K. Pardali et al., Role of smad proteins and transcription factor Sp1 in p21(Wafl/Cip1) regulation by transforming growth factor-beta, J BIOL CHEM, 275(38), 2000, pp. 29244-29256
Transforming growth factor-beta (TGF-beta) inhibits cell cycle progression,
in part through up-regulation of gene expression of the p21(WAF1/Cip1) (p2
1) cell cycle inhibitor. Previously we have reported that the intracellular
effecters of TGF-beta, 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 HaCaT p21 gene by TGF-beta 1 is further enhanced after o
verexpression of Smad3 and Smad4, whereas dominant negative mutants of Smad
3 and Smad4 and the inhibitory Smad7 all inhibit p21 induction by TGF-beta
1 in a dose-dependent manner. We show that Sp1 expressed in the Sp1-deficie
nt Drosophila SL-2 cells binds to the proximal 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 pr
omoter as efficiently as wild type Smads. Co-expression of Smad3 with Smad4
and Sp1 in SL-2 cells or coincubation of phosphorylated Smad3, Smad4, and
Sp1 in vitro results in enhanced binding of Sp1 to the p21 proximal promote
r 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 glutami
ne-rich sequences in the transactivation domain of Sp1 contribute to the co
operativity with Smad proteins. In conclusion, Smad proteins play important
roles in regulation of the p21 gene by TGF-beta, and the functional cooper
ation of Smad proteins with Sp1 involves the physical interaction of these
two types of transcription factors.