Keratinocytes suppress transforming growth factor-beta 1 expression by fibroblasts in cultured skin in substitutes

Transforming growth factor (TGF)-beta 1 is a multipotent growth factor with an important role in tissue homeostasis. This growth factor regulates cell proliferation, adhesion, migration and differentiation, as well as extrace llular matrix deposition. The temporal secretion and activation of latent T GF-beta 1 is thus of major importance to physiological and pathological pro cesses and in wound healing and tumour formation, Cultured skin substitutes , as used to treat extensive acute or chronic skin wounds, offer an attract ive model to investigate cellular interactions in cytokine and growth facto r expression and response in vitro. In the present investigation, expressio n of TGF-beta 1 was analysed in keratinocyte, fibroblast and melanocyte mon olayer cultures, as well as in the dermal vs, epidermal components of recon stituted human skin. Immunohistology, enzyme-linked immunosorbent assay (EL ISA) and Northern blotting were used to demonstrate expression at the RNA a nd protein level. In the monolayer cultures, levels of TGF-beta 1 synthesiz ed by melanocytes were observed to be considerably elevated when compared w ith keratinocytes. Most TGF-beta 1, however, was secreted by fibroblasts. T he relative contribution of the epidermal and dermal components of the skin substitutes to overall TGF-beta 1 levels was determined by comparing resul ts obtained for either component in the presence and absence of fibroblasts and keratinocytes. From results obtained by ELISA it was apparent that TGF -beta 1 levels generated predominantly by fibroblasts within the skin subst itutes were greatly reduced over time in the presence of keratinocytes. Sup pression of fibroblast TGF-beta 1 expression in the presence of keratinocyt es was also demonstrable at the RNA level by Northern blotting. Results obt ained by immunohistochemistry suggest that most, if not all, of the growth factor was present in the latent form. It is therefore most likely that the observed effect results from a factor secreted by keratinocytes, which is capable of suppressing TGF-beta 1 synthesis by fibroblasts. These results s uggest that expression of TGF-beta 1 by fibroblasts is downregulated by par acrine actions of keratinocytes in healing skin.