Transforming growth factor-beta 1 inhibition of macrophage activation is mediated via Smad3

• Published in: The Journal of biological chemistry 2000-11, Vol.275 (47), p.36653-36658
• Main Authors: Werner, F, Jain, M K, Feinberg, M W, Sibinga, N E, Pellacani, A, Wiesel, P, Chin, M T, Topper, J N, Perrella, M A, Lee, M E
• Format: Article
• Language: English
• Subjects: Animals; Biomarkers; Cell Line; DNA-Binding Proteins - metabolism; lipopolysaccharides; Lipopolysaccharides - pharmacology; Macrophage Activation - drug effects; Matrix Metalloproteinase 12; Metalloendopeptidases - metabolism; Nitric Oxide Synthase - metabolism; Nitric Oxide Synthase Type II; Nuclear Proteins - metabolism; Promoter Regions, Genetic; Smad3 Protein; Structure-Activity Relationship; Trans-Activators - metabolism; Transcription, Genetic - drug effects; Transforming Growth Factor beta - pharmacology; transforming growth factor-b1; Up-Regulation
• ISSN: 0021-9258
• DOI: 10.1074/jbc.M004536200

Activated macrophages are critical cellular participants in inflammatory disease states. Transforming growth factor (TGF)-beta1 is a growth factor with pleiotropic effects including inhibition of immune cell activation. Although the pathway of gene activation by TGF-beta1 via Smad proteins has recently been elucidated, suppression of gene expression by TGF-beta1 remains poorly understood. We found that of Smad1-Smad7, Smad3 alone was able to inhibit expression of markers of macrophage activation (inducible nitric-oxide synthase and matrix metalloproteinase-12) following lipopolysaccharide treatment in gene reporter assays. Transient and constitutive overexpression of a dominant negative Smad3 opposed the inhibitory effect of TGF-beta1. Domain swapping experiments suggest that both the Smad MH-1 and MH-2 domains are required for inhibition. Mutation of a critical amino acid residue required for DNA binding in the MH-1 of Smad3 (R74A) resulted in the loss of inhibition. Transient overexpression of p300, an interactor of the Smad MH-2 domain, partially alleviated the inhibition by TGF-beta1/Smad3, suggesting that inhibition of gene expression may be due to increased competition for limiting amounts of this coactivator. Our results have implications for the understanding of gene suppression by TGF-beta1 and for the regulation of activated macrophages by TGF-beta1.