Dissertations / Theses on the topic 'Osteoclastogenesis'

Lysosomes are acidic, hydrolase-rich vesicles capable of degrading most biological macromolecules. During the past several decades, much has been learned about different aspects of lysosome biogenesis. The selective phosphorylation of mannose residues on lysosomal enzymes, in conjunction with specific receptors for the mannose-6-phosphate recognition marker, has been found to be largely responsible for the targeting of newly synthesized lysosomal enzymes to lyzosomes. It is known that lysosomes receive input from both the endocytotic and biosynthetic pathways. Nevertheless the exact molecular mechanisms responsible for sorting of the biosynthetic imput involved in the lysosome biogenesis is still a matter of debate. Because osteoclast precursors do not secrete their lysosomal enzymes and osteoclasts do, the observation of modifications occuring during osteoclastogenesis is a good model to observe mechanisms responsible for lysosomal enzymes traffic. Osteoclasts are bone-degrading cells. To perform this specific task they have to reorganise the sorting of their lysosomal enzymes to be able to target them toward the bone surface in mature cells. Since few years, the differentiation of osteoclasts in vitro did help to study these cells. Osteoclast morphology has been therefore already well studied, and the nature of their specific membrane domains is now established. Sensing the proximity of a bone-like surface the cell reorganises its cytoskeleton, and creates specific membrane domains: an actin-rich ring-like zone (named actin ring) surrounded by highly ruffled membrane (named the ruffled border) where enzymes are secreted, while subsequent bone degradation products are endocytosed. Endocytosed material is then transported through the cell inside transcytotic vesicles and released at the top of the cell in an area named the functional secretory domain. Several molecular machineries are thought to control these different phenomena. The main purpose of this thesis was to identify the major regulators of lysosomal enzymes secretion and therefore to identify the molecular switches responsible for such a membrane traffic re-organisation.

Osteoclast formation is a complex process requiring the temporal activation of a yet unknown number of transcription factors. Osteoclast differentiation is dependent on two cytokines: macrophage colony stimulating factor (M-CSF) and receptor activator of NF-kB ligand (RANKL). These agents induce gene expression changes during the differentiation process, presumably by inducing transcription factors. A search for genes that are regulated in the developing osteoclast was performed using both differential display and gene arrays. Differential display revealed a novel member of the krüppel-associated box (KRAB) containing transcription repressor, KROCS, which was shown to be down regulated during osteoclast formation. The potential targets for this gene remain unknown, as do the targets of the majority of the other members of the krüppel associated box (KRAB) containing family of transcription repressors. In addition to KROCS, three other genes were also identified including ADO21, PRO1859 and an endogenous retrovirus like gene and the regulation of vitamin D up-regulated protein (VDUP) was also confirmed. Array analysis identified a number of other transcription factors regulated during osteoclast formation including the up-regulated NFATc1, GABP?, FBP, EGR1 and the repressed RelB and KOX31, a KRAB containing transcription repressor. The array also identified calmodulin 1 a member of the NFAT activation pathways as up-regulated by RANKL. The expression of NFATc1 to 4 in human osteoclasts was investigated showing NFATc1 to be the most expressed NFATc in osteoclasts. The transcription variants of NFATc1 were tested for expression differences showing that the mRNAs encoding the protein isoforms B and C were most expressed. The involvement of calmodulin, calcineurin and NFATc1 involvement in osteoclast formation was further studied by the use of inhibitors. BAPTA-AM is an intracellular chelator of calcium that prevents changes in calcium concentration. Phenoxybenzamine irreversibly binds calmodulin in the presence of calcium, inhibiting the action of calmodulin. Cyclosporin A (CsA) is an inhibitor of calcineurin. Use of both BAPTA-AM and phenoxybenzamine resulted in inhibition of osteoclast formation, decreasing the percentage of multinucleated cells from 54% in control cultures to 7.9% and 7.1% respectively. Both BAPTA-AM and phenoxybenzamine treated cells showed a marked reduction in TRAP activity with only 14.5% and 16.8% respectively staining positive for TRAP. This represents an approximate 60% reduction in TRAP positive cells compared to control osteoclasts. Both BAPTA-AM treated and phenoxybenzamine treated cells were negative for bone resorption. Addition of increasing doses of cyclosporin A (CsA) to M-CSF and RANKL treated cells resulted in the inhibition of multinucleated osteoclast formation. At 1000ng/mL CsA the formation of TRAP positive cells with more than one nucleus had reduced to less than 5% from 54% without the presence of CsA. Cells treated with 1000ng/mL CsA were unable to resorb bone, however the percentage of cells that were TRAP positive remained unchanged with CsA treatment. No significant decrease in expression of cathepsin K or TRAP transcripts were observed by real-time quantitative PCR (Q-PCR) in cells treated with CsA. Although all three agents inhibited the formation of multinuclear giant cells, both BAPTA-AM and phenoxybenzamine resulted in TRAP negative cells, whereas CsA resulted in TRAP positive cells. These results implicate the intracellular calcium increase caused by RANKL and calmodulin activation as a regulator of TRAP but place the calcineurin activation of NFATc1 downstream of TRAP induction. The regulation of a series of other genes was tested to determine if some RANKL mediated regulation of osteoclast genes were 'sensitive to CsA while others were 'resistant'. Of 28 genes tested, 13 were significantly affected by CsA and were considered 'sensitive' while the RANKL mediated regulation of 15 genes was unaffected by CsA and these were considered 'resistant'. This is strong evidence for two pathways of gene activation in osteoclasts, a CsA 'sensitive' pathway involving calcineurin, NFAT and possibly other transcription factors and a CsA 'resistant' pathway of gene activation, not dependent on calcineurin. Surprisingly, the RANKL mediated induction of NFATc1 was not inhibited by CsA, suggesting that NFATc1 induction is dependent on the resistant pathway. The identity of the second pathway (or pathways) is yet to be established, however the data indicate that this pathway mediate the RANKL sensitive regulation of at least one half of genes in human osteoclasts. The corollary if that only one half of osteoclast genes are dependent on calcineurin and presumably NFATc1 activation. There was no unifying principle that separated the CsA resistant from sensitive pathways of RANKL regulation. Cell surface markers, chemokines and transcription factors were among those affected by CsA. Even classical osteoclast markers fell neatly into two categories. The RANKL mediated induction of calcitonin receptor (CalcR) was inhibited by more than 100 fold in the presence of CsA implicating NFAT/calcineurin in the regulation of CalcR expression in osteoclasts. In contrast, the RANKL mediated induction of TRAP or cathepsin K, two prominent osteoclast markers, was totally unaffected by CsA. The expression of a series of chemokines and receptors was investigated. MCP-1 and RANTES were RANKL induced, and this induction was sensitive to CsA. The CC chemokines MCP-1 and RANTES were down regulated by around 10 fold in the presence of CsA. In contrast the RANKL mediated induction of MCP-1 receptor was resistant to CsA. The existence of chemokine and receptor in the same cell provides for a RANKL inducible autocrine loop, suggesting that MCP-1 should act directly on osteoclasts. The fact that the RANKL induction of the MCP-1 receptor, CCR2B, is unaffected by CsA suggests that exogenous MCP-1 should still signal in CsA treated osteoclasts. Addition of either MCP-1 or RANTES to CsA treated cultures resulted in a recovery of 70-80% of the multinuclear TRAP positive phenotype. The MCP-1 and RANTES induced multinuclear cell could not overcome the CsA induced inhibition of bone resorption. Surprisingly, MCP-1 and RANTES induced multinucleation in the absence of RANKL (M-CSF and chemokine treated cells) resulting in 50% of the normal multinucleation present in cells treated with RANKL. The data suggest that chemokines produced by osteoclasts are involved in promoting a multinuclear phenotype. When inhibited by CsA, osteoclasts fail to produce both MCP-1 and RANTES, although their respective receptors are present. This failure to produce MCP-1 and RANTES prevents the formation of an autocrine loop. When provided with MCP-1 or RANTES the CsA inhibited osteoclasts are subsequently able to pass through to the stage of a multinucleated giant cell. Similarly, in the absence of RANKL, chemokines promote the formation of TRAP positive osteoclast-like giant cells visually indistinguishable from osteoclasts. However, the multinuclear cells formed by chemokines in the absence of RANKL were also incapable of bone resorption. In order to determine if chemokines were capable of stimulating bone resorption, after osteoclasts had formed, pre-differentiated mature osteoclasts were plated onto bone and treated with a range of cytokines. The results showed that bone resorption occurred only in cultures that were exposed continuously to RANKL. These data indicate that chemokine induction by RANKL is required for multinucleation but that RANKL is required for bone resorption. The functional testing of genes detected by array analysis proved crucial involvement of both the NFAT pathway and CC chemokines in osteoclast formation knd function. Other genes identified such as GABP and FBP, are likely to be key factors in the development of a functional osteoclast. Future works investigating human osteoclast formation should take into strong consideration the genes identified in this thesis as targets for further functional studies.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
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Spondyloarthropathies (or Spondyloarthritides; SpAs) are a group of heterogeneous but genetically related inflammatory disorders in which ankylosing spondylitis (AS) is considered the prototypic form. Among the genes associated with AS, HLA-B27 allele has the strongest association although the cause is still not clear. Rats transgenic for the human HLA-B27 gene (B27 rats) develop a systemic inflammation mirroring the human SpA symptoms and thus provide a useful model to study the contribution of this MHC class I molecule in the disease development. Of particular interest was the observation of absence of arthritis in B27 rats grown in germ-free conditions and a recent theory suggests that microbial dysbiosis and gut inflammation might play a key role in initiating the HLA-B27-associated diseases. Studies in our laboratory have previously demonstrated that HLA-B27 expression alters the development of the myeloid compartment within the bone marrow (BM) in B27 rat and causes loss of a specific dendritic cell (DC) population involved in self-tolerance mechanisms within the gut. The aim of this thesis was to further analyse the myeloid compartment in B27 rats with a particular focus on the osteoclast progenitors and the bone phenotype and to link this to the gut inflammation. In addition, translational studies analysed peripheral monocyte/pre-osteoclasts in AS patients and teased apart the role of cytokines in in vitro human osteoclast differentiation. To understand the dynamics of the myeloid/monocyte compartment within the B27-associated inflammation, monocytes within the bloodstream and BM of B27 rats were characterised via flow cytometry and their ability to differentiate into osteoclast was assessed in vitro. Moreover, an antibiotic regime was used to reduce the B27 ileitis and to evaluate whether this could affect the migration, the phenotype, and the osteoclastogenic potential of B27 monocytes. B27 animals display a systemic and central increase of “inflammatory” CD43low MOs, which are the main contributors to osteoclastogenesis in vitro. Antibiotic treatment reduced ileitis and also reverted the B27 monocyte phenotype. This was also associated with the reduction of the previous described TNFα-enhancement of osteoclast differentiation from B27 BM precursors. These evidences support the idea that in genetically susceptible individuals inflammation in the gut might influence the myeloid compartment within the BM; in other terms, pre-emptively educate precursor cells to acquire specific phenotype end functions after being recruited into the tissue. This might explain the enhanced differentiation of osteoclast from B27 BM progenitors and thus the HLA-B27-associated bone loss. The data shown in this thesis suggest a link between the immunity within the gut and BM haematopoiesis. This provides an attractive and novel research prospective that could help not only to increase the understanding of the HLA-B27-associated aetiopathogenesis but also to unravel the cellular crosstalk that allows the mucosal immunity to program central cell differentiation. Human translational studies on monocyte subsets, cytokines and cytokine network in AS osteoclastogenesis evidenced altered osteoclast differentiation in the presence of IL-22 although no differences in the phenotype and functions of circulating CD14+ monocytes were observed. In addition, studies on the role of TNFα and TNFRs showed a dual role of this inflammatory cytokine in the human OC differentiation. In particular, the activation of TNFR1 in monocytes in early osteoclastogenesis inhibits OC differentiation while TNFα-biasing for TNFR2 on osteoclast precursors mediates the osteoclastogenic effect. Whether similar mechanisms are involved in the TNFα-mediated joint destruction in human rheumatic diseases needs further investigations. This could contribute to the development of novel and more specific anti-TNFα agents for the treatment of bone erosion. In conclusion, taken together my studies support the idea of a crosstalk between the periphery and the central system during the inflammatory response and provide new insights to the mechanisms behind the enhancement of osteoclastogenesis in B27-associated disorders.

During osteoclast differentiation and bone resorption the redox status in the cell display a decrease in reduction and a shift to an oxidized state. Structure, metabolism and function are some of the extensive changes that cells undergo during differentiation which alters both the extra- and intracellular redox environment. Osteoclasts express enzymes such as TRAP and NADPH oxidase which generates reactive oxygen species (ROS). ROS are molecules formed by oxygen reduction which gives these radicals at least one unpaired electron and makes them very reactive and chemically unstable. These are factors which stimulates differentiation of osteoclasts and bone resorption. RAW 264.7 cells will differentiate to osteoclasts when stimulated with RANKL and to activated macrophages when stimulated with LPS.

The aim of this project was to analyze if the redox environment is affected during differentiation of RAW 264.7 cells to osteoclasts and macrophages. The reason for this was that we aimed to se if RAW 264.7 cells could be used as an in vitro system to study the effects of redox changes in osteoclasts and macrophages and their activation.

Results from Western blot showed that protein expression of the Cysteine/Glutamate transporter xCT was up regulated with LPS and downregulated with RANKL. Results from the GSH/Cys assay show that the treatments with redox modulators did not affect the levels of GSH and Cys to a measurable extent. However the levels increased for both intracellular and extracellular GSH and Cys forms at day 4 in the control and stimulated cells. Addition of the disulfide reductant DTT affected differentiation to osteoclasts, leading to smaller osteoclasts probably due to interference with fusion of mononuclear pre-osteoclasts. Thus, down regulation of the xCT transporter could be an important mechanism to maintain a low level of free thiols shown to interfere with the differentiation to osteoclasts.

Osteoporosis is a condition that results from substantially weakened bone, increasing an individual’s risk of fracture. Post-menopausal osteoporosis is the most common form of the condition, affecting 30% of post-menopausal women over the age of 50. Following the menopause, female oestrogen levels decline and this perturbs bone homeostasis by promoting an environment that is biased towards bone erosion. Osteoclasts are the cells responsible for eroding bone and are normally inhibited by oestrogen. However, the decline in oestrogen production results in increased osteoclast differentiation and activity. This rapidly decreases the bone mineral density and results in fracture-prone bone. Osteoclasts are derived from mononuclear myeloid progenitors found in the blood and bone marrow, which fuse to form large multinucleated cells that reside in the bone cavity. These progenitor cells are also responsible for replenishing monocytes, macrophages and dendritic cells. One class of receptors present on the surface of these cells, which are capable of dictating a cells function, are Fcγ receptors and modulation of Fcγ receptors has been shown to inhibit the differentiation of human monocytes to osteoclasts. This thesis investigates Fcγ receptor modulation on murine osteoclastogenesis and in order to stimulate Fcγ receptors, both IgG and IgG complexes were used. IgG complexes were generated using Staphylococcus aureus Protein A (SpA) in combination with IgG to form SpA-IgG complexes (SIC). We show that IgG and SIC are capable of engaging with Fcγ receptors resulting in the inhibition of osteoclast differentiation. Furthermore, both IgG and SIC inhibit the transcription of mRNA essential for the fusion of progenitors and enzymes for the erosion of bone matrix. Therefore, IgG and SIC are capable of inhibiting murine osteoclastogenesis. The murine model of osteoporosis was used to further investigate the ability of SIC to inhibit murine osteoclast differentiation. Previous studies have shown that when SpA is administered in vivo it is capable of binding circulating IgG to form SIC. We used this property to test the ability of SpA to bind to the surface of monocytes. SpA was found to bind with highest affinity to blood Ly6Chigh monocytes, which are known to differentiate in vitro to OCs. IgG and SIC were also able to inhibit the in vitro osteoclastogenesis of Ly6Chigh monocytes. It was hypothesised that SpA would co-opt IgG and inhibit the in vivo differentiation of progenitors to osteoclasts in the ovariectomy model of osteoporosis. To generate this animal model the ovaries were removed from the mice in order to simulate the menopause and induce bone loss. To assess the percentage of bone present after ovariectomy, we used micro-computer tomography and discovered that SpA was unable to prevent bone loss associated with ovariectomy. Therefore, SpA can bind to the surface of osteoclast progenitors but is unable to inhibit bone loss in the model of osteoporosis. In addition to studying the role of Fcγ receptor modulation of osteoclastogenesis, the role of Bcl-3 (a negative regulator of NF-κB) in osteoclast differentiation and bone remodelling was also investigated. NF-κB is an essential signalling molecule and transcription factor involved in osteoclast differentiation. Previous research has shown that in the absence of Bcl-3 (Bcl-3-/-) aberrant cytokine responses to LPS and TNF- occur. Therefore, RANKL stimulation of WT and Bcl-3-/- osteoclast precursors was done to determine whether Bcl 3 /- animals responded aberrantly to RANKL. WT and Bcl-3-/- animals were able to generate in vitro osteoclasts, which were phenotypically and transcriptionally similar. However, comparison of in vivo osteoclast progenitors revealed that Bcl-3-/- animals had reduced CD115+ osteoclast progenitors compared to WT animals. Examination of the trabecular bone present in the proximal tibia revealed that Bcl-3-/- animals had a higher percentage of bone present that WT controls. Therefore, Bcl-3 does not effect in vitro osteoclast differentiation but further work needs to be done to understand the role of Bcl 3 in bone remodelling. This thesis aimed to investigate whether SpA-IgG complexes or Bcl-3 could represent a novel avenue of therapeutic intervention in osteoporotic disease. In summation, SpA is able to form IgG complexes that can inhibit the differentiation of OCs in vitro; however, treatment of osteoporotic animals with SpA was unable to halt bone loss. This suggests that SpA-IgG complexes are able to modulate Fcγ receptors in vitro and skew progenitors from differentiation into osteoclasts but cannot overcome the prevailing pro-osteoclastogenic environment that results from ovariectomy. The presence of osteoclast progenitors was also shown to be partially dependent on Bcl-3 and as such Bcl-3 may be a novel target for therapeutic agents to target osteoclast progenitors in diseases like osteoporosis. However, the role of Bcl-3 in bone remodelling requires further investigation.

The present PhD thesis is a compendium of four publications broadening the knowledge on osteoclastogenesis under simulated bone augmentation, more especially about the effects of saliva and bone-conditioned medium on osteoclastogenesis. Resorption of bone grafts and host bone, can be a challenge especially when a bonny defect has to be regenerated or there is a lack of host bone due to a trauma, pathology, aging or tooth extraction among others. In the oral cavity, saliva is present and can reach mineralized surfaces, however, the relationship between saliva and bone resorption is yet unknown. Herein, we examined whether saliva affects the process of osteoclastogenesis in vitro, possibly affecting bone healing and bone regeneration. Bone regeneration is a common procedure in traumatology, periodontology, oral and maxillofacial surgery that involves the use of bone fillers. Bone autograft is considered to be the gold standard bone substitute due to its trinity of properties: osteoinductivity, osteoconductivity and osteogenesis. Paracrine factors released from bone autografts might contribute to the overall process of graft consolidation, however the underlying mechanisms are unknown. Here, we determined the protein spectrum released from porcine bone chips into the conditioned medium (BCM) to mimic the paracrine environment of cortical bone grafts. Some of the factors released by bone autografts could maybe influence on the autograft resorption and therefore explain why osteoclasts rapidly form on the surface of bone chips at augmentation sites. The underlying molecular mechanism, however, is unclear. Soluble factors released from bone chips in vitro have a robust impact on mesenchymal cell differentiation. Here we determined whether these soluble factors change the differentiation of hematopoietic cells into osteoclasts, still unknown. Based on the in vitro results here presented, it can be observed that saliva suppresses osteoclastogenesis and leads to the development of a phagocytic cell phenotype, therefore affecting function of osteoclasts, the bone resorbing cells. Resorption of bone autografts could be attributed to some of the proteins detected on the secretions of bone autografts, termed bone conditioned medium (BCM). Proteomic analysis showed that BCM contains more than 150 proteins, among which, 43 were categorized into “secreted” and “extracellular matrix”. We discovered growth factors that are not only detectable in BCM, but potentially also target cellular processes involved in bone regeneration e.g. pleiotrophin, galectin-1, TGF-β-induced gene (TGFBI), latency-associated peptide forming a complex with TGF-β1, and TGF-β2. Results here presented on the influence of BCM on osteoclastogenesis demonstrated that activated BCM by heat is able to stimulate osteoclastogenesis in vitro. These in vitro results support the notion that the resorption of autografts may be supported by as yet less defined regulatory mechanisms. Moreover the presented protocols on the use of BCM should encourage to further reveal the paracrine effects of bone grafts during bone regeneration and open a path for translational research in the broad field of reconstructive surgery. Taking everything together, it can be concluded that saliva affects bone resorption towards the development of a phagocytic cell line, and that not only saliva affects bone resorption but also the secretions from autologous bone grafts. There is enough evidence to conclude that bone autografts not only have three properties, but one more: a regulation property, the fourth dimension of autologous bone grafts.
La present tesis doctoral és un compendi de quatre publicacions ampliant el coneixement de l’osteoclastogènesis en les regeneracions òssies, més especialment sobre els efectes de la saliva i el medi condicionat ossi en l’osteoclastogènesis. La reabsorció dels empelts ossis i de l’os de l’hoste, pot ser un repte especialment quan un defecte ossi ha de ser regenerat en condicions desfavorables o grans atròfies com per exemple després de traumatismes, diverses patologies, edat avançada o extraccions múlti¬ples. En la cavitat oral, la saliva pot entrar en contacte amb superfícies mineralitzades, tot i això la relació entre saliva i reabsorció òssia és encara desconeguda. En la present tesis hem examinat si la saliva afecta el procés de l’osteoclastogènesis in vitro, possiblement afectant a la regeneració i cicatrització òssia. La regeneració òssia és un procés comú en traumatolo¬gia, periodòncia, cirurgia oral i maxil•lofacial que involucra l’ús de substituts ossis. Els empelts d’os autòleg són considerats l’estàndard d’or dels sub¬stituts ossis degut a la seva trinitat de propietats: osteoconductivitat, oste¬oinducció i osteogènesis. Els factors paracrins alliberats pels empelts d’os autòleg podrien contribuir en el conjunt de processos que donen com a re-sultat la consolidació del empelts, tanmateix els mecanismes que regeixen aquest processos no són coneguts. En el present treball hem pogut carac¬teritzar un conjunt de proteïnes alliberades per partícules d’os cortical porcí en el medi condicionat ossi (BCM) per imitar l’ambient paracrí dels em¬pelts d’os cortical. Alguns dels factors alliberats pels empelts d’os autòleg podrien influenciar la reabsorció òssia explicant per què els osteoclasts es formen ràpidament a la superfície de les partícules d’os autòleg en els llocs regenerats. Tot i això els mecanismes moleculars que regeixen aquest pro¬cés, encara son desconeguts. Factors solubles alliberats pels empelts d’os autòleg in vitro tenen un impacte robust a la diferenciació de cèl•lules mes¬enquimals. En la present tesis doctoral, hem determinat si aquests factors solubles son capaços de canviar la diferenciació de cèl•lules mare hemat¬opoètiques a osteoclasts, desconegut abans de realitzar els estudis aquí presentats. Basant-nos en els resultats in vitro aquí presentats, es pot observar que la saliva suprimeix l’osteoclastogènesis i promociona el desenvolupament de cèl•lules amb un fenotip fagocític, afectant a la funció dels osteoclasts, les cèl•lules encarregades de reabsorbir l’os. La reabsorció dels empelts d’os autòleg es pot atribuir a l’efecte d’algunes de les proteïnes detecta¬des en les secrecions dels auto-empelts, anomenant aquestes secrecions Medi Condicionat d’Os (BCM). Un estudi proteòmic del BCM va mostrar que aquest medi condicionat conté més de 150 proteïnes, de les quals 43 es van caracteritzar com “secretades” i presents en la matriu extracel•lular. Vàrem descobrir que alguns dels factors continguts en el BCM com per exemple pleiotropina, galectina-1 o TGF-β1 poden afectar processos cel•lulars involucrats en la regeneració òssia. El resultats presentats en aquesta tesis sobre l’influencia del BCM en l’osteoclastogènesis demostra que el BCM termo-activat és capaç d’estimular l’osteoclastogènesis in vitro. Aquests resultats in vitro suporten la noció que la reabsorció dels auto-empelts ossis pot ser que estigui estimulada per mecanismes reguladors encara no definits. En aquesta línia, els protocols presentats sobre l’ús del BCM haurien d’animar a revelar els efectes paracrins dels empelts d’os autòleg durant el procés de regeneració òssia i obrir nous camins a investi¬gacions translacionals en l’ampli camp de la cirurgia reconstructora. Resumint-ho tot, podem concloure que la saliva afecta la reabsorció òssia promocionant el desenvolupament de cèl•lules amb un fenotip fagocític, i que no només la saliva pot afectar a la reabsorció òssia, sinó que també les se¬crecions dels injerts d’os autòleg. En aquest punt, hi ha suficient evidencia per concloure que els auto-empelts d’os no només tenen tres propietats, sinó una més: la propietat reguladora, la quarta dimensió dels empelts d’os autòleg.

Glycolaldehyde (Glycol) and glyceraldehyde (Glycer) derived glycated human serum albumin (HSA) significantly reduced RANKL-induced osteoclastic TRAP activity, while glucose, fructose, glyoxal derived glycated HSA have no effect. Secreted high mobility group box protein1 (HMGB1) bind to receptor for AGE (RAGE) and play an important role for osteoclastogenesis. Next, we investigated that the effect of glycated HSA on HMGB1 secretion and RAGE expression. Both Glycol-AGE and Glycer-AGE inhibited RANKL-induced HMGB1 secretion but not altered RAGE expression levels. These result indicated that some kinds of AGEs altered osteoclastic TRAP activity through inhibition of HMGB1 secretion from the cell.
博士(理学)
Doctor of Philosophy in Science
同志社大学
Doshisha University

Paget’s disease (PDB) is the second most common metabolic bone disease after osteoporosis, affecting up to 3% of adults over age 55. It is characterized by focal lesions of bone resorbed by hyperactive osteoclasts coupled with rapid formation of highly disorganized, low quality bone formed by osteoblasts. Such lesions cause skeletal deformity, fractures, and other symptoms that significantly decrease quality of life. In 2001, mutations in the SQSTM1/p62 gene were found in a subset of Paget’s patients. The work summarized in this dissertation sought to answer two broad questions: what is the function of p62 in normal bone homeostasis and how do PDB-associated mutations alter it? These studies took advantage of two mouse models: p62 knock-out (KO) mice, and p62P394L “knock-in” (KI) mice carrying the most common PDB-associated mutation. KO, KI, and wildtype (WT) controls were aged to one year for skeletal-histological characterization. No differences were observed in a variety of bone parameters between WT and KO bones, while bones from age-matched KI mice exhibited a 33% decrease in bone volume and a 25% increase in osteoclast formation. In vivo, TNF-α caused a potent induction of osteoclastogenesis in calvariae of WT and KI, but not KO, mice. In vitro, RANKL induced osteoclast formation in a dose-dependent manner in WT and KI, but not KO, cultures. Gene expression profiling of RANKL-treated osteoclast progenitors from WT, KO, and KI mice was then performed to identify the changes in signaling pathways responsible for these effects. Surprisingly, gene expression patterns from all three groups were consistent with robust activation of NFκB signaling in RANKL-treated samples, indicating that p62 is dispensable for RANKL activation of NFκB. Interestingly, gene expression patterns in KO cells suggested impaired proliferation and response to reactive oxygen species (ROS), a finding which was confirmed in cell culture experiments. In contrast, KI cells displayed enrichment for genes associated with the unfolded protein response, consistent with p62’s role in ubiquitin-mediated protein degradation via proteolysis and autophagy. These studies have therefore generated several novel hypotheses concerning the role of p62 in both normal bone homeostasis and Paget’s disease of bone.

La polyarthrite rhumatoïde est une maladie auto-immune caractérisée par une inflammation chronique qui entraîne la destruction progressive des articulations et des os. Les dommages articulaires observés dans cette pathologie sont causés principalement par les ostéoclastes, des cellules spécialisées dans la résorption de la matrice osseuse. Ce processus de résorption dépend de la capacité à générer des ostéoclastes, de leur activité individuelle et de leur survie. De plus, certaines cytokines inflammatoires peuvent avoir un effet sur la différenciation et l'activité des ostéoclastes. Parmi celles-ci, on retrouve notamment le TNF-?, un médiateur pathologique majeur dans la polyarthrite rhumatoïde. En effet, celui-ci peut agir de façon directe sur la résorption osseuse en stimulant l'ostéoclastogénèse, ou de façon indirecte en augmentant l'expression du RANKL par les ostéoblastes. Subséquemment à ces découvertes, plusieurs agents anti-TNF-? ont été développés pour traiter la polyarthrite rhumatoïde. Ces agents s'avèrent être très efficaces pour réduire les dommages articulaires chez les patients atteints de la maladie. Cependant, leurs mécanismes exacts ainsi que leurs effets sur la biologie des ostéoclastes humains sont encore mal définis. Ainsi, l'objectif principal de cette étude est d'étudier l'effet d'une thérapie anti-TNF-? sur le nombre de précurseurs ostéoclastiques dans le sang périphérique de patients atteints de la polyarthrite rhumatoïde, sur le nombre d'ostéoclastes générés in vitro ainsi que leur activité avant et pendant le traitement avec l'adalimumab, un agent anti-TNF-?. Pour ce faire, 25 patients atteints de cette maladie et ayant reçu une prescription d'adalimumab ont été recrutés pour participer à trois visites consécutives, soit les visites d'inclusion (avant traitement) ainsi que les visites 3 mois et 6 mois après le début du traitement. Pour chaque visite, le nombre de précurseurs ostéoclastiques, le nombre d'ostéoclastes et la résorption osseuse générés in vitro ont été évalués. Les mêmes paramètres ont également été vérifiés pour les cellules incubées en présence d'adalimumab exogène. L'activité de la maladie et le statut fonctionnel du patient, mesurés respectivement avec le Disease Activity Score 28 et le Health Assessment Questionnaire ont été évalués à chaque visite de la présente étude. La collecte de ces données a permis de conclure que le traitement avec l'adalimumab pendant 6 mois n'a pas d'impact statistiquement significatif sur le nombre de précurseurs ostéoclastiques, l'ostéoclastogénèse et la résorption osseuse in vitro , même si nous pouvons observer une tendance vers une diminution pour les deux derniers paramètres. En ce qui concerne les résultats cliniques, l'adalimumab a un effet statistiquement significatif sur le score DAS28 et le questionnaire HAQ, tous deux ayant diminué 6 mois après l'initiation du traitement.

O tecido ósseo possui alto grau de rigidez e resistência à pressão, característica relacionada às suas funções de proteção, sustentação e locomoção. Patologias como periodontite, artrite reumatóide e osteoporose decorrem do desequilíbrio da dinâmica óssea, que ocorre quando os osteoclastos estão em maior atividade em relação aos osteoblastos. O fluoreto (F-) é incorporado nos tecidos mineralizados após ingestão e a sua administração aumenta a formação óssea in vivo, de maneira dependente da dose e da linhagem dos animais. As linhagens A/J e 129P3/J possibilitam a análise dos fenômenos moleculares envolvidos na suscetibilidade e resistência de células ósseas aos efeitos do F- no osso. Neste estudo, avaliou-se a administração in vivo de F- na osteoclastogênese, através da análise da atividade da enzima fosfatase ácida resistente ao tartarato (TRAP), contagem de células TRAP- positivas e quantificação da atividade das metaloproteinases MMP-2 e -9. Para tal, células hematopoiéticas provenientes da medula óssea de camundongos das linhagens 129P3/J e A/J foram cultivadas com M-CSF e RANKL em associação ou não com F-, nas concentrações de 10- 9, 10-7, 10-5, 5x10-5, 5x10-5, 10-4 e 10-3 M. Os dados mostraram que a atividade da TRAP foi semelhante entre as células das duas linhagens de animais, independente do F-. Concomitante à diferenciação osteoclástica, o tratamento das células com F-, independente da concentração, aumentou significativamente a atividade da TRAP em células da linhagem A/J. O aumento da atividade de TRAP foi associada com o aumento do número de osteoclastos formados, na concentração de 10-3 M F-. Já em células dos animais 129P3/J, o F- não alterou a atividade da TRAP bem como a osteoclastogênese. A atividade de MMP-9 foi aumentada em relação à da MMP-2, porém ambas não foram moduladas pelo F-, independente da linhagem. Assim, concluiu-se que as células de ambas as linhagens não diferem com relação à diferenciação de osteoclastos, mas respondem diferentemente ao F-, sendo as células dos animais A/J e 129P3/J sensíveis e resistentes à osteoclastogênese induzida pelo F-, respectivamente.
The bone tissue has a high degree of rigidity and pressure resistance, characteristic related to its protection, support and locomotion functions. Diseases such as periodontitis, osteoporosis and rheumatoid arthritis arise from dynamic bone imbalance that occurs when osteoclasts present increased activity compared to osteoblasts. Fluoride (F-) is incorporated in mineralized tissues after ingestion and its administration increases in vivo bone formation in dose and strain-dependent manner. The strains A/J and 129P3/J make possible the analysis of molecular phenomena involved in susceptibility and resistance of bone cells to the effect of F- in bone. In this study, we evaluated the effect of F- on the in vitro osteoclastogenesis, by analyzing the fosfatase tartrate-resistant acid (TRAP) activity, TRAP positive cell counts and the activity of MMP-2 and -9 measurements in A/J and 129P3/J differentiated hematopoietic stem cells. Hematopoietic cells were differentiated with M-CSF and RANKL and treated or not with F- in the concentrations of 10-9, 10-7, 10-5, 5x10-5, 5x10-5, 10-4 and 10-3 M.The data show that TRAP activity, an osteoclast marker, was similar between cells from both strains of mice, regardless of F-. In addition, the treatment of A/J cells with F- significantly increased TRAP activity, in a dose-independent manner. This was accompained by the increased TRAP+-osteoclast counts, at 10-3 M F- dose. However, while the MMP-9 activity was aumented when compared to MMP-2, it was not altered by F-, regardless of the strain. Thus, we concluded that cells from both strains do not differ regarding the osteoclast differentiation potential, but they respond differently to F-, whereas A/J and 129P3/J cells are sensitive and resistant to F--induced osteoclastogenesis, respectively.

Metastatic bone cancer is the most common cause of pain in patients with malignant tumors. Prolonged opioid treatment remains the primary method to treat pain in these patients. Sustained morphine exposure enhances both bone cancer-induced pain and bone loss in mice implanted with sarcoma cells. Sustained treatment of bone marrow cultures with morphine results in COX-2 dependent upregulation of RANKL and PGE2, and suppression of OPG. This results in increased osteoclastogenesis which was dependent on COX-2 and OPG/RANKL regulatory axis. Treatment with morphine does not induce any direct changes in osteoclasts or sarcoma cells. The in vitro data was validated in the animals where morphine induces an increase in the osteoclastogenesis and RANKL, and suppresses OPG. These data indicate that morphine enhances osteoclastogenesis by modulating the OPG/RANKL regulatory axis in osteoblasts through a COX-2 dependent mechanism.Prolonged opioid exposure induces an opioid-receptor dependent hyperalgesia in humans and in animals. Studying the direct effect of opioids on primary sensory neurons we demonstrate a modest increase in CGRP cellular content that was not opioid-receptor dependent. Although dynorphin A (2-13) and PGE2 enhanced the release of the neuropeptide, pretreatment with opioids does not influence the capsaicin or KCl evoked CGRP release. These date indicate that the neurochemical changes seen in vivo may be dependent on factors upregulated in the periphery and/or the CNS.It has been demonstrated that sensory neurons innervating the femur express markers of neuronal injury and the intramedullary region of the femur becomes devoid of nerve fibers as the tumor expands. In this study we demonstrate that the sarcoma cells generate high levels of ROS and release hydrogen peroxide into the surrounding space, which induces death and injury to both sensory neurons and glia. This death was prevented by the anti-oxidants BHA and catalase. The present study provides evidence that ROS released by cancer cells can directly lead to injury and death of sensory neurons. ROS induced injury may be one of the mechanism through which sensory neurons are injured in the murine bone cancer pain model.

Abstract Osteoclasts are multinuclear bone degrading cells differentiated from monocytes which can be isolated from bone marrow and peripheral blood. Complex signaling between osteoclast precursors and other bone cells, such as mesenchymal stromal cells (MSC) occurs during the differentiation. Gap junctional communication (GJC) is one of the mechanisms in the cell fusion. GJC can be modulated with several substances such as the specific GJC stimulators, antiarrhythmic peptides (AAP). Due to their promising clinical value in the treatment of cardiac disorders, the effects of AAPs in cardiac tissue are studied extensively. This study was conducted in order to investigate the roles of GJC and AAPs in bone cell cultures. Further, the contribution of the MSCs on the effects of AAPs was studied along with comparison of two types of osteoclastogenesis cultures with differing quantities of MSCs. GJC in osteoclastogenesis was studied with both GJC inhibitors and stimulators in mouse monocyte line RAW 264.7 cells and primary cultures with bone marrow hematopoietic cells. The following studies were made with human monocytes from peripheral blood and bone marrow where the effects of AAP10 were investigated in normal and acidic environments. In addition, comparison of osteoclastogenesis from bone marrow and peripheral blood monocytes was carried out in in vitro cell cultures on bovine or human bone slices. The cells were analyzed with regard to multinuclearity, bone resorption and the expression of several osteoclast markers. The results show that GJC is utilized in osteoclastogenesis, but it is not indispensable. GJC in monocytes can be stimulated with the AAPs during osteoclastogenesis, but the effects depend on the culture conditions as well as on the presence of MSCs in the culture. The AAPs can also activate the MSCs leading to indirect regulation of osteoclastogenesis, as the MSCs produce several molecules affecting the differentiation. Further, monocytes from peripheral blood showed increased potential for osteoclastogenic differentiation compared to bone marrow derived monocytes. This can be explained by the presence of the osteoclastogenesis-controlling MSCs in the bone marrow culture, while the peripheral blood cultures contain only few of these cells and thus lack their regulatory effects
Tiivistelmä Osteoklastit ovat monitumaisia luuta hajottavia soluja, jotka ovat erilaistuneet monosyyteistä. Monosyyttejä voidaan eristää luuytimestä tai perifeerisestä verestä. Erilaistumisen aikana osteoklastien esiastesolujen sekä muiden luusolujen, kuten mesenkymaalisten stroomasolujen (MSC) välillä tapahtuu monimutkaista signalointia. Aukkoliitoskommunikointi (GJC) on eräs solufuusiossa tapahtuvista mekanismeista. GJC:tä voidaan muunnella useilla aineilla, esimerkiksi spesifisillä stimulaattoreilla, antiarytmisillä peptideillä (AAP). AAP-yhdisteiden vaikutuksia on tutkittu laajalti sydänkudoksessa johtuen niiden lupaavista kliinisistä ominaisuuksista sydänperäisten oireiden hoidossa. Tämän tutkimuksen tarkoituksena oli selvittää GJC:n ja AAP-yhdisteiden roolia luusoluviljelmissä. Lisäksi tutkittiin MSC-solujen osallistumista AAP-yhdisteiden vaikutuksiin sekä vertailtiin kahta erilaista osteoklastogeneesiviljelmää, joissa oli eri määrä MSC-soluja. GJC:tä osteoklastogeneesissä tutkittiin sekä sitä estävillä että stimuloivilla yhdisteillä hiiren monosyyttilinjan RAW 264.7 -soluissa sekä luuytimen hematopoieettisten solujen primääriviljelmissä. Seuraavat tutkimukset tehtiin ihmisen luuytimen ja perifeerisen veren monosyyteillä, ja niissä selvitettiin AAP10-yhdisteen vaikutuksia fysiologisissa sekä happamissa olosuhteissa. Lisäksi vertailtiin luuytimen ja perifeerisen veren monosyyttien osteoklastogeneesiä. In vitro -soluviljelmät tehtiin naudan tai ihmisen luulastujen päällä, ja soluista analysoitiin monitumaisuus, luun resorptio sekä useiden osteoklastimarkkereiden ilmentyminen. Tulokset osoittavat, että GJC:tä hyödynnetään osteoklastogeneesissä, mutta se ei ole korvaamaton mekanismi. GJC:tä voidaan stimuloida AAP-yhdisteillä osteoklastogeneesin aikana, mutta vaikutukset riippuvat viljelyolosuhteista sekä MSC-solujen läsnäolosta. AAP-yhdisteet voivat aktivoida myös MSC-soluja johtaen osteoklastogeneesin epäsuoraan säätelyyn, kun MSC-solut tuottavat useita erilaistumiseen vaikuttavia molekyylejä. Lisäksi perifeerisen veren monosyyteillä havaittiin korkeampi osteoklastogeeninen erilaistumispotentiaali verrattuna luuytimen monosyytteihin. Tulokset voidaan selittää osteoklastogeneesiä säätelevien MSC-solujen läsnäololla luuydinviljelmissä, kun taas perifeerisen veren monosyyttiviljelmissä näitä soluja on vain vähän, jolloin myös niiden säätelyominaisuudet puuttuvat

Os leucotrienos (LTs) são mediadores inflamatórios derivados da via 5- lipoxigenase (5-LO), com contribuição relevante na reabsorção óssea. Neste estudo investigamos o papel dos LTs na diferenciação osteogênica e o seu impacto na osteoclatogênese. Assim, foi avaliado o perfil ósseo dos camundongos 129/Sv (WT) e 5-LO Knockout (5-LO KO) por meio de microtomografia computadorizada, evidenciando maior densidade óssea vertebral e trabéculas mais espessas em machos 5-LO KO. Após isso, osteoblastos primários (OBL) foram isolados e cultivados para determinar a atividade de fosfatase alcalina (ALP) e o potencial de mineralização. Resultados mostraram que OBL KO possui maior atividade de ALP e mineralização, em todos os períodos quando comparados com WT. Em adição, o tratamento com os LTs B4 e D4 inibiu a deposição de cálcio. Os inibidores da síntese de LTs e os antagonistas do BLT1/2 foram efetivos em recuperar a formação dos nódulos mineralizados. A cinética do Alox5 apresentou um aumento da expressão nos períodos de maior diferenciação celular em OBL WT. Além disso, a expressão de OCN, MMPs 2 e 9 e RANKL foram aumentadas em células 5-LO KO em quase todos os períodos avaliados. Em geral, o estímulo com LTs, seus inibidores e antagonistas diminuiu a expressão de Sp7, Col1a1, Opg e MMP-9 e aumentou RANKL em células KO. A sinalização por meio de segundos mensageiros também foi avaliada. Células 5-LO KO apresentam menor concentração de cálcio intracelular (Ca2+i) em relação ao WT. No período de 14 dias, o estímulo com LTD4 inibiu a liberação Ca2+i independente da linhagem, em relação ao controle. Os níveis de cAMP foram menores em OBL 5- LO KO, em todos os grupos tratados ou controle. LTD4 diminuiu a concentração de cAMP, mas não LTB4, em OBL 5-LO KO. O estudo também quantificou a produção de LTB4 e outros eicosanoides em osteoblastos mostrando a sua capacidade de síntese. A análise proteômica revelou 89 proteínas com expressão diminuída em OBL 5-LO KO, de um total de 154, sendo a maioria relacionada ao citoesqueleto e ao metabolismo energético. Também foram identificadas 59 proteínas exclusivas em OBL 5-LO KO e 06 unicamente expressas em células WT, revelando as diferenças intrínsecas de cada animal. O perfil osteoclastogênico de camundongos WT vs. 5-LO KO mostrou diferenças significativas na análise fenotípica, TRAP e na expressão gênica de células derivadas da linhagem monocítica-macrofágica. Após o estímulo com M-CSF e RANKL, as células WT apresentaram osteoclastos gigantes multinucleados, porém, células 5-LO KO apresentaram uma população de células com formas e tamanhos variáveis, e menor grau de maturação. Em adição, os LTsexógenos não modularam a atividade da TRAP. O meio condicionado proveniente dos OBL WT e KO, retardaram o processo de formação dos osteoclastos. A análise da expressão gênica em osteoclastos mostrou diminuição da expressão de Alox5, Il- 1b, Il-6 e TNFa em células 5-LO KO. BLT1/2, CysLt1 e os marcadores da diferenciação Acp5, Ctsk e Nfact1 não apresentaram diferenças entre os animais. Em adição, o LTB4 diminuiu a expressão do Alox5 e a Il-1b foi aumentada em osteoclastos WT. Assim, os resultados demonstram que os LTs são capazes de modular o metabolismo ósseo, e a ausência do gene da 5-LO está relacionada ao maior perfil osteogênico.
Leukotrienes (LTs) are inflammatory mediators derived from the 5-lipoxygenase (5-LO) pathway, with a relevant contribution in bone resorption. In this study we investigated the role of LTs in osteogenic differentiation and its impact on osteoclastogenesis.Thus, the bone profile of the 129/Sv (WT) and 5-LO Knockout mice (5-LO KO) was evaluated by computerized microtomography, showing higher vertebral bone density and thicker trabeculae in 5-LO KO males. After that, primary osteoblasts (OBL) were isolated and cultured to determine alkaline phosphatase activity (ALP) and mineralization potential. Results showed that OBL KO has higher ALP activity and mineralization, in all periods when compared with WT. In addition, the treatment with LTB4 and LTD4 inhibited calcium deposition. Inhibitors of LT synthesis and BLT1/2 antagonists were effective to recover the mineralized nodules formation. The kinetics of Alox5 showed an increase in expression during cellular differentiation period in WT OBL. In addition, expression of OCN, MMPs 2 and 9 and RANKL were increased in 5- LO KO cells in almost all evaluated periods. In general, the stimulation with LTs, their inhibitors and antagonists decreased the expression of Sp7, Col1a1, Opg and MMP- 9. But it increased the RANKL expression in KO cells. The second messengers signaling was also evaluated. 5-LO KO cells showed lower concentration levels of intracellular calcium (Ca2+ i) when compared to WT cells. In the 14-day period, the LTD4 treatment inhibited the Ca2+i independent of the murine lineage, relative to the control. cAMP levels were lower in OBL 5-LO KO, in all treated or control groups. LTD4 decreased the concentration of cAMP, but not LTB4, in KO cells. The study also quantified the production of LTB4 and other eicosanoids in osteoblasts showing their ability to synthesize those metabolites. The proteomic analysis revealed 89 downregulated proteins in OBL KO, out of a total of 154, most of them related to cytoskeleton and energy metabolism. Also 59 identified proteins were unique in OBL 5-LO KO and 06 exclusively expressed in WT cells, revealing the intrinsic differences of each strain. The osteoclastogenic profile of WT vs. 5-LO KO showed significant differences in phenotypic analysis, TRAP and in the gene expression of cells derived from the monocyte-macrophage-lineage. After M-CSF and RANKL stimulation, WT cells showed multinucleated giant osteoclasts. However, 5-LO KO cells presented a population of cells with variable shapes and sizes, and a lower maturation stage. In addition, exogenous LTs did not modulate TRAP activity. The conditioned medium from OBL WT and 5-LO KO delayed the formation process of osteoclasts. Gene expression analysis in osteoclasts showed decreased expression of Alox 5, Il-1b, Il-6 and TNF in 5-LO KO cells. BLT1/2, CysLt1 and the osteoclast differentiation markers Acp5, Ctsk and Nfact1 showed no differences between the strains. In addition, LTB4 decreased the expression of Alox5, and IL-1b was increased in WT osteoclasts. Thus, the results demonstrate that the LTs are able to modulate the bone metabolism, and the absence of the 5-LO gene is related to the greater osteogenic profile.

Bone mineral density (BMD) is a reflection of the action of osteoblasts compared to osteoclasts. An imbalance in the activity of osteoblasts or osteoclasts, results in bone disease such as osteoporosis caused by overactive osteoclasts. BMD is influenced by genetic and environmental factors as demonstrated through twin studies, association studies and linkage analysis (Ralston, 1999). Several polymorphisms involved in the determination of BMD have been identified, with Vitamin D receptor and Collagen Type 1 showing reproducible associations. To identify genes influencing BMD two distinct strategies have been employed: 1) To determine if DNA polymorphism within the runt related transcription factor (RUNX2) gene is a determinant of BMD and fracture in women. 2) The identification of RANKL target genes in osteoclastogenesis. RUNX2 is a runt domain transcription factor (Werner et al., 1999) essential for osteoblast differentiation (Lee et al., 1997). RUNX2 gene knock-out mice have no osteoblasts due to a failure in osteoblast differentiation and consequently unmineralised skeletons, (Komori et al., 1997; Otto et al., 1997). In humans, mutations in RUNX2 cause cleidocranial dysplasia (CCD), a disorder characterised by hypoplasia or aplasia of the clavicles, short stature, supernumerary teeth, patent fontanelles and other changes in skeletal patterning and growth (Mundlos et al., 1997). RUNX2 contains a poly-glutamine poly-alanine (polyQ/polyA) repeat where mutations causing cleidocranial dysplasia have been observed. BMD has not been routinely examined in CCD, two studies have identified CCD patients with lower BMD with one fracture case identified (Quack et al., 1999; Bergwitz et al., 2001). The central role of RUNX2 in determining osteoblast differentiation makes RUNX2 a prime candidate gene for regulating adult bone density. To determine if polymorphism was present in the polyQ/polyA tract the repeat was amplified within the upper and lower deciles of femoral neck (FN) BMD in the Geelong Osteoporosis study (GOS). The upper and lower deciles of FN BMD acted as a surrogate for genotyping the entire cohort. This study identified two common variants within the polyA repeat: an 18 base pair deletion (11Ala) and a synonymous alanine codon polymorphism with alleles, GCA and GCG (noted as A and G alleles, respectively). The 11Ala and SNP polymorphism are found on codon 64 and 66 respectively (RUNX2 MRIPV variant). A allele frequencies were significantly different in a comparison of the upper and lower deciles of FN BMD (p=0.019). In 495 randomly selected women of the Geelong Osteoporosis Study (GOS), the A allele was associated with higher BMD at all sites tested. The association was maximal at the ultra-distal radius (p=0.001). In a separate fracture study, the A allele was significantly protective against Colles' fracture in elderly women but not spine and hip fracture. The 11Ala polymorphism was not related to BMD in GOS. To further decipher the role of the RUNX2 A allele we genotyped 992 women from a Scottish cohort. The alleles of RUNX2 within the glutamine/alanine repeat were determined by MspA1I restriction digest. To examine the possible influence on estrogen related therapies or estrogen status on the potential genetic effect conferred by RUNX2, we divided the cohort by menopausal and hormone replacement therapy status. Within postmenopausal Scottish women the RUNX2 A allele was associated with significantly higher FN BMD (p=0.028, n=312) but not lumbar spine (LS) BMD. The A allele was associated with higher FN BMD (p=0.035) within a postmenopausal subgroup of the population (n=312). To investigate the effect of weight on the RUNX2 alleles the Scottish cohort was segregated into thin/normal (BMI ≥ 25 kg/m2) and overweight /obese (BMI > 25 kg/m2). RUNX2 A allele showed a stronger effect on FN BMD in postmenopausal women above the median BMI. The 11Ala RUNX2 deletion allele was significantly associated with decreased LS BMD (p=0.018) within overweight/obese women (n=546). The 11Ala allele was significantly associated with increased levels of pyridinoline (p=0.014) and deoxypyridinoline (p=0.038) in the HRT treated subgroup of the population (n=492). Glutamine variants and an alanine insertion were identified within the group. These data suggest that the RUNX2 11Ala and A alleles exert differing affects on BMD showing preference for different skeletal sites in a weight dependent manner. We genotyped 78 individuals from an osteoarthritic population to elucidate the role of the RUNX2 alleles on markers of bone turnover and inflammation. The RUNX2 11Ala allele was significantly associated with decreased osteocalcin (OC) serum levels (p = 0.01). The RUNX2 A allele was significantly related to reduced tumor necrosis factor alpha (TNF-alpha) serum levels (p = 0.004). RUNX2 is known to bind to the OC promoter. An OC promoter polymorphism is found 7bp upstream from a putative RUNX2 binding site. We hypothesized that OC polymorphism may effect the RUNX2 transactivation of the OC gene and thus affect OC serum levels. OC promoter polymorphism was not related to OC serum levels (n=78). These data present a novel link between RUNX2 alleles and OC and TNF serum levels, providing putative mechanisms of action for the RUNX2 alleles. Further studies in larger populations are required to confirm these findings. Ten individuals within the GOS and the Scottish cohort were found to carry rare mutations of the polyQ/polyA repeat. All polyQ variants had a normal polyA repeat (17 amino acids) and were heterozygous for a normal 23Q/17A allele. Variants observed were 15, 16, 24 and 30Q. One individual was observed with an extended polyA repeat (24A). Patient records indicated otherwise unremarkable clinical history except for fracture in 4/10 individuals from GOS (hip and spine). BMD data from the LS and the FN were expressed as T-scores, a measure that relates BMD in terms of standard deviations below the young normal value. In addition, BMD data were also expressed as Z-scores around the age-mean. Under the null hypothesis, where RUNX2 Q repeat variation has no effect on BMD, Z scores would be expected to be distributed around a mean of zero. However, when all variants were pooled the BMD was significantly lower than expected. This effect persisted when deletion variants were considered alone. The effect was stronger on FN BMD (p=0.001) rather than LS BMD (p=0.096), reflecting either difference in precision of BMD measurements at these sites or perhaps a differential genetic effect on different skeletal sites. These data suggest that polyQ and polyA variants are associated with significantly lower BMD, and may be an important determinant for fracture. Glutamine variants exist at high frequency (~0.7%): this rate of mutation could be important when considering large populations at risk of age related osteoporosis. Considering that these subjects are heterozygous for a normal allele, it suggests that a more severe phenotype might be expected in rare subjects homozygous for glutamine repeat variants. In summary, this study investigated the role of novel polymorphisms and rare variants of the RUNX2 gene in influencing BMD, fracture and markers of bone turnover. Two common polymorphisms were identified within the polyA repeat: an 18 base pair deletion and a synonymous alanine codon polymorphism with alleles, A and G. The A allele was associated with increased BMD and was protective against a common form of osteoporotic fracture within a Geelong population. To verify these findings the RUNX2 alleles were genotyped in 992 women from a Scottish cohort. The magnitude and the direction of the effect of the A allele was maintained in the Scottish cohort. Interestingly, the A allele was shown to exert a menopause specific effect, with postmenopausal women showing the strongest effect. On re-analysis of the GOS data the post-menopausal women were found to drive the significance identified in the cohort. The magnitude of the effect of the A allele on BMD was greater in overweight/obese postmenopausal women indicating a gene-weight interaction for RUNX2. The RUNX2 11Ala allele showed a significant relationship with decreased LS BMD in overweight/obese Scottish women. The 11Ala allele was also associated with higher levels of urinary PYD and DPD in women treated with HRT, indicating higher levels of bone turnover in carriers of the 11Ala allele. In contrast to the Scottish cohort, no significant association with heterozygous carriers of 11Ala was observed in GOS, although a significant association was detected for homozygous carriers and LS BMAD. The 11 Ala RUNX2 allele was significantly associated with decreased serum osteocalcin levels and the A allele was significantly associated with TNF in OA patients. Glutamine variants and an alanine insertion were identified within Geelong and Scottish cohorts, which showed low Z and T scores suggesting that RUNX2 variants may be related to genetic effects on BMD and osteoporosis. Polymorphism of the polyQ/polyA region of RUNX2 were identified within this study were shown to associate with significant differences in BMD. The A allele showed a significant association with increased BMD in postmenopausal women from a Geelong and Scottish cohort, with a decreased frequency of the A allele observed in Colles' fracture patients from Geelong. The 11Ala deletion allele was significantly associated with decreased LS BMD and increases in markers of bone turnover in the Scottish cohort. A significant decrease in OC serum levels was observed in OA patients suggesting a direct effect of the allele on the transactivation of the RUNX2 gene. Rare variants of RUNX2 were identified which showed low BMD. These studies have provided insight into the role of RUNX2 in influencing BMD, further studies are required to verify the role of the A allele on BMD and fracture, the role of the rare variants and to identify the precise mechanisms behind the observed changes in BMD. - 2) The identification of RANKL target genes in osteoclastogenesis. Osteoclastogenesis is regulated in vivo by the action of osteoblast/stromal cells that express membrane bound, receptor activator of NF-kB ligand (RANKL). Monocytes treated in vitro with a soluble form of RANKL and macrophage colony stimulating factor (M-CSF) differentiate to osteoclasts, whereas monocytes treated with M-CSF alone differentiate to macrophage-like cells. The gene expression profile of human osteoclasts has not been extensively explored. Genes highly expressed by rabbit osteoclasts were identified through random sequencing of an osteoclast cDNA library (Sakai et al., 1995). Differential gene expression of mouse osteoclastogenesis was elucidated by array analysis (Cappellen et al., 2002). To identify genes important for human osteoclastogenesis, total RNA was isolated from monocytes treated for three weeks with either M-CSF alone or with RANKL and M-CSF. RANKL treatment for 3 weeks and 12 hours was investigated in this study, to complement previous data. Differential display was performed on RNA (12 hour treatment with RANKL) and differential gene expression profiles examined. The differential display products were pooled to generate a probe for screening a gene array system derived from a human osteoclast cDNA library. cDNA (3 week treatment with RANKL) hybridisation experiments against the array revealed additional regulated genes. Gene clones that showed significant regulation in M-CSF and RANKL treated cells compared M-CSF treated cells represent genes that are targets for RANKL-specific regulation. Osteopontin, creatine kinase and various mitochondrial genes were up regulated by the treatment of RANKL. Changes in gene expression observed in the array data were confirmed with real-time PCR using mRNA derived from in vitro induced osteoclasts. Cathepsin K gene expression was more than 300 fold greater in osteoclasts compared to macrophage-like cells after one week treatment with RANKL and M-CSF. Cystatin C expression showed a six-fold induction at two weeks of RANKL and M-CSF treatment and cystatin B showed a steady increase in expression. Some of these regulated genes may provide useful targets for influencing BMD.

O processo de diferenciação e ativação de osteoclastos, essencial para a manutenção da homeostasia do tecido ósseo e também envolvido na patogênese de diversas patologias caracterizadas pela atividade osteolítica, depende de um sistema central de controle que envolve a ligação das moléculas RANK/RANKL. Além do sistema RANK/RANKL, moléculas co-estimulatórias de osteoclastos, tais como os complexos DAP-12, TREM-2 e SIRP1, e FcR, OSCAR e PIR-A, também apresentam um papel importante na geração e ativação de osteoclastos. Entretanto, a possível contribuição de tais moléculas para a progressão da doença periodontal (DP) permanece desconhecida, assim como o possível impacto de citocinas na modulação de sua expressão no microambiente periodontal. Nosso objetivo foi investigar, por RealTimePCR, o padrão de expressão de moléculas co-estimulatórias de osteoclastos (DAP-12, TREM-2 e SIRP1, e FcR, OSCAR e PIR-A) na periodontite crônica em humanos, além de avaliar a cinética de expressão destas moléculas e a sua modulação por citocinas (TNF-, IFN-, IL-17 e IL-10) ao longo do curso da DP em camundongos em camundongos C57Bl/6 wild-type (WT) e geneticamente modificados (TNFp55KO, IFNKO, IL17KO, IL10KO. Nossos resultados demonstram que nas lesões periodontais crônicas a expressão de todas as moléculas co-estimulatórias de osteoclastos apresentaram-se significativamente aumentadas quando comparadas às amostras controle. Com relação à periodontite experimental, verificamos que todas as moléculas co-estimulatórias alvo apresentavam aumento em sua expressão após a indução de doença quando comparado aos controles. Nos camundongos para TNFp55KO, IFNKO e IL17KO, observamos uma redução na severidade da DP (reabsorção óssea e quantidade de células inflamatórias) e na expressão de moléculas co-estimulatórias, ao contrário do observado nos camundongos IL10KO. Entretanto, ao normalizarmos os níveis de expressão das moléculas co-estimulatórias de osteoclastos pelo número de células inflamatórias, verificamos que TNF- e IL-17 se mostram associados a uma maior expressão de moléculas co-estimulatórias, enquanto IFN- e IL-10 parecem regular negativamente a expressão de tais moléculas. Em termos gerais, demonstramos que a expressão de moléculas co-estimulatórias de osteoclastos se mostra aumentada na DP humana e experimental, e que citocinas parecem modular sua expressão por mecanismos diretos e indiretos, tais como a migração de células inflamatórias para os sítios de doença periodontal.
The osteoclast differentiation and activation are essential to bone tissue homeostasis and in the development of bone pathologies, which RANK/RANKL signaling molecules are the major osteoclastogenic factor. However, osteoclast co-stimulatory molecules, such as DAP-12, TREM-2, SIRP1, FcR, OSCAR and PIR-A, also present an important role in the osteoclastogenesis. However, the exact role and regulation of these molecules in human and mice periodontal diseases (PD) development have not completely known. Our aim was to investigate the pattern of osteoclast co-stimulatory expression (DAP-12, TREM-2, SIRP1, FcR, OSCAR and PIR-A) in human chronic periodontitis (CP), apart from analyze the kinetic of these molecules and their regulation by cytokines (TNF-, IFN-, IL-17 and IL-10) in the development of experimental periodontal disease in mice C57Bl/6 and knockout. Our results demonstrated that all osteoclast co-stimulatory molecules presented highly expressed in CP patients when compared with control. Similar results are presented about experimental PD, where all co-stimulatory molecules was presented highly expressed in infected mice when compared with control mice. We observed in TNFp55KO, IFNKO and IL17KO mice a decrease in PD scores and co-stimulatory molecules expression, the opposite of IL10KO mice. However, when we standardized the co-stimulatory molecules levels by the number of inflammatory cells, we found that TNF- and IL-17 are associated with increased expression of co-stimulatory molecules, while IFN- and IL-10 appear to negatively regulate the expression of such molecules. In conclusion, we demonstrated that osteoclast co-stimulatory molecules shown increased in human and experimental PD, and cytokines appear to modulate their expression by direct and indirect mechanisms, such as inflammatory cells migration to the PD infected tissue.

In summary, this study investigated the role of novel polymorphisms and rare variants of the RUNX2 gene in influencing BMD, fracture and markers of bone turnover. Two common polymorphisms were identified within the polyA repeat: an 18 base pair deletion and a synonymous alanine codon polymorphism with alleles, A and G. The A allele was associated with increased BMD and was protective against a common form of osteoporotic fracture within a Geelong population. To verify these findings the RUNX2 alleles were genotyped in 992 women from a Scottish cohort. The magnitude and the direction of the effect of the A allele was maintained in the Scottish cohort. Interestingly, the A allele was shown to exert a menopause specific effect, with postmenopausal women showing the strongest effect. On re-analysis of the GOS data the post-menopausal women were found to drive the significance identified in the cohort. The magnitude of the effect of the A allele on BMD was greater in overweight/obese postmenopausal women indicating a gene-weight interaction for RUNX2. The RUNX2 11Ala allele showed a significant relationship with decreased LS BMD in overweight/obese Scottish women. The 11Ala allele was also associated with higher levels of urinary PYD and DPD in women treated with HRT, indicating higher levels of bone turnover in carriers of the 11Ala allele. In contrast to the Scottish cohort, no significant association with heterozygous carriers of 11Ala was observed in GOS, although a significant association was detected for homozygous carriers and LS BMAD. The 11 Ala RUNX2 allele was significantly associated with decreased serum osteocalcin levels and the A allele was significantly associated with TNF in OA patients. Glutamine variants and an alanine insertion were identified within Geelong and Scottish cohorts, which showed low Z and T scores suggesting that RUNX2 variants may be related to genetic effects on BMD and osteoporosis. Polymorphism of the polyQ/polyA region of RUNX2 were identified within this study were shown to associate with significant differences in BMD. The A allele showed a significant association with increased BMD in postmenopausal women from a Geelong and Scottish cohort, with a decreased frequency of the A allele observed in Colles’ fracture patients from Geelong. The 11Ala deletion allele was significantly associated with decreased LS BMD and increases in markers of bone turnover in the Scottish cohort. A significant decrease in OC serum levels was observed in OA patients suggesting a direct effect of the allele on the transactivation of the RUNX2 gene. Rare variants of RUNX2 were identified which showed low BMD. These studies have provided insight into the role of RUNX2 in influencing BMD, further studies are required to verify the role of the A allele on BMD and fracture, the role of the rare variants and to identify the precise mechanisms behind the observed changes in BMD.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Health Sciences
Full Text

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Periodontal disease is an infection initiated by oral periodontal pathogens that trigger an immune response culminating in tissue destruction. This destruction is mediated by the host by inducing the production and activation of lytic enzymes, cytokines and the stimulation of osteoclastogenesis. The aim of this study was to compare the immunohistochemical expression of factors involved in bone resorption, RANKL (Ligand Receptor Activator of Nuclear Factor kappa B), OPG (Osteoprotegerin) and TNF-α (tumor necrosis factor alpha) between the gingival healthy, gingivitis and chronic periodontitis and correlate them with clinical parameters. The sample consisted of 83 cases and 12 clinically healthy gums, 42 gingivitis and 29 periodontitis, from 74 adolescent and adult patients with a mean age of 35 years, without systemic changes and non-smokers, predominantly female and race brown. There was no statistically significant difference for the expression of anti-RANKL (p = 0.581) and RANKL / OPG ratio (p = 0.334) when comparing the three conditions, but the anti-OPG and anti-TNF-α showed statistically significant between the types of injury (p = 0.001 and p <0.001, respectively), showing greatest expression in periodontitis. In cases of periodontitis, the variable clinical attachment loss (PIC) was statistically significant and positive correlation, respectively, with immunostaining of anti-RANKL (p = 0.002, p = 0.001 and r = 0.642), anti-OPG (p = 0.018, p = 0.014 and r = 0.451), anti-TNF-α (p = 0.032, p = 0.014 and r = 0.453) and the percentage ratio of RANKL / OPG (p = 0.018, p = 0.002 and r = 0.544). The tooth mobility (MB) showed a statistically significant difference only with immunohistochemical anti-RANKL (p = 0.026), and probing depth (PD) was positively correlated with anti-RANKL (p = 0.028 and r = 0.409), both in cases of periodontitis. Only in cases of gingivitis TNF-α was positively correlated with RANKL (p = 0.012 and r = 0.384) and the RANKL / OPG ratio (p = 0.027 and r = 0.341). Given these results, we conclude that the greatest expression of TNF-α in periodontitis demonstrates a relationship with the progression and severity of periodontal disease and the correlation between all antibodies and clinical attachment loss demonstrates their involvement in periodontal bone resorption
A doen?a periodontal ? uma infec??o oral iniciada por periodontopat?genos que desencadeiam a resposta imune culminando com a destrui??o tecidual. Essa destrui??o ? mediada pelo hospedeiro atrav?s da indu??o da produ??o e ativa??o de enzimas l?ticas, citocinas e da estimula??o da osteoclastog?nese. O objetivo deste estudo foi comparar a express?o imuno-histoqu?mica dos fatores envolvidos na reabsor??o ?ssea, RANKL (Ligante do Receptor Ativador do Fator Nuclear kappa B), OPG (Osteoprotegerina) e TNF-α (Fator de Necrose Tumoral Alfa) entre a gengiva clinicamente saud?vel, a gengivite e a periodontite cr?nica, correlacionando-os com os par?metros cl?nicos periodontais. A amostra consistiu de 83 casos, sendo 12 de gengivas clinicamente saud?veis, 42 de gengivite e 29 de periodontite, oriundos de 74 pacientes adolescentes e adultos com idade m?dia de 35 anos, sem altera??es sist?micas e n?o fumantes, predominantemente do sexo feminino e da ra?a parda. N?o houve diferen?a estatisticamente significativa para express?o do anticorpo anti-RANKL (p=0,581) e da raz?o RANKL/OPG (p=0,334) quando se comparou as tr?s condi??es cl?nicas, mas o anti- OPG e anti-TNF-α mostraram diferen?as estatisticamente significativas entre os tipos de les?o (p=0,001 e p<0,001, respectivamente), revelando maior imunoexpress?o na periodontite. Nos casos de periodontite, a vari?vel perda de inser??o cl?nica (PIC) mostrou diferen?a estatisticamente significativa e correla??o positiva, respectivamente, com a imunomarca??o dos anticorpos anti-RANKL (p=0,002; p=0,001 e r=0,642), anti-OPG (p=0,018; p=0,014 e r=0,451), anti-TNF-α (p=0,032; p=0,014 e r=0,453) e com a raz?o percentual de RANKL/OPG (p=0,018; p=0,002 e r=0,544). A mobilidade dent?ria (MB) apresentou diferen?a estatisticamente significativa somente com a imunoexpress?o do anti-RANKL (p=0,026), e a profundidade de sondagem (PS) apresentou correla??o positiva com o anti- RANKL (p=0,028 e r=0,409), ambos nos casos de periodontite. Somente nos casos de gengivite o TNF-α apresentou correla??o positiva com o RANKL (p=0,012 e r=0,384) e com a raz?o RANKL/OPG (p=0,027 e r=0,341). Diante desses resultados, conclui-se que a maior imunoexpress?o do TNF-α na periodontite demonstra uma rela??o com a progress?o e severidade da doen?a periodontal e a correla??o entre todos os anticorpos e a perda de inser??o cl?nica demonstra o envolvimento destes na reabsor??o ?ssea periodontal

Osteoclasts are multinucleated cells found exclusively in bone and are derived from the haematopoietic cells of monocytes/macrophage lineage. The cell-to-cell interaction between osteoblastic/stromal cells and osteoclast precursor cells is necessary for osteoclastogenesis. Receptor Activator of NF-κB ligand (RANKL) was identified as a membrane-bound TNF ligand family member that is the ‘master’ cytokine expressed on osteoblastic/stromal cells, which stimulate osteoclastogenesis through cell-to-cell contact with osteoclast precursors. RANKL is considered to be a factor that is necessary and sufficient for the induction of osteoclastogenesis (Lacey, et al., 1998). RANKL is a type II transmembrane cytokine of the TNF ligand superfamily and has an active TNF-like core domain at the extracellular domain. This active TNF-like core domain is thought to be the region through which it binds to it’s active receptor, RANK, for the activation of signal transduction pathways for the initiation of processes leading to osteoclastogenesis (Lacey, et al., 1998; Li, et al., 1999). It was hypothesized that any change in the active TNF-like core domain might affect the ability of RANKL binding to RANK and consequently affect the activation of signal transduction pathways and osteoclastogenesis. Hence, this thesis sought to investigate the effects of changes in the active TNF-like core domain by truncation mutation on the ability of RANKL binding to RANK and consequently affect the activation of signal transduction pathways and osteoclastogenesis. A cDNA fragment encoding the full-length TNF-like core domain of rat RANKL (rRANKL) (aa160-318) was cloned into the bacterial expression pGEX vectors and stably expressed in Eschechia coli as a fusion protein with the C-terminus of glutathione S-transferase (GST). Four mutants (aa160-302, aa160-268, aa239-318 and aa246-318) were also generated by truncation mutation in the TNF-like core domain, and cloned into the pGEX vector to produce GST-rRANKL mutants. The proteins were over-expressed and affinity purified to 95% in purity. GST-rRANKL (160-318) containing the full length TNF-like core domain was able to induced osteoclastogenesis in spleen cells in the presence of M-CSF and in RAW264.7 cells in the absence of M-CSF. It was also found to activate mature osteoclast activity in vitro, ex vivo and in vivo. It has the highest binding affinity to RANK and the greatest potency for NF-κB activation as well as the induction of osteoclastogenesis compared to the truncated mutants. Mutants generated by truncation of the TNF-like core domain revealed that the TNF-like core domain is important for the interaction with the RANK, for high binding affinity, NF-κB activation and induction of osteoclastogenesis. In general, the truncated mutants not only displayed a reduction in the binding affinity to RANK, but also a reduction in NF-κB activation, and significantly reduced potency in the induction of osteoclastogenesis. Interestingly, mutant GST-rRANKL (160-268) showed a higher affectivity than the other mutants did, in that it had greater binding affinity to RANK, and in NF-κB activation than the rest of the truncated mutants. Mutants GST-rRANKL (239-318) and GST-rRANKL (246-318) on the other hand, showed little potency in the induction of osteoclast formation, however, might have an inhibitory effect through competition with full length GST-rRANKL (160-318) as well as inducing a response in vivo resulting in an increase in the serum calcium level. In conclusion, this thesis demonstrated that the TNF-like core domain of RANKL is active, and imperative in the binding to RANK, activating signal transduction pathways and induction of osteoclastogenesis. Changes in the active TNF-like core domain affected the ability, affinity and efficiency of RANKL binding to the receptor, RANK and consequently affected the activation of signal transduction pathways and osteoclastogenesis.

Les eicosanoïdes sont des médiateurs importants qui encadrent et régulent les fonctions osseuses. Leur production est sous la tutelle des phospholipases A[indice inférieur 2] qui permettent la relâche d’acide arachidonique et de lysophospholipides puis de leur métabolisme subséquent des membranes cellulaires. Les PLA[indice inférieur 2] sécrétées ont également comme particularité de pouvoir exercer leurs effets directement via leurs récepteurs membranaires comme ligand. Malgré l’implication connue des prostaglandines sur les fonctions ostéoclastiques et dans plusieurs processus pathologiques résultants en érosion osseuse, les phospholipases A[indice inférieur 2] ostéoclastiques restent inconnues et leurs rôles, spéculatifs. Les études présentées démontrent la présence de la cPLA[indice inférieur 2]-α et de la sPLA[indice inférieur 2] IIA chez les ostéoclastes humains. Par contre, leur expression semble différer selon l’état de l’os. En effet, la cPLA[indice inférieur 2]-α semble exprimée ubiquitairement, mais la sPLA[indice inférieur 2] IIA n’est détectable que dans l’os fœtal ou atteint de la maladie de Paget et n’est pas exprimée dans l’os sain, ostéoporotique ou arthrosique. La sPLA[indice inférieur 2] IIA semble donc exprimée en condition de fort remodelage osseux. Dans notre modèle, la cPLA[indice inférieur 2]-α revêt un rôle anti-ostéoclastogénique. En effet, la cPLA[indice inférieur 2]- α produit des écosanoïdes qui inhibent l’ostéoclastogenèse. Ces derniers sensibilisent les ostéoclastes à l’apoptose. En revanche, un certain degré d’activation de la cPLA[indice inférieur 2]-α est requis pour la résorption osseuse, car son inhibition bloque la résorption osseuse en désorganisant les anneaux d’actine requis pour la résorption, et ce, de façon dépendante de la production d’acide arachidonique. En ce qui a trait à la sPLA[indice inférieur 2] IIA, elle stimule l’ostéoclastogenèse indépendamment de son activité catalytique, probablement via l’un de ses récepteurs membranaires. D’autre part, elle confère une résistance à l’apoptose autant chez les ostéoclastes matures que chez leurs précurseurs CD14+. Par contre, l’inhibition de la sPLA[indice inférieur 2] IIA bloque la résorption osseuse indépendamment du remodelage du cytosquelette d’actine, probablement via leur apoptose. Les résultats inclus dans cette thèse semblent donc démontrer la présence de deux PLA[indice inférieur 2]s chez les ostéoclastes humains ainsi que leur attribuer des rôles en physiologie et pathologie osseuse. Ces évidences pourraient faire des PLA[indice inférieur 2] de nouvelles cibles thérapeutiques pour le traitement de pathologies ostéo-articulaires, dont la maladie osseuse de Paget. // Abstract : Eicosanoïds are important mediators of bone biology. The first regulated enzymes in the biosynthetic pathway of eicosanoids are the PLA[subscript 2]s, which release arachidonic acid and lysophospholipids from membrane phospholipids. Further metabolism of arachidonic acid will lead, among other things, to the synthesis of prostaglandins, which deeply influence bone metabolism. Secreted PLA[subscript 2]s (sPL[subscript 2]) may also act independently of their catalytic activity, as a ligand to their membrane bound receptors. Even though PLA[subscript 2]s have been associated with joint and bone pathologies, their presence and functions were never investigated in osteoclasts (OCs), the principal cell responsible for bone destruction. Our study established the presence of cPLA[subscript 2] and sPLA[subscript 2] in human OCs, but demonstrated a contrast in their expression. cPLA[subscript 2] seems to be expressed ubiquitously, contrary to sPLA[subscript 2] whose expression is restricted to OCs from foetal bone and bone suffering from Paget disease. There is no trace of sPLA[subscript 2] in healthy bone or bone suffering from osteoarthrosis or osteoporosis, thus sPLA[subscript 2] seems tightly linked to high bone turnover. In our model, cPLA[subscript 2] exerted an anti-osteoclastogenic effect. Indeed, cPLA[subscript 2] produces eicosanoïds that inhibit osteoclastogenesis and sensitize OCs to apoptosis. Nevertheless, a minimum cPLA[subscript 2] activity is required since complete cPLA[subscript 2] inhibition blocks osteoclast mediated bone resorption. Its inhibition leads to disorganization of the OC cytoskeleton and inhibits the actin ring formation required for bone resorption in an arachidonic acid dependent fashion. On the other hand, sPLA[subscript 2] stimulates osteoclastogenesis independently of its catalytic activity; probably via interaction with one of its membrane bound receptors. sPLA[subscript 2] decreases OC and their CD14+ precursors’ sensibility to apoptosis. Moreover, sPLA[subscript 2] inhibition inhibits bone resorption independently of actin cytoskeleton remodeling, probably by inducing mature OC apoptosis. Together, these results demonstrate the presence of two PLA[subscript 2]s in human OCs and highlight their important roles in bone physiology and pathophysiology. Highlighting those functions could eventually lead to the elaboration of new strategies to control hyperosteoclastic states by targeting PL[subscript 2]s.

Durante a resposta inflamatória, diversos mediadores são liberados localmente com o objetivo de estimular a resposta imune celular e humoral. Por meio da ação das enzimas ciclooxigenases e lipoxigenases ocorrerão modificações estruturais na cadeia do ácido araquidônico levando a síntese de prostaglandinas ou leucotrienos e lipoxinas, respectivamente. Tais mediadores são responsáveis pela regulação da expressão dos genes RANK, RANKL e OPG, moduladores da osteoclastogênese. Dessa maneira, o objetivo deste estudo foi avaliar a expressão do RNA mensageiro (RNAm) para as enzimas envolvidas no metabolismo do ácido araquidônico, ciclooxigenase-2 (COX-2) e 5- lipoxigenase (5-LO), e para os mediadores da osteoclastogênese (RANK, RANKL e OPG) no tecido ósseo, após inoculação de lipopolissacarídeo bacteriano (LPS) nos canais radiculares de molares de camundongos. Posteriormente foi investigado o efeito do bloqueio farmacológico da via COX-2 induzida pelo LPS, na expressão de mediadores da osteoclastogênese e de genes envolvidos no metabolismo ósseo. Foram utilizados 144 camundongos C57BL/6, com 6 semanas de idade, pesando de 18 a 20 gramas, nos quais os canais radiculares dos primeiros molares foram inoculados com uma solução contendo lipopolissacarídeo bacteriano de E. coli (0,1, 1,0 e 10mg/ml). Decorridos os períodos experimentais de 7, 14, 21 e 28 dias, os animais foram submetidos à eutanásia e os blocos contendo dente e osso foram removidos para extração do RNA total. Em seguida, foi realizada a avaliação da expressão gênica por meio de transcrição reversa e reação da polimerase em cadeia em tempo real (qRT-PCR). A análise global da expressão de RNAm para proteínas envolvidas no metabolismo ósseo foi realizada por meio de um ensaio de PCR Array (Osteogenesis RT² Profiler PCR Array). Os valores de expressão relativa de cada RNAm, para cada grupo, foram comparados por meio da análise de variância (ANOVA) de duas vias seguido pelo pós-teste de Bonferroni ou por ANOVA de uma via seguido pelo pós-teste de Dunnett (&alpha = 0,05). A inoculação de LPS nos canais radiculares de molares de camundongos foi capaz de induzir a expressão dos genes PTGS2 e ALOX5, responsáveis pela codificação das enzimas COX-2 e 5-LO, envolvidas no metabolismo do ácido araquidônico, concomitantemente à modulação da expressão dos genes TNFRSF11A, TNFSF11 e TNFRSF11B, responsáveis pela codificação dos moduladores da osteoclastogênese RANK, RANKL e OPG, respectivamente. A administração de Indometacina, um inibidor não seletivo de COX-2, inibiu a expressão de RNAm para RANK e RANKL e estimulou a expressão de OPG durante os períodos iniciais de resposta à inoculação de LPS nos canais radiculares. A inibição da via COX-2 de metabolismo do ácido araquidônico nos períodos iniciais de resposta à inoculação de LPS nos canais radiculares modulou diferencialmente a expressão de genes envolvidos no catabolismo e anabolismo ósseo, indicando possíveis papéis para os mediadores derivados no ácido araquidônico na regulação do metabolismo ósseo. Estes resultados sugerem alvos terapêuticos importantes para intervenção precoce em doenças inflamatórias, como lesões periapicais para evitar a reabsorção do tecido ósseo.
During an inflammatory response, several mediators are locally released in order to stimulate cellular and humoral immune response. Through the action of cyclooxygenase and lipoxygenase enzymes structural changes occur in the arachidonic acid chain leading to synthesis of prostaglandins or leukotrienes and lipoxins, respectively. Such mediators are responsible for the regulation of RANK, RANKL and OPG gene expression, osteoclastogenesis modulators. Thus, the objective of this study was to evaluate the expression of messenger RNA (mRNA) for the enzymes involved in arachidonic acid metabolism, cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LO), and the osteoclastogenesis mediators (RANK, RANKL and OPG) in bone tissue after injection of bacterial lipopolysaccharide (LPS) in murine dental root canals. Then, COX-2 pathway was pharmacologically blocked for investigation of expression of osteoclastogenesis mediators and genes involved in bone metabolism. We used 144 C57BL/6 mice, 6 weeks-old, weighing 18-20 grams, which had the first molars root canals inoculated with a solution containing LPS from E. coli (0.1, 1.0 and 10 mg/ml). After 7, 14, 21 and 28 days the animals were euthanized and the tooth-and-bone blocks were removed for total RNA extraction. Subsequently, the evaluation of gene expression was performed by reverse transcription and polymerase chain reaction in real time (qRT-PCR). Global analysis of mRNA expression for proteins involved in bone metabolism was performed using PCR arrays (Osteogenesis RT² Profiler PCR Array). The values for relative expression of each mRNA for each group were compared using two-way analysis of variance (ANOVA) followed by Bonferroni post-test or one-way ANOVA followed by Dunnett\'s test (α=0.05). The injection of LPS into the root canals was induced expression of genes PTGS2 and ALOX5, responsible for encoding COX-2 and 5-LO enzymes, involved in the metabolism of arachidonic acid, simultaneously to the modulation of gene expression of TNFRSF11A, TNFSF11 and TNFRSF11B, responsible for encoding the osteoclastogenesis modulators RANK, RANKL and OPG, respectively. Administration of Indomethacin, a non-selective inhibitor of COX-2, inhibited the expression of mRNA for RANK and RANKL and stimulated the expression of OPG during the initial response to the root canals contamination with LPS. Inhibition of the COX-2 pathway from arachidonic acid metabolism in the initial periods of response to LPS injection into the root canals differentially modulated the expression of genes involved in bone catabolism and anabolism, indicating possible roles for mediators derived from arachidonic acid in the regulation of bone metabolism. These results suggest important therapeutic targets for early intervention in inflammatory diseases such as apical periodontitis to avoid resorption of bone tissue.

Orientadores: Pedro Luiz Rosalen, Francisco Carlos Groppo, Toshihisa Kawai
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: Doxiciclina (Dox) é um antimicrobiano pertencente à família das tetraciclinas com um amplo espectro de ação contra bactérias Gram-positivas e Gram-negativas. Além de suas propriedades antimicrobianas, Dox é atualmente empregada na periodontia como um modulador da resposta do hospedeiro (MRH), ao inibir a atividade da enzima metaloproteinase de matriz (MMP), a qual está relacionada ao processo de destruição tecidual. Neste contexto, este trabalho teve os seguintes objetivos: 1-determinar os parâmetros farmacocinéticos e integrar os índices PK/PD da Dox para o plasma, fluido gengival (FG) e saliva; 2-analisar os efeitos in vitro e in vivo da Dox sobre a osteoclastogênese com a finalidade de elucidar possíveis propriedades biológicas adicionais deste fármaco como MRH. Para a análise farmacocinética, 12 voluntários receberam dose oral única de 100 mg de Dox. Sangue, FG e saliva foram coletados em tempos pré-determinados e a concentração da Dox nestes fluidos foi determinada por bioensaio. A análise dos principais índices PK/PD da Dox foi realizada considerando o CIM para P. gingivalis. Para o segundo objetivo, o efeito da Dox sobre os processos de diferenciação e ativação osteoclástica foi verificado, respectivamente, pela contagem de células TRAP+ multinucleadas geradas a partir de células precursoras estimuladas com sRANKL na presença ou ausência de Dox e pela análise das lacunas de reabsorção formadas por estas células, quando cultivadas sobre discos de dentina. In vivo, o efeito da Dox sobre a osteoclastogênese foi determinado através da indução deste processo em calvária de camundongo. Solução de sRANKL/LPS foi injetada na região da calvária e os animais receberam, por gavagem, Dox ou placebo diariamente. Após 10 dias, a calvária foi removida para análise histoquímica. Em acréscimo, a atividade da Dox sobre a expressão de genes responsáveis pelos processos de diferenciação e ativação osteoclástica foi analisada por RT-PCR. Durante os experimentos in vitro e in vivo, a produção e atividade da MMP foram verificadas através de Western-blot e Zimograma respectivamente. Os resultados demonstraram que as maiores concentrações de Dox foram observadas no plasma, seguido pelo FG e saliva. A análise dos índices PK/PD da Dox indicou que a dose de 100 mg foi insuficiente para se obter os valores ideais antimicrobianos preconizados na /CIM. Os experimentos in vitro e in vivo sobre o efeito da Dox como MRH demonstraram que este fármaco inibiu os processos de diferenciação e ativação dos osteoclastos. Dox também modulou a expressão de proteínas diretamente relacionadas a osteoclastogênese, incluindo TRAP, Catepsina K e c-Myc. Finalmente, embora a síntese da MMP não tenha sido afetada, a atividade da MMP foi reduzida na presença de Dox. Portanto, os resultados do presente estudo sugerem que uma dose inicial maior do que 100 mg é necessária para alcançar o valor preconizado para ASC/CIM e Cmax/CIM, com a finalidade de se obter os melhores resultados clínicos antimicrobianos. A análise da Dox como MRH indicou que este fármaco pode atuar neste processo não somente pela sua capacidade de inativar a MMP, e sim, por apresentar a propriedade de inibir a diferenciação e ativação osteoclástica, incluindo a modulação de sua expressão gênica. literatura para os parâmetros ASC/CIM e Cmax
Abstract: Doxycycline (Dox), a member of the tetracycline family, is an antimicrobial agent with a broad-spectrum of activity against Gram-positive and Gram-negative bacteria. In addition to its antimicrobial properties, Dox is used in the treatment of periodontal diseases as a host response modulator by inhibiting the activity of an important enzyme, matrix metalloproteinase (MMP), which is related to the process of tissue destruction. In this context, this study had the following aims: 1-to determine the pharmacokinetic parameters of Dox and to integrate the PK/PD indices for plasma, gingival crevicular fluid (GCF) and saliva; 2-to analyze the effects in vitro and in vivo of Dox on the osteoclastogenesis and on the osteoclast activation in order to elucidate additional biological properties of Dox on the host response modulation (HRM). Twelve volunteers received single oral administration of Dox (100 mg). Blood, GCF and saliva were collected and the concentrations were measured by bioassay technique. The PK/PD analyses were carried out using the MIC for P. gingivalis. For the second objective, the effect of Dox on the osteoclast differentiation and activation processes was determined, respectively, by the counting of TRAP+ multinuclear cells derived from osteoclast precursory cells sRANKL-stimulated in the presence or absence of Dox and by the analysis of the resorption areas formed by these cells when cultured on dentin discs. In vivo, Dox¿s effect on the osteoclastogenesis was verified using the model of osteoclastogenesis induction in mouse calvaria. sRANKL/LPS was injected in the supra-calvaria area and the animals received Dox or placebo daily by gavage. After the experimental period of 10 days, the calvariae were removed for histochemistry analyses. In addition, the effect of Dox on the expression of genes related to the osteoclast differentiation and activation processes was carried out using RT-PCR technique. MMP production and activity were ensured during in vitro and in vivo experiments by Western-blot and Zymography, respectively. The results demonstrated that Dox achieved the highest concentration in the plasma, following by GCF and saliva. PK/PD analyses showed that the dose of 100 mg was insufficient to get the antimicrobial levels indicated in the literature for AUC/MIC and Cmax/MIC indices. In vitro and in vivo studies of Dox¿s effects on the HRM demonstrated that this drug could inhibit the osteoclast differentiation and activation process. Dox also showed an important property of down-regulation in the expression of proteins directly related to osteoclastogenesis, including TRAP, Cathepsin K and c-Myc. Finally, although Dox did not affect the expression of MMP protein, MMP activity was remarkably decreased by Dox. Therefore, the present study suggests that higher doses than 100 mg would be necessary to obtain effective antimicrobial levels and the effect of DOX on the HRM can be due to not only by MMP inhibition but also by the direct effect on RANKL-mediated osteoclast differentiation and activation, including its gene regulation
Doutorado
Farmacologia, Anestesiologia e Terapeutica
Doutor em Odontologia

Ogni anno in Italia si registrano trentacinquemila nuovi casi di metastasi ossee e l’osso è il terzo sito di metastatizzazione più frequente al mondo. Non avendo a disposizione cure efficaci contro la malattia, il progetto “Dinamica” si impegna a sviluppare dei materiali biomimetici medicati che svolgono la duplice azione antitumorale e inibente il riassorbimento osseo. Obiettivo principale del progetto è la validazione in vitro ed ex vivo dei dispositivi, con l’intento di poterli utilizzare come cura per i pazienti colpiti da metastasi ossee. Per ricreare l’ambiente metastatico in laboratorio, utile per la validazione in vitro dei device, gli studiosi sono partiti ottenendo e analizzando le varie cellule costituenti il microambiente metastatico, tra cui gli osteoclasti. Da prelievi sanguigni di pazienti, sono stati estratti dei monociti e attraverso un trattamento chimico sono stati fatti differenziare in osteoclasti. Dopo quindici giorni, sono state ricavate delle immagini attraverso microscopia confocale in fluorescenza sia delle colture cellulari sottoposte al trattamento che no. Per valutare l’efficienza del processo di differenziazione e il comportamento cellulare in modo automatico e ripetibile, è stato creato un codice in Matlab che analizza i vari campioni di pre-osteoclasti. Lo sviluppo di un codice in ambiente Matlab permette di esaminare le immagini in maniera più accurata e consistente rispetto ad una mera analisi visiva dell’operatore, a cui verrà mostrato il risultato finale dopo che le immagini sono state processate. I risultati ottenuti sono di notevole supporto all’operatore sotto molteplici aspetti. L’affaticamento visivo dell’operatore è notevolmente ridotto, infatti l’elaborazione svolta mette in luce le cellule cercate, focalizzando l’attenzione dell’esperto sulle suddette cellule e non sull’intero campione. Chiaramente è sempre necessario un controllo finale che possa valutare l’apprezzabilità del risultato.

碩士
國立成功大學
生物化學暨分子生物學研究所
101
Backgrounds:Thrombomodulin (TM), a glycoprotein on the cell surface, is highly expressed in endothelial cells and other numerous cell types such as keratinocyes, osteoblasts and myeloid mononuclear cells. It is first discovered as an anticoagulant factor, and recent studies demonstrate that it has multi-functions including angiogenesis, cell proliferation and complement activation. TM in monocytes/macrophages is associated with inflammation and the expression level is altered in physiological and pathological conditions. In osteoclastogenesis, monocytes /macrophages obtained from bone marrow or vascular-blood system can differentiate to osteoclasts by cell-cell fusion to form multinucleated cells, which are involved in bone resorption. Until now, the role of TM in macrophages in osteoclastogenesis has not been investigated. Materials and Methods:Osteoclast precursors were isolated from human peripheral blood mononuclear cells (PBMCs) and mouse bone marrow derived macrophages (BMMs). Myeloid-specific TM-deficent mice (LysMcre/TMflox/flox) and mice lacking lectin-like domain of TM (TMLeD/LeD) were used. The differentiation was induced by macrophage-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-light-chain-enhancer of activated B cells ligand (RANKL). Results:TM expression was decreased during osteoclastogenesis. Results of osteoclast formation from LyMCreTMflox/flox BMMs showed that TM deficiency caused enhancement of osteoclastogenesis. TMLeD/LeD mice BMMs also had higher activity of osteoclast formation, indicating that TM lectin-like domain plays a role in osteoclastogenesis. Finallly, the proinflammatory cytokine high mobility group box 1 (HMGB1) production in LysMCreTMflox/flox BMMs was much more than TMflox/flox group after RANKL treatment, which suggested that TM regulates osteoclastogenesis by interfering the function of HMGB1 in osteoclasts. Conclusion:Taken together, we showed that TM expression was inversely correlated with the differentiation of osteoclasts. It was speculated that TM may participate in osteoclastogenesis through modulating inflammatory response and which may provide a novel application on osteometabolic diseases.

碩士
國立臺灣大學
免疫學研究所
96
Osteoclasts are multinucleated cells (MNCs) that are differentiated from macrophage/ monocyte lineage of hematopoietic precursors. Receptor activator of nuclear factor kappaB (NF-κB) ligand (RANKL), a tumor necrosis factor (TNF) family cytokine, plays a key role in osteoclast differentiation. RANKL binds to its receptor RANK, which recruits TNF receptor associated factor 6 (TRAF6) and activates NF-κB, Akt, and mitogen-activated protein kinase (MAPK) pathways finally leading to osteoclast-specific genes expression. In addition to RANKL, more and more studies indicated that some TNF family members including TNF-α, FasL and LIGHT are involved in differentiation and function of osteoclasts. These studies suggested that some TNF-family cytokines may regulate osteoclast differentiation. In addition to induction of apoptosis, TNF-related apoptosis inducing ligand (TRAIL) exerts different function that includes survival, proliferation and maturation. Our previous studies have demonstrated that TRAIL can induce osteoclasts differentiation from murine monocytic cell line, RAW 264.7 and human peripheral mononuclear cells (PBMCs), and this signaling pathway is distinct from apoptosis signaling. TRAIL could activate NF-κB and MAPKs, which are important for RANKL-induced osteoclastogenesis. However, the signaling pathway that leads to downstream molecules activation and resulting in osteoclastogenesis remains unclear. TRAF6 is critical in RANKL-induced osteoclastogenesis. Cells from TRAF6-deficient mice can not differentiate into osteoclasts in the presence of RANKL. Therefore, we hypothesize that TRAIL stimulation activates MAPKs and NF-κB pathways to induce osteoclastogenesis through adaptor molecule, TRAF6. To determine whether TRAF6 is involved in TRAIL-induced osteoclastogenesis, we used TRAF6 siRNA to inhibit TRAF6 expression. After knockdown TRAF6 expression by transfection of TRAF6 siRNA, we found that similar to RANKL, osteoclast formation was reduced after TRAIL stimulation. We further investigated whether suppression of TRAF6 expression might affect TRAIL-induced NF-κB and MAPKs activation. Our results demonstrated that knockdown TRAF6 expression reduced TRAIL-induced MAPK activation. In conclusion, TRAIL induces osteoclast differentiation via activation of MAPK which is dependent on TRAF6. Our results provide a novel mechanism that TRAIL can transduce a non-apoptotic signal mediated by TRAF6 to induce osteoclast formation.

碩士
慈濟科技大學
放射醫學科學研究所
106
Abstract Shampinon belongs to agaricus mushrooms, of which the extract is rich in beta glucan and represents anti-oxidation and anti-inflammatory effects. Another extract, ergosterol, will be converted to vitamin D2 via radiation exposure. Some factors such as free radicals and inflammatory cytokines can induce osteoclast differentiation. Clinically, chronic inflammation often leads to loss of bone mass, while vitamin D2 has the role of slowing osteoclastogenesis. It is not known whether giving Shampinon extracts affects osteoclastogenesis, and there is no relevant literature to explore. The purpose of this experiment is to study the Shampinon (by radiation 5 Gy or no radiation treatment) biological effects of extracts on osteoclast differentiation and its possible mechanism. In this experiment, Raw264.7 cells from mouse mononuclear cell line were taken, and osteoclast-inducing agent RANKL was added to extracts. The toxicity of extracts on cells was evaluated by MTT assay. Safe dose of Shampinon extracts were added into the culture medium to investigate the osteoclastogenesis. Shampinon extracts were taken to assess the expression of oxygen free radicals (ROS) and CD51 (a cell marker of osteoclast precursor). To assess osteoclast activity, the number of osteoclasts and the amount of enzyme of tartrate-resistant acid phosphatase (TRAP) were analyzed. Results show that the extracts of Shampinon exposed to radiation will inhibit the CD51 marker expression and the number of osteoclast significantly less than the control group. The osteoclast activity of TRAP and ROS in cells were also inhibited. After exposed to radiation, the ergosterol was converted to vitamin D2 using High-performance liquid chromatography (HPLC) assay. The results suggest that Shampinon extracts have inhibition effects on osteoclastogenesis, which is probably via inhibiting the number of preosteoclast.

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.

博士
國立東華大學
生命科學系
107
Bone is a dynamic tissue that is continuously formed by osteoblasts and resorbed by osteoclasts in response to changes in mechanical loading, altered serum calcium levels and a wide range of paracrine, endocrine and neuronal factors. Any unbalances between bone formation and resorption will lead to diseases such as osteopetrosis or osteoporosis. The principle bone-resorbing osteoclasts are giant multinucleated cells derived from hematopoietic precursors of the monocyte-macrophage lineage. Bone loss due to osteoclasts over activity is very common in many inflammatory bone disorders such as rheumatic arthritis (RA), septic arthritis and periprosthetic osteolysis. Thus, understanding of the regulation of osteoclasts differentiation and function is central to the treatment these diseases. Formation of functional and mature osteoclasts requires many factors such as macrophage colony stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). The expression of RANKL is regulated by many factors including inflammatory cytokines. Carbon monoxide (CO), a ubiquitous air pollutant, has been well known as toxic because its affinity to hemoglobin is 240 times compares with oxygen thus causing hypoxia and death. However, low concentration CO has been proved to be useful in anti-oxidative stress, anti-proliferation and anti-inflammation in vivo and in vitro. CO releasing molecule can also reduce RANKL expression and bone erosion in murine collagen-induced arthritis model. These previous results imply that CO might affect osteoclastogenesis to a certain extent. The aims of this proposal are: (1) to set up an osteoclastogenesis model by the murine monocytic cell line, RAW 264.7 cells, stimulated with RANKL, (2) to investigate the effects of CO on osteoclastogenesis, (3) to explore the possible molecules mediating the effects of CO. We expect this study will probably reveal some novel therapeutic implication of CO.

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.

博士
高雄醫學大學
藥學研究所
97
Abstract Osteoporosis is a significant disease in geriatrics. A nutritional approach to reduce bone loss would be a future goal to achieve an inexpensive way for managing osteoporosis. Previous epidemiological studies found that people with a habit of tea drinking have higher bone mineral density and less chance to get hip fracture. Green tea catechin, (−)-epigallocatechin gallate (EGCG), increased the osteogenic function in mesenchymal stem cells was reported. The effect of EGCG on osteoclastogenesis remains to be elucidated. In this study, we investigated the influence of EGCG on osteoclasts and found that treatment of EGCG (10–100 μM) significantly suppressed nuclear factor-kB ligand-induced osteoclast differentiation and pit formation in murine RAW 264.7 cells, the precursor cells of osteoclasts, and primary bone marrow macrophages cells (BMMs). We also identified that EGCG targets at the early stage of osteoclastic differentiation while having no cytotoxic effect on osteoclast precursors. Analysis of the inhibitory mechanisms revealed that EGCG significantly inhibited RANKL-induced NF-κB transcriptional activity by reducing nuclear transport of NF-κB. The results demonstrate that EGCG is a potent inhibitor of osteoclastogenesis via a mechanism involving NF-κB and TRAP gene. Our results also indicate that the possibility of EGCG to become a new strategy against bone resorption. Ipriflavone is a synthetic isoflavone derivative, which has been suggested to be an inhibitor of bone resorption in vitro and in vivo. Several clinical studies suggested that ipriflavone is able to prevent bone loss. We have screened over 150 ipriflavone derivatives by the tartrate resistant acid phosphatase (TRAP) activity of RAW 264.7 cells after RANKL induction for osteoclastogenesis. The results indicated that 3-(3,4-dimethoxyphenyl)-7-(oxiran-2-ylmethoxy)-4H-chromen-4- one (19) and 3-{4-[3-(cyclohexylamino)-2-hydroxypropoxy]phenyl}- 7-methoxy- 4H-chromen-4-one (20a) exhibited significant inhibitory effects on osteoclast activity (TRAP activity in RAW 264.7 with an IC50 of 0.56 and 2.28 μM, respectively). The results imply that these two compounds can be candidates for the discovery of anti-osteoporotic drug.

碩士
高雄醫學大學
醫學系生理學科碩士班
103
Osteoporosis is the common diseases in menopausal women and elderly people, and its incidence just lower than cardiovascular disease. Excessive osteoclastogenesis is one of the main reasons to cause osteoporosis. Recent clinical researches indicated that the patients’ long-term use of anti-depression drugs (Selective Serotonin Reuptake Inhibitor, SSRI) usually with higher risk to suffer osteoporosis. This observation means serotonin, 5-HT, should involve in the regulation of bone metabolism. Previous reports showed that 5-HT 2B receptor (5-HT2BR) is expressed in both osteoblasts and osteoclasts, and which modulate the proliferation and differentiation in osteoblasts. However, the effects of 5-HT2BR in osteoclasts remain unclear. Using RANKL-induced osteoclastogenesis in RAW 264.7 cells as the model, our results presented a positive correlation between the osteoclastogenesis with 5-HT and 5-HT2BR agonist treatment and that decrease SirT1 expression. On the contrary, treatment with specific 5-HT2BR antagonist significantly reduced the osteoclastogenesis and increase SirT1 expression. Treatment with SirT1 agonist significantly reduced the osteoclastogenesis. Taken together, this investigation suggested that 5-HT2BR play an essential role in osteoclastogenesis and may be involved in regulating osteoporosis. Our observations might also provide another direction of prophylaxis and therapy for osteoporosis.

RESUMO:A artrite psoriática (AP) é uma doença inflamatória crónica caracterizada por várias manifestações nas articulações, nas enteses e na pele. A formação de novo osso após inflamação nas enteses é um dos aspetos mais intrigantes desta doença. Os mecanismos celulares e moleculares deste processo ainda não são completamente conhecidos. Este estudo tem como objetivo compreender melhor os mecanismos subjacentes à formação e reabsorção óssea, bem como o efeito de anti-inflamatórios não esteroides (AINEs) nestes processos. Para atingir este objetivo foram quantificados biomarcadores do metabolismo ósseo e citocinas inflamatórias em doentes AP, antes e após terapêutica com AINEs. Os biomarcadores selecionados foram marcadores de remodelação óssea como CTX-I e P1NP, fatores de diferenciação e ativação de osteoclastos como o RANKL e a OPG, inibidores da via de sinalização Wnt, nomeadamente o DKK-1 e a SOST e ainda citocinas pro-inflamatórias como a IL-22 e a IL-23 e a prostaglandina PGE2. Neste contexto foram também estabelecidas culturas celulares de monócitos, isoladas de doentes AP e de controlos saudáveis. Os monócitos foram cultivados in vitro em condições não estimuladas e estimuladas e realizados dois ensaios funcionais: coloração com TRAP e ensaio de reabsorção. Foi observada uma diminuição nos níveis séricos de CTX-I e OPG em doentes AP em relação aos controlos. De igual forma os níveis séricos de SOST encontram-se significativamente mais baixos, em comparação com os controlos saudáveis. Estes valores de SOST são semelhantes aos dos doentes com espondilite anquilosante (EA), documentados anteriormente. Os ensaios com osteoclastos confirmaram a necessidade da presença de RANKL para estimulação da osteoclastogénese e que o celecoxib parece ter um papel inibitório neste processo. Os resultados obtidos sugerem que a população de doentes com AP analisados têm baixos níveis de reabsorção óssea e alguma atividade na formação óssea. --------------------------- ABSTRACT: Psoriatic arthritis (PsA) is a chronic inflammatory disease characterized by several manifestations involving the joints, enthesis and the skin. New bone formation after inflammation at enthesis site has been one of the most intriguing aspects of the disease. Cellular and molecular mechanisms in this process are still not completely understood. This study aims to understand better the mechanisms underlying bone formation and resorption and the effect of non-steroid anti-inflammatory drugs (NSAIDs) in these processes. To access that, biomarkers of bone metabolism and inflammatory cytokines were measured in PsA patients’ serum before and after NSAID therapy. These selected biomarkers were bone turnover markers such as CTX-I and P1NP, osteoclast differentiation and activation factors RANKL and OPG, Wnt pathway inhibitors DKK-1 and SOST and pro-inflammatory cytokines IL-22, IL-23 and prostaglandin PGE2. In this context monocyte cell culture was also established after PBMC isolation from PsA patients and healthy controls. Monocytes were cultured in vitro under unstimulated and stimulated conditions and two functional assays were performed: TRAP staining and resorption pit assay. It was demonstrated that CTX-I and OPG serum levels in PsA patients were lower than controls. SOST levels were extremely decreased in comparison with controls, resembling the ankylosing spondylitis patients results already documented. Osteoclast assays confirmed the need of RANKL in stimulating osteoclastogenesis and that celecoxib seems to have an inhibitor role in this process. The results obtained suggest that PsA patient population analyzed in this study have low bone resorption levels and some bone formation activity.

碩士
國立陽明大學
藥理學研究所
97
Osteoporosis is common in menopause women and old people. When the bone resorption exceeds the bone formation, a net loss of bone occurs. Inhibition of the differentiation of osteoclasts is a potential strategy to treat the osteoporosis. Previous studies showed that the binding of the receptor activator of nuclear factor �羠 ligand (RANKL) to its receptor RANK can activate mitogen-activated protein kinases (MAPKs) and trigger activation of the transcription factors, such as NF-�羠, activating protein-1 (AP-1) and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), to promote osteoclastogenesis, the generation of the tartrate-resistant acid phosphatase (TRAP) as well as bone resorption. 2-Methoxystypandrone (2MS) is a naphthoquinone compound isolated from Polygonum cuspidatum. The present results showed that 2MS (1-7.5 �嵱) concertration-dependently inhibited RANKL-induced the differentiation of RAW 264.7 macrophages into osteoclasts and the TRAP activity with an IC50 value of 6.8 �嵱 without significant cytotoxicity. Incubation the cells with RANKL significantly increased the nuclear expression of NF-�羠, c-Fos, c-Jun and NFATc1, the phosphorylation of p38, ERK and JNK, and the degradation of I�羠. All these phenomena were concentration-dependently suppressed by 2MS. Metalloproteinase-9 (MMP-9) is a digest enzyme participated in bone resorption. RT-PCR analysis revealed that RANKL-induced MMP-9 mRNA expression was inhibited by 2MS. In conclusion, the present results indicated that 2MS can inhibit RANKL-induced osteoclastogenesis probably via attenuating RANKL-induced phosphorylation of p38, ERK and JNK, the degradation of I�羠, and interfering with the activation of transcription factors (NF-κB, c-Jun, c-Fos and NFATc1).

碩士
國立臺灣大學
藥理學研究所
103
As human beings live longer, age-related diseases such as osteoporosis will become more prevalent. Intolerant side effects and poor responses to current treatments are observed. Therefore, novel effective therapeutic agents are greatly needed. Here, pyrazole derivatives were designed and synthesized, and their osteoclastogenesis inhibitory effects both in vitro and in vivo were evaluated. The most promising compound 13 with a 2- (dimethylamino)ethyl group inhibited markedly in vitro osteoclastogenesis as well as the bone resorption activity of osteoclasts. Compound 13 affected osteoclast’s early proliferation and differentiation more than later fusion and maturation stages. In ovariectomized (OVX) mice, compound 13 can inhibit the loss of trabecular bone volume, trabecular bone number, and trabecular thickness. Moreover, compound 13 can antagonize OVX-induced reduction of serum bone resorption marker and then compensatory increase of the bone formation marker. To sum up, compound 13 has high potential to be developed into a novel therapeutic agent for treating osteoporosis in the future.

博士
國防醫學院
生命科學研究所
102
Inhibition of osteoclast formation is a potential strategy to prevent inflammatory bone resorption and treat bone disease. The development of potential antiosteoclastogenic agent is the focus of the present study. Herein, we designed and synthesized the modified salicylanilides and 3-phenyl-2H-benzo[e][1,3]oxazine-2,4(3H)-dione analogues as osteoclastogenesis inhibitors. To investigate the biological activities of these synthetic compounds, we initially evaluated their effects on RANKL-induced osteoclastogenesis from osteoclast precursor cells RAW264.7 by TRAP stain as well as cell cytotoxicity by MTT assay. The most potent compounds 1d and 5d exhibited the strongest antiosteoclastogenic effects, which were selected as candidates for further antiosteoclastogenic evaluation. Both compounds 1d and 5d suppressed RANKL-induced osteoclast differentiation and TRAP activity in a dose-dependent manner. The mechanism study indicated that 1d and 5d could reduce the level of transcription factors NF-κB and NFATc1 in nucleus, and suppress the gene expression levels of osteoclastogenesis-related marker genes during osteoclastogenesis. In addition, 1d and 5d prevented osteoclastic bone resorption dose-dependently, but did not impair osteoblast differentiation in MC3T3-E1. The preliminary structure-activity relationships (SARs) of our novel synthetic compounds described the optimization and modification to a new more potent antiosteoclastogenic inhibitor. Also, salicylanilide and 3-phenyl-2H-benzo[e][1,3]oxazine-2,4(3H)-dione, especially 1d and 5d, could be the novel lead structures for the development of antiresorptive agents.