SummaryOxidative stress influences stem cell behavior by promoting the differentiation, proliferation, or apoptosis of stem cells. Thus, characterizing the effects of reactive oxygen species (ROS) on stem cell behavior provides insights into the significance of redox homeostasis in stem cell-associated diseases and efficient stem cell expansion for cellular therapies. We utilized the Drosophila testis as an in vivo model to examine the effects of ROS on germline stem cell (GSC) maintenance. High levels of ROS induced by alteration in Keap1/Nrf2 activity decreased GSC number by promoting precocious GSC differentiation. Notably, high ROS enhanced the transcription of the EGFR ligand spitz and the expression of phospho-Erk1/2, suggesting that high ROS-mediated GSC differentiation is through EGFR signaling. By contrast, testes with low ROS caused by Keap1 inhibition or antioxidant treatment showed an overgrowth of GSC-like cells. These findings suggest that redox homeostasis regulated by Keap1/Nrf2 signaling plays important roles in GSC maintenance.

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.

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Dysregulated JAK/STAT signaling has been implicated in the molecular pathogenesis of gastric cancer. However, downstream effectors of STAT signaling that facilitate gastric carcinogenesis remain to be explored. We previously identified the Drosophila ortholog of human GRAMD1B in our genome-wide RNAi screen to identify novel components of the JAK/STAT signaling pathway in Drosophila. Here, we examined the involvement of GRAMD1B in JAK/STAT-associated gastric carcinogenesis. We found that GRAMD1B expression is positively regulated by JAK/STAT signaling and GRAMD1B inhibition decreases STAT3 levels, suggesting the existence of a positive feedback loop. Consistently, GRAMD1B and JAK/STAT signaling acted synergistically to promote gastric cancer cell survival by upregulating the expression of the anti-apoptotic molecule Bcl-xL. Interestingly, our immunohistochemical analysis for GRAMD1B revealed a gradual loss of cytoplasmic staining but an increase in the nuclear accumulation of GRAMD1B, as gastric tissue becomes malignant. GRAMD1B expression levels were also found to be significantly associated with clinicopathological features of the gastric cancer patients, particularly the tumor grades and lymph node status. Moreover, GRAMD1B and pSTAT3 (Tyr705) showed a positive correlation in gastric tissues, thereby confirming the existence of a close link between these two signaling molecules in vivo. This new knowledge about JAK/STAT-GRAMD1B regulation deepens our understanding of JAK/STAT signaling in gastric carcinogenesis and provides a foundation for the development of novel biomarkers in gastric cancer.

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