Reduced S-nitrosylation of TGFβ1 elevates its binding affinity towards the receptor and promotes fibrogenic signaling in the breast

Abstract

Transforming Growth Factor {beta} (TGF{beta}) is a pleiotropic cytokine closely linked to tumors. TGF{beta} 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 TGF{beta} activity. In the present study, we tested our hypothesis that NO directly S-nitrosylates (forms an NO-adduct at a cysteine residue) TGF{beta} to inhibit the activity, whereas the reduction of NO denitrosylates TGF{beta} and de-represses the activity. We introduced mutations to three C-terminal cysteines of TGF{beta}1 which were predicted to be S-nitrosylated. We found that these mutations indeed impaired S-nitrosylation of TGF{beta}1 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 TGF{beta}1 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 TGF{beta} regulation based on S-nitrosylation or denitrosylation of the protein. Significance statementTransforming Growth Factor {beta} (TGF{beta}) is a widely studied cytokine associated with tumors. Because of its pleiotropic functions and dichotomous roles in tumorigenesis, the development of therapeutics targeted to TGF{beta} for cancer treatment has been challenging. In the present study, we report that TGF{beta} is indeed S-nitrosylated at specific sites for repressing its functions, whereas it is denitrosylated to derepress its activity. Such covalent modification-based regulation of TGF{beta} activity could potentially be utilized to design a new type of inhibitor or activator of the protein.