Abstract
To identify possible genetic variants influencing expression of EPHA2 (Ephrin-receptor Type-A2), a tyrosine kinase receptor that has been shown to be important for lens development and to contribute to both congenital and age related cataract when mutated, the extended promoter region of EPHA2 was screened for variants. SNP rs6603883 lies in a PAX2 binding site in the EPHA2 promoter region. The C (minor) allele decreased EPHA2 transcriptional activity relative to the T allele by reducing the binding affinity of PAX2. Knockdown of PAX2 in human lens epithelial (HLE) cells decreased endogenous expression of EPHA2. Whole RNA sequencing showed that extracellular matrix (ECM), MAPK-AKT signaling pathways and cytoskeleton related genes were dysregulated in EPHA2 knockdown HLE cells. Taken together, these results indicate a functional non-coding SNP in EPHA2 promoter affects PAX2 binding and reduces EPHA2 expression. They further suggest that decreasing EPHA2 levels alters MAPK, AKT signaling pathways and ECM and cytoskeletal genes in lens cells that could contribute to cataract. These results demonstrate a direct role for PAX2 in EPHA2 expression and help delineate the role of EPHA2 in development and homeostasis required for lens transparency.
Highlights
Cataract is an opacity of the crystalline lens[1]
We show that rs6603883 in the promoter region of EPHA2 is located in a binding motif of PAX2, and the minor allele decreases PAX2 binding reducing the transcriptional activity of EPHA2
Polymorphisms in the EPHA2 region have been shown to be associated with ARC9, 12, 13, 16, but the mechanisms through which these polymorphisms and EPHA2 itself affect ARC are still largely unknown
Summary
While genetic influences on ARC are well documented[33], the specific genes and mechanisms of these effects are only beginning to be elucidated. The extracellular matrix (ECM) plays an important role in lens structure and function, and mutations in ECM genes have been shown to be associated with cataract[49, 50] Consistent with this result, in addition to the MAPK and AKT signaling pathways, knocking down EPHA2 levels resulted in significant changes in the expression of 11 genes related to the ECM, cell membrane, cell surface, or basement membrane. Among the 4 ECM related genes, an NID1 mutation in Romagnola Cattle caused inherited cataracts[51], and the VEGF-KDR signaling pathway has been reported to play important roles in cataractogenesis[52] These results are consistent with the hypothesis that EPHA2 may act through the EMC and cell membrane to alter the MAPK and AKT signaling pathways, affecting the cytoskeleton and increasing susceptibility of the aging lens to cataract. These results will help us to understand the mechanisms of age related cataract, which potentially will allow development of potential methods to delay or even prevent ARC
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