Paget’s disease of bone (PDB) shows a strong genetic component and mutations in SQSTM1 (Sequestosome 1) are observed in about 10% of sporadic PDB patients. My PhD investigated the RIN3 gene (Ras and Rab interactor protein 3), previously implicated in the pathogenesis of PDB by GWAS. The RIN3 gene encodes a guanine exchange factor (GEF), involved in the activation of GTPases which are crucial in osteoclast activity. It also has a role in endocytosis and recycling of tyrosine kinase receptor. The role of RIN3 in bone remodelling is unclear, however some investigations revealed some associations with bone: RIN3 has been associated with high lower limb bone mineral density in children in a meta-analysis of GWAS studies, and was shown to be expressed in primary calvarial osteoblasts. The expression of RIN3 was down regulated during human primary osteoclast differentiation, and also in iliac bone biopsies from osteoporotic patients compared to healthy postmenopausal donors (Kemp et al, 2014).   In Chapter 1, I present normal bone structure, composition and remodelling before detailing PDB and its genetics. I then introduce RIN3 as a candidate gene for PDB. In Chapter 2, I describe all methods performed and materials used for the completion of this project. This includes primary cell cultures, RNA and protein work, immunostaining, immunochemistry and phenotype analysis on Rin3-/- mice. Chapter 3 presents the fine mapping of RIN3 using Sanger and next generation sequencing performed on PDB cases and controls. 18 variants were detected and one common variant (p.R279C) showed a strong association with PDB. Rare variants were also over-represented in cases, and many were shown to be on the same haplotype as p.R279C. Chapter 4 details the association study performed on a UK cohort and includes the investigation of the clinical phenotype severity in patients against the RIN3 mutations. Chapter 5 presents the expression pattern of RIN3 in bone cells and bone microenvironment. Important variations of RIN3 mRNA and protein were detected during the differentiation of bone marrow derived osteoclasts. Protein levels of RIN3 were also found in osteoclasts from human osteoclastoma, human osteosarcoma, PDB patients, giant cell tumour (GCT) and healthy controls. Within all the mouse tissues analysed, Rin3 mRNA was expressed the highest in bone after lung. Chapter 6 focuses on the work performed on mice deficient of the Rin3 gene. They showed a higher trabecular bone volume and a smaller active resorption surface occupied by osteoclasts in trabecular bone. In conclusion, the combined in vitro and in vivo analyses have uncovered that RIN3 plays a role in bone metabolism and is a strong gene candidate for PDB.

Made available in DSpace on 2014-06-11T19:32:50Z (GMT). No. of bitstreams: 0 Previous issue date: 2004Bitstream added on 2014-06-13T19:22:31Z : No. of bitstreams: 1 andrade_jc_dr_botfm.pdf: 2650759 bytes, checksum: 3247fedde23c0a35497a64d9285fa833 (MD5) / Universidade Estadual Paulista (UNESP) / O objetivo deste trabalho foi estudar através do exame Imunoistoquimico, a correlação entre alguns fatores Anatomopatológicos (tipo histológico e grau nuclear) e alguns fatores biológicos (receptores hormonais de estrógeno e de progesterona, proteína c-erbB-2 e proteína p53, no carcinoma de Paget da mama, doença rara e especial. No período entre Janeiro de 1980 a Dezembro de 1998 foram tratados no Instituto Brasileiro de Controle do Câncer, 6.303 casos de câncer de mama, deste total 98 casos foram diagnosticados como carcinoma de Paget, cuja incidência foi de 1,55%. Estudamos retrospectivamente 60 casos, sendo que 38 deles foram excluídos da análise, devido a limitação e escassez da amostra no material disponível, a idade das pacientes variou entre 26 e 84 anos, com média de 55,4 anos, as informações clínicas e terapêuticas foram obtidas dos prontuários das pacientes considerando, idade na época do diagnóstico, tamanho do tumor quando palpável, estadiamento clínico, e o tipo de cirurgia realizada. As amostras de tecido mamário foram recuperadas dos arquivos do Departamento de Anatomia Patológica do I.B.C.C. / MATTOSINHO. Após a revisão histológica, realizada por dois patologistas os casos foram classificados em quatro grupos: Grupo A- Doença de Paget (forma pura) n = 09 Grupo B- Doença de Paget + neoplasia ductal “in situ”, n = 12 Grupo C- Doença de Paget, neoplasia ductal invasora, n = 30 Grupo D- Doença de Paget + neoplasia ductal “in situ”+ neoplasia ductal invasora, n = 09. Entre as variáveis anatomopatológicas, o grupo C prevaleceu com 30 casos (50%). O grau nuclear (GN-II) predominou com 45 casos (75%). Em relação as variáveis biológicas o receptor de estrógeno negativo predominou com 41 casos (68,3%), seguido pelo receptor de progesterona negativo com 40 casos (66,7%), podemos dizer que... / The objective of this paper was to study the immunohistochemical efects of Paget’s disease, a rare and special carcinoma of the breast, by correlating anatomopathological (histological and nuclear grade) and biological (estrogen and progesterone hormonal receptors, c-erbB-2 protein and p53 protein) factors. Between January 1980 and December 1998, 6303 cases of breast cancer were treated at the Brazilian Institute of Cancer Control; 98 of these were diagnosed with Paget’s carcinoma, an incidence of 1.55%. We retrospectively studied 60 of these cases; 38 were excluded due to lack of available sample material. Patient age varied between 26 and 84 years (mean 55.4), clinical and therapeutic information were obtained from patient’s medical records considering age at time of diagnosis, tumor size when palpable, clinical stage, and type of surgery performed. Samples of breast tissue were retrieved the Anatomical Pathology Department, I.B.C.C. / MATTOSINHO. After histological review, by two different pathologists, they were classified into four groups: Group A- Paget’s disease (pure form) n = 09 Group B- Paget’s disease + “in situ” duct neoplasia n = 12 Group C- Paget’s disease, invasive duct neoplasia n = 30 Group D- Paget’s disease + “in situ” duct neoplasia + invasive duct neoplasia n = 09.Invasive duct neoplasia was the most prevalent anatomopathological variable (Group C, n = 30, 50%). Nuclear grade (GNII) was found in 45 cases (75%). In relation to the biological variables, the negative estrogen receptor was predominant with 41 cases (68.3%), followed by the negative progesterone receptor with 40 cases (66.7%); this correlation had good concordance by the Kappa test. The c-erbB-2 protein was positive in 53 cases (88.3%) and p53 was negative in 47 cases (78.3%). From these results, we concluded that there was no statistical... (Complete abstract click electronic address below)

SUMOylation and ubiquitination are important post-translational modifications. While ubiquitination is well known for targeting proteins for degradation, SUMOylation often regulates the intracellular localization of substrates. In the first project of this dissertation, we developed proteomic strategies to identify novel SUMOylated proteins in mammalian cells. In the second project, we investigated the regulation of protein ubiquitination in the NF-κB signaling pathway in the context of Paget’s disease of bone (PDB). Identification of SUMOylated proteins has been a challenge because of low abundance of SUMOylation substrates. Here, we utilized a mass spectrometry (MS)-based proteomic approach to identify novel SUMOylated proteins in mammalian cells. Seventy-four unique proteins were commonly identified in the collection of four SUMO-1 plasmids, thus considered candidate SUMOylated proteins. Many of these proteins are associated with the nucleus. The results were validated by confirming SUMOylation of a novel substrate Drebrin and a well known substrate Ran-GAP1. Furthermore, the potential SUMOylation sites in Drebrin have been identified and confirmed using site-directed mutagenesis. PDB is a disorder characterized by increased bone turnover containing hyperactive osteoclasts. Mutations in Sequestosome 1 (p62) are associated with 40% of familial PDB. P62 is a scaffold protein and plays a critical role in regulating ubiquitination of TRAF family signaling molecules and mediating the activation of NF-κB by RANK and TNFα ligands. P62 also plays a critical role in shuttling substrates for autophagic degradation. The objective of this project is to determine the effects of PDB-associated p62 mutants on NF-κB signaling and autophagy. We compared the effect of wild-type (WT) p62 and PDB mutations (A381V, M404V and P392L) on the TNFα-induced NF-κB signaling using an NF-κB luciferase assay. Our results show that these p62 mutations increased the NF-κB signaling. In addition, we found that the PDB mutations did not change the interaction between p62 and the autophagy marker protein LC3. In summary, the PDB mutations in p62 are likely gain-of-function mutations that can increase NF-κB signaling and potentially contribute to disease progression. Based on the results, we proposed a model to speculate the synergetic role of p62 PDB mutant on NF-κB signaling and autophagy.

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