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TNFR2 Stimulation Promotes Mitochondrial Fusion via Stat3- and NF-kB-Dependent Activation of OPA1 Expression.

ABSTRACT: Mitochondria are important cellular organelles and play essential roles in maintaining cell structure and function. Emerging evidence indicates that in addition to having proinflammatory and proapoptotic effects, TNFα (tumor necrosis factor α) can, under certain circumstances, promote improvements in mitochondrial integrity and function, phenomena that can be ascribed to the existence of TNFR2 (TNFα receptor 2).The present study aimed to investigate whether and how TNFR2 activation mediates the effects of TNFα on mitochondria.Freshly isolated neonatal mouse cardiac myocytes treated with shRNA targeting TNFR1 were used to study the effects of TNFR2 activation on mitochondrial function. Neonatal mouse cardiac myocytes exhibited increases in mitochondrial fusion, a change that was associated with increases in mitochondrial membrane potential, intracellular ATP levels, and oxygen consumption capacity. Importantly, TNFR2 activation-induced increases in OPA1 (optic atrophy 1) protein expression were responsible for the above enhancements, and these changes could be attenuated using siRNA targeting OPA1. Moreover, both Stat3 and RelA bound to the promoter region of OPA1 and their interactions synergistically upregulated OPA1 expression at the transcriptional level. Stat3 acetylation at lysine 370 or lysine 383 played a key role in the ability of Stat3 to form a supercomplex with RelA. Meanwhile, p300 modulated Stat3 acetylation in HEK293T (human embryonic kidney 293T) cells, and p300-mediated Stat3/RelA interactions played an indispensable role in OPA1 upregulation. Finally, TNFR2 activation exerted beneficial effects on OPA1 expression in an in vivo transverse aortic constriction model, whereby TNFR1-knockout mice exhibited better outcomes than in mice with both TNFR1 and TNFR2 knocked out.TNFR2 activation protects cardiac myocytes against stress by upregulating OPA1 expression. This process was facilitated by p300-mediated Stat3 acetylation and Stat3/RelA interactions, leading to improvements in mitochondrial morphology and function.

SUBMITTER: Nan J

PROVIDER: S-EPMC5542782 | biostudies-other | 2017 Aug

REPOSITORIES: biostudies-other

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Project description:Brain inflammation, a common feature in neurodegenerative diseases, is a complex series of events, which can be detrimental and even lead to neuronal death. Nonetheless, several studies suggest that inflammatory signals are also positively influencing neural cell proliferation, survival, migration and differentiation. Recently, correlative studies suggested that astrocytes are able to dedifferentiate upon injury, and may thereby re-acquire neural stem cells (NSC) potential. However, the mechanism underlying this dedifferentiation process upon injury remains unclear. In this study, we find that during the early response of reactive gliosis, inflammation induces a conversion of mature astrocytes into neural progenitors. A TNF treatment induces the decrease of specific astrocyte markers, such as GFAP or genes related to glycogen metabolism, while a subset of these cells re-express immaturity markers, such as CD44, Musashi-1 and Oct4. Thus, TNF treatment results in the appearance of cells that exhibit a neural progenitor phenotype and are able to proliferate and differentiate into neurons and/or astrocytes. This dedifferentiation process is maintained as long as TNF is present in the culture medium. In addition, we identify a role for Oct4 in this process, since the TNF-induced dedifferentiation can be prevented by inhibiting Oct4 expression. Our results show that activation of the NF-kB pathway through TNF plays an important role in the dedifferentiation of astrocytes via the re-expression of Oct4. These findings indicate that the first step of reactive gliosis is in fact a dedifferentiation process of resident astrocytes mediated by the NF-kB pathway. This dedifferentiation process is maintained as long as TNF is present in the culture medium. In addition, we identify a role for Oct4 in this process, since the TNF-induced dedifferentiation can be prevented by inhibiting Oct4 expression. Our results show that activation of the NF-kB pathway through TNF plays an important role in the dedifferentiation of astrocytes via the re-expression of Oct4. These findings indicate that the first step of reactive gliosis is in fact a dedifferentiation process of resident astrocytes mediated by the NF-kB pathway. Cultures of primary mouse astrocytes were treated with TNF (50 ng/mL) for 24 hours. Cells were collected and immediately homogenized in cooled down RNA NOW reagent (OZYME). Total RNA was extracted according to RNA NOW manufacturer's recommendations with -20°C overnight incubation for small RNA precipitation. Total RNA integrity and purity were assessed using the Agilent 2100 Bioanalyzer and RNA 6000 Nano LabChip kits (Agilent Technologies). Only good-quality RNA (no contamination or degradation, RIN > 9) was used and further processed. Total RNA samples were reverse-transcribed to double-stranded cDNA using specific primers, which reduce the priming of rRNA. cRNA was generated by in vitro transcription and reverse transcribed into a sense single-stranded cDNA. The cDNA was fragmented, labeled, and hybridized onto Affymetrix GeneChip Mouse Gene 1.0 ST Arrays according to the Ambion Whole Transcript Expression kit for Affymetrix GeneChip Whole Transcript Expression Array Protocol (P/N 4425209 Rev.B 05/2009) and GeneChip WT Terminal Labeling and Hybridization User Manual for use with the Ambion Whole Transcript Expression kit (P/N 702808 Rev.6). Microarrays were then washed, stained, and scanned according to the manufacturer's instructions. The samples cover combinations of 7 time points (0H, 6H, 24H, 48H, 72H, 1W, 2W) and two conditions (TNF and control), with multiple replicates per condition and time point.

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TNFR2 Stimulation Promotes Mitochondrial Fusion via Stat3- and NF-kB-Dependent Activation of OPA1 Expression.

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