Activation Mechanisms of the Urokinase-type Plasminogen Activator Promoter by Hepatocyte Growth Factor/Scatter Factor

Sabine Ried,Claudia Jäger,Michael Jeffers,George F Vande Woude,Henner Graeff,Manfred Schmitt,Ernst Lengyel

doi:10.1074/jbc.274.23.16377

Abstract

Hepatocyte growth factor/scatter factor (HGF/SF) is a pleiotropic effector inducing invasion and metastasis of tumor cells that express the Met tyrosine kinase receptor. One of the effectors of HGF/SF is the urokinase-type plasminogen activator, a serine protease that facilitates tumor progression and metastasis by controlling the synthesis of the extracellular matrix degrading plasmin. Stimulation of NIH 3T3 cells that were stably transfected with the human Met receptor (NIH 3T3-Methum) with HGF/SF induced a trans-activation of the urokinase promoter and urokinase secretion. Induction of the urokinase promoter by HGF/SF via the Met receptor was blocked by co-expression of a dominant-negative Grb2 and Sos1 expression construct. Further, the expression of the catalytically inactive mutants of Ha-Ras, RhoA, c-Raf, and Erk2 or addition of the Mek1-specific inhibitor PD 098059 abrogated the stimulation of the urokinase promoter by HGF/SF. A sequence residing between -2109 and -1870 base pairs (bp) was critical for stimulation of the urokinase gene by HGF/SF. Mobility shift assays with oligonucleotides spanning an AP-1 site at -1880 bp or a combined PEA3/AP-1 site at -1967 bp showed binding of nuclear factors from NIH 3T3-Methum cells. Expression of an expression plasmid that inhibits DNA binding of AP-1 proteins (A-Fos) abrogated inducible and basal activation of the urokinase promoter. Nuclear extract from unstimulated NIH 3T3-Methum cells contained more JunD and showed a stronger JunD supershift with the AP-1 oligonucleotides, compared with HGF/SF-stimulated cells. Consistent with the levels of JunD expression being functionally important for basal expression of the urokinase promoter, we found that overexpression of wild type JunD inhibited the induction of the urokinase promoter by HGF/SF. These data suggest that the induction of urokinase by HGF/SF is regulated by a Grb2/Sos1/Ha-Ras/c-Raf/RhoA/Mek1/Erk2/c-++ +Jun-dependent mitogen-activated protein kinase pathway.

Highlights

Hepatocyte growth factor/scatter factor (HGF/SF) is a pleiotropic effector inducing invasion and metastasis of tumor cells that express the Met tyrosine kinase receptor
Met-HGF/SF Signaling Activates the Urokinase Promoter and Stimulates Urokinase Secretion—To study the regulation of the urokinase-type plasminogen activator by HGF/SF, we used a cell clone that is derived from an NIH 3T3 cell line stably transfected with the human Met receptor [31]
Deletion of plasminogen from the gel abolished the band, which indicated that the proteolytic activity could be ascribed to a plasminogen activator (Fig. 2E)

Summary

Introduction

Hepatocyte growth factor/scatter factor (HGF/SF) is a pleiotropic effector inducing invasion and metastasis of tumor cells that express the Met tyrosine kinase receptor. Consistent with the levels of JunD expression being functionally important for basal expression of the urokinase promoter, we found that overexpression of wild type JunD inhibited the induction of the urokinase promoter by HGF/SF These data suggest that the induction of urokinase by HGF/SF is regulated by a Grb2/Sos1/Ha-Ras/c-Raf/RhoA/Mek1/ Erk2/c-Jun-dependent mitogen-activated protein kinase pathway. Upon HGF/SF binding, the Met receptor dimerizes and becomes phosphorylated on tyrosines located in the cytoplasmatic region of the ␤-chain These tyrosine residues act as specific binding sites for adaptor proteins [6] with Src homology 2 domains, which transmit signals intracellularly, one effector being Ras [7]. Urokinase [15] converts plasminogen into plasmin, a serine protease with broad substrate specificity toward components of the extracellular matrix including laminin, vitronectin, and fibronectin (16 –18) Together, these proteolytic functions facilitate the migration of tumor cells through the extracellular matrix and basement membrane barriers. High urokinase expression is correlated with a poor prognosis of patients suffering from a variety of different types of cancer including that of the breast, ovary, and lung [21, 22]

Methods

Results

Conclusion