Overexpression of the full-length human LOXL1 (allele 153G/G) in stably transfected epithelial cells (HEK293), resulted in high levels of the LOXL1 protein. The hLOXL1-transfected (pIND-LOXL1) cells showed robust expression of both the 65 kD full-length protein and processed forms immunolocalized in the cytoplasm and nuclei and less prominently in the ECM. In activity assays of 3-day postconfluent pIND-LOXL1 stable overexpressing HEK293 cells, LOXL1 proved to have increased catalytic activity compared with native and empty vector (pIND)-transfected cells (Fig. 3).
Cellular overexpression of LOXL1, or exogenous LOXL1 in HEK293 or ARPE-19 cells treated for 48 hours with LOXL1 in conditioned culture media derived from either high-expressing ARPE-19 or overexpressing HEK293 cells cultures, did not result in visible cell phenotype changes. In cell migration assays, the number of migrating cells (the average number of migrated cells in 6 randomly chosen field in 3 parallel experiments per conditions) in pIND-LOXL1 stably transfected HEK293 cells, did not change significantly (P=0.42) compared with native (HEK) or empty vector-transfected (pIND HEK) cells (data not shown). To test for LOXL1 activity-dependent or activity-independent functions, cultured epithelial cells were treated with BAPN, an irreversible inhibitor of LOXL1 activity, or catalase that promotes the degradation of hydrogen peroxide, a byproduct of the LOXL1 cross-linking reaction and itself an important regulator of cellular functions. There were no significant differences in the number of filopodia formed under different culture conditions, suggesting that LOXL1 activity-related function did not have a significant effect on cell mobility/spindling, a phenotypic marker of epithelial to mesenchymal transition (EMT).
Among epithelial features affected by LOXL1, the epithelial marker and cell adhesion protein E-cadherin, highly expressed in ARPE-19 cells, was reduced to less than 50% by LOXL1 overexpression in stable transfected HEK293 cells (Figs. 4A, B). E-cadherin expression is controlled by Snail, a transcriptional regulator of EMT. Therefore, we considered the possibility that reduced E-cadherin was an indicator for the initiation of LOXL1-mediated EMT in these cells. The basal levels of Snail expression did not increase significantly in response to LOXL1 overexpression. However, in immunoprecipitation experiments, we detected direct interaction between Snail and the full-length LOXL1 in overexpressing cells, an interaction that was not detectable in native HEK293 cells or in vector-transfected cells (Fig. 4C). These results confirmed our earlier data reported for various epithelial cell types12–14 that LOXL1 in interactions with Snail may be directly involved in the induction of EMT and the generation of fibrogenic cell types, a mechanism that in ocular epithelial cells, may contribute to disorderly ECM and the pathomechanism of XFG.
To date, pathomechanistic explanations for XFG have largely focused on downstream scenarios, deduced from the components of the irreversible aberrant fibrillogranular aggregates in eye compartments. Attempts to identify the cell type-specific mechanisms in the initiating pathologic steps, meanwhile, have remained extremely difficult due to limited access to tissue and cell specimens suitable to experimentally test and challenge these hypotheses and lack of animal models that would recapture the XFG process. In this study, therefore, we have established a cell model to address firstly, the contribution of epithelial cells to LOXL1-associated pathologic mechanisms most relevant to its aberrant regulation and cross-linking function of ECM aggregates in XFG and, secondly, to explore cellular roles for LOXL1 within regulatory functions that may additionally contribute to the widespread ECM disturbance and aberrant production of fibrotic material that occurs in XFG.
Results from the current study demonstrated that, compared with mesenchymal cells considered as major sources of LOXL1, E-cadherin-expressing polarized epithelial cells produce higher levels of LOXL1, express abundant amounts of BMP-1 critical for LOXL1 activation, and process and activate LOXL1, supporting high levels of epithelia-derived LOXL1 cross-linking activity in the ECM. The broader epithelial cell type specificity and functional significance of some of the processed forms unique to retinal epithelial cells needs further study. Although there are variations in LOXL1 expression levels among the cell lines analyzed, this is unlikely due to allelic differences at R141L or G153D (Arg/Leu, Gly/Asp) associated with risk for XFS and/or XFG, similar to an earlier report on LOXL1 haplotypes (GG, GA, TG, TA) at these loci that did not affect LOXL1 catalytic activity in vitro.15
Overexpression or inhibition of cellular or exogenous LOXL1 did not induce cell phenotype changes in terms of migratory ability, filopodia, or cell mobility/spindling, visible signs of mesenchymal features. LOXL1, however, significantly downregulated the epithelial marker E-cadherin, suggesting the initiation of LOXL1-mediated EMT in these cells. Consistent with this novel cellular/nuclear LOXL1 function, LOXL1 localized not only to the ECM, but was also prominently present in nuclear compartments, in agreement with our earlier in vivo16 and in vitro12 observations.
EMT has been reported to occur, as part of various eye pathologies, in eye epithelial compartments,17 in the trabecular meshwork,18 under conditions such as ocular tissue fibrosis and injury,19 and with the involvement of Snail1.20 EMT-like phenomenon has been also proposed to directly result in aberrant conditions in the aqueous outflow pathway in glaucomatous eyes.18
We have previously reported the involvement of LOX family members, LOXL2 and LOXL3, in EMT including interactions with Snail1 to downregulate E-cadherin expression. Furthermore, we have also noted that Snail1 lysine residues 98 and 137 are required for stability, functional cooperation with LOXs, and induction of EMT.12,13 We have also described an in vivo role for LOXL1 in cooperation with Snail1 during the transition of liver epithelial cells towards a fibrogenic phenotype.14 Results of the current study further support interactions between LOXL1 and Snail1 in epithelial cells, and LOXL1-induced repression of E-cadherin, a hallmark of EMT, and a mechanism that in eye epithelial cells, may contribute to disorderly ECM production during the pathogenesis of XFG.
Transdifferentiation reported in retinal epithelial cells induced by TGF-β1 and EGF, included gain of α-smooth muscle actin, an indicator of a transition towards a myofibroblast phenotype, and enhanced migration.21 Although the progression of LOXL1-induced EMT including expression patterns of early (vimentin) and late (α-smooth muscle actin) markers need to be defined, enhanced epithelial cell migration does not appear to be a LOXL1-associated feature.
Not only epitihelial, but mesenchymal cells also become activated in XFG and under reactive conditions including injury, repair, and inflammation, and differentiate toward profibrotic myofibroblast phenotypes resulting in persistent and excessive production and disorderly deposition of fibrotic material. Under these conditions, there is an additional, TGF-β-driven, expansion of connective tissue cell population.22 In XFG, the profibrotic TGF-β has been noted to induce LOXL1 in Tenon’s capsule cells.23 In cells of the trabecular meshwork, TGF-βI induced LOXL1 expression involving both Smad and non-Smad signaling pathways, and the resulting increase in LOXL1 cross-linking activity was proposed to be partly responsible for TGF-β-mediated intraocular pressure elevation.24 Of interest is the fact that in the characteristic XFG fibrotic profile, although numerous fibrotic elements are present, unlike in tissue fibrosis, there is no increase in fibrillar and type IV collagens. In epithelial cells; however, TGF-β did not appear to affect LOXL1 expression (A546 cells, NCBI GEO array data). Thus alterations in multiple epithelial and mesenchymal cell-specific regulatory pathways may converge and contribute to the XFG phenotype.
Data collectively support that in XFG, LOXL1-associated pathology involves dysregulated ECM activity of the secreted LOXL1 produced by both mesenchymal and epithelial cells that may directly contribute to irreversible cross-linking of fibrillar ECM aggregates, and additionally, in eye epithelia, a cellular mechanism with a role for LOXL1 in interactions with Snail1, in the regulation of EMT, a process that activates multiple regulatory networks to promote fibrogenic cell phenotypes.
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Keywords:© 2014 by Lippincott Williams & Wilkins.
exfoliation syndrome; Lysyl oxidase-like 1; exfoliation glaucoma; epithelial to mesenchymal transition