Browsing by Subject "Osteoclasts"
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Item Analysis of Skeletal Phenotypes of Matrix-Gla Protein (MGP) and Twisted Gastrulation (Twsg1) Deficient Mice.(2010-08) Emery, Ann ElizabethMatrix Gla-Protein (MGP) and Twisted Gastrulation (Twsg) are secreted proteins that reside in the extracellular matrix. MGP is a potent inhibitor of mineralization in both softand hard- tissues. MGP-/- mice exhibit numerous abnormalities, the most severe being aortic calcification that leads to death by approximately two months of age. Despite extensive studies using cartilage and cardiovascular tissues in these mice, little is known about how MGP deficiency affects bone development, specifically the impact on osteoblasts and osteoclasts. Twisted Gastrulation is a modulator of BMP signaling in many tissues. Twsg1 deficient mice exhibit several phenotypes including craniofacial, salivary gland defects, and skeletal defects. Twsg1 has also been implicated in embryo patterning and cartilage development but to date, there has been no study of the bone in these mice. Preliminary analysis using faxitron (x-ray) analysis of MGP and Twsg1 wild type and homozygous deficient mice revealed reduced bone and an osteopenic bone phenotype. The goal of my thesis project was to characterize 1) the in vivo (both dynamic and static) phenotype of Twsg-/- mice and 2) both in vivo and in vitro (static) skeletal phenotype of MGP-/- mice. Using microcomputer tomography (μCT), both static, and in the case of Twsg1, dynamic, histomorphometric analysis was performed to characterize the skeletal phenotype in the animals. In both mouse models there was a significant decrease in both cortical and trabecular bone present. Primary osteoblasts and osteoclasts were used to further characterize the in vitro phenotype in the MGP-/- mouse model. Primary osteoblasts showed premature mineralization and differentiation. Bone sialoprotein (BSP) and osteoclacin (OCN) mRNA levels were both elevated. Surprisingly, osteoclast differentiation also showed enhancement with increased number and larger multinuclear TRAP+ osteoclasts. They also showed increases in DC-STAMP and TRAP mRNA levels compared to the wild type animals. The data collected in this study was used to evaluate the skeletal phenotype of the MGP and Twsg1-knockout mouse models to better understand the relationship of osteoporosis, BMP signaling, and mineral metabolism.Item Bone Morphogenetic Protein-2 (BMP2) upregulates osteoclast gene expression.(2009-11) Espe, Kelly ChristineBone Morphogenetic Proteins (BMPs) induce bone formation by osteoblasts, but their direct role in bone resorption by osteoclasts remains to be characterized. Twisted Gastrulation (Twsg1) is a secreted BMP binding protein that inhibits BMPs from binding to their receptors. Mice lacking the Twsg1 gene (Twsg1-/-) exhibit an osteopenic skeletal defect. Previous studies indicate that the osteopenic phenotype in Twsg1-/- mice is due to increased osteoclastogenesis and not due to reduced osteoblast function. This study hypothesizes that treatment of wild-type osteoclasts with BMP2 will increase osteoclast gene expression and that this gene expression will decrease with the addition of the known BMP inhibitor, Noggin. The results of this investigation show that the addition of BMP2 to RANKL upregulates Cathepsin K, Nfatc1, Acp5, DCSTAMP, and ATP6v6d02 gene expression levels. The addition of the more well understood BMP inhibitor, Noggin, downregulates these gene expression levels. These results indicate a possible direct mechanism of action for BMP2 on osteoclast activation.Item Elucidating the Role of Protein Kinase D in Osteoclasts(2022-11) Kazemi, ParandisThe Protein Kinase D (PRKD) family of serine/threonine kinases have been shown to contribute to a complex set of biological functions such as signal transduction, cell proliferation, differentiation, cell vesicle trafficking, secretion, polarity, and locomotion. Our lab has demonstrated that PRKD affects multiple aspects of osteoclast formation, such as cell-to-cell fusion, migration, and differentiation. Nevertheless, the specific targets of PRKD phosphorylation in osteoclasts remain poorly characterized. In this thesis, we aim to identify the targets of PRKD in osteoclast cell culture and to study the network effect of dephosphorylating those identified targets in osteoclasts. We utilized two complementary phospho-proteomic approaches: 1) Quantitative phospho-proteomics approach to establish a global view of changes in thousands of phospho-sites upon PRKD inhibition, followed by bioinformatic analysis of the functional enrichment clusters and phospho-motif enrichment analysis to identify candidate targets, 2) Targeted phospho-proteomics using PRKD recombinant kinase, followed by bioinformatic analysis of the phospho-motif enrichment and assessment of candidate targets in the context of known cellular signaling. Using the quantitative proteomics analysis of the PRKD inhibitor-treated osteoclasts cell culture, we showed that the main cellular pathways influenced by PRKD inhibition are mRNA processing, transcription, and regulation of transcription, which corresponded to the candidate targets: SRRM2, HDAC5, and JUND. Additionally, by using the targeted phospho-proteomics method, we identified two novel PRKD targets: SIPA1L3 and LSP1 which are associated with the actin cytoskeleton and cell membrane and contribute to cell motility. Subsequent validations of these findings would play an important role in better understanding the cellular regulators of osteoclast formation and bone resorption and establishing new therapeutic modalities to limit bone resorption in large numbers of patients afflicted with osteolytic conditions.Item Smad 1/5 and Smad 4 expression are necessary for osteoclast differentiation(2014-06) Huang, Brandon Pei HanThe human skeleton is structurally unyielding due to its high mineral content, yet highly adaptive as it is dynamically remodeled throughout life to meet our daily needs. The process of bone remodeling is primarily carried out by two types of cells, osteoblasts and osteoclasts. Osteoblasts synthesize the protein matrix that mineralizes to become bone; whereas osteoclasts demineralize and resorb bone. The intricate balance between their activities is critical for bone homeostasis, and any disruption of this harmonious relationship can lead to both systemic and localized diseases, such as osteoporosis, osteolytic malignancies, and periodontitis (Zaidi, 2007). Osteoblasts and osteoclasts are tightly regulated by systemic hormones and locally produced cytokines, such as bone morphogenetic proteins (BMPs) (Biver, Hardouin, & Caverzasio, 2013; Giannoudis, Kanakaris, & Einhorn, 2007). BMPs have long established themselves as direct positive regulators of osteoblastic activity; however, their influences on osteoclasts have been controversial. Recent work from the Gopalakrishnan/Mansky lab has provided convincing evidence that BMP signaling directly upregulates osteoclastogenesis induced by RANKL and M-CSF (Broege et al., 2013; Jensen et al., 2010; Sotillo Rodriguez et al., 2009). Upon binding of the cell surface BMP receptors, BMPs exert their regulatory effects through the activation of two distinct intracellular pathways: the canonical pathway involving Smad proteins and the non-canonical pathway involving the MAP kinases (Biver et al., 2013). The goal of this project is to further investigate the specific role of the canonical Smad pathway of BMP signaling in osteoclastogenesis.