Connexin 43 (Cx43) Serine Residue Phosphorylation (pser) in the Corneal Epithelium During Homeostasis and Wound Repair

Abstract

Abstract: : Purpose: This study aims to characterise the phosphorylation state of Cx43 during corneal epithelial homeostasis, and the changes that occur during wound repair. Methods: Bovine corneas were maintained in organ culture for up to 7 days, cryopreserved and sectioned. Immunofluorescence detection of 5 antibodies i) Cx43 (phosphorylation–state independent), ii) Cx43npcterm, iii) Cx43pser255, iv)Cx43pser262, v) Cx43pser279/282 was performed using Alexafluor conjugates and analysed. Positive controls were bovine heart and liver. Results: In the unwounded cornea, Cx 43 was immunolocalised to a single basal layer of cells in the limbus, with both intensity and number of cell layers staining increasing with proximity to the centre. Cx43npcterm, Cx43pser255 and Cx43pser262 showed similar staining patterns to the phosphorylation–independent antibody, increasing toward the central epithelium, although Cx43pser 255 showed additional strong nuclear staining in the more superficial cell layers.Cx43pser 279/282 staining was absent. Following wounding, Cx43 was lost at the leading edge, and Cx43pser262 staining decreased, co–ordinated with an increase in nuclear pser255. Transient location of a) Cx43pser279/282 was seen in the basal cells within the wound–site immediately following closure, and b) Cx43pser262 was observed, associated with sites of proliferation. C–terminus phosphorylation was lost during migration over the cut edge of the wound Conclusions: Cx43 appears to be phosphorylated at serine residues 255 and 262 on the carboxyl tail, but not at 279/282 or any of the c–terminus cluster serines during homeostasis. During repair phospho–isoform changes may regulate both proliferation and differentiation of corneal cells.

Keywords: cell-cell communication • cornea: epithelium • phosphorylation