Orientadores: Silvana Pereira Barros, Darcy de Oliveira Tosello / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-06T12:25:34Z (GMT). No. of bitstreams: 1 Silva_EduardodePaulae_M.pdf: 780892 bytes, checksum: 36e3cb799e53830c091d9721116dc937 (MD5) Previous issue date: 2006 / Resumo: O hormônio paratireóideo (PTH), quando administrado de maneira intermitente, promove anabolismo ósseo. Modelos experimentais em ratos, utilizando PTH intermitente, demonstraram que o anabolismo ósseo promovido diminui a perda óssea ocasionada por periodontite induzida, e acelera o reparo ósseo ao redor de implantes osseointegrados. Neste trabalho, nosso objetivo foi analisar a reparação óssea de defeitos em calotas cranianas de ratos que receberam PTH de maneira intermitente. Nas calotas cranianas de 32 ratos Wistar foram feitos defeitos de 4 mm de diâmetro. Após a cirurgia, 16 ratos receberam 40µg/kg de PTH três vezes por semana, enquanto 16 ratos (controle) receberam 40µg/kg do veículo do PTH com a mesma freqüência. Os animais foram sacrificados após 4 semanas e suas calotas foram removidas, radiografadas e processadas para análise histomorfométrica. As radiografias foram analisadas por meio de um fotodensitômetro, e os valores obtidos, tanto na morfometria quanto da densidade radiográfica, foram submetidos a teste estatístico (teste t). Os resultados mostraram que no grupo tratado com PTH, o tamanho do defeito residual foi menor ( 1,5 mm± 0,73) em relação ao grupo controle (1,98 mm ± 0,68) (p<0,05), e também que o grupo tratado apresentou uma maior densidade radiográfica do que o controle (p<0,01). A administração intermitente de PTH acelerou o processo de reparação dos defeitos na calota craniana / Abstract: The study aimed to investigate the effects ofintermittent parathyroid hormone PTH(1-34) treatment on bone regeneration of critically sized rat calvarial bone defects. A fullthickness bone defect (diameter 4 mm) was trephined in the central part of the parietal bones in ten-week old male Wistar rats. The animaIs were then divided into 2 groups of 16. The PTH group was subcutaneously administered with 40 microg PTH(1-34) /kg 3 times a week and to the control group it was given the vehicle. The rats were sacrificed at 28 days post surgery, the parietal bones were extracted, X- rayed and samples were prepared to obtain decalcified histological sections. The results were evaluated radiodensitometrically and histomorphometrically. The histomorphometric analysis for residual bone defect, using an image analyzer software, showed the mean values of 1.5mm for PTH group and 1.98mm for control group; indicating statistical significant differences among groups p<0.05 (test t). The photodensitometer analysis demonstrated a lower bone density in the group that did not receive PTH treatment (p<0.01). The experiment demonstrated that intermittent PTH (1-34) treatment could acelerate healing of rat calvarial defects / Mestrado / Histologia e Embriologia / Mestre em Biologia Buco-Dental

Orientadores: Pedro Duarte Novaes, Cristiane Ribeiro Salmon / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-20T00:19:31Z (GMT). No. of bitstreams: 1 Neves_JulianadosSantos_D.pdf: 4044530 bytes, checksum: 22df8d6b0c562b9ece71cf5970d118cc (MD5) Previous issue date: 2012 / Resumo: A administração intermitente do hormônio paratireóideo (PTH) promove um aumento da massa óssea, sendo atualmente utilizado no tratamento de osteoporose. Entretanto, poucos estudos têm avaliado os efeitos do PTH sobre o metabolismo do osso alveolar, e mais especificamente, no processo de reparo desta estrutura, podendo este surtir um efeito favorável na cicatrização da ferida alveolar. Considerando-se que o padrão de cicatrização da ferida do alvéolo de molares de ratos é similar ao de humanos e a sua relevância clínica, este estudo teve como objetivo analisar o efeito da administração intermitente do PTH sobre o reparo alveolar após exodontia de molares de ratos. Ratas Wistar de 200g tiveram seus primeiros molares inferiores extraídos e receberam injeções subcutâneas de 40 ?g/kg de hPTH(1-34) (grupo PTH) ou solução veículo (grupo controle) em dias intemitentes após a exodontia. Os animais foram sacrificados aos 3, 5, 7 e 11 dias após a cirurgia. Análises histomorfométricas do volume de osso recém-formado, número de osteoblastos e osteoclastos TRAP-positivos foram realizadas em cortes histológicos seriados. O Ligante do receptor ativador do fator nuclear ?? (RANKL), Sialoproteína óssea (BSP) e Osteopontina (OPN) foram localizadas no alvéolo em cicatrização por imunohistoquímica. A expressão dos genes de Colágeno 1 (Col1?1), Fosfatase alcalina (Alp), Fator de crescimento insulínico (Igf-1), Osteocalcina (Ocn), Bsp, Opn, Osteoprotegerina (Opg) e Rankl, foi analisada por qRT-PCR. A presença de osso recém-formado foi notada em alvéolos a partir de 5 dias de reparo em ambos os grupos. O volume de osso recém-formado foi significativamente maior no grupo PTH nos períodos de 5, 7 e 11 dias (p<0.05), acompanhado de um número maior de osteoblastos aos 5 dias de reparo (p<0.05). No grupo controle houve um aumento gradual na expressão de todos os genes estudados, com exceção de OPN, nos períodos após a cirurgia. De um modo geral, a expressão de Col1?1, Alp, Igf-1 e Ocn foi maior no grupo PTH nos períodos iniciais, seguido por um decréscimo significativo aos 7 dias de reparo comparado ao grupo controle. A expressão de Bsp foi similar em ambos os grupos, enquanto a expressão de Opn foi menor vi no grupo PTH aos 7 e 11 dias de reparo, com estas proteínas apresentando a mesma distribuição nas trabéculas ósseas em formação no interior do alvéolo. O número de osteoclastos TRAP-positivos foi similar em ambos os grupos em todos os períodos avaliados, assim como o padrão de marcação para RANKL. Entretanto, a razão dos genes Rankl:Opg foi maior no grupo PTH aos 11 dias de reparo, sugerindo que o PTH pode favorecer a osteoclastogênese a partir deste período. Conclui-se que o tratamento intermitente com hPTH (1-34) teve um efeito anabólico sobre o processo de reparo do osso alveolar de forma pontual aos 5 dias, estimulando o aumento no número de osteoblastos e da expressão de genes relacionados a diferenciação e produção de matriz por estas células, causando consequentemente, um aumento no volume de osso recém-formado no interior do alvéolo / Abstract: The intermittent administration of parathyroid hormone (PTH) promotes an increase in bone mass, and it is currently used to treat osteoporosis. However, few studies have evaluated the effects of PTH on the metabolism of the alveolar bone, and more specifically in the repair process of this structure, which may have a positive effect on alveolar wound healing. Considering that the pattern of the alveolar wound healing post molar extraction in rats is similar to that of the humans and its clinical relevance, this study aimed to analyze the effect of intermittent PTH administration on alveolar wound healing after extraction of rat molars. Wistar rats (200g) had their first molars extracted and received subcutaneous injections of 40 ?g/kg of hPTH (1-34) (PTH group) or vehicle solution (control group) in alternated days after the extraction. The animals were sacrificed at 3, 5, 7 and 11 days after surgery. Histomorphometric analysis of the volume of newly formed bone, number of osteoblasts and TRAP-positive osteoclasts were performed on serial histological sections. Receptor activator of nuclear factor ?? ligand (RANKL), bone sialoprotein (BSP) and osteopontin (OPN) were localized at the healing socket by immunohistochemistry. Collagen 1 (Col1?1), alkaline phosphatase (Alp), insulin growth factor (Igf-1), osteocalcin (Ocn), Bsp, Opn, osteoprotegerin (Opg) and Rankl gene expression was assessed by qRT-PCR. Newly formed bone was observed in sockets from 5 days of repair in both groups. The volume of newly formed bone was significantly higher in PTH group at 5, 7 and 11 days of repair (p <0.05). In the control group there was a gradual increase in the expression of all genes studied, except for Opn, through the periods of healing. Overall, the expression of Col1?1, Alp, Igf-1 and Ocn in the PTH group was higher at the initial periods, followed by a significant down-regulation at 7 days of repair compared to the control group. Bsp expression was similar in both groups, while Opn expression was down-regulated by PTH at 7 and 11 days of repair. Both proteins showed the same distribution on the forming trabeculae in the socket. The number of TRAP-positive osteoclasts was similar in both groups in all periods of repair, as well as the labeling pattern for RANKL. However, viii Rankl:Opg ratio was higher in the PTH group at 11 days of repair, suggesting that PTH may promote osteoclastogenesis from this period. In conclusion, the intermittent treatment with hPTH-(1-34) had an anabolic effect on the alveolar wound healing specifically at 5 days of repair, stimulating an increase on the osteoblast number and on the expression of genes related to the differentiation and production of bone matrix by these cells, thereby resulting in increased volume of newly formed bone in the healing socket / Doutorado / Histologia e Embriologia / Doutor em Biologia Buco-Dental

Parathyroid hormone (PTH) analogs are the main anabolic pharmacological agent for osteoporosis. PTH is an endogenous hormone, of which amino acids 1-34 bind the parathyroid hormone receptor (PTH1r), a G-coupled protein receptor expressed in kidney, fat, and bone. PTH increases trabecular bone mass by promoting the differentiation of the mesenchymal stem cell (MSC) into the osteogenic lineage. The Zinc Finger Protein 467 (Zfp467) is a potential downstream target of PTH1r and an important mediator of the MSC into the adipogenic lineage. Taken together, we ask whether Zfp467 knockout cells will show greater osteogenic potential and increased sensitivity to PTH treatment. We also seek to investigate a mechanistic signaling pathway of PTH1r involving Zfp467. Calvarial osteoblast (COB) and bone marrow stromal cells (BMSCs) from Zfp467 wild type (WT) and knockout (KO) mice were osteogenically differentiated and treated with either continuous (48h) or intermittent (6h/42h) PTH for 7-14 days. At 7 and 14 days, alkaline phosphatase (ALP) and von Kossa staining were conducted, respectively. At 7 days after differentiation, qPCR was used to analyze genes involved in osteogenesis, adipogenesis, WNT signaling, and mitochondrial respiration. ELISA was used to measure cAMP levels. Seahorse XF96 assays were used to measure oxygen consumption rates (OCRs) and extracellular acidification rates (ECARs). Western blot was used to measure PTH1r. Additionally, adipogenic differentiation and Oil Red O staining were performed on BMSCs. ALP and von Kossa results showed that Zfp467 KO cells exhibited increased osteogenesis and an increased response to PTH treatment (continuous and intermittent) as compared to WT controls. qPCR analysis of Alp, Rankl, and Sp7 further supported an increased osteogenic potential of the KO. Also, Oil Red O staining revealed suppressed adipogenesis in KO BMSCs and qPCR analysis showed suppressed Adiponectin and Ppary in KO COBs. Additionally, Pth1r and PTH1r expressions were significantly higher in KO and short PTH treatments (~10m) induced a remarkable reduction in Zfp467 of WT cells. Furthermore, the KO showed suppressed Pgc1a, similar OCR, and increased ECAR as compared to WT. The KO also exhibited higher cAMP levels and was more responsive to PTH-induced increases of cAMP at 10 minutes of PTH exposure. However, qPCR analysis of Lef1 and Sost showed no difference regarding the WNT pathway. Our data support an anti-osteogenic and pro-adipogenic role for Zfp467. The KO displays less adipogenesis, more osteogenesis, and is consistently more sensitive to the osteogenic effects of PTH. The upregulation of Pth1r and PTH1r in KO cells offers an explanation for this increased sensitivity. We propose a mechanism where the suppression of Zfp467 upregulates Pth1r and PTH1r activation suppresses Zfp467, resulting in a constitutively active positive feedback loop. Further still, the KO shows potentially suppressed mitochondrial biogenesis (through Pgc1a analysis), similar oxidative phosphorylation (through OCRs), increased glycolysis (through ECARs), and increased PKA signaling (through cAMP assays), yet their exact connections to the PTH1r-Zfp467 signaling pathway have yet to be investigated.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21019号 / 医博第4365号 / 新制||医||1028(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 別所 和久, 教授 安達 泰治, 教授 妻木 範行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Bone is a metabolically active tissue that is continuously remodeled throughout life. Osteocytes, the most abundant cells in bone, regulate bone homeostasis in response to hormonal and mechanical cues. Parathyroid hormone (PTH), a calciotropic hormone secreted from the parathyroid glands, has been widely used in the clinic to treat age-related osteoporosis. PTH acts on cells of the osteoblast lineage, including osteocytes, by signaling via the PTH receptor (PPR) to promote bone formation. However, the role of PPR signaling in osteocytes during aging has not been investigated. The hypothesis of this study is that PPR signaling in osteocytes plays a key role in maintaining skeletal health in aging mice. To address this hypothesis, mice in which the PPR was ablated in mature osteoblasts/osteocytes (Dmp1-Cre+;PPRfl/fl or Dmp1-PPRKO) were used to study their skeletal phenotype at 4 and 13 months of age. Compared to control littermates (Dmp1-Cre–;PPRfl/fl), Dmp1-PPRKO animals displayed age-dependent osteopenia due to reduced osteoblast activity and increased osteoclast numbers and activity. These changes were associated with a significant decrease in osteoprogenitors and an increase in marrow adiposity. At the molecular level, the absence of PPR signaling in mature osteoblasts/osteocytes was accompanied by a marked increase in serum sclerostin, RANKL-expressing marrow adipocytes, and early onset of oxidative stress in osteocytes. In vitro studies demonstrated that PTH protected osteocytes from oxidative stress-induced cell death by suppressing the intracellular accumulation of reactive oxygen species. Mechanical forces are also important regulators of bone mass and quality. For instance, immobilization and reduced mechanical loading, such as prolonged bed rest or long-duration spaceflight, lead to bone loss or osteopenia due to reduced bone formation and increased bone resorption. Osteocytes are known to sense and transduce mechanical forces applied to the skeleton into biochemical signals. However, the exact molecular mechanism remains unclear. To unravel the mechanism by which osteocytes sense and respond to mechanical unloading, an osteocytic cell line, Ocy454, was exposed to microgravity (µG) conditions for 2, 4, or 6 days onboard the SpaceX Dragon-6 and the International Space Station. Global transcriptomic analysis demonstrated that µG leads to downregulation of key osteocytic marker genes compared to ground controls (1G), suggesting the impaired differentiation of osteocytes. Importantly, glycolysis was the most activated signaling pathway in osteocytes subjected to µG compared to 1G. Gene comparison analysis further identified a set of mechano-sensitive genes that are consistently regulated in multiple types of cells exposed to µG, suggesting a common, yet to be fully elucidated, genome-wide response to µG. In summary, these studies demonstrated that osteocytes are highly regulated by PTH and mechanical forces. We found that PPR signaling in osteocytes is important for protecting the skeleton from age-induced osteopenia by promoting osteoblast’s bone-forming activity and mitigating osteoclast’s bone resorption. We also demonstrated that PTH protects osteocytes from oxidative stress. Finally, we showed that osteocytes respond to µG with an increase in glucose metabolism and oxygen consumption.

No description available.

We have shown that nuclear matrix protein 4 (Nmp4) attenuates the response to intermittent parathyroid hormone (PTH) in healthy and ovariectomized (OVX) female mice using a global knockout of the Nmp4 gene. Additionally, these mice have increased bone marrow osteoprogenitors and CD8+ T-cells which support osteoblast differentiation. The animals were not protected from bone loss following OVX, but retained the hypersensitivity seen in the intact mice. Mesenchymal stem/progenitor cells (osteoprogenitors) demonstrated increased growth rate in culture and showed more robust differentiation into mineralizing bone cells. Chromosome precipitation followed by next generation sequencing and bioinformatics analysis characterized Nmp4 as a negative regulator of synthetic processes and suggested the IGF1/Akt and BMP2/Smad biochemical pathways which are likely targets for Nmp4 regulation. We have experimentally verified these pathways in immortalized bone marrow mesenchymal cells from wild type and Nmp4-KO mice. Disabling Nmp4 in estrogen replete or depleted mice confers an enhanced bone formation from intermittent parathyroid hormone.

Thesis (M.Sc.)--Memorial University of Newfoundland, Faculty of Medicine, 1999. / Bibliography: leaves 118-138.

Indiana University-Purdue University Indianapolis (IUPUI) / PTH increases osteoclasts by upregulating RANKL in cells of the osteoblastic lineage, but the precise differentiation stage of the PTH target cell remains undefined. Recent findings demonstrate that PTH regulates gene expression in osteocytes and that these cells are an important source of RANKL. We therefore investigated whether direct regulation of the RANKL gene by PTH in osteocytes is required to stimulate osteoclastic bone resorption. To address this question, we examined bone resorption and RANKL expression in transgenic mice in which PTH receptor signaling is activated only in osteocytes (DMP1-caPTHR1) crossed with mice lacking the distal control region regulated by PTH in the RANKL gene (DCR -/-). Longitudinal analysis of circulating C-terminal telopeptide (CTX) in male mice showed elevated resorption in growing mice that progressively decreased to plateau at 3-5 month of age. Resorption was significantly higher (~100%) in DMP1-caPTHR1 mice and non-significantly lower (15-30%) in DCR -/-mice, versus wild type littermates (WT) across all ages. CTX in compound DMP1-caPTHR1; DCR -/-mice was similar to DMP1-caPTHR1 mice at 1 and 2 months of age, but by 3 months of age, was significantly lower compared to DMP1-caPTHR1 mice (50% higher than WT), and by 5 months, it was undistinguishable from WT mice. Micro-CT analysis revealed lower tissue material density in the distal femur of DMP1-caPTHR1 mice, indicative of high remodeling, and this effect was partially corrected in compound vi mice. The increased resorption exhibited by DMP1-caPTHR1 mice was accompanied by elevated RANKL mRNA in bone at 1 and 5 months of age. RANKL expression levels displayed similar patterns to CTX levels in DMP1-caPTHR1; DCR -/-compound mice at 1 and 5 month of age. The same pattern of expression was observed for M-CSF. We conclude that resorption induced by PTH receptor signaling requires direct regulation of the RANKL gene in osteocytes, but this dependence is age specific. Whereas DCR-independent mechanisms involving gp130 cytokines or vitamin D 3 might operate in the growing skeleton, DCR-dependent, cAMP/PKA/CREB-activated mechanisms mediate resorption induced by PTH receptor signaling in the adult skeleton.

Development of chimeric, recombinant peptides, proteins and enzymes expands the availability of protein/enzyme–based tools for cellular analysis and new assay platforms. Ideal protein reagents for cellular analysis must translocate into a variety of cells with minimum cell damage, retain stability and biological activity within the cell during analysis, and provide a reliable, measurable signal. This work focused on development, characterization and utilization of chimeric recombinant peptide, protein and enzyme reagents for cellular analysis and immunoassays. A cell-penetrating, fluorescent protein substrate (PKAS) was developed to monitor intracellular protein kinase A activity in cells without the need for cellular transfection. PKAS translocated into HeLa cells, βTC-3 cells and pancreatic islets with minimal toxicity. Upon cellular loading, glucose dependent phosphorylation of PKAS was observed in both βTC-3 and pancreatic islets via capillary zone electrophoresis. In pancreatic islets, maximal PKAS phosphorylation (83 ± 6 %) was observed at 12 mM glucose, whereas maximal PKAS phosphorylation (86 ± 4 %) in βTC-3 cells was with 3 mM glucose indicating a left-shifted glucose sensitivity. A cell-penetrating luciferase chimera (Luc-TAT) and a cell-penetrating phospholipid nanoshell entrapped luciferase (Luc-PPN) was constructed to monitor dynamic changes in intracellular ATP levels in mammalian cells. Upon cellular loading, the activity of Luc-TAT and Luc-PPN was monitored with time. Luc-TAT lost approximately 50% activity within one hour, and decreased rapidly over time. In contrast Luc-PPNs retain approximately 95% activity in 1 hour and 77% after 12 hours showing longer biological lifetime. Luc-PPNs were able to detect dynamic ATP changes in intact HeLa cells in the presence of KCN and NaN3. The bioluminescence returned to background levels within 8-10 minutes after treatment with KCN, whereas NaN₃ showed ~ 40% reduction. Two novel recombinant human parathyroid hormone (hPTH) analogs hPTHEGFP and hPTH-Cys were prepared to develop immunoassays for PTH detection in clinical samples. Initial experiments show promise for these analogs for use in CZELIF based immunoassays. The analogs present a number of distinctive advantages for clinical assays and can be used to develop several immunoassay platforms.