Interaction of Integrin alpha-3/beta-1 and Laminin-5 Modulates Alveolar Epithelial Barrier Formation

Overview

Objectives/Goals This study investigated the interaction between integrin a3/b1and its ligand, laminin-5 in modulating alveolar epithelial barrier. Methods/Materials Cell Culture and preparation of slides- Primary type II cells were harvested from male Sprague Dawley rats. Procedures for cell extraction and animal sacrifice were pre-approved by review board. Culture media was changed every day. Slides that had an Ln-5 rich matrix were coated in Ln-5 for two hours and cells placed thereafter. Slides with extracellular matrix protein coating were also coated for two hours and cells placed thereafter. Western Blot- Western blots were done on SDS-page using a 7.5% stacking gel. The protein concentration of samples was determined through Bio Rad DC protein assay kit. Standard Western Blot procedure was followed. Immunofluorescence- Standard immunoflorescence procedure was followed. Results Cells plated on Ln-5 developed confluence at <24 h, those plated on plastic without Ln-5 developed confluence at 24-48 h, those plated on anti-integrin antibodies didn#t develop confluence. Uniform scratch made with needle on confluent monolayers of AECs. Wound closure measured serially +/- anti-integrin a3 mAb, or either anti-Ln-5 mAb with/without blocking activity. Closure faster on Ln-5-coated (at <48 h) to uncoated plastic (50% at 48 h). Presence of anti-integrin a3 mAb reduced rate of closure for cells plated on Ln-5, and prevented closure for cells on plastic Conclusions/Discussion . Interaction is likely important for alveolar epithelial cell migration and spreading during re-epithelialization of alveolar surface following lung injury in vivo. Cells coated on Laminin-5 had higher levels of Integrin a3-b1, which was probably attributed to the surface affinity of Laminin-5 or Laminin-5#s ability to up regulate levels of Integrin a3-b1. Interaction is likely to be important for epithelial barrier formation, which consists of cell adhesion, monolayer formation, migration and wound healing. Cell adhesion and spreading either induces Integrin a3-b1, is promoted by Integrin a3-b1 or both. The Laminin-5 coated filters bind cells at their a3b1 receptors, which accelerates attachment and spreading, and probably induces the expression of more a3b1, which further hastens the development of monolayers.

Awards (1)

• Category Award

Competition history

• CSEF 2002 Biochemistry/ Molecular Biology · Entry S0416

Resources

• CSEF project summary ↗

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