Involvement of Smads in TGFβ1‐induced furin (fur) transcription

Abstract

Furin is recognized as being one of the main convertases of the cellular constitutive secretion pathway but the mechanisms regulating its expression are still unknown. We have previously demonstrated that TGFbeta1 up-regulates its own converting enzyme, furin, creating a novel activation/regulation cycle of potential importance in a variety of physiological and pathophysiological conditions. The fur (fes upstream region) gene is regulated via three alternative promoters; P1, P1A, and P1B. To gain insight into the molecular mechanism(s) underlying this up-regulation, we performed transient cell transfections with P1, P1A, and P1B promoter luciferase constructs. Transfection experiments in HepG2 cells revealed that fur P1 promoter is the strongest and the most sensitive to TGFbeta1 stimulation (5 ng/ml) (3.2-fold vs. 2.4-fold for P1A and 2.1-fold for P1B). Cotransfection with either a dominant negative mutant form of Smad2 [Smad2(3SA)] or a known Smad inhibitor [Smad7] inhibit constitutive and TGFbeta1-induced luciferase activity indicating the participation of endogenous Smads. Increased levels of TGFbeta1-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 Smad2/Smad4 complexes. Furthermore, the fork-head activin signal transducer (FAST-1), known to interact with Smad2/Smad4 complexes, is a potent stimulator of TGFbeta1-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 present data demonstrate that Smad2 and Smad4 possibly in complex with FAST-1 or other DNA binding partners participate in the constitutive and inducible transactivation of the fur P1 promoter. This represents the first detailed study of the transcriptional regulation of the fur gene.

Platelet-Derived Growth Factor Receptor Activation Promotes the Prodestructive Invadosome-Forming Phenotype of Synoviocytes from Patients with Rheumatoid Arthritis

“…In arthritic synoviocytes, evidence suggests that at least two pathways may be involved in PDGFR phosphorylation and activation. First, RA or growth factor-activated synoviocytes were shown to secrete PDGF ligands and to express the intracellular (e.g., furin) or extracellular (e.g., tPA, uPA) proteases required for their activation (59,60,(64)(65)(66). In this study, the findings that RA FLS overexpressed PDGF-B and that the invadosome-forming phenotype of RA cells was inhibited using PDGF-BB-specific or PAN-PDGF-neutralizing Abs suggest direct phosphorylation of PDGFR as a result of ligation by autocrine PDGF-B.…”

Furin gene (fur) regulation in differentiating human megakaryoblastic Dami cells: involvement of the proximal GATA recognition motif in the P1 promoter and impact on the maturation of furin substrates

“…Plates were incubated overnight, and cell lysates were assayed for luciferase activity as previously described. 23 Data were from at least 3 independent experiments performed in duplicate. Values were normalized for transfection efficiency with either green fluorescence protein (GFP) mean fluorescence or ␤-galactosidase activity.…”

“…42 Northern blot analysis was performed as previously described using a rat cDNA probe (generously provided by Dr Robert Day, University of Sherbrooke, Canada). 2,23 Immunocytochemistry Dami cells were grown on Goldline microscope slides (VWR Canlab, ville Mont-Royal, QC, Canada) in the presence or absence of 100 nM PMA for 3 days. Cells were fixed in cold methanol and permeabilized in cold phosphate-buffered saline-0.1% Triton.…”

“…22 Recent observations indicated that in HepG2 hepatic cells, the P1 promoter is the strongest and the most sensitive to TGF-␤. 23 Studies of the transcriptional activity involved indicate that the Smad signal transducers are central participants of TGF-␤-induced fur regulation in these cells. However, the transcriptional machinery involved in the regulation of furin in other cell types, including megakaryocytes, remains unknown.…”

“…In the nonhematopoietic HepG2 cell, for example, the transcriptional activity of the P1 promoter is in part under the control of endogenous C/EBP-␤ and of members of the Smad transducers. 22,23 The known capacity and yet to be uncovered capacity of the fur promoters to respond to various transcriptional contexts would likely help explain the temporal and spatial expression of furin in megakaryocytes and in other tissues and organs.The proximal region of the fur P1 promoter contains 2 potential GATA-binding sites at positions Ϫ66 and ϩ62. The relative contribution of each GATA-1-binding site to the transcriptional activity of the promoter was examined.…”