Tenascin-C upregulation by transforming growth factor-beta in human dermal fibroblasts involves Smad3, Sp1, and Ets1.
In cultured human dermal fibroblasts, transforming growth factor (TGF)-beta induced the mRNA expression of tenascin-C (TN-C). The molecular mechanism(s) underlying this process is not presently understood. In this study, we performed serial 5' deletion and a transient transfection analysis to define a region in the TN-C promoter mediating the inducible responsiveness to TGF-beta. This region contains an atypical nucleotide recognition element for the Smad family of transcriptional regulators. A DNA affinity precipitation assay revealed that Smad2/Smad3 bound to this site in a transient and specific manner. Overexpression of Smad3 or Smad4 activated the TN-C promoter activity and superinduced the TN-C promoter activity stimulated by TGF-beta. Moreover, simultaneous cotransfection of Smad3 and Smad4 activated the TN-C promoter activity in a synergistic manner. Mutation of the Smad-binding sites, the Ets-binding sites, or Sp1/3-binding sites in the TN-C promoter abrogated the TGF-beta/Smad-inducible promoter activity. Immunoprecipitation analysis revealed that Smad3, Sp1, and Ets1 form a transcriptionally active complex. Furthermore, the interaction between Smads and CBP/p300 in TGF-beta signaling was confirmed. These findings demonstrate the existence of a novel, functional binding element in the proximal region of the TN-C promoter mediating responsiveness to TGF-beta involving Smad3/4, Sp1, Ets1, and CBP/p300.
Department of Dermatology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
Masatoshi
M
Jinnin
Hironobu
H
Ihn
Yoshihide
Y
Asano
Kenichi
K
Yamane
Maria
M
Trojanowska
Kunihiko
K
Tamaki
D001483:Base Sequence; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism*; D020405:Dermis / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000378:metabolism; D008926:Plicamycin / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D050783:Proto-Oncogene Proteins c-ets; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects; D051900:Smad3 Protein; D051901:Smad4 Protein; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D019063:Tenascin / Q000235:genetics*; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D015854:Up-Regulation / Q000187:drug effects*
D004268:DNA-Binding Proteins; C494041:ETS1 protein, human; D009687:Nuclear Proteins; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D012333:RNA, Messenger; C497790:SMAD3 protein, human; C497796:SMAD4 protein, human; D051900:Smad3 Protein; D051901:Smad4 Protein; D016329:Sp1 Transcription Factor; D019063:Tenascin; D015534:Trans-Activators; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D008926:Plicamycin