Transforming growth factor-beta1-dependent activation of Smad2/3 and up-regulation of PAI-1 expression is negatively regulated by Src in SKOV-3 human ovarian cancer cells

J Cell Biochem. 2004 Oct 15;93(3):437-53. doi: 10.1002/jcb.20160.


The net balance between urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) has been implicated in tumor cell invasion and metastasis. To elucidate the mechanism of the transforming growth factor-beta1 (TGF-beta1)-dependent up-regulation of PAI-1 expression, we investigated which signaling pathway transduced by TGF-beta1 is responsible for this effect. Here, we show (1) nontoxic concentrations of TGF-beta1 up-regulates uPA expression in HRA and SKOV-3 human ovarian cancer cells, (2) TGF-beta1 activates Smads (phosphorylation of Smad2 and nuclear translocation of Smad3) and subsequently up-regulates PAI-1 expression in HRA cells, whereas TGF-beta1 neither activates Smads nor up-regulates PAI-1 in SKOV-3 cells, (3) pharmacological Src inhibitor PP2 or antisense (AS) c-Src oligodeoxynucleotide (ODN) treatment significantly induces TGF-beta1-dependent activation of Smads, leading to PAI-1 synthesis, compared with controls, in SKOV-3 cells, (4) combination of TGF-beta1 and PP2, which activates PAI-1 expression and reduces uPA expression in SKOV-3, results in decreased invasiveness, (5) pharmacological inhibitors for mitogen-activated protein kinase (MAPK) (PD98059) and phosphoinositide-3-kinase (PI3K) (LY294002 and wortmannin) or AS-PI3K ODN transfection do not affect TGF-beta1-induced Smad signaling and up-regulation of PAI-1 expression in SKOV-3 cells pretreated with PP2, and (6) the induction of PAI-1 protein was partially inhibited by an inhibitor of Sp1-DNA binding, mithramycin, implicating, at least in part, Sp1 in the regulation of this gene by TGF-beta1. In conclusion, TGF-beta1-dependent activation of Smad2/3, leading to PAI-1 synthesis, may be negatively regulated by Src, but not its downstream targets MAPK and PI3K in SKOV-3 cells. These data also reflect the complex biological effect of uPA-PAI-1 system.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA-Binding Proteins / metabolism*
  • Female
  • Flavonoids / pharmacology
  • Humans
  • Intracellular Signaling Peptides and Proteins / pharmacology
  • MAP Kinase Signaling System / physiology
  • Ovarian Neoplasms / metabolism
  • Plasminogen Activator Inhibitor 1 / metabolism*
  • Protein Kinases / metabolism
  • Pyrimidines / pharmacology
  • Signal Transduction / physiology
  • Smad2 Protein
  • Smad3 Protein
  • Trans-Activators / metabolism*
  • Transforming Growth Factor beta / metabolism*
  • Transforming Growth Factor beta1
  • Tumor Cells, Cultured
  • Up-Regulation / physiology
  • Urokinase-Type Plasminogen Activator / metabolism
  • src-Family Kinases / antagonists & inhibitors
  • src-Family Kinases / metabolism*


  • AG 1879
  • DNA-Binding Proteins
  • Flavonoids
  • Intracellular Signaling Peptides and Proteins
  • Plasminogen Activator Inhibitor 1
  • Pyrimidines
  • SMAD2 protein, human
  • SMAD3 protein, human
  • Smad2 Protein
  • Smad3 Protein
  • TGFB1 protein, human
  • Trans-Activators
  • Transforming Growth Factor beta
  • Transforming Growth Factor beta1
  • protein kinase modulator
  • Protein Kinases
  • Sp1 kinase
  • src-Family Kinases
  • Urokinase-Type Plasminogen Activator
  • 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one