title: Plasmin induces degradation and dysfunction of laminin 332 (laminin 5) and impaired assembly of basement membrane at the dermal-epidermal junction.
abstract: 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.
  
  
  OBJECTIVE
  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 micromol 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 alpha3, beta3 and gamma2 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.
  
  
  RESULTS
  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 alpha3 chain, and the LN domain, which binds to the noncollagenous 1 domain in type VII collagen, from the beta3 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.
authors.affiliation: Shiseido Life Science Research Center, 2-2-1 Hayabuchi, Tsuzuki-ku, Yokohama 224-8558, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Ogura
authors.forename: Y
authors.initials: Y
authors.lastname: Matsunaga
authors.forename: T
authors.initials: T
authors.lastname: Nishiyama
authors.forename: S
authors.initials: S
authors.lastname: Amano
mesh_terms: D001485:Basement Membrane / Q000378:metabolism* / Q000648:ultrastructure; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002478:Cells, Cultured; D024143:Collagen Type VII / Q000378:metabolism; D020405:Dermis / Q000378:metabolism* / Q000648:ultrastructure; D004817:Epidermis / Q000378:metabolism* / Q000648:ultrastructure; D005341:Fibrinolysin / Q000378:metabolism*; D005343:Fibrinolytic Agents / Q000378:metabolism*; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D008854:Microscopy, Electron; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000378:metabolism; D000097794:Kalinin
chemical_list: D015815:Cell Adhesion Molecules; D024143:Collagen Type VII; D005341:Fibrinolysin; D005343:Fibrinolytic Agents; D020778:Matrix Metalloproteinase 2; D019716:Tissue Inhibitor of Metalloproteinase-2; D000097794:Kalinin