― 264 ―The sulfur oxidation pathway is required for persulfide-mediated mitochondrial activation.To clarify the significance of the sulfur oxidation pathway in mitochondrial function, we inhibited the sulfur oxidation pathway by knocking down genes encoding SQOR, ETHE1 and SUOX and examined their effects on MMP in Hepa1c1c7. CARS2 knockdown was used as a positive control because CARS2 is one of the major persulfide synthesizing enzymes. The MMP was decreased by the reduction of these enzymes, except for SUOX (Fig. 3A). These results suggested that CARS2-mediated persulfide production and SQOR- and ETHE1-mediated sulfur oxidation are required for mitochondrial activity, which is consistent with previous reports3,6). Importantly, GSSSG-induced mitochondrial activation, i.e., elevation of MMP and relative ATP amount vs. ADP, which was not observed in Sqor-knockdown Hepa1c1c7 cells (Fig. 3B and 3C), indicating that the sulfur oxidation pathway, especially SQOR, is required for GSSSG-induced mitochondrial activation.NRF2 activation increases intracellular persulfides and promotes mitochondrial activity.To investigate the impacts of NRF2 activity on mitochondrial function in this experimental setting, MMP was examined in Hepa1c1c7 with different NRF2 activities. Hepa1c1c7 cells were treated with siRNAs against Nrf2 and Keap1 and used for MMP analyses. NRF2 inhibition decreased MMP, whereas NRF2 activation by KEAP1 inhibition increased MMP (Fig. 4A). Consistent with a positive correlation between persulfide levels and mitochondrial activities, intracellular persulfides were decreased and increased in Nrf2-knockdown and Keap1-knockdown Hepa1c1c7 cells, respectively (Fig. 4B). These results suggested that NRF2 activation promotes mitochondrial function by increasing intracellular persulfides.NRF2-mediated activation of mitochondrial function requires cystine uptake via xCT.We wanted to know how NRF2 increases intracellular persulfides. Because Slc7a11, which encodes a cystine transporter xCT, is a well-established NRF2 target gene4) and because persulfide-synthesizing enzymes, including CARS2, did not seem to be directly regulated by NRF2, we hypothesized that NRF2 increases persulfides by promoting cystine uptake through xCT upregulation. Sulfasalazine (SASP), an inhibitor of xCT, was used. Increased persulfides and elevated MMP by Keap1 knockdown were reversed by SASP treatment in Hepa1c1c7 cells (Fig. 5A and 5B), suggesting that the NRF2-mediated persulfide increase and mitochondrial activation depend on increased cystine uptake.NRF2-mediated activation of mitochondrial function requires the mitochondrial sulfur oxidation pathway.To further examine whether NRF2-mediated mitochondrial activation requires persulfide production and sulfur oxidation pathway activity, we conducted simultaneous inhibition of KEAP1 and mitochondrial sulfur-metabolizing enzymes. The simultaneous knockdown of Keap1 and mitochondrial sulfur metbolzing enzymes, including CARS2, SQOR, and ETHE1, reversed the MMP elevation induced by Keap1 knockdown in Hepa1c1c7 cells (Fig. 6A-6C), suggesting that NRF2-mediated mitochondrial activation depends on mitochondrial sulfur metabolism, persulfide production and oxidation.
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