Involvement of Smads in TGF beta 1-induced furin (fur) transcription

Furin is recognized as being one of the main convertases of the cellular co nstitutive secretion pathway but the mechanisms regulating its expression a re still unknown. We have previously demonstrated that TGF beta1 up-regulat es its own converting enzyme, furin, creating a novel activation/regulation cycle of potential importance in a variety of physiological and pathophysi ological conditions. The fur (fes upstream region) gene is regulated via th ree alternative promoters; P1, P1A, and P1B. To gain insight into the molec ular mechanism(s) underlying this up-regulation, we performed transient cel l transfections with P7, P1A, and P1B promoter luciferase constructs. Trans fection experiments in HepG2 cells revealed that fur PI promoter is the str ongest and the most sensitive to TGF beta1 stimulation (5 ng/ml) (3.2-fold vs. 2.4-fold for P1A and 2.1-fold for P7 B). Cotransfection with either a d ominant negative mutant form of Smad2 [Smad2(3SA)] or a known Smad inhibito r [Smad7] inhibit constitutive and TCF beta1-induced luciferase activity in dicating the participation of endogenous Smads. increased levels of TGF bet a1-induced transcriptional activation of the P1 promoter by overexpression of Smad2 and/or Smad4 is greatly reduced in the presence of Smad2(3SA) and completely inhibited by Smad7, suggesting the participation of endogenous S mad2/Smad4 complexes. Furthermore, the fork-head activin signal transducer (FAST-1), known to interact with Smad2/Smad4 complexes, is a potent stimula tor of TGF beta1-induced transactivation of the fur P1 promoter. Five prime -deletion analysis of this promoter identified the proximal region (between positions -8734 and -7925), as the nucleotide stretch that carries most of the transcriptional activation of fur P1 promoter by Smad2. Overall, the p resent data demonstrate that Smad2 and Smad4 possibly in complex with FAST- 1 or other DNA binding partners participate in the constitutive and inducib le transactivation of the fur P1 promoter. This represents the first detail ed study of the transcriptional regulation of the fur gene. (C) 2001 Wiley- Liss, Inc.