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Short Exposure to Ethanol Diminishes Caspase-1 and ASC Activation in Human HepG2 Cells In Vitro
Jason-Alexander Hörauf1, Shinwan Kany2,3, Andrea Janicova2
1Department of Trauma, Hand and Reconstructive Surgery, Goethe University Frankfurt, 60438 Frankfurt am Main, Germany.
Abstract:
This paper discusses how the assembly of pro-caspase-1 and apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) in macromolecular protein complexes, inflammasomes, activates caspase-1. The present study investigates the molecular mechanisms of inflammasome activation in HepG2 cells and examines how short exposures to ethanol (EtOH) affect inflammasome activation. HepG2 cells were treated with lipopolysaccharide (LPS), ATP or nigericin (NIG) in a two-step model. After LPS priming, ATP or NIG were added. As inhibitors, sodium orthovanadate (general inhibitor of tyrosine phosphatases), AC-YVAD-CMK (caspase-1 inhibitor) or AZ10606120 (purinergic receptor P2X7R inhibitor) were applied after LPS priming. To monitor the inflammasome activation, the caspase-1 activity, ASC speck formation, reactive oxygen species (ROS) production and cell death were analyzed. To elucidate the mechanistical approach of EtOH to the inflammasome assembly, the cells were treated with EtOH either under simultaneous LPS administration or concurrently with ATP or NIG application. The co-stimulation with LPS and ATP induced a significant ASC speck formation, caspase-1 activation, cell death and ROS generation. The inhibition of the ATP-dependent purinoreceptor P2X7 decreased the caspase-1 activation, whereas sodium orthovanadate significantly induced caspase-1. Additional treatment with EtOH reversed the LPS and ATP-induced caspase-1 activation, ASC speck formation and ROS production. The ASC speck formation and caspase-1 induction require a two-step signaling with LPS and ATP in HepG2 cells. Inflammasome activation may depend on P2X7. The molecular pathway of an acute effect of EtOH on inflammasomes may involve a reduction in ROS generation, which in turn may increase the activity of tyrosine phosphatases.
Insights
Ethanol (EtOH) exposure reduces inflammasome activation in HepG2 cells by decreasing reactive oxygen species (ROS) and potentially increasing tyrosine phosphatase activity. This impacts caspase-1 activation and ASC speck formation.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Inflammasomes are crucial protein complexes that activate caspase-1, a key mediator of inflammation and cell death.
- HepG2 cells are a human hepatoma cell line frequently used to study cellular responses to stimuli.
- Ethanol (EtOH) is known to affect cellular processes, but its precise impact on inflammasome activation requires elucidation.
Purpose of the Study:
- To investigate the molecular mechanisms of inflammasome activation in HepG2 cells.
- To examine the effects of short-term ethanol (EtOH) exposure on inflammasome activation pathways.
- To elucidate the role of purinergic receptor P2X7 and reactive oxygen species (ROS) in EtOH-mediated inflammasome modulation.
Main Methods:
- HepG2 cells were stimulated using a two-step model involving lipopolysaccharide (LPS) priming followed by ATP or nigericin (NIG) stimulation.
- Inhibitors including sodium orthovanadate, AC-YVAD-CMK (caspase-1 inhibitor), and AZ10606120 (P2X7R inhibitor) were used to probe signaling pathways.
- Measurements included caspase-1 activity, ASC speck formation, ROS production, and cell death assays.
- Cells were treated with EtOH during various stages of stimulation to assess its acute effects.
Main Results:
- Co-stimulation with LPS and ATP significantly induced ASC speck formation, caspase-1 activation, cell death, and ROS generation.
- Inhibition of P2X7R decreased caspase-1 activation, while sodium orthovanadate treatment enhanced it.
- Acute ethanol (EtOH) exposure reversed LPS/ATP-induced caspase-1 activation, ASC speck formation, and ROS production.
- Ethanol's effect appears linked to reduced ROS generation and potentially increased tyrosine phosphatase activity.
Conclusions:
- Inflammasome activation in HepG2 cells requires a two-step signaling process involving LPS and ATP, potentially mediated by P2X7.
- Acute ethanol exposure modulates inflammasome activation, characterized by decreased ROS production and altered tyrosine phosphatase activity.
- These findings shed light on the molecular mechanisms underlying ethanol's impact on innate immune signaling pathways.
