Hypertrophy-Reduced Autophagy Causes Cardiac Dysfunction by Directly Impacting Cardiomyocyte Contractility.

Cardiac remodeling and contractile dysfunction are leading causes in hypertrophy-associated heart failure (HF), increasing with a population's rising age. A hallmark of aged and diseased hearts is the accumulation of modified proteins caused by an impaired autophagy-lysosomal-pathway. Although, autophagy inducer rapamycin has been described to exert cardioprotective effects, it remains to be shown whether these effects can be attributed to improved cardiomyocyte autophagy and contractility. In vivo hypertrophy was induced by transverse aortic constriction (TAC), with mice receiving daily rapamycin injections beginning six weeks after surgery for four weeks. Echocardiographic analysis demonstrated TAC-induced HF and protein analyses showed abundance of modified proteins in TAC-hearts after 10 weeks, both reduced by rapamycin. In vitro, cardiomyocyte hypertrophy was mimicked by endothelin 1 (ET-1) and autophagy manipulated by silencing Atg5 in neonatal cardiomyocytes. ET-1 and siAtg5 decreased Atg5-Atg12 and LC3-II, increased natriuretic peptides, and decreased amplitude and early phase of contraction in cardiomyocytes, the latter two evaluated using ImageJ macro Myocyter recently developed by us. ET-1 further decreased cell contractility in control but not in siAtg5 cells. In conclusion, ET-1 decreased autophagy and cardiomyocyte contractility, in line with siAtg5-treated cells and the results of TAC-mice demonstrating a crucial role for autophagy in cardiomyocyte contractility and cardiac performance.

Punicalagin Attenuates Palmitate-Induced Lipid Droplet Content by Simultaneously Improving Autophagy in Hepatocytes.

SCOPE: Several studies show that excessive lipid intake can cause hepatic steatosis. To investigate lipotoxicity on cellular level, palmitate (PA) is often used to highly increase lipid droplets (LDs). One way to remove LDs is autophagy, while it is controversially discussed if autophagy is also affected by PA. It is aimed to investigate whether PA-induced LD accumulation can impair autophagy and punicalagin, a natural autophagy inducer from pomegranate, can improve it. METHODS AND RESULTS: To verify the role of autophagy in LD degradation, HepG2 cells are treated with PA and analyzed for LD and perilipin 2 content in presence of autophagy inducer Torin 1 and inhibitor 3-Methyladenine. PA alone seems to initially induce autophagy-related proteins but impairs autophagic-flux in a time-dependent manner, considering 6 and 24 h PA. To examine whether punicalagin can prevent autophagy impairment, cells are cotreated for 24 h with PA and punicalagin. Results show that punicalagin preserves expression of autophagy-related proteins and autophagic flux, while simultaneously decreasing LDs and perilipin 2. CONCLUSION: Data provide new insights into the role of PA-induced excessive LD content on autophagy and suggest autophagy-inducing properties of punicalagin, indicating that punicalagin can be a health-beneficial compound for future research on lipotoxicity in liver.

Selenoprotein H controls cell cycle progression and proliferation of human colorectal cancer cells.

Selenoprotein H (SELENOH) is supposed to be involved in redox regulation as well as in tumorigenesis. However, its role in healthy and transformed cells of the gastrointestinal tract remains elusive. We analyzed SELENOH expression in cells depending on their selenium supply and differentiation status and found that SELENOH expression was increased in tumor tissue, in undifferentiated epithelial cells from mice and in colorectal cancer lines as compared to more differentiated ones. Knockdown studies in human colorectal cancer cells revealed that repression of SELENOH decreased cellular differentiation and increased proliferation and migration. In addition, SELENOH knockdown cells have a higher competence to form colonies or tumor xenografts. In parallel, they show a faster cell cycle transition. The high levels of SELENOH in tumors as well as in undifferentiated, proliferative cells together with its inhibitory effects on proliferation and G1/S phase transition suggest SELENOH as a key regulator for cell cycle progression and for prevention of uncontrolled proliferation. As SELENOH expression is highly dependent on the selenium status, effects of selenium supplementation on cancer initiation and progression appear to involve SELENOH.

Time- and cell-resolved dynamics of redox-sensitive Nrf2, HIF and NF-κB activities in 3D spheroids enriched for cancer stem cells.

Cancer cells have an altered redox status, with changes in intracellular signaling pathways. The knowledge of how such processes are regulated in 3D spheroids, being well-established tumor models, is limited. To approach this question we stably transfected HCT116 cells with a pTRAF reporter that enabled time- and cell-resolved activity monitoring of three redox-regulated transcription factors Nrf2, HIF and NF-κB in spheroids enriched for cancer stem cells. At the first day of spheroid formation, these transcription factors were activated and thereafter became repressed. After about a week, both HIF and Nrf2 were reactivated within the spheroid cores. Further amplifying HIF activation in spheroids by treatment with DMOG resulted in a dominant quiescent stem-cell-like phenotype, with high resistance to stress-inducing treatments. Auranofin, triggering oxidative stress and Nrf2 activation, had opposite effects with increased differentiation and proliferation. These novel high-resolution insights into spatiotemporal activation patterns demonstrate a striking coordination of redox regulated transcription factors within spheroids not occurring in conventional cell culture models.

GPx2 Induction Is Mediated Through STAT Transcription Factors During Acute Colitis.

BACKGROUND: The selenoprotein glutathione peroxidase 2 (GPx2) is highly expressed in the gastrointestinal epithelium. During inflammatory bowel disease and colorectal cancer, GPx2 expression is enhanced. METHODS: We analyzed GPx2 expression and transcriptional regulation during the different phases of dextran sulfate sodium (DSS)-induced colitis in mice and in cytokine-treated colorectal cancer cells. RESULTS: In the colon of DSS-treated mice, GPx2 was upregulated during the acute and recovery phase. In the latter, it was specifically localized in regenerating ki67-positive crypts next to ulcerations. In cultured cells, endogenous GPx2 expression and GPx2 promoter activity were enhanced by the anti-inflammatory mediators 15-deoxy-Δ(12,14)-prostaglandin J2 (15d-PGJ2) and interleukin-22 (IL-22), while it was unaffected by classical proinflammatory cytokines like IL-1ß. Induction of GPx2 expression by 15d-PGJ2 was mediated through Nrf2. In contrast, in DSS-treated Nrf2-KO mice GPx2 expression remained upregulated during recovery, which appeared to be independent of Nrf2. IL-22 activates transcription factors of the signal transducers and activators of transcription (STAT) family. Therefore, we analyzed the GPx2 promoter for putative STAT-responsive elements and identified 4 of them. Point mutation of the binding element next to the transcription start completely abolished promoter activation after IL-22 treatment and after cotransfection of STAT expression plasmids. To show in vivo relevance of the obtained results, we performed immunohistochemistry for phospho-STAT3 and GPx2. Especially during acute colitis, GPx2 and nuclear STAT3 colocalized in inflamed areas. CONCLUSIONS: GPx2 is a novel target of STAT transcription factors. The upregulation of GPx2 by IL-22 indicates that GPx2 might be important for the resolution of inflammation.

Selenoprotein W as biomarker for the efficacy of selenium compounds to act as source for selenoprotein biosynthesis.

Selenium is an essential trace element and, like all elements, present in many different compounds with unequivocal functions. This fact is only sporadically mentioned when recommended intake or supplementation is indicated just as "selenium." In mammals, selenium is an integral part of selenoproteins as selenocysteine. Selenocysteine is formed from serine at the respective tRNA((ser)sec), a reaction that requires selenophosphate formed from selenide and ATP. Thus, only compounds that can be metabolized into selenide can serve as sources for selenoprotein biosynthesis. We therefore tested the ability of selenium compounds such as sodium selenite, methylseleninic acid (MeSeA), Se-methyl selenocysteine, and selenomethionine to increase the activity, protein, or mRNA levels of commonly used biomarkers of the selenium status, glutathione peroxidase-1 (GPx1) and thioredoxin reductase, and of putatively new biomarkers, selenoprotein W1 (SepW1), selenoprotein H, and selenoprotein 15 in three different cell lines. Selenite and MeSeA were most efficient in increasing all markers tested, whereas the other compounds had only marginal effects. Effects were higher in the noncancerous young adult mouse colon cells than in the cancer cell lines HepG2 and HT-29. At the protein level, SepW1 responded as well as GPx1 and at the mRNA level, even better. Thus, the outcome of selenium treatment strongly depends on the chemical form, the cell type, and the biomarker used for testing efficacy.

The selenoproteins GPx2, TrxR2 and TrxR3 are regulated by Wnt signalling in the intestinal epithelium.

BACKGROUND: The glutathione peroxidase 2 (GPx2) is expressed at crypt bases of the intestinal epithelium and in tumour tissue. The GPx2 promoter is activated by the Wnt pathway, which might be the reason for the specific expression pattern of GPx2. Together with additional selenoproteins, thioredoxin reductases TrxR2 and TrxR3, which are putative Wnt targets based on microarray analysis, Wnt-dependent GPx2 expression was analysed. METHODS: Two cell culture models for either an activated (3T3 cells with Wnt3a overexpression) or an inhibited Wnt pathway (HT-29 APC cells) were analysed. To provide physiological relevance, crypt base epithelial cells of the jejunum and colon of mice were compared to cells of the villus or crypt table, respectively. In addition, ß-catenin was deleted in crypt base cells ex vivo. RESULTS: In cancer cell lines, the endogenous expression of all three selenoproteins was consistently dependent on Wnt pathway activity. Expression was higher in the proliferative crypt compartment, where also the Wnt pathway is active. An inducible knockout of ß-catenin in isolated colonic crypt base cells reduced basal GPx2 expression. We, thus, demonstrated the regulation of GPx2 expression by the Wnt pathway in vitro and in vivo. Furthermore, the selenoproteins TrxR2 and TrxR3 have been identified as novel Wnt targets. This may imply a role of GPx2, TrxR2 and TrxR3 in proliferation, apoptosis and, therefore, also during cancer development. GENERAL SIGNIFICANCE: Selenium which is essential for the biosynthesis of Wnt-dependent selenoproteins might be important for the renewal of the intestinal epithelium and during carcinogenesis.

alpha-Tocopherol enhances degranulation in RBL-2H3 mast cells.

Based on the observation that 3 months alpha-tocopherol supplementation caused an up-regulation of the mRNA of vesicular transport proteins in livers of mice, the functional relevance was investigated in RBL-2H3 cells, a model for mast cell degranulation. In total, 24 h incubation with 100 muM alpha-tocopherol enhanced the basal and phorbol-12-myristyl-13-acetate/ionomycin-stimulated release of beta-hexosaminidase and cathepsin D as measured by enzymatic analysis as well as Western blotting and immunocytochemistry, respectively. beta-Tocopherol exerted the same effect, whereas alpha-tocopheryl phosphate and trolox were inactive, indicating that both the side chain and the 6-OH group at the chroman ring are essential for activation of degranulation. alpha-Tocopherol did not induce mRNA expression of soluble NSF-attachment protein receptor (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) proteins, such as N-ethylmaleimide sensitive fusion protein, complexin-2, SNAP23 or syntaxin-3, in the RBL-2H3 cell model. In view of the well known alpha-tocopherol-mediated activation of protein phosphatases, which regulate soluble NSF-attachment protein receptor activities by dephosphorylation, underlying mechanisms are discussed in terms of preventing oxidative inactivation of protein phosphatases and so far unknown functions in certain membrane domains.

GPx2 counteracts PGE2 production by dampening COX-2 and mPGES-1 expression in human colon cancer cells.

GPx2, the gastrointestinal glutathione peroxidase, is a selenoprotein predominantly expressed in the intestine. An anti-inflammatory and anticarcinogenic potential has been inferred from the development of colitis and intestinal cancer in GPx1 and GPx2 double knockout mice. Further, induction by Nrf2 activators classifies GPx2 as a protective enzyme. In contrast, enhanced COX-2 expression is consistently associated with inflammation. The antagonistic roles and an intriguing co-localization of GPx2 and COX-2 prompted us to investigate their possible mutual regulation. Both enzymes were upregulated in tissues of patients with colorectal cancer and colitis, and co-localized in the endoplasmic reticulum. A stable knockdown of GPx2 in HT-29 cells by siRNA resulted in a high basal and IL-1-induced expression of COX-2 and mPGES-1, enzymes required for the production of the pro-inflammatory PGE(2). Accordingly, si-GPx2 cells released high concentrations of PGE(2). Observed effects were specific for GPx2, since COX-2 and mPGES-1 expression was not affected by selenium-deprivation which resulted in the disappearance of GPx1. It is concluded that GPx2 by compartmentalized removal of hydroperoxides silences COX-2 activity and suppresses PGE(2)-dependent COX-2 expression. Thus, GPx2 may prevent undue responses to inflammatory stimuli and, in consequence, inflammation-driven initiation of carcinogenesis.

Bioactivation of selenocysteine derivatives by beta-lyases present in common gastrointestinal bacterial species.

Studies in cell cultures and animal models have demonstrated cancer chemopreventive effects of certain selenium compounds. Here we describe the screening of cysteine S-conjugate beta-lyase activity in bacterial species that are implicated in the bio-activation of sulfur- and selenocysteine derivatives. We screened a range of bacterial species commonly found in the human intestine for beta-lyase activity on Se-p-methoxybenzylselenocysteine and the natural occurring S-methylcysteine and Se-methylselenocysteine conjugates. A high-performance liquid chromatography (HPLC)-assisted assay was established to determine specific activities of each strain. Of the 29 tested bacterial species, 22 showed specific activities towards the test compound reaching up to 10.1 U/mg protein, thereby accounting for 75% of total fecal activity (13.3. U/mg protein). Lysates of four bacterial strains (Bacteroides distasonis, bacteroides vulgatus, Enterococcus faecalis, and Enterococcus faecium), which exhibited high specific activities towards the test compound and which are known to be present at high numbers in the human intestine, were characterized further. Our results indicate that beta-lyase activity is widely distributed in human intestinal bacteria and might play a key role in the bioactivation of selenocysteine derivatives.

The GI-GPx gene is a target for Nrf2.

The gastrointestinal glutathione peroxidase (GI-GPx, GPx2) is a selenoprotein that was suggested to act as barrier against hydroperoxide absorption but has also been implicated in the control of inflammation and malignant growth. In CaCo-2 cells, GI-GPx was induced by t-butyl hydroquinone (tBHQ) and sulforaphane (SFN), i.e., "antioxidants" known to activate the "antioxidant response element" (ARE) via electrophilic thiol modification of Keap1 in the Nrf2/Keap1 system. The functional significance of a putative ARE in the GI-GPx promoter was validated by transcriptional activation of reporter gene constructs upon exposure to electrophiles (tBHQ, SFN, and curcumin) or overexpression of Nrf2 and by reversal of these effects by mutation of the ARE in the promoter and by overexpressed Keap1. Binding of Nrf2 to the ARE sequence in authentic gpx2 was corroborated by chromatin immunoprecipitation. Thus, the presumed natural antioxidants sulforaphane and curcumin may exert their anti-inflammatory and anticarcinogenic effects not only by induction of phase 2 enzymes but also by the up-regulation of the selenoprotein GI-GPx.