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

Abstract

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.

Time course of skin features and inflammatory biomarkers after liquid sulfur mustard exposure in SKH-1 hairless mice

“…After SM exposure, previous studies have revealed a reduction of its immunoreactivity (Greenberg et al, 2006;Smith et al, 1997bSmith et al, , 1998. Moreover in an in vitro model, it has been demonstrated that this process is associated with the degradation of the chain gamma-2 in a SM dose-and a time-dependent manner and that this degradation can be prevented by the use of protease inhibitor (Jin et al, 2008). Similarly to our results, an increase of Lamc2 mRNA was observed in mouse skin after SM exposure (Chang et al, 2009;Vallet et al, 2012).…”

“…Interestingly a key candidate is the anchoring filament protein of the basement membrane, laminin-332. Some studies have shown that SM exposure causes a significant reduction of the immunoreactivity of the latter protein (Greenberg et al, 2006;Smith et al, 1997bSmith et al, , 1998 which has been recently characterized by the degradation of the chain gamma-2 (Jin et al, 2008). In addition, some studies have suggested that proteases such as matrix metalloproteinases (MMP) are main players in SM-induced skin blister formation (Lindsay and Rice, 1995;Powers et al, 2000;Sabourin et al, 2002).…”

Sulfur Mustard Exposure and Cardiovascular Effects: A Review

“…Prolonged inflammatory status, defects in adhering molecules such as laminine and fibronectin and impaired repair processes can predispose injured vessels to dilation and ectasia. This defect in the repair process of connective tissue is notable after exposure with alkylating agents, such as SM (64,65).…”

Sulfur mustard-stimulated proteases and their inhibitors in a cultured normal human epidermal keratinocytes model: A potential approach for anti-vesicant drug development

“…The major target of SM in the skin are the replicating basal epidermal keratinocytes, which remain attached to the sub epithelial layer (dermis) via some attachment proteins e.g., laminin-5, integrin etc. Protease activation and release at this epidermal-dermal junction degrades these attachment proteins and as a consequence the epithelial layer detaches from the substratum and eventually micro blister formation [27] . The above discussion suggests that SM-induced protease activation may contribute to the pathology of blister formation; therefore, inhibitors of SM-stimulated proteases may serve as prospective therapeutics for intervention for sulfur mustard toxicity.…”