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Stress-induced endocytosis and degradation of epidermal growth factor receptor are two independent processes.

Peng K, Dai Q, Wei J, Shao G, Sun A, Yang W, Lin Q - Cancer Cell Int. (2016)

Bottom Line: However, it is not clear whether stress-induced endocytosis and degradation are consequential or two independent events.Endocytosis and degradation of EGFR in response to stress treatment and effects of the p38 inhibitor, the Caspase-3 inhibitor and the proteasomal inhibitor in cervical cancer HeLa cells were determined using immunoblotting and immunofluorescent staining assays.Inhibiting p38 kinase impairs only the endocytosis but not the degradation, while inhibiting Caspase-3 results in the opposite effect to inhibiting p38.

View Article: PubMed Central - PubMed

Affiliation: School of Medicine, Jiangsu University, 301 Xuefu Road, Zhenjiang, China.

ABSTRACT

Background: Epidermal growth factor receptor (EGFR) is an important oncogenic protein in multiple types of cancer. Endocytosis and degradation of epidermal growth factor receptor (EGFR) are two key steps for down-regulation of cell surface level of EGFR and modulation of EGFR signaling. Stress conditions induce ligand-independent endocytosis and degradation of EGFR. However, it is not clear whether stress-induced endocytosis and degradation are consequential or two independent events.

Methods: Endocytosis and degradation of EGFR in response to stress treatment and effects of the p38 inhibitor, the Caspase-3 inhibitor and the proteasomal inhibitor in cervical cancer HeLa cells were determined using immunoblotting and immunofluorescent staining assays.

Results: Stress conditions, such as protein biosynthesis inhibition, UV light irradiation, and hyper-osmosis, induced both ligand-independent endocytosis and degradation of EGFR. Stress-induced endocytosis of EGFR relies on p38 kinase activity, while stress-induced degradation of EGFR is catalyzed by Caspase-3 activity. Inhibiting p38 kinase impairs only the endocytosis but not the degradation, while inhibiting Caspase-3 results in the opposite effect to inhibiting p38. Furthermore, proteasomal activity is required for stress-induced degradation of EGFR and cell death, but not for endocytosis.

Conclusions: The results indicate that stress-induced endocytosis and degradation are two independent events and suggest stress signaling may utilize a double-secure mechanism to down-regulate cell surface EGFR in cancer cells.

No MeSH data available.


Related in: MedlinePlus

Proteasomal activity is required for stress-induced degradation of EGFR and cell death, but not endocytosis of EGFR. HeLa cells were cultured and serum-starved the same as described in Fig. 1. The proteasome inhibitor MG-132 (10 μM) was added to the cells 30 min prior to the treatment of EGF, anisomycin, UV irradiation or 0.7 M NaCl. A The cells were lysed and EGFR was immunoprecipitated and detected by immunoblotting with anti-EGFR(1005) (the top panel). B The immunoblotting data of EGFR from three repetitions of experiments was quantified by Kodak EDAS290 image system. ***p < 0.001. C Examination of effect of MG-132 on stress-induced endocytosis of EGFR by immunofluorescent staining of EGFR. The experimental procedures were the same as described in Fig. 3A–C Bar 20 μm. D MG-132 protects stress-induced cell death. The cells were incubated in MG-132 (10 μM)-contained medium 30 min prior to the treatment of anisomycin or UV irradiation. The viable cell numbers were counted under a microscope with a hemacytometer. The relative cell survival was plotted as percentage of the control cell numbers. In statistical analysis, the samples treated with MG-132 alone are used as the control data set. *p < 0.05, **p < 0.01, ***p < 0.001
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Fig4: Proteasomal activity is required for stress-induced degradation of EGFR and cell death, but not endocytosis of EGFR. HeLa cells were cultured and serum-starved the same as described in Fig. 1. The proteasome inhibitor MG-132 (10 μM) was added to the cells 30 min prior to the treatment of EGF, anisomycin, UV irradiation or 0.7 M NaCl. A The cells were lysed and EGFR was immunoprecipitated and detected by immunoblotting with anti-EGFR(1005) (the top panel). B The immunoblotting data of EGFR from three repetitions of experiments was quantified by Kodak EDAS290 image system. ***p < 0.001. C Examination of effect of MG-132 on stress-induced endocytosis of EGFR by immunofluorescent staining of EGFR. The experimental procedures were the same as described in Fig. 3A–C Bar 20 μm. D MG-132 protects stress-induced cell death. The cells were incubated in MG-132 (10 μM)-contained medium 30 min prior to the treatment of anisomycin or UV irradiation. The viable cell numbers were counted under a microscope with a hemacytometer. The relative cell survival was plotted as percentage of the control cell numbers. In statistical analysis, the samples treated with MG-132 alone are used as the control data set. *p < 0.05, **p < 0.01, ***p < 0.001

Mentions: Previous studies have shown that the proteasome inhibitor MG-132 completely blocked anisomycin-induced degradation of EGFR and proposed that proteasomes may execute the anisomycin-induced degradation of EGFR [22]. To examine the role of proteasome in stress-induced endocytosis and degradation, we treated HeLa cells with MG-132 prior to stress treatments. As shown in Fig. 4A, B, MG-132 completely blocked anisomycin-, 0.7 M NaCl- and UV irradiation-induced degradation of EGFR (lanes 2–7, Fig. 4A, B), but not ligand (EGF)-induced degradation of EGFR (lanes 8 and 9; Fig. 4A, B). This data suggest that the proteasome activity is required for stress-induced degradation of EGFR.Fig. 4


Stress-induced endocytosis and degradation of epidermal growth factor receptor are two independent processes.

Peng K, Dai Q, Wei J, Shao G, Sun A, Yang W, Lin Q - Cancer Cell Int. (2016)

Proteasomal activity is required for stress-induced degradation of EGFR and cell death, but not endocytosis of EGFR. HeLa cells were cultured and serum-starved the same as described in Fig. 1. The proteasome inhibitor MG-132 (10 μM) was added to the cells 30 min prior to the treatment of EGF, anisomycin, UV irradiation or 0.7 M NaCl. A The cells were lysed and EGFR was immunoprecipitated and detected by immunoblotting with anti-EGFR(1005) (the top panel). B The immunoblotting data of EGFR from three repetitions of experiments was quantified by Kodak EDAS290 image system. ***p < 0.001. C Examination of effect of MG-132 on stress-induced endocytosis of EGFR by immunofluorescent staining of EGFR. The experimental procedures were the same as described in Fig. 3A–C Bar 20 μm. D MG-132 protects stress-induced cell death. The cells were incubated in MG-132 (10 μM)-contained medium 30 min prior to the treatment of anisomycin or UV irradiation. The viable cell numbers were counted under a microscope with a hemacytometer. The relative cell survival was plotted as percentage of the control cell numbers. In statistical analysis, the samples treated with MG-132 alone are used as the control data set. *p < 0.05, **p < 0.01, ***p < 0.001
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Related In: Results  -  Collection

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Fig4: Proteasomal activity is required for stress-induced degradation of EGFR and cell death, but not endocytosis of EGFR. HeLa cells were cultured and serum-starved the same as described in Fig. 1. The proteasome inhibitor MG-132 (10 μM) was added to the cells 30 min prior to the treatment of EGF, anisomycin, UV irradiation or 0.7 M NaCl. A The cells were lysed and EGFR was immunoprecipitated and detected by immunoblotting with anti-EGFR(1005) (the top panel). B The immunoblotting data of EGFR from three repetitions of experiments was quantified by Kodak EDAS290 image system. ***p < 0.001. C Examination of effect of MG-132 on stress-induced endocytosis of EGFR by immunofluorescent staining of EGFR. The experimental procedures were the same as described in Fig. 3A–C Bar 20 μm. D MG-132 protects stress-induced cell death. The cells were incubated in MG-132 (10 μM)-contained medium 30 min prior to the treatment of anisomycin or UV irradiation. The viable cell numbers were counted under a microscope with a hemacytometer. The relative cell survival was plotted as percentage of the control cell numbers. In statistical analysis, the samples treated with MG-132 alone are used as the control data set. *p < 0.05, **p < 0.01, ***p < 0.001
Mentions: Previous studies have shown that the proteasome inhibitor MG-132 completely blocked anisomycin-induced degradation of EGFR and proposed that proteasomes may execute the anisomycin-induced degradation of EGFR [22]. To examine the role of proteasome in stress-induced endocytosis and degradation, we treated HeLa cells with MG-132 prior to stress treatments. As shown in Fig. 4A, B, MG-132 completely blocked anisomycin-, 0.7 M NaCl- and UV irradiation-induced degradation of EGFR (lanes 2–7, Fig. 4A, B), but not ligand (EGF)-induced degradation of EGFR (lanes 8 and 9; Fig. 4A, B). This data suggest that the proteasome activity is required for stress-induced degradation of EGFR.Fig. 4

Bottom Line: However, it is not clear whether stress-induced endocytosis and degradation are consequential or two independent events.Endocytosis and degradation of EGFR in response to stress treatment and effects of the p38 inhibitor, the Caspase-3 inhibitor and the proteasomal inhibitor in cervical cancer HeLa cells were determined using immunoblotting and immunofluorescent staining assays.Inhibiting p38 kinase impairs only the endocytosis but not the degradation, while inhibiting Caspase-3 results in the opposite effect to inhibiting p38.

View Article: PubMed Central - PubMed

Affiliation: School of Medicine, Jiangsu University, 301 Xuefu Road, Zhenjiang, China.

ABSTRACT

Background: Epidermal growth factor receptor (EGFR) is an important oncogenic protein in multiple types of cancer. Endocytosis and degradation of epidermal growth factor receptor (EGFR) are two key steps for down-regulation of cell surface level of EGFR and modulation of EGFR signaling. Stress conditions induce ligand-independent endocytosis and degradation of EGFR. However, it is not clear whether stress-induced endocytosis and degradation are consequential or two independent events.

Methods: Endocytosis and degradation of EGFR in response to stress treatment and effects of the p38 inhibitor, the Caspase-3 inhibitor and the proteasomal inhibitor in cervical cancer HeLa cells were determined using immunoblotting and immunofluorescent staining assays.

Results: Stress conditions, such as protein biosynthesis inhibition, UV light irradiation, and hyper-osmosis, induced both ligand-independent endocytosis and degradation of EGFR. Stress-induced endocytosis of EGFR relies on p38 kinase activity, while stress-induced degradation of EGFR is catalyzed by Caspase-3 activity. Inhibiting p38 kinase impairs only the endocytosis but not the degradation, while inhibiting Caspase-3 results in the opposite effect to inhibiting p38. Furthermore, proteasomal activity is required for stress-induced degradation of EGFR and cell death, but not for endocytosis.

Conclusions: The results indicate that stress-induced endocytosis and degradation are two independent events and suggest stress signaling may utilize a double-secure mechanism to down-regulate cell surface EGFR in cancer cells.

No MeSH data available.


Related in: MedlinePlus