Enhanced formation of reactive oxygen species (ROS), superoxide ( O2· -), and hydrogen peroxide ( H2 O2) may result in either apoptosis or other forms of cell death. Here, we studied the mechanisms ...underlying activation of the apoptotic machinery by ROS. Exposure of permeabilized HepG2 cells to O2· - elicited rapid and massive cytochrome c release (CCR), whereas H2 O2 failed to induce any release. Both O2· - and H2 O2 promoted activation of the mitochondrial permeability transition pore by Ca2+, but Ca2+-dependent pore opening was not required for O2· --induced CCR. Furthermore, O2· - alone evoked CCR without damage of the inner mitochondrial membrane barrier, as mitochondrial membrane potential was sustained in the presence of extramitochondrial ATP. Strikingly, pretreatment of the cells with drugs or an antibody, which block the voltage-dependent anion channel (VDAC), prevented O2· --induced CCR. Furthermore, VDAC-reconstituted liposomes permeated cytochrome c after O2· - exposure, and this release was prevented by VDAC blocker. The proapoptotic protein, Bak, was not detected in HepG2 cells and O2· --induced CCR did not depend on Bax translocation to mitochondria. O2· --induced CCR was followed by caspase activation and execution of apoptosis. Thus, O2· - triggers apoptosis via VDAC-dependent permeabilization of the mitochondrial outer membrane without apparent contribution of proapoptotic Bcl-2 family proteins.
Abstract
The mitochondrial calcium (Ca
2+
) uniporter (MCU) channel is responsible for mitochondrial Ca
2+
influx. Its expression was found to be upregulated in endothelial cells (ECs) under ...cardiovascular disease conditions. Since the role of MCU in regulating cytosolic Ca
2+
homeostasis in ECs exposed to shear stress (SS) is unknown, we studied mitochondrial Ca
2+
dynamics (that is known to decode cytosolic Ca
2+
signaling) in sheared ECs. To understand cause-and-effect, we ectopically expressed MCU in ECs. A higher percentage of MCU-transduced ECs exhibited mitochondrial Ca
2+
transients/oscillations, and at higher frequency, under SS compared to sheared control ECs. Transients/oscillations correlated with mitochondrial reactive oxygen species (mROS) flashes and mitochondrial membrane potential (ΔΨ
m
) flickers, and depended on activation of the mechanosensitive Piezo1 channel and the endothelial nitric oxide synthase (eNOS). A positive feedback loop composed of mitochondrial Ca
2+
uptake/mROS flashes/ΔΨ
m
flickers and endoplasmic reticulum Ca
2+
release, in association with Piezo1 and eNOS, provided insights into the mechanism by which SS, under conditions of high MCU activity, may shape vascular EC energetics and function.
Calcium (Ca2+) transport by mitochondria is an important component of the cell Ca2+ homeostasis machinery in metazoans. Ca2+ uptake by mitochondria is a major determinant of bioenergetics and cell ...fate. Mitochondrial Ca2+ uptake occurs via the mitochondrial Ca2+ uniporter (MCU) complex, an inner mitochondrial membrane protein assembly consisting of the MCU Ca2+ channel, as its core component, and the MCU complex regulatory/auxiliary proteins. In this review, we summarize the current knowledge on the molecular nature of the MCU complex and its regulation by intra- and extramitochondrial levels of divalent ions and reactive oxygen species (ROS). Intracellular Ca2+ concentration (Ca2+i), mitochondrial Ca2+ concentration (Ca2+m) and mitochondrial ROS (mROS) are intricately coupled in regulating MCU activity. Here, we highlight the contribution of MCU activity to vascular endothelial cell (EC) function. Besides the ionic and oxidant regulation, ECs are continuously exposed to haemodynamic forces (either pulsatile or oscillatory fluid mechanical shear stresses, depending on the precise EC location within the arteries). Thus, we also propose an EC mechanotransduction-mediated regulation of MCU activity in the context of vascular physiology and atherosclerotic vascular disease.
Mitochondrial Ca2+ (mCa2+) uptake mediated by the mitochondrial calcium uniporter (MCU) plays a critical role in signal transduction, bioenergetics, and cell death, and its dysregulation is linked to ...several human diseases. In this study, we report a new ruthenium complex Ru265 that is cell-permeable, minimally toxic, and highly potent with respect to MCU inhibition. Cells treated with Ru265 show inhibited MCU activity without any effect on cytosolic Ca2+ dynamics and mitochondrial membrane potential (ΔΨm). Dose-dependent studies reveal that Ru265 is more potent than the currently employed MCU inhibitor Ru360. Site-directed mutagenesis of Cys97 in the N-terminal domain of human MCU ablates the inhibitory activity of Ru265, suggesting that this matrix-residing domain is its target site. Additionally, Ru265 prevented hypoxia/reoxygenation injury and subsequent mitochondrial dysfunction, demonstrating that this new inhibitor is a valuable tool for studying the functional role of the MCU in intact biological models.
IL-6 plays an important role in determining the fate of effector CD4 cells and the cytokines that these cells produce. Here we identify a novel molecular mechanism by which IL-6 regulates CD4 cell ...effector function. We show that IL-6-dependent signal facilitates the formation of mitochondrial respiratory chain supercomplexes to sustain high mitochondrial membrane potential late during activation of CD4 cells. Mitochondrial hyperpolarization caused by IL-6 is uncoupled from the production of ATP by oxidative phosphorylation. However, it is a mechanism to raise the levels of mitochondrial Ca(2+) late during activation of CD4 cells. Increased levels of mitochondrial Ca(2+) in the presence of IL-6 are used to prolong Il4 and Il21 expression in effector CD4 cells. Thus, the effect of IL-6 on mitochondrial membrane potential and mitochondrial Ca(2+) is an alternative pathway by which IL-6 regulates effector function of CD4 cells and it could contribute to the pathogenesis of inflammatory diseases.
Transient receptor potential melastatin 2 (TRPM2) ion channel has an essential function in maintaining cell survival following oxidant injury. Here, we show that TRPM2 is highly expressed in acute ...myeloid leukemia (AML). The role of TRPM2 in AML was studied following depletion with CRISPR/Cas9 technology in U937 cells. In in vitro experiments and in xenografts, depletion of TRPM2 in AML inhibited leukemia proliferation, and doxorubicin sensitivity was increased. Mitochondrial function including oxygen consumption rate and ATP production was reduced, impairing cellular bioenergetics. Mitochondrial membrane potential and mitochondrial calcium uptake were significantly decreased in depleted cells. Mitochondrial reactive oxygen species (ROS) were significantly increased, and Nrf2 was decreased, reducing the antioxidant response. In TRPM2-depleted cells, ULK1, Atg7, and Atg5 protein levels were decreased, leading to autophagy inhibition. Consistently, ATF4 and CREB, two master transcription factors for autophagosome biogenesis, were reduced in TRPM2-depleted cells. In addition, Atg13 and FIP200, which are known to stabilize ULK1 protein, were decreased. Reconstitution with TRPM2 fully restored proliferation, viability, and autophagy; ATF4 and CREB fully restored proliferation and viability but only partially restored autophagy. TRPM2 expression reduced the elevated ROS found in depleted cells. These data show that TRPM2 has an important role in AML proliferation and survival through regulation of key transcription factors and target genes involved in mitochondrial function, bioenergetics, the antioxidant response, and autophagy. Targeting TRPM2 may represent a novel therapeutic approach to inhibit myeloid leukemia growth and enhance susceptibility to chemotherapeutic agents through multiple pathways.
Calcium oscillations exert physiological control on mitochondrial energy metabolism and can also lead to mitochondrial membrane permeabilization and cell death. The outcome of the mitochondrial ...calcium signaling is altered by stress factors such as ceramide or staurosporine. However, the mechanism of this proapoptotic switch remains unclear. Using genetic, biochemical, pharmacological, and functional approaches, we here show that ceramide and staurosporine target PP2A and protein kinases A and C, respectively, in a mitochondria-associated signaling complex to induce dephosphorylation of the BH3-only protein Bad. Dephosphorylated Bad sensitizes the mitochondrial permeability transition pore (PTP) to Ca2+ through a Bcl-xL-sensitive and VDAC-mediated process. Furthermore, the Bad-induced sensitization of the PTP to Ca2+ does not require Bax or Bak. Thus, phospho-regulatory mechanisms converge on Bad to switch between the survival and apoptotic functions of mitochondrial calcium signaling by activating a mechanism whereby a BH3-only protein bypasses Bax/Bak and engages the PTP.
RATIONALE:The cellular and molecular basis for post–myocardial infarction (MI) structural and functional remodeling is not well understood.
OBJECTIVE:Our aim was to determine if Ca influx through ...transient receptor potential canonical (TRPC) channels contributes to post-MI structural and functional remodeling.
METHODS AND RESULTS:TRPC1/3/4/6 channel mRNA increased after MI in mice and was associated with TRPC-mediated Ca entry. Cardiac myocyte–specific expression of a dominant-negative (loss-of-function) TRPC4 channel increased basal myocyte contractility and reduced hypertrophy and cardiac structural and functional remodeling after MI while increasing survival in mice. We used adenovirus-mediated expression of TRPC3/4/6 channels in cultured adult feline myocytes to define mechanistic aspects of these TRPC-related effects. TRPC3/4/6 overexpression in adult feline myocytes induced calcineurin (Cn)-nuclear factor of activated T-cells (NFAT)–mediated hypertrophic signaling, which was reliant on caveolae targeting of TRPCs. TRPC3/4/6 expression in adult feline myocytes increased rested state contractions and increased spontaneous sarcoplasmic reticulum Ca sparks mediated by enhanced phosphorylation of the ryanodine receptor. TRPC3/4/6 expression was associated with reduced contractility and response to catecholamines during steady-state pacing, likely because of enhanced sarcoplasmic reticulum Ca leak.
CONCLUSIONS:Ca influx through TRPC channels expressed after MI activates pathological cardiac hypertrophy and reduces contractility reserve. Blocking post-MI TRPC activity improved post-MI cardiac structure and function.
Cold physical plasma is a partially ionized gas investigated as a new anticancer tool in selectively targeting cancer cells in monotherapy or in combination with therapeutic agents. Here, we ...investigated the intrinsic resistance mechanisms of tumor cells towards physical plasma treatment. When analyzing the dose-response relationship to cold plasma-derived oxidants in 11 human cancer cell lines, we identified four ‘resistant’ and seven ‘sensitive’ cell lines. We observed stable intracellular glutathione levels following plasma treatment only in the ‘resistant’ cell lines indicative of altered antioxidant mechanisms. Assessment of proteins involved in GSH metabolism revealed cystine-glutamate antiporter xCT (SLC7A11) to be significantly more abundant in the ‘resistant’ cell lines as compared to ‘sensitive’ cell lines. This decisive role of xCT was confirmed by pharmacological and genetic inhibition, followed by cold physical plasma treatment. Finally, microscopy analysis of ex vivo plasma-treated human melanoma punch biopsies suggested a correlation between apoptosis and basal xCT protein abundance. Taken together, our results demonstrate that xCT holds the potential as a biomarker predicting the sensitivity of tumor cells towards plasma treatment.
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Mitochondria house evolutionarily conserved pathways of carbon and nitrogen metabolism that drive cellular energy production. Mitochondrial bioenergetics is regulated by calcium uptake through the ...mitochondrial calcium uniporter (MCU), a multi-protein complex whose assembly in the inner mitochondrial membrane is facilitated by the scaffold factor MCUR1. Intriguingly, many fungi that lack MCU contain MCUR1 homologs, suggesting alternate functions. Herein, we characterize Saccharomyces cerevisiae homologs Put6 and Put7 of MCUR1 as regulators of mitochondrial proline metabolism. Put6 and Put7 are tethered to the inner mitochondrial membrane in a large hetero-oligomeric complex, whose abundance is regulated by proline. Loss of this complex perturbs mitochondrial proline homeostasis and cellular redox balance. Yeast cells lacking either Put6 or Put7 exhibit a pronounced defect in proline utilization, which can be corrected by the heterologous expression of human MCUR1. Our work uncovers an unexpected role of MCUR1 homologs in mitochondrial proline metabolism.
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