Pannexin channels regulate cell‐cell calcium mobilization and motility during epithelial wound repair

Abstract

The cornea is a transparent avascular tissue that transmits light to the retina and covers the lens and iris. According to the World Health Organization, blindness due to injury and disease in the cornea is the fourth leading cause of preventable blindness. Previous research demonstrates that epithelial cells release nucleotides that activate purinergic receptors P2X7 and P2Y2, which induces cell‐cell calcium mobilization. This calcium mobilization is reported to potentially play a role in collective cell migration and wound healing. To better understand this specific mobilization, we studied pannexin, a channel forming glycoprotein. ATP is able to move through the Pannexin channel and binds with P2X7. We want to investigate the mechanism of the calcium signal propagation and whether pannexins are involved. We hypothesize that pannexin channels mediate cell‐cell calcium mobilization and regulates actin activity in response to injury or agonist stimulation. Experiments were performed using 10Panx, an inhibitory peptide for pannexin channels, and scramble Panx, a control peptide. Human Corneal Limbal Epithelial (HCLE) cells were cultured to confluence on glass bottom dishes. Twenty‐four hours prior to experimentation, growth supplements were removed. Cells were preincubated with either 100uM 10Panx or 100uM scramble Panx to inhibit pannexin channels. In order to visualize the change in calcium, cells were pre‐loaded with 5 μM Fluo‐3AM, a fluorescent dye, at a final concentration of 1% (v/v) DMSO and 0.02% (w/v) pluronic acid for 20 minutes at 37°C and 5% CO2. Images were collected every 3 seconds on a Zeiss Axiovert LSM 880 confocal microscope for a period of 45 minutes after either agonist stimulation or injury. The agonist used for stimulation was BzATP, a synthetic nucleotide that selectively activates P2X7 and UTP, an agonist for P2Y2. Baseline and post‐wound frames were captured. Analysis was performed using FIJI/ImageJ and MATLAB programs. We found that 10PanX correlated with dampened calcium mobilization after injury. In uninjured cultures the decrease was only detected in response to BzATP and not to UTP. Actin activity was also inhibited by 10PanX after injury. MATLAB analysis using heatmap indicates that 10PanX reduces correlation of cells during calcium mobilization (Scr BzATP mean: 0.030 10PanXBzATP mean:0.079). Our data indicates that P2X7‐mediated Pannexin channels play a role in the injury response. This provides greater insight as to the level of involvement of pannexin in calcium signal propagation in response to injury in HCLE cells.Support or Funding InformationNIH 5R25HL118693‐05, NIH RO1 EY06000, NIH R21 EY024392, The Mass Lions Foundation New England Corneal Transplant FundThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.