title: M1 muscarinic receptor activation mediates cell death in M1-HEK293 cells.
abstract: HEK293 cells have been used extensively to generate stable cell lines to study G protein-coupled receptors, such as muscarinic acetylcholine receptors (mAChRs). The activation of M1 mAChRs in various cell types in vitro has been shown to be protective. To further investigate M1 mAChR-mediated cell survival, we generated stable HEK293 cell-lines expressing the human M1 mAChR. M1 mAChRs were efficiently expressed at the cell surface and efficiently internalised within 1 h by carbachol. Carbachol also induced early signalling cascades similar to previous reports. Thus, ectopically expressed M1 receptors behaved in a similar fashion to the native receptor over short time periods of analysis. However, substantial cell death was observed in HEK293-M1 cells within 24 h after carbachol application. Death was only observed in HEK cells expressing M1 receptors and fully blocked by M1 antagonists. M1 mAChR-stimulation mediated prolonged activation of the MEK-ERK pathway and resulted in prolonged induction of the transcription factor EGR-1 (>24 h). Blockade of ERK signalling with U0126 did not reduce M1 mAChR-mediated cell-death significantly but inhibited the acute induction of EGR-1. We investigated the time-course of cell death using time-lapse microscopy and xCELLigence technology. Both revealed the M1 mAChR cytotoxicity occurs within several hours of M1 activation. The xCELLigence assay also confirmed that the ERK pathway was not involved in cell-death. Interestingly, the MEK blocker did reduce carbachol-mediated cleaved caspase 3 expression in HEK293-M1 cells. The HEK293 cell line is a widely used pharmacological tool for studying G-protein coupled receptors, including mAChRs. Our results highlight the importance of investigating the longer term fate of these cells in short term signalling studies. Identifying how and why activation of the M1 mAChR signals apoptosis in these cells may lead to a better understanding of how mAChRs regulate cell-fate decisions.
authors.affiliation: Department of Pharmacology and Clinical Pharmacology, Centre for Brain Research, School of Medical Sciences, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand.
authors.forename: E Scott
authors.initials: ES
authors.lastname: Graham
authors.forename: Kerhan K
authors.initials: KK
authors.lastname: Woo
authors.forename: Miranda
authors.initials: M
authors.lastname: Aalderink
authors.forename: Sandie
authors.initials: S
authors.lastname: Fry
authors.forename: Jeffrey M
authors.initials: JM
authors.lastname: Greenwood
authors.forename: Michelle
authors.initials: M
authors.lastname: Glass
authors.forename: Mike
authors.initials: M
authors.lastname: Dragunow
mesh_terms: D015374:Biosensing Techniques; D002070:Butadienes / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D057809:HEK293 Cells; D006801:Humans; D007150:Immunohistochemistry; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000235:genetics; D009570:Nitriles / Q000494:pharmacology; D043643:Receptor, Muscarinic M1 / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D002070:Butadienes; D009570:Nitriles; D043643:Receptor, Muscarinic M1; C113580:U 0126
references.citation: J Biol Chem. 2000 Sep 15;275(37):28929-36
references.pmid: 10852911
references.citation: Arch Biochem Biophys. 2008 Feb 1;470(1):93-102
references.pmid: 18061571
references.citation: Cancer Res. 2003 Oct 15;63(20):6744-50
references.pmid: 14583469
references.citation: Biochem Pharmacol. 2003 Dec 1;66(11):2099-105
references.pmid: 14609734
references.citation: Annu Rev Pharmacol Toxicol. 2004;44:423-50
references.pmid: 14744253
references.citation: J Neurochem. 2012 Jan;120 Suppl 1:22-33
references.pmid: 22122190
references.citation: Nat Rev Drug Discov. 2008 Feb;7(2):1 p following p185
references.pmid: 18246605
references.citation: Cell Mol Neurobiol. 2008 Feb;28(2):263-75
references.pmid: 18172757
references.citation: Mol Pharmacol. 1995 Feb;47(2):248-57
references.pmid: 7870032
references.citation: J Pharmacol Toxicol Methods. 2005 May-Jun;51(3):187-200
references.pmid: 15862464
references.citation: J Cell Physiol. 2010 Mar;222(3):640-7
references.pmid: 19927300
references.citation: J Neurochem. 2008 Jul;106(1):70-82
references.pmid: 18331587
references.citation: J Biol Chem. 1991 Apr 5;266(10):6031-4
references.pmid: 2007563
references.citation: J Biol Chem. 1998 Jun 5;273(23):14538-44
references.pmid: 9603968
references.citation: Jpn J Pharmacol. 2000 Oct;84(2):101-12
references.pmid: 11128032
references.citation: J Neurosci Methods. 2008 Jun 15;171(1):78-86
references.pmid: 18406468
references.citation: Neuron. 2006 Mar 2;49(5):671-82
references.pmid: 16504943
references.citation: Nat Rev Neurosci. 2008 Oct;9(10):779-88
references.pmid: 18784656
references.citation: Cancer Res. 2003 Jan 1;63(1):214-21
references.pmid: 12517800
references.citation: Life Sci. 2003 Dec 5;74(2-3):217-24
references.pmid: 14607249
references.citation: Mol Pharmacol. 2004 May;65(5):1080-91
references.pmid: 15102936
references.citation: Biochem J. 2004 Jul 1;381(Pt 1):43-9
references.pmid: 15068397
references.citation: J Neurosci Methods. 2011 Sep 15;200(2):173-80
references.pmid: 21781988
references.citation: Glia. 2001 Aug;35(2):111-20
references.pmid: 11460267
references.citation: J Neurosci. 2007 Apr 11;27(15):4083-92
references.pmid: 17428986
references.citation: Int J Dev Neurosci. 2001 Oct;19(6):559-67
references.pmid: 11600318
references.citation: J Biol Chem. 1998 May 22;273(21):12967-72
references.pmid: 9582330
references.citation: Toxicol Sci. 2004 Jul;80(1):60-8
references.pmid: 15141107
references.citation: J Cell Biochem. 2008 Aug 1;104(5):1818-31
references.pmid: 18348264
references.citation: J Biol Chem. 2010 Oct 15;285(42):32539-48
references.pmid: 20634292
references.citation: J Neurochem. 2002 Jul;82(2):389-97
references.pmid: 12124440
references.citation: Jpn J Pharmacol. 1997 Apr;73(4):311-6
references.pmid: 9165367
references.citation: J Toxicol Environ Health A. 2008;71(16):1109-23
references.pmid: 18569623
references.citation: J Biol Chem. 2003 Mar 28;278(13):11086-93
references.pmid: 12538580
references.citation: Clin Exp Pharmacol Physiol. 2008 Nov;35(11):1377-82
references.pmid: 18565191
references.citation: Ann N Y Acad Sci. 1993 Sep 24;695:300-3
references.pmid: 8239299
references.citation: Prostate. 1997 Feb 15;30(3):160-6
references.pmid: 9122040
references.citation: Ann N Y Acad Sci. 2000;920:315-20
references.pmid: 11193170
references.citation: J Biol Chem. 2006 Sep 29;281(39):29085-95
references.pmid: 16864584
references.citation: Cell Death Differ. 2000 Sep;7(9):825-33
references.pmid: 11042677
references.citation: J Biol Chem. 2004 Oct 1;279(40):41521-8
references.pmid: 15280370
references.citation: J Neurochem. 2009 Apr;109(2):525-38
references.pmid: 19200344
references.citation: J Biol Chem. 2003 May 23;278(21):19565-73
references.pmid: 12649280
references.citation: Mol Pharmacol. 2008 Aug;74(2):338-47
references.pmid: 18388243