Pannexin1: Role as a Sensor to Injury Is Attenuated in Pretype 2 Corneal Diabetic Epithelium

Abstract

Epithelial wound healing is essential to repair the corneal barrier function after injury and requires coordinated epithelial sheet movement over the wounded region. The presence and role of pannexin1 on multilayered epithelial sheet migration was examined in unwounded and wounded corneal epithelium from C57BL/6J (B6) control and diet-induced obese (DiO) mice, a pretype 2 diabetic model. We hypothesize that pannexin1 is dysregulated, and the interaction of two ion-channel proteins (P2X7 and pannexin1) is altered in pretype 2 diabetic tissue. Pannexin1 was found to be present along cell borders in unwounded tissue, and no significant difference was observed between DiO and B6 control. However, an epithelial debridement induced a striking difference in pannexin1 localization. The B6 control epithelium displayed intense staining near the leading edge, which is the region where calcium mobilization was detected, whereas the staining in the DiO corneal epithelium was diffuse and lacked distinct gradation in intensity back from the leading edge. Cells distal to the wound in the DiO tissue were irregular in shape, and the morphology was similar to that of epithelium inhibited with 10Panx, a pannexin1 inhibitor. Pannexin1 inhibition reduced mobilization of calcium between cells near the leading edge, and MATLAB scripts revealed a reduction in cell-cell communication that was also detected in cultured cells. Proximity ligation was performed to determine if P2X7 and pannexin1 interaction was a necessary component of motility and communication. While there was no significant difference in the interaction in unwounded DiO and B6 control corneal epithelium, there was significantly less interaction in the wounded DiO corneas both near the wound and back from the edge. The results demonstrate that pannexin1 contributes to the healing response, and P2X7 and pannexin1 coordination may be a required component of cell-cell communication and an underlying reason for the lack of pathologic tissue migration.

Age Dependent Changes in Corneal Epithelial Cell Signaling

“…We will also examine changes in ectonucleotidases, decreased expression of pannexin-1 and other potential candidate proteins, or a decrease in the interaction between P2X7 and pannexin-1. The latter was shown in wounded corneas of an obese mouse ( Rhodes et al, 2021 ).…”

“…Pannexin-1 is a mechanosensitive ion channel that binds to F-actin in the carboxy terminus ( Bao et al, 2004 ; Bhalla-Gehi et al, 2010 ). Inhibition of pannexin-1 in corneas from 12-week mice with 10PanX caused decreases in calcium signaling after injury and alterations at the leading edge with a reduction in lamellipodia ( Rhodes et al, 2021 ). Pannexin-1 localization near the wound edge is elevated in younger mice when compared to older mice 5 h after injury.…”

“…Calcium mobilization experiments were performed as described previously ( Klepeis et al, 2001 ; Weinger et al, 2005 ; Lee et al, 2019 ). The imaging of intact murine eyes was modified from that of corneas as described ( Lee et al, 2019 ; Rhodes et al, 2021 ). Globes were enucleated and were preincubated in Fluo-4, AM (1:100) and CellMask™ Deep Red (Thermo Fisher, Waltham, MA, United States) (1:10,000) with a final concentration of 1% (v/v) DMSO and 0.1% (w/v) pluronic acid for 2 h at 20°C.…”

“…Globes were fixed in freshly prepared 4% paraformaldehyde in PBS for 20 min at room temperature. Immunofluorescent staining was performed ( Minns et al, 2016 ; Rhodes et al, 2021 ). The cornea was removed and cut into radial sections.…”

“…Furthermore, the localization and regulation of two ion channel proteins, P2X7 and pannexin-1, have been of great interest in respect to their response to injury. Both proteins exhibit injury induced changes in localization, and the intense staining that is adjacent to the injury corresponds to the region of intense calcium mobilization or signaling between cells ( Lee et al, 2019 ; Rhodes et al, 2021 ). Inhibition of either P2X7 or pannexin-1 greatly attenuates calcium signaling events, cellular motility, and migration of the epithelial sheet ( Lee et al, 2019 ).…”