Abstract
Insulin-like growth factor 1 (IGF-1) not only regulates neuronal function and development but also is neuroprotective in the setting of acute ischemic stroke. G-protein-coupled receptor 17 (GPR17) expression in brain tissue serves as an indicator of brain damage. As whether IGF-1 regulates GPR17 expression remains unknown, the aim of this study is to investigate how IGF-1 regulates GPR17 expression in vitro. Human neuroblastoma SK-N-SH cells were used. Lentivirus-mediated short hairpin RNA (shRNA) was constructed to mediate the silencing of FoxO1, while adenoviral vectors were used for its overexpression. Verification of the relevant signaling cascade was performed using a FoxO1 inhibitor (AS1842856), a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002), and a GPR17 antagonist (cangrelor). Cell proliferation was analyzed using EdU staining; immunofluorescence staining was used to detect the expression and subcellular localization of FoxO1. Chromatin immunoprecipitation was used to analyze the binding of FoxO1 to the GPR17 promoter in SK-N-SH cells. The expression of FoxO1, GPR17, and protein kinase B (also known as Akt) mRNA and protein as well as the levels of FoxO1 and Akt phosphorylation were investigated in this study. IGF-1 was found to downregulate FoxO1 and GPR17 expression in SK-N-SH cells while promoting cell viability and proliferation. Inhibition of FoxO1 and antagonism of GPR17 were found to play a role similar to that of IGF-1. Silencing of FoxO1 by lentivirus-mediated shRNA resulted in the downregulation of FoxO1 and GPR17 expression. The overexpression of FoxO1 via adenoviral vectors resulted in the upregulation of FoxO1 and GPR17 expression. Blocking of PI3K signaling by LY294002 inhibited the effect of IGF-1 on GPR17 suppression. Results from chromatin immunoprecipitation revealed that IGF-1 promotes FoxO1 nuclear export and reduces FoxO1 binding to the GPR17 promoter in SK-N-SH cells. Here, we conclude that IGF-1 enhances cell viability and proliferation in SK-N-SH cells via the promotion of FoxO1 nuclear export and reduction of FoxO1 binding to the GPR17 promoter via PI3K/Akt signaling. Our findings suggest that the enhancement of IGF-1 signaling to antagonize GPR17 serves as a potential therapeutic strategy in the management of acute ischemic stroke.
Highlights
Acute ischemic stroke (AIS) is defined as sudden focal ischemia of brain tissue lasting for over 24 h and resulting in neurologic dysfunction [1]
Results from Western blot (WB) analysis indicated that blocking the phosphatidylinositol 3-kinase (PI3K)/Akt/forkhead box protein O1 (FoxO1) signaling pathway results in the effects of Insulin-like growth factor 1 (IGF-1) on PI3K/Akt/FoxO1 signaling to be muted (Figure 4e,f,c)
LY294002, and no significant difference was found (Supplementary Figure S5c,d). These results indicate that IGF-1 governs G-protein-coupled receptor 17 (GPR17) expression via PI3K/Akt/FoxO1 signaling in SK-N-SH cells
Summary
Acute ischemic stroke (AIS) is defined as sudden focal ischemia of brain tissue lasting for over 24 h and resulting in neurologic dysfunction [1]. Numerous conditions, such as embolism, atherosclerotic disease, vessel dissection, and vasculitis, lead to focal cerebral hypoperfusion, causing AIS [1]. IGF-1 can be produced by all neuronal cell types and functions in both autocrine and paracrine manners. It possesses neuroprotective, neuroregenerative, and vasculoprotective properties following brain injury [5,6,7]. The PI3K/Akt and Ras/ERK cascades, are activated by IGF-1 [4]
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