Kana Unuma

Cobalt Protoporphyrin Accelerates TFEB Activation and Lysosome Reformation during LPS-Induced Septic Insults in the Rat Heart

Lipopolysaccharide (LPS)-induced myocardial dysfunction is caused, at least in part, by mitochondrial dysfunction. Mitochondrial dysfunction and the oxidative damage associated with it are scavenged through various cellular defense systems such as autophagy to prevent harmful effects. Our recent study has demonstrated that cobalt protoporphyrin IX (CoPPIX), a potent inducer of heme oxygenase-1 (HO-1), ameliorates septic liver injuries by enhancing mitochondrial autophagy in rats. In our current study, we show that CoPPIX (5 mg/kg s.c.) not only accelerates the autophagic response but also promotes lysosome reformation in the rat heart treated with LPS (15 mg/kg i.p.). Lysosomal membrane-associated protein-2 (LAMP2), which is essential to the maintenance of lysosomal functions in the heart, is depleted transiently but restored rapidly during LPS administration in the rat. Activation of transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy, was also observed, indicating a hyper consumption and subsequent reformation of the lysosome to meet the increased demand for autophagosome cleaning. CoPPIX was found to promote these processes and tended to restore the LPS-induced suppression of cardiac performances whilst chloroquine (CQ; 20 mg/kg i.p.), an inhibitor of lysosomes and autophagic protein degradation, abrogates these beneficial effects. The cardioprotective effect of CoPPIX against LPS toxicity was also observed via decreased levels of cardiac releasing enzymes in the plasma. Taken together, our current data indicate that lysosome reformation mediated by TFEB may be involved in cardioprotection against LPS-induced septic insults, and serve as a novel mechanism by which CoPPIX protects the heart against oxidative stress.

Extrusion of mitochondrial contents from lipopolysaccharide-stimulated cells: Involvement of autophagy

Sepsis/endotoxemia is elicited by the circulatory distribution of pathogens/endotoxins into whole bodies, and causes profound effects on human health by causing inflammation in multiple organs. Mitochondrial damage is one of the characteristics of the cellular degeneration observed during sepsis/endotoxemia. Elimination of damaged mitochondria through the autophagy-lysosome system has been reported in the liver, indicating that autophagy should play an important role in liver homeostasis during sepsis/endotoxemia. An increased appearance of mitochondrial DNA and proteins in the plasma is another feature of sepsis/endotoxemia, suggesting that damaged mitochondria are not only eliminated within the cells, but also extruded through currently unknown mechanisms. Here we provide evidence for the secretion of mitochondrial proteins and DNA from lipopolysaccharide (LPS)-stimulated rat hepatocytes as well as mouse embryonic fibroblasts (MEFs). The secretion of mitochondrial contents is accompanied by the secretion of proteins that reside in the lumenal space of autolysosomes (LC3-II and CTSD/cathepsin D), but not by a lysosomal membrane protein (LAMP1). The pharmacological inhibition of autophagy by 3MA blocks the secretion of mitochondrial constituents from LPS-stimulated hepatocytes. LPS also stimulates the secretion of mitochondrial as well as autolysosomal lumenal proteins from wild-type (Atg5+/+) MEFs, but not from atg5−/− MEFs. Furthermore, we show that direct exposure of purified mitochondria activates polymorphonuclear leukocytes (PMNs), as evident by the induction of IL1B/interlekin-1β, a pro-inflammatory cytokine. Taken together, the data suggest the active extrusion of mitochondrial contents, which provoke an inflammatory response of immune cells, through the exocytosis of autolysosomes by cells stimulated with LPS.

Inducer of heme oxygenase-1 cobalt protoporphyrin accelerates autophagy and suppresses oxidative damages during lipopolysaccharide treatment in rat liver

Aim:  Mitochondrial damage and subsequent oxidative stresses play important roles in the pathogenesis of sepsis‐induced organ failure. Recently, autophagy, the major degradation pathway involved in mitochondrial quality control, was reported as a cellular adaptive response to oxidative stresses. The aim of the present study was to elucidate the molecular mechanism that underlies hepatic damage during lipopolysaccharide (LPS) treatment. We also try to determine if the damage can be attenuated by administration of cobalt protoporphyrin (CoPP), a potent heme oxygenase‐1 (HO‐1) inducer.

Impairment of autophagy: from hereditary disorder to drug intoxication.

At first, the molecular mechanism of autophagy was unveiled in a unicellular organism Saccharomyces cerevisiae (budding yeast), followed by the discovery that the basic mechanism of autophagy is conserved in multicellular organisms including mammals. Although autophagy was considered to be a non-selective bulk protein degradation system to recycle amino acids during periods of nutrient starvation, it is also believed to be an essential mechanism for the selective elimination of proteins/organelles that are damaged under pathological conditions. Research advances made using autophagy-deficient animals have revealed that impairments of autophagy often underlie the pathogenesis of hereditary disorders such as Danon, Parkinsons, Alzheimers, and Huntingtons diseases, and amyotrophic lateral sclerosis. On the other hand, there are many reports that drugs and toxicants, including arsenic, cadmium, paraquat, methamphetamine, and ethanol, induce autophagy during the development of their toxicity on many organs including heart, brain, lung, kidney, and liver. Although the question as to whether autophagic machinery is involved in the execution of cell death or not remains controversial, the current view of the role of autophagy during cell/tissue injury is that it is an important, often essential, cytoprotective reaction; disturbances in cytoprotective autophagy aggravate cell/tissue injuries. The purpose of this review is to provide (1) a gross summarization of autophagy processes, which are becoming more important in the field of toxicology, and (2) examples of important studies reporting the involvement of perturbations in autophagy in cell/tissue injuries caused by acute as well as chronic intoxication.

Immunohistochemical study of the autophagy marker microtubule-associated protein 1 light chain 3 in normal and steatotic human livers.

Autophagy has been implicated in lipid droplet (LD) turnover. Adipose differentiation‐related protein (ADRP) and microtubule‐associated protein 1 light chain 3 (LC3) monitor LD and autophagosomes, respectively. We examined whether immunohistochemical staining of ADRP and LC3 can monitor LD and autophagy, and if so, whether autophagy is related to LD turnover in post‐mortem human livers.

Distinct effects of methamphetamine on autophagy–lysosome and ubiquitin–proteasome systems in HL-1 cultured mouse atrial cardiomyocytes

The aim of this study is to investigate the molecular mechanism underling the cardiotoxicity of methamphetamine, a psychostimulant drug that is currently abused in the world. A mouse atrial cardiac cell line, HL-1, which retains phenotypes of cardiac cells and serves as a useful model for examining cardiac pathophysiology, was used for this purpose. During treatment with 1mM methamphetamine (MAP) for 3-48h, massive but transient cytoplasmic vacuolization (3-12h) followed by an intracellular accumulation of granules (24-48h) was observed under light microscopy. The vacuoles were surrounded by the lysosome membrane marker LAMP1, while the granules colocalized with the autophagy markers LC3 and p62 as well as ubiquitinated proteins. Western blot analysis showed that LC3 was activated during MAP administration, although p62 was not degraded but rather accumulated. Concordant with p62 accumulation, the nuclear translocation of an anti-oxidative transcription factor, Nrf2, and the subsequent induction of its target gene, HO-1, was observed, suggesting an impairment of autophagic protein degradation and the subsequent activation of the p62/Nrf2/HO-1 pathway. In addition, proteomic analysis revealed a reduction in myosin heavy chain (MHC) protein levels during MAP administration. The ubiquitination of MHC and the induction of the muscle sarcomere protein-specific E3 ubiquitin ligases MuRF1 and atrogin-1 were proved by immunoprecipitation and quantitative real-time PCR, respectively. Taken together, the vacuolization of lysosomes and the subsequent accumulation of autophagosomes indicate an impairment of autophagic protein degradation during MAP administration; on the other hand, the ubiquitination and subsequent degradation of MHC indicate the proper progression of proteasomal degradation.

Hyperstimulation of macropinocytosis leads to lysosomal dysfunction during exposure to methamphetamine in SH-SY5Y cells

Although various cytotoxic effects on neuronal cells caused by methamphetamine (METH) have been investigated, the cellular and molecular mechanisms of METH-induced neurotoxicity remain to be elucidated. We previously reported that METH-induced cytomorphological effects on retinoic acid (RA)-differentiated SH-SY5Y human neuroblastoma cells involved macropinocytosis, which is an actin-dependent endocytic pathway. We also noted that hyperstimulation of this process might play an important role in the cytotoxicity of METH in neuronal cells. In this study, we investigated the molecular mechanisms as well as subsequent outcomes of macropinocytosis during METH treatment. It was found that macropinosomes formed upon exposure to METH were colocalized with constitutively active GFP-Ras (G12V) and GFP-Rac1 (Q61L). Furthermore, both Ras inhibitor, farnesylthiosalicylic acid (FTS), and Rac1 inhibitor, EHT1864, significantly inhibited the formation of macropinosomes, suggesting the involvement of these molecules. The expressions of lysosome-associated membrane proteins (lamps) gradually increased by METH in a time-dependent manner. In contrast, the proteolytic activation of cathepsin L, a marker of lysosomal function, was markedly suppressed by METH. METH-induced dysfunction of lysosomal enzyme as well as cell death was significantly attenuated by nocodazole, a microtube interfering reagent that inhibits the transport of vesicles, including macropinosome, to lysosomes. Our results indicate that METH has cytotoxic effects, at least in part, by inhibiting normal lysosomal function through Ras- and Rac1-mediated macropinocytosis in RA-differentiated SH-SY5Y cells.

Paraquat Induces Epithelial-Mesenchymal Transition-Like Cellular Response Resulting in Fibrogenesis and the Prevention of Apoptosis in Human Pulmonary Epithelial Cells

The aim of this study is to investigate the molecular mechanisms underlying delayed progressive pulmonary fibrosis, a characteristic of subacute paraquat (PQ) poisoning. Epithelial-mesenchymal transition (EMT) has been proposed as a cause of organ fibrosis, and transforming growth factor-β (TGF-β) is suggested to be a powerful mediator of EMT. We thus examined the possibility that EMT is involved in pulmonary fibrosis during PQ poisoning using A549 human alveolar epithelial cells in vitro. The cells were treated with various concentrations of PQ (0–500 μM) for 2–12 days. Short-term (2 days) high-dose (>100 μM) treatments with PQ induced cell death accompanied by the activation of caspase9 as well as a decrease in E-cadherin (an epithelial cell marker), suggesting apoptotic cell death with the features of anoikis (cell death due to the loss of cell-cell adhesion). In contrast, long-term (6–12 days) low-dose (30 μM) treatments with PQ resulted in a transformation into spindle-shaped mesenchymal-like cells with a decrease of E-cadherin as well as an increase of α-smooth muscle actin (α-SMA). The mesenchymal-like cells also secreted the extracellular matrix (ECM) protein fibronectin into the culture medium. The administration of a TGF-β1 receptor antagonist, SB431542, almost completely attenuated the mesenchymal transformation as well as fibronectin secretion, suggesting a crucial role of TGF-β1 in EMT-like cellular response and subsequent fibrogenesis. It is noteworthy that despite the suppression of EMT-fibrogenesis, apoptotic death was observed in cells treated with PQ+SB431542. EMT-like cellular response and subsequent fibrogenesis were also observed in normal human bronchial epithelial (NHBE) cells exposed to PQ in a TGF-β1-dependent manner. Taken together, our experimental model reflects well the etiology of PQ poisoning in human and shows the involvement of EMT-like cellular response in both fibrogenesis and resistance to cell death during subacute PQ poisoning of pulmonary epithelial cells.

Autopsy report on pseudo-Bartter syndrome with renal calcification induced by diuretics and diet pills.

A woman in her mid-forties had repeated vomiting and diarrhoea accompanied by muscle weakness soon after she started taking seven different diet pills imported from Thailand. After she had taken the pills for 8 days, respiratory depression progressed rapidly to arrest. Blood tests at the Emergency Department showed severe hypokalaemia with metabolic alkalosis. We diagnosed that she had developed pseudo-Bartter syndrome from the findings based on ionic abnormalities and high renin and aldosterone levels, and hyperplasia of the juxtaglomerular apparatus. A postmortem blood analysis indicated subtherapeutic levels of furosemide. We concluded that the patient died from pseudo-Bartter syndrome, which was triggered by chronic self-administration of furosemide and aggravated by the diet pills. This is the first pseudo-Bartter syndrome autopsy report to show histological localisation of calcification in the kidneys.

Activation of the ubiquitin-proteasome system against arsenic trioxide cardiotoxicity involves ubiquitin ligase Parkin for mitochondrial homeostasis.

Parkin is an E3 ubiquitin ligase involved in the elimination of damaged mitochondria. Ubiquitination of mitochondrial substrates by Parkin results in proteasomal as well as lysosomal degradation of mitochondria, the latter of which is executed by the autophagy machinery and is called as mitophagy (mitochondrial autophagy). The aim of this study is to examine the possible role of Parkin against cardiotoxicity elicited by arsenic trioxide (ATO) exposure in HL-1 mouse atrial cardiomyocytes. HL-1 cells were administered 1-10μM ATO for up to 24h, and the involvements of apoptosis, and the ubiquitin-proteasome and autophagy-lysosome systems (UPS and ALS) were examined. ATO dose-dependently reduced mitochondrial membrane potentials (ΔΨm) in HL-1 cells, indicating that ATO works as a mitochondrial toxin in these cells. Apoptosis was evident in cells exposed to more than 6μM ATO for 24h. Levels of Parkin in mitochondria-rich fractions were increased, suggesting the recruitment of Parkin to mitochondria. Ubiquitination of the voltage-dependent anion channel1 (VDAC1), a substrate of Parkin, was also proved by immunoprecipitation. Accumulation of ubiquitinated proteins including both K48- and K63-lineages was observed in HL-1 cells after ATO exposure, implying an increased demand for proteasomal as well as lysosomal degradation of cellular proteins. Although UPS was activated by ATO as proved by increased proteasomal activity, only slight activation of the ALS marker LC3 was observed, suggesting differential reactions of UPS and ALS to ATO toxicity. The abrogation of UPS by the proteasome inhibitor bortezomib significantly sensitized HL-1 cells to ATO toxicity, showing the contribution of UPS to the maintenance of cellular homeostasis during ATO exposure. Taken together, our results reveal the activation of Parkin as well as UPS during ATO exposure in HL-1 cardiomyocytes, which contributes to the maintenance of mitochondrial as well as cellular homeostasis.