Transforming Growth Factor-β1-dependent Urokinase Up-regulation and Promotion of Invasion Are Involved in Src-MAPK-dependent Signaling in Human Ovarian Cancer Cells

Abstract

Urokinase-type plasminogen activator (uPA) has been implicated in tumor cell invasion and metastasis. We reported previously that transforming growth factor (TGF)-β1 induces a dose- and time-dependent up-regulation of uPA mRNA and protein in highly invasive human ovarian cancer cell line HRA, leading to invasion. To further elucidate the mechanism of the invasive effect of TGF-β1, we investigated which signaling pathway transduced by TGF-β1 is responsible for this effect. Here, we show that 1) nontoxic concentrations of TGF-β1 activated Src kinase; 2) TGF-β1 rapidly phosphorylates ERK1/2 and Akt, but not p38; 3) pharmacological Src inhibitor PP2 or antisense (AS) c-Src oligodeoxynucleotide (ODN) treatment reduced TGF-β1-induced phosphorylation of ERK1/2 and Akt by 85-90% compared with controls; 4) pharmacological inhibition of MAPK by PD98059 abrogated TGF-β1-mediated Aht stimulation, whereas TGF-β1-induced ERK1/2 stimulation was not inhibited by PI3K inhibitor LY294002 or AS-PI3K ODN transfection; 5) up-regulation of uPA mRNA in response to TGF-β1 was almost totally blocked by PP2 and PD98059 and partially (∼55%) by LY294002; 6) TGF-β1-induced uPA mRNA up-regulation was inhibited by treatment with AS ODNs to c-Src of PI3K by 90 of 60%, respectivey, compared with control ODN treatment; and 7) blockade of the release of the transcription factor NF-κB by pyrrolidinedithiocarbamate reduced the TGF-β1-induced activation of the uPA gene by ∼65%. In addition, curcumin, a blocker of the transcriptional factor AP-1, partially (35%) canceled this effect. Taken together, these data support a role for TGF-β1 activation of two distinct pathways (Src-MAPK-PI3K-NF-κB-dependent and Src-MAPK-AP-1-dependent) for TGF-β1-dependent uPA up-regulation and promotion of invasion.