It is known that the teleost scale regenerates after being removed. We previously reported that the osteogenesis in regenerating scales was very similar to that in calvarial bone, which suggests that regenerating scale can be used as a model for osteogenesis to analyze the inte raction b e t we e n oste oblasts and osteoclasts. In the present study, we developed an assay system using regenerating scales by modifying our assay system with normal scales. The weight of one regenerating scale cannot be measured precisely because the rate of calcification in regenerating scales is lower than that in normal scales. In regenerating scales, thus, the respective marker enzyme activit y, i.e ., alkaline phosphatase (ALP) for osteoblasts and tartrate-resistant acid phosphatase (TRAP) for osteoclasts, was normalized by the surface area (mm 2) of each goldfish scale. With this modified method, we were able to detect ALP and TRAP activities with no variation in the lines of regenerating scales. In addition, we found that the ALP activity in the regenerating scales significantly increased under 3G acceleration loading by vibration, while the TRAP activity in the loaded regenerating scales significantly decreased. We strongly believe that the regenerating scale is a good material for the analysis of bone me tabolism unde r dif fe re nt gravit y environments.
Osteocytes, osteoblasts (bone-forming cells), and osteoclasts (bone-resorbing cells) are the primary types of cells that regulate bone metabolism in mammals. Sclerostin produced in bone cells activates osteoclasts, inhibiting bone formation; excess production of sclerostin, therefore, leads to the loss of bone mass. Fish scales have been reported to have morphological and functional similarities to mammalian bones, making them a useful experimental system for analyzing vertebrate bone metabolism in vitro. However, whether fish scales contain cells producing sclerostin and/or osteocytes has not been determined. The current study demonstrated, for the first time, that sclerostin-containing cells exist in goldfish scales. Analysis of the distribution and shape of sclerostin-expressing cells provided evidence that osteoblasts produce sclerostin in goldfish scales. Furthermore, our results found that osteocyte-like cells exist in goldfish scales, which also produce sclerostin. Finally, we demonstrated that microgravity in outer space increased the level of sclerostin in the scales of goldfish, a finding suggesting that the induction of sclerostin is the mechanism underlying the activation of osteoclasts under microgravity.
We have previously reported that microgravity promotes the activation of osteoclasts in cultured regenerating scales. This osteoclastic activation was induced by increased levels of receptor activator of the nuclear factor-κB ligand (RANKL). Therefore, we determined that RANKL is an important factor in evaluating osteoclastogenesis in bone tissue. However, the role of RANKL in fish scales is poorly understood. In the present study, we prepared antiserum against goldfish RANKL in rabbits and detected RANKL-producing cells in regenerating goldfish scales. Furthermore, we studied osteoclastic activation by the addition of RANKL to examine exogenous RANKL on osteoclastogenesis in regenerating goldfish scales. As a result, RANKL immunepositive cells were detected in grooves of regenerating scales. In addition, treating the regenerating scales with mammalian RANKL for 3 h significantly increased the expression of the nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), which is essential for osteoclast differentiation. After 6 h of incubation with RANKL, the expression of cathepsin K, a functional osteoclastic gene, significantly increased. Furthermore, the molecules for osteoclast multinucleation and differentiation significantly increased following treatment with mammalian RANKL. Therefore, in fish scales as well as mammalian bone, we concluded that RANKL plays an important role in osteoclastogenesis. ©2020 Jpn. Soc. Biol. Sci.
Fish scale is a calcified tissue which is similar to those found in human bones. Recently, we developed a scale culture system in which osteoblasts and osteoclasts remain intact after being prepared. Using this system, we analyzed the bone metabolism under 2-, 4-, and 7-gravity (G) loading by centrifuge and compared with control (1-G). After loading for 5 and 10 min, the scales were incubated for 6 and 24 h. The osteoblastic and osteoclastic activities were then measured. The osteoblastic activity significantly increased under 2-and 4-G loading. This activity increased remarkably under 7-G loading. On the other hand, we found that the osteoclastic activity significantly decreased under 2-G loading. Under 4-G loading, there was no significant difference between G-loaded scales and control scales. The osteoclastic activity tended to increase under 7-G loading. These results were similar to our previous study of acceleration by vibration. Bone consists of osteoblasts, osteoclasts, and bone matrix. In our culture system including bone matrix, osteoblasts and osteoclasts reacted precisely to G loading by centrifuge. Therefore, we strongly believe that our culture system is useful for the analysis of bone metabolism under G loading.
The effects of low-intensity pulsed ultrasound (LIPUS) on osteoclastogenesis were examined using fish scales that had both osteoclasts and osteoblasts. The binding of the receptor activator of NF-κB ligand (RANKL) in osteoblasts to the receptor activator of NF-κB (RANK) in osteoclasts induced osteoclastogenesis. Therefore, we focused on RANK/RANKL signaling. After 6 h of incubation following LIPUS treatment, mRNA expression of RANKL increased significantly. Resulting from the increased RANKL mRNA level, the expression of transcription-regulating factors significantly increased after 6 h of incubation, and then the mRNA expression of functional genes was significantly up-regulated after 12 h of incubation. However, the mRNA expression of osteoprotegerin (OPG), which is known as an osteoclastogenesis inhibitory factor, also significantly increased after 6 h of incubation and tended to further increase after 12 h of incubation. At 24 h of incubation, osteoclastic functional genes' mRNA expression decreased to the level of the control. Furthermore, we performed an in vivo experiment with goldfish. Two weeks after daily LIPUS exposure, osteoclastic marker enzymes tended to decrease while osteoblastic marker enzymes were activated. The regeneration rate of the LIPUS-treated scales was significantly higher than that of the control scales. Thus, LIPUS moderately activates osteoclasts and induces bone formation.Low-intensity pulsed ultrasound (LIPUS), a noninvasive remedial measure, promotes the repair of bone fracture and distraction osteogenesis (see a review, 25). Until now, the study of LIPUS has focused on osteoblastic growth and differentiation (3,8,10). However, the details of the direct effect of
Mechanical stress promotes osteoblast proliferation and differentiation from mesenchymal stem cells (MSCs). Although numerous growth factors and cytokines are known to regulate this process, information regarding the differentiation of mechanically stimulated osteoblasts from MSCs in in vivo microenvironment is limited. To determine the significant factors involved in this process, we performed a global analysis of differentially expressed genes, in response to tensile stress, in the mouse cranial suture wherein osteoblasts differentiate from MSCs. We found that the gene expression levels of several components involved in bone morphogenetic protein, Wnt, and epithelial growth factor signalings were elevated with tensile stress. Moreover gene expression of some extracellular matrices (ECMs), such as cysteine rich protein 61 (Cyr61)/CCN1 and galectin-9, were upregulated. These ECMs have the ability to modulate the activities of cytokines and are known as matricellular proteins. Cyr61/CCN1 expression was prominently increased in the fibroblastic cells and preosteoblasts in the suture. Thus, for the first time we demonstrated the mechanical stimulation of Cyr61/CCN1 expression in osteogenic cells in an ex vivo system. These results suggest the importance of matricellular proteins along with the cytokine-mediated signaling for the mechanical regulation of MSC proliferation and differentiation into osteoblastic cell lineage in vivo.Mechanical stress controls bone mass by affecting recruitment of osteoprogenitors as well as osteoblast differentiation from mesenchymal stem cells (MSCs) in the bone tissue (7,8). Elucidation of the precise mechanisms involved in the osteoblastogenesis stimulated by mechanical stress, especially the mechanism for osteogenic differentiation of MSCs, will contribute to the progress of regenerative medicine for effective production of bone tissue from MSC sources. Increasing evidence has revealed that the process of mechanical stress-stimulated osteoblastogenesis is controlled by many cytokines/growth factors (9,32,34,36). Especially bone morphogenetic protein (BMP) signaling and Wnt signaling play important roles in the development of osteoblasts from MSCs (5), but the more relevant factors among these or
We have developed an immortalized oral epithelial cell line, ROE2, from fetal transgenic rats harboring temperature-sensitive simian virus 40 large T-antigen gene. The cells grew continuously at either a permissive temperature of 33°C or an intermediate temperature of 37°C. At the nonpermissive temperature of 39°C, on the other hand, growth decreased significantly, and the Sub-G1 phase of the cell cycle increased, indicating that the cells undergo apoptosis at a nonpermissive temperature. Histological and immunocytochemical analyses revealed that ROE2 cells at 37°C had a stratified epithelial-like morphology and expressed cytokeratins Krt4 and Krt13, marker proteins for oral nonkeratinized epithelial cells. Global-scale comprehensive microarray analysis, coupled with bioinformatics tools, demonstrated a significant gene network that was obtained from the upregulated genes. The gene network contained 16 genes, including Cdkn1a, Fos, Krt13, and Prdm1, and was associated mainly with the biological process of skin development in the category of biological functions, organ development. These four genes were validated by quantitative real-time polymerase chain reaction, and the results were nearly consistent with the microarray data. It is therefore anticipated that this cell line will be useful as an in vitro model for studies such as physiological functions, as well as for gene expression in oral epithelial cells.
Tight junction (TJ) is one of the cell-cell junctions and known to have the barrier and fence functions between adjacent cells in both simple and stratified epithelia. We examined the distribution pattern, constitutive proteins, and permeability of TJ in the stratified squamous epithelium of the palatal mucosa of mice. Ultrastructural observations based on the ultrathin section and freeze-fracture methods revealed that poorly developed TJs are located at the upper layer of the stratum granulosum. The positive immunofluorescence of occludin (OCD), claudin (CLD)-1 and -4 were localized among the upper layer of the stratum granulosum showing a dot-like distribution pattern. And CLD-1 and -4 were localized among the stratum spinosum and the lower part of stratum granulosum additionally showed a positive reaction along the cell profiles. Western blotting of TJ constitutive proteins showed OCD, CLD-1, -2, -4, and -5 bands. The permeability test using biotin as a tracer revealed both the areas where biotin passed through beyond OCD positive points and the areas where biotin stopped at OCD positive points. These results show that poor TJs localize at the upper layer of the stratum granulosum of the palatal epithelium, and the TJs are leaky and include at least CLD-1 and -4.
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