Mutation of the EPHA2 Tyrosine-Kinase Domain Dysregulates Cell Pattern Formation and Cytoskeletal Gene Expression in the Lens

Abstract

Genetic variations in ephrin type-A receptor 2 (EPHA2) have been associated with inherited and age-related forms of cataract in humans. Here, we have characterized the eye lens phenotype and transcript profile of germline Epha2 knock-in mutant mice homozygous for either a missense variant associated with age-related cataract in humans (Epha2-Q722) or a novel insertion-deletion mutation (Epha2-indel722) that were both located within the tyrosine-kinase domain of EPHA2. Confocal imaging of ex vivo lenses from Epha2-indel722 mice on a fluorescent reporter background revealed misalignment of epithelial-to-fiber cell meridional-rows at the lens equator and severe disturbance of Y-suture formation at the lens poles, whereas Epha2-Q722 lenses displayed mild disturbance of posterior sutures. Immunofluorescent labeling showed that EPHA2 was localized to radial columns of hexagonal fiber cell membranes in Epha2-Q722 lenses, whereas Epha2-indel722 lenses displayed disorganized radial cell columns and cytoplasmic retention of EPHA2. Immunoprecipitation/blotting studies indicated that EPHA2 formed strong complexes with Src kinase and was mostly serine phosphorylated in the lens. RNA sequencing analysis revealed differential expression of several cytoskeleton-associated genes in Epha2-mutant and Epha2-null lenses including shared downregulation of Lgsn and Clic5. Collectively, our data suggest that mutations within the tyrosine-kinase domain of EPHA2 result in lens cell patterning defects and dysregulated expression of several cytoskeleton-associated proteins.

Roles of Eph-Ephrin Signaling in the Eye Lens Cataractogenesis, Biomechanics, and Homeostasis

“…EphA2 signals through the Src and cortactin pathway to influence actin cytoskeleton in equatorial lens epithelial cells to regulate hexagon cell shape and cell organization ( Cheng et al, 2013 ), similar to interactions previously shown in other tissues between EphA2 and Src ( Baldwin et al, 2006 ; Parri et al, 2007 ; Faoro et al, 2010 ). A recent study has confirmed complex formation between EphA2 and Src kinase thereby supporting their direct downstream interaction ( Zhou et al, 2021 ). The development and maintenance of the lens’ unique structure are determined by growth factors and signaling pathways ( Beebe et al, 1987 ; Belecky-Adams et al, 1997 ; Faber et al, 2002 ; Lovicu and McAvoy, 2005 ).…”

“…Recently, disruption of Eph-ephrin signaling in human patients has been associated with congenital and age-related cataracts, defined as any opacity in the transparent eye lens ( Shiels et al, 2008 ; Jun et al, 2009 ; Zhang et al, 2009 ; Kaul et al, 2010 ; Tan et al, 2011 ; Sundaresan et al, 2012 ; Dave et al, 2013 ; Patel et al, 2017 ; Berry et al, 2018 ; Zhai et al, 2019 ). Mouse models are now being used to understand the mechanism of cataractogenesis ( Cooper et al, 2008 ; Jun et al, 2009 ; Cheng and Gong, 2011 ; Shi et al, 2012 ; Cheng et al, 2013 ; Son et al, 2013 ; Biswas et al, 2016 ; Cheng et al, 2017 ; Cheng et al, 2021 ; Zhou et al, 2021 ; Cheng et al, 2022 ). In this review, we highlight the different functions of Eph-ephrin signaling in the lens, describe how genetic background influences cataract phenotypes, as well as provide some insights into future directions and potential therapeutic strategies that can be tested to understand the pathogenesis of age-related cataracts.…”

“…Mice containing an EphA2 mutation (p.R722Q), which is homologus to the human EPHA2 mutation (p.R721Q) associated with age-related cataracts ( Jun et al, 2009 ), were generated using CRISPR/Cas9 technology ( Zhou et al, 2021 ). The generation of mutant EphA2-R722Q mice also resulted in a separate off-target insertion-deletion mutant allele in exon 13 (EphA2-indel722).…”

“…The generation of mutant EphA2-R722Q mice also resulted in a separate off-target insertion-deletion mutant allele in exon 13 (EphA2-indel722). EphA2-R722Q mutant lenses (homozygous and heterozygous) were similar in size and transparency to control wild-type lenses, but EphA2-indel722 mutant lenses exhibited translucent regions, disrupted alignment of equatorial hexagonal epithelial and fiber cells, and polar axis shift of the fiber cell tips with severely disrupted suture pattern at the posterior pole ( Zhou et al, 2021 ). There was no significant manifestation of cataract in young (3 weeks old) and old (12 months old) heterozygous and homozygous EphA2-R722Q and EphA2-indel722 mutant mice ( Zhou et al, 2021 ).…”

“…EphA2-R722Q mutant lenses (homozygous and heterozygous) were similar in size and transparency to control wild-type lenses, but EphA2-indel722 mutant lenses exhibited translucent regions, disrupted alignment of equatorial hexagonal epithelial and fiber cells, and polar axis shift of the fiber cell tips with severely disrupted suture pattern at the posterior pole ( Zhou et al, 2021 ). There was no significant manifestation of cataract in young (3 weeks old) and old (12 months old) heterozygous and homozygous EphA2-R722Q and EphA2-indel722 mutant mice ( Zhou et al, 2021 ). These results strengthen the hypothesis that EphA2 is required for the precise alignment of fiber cells at the equator and the formation of suture at the posterior pole.…”