Abstract
Urokinase-type plasminogen activator (uPA) has been implicated in tumor cell invasion and metastasis. We reported previously that transforming growth factor (TGF)-beta1 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-beta1, we investigated which signaling pathway transduced by TGF-beta1 is responsible for this effect. Here, we show that 1) nontoxic concentrations of TGF-beta1 activated Src kinase; 2) TGF-beta1 rapidly phosphorylates ERK1/2 and Akt, but not p38; 3) pharmacological Src inhibitor PP2 or antisense (AS) c-Src oligodeoxynucleotide (ODN) treatment reduced TGF-beta1-induced phosphorylation of ERK1/2 and Akt by 85-90% compared with controls; 4) pharmacological inhibition of MAPK by PD98059 abrogated TGF-beta1-mediated Akt stimulation, whereas TGF-beta1-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-beta1 was almost totally blocked by PP2 and PD98059 and partially ( approximately 55%) by LY294002; 6) TGF-beta1-induced uPA mRNA up-regulation was inhibited by treatment with AS ODNs to c-Src or PI3K by 90 or 60%, respectively, compared with control ODN treatment; and 7) blockade of the release of the transcription factor NF-kappaB by pyrrolidinedithiocarbamate reduced the TGF-beta1-induced activation of the uPA gene by approximately 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-beta1 activation of two distinct pathways (Src-MAPK-PI3K-NF-kappaB-dependent and Src-MAPK-AP-1-dependent) for TGF-beta1-dependent uPA up-regulation and promotion of invasion.
Highlights
The processes of ovarian cancer dissemination are characterized by altered local proteolysis, cellular proliferation, cell attachment, and invasion, suggesting that the urokinase-type plasminogen activator1 could be involved in the patho
General tyrosine kinase inhibition with genistein or herbimycin A significantly attenuated transforming growth factor- (TGF-)1-induced extracellular signalregulated kinase 1/2 (ERK1/2) or Akt phosphorylation as did the specific Src family kinase inhibitor PP2 (Fig. 3). These results show that ERK1/2 and Akt down-regulation via pharmacological inhibition of Src and c-Src ODN treatment supports the critical position of Src upstream of mitogen-activated protein kinase (MAPK) and PI3K pathways
We found that AS-Src ODN transfection had almost complete inhibition of expression of the 50-kDa band corresponding to urokinase-type plasminogen activator (uPA) in TGF-1-stimulated HRA cells (Fig. 6, C and D)
Summary
The processes of ovarian cancer dissemination are characterized by altered local proteolysis, cellular proliferation, cell attachment, and invasion, suggesting that the urokinase-type plasminogen activator (uPA)1 could be involved in the patho-. We investigated the AS c-Src ODN transfection on TGF-1-induced uPA mRNA up-regulation in HRA cells. A and B, TGF-1 treatment of HRA cells transfected with the AS c-Src ODN (lane 3) resulted in abrogation of uPA mRNA up-regulation by ϳ90%.
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