Transforming growth factor-beta stimulates collagen VII expression by cutaneous cells in vitro.

Abstract

. Collagen VII, the major component of cutaneous anchoring fibrils is expressed at a low level by normal human keratinocytes and fibroblasts in vitro. In cocultures of these two cell types, signals from fibroblasts enhance expression of collagen VII by keratinocytes and vice versa . In this study, the effects of a possible mediator of such a stimulation, transforming growth factor-ß (TGF-ß), were investigated . Its effect on the expression and deposition of the highly insoluble collagen VII was assessed in a semiquantitative manner by a newly developed enzyme-linked immunoassay which is based on immunoblotting . In keratinocyte monocultures, 0.5-20 ng/ml of TGF-ß2 induced a dose-dependent stimulation of collagen VII expression as measured per microgram of DNA . The maximal enhancement was about sevenfold compared to con-HE dermo-epidermal junction of the skin represents an epithelial-mesenchymal interface with a unique structure and highly specialized functions (Palade and Farquhar, 1965 ;Bruns, 1969 ; Briggaman and Wheeler, 1975x ;Tidman and Eady, 1984 ;Burgeson, 1987). One ofits main functions is to provide the resistance ofthe skin against shearing forces which requires strong cohesion of the skin layers . This is achieved by several interconnected macromolecular networks which attach the epidermis to the basement membrane and to the underlying dermal connective tissue (Timpl, 1989;Burgeson et al ., 1990 ; Yurchenko and Schittny, 1990) . One of the major structures mediating the attachment is the anchoring fibril network that extends from the basement membrane to the anchoring plaques in thepapillary dermis Keene et al., 1987) . Abnormalities ofthe anchoring fibrils lead to separation of the epidermis from the dermis and to clinical blistering of the skin, as seen in dystrophic epidermolysis bullosa, a group of inherited blistering disorders (Briggaman and Wheeler, 1975b ;Hashimoto et al ., 1976;Tidman and Eady, 1985 ;Heagerty et al ., 1986 ;Bruckner-Tuderman et al., 1989,

Proteinases of the Bone Morphogenetic Protein-1 Family Convert Procollagen VII to Mature Anchoring Fibril Collagen

“…Extraction of proteins from normal or DEB human skin biopsies and normal or Bmp1 Ϫ/Ϫ mouse embryo skin (41) was performed as described (42).…”

Some, but Not All, Glycine Substitution Mutations inCOL7A1 Result in Intracellular Accumulation of Collagen VII, Loss of Anchoring Fibrils, and Skin Blistering

“…The reaction was stopped by adding soybean trypsin inhibitor (Sigma) to a final concentration of 10 g/ml (34). Proteins were precipitated with ethanol, redissolved in sample buffer, separated by SDS-polyacrylamide gel electrophoresis, electrotransferred to nitrocellulose, and analyzed by immunoblotting using domain-specific collagen VII antibodies (3,28).…”

“…The medium was precipitated with ethanol in the presence of proteinase inhibitors (28), and the cell layer was extracted with a neutral buffer containing 0.1 M NaCl, 0.020 M Tris-HCl, pH 7.4, 1% Nonidet P-40, and a mixture of proteinase inhibitors (32). The samples were subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting using domain-specific collagen VII antibodies (3).…”

“…Keratinocyte Cultures-Keratinocytes obtained by trypsinization of skin biopsies were cultured in serum-free, low calcium keratinocyte growth medium (28) supplemented with bovine pituitary extract and epidermal growth factor (keratinocyte growth medium, Life Technologies, Inc.). Prior to analyses, cells at an early passage received 50 g/ml L-ascorbate for 48 h (28,29).…”

“…The samples were subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting using domain-specific collagen VII antibodies (3). Semiquantitative immunoassays were used to determine relative amounts of collagen VII in the cells and the medium (28).…”