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Optineurin Regulates Nrf2-mediated Antioxidant Response in Bone

Year: 2024
Type: dissertation
Abstract: Age-dependent tissue degeneration leads to many diseases that threatens the lives of elderly people. Recently, Optineurin (OPTN), a multi-functional protein previously associated with numerous neurodegenerative diseases, was genetically implicated in Paget’s disease of bone (PDB), a degenerative bone disease initiated by hyperactivation of osteoclasts. It has been well-documented that excessive reactive oxygen species (ROS), if not counterbalanced by erythroid-derived factor 2-related factor 2 (NRF2)-induced antioxidant, leads to increased osteoclasts in bone and various tissue degenerations. In this dissertation, I test the hypothesis that compromised Nrf2-induced antioxidant response leads to age-dependent bone loss in OPTN deficiency mice. In Chapter 1, an introduction to my thesis is given. In Chapter 2, effects of numerous NRF2 activators on osteoclastogenesis are presented. In Chapter 3, how OPTN regulates NRF2-induced antioxidant response in osteoclasts in-vitro is discussed. Finally in Chapter 4, how OPTN affects NRF2-induced antioxidant response in bone in-vivo is discussed.In Chapter 2 “CDDO-Me, Sulforaphane and tBHQ attenuate the RANKL-induced osteoclast differentiation via activating the NRF2-mediated antioxidant response”, I investigated the effects of three NRF2 inducers on osteoclastogenesis, including Bardoxolone methyl (CDDO-Me), Sulforaphane (SFN), and tert-butylhydroquinone (tBHQ). By treating RAW cells with three compounds, I found that NRF2 was activated and its downstream antioxidant genes were upregulated, and the RANKL-induced intracellular ROS production and osteoclastogenesis were impaired. Taken together, these results suggest that NRF2-mediated antioxidant response regulates RANKL-induced osteoclastogenesis through modulating intracellular ROS, which is the basis of my hypothesis.In Chapter 3 “Deficiency of optineurin enhances osteoclast differentiation by attenuating NRF2-mediated antioxidant response”, I primary cultured osteoclasts from either wildtype mice or OPTN global knockout mice whose cells are genetically lacking OPTN. I first compared the two types of cells in osteoclastogenic activity and intracellular ROS level using TRAP staining and various fluorescence-based assays. Further, I compared the two types of cells regarding the expressions of NRF2-antioxidants using qPCR and Western Blotting. Finally, I revealed the OPTN-NRF2 binding by co-IP and confocal fluorescence microscopy. Collectively, the study tests my hypothesis in-vitro by showing a potential regulatory mechanism through which OPTN binds with NRF2 to mediate antioxidant response during osteoclastogenesis.In Chapter 4 “Optineurin regulates NRF2-mediated antioxidant response in a mouse model for Paget’s disease of bone”, I utilized a variety of flow-cytometry and confocal fluorescence imaging-based techniques to uncover how ROS and NRF2-induced antioxidant response changed over time to maintain the redox homeostasis of bone, and how deletion of OPTN breaks the balance in aged bones. In this study, I found age related increase in OPTN and NRF2 in-vivo. At the molecular level, OPTN could directly interact with both NRF2 and its negative regulator Kelch-like ECH-associated protein 1 (KEAP1) for upregulating antioxidant response. Furthermore, young (2-month-old) and old (24-month-old) global OPTN knockout mice were compared to wildtype mice. At the cellular level, deletion of OPTN resulted in increased intracellular ROS and increased osteoclastogenic potential. At the tissue level, deletion of OPTN resulted in significantly increased oxidative stress derived from leukocytes that further stimulate osteoclastogenesis. These differences were only pronounced in aged mice. Collectively, this study tests my hypothesis in-vivo by revealing a novel OPTN-NRF2 axis in maintaining ROS and bone homeostasis. Beyond pathogenesis, my work also suggests antioxidants have therapeutic potential for oxidative stress associated with PDB.
DOI: 10.17615/cacc-0246
Author: Hu, Xiangxiang
Language: English
Open Access status: green
OpenAlex ID: W6962731705
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