The alpha 3 laminin subunit, alpha 6 beta 4 and alpha 3 beta 1 integrin coordinately regulate wound healing in cultured epithelial cells and in the skin

Previously, we demonstrated that proteolytic processing within the globular domain of the alpha 3 subunit of laminin-5 (LN5) converts LN5 from a cell motility-inducing factor to a protein complex that can trigger the formatio n of hemidesmosomes, certain cell-matrix attachment sites found in epitheli al cells. We have prepared a monoclonal antibody (12C4) whose epitope is lo cated toward the carboxy terminus of the globular domain of the alpha 3 lam inin subunit. This epitope is lost from the alpha 3 subunit as a consequenc e of proteolytic processing. Antibody 12C4 stains throughout the matrix of cells that fail to process the alpha 3 laminin subunit, but does not recogn ize the matrix of confluent cultures of MCF-10A cells, which efficiently pr ocess their alpha 3 laminin chain. In subconfluent populations of MCF-10A c ells, 12C4 only stains matrix deposited at the outer edges of cell colonies . In these cells, integrin alpha 3 beta 1 occasionally colocalizes with the staining generated by the 12C4 antibody but alpha 6 beta 4 integrin does n ot. In wounded MCF-10A cell cultures, the 12C4 antibody stains the extracel lular matrix beneath those cells at the very edge of the cellular sheet tha t moves to cover the wound site. A similar phenomenon is observed in human skin wounds, since we also detect expression of the unprocessed alpha 3 lam inin subunit at the leading tip of the sheet of epidermal cells that epithe lializes skin wounds in vivo. In addition, using alpha 3 laminin subunit an d integrin function-inhibiting antibodies, we provide evidence that LN5 and its two integrin receptors (alpha 6 beta 4 and alpha 3 beta 1) appear nece ssary for wound healing to occur in MCF-10A cell culture wounds. We propose a model for healing of wounded epithelial tissues based on these results.