Regulation of Smad7 Promoter by Direct Association with Smad3 and Smad4*
- From the Department of Medical and Molecular Genetics and the Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana 46202
Abstract
Smad7 is a regulatory Smad protein that is able to antagonize signal transduction by transforming growth factor-β (TGF-β) and activin receptors. To characterize the regulation of Smad7 at the transcriptional level, we isolated the promoter region of the mouse Smad7 gene. When the Smad7 promoter luciferase reporter gene (−408 and +112 bp) was expressed in human hepatoma (HepG2) cells, its transcriptional activity was increased following TGF-β or activin treatment. In addition, this region of the Smad7 promoter was stimulated by ectopic expression of Smad3 as well as constitutively active TGF-β and activin receptors, indicating that Smad7 transcription was modulated by the signaling downstream those two receptors. A gel mobility shift assay indicated that a DNA fragment spanning −408 to −126 base pairs (bp) was able to directly bind purified Smad4. Furthermore, a consensus Smad3-Smad4 binding element (SBE) was discovered in this region of the promoter with a palindromic sequence of GTCTAGAC. A 33-bp Smad7 promoter fragment containing this SBE was able to bind Smad3 and Smad4. In human embryonic kidney 293 cells, the expression of constitutively active TGF-β type I receptor was able to induce the formation of a Smad3- and Smad4-containing nuclear protein complex that bound the SBE. In HepG2 cells, TGF-β1 treatment could induce the formation of an endogenous SBE-binding complex. Taken together, these data provided the first evidence that Smad7 transcription is regulated by TGF-β and activin signaling through direct binding of Smad3 and Smad4 to the Smad7 promoter.
Footnotes
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↵* This work was supported by the Foundation for Medical Research, Inc., Indiana University School of Medicine and by Grant-in-aid 9951372Z from the American Heart Association Midwest Affiliate (to Y. C.).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) .
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↵‡ To whom correspondence should be addressed: Dept. of Medical and Molecular Genetics, Indiana University School of Medicine, IB130, 975 West Walnut St., Indianapolis, IN 46202. Tel.: 317-278-0275; Fax: 317-274-2387; E-mail: ychen3@iupui.edu.
- Abbreviations:
- TGF-β
-
transforming growth factor-β
- ALK
-
activin receptor-like kinase
- BMP
-
bone morphogenetic protein
- FAST
-
forkhead activin signal transducer
- HEK293
-
human embryonic kidney 293 cells
- HepG2 cells
-
human hepatoma cells
- Smad
-
Sma- and Mad-related protein
- SBE
-
Smad binding element
- PCR
-
polymerase chain reaction
- bp
-
base pair(s)
- kb
-
kilobase pair(s)
- GST
-
glutathione S-transferase
- JAK
-
Janus kinase
- STAT
-
signal transducers and activators of transcription
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- Received July 15, 1999.
- Revision received September 3, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
