Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Activin Receptors, Type I*
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Blotting, Northern
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Carrier Proteins / biosynthesis
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Carrier Proteins / genetics*
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Carrier Proteins / metabolism
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Cell Cycle
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Cell Cycle Proteins*
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Cell Line
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Cyclin-Dependent Kinase Inhibitor p15
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Cyclin-Dependent Kinase Inhibitor p16*
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DNA-Binding Proteins / genetics
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DNA-Binding Proteins / metabolism*
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Genes, Tumor Suppressor
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Glutamine / chemistry
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Promoter Regions, Genetic
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Protein Binding
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Protein Serine-Threonine Kinases / physiology
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Receptor, Transforming Growth Factor-beta Type I
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Receptor, Transforming Growth Factor-beta Type II
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Receptors, Transforming Growth Factor beta / physiology
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Reverse Transcriptase Polymerase Chain Reaction
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Signal Transduction
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Smad2 Protein
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Smad3 Protein
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Sp1 Transcription Factor / chemistry
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Sp1 Transcription Factor / metabolism*
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Trans-Activators / genetics
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Trans-Activators / metabolism*
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Transcriptional Activation
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Transforming Growth Factor beta / metabolism*
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Tumor Suppressor Proteins*
Substances
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Carrier Proteins
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Cell Cycle Proteins
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Cyclin-Dependent Kinase Inhibitor p15
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Cyclin-Dependent Kinase Inhibitor p16
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DNA-Binding Proteins
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Receptors, Transforming Growth Factor beta
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Smad2 Protein
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Smad3 Protein
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Sp1 Transcription Factor
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Trans-Activators
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Transforming Growth Factor beta
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Tumor Suppressor Proteins
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Glutamine
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Protein Serine-Threonine Kinases
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Activin Receptors, Type I
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Receptor, Transforming Growth Factor-beta Type I
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Receptor, Transforming Growth Factor-beta Type II