人角质形成细胞株HaCaT细胞表皮生长因子受体内化和下调的研究

Internalization and down-regulation of the epidermal growth factor receptor in a human kera- tinocyte cell line HaCaT

目的了解表皮生长因子受体（EGFR）信号转导与负反馈调节的分子生物学机制。方法采用免疫沉淀、免疫印迹、间接免疫荧光结合激光扫描共聚焦显微镜等技术，研究HaCaT和CHOwt细胞株中多种配体诱导的EGFR内化和下调情况。结果免疫印迹检测表明，表皮生长因子（EGF）与肝素结合的EGF（HB—EGF）可引起EGFR总量的快速下调，转化生长因子α（TGFα）及Heregulin则未明显促进受体的降解。EGF、HB—EGF和TGFα处理HaCaT细胞较长时间后活化型受体数量亦不同程度减少。问接免疫荧光染色显示，未处理细胞的EGFR主要分布在胞膜，胞质亦见少量分布。经10min EGF处理后EGFR聚集成斑状结构（HaCaT细胞明显），并形成内吞体（CHOwt细胞明显），而此时EGFR总量尚未发生明显改变。经过4hEGF处理后，HaCaT和CHOwt细胞内EGFR信号明显减弱，说明此时受体已经下调。结论不同配体对HaCaT细胞的EGFR总量及活化型受体的下调作用不同。HaCaT和CHOwt细胞EGFR内化和下调的信号转导机制可能存在差异。

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 scanning 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 signifi- cant 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 immunofluorescence 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 Heregnlin, could down-regulate the amount of total and active EGFRs. There might be different mechanisms for the signal transduction related to EGFRs internalization and down-regulation between HaCaT and CHOwt cells.