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The intricate processes of osteoclastogenesis are highly dependent on the dynamic regulation of the actin cytoskeleton. Adseverin, a member of the gelsolin superfamily of actin binding proteins, regulates actin remodeling by severing and capping actin filaments in a calcium dependent manner. The objective of this project was to characterize the role(s) of adseverin during osteoclastogenesis, by assessing adseverin expression throughout osteoclastogenesis and through differentiation assays using a knockdown strategy. Methods: qRT-PCR and immunoblot analyses were used to examine adseverin expression during osteoclastogenesis. A stable adseverin knockdown macrophage cell line was generated using a retroviral shRNA construct. Results: Adseverin expression increased significantly in response to RANKL during the early phases of osteoclastogenesis, and adseverin was highly expressed in mature osteoclasts. Adseverin knockdown macrophages experienced a major osteoclastogenesis defect, most likely caused by a defect in pre-osteoclast fusion. Conclusion: Adseverin is a RANKL induced early and pro-fusion marker of osteoclastogenesis.

The intricate processes of osteoclastogenesis are highly dependent on the dynamic regulation of the actin cytoskeleton. Adseverin, a member of the gelsolin superfamily of actin binding proteins, regulates actin remodeling by severing and capping actin filaments in a calcium dependent manner. The objective of this project was to characterize the role(s) of adseverin during osteoclastogenesis, by assessing adseverin expression throughout osteoclastogenesis and through differentiation assays using a knockdown strategy. Methods: qRT-PCR and immunoblot analyses were used to examine adseverin expression during osteoclastogenesis. A stable adseverin knockdown macrophage cell line was generated using a retroviral shRNA construct. Results: Adseverin expression increased significantly in response to RANKL during the early phases of osteoclastogenesis, and adseverin was highly expressed in mature osteoclasts. Adseverin knockdown macrophages experienced a major osteoclastogenesis defect, most likely caused by a defect in pre-osteoclast fusion. Conclusion: Adseverin is a RANKL induced early and pro-fusion marker of osteoclastogenesis.

Osteoclasts are multinucleated, bone resorbing cells that carry out their function using specialized actin-based structures called actin rings and podosomes. Rho GTPases function as molecular switches that regulate the actin cytoskeleton in osteoclasts and many other cell types. Filamin A (FLNa) and SBDS are two proteins that have the potential to interact with both F-actin and Rho GTPases, and thus regulate osteoclast formation, differentiation, or function. We found that in FLNa-null pre-osteoclasts, activation of RhoA, Rac1, and Cdc42 was perturbed, leading to defective pre-osteoclast migration prior to fusion. Ablation of SBDS resulted in the blockage of osteoclast differentiation downstream of RANK and defective RANKL-mediated upregulation of Rac2 that is required for pre-osteoclast migration. Therefore, both FLNa and SBDS are required to coordinate Rho GTPase activation during osteoclastogenesis, in addition to a role for SBDS in osteoclast differentiation downstream of RANK.

Osteoclasts are multinucleated, bone resorbing cells that carry out their function using specialized actin-based structures called actin rings and podosomes. Rho GTPases function as molecular switches that regulate the actin cytoskeleton in osteoclasts and many other cell types. Filamin A (FLNa) and SBDS are two proteins that have the potential to interact with both F-actin and Rho GTPases, and thus regulate osteoclast formation, differentiation, or function. We found that in FLNa-null pre-osteoclasts, activation of RhoA, Rac1, and Cdc42 was perturbed, leading to defective pre-osteoclast migration prior to fusion. Ablation of SBDS resulted in the blockage of osteoclast differentiation downstream of RANK and defective RANKL-mediated upregulation of Rac2 that is required for pre-osteoclast migration. Therefore, both FLNa and SBDS are required to coordinate Rho GTPase activation during osteoclastogenesis, in addition to a role for SBDS in osteoclast differentiation downstream of RANK.

The extensive infiltration of immune cells in the joints of patients affected by rheumatoid arthritis (RA), and the subsequent production of pro-inflammatory cytokines triggers bone erosion through the extensive stimulation of bone resorbing osteoclasts (OCs). The activity of γδ T cells has been implicated to influence the onset and severity of the disease pathology in murine models of human RA. With this study the effects of γδ T cells for influencing OC differentiation and resorptive activity were assessed in vitro. Activated γδ T cells exerted inhibitory effects on OC differentiation and resorptive activity, these effects were mediated by the release of soluble factors, since similar inhibitory effects were obtained using conditioned medium (CM) from activated γδ T cells. The primary mediator of such effects was determined to be IFN, since neutralisation markedly restored OC differentiation and resorptive activity. γδ T cell proliferation, activation and survival following culture with autologous mature OCs were assessed by flow cytometry. Interestingly, OCs and OC-derived CM induced activation of γδ T cells as determined by the expression of the early activation marker CD69. A mediator of this stimulatory effect on T cells was found to be TNF, since neutralisation of TNFα decreased the stimulatory effect of OCs on CD69 expression. Consistently, OCs, but not OC-derived CM, increased the proliferation of IL-2-stimulated γδ T cells and also supported the survival of resting γδ T cells. This study provides new insights into the in vitro interactions between human γδ T cells and OCs, moreover it defines osteoclasts as immune competent cells capable of influencing the activation status and the viability of T lymphocytes, and provide evidence for a novel stimulatory effect of OCs on γδ T cells.

Inflammatory bone diseases like rheumatoid arthritis and periodontal disease lead to increased bone loss in the inflamed areas. The multinucleated bone resorbing cells, the osteoclasts, present in these diseases are larger than normal, and these larger osteoclasts (10+ nuclei) resorb more bone and more often than smaller osteoclasts (2-5 nuclei). Thus, the focus of this thesis was to determine if there are differences in mechanisms of osteoclast activation between large and small osteoclasts. Experiments using authentic rabbit osteoclasts and RAW 264.7-derived osteoclasts revealed differences in the expression of a number of activating factors; with large osteoclasts expressing higher levels of activating receptors (RANK, IL-1RI, TNFR1 and integrins αv and β3), as well as enzymes involved in cellular resorption, while small osteoclasts expressed higher levels of an alleged fusion receptor and the inhibitory receptor, IL-1RII. Further studies revealed that large osteoclasts more readily responded to stimulation by IL-1 compared to small osteoclasts and at lower concentrations suggesting this is a result of their higher expression of activating receptors. Differences in responses to the IL-1 isoforms, IL-1α and IL-1β, were also seen in large osteoclasts: IL-1α generated more large osteoclasts over the course of differentiation, while IL-1β induced changes in cell morphology and in the induction of integrin β3 phosphorylation. These observations suggested that differences in osteoclast responses are induced by IL-1α and IL-1β and it led to the hypothesis that there are differences in signaling between large and small osteoclasts. To elucidate differences in signaling mechanisms a signaling pathway microarray was used which revealed higher expression of Vegfa in large compared to small osteoclasts. Osteoclast differentiation with RANKL increased Vegfa gene expression in a time-dependent manner and VEGF-A secretion was elevated in populations enriched for large osteoclasts. Furthermore, mechanistic studies with inhibitors of transcription factors involved in differentiation revealed that RANKL-mediated Vegfa expression in large osteoclasts was regulated by the NF-κB pathway via induction of Hif1α. These results support the hypothesis that signaling differences exist between large and small osteoclasts and implicates VEGF-A in osteoclast hyperactivity in inflammatory conditions.

The fall in circulating oestrogen (E2) after the menopause leads to an increased rate of bone remodelling, excessive osteoclast activity and a greater fracture risk. Until recently hormone replacement therapy (HRT) was prescribed to post-menopausal women to prevent bone loss, however HRT is associated with an elevated incidence of cardiovascular disease, stroke and cancer. These side-effects led to an interest in naturally occurring compounds with oestrogenic action such as phytoestrogens (PEs), which are non-steroidal-plant derived compounds. Human trials and animal studies suggest a beneficial effect of PEs on bone mass, although their ability to modify osteoclast formation in response to key inflammatory cytokines has not been examined. The aim of the following studies was to determine the effect of physiologically relevant concentrations of genistein, coumestrol and daidzein on TNF-α-induced osteoclast formation, osteoblasts differentiation and T cell activity. Genistein (10-7 M), daidzein (10-5 M), and coumestrol (10-7 M) significantly reduced TNF-α-induced TRAP positive osteoclast formation and bone resorption, which was prevented by the E2 antagonist ICI 182,780. The suppressive action on osteoclast formation was associated with a significant reduction in TNF-α-induced c-fos and NFATc1 mRNA expression and NFATc1 nuclear translocation. Constitutive c-fos expression prevented the inhibitory action of PEs on osteoclast differentiation, resorption and NFATc1 expression. The effect of PEs, in the presence or absence of the anabolic nutritional factor zinc, on osteoblasts differentiation and bone nodule formation was examined in-vitro. Coumestrol (10-5 to 10-7 M), daidzein (10-5 to 10-6 M) and genistein (10-5 M) enhanced bone nodule formation and ALP activity in human osteoblasts, and this effect was significantly augmented in the presence of zinc (10-5 M). Furthermore, PEs and zinc increased Runx2 mRNA expression and Zn2+ augmented the inhibitory effect of PEs on RANKL/OPG ratio. This suggests that in addition to the direct inhibitory effect on osteoclast formation PEs also in-directly reduce the osteoblastsic stimulus for osteoclast formation and promote bone formation. E2 deficiency is thought to promote osteoclastogenesis by modifying Thelper1 (Th1) cell proliferation and inflammatory cytokine production in particular TNF-α. I therefore examined the effect of PEs on T cell proliferation and inflammatory cytokine production. All PEs prevented the augmentative effect of con A stimulated T cells on osteoclast formation in co-culture. However the mechanism of action varied, genistein reduced con A stimulated TNF-α, IL-1β and RANKL expression with little effect on viability, coumestrol decreased cell viability and TNF-α expression whereas the inhibitory effect of daidzein was mediated via suppression of viable T cell number. This study provides novel evidence that PEs have multiple effects on bone cell activity, directly inhibiting TNF-α-induced osteoclast formation, reducing the osteoblasts and T cell derived stimulus for osteoclast formation and augmenting osteoblasts differentiation and bone formation. Thus, PEs have a potential role in the treatment of post-menopausal osteoporosis and inflammatory skeletal disorders and that the beneficial effect noted in previous studies is mediated through multiple mechanisms.

The skeletal system is a dynamic organ that provides support, protection, aids in the production of all blood cells, and serves as a calcium ion reservoir. It is constantly undergoing a process called ‘remodeling’, which occurs through the actions of osteoclast and osteoblast cells. The former is responsible for breaking down bone whereas the latter secretes an organic matrix for bone synthesis. Two experiments were conducted to analyze the effects of physiological perturbations on the bone remodeling process. Specifically, the impact of aging and selective serotonin re-uptake inhibitor administration coupled with lactation on bone morphology and composition were observed. The bony skeleton is not a stagnant organ, rather it undergoes functional, mechanical, and compositional changes throughout life. Thus, we wanted to determine the consequences age had on various bone parameters and found a decrease in bone volume fraction (BV/TV), trabecular number (Tb.N), cortical thickness (C.Th), and an increase in periosteal area, endosteal area, and cortical porosity (C.Po). In regards to pregnancy, post-partum depression is a common condition. As a result, many women utilize selective serotonin re-uptake inhibitors (SSRIs) to combat the negative symptoms associated with it. Serotonin is an important hormone involved in mood regulation and the mammary-derived form has a role in lactation. It induces the production of parathyroid hormone-related protein, which is essential for regulating maternal calcium. Because the calcium source for milk production is derived from the maternal bone, we were interested in the impact peri-partum usage of SSRIs had on maternal bone mineral density. Additionally, we sought to understand the effect that circulating SSRIs had on the bone formation of pups. Analysis revealed age-related decreases in BV/TV, Tb.N, trabecular thickness (Tb.Th), C.Th and increases in trabecular spacing (Tb.Sp) and C.Po, with the effects being exacerbated in cohorts treated with a SSRI during lactation. We did not observe, however, any change in trabecular bone mineral density (Tb.BMD) or cortical bone mineral density (C.BMD) over time. In regards to the pups, we observed similar results to those of the dams in addition to a significant reduction in femur length. With the data obtained from the two projects, we hope that they bring awareness to potential consequences that physiological perturbations may have on the bone remodeling process. Both experiments have clinical correlations to humans. Further understanding the relationship between aging and bone may aid in developing methodologies to prevent age-related changes. On the other hand, analyzing the effects that SSRI usage has on maternal and child bone density may result in alternative methods to combat post-partum depression.

歯科基礎医学会, 飯塚 正 = Tadashi Iizuka, 放射線照射ラットの抜歯創治癒過程に関する形態学的研究 = Morphological studies on the healing process of extraction wound in irradiated rats, 歯科基礎医学会雑誌, 26(3), SEP 1984, pp.745-785 / Hokkaido University (北海道大学) / 博士 / 歯学