1. ¹Department of Dermatology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan
2. ²Division of Rheumatology and Immunology, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425-2229, USA

Correspondence: H Ihn, E-mail: IN-DER@h.u-tokyo.ac.jp

Abstract

In cultured human dermal fibroblasts, transforming growth factor (TGF)- 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-. 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-. 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-/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- 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- involving Smad3/4, Sp1, Ets1, and CBP/p300.