Knockdown of GnT-Va expression inhibits ligand-induced downregulation of the epidermal growth factor receptor and intracellular signaling by inhibiting receptor endocytosis
Knockdown of GnT-Va expression inhibits ligand-induced downregulation of the epidermal growth factor receptor and intracellular signaling by inhibiting receptor endocytosis
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DOI：
10.1093/glycob/cwp023
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发表时间：
2009-05-01
期刊：
影响因子：
4.3
通讯作者：
Pierce, Michael
中科院分区：
文献类型：
--
作者：
Guo, Hua-Bei;Johnson, Heather;Pierce, Michael
N-葡聚糖分支糖基转移酶表达的变化可以改变细胞表面受体的功能，包括细胞表面保留的水平、内化到内体的速率以及随后的细胞内信号转导。为了详细研究GlcNAcβ(1，6)Man分支对表皮生长因子受体(EGFR)信号的调节，我们利用特异的siRNA选择性地敲除高侵袭性人乳腺癌细胞株MDA-MB231中GnT-Va的表达，从而导致其侵袭性相关表型的减弱。与对照细胞相比，在GnT-Va抑制的细胞中，配体诱导的EGFR下调明显受到抑制。这种作用可以被GnT-Va的重新表达逆转，这表明配体诱导的受体下调的变化依赖于GnT-Va的活性。GnT-Va基因敲除对c-Cbl介导的受体泛素化和降解没有显著影响，但确实引起受体内化的抑制，表明信号的改变和延迟的配体诱导的EGFR表达下调是由于EGFR内吞减少所致。用GnT-Va siRNA处理HT1080纤维肉瘤细胞也得到了类似的结果。GnT-Va siRNA表达引起的受体内化抑制似乎与Galectin结合无关，因为GnT-Va siRNA抑制的EGFR内化减少不受Galectin抑制剂乳糖的影响。我们的结果表明，由于siRNA在人类癌细胞中表达的GnT-Va活性降低，抑制了配体诱导的EGFR内化，从而导致下游信号转导延迟，并抑制了EGF诱导的侵袭性相关表型。
Changes in the expression of N-glycan branching glycosyltransferases can alter cell surface receptor functions, involving their levels of cell surface retention, rates of internalization into the endosomal compartment, and subsequent intracellular signaling. To study in detail the regulation of signaling of the EGF receptor (EGFR) by GlcNAc beta(1,6)Man branching, we utilized specific siRNA to selectively knockdown GnT-Va expression in the highly invasive human breast carcinoma line MDA-MB231, which resulted in the attenuation of its invasiveness-related phenotypes. Compared to control cells, ligand-induced downregulation of EGFR was significantly inhibited in GnT-Va-suppressed cells. This effect could be reversed by re-expression of GnT-Va, indicating that changes in ligand-induced receptor downregulation were dependent on GnT-Va activity. Knockdown of GnT-Va had no significant effect on c-Cbl mediated receptor ubiquitination and degradation, but did cause the inhibition of receptor internalization, showing that altered signaling and delayed ligand-induced downregulation of EGFR expression resulted from decreased EGFR endocytosis. Similar results were obtained with HT1080 fibrosarcoma cells treated with GnT-Va siRNA. Inhibited receptor internalization caused by the expression of GnT-Va siRNA appeared to be independent of galectin binding since decreased EGFR internalization in the knockdown cells was not affected by the treatment of the cells with lactose, a galectin inhibitor. Our results show that decreased GnT-Va activity due to siRNA expression in human carcinoma cells inhibits ligand-induced EGFR internalization, consequently resulting in delayed downstream signal transduction and inhibition of the EGF-induced, invasiveness-related phenotypes.