M1 muscarinic receptor activation mediates cell death in M1-HEK293 cells
E Scott Graham1, Kerhan K Woo, Miranda Aalderink
1Department 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.
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.
Insights
Activation of muscarinic acetylcholine receptors (M1 mAChRs) in HEK293 cells unexpectedly caused cell death, despite initial protective signaling. This highlights the need to consider long-term effects in G protein-coupled receptor studies.
Area of Science:
- Cell biology
- Pharmacology
- Neuroscience
Background:
- HEK293 cells are widely used for studying G protein-coupled receptors (GPCRs).
- Muscarinic acetylcholine receptors (mAChRs), particularly M1 subtype, are implicated in protective cellular responses.
- Previous studies suggest M1 mAChR activation can be protective in certain cell types.
Purpose of the Study:
- To investigate M1 mAChR-mediated cell survival and death in stable HEK293 cell lines.
- To characterize the signaling pathways involved in M1 mAChR activation in HEK293 cells.
- To assess the long-term consequences of M1 mAChR stimulation.
Main Methods:
- Generation of stable HEK293 cell lines expressing human M1 mAChR.
- Carbachol stimulation to activate M1 mAChRs.
- Analysis of receptor internalization, early signaling cascades (MEK-ERK pathway), and transcription factor EGR-1 induction.
- Time-lapse microscopy and xCELLigence technology to monitor cell death.
- Assessment of cleaved caspase 3 expression.
Main Results:
- M1 mAChRs were expressed at the cell surface and internalized upon carbachol stimulation.
- Carbachol induced early signaling cascades and prolonged MEK-ERK activation and EGR-1 induction (>24h).
- Substantial cell death was observed in HEK293-M1 cells within 24h of carbachol application, blocked by M1 antagonists.
- ERK pathway was not directly involved in cell death, but MEK inhibition reduced carbachol-mediated cleaved caspase 3 expression.
Conclusions:
- M1 mAChR stimulation in HEK293 cells leads to cytotoxicity, contradicting protective roles observed elsewhere.
- The MEK-ERK pathway is not the primary driver of M1 mAChR-induced cell death but influences apoptosis markers.
- These findings underscore the importance of evaluating long-term cellular fate in GPCR studies using cell lines like HEK293.
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