Upregulation of α₉Integrin and Tenascin During Epithelial Regeneration After Debridement in the Cornea

Abstract

SUMMARY Stratified epithelia are exposed to abrasive forces and are required to respond rapidly to injury to minimize fluid loss and the risk for microbial infection. Healing involves a cell migratory phase to reestablish barrier function and cell proliferation to restratify the epithelium. Cell migration during re-epithelialization involves cell sliding, termed sheet movement, during which cells retain their cell-cell junctions while dynamically altering their shape and cell-substrate interactions to permit movement across the exposed wound bed. Proteins of the integrin family of receptor molecules modulate cell shape, cell migration, and signal transduction in many cell types. In epithelial cells, integrins of the ␤ 1 family have been implicated in regulating cell proliferation and differentiation. ␣ 9 ␤ b 1 is one of the newer members of the integrin ␤ 1 family and has been recently shown to function as a tenascin receptor. Although little is known about its function in vivo, studies in developing mouse cornea and eyelid suggest that it may play a role in epithelial differentiation. Using a debridement wound model in the mouse cornea, we show in this study that (a) in response to small debridement wounds that close without cell proliferation, ␣ 9 integrin protein and mRNA are not induced during migration but are induced during restratification, (b) larger debridement wounds that require cell proliferation to generate the cells necessary for sheet movement result in a dramatic induction of ␣ 9 protein and its mRNA during both migration and restratification, and (c) tenascin, an ␣ 9 ␤ 1 ligand, accumulates beneath epithelial cells during restratification but not during cell migration. Therefore, ␣ 9 integrin protein production and tenascin accumulation are dynamically regulated in response to corneal epithelial injury.

Defects in keratinocyte activation during wound healing in the syndecan-1-deficient mouse

“…Both α3β1 and α6β4 laminin-binding integrins have been implicated in mediating epithelial cell migration in response to injury (Belkin and Stepp, 2000), and their ligand, laminin-5, and its cleavage play a role in facilitating stable or transient integrin-mediated cellular attachment (Gianelli et al, 1997;Goldfinger et al, 1998;Goldfinger et al, 1999). α6β4 protein expression increases in wild-type mice after corneal scrape wounding (Stepp et al, 1997), and in this study, we show a comparable increase in the wounded synd1ko mouse corneas. However, after induction of cell proliferation in skin by dermabrasion, β4 integrin localization was unaltered in the wild-type and synd1ko basal cells, suggesting that the enhanced expression of α6β4 after full thickness skin wounding maybe related more to the disassembly of hemidesmosomes and induction of cell migration than to induction of cell proliferation.…”

“…Manual debridement wounds were created on the corneas of 8-week-old synd1ko and wild-type mice, as described previously (Stepp and Zhu, 1997). Mice were anaesthetized with general anesthesia and their eyes numbed with a topical anesthetic.…”

“…Corneal or skin tissues were sectioned (8 µm), and unfixed frozen sections used for immunofluorescence microscopy as described previously (Stepp and Zhu, 1997). Synd1 localization was evaluated using a rat monoclonal antibody against the synd1 core protein (281-2, now commercially available from Pharmingen, cat # 09341D; San Diego, CA) and the synd4 core protein.…”

“…Corneas were wounded as described above, and after sacrifice, eyes were immediately placed in a BrdU-containing labeling solution consisting of complete MEM (Stepp et al, 1997). After 30 minutes in labeling solution, the eyes were washed three times and allowed to incubate for an additional 15 minutes in MEM without BrdU.…”

Cdk5 regulates activation and localization of Src during corneal epithelial wound closure

“…Manual debridement wounds were created on the corneas of 8-10-week-old wild-type and ALDH3-Cdk5 transgenic mice as described (Stepp and Zhu, 1997). Mice were anesthetized with general anesthesia and their eyes numbed with a topical anesthetic.…”