TGF-beta 1-induced PAI-1 expression is E box/USF-dependent and requires EGFR signaling

Exp Cell Res. 2006 Apr 15;312(7):1093-105. doi: 10.1016/j.yexcr.2005.12.027. Epub 2006 Feb 7.

Abstract

Transforming growth factor-beta1 (TGF-beta1) transcriptionally regulates the expression of genes that encode specific proteins (e.g., plasminogen activator inhibitor-1; PAI-1) important in stromal remodeling and cellular invasion. Definition of molecular events underlying TGF-beta1-initiated PAI-1 transcription, therefore, may lead to the identification of new therapeutic targets for diseases associated with elevated PAI-1 synthesis (e.g., tissue fibrosis, vascular disorders, tumor progression). An intact upstream stimulatory factor (USF)-binding E box motif (5'-(-165)CACGTG(-160)-3') at the HRE-2 site in the rat PAI-1 gene was required for PAI-1 transcription in TGF-beta1-treated cells. Mutation of the CA dinucleotide to TC at position -165/-164 in a reporter construct driven by 764 bp of PAI-1 promoter sequence decreased TGF-beta1-dependent CAT activity by >80% indicating the necessity for a consensus hexanucleotide E box motif in induced expression. The same CA --> TC substitution eliminated USF binding to an 18-bp HRE-2 DNA target highlighting the importance of site occupancy to transcriptional activation. Transfection of a dominant-negative USF construct, moreover, completely inhibited formation of USF/HRE-2 probe complexes, attenuated PAI-1 promoter-driven luciferase activity and reduced the response of the endogenous PAI-1 gene to TGF-beta1 (to that approximating quiescent controls). Maximal immediate-early PAI-1 induction upon exposure to TGF-beta1 required EGFR, p21ras, MEK and pp60(c-src) signaling as pharmacologic or dominant-negative inhibition of any of the four intermediates (EGFR, p21ras, MEK, pp60(c-src)) virtually eliminated TGF-beta1-augmented PAI-1 levels. U0126 titering experiments, furthermore, revealed that the same MEK inhibitor concentration that blocked the TGF-beta1 increase in ERK1/2 phosphorylation (20 microM) also effectively attenuated the PAI-1 inductive response suggesting a requirement for stimulated ERK signaling in TGF-beta1-mediated PAI-1 expression. These data suggest a model whereby TGF-beta1 activates a complex signaling cascade to affect PAI-1 gene control and involves USF occupancy of a critical E box motif at the HRE-2 site in the PAI-1 gene.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Cell Line
  • E-Box Elements / physiology*
  • Enzyme Inhibitors / pharmacology
  • ErbB Receptors / antagonists & inhibitors
  • ErbB Receptors / physiology*
  • Humans
  • MAP Kinase Kinase Kinases / physiology
  • Mutagenesis, Site-Directed
  • Plasminogen Activator Inhibitor 1 / biosynthesis*
  • Plasminogen Activator Inhibitor 1 / genetics
  • Promoter Regions, Genetic
  • Proto-Oncogene Proteins pp60(c-src) / physiology
  • Quinazolines
  • Rats
  • Signal Transduction / physiology*
  • Transforming Growth Factor beta / physiology*
  • Transforming Growth Factor beta1
  • Tyrphostins / pharmacology

Substances

  • Enzyme Inhibitors
  • Plasminogen Activator Inhibitor 1
  • Quinazolines
  • TGFB1 protein, human
  • Tgfb1 protein, rat
  • Transforming Growth Factor beta
  • Transforming Growth Factor beta1
  • Tyrphostins
  • RTKI cpd
  • ErbB Receptors
  • Proto-Oncogene Proteins pp60(c-src)
  • MAP Kinase Kinase Kinases