Transforming growth factor-beta 1 inhibition of macrophage activation is mediated via Smad3.
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.
Werner
F
F
Program of Developmental Cardiovascular Biology, Harvard Medical School, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.
Jain
M K
MK
Feinberg
M W
MW
Sibinga
N E
NE
Pellacani
A
A
Wiesel
P
P
Chin
M T
MT
Topper
J N
JN
Perrella
M A
MA
Lee
M E
ME
D000818:Animals; D015415:Biomarkers; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation / Q000187:drug effects*; D053508:Matrix Metalloproteinase 12; D008666:Metalloendopeptidases / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D051900:Smad3 Protein; D013329:Structure-Activity Relationship; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D015854:Up-Regulation
D015415:Biomarkers; D004268:DNA-Binding Proteins; D008070:Lipopolysaccharides; D009687:Nuclear Proteins; D051900:Smad3 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D008666:Metalloendopeptidases; D053508:Matrix Metalloproteinase 12