論文の公開元へSTAT3 induces hypoxic preconditioning against oxidative stress in neural stem cells
概要
Introduction
Transplantation of neural stem cells (NSCs) which can differentiate into neurons, astrocytes and oligodendrocytes has been proposed as a promising therapeutic strategy in stroke. However, a hostile microenvironment reduced remarkably the number of surviving transplanted cells. One of the approaches to improve the ability of NSCs to survive in the harsh environment and enhance the therapeutic efficacy is hypoxic preconditioning (HP). HP activates endogenous defense mechanisms that show marked protective effects against multiple insults. Clarification of the molecular mechanisms of HP in NSCs would bring new insights for the improvement of NSCs transplantation therapy; however, the mechanisms have not been completely elucidated. Signal transducer and activator of transcription 3 (STAT3) is a transcription factor activated by various stimulations, which phosphorylate STAT3 on tyrosine residue (Tyr 705) resulting in the induction of various gene expression such as vascular endothelial growth factor (VEGF) and cyclin D1. Previous studies that show the pivotal roles of STAT3 in several models of preconditioning suggest that STAT3 would also play an important role in HP of NSCs; however, it has not been investigated so far. The purpose of present study is to investigate the effect of HP on NSCs against oxidative stress, and to test the hypothesis that STAT3 and its related signals have pivotal roles in HP of NSCs.
Materials and Methods
NSCs were isolated from the subventricular zones of postnatal day1 mice (C57BL/6). The cells were collected and suspended in the medium and grown as neurospheres or adherent monolayers. Cell viability was assessed with a cell proliferation reagent using a water-soluble tetrazolium (WST-1) assay kit. To observe the differentiation of NSCs, differentiated cells were stained with the neuronal marker mitogen-activated protein 2 (MAP2), the astrocytic marker glial fibrillary acidic protein (GFAP), the oligodendrocytic marker neuron-glial antigen 2 (NG2), the immature neural stem cell’s marker nestin.
For the induction of HP, NSCs were incubated under hypoxic conditions (5% O2) for 24 hours. As lethal oxidative stress, cultured NSCs were exposed to H2O2 for 12 hours.
To investigate whether hypoxic conditions enhance cell proliferation, the viability was assessed 0, 24 and 48 hours after HP. After 12 hours oxidative stress, cell injury and viability were investigated by terminal deoxynucleotidyl transferase-mediated uridine 5’-triphosphate-biotin nick end labeling (TUNEL) and WST-1 assays. The phospho-STAT3 (pSTAT3) expression in NSCs after HP was investigated by western blotting. The STAT3 inhibition experiment was performed in order to confirm reduction in the protective effect of NSCs by HP. For the detection of VEGF as a paracrine factor, the concentration of VEGF in the culture media was measured by ELISA. The expression changes of STAT3 and its related signals (JAK2 and cyclin D1) in NSCs after HP with/without inhibition of STAT3 were analyzed using western blotting.
Results
H2O2 significantly increased NSC injury, indicated by TUNEL staining. Conversely, WST-1 assay revealed that cell viability of NSCs was significantly reduced by exposure to H2O2 (n=6, p<0.05 or 0.01). HP significantly increased cell viability of NSCs in WST-1 assay (n=5, p<0.05 or 0.01). TUNEL assay revealed that HP decreased cell death 12 hours after H2O2 exposure (n=5, p<0.05 or 0.01). Further, WST-1 assay showed that HP significantly protected the decrease of cell viability due to H2O2 exposure (n=5, p<0.05 or 0.01). Western blot analysis revealed that the expression of pSTAT3 (Tyr705) was increased 0 to 48 hours after HP (n=5, p<0.05 or 0.01). STAT3 inhibitor peptide reversed protective effects of HP on NSC injury due to lethal oxidative stress. The expression levels of JAK2 and pJAK2 were significantly elevated 24 hours after HP, and STAT3 inhibitor further increased the total expression level of JAK2 (n=5, p<0.05). Cyclin D1 expression was increased after HP, which was significantly inhibited by the STAT3 inhibitor (n=5, p<0.05 or 0.01). Moreover, HP increased VEGF concentration in the culture media investigated by ELISA, and the STAT3 inhibitor suppressed this increase (n=5, p<0.05). Neither HP nor STAT3 inhibitor peptide did not affect the differentiation of NSCs.
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