Motogenic and biosynthetic response of adult skin fibroblasts to TGF-beta isoforms (-1, -2 and -3) determined by 'tissue response unit': role of cell density and substratum.

We have recently demonstrated that the three principal mammalian isoforms of transforming growth factor beta (TGF-beta) exert distinct effects upon: (1) the migration of confluent adult fibroblasts into 3D gels of native type I collagen fibres (i.e. TGF-beta-1 and -2 had no apparent motogenic activity, whilst TGF-beta-3 induced a dose-dependent stimulation of cell migration); and (2) the synthesis of hyaluronan (HA) by these cells is also affected by the TGF-beta isoforms in a manner which parallels their effect on cell migration. The objective of the present study is to elucidate the manner in which this differential activity of the TGF-beta-1, -2 and -3 may be modulated by experimental parameters. Data presented in this communication indicate that cytokine bioactivity is determined by a combination of cell density and the nature of the macromolecular substratum. Thus, we now report that all three TGF-beta isoforms inhibit the migration of subconfluent cells in the collagen gel assay. Our data confirm that the migration of confluent cells is stimulated by TGF-beta-3 and further indicate that this motogenic activity is completely abrogated by either TGF-beta-1 or -2 when these are co-incubated with TGF-beta-3. In contrast to these results obtained using a native type I collagen substratum, all three isoforms stimulated adult fibroblast migration in the transmembrane assay (in which cells are adherent to a 2-D porous polycarbonate substratum). The precise effect of TGF-beta isoforms on HA synthesis was also affected by cell density and the nature of the substratum in a manner which paralleled their diverse effects on cell migration (i.e. stimulation, inhibition or no effect). Streptomyces hyaluronidase completely neutralized the TGF-beta-3-induced stimulation of confluent fibroblast migration, thus suggesting a mechanistic link between the cytokine-induced cell migration and HA synthesis under these conditions. Taken together, these data indicate that: (1) the bioactivity of TGF-beta-1, -2 and -3 are determined by cell density, the macromolecular substratum and the presence of other cytokines; and (2) it is therefore necessary to define cytokine bioactivity within the context of a larger 'tissue response unit' which more fully defines the activity state of the target cell and its microenvironment.