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Mallika Valapala, Jian Fei Hu, J Samuel Zigler, Stacey L Hose, Eric F Wawrousek, Jiang Qian, Debasish Sinha; βA3/A1-crystallin is a local mediator in astrocytes, regulating the Notch/STAT3 signaling axis. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2275.
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We have shown that decreased Notch signaling in Nuc1 (a spontaneous mutation in the Cryba1 gene) astrocytes accounts for the reduced promoter activity for glial fibrillary acidic protein (GFAP). This has led us to explore the signaling mediators that regulate GFAP expression. It is known that the signal transducer and activator of transcription 3 (STAT3) operates downstream of Notch. The objective of this study was to determine if βA3/A1-crystallin is required for the phosphorylation of STAT3 and the subsequent dimerization and translocation to the nucleus, activating transcription of Cryba1 and concomitantly, GFAP.
Optic nerve astrocytes were isolated from postnatal day 1-2 Cryba1-floxed mice. A lentiviral vector expressing Cre recombinase was used to delete Cryba1. The activity of STAT3 was upregulated by IL-6 treatment and inhibited by a STAT3-specific inhibitor, Stattic. Immunoblotting and ELISA were used to quantify the phosphorylation of STAT3. ENCODE and synteny mapping of human to mouse genome were used to identify STAT3 binding sites in the mouse Cryba1 gene. Quantitative real time PCR and luciferase assays were used to study the expression of Cryba1 and to validate the potential STAT3-binding sites in Cryba1.
Our studies show that phosphorylation of STAT3 is inhibited in the Cryba1-floxed cells upon lentiviral-mediated knockout of Cryba1. Our data also show that STAT3 could be activated by IL-6 and inhibited by Stattic. The expression of βA3/A1-crystallin was elevated by IL-6 and inhibited upon treatment with Stattic. Furthermore, ENCODE search for STAT3-binding sites in Cryba1, suggested two potential STAT3-binding sites, one in the promoter and another within the second intron. Our promoter-based luciferase assay confirmed that Cryba1 possesses a functional STAT3-binding site in its promoter. The Notch pathway inhibitor, DAPT also inhibits STAT3 activity.
Our data suggest that in astrocytes, STAT3 and βA3/A1-crystallin are co-regulated. This leads to a positive feedback loop in astrocytes, with βA3/A1-crystallin participating in the phosphorylation of STAT3 in the cytosol and in turn, STAT3 regulating the transcription of Cryba1 in the nucleus. βA3/A1-crystallin modulates the Notch/STAT3 signaling axis in astrocytes and is involved in regulating GFAP, which might potentiate the abnormal astrocyte template formation in Nuc1.
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