Plasmin induces degradation and dysfunction of laminin 332 (laminin 5) and impaired assembly of basement membrane at the dermal-epidermal junction
Plasmin induces degradation and dysfunction of laminin 332 (laminin 5) and impaired assembly of basement membrane at the dermal-epidermal junction
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DOI：
10.1111/j.1365-2133.2008.08576.x
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发表时间：
2008-07-01
影响因子：
10.3
通讯作者：
Amano, S.
中科院分区：
文献类型：
--
作者：
Ogura, Y.;Matsunaga, Y.;Amano, S.
背景表皮基底膜（BM）位于真皮-表皮交界处（DEJ），不仅在表皮与真皮的粘附中起重要作用，而且在控制皮肤功能方面也起重要作用。在暴露于阳光下的皮肤中，BM变得破坏和多层。为了探索BM组装的损伤，我们使用皮肤等效物（SE）作为BM损伤的模型，并且先前阐明了基质金属蛋白酶（MMPs）参与BM组装的损伤。目的在这项工作中，我们通过使用SE检查了尿激酶型纤溶酶原激活物（uPA）和纤溶酶在DEJ的BM组装损伤中的作用，紫外线照射皮肤可引起uPA和MMPs的升高。采用酶联免疫吸附试验和酶谱法检测uPA，采用酶谱法检测明胶酶MMP-2和MMP-9。以0.06 μ mol L-1（约为血浆水平的3%）加入人纤溶酶原，使纤溶酶增加至病理水平。进行纤溶酶处理的层粘连蛋白332的N-末端肽序列分析，以鉴定层粘连蛋白332的α 3、β 3和γ 2链及其每条链的切割位点。结果除MMP-2和MMP-9外，在SE的条件培养液中还检测到人uPA。虽然在单独的MMP抑制剂的存在下，BM组织良好，但即使在抑制剂的存在下，活化的纤溶酶也使BM紊乱。与MMP抑制剂存在下培养的对照相比，BM组装的损伤使表皮变薄，表明BM影响表皮的极性和分化。加入抑肽酶（一种丝氨酸蛋白酶抑制剂）和氨甲环酸（一种uPA-纤溶酶抑制剂）可抑制纤溶酶诱导的BM组装损伤，促进BM重组，从而改善表皮结构。纤溶酶处理的层粘连蛋白332的N末端肽序列分析显示，从α 3链的G3结构域和LN结构域中去除了5或10 kDa的片段，包括细胞粘附区，LN结构域与VII型胶原中的非胶原1结构域结合，从β 3链。纤溶酶处理的层粘连蛋白332表现出较低的角质形成细胞粘附活性和减少结合到VII型collagen.Conclusions这些结果表明，uPA和纤溶酶参与损害BM组装和表皮分化，这些影响至少部分通过层粘连蛋白332的直接降解。
Background The epidermal basement membrane (BM), located at the dermal-epidermal junction (DEJ), plays important roles not only in adhesion between epidermis and dermis, but also in controlling skin functions. In sun-exposed skin, the BM becomes disrupted and multilayered. In order to explore the impairment of BM assembly, we have used a skin-equivalent (SE) as a model of BM damage and previously clarified the involvement of matrix metalloproteinases (MMPs) in impairment of BM assembly.Objectives In this work, we examined the role of urokinase-type plasminogen activator (uPA) and plasmin in impairment of BM assembly at the DEJ by using the SE, as ultraviolet irradiation to the skin increases uPA as well as MMPs.Methods SEs were used as a model of formation and damage of BM. Human uPA was detected by enzyme-linked immunosorbent assay and zymography, and gelatinases such as MMP-2 and MMP-9 were detected by zymography. Human plasminogen was added at 0.06 mu mol L-1 (about 3% of plasma level) to increase plasmin to a pathological level. N-terminal peptide sequence analysis of plasmin-treated laminin 332 was carried out to identify alpha 3, beta 3 and gamma 2 chains of laminin 332 and their cleavage sites of each chain. Plasmin-treated laminin 332 was analysed in keratinocyte adhesion activity and binding to type VII collagen.Resultls Human uPA was detected in addition to MMP-2 and MMP-9, in conditioned medium of SE. Although the BM was well organized in the presence of an MMP inhibitor alone, the activated plasmin disorganized the BM even in the presence of the inhibitor. The impairment of BM assembly made the epidermis thinner as compared with that of a control cultured in the presence of MMP inhibitor, indicating that the BM affects the polarity and differentiation of the epidermis. The addition of aprotinin, a serine proteinase inhibitor, and tranexamic acid, a uPA-plasmin inhibitor, inhibited the plasmin-induced impairment of BM assembly and facilitated BM reorganization, thereby improving the epidermal structure. N-terminal peptide sequence analysis of plasmin-treated laminin 332 revealed the removal of a 5- or 10-kDa fragment, including the cell adhesion region, from the G3 domain of the alpha 3 chain, and the LN domain, which binds to the noncollagenous 1 domain in type VII collagen, from the beta 3 chain. Plasmin-treated laminin 332 showed lower keratinocyte adhesion activity and reduced binding to type VII collagen.Conclusions These results suggest that uPA and plasmin are involved in the impairment of BM assembly and epidermal differentiation, and that these effects arise at least partly through direct degradation of laminin 332.