Bone is a dynamic, living tissue that constantly undergoes remodeling through the coordinated actions of osteoblasts (cells that build bone) and osteoclasts (cells that break down bone). Parathyroid hormone (PTH) is used clinically to treat osteoporosis due to its anabolic actions on bone. PTH binds to its receptor (PPR), which is highly expressed in cells of the osteoblastic lineage, including osteocytes. Radiotherapy, a common cancer treatment, adversely impacts bone health by impairing osteoblast and osteoclast functions, leading to conditions such as osteopenia and osteoporosis. Pre-clinical and cell culture studies have indicated that radiotherapy damages bone. This study investigates whether pretreatment with parathyroid hormone (PTH1-34) can protect osteocytes from radiation-induced DNA damage and cell death. We utilized a conditionally immortalized murine osteocytic cell line, Ocy454-12H, treated it with PTH, and exposed it to varying radiation doses. Results showed that PTH significantly reduced γH2AX foci, indicating decreased DNA damage. qPCR analysis revealed modulated expression of DNA damage-related genes (FOXO1, P53, MKi67), suggesting enhanced DNA repair and reduced apoptosis. Additionally, PTH mitigated radiation-induced bystander effects, reducing osteoclast activity. These findings highlight PTH's potential as a therapeutic agent to protect bone health in radiotherapy patients.

Paget’s disease of bone (PDB) is a common disease with a strong genetic component. Approaches such as linkage analysis and candidate gene studies have shown that mutations in Sequestosome 1 (SQSTM1) explain up to 40% of familial cases and 10% of sporadic cases, however the majority of PDB patients have no mutations in this gene. Genome-wide association studies (GWAS) have recently identified new susceptibility loci for PDB including variants at CSF1, TNFRSF11A, OPTN, TM7SF4, PML, NUP205 and RIN3 loci. These loci were confirmed to be associated with PDB in various European populations. OPTN encodes optineurin, a widely expressed protein involved in many cellular processes but its role in bone metabolism is yet unknown. The aim of this PhD thesis was to investigate the role of OPTN in bone metabolism and PDB using in vitro and in vivo studies. In chapter 3, the OPTN rs1561570 identified by previous GWAS was examined for its association with the severity and clinical outcome of PDB in patients without SQSTM1 mutations. The results showed that rs1561570 was significantly associated with total disease severity score so that carriers of the risk allele “T” had higher severity score compared to non-carriers (P < 0.05). A trend for reduced quality of life physical scores (SF36) was also associated with the rs1561570 risk allele, but the relationship was not statistically significant. In order to identify functional variants within OPTN, the coding regions as well as the exon-intron boundaries were sequenced in 24 familial PDB cases and 19 controls. No mutation was found that could be predicted as pathogenic suggesting that disease susceptibility could be mediated by regulatory polymorphisms that influence gene expression. In chapter 4, the role of OPTN was investigated in osteoclast development using in vitro knockdown experiments. Optn was expressed in mouse bone marrow derived macrophages (BMDMs) as well as all stages of osteoclast development and it was significantly increased three days post RANKL treatment. Optn expression was knocked down in BMDMs and cells were induced to form osteoclast in the presence of RANKL and M-CSF. Compared to non-targeted cells, Optn depleted cells formed significantly more and larger osteoclasts (P< 0.05). Optn knockdown was also found to enhance osteoclast survival as well as RANKL-induced NFκB activation. In chapter 5, the role of OPTN was investigated in vitro from cells obtained from knock in mice with a loss-of-function mutation in Optn (OptnD477N/D477N). In agreement with the in vitro knockdown experiments, osteoclasts were significantly higher and larger in mutant mice compared to WT and the NF-B activity measured by luciferase reporter assay was significantly higher in cells from OptnD477N/D477N compared to WT during most stages of osteoclast development. OPTN from mutant and WT mice was co-precipitated with its CYLD binding-partner, which acts as a negative regulator to RANK signalling by inhibiting the TRAF6 downstream signalling. The data from this immunoprecipitation (IP) experiment revealed that defective OPTN interacted less with CYLD from mutant mice compared to WT. This study also showed that OPTN was expressed in osteoblasts and the expression rate did not change during osteoblast development. The data obtained from the mineralization assay revealed no significant difference between OptnD477N/D477N and WT. In chapter 6, I investigated the effect of the D477N loss of function mutation in Optn on bone metabolism. Bone Histomorphometrical analysis of OptnD477N/D477N mice showed higher bone resorption parameters (Oc.N/BS and Oc.S/BS) compared to wild type (WT). Osteoid analysis showed evidence of increased bone formation parameters (OS/BS and OV/BV) in mutant mice compared to WT. Calcein labelling showed a significant difference in mineral apposition rate (MAR) from mutant mice compared to WT. Analysis of serum biomarkers of bone turnover showed evidence of enhanced bone turnover in mutant mice compared to WT. Micro computed tomography (μCT) analysis of 4 and 14 months old mice showed no significant differences in bone morphology between WT and OptnD477N/D477N mice of both sexes. In conclusion, this study has shown for the first time that OPTN plays a role in regulating bone turnover by acting as a negative regulator of osteoclast differentiation. The data obtained from this study strongly suggest the crucial role of OPTN in RANK signalling. The effect of OPTN on osteoblast activity may be direct or indirect compensation for increased osteoclast activity. Further detailed studies will be required to explore the underlying mechanism of OPTN including downstream RANK signalling and a complete knockout model to corroborate these findings.

The studies presented in this dissertation offer evidence to support the hypothesis that polyphenolic curcuminoids isolated from the plant turmeric (Curcuma longa L.) are bone-protective in metabolic bone disorders characterized by excessive osteoclastic bone resorption. Activation of the critical transcription factor NF-kappaB in osteoclast precursor RAW 264.7 cells and osteoclastogenesis in rat primary bone marrow cell culture were directly inhibited by curcuminoids. Loss of bone mineral density and impaired structural connectivity of the trabecular bone microarchitecture associated with ovariectomy and estrogen deficiency were attenuated by curcuminoids in a rat model of postmenopausal osteoporosis. Additionally, the studies presented in this dissertation offer evidence for bone-protection conferred by curcuminoids by demonstrating for the first time their potent inhibitory effect on breast cancer cell secretion of the osteolytic peptide parathyroid hormone-related protein (PTHrP) via inhibition of Smad-dependent TGF-beta signaling in MDA-MB-231 cells. Finally, curcuminoids inhibited osteolytic lesion formation in a PTHrP-mediated murine model of breast cancer bone metastasis. Taken together, these novel and encouraging findings justify the need for further studies to determine whether curcuminoids may hold promise for the prevention of bone loss and associated complications in resorptive bone diseases in women.

Osteocytes (resident bone cells) are believed to sense loading-induced interstitial fluid flow in bone and transduce the signals to osteoclasts (bone resorption cells) and osteoblasts (bone formation cells) to regulate bone remodeling. Recent studies have shown that bone disuse causes osteocyte apoptosis, which precedes osteoclast activity at the local remodeling site. Although osteoclast precursors are known to travel via the circulation, the specific mechanism by which they are transported to the remodeling site is unclear. We hypothesized that lack of fluid flow induces osteocyte apoptosis. Furthermore, we hypothesized that osteocyte populations containing apoptotic osteocytes secrete cytokines that: 1) promote angiogenesis, and 2) activate the endothelium to promote osteoclast precursor adhesion to the endothelium such that osteoclast precursors can be delivered closer and directly to the remodeling site. In our in vitro studies, we found that lack of oscillatory fluid flow (mimicking mechanical disuse) promotes osteocyte apoptosis. In addition, osteocyte populations containing apoptotic cells promote endothelial cell proliferation, migration, and tubule formation. Inhibition of the potent angiogenic cytokine, vascular endothelial growth factor (VEGF), abrogated osteocyte apoptosis-mediated angiogenesis. Furthermore, we found that osteocyte populations containing apoptotic cells secrete cytokines that promoted osteoclast precursor adhesion. Upon further investigation, we found that apoptotic osteocytes secreted elevated levels of inflammatory cytokine interleukin 6 (IL-6), and its soluble receptor, sIL-6R. We demonstrated that both IL-6 and sIL-6R are required to activate the endothelium to express ICAM-1. Inhibition of ICAM-1 and IL-6 by blocking antibodies abolished apoptotic osteocyte-mediated osteoclast precursor adhesion. Our findings suggest for the first time that osteocytes communicate to endothelial cells directly to mediate angiogenesis and osteoclast precursor adhesion. Results from this study may assist in a better understanding of osteoclast precursor recruitment at the initial onset of bone resorption.

Millions of people around the world suffer from the debilitating effects of inflammatory bone diseases characterized by excessive bone loss due to an increase in osteoclast formation and activity. Osteoclasts are multinucleated cells responsible for bone resorption in health and disease. Arthritic joints also have elevated levels of extracellular matrix proteins affecting the disease progression. The interaction between osteoclasts and the external milieu comprised of extracellular matrix proteins through integrins is essential for modulating the formation and activity of osteoclasts. The focus of this thesis was to elucidate how the interaction between the extracellular matrix proteins and osteoclasts regulates osteoclast formation and activity and the role of alphavbeta3 in this process. In primary rabbit osteoclast cultures, blocking the integrin alphavbeta3 using Vitaxin, an anti-human alphavbeta3 antibody, decreased osteoclast resorption by decreasing osteoclast attachment. Vitaxin’s inhibitory effect on osteoclast attachment was enhanced when osteoclasts were pretreated with M-CSF, a growth factor known to induce an activated conformation of the integrin alphavbeta3. Using the RAW264.7 cell line, the effects of the matrix proteins fibronectin and vitronectin on osteoclast activity were compared to those of osteopontin. Both fibronectin and vitronectin decreased the number of osteoclasts formed compared to osteopontin. Fibronectin’s effect on osteoclastogenesis was through decreasing pre-osteoclast migration and/or fusion but not through inhibiting their recruitment. In contrast, fibronectin induced resorption through increasing resorptive activity per osteoclast in comparison to vitronectin and osteopontin. These stimulatory effects were accompanied by an increase in the pro-inflammatory cytokines nitric oxide and IL-1beta Crosstalk between the signalling pathways of nitric oxide and IL-1betawas suggested by the ability of the nitric oxide inhibitor to decrease the level of IL-1beta which occurred exclusively on fibronectin. Osteoclasts on fibronectin also had a compact morphology with the smallest planar area while vitronectin increased the percentage of osteoclast with migratory morphology and osteopontin induced osteoclast spreading. The increase in compact morphology on fibronectin was associated with a decrease in extracellular pH. Low extracellular pH was found to increase the total time osteoclasts spend in a compact phase. These results show that matrix proteins differentially regulate osteoclast formation, activity and morphology.

A periodontite é uma doença de natureza multifatorial e infecciosa, que resulta na inflamação e perda dos tecidos de suporte dos dentes. Essa inflamação é causada por bactérias associadas ao biofilme, causando a perda progressiva de inserção. Os bisfosfonatos são fármacos com capacidade de inibir a reabsorção óssea, atuando nas células clásticas. O presente estudo teve como objetivo investigar os efeitos do alendronato, um bisfosfonato nitrogenado com grande potência antireabsortiva, na evolução da doença periodontal induzida em ratos, bem como a possível presença de necrose óssea no processo alveolar. Foram utilizados 48 ratos Wistar albinos, do sexo masculino, com 3 meses de vida e peso médio de 250g. Os animais foram divididos aleatoriamente em dois grupos: Alendronato (ALN) e Controle (CON). A periodontite foi induzida com a inserção de um fio de seda 4.0 no sulco gengival do segundo molar superior. Os ratos do grupo ALN, receberam doses diárias de 2,5 mg/kg durante 7 dias antes e 7, 14, 21 e 30 dias após a indução da doença; o grupo CON recebeu solução salina estéril. Nos tempos citados as maxilas foram fixadas, descalcificadas e incluídas em parafina ou resina Spurr. Os cortes foram corados com HE, para análise morfológica, e histomorfométrica. Alguns cortes foram submetidos à imuno-histoquímica para detecção de RANKL e OPG. Foi utilizado o método TRAP, marcador de osteoclastos e microscopia eletrônica de transmissão para análise ultraestrutural. O ALN inibiu a reabsorção da crista alveolar de todos os grupos tratados. As células clásticas apresentaram-se em estado latente. No grupo controle a crista alveolar foi reabsorvida e o TRAP revelou clastos ativos, achados confirmados pela microscopia eletrônica de transmissão. A expressão de RANKL, molécula ativadora da célula clástica, não foi inibida pela droga. A expressão de OPG foi aumentada nos animais tratados. Os animais do grupo tratado durante 21 e 30 dias, apresentaram sinais de osteonecrose na crista alveolar, como lacunas de osteócitos vazias e regiões exposta de osso. Os resultados demonstraram que o uso de alendronato durante a doença periodontal inibe a reabsorção óssea e que durante tempos prolongados pode gerar osteonecrose na região da crista óssea. / Periodontitis is an infectious disease of multifactor nature that results in the inflammation of the tissues supporting the teeth. This inflammation is caused by accumulation of biofilm and causes progressive insertion and bone loss. The bisphosphonates are drugs with the capability to inhibit the activity of clastic cells. The aim of this study was to investigate the effects of alendronate, a nitrogenated bisphosphonate with high antiresorptive power on experimental periodontal disease, and to analyze the possible presence of osteonecrosis in the rat alveolar process. Forty-eight male Wistar rats, three months old, with 250g weight were used. The animals were randomly divided into two groups: Alendronate (ALN) and Control (CON). The periodontitis was induced with a 4.0 silk wire inserted into the gingival sulcus around the right upper second molar. The ALN rats, received daily doses of 2.5 mg/kg alendronate (ALN) for 7 days before the induction of periodontitis; the treatment continued for additional 7, 14, 21 or 30 days. The CON rats, received sterile saline solution. In the time points cited, the maxillae were fixed, decalcified and embedded in Spurr resin or paraffin. The specimens were morphologically analyzed in HE stained sections, after which histomorphometry was carried out. Some stained sections were used for immunolabeling for RANKL and OPG. The osteoclasts were marker by tartrate-resistant acid phosphatase (TRAP) histochemistry. The ultrathin sections were examined in a transmission electron microscope. ALN reduced the activity of osteoclasts and significantly decreased the resorption of the alveolar crest. In the control group the alveolar crest appeared resorbed, while TRAP showed active osteoclasts, findings confirmed by transmission electron microscopy. The expression of RANKL, an osteoclast-activating molecule, was not inhibited by the drug. The expression of OPG was increased in the treated animals. The animals of the group treated for 21 and 30 days showed signs of osteonecrosis of the alveolar crest, as empty osteocyte lacunae in the exposed bone regions. The results showed that the use of ALN for periodontal disease inhibited bone resorption; when it was administered for prolonged periods it can cause osteonecrosis in the bone crest area.

Breast cancer most commonly metastasizes to the bone, where it perpetuates the vicious cycle leading to osteolytic lesions. This occurs when secreted factors from breast cancer cells disrupt bone homeostasis by deregulation of osteoblast bone formation, and enhance osteoclast bone degradation thereby releasing bone matrix bound growth factors leading to further tumor growth. Although the use of osteoclast targeting agents, such as bisphosphonates and RANK-L inhibitors, are common practice for the treatment of bone metastasis, they have not been shown to increase patient survival. We therefore sought to investigate the role of focal adhesion kinase (FAK), a potential therapeutic target, in the treatment of breast cancer mediated osteolysis. FAK is a non-receptor tyrosine kinase known to directly regulate tumor progression and metastasis; it is also expressed in all of the cell types involved in breast cancer mediated osteolysis. Thus, we hypothesized that the inhibition of FAK would restore normal bone homeostasis, as well as mediate direct anti-tumor activity. FAK depletion resulted in the decrease of expression of several osteolytic factors secreted by breast cancer cells. However, the use of FAK depleted breast cancer conditioned media did not prevent breast cancer mediated osteoclastogenesis in an osteoblast/osteoclast coculture. In monoculture however, using the FAK inhibitor PF-271, we have shown that FAK inhibition leads to increased apoptosis of mature osteoclasts, and their decreased ability to degrade mineralized bone matrix, perhaps in part due to reduced expression of lytic factors such as tartrate resistant acid phosphatase and cathepsin K. Further, FAK inhibition in osteoblast monoculture led to a decrease in their ability to express the maturation factor alkaline phosphatase, and also inhibited their ability to induce mineralization. This inhibition may be due in part to the specific effects of FAK inhibition using PF-271, which may result in decreased levels of p53 in treated osteoblasts. These results suggests that the pharmacological inhibition of FAK can effect all three cell types involved in the vicious cycle of bone metastasis, and as such could be a beneficial therapeutic for patients with bone metastasis resulting in prevention of bone degradation along with direct inhibition of tumor growth. However, it may require further evaluation in animal models to determine if observed effects on osteoblast activity in vitro also occurs in vivo with possible detrimental effects on restoration of damaged bone.

歯科基礎医学会, 日下部 豊寿 = Toyohisa Kusakabe, 実験的歯の移動時における圧迫側歯槽骨に生じる背部骨吸収と血管分布 = Rear resorption at the pressure side incident in orthodontic tooth movement, 歯科基礎医学会雑誌, 39(6), DEC 1997, pp.623-640 / Hokkaido University (北海道大学) / 博士 / 歯学

For bone resorption, vacuolar-type H+-ATPases (V-ATPases) on the plasma membranes of osteoclasts acidifies the extracellular millieu adjacent to the bone surface. The V-ATPase a3 and d2 subunits are enriched in osteoclasts. B2 subunit is also expressed on the osteoclast plasma membrane. Disruption of genes encoding subunits a3 and d2 impairs bone resorption. In this study, we have shown an interaction between the a3-B2 and a3-d2 subunits. Luteolin and KM91104 were found to be effective inhibitors of the a3-d2 and a3-B2 interactions respectively. Secondary assays revealed luteolin and KM91104 were not toxic to cells, did not affect osteoclastogenesis yet inhibited bone resorption. Furthermore luteolin did not affect V-ATPase subunit formation or assembly. Inhibitors of osteoclast resorption that do not affect osteoclast viability, preserve osteoclast–osteoblast signalling are desirable than existing anti-resorptives. Therefore, V-ATPase a3–d2 and a3-B2 interactions are viable targets for anti-resorptive therapeutics for osteoporosis.

For bone resorption, vacuolar-type H+-ATPases (V-ATPases) on the plasma membranes of osteoclasts acidifies the extracellular millieu adjacent to the bone surface. The V-ATPase a3 and d2 subunits are enriched in osteoclasts. B2 subunit is also expressed on the osteoclast plasma membrane. Disruption of genes encoding subunits a3 and d2 impairs bone resorption. In this study, we have shown an interaction between the a3-B2 and a3-d2 subunits. Luteolin and KM91104 were found to be effective inhibitors of the a3-d2 and a3-B2 interactions respectively. Secondary assays revealed luteolin and KM91104 were not toxic to cells, did not affect osteoclastogenesis yet inhibited bone resorption. Furthermore luteolin did not affect V-ATPase subunit formation or assembly. Inhibitors of osteoclast resorption that do not affect osteoclast viability, preserve osteoclast–osteoblast signalling are desirable than existing anti-resorptives. Therefore, V-ATPase a3–d2 and a3-B2 interactions are viable targets for anti-resorptive therapeutics for osteoporosis.