Inhibition of ALK3-mediated signalling pathway protects against acetaminophen-induced liver injury.

Acetaminophen (APAP)-induced liver injury is one of the most prevalent causes of acute liver failure (ALF). We assessed the role of the bone morphogenetic protein (BMP) type I receptors ALK2 and ALK3 in APAP-induced hepatotoxicity. The molecular mechanisms that regulate the balance between cell death and survival and the response to oxidative stress induced by APAP was assessed in cultured human hepatocyte-derived (Huh7) cells treated with pharmacological inhibitors of ALK receptors and with modulated expression of ALK2 or ALK3 by lentiviral infection, and in a mouse model of APAP-induced hepatotoxicity. Inhibition of ALK3 signalling with the pharmacological inhibitor DMH2, or by silencing of ALK3, showed a decreased cell death both by necrosis and apoptosis after APAP treatment. Also, upon APAP challenge, ROS generation was ameliorated and, thus, ROS-mediated JNK and P38 MAPK phosphorylation was reduced in ALK3-inhibited cells compared to control cells. These results were also observed in an experimental model of APAP-induced ALF in which post-treatment with DMH2 after APAP administration significantly reduced liver tissue damage, apoptosis and oxidative stress. This study shows the protective effect of ALK3 receptor inhibition against APAP-induced hepatotoxicity. Furthermore, findings obtained from the animal model suggest that BMP signalling might be a new pharmacological target for the treatment of ALF.

TMEM65 regulates NCLX-dependent mitochondrial calcium efflux.

The balance between mitochondrial calcium (mCa2+) uptake and efflux regulates ATP production, but if perturbed causes energy starvation or mCa2+ overload and cell death. The mitochondrial sodium-calcium exchanger, NCLX, is a critical route of mCa2+ efflux in excitable tissues, such as the heart and brain, and animal models support NCLX as a promising therapeutic target to limit pathogenic mCa2+ overload. However, the mechanisms that regulate NCLX activity remain largely unknown. We used proximity biotinylation proteomic screening to identify the NCLX interactome and define novel regulators of NCLX function. Here, we discover the mitochondrial inner membrane protein, TMEM65, as an NCLX-proximal protein that potently enhances sodium (Na+)-dependent mCa2+ efflux. Mechanistically, acute pharmacologic NCLX inhibition or genetic deletion of NCLX ablates the TMEM65-dependent increase in mCa2+ efflux. Further, loss-of-function studies show that TMEM65 is required for Na+-dependent mCa2+ efflux. Co-fractionation and in silico structural modeling of TMEM65 and NCLX suggest these two proteins exist in a common macromolecular complex in which TMEM65 directly stimulates NCLX function. In line with these findings, knockdown of Tmem65 in mice promotes mCa2+ overload in the heart and skeletal muscle and impairs both cardiac and neuromuscular function. We further demonstrate that TMEM65 deletion causes excessive mitochondrial permeability transition, whereas TMEM65 overexpression protects against necrotic cell death during cellular Ca2+ stress. Collectively, our results show that loss of TMEM65 function in excitable tissue disrupts NCLX-dependent mCa2+ efflux, causing pathogenic mCa2+ overload, cell death and organ-level dysfunction, and that gain of TMEM65 function mitigates these effects. These findings demonstrate the essential role of TMEM65 in regulating NCLX-dependent mCa2+ efflux and suggest modulation of TMEM65 as a novel strategy for the therapeutic control of mCa2+ homeostasis.

Measurement of Superoxide Production in Acute Hypoxia by Fixed-Cell Microscopy.

Redox signaling implication in cell adaptation to hypoxia has been studied for a long time, both in long-term and acute responses. However, measurement of superoxide and other reactive oxygen species (ROS) in acute hypoxia is technically challenging, for example, because of the need to overcome the effect of cell reoxygenation before measurement.Here we describe a method we have developed for measuring superoxide production in acute hypoxia using the fluorescent probe dihydroethidine in fixed-cell microscopy. The method allows measuring the kinetics of superoxide production (or other ROS with the appropriate probes) by incubating the probe in different time windows during hypoxia incubation.

Na+ controls hypoxic signalling by the mitochondrial respiratory chain.

All metazoans depend on the consumption of O2 by the mitochondrial oxidative phosphorylation system (OXPHOS) to produce energy. In addition, the OXPHOS uses O2 to produce reactive oxygen species that can drive cell adaptations1-4, a phenomenon that occurs in hypoxia4-8 and whose precise mechanism remains unknown. Ca2+ is the best known ion that acts as a second messenger9, yet the role ascribed to Na+ is to serve as a mere mediator of membrane potential10. Here we show that Na+ acts as a second messenger that regulates OXPHOS function and the production of reactive oxygen species by modulating the fluidity of the inner mitochondrial membrane. A conformational shift in mitochondrial complex I during acute hypoxia11 drives acidification of the matrix and the release of free Ca2+ from calcium phosphate (CaP) precipitates. The concomitant activation of the mitochondrial Na+/Ca2+ exchanger promotes the import of Na+ into the matrix. Na+ interacts with phospholipids, reducing inner mitochondrial membrane fluidity and the mobility of free ubiquinone between complex II and complex III, but not inside supercomplexes. As a consequence, superoxide is produced at complex III. The inhibition of Na+ import through the Na+/Ca2+ exchanger is sufficient to block this pathway, preventing adaptation to hypoxia. These results reveal that Na+ controls OXPHOS function and redox signalling through an unexpected interaction with phospholipids, with profound consequences for cellular metabolism.

Downregulation of thioredoxin-1-dependent CD95 S-nitrosation by Sorafenib reduces liver cancer.

Hepatocellular carcinoma (HCC) represents 80% of the primary hepatic neoplasms. It is the sixth most frequent neoplasm, the fourth cause of cancer-related death, and 7% of registered malignancies. Sorafenib is the first line molecular targeted therapy for patients in advanced stage of HCC. The present study shows that Sorafenib exerts free radical scavenging properties associated with the downregulation of nuclear factor E2-related factor 2 (Nrf2)-regulated thioredoxin 1 (Trx1) expression in liver cancer cells. The experimental downregulation and/or overexpression strategies showed that Trx1 induced activation of nitric oxide synthase (NOS) type 3 (NOS3) and S-nitrosation (SNO) of CD95 receptor leading to an increase of caspase-8 activity and cell proliferation, as well as reduction of caspase-3 activity in liver cancer cells. In addition, Sorafenib transiently increased mRNA expression and activity of S-nitrosoglutathione reductase (GSNOR) in HepG2 cells. Different experimental models of hepatocarcinogenesis based on the subcutaneous implantation of HepG2 cells in nude mice, as well as the induction of HCC by diethylnitrosamine (DEN) confirmed the relevance of Trx1 downregulation during the proapoptotic and antiproliferative properties induced by Sorafenib. In conclusion, the induction of apoptosis and antiproliferative properties by Sorafenib were related to Trx1 downregulation that appeared to play a relevant role on SNO of NOS3 and CD95 in HepG2 cells. The transient increase of GSNOR might also participate in the deactivation of CD95-dependent proliferative signaling in liver cancer cells.

The Metabolite Urolithin-A Ameliorates Oxidative Stress in Neuro-2a Cells, Becoming a Potential Neuroprotective Agent.

Urolithin A is a metabolite generated from ellagic acid and ellagitannins by the intestinal microbiota after consumption of fruits such as pomegranates or strawberries. The objective of this study was to determine the cytoprotective capacity of this polyphenol in Neuro-2a cells subjected to oxidative stress, as well as its direct radical scavenging activity and properties as an inhibitor of oxidases. Cells treated with this compound and H2O2 showed a greater response to oxidative stress than cells only treated with H2O2, as mitochondrial activity (MTT assay), redox state (ROS formation, lipid peroxidation), and the activity of antioxidant enzymes (CAT: catalase, SOD: superoxide dismutase, GR: glutathione reductase, GPx: glutathione peroxidase) were significantly ameliorated; additionally, urolithin A enhanced the expression of cytoprotective peroxiredoxins 1 and 3. Urolithin A also acted as a direct radical scavenger, showing values of 13.2 µM Trolox Equivalents for Oxygen Radical Absorbance Capacity (ORAC) and 5.01 µM and 152.66 µM IC50 values for superoxide and 2,2-diphenyss1-picrylhydrazyl (DPPH) radicals, respectively. Finally, inhibition of oxidizing enzymes, such as monoamine oxidase A and tyrosinase, was also detected in a dose-dependent manner. The cytoprotective effects of urolithin A could be attributed to the improvement of the cellular antioxidant battery, but also to its role as a direct radical scavenger and enzyme inhibitor of oxidases.