Efficient TGF-β Induction of the Smad7 Gene Requires Cooperation between AP-1, Sp1, and Smad Proteins on the Mouse Smad7 Promoter*
- From the ‡Ludwig Institute for Cancer Research, Box 595, Biomedical Center, S-751 24 Uppsala, Sweden and ¶The Netherlands Cancer Institute, Division of Cellular Biochemistry, H3, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
Abstract
Sma- and Mad-related protein 7 (Smad7) is an antagonist of transforming growth factor-β (TGF-β) signaling, which has been shown to be induced by TGF-β itself and also by other stimuli. In an effort to understand the molecular mechanisms underlying the transcriptional regulation of the Smad7 gene by TGF-β, we cloned and functionally characterized a mouse genomic DNA fragment encompassing the mouse Smad7 proximal promoter. This region was found to contain a CpG island and to be devoid of a classical TATA box. Cloned upstream of a promoter-lacking luciferase reporter gene, this region conferred robust TGF-β-induced transcription. Point mutations in a palindromic Smad binding element, abolished TGF-β inducibility completely. Through the use of electrophoretic mobility shift assays, we showed the presence of Smad2, Smad3, and Smad4 in complexes binding to the Smad binding element. Interestingly, we also found that point mutation and/or deletion of binding sites for the transcription factors activator protein-1 and Sp1 led to an attenuation of the basal promoter activity, as well as of the TGF-β-mediated induction of Smad7. Taken together, our data imply that Smads, together with activator protein-1 and Sp1 transcription factors, are essential for efficient Smad7 promoter activity.
Footnotes
-
↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) .
-
↵§ These authors both contributed equally to this work.
-
↵‖ Supported by the Dutch Cancer Foundation.
-
↵** To whom correspondence should be addressed: Ludwig Inst. for Cancer Research, Box 595, S-751 24 Uppsala, Sweden. Tel.: 46 18 16 04 08; Fax: 46 18 16 04 20; E-mail: Rainer.Heuchel@LICR.uu.se.
-
Published, JBC Papers in Press, June 7, 2000, DOI 10.1074/jbc.M002815200
- Abbreviations:
- TGF-β
-
transforming growth factor-β
- Smad
-
Sma- and Mad-related protein
- SBE
-
Smad binding element
- AP-1
-
activator protein-1
- bp
-
base pair(s)
- PCR
-
polymerase chain reaction
- CREB
-
cAMP-responsive element-binding protein
- HepG2 cells
-
human hepatoma cells
-
- Received April 3, 2000.
- Revision received June 7, 2000.
- The American Society for Biochemistry and Molecular Biology, Inc.
