Osteoclasts are cells that degrade bone, by forming a ruffled border (RB) membrane, contained within an actin-rich attachment site (the sealing zone; SZ). Lysosomal vesicles fuse to the RB, and release their contents into the extracellular space to degrade bone matrix. LC3, a marker of autophagosomes, localises to the RB, implying that either canonical autophagy (i.e. autophagosomes) or non-canonical autophagy (a process where LC3 localises to non-autophagic membrane) is involved in the resorptive function of osteoclasts. To examine this in detail, this study used a model with reduced canonical autophagy (FIP200 conditional knockout mouse), and two non-canonical autophagy deficient models (Rubicon knockdown in RAW 264.7-cell derived osteoclasts and an Atg16L1 WD40 domain knockout mouse). Using advanced imaging and molecular techniques I examined whether impairing either process affected LC3 RB localisation and resorption. Reducing canonical autophagy through FIP200 deficiency did not significantly affect GFP-LC3 RB localisation or bone homeostasis. However, impairing non-canonical autophagy resulted in a trend towards increased resorption in vitro. In Atg16L1 WD40 domain-deficient osteoclasts, this may be due to the significantly larger SZs formed in the mutants, which were often stable and contained LysoTracker-positive acidic vesicles within them, putatively signalling increased resorption. As LC3 was frequently observed at the RB, I then examined the LC3-interacting lysosomal adaptor protein, PLEKHM1. I showed that in the PLEKHM1 functional knockout mouse model (R714 STOP), osteoclasts still form RBs but have impaired resorption in vitro. Detailed analysis of multiple aspects of resorption in PLEKHM1 deficient osteoclasts, was required to uncover these defects which may underlie the osteopetrotic phenotype observed in PLEKHM1 deficient mice. Overall this work reveals the potential role of non-canonical autophagy in osteoclast function. Additional dissection of this pathway in osteoclasts may uncover further new insights regarding the regulation of bone resorption and defects underlying bone disorders.

Nitric oxide (NO) is a short lived free radical regulating bone turnover and bone cell function (1, 2). Osteoclasts are multinucleated bone resorbing cells which form by fusion of pre-osteoclasts. In addition, NO is a signaling molecule in mechanical loading of the bone (3), and in orthodontic tooth movement (OTM) (4). In OTM, force is applied to the tooth and transferred to the bone resulting in bone remodeling leading to tooth movement. This project has two parts: 1) NO in osteoclastogenesis: a) An intense NO signal was observed in pre-osteoclasts preceding cell fusion. b) Osteoclastogenesis increased when cells were exposed to the NOS inhibitor, L-NMMA, during their differentiation phase. c) In contrast, pre-osteoclast fusion decreased in presence of to L-NMMA during the fusion phase. d) NOS inhibitors, decreased osteoclast formation. e) The inhibitory effect of L-NMMA on osteoclast formation was abolished with increasing concentrations of sRANKL. f) NO donors increased osteoclast formation. g) An increase in NO production coincided with pre-osteoclasts fusion. h) Inhibiting fusion decreased osteoclast formation and NO production. i) L-NMMA decreased, while NO donors increased actin free barbed ends. Conclusion: While NO initially negatively regulates pre-osteoclast differentiation, it later facilitates the fusion of mononuclear pre-osteoclasts, possibly by up regulating actin remodeling. 2) Involvement of NO in OTM: Differential expression of NOS isoforms was investigated in periodontal ligament (PDL) and bone in tension and pressure sides using immunohistochemistry with NOS isoforms in a rat model of OTM. a) Expression of all isoforms was increased in the tension side. b) iNOS and nNOS expressions in the pressure side with the cell free zone were decreased while in the pressure side without the cell free zone were increased. c) The intensity of eNOS staining was increased in the tension side. d) Duration of force only changed the pattern of nNOS expression. e) Osteocyte NOS expression did not change. Conclusion: All NOS isoforms are involved in OTM with different expression patterns between the tension and pressure with nNOS being more involved in early OTM. PDL cells, rather than osteocytes are the mechanosensors in early OTM with regards to NO signaling.

Nitric oxide (NO) is a short lived free radical regulating bone turnover and bone cell function (1, 2). Osteoclasts are multinucleated bone resorbing cells which form by fusion of pre-osteoclasts. In addition, NO is a signaling molecule in mechanical loading of the bone (3), and in orthodontic tooth movement (OTM) (4). In OTM, force is applied to the tooth and transferred to the bone resulting in bone remodeling leading to tooth movement. This project has two parts: 1) NO in osteoclastogenesis: a) An intense NO signal was observed in pre-osteoclasts preceding cell fusion. b) Osteoclastogenesis increased when cells were exposed to the NOS inhibitor, L-NMMA, during their differentiation phase. c) In contrast, pre-osteoclast fusion decreased in presence of to L-NMMA during the fusion phase. d) NOS inhibitors, decreased osteoclast formation. e) The inhibitory effect of L-NMMA on osteoclast formation was abolished with increasing concentrations of sRANKL. f) NO donors increased osteoclast formation. g) An increase in NO production coincided with pre-osteoclasts fusion. h) Inhibiting fusion decreased osteoclast formation and NO production. i) L-NMMA decreased, while NO donors increased actin free barbed ends. Conclusion: While NO initially negatively regulates pre-osteoclast differentiation, it later facilitates the fusion of mononuclear pre-osteoclasts, possibly by up regulating actin remodeling. 2) Involvement of NO in OTM: Differential expression of NOS isoforms was investigated in periodontal ligament (PDL) and bone in tension and pressure sides using immunohistochemistry with NOS isoforms in a rat model of OTM. a) Expression of all isoforms was increased in the tension side. b) iNOS and nNOS expressions in the pressure side with the cell free zone were decreased while in the pressure side without the cell free zone were increased. c) The intensity of eNOS staining was increased in the tension side. d) Duration of force only changed the pattern of nNOS expression. e) Osteocyte NOS expression did not change. Conclusion: All NOS isoforms are involved in OTM with different expression patterns between the tension and pressure with nNOS being more involved in early OTM. PDL cells, rather than osteocytes are the mechanosensors in early OTM with regards to NO signaling.

CCN protein family members (CYR61, CTGF, Nov, Wisp-1, Wisp-2 and Wisp-3) have important roles in many different processes including angiogenesis, inflammation, remodelling of extracellular matrix and tumorigenesis. In bone, CCN1 increases osteoblastogenesis via Wnt3A signalling and activation of -catenin which, in turn, upregulates CCN1 expression. The exact role of CCN1, CCN2, and CCN6 in osteoclast physiology are not known but we have recently shown that recombinant human (rh)CCN1 inhibits osteoclastogenesis in vitro. The aim of this study was to determine: 1) the expressions of all six members of the CCN protein family in osteoblasts and osteoclasts; 2) the functions of recombinant human CCN2, CCN6 in osteoclastogenesis; 3) whether osteoblast-derived CCN1 may mediate the effect of CCN1 on osteoclast formation and the roles of osteoblast-derived CCN1 and/or osteoclast-derived CCN1 in osteoblast and/or osteoclast differentiation; 4) which signalling pathways are involved in the function of CCN1 in osteoclasts and osteoblasts. We found CCN1-5 but not CCN6 expressed in murine osteoclasts and osteoblasts. All six members were expressed in human OA osteoblasts but only CCN1-3 were detected in human osteoclasts using quantitative RT-PCR. rhCCN1 (in agreement with our previous observations), and also 2 and 6 inhibited human and mouse osteoclast formation in a concentration-dependent manner. We generated and validated an expression construct to specifically overexpress CCN1 in osteoblasts. Incorporation of CCN1-specific siRNA reduced CCN1 expression to between 12.5% and 50% of control osteoblast cultures. In both co-cultures with direct contact between osteoblasts and osteoclast co-cultures as well as Transwell cultures, overexpression of CCN1 in osteoblasts decreased the formation of TRAP positive multinucleated osteoclast-like cells, while siRNA mediated knockdown of CCN1 in the osteoblasts resulted in increased osteoclast-like cell formation. These data suggest that osteoblast-derived CCN1 is a secreted negative regulator of osteoclastogenesis. Moreover, overexpression or knockdown of CCN1 in osteoclast precursors inhibited or increased osteoclast differentiation whilst overexpression or knockdown CCN1 in osteoblasts increased or inhibited osteoblast mineralization respectively. Further investigation found that CCN1 increased Wnt and MAPK signalling in osteoblasts cultured in mineralization medium and inhibited Wnt and IGF-1 signalling during osteoclast differentiation. In conclusion, paracrine and autocrine effects of CCN1 have been demonstrated in osteoclasts and osteoblasts in this study and Wnt, MAPK, amd IGF-1 signalling pathways, may be involved in these effects.

Osteoclasts are multinucleated, terminally differentiated cells found in bone tissue, which have the unique ability to resorb calcified substrates. The study of human osteoclasts has been restricted in the past due to difficulties in obtaining these cells. Recently, however, cell culture techniques have been devised that allow human osteoclasts to be grown in culture from their mononuclear precursors found in peripheral blood, therefore providing a constant supply of these cells. These cultures are based on the discovery of RANKL (receptor activator for NFJ(B ligand), which has recently been shown to playa central role in osteoclast formation and activation. This thesis has initially characterised cells grown in such cultures to confirm that they are authentic human osteoclasts, possessing osteoclast markers and with the ability to resorb calcified substrates. Once these cultures were established, and the authenticity of human osteoclasts grown in these cultures was confirmed, these cells were used to study the effects of extracellular nucleotides on human osteoclast acti vity. Adenosine-5' -triphosphate (ATP) is well known as an intracellular energy source. This and other nucleotides, however, also exist extracellularly, where they are agonists at a group of receptors termed P2 receptors. This receptor family is subdivided into P2X ligand gated ion channels, and P2Y G-protein coupled receptors. Bone-forming osteoblast cells, and boneresorbing osteoclast cells both express multiple SUbtypes of these receptors. Studies examining the effects of extracellular nucleotides on osteoclasts have been largely limited to non-human cells in the past due to the difficulty in obtaining human osteoclasts. This thesis, therefore, has examined the effects of extracellular nucleotides on human osteoclast activity. Human osteoclasts and their precursors were shown to express mRNA for nearly all of the P2Y and P2X receptor subtypes. ATP was found to both activate human osteoclast formation, by acting directly on P2X receptors expressed by osteoclast precursors, and to stimulate osteoclast resorption indirectly by acting at osteoblast-expressed P2Y) receptors to induce elevated RANKL expression by these cells. Activation of P2Y receptors expressed by osteoclasts was shown to result in downstream activation of MAPKinase pathways. Parathyroid hormone (PTH) is considered to be one of, if not the most important systemic factor in the regulation of bone. Co-stimulation of UMR-I 06 osteoblast-like cells with this hormone and P2Y) agonists resulted in potentiation of P2Y) agonist-induced [Ca2+Ji release by PTH, while PTH alone produced no [Ca2+]j elevation at all. The mechanism of this potentiation was attributed to Gs activation following PTH receptor stimulation, Gq having no involvement. Co-stimulation of these cells by PTH and P2Y) agonists also resulted in synergistic immediate early gene expression. These findings suggested that extracellular nucleotides are able to sensitize osteoblasts to the actions of PTH, providing a mechanism for localizing the response to this systemic hormone in bone, consistent with the discrete pattern of remodelling observed in the skeleton. Finally, the involvement of PTH on osteoclast formation was investigated. PTH was found to inhibit this process via activation of contaminating T lymphocytes present in osteoclast cultures. In conclusion this thesis presents evidence to suggest that extracellular nucleotides are important local signaling molecules in bone, affecting both osteoclast and osteoblast activity, alone and in combination with systemic factors such as PTH. Additionally, a novel action of PTH acting via lymphocytes to affect osteoclast formation is described.

No description available.

Osteoporosis is a skeletal disorder that affects millions of people worldwide, and is characterized by the accelerated loss of bone mass. Current anti-resorptive drug approaches such as bisphosphonates and salmon calcitonin exhibit severe side effects and very low bioavailability, respectively. In this study, we have designed, synthesized, and performed preliminary tests on a novel conjugate that targets the RANK receptor on bone resorbing cells (osteoclasts) in vitro with one arm, while delivering a specific effector molecule, calcitonin, to osteoclasts with the other arm. First, we successfully generated osteoclasts from precursor RAW 264.7 cells and confirmed that they were functional. We also designed a resorption assay that can be used to test the efficacy of new and existing anti-resorptive drugs. RAW 264.7 cells were then treated with an antibody to RANK to prove that anti-RANK could be used as a targeting mechanism. We then showed that delivery of calcitonin-loaded anti-Calcitonin antibodies onto osteoclasts results in the association of calcitonin onto its receptors on osteoclasts. Finally, we constructed a novel conjugate: calcitonin–Streptavidin–anti-RANK, and showed that it can be used to introduce calcitonin into an osteoclast-like microenvironment.

Thesis (M. Med. Sc.)--University of Adelaide, Dept. of Orthopaedics and Trauma, 1998? / Includes bibliographical references (leaves 115-133).

Thesis (doctoral)--Karolinska Institutet, Stockholm, 1995. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (doctoral)--Karolinska Institutet, Stockholm, 1995. / Added t.p. with thesis statement inserted. Includes bibliographical references.