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Hydrogen peroxide reversibly inhibits epidermal growth factor (EGF) receptor internalization and coincident ubiquitination of the EGF receptor and Eps15¹

Institute of Biomembranes, Department of Molecular Cell Biology, Utrecht University, Utrecht, The Netherlands

²Correspondence: Institute of Biomembranes, Department of Molecular Cell Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, the Netherlands; Tel: +31 (0) 30–2532859; Fax: +31 (0) 30–2513655, E-mail: R.deWit{at}bio.uu.nl

SPECIFIC AIMS

Previously, we described that hydrogen peroxide (H₂O₂) inhibited the internalization of the epidermal growth factor (EGF) receptor and coincident EGF-induced mono-ubiquitination of Eps15 in fibroblasts. We suggested that EGF receptor internalization was inhibited by H₂O₂ by inhibition of ubiquitination of proteins that are involved in EGF receptor-mediated endocytosis. To gain further insight in the mechanism underlying this inhibition of internalization of the EGF receptor, the direct effect of H₂O₂, and its reversibility on EGF receptor internalization, ubiquitination of the EGF receptor and Eps15 and coincident changes in the cellular ratio of GSSG:GSH were investigated in the present study.

PRINCIPAL FINDINGS

1. H₂O₂ inhibits the polyubiquitination of the EGF receptor
Because EGF-induced polyubiquitination of the EGF receptor occurs at the plasma membrane, and thus prior to internalization, the effect of H₂O₂ on the ubiquitination of the EGF receptor was investigated. Therefore, HUB1 cells—HER14 cells stably transfected with cDNA encoding HA-tagged ubiquitin—were treated with EGF ± 5 mM H₂O₂ for 10, 20, or 30 min., followed by immunoprecipitation of the EGF receptor. Detection on Western blot by using anti-HA antibody (Fig. 1 ) demonstrated that EGF induced a transient ubiquitination of the EGF receptor. This polyubiquitination was completely inhibited in the presence of 5 mM H₂O₂, indicating that H₂O₂ inhibits EGF-induced polyubiquitination of the EGF receptor under the same conditions that blocked EGF receptor internalization. Inhibition of EGF receptor endocytosis at t = 10 min is presented in Fig. 2 .

View larger version (38K): [in this window] [in a new window]   Figure 1. Effect of H2O2 on the polyubiquitination of the EGF receptor. HUB1 cells were washed with PBS and incubated with EGF (50 ng/ml) in the absence or presence of 5 mM H2O2 for 10, 20, or 30 min at 37°C. Subsequently, EGF receptor was precipitated with monoclonal anti-EGF receptor antibody and immunoprecipitates were separated by 8% SDS-PAGE. The ubiquitinated EGF receptors were detected with polyclonal anti-HA antibody. The results of a representative experiment out of four independent experiments are shown.

View larger version (13K): [in this window] [in a new window]   Figure 2. Effect of increasing concentrations of H2O2 on the cellular ratio of GSSG:GSH and EGF receptor internalization. To determine the ratio of GSSG:GSH (--), HER14 cells were washed with PBS and incubated with 0, 1, 2, or 5 mM H2O2 for 10 min at 37°C, and the cellular ratio of GSSG:GSH was determined subsequently. Results +/- SEM of a representative experiment out of three independent experiments are shown with n = 3. To determine the effect of H2O2 on EGF receptor internalization (--), HER14 cells were washed with PBS and incubated with 125I-EGF in the absence or presence of 1, 2, or 5 mM H2O2 for 10 min at 37°C. This procedure was followed by treatment with acid wash and, after lysis of the cells, the amount of internalized EGF was determined. Results +/- SEM of five independent experiments are shown.

2. H₂O₂ reversibly inhibits the internalization of the EGF receptor
To establish whether EGF receptor-mediated endocytosis recovered upon removal of H₂O₂, HER14 cells were treated with EGF-biotin ± 2 or 5 mM H₂O₂ for 20 min followed by removing EGF-biotin and H₂O₂ and recovering for 10, 20, or 30 min. Then, the amount of internalized EGF-biotin was determined as previously described. Recovery after incubation with H₂O₂ resulted in a time-dependent re-establishment of EGF receptor internalization and the time of complete restoration was dependent on the concentration of H₂O₂ that was used before removal of the stress. Recovery after treatment of cells with 2 mM of H₂O₂ resulted in an almost complete re-establishment of EGF receptor internalization within 20 min., whereas recovery after treatment with 5 mM H₂O₂ reached control levels approximately after 30 min. of recovery.

3. Ubiquitination of Eps15 and EGF receptor are reversibly inhibited by H₂O₂
To determine the reversibility of the inhibitory effect of H₂O₂ on ubiquitination of the EGF receptor and Eps15, HUB1 cells were treated with EGF ± 5 mM H₂O₂ for 20 min, followed by recovery for 10, 20, or 30 min. This revealed that upon removal of H₂O₂, recovery of the ubiquitination of both the EGF receptor Eps15 started within 10 min. and was comparable with control levels (no H₂O₂) approximately after 20 min of recovery.

4. Blocking of intracellular sulphydryl groups results in inhibition of EGF receptor internalization
Because oxidative stress alters the cellular redox (and SH) status, the effect of the membrane-permeable thiol blocking agent N-ethylmaleimide (NEM), was investigated. In addition, the effect of the membrane-impermeant SH reagent 5–5‘-dithiobis (2-nitrobenzoic acid) (DTNB) on EGF receptor internalization was determined as well. This determination revealed that EGF receptor internalization was inhibited in the presence of NEM in a dose-dependent manner but not in the presence of DTNB. This finding suggests that EGF receptor internalization is dependent on intracellular SH groups, whereas extracellular SH groups are not involved.

5. H₂O₂ and H₂O₂ removal rapidly change the cellular ratio of GSSG:GSH
A major cellular compound that regulates the cellular redox (and SH) status is glutathione and thus, the effect of H₂O₂ on cellular glutathione was investigated Therefore, HER14 cells were treated with increasing concentrations of H₂O₂ for 10 min. followed by determination of the GSSG:GSH ratio. This finding revealed that H₂O₂ induced a dose-dependent increase in the ratio of GSSG:GSH (Fig. 2) . As also shown in Fig. 2 , the increase in the cellular ratio of GSSG:GSH correlated with a concentration-dependent decrease in EGF receptor internalization after 10 min of incubation.

To study the effect of recovery on the cellular levels of GSSG and GSH, HER14 cells were treated with 5 mM H₂O₂ for 10 or 20 min, followed by removal of the stress and recovery for 10, 20, or 30 min. This demonstrated that removal of the stress both after 10 and 20 min of incubation resulted in a rapid re-establishment of the cellular ratio of GSSG:GSH. Recovery resulted in a decline of the ratio almost to control levels within 10 min, which led to a complete restoration after recovery for 20 min.

CONCLUSIONS

Oxygen free radicals are generated under both normal and pathological circumstances and have been implicated in the pathogenesis of diseases such as atherosclerosis and cancer, as well as in aging and in some inflammatory disorders. It has been suggested that oxygen free radicals act as second messengers in signal transduction and are involved in increased receptor phosphorylation. Increased phosphorylation of signaling proteins by H₂O₂ is probably accomplished by a reversible inactivation of tyrosine phosphatases, via oxidation of essential SH groups within their active site cysteines. This finding indicates that oxidative stress has an inhibitory effect on this negative-feedback mechanism of the cell.

Another feedback mechanism to attenuate EGF-induced signaling is the internalization and subsequent degradation of activated receptors. This down-regulation is important, because the inability of cells to undergo this ligand-induced receptor-mediated endocytosis might lead to cellular transformation or tumor formation. In a previous study, we described that H₂O₂ inhibits the internalization of the EGF receptor in fibroblasts in a concentration-dependent manner. Here we report the effect of H₂O₂ and recovery upon H₂O₂ removal on EGF receptor internalization, ubiquitination, and the cellular ratio of GSSG:GSH in fibroblasts. We found that the inhibition of EGF receptor internalization by H₂O₂ was reversible and that complete re-establishment of internalization after removal of 5 mM H₂O₂ required a recovery time of at least 30 min.

We have suggested previously that H₂O₂ inhibits EGF receptor internalization by inhibition of ubiquitination of proteins involved in the EGF receptor-mediated endocytosis. Although the precise role of ubiquitination in the endocytosis of the EGF receptor has not been elucidated, ubiquitin-dependent internalization of other receptors has been described. It is interesting that recent studies revealed that both the mono-ubiquitination of Eps15 and the polyubiquitination of the EGF receptor occur at the plasma membrane, which suggests a role for ubiquitin in EGF receptor-mediated endocytosis. In this study, we show that both the mono-ubiquitination of Eps15 and the polyubiquitination of the EGF receptor are inhibited by H₂O₂ and that inhibition of ubiquitination was accompanied by an inhibition of EGF receptor internalization. Therefore, although these results do not establish the exact role for ubiquitin in EGF receptor-mediated endocytosis, the results strongly suggest that the internalization of the EGF receptor is ubiquitin-dependent.

Others have described a rapid and dose-dependent loss of endogenous ubiquitin-protein conjugates upon exposure of cells to H₂O₂. These decrements were consistent with reductions in ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzyme (E2) activities and were inversely related with the cellular ratio of GSSG:GSH. It has been suggested that GSSG induces thiolation of the active SH group of ubiquitination enzymes, which leads to the inability of these enzymes to activate and transfer ubiquitin to a substrate protein. In this study, a dose-dependent increase in the ratio of GSSG:GSH was determined as well after treatment of cells with H₂O₂ (Fig. 2) , which correlated with a dose-dependent inhibition of EGF receptor internalization (Fig. 2) and a coincident inhibition of ubiquitination of Eps15 and the EGF receptor (Fig. 1) , which suggests a causal relation between inhibition of EGF receptor internalization, ubiquitination, and increased ratio of GSSG:GSH.

We demonstrated that the increased ratio of GSSG:GSH re-established within 10 min upon removal of H₂O₂ and restored to control levels after 20 min of recovery. In addition, both the inhibition of ubiquitination of the EGF receptor and of Eps15 recovered upon stress removal and reached control levels after approximately 20–30 min. This finding indicates that both ubiquitination and cellular ratio of GSSG:GSH were reversibly affected by H₂O₂ and revealed that GSSG:GSH levels restored prior to recovery of ubiquitination. Furthermore, because complete restoration of internalization after removal of 5 mM H₂O₂ required a recovery time of at least 30 min., these data imply that recovery of the cellular ratio of GSSG:GSH might be required for re-establishment of ubiquitination and of subsequent EGF receptor internalization. We conclude therefore that although the precise role of ubiquitination in EGF receptor internalization remains unsolved, the results shown here support the hypothesis that the internalization of the EGF receptor might be inhibited by an inhibition of ubiquitination of proteins that are involved in EGF receptor-mediated endocytosis, probably regulated by the cellular GSSG:GSH ratio. A schematic model is drawn in Fig. 3 .

View larger version (22K): [in this window] [in a new window]   Figure 3. Schematic model of the hypothesized mechanism underlying inhibition of EGF receptor internalization by H2O2. In the absence of oxidative stress, both ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzyme (E2) can bind ubiquitin (Ub) via an active sulphydryl (SH) group, which leads to the activation and transfer of ubiquitin to a substrate protein (Ub-protein). Although the exact role of ubiquitin has not been established, ubiquitination seems to be required for EGF receptor-mediated endocytosis. During oxidative stress, induced by exposure of cells to H2O2, the cellular GSSG:GSH ratio will be increased. Subsequently, GSSG reversibly induces thiolation of the active sulphydryl of E1 and E2, which leads to the inactivation of these enzymes. As a result, ubiquitination of proteins, which is are required for EGF receptor internalization, is inhibited in the presence of H2O2 and results in an inhibition of the internalization of the EGF receptor. Removal of H2O2 results in recovery of the GSSG:GSH ratio, followed by dethiolation of the ubiquitination enzymes and restoration of their activities.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0454fje To cite this article, use (December 8, 2000) FASEB J. 10.1096/fj.00–0454fje

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