Regulation of transforming growth factor β1 by nitric oxide

Many tumor cells or their secreted products suppress the function of tumor-infiltrating macrophages. Tumor cells often produce abundant transforming growth factor β1 (TGF-β1), which in addition to other immunosuppressive actions suppresses the inducible isoform of NO synthase. TGF-β1 is secreted in a latent form, which consists of TGF-β1 noncovalently associated with latency-associated peptide (LAP) and which can be activated efficiently by exposure to reactive oxygen species. Coculture of the human lung adenocarcinoma cell line A549 and ANA-1 macrophages activated with IFN- γ, plus lipopolysaccharide resulted in increased synthesis and activation of latent TGF-β1 protein by both A549 and ANA-1 cells, whereas unstimulated cultures of either cell type alone expressed only latent TGF-β1. We investigated whether exposure of tumor cells to NO influences the production, activation, or activity of TGF-β1. A549 human lung adenocarcinoma cells exposed to the chemical NO donor diethylamine-NONOate showed increased immunoreactivity of cell-associated latent and active TGF-β1 in a time- and dose-dependent fashion at 24-48 h after treatment. Exposure of latent TGF- β1 to solution sources of NO neither led to recombinant latent TGF-β1 activation nor modified recombinant TGF-β1 activity. A novel mechanism was observed, however: treatment of recombinant LAP with NO resulted in its nitrosylation and interfered with its ability to neutralize active TGF-β1. These results provide the first evidence that nitrosative stress influences the regulation of TGF-β1 and raise the possibility that NO production may augment TGF-β1 activity by modifying a naturally occurring neutralizing peptide.