The human skeleton is a dynamic organ that serves multiple functions to maintain normal physiology and health. It protects vital organs, provides support for movement, houses marrow and maintains calcium homeostasis. The skeleton is maintained by the work of two cells with opposing functions: osteoblasts, cells that synthesize organic bone matrix and osteoclasts that degrade and resorb it. These cells interact with one another in a tightly regulated process known as the bone remodeling cycle. This cycle maintains the health of bone by removing and replacing weak or damaged bone and responding to stress loads by remodeling portions of the skeleton that require reinforcement. Osteoblasts differentiate from mesenchymal stem cells and respond to hormonal stimuli by synthesizing and secreting cytokines necessary for osteoclast differentiation. Osteoblasts may become embedded within mineralized matrix, becoming osteocytes, cells that can sense changes in mechanical loading and facilitate localization of the remodeling cycle. Osteoclasts differentiate from hematopoietic stem cells (HSC) when the cell surface receptors, c-FMS and RANK, are activated by ligands produced by osteoblasts, M-CSF and RANKL respectively. In addition to c-FMS and RANK stimulation, another calcium-mediated, co-stimulatory pathway must be activated to ensure proper osteoclast differentiation. This pathway is activated by two immunoreceptors, OSCAR and TREM-2 that interact with adaptor proteins termed FcRγ and DAP12 respectively. These adaptor proteins harbor immunoreceptor tyrosine-based activation motifs (ITAM), which exist on their cytoplasmic tail. Once the immunoreceptors are triggered, specific tyrosines within the ITAM motifs become phosphorylated and act as docking points for the tyrosine kinase, Syk. Once bound, Syk autophosphorylates and acts on its downstream targets. Syk dephosphorylation is, therefore, necessary to attenuate this signal to prevent over activation of osteoclasts. Recently, a novel tyrosine phosphatase, T-cell Ubiquitin ligand -2 (TULA-2) has been shown to dephosphorylate specific phosphotyrosine residues on Syk in various systems and has shown an increased specificity to dephosphorylate tyrosine 352. The goal of this project is to determine how TULA-2 mediated dephosphorylation of Syk regulates osteoclast differentiation and function. TULA-2 is a member of the TULA family of proteins, TULA and TULA-2. In spite of a significant homology and similar domain organization between TULA and TULA-2, only TULA-2 has significant phosphatase activity. Furthermore, whereas TULA is expressed only in lymphocytes, TULA-2 is expressed in most tissues albeit a higher level of expression is seen in cells of hematopoietic origin. In vivo analysis including Micro-computed tomography (Micro CT) and histomorphometry indicated that mice that lack both TULA and TULA-2 (DKO) have decreased bone mass compared to wild-type (WT) counterparts. An in vitro cell differentiation assay revealed that a larger population of osteoclast-like cells (OCL) could be cultivated from bone marrow isolated from DKO mice compared to OCL derived from WT bone marrow. An in vitro resorption pit assay revealed that DKO osteoclasts could resorb bone at a faster rate than WT counterparts. Additionally, over-expression of phosphatase-dead TULA-2 in WT osteoclasts increased the ability of the cells to resorb bone. At the molecular level, activation of the co-stimulatory pathway revealed increased tyrosine phosphorylation of Syk 352 in DKO pre-osteoclasts when compared to phosphorylation of Syk isolated from WT pre-osteoclasts. Cumulatively, the above data indicates that the absence of TULA-2 results in an increased signaling response leading to a larger population of hyperactive osteoclasts, which contributes to decreased bone mass in mice. These data suggest that the phosphatase activity of TULA-2 is required for negative regulation of bone resorption. / Cell Biology

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.

Considerando-se que o tabagismo afeta a maioria dos sistemas do corpo humano, que os estudos concentram-se mais sobre os efeitos deletérios do tabaco em doenças cujos órgãos vitais são afetados e que seu impacto na formação e na consolidação óssea ainda não está estabelecido, a proposta neste estudo foi avaliar os efeitos da exposição à fumaça de cigarro no mecanismo de consolidação e remodelamento da matriz óssea e relacioná-los ao processo inflamatório desencadeado por fratura em modelo desenvolvido em camundongos. Para isso, camundongos C57BL6 machos foram distribuídos em quatro grupos: C (n=29) expostos ao ar ambiente; F (n=23) expostos ao ar ambiente e submetidos à osteotomia da tíbia direita; CS (n=29) expostos à fumaça de cigarro; FCS (n=23) expostos à fumaça de cigarro e submetidos à osteotomia da tíbia direita. Nossos dados indicaram diminuição do volume trabecular, da mineralização, MS/BS% (p < 0,05), e da formação óssea, MAR (p=0,0004), bem como aumento de osteoclastos, Oc.S/BS% (p=0,0361), e de reabsorção óssea, ES/BS% (p=0,0114), no grupo CS, quando comparado ao grupo C. Identificamos uma elevação significativa de IL-6 nos grupos CS e FCS (p < 0,001) na comparação com os grupos C e F (p < 0,05). Observamos diminuição de VEGF nos grupos CS e FCS, quando comparados com os grupos C e F (p=0,01). O índice de IGF demonstrou-se diminuído nos grupos CS e FCS, quando comparados com os grupos C e S (p < 0,05). A matriz fibrilar apresentou um intenso remodelamento, caracterizado pelo aumento da expressão de Colágeno V na avaliação por imuno-histoquímica nos grupos CS, F e FCS, comparados com C (p < 0,05), e da expressão dos genes COL5A1 (p < 0,0001) e COL5A2 nos grupos F e FCS (p < 0,0001). Identificamos também diminuição significativa de Colágeno I nos grupos CS, F e FCS, comparados com C (p < 0,001), e da expressão dos genes COL1A1 no grupo CS (p=0,008) e COL1A2 nos grupos CS, F e FCS (p=0,005). Nosso estudo mostra que a exposição à fumaça de cigarro altera a composição das fibras de colágeno pertencentes à matriz fibrilar óssea, retarda a mineralização e modifica a interação entre os diferentes tipos de colágeno, importantes no desempenho funcional do osso / Introduction: The impact of cigarette smoke on bone metabolism is not yet fully understood. This study aimed to verify the effects of cigarette smoke exposure in bone healing in an experimental tibial fracture model, evaluating the mineralization process, bone cellular differentiation/function and collagen types deposition. Materials and Methods: C57BL/6 male mice were assigned into four groups: C(n=29): exposure to room air; F(n=23): exposure to room air and right tibia osteotomy; CS(n=29): exposure to cigarette smoke; FCS(n=23): exposure to cigarette smoke and right tibia osteotomy. Results: Histomorphometry of Bone Mineral Matrix revealed that cigarette smoke exposure significantly reduced thickness of bone trabeculae, associated with a decrease in mineralizing surface and in rate of mineral apposition, which was reflected in lower bone formation rate and consequently in a longer time for mineralization. Both resorption surface and osteoclastic surface were higher in the CS group, evidencing the increase of the resorptive action of cigarette smoke. Histomorphometry of Bone Fibrillar Matrix: Type I collagen demonstrated a decrease in CS and FCS compared to C (p < 0.01); Type V collagen demonstrated an increase in CS, FC and FSC compared to C (p < 0.0001). The cytokines expression evaluation demonstrated that only CS exposure has already induced a VEGF and IGF decrease with a concomitant increase in IL-6 and these changes were intensified under fracture conditions. COL1A1 gene expression was reduced in the CS and FCS groups, similar result was observed in COL1A2 evaluation. COL5A1 gene expression, showed an increase in CS and Fracture groups while the COL5A2 gene expression increase was detected only in Fracture groups. Conclusion: Cigarette smoke exposure alters bone matrix composition and worsens bone mineralization, leading to bone fragility by increasing collagen V synthesis and deposition and impairing collagen I fibril forming and assembling

A erupção dentária depende de uma coordenada interação entre o germe dentário e o tecido ósseo da cripta que o envolve. Para que seja formada a via eruptiva, a reabsorção da porção oclusal da cripta óssea por osteoclastos é indispensável. Os bisfosfonatos são drogas com reconhecida capacidade de inibir a atividade clástica e foram empregados no presente estudo a fim de interferir no tecido ósseo da cripta alveolar durante a formação e erupção de molares de ratos. Doses diárias dos bisfosfonatos alendronato ou etidronato de 2,5 e 8 mg/kg, respectivamente, foram administradas a ratos recém nascidos. Os controles foram injetados com solução salina. Nos períodos de 4, 8, 14, 21 e 28 dias, as maxilas foram fixadas em 2,5% de formaldeído + 2% de glutaraldeído, 4% de formaldeído + 0,1% de glutaraldeído ou fixador Zamboni, descalcificadas em EDTA a 4,13% e processadas para análise em microscopias de luz, eletrônica de transmissão e confocal, histoquímica TRAP, imunocitoquímica para OPN, BSP, RANK, RANKL e OPG. Alguns espécimes não foram descalcificados para análise em microscopia eletrônica de varredura, ou congelados em nitrogênio líquido para extração e análise e da expressão de proteínas por Western Blotting. O etidronato ocasionou alterações no metabolismo ósseo da cripta que resultaram no atraso da erupção e da formação radicular em relação ao controle. O alendronato aumentou o número de osteoclastos no osso alveolar, porém a maioria apresentou estado latente, o que diminuiu a reabsorção óssea da cripta ao redor do germe dentário e impediu a erupção dos molares e a formação radicular. A expressão de RANKL, molécula ativadora dos osteoclastos, durante o início do processo eruptivo, diminuiu em comparação ao controle. Com a diminuição da remodelação óssea, o tecido apresentou distribuição de OPN e BSP típica de osso primário. Os resultados demonstram que a reabsorção óssea é importante em todos os pontos da cripta e não apenas em sua porção oclusal durante a formação da via eruptiva, para que a formação e erupção dentária ocorram adequadamente. / Tooth eruption depends on coordinated interactions between the tooth germ and the surrounding bony crypt. The formation of the eruptive pathway requires the resorption of the occlusal portion of the bony crypt by osteoclasts. The bisphosphonates are drugs with known capability to inhibit clastic activity, and were employed in the present study with the aim of interfering in the alveolar bony crypt during the formation and eruption of rat molars. Daily alendronate or etidronate 2.5 and 8 mg/kg doses, respectively, were administered to newborn rats. The controls were injected with saline solution. At 4, 8, 14, 21 and 28 days, the maxillae were fixed in 2.5% formaldehyde + 2% glutaraldehyde, 4% formaldehyde + 0,1% glutaraldehyde or Zambonis fixative, decalcified in 4.13% EDTA and processed for light, confocal and transmission electron microscopy analyzes, TRAP histochemistry, and immunocytochemistry for OPN, BSP, RANK, RANKL and OPG. Some specimens were left undecalcified for scanning electron microscopy analyzes, or frozen in liquid nitrogen for protein extraction and Western Blotting protein expression analyzes. Etidronate occasioned alterations in the alveolar bony crypt metabolism that resulted in the delay of tooth eruption. Alendronate increased osteoclast number in the alveolar bone; however, most of them were latent, which decreased the resorption of the bony crypt surrounding the tooth germ and impeded the eruption and root formation of the molars. The expression of RANKL, an osteoclast-activating molecule, was decreased. The inhibition of the bone remodeling resulted in typical primary bone OPN and BSP labeling pattern. These results demonstrate that bone resorption at the entire surface of the crypt, and not only during the eruptive pathway formation, is crucial for adequate tooth eruption and formation.

A citocina IL-33 apresenta papel dual e está envolvida com a resolução ou progressão de inúmeras doenças, além disso, acredita-se que a via IL-33/ST2 esteja envolvida no equilíbrio entre a atividade de osteoclastos e osteoblastos. O objetivo deste estudo foi avaliar o papel do receptor ST2 no desenvolvimento e progressão de lesões periapicais experimentalmente induzidas em camundongos. Lesões periapicais foram induzidas em primeiros molares inferiores de camundongos WT e ST2 knockout (KO). Decorridos 7 e 14 dias, as amostras de mandíbula foram submetidas às análises: determinação da área de lesão periapical em cortes histológicos e do volume por microtomografia computadorizada (&mu;CT); contagem de osteoclastos submetidos ao ensaio de histoenzimologia (TRAP); expressão gênica de marcadores osteogênicos e osteoclastogênicos por q-PCR; quantificação de neutrófilos por ensaio de mieloperoxidases. Os linfonodos foram submetidos à análise da expressão dos fatores transcricionais T-bet, GATA-3, RORc e Foxp-3 por q-PCR. Análise estatística utilizada foi One-way ANOVA, seguido de pós-teste de Bonferroni. Aos 14 dias, observou-se maior extensão da lesão periapical em animais WT que em ST2KO (p<0,05). O tamanho da lesão nos animais ST2KO permaneceu igual em função do tempo. Foi observada maior quantidade de neutrófilos na lesão do grupo WT aos 7 dias, em comparação aos animais ST2KO (p<0,05). Na expressão de T-bet, GATA-3, RORc e Foxp-3 não foram observadas diferenças estatisticamente significantes. O número de osteoclastos contados nos animais ST2KO foi maior que o observado em WT aos 7dias e aos 14 dias (p<0,05). A expressão de Runx2 foi maior no grupo lesão dos animais ST2KO quando comparado a seu respectivo controle. Os outros marcadores relacionados com a formação óssea não apresentaram diferenças estatisticamente significantes. Dentre os marcadores relacionados com a reabsorção óssea, a catepsina K e o MMP-9 apresentaram maior expressão aos 14 dias, na lesão dos animais WT quando comparada à expressão na lesão dos animais ST2KO (p<0,05). Com base nos resultados obtidos no presente estudo, pode-se concluir que na ausência do receptor ST2 as lesões periapicais são menos extensas e embora em maior quantidade, os osteoclastos são menos ativos. Nossos resultados sugerem um importante papel da via IL-33/ST2 na ativação dos osteoclastos e desenvolvimento da lesão periapical. / The IL -33 cytokine presents a dual role and is involved either in the resolution and progression of many diseases. Furthermore, it is believed that this pathway is involved between osteoclast and osteoblast activity balance. The aim of this study was to evaluate the role of ST2 receptor in the development and progression of experimentally induced periapical lesions in mice. Periapical lesions were induced in first molars of WT and ST2 knockout (KO) mice. After 7 and 14 days, jaw samples were subjected to various analysis: determination of periapical lesions area by histology and volume by computed microtomography (&mu;CT); osteoclasts number by TRAP histoenzymology; osteogenic and osteoclastogenic markers expression by q-PCR; neutrophil quantification by myeloperoxidase activity. The expression of transcription factors T-bet, GATA-3, RORC and Foxp-3 in lymph nodes were analysed by q-PCR. Statistical analysis was done by One-way ANOVA and Bonferroni post-test. It was observed a greater extent in periapical lesions of WT compared to ST2KO animals at 14 days (p<0.05). There is no progression in the lesion of ST2KO mice with the time. A larger number of neutrophils in WT group was observed, compared to ST2KO mice evaluated at 7 days (p<0.05). The expression of T-bet, GATA-3, RORc and Foxp-3 were not statistically significant different among the groups. The number of osteoclasts in lesions of ST2KO animals were greater than the observed in WT, at 7 and 14 days (p<0.05). Although, other osteogenic markers showed no statistically significant difference, Runx2 expression in ST2KO was higher in lesion side compared to control side at 14 days. The markers related to bone resorption, cathepsin K and MMP-9, were significantly abrogated in the lesion side of ST2KO mice, at 14 days (p<0.05). Based on the results, it can be concluded that although larger amounts of osteoclast were counted in ST2KO, the lesion was less extensive and osteoclasts less active. It all suggests that the IL-33/ST2 pathway play an important role in osteoclasts activation and periapical lesion development.

A citocina IL-33 apresenta papel dual e está envolvida com a resolução ou progressão de inúmeras doenças, além disso, acredita-se que a via IL-33/ST2 esteja envolvida no equilíbrio entre a atividade de osteoclastos e osteoblastos. O objetivo deste estudo foi avaliar o papel do receptor ST2 no desenvolvimento e progressão de lesões periapicais experimentalmente induzidas em camundongos. Lesões periapicais foram induzidas em primeiros molares inferiores de camundongos WT e ST2 knockout (KO). Decorridos 7 e 14 dias, as amostras de mandíbula foram submetidas às análises: determinação da área de lesão periapical em cortes histológicos e do volume por microtomografia computadorizada (&mu;CT); contagem de osteoclastos submetidos ao ensaio de histoenzimologia (TRAP); expressão gênica de marcadores osteogênicos e osteoclastogênicos por q-PCR; quantificação de neutrófilos por ensaio de mieloperoxidases. Os linfonodos foram submetidos à análise da expressão dos fatores transcricionais T-bet, GATA-3, RORc e Foxp-3 por q-PCR. Análise estatística utilizada foi One-way ANOVA, seguido de pós-teste de Bonferroni. Aos 14 dias, observou-se maior extensão da lesão periapical em animais WT que em ST2KO (p<0,05). O tamanho da lesão nos animais ST2KO permaneceu igual em função do tempo. Foi observada maior quantidade de neutrófilos na lesão do grupo WT aos 7 dias, em comparação aos animais ST2KO (p<0,05). Na expressão de T-bet, GATA-3, RORc e Foxp-3 não foram observadas diferenças estatisticamente significantes. O número de osteoclastos contados nos animais ST2KO foi maior que o observado em WT aos 7dias e aos 14 dias (p<0,05). A expressão de Runx2 foi maior no grupo lesão dos animais ST2KO quando comparado a seu respectivo controle. Os outros marcadores relacionados com a formação óssea não apresentaram diferenças estatisticamente significantes. Dentre os marcadores relacionados com a reabsorção óssea, a catepsina K e o MMP-9 apresentaram maior expressão aos 14 dias, na lesão dos animais WT quando comparada à expressão na lesão dos animais ST2KO (p<0,05). Com base nos resultados obtidos no presente estudo, pode-se concluir que na ausência do receptor ST2 as lesões periapicais são menos extensas e embora em maior quantidade, os osteoclastos são menos ativos. Nossos resultados sugerem um importante papel da via IL-33/ST2 na ativação dos osteoclastos e desenvolvimento da lesão periapical. / The IL -33 cytokine presents a dual role and is involved either in the resolution and progression of many diseases. Furthermore, it is believed that this pathway is involved between osteoclast and osteoblast activity balance. The aim of this study was to evaluate the role of ST2 receptor in the development and progression of experimentally induced periapical lesions in mice. Periapical lesions were induced in first molars of WT and ST2 knockout (KO) mice. After 7 and 14 days, jaw samples were subjected to various analysis: determination of periapical lesions area by histology and volume by computed microtomography (&mu;CT); osteoclasts number by TRAP histoenzymology; osteogenic and osteoclastogenic markers expression by q-PCR; neutrophil quantification by myeloperoxidase activity. The expression of transcription factors T-bet, GATA-3, RORC and Foxp-3 in lymph nodes were analysed by q-PCR. Statistical analysis was done by One-way ANOVA and Bonferroni post-test. It was observed a greater extent in periapical lesions of WT compared to ST2KO animals at 14 days (p<0.05). There is no progression in the lesion of ST2KO mice with the time. A larger number of neutrophils in WT group was observed, compared to ST2KO mice evaluated at 7 days (p<0.05). The expression of T-bet, GATA-3, RORc and Foxp-3 were not statistically significant different among the groups. The number of osteoclasts in lesions of ST2KO animals were greater than the observed in WT, at 7 and 14 days (p<0.05). Although, other osteogenic markers showed no statistically significant difference, Runx2 expression in ST2KO was higher in lesion side compared to control side at 14 days. The markers related to bone resorption, cathepsin K and MMP-9, were significantly abrogated in the lesion side of ST2KO mice, at 14 days (p<0.05). Based on the results, it can be concluded that although larger amounts of osteoclast were counted in ST2KO, the lesion was less extensive and osteoclasts less active. It all suggests that the IL-33/ST2 pathway play an important role in osteoclasts activation and periapical lesion development.

La Cathepsine K est parmi la plus efficace des collagénases de mammifère pour cliver la triple hélice de collagène de type-1. Nous avons développé une série d'azanitriles, (CKI-8 and CKI-13) inhibiteurs de cathepsine K. CKI-8 (un isomère de CKI-13) et CKI-13 ne sont pas toxiques sur les osteoblastes Saos-2 et les cellules RAW 264.7 jusqu' à une concentration de 1000 nM, tandis qu'ils ne le sont pas jusqu'à une concentration de 100 nM sur les osteoclastes. CKI-8 n'affecte pas l'activité de la phosphatase alkaline ainsi que la minéralisation induite par les Saos-2 et par les osteoblastes primaires. CKI-13 diminue de 35 % la minéralisation induite par les Saos-2 tandis qu'il n'affecte pas la minéralisation induite par les osteoblastes primaires. L'addition de CKI-13 diminue l'activité de la phosphatase alkaline d'environ 20% (Saos-2) et de 40 % (osteoblastes primaires). La résorption osseuse sur des tranches d'os d'origine bovine est diminuée avec 10 nM de CKI-13, 100 nM de CKI- 8 et 100 nM d'inhibiteur commercial E64. CKI-8 et CKI-13 diminuent la mobilité des osteoclastes. Nous avons développé un dosage d'hydrolyse de PPi par la phosphatase alkaline au moyen de l'IR, ayant l'avantage de fonctionner sur des vésicules matricielles et des cellules avec des substrats naturels à un pH physiologique. La bande de PPi localisée à 1107 cm-1 (∑= 2158 ± 211 M-1.cm-1) et celles de Pi localisées à 1076 cm-1 (∑= 1346 ± 116 M-1.cm- 1) et à 991 cm-1 (∑= 493 ± 49 M-1.cm-1) ont servis à mesurer les concentrations du substrat et du produit / Cathepsin K is among the most potent mammalian collagenase, capable of cleaving the triple helix in type-I collagen. We developed a series of azanitriles (CKI-8 and CKI-13) which are inhibitors of cathepsin K. CKI-8 (an isomer of CKI-13) and CKI-13 did not induce significant toxicity on osteoblasts Saos-2 and RAW 264.7 cells up to 1000 nM, while they were not toxic on mature osteoclasts up to 100 nM. Commercial E64 inhibitor was not toxic in primary osteoclast cells up to 1000 nM. CKI-8 did not affect alkaline phosphatase activity as well the mineralization induced by Saos-2 cells and by primary osteoblasts. CKI-13 decreased by 35% the mineralization induced by Saos-2 cells while it did not on mineralization induced by primary osteoblasts. Addition of CKI-13 decreased alkaline phosphatase activity by around 20% (Saos-2 cells) and 45% (primary osteoblasts). Bone resorption on bovine slices decreased significantly with 10 nM of CKI-13, with 100 nM of CKI-8 and commercial inhibitor E64. Our findings indicated that CKI-8 and CKI-13 inhibited bone resorption and affected the mobility of osteoclast. To monitor directly the PPi hydrolytic activity by alkaline phosphatase, we developed an infrared (IR) assay taking the advantage to use natural substrate under physiological pH in matrix vesicles and in living cells. PPi band located at 1107 cm-1 (∑= 2158 ± 211 M-1.cm-1) and Pi bands located at 1076 cm-1 (∑= 1346 ± 116 M-1.cm-1) and at 991 cm-1 (∑= 493 ± 49 M-1.cm-1) served to measure the substrate and the product concentrations

[Truncated abstract] Intracellular communication between osteoclasts and osteoblasts is imperative to maintain bone integrity. A myriad of molecules are responsible for regulating osteoblast and osteoclast activity. In particular, it is well documented that osteoblast-derived factors are crucial in directly controlling osteoclast formation and function. Since bone formation is coupled to bone resorption, it would be expected that osteoclasts also have some role in regulating the growth and function of osteoblast cells. However, despite extensive research upon osteoclast and osteoblast biology, the mechanisms by which osteoclasts regulate osteoblast growth and function is not well understood. In an attempt to further elucidate the mechanisms by which osteoclasts and osteoblasts communicate, the technique of subtractive hybridisation was used to identify a novel osteoclastderived factor identical to that of mouse Seminal Vesicle Secretion VII (SVS VII). Previous characterisation of the gene in bone demonstrated that SVS VII was abundantly and specifically expressed by mature osteoclasts (Phan, 2004). Additional research hinted that SVS VII acted as a novel osteoclast-derived factor, that by paracrine mechanisms, targeted osteoblast function (Phan, 2004). However, it remained open as to whether the SVS VII molecule did uniquely target the osteoblast, and whether this interaction influenced bone formation in vivo. Therefore, this thesis endeavoured to functionally characterise the role of the SVS VII molecule in the bone environment. ... Further work is needed to identigy a clear consensus binding sequence, to determine the specificity of the interaction between SVS VII protein and each phage clone, and to isolate a specific binding partner for SVS VII. In conclusion, the studies of this thesis sought to characterise the significance of SVS VII expression by mature osteoclasts, relative to its effects on osteoblast behaviour, but failed to conclusively determine a role for SVS VII in bone. Given that the effects of SVS VII on in vitro osteoblast activity and function are minimal, it is doubtful that SVS VII primarily acts as a paracrine factor integral to osteoblast function. Therefore, these findings conflict with those presented previously (Phan, 2004). However, it was demonstrated that SVS VII treatment was associated with in vivo effect on the skeleton, suggesting that SVS VII may target other elements of the bone microenvironment. Via mechanisms not yet understood, which possibly involves additional factors of the bone 11 extracellular matrix, SVS VII may target a subset of osteoprogenitor cells within the bone environment and act to regulate their proliferation. Therefore, SVS VII may enhance osteogenic precursor cell number at sites of bone formation which would increase the pool of cells that can differentiate down the osteoblast linage and contribute to bone formation. In this regard, SVS VII might function in a manner homologous to the Ly-6 molecule Sca-1 and act as an important factor that maintains a balance between the bone formation and resorption process. Clearly, more work focusing on alternative facets of bone biology is needed to identify whether there is a significant role for SVS VII in skeletal tissue.

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.

Το οστό αποτελεί ένα ιδανικό περιβάλλον για μετάσταση, καθώς η συνεχής και δυναμική ανάπλασή του παρέχει μια γόνιμη βάση για την παλιννόστηση και τον πολλαπλασιασμό των καρκινικών κυττάρων. Ο μεταβολισμός του οστού αποτελείται από το στάδιο της απορρόφησης (bone resorption), το στάδιο του σχηματισμού (bone formation) και το στάδιο της ηρεμίας. Το 65-75% των γυναικών με καρκίνο του μαστού εμφανίζουν οστικές μεταστάσεις, καθώς τα καρκινικά κύτταρα του μαστού εμφανίζουν αυξημένο οστεοτροπισμό. Τα καρκινικά κύτταρα του μαστού όταν μεταναστεύουν στο οστό εκκρίνουν διάφορους αυξητικούς παράγοντες και κυτταροκίνες, οι οποίοι μέσω των οστεοβλαστών, υπερενεργοποιούν τους οστεοκλάστες με αποτέλεσμα την διατάραξη της φυσιολογικής ομοιοστασίας του οστού. Η αυξημένη οστεολυτική δραστηριότητα έχει ως αποτέλεσμα την απελευθέρωση αυξητικών παραγόντων που βρίσκονται εγκλωβισμένοι στο οστό, οι οποίοι συμβάλλουν στην επιβίωση και τον πολλαπλασιασμό των καρκινικών κυττάρων στο περιβάλλον του οστού. Έτσι εγκαθιδρύεται ένας ‘φαύλος κύκλος’ μεταξύ την ενεργοποίησης των οστεοκλαστών και την ανάπτυξης του όγκου στο περιβάλλον του οστού. Η οστική νόσος στον καρκίνο έχει σημαντικές κλινικές εκδηλώσεις που επηρεάζουν την ποιότητα ζωής του ασθενή. Αυτές περιλαμβάνουν πόνο στον σκελετό, εξασθένηση της κινητικότητας, υπερασβεστιαιμία, συχνά κατάγματα, συμπίεση των νευρικών ριζών και του νωτιαίου μυελού, καθώς και διαταραχές αιμοποίησης. Για την αντιμετώπιση των εκδηλώσεων της οστικής νόσου και γενικότερα των ασθενειών με αυξημένη οστεοκλαστική δραστηριότητα έχει εγκριθεί από τον FDA η χορήγηση διφωσφονικών. Τα διφωσφονικά είναι συνθετικά ανάλογα του πυροφωσφορικού (PPi), όπου οι δύο φωσφονικές ομάδες ενώνονται μέσω φωσφοαιθερικού δεσμού με ένα κεντρικό άτομο άνθρακα (P–C–P) στο οποίο είναι ομοιοπολικά συνδεδεμένες δύο πλευρικές αλυσίδες. Χωρίζονται σε δύο κατηγορίες τα αμινο-διφωσφονικά και τα μη αμινο-διφωσφονικά με διαφορετικούς μηχανισμούς δράσης. Το ζολενδρονικό ανήκει στην κατηγορία των αμινο-διφωσφονικών και δρα αναστέλλοντας την συνθάση του πυροφωσφορικού φαρνεσυλίου (FPP) στο μονοπάτι του μεβαλονικού. Το γεγονός αυτό έχει ως αποτέλεσμα την αναστολή της σύνθεσης των ισοπρενοϊδών λιπδίων FPP και πυροφωσφορικό γερανυλγερανύλιο (GGPP). Τα FPP και GGPP είναι απαραίτητα για την πρενυλίωση δηλαδή την λιπιδική τροποποίηση των μικρών GTPασων. Η πρενυλίωση είναι απαραίτητη για την «αγκυροβόληση» τους στην κυτταροπλασματική πλευρά τις κυτταρικής μεμβράνης 274 και ως εκ τούτου στην λειτουργία των σηματοδοτικών αυτών πρωτεϊνών που είναι επίσης απαραίτητη για την λειτουργία των οστεοκλαστών. Αρχικός σκοπός της διατριβής ήταν να εξετάσει την επίδραση του ζολενδρονικού σε βασικές ιδιότητες των καρκινικών κυττάρων, όπως είναι ο ανεξέλεγκτος πολλαπλασιασμός και η διήθηση σε γειτονικούς ιστούς. Για τα πειράματα που πραγματοποιήθηκαν χρησιμοποιήθηκαν δύο καρκινικές επιθηλιακές κυτταρικές σειρές από ανθρώπινο μαστό, με διαφορετική μεταστατική ικανότητα και διαφορετική έκφραση οιστρογοϋποδοχέων. Οι πειραματικές μελέτες που πραγματοποιήθηκαν σε θρεπτικό υλικό παρουσία ορού, ώστε να υπάρχει διέγερση των κυττάρων από μίγμα αυξητικών παραγόντων που υπάρχει στον ορό, έδειξαν ότι το ζολενδρονικό ανέστειλε στατιστικώς σημαντικά των κυτταρικό πολλαπλασιασμό και των δύο κυτταρικών σειρών. Επίσης αξιοσημείωτο είναι ότι το ζολενδρονικό ανέστειλε σε σημαντικό βαθμό την διήθηση και των δύο καρκινικών σειρών είτε χρησιμοποιήθηκε ως χημειοτακτικό ορός είτε εθισμένο υλικό καλλιέργειας από ινοβλάστες μαστού. Για την περαιτέρω μελέτη της ανασταλτικής δράσης του ζολενδρονικού στην διηθητική ικανότητα των καρκινικών κυττάρων, μελετήθηκε η δράση του σε βασικές διεργασίες των καρκινικών κυττάρων που συμβάλλουν στην διηθητική τους ικανότητα, την ικανότητα προσκόλλησης τους στον ECM και την κινητικότητά τους. Η επώαση των καρκινικών κυττάρων με ζολενδρονικό είχε ως αποτέλεσμα την σημαντική αναστολή της ικανότητας προσκόλλησης τους σε όλα τα συστατικά του ECM για την MDA-MB-231 σειρά, ενώ για την MCF-7 παρατηρήθηκε αναστολή της προσκόλλησης σε όλα τα συστατικά του ECM με μόνη διαφορά ότι η αναστολή αυτή δεν ήταν στατιστικώς σημαντική για τα κολλαγόνα τύπου Ι και IV. Η μελέτη της κινητικότητας των καρκινικών κυττάρων μελετήθηκε με δύο τρόπους, έτσι ώστε να έχουμε μια πιο ξεκάθαρη εικόνα για την δράση του ζολενδρονικού. Και από τις δύο πειραματικές τεχνικές παρατηρήθηκε ότι η κυτταρική κινητικότητα αναστέλλεται σημαντικά υπό την επίδραση του ζολενδρονικού για τα MDA-MB-231 κύτταρα. Αξιοσημείωτο είναι το γεγονός ότι η ανασταλτική δράση του ζολενδρονικού στην κυτταρική κινητικότητα παρατηρήθηκε και στην συγκέντρωση των 3μΜ. Ο εξωκυττάριος χώρος δεν είναι ένα απλό υποστηρικτικό σύστημα μορίων του συνδετικού ιστού, αλλά αποτελεί μια δομική μονάδα η οποία λαμβάνει μέρος σε φυσιολογικές αλλά και παθολογικές διεργασίες. Η σημαντικότητα της δομής και Περίληψη 275 λειτουργικότητας των μορίων του εξωκυττάριου χώρου στις παθοφυσιολογικές διαδικασίες τον καθιστούν ως ένα από τα σημαντικότερα προς μελέτη πεδία. Είναι ευρέως γνωστό ότι οι αλληλεπιδράσεις μεταξύ καρκινικών κυττάρων και των συστατικών του εξωκυττάριου χώρου, εκτός από την επαγωγή του κυτταρικού πολλαπλασιασμού, είναι σημαντικές για την πρόοδο του καρκίνου, την αγγειογέννεση και τη μετάσταση. Με βάση τη σύγχρονη βιβλιογραφία, αλλά και την μακροχρόνια εμπειρία και το δημοσιευμένο έργο του εργαστηρίου μας, είναι γνωστό ότι αλλαγές στην έκφραση τριών ο σημαντικών κατηγοριών μορίων του εξωκυττάριου χώρου, των PGs των MMPs και των ιντεγκρινών, μπορεί να έχουν σημαντικές συνέπειες στην ανάπτυξη και την πρόοδο του καρκίνου του μαστού. Για το σκοπό αυτό έγινε εκτενής μελέτη στο επίπεδο της δράσης του ζολενδρονικού στις κατηγορίες αυτών των μορίων. Η χορήγηση του ζολενδρονικού στα κύτταρα είχε ως αποτέλεσμα την αναστολή της έκφρασης, ενός σημαντικού για την οστική μετάσταση μορίου, της συνδεκάνης-1 τόσο σε γονιδιακό όσο και σε πρωτεϊνικό επίπεδο και στις δύο καρκινικές σειρές. Επίσης ανέστειλε την γονιδιακή έκφραση της συνδεκάνης -2 στην MCF-7 και MDAMB- 231 παρατηρήθηκε μια τάση αναστολής της. Για την συνδεκάνη-4 δεν επηρέασε την γονιδιακή της έκφραση στην MDA-MB-231, ενώ στην MCF-7 την ανέστειλε. 6στόσο σε πρωτεϊνικό επίπεδο αύξησε την έκφραση της και στις δύο καρκινικές σειρές, γεγονός που υποδεικνύει πως καταστέλλει τον μηχανισμό αποικοδόμησής της. Βάσει πρόσφατων μελετών η αύξηση της έκφρασης της συνδεκάνης-4 έχει ως αποτέλεσμα την καταστολή της κινητικότητας των κυττάρων, γεγονός που εξηγεί την παρατηρούμενη από το ζολενδρονικό μείωση στην κινητικότητα των κυττάρων. Η μελέτη της δράσης του ζολενδρονικού στην έκφραση των MMPs και των TIMPs έδωσε σημαντικά στοιχεία για την παρατηρούμενη αναστολή της διηθητικής ικανότητας των καρκινικών κυττάρων, καθώς η έκφραση της των MMPs που μελετήθηκαν και στις δύο καρκινικές σειρές μειώθηκε σημαντικά, ενώ η έκφραση των TIMPs αυξήθηκε σημαντικά. Όσον αφορά τις ιντεγκρίνες, των οποίων ο ρόλος στην κυτταρική προσκόλληση είναι γνωστός, στα κύτταρα MDA-MB-231 το ζολενδρονικό προκάλεσε στατιστικά σημαντική αναστολή στην έκφραση των υπομονάδων α1, α4, α5, β1, β2, β6 των ιντεγκρινών, καθώς και στις ιντεγκρίνες αvβ3 και α5β1, προκάλεσε σχεδόν πλήρη αναστολή στην έκφραση των β3 και αvβ5, επαγωγή στις αv και β4 υπομονάδες και δεν επηρέασε την έκφραση των α2 και α3. Αντίστοιχα, στα MCF-7 το ζολενδρονικό προκάλεσε αναστολή στην έκφραση των 276 υπομονάδων α2, α3, α5, αν, αvβ3, β1, β2, β3, β6 και των ιντεγκρινών αvβ5 και α5β1. Προκάλεσε επίσης επαγωγή στην υπομονάδα α1, ενώ δεν επηρέασε την έκφραση των υπομονάδων α4 και β4. Αξιοσημείωτη είναι η αναστολή της αvβ3, καθώς το ετεροδιμερές αυτό παίζει σημαντικό ρόλο στην μετάσταση των καρκινικών κυττάρων στο οστό. Συγκρίνοντας τα πειράματα της κυτταρικής προσκόλλησης και της έκφρασης των ιντεγκρινών παρατηρείται συμφωνία μεταξύ τους. Σημαντική επίσης για την αντικαρκινική και αντιμετασταστική δράση του ζολενδρονικού είναι η παρατηρούμενη αναστολή της έκκρισης του HA και της έκφρασης του υποδοχέα του, του CD44, καθώς το και τα δύο μόρια ξεχωριστά αλλά και ως σύμπλοκο διαδραματίζουν σημαντικό ρόλο στην πρόοδο του καρκίνου και στην μετάσταση του στα οστά. Επόμενος σκοπός της διατριβής ήταν να μελετηθεί η επίδραση του ζολενδρονικού εκεί που κυρίως δίνει μετάσταση ο καρκίνος του μαστού, δηλαδή στα οστά. Για το λόγο μελετήθηκε η δράση του στην ενεργοποίηση των πρώιμων οστεοκλαστων σε υπόστρωμα οστού, χρησιμοποιώντας ως δείκτες την TRAP 5b και το NTx, και διαπιστώθηκε πως το ζολενδρονικό μπορεί να επάγει την αναστολή της οστεοκλαστογένεσης με μηχανισμό ο οποίος δεν βασίζεται στην υπερέκκριση της OPG. Επίσης ανέστειλε την έκφραση της ΜΜP-9, η οποία πέρα από την αποικοδομητική της δράση, αποτελεί και δείκτη της οστεοκλαστικής σειράς. Στην συνέχεια μελετήθηκε η ενεργοποίηση των οστεοκλαστών σε ένα πειραματικό μοντέλο που περιλάμβανε την συν-καλλιέργεια πρώιμων οστεοκλαστών και καρκινικών κυττάρων της σειράς MDA-MB-231 παρουσία και απουσία αυξητικών παραγόντων που επάγουν την ενεργοποίηση των οστεοκλαστών. Και στις δύο περιπτώσεις η ωρίμανση των οστεοκλαστών αναστάλθηκε δοσο-εξαρτώμενα. Αξιοσημείωτο είναι το γεγονός ότι τα καρκινικά κύτταρα μπορούν από μόνα τους να επάγουν την ενεργοποίηση των οστεοκλαστών καθώς και ότι ζολενδρονικό αναστέλλει την ενεργοποίηση των οστεοκλαστων στην χαμηλή συγκέντρωση των οστεοκλαστών. Επίσης και στις δύο περιπτώσεις το ζολενδρονικό μείωσε σημαντικά το φορτίο των ζελατινασών στο περιβάλλον του οστού μειώνοντας έτσι και την λυτική τους δραστηριότητα. Όπως είναι γνωστό όταν τα καρκινικά κύτταρα μεταναστεύουν στο περιβάλλον του οστού ουσιαστικά εκμεταλλεύονται του μηχανισμούς αποικοδόμησης έτσι ώστε να δημιουργήσουν ένα περιβάλλον ευνοϊκό για την επιβίωσή τους. Για το λόγο αυτό μελετήθηκε η δράση του ζολενδρονικού στην δραστικότητα ενεργών οστεοκλαστών Περίληψη 277 καθώς και σε μόρια, όπως η TRAP, η β3 ιντεγκρίνη, η καθεψίνη Κ και οι MMP-1, -9, -14, που παίζουν σημαντικό ρόλο στην λειτουργία τους. Τα αποτελέσματα από την μέτρηση του NTx έδειξαν πως το ζολενδρονικό καταστέλλει την δραστικότητα των οστεοκλαστών. Όσον αφορά τα μόρια που μελετήθηκαν το ζολενδρονικό ανέστειλε την έκφραση τους στην συγκέντρωση των 15 μΜ, ενώ στην συγκέντρωση των 3 μΜ είτε ανέστειλε την έκφραση τους (ΜΜP-1, MT1-MMP, ιντεγκρίνη β3) είτε δεν την επηρέασε (καθεψίνη Κ, TRAP) είτε την αύξησε (MMP-9). Η μελέτη της πρωτεϊνικής έκφρασης της ΜΜP- 9 εμφάνισε το ίδιο μοτίβο με την γονιδιακή της έκφραση. Όπως είναι γνωστό η χημειοθεραπεία στον καρκίνο δεν έγκειται στην χορηγία ενός μόνο φαρμάκου αλλά σε συνδυασμούς φαρμάκων. Για το λόγο αυτό αξιολογήθηκε η δράση του ζολενδρονικού με άλλα γνωστά αντικαρκινικά μόρια (λετροζόλη και STI571), των οποίων η δράση τους έχει μελετηθεί και αξιολογηθεί από το εργαστήριο Βιοχημείας. Τα μόρια αυτά είναι η λετροζόλη και ο μοριακός αναστολέας STI571. Παρουσία των δύο ειδικών αναστολέων παρατηρήθηκε ενίσχυση της ανασταλτικής δράσης του ζολενδρονικού στον κυτταρικό πολλαπλασιασμό, χωρίς ωστόσο να παρατηρείται συνεργιστική δράση τους. Συνοψίζοντας το ζολενδρονικό φαίνεται να έχει σημαντική in vitro αντικαρκινική δράση, καθώς εμφανίζει ανασταλτική δράση στον πολλαπλασιασμό των καρκινικών κυττάρων και στα επιμέρους βήματα της μεταστατικής διαδικασίας. Επιδρά σε μόρια που παίζουν σημαντικό ρόλο στην εξάπλωση του όγκου και φαίνεται να μπορεί να διακόψει το ‘φαύλο κύκλο’ της οστικής νόσου στον καρκίνο του μαστού. Η χρησιμοποίηση του μαζί με άλλα αντικαρκινικά μπορεί να αυξήσει την ανασταλτική του ικανότητα. Περαιτέρω μελέτες θα βοηθήσουν στην in vivo επιβεβαίωση των παραπάνω και συγκεκριμένα ότι το ζολενδρονικό εν δυνάμει θα μπορούσε να χρησιμοποιηθεί ως αντικαρκινικός παράγοντας σε γυναίκες με καρκίνο του μαστού, έτσι ώστε να αποφευχθούν οι πιθανές μεταστάσεις στα οστά. / In this doctoral thesis the effect of zoledronic acid (zol) on human MDA-MB- 231 and MCF-7 breast cancer cell lines on cell proliferation, invasion, adhesion and migration were examine. The obtained results demonstrated that zoledronate effectively reduced cell growth for both cancer cell lines. Moreover the effect of zol on key matrix molecules that implicated in cell migration, invasion and metastasis, such as Proteoglycans, Metalloproteinase and integrins were studied. In both cell lines the inhibition of expression of both protein and gene levels were demonstrated. In order to evaluated the effect of zol on the activation of osteoclasts, bone particles with the appropriate growth factors were co-cultured with MDA-MB-231 cells. Conclusively the result of this thesis demonstrated that zol inhibits migration and invasiveness of breast cancer cells, which is of crucial importance in preventing the spread in bone inviroment.