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Originally published In Press as doi:10.1074/jbc.M010485200 on February 5, 2001

J. Biol. Chem., Vol. 276, Issue 19, 16573-16579, May 11, 2001
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Interaction between GC Box Binding Factors and Smad Proteins Modulates Cell Lineage-specific alpha 2(I) Collagen Gene Transcription*

Yutaka InagakiDagger §, Tomoyuki Nemoto||, Atsuhito Nakao**, Peter ten DijkeDagger Dagger , Kenichi Kobayashi, Kazuhiko Takehara§§, and Patricia Greenwel¶¶

From the Dagger  Department of Internal Medicine and Division of Clinical Research, National Kanazawa Hospital, Kanazawa 920-8650, Japan, the  First Department of Internal Medicine and §§ Department of Dermatology, Kanazawa University School of Medicine, Kanazawa 920-8640, Japan, the ** Allergy Research Center, Juntendo University School of Medicine, Tokyo 113-8421, Japan, the Dagger Dagger  Division of Cellular Biochemistry, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands, and the ¶¶ Brookdale Center in the Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, New York, New York 10029

Type I collagen is produced predominantly in mesenchymal cells, but molecular mechanisms responsible for cell type-specific expression are virtually unknown. During fibrogenic process in the liver, activated hepatic stellate cells (HSC) are the main producers of type I collagen, whereas parenchymal hepatocytes produce little, if any, of this protein. We have previously reported that Sp1 and an interacting unknown factor(s) bind to the -313 to -255 sequence of the alpha 2(I) collagen gene (COL1A2) and play essential roles for basal and TGF-beta -stimulated transcription in skin fibroblasts and HSC. Recently, Smad3 has been shown to bind to this region, and its interaction with Sp1 has been implicated in TGF-beta -elicited COL1A2 stimulation. The present study demonstrates predominant binding of Sp3 rather than Sp1 to this regulatory element in parenchymal hepatocytes. In these cells, this region did not exhibit strong enhancer activity or mediate the effect of TGF-beta . Transfection of HSC with an Sp3 expression plasmid abolished the COL1A2 response to TGF-beta , whereas overexpression of Sp1 in hepatocytes increased basal COL1A2 transcription and conferred TGF-beta responsiveness. Functional and physical interactions between Sp1 and Smad3, but not between Sp3 and Smad3, were demonstrated using the bacterial GAL4 system and immunoprecipitation-Western blot analyses. These results indicate that cell lineage-specific interactions between GC box binding factors and Smad protein(s) may account, at least in part, for differential COL1A2 transcription and TGF-beta responsiveness in HSC and parenchymal hepatocytes.

* This work was supported in part by a research grant from the Scleroderma Research Committee of the Ministry of Health and Welfare, Japan (to Y. I.), by a grant-in-aid for Cancer Research from the Ministry of Health and Welfare, Japan (to Y. I.), by a grant from The Netherlands Organization for Scientific Research (to P. t. D.), and by Grant R01 AA12196 from the National Institute on Alcohol Abuse and Alcoholism (to P. G.).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.

§ To whom correspondence should be addressed: Dept. of Internal Medicine and Division of Clinical Research, National Kanazawa Hospital, 1-1 Shimoishibiki-machi, Kanazawa 920-8650, Japan. Tel.: 81-76-262-4161; Fax: 81-76-263-3450; E-mail: inagaki@kinbyou.hosp.go.jp.

|| Present address: The Fourth Division, Osaka Bioscience Inst., Suita, Osaka 565-0874, Japan.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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