Smad2, Smad3 and Smad4 cooperate with Sp1 to induce p15(Ink4B) transcription in response to TGF-beta.
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 Sp1. 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 Sp1 reduced the basal and TGF-beta-induced p15(Ink4B) 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 p15(Ink4B) 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 p15(Ink4B) gene. These findings explain the tumor suppressor roles of Smad2 and Smad4 in growth arrest signaling by TGF-beta.
Feng
X H
XH
Departments of Growth and Development and Anatomy, and Programs in Cell Biology and Developmental Biology, University of California, San Francisco, CA 94143-0640, USA. xfeng@bcm.tmc.edu
Lin
X
X
Derynck
R
R
D030201:Activin Receptors, Type I; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D002460:Cell Line; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D016147:Genes, Tumor Suppressor; D005973:Glutamine / Q000737:chemistry; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000502:physiology; D000077293:Receptor, Transforming Growth Factor-beta Type I; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D051899:Smad2 Protein; D051900:Smad3 Protein; D016329:Sp1 Transcription Factor / Q000737:chemistry / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D025521:Tumor Suppressor Proteins
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004268:DNA-Binding Proteins; D018125:Receptors, Transforming Growth Factor beta; D051899:Smad2 Protein; D051900:Smad3 Protein; D016329:Sp1 Transcription Factor; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D025521:Tumor Suppressor Proteins; D005973:Glutamine; D017346:Protein Serine-Threonine Kinases; D030201:Activin Receptors, Type I; D000077293:Receptor, Transforming Growth Factor-beta Type I; D000077294:Receptor, Transforming Growth Factor-beta Type II