Latent and active transforming growth factor β1 released from genetically modified keratinocytes modulates extracellular matrix expression by dermal fibroblasts in a coculture system

Transforming growth factor beta1 is a multifunctional cytokine involved in many aspects of wound healing. Here we report the effects of both latent and active transforming growth factor beta1 released from genetically modified keratinocytes on extracellular matrix expression by dermal fibroblasts in a coculture system. Human keratinocytes were genetically modified with adenovirus containing cDNA for latent transforming growth factor beta1 (AdTGF-beta1) or active transforming growth factor beta1 (AdTGF-beta1(223/225) ) or LacZ and cultured with human dermal fibroblasts. Northern blotting for mRNA confirmed that keratinocytes were successfully transduced with the adenoviruses as the cDNA transcripts are smaller than native transforming growth factor beta1 mRNA. An enzyme-linked immunosorbent assay specific for transforming growth factor beta1 demonstrated that the transforming growth factor beta1 produced by the genetically modified keratinocytes was able to pass through the membrane separating the two cell layers. Levels of transforming growth factor beta1 were significantly higher for both latent (p < 0.0001) and active (p < 0.0001) transforming growth factor beta1 compared to the LacZ control. Without acid activation of samples, keratinocytes transduced with the active transforming growth factor beta1 construct exhibited significantly higher levels of transforming growth factor beta1 than either the latent construct or the LacZ control (p < 0.0001). The transforming growth factor beta1 produced was biologically active, as shown by the plasminogen activator inhibitor assay (p < 0.0001). To demonstrate that transforming growth factor beta1 had an effect on underlying fibroblasts, mRNA was extracted and analyzed using Northern analysis. Latent transforming growth factor beta1 significantly increased the expression of type I collagen mRNA (p < 0.05) but did not significantly affect collagenase mRNA. Active transforming growth factor beta1 significantly increased type I collagen mRNA (p < 0.005) while also decreasing collagenase mRNA (p < 0.05). These results illustrate the ability of increased levels of transforming growth factor beta1 to override the effects of normal keratinocytes on the behavior of dermal fibroblasts.