Cross-talk between the p42/p44 MAP Kinase and Smad Pathways in Transforming Growth Factor β1-induced Furin Gene Transactivation

François Blanchette,Nathalie Rivard,Penny Rudd,Francine Grondin,Liliana Attisano,Claire M Dubois

doi:10.1074/jbc.m100093200

Abstract

Furin, a predominant convertase of the cellular constitutive secretory pathway, is known to be involved in the maturation of a number of growth/differentiation factors, but the mechanisms governing its expression remain elusive. We have previously demonstrated that transforming growth factor (TGF) beta 1, through the activation of Smad transducers, regulates its own converting enzyme, furin, creating a unique activation/regulation loop of potential importance in a variety of cell fate and functions. Here we studied the involvement of the p42/p44 MAPK pathway in such regulation. Using HepG2 cells transfected with fur P1 LUC (luciferase) promoter construct, we observed that forced expression of a dominant negative mutant form of the small G protein p21(ras) (RasN17) inhibited TGF beta 1-induced fur gene transcription, suggesting the involvement of the p42/p44 MAPK cascade. In addition, TGF beta induced sustained activation/phosphorylation of endogenous p42/p44 MAPK. Further-more, the role of MAPK cascade in fur gene transcription was highlighted by the use of the MEK1/2 inhibitors, PD98059 or U0126, or co-expression of a p44 antisense construct that repressed the induction of fur promoter transactivation. Conversely, overexpression of a constitutively active form of MEK1 increased unstimulated, TGF beta 1-stimulated, and Smad2-stimulated promoter P1 transactivation, and the universal Smad inhibitor, Smad7, inhibited this effect. Activation of Smad2 by MEK1 or TGF beta 1 resulted in an enhanced nuclear localization of Smad2, which was inhibited upon blocking MEK1 activity. Our findings clearly show that the activation of the p42/p44 MAPK pathway is involved in fur gene expression and led us to propose a co-operative model whereby TGF beta 1-induced receptor activation stimulates not only a Smad pathway but also a parallel p42/p44 MAPK pathway that targets Smad2 for an increased nuclear translocation and enhanced fur gene transactivation. Such an uncovered mechanism may be a key determinant for the regulation of furin in embryogenesis and growth-related physiopathological conditions.

Highlights

Furin is a mammalian subtilisin/Kex2p-like Ca2ϩ-dependant endoprotease involved in the processing of various types of higher molecular mass precursor substrates, containing the minimal basic amino acid RXXR recognition motif
Our findings clearly show that the activation of the p42/p44 MAPK pathway is involved in fur gene expression and led us to propose a co-operative model whereby transforming growth factor (TGF)␤1-induced receptor activation stimulates a Smad pathway and a parallel p42/p44 MAPK pathway that targets Smad2 for an increased nuclear translocation and enhanced fur gene transactivation
Because TGF␤1 is widely documented to be implicated in the regulatory mechanisms of cellular growth and differentiation (39 – 41), it was of interest to determine whether the classical proliferation/differentiation module Ras/Raf/MEK/p42/p44 MAPK is involved in fur P1 promoter transactivation

Summary

Introduction

Furin is a mammalian subtilisin/Kex2p-like Ca2ϩ-dependant endoprotease involved in the processing of various types of higher molecular mass precursor substrates, containing the minimal basic amino acid RXXR recognition motif. Using HepG2 cells transfected with fur P1 LUC (luciferase) promoter construct, we observed that forced expression of a dominant negative mutant form of the small G protein p21ras (RasN17) inhibited TGF␤1-induced fur gene transcription, suggesting the involvement of the p42/p44 MAPK cascade.

Results

Conclusion