Deubiquitinase activities required for hepatocyte growth factor-induced scattering of epithelial cells

Abstract

The scattering response of epithelial cells to activation of the Met receptor tyrosine kinase represents one facet of an "invasive growth" program. It is a complex event that incorporates loss of cell-cell adhesion, morphological changes, and cell motility. Ubiquitination is a reversible posttranslational modification that may target proteins for degradation or coordinate signal transduction pathways. There are approximately 79 active deubiquitinating enzymes (DUBs) predicted in the human genome. Here, via a small interfering RNA (siRNA) library approach, we have identified 12 DUBs that are necessary for aspects of the hepatocyte growth factor (HGF)-dependent scattering response of A549 cells. Different phenotypes are evident that range from full loss of scattering, similar to receptor knockdown (e.g., USP30, USP33, USP47), to loss of cell-cell contacts even in the absence of HGF but defective motility (e.g., USP3, ATXN3L). The knockdowns do not incur defective receptor, phosphatidylinositol 3-kinase, or MAP kinase activation. Our data suggest widespread involvement of the ubiquitin system at multiple stages of the Met activation response, implying significant crosstalk with phosphorylation-based transduction pathways. Development of small-molecule inhibitors of particular DUBs may offer a therapeutic approach to contain metastasis.

Figure 1

Inhibition of HGF-Induced Scattering Response of A549 Cells by siRNA Knockdown of the Met Receptor (A) Reduction in cellular Met receptor levels following incubation with siRNA oligos directed against the Met receptor. (B) A549 cells treated with vehicle (Oligofectamine, left panels) or Met siRNA (right panels) and stimulated with 50 ng/ml hepatocyte growth factor (HGF) for 12 hr. Cells were fixed and stained with crystal violet (top panels) or DAPI (bottom panels). Scale bars represent 100 μm.

Figure 2

Morphological Features of A549 Cells following Selected DUB Knockdown Twelve deubiquitinating enzymes (DUBs) identified in our screen as required for HGF-mediated scattering of A549 cells were knocked down with pooled oligonucleotides from a siGenome library. One set of cells was treated with 50 ng/ml HGF for 12 hr (+HGF panels), while the other set was left untreated (−HGF panels). Cells were stained with crystal violet. Scale bars represent 50 μm.

Figure 3

Correlation of USP33 and USP30 Knockdown Efficiency with HGF-Induced Scattering Response in A549 Cells (A) Estimation by western blotting of endogenous protein of the knockdown efficiency for eight individual siRNA oligonucleotides directed against USP33 (top) and their respective effects on HGF-mediated cell scattering of A549 cells (bottom) (IS, inhibition of scattering; S, scattering). (B) No antibody is currently available for USP30, but each of the four individual oligonucleotides effectively represses transient expression of USP30-GFP in HeLa cells as judged by western blotting with GFP-antibody, as well as inhibiting HGF-mediated scattering of A549 cells (IS).

Figure 4

DUB Requirements for HGF-Dependent Wound Healing in A549 Cells Confluent monolayers of A549 cells were pretreated for 72 hr with siRNA oligonucleotides (40 nM) consisting of pools from the siGenome DUB library targeting specific DUBs. After introducing a scratch, cells were incubated in full medium supplemented with 50 ng/ml HGF and visualized 20 hr later.