Tadayoshi Kagiya

Expression profiling of microRNAs in RAW264.7 cells treated with a combination of tumor necrosis factor alpha and RANKL during osteoclast differentiation

BACKGROUND AND OBJECTIVE Tumor necrosis factor alpha (TNF-α), a cytokine involved in the pathogenesis of periodontal disease, induces osteoclast differentiation and indirectly promotes alveolar bone resorption. We investigated TNF-α-regulated osteoclast differentiation, focusing on microRNAs. MicroRNAs are small, noncoding RNAs that are involved in various biological processes, including cellular differentiation, proliferation and apoptosis. Aside from miR-21, miR-155 and miR-223, the identities of the microRNAs that play roles in osteoclast differentiation are unknown. Notably, no previous studies have reported the expression profiling of microRNAs during TNF-α-regulated osteoclast differentiation. MATERIAL AND METHODS We used microarrays to screen the levels of expression of mature microRNAs in RAW264.7 cells treated with a combination of TNF-α and RANKL, or RANKL alone for 0, 24 or 82 h during osteoclast formation. We validated the results of the microarray analyses through quantitative RT-PCR analyses of representative microRNAs in RAW264.7 cells and murine bone marrow macrophages. RESULTS During osteoclast formation, the expression of 44 mature microRNAs differed by more than twofold between untreated cells and cells treated with a combination of TNF-α and RANKL, and the expression of 52 mature microRNAs differed upon RANKL treatment. According to quantitative RT-PCR analyses, miR-378 was upregulated and miR-223 was downregulated during osteoclast formation. Furthermore, miR-21, miR-29b, miR-146a, miR-155 and miR-210 were highly expressed during osteoclast differentiation in TNF-α/RANKL-treated cells compared with RANKL-treated cells. CONCLUSIONS These results suggest that miR-223 and miR-378 may play important roles in osteoclastogenesis, and that miR-21, miR-29b, miR-146a, miR-155 and miR-210 are involved in TNF-α-regulated osteoclast differentiation.

Establishment of Hertwig's epithelial root sheath cell line from cells involved in epithelial-mesenchymal transition.

The epithelial-mesenchymal transition (EMT) is an important event in the developmental process of various organs. In periodontal development during root formation of a tooth, this EMT has been a subject of controversy. Hertwigs epithelial root sheath (HERS), consisting of two epithelial layers, plays a role of inducing odontogenesis during root development and thereafter becomes fragmented. Some researchers have maintained that in the process of this fragmentation, some HERS cells change from epithelial to mesenchymal cells. Here, we established a HERS cell line (HERS01a) and examined its gene and protein expression. Immunohistochemical staining and real-time PCR analysis showed that HERS01a cells expressed vimentin and N-cadherin as mesenchymal markers as well as cytokeratin14, E-cadherin, and p63 as epithelial stem cell markers. In the presence of TGF-β, HERS01a cells also expressed many more mesenchymal markers, as well as snail1 and 2 as EMT markers. Taken together, our data show that HERS01a displayed unique features associated with EMT in the root formation process, and will thus be useful for analyzing the biological characteristics of HERS and the molecular mechanism underlying the EMT.

Reduction of Egf Signaling Decides Transition From Crown to Root in the Development of Mouse Molars

Mouse, rat, and human molars begin to form their roots after the completion of crown morphogenesis. Though several signaling pathways and transcription factors have been implicated in the regulation of molar crown development, relatively little is known about the regulatory mechanisms involved in the transition from crown to root development. Tooth root formation is initiated by the development of Hertwigs epithelial root sheath (HERS) from the cervical loop in the enamel organ. In this study we examined the change in epidermal growth factor (Egf) signaling during this transition process. Immunohistochemical studies showed that the expression of Egf receptors in the enamel organ disappear gradually in the process and are not observed in HERS. Here, to examine the effect of Egf on the transition, we used the organ culture method to examine the root development. In the presence of Egf, stellate reticulum (SR) cells between the inner and outer epithelial layers in the enamel organ actively proliferated and maintained the enamel organ, and the formation of HERS was not observed. On the other hand, in either the absence of Egf or the presence of the inhibitor of Egf receptors, the SR cells disappeared and HERS formation started. Subsequently, root formation proceeded in the culture period. Therefore, disappearance of SR area may be a key event that controls the timing of onset of HERS formation, and Egf may be one of regulatory factors involved in the change from cervical loop epithelium to HERS during root development.

MicroRNAs and Osteolytic Bone Metastasis: The Roles of MicroRNAs in Tumor-Induced Osteoclast Differentiation.

Osteolytic bone metastasis frequently occurs in the later stages of breast, lung, and several other cancers. Osteoclasts, the only cells that resorb bone, are hijacked by tumor cells, which break down bone remodeling systems. As a result, osteolysis occurs and may cause patients to suffer bone fractures, pain, and hypercalcemia. It is important to understand the mechanism of bone metastasis to establish new cancer therapies. MicroRNAs are small, noncoding RNAs that are involved in various biological processes, including cellular differentiation, proliferation, apoptosis, and tumorigenesis. MicroRNAs have significant clinical potential, including their use as new therapeutic targets and disease-specific biomarkers. Recent studies have revealed that microRNAs are involved in osteoclast differentiation and osteolytic bone metastasis. In this review focusing on microRNAs, the author discusses the roles of microRNAs in osteoclastogenesis and osteolytic bone metastasis.

Preparation and in vivo evaluation of apatite/collagen packed composite by alternate immersion method and Newton press

Further development of bio-compatible, bio-absorbable, and osteo-conductive bio-materials is desired for bone grafts in dental and medical clinics. One candidate material might be a high-density apatite/collagen composite, which cures relatively large bone defects. To produce such a composite, we freeze-dried type I collagen solution, cross-linked the formed sponge by 2 wt % glutaraldehyde, immersed the insoluble sponge in CaCl(2) and Na(2)HPO(4) solutions alternately five times, and compacted the sponge by Newton press at 5000 kgf. For comparison, cross-linked collagen without alternate immersion was also pressed. SEM/EPMA, XRD, and FTIR analyses clarified that alternate immersion successfully coated the collagen sponge with hydroxyapatite. Packed apatite/collagen composite and collagen disks 6 mm in diameter and 0.5 mm in height were implanted in the subperiostea of rabbit tibiae for 2, 4, 8, and 12 weeks to assess bio-compatibility, bio-absorbability, and osteo-conductivity. Histological observations showed that the packed apatite/collagen composite was biocompatible, osteo-conductive for up to 8 weeks, and largely bio-absorbed at 12 weeks, while the packed collagen sponge caused an undesirable foreign body reaction, which worsened with the implantation period. The overall findings suggest that this packed apatite/collagen composite might be used as a new bio-absorbable bone graft material.

Molecular Mechanisms Regulating Transition from Crown to Root Formation in the Development of Mouse Molars

Abstract The tooth root is a foundation structure embedded in the alveolar bone of the jaws. Root formation starts after the completion of crown morphogenesis and proceeds slowly while coordinating the formation of the periodontal tissue that supports the tooth. Although several signaling pathways and transcription factors have been implicated in the regulation of molar crown development, relatively little is known about the regulatory mechanisms governing the transition from crown to root development. The formation of Hertwigs epithelial root sheath (HERS), consisting of 2 epithelial layers, is a key event in the initiation of root development. Recently, a new hypothesis about the mechanism of HERS formation was proposed based on the results of experiments using Fibroblast growth factor ( Fgf ) -10-deficient mice. In this review, we discuss this new hypothesis of HERS development and the signaling change for HERS formation during the transition process from crown to root development.

MicroRNAs: Potential Biomarkers and Therapeutic Targets for Alveolar Bone Loss in Periodontal Disease

Periodontal disease is an inflammatory disease caused by bacterial infection of tooth-supporting structures, which results in the destruction of alveolar bone. Osteoclasts play a central role in bone destruction. Osteoclasts are tartrate-resistant acid phosphatase (TRAP)-positive multinucleated giant cells derived from hematopoietic stem cells. Recently, we and other researchers revealed that microRNAs are involved in osteoclast differentiation. MicroRNAs are novel, single-stranded, non-coding, small (20–22 nucleotides) RNAs that act in a sequence-specific manner to regulate gene expression at the post-transcriptional level through cleavage or translational repression of their target mRNAs. They regulate various biological activities such as cellular differentiation, apoptosis, cancer development, and inflammatory responses. In this review, the roles of microRNAs in osteoclast differentiation and function during alveolar bone destruction in periodontal disease are described.

Biological Significance of Site-specific Transformation of Chondrocytes in Mouse Meckel's Cartilage

Abstract Meckels cartilage (MC), which develops as a template of the first skeleton of the mandible, undergoes site-specific fates. The anterior portion contributes to the formation of the rostral part of the mandible by endochondral ossification, while the posterior portion is involved in the formation of the ossicle bones. In contrast, the posterior part of the midportion (PM portion; located in the soft tissue) forms the sphenomandibular ligament, but its formation mechanism remains unclear. We found that chondrocytes in the PM portion have the potential to transform into other phenotypic cells under in vivo and in vitro conditions. In the present review, we focus on the results of cellular transformation based on our investigations. It has been clarified that MC chondrocytes in the PM portion transform into fibroblastic cells by phenotypic switching of the chondrocytes themselves in response to epidermal growth factor. In contrast to their conversion to fibroblastic cells in vivo , MC chondrocytes subjected to long-term culture or transplanted into ectopic sites, such as the spleen, expressed bone-type proteins accompanied by transformation into osteocytic cells. Since phenotypic transformation did not occur in mesoderm-derived costal chondrocytes, the findings suggest that neural crest-derived MC chondrocytes have high potential for transformation in response to altered stimuli.