Ethanol-induced Stress Leads to Apoptosls Via Endoplasmic Reticulum Stress in SK-Hepl Cells

Alcoholic liver disease causes oxidative stress and induces apoptosis during alcohol metabolism. Ethanol causes endoplasmic reticulum (ER) stress in hepatocytes, stimulating the unfolded protein response (UPR) pathway and/or Ca2+-dependent calpain and caspase-4 activities. However, it is poorly understood whether ethanol-induced oxidative stress directly leads to apoptosis promoted by ER stress-associated pathways. This study investigated this question in human liver adenocarcinoma (SK-Hep1) cells, which were treated with 200 mM ethanol for 5 hours in the presence or absence of the antioxidant N-acetyl-cysteine (NAC). We found that treatment with ethanol significantly increased ROS production and cellular apoptosis in the SK-Hep1 cells, and that this response was significantly suppressed by pretreatment with NAC. Furthermore, pretreatment with NAC significantly reduced the observed increases in the mRNA expressions of Bip, Chop, and sXbp-1, and the activity of caspase-3 in ethanol-induced apoptotic cells. However, pretreatment with NAC did not attenuate the transient rise in cytosolic Ca2+ nor the activities of caspase-4 and calpain induced by ethanol. Together, these results revealed that ethanol-induced stress promotes apoptosis not only through mitochondria-mediated pathways, but also via ER stress. The findings further suggested that ethanol-induced oxidative stress and non-oxidative stress both stimulate the pathway regulating ER stress-mediated apoptosis

Regulation of NR4A nuclear receptors by p38

In Drosophila, the melanization reaction is an important defense mechanism against injury and invasion of microorganisms. Drosophila tyrosine hydroxylase (TH, also known as Pale) and dopa decarboxylase (Ddc), key enzymes in the dopamine synthesis pathway, underlie the melanin synthesis by providing the melanin precursors dopa and dopamine, respectively. It has been shown that expression of Drosophila TH and Ddc is induced in various physiological and pathological conditions, including bacterial challenge; however, the mechanism involved has not been fully elucidated. Here, we show that ectopic activation of p38 MAPK induces TH and Ddc expression, leading to upregulation of melanization in the Drosophila cuticle. This p38-dependent melanization was attenuated by knockdown of TH and Ddc, as well as by that of Drosophila HR38, a member of the NR4A family of nuclear receptors. In mammalian cells, p38 phosphorylated mammalian NR4As and Drosophila HR38 and potentiated these NR4As to transactivate a promoter containing NR4A-binding elements, with this transactivation being, at least in part, dependent on the phosphorylation. This suggests an evolutionarily conserved role for p38 MAPKs in the regulation of NR4As. Thus, p38-regulated gene induction through NR4As appears to function in the dopamine synthesis pathway and may be involved in immune and stress responses

Increase in Matrix Metalloproteinase-2 and 9 in the Liver of Nonalcoholic Steatohepatitis (NASH) Model Rats

Nonalcoholic steatohepatitis (NASH) is regarded as a hepatic manifestation of the metabolic syndrome which can progress to hepatic cirrhosis and hepatocellular carcinoma. It is thought that matrix metalloproteinases (MMPs) play an important role in hepatic fibrosis and we previously reported a correlation between oxidative stress and MMP-9 expression. However, the expression of MMPs and tissue inhibitors of metalloproteinases (TIMPs) in the progression of NASH is unclear. In this study we used spontaneously hypertensive and hyperlipidemic rats (SHHR) fed a high-fat diet and 30% sucrose solution (HFDS) as a model for NASH, in order to clarify the relationships between oxidative stress, liver weight (LW), MMPs and TIMPs at various time-points in the progression of NASH. Male SHHR and Sprague-Dawley (SD) rats were divided into four groups: SHHR-normal diet (ND), SHHR-HFDS, SD-ND and SD-HFDS. Hepatic fibrosis was clearly increased at 13 months in SHHR-HFDS, resembpling NASH. LW and oxidative stress markers in plasma were increased in SHHR-HFDS compared to the other groups. Oxidative stress was correlated with LW in all rats. Expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 mRNA, measured by real-time polymerase chain reaction, was increased in the liver of SHHR-HFDS at 13 months. This study suggests that oxidative stress, MMPs and TIMPs may play an important role in the progression of NASH

Nitrogen oxide cycle regulates nitric oxide levels and bacterial cell signaling

Nitric oxide (NO) signaling controls various metabolic pathways in bacteria and higher eukaryotes. Cellular enzymes synthesize and detoxify NO; however, a mechanism that controls its cellular homeostasis has not been identified. Here, we found a nitrogen oxide cycle involving nitrate reductase (Nar) and the NO dioxygenase flavohemoglobin (Fhb), that facilitate inter-conversion of nitrate, nitrite, and NO in the actinobacterium Streptomyces coelicolor. This cycle regulates cellular NO levels, bacterial antibiotic production, and morphological differentiation. NO down-regulates Nar and up-regulates Fhb gene expression via the NO-dependent transcriptional factors DevSR and NsrR, respectively, which are involved in the auto-regulation mechanism of intracellular NO levels. Nitrite generated by the NO cycles induces gene expression in neighboring cells, indicating an additional role of the cycle as a producer of a transmittable inter-cellular communication molecule

Prevention of Apoptosis by Mitochondrial Phosphatase PGAM5 in the Mushroom Body Is Crucial for Heat Shock Resistance in Drosophila melanogaster

The heat shock (HS) response is essential for survival of all organisms. Although the machinery of the HS response has been extensively investigated at the cellular level, it is poorly understood at the level of the organism. Here, we show the crucial role of the mushroom body (MB) in the HS response in Drosophila. Null mutants of the mitochondrial phosphatase Drosophila PGAM5 (dPGAM5) exhibited increased vulnerability to HS, which was reversed by MB-specific expression of the caspase inhibitor p35, and similar vulnerability was induced in wild-type flies by knockdown of MB dPGAM5. Elimination of the MB did not affect the HS response of wild-type flies, but did increase the resistance of dPGAM5-deficient flies to HS. Thus, the MB may possess an apoptosis-dependent toxic function, the suppression of which by dPGAM5 appears to be crucial for HS resistance

Protective Effects of Fucoidan Against Interleukin-1β-induced Inflammation in SW982 Human Synovial Cells

Fucoidan, a natural sulfated polysaccharide found in the extracellular matrix of brown algae, is rich in L-fucose and sulfate. Fucoidan has a variety of biological actions, including anti-oxidative, anti-coagulative, anti-cancer, and anti-inflammatory activity. However, the cellular molecular mechanism underlying the anti-inflammatory effects of fucoidan remains poorly understood. Rheumatoid arthritis (RA) is a chronic inflammatory disease that causes synovitis and progressive joint destruction. Interleukin (IL) -1β, one of the important mediators involved in the pathogenesis of RA, is known to activate various intracellular signaling pathways. Therefore, in the present study we investigated the inhibitory effects of fucoidan on IL-1β-induced inflammation in human synovial (SW982) cells. SW982 cells were pretreated with fucoidan (100µg/mL) for 1h before cotreatment with 5ng/mL IL-1β plus fucoidan for periods ranging from 20min to 24h. Levels of the proinflammatory mediators IL-6, tumor necrosis factor-α, and cyclooxygenase-2 were then determined. We also assayed translocation of nuclear factor (NF) -κB into the nucleus and activation of mitogen-activated protein kinase (MAPK). Significant increases in the production of proinflammatory mediators were observed from 6 to 24h of IL-1β treatment. The translocation of NF-κB into the nucleus peaked after approximately 6h incubation. After 20min incubation, IL-1β activated c-jun N-terminal kinase and p38 MAPK in SW982 cells. This effect was ameliorated by the coincubation of cells with fucoidan. These results suggest that fucoidan exerts its anti-inflammatory effect by regulating the gene expression of proinflammatory mediators by suppressing the activity of transcription factors and MAPK. Thus, fucoidan may have therapeutic potential for the treatment of RA

(-)-Epigallocatechin-3-gallate Inhibits Differentiation and Matrix Metalloproteinases Expression in Osteoclasts

The osteoclast is a multinucleated giant cell differentiated from monocyte macrophages that has an important role in bone resorption. Several studies have reported a relationship between tea consumption and decreased risk of bone fracture. Matrix metalloproteinases (MMPs) play an important role in the degeneration of bone and cartilage matrix. Regulation of osteoclast activity is essential in the treatment of bone disease. Moreover, MMPs are associated with osteoclast formation and differentiation. We have reported previously that (-) -epigallocatechin-3-gallate (EGCG) inhibits MMP-2 and MMP-9 expression and activity. However, the effects of EGCG on osteoclasts and other MMPs are not clear. Therefore, in the present study we examined whether EGCG affects MMP expression, as well as osteoclast formation, differentiation and activity, in vitro. We used bone marrow cells from the femur and tibial bones of male ddY mice. Bone marrow cells were cultured in the presence of 1-100µM EGCG for 6 or 8 days. EGCG decreased the number of mature osteoclasts, as determined by tartrate-resistant acid phosphatase staining. Concentrations as low as 1µM EGCG clearly inhibited the differentiation of osteoclasts from bone marrow cells. EGCG also inhibited the number of osteoclasts with an actin-ring, as determined by rhodamine phalloidin staining, as well as osteoclast activity, as determined by the pit formation assay. Furthermore, EGCG concentration-dependently decreased MMP-9 and membrane type 1-MMP mRNA expression in mouse osteoclasts. However, EGCG had no changing on mRNA levels of tissue inhibitor of metalloprotease (TIMP)-1 and TIMP-3. Together, the results suggest that EGCG may be a suitable agent or lead compound for the development of treatments for bone resorption diseases associated with MMPs

Phenotypic Analysis of Growth and Morphological Traits in Miniature Breeds of Japanese Indigenous Chickens

Japanese indigenous chickens include approximately 50 breeds exhibiting various morphological traits, such as a long tail. These genetic resources will be important for revealing the genetic basis of morphological traits in the future. However, little is known about the phenotypic characteristics of each breed during the growth stages. To understand age-dependent changes in growth and morphological traits, we investigated tail length, tail number, body weight, and shank length at several time points using three genetically distinct Japanese indigenous chicken breeds. A total of 155 birds from the Tosa-jidori, Chabo, and Minohikichabo breeds were used for trait measurements from 1 to 36 weeks of age to reveal breed and sex effects. Significant sex differences through the growth stages were observed for all traits except for tail number. Although there were no clear breed differences in tail length traits at the 6- and 20-week stages, Minohikichabo ultimately had a significantly longer tail due to extended tail feather growth at later stages (28 and 36 weeks). By measuring two tail length variables (central and maximum), it was revealed that the shape of the tail feathers varies with the growth stage. Minohikichabo’s tail number was higher than that of Tosa-jidori and Chabo at earlier ages (8 and 16 weeks), which leads to an elegant visual in Minohikichabo. Tosa-jidori’s body weight was higher than that of Chabo and Minohikichabo, whereas the shank lengths of Chabo and Minohikichabo were shorter than those of Tosa-jidori. These differences in body weight and shank length were consistent from the early to late growth stages. These results revealed the age-dependency of growth and morphological trait breed characteristics

dPGAM5 exerts its protective effect against HS by preventing apoptosis in the MB.

<p>(<b>A–F</b>) TUNEL-positive KCs are detected in the MB of HS-treated <i>PGAM5¹</i> flies, but not in that of control flies. TUNEL staining of the MB of control flies (<b>A</b>) and <i>PGAM5¹</i> flies (<b>B</b>) treated with HS for 75 min is shown. Counter nuclear staining with Hoechst33258 (<b>C</b> and <b>D</b>) and merged images of TUNEL and Hoechst 33258 staining (<b>E</b> and <b>F</b>) are also shown. Scale bar (<b>A</b>–<b>F</b>) = 20 µm. The genotypes are <i>c739-GAL4/UAS-Histone2B::ECFP</i> (Control) and <i>PGAM5¹/Y; c739-GAL4/UAS-Histone2B::ECFP</i> (<i>PGAM5¹</i>). (<b>G</b>) Expression of p35 in the MB attenuates the vulnerability of <i>PGAM5¹</i> flies to HS. Survival curves of the indicated adult male flies subjected to HS are shown (<i>n</i> = 105). <i>PGAM5¹</i> vs. <i>PGAM5¹; c739>p35</i>, <i>p</i><0.0001 by the log-rank test. The genotypes are <i>c739-GAL4/+</i> (Control), <i>PGAM5¹/Y; c739-GAL4/+</i> (<i>PGAM5¹</i>) and <i>PGAM5¹/Y; c739-GAL4/+; UAS-p35/+</i> (<i>PGAM5¹; c739>p35</i>). (<b>H</b>) Expression of p35 in the MB does not affect the response of control flies to HS. Survival curves of the indicated adult male flies subjected to HS are shown (<i>n</i> = 130). The genotypes are <i>c739-GAL4/+</i> (Control) and <i>c739-GAL4/+; UAS-p35/+</i> (Control; <i>c739>p35</i>).</p