Urokinase Receptors Are Required for α5β1 Integrin-mediated Signaling in Tumor Cells

Ying Wei,Chi-Hui Tang,Young Kim,Liliane Robillard,Feng Zhang,Matthias C Kugler,Harold A Chapman

doi:10.1074/jbc.m607989200

Ying Wei, Chi-Hui Tang + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m607989200

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Abstract

Up-regulation of urokinase receptors is common during tumor progression and thought to promote invasion and metastasis. Urokinase receptors bind urokinase and a set of beta1 integrins, but it remains unclear to what degree urokinase receptor/integrin binding is important to beta1 integrin signaling. Using site-directed mutagenesis, single amino acid mutants of the urokinase receptor were identified that fail to associate with either alpha3beta1 (D262A) or alpha5beta1 (H249A) but associate normally with urokinase. To study the effects of these mutations on beta1 integrin function, endogenous urokinase receptors were first stably silenced in tumor cell lines HT1080 and H1299, and then wild type or mutant receptors were expressed. Knockdown of urokinase receptors resulted in markedly reduced fibronectin and alpha5beta1-dependent ERK activation and metalloproteinase MMP-9 expression. Re-expression of wild type or D262A mutant receptors but not the alpha5beta1 binding-deficient H249A mutant reconstituted fibronectin responses. Because urokinase receptor.alpha5beta1 complexes bind in the fibronectin heparin-binding domain (Type III 12-14) whereas alpha5beta1 primarily binds in the RGD-containing domain (Type III 7-10), signaling pathways leading to ERK and MMP-9 responses were dissected. Binding to III 7-10 led to Src/focal adhesion kinase activation, whereas binding to III 7-14 caused Rac 1 activation. Tumor cells engaging fibronectin required both Type III 7-10- and 12-14-initiated signals to activate ERK and up-regulate MMP-9. Thus urokinase receptor binding to alpha5beta1 is required for maximal responses to fibronectin and tumor cell invasion, and this operates through an enhanced Src/Rac/ERK signaling pathway.

Highlights

One mechanism by which uPAR is reported to influence cellular behavior is by associating with signaling molecules and initiating signal transduction [3, 13,14,15]
We demonstrate that suppression of uPAR expression in tumor cells reduces ␣5␤1/Fn-dependent induction of ERK activation and MMP-9 secretion
A recent study indicates that uPAR1⁄7␤1 integrin complexes mediate urokinase-type plasminogen activator (uPA)-induced ERK signaling that leads to an increase in pro-MMP-9 secretion and colon cancer cell invasion [10]

Summary

Introduction

One mechanism by which uPAR is reported to influence cellular behavior is by associating with signaling molecules and initiating signal transduction [3, 13,14,15]. In some systems both CBD and HepII cooperatively regulate p125FAK activity, affecting cell survival [52] These observations invite the hypothesis that the second binding site on Fn created by uPAR/␣5␤1 association is critical for Fn signaling and enhanced protease expression and raise the possibility that invasive tumor cells are addicted to this pathway for their malignant phenotype. We tested these hypotheses by generating stable knockdown of uPAR in invasive tumor cell lines of different origins and reintroducing point mutants of uPAR with selective defects in ␣5␤1 binding This allowed us to dissociate the capacity of uPAR to bind uPA from its direct binding of ␣5␤1 and to dissect the signaling pathways initiated by direct binding of uPAR to the integrin. We show that uPAR is required for maximal ␣5␤1-dependent responses to Fn that promote tumor cell invasion, and this operates through integration of two signals initiated by cell engagement to the HepII and RGD sites on Fn by uPAR-bound and unbound ␣5␤1

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