Research ArticleIn vivo inhibition of epidermal growth factor receptor autophosphorylation prevents receptor internalization
Introduction
Epidermal growth factor receptor (EGFR) expression has been shown to be up-regulated in chronic hypersecretory airway diseases such as asthma, chronic bronchitis, and chronic obstructive pulmonary disease (COPD) [1]. Oxidative stress associated with cigarette smoke is known to activate EGFR [2]. Activated EGFR in turn induces airway goblet cell hyperplasia and mucin production from these goblet cells; both effects could be prevented by treatment with specific EGFR tyrosine kinase (TK) inhibitors [2], [3], [4].
The question whether the auto-/transphosphorylation of the EGFR kinase domains is a prerequisite for EGF-induced receptor endocytosis has been contentious for more than two decades [5], [6], [7], [8], [9], [10], [11]. One reason for the partially contradictory experimental results may be the use of different model cell lines, sometimes recombinantly overexpressing the EGFR to non-physiologically high levels. With the endogenous expression levels of EGFR in NCI-H292 airway epithelial cells, we demonstrate that both BIBW 2948 BS and AG1478, specific inhibitors of the EGFR TK activation, suppressed both EGFR autophosphorylation and internalization with virtually identical IC50 values. Secondly, we found that BIBW 2948 BS, when inhalatively administered to COPD patients in the course of a double-blind clinical phase II study [12], prevented the subsequent EGF-stimulated EGFR internalization in an ex vivo assay using airway biopsies.
Section snippets
EGFR antibody co-internalization
NCI-H292 (ATCC®, #CRL-1848) cells were cultured on 384 well imaging-suitable collagen I-coated microtiter plates (Falcon BD) in an RPMI 1640 medium with l-Glutamine (BioWhittaker, #BE12-702F), 1% Pen/Strep (Biochrom, #A2213), and 10% fetal bovine serum (FBS; Gibco, #10500-064), hereafter referred to as the "culture medium", and incubated at 37 °C, 5% CO2 overnight. EGFR antibody co-internalization experiments were performed using an FITC-conjugated antibody directed against an extracellular
EGFR-specific kinase inhibitors suppress EGF-driven receptor autophosphorylation and internalization
EGF-induced phosphorylation and internalization of endogenously expressed EGFR was analyzed in human bronchoepithelial NCI-H292 cells by fluorescence microscopic imaging. Binding of EGF to EGFR activates tyrosine kinase activity of the receptor [14], resulting in the autophosphorylation of several tyrosine residues in the carboxy-terminal domain of EGFR [15]. Representatively, the autophosphorylated EGFR-Tyr1086 [16], [17] was detected with a specific rabbit monoclonal antibody. The
Discussion
Our results are consistent with previous findings that the in vitro inhibition of the EGFR kinase activity by mutation of the catalytic Lys 721 caused a reduction of the EGFR endocytic rate under conditions of recombinant receptor overexpression in B82 mouse L cells [5], [6]. Furthermore, AG1478 had been found to suppress EGF-driven EGFR internalization when the receptor was overexpressed in CHO cells [9], an observation identical to our findings for AG1478 in the NCI-H292 cells endogenously
Acknowledgments
We thank Dres. Ulrike Küfner-Mühl, Martin Valler and Wolfgang Seibold for helpful advice and discussions. We are grateful to Dres. Tamara Manuelian, Thomas Haberichter and Susanne Heim for support in applying the Definiens Cellenger software and to Claudia Huber, Martin Krell, Eberhard Lemmer, Clemens Schütterle, Gabriele Böckenholt, Beate Rychlowski, Dipl-Biol. Annette Charra, Dipl.-Biol. Kerstin Höhne, Taylor Moss, Gretel Larese Ortiz, Margaret Solon and Suzanne B. Parker for perfect
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