Abstract
Transforming Growth Factor β (TGFβ) is a pleiotropic cytokine closely linked to tumors. TGFβ is often elevated in precancerous breast lesions in association with epithelial-to-mesenchymal transition (EMT), indicating its contribution to precancerous progression. We previously reported that basal nitric oxide (NO) levels declined along with breast cancer progression. We then pharmacologically inhibited NO production in healthy mammary glands of wild-type mice and found that this induced precancerous progression accompanied by desmoplasia and upregulation of TGFβ activity. In the present study, we tested our hypothesis that NO directly S-nitrosylates (forms an NO-adduct at a cysteine residue) TGFβ to inhibit the activity, whereas the reduction of NO denitrosylates TGFβ and de-represses the activity. We introduced mutations to three C-terminal cysteines of TGFβ1 which were predicted to be S-nitrosylated. We found that these mutations indeed impaired S-nitrosylation of TGFβ1 and shifted the binding affinity towards the receptor from the latent complex. Furthermore, in silico structural analyses predicted that these S-nitrosylation-defective mutations strengthen the dimerization of mature protein, whereas S-nitrosylation-mimetic mutations weaken the dimerization. Such differences in dimerization dynamics of TGFβ1 by denitrosylation/S-nitrosylation likely account for the shift of the binding affinities towards the receptor vs. latent complex. Our findings, for the first time, unravel a novel mode of TGFβ regulation based on S-nitrosylation or denitrosylation of the protein.