Plasma membrane calcium-ATPase isoform four distribution changes during corneal epithelial wound healing

Abstract

Purpose: Plasma Membrane Calcium-ATPases (PMCAs) are integral membrane proteins essential to the control of intracellular Ca2+ concentration. In humans, four genes encode PMCA proteins termed PMCA1-PMCA4. PMCA4 is the major PMCA isoform expressed in human corneal epithelium (hCE); however, little is known about its role. The present study documented expression of PMCA4 in rabbit CE (rbCE) and followed the distribution of PMCA4 during CE wound healing in a rabbit (rb) model. Methods: Reverse transcriptase PCR using PMCA4 isoform gene-specific primers that flanked alternative splice site A was used to examine the presence of PMCA4 mRNA in rbCE. Protein expression was assessed by immunoblotting using panPMCA- and PMCA4-specific antibodies. Immunocytochemistry was employed to examine PMCA immunolocalization in frozen, formaldehyde-fixed sections of control and wounded rb corneas. In wound healing studies, circular, 6-mm diameter corneal wounds were produced in the central CE using the n-heptanol technique. The distribution of PMCA4 in CE was examined by immunohistochemical staining of frozen sections using PMCA4 isoform-specific antibody at 6-, 24-, 36-, and 48 h post-injury. siRNA(PMCA4) was used to transfect telomerase-immortalized human corneal epithelial (hTCEpi) cells. Cell cultures were wounded 48 h after transfection, and the wound area was measured at 0 h and at 3 h intervals post-wounding. Results: Direct sequencing of PCR DNAs documented the presence of PMCA4 transcripts in rbCE and showed that the splice variant at site A was PMCA4x. Immunoblot analysis for PMCA4 detected an intense band at approximately 160 kDa and a faint band at approximately 142 kDa. Immunohistochemistry with the panPMCA antibody demonstrated strong immunoreactivity (IR) in all layers of uninjured rbCE. Immunohistochemistry with a PMCA4-specific antibody demonstrated a similar pattern of intense IR along the plasma membrane of cells in all layers of CE, except for the notable absence of PMCA4 IR along the basal cell membranes adjacent to the stroma. The pattern of PMCA4 IR changed following wound healing. During the lag phase of corneal epithelial wound healing, PMCA4 IR was seen mostly on apical plasma membranes of basal cells near the wound margin, with little staining of basal plasma membranes. During the migration phase (24 h), PMCA4 IR was found mostly on basal cell membranes adjacent to the stroma. At 6 h and 24 h following wounding, PMCA4 IR of the cytoplasm was increased compared to control eyes. After closure of the denuded area and stratification, PMCA4 IR was again primarily found along the apical and lateral plasma membranes of basal cells and was again absent from basal cell membranes adjacent to the stroma; PMCA4 IR of the cytoplasm was also similar to that observed in control eyes. siRNA(PMCA4) transfected hTCEpi cells failed to seal the wound area, whereas wounds in control cultures transfected with a scrambled construct were completed healed. Conclusions: PMCA4 is strongly expressed in rabbit CE and its immunolocalization exhibits marked changes in distribution during the wound healing process. Knockdown of PMCA4 expression in hTCEpi cells decreases wound healing. Present findings suggest that PMCA4 redistribution could function as one factor in mediating calcium-regulated events necessary for cell migration in regenerating CE.