Molecular determination of laminin-5 degradation: a biomarker for mustard gas exposure diagnosis and its mechanism of action.

Laminin-5, a heterotrimer of laminin alpha3, beta3 and gamma2 subunits, is a component of epithelial cell basement membranes. Laminin-5 functions as a ligand of the alpha3beta1 and alpha6beta4 integrins to regulate cell adhesion, migration and morphogenesis. In the skin, laminin-5 facilitates the assembly of basement membranes; thus it is essential for a stable attachment of the epidermis to the dermis and recovery of damaged skin. Sulphur mustard (SM), also known as mustard gas, is a vesicant that has been employed as a chemical weapon in various conflicts during the twentieth century. Skin exposure to SM results in fluid-filled blisters; proposed mechanisms are inflammation, protease stimulation, basal cell death and separation of the epidermis from the dermis apparently because of the degradation of attachment proteins like laminin-5. Therefore, we investigated the effects of SM exposure on the degradation of laminin-5 and its three subunits, alpha3, beta3 and gamma2 by exposing normal human epidermal keratinocytes (NHEK) to SM (0-300 microM, 1-24 h). We found that SM degraded laminin-5 and its two subunits beta3 and gamma2, but not alpha3. Preincubation of cells with a serine protease inhibitor (PMSF), or a metalloprotease inhibitor (1,10-phenanthroline) prior to SM exposure partially prevented SM-induced degradation of laminin-5 subunits, beta3 and gamma2. Specificity studies showed that the degradation of laminin-5 gamma2 was due to a bifunctional mustard compound such as SM, but not due to the other alkylating agents tested. Our results support that laminin-5 degradation is an important mechanism of SM injury as well as a useful biomarker of SM exposure. The knowledge of the mechanisms of laminin-5 degradation in SM-exposed NHEK has potential application in developing cutaneous therapeutics against SM.