Differences in Integrin Expression and Function in Epithelial Cells Derived from the Syndecan-1 Knockout Mouse

Abstract

Abstract: : Purpose: Our goal is to study, at the molecular level, the mechanisms controlling corneal epithelial cell migration during wound healing. We have shown that syndecan-1 knockout mice show signs of delayed corneal and epidermal healing and that the mechanisms behind the healing defects in both tissues appear similar (Stepp, et al., J. Cell Science, 115, 4517-4531, 2002). Because of difficulties culturing primary mouse ocular surface epithelial cells, we decided to use epidermal keratinocytes derived from the syndecan-1 ko mouse to assess the mechanisms underlying the corneal and skin healing defects. Methods: Primary mouse keratinocytes from Balb/c wt and syndecan-1 knockout 1-3 day old pups were isolated and cultured as described by Yuspa and colleagues (J. Invest. Dermatol. 76, 144-146, 1981). Cells were plated in fibronectin/collagen coated dishes and cultured in standard low Ca²⁺ (0.05mM) medium. On day 4, cells were either shifted to high Ca²⁺ (0.12mM) medium or maintained in standard medium. 24 hr after shifting cells to high calcium, cell lysates were prepared and equal micrograms total protein analyzed by immunoblotting for differences in α3 and ß1 integrin expression. Results: Preliminary data indicates that when cultured using standard media conditions of 0.05mM Ca²⁺, syndecan-1 ko cells have more α3 integrin than wt cells; it is unclear whether ß1 and/or other epithelial integrins are also overexpressed in the syndecan-1 ko cells. It is known that when the calcium concentration is increased, cultured epithelial cells are stimulated to undergo terminal differentiation over a 24-48 hour time period and this process is known to involve reduced expression of ß1 family integrins. While both wt and syndecan-1 ko cells reduced their expression of ß1 and α3 integrins 24 hr after the calcium shift, knockout cells appeared to reduce their expression to a greater extent than did wt cells. Conclusions: Our in vivo wound healing data showed delayed epithelial healing in syndecan-1 ko mouse. The results presented above using primary epithelial cells cultured from the syndecan-1 ko mouse indicate that differences in integrin expression and sensitivity to calcium are present in cells derived from the syndecan-1 knockout mouse. These differences might well impact corneal and skin healing in the these mice.

Keywords: cell adhesions/cell junctions • cornea: epithelium • extracellular matrix