Role of Smad Proteins and Transcription Factor Sp1 in p21Waf1/Cip1 Regulation by Transforming Growth Factor-β

doi:10.1074/jbc.M909467199

Role of Smad Proteins and Transcription Factor Sp1 in p21^Waf1/Cip1 Regulation by Transforming Growth Factor-β*

From the Ludwig Institute for Cancer Research, Box 595, SE-751 24 Uppsala, Sweden, ^§The Netherlands Cancer Institute, Division of Cellular Biochemistry, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands, and ^¶Department of Basic Sciences, University of Crete Medical School and Institute of Molecular Biology and Biotechnology, F.O.R.T.H., Heraklion GR-711 10, Greece

Abstract

Transforming growth factor-β (TGF-β) inhibits cell cycle progression, in part through up-regulation of gene expression of the p21^WAF1/Cip1(p21) cell cycle inhibitor. Previously we have reported that the intracellular effectors of TGF-β, Smad3 and Smad4, functionally cooperate with Sp1 to activate the human p21 promoter in hepatoma HepG2 cells. In this study we show that Smad3 and Smad4 when overexpressed in HaCaT keratinocytes lead to activation of the p21 promoter. Activation requires the binding sites for the ubiquitous transcription factor Sp1 on the proximal promoter. Induction of the endogenous HaCaTp21 gene by TGF-β1 is further enhanced after overexpression of Smad3 and Smad4, whereas dominant negative mutants of Smad3 and Smad4 and the inhibitory Smad7 all inhibit p21induction by TGF-β1 in a dose-dependent manner. We show that Sp1 expressed in the Sp1-deficient Drosophila SL-2 cells binds to the proximal p21 promoter sequences, whereas Smad proteins do not. In support of this finding, we show that DNA-binding domain mutants of Smad3 and Smad4 are capable of transactivating the p21 promoter as efficiently as wild type Smads. Co-expression of Smad3 with Smad4 and Sp1 in SL-2 cells or co-incubation of phosphorylated Smad3, Smad4, and Sp1 in vitro results in enhanced binding of Sp1 to the p21 proximal promoter sequences. We demonstrate that Sp1 physically and directly interacts with Smad2, Smad3, and weakly with Smad4 via their amino-terminal (Mad-Homology 1) domain. Finally, by using GAL4 fusion proteins we show that the glutamine-rich sequences in the transactivation domain of Sp1 contribute to the cooperativity with Smad proteins. In conclusion, Smad proteins play important roles in regulation of the p21 gene by TGF-β, and the functional cooperation of Smad proteins with Sp1 involves the physical interaction of these two types of transcription factors.

Footnotes

↵* This research was supported in part by a grant from the Human Frontier Science Program (to D. K. and A. M.), Institute of Molecular Biology and Biotechnology for internal funds (to D. K.), and a grant from the Dutch Cancer Society (to P. t. D.).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.
↵‡ Recipient of a STINT post-doctoral fellowship from the Swedish Foundation for International Cooperation in Research and High Education.
↵‖ To whom correspondence should be addressed: Ludwig Institute for Cancer Research, Box 595, SE-751 24 Uppsala, Sweden. Tel.: 46-18-160411/2; Fax: 46-18-160420; E-mail: aris.moustakas@licr.uu.se.
Published, JBC Papers in Press, June 30, 2000, DOI 10.1074/jbc.M909467199
↵2 Morén, A., Itoh, S., Moustakas, A., ten Dijke, P., and Heldin, C.-H. (2000) Oncogene 19, in press.
Abbreviations:

TGF-β

transforming growth factor β

CKI

cyclin-dependent kinase inhibitor

Smad

Sma and Mad-related protein

MH

Mad-Homology

SBE

Smad-binding element, ALK, activin receptor-like kinase

CA

constitutively active

HaCaT

human keratinocyte cell line

HepG2

human hepatoma cell line

m.o.i.

multiplicity of infection

GEMSA

gel electrophoretic mobility shift assay

PAGE

polyacrylamide gel electrophoresis

CAT

chloramphenicol acetyltransferase

GST

glutathione S-transferase

DMEM

Dulbecco's modified Eagle medium

PMSF

phenylmethylsulfonyl fluoride

CDKs

cyclin-dependent kinases

HA

hemagglutinin

DN

dominant negative

DTT

dithiothreitol
- Received November 30, 1999.
- Revision received May 25, 2000.
The American Society for Biochemistry and Molecular Biology, Inc.