Identification of SIRT1 as a novel regulator of NFAT5 dependent aldose reductase expression under osmatic stress
Timuçin, Ahmet Can (2015) Identification of SIRT1 as a novel regulator of NFAT5 dependent aldose reductase expression under osmatic stress. [Thesis]
Until now, numerous diverse molecules modulating NFAT5 and its targets have been characterized. Among these widespread NFAT5 modifiers, SIRT1 has been proposed to be a promising candidate to play a role in NFAT5 dependent events, yet the exact machinery still remains inconclusive. Hence, in this thesis, we aimed to delineate the link between SIRT1 and NFAT5-Aldose Reductase (AR) axis under osmotic stress. A unique osmotic stress model was generated and its mechanistic components were deciphered in U937 monocytes. By utilization of pharmacological modulators in this model, we showed that AR expression and stabilization of nuclear NFAT5, were revealed to be positively regulated by SIRT1. Overexpression and co-transfection studies of NFAT5 and SIRT1, further validated contribution of SIRT1 on NFAT5 dependent AR expression. Involvement of SIRT1 activity in these events was mediated via modification of DNA binding of NFAT5 to AR ORE region. Besides, NFAT5 and SIRT1 were also shown to co-immunoprecipitate under isosmotic conditions and this interaction was disrupted by osmotic stress. Subsequently, in silico experiments were conducted for investigating if SIRT1 directly targets NFAT5. In this regard, certain lysine residues of NFAT5, when kept deacetylated, were found to contribute to its DNA binding and SIRT1 was shown to target acetylated lysine 282 of NFAT5. Based on in vitro and in silico findings, we identified SIRT1, for the first time, as a novel contributor to NFAT5 dependent AR expression under osmotic stress.
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