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Yichen Wang, Anne M Terrell, Deepti Anand, Salil Anil Lachke, Melinda K Duncan; β1-integrin deletion from lens activates AKT signaling and EGR1 expression in the epithelium leading to fibrosis and apoptosis. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© ARVO (1962-2015); The Authors (2016-present)
β1-integrin is the major β-integrin subunit expressed in both lens epithelial and fiber cells. Previous research has shown that β1-integrin is essential for the maintenance of lens epithelial integrity and survival in late embryonic lens development. Lack of β1-integrin in the mouse lens after E13.5 (β1-cKO) will lead to fibrosis in lens epithelial cells and massive apoptosis in newborn (NB) lenses, which contributes to severe microphthalmia and lack of lens in adult mice (Simiriski et al, 2007). However, the molecular connections between β1-integrin deletion and these phenotypic changes remain unclear.
The activation status of some known signaling pathways (FAK/Erk, TGF-β and AKT signaling) were studied via Western Blot and immunohistochemistry. Then, an unbiased approach, RNA-sequencing, was performed to identify differentially expressed genes in β1-cKO lenses at E15.5. By using motif enrichment analysis and literature searching, EGR1 (early growth response 1), a major regulator of fibrosis and apoptosis, was selected for further study. To better elucidate this, mice lacking both β1-integrin and EGR1 genes from the lenses were created (β1-cKO/EGR1 double null).
Signaling pathway analysis revealed that: (1) β1-cKO lenses lacked FAK activation, but did not exhibit any changes in ERK signaling; (2) TGF-β signaling was down-regulated in β1-cKO lenses; (3) higher levels of pAkt was detected in β1-cKO lenses. RNAseq revealed 120 genes were differentially expressed (DRGs) in β1-cKO lenses, many of which were involved in EMT and stress responses. One of these, the transcription factor, EGR1, was upregulated more than 10 fold in β1-cKO lenses, and motif enrichment analysis revealed EGR1 binding sites in the promoters of 19% of up-regulated DRGs. More importantly, β1-cKO/EGR1 double null mice showed larger eyes than β1-cKOs and identifiable lens tissue as adults, accompanied with alleviated apoptosis at NB stage. However, they maintained the previously reported lens epithelial fibrosis at E16.5.
These data suggest that loss of β1-integrin from the lens leads to aberrant AKT phosphorylation, which results in EGR1 overexpression. This EGR1 upregulation appears to result in some of the phenotypic abnormalities found in β1-cKO lenses, which could be partially reversed by deletion of EGR1.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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