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In order to compare its characteristics with those of jaw tooth collar enamel, normally developing and experimentally regenerating ganoine from ganoid scales of Lepisosteus oculatus (spotted gar), an actinopterygian fish species, was examined by Western blotting and immunohistochemistry. Amelogenin, a major enamel matrix protein (EMP), is widely found from sarcopterygian fish to mammals. Therefore, we used antimammalian amelogenin antibodies and antisera: an antibody against bovine amelogenin; antiserum against porcine amelogenin; and region-specific antibodies or antiserum against the C-terminus, middle region, or N-terminus of porcine amelogenin in this study. Positive immunoreactivity with the antibody against bovine amelogenin, antiserum against porcine amelogenin, and the middle and C-terminal region-specific antibodies was detected in both normally developing and regenerating ganoine matrix, as well as in granules found within inner ganoine epithelial cells. These immunohistochemical analyses indicated that the Lepisosteus ganoine matrix contains EMP-like proteins with epitopes similar to mammalian amelogenins. In Western blotting analyses of regenerating ganoid scales with the antibovine amelogenin antibody, two protein bands with molecular weights of approximately 78 and 65 kDa were detected, which were similar to those found in Lepisosteus tooth enamel. Our study suggests that in Lepisosteus, EMP-like proteins in the ganoine matrix corresponded to those in tooth enamel. However, it was revealed that the 78 and 65 kDa EMP-like proteins were different from 27 kDa bovine amelogenin.

Enameloid is a well-mineralized tissue covering the tooth surface in fish and it corresponds to the outer-most layer of dentin. It was reported that both dental epithelial cells and odontoblasts are involved in the formation of enameloid. Nevertheless, the localization and timing of secretion of ectodermal enamel matrix proteins in enameloid are unclear. In the present study, the enameloid matrix during the stages of enameloid formation in spotted gar, Lepisosteus oculatus, an actinopterygian, was examined mainly by transmission electron microscopy-based immunohistochemistry using an anti-mammalian amelogenin antibody and antiserum. Positive immunoreactivity with the antibody and antiserum was found in enameloid from the surface to the dentin-enameloid junction just before the formation of crystallites. This immunoreactivity disappeared rapidly before the full appearance of crystallites in the enameloid during the stage of mineralization. Immunolabelling was usually found along the collagen fibrils but was not seen on the electron-dense fibrous structures, which were probably derived from matrix vesicles in the previous stage. In inner dental epithelial cells, the granules in the distal cytoplasm often showed positive immunoreactivity, suggesting that the enamel matrix protein-like proteins originated from inner dental epithelial cells. Enamel matrix protein-like proteins in the enameloid matrix might be common to the enamel matrix protein-like proteins previously reported in the collar enamel of teeth and ganoine of ganoid scales, because they exhibited marked immunoreactivity with the same anti-mammalian amelogenin antibodies. It is likely that enamel matrix protein-like proteins are involved in the formation of crystallites along collagen fibrils in enameloid.

Although most fish have no enamel layer in their teeth, those belonging to Lepisosteus (gars), an extant actinopterygian fish genus, do and so can be used to study amelogenesis. In order to examine the collar enamel matrix in gar teeth, we subjected gar teeth to light and electron microscopic immunohistochemical examinations using an antibody against bovine amelogenin (27 kDa) and antiserum against porcine amelogenin (25 kDa), as well as region-specific antibodies and antiserum against the C-terminus and middle region, and N-terminus of porcine amelogenin, respectively. The enamel matrix exhibited intense immunoreactivity to the anti-bovine amelogenin antibody and the anti-porcine amelogenin antiserum in addition to the C-terminal and middle region-specific antibodies, but not to the N-terminal-specific antiserum. These results suggest that the collar enamel matrix of gar teeth contains amelogenin-like proteins and that these proteins possess domains that closely resemble the C-terminal and middle regions of porcine amelogenin. Western blot analyses of the tooth germs of Lepisosteus were also performed. As a result, protein bands with molecular weights of 78 kDa and 65 kDa were clearly stained by the anti-bovine amelogenin antibody as well as the antiserum against porcine amelogenin and the middle-region-specific antibody. It is likely that the amelogenin-like proteins present in Lepisosteus do not correspond to the amelogenins found in mammals, although they do possess domains that are shared with mammalian amelogenins.

Previously, we have demonstrated that the extracellular matrix from dentin affects osteoclastic activity in co-culture between osteoclast and osteoblast-rich fraction from mouse marrow cells. In the present study, we aimed to investigate the mechanisms of dentin matrix extract-induced osteoclastogenesis in mouse bone marrow macrophages (BMMs). Dentin proteins were extracted from bovine incisor root dentin using 0.6 M HCl. BMMs were cultured in α-MEM containing macrophage colony-stimulating factor/receptor activator of nuclear factor kappa-B ligand in the presence or absence of dentin matrix extract. Tartrate-resistant acid phosphatase (TRAP)-positive cell number, total TRAP activity, and the mRNA levels of osteoclast-related genes, assayed by real-time RT-PCR, were determined as markers of osteoclastogenesis. A neutralizing antibody against transforming growth factor-β1 (TGF-β1), SB431542, a TGF-β receptor inhibitor, and ELISA were used to determine the role of TGF-β1. We observed increases in TRAP-positive cell number, TRAP activity, and the mRNA levels of osteoclast-related genes of BMMs cultured with dentin extract. The use of a neutralizing antibody against TGF-β1 or SB431542 inhibited the inductive effect of dentin extract, suggesting TGF-β1 involvement. The addition of exogenous TGF-β1, but not bone morphogenic protein-2, also increased osteoclastogenesis, corresponding to the ELISA determination of TGF-β1 in the dentin extract. In conclusion, our results indicate that proteins from dentin matrix have an inductive effect in osteoclastogenesis, which is mediated, in part, by TGF-β1.

Bone degenerative diseases, including osteoporosis, impair the fine balance between osteoclast bone resorption and osteoblast bone formation. Single-agent therapy for anabolic and anticatabolic effects is attractive as a drug target to ameliorate such conditions. Inhibition of nuclear factor (NF)-κB reduces the osteoclast bone resorption. The role of NF-κB inhibitors on osteoblasts and bone formation, however, is minimal and not well investigated. Using an established NF-κB inhibitor named S1627, we demonstrated that inhibition of NF-κB increases osteoblast differentiation and bone formation in vitro by up-regulating the mRNAs of osteoblast-specific genes like type I collagen, alkaline phosphatase, and osteopontin. In addition, S1627 was able to increase bone formation and repair bone defect in a murine calvarial defect model. To determine the effect of NF-κB on a model of osteoporosis, we injected two doses of inhibitor (25 and 50 mg/kg·d) twice a day in sham-operated or ovariectomized 12-wk-old mice and killed them after 4 wk. The anabolic effect of S1627 on trabecular bone was determined by micro focal computed tomography and histomorphometry. Bone mineral density of inhibitor-treated ovariectomized animals was significantly increased compared with sham-operated mice. Osteoblast-related indices like osteoblast surface, mineral apposition rate, and bone formation rate were increased in S1627-treated animals in a dose-dependent manner. NF-κB inhibition by S1627 increased the trabecular bone volume in ovariectomized mice. Furthermore, S1627 could inhibit the osteoclast number, and osteoclast surface to bone surface. In vitro osteoclastogenesis and bone resorbing activity were dose-dependently reduced by NF-κB inhibitor S1627. Taken collectively, our results suggest that NF-κB inhibitors are effective in treating bone-related diseases due to their dual anabolic and antiresorptive activities.

In the rat tibial bone marrow cavity, following colchicine injection, there is a phase of osteogenesis in which bone trabeculae replace the necrotic bone marrow tissues and fill the marrow cavity. The newly formed bone is subsequently resorbed by osteoclasts and normal bone marrow is restored. In this study, we correlated these morphologic events with the pattern of gene expression of bone sialoprotein (BSP), an extracellular matrix protein in mineralized tissues, to elucidate the possible functions of BSP in bone formation and resorption in vivo. The expressions of osteopontin (OPN) and type I collagen were also examined. Northern hybridization of the tibia demonstrated that OPN mRNA was gradually increased and expressed at a maximal level 10 days after colchicine injection (during the bone resorption process), while BSP mRNA expression already reached a maximal level at day 6 (during the initial process of bone formation). Its expression was, thus, quite temporary at the beginning of bone formation and different from that of type I collagen, which was continually elevated from days 6 to 10. In situ hybridization of the newly formed bone induced in the tibia revealed that BSP mRNA was evenly expressed in most osteoblasts and osteocytes, moreover in interconnecting colonies of spindle-shaped cells, possibly preosteoblasts, at day 6. At day 10, however, its expression became restricted to some cells on the bone surfaces, some osteoblasts, and most osteoclasts. These observations suggest that BSP may play an important role mainly in the initiation of bone formation and is also associated with the functions of osteoclast in vivo.

Rat osteoblasts were cultured for 4 or 5 days aboard the Space Shuttle and solubilized during spaceflight. Post-flight analyses by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) determined the relative mRNA levels of matrix proteins, adhesion molecules, and cytoskeletal proteins including osteopontin (OP), osteonectin (ON), CD44, alpha-tubulin, actin, vimentin, fibronectin (FN), and beta1-integrin. The mRNA levels of OP and alpha-tubulin in the flight cultures were decreased by 30% and 50% on day 4 and day 5 of flight, as compared to the ground controls. In contrast, the CD44 mRNA levels in the flight cultures increased by 280% and 570% of the ground controls on day 4 and day 5. The mRNA levels of ON and FN in the flight cultures were slightly increased as compared to ground controls. The mRNA levels of actin, vimentin, or beta1-integrin did not change in spaceflight conditions. The matrix proteins, adhesion molecules, and cytoskeletal proteins may form dynamic network complexity with signaling molecules as an adaptive response to perturbation of mechanical stress under microgravity.

Membrane type 1-matrix metalloproteinase (MT1-MMP) is a membrane-bound matrix metalloproteinase capable of mediating pericellular proteolysis of extracellular matrix components. In osteoclasts, the localization of MT1-MMP has been reported at the tips of specialized membrane protrusions (podosomes and lamellipodia) so that osteoclasts might use MT1-MMP to perform focal proteolysis and move through the extracellular matrix to the bone surface. The objectives of this study were to investigate an association of MT1-MMP in physiological root resorption of the deciduous tooth by reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blot analysis, and to identify MT1-MMP-producing cell during deciduous tooth resorption by in situ hybridization and immunohistochemistry. RT-PCR and Northern blot analysis revealed the exclusively high expression of MT1-MMP mRNA in bovine root-resorbing tissue, which lies between the root of the deciduous tooth and its permanent successor. Expression of MT1-MMP mRNA was seen in odontoclasts aligning in the surface layer of the root-resorbing tissue at sites of root resorption. Furthermore, immmunohistochemistry also confirmed the localization of MT1-MMP protein to the odontoclasts. The present identification of MT1-MMP in odontoclasts during deciduous tooth resorption might be relevant to the migration activity that these cells have to gain access to the root surface.

The objective of this study is to enhance in vivo ectopic bone formation by combination of plasmid DNA impregnation into three-dimensional (3-D) cell scaffolds and a developed in vitro culture method. Gelatin was cationized by introducing spermine (Sm) to the carboxyl groups for complexation with the plasmid DNA. As the MSC scaffold, collagen sponge reinforced by incorporation of poly(glycolic acid) (PGA) fibers was used. A complex of the cationized gelatin and plasmid DNA of BMP-2 was impregnated into the scaffold. MCS were seeded into each scaffold and cultured by a static and perfusion methods. When MSC were cultured in the PGA-reinforced collagen sponge, the level of BMP-2 expression was significantly enhanced by the perfusion culture compared with static method. When the osteoinduction activity of the PGA-reinforced collagen sponges seeded with PBS, MSC, naked plasmid DNA-BMP-2, cationized gelatin-plasmid DNA-BMP-2 complex, and transfected MSC by static and perfusion method, were studied following the implantation into the back subcutis of rats in terms of histological and biochemical examinations, homogeneous bone formation was histologically observed throughout the sponges seeded with cationized gelatin-plasmid DNA of BMP-2 complex and transfected MSC by perfusion method, although the extent of bone formation was higher for the later one. The level of alkaline phosphatase activity and osteocalcin content at the implanted sites of sponges seeded with transfected MSC by perfusion method were significantly high compared with those seeded with other agents. We conclude that combination of plasmid DNA-impregnated PGA-reinforced collagen sponge and the perfusion method was promising to promote the in vitro gene expression for MSC and in vivo ectopic bone formation.

Differentiation of mesenchymal stromal cells into osteoblasts is regulated by many factors including growth factors, cytokines, and hormones. Mechanical stress has been considered to be an important factor in bone modeling and remodeling. However, biological responses of stromal cells to mechanical stimuli are still unknown. To show the correlation between magnitude of mechanical strain and differentiation of stromal cells into osteoblasts, we investigated the proliferation and the expression of osteoblast-related genes in stromal cell line ST2 that is in the process of osteoblastic differentiation by treatment with ascorbic acid and beta-glycerophosphate, under 0.8%-15% elongation using the Flexercell Strain system. The expression of osteoblast-related genes was analyzed by real-time quantitative polymerase chain reaction (PCR). Cell proliferation significantly increased at 5%, 10%, and 15% elongation compared to that of unloaded controls. Alkaline phosphatase (ALPase) activity significantly increased at 0.8% and 5% elongation but decreased at 10% and 15% elongation. At 1 h and 6 h, mRNA level of Cbfa1/Runx2 increased at lower magnitudes of strain (0.8% and 5% elongation) but decreased at higher magnitude of strain (15% elongation). At 24 and 48 h, Cbfa1/Runx2 and osteocalcin mRNAs decreased at 5%, 10%, and 15% elongation, whereas cell proliferation and expression of type I collagen mRNA increased at the same elongation. These results indicate that mechanical strain stimulates osteoblastic differentiation of stromal cells at low magnitudes of strain.

Physiological actions of osteoblasts are disordered by gravity unloading. We investigated the possibility that the appropriate level of hypergravity could improve osteoblast functions that are susceptible to mechanical unloading. We evaluated hypergravity effects on the 1alpha,25-dihydroxyvitamin D(3) (VD)-inducible osteocalcin expression of primary rat osteoblasts. Cell culture plates were centrifuged for 24 h at 3, 6, 12, 24, and 48 g in a 37 degrees C incubator. The mRNA levels were analyzed by quantitative RT-PCR. The mRNA levels for osteocalcin and vitamin D receptor (VD-R) at 12 g were enhanced to 187% and 228% of the 1 g control, respectively. However, the excess hypergravity conversely decreased osteocalcin expression. Osteocalcin gene expression was enhanced by VD/VD-R through the vitamin D-responsive element in the promoter. The increased osteocalcin expression might reflect the augmented VD-R expression. Alternatively, Runx2, a master gene of osteoblast differentiation, might be responsible for the osteocalcin induction, since the Runx2 mRNA levels were also increased to 247% of control at 12 g. Another VD-inducible osteoblast phenotype, alkaline phosphatase, was also upregulated at 12 g and 24 g. The appropriate level of hypergravity enhanced the VD-inducible expression of osteocalcin, a typical phenotype of osteoblast differentiation. These data suggest molecular features to prevent disuse bone atrophy of long-term bed-rest patients.

Recently, the involvement of immune responses in metabolic bone disease and/or local bone destruction has received much attention. Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), a member of the immunoglobulin (Ig) superfamily, negatively regulates T cell activation. The deficiency of CTLA-4 induces profound osteopenia with an increase in osteoclastogenesis, suggesting the important role of activated T cells in osteoclastogenesis. Programmed death-1 (PD-1) is the newly identified immunoregulatory receptor, which also belongs to the Ig superfamily. Both CTLA-4 and PD-1 are induced on activated T cells, however, there are no reports linking PD-1 with osteoclasts. In the present study, we have examined the bone phenotype in PD-1-deficient mice PD-1-/- and the role of PD-1 in osteoclastogenesis and osteoclast function. Both trabecular and cortical bone mineral densities of tibia were significantly increased, as observed in peripheral quantitative computed tomography (pQCT), at 12 weeks of age in PD-1-/- mice. Histomorphometric analysis of the PD-1-/- mice and the age-matched controls at 12 weeks of age showed a 2-fold increase in bone volume (BV/TV) with a 55% decrease in osteoclast number (N.Oc/BS). Bone formation indices were similar in both groups. The number of soluble receptor activator of nuclear factor kappaB ligand (sRANKL)-induced osteoclast-like cells (OCLs) derived from the PD-1-deficient splenocytes was significantly decreased (by 25%). On the other hand, PD-1 deficiency did not affect the bone-resorbing activity of mature osteoclasts. Our results suggest that PD-1 deficiency reduces osteoclastogenesis resulting in an osteopetrotic phenotype. Identical members of the Ig superfamily, CTLA-4 and PD-1, which negatively regulate immune responses, may differentially affect osteoclastogenesis and bone remodeling.

Rat osteoblasts were cultured aboard a space shuttle for 4 or 5 days. Cells were exposed to 1alpha, 25 dihydroxyvitamin D(3) during the last 20 h and then solubilized by guanidine solution. The mRNA levels for molecular chaperones were analyzed by semi-quantitative RT-PCR. ELISA was used to quantify TGF-beta1 in the conditioned medium. The HSP70 mRNA levels in the flight cultures were almost completely suppressed, as compared to the ground (1 x g) controls. The inducible HSP70 is known as the major heat shock protein that prevents stress-induced apoptosis. The mean mRNA levels for the constitutive HSC73 in the flight cultures were reduced to 69%, approximately 60% of the ground controls. HSC73 is reported to prevent the pathological state that is induced by disruption of microtubule network. The mean HSP47 mRNA levels in the flight cultures were decreased to 50% and 19% of the ground controls on the 4th and 5th days. Concomitantly, the concentration of TGF-beta1 in the conditioned medium of the flight cultures was reduced to 37% and 19% of the ground controls on the 4th and 5th days. HSP47 is the collagen-specific molecular chaperone that controls collagen processing and quality and is regulated by TGF-beta1. Microgravity differentially modulated the expression of molecular chaperones in osteoblasts, which might be involved in induction and/or prevention of osteopenia in space.

The condylar cartilage, an important growth site in the mandible, shows characteristic modes of growth and differentiation, unlike the limb bud cartilage. To elucidate the mechanism of chondrogenesis at the condylar cartilage, we analyzed the effects of basic fibroblast growth factor (bFGF) on the growth and development of mouse mandibular condyle using serum-free organ culture and on the expression of genes related to the chondrogenesis. Further, we investigated the localization of bFGF in cultured condyle by immunohistochemistry. The present immunohistochemical observations showed that bFGF is localized in the extracellular matrix of the mesenchymal condylar anlage, the perichondrium and the proliferative cell zone, and that immunostaining was diminished in the metachromatically stained area. In the condyle culture with added recombinant human bFGF (rhbFGF) for 5 days, the area occupied by hypertrophic chondrocytes in the mandibular condylar cartilage was reduced. A reverse transcription-polymerase chain reaction (RT-PCR) assay also showed that the mRNA levels of aggrecan and type X collagen were reduced compared with nontreated tissues. Treatment with rhbFGF for 2 days decreased cell proliferation in the perichondrium, and bFGF downregulated the Indian hedgehog (Ihh), parathyroid hormone-related protein (PTHrP), bone morphogenetic protein 4 (BMP4), and core-binding factor alpha1 (Cbfa1) expression in the RT-PCR assay. These findings suggest that bFGF has the ability for inhibitory regulation of condylar growth, via the inhibition of proliferation and differentiation of chondrocytes, and that this inhibitory regulation is related to the downregulation of growth factors and transcription factors.

Matrix metalloproteinase-9 (MMP-9, or gelatinase B) is an extracellular proteinase that is highly expressed in osteoclasts and has been postulated to play an important role in their resorptive activity. Although MMP-9 has been reported to play a role in bone resorption, the association of this enzyme during deciduous tooth resorption has not yet been clarified. The purpose of the present study was to increase our understanding of the role of MMP-9 during deciduous tooth resorption. Reverse transcription-polymerase chain reaction (RT-PCR) and northern blot analysis of total RNAs extracted from bovine root-resorbing tissues, which lie between the root of a deciduous tooth and its permanent successor, revealed the expression of mRNA for MMP-9 in the tissue. These results indicate that MMP-9 may be involved in the process of deciduous tooth resorption. In addition, in situ hybridization and immunohistochemistry were also performed to identify the cells that produced MMP-9 in bovine root-resorbing tissue. MMP-9 mRNA was highly expressed in odontoclasts that were aligned along the surface of the tissue. Immunohistochemistry confirmed the predominant localization of MMP-9 in odontoclasts. The present data demonstrate that odontoclasts in deciduous root resorption express MMP-9, which may participate in proteolysis during root resorption of deciduous tooth.

Background: Odontoma-like structures are formed in the jaw bone of osteopetrotic (op/op) mice, which have a congenital deficiency in osteoclastic differentiation due to the absence of functional macrophage colony-stimulating factor (M-CSF). Methods: To clarify the histopathogenesis of tooth malformation and odontoma-like structures, a 2-year postnatal process of development of the op/op mandibular incisor was examined radiologically and histologically. At the same time, extracellular matrix (ECM) remodeling around tooth germs was analyzed immunohistochemically. Results: Abnormal forms of op/op tooth germ were noticeable even at 3 days after birth on a radiogram. Histologically, op/op mice were clearly distinguished by the disappearance of dental follicular space at 3 days. With aging, bone trabeculae, which were not remodeled, penetrated into op/op tooth germs and divided them into several daughter germs, which were recognized as odontomas. In mandibular incisor bodies, the immature ECM components, such as heparan sulfate proteoglycan and tenascin, were preserved diffusely in the dental papilla/pulp, which indicates that maturation of the stroma does not take place in op/op mandibular incisors. Conclusion: The observation suggests that the disturbed morphogenesis of op/op tooth germs is functionally explained by the disordered immunolocalization of ECM molecules, and that the dental follicular space is essential for normal tooth development because it prevents bone penetration into the tooth germs.

Purpose: To investigate the role of mechanical stretch in the regulation of matrix metalloproteinase-2 (MMP-2) in scleral fibroblasts of chick embryos. Methods: Scleral fibroblasts derived from chick embryos were seeded onto flexible bottom culture plates, and subjected to a pulsatile stretch when the cells became subconfluent. After stretching, the cells and the conditioned medium were harvested. One portion of the conditioned medium was activated by reduction, alkylation, and 4-aminophenylmercuric acetate (APMA) treatment. The conditioned medium, with and without the treatment, was subjected to gelatin zymography and quantitative assays. Total cytoplasmic RNA was extracted from the cells, and the expression of MMP-2 and the tissue inhibitor of metalloproteinase-2 (TIMP-2) mRNA was examined by Northern blot analysis. Results: The predominant gelatinolytic enzyme secreted by scleral fibroblasts was MMP-2. The mechanical stretch increased the gelatinolytic activities significantly in the conditioned medium with reduction, alkylation, and APMA treatment. Mechanical stretch also enhanced the expression of MMP-2 and TIMP-2 mRNA in scleral fibroblasts significantly. Conclusions: These results suggest that mechanical stretch may be involved in the regulation of the extracellular matrix in the fibrous sclera of chicks in vivo.

Cathepsin K is a cysteine proteinase, which is abundantly and selectively expressed in osteoclasts. It is believed to play an important role in the proteolysis of bone resorption by osteoclasts. The objectives of this study were to investigate the association of cathepsin K in the physiological root resorption of deciduous teeth and to identify the cathepsin K-producing cells in deciduous root resorption. RT-PCR and Northern blot analysis of the total RNAs extracted from bovine active and resting root-resorbing tissues, which lie between the root of deciduous tooth and its permanent successor, were performed. The active root-resorbing tissue, which has a high population of odontoclasts on its surface that is attached to resorbing root surface, showed an extremely high expression of cathepsin K in comparison with the resting root-resorbing tissue. By in situ hybridization, cathepsin K mRNA was highly and selectively expressed in multinucleated odontoclasts that aligned along the surface of the tissue and apposed to the resorbing root surface of the deciduous tooth. Western blot analysis of the active root-resorbing tissue was used to characterize the anti-cathepsin K antibody. A band of 27 kDa, corresponding with the predicted size for mature cathepsin K, was demonstrated. Immunohistochemistry confirmed the specific localization of cathepsin K protein to the odontoclasts. These results demonstrate that odontoclasts in the deciduous root resorption express cathepsin K mRNA and protein that may participate in the proteolysis of root resorption of the deciduous tooth.

In order to examine the functional differentiation of tumor cells of adenomatoid odontogenic tumor (AOT) as ameloblasts and to determine the participation of the extracellular matrix (ECM) in the formation of its characteristic histologic architecture, tissue samples from five cases of adenomatoid odontogenic tumor were examined by immunohistochemical staining for enamel proteins and ECM molecules. Amelogenin, enamelin, laminin, heparan sulfate proteoglycan, fibronectin, collagen type IV and type V were immunolocalized within the luminal space and along the inner rim of duct-like structures. Eosinophilic hyaline droplets within the whorled or rosette masses of tumor cells showed basically the same staining pattern as the luminal contents. High columnar tumor cells that formed duct-like structures were immunopositive for amelogenin, while the staining intensity decreased with flattening of the cells, which was a result of luminal growth. The findings suggest that the constituent cells of duct-like structures are differentiated once to ameloblasts but fail to mature further due instead to increased production of ECM molecules and due to their retention in the lumina. It is possible to regard these special structures in AOT as stromal pseudocysts.

The movement of teeth during orthodontic treatment occasionally induces undesirable root resorption. Although high collagenolytic activity has been detected in resorbing tissue of deciduous teeth, the cellular origin of collagenolytic enzymes in root-resorbing tissue caused by tooth movement has not been identified. Here, rats were subject to 7 days of experimental tooth movement to induce root resorption. In situ hybridization with digoxigenin-labelled RNA probes was performed on sections of the maxillary bone to detect the mRNAs that encode matrix metalloproteinase-1 (MMP-1) and cathepsin K in root-resorbing tissue. MMP-1 mRNA was detected in fibroblastic cells, cementoblasts and osteoblasts, but not in odontoclasts nor osteoclasts. Moreover, MMP-1 mRNA was highly expressed in some cementocytes located near odontoclasts and in many osteocytes. In contrast, cathepsin K mRNA was expressed only in odontoclasts and osteoclasts. These results suggest that MMP-1 and cathepsin K are important in root resorption during tooth movement in a mode similar to bone resorption.

Previous studies have demonstrated the morphological similarity of the enamel-like layer found in the teeth of the coelacanth, lungfish and gar-pike to the enamel of tetrapods. In order to clarify the phylogenetic continuity between both structures, tooth germs of the gar-pike were immunocytochemically studied using an anti-bovine amelogenin polyclonal antibody. Intense immunoreaction was shown over the enamel-like matrix layer. Certain cell organelles associated with the secretory pathway of the ameloblasts were recognized as immunoreactive. These results indicate that the enamel-like layer of the gar-pike is a tissue homologous with the mammalian enamel because both possess a common, amelogenin-like substance.

A number of studies have indicated that microgravity induces osteopenia and modulates functions of mammalian cells. However, the molecular mechanisms underlying these effects of microgravity are still unknown. Rat osteoblasts were cultured for 4 and 5 days during Shuttle-Spacelab flight, and fixed by guanidine isothiocyanate solution on board after treatment with 1α, 25 (OH)2 vitamin D3. The mRNA levels for platelet-derived growth factor (PDGF)-β receptor, epidermal growth factor (EGF) receptor, the growth factor receptor adaptor protein Shc, and c-fos were determined using the method of quantitative reverse transcription-polymerase chain reaction. The mRNA levels for EGF receptor were not altered by microgravity. However, the mRNA levels for PDGF-β receptor, Shc, and c-fos were decreased to 62, 55 and 25% on the 4th day of flight, and 47, 40, and 43% on the 5th day, respectively, as compared to the corresponding ground controls. Expression of the growth factor receptor and the receptor adaptor protein was modulated in rat osteoblasts during spaceflight. Data suggest that signal transduction via growth factor receptors in rat osteoblasts is impaired by microgravity. Dysfunction of osteoblasts might be involved in spaceflight-induced osteopenia.

In the snake, Elaphe quadrivirgata, the occurrence of amelogenin was immunohistochemically demonstrated in the enamel of developing tooth germs. Teeth of the snake are covered with a thin true enamel layer about 1-2 microns in thickness. Light and electron microscopic immunohistochemistry indicated an intense amelogenin immunoreactivity occurring in the enamel layer during the secretory stage of tooth development. Cloning and cDNA sequence of snake amelogenin was performed by RT-PCR. The amino acid sequence of the snake amelogenin cDNA--in its portion corresponding to the area from exon 5 to exon 7 of human X189 amelogenin gene--showed 45% homology with humans. Regions of both the N-terminus and C-terminus were well conserved. Furthermore, the positions of prolin in the amino acid alignment of the snake amelogenin corresponded well with those of human amelogenin. It is suggested that prolin is an essential constituent of amelogenin and therefore its positions in the molecule have been conserved after the evolutionary divergence of reptiles and mammals. This study using reptiles is the first detection of specific amelogenin immunoreactivity by high resolutional immunoelectron microscopy and the first cloning of amelogenin cDNA in a non-mammalian animal.

Both the expression of amelogenin mRNA and secretion of amelogenin were investigated in rat molars by in situ hybridization and immunohistochemistry. Probes were designed by multiple-labeling of oligonucleotide probes for in situ hybridization. Amelogenin mRNA first appeared in differentiating ameloblasts of the distal region and some inner enamel epithelial cells of enamel-free area (EFA cells) of the second cusp at postnatal day 0. At the same time, amelogenin protein was detected in the extracellular matrix between dentin and differentiating ameloblasts and in some EFA cells of the second cusp. At postnatal day 1–3, amelogenin was expressed in the secretory ameloblasts, and in the matrix beneath these cells. Both amelogenin mRNA and amelogenin were detected in the EFA cells and their extracellular matrix. After postnatal day 5, amelogenin mRNA and amelogenin were detected in the secretory ameoloblasts and extracellular matrix in the enamel-forming region, respectively. At this time, amelogenin mRNA was not detected in the EFA cells, but a small amount of amelogenin was found in the matrix beneath the EFA cells. These findings suggest that EFA cells differentiate into amelogenin-secreting cells, i.e. ameloblasts, but that the secretion lasts for only a short period at the early stage of tooth development.

Tooth abnormalities occur in microphthalmic (mi/mi) mice. The elongated odontogenic epithelium is interrupted by unresorbed bone at the basal end of the mi/mi incisor, with the epithelium gathered into cell clusters. These clusters develop to odontoma-like masses. To identify the origin of the cell types of these odontoma-like masses, the localization of osteonectin (Osn), osteocalcin (Osc), osteopontin (Osp), matrix Gla protein (MGP) and amelogenin (Am) mRNA in the process of tooth development in mi/mi and +/+ mice was investigated by means of in situ hybridization. Decalcified mandibles of neonatal, 5-, 10-, 14-day-old mice were examined. Osn and Osc mRNA, which localized in osteoblasts and odontoblasts, were also detected in the cells of odontoma-like masses in mi/mi mice. The cells expressing these mRNA were short, columnar and odontoblast-like. Am mRNA was detected in ameloblasts. In mi/mi mice, Am mRNA was also detected in ameloblastic cell clusters, which were formed by the tall columnar cells in the odontoma-like masses. No apparent Osp mRNA expression was detected in the masses. These results indicated that even in odontogenic abnormal cells resulting from physical obstruction in mi/mi mice, the genes that are involved in normal tooth development were still expressed.

The purpose of this study is to clarify the role of retinoic acid (RA) in the mechanism of form-deprivation myopia (FDM) in the chick. FDM was induced in two-day old chicks by placement of a translucent plastic goggle over one eye, with the contralateral eye used as a control. After 12 days, the chicks were euthanized. RNA was extracted from scleras in the posterior segments, transcribed into cDNA, and amplified by PCR with primers specific for retinoic acid receptor (RAR) beta. G3PDH was used as a reference gene for normalization. The effects of RA, with or without TGF-beta, on the proliferation of scleral chondrocytes and scleral fibroblasts from 17-day chick embryos were studied by use of a colorimetric assay, and the alkaline phosphatase activities of those cells also studied. Furthermore, RAR beta expression in response to RA in cultured scleral cells was studied. As a result, RT-PCR products of the expected sizes were obtained from scleras from the myopic and control eyes. Expression of RAR beta in the myopic scleras was significantly higher than that in the controls. The proliferation of scleral chondrocytes and scleral fibroblasts was inhibited by treatment with RA in a dose-dependent manner (in 10% FBS). In the presence of TGF-beta (in 0.5% FBS), RA treatment stimulated the proliferation of scleral chondrocytes but inhibited the proliferation of scleral fibroblasts. RA induced alkaline phosphatase activities in both the scleral chondrocytes and scleral fibroblasts. RAR beta expression was induced by RA in cultured scleral cells. These results demonstrate that RA appears to play a role in the mechanism of FDM in the chick. However, it is also possible that the changes in the expression of RAR beta were secondary events related to other mechanisms responsible for ocular enlargement.

It has been suggested that microgravity alters bone metabolism. Evidence for this phenomenon includes the negative calcium balance and decreased bone density in astronauts, as well as, inhibition of bone formation in rats flown for 2 to 3 weeks. However, the specific mechanisms that modulate these changes in microgravity are unknown. The purpose of this study was to clarify the mechanism of microgravity-induced bone demineralization using normal rat osteoblasts obtained from femur marrow cultures. The osteoblasts were cultured for 5 days during a Shuttle-Spacelab flight (STS-65). After collection of the culture medium, the cellular DNA and RNA were fixed on board. Enzyme-immunoassay of the culture medium for prostaglandin E2 (PGE2) indicated that microgravity induced a 4.5-to 136-fold increase in flight samples as compared to the ground control cultures. This increase of PGE2 production was consistent with a 3.3-to 9.5-fold elevation of inducible prostaglandin G/H synthase-2 (PGHS-2) mRNA, quantitated by reverse transcription-polymerase chain reaction (RT-PCR). The mRNA induction for the constitutive isozyme PGHS-1 was less than that for PGHS-2. The interleukin-6 (IL-6) mRNA was also increased (6.4-to 9.3-fold) in microgravity as compared to the ground controls. Since PGE2 and IL-6 are both known to play a role in osteoclast formation and bone resorption, these data provide molecular mechanisms that contribute to our understanding of microgravity-induced alterations in the bone resorption process.

Fetuin, an abundant protein in fetal bovine plasma, is the bovine homolog of human alpha 2HS glycoprotein (alpha 2HS). In spite of numerous studies, the biological functions of both proteins remain elusive. We now report the remarkable 45Ca-binding activity of fetuin in fetal bovine serum that has been blotted on a membrane after electrophoresis. The Ca-binding ability of the purified protein was analyzed by equilibrium dialysis, which revealed that bovine fetuin had multiple Ca-binding sites, one of which had a Kd of 0.95 x 10(-4) M. It was also shown that the Ca-binding activity of fetuin was greater than that of albumin in serum of the bovine fetus at the late gestational stage. Since fetal bovine serum contains not only a high concentration of fetuin but also a high concentration of Ca, it is possible that fetuin functions to maintain high levels of Ca in fetal serum via normalization of the concentration of Ca2+ ions.

Interstitial collagenase (EC 3.4.24.7, MMP-1) is a member of a family of metalloproteinases and is thought to play a role in extracellular matrix remodeling. We have isolated and sequenced a cDNA for bovine interstitial collagenase from a periodontium fibroblast cDNA library. An insert of the cDNA we isolated was 2,025 bp containing an open reading frame which encodes a sequence of 469 amino acids. The identity at the amino acid level between bovine and human interstitial collagenase was 88%, between bovine and rabbit 85% and between bovine and porcine 87%. However, sequence similarity of mouse and rat interstitial collagenases to that of bovine was revealed 55%.

A cDNA clone of murine bone morphogenetic protein (BMP) was obtained by polymerase chain reaction, using oligonucleotides as primers derived from human BMP2 and BMP4. Messenger RNAs were isolated from neonatal murine tissue and used for the synthesis of cDNA with reverse transcriptase.Specifically amplified cDNAs were cloned into bluescript plasmid and subjected to sequencing. One clone which partially encoded murine BMP2 was obtained. The nucleotide sequence, 554 bp in length, shared 88.6% identity with that of human BMP2 and the deduced amino acids including the mature, biologically active protein exhibited 96.2% homology. Within the C-terminal 114 amino acids of the protein, seven cysteine residues were completely conserved which is characteristic of TGF-β superfamily. Substitution of only three amino acids was found when compared with the corresponding human sequence. Isolation of the full length cDNA is now in progress.

Immunohistochemical localization of two enamel proteins, amelogenin and enamelin, in comparison with that of keratin, was determined in odontogenic tumors and the allied lesions in order to verify functional differentiation of the tumor cells as ameloblasts. Amelogenin and enamelin were demonstrated in small mineralized foci and in the tumor cells surrounding them in adenomatoid odontogenic tumor (AOT), calcifying epithelial odontogenic tumor (CEOT), and calcifying odontogenic cyst (COC). Hyaline droplets in AOT showed positive staining for both enamel proteins. These mineralized and hyaline materials were not positive for keratin, although tumor cells were positive. On the other hand, no immunoreaction for enamel proteins was obtained in ameloblastoima and odontogenic epithelial cell nests within myxoma and epulis. The results suggest that tumor cells of AOT and CEOT and lining epithelial cells of COC show ameloblastic differentiation in part, but that ameloblastoma cells do not attain functional matauration as secretory phase ameloblasts.

The alternative NF-kappaB pathway consists predominantly of NF-kappaB-inducing kinase (NIK), IkappaB kinase alpha (IKKalpha), p100/p52, and RelB. The hallmark of the alternative NF-kappaB signaling is the processing of p100 into p52 through NIK, thus allowing the binding of p52 and RelB. The physiologic relevance of alternative NF-kappaB activation in bone biology, however, is not well understood. To elucidate the role of the alternative pathway in bone homeostasis, we first analyzed alymphoplasic (aly/aly) mice, which have a defective NIK and are unable to process p100, resulting in the absence of p52. We observed increased bone mineral density (BMD) and bone volume, indicating an osteopetrotic phenotype. These mice also have a significant defect in RANKL-induced osteoclastogenesis in vitro and in vivo. NF-kappaB DNA-binding assays revealed reduced activity of RelA, RelB, and p50 and no binding activity of p52 in aly/aly osteoclast nuclear extracts after RANKL stimulation. To determine the role of p100 itself without the influence of a concomitant lack of p52, we used p100(-/-) mice, which specifically lack the p100 inhibitor but still express p52. p100(-/-) mice have an osteopenic phenotype owing to the increased osteoclast and decreased osteoblast numbers that was rescued by the deletion of one allele of the relB gene. Deletion of both allele of relB resulted in a significantly increased bone mass owing to decreased osteoclast activity and increased osteoblast numbers compared with wild-type (WT) controls, revealing a hitherto unknown role for RelB in bone formation. Our data suggest a pivotal role of the alternative NF-kappaB pathway, especially of the inhibitory role of p100, in both basal and stimulated osteoclastogenesis and the importance of RelB in both bone formation and resorption.

Various growth factors are known to play important roles in wound healing, especially in an early inflammatory phase. However, their roles in subsequent scar formation phase are relatively unexplored. The aim of this study is to investigate the mechanisms of regulation in scar formation by these factors. Scar fibroblasts (SF) were obtained from immature scar tissue made at rat hard palate 1 month after excision and normal fibroblasts (NF) were obtained from the palatal mucosa of untreated control animals. SF showed a longer doubling time, and increased level of protein synthesis when compared to NF. Platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) stimulated [ 3 H]-thymidine uptake less effectively in SF than in NF. In both cells, transforming growth factor-β1 (TGF-β1) inhibited EGF-induced stimulation of [ 3 H]-thymidine uptake, but had no effects when it was added alone. TGF-β1 increased collagen synthesis more effectively in SF than in NF. These data indicate that the growth factors may play key roles in regulating proliferation and metabolic activity of fibroblasts during scar formation.

The polymerase chain reaction was used to amplify bovine tooth amelogenin cDNA, resulting in several products which were separated by agarose gel electrophoresis. Sequence determination of one of the products revealed that it encoded an amino acid sequence identical to that of a small leucine-rich amelogenin polypeptide (LRAP) previously characterized by protein sequencing. Comparison of the nucleotide sequence of this cDNA with that determined for the cloned bovine amelogenine gene strongly suggested that the LRAP transcript resulted from alternative splicing of the primary transcript of this gene, thus explaining the origin of the puzzling LRAP sequence. Analysis of the structure of LRAP suggests that the polypeptide might exhibit interesting properties relative to hydroxy apatite crystal formation.

The classical NF-kappaB pathway plays an important role in osteoclast formation and differentiation; however, the role of NF-kappaB in osteoclast bone-resorbing activity is not well understood. To elucidate whether NF-kappaB is important for osteoclast bone-resorbing activity, we used a selective peptide inhibitor of the classical NF-kappaB pathway named the NBD peptide. Osteoclasts were generated using bone marrow macrophages in the presence of M-CSF and RANKL. The NBD peptide dose-dependently blocked the bone-resorbing activity of osteoclasts by reducing area, volume (p0.001) and depths (p0.05) of pits. The reduced resorption by the peptide was due to reduced osteoclast bone-resorbing activity, but not reduced differentiation as the number of osteoclasts was similar in all groups. The peptide inhibited bone resorption by reducing TRAP activity, disrupting actin rings and preventing osteoclast migration. Gene expressions of a panel of bone resorption markers were significantly reduced. The NBD peptide dose-dependently reduced the RANKL-induced c-Src kinase activity, which is important for actin ring formation and osteoclast bone resorption. Therefore, these data suggest that the classical NF-kappaB pathway plays a pivotal role in osteoclast bone-resorbing activity.

Monoclonal antibody against bovine serum α2HS glycoprotein was raised in mice and the localization of this glycoprotein in bovine and human teeth was examined by immunohistochemical techniques on undemineralized ground sections after surface etching with dilute HCl. Positive reactions were found in both bovine and human peritubular dentin, whereas virtually negative staining was detected in the intertubular dentin. When the ground section was examined without surface etching, no reaction was found in any area of mineralized dentin. These results suggest that α2HS glycoprotein is present mainly in the peritubular dentin associated with the mineral phase. The presence of this component in dentin may not be due simply to nonspecific binding to calcium phosphate crystals.

Rat osteoblasts were cultured aboard a space shuttle for 4 and 5 days. Cells were treated with 1 nM 1alpha,25-dihydroxyvitamin D(3) (VD) for the last 1 day. The conditioned media were harvested. Cells were solubilized with guanidine solution on board. We examined microgravity effects on the production/expression of osteocalcin, bone sialoprotein (BSP), and VD receptor (VD-R) in osteoblasts. Under VD treatment, the osteocalcin protein level was 243 +/- 117 and 1,718 +/- 534 pg/microg cellular DNA in flight cultures and ground controls, respectively. Without VD treatment, the osteocalcin protein level was not different between flight cultures and ground controls. The osteocalcin mRNA level in the VD-treated flight cultures was as low as 16% of that in ground controls. The VD-R mRNA level in the VD-treated flight cultures was also decreased to 16% of that in ground controls. Microgravity would suppress the VD-inducible production of osteocalcin but not the basal productivity. The BSP mRNA level was increased by microgravity. VD/VD-R binds to the vitamin D-responsive element (VDRE) on the target genes. The rat osteocalcin gene is positively regulated via "enhancer" VDRE, whereas the rat BSP gene is negatively regulated via "repressor " VDRE. Microgravity might modulate osteoblast responsiveness to VD through the suppression of VD-R.

The platelet-activating factor (PAF) is a lipid mediator. The G-protein-coupled receptor of PAF (PAF-R) is activated by inflammatory and stressful conditions in numerous cell types. PAF/PAF-R is involved in apoptotic and antiapoptotic processes. We examined microgravity effects on the expression of PAF-R and second messengers in rat osteoblasts. The PAF-R signals are transmitted via arachidonic acid, phospholipase C (PLC), protein kinase C (PKC), and mitogen-activated protein kinase. Rat osteoblasts were cultured for 4 and 5 days aboard a space shuttle and solubilized on board. PAF-R gene expression in flight cultures increased to 2-6-fold higher than in ground controls. Gene expression of the G-protein alpha subunit Galphaq in flight cultures increased to 3-fold and higher than in ground controls. It is known that Galphaq stimulates the effecter PLCbeta, activating PKC. The mRNA levels of PKCdelta and PKCtheta in flight cultures were increased to 2-5-fold higher than in ground controls. The PKCalpha mRNA level in flight cultures was increased to 3-fold higher than in ground controls on the 4th day. Gene expression of catalytic and regulatory subunits of protein kinase A was suppressed in flight cultures. PKCdelta and PKCtheta are novel PKCs that can be target substrates of caspases. The PAF-R gene may act as a mechano-sensitive gene that is involved in the apoptotic and antiapoptotic processes of osteoblasts under microgravity.

The mechanism underlying space flight-induced osteopenia is unknown. In osteoblasts, the inducible nitric oxide (NO) synthase (iNOS) is involved in the early response to mechanical strain and induction of apoptosis. GTP cyclohydrolase I (GTPCH) is a key enzyme that is essential for iNOS activity. The coordinate expression of GTPCH prevents apoptosis that is induced by iNOS/NO. The purpose of this study was to investigate the effects of space flight on the expression of apoptotic/anti-apoptotic molecules iNOS and GTPCH in rat osteoblasts. Rat osteoblasts were cultured aboard a space shuttle and solubilized on the 4th and 5th days of the mission. The mRNA levels for iNOS and GTPCH in the flight cultures were increased to at least 120-fold and threefold higher than the ground (1 × g) controls, respectively. The amount of cellular DNA per flight culture vessel was 53% and 58% of the ground controls on the 4th and 5th days, respectively. However, the increasing rate of the DNA amount from the 4th to the 5th day was not different between the flight cultures and the ground controls. Morphologically, the cells grew in space as well as on the ground. Co-expression of GTPCH and iNOS may indicate a self-protective mode of action in osteoblasts against the harmful stress under microgravity.

A functional disorder associated with weightlessness is well documented in osteoblasts. The apototic features of this disorder are poorly understood. Harmful stress induces apoptosis in cells via mitochondria and/or Fas. The Bax triggers cytochrome c release from mitochondria, which can be blocked by the Bcl-2. Released cytochrome c then activates the initiator caspase, caspase-9, which can be blocked by the anti-apototic (IAP) family of molecules. The effector caspase, caspase-3, finally exerts DNA fragmentation. We conducted this study to examine the apoptotic effects of vector-averaged gravity on normal human osteoblastic cells. Cell culture flasks were incubated on the clinostat, which generated vector-averaged gravity condition (simulated microgravity) for 12, 24, 48, and 96 hours. Upon termination of clinostat cultures, the cell number and cell viability were assessed. DNA fragmentation was analyzed on the agarose-gel electrophoresis. The mRNA levels for Bax, Bcl-2, XIAP, and caspase-3 genes were analyzed by semi-quantitative RT-PCR. Twenty-four hours after starting clinostat rotation, the ratios of Bax/Bcl-2 mRNA levels (indicator of apoptosis) were significantly increased to 136% of the 1G static controls. However, the XIAP mRNA levels (anti-apoptotic molecule) were increased concomitantly to 138% of the 1G static controls. Thus, cell proliferation or cell viability was not affected by vector-averaged gravity. DNA fragmentation was not observed in clinostat group as well as in control group. Finally, the caspase-3 mRNA levels were not affected by vector-averaged gravity. Simulated microgravity might modulate some apoptotic signals upstream the mitochondrial pathway.

The primary culture of rat osteoblasts was treated with 1alpha,25 dihydroxyvitamin D(3) and fixed with guanidine isothiocyanate solution during a space shuttle flight. The mRNA levels were analyzed by quantitative reverse transcription-polymerase chain reaction. Microgravity decreased the mRNA levels of insulin-like growth factor-I (IGF-I) and increased reciprocally the IGF-I receptor mRNA levels, as compared to the ground (1 x g) control. Microgravity completely suppressed the mRNA expression of the insulin receptor substrate-1, the postreceptor signaling molecule of IGF-I. Microgravity might deteriorate the action and signaling of IGF-I in rat osteoblasts.

The tooth in the gar-pike, Lepisosteus oculatus, an actinopterygian fish, is characterized by the occurrence of both enamel and enameloid, the former covering the tooth shaft and the latter, the tooth cap. Our previous research demonstrated that the enamel in this species was, as in the lungfish, immunoreactive for amelogenin, indicating its homologous nature with the mammalian tooth enamel, whereas the enameloid was completely immunonegative. The present study demonstrates that, during the early maturation stage of the enameloid formation, the inner enamel epithelial cells (IEECs) synthesize through a well-developed Golgi apparatus a fine-granular substance which is intensely immunoreactive for amelogenin. This substance was accumulated in a large saccule extended in a suprabasal zone of the cell; we were unable to find any morphological sign indicating a connection of the substance with the enameloid matrix. The abortive secretion of the enamel matrix-like substance in the IEEC during an enameloid formation was considered to be an instance of rudimental enamel formation. In the gar-pike, the synthesis of amelogenin in the IEEC has been demonstrated to occur independently from that of the enameloid matrix. The present findings demonstrate a prominent difference between the tooth enamel and enameloid.

Interferon-gamma (IFN-gamma), a multifunctional cytokine, has been noted as a potential therapeutic agent for various fibrotic disorders, including excessive scar tissue formation. We previously reported that transforming growth factor-beta1 (TGF-beta1) induced the myofibroblastic phenotype in palatal fibroblasts derived from palatal mucosa, and that such effects might have a close link to palatal scar formation. In the present study, we examined the effects of IFN-gamma on TGF-beta1-pretreated palatal fibroblasts for the purpose of clarifying the suppressive potency against myofibroblastic phenotype expression in vitro. IFN-gamma significantly altered the spindle morphology of TGF-beta1-pretreated palatal fibroblasts into the polygonal one that was similar to the non-treated palatal fibroblasts. This change was parallel with a decrease in the expression of alpha-smooth muscle actin protein, a marker for myofibroblast, as determined by immunoblot analysis. Northern blot analysis showed that IFN-gamma inhibited proalpha2(I) collagen mRNA expression that was stimulated by TGF-beta1 pretreatment for 24 h. Furthermore, IFN-gamma decreased the cell contractility enhanced by TGF-beta1 pretreatment for 24 h in a three-dimensional collagen gel culture system. These results suggest that IFN-gamma may have negative effects with regard to controlling the myofibroblastic phenotype induced by TGF-beta1 in palatal fibroblasts.

Rat osteoblasts were cultured for 4 or 5 days during a Space Shuttle mission. After 20-h treatment with 1α,25-dihydroxyvitamin D3, conditioned media were harvested and cellular DNA and/or RNA were fixed on board. The insulin-like growth factor binding protein (IGF BP)-3 levels in the media were three- and tenfold higher than in ground controls on the fourth and fifth flight days, as quantitated by Western ligand blotting and radioimmunoassay, respectively. The increased IGF BP-3 protein levels correlated with two- to threefold elevation of IGF BP-3 mRNA levels, obtained by reverse transcription-polymerase chain reaction. The IGF BP-5 mRNA levels in flight cultures were 33–69% lower than in ground controls. The IGF BP-4 mRNA levels in flight cultures were 75% lower than in ground controls on the fifth day but were not different on the fourth day. The glucocorticoid receptor mRNA levels in flight cultures were increased by three- to eightfold on the fourth and fifth days compared with levels in ground controls. These data suggest potential mechanisms underlying spaceflight-induced osteopenia.

The effect of basic fibroblast growth factor (bFGF) on apoptosis in normal rat palatal fibroblasts and rat palatal scar fibroblasts was examined by the TUNEL method in order to clarify the mechanism of apoptosis induction in myofibroblasts during the scar formation process. A percentage of scar fibroblasts undergoing apoptosis was significantly higher than that of palatal fibroblasts when they were treated with bFGF succeeding to serum starvation. Palatal fibroblasts, phenotypically modulated into myofibroblasts by the pretreatment with transforming growth factor-beta 1 (TGF-beta 1), similarly showed a higher level of apoptosis induction by bFGF-treatment. TGF-beta 1 elevated protein and mRNA level of FGF receptor (FGFR) in palatal fibroblasts. Tyrosine autophosphorylation of FGFR upon stimulation by bFGF was significantly higher in scar fibroblasts than in normal palatal fibroblasts. These findings suggested that bFGF may be a potential stimulator of apoptosis in myofibroblasts during palatal scar formation and that FGFR may be responsible for this process.

Gene expression and localization of amelogenin were studied in the developing rat incisor by the methods of in situ hybridization and immunohistochemistry. ISH revealed the first expression of amelogenin mRNA in the inner enamel epithelium of the cervical loop. The signals were clearly observed in pre-ameloblasts in the region bordering on predentin formation and became more intense toward the cells on the initial enamel matrix secretion. The maximal signals were found in the cytoplasm of secretory ameloblasts. From the terminal secretion zone, the signals then became gradually weaker toward the incisal edge but were still evident in the cytoplasm of shortening, transitional ameloblasts and those at the early maturation stage. No signals were found in the cells of the stratum intermedium and stellate reticulum throughout amelogenesis. Immunohistochemistry by means of an antibody against amelogenin C-telopeptide consisting of 12 amino acids revealed immunoreaction in the secretory ameloblasts reacting to the ISH. When a polyclonal antibody against amelogenin was used, immunoreaction was found in the distal ends of ruffle-ended ameloblasts (RA) in the maturation zone. Those results indicated that amelogenin is synthesized by ameloblastic cells from the inner enamel epithelium to the early maturation stage and is then resorbed by the RA.

A genomic DNA clone (14.5 kbp) for amelogenin, a tooth enamel specific protein, has been isolated from a mouse genomic library. The nucleotide structure of this DNA was determined by the polymerase chain reaction and sequence analysis. The result indicated that the cloned DNA contained all the exons in addition to approximately 5.5 kbp of upstream sequence.

PURPOSE To determine factors that control ocular enlargement in experimental form-deprivation myopia and to clarify the mechanism of form-deprivation myopia. METHODS After the left eyes of 20 chicks were monocularly occluded for 2 weeks, protein, basic fibroblast growth factor (bFGF) and transforming growth factor (TGF)-beta 2 contents in samples of constant area (circular button, diameter = 8.5 mm) in the retina-retinal pigment epithelium (RPE)-choroid and the sclera in the posterior region of control and myopic eyes were determined by enzyme-linked immunosorbent assay. RESULTS The bFGF content (per circular button) and bFGF concentration (per mg protein) were significantly lower in the sclera in the posterior region of the myopic eyes than in control eyes. The bFGF content and concentration were similar in the retina-RPE-choroid in myopic and control eyes. The TGF-beta 2 content and concentration were significantly higher in myopic eyes in both the retina-RPE-choroid and the sclera (P < 0.05). CONCLUSIONS These results are consistent with the possibility that bFGF and TGF-beta 2 regulate ocular enlargement or respond to myopiagenic mechanisms in form-deprivation myopia.