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In the current work we show that the profibrotic actions of TGF-β are mediated, at least in part, through a metabolic maladaptation in glutamine metabolism and how the inhibition of glutaminase 1 (GLS1) reverses pulmonary fibrosis. GLS1 was found to be highly expressed in fibrotic vs normal lung fibroblasts and the expression of profibrotic targets, cell migration, and soft agar colony formation stimulated by TGF-β required GLS1 activity. Moreover, knockdown of SMAD2 or SMAD3 as well as inhibition of PI3K, mTORC2, and PDGFR abrogated the induction of GLS1 by TGF-β. We further demonstrated that the NAD-dependent protein deacetylase, SIRT7, and the FOXO4 transcription factor acted as endogenous brakes for GLS1 expression, which are inhibited by TGF-β. Lastly, administration of the GLS1 inhibitor CB-839 attenuated bleomycin-induced pulmonary fibrosis. Our study points to an exciting and unexplored connection between epigenetic and transcriptional processes that regulate glutamine metabolism and fibrotic development in a TGF-β-dependent manner.

Pathogenic fibrotic diseases, including Idiopathic Pulmonary Fibrosis (IPF), have some of the worst prognoses and affect millions of people worldwide. With unclear etiology and minimally effective therapies, two-thirds of IPF patients die within 2-5 years from this progressive interstitial lung disease. Transforming Growth Factor Beta (TGFβ) and Insulin-like Growth Factor-1 (IGF-1) are known to promote fibrosis; however, myofibroblast specific upregulation of IGF-1 in the initiation and progression of TGFβ-induced fibrogenesis and IPF have remained unexplored. To address this, the current study (1) documents the upregulation of IGF-1 via TGFβ in myofibroblasts and fibrotic lung tissue, as well as its correlation with decreased pulmonary function in advanced IPF; (2) identifies IGF-1’s C1 promoter as mediating the increase in IGF-1 transcription by TGFβ in pulmonary fibroblasts; (3) determines that SMAD2 and mTOR signaling are required for TGFβ-dependent Igf-1 expression in myofibroblasts; (4) demonstrates IGF-1R activation is essential to support TGFβ-driven profibrotic myofibroblast functions and excessive wound-healing; and (5) establishes the effectiveness of slowing the progression of murine lung fibrosis with the IGF-1R inhibitor OSI-906. These findings expand our knowledge of IGF-1’s role as a novel fibrotic-switch, bringing us one step closer to understanding the complex biological mechanisms responsible for fibrotic diseases and developing effective therapies.

Metabolic dysregulation in fibroblasts is implicated in the profibrotic actions of transforming growth factor-β (TGF-β). Here, we present evidence that hexokinase 2 (HK2) is important for mediating the fibroproliferative activity of TGF-β both in vitro and in vivo. Both Smad-dependent and Smad-independent TGF-β signaling induced HK2 accumulation in murine and human lung fibroblasts through induction of the transcription factor c-Myc. Knockdown of HK2 or pharmacological inhibition of HK2 activity with Lonidamine decreased TGF-β-stimulated fibrogenic processes, including profibrotic gene expression, cell migration, colony formation, and activation of the transcription factors YAP and TAZ, with no apparent effect on cellular viability. Fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) exhibited an increased abundance of HK2. In a mouse model of bleomycin-induced lung fibrosis, Lonidamine reduced the expression of genes encoding profibrotic markers (collagenΙα1, EDA-fibronectin, α smooth muscle actin, and connective tissue growth factor) and stabilized or improved lung function as assessed by measurement of peripheral blood oxygenation. These findings provide evidence of how metabolic dysregulation through HK2 can be integrated within the context of profibrotic TGF-β signaling.

Transforming growth factor-beta (TGFβ) is an enigmatic protein with various roles in healthy tissue homeostasis/development as well as the development or progression of cancer, wound healing, fibrotic disorders, and immune modulation, to name a few. As TGFβ is causal to various fibroproliferative disorders featuring localized or systemic tissue/organ fibrosis as well as the activated stroma observed in various malignancies, characterizing the pathways and players mediating its action is fundamental. In the current study, we found that TGFβ induces the expression of the immunoinhibitory molecule Programed death-ligand 1 (PD-L1) in human and murine fibroblasts in a Smad2/3- and YAP/TAZ-dependent manner. Furthermore, PD-L1 knockdown decreased the TGFβ-dependent induction of extracellular matrix proteins, including collagen Iα1 (colIα1) and alpha-smooth muscle actin (α-SMA), and cell migration/wound healing. In addition to an endogenous role for PD-L1 in profibrotic TGFβ signaling, TGFβ stimulated-human lung fibroblast-derived PD-L1 into extracellular vesicles (EVs) capable of inhibiting T cell proliferation in response to T cell receptor stimulation and mediating fibroblast cell migration. These findings provide new insights and potential targets for a variety of fibrotic and malignant diseases.

Chronic alcohol consumption causes hepatic steatosis, which is characterized by a considerable increase in free fatty acid (FFA) and triglyceride levels. To identify the possible proteins involved in the progression to alcoholic hepatosteatosis, we performed proteomic analysis on livers of mice exposed to alcohol. 2D-based proteomic analysis revealed that EtOH exposure in mice changed the expression of 43 proteins compared with that in mice fed a normal diet (ND). The most notable protein changes were proteins involved in Met metabolism and oxidative stress, most of which were significantly downregulated in alcohol-exposed animals. Although non-alcoholic fatty liver disease (NAFLD) and alcoholic liver disease (ALD) seem to share the same molecular processes, the difference between these conditions is still unclear. To address this question, we explored the features of alcoholic hepatosteatosis that were different compared with those of methionine and choline deficient (MCD) diet-induced mice with nonalcoholic liver damage. Although most of the differentially expressed proteins associated with ALD did not significantly differ from those of NAFLD, nine proteins showed considerably different patterns. Of these, ornithine aminotransferase, vitamin D binding protein, and phosphatidylethanolamine-binding protein were considerably upregulated in ALD mice, compared to that in NAFLD and ND mice. However, other proteins including inorganic pyrophosphatase were differentially regulated in MCD mice; however, they did not differ significantly between the alcoholic model and ND control mice. These results suggested that the identified proteins might be useful candidate markers to differentiate ALD from NAFLD. J. Cell. Biochem. 118: 1189-1200, 2017. © 2016 Wiley Periodicals, Inc.

TGF-β plays a central role in the pathogenesis of fibroproliferative disorders. Defining the exact underlying molecular basis is therefore critical for the development of viable therapeutic strategies. Here, we show that expression of the facilitative glucose transporter 1 (GLUT1) is induced by TGF-β in fibroblast lines and primary cells and is required for the profibrotic effects of TGF-β. In addition, enhanced GLUT1 expression is observed in fibrotic areas of lungs of both patients with idiopathic pulmonary fibrosis and mice that are subjected to a fibrosis-inducing bleomycin treatment. By using pharmacologic and genetic approaches, we demonstrate that up-regulation of GLUT1 occurs via the canonical Smad2/3 pathway and requires autocrine activation of the receptor tyrosine kinases, platelet-derived and epidermal growth factor receptors. Engagement of the common downstream effector PI3K subsequently triggers activation of the MEK and mammalian target of rapamycin complex 2, which cooperate in regulating GLUT1 expression. Of note, inhibition of GLUT1 activity and/or expression is shown to impair TGF-β-driven fibrogenic processes, including cell proliferation and production of profibrotic mediators. These findings provide new perspectives on the interrelation of metabolism and profibrotic TGF-β signaling and present opportunities for potential therapeutic intervention.-Andrianifahanana, M., Hernandez, D. M., Yin, X., Kang, J.-H., Jung, M.-Y., Wang, Y., Yi, E. S., Roden, A. C., Limper, A. H., Leof, E. B. Profibrotic up-regulation of glucose transporter 1 by TGF-β involves activation of MEK and mammalian target of rapamycin complex 2 pathways.

Previously our research found that caffeic acid (CA) has antioxidant activity in vitro and in vivo. However, the antioxidant mechanism of CA has not been clearly demonstrated. We investigated the protective mechanism of CA on oxidative stress in HepG2 cells and primary hepatocytes. We focused on the effects of CA on glutathione (GSH) synthesis and its mechanisms. Reporter gene assay, transient transfection, quantitative reverse transcription-polymerase chain reaction and western blot were performed to confirm the mechanisms of antioxidant capacity. CA increased GSH level and γ-glutamylcysteine ligase (γ-GCL) activity. γ-Glutamylcysteine ligase catalytic subunit (GCLC) mRNA and protein levels were significantly increased following treatment with CA, whereas no changes were observed in the γ-GCL modifier subunit levels in HepG2 and rat hepatocytes. CA phosphorylated JNK, and activated the AP-1 transcription factor. These results show that the intracellular GSH levels and GCLC increased through the JNK/AP-1 pathways by CA treatment.

Evidence is provided that the fibroproliferative actions of TGF-β are dependent on a metabolic adaptation that sustains pathologic growth. Specifically, profibrotic TGF-β signaling is shown to require fatty acid synthase (FASN), an essential anabolic enzyme responsible for the de novo synthesis of fatty acids. With the use of pharmacologic and genetic approaches, we show that TGF-β-stimulated FASN expression is independent of Smad2/3 and is mediated via mammalian target of rapamycin complex 1. In the absence of FASN activity or protein, TGF-β-driven fibrogenic processes are reduced with no apparent toxicity. Furthermore, as increased FASN expression was also observed to correlate with the degree of lung fibrosis in bleomycin-treated mice, inhibition of FASN was examined in a murine-treatment model of pulmonary fibrosis. Remarkably, inhibition of FASN not only decreased expression of profibrotic targets, but lung function was also stabilized/improved, as assessed by peripheral blood oxygenation.-Jung, M.-Y., Kang, J.-H., Hernandez, D. M., Yin, X., Andrianifahanana, M., Wang, Y., Gonzalez-Guerrico, A., Limper, A. H., Lupu, R., Leof, E. B. Fatty acid synthase is required for profibrotic TGF-β signaling.

B7-1 proteins are routinely expressed on the surface of antigen presenting cells (APC) and within the innate immune system. They function to establish a biologically optimal and dynamic balance between immune activation and inhibition or self-tolerance. Interactions between B7-1 and its receptors, which include CD28, CTLA4 and PD-L1, contribute to both stimulatory as well as inhibitory or homeostatic regulation. In the current study, we investigated whether the tumor-promoting actions of transforming growth factor beta (TGF-β) disrupted this equilibrium in pancreatic cancer to promote malignant progression and an enhanced means to evade immune detection. The data show that B7-1 is (i) upregulated following treatment of pancreatic carcinoma cells with TGF-β; (ii) induced by TGF-β via both Smad2/3-dependent and independent pathways; (iii) required for pancreatic tumor cell in vitro migration/invasion; and (iv) necessary for TGF-β regulated epithelial-mesenchymal transition (EMT) through induction of Snail family members. Results from the proposed studies provide valuable insights into mechanisms whereby TGF-β regulates both the innate immune response and intrinsic properties of pancreatic tumor growth.

CTGF (connective tissue growth factor) is widely regarded as an important amplifier of the profibrogenic action of TGF-β (transforming growth factor β) in a variety of tissues, although the precise mechanism of how the TGF-β signalling pathways modulate CTGF expression remains unclear. In the present study, the role of PKCδ (protein kinase Cδ) in TGF-β1-mediated CTGF expression was investigated using HepG2 cells. TGF-β1 treatment specifically elevated PKCδ activation and CTGF expression. In contrast, blockade of PKCδ by the selective inhibitor Rottlerin or by siRNA knockdown significantly reduced TGF-β1-induced CTGF production. The regulatory mechanism was further demonstrated in HepG2 cells whereby TGF-β1-induced PKCδ activation negatively regulated the nuclear levels of PPM1A (protein phosphatase, Mg2+/Mn2+ dependent, 1A) through the RhoA/ROCK (Rho-associated kinase) pathway. Moreover, we showed that both Smad signalling and the PKCδ pathway appeared to be stimulated by TGF-β1 in parallel. Time course assessments indicated that PKCδ signalling may have a function in maintaining nuclear phospho-Smads at a maximal level. The collective results of the present study demonstrated that PKCδ-stimulated RhoA/ROCK activation resulted in a reduction in PPM1A, thereby up-regulating Smad-dependent gene induction for extended periods. These findings indicated that PKCδ plays a critical role in TGF-β1-induced CTGF production in HepG2 cells.

Perilla frutescens usually dieted in East Asian country such as Korea and Japan. Antioxidant, anti- inflammatory and anticancer activities of perilla leaves have been founded. In previous study, we confirmed that caf- feic acid, major compound of perilla, was accumulation by sucrose aqueous solution and thus antioxidant effect of perilla was enhanced. In this study, we investigated the protective effect of functional perilla leaves extract (PLE) against tert-butyl hydroperoxide(t-BHP) induced-oxidative hepatotoxicity. The pretreatment with PLE (250, 500 and 1000 mg/kg b.w.) for 5 days before a single dose of t-BHP (i.p.; 0.5 mmol/kg) significantly lowered the serum levels of aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase dose-dependently. And we con- firmed that the indicators of oxidative stress were remarkably reduced in the liver, such as the glutathione contents and malondialdehyde, marker of lipid peroxidation. Pathological histology of the rat livers tissues showed that PLE reduced the hepatocyte degeneration and neutrophilic infiltration of liver induced by t-BHP. These results suggest that functional perilla frutescens has the protective effect of liver against t-BHP-induced oxidative hepatic stress in rats.

Macroautophagy is involved in the bulk degradation of long-lived cytosolic proteins and subcellular organelles, which is important for the survival of cells during starvation. To identify potential players of the autophagy process, we subjected HCT116 cells cultured in complete medium and in Earle's balanced salt solution to proteomics analysis. In approximately 1500 protein spots detected, we characterized 52 unique proteins, whose expression levels were significantly changed following starvation. Notably, we found that Annexin A1 was significantly upregulated following starvation at both mRNA and protein levels. Inhibition of Annexin A1 expression with specific siRNA did not alter starvation-induced autophagy as measured by the level of lipidated LC3, but significantly reversed autophagy degradation as measured by the level of p62/SQSTM 1. Thus Annexin A1 seemed to be positively upregulated during starvation to promote autophagic degradation. Overall, the data presented in this study established a expression profile of the proteome in starved cells, which allowed the identification of proteins with potential significance in starvation-induced autophagy.

Chemopreventive or anticancer agents induce cancer cells to apoptosis through the activation of adenosine AMP-activated protein kinase (AMPK), which plays a major role as energy sensors under ATP-deprived condition or ROS generation. In this study, we compared the effects of ascochlorin (ASC), from the fungus Ascochyta viciae, and its derivatives on AMPK activity. We also examined a regulatory mechanism for hypoxia-inducible factor-1α (HIF-1α) stabilization in response to 4-O-methylascochlorin (MAC). We found that AMPK activation was mainly involved with MAC, but not ASC and 4-O-carboxymethylascochlorin (AS-6), indicating that the substitution of 4-O-methyl group from 4-O-hydroxyl group of ASC is important in the activation of AMPK and the expression of HIF-1α. MAC-stabilized HIF-1α via AMPK activation triggered by lowering the intracellular ATP level, not by ROS generation, increases glucose uptake and the expression of vascular endothelial growth factor (VEGF) and glucose transporter 1 (GLUT-1), major target genes of HIF-1α. Moreover, MAC-induced AMPK activity suppressed survival factors, including mTOR and ERK1/2 or translational regulators, including p70S6K and 4E-BP1. Our data suggest that AMPK is a key determinant of MAC-induced HIF-1α expression in response to energy stress, further implying its involvement in MAC-induced apoptosis.

The combination of bioceramics with biopolymers are playing major role in the construction of artificial bone. Hydroxyapatite (HA) has been extensively studied as a material in bone repair and replacement in last two decades. In the present study, we have prepared the hydroxyapatite-fucoidan (HA-Fucoidan) nanocomposites by in situ chemical method and biologically characterized them for bone graft substitute. Biological results inferred that mineralization effect of HA-F nanocomposites shows significant enhancement compared to HA in adipose derived stem cell (ADSC). It may be due to the addition of fucoidan in the nanocomposites. The important gene expression such as osteocalcin, osteopontin, collagen and runx-2 were checked using ADSC with HA and HA-fucoidan nanocomposites and the results show that the enhancements were found at 7th day. Furthermore, we have performed in vivo study of HA-fucoidan nanocomposites with rabbit model and a slight amount of bone formation was observed in HA-fucoidan nanocomposites. Herewith, we suggest that HA-fucoidan nanocomposites will be good biomaterials for bone repair/replacement in future.

Ascochlorin is a prenyl-phenol compound that was isolated from the fungus Ascochyta viciae. Ascochlorin reduces serum cholesterol and triglyceride levels, suppresses hypertension and tumor development, and ameliorates type I and II diabetes. Here, to better understand the mechanisms by which ascochlorin regulates physiological or pathological events and induces responses in the pharmacological treatment of cancer, we performed differential analysis of the proteome of the human osteosarcoma cells U2OS in response to ascochlorin. In addition, we established the first two-dimensional map of the U2OS proteome. The U2OS cell proteomes with and without treatment with ascochlorin were compared using two-dimensional electrophoresis, matrix-assisted laser desorption/ionization mass spectrometry and bioinformatics. The largest differences in expression were observed for the epidermal growth factor receptor (4-fold decrease), ribulose-5-phosphate-epimerase (13-fold decrease), ATP-dependent RNA helicase (8-fold decrease), and kelch-like ECH-associated protein 1 (6-fold decrease). The abundance of heterogeneous nuclear ribonucleoprotein L and minichromosome maintenance protein 7 increased 12- and 8.2-fold, respectively. In addition, Erk 2 was increased 3-fold in U2OS cells treated with ascochlorin. The expression of some selected proteins was confirmed by western blotting, zymography and RT-PCR analysis.

Native low-density lipoprotein (LDL) and oxidized LDL (oxLDL) possess a wide variety of biological properties, and play a central role in atherogenesis. In this study, we used a proteomic analysis of human monocyte THP-1 cells induced with oxLDL or with LDL, to identify proteins potentially involved in atherosclerotic processes. Of the 2500 proteins detected, 93 were differentially expressed as a result of priming with LDL or oxLDL. The proteins were unambiguously identified by comparing the masses of their tryptic peptides with those of all known proteins using MALDI-TOF MS and the NCBI database. The largest differences in expression were observed for vimentin (94-fold increase), meningioma-expressed antigen 6 (48-fold increase), serine/threonine protein phosphatase 2A (40-fold increase), and beta-1,3-galactosyltransferase (15-fold increase). In contrast, the abundance of an unnamed protein product and phosphogluconate dehydrogenase decreased 30-fold and 25-fold, respectively. The expression of some selected proteins was confirmed by Western blot and RT-PCR analyses. The proteins identified in this study are attractive candidates for further biomarker research. This description of the altered protein profiles induced by oxLDL in human monocytes will support functional studies of the macrophage-derived foam cells involved in the pathogenesis of atherosclerosis.

The succinic semialdehyde dehydrogenase gene (SSADH; EC 1.2.1.24) from human brain was cloned and overexpressed in Escherichia coli . Based on SDS–PAGE, the apparent molecular mass of subunit was 54 kDa, in good agreement with the theoretical size. The purified SSADH appears to be a tetramer of identical subunits. The specific activity of the recombinant protein was 1.82 μmol NADH formed min −1 mg −1 and the optimal pH was found to be 8.5. The Michaelis constants K m for succinic semialdehyde and NAD + were 6.3 and 125 μM, respectively. Initial velocity studies show NADH to be a competitive inhibitor with respect to NAD + , but to be non-competitive inhibitor with respect to succinic semialdehyde. The overexpression of SSADH in E. coli and one-step purification of the highly active SSADH will facilitate further biochemical studies on this enzyme. In addition, an mRNA master dot-blot for multiple human tissues provided a complete map of the tissue distribution for SSADH. The major sites of SSADH expression are liver, skeletal muscle, kidney, and brain. The data indicate that mRNA expression of SSADH is ubiquitous, but highly regulated at the level of transcription in a tissue-specific manner.

We used a combined computer and biochemical approach to characterize human pyridoxine 5'-phosphate oxidase (PNPO). The human PNPO gene is composed of seven exons and six introns, and spans approximately 8 kb. All exon/intron junctions contain the gt/ag consensus splicing site. The absence of TATA-like sequences, the presence of Sp1-binding sites and more importantly, the presence of CpG islands in the regulatory region of the PNPO gene are characteristic features of housekeeping genes. Northern blot analyses showed two species of poly(A)(+) RNA of approximately 2.4 and approximately 3.4 kb at identical intensity, whereas Western blot analysis showed that no protein isoform exists in any of the tissues examined. PCR-based analysis led to the idea that two messages are transcribed from a single copy gene, and that the size difference is due to differential usage of the polyadenylation signal. The major sites of PNPO expression are liver, skeletal muscle and kidneys while a very weak signal was detected in lung. The mRNA master dot-blot for multiple human tissues provided a complete map of the tissue distribution not only for PNPO but also for pyridoxal kinase and pyridoxal phosphatase. The data indicate that mRNA expression of all three enzymes essential for vitamin B(6) metabolism is ubiquitous but is highly regulated at the level of transcription in a tissue-specific manner. In addition, human brain PNPO cDNA was expressed in Escherichia coli, and the roles of both the N- and C-terminal regions were studied by creating sequential truncation mutants. Our results showed that deletion of the N-terminal 56 residues affects neither the binding of coenzyme nor catalytic activity.

The mechanisms underlying the progression of simple steatosis to steatohepatitis are yet to be elucidated. To identify the proteins involved in the development of liver tissue inflammation, we performed comparative proteomic analysis of non-alcoholic steatohepatitis (NASH). Mice fed a methionine and choline deficient diet (MCD) developed hepatic steatosis characterized by increased free fatty acid (FFA) and triglyceride levels as well as alpha-SMA. Two-dimensional proteomic analysis revealed that the change from the normal diet to the MCD diet affected the expressions of 50 proteins. The most-pronounced changes were observed in the expression of proteins involved in Met metabolism and oxidative stress, most of which were significantly downregulated in NASH model animals. Peroxiredoxin (Prx) is the most interesting among the modulated proteins identified in this study. In particular, cross-regulated Prx1 and Prx6 are likely to participate in cellular defense against the development of hepatitis. Thus, these Prx isoforms may be a useful new marker for early stage steatohepatitis. Moreover, curcumin treatment results in alleviation of the severity of hepatic inflammation in steatohepatitis. Notably, curcumin administration in MCD-fed mice dramatically reduced CYP2E1 as well as Prx1 expression, while upregulating Prx6 expression. These findings suggest that curcumin may have a protective role against MCD fed-induced oxidative stress.

One of causes of skin wrinkles may be the nonenzymatic glycation followed formation of browning products called advanced glycation endproducts (AGEs), an irreversible cross-linked protein. The accu...

Niclosamide is a salicylanilide derivative used as antihelminthic agent. Recently, Nic has been screened out as an autophagy modulator by us and other groups and was identified to be a structurally distinct mTORC1 signaling inhibitor. In this work, we measured the long-lived protein degradation activity and found Niclosamide could inhibit the autophagic process. To elucidate the inhibition of Niclosamide on autophagy, we further tested the lysosomal activity using DQ-BSA assay and measured protease activity such as Cathepsin B and Cathepsin D with fluorescent substrates. The lower degradation of DQ-BSA and decreased protease activity after Niclosamide treatments indicated the impaired lysosome function. In addition, both LC-II and P62 proteins accumulated significantly when treated by Niclosamide suggesting the late stage of autophagy was blocked. The increased pH of lysosome and release of Cathepsins from lysosome to cytosol demonstrated that Niclosamide might affect lysosome resulted from lysosomal memb...

Autophagy can be activated via MTORC1 down-regulation by amino acid deprivation and by certain chemicals such as rapamycin, torin, and niclosamide. Lysosome is the degrading machine for autophagy but has also been linked to MTORC1 activation through the Rag/RRAG GTPase pathway. This association raises the question of whether lysosome can be involved in the initiation of autophagy. Toward this end, we found that niclosamide, an MTORC1 inhibitor, was able to inhibit lysosome degradation and increase lysosomal permeability. Niclosamide was ineffective in inhibiting MTORC1 in cells expressing constitutively activated Rag proteins, suggesting that its inhibitory effects were targeted to the Rag-MTORC1 signaling system. This places niclosamide in the same category of bafilomycin A1 and concanamycin A, inhibitors of the vacuolar H(+)-ATPase, for its dependence on Rag GTPase in suppression of MTORC1. Surprisingly, classical lysosome inhibitors such as chloroquine, E64D, and pepstatin A were also able to inhibit MTORC1 in a Rag-dependent manner. These lysosome inhibitors were able to activate early autophagy events represented by ATG16L1 and ATG12 puncta formation. Our work established a link between the functional status of the lysosome in general to the Rag-MTORC1 signaling axis and autophagy activation. Thus, the lysosome is not only required for autophagic degradation but also affects autophagy activation. Lysosome inhibitors can have a dual effect in suppressing autophagy degradation and in initiating autophagy.

Background Bee venom has been used to relieve pain and to treat inflammatory diseases, including rheumatoid arthritis, in humans. To better understand the mechanisms of the anti-inflammatory and anti-atherosclerosis effect of bee venom, gel electrophoresis and mass spectrometry were used to identify proteins whose expression was altered in human Vascular Smooth Muscle Cells (hVSMCs) stimulated by tumor necrosis factor alpha after 12 h in the presence of melittin. Results To obtain valuable insights into the anti-inflammatory and anti-atherosclerosis mechanisms of melittin, two-dimensional (2-D) gel electrophoresis and MALDI-TOF/TOF were used. The proteome study, we showed 33 significant proteins that were differentially expressed in the cells treated with tumor necrosis factor alpha and melittin. Thirteen proteins were significantly increased in the cells treated with tumor necrosis factor alpha, and those proteins were reduced in the cells treated with melittin. Five of the proteins that showed increased expression in the cells treated with tumor necrosis factor alpha are involved in cell migration, including calreticulin, an essential factor of development that plays a role in transcription regulation. The proteins involved in cell migration were reduced in the melittin treated cells. The observed changes in the expression of GRP75, prohibitin, and a select group of other proteins were validated with reverse transcribed-PCR. It was confirmed that the observed change in the protein levels reflected a change in the genes level. In addition, the phosphorylation of EGFR and ERK was validated by analyzing the protein pathway. Conclusion Taken together, these data established that the expression of some proteins was significantly changed by melittin treatment in tumor necrosis factor alpha stimulated the cells and provided insights into the mechanism of the melittin function for its potential use as an anti-inflammatory agent.

The synthetic derivative of ascochlorin, 4-O-carboxymethyl ascochlorin (AS-6) is an agonist of the nuclear hormone receptor PPARγ and has been shown to induce differentiation in mouse pre-adipocytes and to ameliorate type II diabetes in a murine model. AS-6 was cytotoxic when added at micromolar concentrations to cultures of three different human cancer cell lines. We used gel electrophoresis and mass spectrometry to identify proteins with altered expression in human hepatocarcinoma cells (HepG2) cells after 12 h in the presence of AS-6 and found 58 proteins that were differentially expressed. Many of the proteins showing increased expression in cells treated with AS-6 are involved in protein quality control, including glucose-regulated protein 78 (GRP78/BiP), a regulator of ER stress responses, and the transcriptional regulator CHOP, which mediates ER stress-induced apoptosis. Cells treated with AS-6 undergo an autophagic response accompanied by increased expression of beclin1, ATG5, and LC3-II and autophagosome formation marked by the appearance of large vesicles containing LC3-II. Grp78 induction was inhibited when the PPARγ antagonist, GW9662, was added together with AS-6, and autophagy and cell death were partially blocked. 3-methyl-adenine (3-MA), an inhibitor of phosphatidyl inositol 3-kinase (PI3-kinase) prevented induction of ATG5 and activation of LC3-II and blocked autophagosome formation. 3-MA also blocked induction of GRP78 and CHOP, suggesting that PI3-kinase, which is known to mediate ER stress-induced autophagy, also plays a role in initiating apoptosis in response to ER stress. Together these data establish that the cytotoxicity of AS-6 operates by a mechanism dependent on ER stress-induced autophagy and apoptosis.

Obesity causes changes in fatty acid metabolism that consequently leads to fatty liver. To identify the possible proteins involved in the processes of obesity, we performed a proteomic analysis of obesity-induced mouse liver. Male C57BL/6J mice that were fed a high-fat diet (HFD) for 24wk, developed hepatic steatosis characterized by considerable increase in free fatty acid (FFA) and triglyceride levels. Body weights were measured weekly and other measurements at weeks 2, 6, 12, 16, and 24. 2-D-based proteomic analysis revealed that, compared with the normal diet (ND) (n 550), high-fat diet (n 550) changed the expression of 12 protein (8 up and 4 downregulated, by a 1.5 fold change and more, po0.05). The most pronounced difference was observed in intermediate microfilament (IF) cytoskeleton proteins. In particular, vimentin (vim) as well as cytokeratins (CK-8 and CK-18) were significantly upregulated in obese animals. Moreover, the level of caspase-generated IF fragment was also positively correlated with the degree of steatosis. The results suggest a significant alteration in IF organization during the development of hepatic steatosis leading to inflammation. The expression profile of selected proteins including vim was validated by Western blot, microarray analysis, and hepatocyte morphology by immunohistochemistry. Our results suggest that vim, like CK-18, may be a useful marker for predicting obesity and liver disease.

Autophagosomes and their precursors are best defined by electron microscopy but may also be traced in living cells based on the distribution of specific autophagy molecules. LC3, the most commonly examined autophagy marker in mammalian cells, labels structures that are frequently manifested as dots or rings using light microscopy; however, the nature of these structures is not entirely clear. We reported here a novel approach to examine the LC3-positive compartment in cell-free lysates, which revealed that they were actually tubulovesicular structures with considerable heterogeneity. Using affinity purification, we isolated these membranes for electron microscopy, which indicated that they possessed ultrastructural features consistent with autophagosomal membranes at various maturation stages. Further biochemical and proteomics analyses demonstrated the presence of multiple autophagy-related and other functional molecules. The different distribution patterns of Atg5, Atg16, Atg9, and p62/SQSTM1 on the LC3-positive compartment provided new clues on how these molecules might be involved in the dynamics of the autophagosomal membranes. Finally, several morphologically unique groups of LC3-positive membranes were categorized. Their topological configurations suggested that double-membrane vesicles could be derived from single membrane compartments via different means, including tubule-to-vesicle conversion, whose presence was supported by live cell imaging. These findings thus provide new information on the dynamics of the autophagosomal compartment.

Transforming growth factor-beta1 (TGF-beta1) has a wide range of biological functions such as the regulation of cell growth, differentiation, and immunological response in various types of cells. Particularly, TGF-beta1 induces plasminogen activator inhibitor-1 (PAI-1) as a major target protein. PAI-1 is associated with fibrosis, thrombosis, and metabolic disorders. In this study, to identify proteins potentially involved in TGF-beta1-induced fibrosis processes, we performed a proteomic analysis of TGF-beta1-induced normal rat kidney cells exposed to ascofuranone (AF). In these cells, we detected 1500 proteins, with 74 differentially expressed proteins identified by MALDI-TOF and reference to the NCBI and Swiss-Prot databases, including PAI-1, peroxisome prdifesator-activated receptor, prohibitin, glutamate formyltransferase, LIM domain protein 1, LASP-1, porphobilinogen deaminase, and peroxiredoxin 2. We also found that AF suppresses expression of profibrotic factors induced by TGF-beta in renal fibroblasts, including matrix proteins and PAI-1. AF was also shown to inhibit selectively phosphorylation of epidermal growth factor receptor, and downstream kinases such as extracellular signal-regulated kinase 1/2 (ERK-1/2). Further ongoing analysis of fibrosis-related proteins will determine AF's potential for application in fibrotic diseases and therapeutics.

Fibrosis in glomerulosclerosis causes progressive loss of renal function. Transforming growth factor (TGF)-beta, one of the major profibrotic cytokines, induces the synthesis of plasminogen activator inhibitor (PAI)-1, a factor that plays a crucial role in the development of fibrosis. Here, we found that an isoprenoid antibiotic, ascofuranone, suppresses expression of profibrotic factors including matrix proteins and PAI-1 induced by TGF-beta in renal fibroblasts. Ascofuranone selectively inhibits phosphorylation of epidermal growth factor receptor (EGFR), and downstream kinases such as Raf-1, MEK-1/2, and ERK-1/2. PAI-1 transcription also is suppressed by treatment with kinase inhibitors for MEK-1/2 or EGFR, and with small interfering RNA for EGFR. Ascofuranone inhibits cellular metalloproteinase activity, and an inhibitor of metalloproteinases suppresses EGFR phosphorylation and PAI-1 transcription. These results suggest that ascofuranone suppresses expression of profibrotic factors through the inhibition of an EGFR-dependent signal transduction pathway activated by metalloproteinases.

Proteomics is a novel molecular profiling technology. It is mainly concerned with determining the structure, expression, localization, biochemical activity, interactions, and cellular roles of any proteins. Clinical research hopes to benefit from proteomics in the identification of new drug targets and the development of new diagnostic markers. Better to understand the mechanisms by which ascofuranone (AF), an isoprenoid antibiotic, regulates physiological or pathological events and induces responses in the pharmacological treatment of cancer, we performed differential analysis of the human osteosarcoma cells U2OS proteomes in response to this agent. The U2OS cell proteomes with and without treatment with AF were compared using two-dimensional electrophoresis, matrix-assisted laser desorption/ionization mass spectrometry, and bioinformatics. The largest differences in protein expression were observed for hydroxyindole O-methyltransferase, syntaxin-binding protein 1, the matrix metalloproteinase (MMP)-2, urokinase receptor, and endothelial protein C receptor. Changes in expression and activity of some selected proteins were confirmed by Western blotting, zymography, and reverse transcription-polymerase chain reaction analysis. In particular, we observed downregulated tumor growth related-proteins such as MMP-2 and endothelial protein C receptor. According to these results, AF might be useful as a potent chemotherapeutic agent.

The invasion of monocytes through the endothelial wall of arteries and their transformation from macrophage into form cells has been implicated as a critical initiating event in atherogenesis. Human THP-1 monocytic cells can be induced to differentiate into macrophages by phorbol myristate acetate (PMA) treatment, and can be converted into foam cells by exposure to oxidized low-density lipoprotein (oxLDL). To identify proteins potentially involved in atherosclerotic processes, we performed a proteomic analysis of THP-1 macrophages exposed to oxLDL generated by treatment with native LDL with hypochlorous acid/hypochlorite (HOCl/OCl(-)). We detected more than a thousand proteins, of which 104 differentially expressed proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and the NCBI database. The largest differences in expression were observed for bifunctional purine biosynthesis protein, vacuolar protein sorting 33A, breast carcinoma amplified sequence, adenine phosphoribosyltransferase, and tropomyosin alpha 3 chain. Interestingly, many apoptotic proteins such as lamin B1, poly (ADP-ribose) polymerase, Bcl-2 related protein A1 and vimentin were identified by MALDI-TOF analysis. Identities were confirmed by matching the sequence of several tryptic peptides using MALDI-TOF/TOF MS, Western blot analyses and immunofluorescent microscopy. The data described here will contribute to establishing a functional profile of the human macrophage proteome. Furthermore, the proteins identified in this study are attractive candidates for further biomarkers involved in the pathogenesis of atherosclerosis.

Transcription factor Sp-1 is an important fibrogenic factor that is involved in the pathogenesis of diabetic nephropathy. In this study, we examined the effect of Sp1 decoy oligodeoxynucleotides (ODNs) on the extracellular matrix (ECM) gene expression in cultured rat mesangial cells (RMC) and streptozotocin (STZ)-induced diabetic rats. The ring-type Sp1 decoy ODNs significantly decreased ECM mRNA expression and Sp1 binding to the promoter region of these PDGF-induced genes in RMC. In addition, the decoy ODNs was introduced into the left renal artery of diabetic rat using the hemagglutinating virus of Japan (HVJ)-liposome mediated gene transfer method and effectively delivered to the kidney. On 14 days after ring-type Sp1 decoy ODNs injection, type IV collagen, fibronectin mRNA, and protein expression were markedly decreased, and the rate of urinary creatinine excretion was reduced in the ring-type Sp1 decoy ODNs-treated diabetic rats. These results indicated that the ring-type Sp1 decoy ODNs would be superior to P-Sp1 ODNs. Also, the R-Sp1 decoy ODN when introduced in vivo, effectively reduced ECM production during the progression of nephropathy. Therefore, ring-type Sp1 decoy is a promising tool for developing new therapeutic applications for progressive diabetic nephropathy. J. Cell. Biochem. 103: 663–674, 2008. © 2007 Wiley-Liss, Inc.

The critical initiating event in atherogenesis involves the invasion of monocytes through the endothelial walls of arteries and the transformation of monocytes from macrophages into foam cells. Human THP-1 monocytic cells can be induced to differentiate into macrophages by phorbol myristate acetate (PMA) and can then be converted into foam cells by exposure to oxidized low-density lipoprotein (oxLDL). Also, during a chronic inflammatory response, monocytes/macrophages produce the 92-kDa matrix metalloproteinase-9 (MMP-9) that may contribute to the extravasation, migration, and tissue remolding capacities of the phagocytic cells. Here, we investigate the effect of ascochlorin (ASC), a prenylphenol antiviral compound from the fungus Ascochyta viciae, on oxLDL-induced MMP-9 expression and activity in human THP-1 macrophages. ASC reduced oxLDL-induced MMP-9 expression and activity in a time-dependent and dose-dependent manner. Also, an analysis of MMP-9 activity using pharmacologic inhibitors showed that ASC inhibits MMP-9 activity via the extracellular signal-regulated kinase 1 and kinase 2 pathways. Our results suggest that ASC may be useful as a potent clinical antiatherogenic agent, a topic of considerable interest in the biological chemistry of chemotherapeutic agents.