2. Academic Validation
  3. Demonstration of P-selectin expression and potential function in human corneal epithelial cells

Demonstration of P-selectin expression and potential function in human corneal epithelial cells

  • Exp Eye Res. 2018 Nov;176:196-206. doi: 10.1016/j.exer.2018.07.018.
Peter J Gillies 1,  Neil A Richardson 2,  Jennifer Walshe 3,  Sally-Anne Stephenson 4,  Rebecca A Dawson 5,  Damien G Harkin 6
Affiliations

Affiliations

  • 1 School of Biomedical Sciences, Faculty of Health and the Institute of Health & Biomedical Innovation, Queensland University of Technology, 2 George Street, Brisbane, Queensland, 4000, Australia; Queensland Eye Institute, 140 Melbourne Street, South Brisbane, Queensland, 4101, Australia. Electronic address: peter.gillies2@health.qld.gov.au.
  • 2 School of Biomedical Sciences, Faculty of Health and the Institute of Health & Biomedical Innovation, Queensland University of Technology, 2 George Street, Brisbane, Queensland, 4000, Australia; Queensland Eye Institute, 140 Melbourne Street, South Brisbane, Queensland, 4101, Australia. Electronic address: n.richardson@qut.edu.au.
  • 3 Queensland Eye Institute, 140 Melbourne Street, South Brisbane, Queensland, 4101, Australia. Electronic address: Jennifer.young@qei.org.au.
  • 4 School of Biomedical Sciences, Faculty of Health and the Institute of Health & Biomedical Innovation, Queensland University of Technology, 2 George Street, Brisbane, Queensland, 4000, Australia; Translational Research Institute, 37 Kent Street, Woolloongabba, Queensland, 4102, Australia. Electronic address: s.stephenson@qut.edu.au.
  • 5 School of Biomedical Sciences, Faculty of Health and the Institute of Health & Biomedical Innovation, Queensland University of Technology, 2 George Street, Brisbane, Queensland, 4000, Australia; Queensland Eye Institute, 140 Melbourne Street, South Brisbane, Queensland, 4101, Australia. Electronic address: r2.dawson@qut.edu.au.
  • 6 School of Biomedical Sciences, Faculty of Health and the Institute of Health & Biomedical Innovation, Queensland University of Technology, 2 George Street, Brisbane, Queensland, 4000, Australia; Queensland Eye Institute, 140 Melbourne Street, South Brisbane, Queensland, 4101, Australia. Electronic address: d.harkin@qut.edu.au.
PMID: 30025918 DOI: 10.1016/j.exer.2018.07.018
Abstract

In response to an unexpected observation of apparent localisation by immunocytochemistry, we have investigated the potential expression and function of P-selectin (CD62P) in human corneal epithelial cells. The SV40 immortalised cell line, HCE-T (validated by STR profiling), along with multiple donor corneal-limbal tissue samples, were examined for P-selectin expression using a combination of immunocytochemistry, Western blotting, RT-PCR and immunohistochemistry. Potential expression of the major ligand for P-selectin (P-selectin glycoprotein ligand-1; PSGL-1; CD162) was also examined by immunocytochemistry and RT-PCR. A selective inhibitor of P-selectin-PSGL-1 binding (KF38789) was subsequently tested for effects on HCE-T cells using a Cell Culture gap-closure assay. HCE-T cells as well as primary epithelial cultures derived from donor corneal-limbal tissue, displayed positive immunostaining for P-selectin. Staining was particularly evident at cell-cell boundaries and at the outer edge of expanding epithelial islands. P-selectin expression was confirmed by Western blotting and RT-PCR (validated by product Sequencing), as well as by immunohistochemistry performed on serial sections of corneal-limbal tissue stained for P-selectin, keratin 3 and p63. PSGL-1 was detected by RT-PCR and immunocytochemistry in both corneal epithelial cells as well as human limbal fibroblasts (HLF). KF38789 (5 μM) significantly reduced closure of a 500-μm gap between confluent sheets of HCE-T cells over an 8-hr period (by ∼40%, p < 0.01; paired two-tailed T test), but had no effect on culture gap-closure by either HLF or murine 3T3 fibroblasts. These results provide evidence of P-selectin expression in human corneal epithelial cells and suggest a potential role for this glycoprotein in facilitating the net movement of confluent sheets of human corneal epithelial cells.

Keywords

CD162; CD62P; Corneal epithelium; Corneal limbus; P-selectin; PSGL-1.

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