Suppression of Urokinase Expression and Invasiveness by Urinary Trypsin Inhibitor Is Mediated through Inhibition of Protein Kinase C- and MEK/ERK/c-Jun-dependent Signaling Pathways

Abstract

Urinary trypsin inhibitor (UTI), a Kunitz-type protease inhibitor, interacts with cells as a negative modulator of the invasive cells. Human ovarian cancer cell line, HRA, was treated with phorbol ester (PMA) to evaluate the effect on expression of urokinase-type plasminogen activator (uPA), since the action of uPA has been implicated in matrix degradation and cell motility. Preincubation of the cells with UTI reduced the ability of PMA to trigger the uPA expression at the gene level and at the protein level. UTI-induced down-regulation of PMAstimulated uPA expression is irreversible and is independent of a cytotoxic effect. Down-regulation of uPA by UTI is mediated by its binding to the cells. We next asked whether the mechanism of inhibition of uPA expression by UTI was due to interference with the protein kinase C second messenger system. An assay for PKC activity demonstrated that UTI does not directly inhibit the catalytic activity of PKC and that PMA translocation of PKC from cytosol to membrane was inhibited by UTI, indicating that UTI inhibits the activation cascade of PKC. PMA could also activate a signaling pathway involving MEK1/ERK2/c-Jun-dependent uPA expression. When cells were preincubated with UTI, we could detect suppression of phosphorylation of these proteins. Like several types of PKC inhibitor, UTI inhibited PMA-stimulated invasiveness. We conclude that UTI markedly suppresses the cell motility possibly through negative regulation of PKC-and MEK/ERK/c-Jun-dependent mechanisms, and that these changes in behavior are correlated with a coordinated down-regulation of uPA which is likely to contribute to the cell invasion processes.

Reproductive failure in mice lacking inter-alpha-trypsin inhibitor (ITI) – ITI target genes in mouse ovary identified by microarray analysis

“…In addition, tumor necrosis factor (TNF)-is a cytotoxic cytokine that is produced in progressive amounts during the secretory phase. It has been reported that bikunin has an ability to inhibit expression of several cytokines including TNFby inflammatory cells or neoplastic cells (Kobayashi et al 2001). Here, we show that TNF-receptor (osteoprotegerin; see below) is constitutively overexpressed in bik / mouse ovary.…”

“…Interestingly, in the case of neoplastic cells, exposure of bikunin or bikunin gene transfection to cancer cells induces suppression of invasion and metastasis (Kobayashi et al 2002, Suzuki et al 2003a. Bikunin may modulate different intracellular signaling pathways including MAP (ERK) and phosphoinositide-3 (PI3) kinases (Akt), which lead to the down-regulation of the expression of urokinasetype plasminogen activator (uPA) (Kobayashi et al 2001(Kobayashi et al , 2003 and its receptor, uPAR (Nakamura et al 1997).…”

Met and the microenvironment

“…For instance, one can ask what the roles of PI3K/Akt and Erk pathways during early stage invasion of ovarian cancer cells are. Although those pathways have been previously identified as therapeutic targets, 9,10 in this report, activation of Met by fibronectin, unlike HGF/SF, did not activate Akt or Erk. We can then wonder whether activation of Akt/Erk pathways would be beneficial or not to the early stage attachement/invasion of those cells.…”

Suppression of Urokinase Receptor Expression by Thalidomide Is Associated with Inhibition of Nuclear Factor κB Activation and Subsequently Suppressed Ovarian Cancer Dissemination

“…4C and D, TGF-h1 activation induced increased nuclear NF-nB The effect of thalidomide as well as neutralizing antibodies against urokinase-type plasminogen activator and urokinasetype plasminogen activator receptor on cell invasiveness. Our previous experiments showed that treatment with TGF-h1 produced a significant stimulation of the invasiveness of HRA cells in a dose-dependent manner, with a maximum stimulation at 10 ng/mL TGF-h1 (29). The uPA inhibitor amiloride (SigmaAldrich, Co.) was added to HRA cells in the upper chamber of the transwells at 100 Amol/L to study the effect of uPA inhibition on invasion.…”

“…Invasion assays were done essentially as described previously (29). The ability of cells to migrate across a Matrigel barrier (invasion) was determined by the modified Boyden chamber method.…”