Abstract
Reactive oxygen species (ROS) play important roles in fundamental cellular processes such as proliferation and survival. Here we investigated the effect of oxidative stress on stem cell maintenance and neuronal differentiation in a human embryonic stem cell (hESC) model, Ntera2 (NT2). CM-H2DCFDA and DHE assays confirmed that the oxidizing agent paraquat could induce a high level of ROS in NT2 cells. Quantitative PCR, Western blotting and immunocytochemistry showed that paraquat-induced oxidative stress suppressed the expression of stemness markers, including NANOG, OCT4 and TDGF1, whereas it enhanced the spontaneous expression of neuronal differentiation markers such as PAX6, NEUROD1, HOXA1, NCAM, GFRA1 and TUJ1. The treated cells even exhibited a strikingly different morphology from control cells, extending out long neurite-like processes. The neurogenic effect of ROS on stem cell behaviour was confirmed by the observations that the expression of neuronal markers in the paraquat-treated cells was suppressed by an antioxidant while further enhanced by knocking down Nrf2, a key transcription factor associated with antioxidant signaling. Lastly, paraquat dose-dependently activated the neurogenic MAPK-ERK1/2, which can be reversed by the MEK1/2 inhibitor SL327. Our study suggests that excessive intracellular ROS can trigger the exit from stem cell state and promote the neuronal differentiation of hESCs, and that MAPK-ERK1/2 signaling may play a proactive role in the ROS-induced neuronal differentiation of hESCs.
Highlights
Reactive oxygen species (ROS), including superoxide (O-), hydroxyl free radicals (HO) and hydrogen peroxide (H2O2), are constantly generated as the result of normal cellular metabolism
Quantitative PCR, Western blotting and immunocytochemistry showed that paraquat-induced oxidative stress suppressed the expression of stemness markers, including NANOG, OCT4 and TDGF1, whereas it enhanced the spontaneous expression of neuronal differentiation markers such as PAX6, NEUROD1, HOXA1, NCAM, GFRA1 and TUJ1
Our study suggests that excessive intracellular ROS can trigger the exit from stem cell state and promote the neuronal differentiation of human embryonic stem cell (hESC), and that mitogen-activated protein kinase (MAPK)-ERK1/2 signaling may play a proactive role in the ROS-induced neuronal differentiation of hESCs
Summary
Reactive oxygen species (ROS), including superoxide (O-), hydroxyl free radicals (HO) and hydrogen peroxide (H2O2), are constantly generated as the result of normal cellular metabolism. Nrf is a transcription factor that controls the expression of a large pool of antioxidant and detoxifying genes in response to oxidative stress [8] It is negatively regulated by its physical association with the repressor Keap in cytoplasm [9]. Nrf engages in the transcriptional www.impactjournals.com/oncotarget regulation of many antioxidant and detoxification genes, which are involved in the cellular response to oxidative and electrophilic stress, via antioxidant response element (ARE) [8, 10] The antioxidant enzymes, such as superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and peroxiredoxin (Prx), participate in various enzymatic reactions to convert O- to H2O and dioxide (O2) [11,12,13]
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