Introduction: Axial spondyloarthritis (ax-SpA) is an inflammatory rheumatic disease. It is a unique model of bone remodeling disorders because, although one of the main diagnostic parameters is the rate of bone formation, inflammation present in patients' bodies increases the risk of pathological bone resorption, which can lead to osteoporosis. The processes of pathological resorption in ax-SpA have not been fully investigated, both in the disease as such and in the individual forms of the disease, i.e. non-radiographic (nr-axSpA), radiographic axial spondyloarthritis (r-axSpA) and ankylosing spondylitis (AS). This work deals with the influence of inflammatory serum of patients on the process of osteoclast differentiation from peripheral precursors of patients and healthy donors. Material and methods: Monocytes separated from the peripheral blood of either axSpA patients or healthy donors were stimulated for 14 days in vitro with serum from patients and in parallel with serum of age and sex of the corresponding healthy donors. Osteoclasts were evaluated as multinucleated, TRAP positive cells. Their numbers were statistically processed. Results: The inflammatory serum environment of patients with axSpA stimulated the osteoclastogenesis of axSpA monocytes significantly more (P <0,05) than the...

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.

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.

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.

Please read the abstract on page 3 in the dissertation. / Dissertation (MSc)--University of Pretoria, 2011. / Chemical Pathology / unrestricted

Introduction: Extracellular DNA (ecDNA) is a common component of blood plasma. Increased levels of ecDNA in plasma can be found in some autoimmune diseases like systemic lupus erythematosus (SLE), rheumatoid arthritis or celiac disease which are associated with inflammatory processes. These diseases are also associated with an increased risk of osteoporosis. Bone is a dynamic structure undergoing constant modelling caused by osteoblasts, osteocytes and osteoclasts. Shifting their equilibrium can lead to pathological conditions such as osteoporosis. In this thesis we focused on elucidating whether ecDNA, an inflammatory agent with proven immunoregulatory effects can alter differentiation potential of monocytes and alternatively lead to osteoclastogenesis via TLR9. Material and methods: We obtained monocytes from peripheral blood of healthy donors and cultivated them with four types of ODNs control (CO), stimulatory (ST), inhibitory (INH, telomeric (TLM) with phosphodiester (-pO) or phosphorothioate (-pS) backbone for two weeks to establish their effect on differentiation potential of monocytes into osteoclasts. Osteoclastogenesis was evaluated by number of yielded osteoclasts observed on a light microscope. To establish the effect of ODNs on osteoclast activity samples were analysed by qPCR for...

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 (&alpha;=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.

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 (&alpha;=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.

Introduction: Bone is a highly active tissue throughout life and is a subject to constant remodelling. Main cells responsible for continuous resorption and de novo synthesis of bone matrix are osteoclast, osteoblasts and osteocytes. Osteoclasts are the only known type of cells able to resorb bone. These cells are formed by fusion of precursor cells in bone marrow or peripheral blood in a process called osteoclastogenesis. Formation of osteoclasts may be of importance concerning chronic inflammatory diseases that are linked with higher risk of developing osteoporosis during lifespan. Celiac disease is one of those diseases, which is characterized by destruction of intestinal mucosa after ingestion of gluten by susceptible individuals followed by induction of chronic inflammation. In this work, we focused on the potential role of osteoclastogenesis in the development of osteoporosis in patients with celiac disease and we studied roles of selected inflammatory agents (TNF-α, IL-6, IFN-γ a cfDNA) with supposed or hypothesised effects on osteoclastogenesis. Material & Methods: We obtained plasma and serum samples from newly diagnosed patients with celiac disease, patients on gluten free diet and healthy controls and analysed concentrations of cfDNA and inflammatory cytokines TNF-α, IL-6 and IFN-γ in...