Browsing by Subject "Osteoclast"

Now showing 1 - 6 of 6
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    Investigating HDAC-MEF2 Roles in Osteoclastogenesis
    (2019-02) Blixt, Nicholas
    Bone remodeling is the process of removing damaged or old bone and replacing it with new bone. This process occurs in the adult skeleton in order to maintain structural integrity and accomplish other necessary functions. Osteoclasts degrade bone in a localized and controlled manner, and osteoblasts deposit new bone in response to osteoclast activity. The activity of each cell type can be controlled using multiple mechanisms, including osteoclast-mediated regulation of bone formation. In order to determine how osteoclast activity can be regulated in order to manage pathological bone loss, it is necessary to understand transcriptional mechanisms that control their development. With this goal, I expanded on previous research showing histone deacetylase 7 (HDAC7) repressed osteoclast differentiation. I found that overexpression of a fragment of HDAC7 that interacts with the myocyte enhancer factor 2 (MEF2) family of transcription factors repressed osteoclast differentiation to a similar degree as the full-length HDAC7. This led me to investigate the roles of MEF2 in osteoclast differentiation. Mef2a and Mef2d were more highly expressed than Mef2b and Mef2c. Osteoclast-specific deletion of Mef2a (A-KO) or Mef2d (D-KO) in mice compromised in vitro osteoclast differentiation and activity. A-KO and D-KO male mice and D-KO female mice presented no overt skeletal phenotype, but female A-KO mice were osteopetrotic due to increased trabecular number. To determine whether MEF2A compensated for loss of MEF2D and vice versa, I generated mice with osteoclast-specific deletion of both Mef2a and Mef2d (AD-KO). In vitro assays demonstrated a complete block in osteoclast development prior to fusion. However, AD-KO mice presented with osteopenia from reduced cortical and trabecular bone thickness relative to WT mice but unchanged levels of osteoclast activity markers. These experiments suggest specific signals present in vivo but not in vitro rescue osteoclast differentiation of AD-KO osteoclasts. Additionally, MEF2A/D potentially regulates expression of an osteoclastderived factor that regulates osteoblast activity.
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    Mechanisms underlying the regulation and functions of HDAC7 in osteoclast differentiation
    (2013-06) Cho, JangYeun Janice
    The purpose of this research is to characterize the regulation and functions of Histone Deacetylase 7 (HDAC7) in osteoclast differentiation. Histone Deacetylases (HDACs) are negative regulators of transcription.1 Endochondral bone formation including maturation of chondrocytes and osteoblasts is regulated by HDACs.2 It has been shown that the suppression of HDAC7 enhanced osteoclast formation while the overexpression of myc-HDAC7 inhibits osteoclast formation.3 Hence, it has been suggested that the stimulation of HDAC7 might be a unique therapeutic strategy to reduce osteoclastic bone loss.3 However, the mechanism and the molecular pathways regulating how HDAC7 inhibits osteoclast formations have not been examined. In this study, we hypothesized that the deacetylase catalytic activity of HDAC7 is necessary for suppression of osteoclast formation. It is also hypothesized that the subcellular localization of HDAC7 in nucleus facilitated by the phosphorylation of serine residues at N- terminal with Receptor activator of nuclear factor-kappaB ligand (RANKL) stimulation is the one of the mechanism that HDAC7 controls osteoclast differentiation. However, from this study, it is found that HDAC7 deacetylase activity is dispensable for HDAC7-mediated inhibition of osteoclastogenesis. It is also concluded that the presence of HDAC7, not necessarily the localization of HDAC7 in the nucleus, is necessary to repress the osteoclastogenesis.
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    The Regulation And Function Of Bmps In Osteoclastogenesis
    (2019-06) Huntley, Raphael
    Bone morphogenetic proteins (BMPs) play a large role in the formation and maintenance of the skeleton, and, in recent years, have become a common clinical adjuvant. The regulatory processes tightly control BMP gradients, which can stimulate both osteoblasts to build bone, and osteoclasts to resorb bone. This thesis discusses data from multiple in-vitro and in-vivo BMP-related knockout models to elucidate the different function BMPs have on osteoclast differentiation and activity. We elucidate the mechanism by which BMP signaling is tightly controlled by Twisted Gastrulation, an extracellular BMP binding protein. Using an murine model we also show osteoclast derived BMP2 acts to regulate bone mineral density in-vivo, and can function to coordinate osteoblast matrix deposition.
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    The Role of Myosin X in BMPs Promotion of SMAD 1/5 in Osteoclast Formation and Function
    (2017-07) Tasca, Amy
    Novel therapeutic strategies that target osteoclasts are needed for many orthopedic, craniofacial and cancer applications. Incomplete understanding of the factors that regulate osteoclasts limits this development. Currently, bone morphogenetic proteins (BMPs) are used to promote healing of open fractures, bone grafts and non-union fractures. Bone healing requires synthesis of new bone by osteoblasts and the removal of old or damaged bone matrix by osteoclasts. Although BMPs have been widely studied as stimulators of osteoblastic bone formation, it has also been shown that BMPs directly increase osteoclast formation, however the mechanism behind this enhancement is not completely understood. Two potential candidates responsible for BMPs enhancement of osteoclast formation could be SMADs, components of the canonical BMP signaling pathway, and MYO10, a regulator of the osteoclast cytoskeleton. The data presented in this thesis demonstrates that loss of SMAD1/5 expression by osteoclasts in in vitro cultures of osteoclasts inhibits osteoclast differentiation and activity. However, the loss of SMAD1/5 expression in osteoclasts and macrophages in a mouse model leads to enhance osteoclast differentiation and activity. Interestingly bone formation is also increased in the mouse model with decreased SMAD1/5 expression suggesting that SMAD1/5 signaling in osteoclasts may regulate coupling between osteoclast and osteoblast activity. Lastly MYO10 besides playing a role in osteoclast activity also plays a role in regulating osteoclast differentiation. Data presented demonstrates that loss of MYO10 expression correlates with changes in the osteoclast cytoskeleton which impacts the ability of osteoclasts to fuse. By gaining knowledge of regulators of osteoclastogenesis and the mechanism by which they modulate osteoclast differentiation and activity, we will better understand the etiology of osteoclast diseases and provide novel insight into future therapies.
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    Role of twisted gastrulation and matrix gamma-carboxyglutamic acid protein in bone homeostasis.
    (2012-05) Sotillo Rodriguez, Julio Eliezer
    The regulation signaling activity of osteoblasts and osteoclasts has been linked to extracellular proteins, including twisted gastrulation (TWSG1) and matrix gamma-carboxyglutamic acid protein (MGP). TWSG1 is a regulator of bone morphogenetic proteins (BMPs) signaling activity. Previous studies have shown that TWSG1 is an essential modulator of not only skeletal development but also soft tissue development (i.e. salivary gland). MGP is a vitamin K-dependent protein previously characterized as a potent inhibitor of biomineralization. Numerous cells types produce MGP, including osteoblasts, chondrocytes, vascular smooth muscle cells and endothelial cells. Despite current research, little is known about the effects of TWSG1 and MGP on the regulation of osteoblasts and osteoclasts. Both of which are cells that maintain bone remodeling. In this dissertation, we assessed the role of both TWSG1 and MGP in postnatal bone homeostasis as well as in vitro functional analysis of TWSG1 and MGP on osteoblasts and osteoclasts. We were able to determine that the C57BL/6 MGP deficient mice and 129Sv/Ev TWSG1 deficient mice exhibited an osteopenic skeletal phenotype. This phenotype was explained by an imbalance in the cellular regulatory patterns associated with the regulation of homeostatic bone remodeling. Further, we characterized cellular pathways which explained how, in both TWSG1 and MGP deficient mice, excessive osteoclastic activity was the name culprit in the osteopenic phenotypes. Taken together, our results highlight the importance in the modulation of osteoblastic and osteoclastic activity by TWSG1 and MGP.
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    Will Topical Application of Alendronate Disodium to Dental Roots Reduce Osteoclast Adherence and Resorption in Vitro?
    (2016-08) Finley, Rhett
    Introduction: The purposes of this study were: 1. To demonstrate the presence of fully differentiated, multinucleated human osteoclasts on bovine and human dentin and cementum in vitro. 2. To comparatively quantify osteoclast presence and distribution on tooth sections and adjacent lab wells following differentiation. 3. To comparatively quantify the amount of resorption that occurs when human root sections are subjected to a bisphosphonate antiresorptive treatment in comparison to no treatment. Materials and Methods: 30 sections of human tooth root and 48 sections of bovine tooth root were created using an IsoMet saw. The tooth sections from both groups were then divided into 2 equal groups of 15 and 24 sections respectively. One group received no treatment and the second group was soaked in 2mM Alendronate Disodium prior to transfer to culture. All sections were then plated in sterile culture wells with osteoclast precursor cells derived from whole human blood. The sections remained in culture until completion of osteoclastic differentiation, which took approximately 24 days. The supernatant was recovered from each well and used in an ELISA to determine the CTX-1 breakdown products from each section. The sections were fixed in paraformaldehyde and TRAP stained for visualization of osteoclasts. TRAP+ cell counts were performed on tooth sections and residual treatment wells. Student t-tests were used to determine differences between groups Results: Osteoclast attachment was observed on all human and bovine samples with the exception of one bacterially contaminated sample. The no treatment group had significantly more TRAP+ cells than the ALN group on both the human and bovine tooth sections. The no treatment group also had significantly more cells per residual well than the ALN group. No evidence of osteoclastic resorption could be demonstrated with the ELISA. Conclusions: Within the limitations of this study, it can be concluded that: 1. Fully differentiated, multinucleated osteoclasts can be successfully isolated from whole human blood and seeded onto human tooth sections in vitro. 2. Topical application of ALN disodium to extracted and sectioned tooth surfaces suppresses osteoclastic proliferation and differentiation in vitro. 3. Bovine teeth and human teeth allow for similar human osteoclast attachment and distribution in an in vitro model. 4. Mononuclear osteoclasts do not exhibit appreciable resorptive potential on sectioned surfaces in vitro.