Differential Distribution of Laminin N-Terminus α31 Across the Ocular Surface: Implications for Corneal Wound Repair

Abstract

PURPOSE. Laminin N-terminus (LaNt) a31 is a relatively unstudied protein derived from the laminin a3 gene but structurally similar to netrins. LaNt a31 has, to date, been investigated only in two-dimensional (2D) keratinocyte culture where it influences cell migration and adhesion, processes integral to wound repair. Here we investigated LaNt a31 distribution in ocular surface epithelium, during limbal stem cell activation, and corneal wound healing.METHODS. Human, mouse, and pig eyes, ex vivo limbal explant cultures, and alkali burn wounds were processed for immunohistochemistry with antibodies against LaNt a31 along with progenitor cell-associated proteins. LaNt a31 expression was induced via adenoviral transduction into primary epithelial cells isolated from limbal explants, and cell spreading and migration were analyzed using live imaging.RESULTS. LaNt a31 localized to the basal layer of the conjunctival, limbal, and corneal epithelial cells. However, staining was nonuniform with apparent subpopulation enrichment, and some suprabasal reactivity was also noted. This LaNt a31 distribution largely matched that of keratin 15, epidermal growth factor receptor, and transformation-related protein 63a (p63a), and displayed similar increases in expression in activated limbal explants. During active alkali burn wound repair, LaNt a31 displayed increased expression in limbal regions and loss of basal restriction within the cornea. Distribution returned to predominately basal cell restricted once the wounded epithelium matured. Cultured corneal epithelial cells expressing LaNt a31 displayed increased 2D area and reduced migration, suggesting a functional link between this protein and key wound repair activities.CONCLUSIONS. These data place LaNt a31 in position to influence laminin-dependent processes including wound repair and stem cell activation.

Laminin N-terminus α31 regulates keratinocyte adhesion and migration through modifying the organisation and proteolytic processing of laminin 332

“…Previously, we reported that increased expression of LaNt α31 led to increased cell spreading and reduced migration of primary limbal-derived corneal epithelial cells (Barrera et al, 2018) . To investigate the mechanism for these effects we turned to the widely-used and well characterised limbal-derived corneal epithelial cell line hTCEpi (Yamamoto et al, 2005;Dreier et al, 2012;Yanez-Soto et al, 2015;Hampel et al, 2018;Kaplan et al, 2019) (Supplemental Figure 1A) and used the same adenoviral construct to drive expression of LaNt α31 with a C-terminal eGFP tag (hereafter +LaNt α31, Figure 1B), comparing results against cells expressing eGFP alone (+eGFP).…”

“…Mouse monoclonal antibodies for IF against LMα3 (clone RG13) (Gonzales et al, 1999) used at 10 μg/ml, rabbit polyclonal antibodies raised against LM332 (J18) (Hopkinson et al, 1992) and cytokeratin 12 {Kurpakus, 1990 #12636} were used at 1:50, and human monoclonal antibodies to BPAG1e (5E) (Hashimoto et al, 1993) used at 1:2, were generous gifts from Jonathan Jones, Washington State University, WA. Mouse monoclonal antibodies raised against human LaNt α31 (clone 3E11) used at 1.9 μg/ml for IB were described previously (Barrera et al, 2018;Troughton et al, 2020a). Rat monoclonal antibodies raised against LMγ1 (clone A5, ab80580) used at 1 μg/ml, and mouse monoclonal antibodies raised against LMβ2 (clone CL2979) used at 1 μg/ml were from Abcam.…”

“…with C-terminal eGFP tag were described previously (Barrera et al, 2018). Adenoviruses inducing CMV driven expression of LMβ3-mCherry (Hopkinson et al, 2008)…”

“…In the ocular anterior surface epithelium, it is highly abundant in the limbal epithelium with comparatively lower levels found in corneal and conjunctiva in intact tissue. LaNt α31 expression also has been shown to increase in the cornea during the active phases of wound healing the and to normal levels once the wound has healed (Barrera et al, 2018). Upregulation of LaNt α31 expression has also been identified in ex vivo models of limbal stem cell activation and been associated with breast cancer (Barrera et al, 2018;Troughton et al, 2020b).…”

“…LaNt α31 expression also has been shown to increase in the cornea during the active phases of wound healing the and to normal levels once the wound has healed (Barrera et al, 2018). Upregulation of LaNt α31 expression has also been identified in ex vivo models of limbal stem cell activation and been associated with breast cancer (Barrera et al, 2018;Troughton et al, 2020b). In vitro functional studies have revealed that knockdown in epidermal keratinocytes reduced cell adhesion strength and slowed the rate of scratch wound closure (Hamill et al, 2009c), while overexpression in primary limbal keratinocytes also caused a reduction in migration rates (Barrera et al, 2018).…”