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Krista M. Beach, Deborah C. Otteson; Differential Phosphorylation of STAT3 at Tyr705 and Ser727 in Differentiation Cultures of ImM10 Mouse Muller Glia are Not Regulated by CNTF. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2241.
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© ARVO (1962-2015); The Authors (2016-present)
Despite evidence of stem-cell characteristics, the neurogenic ability of mammalian Muller glia is limited both in vivo and in vitro. CNTF differentially activates pro-glial JAK/STAT or pro-neuronal MAPK signal transduction cascades in retinal progenitors. To determine if CNTF signaling regulates the neurogenic vs. gliotic response of mouse Muller glia in vitro, we analyzed STAT3 phosphorylation following CNTF treatment in growth and FGF2-induced differentiation cultures.
ImM10 cells were cultured in immortalizing growth conditions. For differentiation, cells were grown in serum-free, non-immortalizing conditions in Neurobasal, B27, G5, 20 ng each EGF+FGF2 for 7 days to generate spheres, followed by 5 days in Neurobasal, G5, FGF2, and 5 days in Neurobasal with B27. ImM10 cells in both growth and differentiation conditions were treated with CNTF (20ng/ml) for 15 min and lysed in Laemmli buffer with phosphatase inhibitors. STAT3 phosphorylation was assessed by western blots, quantified by densitometry and compared by ANOVA with Tamhane's T2 post hoc analysis.
STAT3α-Tyr705 and STAT3β-Tyr705 phosphorylation were significantly decreased in differentiation vs. growth conditions (p=0.013 and p=0.033 respectively). The α/β ratio (activating/inhibiting) of STAT3-PTyr705 also increased in differentiation conditions by 173%. Differences in STAT3α-PSer727 or STAT3β-PSer727 were not statistically significant between conditions. However, the α/β ratio for P-Ser727 increased 213% in differentiation conditions, due to a greater decrease in β-PSer727 than α-PSer727. Unexpectedly, CNTF had no significant effect on STAT3 phosphorylation at either residue in any condition.
In differentiation cultures of ImM10 Muller glia, we previously reported up-regulation of both gliotic and neurogenic genes. Simultaneous increases in relative STAT3α-PSer727 levels, which would activate neurogenic MAPK signaling, and decreases in relative STAT3β-PTyr705 levels, which would lead to de-inhibition of gliogenic JAK/STAT signaling, offers a potential mechanism for these apparently contradictory gene expression changes in differentiation cultures. The inability of CNTF to alter STAT3 phosphorylation indicates that CNTF is unlikely to modulate differentiation or gliosis in ImM10 cells.
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