Transforming growth factor-beta stimulates the expression of fibronectin by human keratinocytes.

Transforming growth factor beta (TGF-beta) is a 25-kD protein which has regulatory activity over a variety of cell types. It is distinct from epidermal growth factor (EGF) and EGF analogs, and exerts its action via a distinct receptor. Its effect on proliferation or differentiation can be positive or negative depending on the cell type and the presence of other growth factors. It also modulates the expression of cellular products. TGF-beta causes fibroblasts to increase their production of the extracellular matrix components, fibronectin and collagen. Human keratinocytes (HK) are known to have TGF-beta receptors. We wished to study the effect of TGF-beta on the production of extracellular matrix proteins by human keratinocytes in culture. Human keratinocytes were grown in serum-free defined medium (MCDB-153) to about 70% confluence. Following a 16-h incubation in medium lacking EGF and TGF-beta, cells were incubated for 12 h in medium containing varying concentrations of EGF and TGF-beta. Cells were then labeled with 35S-methionine for 10 h in the same conditions. Labeled proteins from the medium were analyzed by SDS-PAGE and autoradiography. TGF-beta at 10 ng/ml induced a sixfold increase in the secretion of fibronectin, as well as an unidentified 50-kD protein. Thrombospondin production was also increased, but not over a generalized twofold increase in the production of all other proteins. EGF, at 10 ng/ml, caused a smaller additive effect. TGF-beta may be an important stimulator of extracellular matrix production by human keratinocytes.

Fibronectin extracellular matrix assembly by human epidermal cells implanted into athymic mice.

Human epidermal keratinocytes reorganize into epidermal inclusion cysts when implanted subcutaneously into athymic mice. During the organization and maturation of these cysts, fibronectin accumulates in the surrounding extracellular matrix and the basement membrane proteins bullous pemphigoid antigen and laminin appear at the epithelial-stromal interface. The sequence in which these proteins appear parallels that seen during reepithelialization of a skin wound in vivo. Fibronectin appears during aggregation of the epidermal cells and persists in the area surrounding the cysts for at least 7 days. Bullous pemphigoid antigen and laminin appear later (by 4 and 7 days, respectively) and ultimately become organized into a continuous band at the periphery of the cyst. This distribution of bullous pemphigoid antigen and laminin at the stromal-epithelial interface persists at least 5 weeks, suggesting that the implanted epidermal cells are capable of developing and maintaining a stable basement membrane zone. Fibronectin, which is abundant in the matrix adjacent to the epidermal cysts and in the surrounding stroma during cyst organization and maturation, diminishes to undetectable levels by 5 weeks. While much of the fibronectin derives from the host tissues, species-specific antibodies to human fibronectin reveal that at least a portion of this protein is synthesized and deposited by the implanted epidermal cells.