Functional characterization of transforming growth factor beta signaling in Smad2- and Smad3-deficient fibroblasts.
A prominent pathway of transforming growth factor (TGF)-beta signaling involves receptor-dependent phosphorylation of Smad2 and Smad3, which then translocate to the nucleus to activate transcription of target genes. To investigate the relative importance of these two Smad proteins in TGF-beta1 signal transduction, we have utilized a loss of function approach, based on analysis of the effects of TGF-beta1 on fibroblasts derived from mouse embryos deficient in Smad2 (S2KO) or Smad3 (S3KO). TGF-beta1 caused 50% inhibition of cellular proliferation in wild-type fibroblasts as assessed by [(3)H]thymidine incorporation, whereas the growth of S2KO or S3KO cells was only weakly inhibited by TGF-beta1. Lack of Smad2 or Smad3 expression did not affect TGF-beta1-induced fibronectin synthesis but resulted in markedly suppressed induction of plasminogen activator inhibitor-1 by TGF-beta1. Moreover, TGF-beta1-mediated induction of matrix metalloproteinase-2 was selectively dependent on Smad2, whereas induction of c-fos, Smad7, and TGF-beta1 autoinduction relied on expression of Smad3. Investigation of transcriptional activation of TGF-beta-sensitive reporter genes in the different fibroblasts showed that activation of the (Smad binding element)(4)-Lux reporter by TGF-beta1 was dependent on expression of Smad3, but not Smad2, whereas activation of the activin response element-Lux reporter was strongly suppressed in S2KO fibroblasts but, on the contrary, enhanced in S3KO cells. Our findings indicate specific roles for Smad2 and Smad3 in TGF-beta1 signaling.
Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. pieke@mail.nih.gov
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Piek
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Heyer
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Escalante-Alcalde
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Stewart
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Weinstein
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Deng
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Kucherlapati
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Bottinger
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Roberts
D000818:Animals; D018797:Cell Cycle Proteins; D002455:Cell Division; D050762:Cyclin-Dependent Kinase Inhibitor p15; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D017781:Genes, Immediate-Early; D017930:Genes, Reporter; D016762:Genes, fos; D051379:Mice; D018345:Mice, Knockout; D015398:Signal Transduction; D051899:Smad2 Protein; D051900:Smad3 Protein; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D016212:Transforming Growth Factor beta / Q000096:biosynthesis / Q000378:metabolism*; D025521:Tumor Suppressor Proteins
C493957:Cdkn1a protein, mouse; C493968:Cdkn2b protein, mouse; D018797:Cell Cycle Proteins; D050762:Cyclin-Dependent Kinase Inhibitor p15; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D051899:Smad2 Protein; C497783:Smad2 protein, mouse; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; D015534:Trans-Activators; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D025521:Tumor Suppressor Proteins