formation, readily formed polymers in vitro when incubated with TG2. The ability of glutamine-lacking DPP to form polymers in vitro and in vivo demonstrates that it could act as a lysine donor for crosslinking, potentially having protein crosslinking partner(s) in teeth. Consistent with a role in cell adhesion, the TG2 isoform was co-localized by immunohistochemistry with its substrates at cell-matrix adhesion sites, including along odontoblast tubules (DMP1 and DPP), in the pericellular matrix of cementocytes (DMP1), and in predentin (BSP).     

A comparative study of sialic acid-rich proteins in rat bone and dentin

Four sialic acid-rich (SA-rich) proteins found in bone and dentin, osteopontin (OPN), bone sialoprotein (BSP), bone acidic glycoprotein-75 (BAG-75), and dentin matrix protein 1 (DMP1), share some common features. We used SDS-PAGE and Western immunoblots to analyze and compare SA-rich proteins in bone and dentin extracts from rats with a single chromatographic procedure. OPN was detected in dentin extracts, with a relative level less than one-seventieth of that in bone. Both bone and dentin BSP demonstrated an extremely broad distribution pattern, probably due to a high degree of heterogeneity in post-translational modifications. BAG-75 in both bone and dentin was detected as an 83 kDa band, dramatically distinct from that of DMPI. Using a polyclonal antibody raised against a purified bone 57 kDa protein (a portion of DMPI), we detected 150 kDa protein bands in bone fraction; the same bands were recognized by antirecombinant rat DMPI antibody. Bands from dentin migrating at about 150 kDa in earlier fractions and progressing to 200 kDa in later fractions showed a clear immunoreactivity to the anti-57 kDa antibody. We conclude that the majority of DMPI in rat bone is processed into fragments, whereas that in dentin remains intact.     

Role of the NH2‐terminal fragment of dentin sialophosphoprotein in dentinogenesis

Dentin sialophosphoprotein (DSPP) is a large precursor protein that is proteolytically processed into a NH₂‐terminal fragment [composed of dentin sialoprotein (DSP) and a proteoglycan form (DSP‐PG)] and a COOH‐terminal fragment [dentin phosphoprotein (DPP)]. In vitro studies indicate that DPP is a strong initiator and regulator of hydroxyapatite crystal formation and growth, but the role(s) of the NH₂‐terminal fragment of DSPP (i.e. DSP and DSP‐PG) in dentinogenesis remain unclear. This study focuses on the function of the NH₂‐terminal fragment of DSPP in dentinogenesis. Here, transgenic (Tg) mouse lines expressing the NH₂‐terminal fragment of DSPP driven by a 3.6‐kb type I collagen promoter (Col 1a1) were generated and cross‐bred with Dspp null mice to obtain mice that express the transgene but lack the endogenous Dspp (Dspp KO/DSP Tg). We found that dentin from the Dspp KO/DSP Tg mice was much thinner, more poorly mineralized, and remarkably disorganized compared with dentin from the Dspp KO mice. The fact that Dspp KO/DSP Tg mice exhibited more severe dentin defects than did the Dspp null mice indicates that the NH₂‐terminal fragment of DSPP may inhibit dentin mineralization or may serve as an antagonist against the accelerating action of DPP and serve to prevent predentin from being mineralized too rapidly during dentinogenesis.     

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.     

X连锁磷酸盐调节基因的研究进展

X连锁磷酸盐调节基因在体内与肾磷重吸收以及骨和牙的矿化密切相关。X连锁磷酸盐调节基因突变可引起骨和牙的形态发生严重改变,例如佝偻病、球间牙本质和前期牙本质加宽等。下面就X连锁磷酸盐调节基因参与骨和牙的生物矿化和磷代谢平衡的研究进展作一综述。     

Distribution of non-collagenous dentin matrix proteins and proteoglycans, and their relation to calcium accumulation in bisphosphonate-affected rat incisors

It has been reported that multiple injections of 1-hydroxyethylidene- 1,1-bisphosphonate (HEBP) to rats prevent mineralization of incisor dentin, thereby revealing high concentrations of calcium in the non-mineralized matrix of circumpulpal dentin. To identify the molecules responsible for calcium accumulation in circumpulpal dentin matrix, rats were injected daily with HEBP (8 mg P/kg) for 7 d, and the incisors processed for various histochemical and immunohistochemical staining of non-collagenous matrices of dentin. Cuprolinic blue reactions for proteoglycans (PGs) were equally distributed in non-mineralized matrix of mantle and circumpulpal dentin layers. Dentin sialoprotein (DSP) and osteopontin (OPN) immunoreactions were found in non-mineralized circumpulpal dentin matrix, but not in mantle dentin. In normal incisors, however, predentin matrix showing significant DSP immunoreactivity was negative for Ca-GBHA reactions. HEBP-affected, non-mineralized OPN immunopositive bone matrix was also non-reactive for calcium. From these observations, neither PGs, OPN nor DSP appear to be responsible for calcium accumulation in HEBP-affected circumpulpal dentin. Stains-all reactive component, possibly dentin phosphoprotein (DPP), only showed the same distribution as that of Ca-GBHA in both HEBP-affected and normal dentin matrix, implicating a possible contribution of DPP to calcium accumulation in circumpulpal dentin and, hence, to appositional mineralization of dentin.     

Failure to process dentin sialophosphoprotein into fragments leads to periodontal defects in mice

Dentin sialophosphoprotein (DSPP) plays a vital role in dentinogenesis. Previously, we showed that, in addition to dentin, DSPP is also highly expressed in alveolar bone and cellular cementum, and plays a crucial role in maintaining periodontal integrity; Dspp‐deficient mice demonstrate severe periodontal defects, including alveolar bone loss, decreased cementum deposition, abnormal osteocyte morphology in the alveolar bone, and apical migration of periodontal ligament. Dentin sialophosphoprotein in dentin and bone is cleaved into NH₂‐terminal and COOH‐terminal fragments. Whilst our previous study showed that the proteolytic processing of DSPP is critical for dentinogenesis, it is unclear whether the post‐translational cleavage of DSPP also plays an essential role in maintaining a healthy periodontium. In this study, we analyzed the periodontal tissues from transgenic mice expressing the uncleavable full‐length DSPP in the Dspp knockout (Dspp‐KO) background (named ‘Dspp‐KO/D452A‐Tg mice’), in comparison with those from wild‐type mice, Dspp‐KO mice, and mice expressing the normal Dspp transgene in the Dspp‐KO background (designated ‘Dspp‐KO/normal‐Tg mice’). We found that transgenic expression of the normal DSPP fully rescued the periodontal defects of the Dspp‐KO mice, whereas this was not the case in Dspp‐KO/D452A‐Tg mice. These results indicate that proteolytic processing of DSPP is essential to periodontal integrity.     

A review of the nature,role, and function of dentin non‐collagenous proteins. Part 1: proteoglycans and glycoproteins

Dentin is a collagen‐based mineralized tissue consisting of inorganic apatite crystallites embedded in an extracellular matrix. In addition to type I collagen, the dentin organic matrix contains several proteins and proteoglycans, collectively referred to as non‐collagenous proteins, that play fundamental roles in actively promoting, controlling, and regulating fibrillogenesis, crystal growth, and mineralization during dentinogenesis. Similarly to collagen fibrils, non‐collagenous proteins are synthesized and secreted by odontoblasts and some of them are detectable in both predentin and mineralized dentin while others are only within the dentin layer. Non‐collagenous components include proteoglycans (such as decorin, biglycan, fibromodulin, lumican, osteoadherin, and versican) and several glycoproteins such as osteocalcin, osteonectin, and the SIBLING proteins (i.e. osteopontin, bone sialoprotein, dentin matrix protein‐1, dentin sialophosphoprotein, and matrix extracellular phosphoglycoprotein). The first part of this review offers an overview of the nature of the above‐mentioned non‐collagenous proteins of the dentin organic matrix and their postulated functions, since a better understanding of the biological dynamics of the extracellular macromolecules is fundamental to clarifying the properties and function of the human mature sound dentin.     

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.     

A review of protein structure and gene organisation for proteins associated with mineralised tissue and calcium phosphate stabilisation encoded on human chromosome 4

Several proteins associated with mineralised tissue (teeth and bone) or involved in calcium phosphate stabilisation in the body fluids, milk and saliva have been mapped to the q arm of human chromosome 4. These include the dentine/bone proteins dentine sialophosphoprotein (DSPP), dentine matrix protein 1 (DMP1), bone sialoprotein (BSP), matrix extracellular phosphoglycoprotein, osteopontin (OPN), enamelin, ameloblastin, milk caseins, salivary statherin, and proline-rich proteins. The proposed function of those that are multiphosphorylated is: (i) the stabilisation of calcium phosphate in solution (e.g. casein, statherin) preventing spontaneous precipitation and seeded-crystal growth or (ii) promoting biomineralisation (e.g. the phosphophoryn domain of DSPP), where the protein described as a template macromolecule, is proposed to act as a nucleator/promoter of crystal growth. The genes of these proteins have been subjected to conserved chromosomal synteny during mammalian evolution. The multiphosphorylated proteins statherin, caseins, phosphophoryn, BSP and OPN have been characterised as intrinsically disordered. The codon usage patterns for the amino acid serine reveal a bias for AGC and AGT codons within the human genes dspp, dmp1 and bsp, mouse dspp and dmp1 but not significantly for statherin or caseins. This pattern was also observed in the gene encoding hen phosvitin that also contains stretches of multiphosphorylated serines and in the dmp1 gene sequences of mammalian, reptilian and avian classes. In conclusion, these intrinsically disordered multiphosphorylated proteins are the translation products of genes displaying examples of codon usage bias, internal repeats and conserved chromosomal synteny within the mammalian class.     

核心结合因子α1诱导人牙乳头细胞矿化的相关蛋白表达

目的 在人牙乳头细胞中探讨核心结合因子α1(core binding factor α1，cbfal)能否调控矿化相关蛋白的表达。方法 体外培养人牙乳头细胞，转染peDNA3-cbfal重组质粒，稳定表达cbfal后检测碱性磷酸酶(alkaline phosphatase，ALP)和骨钙素(osteoealcin，OC)含量，同时采用免疫组化、免疫印迹和PCR等方法观察骨桥素(osteopontin，OPN)、骨涎蛋白(bone sialoprotein，BSP)、骨连素(osteonectin，ON)、牙本质基质蛋白(dentin matrix protein 1，DMP1)、牙本质涎蛋白(dentin sialoprotein，DSP)和牙本质涎磷蛋白(dentin sialophosphopmtein，DSPP)的表达。结果 建立了稳定表达cbfal基因和蛋白的人牙乳头细胞模型PC-3，发现转染后细胞的ALP和OC含量明显增高，OPN、BSP、ON、DMP1的表达也明显增高，但未发现牙本质特异性蛋白DSP及其编码基因DSPP的表达。结论 在人牙乳头细胞中，cbfal能上调ALP和OC含量，诱导多种矿化组织特异性蛋白的表达，在牙齿发育矿化中有重要作用，提示它与牙乳头细胞分化为成牙本质细胞的启动有关。     

犬年轻恒牙组织再生后牙本质基质蛋白1的表达

目的：观察牙本质基质蛋白1 （DMP-1）在血管再生治疗犬年轻恒牙根尖周炎后的表达.方法：免疫组化方法检测DMP-1在新生成的矿化物、牙本质、牙骨质中的表达;用天狼星红染色法来判断胶原基质的组成和成熟度.结果：①.DMP-1在新生成矿化组织,牙本质,牙骨质中都有不同程度表达.在新生成矿化物组织中的表达强度明显高于牙本质.②.新生成的矿化物形状不规则,成斑片状,颜色为黄色或橙色.结论：DMP-1在根尖新形成的矿化区有高表达,与新生物矿化有关系.     

牙本质基质蛋白-1与生物矿化

牙本质基质蛋白(DMP)-1是骨、软骨、釉质、牙骨质和牙本质生物矿化所必需的一种酸性非胶原蛋白.蛋白质化学研究显示,DMP-1全基因序列是生物矿化的启动因子,由C末端和N末端组成.体内实验证实DMP-1具有多方面功能,不仅是生物矿化的信号分子,同时也是调节因子,对成骨细胞和成牙本质细胞的成熟至关重要.本文就DMP-1在基因调节、组织细胞中的表达以及成骨、成牙本质中的生物矿化作用作一.