Abstract
ABSTRACTEGF receptor (EGFR) endocytosis is induced by stress in a manner dependent on the p38 MAPK family. Ligand and stresses such as X-rays, reportedly promote nuclear trafficking of endocytosed EGFR for regulation of gene transcription and DNA repair. We fail to detect EGFR endocytosis or nuclear transport following X-ray treatment of HeLa or head and neck cancer cells, despite extensive DNA damage induction. Apparent nuclear staining with EGFR extracellular domain antibody remained present despite reduced/absent EGFR expression, and so did not represent nuclear EGFR. UVB and UVC, but not X-ray or UVA, treatment induced p38 activation and EGFR endocytosis, although all of these stresses induced DNA damage, indicating that DNA damage alone is not sufficient to induce EGFR endocytosis. Increased reactive oxygen species (ROS) levels following UVB treatment, compared to that seen with X-rays, do not alone explain differences in p38 activation. UVB, like UVC, induced EGFR accumulation predominantly in perinuclear endosomes, rather than in the nucleus. Our morphological techniques identifying major changes in receptor distribution do not exclude the possibility that small but biologically relevant amounts of EGFR enter the nucleus. This study highlights the importance and limitations of morphological analyses of receptor distribution in understanding signaling outcome.
Highlights
The ligand-stimulated endocytosis of EGF receptor (EGFR) and endosomal sorting complexes required for transport (ESCRT)dependent delivery to lysosomes is a well-described pathway that is known to regulate EGFR signaling (Tomas et al, 2014)
X-ray treatment does not induce significant nuclear translocation of EGFR in HeLa or SCC47 head and neck cancer cells Immunofluorescence (IF) of HeLa cells using both the extracellular and cytoplasmic domain anti-EGFR antibodies showed that the majority of EGFR staining was localized to the plasma membrane in both untreated controls and cells exposed to 4 Gy X-rays, a typical patient dose (Fig. 1A)
Many of these studies have demonstrated increased levels of full-length EGFR in nuclear fractions following treatment with different stimuli, this approach relies on the purity of these nuclear fractions
Summary
The ligand-stimulated endocytosis of EGF receptor (EGFR) and endosomal sorting complexes required for transport (ESCRT)dependent delivery to lysosomes is a well-described pathway that is known to regulate EGFR signaling (Tomas et al, 2014). The fates of endocytosed EGFR induced by stress are less well-characterized, but have been reported to include import into the nucleus where the receptor can. The transport of full-length EGFR to the nucleus remains controversial despite being widely reported following exposure to DNA damageinducing agents (Dittmann et al, 2008; Hsu et al, 2009; Liccardi et al, 2011; Xu et al, 2009) and to ligand (Liao and Carpenter, 2007; Lin et al, 2001). Other studies report transport of EGFR to the nucleus within minutes of EGF stimulation (Lin et al, 2001)
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