TGF-β (transforming growth factor-β) plays a critical role in modulating the inflammatory response and other biological processes through its regulation of the production of MMPs (matrix metalloproteinases). In both Mono-Mac-6 and RAW264.7 monocyte/macrophage cells, TGF-β abrogated lipopolysaccharide-induced increases in the enzymic activity and mRNA level of MMP-9. A fragment of the human MMP-9 promoter was used to characterize its regulation by TGF-β signalling. In RAW264.7 cells, TGF-β or its downstream signalling protein, Smad3 (Sma- and Mad-related protein 3), inhibited lipopolysaccharide-stimulated promoter activity. The suppressive activity of TGF-β on the MMP-9 promoter was abrogated by an inhibitory Smad, Smad7. The MMP-9 promoter contains a putative TIE (TGF-β inhibitory element). However, neither mutation nor deletion of the TIE had any effect on the inhibitory activity of TGF-β on MMP-9 transcription, indicating that the consensus TIE is not required for this effect of TGF-β. Analysis using a series of deletion mutants of the MMP-9 promoter revealed that a region containing a consensus NF-κB (nuclear factor-κB) site is required for the basal activity and TGF-β-mediated suppression of the promoter. Mutation of the putative NF-κB site not only markedly reduced the basal transcriptional activity of the promoter, but also abrogated the responsiveness of the promoter to TGF-β. In addition, a minimal promoter containing one copy of the NF-κB sequence was responsive to TGF-β treatment. Furthermore, an electrophoretic mobility shift assay was performed with the nuclear extracts from RAW264.7 cells, and it was found that TGF-β treatment did not disrupt the binding of NF-κB p50 and p65 proteins to the NF-κB sequence. Taken together, these studies indicate that the NF-κB site is indispensable for the suppressive activity of TGF-β in the regulation of MMP-9 transcription.
TG-interacting factor (TGIF) is a transcriptional co-repressor that directly associates with Smad (Sma- and Mad-related protein) proteins and inhibits Smad-mediated transcriptional activation. By using Affymetrix (Santa Clara, CA, U.S.A.) oligonucleotide microarray analysis, we found that TGIF mRNA level was elevated by transforming-growth-factor-β (TGF-β) treatment in a human T-cell line, HuT78. Subsequent reverse-transcription PCR assays indicated that TGF-β1 and activin were able to induce a rapid and transient increase in the level of TGIF in both HuT78 and HepG2 hepatoma cells. To analyse whether or not the regulation of TGIF mRNA occurs at the transcriptional level, a 2.4kb human TGIF promoter was isolated. A primer extension assay was performed to localize the putative transcription initiation site of the promoter. When transiently expressed in HepG2 cells, this promoter was stimulated by TGF-β1 and activin treatment in a time-dependent manner. A series of deletion mutants of the TGIF promoter were also generated to further characterize the TGF-β responsive region of the promoter. In addition, expression of TGIF was able to cause a dose-dependent inhibition of TGF-β and activin signalling. Taken together, these experiments indicated that TGIF is a novel transcriptional target of TGF-β and activin signalling and is likely involved in a negative feedback loop to desensitize TGF-β/activin action.