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Mitochondria-targeted paraquat and metformin mediate ROS production to induce multiple pathways of retrograde
Anindya Roy Chowdhury1, Jacek Zielonka2, Balaraman Kalyanaraman2
1Department of Biomedical Sciences, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA.
Abstract:
The mitochondrial electron transport chain is a major source of reactive oxygen species (ROS) and is also a target of ROS, with an implied role in the stabilization of hypoxia-inducible factor (HIF) and induction of the AMPK pathway. Here we used varying doses of two agents, Mito-Paraquat and Mito-Metformin, that have been conjugated to cationic triphenylphosphonium (TPP+) moiety to selectively target them to the mitochondrial matrix compartment, thereby resulting in the site-specific generation of ROS within mitochondria. These agents primarily induce superoxide (O2•-) production by acting on complex I. In Raw264.7 macrophages, C2C12 skeletal myocytes, and HCT116 adenocarcinoma cells, we show that mitochondria-targeted oxidants can induce ROS (O2•- and H2O2). In all three cell lines tested, the mitochondria-targeted agents disrupted membrane potential and activated calcineurin and the Cn-dependent retrograde signaling pathway. Hypoxic culture conditions also induced Cn activation and HIF1α activation in a temporally regulated manner, with the former appearing at shorter exposure times. Together, our results indicate that mitochondrial oxidant-induced retrograde signaling is driven by disruption of membrane potential and activation of Ca2+/Cn pathway and is independent of ROS-induced HIF1α or AMPK pathways.
Insights
Targeting reactive oxygen species (ROS) to mitochondria activates calcineurin signaling. This mitochondrial oxidant-induced pathway is independent of hypoxia-inducible factor (HIF) and AMPK signaling.
Area of Science:
- Cell Biology
- Mitochondrial Biology
- Signaling Pathways
Background:
- Mitochondria are key sources and targets of reactive oxygen species (ROS).
- ROS are implicated in regulating hypoxia-inducible factor (HIF) and AMPK pathways.
- Targeted delivery of ROS to mitochondria can elucidate specific signaling mechanisms.
Purpose of the Study:
- To investigate the effects of mitochondria-targeted oxidants on cellular signaling.
- To determine the role of mitochondrial ROS in activating calcineurin (Cn) and retrograde signaling.
- To differentiate mitochondrial ROS signaling from HIF and AMPK pathways.
Main Methods:
- Conjugation of agents (Mito-Paraquat, Mito-Metformin) to triphenylphosphonium (TPP+) for mitochondrial targeting.
- Induction of ROS (superoxide, hydrogen peroxide) in mitochondria using targeted agents.
- Assessment of mitochondrial membrane potential, calcineurin activation, and HIF1α/AMPK pathways in various cell lines (Raw264.7, C2C12, HCT116).
Main Results:
- Mitochondria-targeted oxidants successfully induced ROS production within mitochondria.
- Disruption of mitochondrial membrane potential and activation of Ca2+/Cn-dependent retrograde signaling were observed.
- Calcineurin activation occurred earlier than HIF1α activation under hypoxic conditions.
Conclusions:
- Mitochondrial ROS can directly activate the Ca2+/Cn retrograde signaling pathway.
- This pathway is initiated by mitochondrial membrane potential disruption.
- Mitochondrial oxidant-induced signaling is independent of ROS-mediated HIF1α or AMPK activation.
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