Kunimichi Soma

Contribution of Nuclear Factor of Activated T Cells c1 to the Transcriptional Control of Immunoreceptor Osteoclast-associated Receptor but Not Triggering Receptor Expressed by Myeloid Cells-2 during Osteoclastogenesis

Bone homeostasis depends on the coordination of osteoclastic bone resorption and osteoblastic bone formation. Receptor activator of NF-κB ligand (RANKL) induces osteoclast differentiation through activating a transcriptional program mediated by the key transcription factor nuclear factor of activated T cells (NFAT) c1. Immunoreceptors, including osteoclast-associated receptor (OSCAR) and triggering receptor expressed by myeloid cells (TREM)-2, constitute the co-stimulatory signals required for RANKL-mediated activation of calcium signaling, which leads to the activation of NFATc1. However, it remains unknown whether the expression of immunoreceptors are under the control of NFATc1. Here we demonstrate that the expression of OSCAR, but not that of TREM-2, is up-regulated during osteoclastogenesis and markedly suppressed by the calcineurin inhibitor FK506, suggesting that OSCAR is transcriptionally regulated by NFATc1. NFATc1 expression results in the activation of the OSCAR promoter, which was found to be further enhanced by co-expression of PU.1 and microphthalmia-associated transcription factor (MITF). We further provide evidence that NFATc1 specifically regulates OSCAR by chromatin immunoprecipitation assay and quantification of OSCAR and TREM-2 mRNA in NFATc1-/- cells. Thus, OSCAR but not TREM-2 is involved in the positive feedback loop of the immunoreceptor-NFATc1 pathway during osteoclastogenesis. Although several immunoreceptors have been identified as co-stimulatory molecules for RANKL, the expression and function are differentially regulated. These mechanisms, possibly together with the delicate regulation of their ligands on osteoblasts, may provide the exquisite machinery for the modulation of osteoclastogenesis in the maintenance of bone homeostasis.

Effects of mechanical strain on proliferation and differentiation of bone marrow stromal cell line ST2

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 β-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.

In situ hybridization for matrix metalloproteinase-1 and cathepsin K in rat root-resorbing tissue induced by tooth movement

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.

Effect of compressive forces on extracellular matrix in rat mandibular condylar cartilage.

To reveal the effect of compressive force on the mandibular condylar cartilage, an appliance was set on 8-week-old Wistar rats to load continuous compressive force. Immunohistochemical and histochemical analyses were performed using toluidine blue, antibodies, and probes for aggrecan, hyaluronan, type II collagen, type X collagen, and 5-bromo-2″-deoxyuridine (BrdU). Histomorphometry and statistical analyses were also performed for aggrecan and BrdU immunostaining. In toluidine blue staining, tissue metachromasia was observed in the transitional zone and the hypertrophic zone of the mandibular condylar cartilage. In histomorphometry and statistical analysis, thickness of the cartilage decreased significantly in all regions in the 3-day experimental group. However, the thickness of the cartilage in the anterior region showed recovery while it decreased continuously in the posterior region. Distributional changes of aggrecan, hyaluronan, type II collagen, and type X collagen in the experimental groups were similar to those for toluidine blue staining. The immunostained area of all these molecules decreased as a result of the decrement of the cartilage area. However, enhanced immunostaining for aggrecan in the proliferative zone was observed only in the 1-day experimental group. BrdU-positive cells, observed in the proliferating zone and the transitional zone, decreased significantly in the experimental group 3 days after force was applied. These results demonstrate that continuous compressive forces on the mandibular condylar cartilage decrease the proliferation of chondrocytes and the amount of extracellular matrices.

Root Resorption Related to Hypofunctional Periodontium in Experimental Tooth Movement

Orthodontic movement of non-occluding teeth may result in undesirable apical root resorption. These teeth present with a histologically altered periodontium and are considered to be hypofunctional. The purpose of this study was to compare the amount of root resorption associated with a normal and a hypofunctional periodontium in rats during experimental tooth movement caused by heavy continuous force. The mandibular first molar was induced into a non-occluding condition in the hypofunctional periodontium group. Mesial orthodontic force was applied by means of 50-gram-force closed-coil springs for 15 days in both groups. The active root-resorption lacunae from histological sections, identified by tartrate-resistant acid phosphatase, were measured in terms of length, depth, and area. The results showed that the amount of root resorption was significantly greater in teeth with a hypofunctional periodontium than in those with a normal periodontium (p < 0.05). These results suggest that orthodontic movement of non-occluding teeth should be performed with caution.

Effect of Nitric Oxide on the Recovery of the Hypofunctional Periodontal Ligament

The relationship between occlusal stimuli and a hypofunctional periodontal ligament (PDL) structure has been reported, though changes in occlusal recovery conditions were still unclear. Nitric oxide (NO) produced by NO synthase (NOS) is considered a factor for vascular and immune system control, and it increases according to mechanical stimuli. The objective of this study was to examine the relationship between NOS and occlusal stimuli in PDL by comparing hypofunction with occlusal recovery. The study focused on the expression of endothelial NOS (eNOS) and inducible NOS (iNOS). Their expression significantly decreased in occlusal hypofunction compared with the control group and increased close to normal in an occlusal recovery group. The change in the immunopositive area was more dramatic than the immunopositive cell number. Moreover, the rate of iNOS increase was higher than that of eNOS. This study suggests that NO plays an important role in the recovery of the hypofunctional PDL.

Effects of Occlusal Stimuli on Alveolar/Jaw Bone Formation

Occlusion is known to influence the growth and development of the craniofacial complex. However, the consequences of occlusal hypofunction, or its recovery, on the amount of formation and development of alveolar bone and the jaw are not fully understood. Therefore, the present study was designed to elucidate the relationship between the occlusal stimuli and alveolar and jaw bone growth by the use of a hypofunction/recovered occlusal function model in growing rats. Bone histomorphometric analyses, including bone apposition rate and mineral apposition rate, were evaluated in double-labeled frontal sections of mandibular second molars. Results showed that occlusal hypofunction significantly suppressed alveolar and jaw bone formation compared with that in animals growing normally (p < 0.05). However, recovered occlusal function induced an enhancement in jaw bone formation. These results indicate the influence of occlusal function on alveolar and jaw bone formation during the growth period.

Innervation analysis of the small muscle bundles attached to the temporalis: truly new muscles or merely derivatives of the temporalis?

Detailed examinations were performed in ten temporal muscles from five cadavers to identify the muscle bundle arrangements of the temporalis and their innervation. Three additional muscle bundles were clearly observed in the main part of the fan-shaped temporalis: the anteromedial, anterolateral, and mid-lateral muscle bundles. Based on the origins, insertions and detailed innervation patterns, these bundles were considered as parts of the temporalis rather than independent muscles, although the anteromedial and anterolateral bundles had been recently reported as newly described muscles. A possible schematic model of the origins of these muscle bundles is proposed. We also report a branch from the posterior deep temporal nerve which was distributed to the temporal fascia and to the skin of the temporal region.

OPN deficiency suppresses appearance of odontoclastic cells and resorption of the tooth root induced by experimental force application.

Osteopontin (OPN) is a major non‐collagenous bone matrix protein implicated in the regulation of cell function. Although OPN is rich in the cementum of the tooth, the significance of OPN in this tissue is not understood. Tooth root resorption is the most frequent complication of orthodontic tooth movement (TM). The objective of this study was to examine the pathophysiological role of OPN in cementum of the tooth root. For this purpose, the upper right first molar (M1) in OPN‐deficient and wild‐type (WT) mice was subjected to mechanical force via 10 gf NiTi coil spring while the left side molar was kept intact to serve as an internal control. Micro‐CT section and the level of tartrate resistant acid phosphatase (TRAP)‐positive cells on the tooth root surface defined as odontoclasts were quantified at the end of the force application. In WT mice, force application to the tooth caused appearance of odontoclasts around the mesial surface of the tooth root resulting in tooth root resorption. In contrast, OPN deficiency significantly suppressed the force‐induced increase in the number of odontoclasts and suppressed root resorption. This force application also induced increase in the number of TRAP‐positive cells in the alveolar bone on the pressure side defined as osteoclasts, while the levels of the increase in osteoclastic cell number in such alveolar bone were similar between the OPN‐deficient and WT mice. These observations indicate that OPN deficiency suppresses specifically tooth root resorption in case of experimental force application. J. Cell. Physiol. 214: 614–620, 2008.

Osteopontin Negatively Regulates Parathyroid Hormone Receptor Signaling in Osteoblasts

Systemic hormonal control exerts its effect through the regulation of local target tissues, which in turn regulate upstream signals in a feedback loop. The parathyroid hormone (PTH) axis is a well defined hormonal signaling system that regulates calcium levels and bone metabolism. To understand the interplay between systemic and local signaling in bone, we examined the effects of deficiency of the bone matrix protein osteopontin (OPN) on the systemic effects of PTH specifically within osteoblastic cell lineages. Parathyroid hormone receptor (PPR) transgenic mice expressing a constitutively active form of the receptor (caPPR) specifically in cells of the osteoblast lineage have a high bone mass phenotype. In these mice, OPN deficiency further increased bone mass. This increase was associated with conversion of the major intertrabecular cell population from hematopoietic cells to stromal/osteoblastic cells and parallel elevations in histomorphometric and biochemical parameters of bone formation and resorption. Treatment with small interfering RNA (siRNA) for osteopontin enhanced H223R mutant caPPR-induced cAMP-response element (CRE) activity levels by about 10-fold. Thus, in addition to the well known calcemic feedback system for PTH, local feedback regulation by the bone matrix protein OPN also plays a significant role in the regulation of PTH actions.