Biomedical subjects

Kazuharu Irie

Publications and source records attributed to Kazuharu Irie.

16 recordsLinked to original sources

PubMed2006

Contributions of matrix metalloproteinases toward Meckel's cartilage resorption in mice: immunohistochemical studies, including comparisons with developing endochondral bones.

The middle portion of Meckel's cartilage (one of four portions that disappear with unique fate) degrades via hypertrophy and the cell death of chondrocytes and via the resorption of cartilage by chondroclasts. We have examined the immunolocalization of matrix metalloproteinase-2 (MMP-2), MMP-9, MMP-13, and MMP-14 (members of the MMP activation cascade) and galectin-3 (an endogenous substrate for MMP-9 and an anti-apoptotic factor) during resorption of Meckel's cartilage in embryonic mice and have compared the results with those of developing endochondral bones in hind limbs. MMP immunoreactivity, except for MMP-2, is present in nearly all chondrocytes in the middle portion of Meckel's cartilage. On embryonic day 15 (E15), faint MMP-2-immunoreactive and intense MMP-13-immunoreactive signals occur in the periosteal bone matrix deposited by periosteal osteoblasts on the lateral surface, whereas MMP-9 and MMP-14 are immunolocalized in the peripheral chondrocytes of Meckel's cartilage. The activation cascade of MMPs by face-to-face cross-talk between cells may thus contribute to the initiation of Meckel's cartilage degradation. On E16, immunopositive signaling for MMP-13 is detectable in the ruffled border of chondroclasts at the resorption front, whereas immunostaining for galectin-3 is present at all stages of chondrocyte differentiation, especially in hypertrophic chondrocytes adjacent to chondroclasts. Galectin-3-positive hypertrophic chondrocytes may therefore coordinate the resorption of calcified cartilage through cell-to-cell contact with chondroclasts. In metatarsal specimens from E16, MMPs are detected in osteoblasts, young osteocytes, and the bone matrix of the periosteal envelope, whereas galectin-3 immunoreactivity is intense in young periosteal osteocytes. In addition, intense MMP-9 and MMP-14 immunostaining has been preferentially found in pre-hypertrophic chondrocytes, although galectin-3 immunoreactivity markedly decreases in hypertrophic chondrocytes. These results indicate that the degradation of Meckel's cartilage involves an activation cascade of MMPs that differs from that in endochondral bone formation.

Acid Phosphatase↗

PubMed2006

Macrophage migration inhibitory factor-deficient mice are resistant to ovariectomy-induced bone loss.

A link between macrophage migration inhibitory factor (MIF) and estrogen has recently emerged. We examined the involvement of MIF in osteoporotic changes in bone after ovariectomy (OVX), and revealed that MIF-deficient mice (MIF-KO) were completely protected from this phenomenon. The increase in osteoclast number per bone surface and serum IL-1beta levels, which were observed in wild-type mice after OVX, did not occur in MIF KO. Our data suggest that MIF plays an important role in the pathogenesis of postmenopausal osteoporosis, and could be a novel target for the treatment of this disease.

Animals↗

PubMed2006

Transgenic mice overexpressing macrophage migration inhibitory factor (MIF) exhibit high-turnover osteoporosis.

UNLABELLED: The bone phenotype of mice overexpressing MIF was studied. These mice showed decreased trabecular bone, increased bone formation rate, and increased MMP-3, -9, and -13 mRNA expression in the femora and tibias. This model provides evidence of the role played by MIF in bone remodeling and balance in vivo. INTRODUCTION: The role of macrophage migration inhibitory factor (MIF) in in vivo bone remodeling remains unelucidated. We describe disordered bone metabolism in transgenic mice overexpressing MIF. MATERIALS AND METHODS: For in vivo study, muCT, bone histomorphometry, blood and urine biochemical data, and gene expression of MIF transgenic (MIF Tg) mice and littermate wildtype (WT) mice were examined. For in vitro study, osteoclastogenesis in the co-culture of bone marrow cells and osteoblasts from MIF Tg and WT were assessed. RESULTS: muCT analyses revealed a significant reduction in the trabecular bone of distal femur in MIF Tg at 8-12 weeks of age. Histomorphometric analysis revealed increase in several measures of bone formation. Osteoclastogenesis was not influenced by the origin of bone marrow cells or osteoblasts. Urine level of deoxypyridinoline/creatinine and the mRNA levels of matrix metalloproteinase (MMP) -3, -9, and -13 in femurs were elevated in MIF Tg. CONCLUSIONS: Overexpression of MIF causes high-turnover osteoporosis in mice. The increased expression of MMPs in bone was suggested, at least in part, as one cause of this phenotype, because MMPs plays important roles for bone resorption without affecting the formation of osteoclasts. This model provides evidence of the role played by MIF in bone remodeling and balance.

Amino Acids↗

PubMed2005

Impaired alveolization in mice deficient in membrane-type matrix metalloproteinase 1 (MT1-MMP).

To clarify the involvement of membrane-type matrix metalloproteinase 1 (MT1-MMP) in lung organogenesis, we studied the lung morphology of 13-day-old MT1-MMP null mice. The lung architecture in MT1-MMP null mice was abnormal, and the airspace compartments were characterized by smooth walls and larger size. Most of the compartment wall consisted of one or two layers of cells and interstitial connective tissue that was thicker than that of normal alveoli. The wall frequently had capillaries on both sides of the interstitial connective tissue. These findings indicate that the lung in MT1-MMP null mice at 13 days of age is comparable to that of neonatal mice, i.e., it represents the stage before alveolization, suggesting that the generation of a large respiratory surface - the final process of lung development - is impaired in MT1-MMP null mice. Moreover, a zymography assay revealed decreased activity of matrix metalloproteinase 2 (MMP-2) in MT1-MMP null mice, suggesting that activation of pro-MMP-2 by MT1-MMP is critical in this process.

Animals↗

PubMed2005

Microfibril-associated glycoprotein-1 and fibrillin-2 are associated with tropoelastin deposition in vitro.

Elastic system fibers consist of microfibrils and tropoelastin. During development, microfibrils act as a template on which tropoelastin is deposited. Microfibril-associated glycoprotein-1 (MAGP-1) and fibrillin-2, the major components of microfibrils, provide the likely template for tropoelastin deposition. In this study, we used the RNA interference (RNAi) technique to establish MAGP-1 and fibrillin-2 gene-specific knock-downs individually in elastin-producing cells (human gingival fibroblasts). We then examined the extracellular deposition of tropoelastin by western blotting. These two genes were specifically suppressed to < 30% of the control level, and this was responsible for the diminution of tropoelastin deposition. An immunofluorescence study also confirmed that RNAi-mediated down-regulation of MAGP-1 or fibrillin-2 led to the loss of tropoelastin immunoreactivity. These results suggest that MAGP-1 and fibrillin-2 are, directly or indirectly, associated with the extracellular deposition of tropoelastin during elastic fiber formation in human gingival fibroblasts in vitro.

Cells, Cultured↗

PubMed2005

Immunolocalization of receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) in Meckel's cartilage compared with developing endochondral bones in mice.

We examined the immunolocalization of receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) in areas of resorption caused by osteoclasts/chondroclasts on embryonic days 14-16 (E14-16) in Meckel's cartilage, and compared the results with those in endochondral bones in mice. Intense RANKL and OPG immunoreactivity was detected in the chondrocytes in Meckel's cartilage. On E15, when the incisor teeth were closest to the middle portion of Meckel's cartilage, tartrate-resistant acid phosphatase (TRAP)-positive cells appeared on the lateral side of the cartilage. Furthermore, the dental follicle showed moderate immunoreactivity for RANKL and OPG, whereas osteoblasts derived from perichondral cells were immunonegative for RANKL and OPG in that area. On E16, cartilage resorption by TRAP-positive cells had progressed at the differential position, and intensely immunoreactive products of RANKL were overlapped on and found to exist next to TRAP-positive cells in the resorption area. In developing metatarsal tissue, OPG immunoreactivity was intense in periosteal osteoblasts, whereas RANKL was only faintly seen in some of the periosteal cells. In epiphyseal chondrocytes of the developing femur, RANKL immunoreactivity was moderate, and OPG scarcely detected. These results indicate a peculiarity of RANKL and OPG immunolocalization in resorption of Meckel's cartilage. Growth of the incisor teeth may be involved in the time- and position-specific resorption of Meckel's cartilage through local regulation of the RANKL/OPG system in dental follicular cells and periosteal osteoblasts, whereas RANKL and OPG in chondrocytes seem to contribute to resorption through regulation of the chondroclast function.

Animals↗

PubMed2004

Periodontal regeneration of transplanted rat molars after cryopreservation.

The effects of cryopreservation on periodontal regeneration of transplanted rat molars were investigated histologically and histochemically in rats. Bilateral first and second maxillary molars of 4-week-old Wistar rats were gently extracted and transplanted into the abdominal subcutaneous connective tissue immediately or after cryopreservation in liquid nitrogen overnight. Donor teeth were slowly frozen by a rate-controlling freezer (program freezer) using 5% dimethylsulfoxide (DMSO) and 6% hydroxyethyl starch (HES) as cryoprotectants. One-four weeks after transplantation, they were carefully excised with the surrounding tissues. Regeneration of acellular cementum, periodontal ligament, and alveolar bone were observed 2 weeks after immediate transplantation. The pulp was repaired by the ingrowth of granulation tissue from the root apex followed by the formation of calcified tissue. The regenerated periodontal ligament was positive for alkaline phosphatase (ALP). Small or mononuclear tartrate resistant acid phosphatase (TRAP) positive cells were scattered on the newly formed alveolar bone and on the hard tissue in the pulp, but there was no external or internal progressive root resorption at 4 weeks. Cryopreserved teeth had acellular cementum with a rough surface at 1 week, but with the increase of cementoblasts and the appearance of periodontal ligament and alveolar bone, the surface became smooth at 3 weeks. Epithelial rests of Malassez (ERM) also revived. After regeneration of the periodontal tissues at 4 weeks, there was no evidence of root resorption. Although the process proceeded slowly, the cryopreserved teeth showed the periodontal regeneration substantially similar to that of the immediately transplanted teeth without progressive root resorption, indicating that they could be applicable for clinical use.

Alkaline Phosphatase↗

PubMed2004

Induction of fibulin-5 gene is regulated by tropoelastin gene, and correlated with tropoelastin accumulation in vitro.

Fibulin-5 (also known as DANCE) is an elastin-binding protein that is thought to play a role in elastogenesis. We examined the relationship between the gene expression of fibulin-5 and the gene expression and accumulation of tropoelastin by comparing elastin-producing cells (human gingival fibroblasts) with non-elastin-producing cells (human periodontal ligament fibroblasts) by Northern blot analysis. Fibulin-5 gene induction was found only in elastin-producing cells. Induction of the fibulin-5 gene in elastin-producing cells occurred after induction of the tropoelastin gene, and the fibulin-5 level was reduced upon RNA interference-mediated down-regulation of tropoelastin. Fibulin-5 gene induction was also correlated with a rapid increase of tropoelastin accumulation within the cell layer. These results may suggest that the fibulin-5 gene induction is directly or indirectly regulated by tropoelastin gene expression and plays a role in the accumulation of elastic fibers within matrices.

Blotting, Northern↗

PubMed2003

Osteoclast differentiation in ectopic bone formation induced by recombinant human bone morphogenetic protein 2 (rhBMP-2).

Osteoclast differentiation in the process of ectopic bone formation induced by recombinant human bone morphogenetic protein 2 (rhBMP-2) was examined to clarify the relationship between osteoclast development and rhBMP-2-induced bone formation. A combination of rhBMP-2 with a porous microsphere (PMS) and blood clot was implanted subcutaneously on the bilateral chest muscles of rats. Tartrate-resistant acid phosphatase (TRAPase) activity, cathepsin K (cath K), and calcitonin receptor (CTR), as markers of osteoclasts and their precursors, were examined using enzyme and immunohistochemical analysis up to 7 days after implantation. Mononuclear cells positive for TRAPase, cath K, and CTR first appeared on day 3 in connective tissue surrounding the PMS after implantation of rhBMP-2. Simultaneously, alkaline phosphatase activity became detectable in mesenchymal cells in the connective tissue. Electron microscopy demonstrated some mononuclear cells with abundant mitochondria and poorly developed rough endoplasmic reticulum in the proximity of mesenchymal cells. However, there was no evidence of cartilage or bone matrix formation on day 3. Osteoclasts in various stages of development, classified by the pattern of immunoreactivity for cath K, were observed by day 7. The polarized intracellular distribution of cath K was found only in osteoclasts attached to bone matrix. In conclusion, we have demonstrated for the first time the appearance of osteoclast precursors before bone matrix formation induced by rhBMP-2, suggesting that bone matrix is not a prerequisite for osteoclast precursor recruitment. Furthermore, we suggest that differentiation into polarized functional osteoclasts is accomplished when the osteoclasts attach to the bone matrix.

Animals↗

PubMed2003

Ultrastructural study of synovitis induced by trauma to the rat temporomandibular joint (TMJ).

BACKGROUND: Electron microscopy was used to examine the histologic effect of trauma on the rat temporomandibular joint synovial membrane. METHODS: Trauma to the TMJ in male Wister rats (100-200 g) was introduced through repeated forced condylar hypermobility. Ultrastructural observations were made 5 days and 6 weeks after the trauma. RESULTS: The early response of the synovial membrane was synovial hyperplasia, type A synovial cell loss, dilation of the r-ER in the type B synovial cells and fibrin deposition on the synovial surfaces. The late response included degeneration of synovial cells with swollen mitochondria and cell projections, and cell fragmentation. Large amount of fibrin deposition on opposing surface layers was also noticed. CONCLUSION: The type A cell loss and fibrin deposition followed by the occurrence of fibrinous materials at opposing surface layers of the synovial membrane suggest that traumatic synovitis causes synovial adhesions.

Animals↗

PubMed2003

Early expression of bone matrix proteins in osteogenic cell cultures.

Osteogenic cells express some matrix proteins at early culture intervals. The aim of this study was to determine if, and in what proportion, cells used for plating contain bone sialoprotein (BSP) and osteopontin (OPN), two matrix proteins associated with initial events in bone formation. Their pattern of expression, as well as that of fibronectin (FN) and type I pro-collagen, was also examined at 6 hr and at 1 and 3 days. The cells were obtained by enzymatic digestion of newborn rat calvariae, and grown on glass coverslips. Cytocentrifuge preparations of isolated cells and coverslips were processed for single or dual immunolabeling with monoclonal and/or polyclonal primary antibodies, followed by fluorochrome-conjugated antibodies. The cell labeling was mainly associated with perinuclear elements. OPN was also distinctively found at peripheral cytoplasmic sites. About 31% of isolated cells were OPN-positive and 18% were BSP-positive. After 1 day, almost 50% of cells were immunoreactive for OPN and for type I pro-collagen, and still less than 20% reacted for BSP. Approximately 7% exhibited peripheral staining for OPN. Almost all cells were associated with extracellular FN. However, only 15% showed intracellular labeling. These results indicate that an important proportion of cells used for plating contain BSP and OPN, a situation that should be taken into consideration in experimental analyses of osteoblast activity in vitro.

Animals↗

PubMed2003

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Journal Article↗

PubMed2002

Calcitonin gene-related peptide (CGRP)-containing nerve fibers in bone tissue and their involvement in bone remodeling.

Bone remodeling is a process of bone renewal accomplished by osteoclastic bone resorption and osteoblastic bone formation. These two activities are regulated by systemic hormones and by local cytokines and growth factors. Moreover, the nervous system and certain neuropeptides seem to be involved in regulation of bone remodeling. In this paper, we focus on the distribution of CGRP-containing nerve fibers and their dynamics, and discuss the role of these fibers as a possible mechanism for nervous system involvement in regulation of bone remodeling. CGRP-immunoreactive nerve fibers are widely distributed in bone tissue, such as periosteum and bone marrow, and show apparent regional distribution with different densities. They are often associated with blood vessels and show a beaded appearance. The wide distribution of CGRP-immunoreactive nerve fibers in bone tissue and the changes in distribution during bone development and regeneration suggest the involvement of these fibers in bone remodeling. The effect of CGRP on bone remodeling could partly be through its action on blood vessels, thereby regulating local blood flow. Moreover, in vitro biochemical data and the localization of CGRP-immunoreactive nerve fibers in the vicinity of bone cells suggest that they are directly involved in local regulation of bone remodeling by elevating the concentration of CGRP in the microenvironment around bone cells, especially during bone growth or repair.

Animals↗

PubMed2002

Evidence that zymogen granules do not function as an intracellular Ca2+ store for the generation of the Ca2+ signal in rat parotid acinar cells.

Rat parotid acinar cells lacking zymogen granules were obtained by inducing granule discharge with the beta-adrenoceptor agonist isoproterenol. To assess whether zymogen granules are involved in the regulation of Ca(2+) signalling as intracellular Ca(2+) stores, changes in cytosolic free Ca(2+) ion concentration ([Ca(2+)](i)) were studied with imaging microscopy in fura-2-loaded parotid acinar cells lacking zymogen granules. The increase in [Ca(2+)](i) induced by muscarinic receptor stimulation was initiated at the apical pole of the acinar cells, and rapidly spread as a Ca(2+) wave towards the basolateral region. The magnitude of the [Ca(2+)](i) response and the speed of the Ca(2+) wave were essentially similar to those in control acinar cells containing zymogen granules. Western blot analysis of the inositol 1,4,5-trisphosphate receptor (IP(3)R) was performed on zymogen granule membranes and microsomes using anti-IP(3)R antibodies. The immunoreactivity of all three IP(3)Rs was clearly observed in the microsomal preparations. Although a weak band of IP(3)R type-2 was detected in the zymogen granule membranes, this band probably resulted from contamination by the endoplasmic reticulum (ER), because calnexin, a marker protein of the ER, was also detected in the same preparation. Furthermore, Western blotting and reverse transcriptase-PCR analysis failed to provide evidence for the expression of ryanodine receptors in rat parotid acinar cells, whereas expression was clearly detectable in rat skeletal muscle, heart and brain. These results suggest that zymogen granules do not have a critical role in Ca(2+) signalling in rat parotid acinar cells.

Animals↗

PubMed2002

Expression of fibrillins and tropoelastin by human gingival and periodontal ligament fibroblasts in vitro.

The elastic system fibers consist of three different types, oxytalan, elaunin and elastic fibers, which differ in the relative content of microfibrils and elastin. In periodontal tissues, oxytalan fibers are known to be distributed in the periodontal ligament and gingiva, while elaunin and elastic fibers are present only in the gingiva. We examined the in vitro synthesis of microfibrils and elastin by human gingival fibroblasts (HGF) and periodontal ligament fibroblasts (HPLF). The two kinds of HGF and HPLF were cultured in MEM containing 10% newborn calf serum for 30 days. Since fibrillin-1 and fibrillin-2 are the major components of microfibrils involved in elastogenesis, we investigated the synthesis of fibrillins and tropoelastin in the conditioned medium of HGF and HPLF. Western blot analysis revealed fibrillin-1 and fibrillin-2 to occur in the HGF and HPLF culture medium, HGF exhibiting a higher level of synthesis than HPLF. Tropoelastin, on the other hand, was detected only in the medium of HGF after day 24. In addition, analysis of RNA extracted from HGF and HPLF on day 30 showed that only HGF expressed mRNA encoding tropoelastin. Immunohistochemically, accumulation of tropoelastin in the perinuclear area was found only in HGF. These results show that HGF expressed microfibrils and elastin, while HPLF expressed only microfibrils for the experimental period, and suggest a biochemical basis for the different distribution of elastic system fibers of the gingiva and periodontal ligament in vivo.

Blotting, Northern↗

PubMed2002

Apoptosis induced by generated OH radicals inside cells after irradiation.

OH radicals play a major role in radiation-induced DNA and cell membrane damage. These types of damage can also induce death by apoptosis through activation of a pro-apoptosis pathway. We attempted to detect OH radicals inside human promyelocytic leukemia (HL60) cells and estimate the relationship between radiation-induced apoptosis and OH radicals generated inside the cells. Electron spin resonance spectroscopy showed that OH radicals were generated by X-rays within irradiated cell pellets and the relative signal intensities of OH radicals increased with the radiation dose. Agarose gel electrophoresis revealed that the death of HL60 cells by apoptosis was accompanied by internucleosomal DNA fragmentation at 2 h after irradiation with 10-30 Gy. On ultrastructure evaluation by transmission electron microscopy, certain irradiated HL60 cells demonstrated condensed chromatin forms at the nuclear membrane and nuclear fragmentation. The frequency of apoptotic cells with condensation and fragmentation of nuclear chromatin increased with radiation dose in semithin sections. The increase of quantitative DNA fragmentation and percentage of non-living cells also correlated with radiation dose. These results suggest that OH radicals are generated inside cells before apoptosis occurs. The amount of OH radicals generated correlates with apoptotic cell death.

Apoptosis↗