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.
Jin
Xiannu
X
Molecular Biology Section, Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA.
Ray
Radharaman
R
Leng
Yan
Y
Ray
Prabhati
P
D015415:Biomarkers / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002478:Cells, Cultured; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003879:Dermatologic Agents / Q000494:pharmacology*; D004781:Environmental Exposure / Q000009:adverse effects; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D007797:Laminin / Q000378:metabolism; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D009151:Mustard Gas / Q000494:pharmacology*; D010618:Phenanthrolines / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D000097794:Kalinin
D015415:Biomarkers; D015815:Cell Adhesion Molecules; D015853:Cysteine Proteinase Inhibitors; D003879:Dermatologic Agents; D007797:Laminin; D007930:Leucine; D009151:Mustard Gas; D010618:Phenanthrolines; D011480:Protease Inhibitors; D015842:Serine Proteinase Inhibitors; D000097794:Kalinin; C400567:LAMC2 protein, human; C095567:laminin alpha 3; C024974:E 64; C025205:1,10-phenanthroline