Lysyl oxidase-like 1-antisense 1 (LOXL1-AS1) lncRNA differentially regulates gene and protein expression, signaling and morphology of human ocular cells

Abstract

Pseudoexfoliation glaucoma (PEXG) is characterized by dysregulated extracellular matrix (ECM) homeostasis that disrupts conventional outflow function and increases intraocular pressure (IOP). Prolonged IOP elevation results in optic nerve head damage and vision loss. Uniquely, PEXG is a form of open angle glaucoma that has variable penetrance, is difficult to treat and does not respond well to common IOP-lowering pharmaceuticals. Therefore, understanding modulators of disease severity will aid in targeted therapies for PEXG. Genome-wide association studies have identified polymorphisms in the long non-coding RNA lysyl oxidase-like 1-antisense 1 (LOXL1-AS1) as a risk factor for PEXG. Risk alleles, oxidative stress and mechanical stretch all alter LOXL1-AS1 expression. As a long non-coding RNA, LOXL1-AS1 binds hnRNPL and regulates global gene expression. In this study, we focus on the role of LOXL1-AS1 in the ocular cells (trabecular meshwork and Schlemm's canal) that regulate IOP. We show that selective knockdown of LOXL1-AS1 leads to cell-type-specific changes in gene expression, ECM homeostasis, signaling and morphology. These results implicate LOXL1-AS1 as a modulator of cellular homeostasis, altering cell contractility and ECM turnover, both of which are well-known contributors to PEXG. These findings support LOXL1-AS1 as a key target for modifying the disease.

Figure 1

LOXL1-AS1 knockdown differentially affects gene expression in TM and SC. Primary TM and SC cells were transduced with Ad5-Scrambled-shRNA-GFP (Scr) or Ad5-LOXL1-AS1-shRNA-GFP (KD) and analyzed at 72 h post-transduction for changes in global gene regulation. (A) In TM cells, there were 165 significantly downregulated and 55 significantly upregulated targets upon RNAseq analysis (P < 0.05), n = 7 cell strains. (B) In SC cells, there were only five significantly downregulated targets and 19 significantly upregulated targets upon RNAseq analysis (P < 0.05), n = 6 cell strains.

Figure 2

LOXL1-AS1 knockdown differentially affects ECM target gene expression. Human immortalized lens epithelial (B3) cells, primary TM cells and primary SC cells underwent knockdown of LOXL1-AS1 via transfection or transduction, respectively, and all cell types were analyzed specifically for ECM gene and protein expression. (A) In TM cells at 72 h post transduction, five gene targets were significantly upregulated, including Integrin beta-3 (ITGB3), and eight gene targets were significantly downregulated, including Collagen type XI, alpha 1 (COL11A1) (P < 0.05), n = 4 cell strains. (B) In SC cells at 72 h post transduction, two gene targets were significantly upregulated, including Integrin alpha-2 (ITGA2), and 11 gene targets were significantly downregulated, including Laminin gamma 1 (LAMC1) (P < 0.05), n = 4 cell strains. (C) In B3 cells at 48 h post transfection, five gene targets were significantly upregulated, including Thrombospondin-2 (THBS2), and two targets were significantly downregulated, including TIMP metallopeptidase inhibitor 1 (TIMP1) (P < 0.05), n = 3 biological replicates. (D) Western blot analysis performed on LOXL1-AS1 knockdown TM cell lysates indicated that Collagen type IX, alpha 1 was significantly downregulated at the protein level (P < 0.05), n = 4 cell strains. ICAM-1 and Integrin beta3 were not significantly different at the protein level from scrambled controls (P > 0.05), n = 7 cell strains. (E) Upon western blot analysis on SC cell lysates for Laminin gamma 1 and Integrin alpha 2, no significant change was detectable at the protein level (P > 0.05), n = 4 cell strains. (F) Lastly, western blot analysis in B3 cell lysates for TIMP-1 and Thrombospondin-2 showed no significant change was detectable at the protein level in LOXL1-AS1 knockdown cells (P > 0.05), n = 4 biological replicates.

Figure 3

LOXL1-AS1 knockdown differentially affects mechanotransduction targets in B3, TM and SC cells. Human primary TM cells, primary SC cells and immortalized lens cells (B3 cells) underwent knockdown of LOXL1-AS1 via transduction or transfection, respectively, and all cell types were analyzed specifically for known mechanotransduction protein expression via western blot. (A) In TM cells at 72 h post transduction, no targets were significantly dysregulated with LOXL1-AS1 knockdown (P > 0.05), n = 3–11 cell strains. (B) In SC cells at 72 h post transduction, pAKT/AKT was significantly upregulated (P < 0.05), n = 4 cell strains, while all other targets showed no significant change with LOXL1-AS1 knockdown (P > 0.05), n = 3–6 cell strains. These data indicate differential mechanotransduction regulation for lens and outflow pathway cell types. (C) In B3 cells at 48 h post transfection, phosphorylated MAPK (pMAPK/MAPK) was upregulated, while pAKT/AKT, VLK, Talin-1 and RhoA were all significantly downregulated with LOXL1-AS1 knockdown (^*P < 0.05, ^**P < 0.01, ^***P < 0.001), n = 4–5 biological replicates.

Figure 4

LOXL1-AS1 knockdown changes B3, TM and SC morphology. Human primary TM cells, primary SC cells and immortalized lens epithelial (B3) cells underwent knockdown of LOXL1-AS1 via viral transduction or plasmid transfection, respectively, and all cell types were analyzed specifically for cell morphology using immunofluorescence of either GFP or labeled TAZ with Al488 secondary antibody. (A–C) TM cells displayed a non-significant trend toward circularity between the Scrambled (A) and the LOXL1-AS1 knockdown (B) cells, as indicated by (C) a decrease in semiaxis ratio (Major axis/Minor axis) (P < 0.05). Scale bar = 10 μm. (D–F) SC cells displayed a significant increase in circularity between the scrambled (D) and the LOXL1-AS1 knockdown (E) cells, as indicated by (F) a decrease in semiaxis ratio (Major axis/Minor axis) (P < 0.05). Scale bar = 10 μm. (G–I) B3 cells displayed no change in circularity between the Scrambled (A) and the LOXL1-AS1 knockdown (B) cells, as indicated by (C) a similar in semiaxis ratio (Major axis/Minor axis). Scale bar = 10 μm. Images were brightened equally to visualize cell borders.

Figure 5

LOXL1-AS1 knockdown decreases myosin light chain activity. Primary human SC cells were transduced with an adenoviral construct to knockdown LOXL1-AS1. 72 h post transduction cells were lysed and levels of phosphorylated MLC and total MLC were measured via western blotting. Results indicated a downward trend in pMLC/MLC following LOXL1-AS1 knockdown, albeit not statistically significant (P = 0.0563), n = 8 biological replicates.