Internalization and down-regulation of the epidermal growth factor receptor in a human keratinocyte cell line HaCaT / 中华皮肤科杂志

ABSTRACT

Objective To understand the molecular mechanism underlying the epidermal growth factor receptors(EGFR)signal transduction and its feed-back regulation.Methods Two human keratinocyte cell lines,HaCaT and CHOwt,were cultured and treated with a certain concentration of different ligands,including epidermal growth factor(EGF),heparin-bounding(HB)-EGF,transforming growth factor α (TGFα)and heregulin(HER),for various durations(2,4,8,16,20 hours).After the treatment,cells were collected and protein was extracted.The amount of total and active EGFR was measured by immunoprecipitation and immunoblot assay.The internalization and down-regulation of EGFR were visualized with immunofluorescence and laser seanning confocal microscopy.Results As shown by immunoblot technique,EGF and HB-EGF continuously down-regulated the total amount of EGFRs,whereas TGFα and HER had no significant effect on the degradation of EGFRs.The activation of EGFRs was also attenuated to different extent after long-time treatment with EGF,HB-EGF and TGFα.As indirect immunofluorescenee revealed,in untreated HaCaT and CHOwt cells,EGFRs were essentially located at the plasma membrane,with a little cytosolic distribution;after ten-minute treatment with EGF,EGFRs clustered into patch-like structures which were particularly obvious in HaCaT cells,and translocated into cytoplasmic vesicles resembling endosomes (relatively apparent in CHOwt cells),while the total amount of EGFRs remained constant in these cells.The fluorescence signal from the total EGFRs decreased evidently after four-hour treatment with EGF,indicating a strong reduction in the receptors.Conclusions EGF and HB-EGF,but not TGFα or Heregulin,could down-regulate the amount of total and active EGFRs.There might be different mechanisms for the signal transduction related to EGFRs intemalization and down-regulation between HaCaT and CHOwt cells.