Disproportionately high levels of HGF induce the degradation of the c-met receptor through the proteasomal degradation pathway

Nayeon Lee a b, Junghun Lee b, Sang Hwan Lee a, Subin Kim b, Sunyoung Kim b

Highlights
•HGF-mediated cell migration increased as concentration of HGF increased, but declined beyond a certain point.
•The c-met receptor was degraded when unnecessarily large amounts of HGF were present.
•Excessive amounts of HGF attenuated downstream signaling even if cells were re-treated with optimal amounts of HGF.
•Excessive amounts of HGF diverted c-met toward the proteasomal degradation pathway through phosphorylation of Tyr 1003.

Abstract
Hepatocyte growth factor (HGF) receptor is a member of the receptor tyrosine kinases (RTKs) and has been reported to perform diverse functions in various cell types during both the developmental and adult stages. Among different roles, HGF is best known for its angiogenic effects of inducing the migration of endothelial cells. Because angiogenesis is one of the prerequisite steps for tumor metastasis, HGF-dependent cell migration has to be tightly controlled. However, the underlying mechanisms regulating the optimum level of HGF/c-met signaling have been poorly understood. In this study, we tested whether the migration of endothelial cells is regulated by a negative feedback mechanism under disproportionately large amounts of HGF.

Data from endothelial cell migration assays showed that HGF activity increased as its concentration increased, but declined beyond a certain point. Under limiting conditions, amounts of phosphorylated Erk and Akt surged, reaching a plateau in which the enhanced level was more or less maintained. The c-met receptor was degraded when unnecessarily large amounts of HGF were present. Under these conditions, HGF could no longer activate downstream signaling pathways even if cells were re-treated with optimal amounts of HGF. Excessive doses of HGF increased the phosphorylation of tyrosine residue 1003 involved in the ubiquitination of c-met, and phosphorylated c-met was diverted toward the proteasomal degradation pathway. Taken together, HGF/c-met signaling is tightly regulated by a negative feedback loop through an ubiquitin-proteasomal degradation pathway.

Introduction
Hepatocyte growth factor (HGF), also known as scatter factor, is one of the representative receptor tyrosine kinases (RTKs) and exerts pleiotropic effects [38]. HGF plays important roles in cell proliferation, survival, and motility [7]. During embryonic development, HGF-null mice have reduced liver size [33], show deficits in the migration of muscle progenitor cells [3], and fail to survive sensory neurons [27] and sympathetic neurons [26]. In the adult stage, HGF plays a decisive role in the regeneration of the liver and kidney [5,19,25].

RTKs regulate numerous cellular processes through physical interactions between specific ligands and respective cellular receptors. The binding of almost all ligands -except that of the insulin receptor family-induces dimerization of their receptors and autophosphorylation followed by recruitment of intracellular substrates [32]. After the binding, ligand and receptor complex is internalized by endocytosis [20], and sorted into recycling endosomes or late endosomes destined for degradation [12,37].

In the case of EGF and EGFR, at rate-limiting concentrations of ligand, the EGF and EGFR complex is internalized through clathrin-mediated endocytosis and preferentially recycled. At unnecessarily higher concentrations of EGF, however, the ligand and receptor complex is internalized via non-clathrin endocytosis subjected to the degradation process [35]. It was reported that the internalized HGF and c-met complex could also be recycled to the plasma membrane or degraded by the lysosome or proteasome [30]. But it remains poorly understood under what situations the internalized HGF and c-met complex is recycled or degraded.

In this study, we found that the HGF-induced migration of human umbilical vein endothelial cells (HUVECs) showed an interesting bell-shaped dose response; at lower concentrations, the number of migrated cells was increased as HGF concentration increased, but was significantly reduced at concentrations higher than a certain point. Data from experiments involving cycloheximide (CHX) indicated that the c-met receptor degraded rapidly at high concentrations. Treatment with MG132, an inhibitor of proteasomes, blocked such c-met degradation. Consistent with this result, the amount of c-met phosphorylation at Y1003 involved in the ubiquitination process was increased. Our results show that the c-met receptor underwent ubiquitin-proteasome associated degradation in the presence of disproportionately large amounts of HGF.

Section snippets
Chemicals, antibodies, and cells
Gelatin, Cycloheximide and MG132 were purchased from Sigma Aldrich (Sigma Aldrich, MO, United States). Recombinant human HGF protein was purchased from R&D Systems (R&D Systems, MN, United States). The following antibodies were purchased from Cell Signaling Technology (Cell Signaling Technology, MA, United States): Erk, pErk (T202/Y204), Akt, pAkt (S473), c-met, pc-met (Y1003), and HRP conjugated anti Rabbit IgG. β-actin peroxidase antibody was purchased from Sigma Aldrich (Sigma Aldrich, MO, HGF-mediated cell migration showed a bell-shaped dose response curve While investigating the effect of HGF on the migration of HUVECs, we found that cells responded to different doses of HGF in a bell-shaped form. HUVECs were plated in the upper chambers, while the lower chambers contained recombinant human HGF proteins. Two hours after culture, cells that had migrated to the lower chambers were stained, visualized, and counted. The number of migrated cells was increased by a HGF treatment in a dose-dependent manner and peaked at 50 ng/ml, but then decreased.

Discussion
In this study, we showed that the migration of HUVECs responded to HGF in a bell-shaped dose response manner, and that when unnecessarily large amounts of HGF were present, its cellular receptor, c-met, underwent degradation through the ubiquitination-proteasome pathway as evidenced by the blocking of this process by MG132 and the increased level of Y1003 of c-met at an excessive dose. Our results are XL092 consistent with previous reports showing that RTK signaling is regulated to a moderate level.

Acknowledgements
This work was supported by grant from The Basic Science Research Program through the National Research Foundation of Korea (No. 2014R1A1A2055890) administered by the Ministry of Education and Viromed Co. Ltd.