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Anne Terrell, Melinda Duncan; β1-integrin may be a direct regulator of EGR1 (Early Growth Response 1) within the lens. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3195.
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© ARVO (1962-2015); The Authors (2016-present)
Integrins are heterodimeric transmembrane extracellular matrix receptors that regulate cell-to-cell and cell-to-ECM communication. Functional integrins require an α and a β subunit; β1-integrin is expressed in both epithelial and the fibers cells within the lens. Conditionally deleting β1-integrin from the lens at 12.5 dpc causes lens epithelial cells to become disorganized by 16.5 dpc with ectopic expression of fiber cell and EMT markers, and aberrant cell death by birth. Analysis of candidate genes based on their known roles in integrin and TGFβ pathways failed to explain the observed phenotype. Here we used an unbiased analysis of gene expression changes to identify the likely regulator of the β1-integrin null phenotype.
Next generation RNA-sequencing was used to determine gene transcript abundance in 15.5 dpc C57BL/6 and β1MLR10-cKO lenses. Reads Per Million per Kilobase (RPMK) was used to determine gene transcript abundance. Differentially expressed genes were determined by filtering criteria: mean RPMK ≥ 1, P-value ≤ 0.05, and fold change ≥ 2. Differentially expressed genes were validated via qRT-PCR and immunohistochemistry.
Comparing wild-type lenses with β1MLR10-cKO lenses revealed that less than 50 genes were differentially expressed three days after the deletion of β1-integrin and one day prior to the onset of the phenotype including: EGR1, MT1, Nab2, Tsc2, FGF12 and αSMA. EGR1, a transcription factor which can be directly regulated by integrin signaling, also directly regulates MT1, Nab2, FGF12, αSMA and Tsc2 in other tissues. At 14.5 dpc, qRT-PCR validated that EGR1 and Nab2 were 7.4 and 5.7 fold increased, respectively. The level of EGR1 remained high in the embryonic lens and was greater than 4 fold increased at 16.5 dpc. Immunolocalization showed that EGR1 was also increased at the protein level at 15.5 and 16.5 dpc.
In the lens, the expression of EGR1, previously shown to negatively regulate the growth of both the eye and lens during the pathogenesis of myopia, appears to be negatively regulated by β1-integrins. Since EGR1 has been found to be a regulator of αSMA, TGFβ1, PTEN, fibronectin and p53 expression in other tissues; it is likely that EGR1 is a major mediator of the β1MLR10-cKO lens phenotype. Future work will investigate the role that EGR1 plays in lens pathology and how β1-integrin regulates its expression levels in the lens.
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