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

Transforming growth factor‐β (TGF‐β) arrests growth of epithelial cells by inducing the transcription of p15Ink4B, a cyclin‐dependent kinase inhibitor. In this study, we demonstrate that p15Ink4B induction was mediated by a TGF‐β‐induced complex of Smad2, Smad3, Smad4 and Sp1. Mutations in the Sp1‐ or Smad‐binding sequences decreased or abolished the TGF‐β responsiveness of the p15Ink4B promoter. Interference with, or deficiency in, Smad2, Smad3 or Smad4 functions also reduced or abolished the TGF‐β‐dependent p15Ink4B induction, whereas the absence of Sp1 reduced the basal and TGF‐β‐induced p15Ink4B transcription. In the nucleoprotein complex, Smad2 interacted through its C‐domain with Sp1 and enhanced the DNA binding and transcriptional activity of Sp1. Smad3 interacted indirectly with Sp1 through its association with Smad2 and/or Smad4, and bound directly to the p15Ink4B promoter. Finally, Smad4 interacted through its N‐domain with Sp1. Our data demonstrate the physical interactions and functional cooperativity of Sp1 with a complex of Smad2, Smad3 and Smad4 in the induction of the p15Ink4B gene. These findings explain the tumor suppressor roles of Smad2 and Smad4 in growth arrest signaling by TGF‐β