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Browsing by Subject "Sirtuin"
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Item The Mechanotransduction of Hydrostatic Pressure by Mesenchymal Stem Cells(2018-12) Hosseini, Seyedeh Ghazaleh; Wagner, Diane R.; Na, Sungsoo; Ji, JulieMesenchymal stem cells (MSCs) are responsive to mechanical stimuli that play an essential role in directing their differentiation to the chondrogenic lineage. A better understanding of the mechanisms that allow MSCs to respond to mechanical stimuli is important to improving cartilage tissue engineering and regenerative medicine. Hydrostatic pressure (HP) in particular is known to be a primary mechanical force in joints. However, little is known about the underlying mechanisms that facilitate HP mechanotransduction. Understanding the signaling pathways in MSCs in transducing HP to a beneficial biologic response and their interrelationship were the focus of this thesis. Studies used porcine marrow-derived MSCs seeded in agarose gel. Calcium ion Ca++ signaling, focal adhesion kinase (FAK) involvement, and sirtuin1 activity were investigated in conjunction with HP application. Intracellular Ca++ concentration was previously shown to be changed with HP application. In our study a bioreactor was used to apply a single application of HP to the MSC-seeded gel structures and observe Ca++ signaling via live imaging of a fluorescent calcium indicator in cells. However, no fluctuations in Ca++ concentrations were observed with 10 minutes loading of HP. Additionally a problem with the biore actor design was discovered. First the gel was floating around in the bioreactor even without loading. After stabilizing the gel and stopping it from floating, there were still about 16 µm of movement and deformation in the system. The movement and deformation was analyzed for the gel structure and different parts of the bioreactor. Furthermore, we investigated the role of FAK in early and late chondrogenesis and also its involvement in HP mechanotransduction. A FAK inhibitor was used on MSCs from day 1 to 21 and showed a dose-dependent suppression of chondrogenesis. However, when low doses of FAK inhibitor added to the MSC culture from day 21 to 42, chondrogenesis was not inhibited. With 4 hour cyclic HP, FAK phosphorylation increased. The beneficial effect of HP was suppressed with overnight addition of the FAK inhibitor to MSC medium, suggesting FAK involvement in HP mechanotransd ucation by MSCs. Moreover, sirtuin1 participation in MSC chondrogenesis and mechanotransduc tion was also explored. The results indicated that overnight sirtuin1 inhibition in creased chondrogenic gene expression (Agc, Col2, and Sox9) in MSCs. Additionally, the activity of sirtuin1 was decreased with both 4 hour cyclic hydrostatic pressure and inhibitor application. These two together demonstrated that sirtuin1 inhibition enhances chondrogenesis. In this research we have investigated the role of Ca++ signaling, FAK involvement, and sirtuin1 activity in the mechanotransduction of HP in MSCs. These understand ings about the mechanisms regulating the chondrogenesis with respect to HP could have important implications for cartilage tissue engineering and regenerative studies.Item SIRT6 controls hepatic lipogenesis by suppressing LXR, ChREBP, and SREBP1(Elsevier, 2021) Zhu, Chaoyu; Huang, Menghao; Kim, Hyeong-Geug; Chowdhury, Kushan; Gao, Jing; Liu, Sheng; Wan, Jun; Wei, Li; Dong, X. Charlie; Biochemistry and Molecular Biology, School of MedicineFatty liver disease is the most prevalent chronic liver disorder, which is manifested by hepatic triglyceride elevation, inflammation, and fibrosis. Sirtuin 6 (Sirt6), an NAD+-dependent deacetylase, has been implicated in hepatic glucose and lipid metabolism; however, the underlying mechanisms are incompletely understood. The aim of this study was to identify and characterize novel players and mechanisms that are responsible for the Sirt6-mediated metabolic regulation in the liver. We generated and characterized Sirt6 liver-specific knockout mice regarding its role in the development of fatty liver disease. We used cell models to validate the molecular alterations observed in the animal models. Biochemical and molecular biological approaches were used to illustrate protein-protein interactions and gene regulation. Our data show that Sirt6 liver-specific knockout mice develop more severe fatty liver disease than wild-type mice do on a Western diet. Hepatic Sirt6 deficiency leads to elevated levels and transcriptional activities of carbohydrate response element binding protein (ChREBP) and sterol regulatory element binding protein 1 (SREBP1). Mechanistically, our data reveal protein-protein interactions between Sirt6 and liver X receptor α (LXRα), ChREBP, or SREBP1c in hepatocytes. Moreover, Sirt6 suppresses transcriptional activities of LXRα, ChREBP, and SREBP1c through direct deacetylation. In conclusion, this work has identified a key mechanism that is responsible for the salutary function of Sirt6 in the inhibition of hepatic lipogenesis by suppressing LXR, ChREBP, and SREBP1.