Minoru Takagi

Dentine proteoglycans: composition, ultrastructure and functions.

SummaryProteoglycans (PGs) have been visualized in the predentine and dentine with cationic dyes by staining thin sections with Alcian Blue, bismuth nitrate, or using Spicers high-iron diamine (HID) method. The precise location may be obtained by adding cationic dyes such as Cuprolinic Blue, ruthenium hexammine trichloride or cationic detergent (cetylpyridinium chloride) to the fixative. These methods induced the formation of aggregates which varied in shape and number according to the method used. Rapid freezing followed by freeze-substitution revealed an amorphous ground substance, homogeneously stained with Alcian Blue, located in the predentine between the collagen fibres. These PGs may be involved in transport and diffusion in predentine. In dentine, small granules and needle-like structures were observed along the collagen fibres. This second group of PGs differs in composition, distribution and functions from the predentine PGs. The same distribution was seen when hyaluronidase-gold labelling was used. Labelling with antibodies and autoradiography also gave evidence of two distinct groups of PGs. In predentine, as an hydrated gel, PGs seems to act as mineral inhibitors, whereas immobilized on a surface, as seen at the dentine edge, they act as nucleating agents. The interaction between PGs and phospholipids seems also to play a role in the mineralization process.

Inductive Effects of Dexamethasone on the Gene Expression of Cbfa1, Osterix and Bone Matrix Proteins During Differentiation of Cultured Primary Rat Osteoblasts

Runx2/core binding factor alpha 1 (Cbfa1) and Osterix (Osx) are osteoblast-specific transcription factors essential for the development of a mature osteoblast phenotype and are thought to activate osteoblast marker genes in vivo to produce a bone-specific matrix. Dexamethasone (Dex) is known to be a potent stimulator of osteoblastic differentiation in vitro, however, the exact role is still unclear. To investigate the mechanisms of the stimulation of osteoblastic differentiation by Dex, we evaluated the effects of Dex on proliferation and mineralization as well as on mRNA expression of Cbfa1, Osx and osteoblast marker genes, osteocalcin (OC) and bone sialoprotein (BSP) mRNAs in differentiating foetal rat calvarial cells (FRCC), which were cultured for 35 days in the presence or absence of 10−7 M Dex. Treatment of FRCC with Dex resulted in the stimulation of cell proliferation and increased the number of cells, which are able to produce bone-like nodules with a mineralized matrix when compared to untreated controls. Northern blot analysis revealed that, in the absence of Dex, Cbfa1 mRNA expressed at day 8, while Osx mRNA expressed at day 15. Subsequently expression of these mRNAs increased up to day 21, followed by constant expression during the culture period. The expression of OC and BSP mRNAs appeared to be synchronous with that of Osx mRNA and was detectable at day 15 with an increase thereafter. The presence of Dex resulted in an induction in Cbfa1 and Osx mRNA expression. The former appeared at day 5 and the latter appeared at day 11. Subsequently expression of Cbfa1 and Osx mRNAs increased up to day 15 with a decrease thereafter. Expression of OC and BSP mRNAs appeared to be coincident with that of Osx mRNA and was detectable at day 11 and reached a maximum at day 15 followed by constant expression. These observations indicate that induction of Cbfa1 and Osx mRNAs by Dex may be followed by activation of osteoblast marker genes such as OC and BSP mRNAs to produce a bone-specific matrix that subsequently becomes mineralized. Thus, it is likely that Dex may promote osteoblastic differentiation and mineralization of FRCC by inducing the expression of Cbfa1 and Osx genes in vitro.

Immunohistochemical localization of glycosaminoglycans and proteoglycans in predentin and dentin of rat incisors

We examined immunocytochemically the type and distribution of glycosaminoglycans and proteoglycans (PG) in predentin and dentin demineralized with EDTA after aldehyde fixation of rat incisors using (a) four monoclonal antibodies (1-B-5,9-A-2,3-B-3, and 5-D-4) which recognize epitopes in unsulfated chondroitin (C0-S), chondroitin 4-sulfate (C4-S), chondroitin 6-sulfate (C6-S), and keratan sulfate (KS) associated with the PG, and (b) monoclonal (5-D-5) and polyclonal antibodies specific for the core protein of large and small dermatan sulfate (DS) PG. Light microscope immunoperoxidase staining after pre-treatment of tissue sections with chondroitinase ABC localized the majority of stainable PG (C4-S, KS, DSPG, C0-S, and C6-S) in predentin and, to a lesser extent (C4-S and small DSPG), in the dentin matrix. The former site demonstrated relatively homogeneous PG distribution, whereas the latter site revealed that strong staining of C4-S and small DSPG was confined mostly to dentinal tubules surrounding odontoblastic processes, with only weak staining in the rest of the dentin matrix. These results indicate that there is not only a definite difference between PG of predentin and dentin but also a selective decrease in the concentration or alteration of these macromolecules during dentinogenesis and mineralization.

Bone morphogenetic protein 2 and dexamethasone synergistically increase alkaline phosphatase levels through JAK/STAT signaling in C3H10T1/2 cells

Alkaline phosphatase (ALP) is generally believed to be a faithful marker of osteoblast differentiation, and its expression is induced by bone morphogenetic protein‐2 (BMP‐2) and dexamethasone (Dex). However, the effects of combined administration of BMP‐2 and Dex on ALP transcription have not been extensively examined. In this study, we found that BMP‐2 and Dex synergistically increase ALP levels in mouse C3H10T1/2 pluripotent stem cells. However, switching from one inducer to the other, by adding BMP‐2 or Dex to cell cultures at different times, was no more effective than continuous treatment with either inducer alone. A significant induction of ALP mRNA expression was observed only in cells continuously treated with both inducers. This result suggests that both BMP‐2 and Dex may act in the same pathway or at the same stage of differentiation. A luciferase assay using ALP promoter deletion constructs showed that a region of the promoter containing a putative signal transducer and activator of transcription 3 (STAT3) response element (SRE) responds to treatment with a combination of BMP‐2 and Dex. Furthermore, a ChIP assay indicated that STAT3 bound to the SRE. In addition, a STAT3 siRNA suppressed the synergistic effect of BMP‐2 and Dex on ALP levels. These results indicate that STAT3 may play an important role in regulating ALP expression. To our knowledge, this is the first time that STAT3 has been implicated in the regulation of ALP expression by BMP‐2 and Dex. These findings raise the possibility of developing new strategies for the enhancement of bone formation using a combination of BMPs and Dex. J. Cell. Physiol. 223: 123–133, 2010.

Ultrastructural localization of complex carbohydrates in odontoblasts, predentin, and dentin.

Glycosaminoglycans (GAGs) and glycoproteins (GPs) are essential components for dentinogenesis. We have examined rat odontoblasts, predentin, and dentin decalcified with EDTA and stained with: 1) Spicers hig-iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP) method for sulfated glycoconjugates, and 2) Thiérys periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method for vicinal glycol-containing glycoconjugates. HIS-TCH-SP stained distended portions of Golgi saccules and secretory granules. The predentin contained three times the number of HID-TCH-SP stain precipitates when compared to the mineralization front of the dentin matrix. PA-TCH-SP weakly stained membranes of Golgi saccules and cisternae of rough endoplasmic reticulum (RER), whereas stronger staining was observed in secretory granules, lysosomes, and multivesicular bodies (MVBs). Collagen fibrils in predentin demonstrated moderate PA-TCH-SP staining. In contrast, strong PA-TCH-SP staining was observed on and between collagen fibrils in the mineralization front of the dentin matrix. TCH-SP controls of unosmicated specimens lacked significant staining, however, osmicated control specimens did contain some TCH-SP stain deposits in the mineralization front. These results indicate that sulfated and vicinal glycol-containing glycoconjugates are packaged in the same type of secretory granule and released into the extracellular matrix; subsequently vicinal glycol-containing glycoconjugates concentrate in the calcification front, whereas sulfated glycoconjugates accumulate in the predentin and are either removed or masked to staining in the dentin.

Biochemical and Immunocytochemical Characterization of Mineral Binding Proteoglycans in Rat Bone

We examined biochemically and immunocytochemically the type and distribution of mineral binding proteoglycans (PGs) in rat mid-shaft subperiosteal bone using three monoclonal antibodies (MAb 1-B-5, 9-A-2, and 3-B-3) which specifically recognize unsulfated chondroitin, chondroitin 4-sulfate (C4-S) and dermatan sulfate (DS), and chondroitin 6-sulfate. Bone proteins were extracted from fresh specimens with a three-step technique: 4 M guanidine HCl (GdnCl), aqueous EDTA without GdnCl (E-extract), followed by GdnCl. Western blot analysis of SDS-polyacrylamide gel electrophoresis revealed that E-extract after chondroitinase ABC digestion reacted strongly with MAb 9-A-2 but not with MAb 1-B-5 or 3-B-3. After adehyde fixation, ethanolic trimethylammonium EDTA was used as a demineralizing agent for light and electron immunocytochemistry. This provided good retention of water-soluble PGs in the specimens. After chondroitinase ABC pre-treatment of tissue sections, MAb 9-A-2 specifically stained C4-S and/or DS in the walls of osteocyte lacunae and bone canaliculi in the mineralized matrix as well as in the unmineralized matrix such as pre-bone, vascular canals, and pericellular matrix surrounding osteocytes; the remainder of the mineralized matrix lacked staining. These results indicate that mineral binding PGs contain C4-S and/or DS and are exclusively localized in the walls of the bone lacuna and canaliculus.

Identification of Stem Cells During Prepubertal Spermatogenesis via Monitoring of Nucleostemin Promoter Activity

The nucleostemin (NS) gene encodes a nucleolar protein found at high levels in several types of stem cells and tumor cell lines. The function of NS is unclear but it may play a critical role in S‐phase entry by stem/progenitor cells. Here we characterize NS expression in murine male germ cells. Although NS protein was highly expressed in the nucleoli of all primordial germ cells, only a limited number of gonocytes showed NS expression in neonatal testes. In adult testes, NS protein was expressed at high levels in the nucleoli of spermatogonia and primary spermatocytes but at only low levels in round spermatids. To evaluate the properties of cells expressing high levels of NS, we generated transgenic reporter mice expressing green fluorescent protein (GFP) under the control of the NS promoter (NS‐GFP Tg mice). In adult NS‐GFP Tg testes, GFP and endogenous NS protein expression were correlated in spermatogonia and spermatocytes but GFP was also ectopically expressed in elongated spermatids and sperm. In testes of NS‐GFP Tg embryos, neonates, and 10‐day‐old pups, however, GFP expression closely coincided with endogenous NS expression in developing germ cells. In contrast to a previous report, our results support the existence in neonatal testes of spermatogonial stem cells with long‐term repopulating capacity. Furthermore, our data show that NS expression does not correlate with cell‐cycle status during prepuberty, and that strong NS expression is essential for the maintenance of germline stem cell proliferation capacity. We conclude that NS is a marker of undifferentiated status in the germ cell lineage during prepubertal spermatogenesis.

In situ Localization and in vitro Expression of Osteoblast/Osteocyte Factor 45 mRNA during Bone Cell Differentiation

The in situ localization of osteoblast/osteocyte factor 45 (OF45) mRNA during bone formation has been examined in the rat mandible from embryonic day 14 (E14) up to postnatal 90-day-old Wistar rats. Gene expression was also examined during cell culture not only in primary rat osteoblast-like cells but also in two clonal rat osteoblastic cell lines with different stages of differentiation, ROB-C26 (C26) and ROB-C20 (C20) using Northern blot analysis. The C26 cell is a potential osteoblast precursor cell line, whereas the C20 cell is a more differentiated osteoblastic cell line. At E15 osteoblast precursor cells differentiated into a group of osteoblasts, some of which expressed the majority of non-collagenous proteins, whereas no expression of OF45 was observed in these cells. Intercellular matrices surrounded by osteoblasts were mineralized at E16. Subsequently, the number of osteoblasts differentiated from osteoblast precursor cells was increased in association with bone formation. At E17, the first expression of OF45 mRNA was observed only in a minority of mature osteoblasts attached to the bone matrix, but not in the rest of less mature osteoblasts. At E20, concomitant with the appearance of osteocytes, OF45 mRNA expression was observed not only in more differentiated osteoblasts that were encapsulated partly by bone matrix but also in osteocytes. Subsequently, osteocytes increased progressively in number and sustained OF45 mRNA expression in up to 90-day-old rats. Northern blot analysis of the cultured cells with or without dexamethasone treatment revealed that the gene expression of OF45 correlated well with the increased cell differentiation. These results indicate that OF45 mRNA is transiently expressed by mature osteoblasts and subsequently expressed by osteocytes throughout ossification in the skeleton and this protein represents an important marker of the osteocyte phenotype and most likely participates in regulating osteocyte function.

Ultrastructural localization of acidic glycoconjugates with the low iron diamine method.

The present study has applied the low iron diamine (LID) method at the ultrastructural level to demonstrate acid glycoconjugates. We have examined rat epiphyseal cartilage, human bone marrow, rat tracheal glands, and mouse sublingual glands stained with LID prior to embedment. The LID staining appeared to require postosmication for adequate visualization at the electron microscope level. Thiocarbohydrazide-silver proteinate (TCH-SP) staining of thin sections variably enhanced LID reactive sites. LID-TCH-SP stained carboxyl and sulfate groups of glycosaminoglycans in the extracellular cartilage matrix, secretory granules, and expanded Golgi saccules of chondrocytes. In human bone marrow, LID-TCH-SP variably stained the cytoplasmic granules, known to contain sulfated glycosaminoglycans, and the external surface of the plasma membrane of leukocytes. Moderately strong LID staining was observed in secretory granules in mucous tubules of rat tracheal glands, known to contain sulfated glycoproteins, and in acinar cells of mouse sublingual glands, known to contain a sialoglycoprotein. The lack of sulfated glycoconjugates in acinar cells of the mouse sublingual gland was confirmed by their failure to stain with the high iron diamine method. Thus these studies indicate that the LID and LID-TCH-SP methods are useful for the ultrastructural localization of carboxylated and sulfated glycoconjugates in extracellular and intracellular sites.

Differential Expression of Dentin Matrix Protein 1, Type I Collagen And Osteocalcin Genes in Rat Developing Mandibular Bone

The expression of dentin matrix protein 1 (Dmp1) mRNA has been compared with that of type I collagen and osteocalcin mRNAs during bone formation in the rat mandible, using in situ hybridization. At embryonic day 15 (E15), type I collagen and osteocalcin mRNAs were expressed by the majority of newly-differentiated osteoblasts attached to unmineralized bone matrices, whereas Dmp1 mRNA expression was confined to only a few osteoblasts. Expression of these genes increased as the number of osteoblasts increased in specimens from E16 to E18. At E20, expression of Dmp1, type I collagen and osteocalcin was also observed in osteocytes. Dmp1 expression continued in osteocytes as they matured up to the 90-day-old specimens, whereas type I collagen and osteocalcin expression in osteocytes almost disappeared at 30 days of postnatal life. In contrast, osteoblasts continued to express type I collagen and osteocalcin in 90-day-old rats, but transiently expressed Dmp1 mRNA, which was seen in the minority of osteoblasts at 14 days of postnatal life. These data show that the developmental expression patterns of Dmp1 in osteogenic differentiation differ from those of type I collagen and osteocalcin, and Dmp1 appears to be expressed by osteocytes throughout ossification in the skeleton. These observations indicate that Dmp1 may serve unique biological functions in osteocyte and bone metabolism.