Smad2, Smad3 and Smad4 cooperate with Sp1 to induce p15Ink4B transcription in response to TGF-β

Transforming growth factor-beta (TGF-beta) arrests growth of epithelial cells by inducing the transcription of p15(Ink4B), a cyclin-dependent kinase inhibitor. In this study, we demonstrate that p15(Ink4B) induction was mediated by a TGF-beta-induced complex of Smad2, Smad3, Smad4 and Spl, Mutations in the Sp1- or Smad-binding sequences decreased or abolished the TGF-beta responsiveness of the p15(Ink4B) promoter. Interference with, or deficiency in, Smad2, Smad3 or Smad4 functions also reduced or abolished the TGF-beta-dependent p15(Ink4B) induction, whereas the absence of Spl reduced the basal and TGF-beta-induced p15(Ink4B) transcription. In the nucleoprotein complex, Smad2 interacted through its C-domain with Spl and enhanced the DNA binding and transcriptional activity of Spl, Smad3 interacted indirectly with Spl through its association with Smad2 and/or Smad4, and bound directly to the p15(Ink4B) promoter. Finally, Smad4 interacted through its N-domain with Spl, Our data demonstrate the physical interactions and functional cooperativity of Spl with a complex of Smad2, Smad3 and Smad4 in the induction of the p15(Ink4B) gene. These findings explain the tumor suppressor roles of Smad2 and Smad4 in growth arrest signaling by TGF-beta.