NADPH-oxidase-dependent reactive oxygen species mediate EGFR transactivation by FPRL1 in WKYMVm-stimulated human lung cancer cells

doi:10.1016/j.freeradbiomed.2011.05.040

Free Radical Biology and Medicine

Volume 51, Issue 6, 15 September 2011, Pages 1126-1136

https://doi.org/10.1016/j.freeradbiomed.2011.05.040 Get rights and content

Abstract

Cross talk between unrelated cell surface receptors, such as G-protein-coupled receptors (GPCR) and receptor tyrosine kinases (RTK), is a crucial signaling mechanism to expand the cellular communication network. We investigated the ability of the GPCR formyl peptide receptor-like 1 (FPRL1) to transactivate the RTK epidermal growth factor receptor (EGFR) in CaLu-6 cells. We observed that stimulation with WKYMVm, an FPRL1 agonist isolated by screening synthetic peptide libraries, induces EGFR tyrosine phosphorylation, p47^phox phosphorylation, NADPH-oxidase-dependent superoxide generation, and c-Src kinase activity. As a result of EGFR transactivation, phosphotyrosine residues provide docking sites for recruitment and triggering of the STAT3 pathway. WKYMVm-induced EGFR transactivation is prevented by the FPRL1-selective antagonist WRWWWW, by pertussis toxin (PTX), and by the c-Src inhibitor PP2. The critical role of NADPH-oxidase-dependent superoxide generation in this cross-talk mechanism is corroborated by the finding that apocynin or a siRNA against p22^phox prevents EGFR transactivation and c-Src kinase activity. In addition, WKYMVm promotes CaLu-6 cell growth, which is prevented by PTX and by WRWWWW. These results highlight the role of FPRL1 as a potential target of new drugs and suggest that targeting both FPRL1 and EGFR may yield superior therapeutic effects compared with targeting either receptor separately.

Section snippets

Reagents and cell culture treatments

The WKYMVm and WRWWWW (WRW4) peptides were synthesized and HPLC-purified by PRIMM (Milan, Italy). SDS–PAGE reagents were from Bio-Rad (Hercules, CA, USA). Protein A/G Plus agarose, anti-active phosphorylated ERK1/2, anti-tubulin, anti-c-Src, anti-p47^phox, anti-FPRL1, anti-EGFR, anti-STAT3, anti-cyclin A, anti-p-Y, and anti-rabbit antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-p-c-Src(Tyr416), anti-p-STAT3(Tyr705), and anti-p-STAT3(Ser727) were from Cell Signaling

FPRL1 is a biologically functional receptor in CaLu-6 cells

We first analyzed the expression of FPR and FPRL1 in the CaLu-6 cell line by RT-PCR analysis, performed with specific primers for the coding sequences of the two receptors, and by Western blot analysis using an α-FPRL1 antibody. We observed that FPRL1 mRNA, but not FPR, is expressed in these cells (Fig. 1A) and we detected the presence of the band corresponding to FPRL1 protein at the expected molecular size in immunoblot experiments (Fig. 1B). The α-FPRL1 antibody recognized the same band in

Discussion

We demonstrate that in serum-deprived CaLu-6 cells, stimulation of FPRL1 by a specific agonist results in NADPH-oxidase-dependent superoxide generation and EGFR transactivation. Furthermore, we show that ROS play a key role in bridging the signals from FPRL1 to EGFR by modulating c-Src kinase activity, as demonstrated by the effects of PP2, apocynin, and a siRNA against p22^phox on c-Src Y416 phosphorylation and EGFR transactivation. Our results also indicate that, as a consequence of the

Acknowledgments

This work was supported by grants from the Ministero dell'Università e della Ricerca Scientifica e Tecnologica PRIN 2007 “Attivazione dei recettori per formil-peptidi e regolazione della NADPH ossidasi in linee cellulari tumorali umane non fagocitiche.” We thank Jean Ann Gilder (Scientific Communication srl) for text editing.

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Original ContributionNADPH-oxidase-dependent reactive oxygen species mediate EGFR transactivation by FPRL1 in WKYMVm-stimulated human lung cancer cells

Abstract

Section snippets

Reagents and cell culture treatments

FPRL1 is a biologically functional receptor in CaLu-6 cells

Discussion

Acknowledgments

Pharmacol. Ther.

Cytokine Growth Factor Rev.

Trends Immunol.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

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Free Radic. Biol. Med.

J. Biol. Chem.

Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

J. Biol. Chem.

Life Sci.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Biochim. Biophys. Acta

J. Biol. Chem.

J. Biol. Chem.

Kidney Int.

Cell. Signal.

J. Biol. Chem.

Exp. Cell Res.

J. Biol. Chem.

Cancer Lett.

J. Biol. Chem.

J. Mol. Cell. Cardiol.

Curr. Opin. Cell Biol.

Free Radic. Biol. Med.

Exp. Hematol.

Blood

Trends Pharmacol. Sci.

Int. Immunopharmacol.

Broad immunocytochemical localization of the formylpeptide receptor in human organs, tissues and cells

Cell Tissue Res.

Intracellular signaling triggered by formyl-peptide receptors in nonphagocytic cells

Curr. Signal Transduct. Ther.

Expression and signaling of formyl peptide receptors in the brain

Neurochem. Res.

Phosphorylation of p47phox sites by PKC alpha, beta II, delta, and zeta: effect on binding to p22phox and on NADPH oxidase activation

Biochemistry

The mitogen-activated protein kinase extracellular signal-regulated kinase 1/2 pathway is involved in formyl-methionyl-leucyl-phenylalanine-induced p47phox phosphorylation in human neutrophils

J. Immunol.

Targeting and regulation of reactive oxygen species generation by Nox family NADPH oxidase

Antioxid. Redox Signal.

Original Contribution
NADPH-oxidase-dependent reactive oxygen species mediate EGFR transactivation by FPRL1 in WKYMVm-stimulated human lung cancer cells