Abstract
δ-Opioid receptor (DOR)-induced activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) is mediated by the transactivation of epidermal growth factor (EGF) receptors. Here we demonstrate that in stably DOR-expressing human embryonic kidney (HEK) 293 (HEK/DOR) cells, down-regulation of EGF receptors by long-term EGF (0.1 μg for 18 h) treatment, but not by small interfering RNA, results in functional desensitization of EGF (10 ng/ml)-stimulated ERK1/2 signaling. In EGF receptor-desensitized (HEK/DOR−EGFR) cells, however, [d-Ala2,d-Leu5]enkephalin (1 μM) and etorphine (0.1 μM) retained their ability to stimulate ERK1/2 activation. The newly acquired signal transduction mechanism is insensitive to the EGF receptor blockers 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478) and N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide (CL-387,785), does not involve DOR internalization and activation of the focal adhesion kinase pp125FAK, but requires matrix metalloproteinase-dependent release of soluble growth factors. A supernatant transfer assay in which conditioned growth media of opioid-treated HEK/DOR and HEK/DOR−EGFR “donor” cells are used to stimulate ERK1/2 activity in DOR-lacking HEK293 wild type and HEK293−EGFR “acceptor” cells revealed that long-term EGF treatment produces a switch in the receptor tyrosine kinase (RTK) system transactivated by opioids. Using microfluidic electrophoresis, chemical inhibitors, phosphorylation-specific antibodies, and EGF receptor-deficient Chinese hamster ovary-K1 cells, we identified the release of an insulin-like growth factor-1 (IGF-1)-like peptide and activation of IGF-1 receptors in HEK/DOR−EGFR cells after DOR activation. A similar switch from a neurotrophic tyrosine kinase receptor type 1 to an IGF-1 receptor-dependent ERK1/2 signaling was observed for chronically nerve growth factor-treated neuroblastoma × glioma (NG108-15) cells. These results indicate that transactivation of the dominant RTK system in a given cellular setting may represent a general feature of opioids to maintain mitogenic signaling.
Footnotes
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.110.064956.
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ABBREVIATIONS:
- RTK
- receptor tyrosine kinase
- Con A
- concanavalin A
- DADLE
- [d-Ala2,d-Leu5]enkephalin
- CHO
- Chinese hamster ovary
- DOR
- δ-opioid receptor
- EGCG
- epigallocatechin-3-gallate
- EGF
- epidermal growth factor
- ERK1/2
- extracellular signal-regulated protein kinases 1/2
- FAK
- focal adhesion kinase
- GPCR
- G protein-coupled receptor
- HB-EGF
- heparin binding epidermal growth factor
- HEK
- human embryonic kidney
- IGF-1
- insulin-like growth factor type 1
- MMP
- matrix metalloproteinase
- NGF
- nerve growth factor
- wt
- wild type
- TrkA
- neurotrophic tyrosine kinase receptor type 1
- CL-387,785
- N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide
- HA
- hemagglutinin
- FCS
- fetal calf serum
- siRNA
- small interfering RNA
- DMEM
- Dulbecco's modified Eagle's medium
- SOCS-3
- suppressor of cytokine signaling 3
- PDGF
- platelet-derived growth factor
- PBS
- phosphate-buffered saline
- AG538
- α-cyano-(3-methoxy,4-hydroxy,5-iodo)cinnamoyl-(3′,4′-dihydroxyphenyl)ketone
- AG1295
- 6,7-dimethyl-2-phenylquinoxaline
- AG1024
- 3-bromo-5-t-butyl-4-hydroxy-benzylidenemalo-nitrile
- AG879
- α-cyano-(3,5-di-t-butyl-4-hydroxy)thiocinnamide
- AG1478
- 4-(3-chloroanilino)-6,7-dimethoxyquinazoline
- GM6001
- N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-l-tryptophan methylamide.
- Received March 22, 2010.
- Accepted November 15, 2010.
- Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics
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