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Krista M Beach, Deborah C Otteson; Culture conditions, but not CNTF, modulate ERK/STAT signaling and gene expression in mouse Müller glia in vitro. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5492.
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© ARVO (1962-2015); The Authors (2016-present)
Understanding mechanisms regulating stem-cell properties of mammalian Müller glia is important to enhance their utility for retinal regeneration. In rat retinal progenitor cells in vitro, differential activation of extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription 3 (STAT3) signaling by ciliary neurotrophic factor (CNTF) promotes neurogenesis vs. gliogenesis respectively. This study tests the effects of culture condition and CNTF on ERK vs. STAT3 activation and on gene expression in mouse Müller glia in vitro.
Mouse Müller glia (ImM10 cell line), cultured in growth (G), sphere (S) and differentiation conditions for 5 days, were treated with CNTF (0 or 20ng/ml) for 15 min. Total and phosphorylated ERK and STAT3 (pERK and pSTAT3) were analyzed by western blot, quantified by grid densitometry, normalized to corresponding phospho/total protein ratios in a reference sample and compared using two-way ANOVA, with post-hoc T-tests (Bonferroni’s correction). mRNA expression in ImM10 cell differentiation cultures (0 or 20 ng/ml CNTF, 24 hours) was measured by quantitative reverse-transcriptase polymerase chain reaction and analyzed with Relative Expression Software Tool.
Without exogenous CNTF, the normalized pERK/total ERK ratio was significantly increased in differentiation cultures: 5.68-fold vs. G (p=0.006), 13.12-fold vs. S (p=0.005). The normalized pSTAT3/total STAT3 ratio was also significantly increased in differentiation cultures: 3.79-fold vs. G (p=0.030), 4.24-fold vs. S (p=0.028). ERK and STAT3 phosphorylation were unaffected by exogenous CNTF. Of 33 genes associated with neurogenesis and gliogenesis, only melanopsin expression was altered by CNTF, decreasing 65.2% vs. no CNTF (p=0.0036).
Increased ERK and STAT3 phosphorylation in ImM10 differentiation cultures is consistent with activation of both pro-neural and pro-glial signaling pathways in vitro. The absence of detectible increases in phosphorylated ERK or STAT3 with CNTF treatment suggests that other factors modulate ERK/STAT3 signaling in this cell line. Simultaneous activation of ERK and STAT3 signaling pathways in differentiation cultures may reflect heterogeneous responses of ImM10 Müller glia, consistent with the modest neurogenic capacity demonstrated to date by this cell line.
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