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Heather Schmitt, William Johnson, Shelby Strickland, Michael A Hauser, William D Stamer; The PEXpress lncRNA/hnRNPL complex regulates signaling and morphology of human Schlemm’s canal cells. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3185. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Pseudoexfoliation glaucoma (PEXG) is the most common secondary form of open angle glaucoma worldwide. PEXpress is a lncRNA located within the LOXL1 locus, which is associated with high risk of PEXG. We have previously shown that (i) the risk variants alter PEXpress promoter strength, (ii) mechanical stretch of Schlemm’s canal (SC) cells increases PEXpress expression, (iii) PEXpress specifically complexes with hnRNPL, a RNA regulation protein and (iv) knockdown of PEXpress changes expression levels of 300+ genes in B3 cells. The goal of the present study was to examine the role of PEXpress in modulating hnRNPL splicing activity and mechanotransduction signaling.
Adenoviruses encoding shRNA targeted to PEXpress or hnRNPL were used to alter their expression in SC cells. At 4 days post-transduction, cell lysates were collected for Western blot analyses. Mechanotransduction and adhesion hub proteins FAK, AKT, and MAPK were monitored for state of activation. We also examined hnRNPL splicing activity on glaucoma relevant substrates, eNOS and CD44 V10. Morphological changes were determined in monolayers of SC cells by immunofluorescence.
Knockdown of PEXpress in SC cells significantly increased the proportion of phosphorylated AKT to total AKT (p=0.008), but no changes were observed with phosphorylation status of FAK or MAPK. Importantly, knockdown of PEXpress significantly shortened the major axis of SC cells (p=0.0006), while knockdown of hnRNPL did not. In parallel experiments, we observed that PEXpress did not affect hnRNPL-mediated eNOS splicing (p=0.956 in HEK293 cells. Experiments examining PEXpress-mediation of CD44 splicing are underway.
PEXpress/hnRNPL directly regulates and is responsive to critical pathways of cellular adhesion and morphology in SC cells. PEXpress/hnRNPL complex represents a viable therapeutic target for treating PEXG.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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