Analysis of TR4 nuclear receptor knockout mouse: Roles of TR4 in mitochondrial function and cellular stress defenses

Testicular nuclear receptor 4 (TR4) is a member of the nuclear receptor superfamily. Via knockout strategy, its physiological roles have been recently reported to be involved in a broad range of biological activities including neural development, fertility, metabolism, and more to be explored. This thesis is focused on TR4 roles in mitochondria function and the cellular defense system.;Mitochondria, the powerhouses of cells, generate most of the ATP for cell needs through oxidative phosphorylation (OXPHOS). Defects in the OXPHOS system result in devastating diseases with diverse clinical presentations in human. We found that TR4 knockout mice (TR4-/-) suffered mitochondrial myopathy. In addition, increased serum lactate level, decreased mitochondrial ATP production, and decreased electron transport chain complex I (NADH:ubquinone oxidoreductase; EC 1.6.5.3) activity were found in TR4 -/- skeletal muscle tissues. Restoration of TR4 into TR4-/- cells could partially rescue mitochondrial ATP generation capacity and complex I enzyme activity. Further mechanistic dissection found TR4 could modulate complex I activity via transcriptionally regulating NDUFAF1 and addition of NDUFAF1 into TR4-/- cells increased mitochondrial ATP generation capacity and complex I activity.;TR4 has also been implicated in mediating cellular responses against oxidative stress, IR irradiation, and UV irradiation, although the detailed mechanisms remain elusive. To investigate the molecular mechanism through which TR4 regulates cellular response to UV, we performed DNA repair assays aiming to specifically assess the DNA repair pathways in TR4-proficient and TR4-deficient cells and found TR4 was ix involved in transcription-coupled nucleotide excision repair (TC-NER) but not global genomic nucleotide excision repair (GG-NER). Further gene screening showed the expression level of cockayne syndrome B protein (CSB), a member of the TC-NER pathway, was reduced in TR4 deficient tissues and cells. CSB 5' promoter studies and ChIP assays confirmed CSB is a TR4 target gene. Remarkably, when CSB was restored in TR4 deficient cells, UV hypersensitivity was attenuated, indicating TR4 regulates cellular UV defense through CSB.;TR4 roles in defending oxidative stress were examined. We found elevated reactive oxygen species (ROS) and increasing genome instability in TR4 -/- mice. At the cellular level, we observed an early onset of cellular senescence in TR4-/- mouse embryonic fibroblasts (MEFs), accompanied by increased DNA damage. TR4-/- MEFs restored with TR4 or treated by an anti-oxidant NAC showed less DNA damage and a slower cellular decay, suggesting TR4 is involved in maintaining cellular redox homeostasis and oxidative stress defense.;In summary, we concluded that TR4 controls mitochondria function via modulating complex I assembly factor NDUFAF1 expression and participates in cellular stress defenses against oxidative stress and UV irradiation.