Transforming growth factor‐β1‐dependent activation of Smad2/3 and up‐regulation of PAI‐1 expression is negatively regulated by Src in SKOV‐3 human ovarian cancer cells

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Abstract
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‐β1 (TGF‐β1)‐dependent up‐regulation of PAI‐1 expression, we investigated which signaling pathway transduced by TGF‐β1 is responsible for this effect. Here, we show (1) nontoxic concentrations of TGF‐β1 up‐regulates uPA expression in HRA and SKOV‐3 human ovarian cancer cells, (2) TGF‐β1 activates Smads (phosphorylation of Smad2 and nuclear translocation of Smad3) and subsequently up‐regulates PAI‐1 expression in HRA cells, whereas TGF‐β1 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‐β1‐dependent activation of Smads, leading to PAI‐1 synthesis, compared with controls, in SKOV‐3 cells, (4) combination of TGF‐β1 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‐β1‐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‐β1. In conclusion, TGF‐β1‐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.

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