Jin Xiannu, Ray Radharaman, Leng Yan, Ray Prabhati
Molecular Biology Section, Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA.
Exp Dermatol. 2008 Jan;17(1):49-56. doi: 10.1111/j.1600-0625.2007.00632.x.
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.
层粘连蛋白-5是由层粘连蛋白α3、β3和γ2亚基组成的异源三聚体，是上皮细胞基底膜的一个组成部分。层粘连蛋白-5作为α3β1和α6β4整合素的配体，发挥调节细胞黏附、迁移和形态发生的作用。在皮肤中，层粘连蛋白-5促进基底膜的组装；因此，它对于表皮与真皮的稳定附着以及受损皮肤的修复至关重要。硫芥（SM），也被称为芥子气，是一种糜烂性毒剂，在20世纪的各种冲突中被用作化学武器。皮肤接触硫芥会导致充满液体的水疱；推测的机制包括炎症、蛋白酶刺激、基底细胞死亡以及表皮与真皮的分离，这显然是由于诸如层粘连蛋白-5等附着蛋白的降解所致。因此，我们通过将正常人表皮角质形成细胞（NHEK）暴露于硫芥（0 - 300微摩尔，1 - 24小时），研究了硫芥暴露对层粘连蛋白-5及其三个亚基α3、β3和γ2降解的影响。我们发现硫芥可降解层粘连蛋白-5及其两个亚基β3和γ2，但不降解α3。在硫芥暴露之前，用丝氨酸蛋白酶抑制剂（苯甲基磺酰氟，PMSF）或金属蛋白酶抑制剂（1,10 - 菲咯啉）对细胞进行预孵育，可部分阻止硫芥诱导的层粘连蛋白-5亚基β3和γ2的降解。特异性研究表明，层粘连蛋白-5γ2的降解是由于双功能芥子化合物如硫芥，而非由于所测试的其他烷基化剂。我们的结果支持层粘连蛋白-5降解是硫芥损伤的一个重要机制以及硫芥暴露的一个有用生物标志物。了解硫芥暴露的NHEK中层粘连蛋白-5降解的机制在开发针对硫芥的皮肤治疗方法方面具有潜在应用价值。
