Abstract
NRF2 (nuclear factor erythroid-derived 2-like 2) plays an important role in defense against oxidative stress at the cellular level. Recently, the roles of NRF2 in embryonic and adult stem cells have been reported, but its role in maintaining self-renewal and differentiation potential remains unknown. We studied the mechanisms of NRF2 action in mesenchymal stem cells (MSCs) derived from human bone marrow. We found that the cellular localization of NRF2 changed during prolonged cell passage and osteogenic differentiation. Blocking the nuclear import of NRF2 using ochratoxin A (OTA) induced the loss of the self-renewal and osteogenic potential of early-passage (EP) MSCs. Conversely, reinforcing the nuclear import of NRF2 using tert-butylhydroquinone (t-BHQ) improved the self-renewal capacity and maintained the differentiation potential in the osteogenic lineage of EP MSCs. Real-time quantitative PCR and western blot analysis showed that NRF2 positively regulates sirtuin 1 (SIRT1) at the mRNA and protein levels via the negative regulation of p53. The self-renewal and osteogenic potential suppressed in OTA-treated or NRF2-targeting small hairpin RNA (shRNA)-infected EP MSCs were rescued by introducing small interfering RNA (siRNA) targeting p53. t-BHQ treatment in late-passage (LP) MSCs, which lost their self-renewal and osteogenic potential, reversed these effects. In LP MSCs treated with t-BHQ for ∼7 days, the phosphorylation and nuclear localization of NRF2 improved and SIRT1 protein level increased, whereas p53 protein levels decreased. Therefore, our results suggest that NRF2 plays an important role in regulating p53 and SIRT1 to maintain MSC stemness. This study is the first to establish a functional link between NRF2 and SIRT1 expression in the maintenance of MSC self-renewal and differentiation potential.
Highlights
These results indicate that Nuclear factor erythroid-derived 2-like 2 (NRF2) phosphorylation and activity may be important factors in the maintenance of the early characteristics of Mesenchymal stem cells (MSCs), whereas the mRNA and protein levels did not change during prolonged cell passages and the osteogenic differentiation process
Our study showed that the inhibition or induction of NRF2 nuclear localization affected cell proliferation and the colonyforming ability of EP MSCs
These results indicate that NRF2 activity is essential for maintaining the self-renewal and osteogenic potentials of MSCs during ex vivo cultivation
Summary
We identify NRF2 as a regulator of sirtuin 1 (SIRT1). NRF2 phosphorylation was decreased and NRF2 was exported from the nucleus in late-passage (LP) and differentiated MSCs. The inhibition of NRF2 activity in early-passage (EP) MSCs suppressed the self-renewal and differentiation capacities. NRF2 activation in EP or LP MSCs enhanced the selfrenewal capacity, but suppressed the differentiation potential. NRF2 increased SIRT1 protein expression by decreasing p53 protein levels. RNA interference targeting p53 in NRF2-knockdown MSCs rescued SIRT1 protein levels as well as the self-renewal and differentiation potentials. Our data indicate that NRF2 plays an important role in the maintenance of MSC stemness via p53–SIRT1 regulation
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