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All-trans retinoic acid and bone morphogenetic protein 2 (BMP2) synergistically induced an alkaline phosphatase (ALP) activity, one of the osteoblastic differentiation markers, and promoted the extracellular matrix calcification in a myoblastic C2C12 cell culture system. The induced ALP mRNA was not suppressed in the presence of a protein synthesis inhibitor, suggesting that the de novo protein synthesis does not influence this induction. There are three isotypes for the retinoic acid receptor (RARα, RARβ, RARγ). Both the ALP activity and the extracellular matrix calcification were inhibited by the addition of the specific siRNA for RARγ, but not by that for RARα or RARβ. When the effects of the RAR subtype-specific agonists on the ALP activity in the presence of BMP2 were examined, the RARγ-specific agonist was the most effective. The ALP activity induced by any RAR subtype-specific agonist was inhibited by the addition of the specific siRNA for RARγ, but not by that for RARα or RARβ. These results suggest that a RARγ-dependent functional crosstalk is present between the retinoic acid and BMP2 signaling to induce osteogenic transdifferentiation in myoblastic C2C12 cells. [ABSTRACT FROM AUTHOR]

Background: Enamel extracts are biologically active and capable of inducing osteogenesis and cementogenesis, but the specific molecules carrying these activities have not been ascertained. The purpose of this study was to identify osteogenic factors in porcine enamel extracts. Methods: Enamel proteins were separated by size-exclusion chromatography into four fractions, which were tested for their osteogenic activity on osteoblast-like cells (ST2) and human periodontal ligament (HPDL) cells. Results: Fraction 3 (Fr.3) and a transforming growth factor-beta 1 (TGF-β1) control reduced alkaline phosphatase (ALP) activity in ST2 but enhanced ALP activity in HPDL cells. The enhanced ALP activity was blocked by anti-TGF-β antibodies. Furthermore, using a dual-luciferase reporter assay, we demonstrated that Fr.3 can induce the promoter activity of the plasminogen activator inhibitor type 1 (PAI-1) gene. Conclusion: These results show that the osteoinductive activity of enamel extracts on HPDL cells is mediated by TGF-β1. [ABSTRACT FROM AUTHOR]

Fukae M, Kanazashi M, Nagano T, Tanabe T, Oida S, Gomi K. Porcine sheath proteins show periodontal ligament regeneration activity. Eur J Oral Sci 2006; 114 (Suppl. 1): 212–218 © Eur J Oral Sci, 2006 --> The purpose of this study was to identify the periodontal regeneration factors of enamel protein extracts that induce cementum and bone regeneration in vivo. Cementum regeneration, one aspect of periodontal ligament regeneration, was examined using a buccal dehiscence model of dogs. Enamel matrix protein fractions were prepared from developing porcine incisors. Cementum‐regeneration activity was found to reside in a protein aggregate composed of amelogenins and sheath proteins extracted from newly formed secretory enamel. Cementum‐regeneration activity was not observed in protein fractions containing only amelogenin or its derivatives. The sheath proteins were purified to homogeneity and tested for alkaline phosphatase (ALP)‐inducing activity on human periodontal ligament (HPDL) cells. The induction of ALP was observed following application of the 17‐kDa sheath protein but not of the lower‐molecular‐weight sheath proteins. Although transforming growth factor‐β1 also shows ALP‐inducing activity, contamination with growth factors was excluded because synthetic peptides (based on the sheath protein's sequence) also showed ALP‐inducing activity. The 17‐kDa sheath protein showed both cytodifferentiation and cementum‐regeneration activity, but it is unclear whether its cementum‐regeneration activity is derived from its ALP‐inducing activity on HPDL cells. [ABSTRACT FROM AUTHOR]

Yamakoshi Y, Hu JC-C, Fukae M, Yamakoshi F, Simmer JP. How do enamelysin and kallikrein 4 process the 32-kDa enamelin? Eur J Oral Sci 2006; 114 (Suppl. 1): 45–51 © Eur J Oral Sci, 2006 --> The activities of two proteases – enamelysin (MMP‐20) and kallikrein 4 (KLK4) – are necessary for dental enamel to achieve its high degree of mineralization. We hypothesize that the selected enamel protein cleavage products which accumulate in the secretory‐stage enamel matrix do so because they are resistant to further cleavage by MMP‐20. Later, they are degraded by KLK4. The 32‐kDa enamelin is the only domain of the parent protein that accumulates in the deeper enamel. Our objective was to identify the cleavage sites of 32‐kDa enamelin that are generated by proteolysis with MMP‐20 and KLK4. Enamelysin, KLK4, the major amelogenin isoform (P173), and the 32‐kDa enamelin were isolated from developing porcine enamel. P173 and the 32‐kDa enamelin were incubated with MMP‐20 or KLK4 for up to 48 h. Then, the 32‐kDa enamelin digestion products were fractionated by reverse‐phase high‐performance liquid chromatography (RP‐HPLC) and characterized by Edman sequencing, amino acid analysis, and mass spectrometry. Enamelysin cleaved the 32‐kDa enamelin only after it was deglycosylated. Kallikrein 4 digestion of the 32‐kDa enamelin generated nine major cleavage products, six of which were successfully characterized. After 12 h of digestion with KLK4, all of the 32‐kDa enamelin had been cleaved, but some cleavage products persisted after 48 h of digestion. [ABSTRACT FROM AUTHOR]

The activities of two proteases – enamelysin (MMP-20) and kallikrein 4 (KLK4) – are necessary for dental enamel to achieve its high degree of mineralization. We hypothesize that the selected enamel protein cleavage products which accumulate in the secretory-stage enamel matrix do so because they are resistant to further cleavage by MMP-20. Later, they are degraded by KLK4. The 32-kDa enamelin is the only domain of the parent protein that accumulates in the deeper enamel. Our objective was to identify the cleavage sites of 32-kDa enamelin that are generated by proteolysis with MMP-20 and KLK4. Enamelysin, KLK4, the major amelogenin isoform (P173), and the 32-kDa enamelin were isolated from developing porcine enamel. P173 and the 32-kDa enamelin were incubated with MMP-20 or KLK4 for up to 48 h. Then, the 32-kDa enamelin digestion products were fractionated by reverse-phase high-performance liquid chromatography (RP-HPLC) and characterized by Edman sequencing, amino acid analysis, and mass spectrometry. Enamelysin cleaved the 32-kDa enamelin only after it was deglycosylated. Kallikrein 4 digestion of the 32-kDa enamelin generated nine major cleavage products, six of which were successfully characterized. After 12 h of digestion with KLK4, all of the 32-kDa enamelin had been cleaved, but some cleavage products persisted after 48 h of digestion. [ABSTRACT FROM AUTHOR]

The purpose of this study was to identify the periodontal regeneration factors of enamel protein extracts that induce cementum and bone regeneration in vivo. Cementum regeneration, one aspect of periodontal ligament regeneration, was examined using a buccal dehiscence model of dogs. Enamel matrix protein fractions were prepared from developing porcine incisors. Cementum-regeneration activity was found to reside in a protein aggregate composed of amelogenins and sheath proteins extracted from newly formed secretory enamel. Cementum-regeneration activity was not observed in protein fractions containing only amelogenin or its derivatives. The sheath proteins were purified to homogeneity and tested for alkaline phosphatase (ALP)-inducing activity on human periodontal ligament (HPDL) cells. The induction of ALP was observed following application of the 17-kDa sheath protein but not of the lower-molecular-weight sheath proteins. Although transforming growth factor- β1 also shows ALP-inducing activity, contamination with growth factors was excluded because synthetic peptides (based on the sheath protein's sequence) also showed ALP-inducing activity. The 17-kDa sheath protein showed both cytodifferentiation and cementum-regeneration activity, but it is unclear whether its cementum-regeneration activity is derived from its ALP-inducing activity on HPDL cells. [ABSTRACT FROM AUTHOR]

Nagano T, Iwata T, Ogata Y, Tanabe T, Gomi K, Fukae M, Arai T, Oida S. Effect of heat treatment on bioactivities of enamel matrix derivatives in human periodontal ligament (HPDL) cells. J Periodont Res 2004; doi: 10.1111/j.1600-0765.2004.00733.x © Blackwell Munksgaard, 2004 It has been shown that Emdogain® (EMD) containing enamel matrix derivative has cementum- and osteo-promotive activities in vivo and in vitro. Nevertheless, the commercial sale of EMD was halted because it has some possible risk for infectiosity. At present, Emdogain® Gel (Emd-Gel) containing the EMD heated to avoid the infectiosity is commercially available. The purpose of this study was to compare the in vitro bioactivities of Emd-Gel and EMD. Healthy human periodontal ligament (HPDL) cells were used to study the effect of Emd-Gel and EMD on cell differentiation. The HPDL cells exposed to Emd-Gel and EMD were evaluated for the following effects: (i) alkaline phosphatase (ALP) activity; (ii) mitogenic (MTT) assay; (iii) biomineralization activity; (iv) gene expressions using reverse transcription-polymerase chain reaction (RT-PCR). The effect of EMD with or without heat treatment was examined for ALP activity on the cell differentiation. The effect of Emd-Gel on ALP activity was greater than that of EMD. It was confirmed from the effect of EMD with heat treatment at 60°C, 80°C and 100°C on the ALP activity. The effect of Emd-Gel on the biomineralization activity was also greater than that of the EMD. The Emd-Gel has a stronger effect for the expression of osteoblast-like phenotype than the EMD. The results indicate that the Emd-Gel has greater bioactivities than the EMD in vitro. [ABSTRACT FROM AUTHOR]

Dentin sialophosphoprotein (DSPP) is a chimeric glycoprotcin with dentin sialoprotein (DSP) on its N-terminus and dentin phosphoprotein (DPP) on its C-terminus. We have constructed and screened a unidirectional cDNA library derived from the pulp organ of developing pig teeth, and isolated cDNA clones encoding DSP-only, as well as two DSPP clones with alternative sequences in their 3' coding regions. The DSP-only transcript has an open reading frame of 386 codons, and is generated through the use of a polyadenylation signal within intron 4, immediately following the DSP coding region, the use of this polyadenylation signal deletes the DPP coding region and places a TGA translation termination signal as the fourth codon following the exon 4-encoded segment, The DSPP cDNAs contain open reading frames of 593 and 600 codons. Northern blots hybridized to radiolabeled DSP probes showed bands at 1.4, 2.5, 4.4, and 4.8 kb, Cloning and characterization of reverse transcriptase polymerase chain reaction products confirmed the existence of mRNA encoding pDSP[sub 386], pDSPP[sub 593], and pDSPP[sub 600] in vivo, but also suggested that DNA sequence redundancies in the DSPP coding region make it prone to cloning artifacts. [ABSTRACT FROM AUTHOR]

There have been many immunohistochemical studies of enamel proteins during root formation. In the present article, the detection and expression of enamel proteins in tissue samples prepared from the apical portion of the forming root (APFR) in porcine permanent incisor tooth germs were studied. Amelogenin, enamelin, and sheathlin were detected by immunoblot analysis, but only in small amounts. The detection of their derivatives indicated their degradation. It is, at present, unclear as to which proteinases are involved in these degradations, because activity of enamel matrix serine proteinase 1 and enamelysin was not detected on gelatin and casein zymograms. The expression of enamel proteins was also proved in the APFR sample by the detection of polymerase chain reaction products of their cDNAs, and this may be related to cells of fragmentized Hertwig's epithelial root sheath. Amelogenin expression was not greater than that of enamelin and sheathlin. It was different from the expression pattern of secretory ameloblasts involved in enamel matrix formation. These results suggest that the amelogenins found in the APFR do not form a three-dimensional structure of amelogenin micelles, which has been proposed for the secretory enamel matrix structure. In this case, the enamel proteins could spread out easily following degradation into the matrix of future cementum. Some of their derivatives may play a role in the formation of the cementum. [ABSTRACT FROM AUTHOR]

Amelogenins are the most abundant constituent in the enamel matrix of developing teeth. Recent investigations of rodent incisors and molar tooth germs revealed that amelogenins are expressed not only in secretory ameloblasts but also in maturation ameloblasts, although in relatively low levels. In this study, we investigated expression of amelogenin in the maturation stage of porcine tooth germs by in situ hybridization and immunocytochemistry. Amelogenin mRNA was intensely expressed in ameloblasts from the differentiation to the transition stages, but was not detected in maturation stage ameloblasts. C-terminal specific anti-amelogenin antiserum, which only reacts with nascent amelogenin molecules, stained ameloblasts from the differentiation to the transition stages. This antiserum also stained the surface layer of immature enamel at the same stages. At the maturation stage, no immunoreactivity was found within the ameloblasts or the immature enamel. These results indicate that, in porcine tooth germs, maturation ameloblasts do not express amelogenins, suggesting that newly secreted enamel matrix proteins from the maturation ameloblast are not essential to enamel maturation occurring at the maturation stage. [ABSTRACT FROM AUTHOR]

When parathyroidectomized (PTX) rats were maintained on a diet containing 0.3% calcium and lacking vitamin D, the forming incisor dentin consisted of two distinct layers that differed in their degree of mineralization. The two layers could be distinguished by hematoxylin staining and by contact microradiography. The layer near the dental pulp was the hypomineralized dentin, different from a predentin, containing a small amount of mineral detected by electron probe and chemical analyses. The mineralization of the outer dentin was almost normal. Biochemical characterization demonstrated that the two dentin layers differed in their phosphophoryn content. The outer dentin contained two phosphophoryns, which is the pattern found in normal dentin. In contrast, the hypomineralized dentin contained only the lower molecular weight and lower phosphorylated phosphophoryn, which may be involved in crystal nucleation. From the distribution of lead, which marked dentin formed at the time of injection, it could be inferred that the mineralized dentin begins as a hypomineralized layer that undergoes secondary mineralization. The secondary mineralization occurs by the active calcium transport of the odontoblasts via odontoblastic processes. [ABSTRACT FROM AUTHOR]

Several proteinase activities associated with different molecular weight proteins were detected by substrate-gel electrophoresis using gelatin as a substrate in the 4M guanidine soluble fraction obtained from the demineralized matrix of the mineralized phase of newborn rat calvaria (mineralized phase matrix proteinases = MPM proteinases). The major gelatinase activity migrated with the molecular weight fraction of 58,000 daltons. This proteinase was active over the pH range 6-9 against gelatin. Based on the behavior against inhibitors and the differences of molecular weight, the 58kD MPM proteinase was thought to be different from the already known matrix metalloproteinases and proteinases such as kallikrain, plasmin and cathepsin B 1. Some of bone MPM proteinases may participate in the degradation of acidic proteins (sialoprotein and phosphorylated proteins) which are stained blue with Stains-all in newborn rat calvaria. [ABSTRACT FROM AUTHOR]

The originally mineralized area of a fixed and demineralized specimen of newborn rat calvarium was stained blue with Stains-all, which stained phosphoprotiens, glycoproteins and Ca-binding proteins blue. The material stained blue in the histological sections was solubilized by an acidic solution and found to be the protein (Stains-all positive protein = SAPP). This SAPP was found to consist of two different types of phosphoproteins, which had the same amino acid sequence at the N-terminal region and a quite similar amino acid composition to each other. The minor one was equal to the 44kD phosphoprotein which has previously been reported by Prince et al (J. Biol. Chem., 262, 2900, 1987). The major one was a 66kD phosphoprotein, it's molecular weight being determined by SDS electrophoresis on a 15% polyacrylamide gel. The result of the amino acid analysis and peptide mapping analysis after CNBr cleavage contained no Met. Based on its solubility characteristics at sequential extractions with three acidic solutions (pH 5.5, pH 3.5 and pH 2.6), the histochemically discovered SAPP was believed to be bound to the matrix by a weak ionic bond and covered with mineral. It is suggested that the Met free phosphoprotein is a new protein in the mineralized phase of newborn rat calvaria and participates in the induction of bone mineralization. [ABSTRACT FROM AUTHOR]

In order to separate only the mineral and collagen binding (MCB) proteins from the mineral compartment of newborn rat calvaria after the mineral unassociated proteins were removed by washing in 4 M guanidine solution, the sequential extractions were carried out by homogenizing in acetic acid and several neutral solutions with a different ion concentration. Three noncollagenous proteins were solubilized as MCB proteins by sequential extractions and each had an affinity for calcium ions. One is the sialoprotein which has an apparent molecular weight (Mr=) of 80,000-115,000 and stains blue with Stains-all but does not stain with Coomassie blue (CBB), this sialoprotein is expected to bind more tightly to the matrix by ionic bonds than the other two proteins. The protein species of Mr=66,000 is a phosphorylated glycoprotein which contains a small amount of sialic acid and appears to bind to the matrix. Partial amino acid sequence analysis suggests that this protein is osteopontin reported by Oldberg et al (Proc. Natl. Acad. Sci. USA. 83, 8819-8823, 1986). The other Mr=49,000 species may have an affinity for the matrix, although part of this protein binds only to the mineral, and by N-terminal amino acid sequence analysis is found to be derived from serum and the analogue of human αHS glycoprotein. [ABSTRACT FROM AUTHOR]

Two kinds of proteinases were found in the mineralized phase matrix of 24-week-old rat calvaria by means of enzymography using gelatin as a substrate. One proteinase was a neutral thiol 58kD proteinase as shown in a previous paper [2]. The other was a neutral metalloproteinase that had a molecular mass of 56kD and was detected only when calcium (Ca) ions were added to the incubation buffer. It is believed that the 56kD proteinase is bound to the insoluble collagen of the bone matrix, as it is solubilized by 4 M guanidine HC1 solution from the insoluble collagen fraction, when prepared by removing extractable proteins of the mineralized phase matrix. The insoluble collagen fraction could also be solubilized and prepared as gelatin by heating at 65 degrees C for 5 minutes. The gelatin was then incubated at 37 degrees C without further treatment and became degraded without an activation of 4-aminophenylmercuric acetate (APMA). This nonactivated degradation was enhanced by adding Ca ions. These results suggest that the 56kD metalloproteinase bound to the insoluble collagen of bone matrix is in an active form and may participate in the rapid degradation of collagen during bone resorption. As partially purified 56kD metalloproteinase degraded cartilage type proteoglycan, but not type I, IV, and V collagens, it is possibly related to the degradation of proteoglycans before it binds to collagen fibers during bone formation. [ABSTRACT FROM AUTHOR]

The enamel protein fraction free from enamel crystals was investigated to determine the existance of nonamelogenin like enamelin. Enamel proteins were extracted by neutral and alkaline buffers from the porcine immature enamel at an early stage of development and resolved into four fractions on Sephadex G-100 gel filtration in a carbonate buffer (pH 10.8). The first eluted fraction contained the aggregate of proteins from 13,000 daltons to 142,000 daltons in molecular size and most of these proteins were found to differ from amelogenin by their different solubility against 25% isopropanol on acrylamide gel, and their amino acid composition. These nonamelogenins showed the property of closely associating with synthetic hydroxyapatite under dissociative conditions, and their electrophoretic properties and amino acid compositions were quite similar to those of the enamelins prepared from porcine immature enamel. [ABSTRACT FROM AUTHOR]

Enamelins comprise an important family of the enamel matrix proteins. Porcine tooth germs were investigated immunochemically and immunocytochemically using two antibodies: a polyclonal antibody raised against the porcine 89-kDa enamelin (89 E) and an affinity purified anti-peptide antibody against the porcine enamelin amino-terminus (EN). Immunochemical analysis of layers of immature enamel from the matrix formation stage detected immunopositive protein bands ranging from 10 kDa to 155 kDa in the outer layer enamel sample irrespective of the antibodies used. In contrast, the middle and inner enamel layer mainly contained lower molecular weight enamelins. In immunocytochemical analyses of the differentiation stage, 89 E stained enamel matrix islands around mineralized collagen fibrils of dentin, while EN stained both enamel matrix islands and stippled material. At the matrix formation stage, both antibodies intensely stained enamel prisms located in the outer layer. In the inner layer, 89 E moderately stained enamel matrix homogeneously, while EN primarily stained the prism sheath. The intense immunoreaction over the surface layer of enamel matrix at the matrix formation stage, following staining with 89 E and EN, disappeared by the end of the transition stage and the early maturation stage, respectively. The Golgi apparatus and secretory granules in the ameloblasts from the late differentiation stage to the transition stage were immunostained by both antibodies. These results suggest that expression of enamelin continues from late differentiation to the transition stage and the cleavage of N-terminal region of enamelin occurs soon after secretion. Some enamelin degradation products, which apparently have no affinity for hydroxyapatite crystals, concentrate in the prism sheaths during enamel maturation. [ABSTRACT FROM AUTHOR]

We investigated expression of ameloblastin and sheathlin, recently cloned enamel matrix proteins from the rat and pig, in forming enamel immunocytochemically and immunochemically, using region-specific antibodies. The results obtained from the rat and pig were essentially the same. Antibodies which recognize the N-terminal region stained the secretory machinery of the secretory ameloblast and the entire thickness of the enamel matrix, especially the peripheral region of the enamel rod. Immunostained protein bands were observed near 65 or 70 kDa and below 20 kDa. C-terminal-specific antibodies stained the secretory machinery of the ameloblast and the immature enamel adjacent to the secretion sites. Immunostained protein bands were found ranging from 25 to 70 kDa. Antibodies which recognize a region in the protein just prior to the C-terminal region stained the cis-side of the Golgi apparatus but not the enamel matrix. Immunostained protein bands were observed of about 55 kDa. These results suggest that post-translational and post-secretory modifications of ameloblastin and sheathlin are similar to each other, and further showed that their cleaved N-terminal polypeptides concentrate in the prism sheath. We propose that sheathlin and ameloblastin share the same role in amelogenesis and should be classified as sheath proteins. [ABSTRACT FROM AUTHOR]