Identification of novel activators of the metal responsive transcription factor (MTF-1) using a gene expression biomarker in a microarray compendium.

Environmental exposure to metals is known to cause a number of human toxicities including cancer. Metal-responsive transcription factor 1 (MTF-1) is an important component of metal regulation systems in mammalian cells. Here, we describe a novel method to identify chemicals that activate MTF-1 based on microarray profiling data. MTF-1 biomarker genes were identified that exhibited consistent, robust expression across 10 microarray comparisons examining the effects of metals (zinc, nickel, lead, arsenic, mercury, and silver) on gene expression in human cells. A subset of the resulting 81 biomarker genes was shown to be altered by knockdown of the MTF1 gene including metallothionein family members and a zinc transporter. The ability to correctly identify treatment conditions that activate MTF-1 was determined by comparing the biomarker to microarray comparisons from cells exposed to reference metal activators of MTF-1 using the rank-based Running Fisher algorithm. The balanced accuracy for prediction was 93%. The biomarker was then used to identify organic chemicals that activate MTF-1 from a compendium of 11 725 human gene expression comparisons representing 2582 chemicals. There were 700 chemicals identified that included those known to interact with cellular metals, such as clioquinol and disulfiram, as well as a set of novel chemicals. All nine of the novel chemicals selected for validation were confirmed to activate MTF-1 biomarker genes in MCF-7 cells and to lesser extents in MTF1-null cells by qPCR and targeted RNA-Seq. Overall, our work demonstrates that the biomarker for MTF-1 coupled with the Running Fisher test is a reliable strategy to identify novel chemical modulators of metal homeostasis using gene expression profiling.

Isobavachalcone attenuates Sephadex-induced lung injury via activation of A20 and NRF2/HO-1 in rats.

The aim of this study is to investigate the protective effect and underlying molecular mechanisms of isobavachalcone on Sephadex-induced lung injury in rats. The result showed isobavachalcone inhibited massive granulomas, decreased inflammatory cells infiltration and oxidative stress markers level, but it can increase antioxidant enzymes level. The ELISA assay exhibited isobavachalcone decreased TNF-α production in BALF and lung tissue. Western blotting analysis showed isobavachalcone can inhibit NF-κB pathway that may be mediated by upregulation of A20. Furthermore, we also found isobavachalcone can activate NRF2/HO-1 pathway and inhibit adhesion molecule expression. Taken together, the present results suggested that isobavachalcone can attenuate Sephadex-induced lung injury that may be related to inhibition of NF-κB mediated by upregulation of A20 and activation of NRF2/HO-1 signaling pathway.

Differential Activation of NLRP3, AIM2, and IFI16 Inflammasomes in Humans with Acute and Chronic Hepatitis B.

Nod-like receptor protein 3 (NLRP3), absent in melanoma 2 (AIM2), and interferon gamma inducible protein 16 (IFI16) are innate immune sensors for intracellular microbes, which can be activated by various dangerous signals and subsequently lead to caspase-1 (CASP1) activation and the maturation cleavage of effector molecules pro-IL-1ß and pro-IL-18. Their roles in immunopathology of acute and chronic hepatitis B virus (HBV) infection are still unclear. In this study, we first investigated the activation of NLRP3, AIM2, and IFI16 inflammasomes in peripheral blood mononuclear cells (PBMCs) from patients infected with acute hepatitis B (AHB) and chronic hepatitis B (CHB) by quantitative real-time PCR and enzyme-linked immunosorbent assay. We next analyzed the impact of hepatitis B e antigen (HBeAg) on activation of AIM2 and IFI16 inflammasomes in PBMCs of CHB patients stimulated in vitro with AIM2 and IFI16 agonist ligands, poly (dA:dT) and VACA-70mer, respectively. The results showed that the mRNA expression levels of AIM2, IFI16, and CASP1 in PBMCs from AHB and CHB patients were both upregulated. Furthermore, the mRNA levels of AIM2 and IFI16 in CHB patients were significantly positively correlated with serum HBV loads. However, only in patients with AHB there was elevation of serum IL-1ß and IL-18. There was no activation of NLRP3, AIM2, and IFI16 inflammasomes in CHB patients. Stimulation of PBMCs of CHB patients in vitro with poly (dA:dT) and VACA-70mer induced the activation of AIM2 and IFI16 inflammasomes, respectively. This ligand-induced activation was suppressed by HBeAg. Our results suggest that there exists activation of the AIM2 and IFI16 inflammasomes, but not the NLRP3 inflammasome, in AHB, and the activation of the AIM2 and IFI16 inflammasomes can be inhibited by HBeAg in CHB, which may contribute to HBV-induced immunotolerance.

Salicylates promote mitochondrial biogenesis by regulating the expression of PGC-1α in murine 3T3-L1 pre-adipocytes.

Mitochondrial dysfunction has been associated with insulin resistance and diabetes. Decreased mitochondrial density and mitochondrial copy numbers have been found in insulin-resistant individuals. Restoration of the number of mitochondria and normal mitochondrial function has become an important therapeutic target of diabetes. Salicylate, the main active ingredient in aspirin, has been in medicinal use since ancient times. Little information regarding the effects of salicylate on mitochondrial function has been reported. In this study, we assessed the effects of salicylate on the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) signaling pathway and mitochondrial biogenesis in pre-adipocytes. Our findings demonstrate that treatment with salicylate promoted the expression of PGC-1α and its downstream targets nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM). Importantly, salicylate treatment significantly increased the number of mDNA, citrate synthase activity, expression of respiratory chain complex I, and mitochondrial mass, which were suppressed by the specific AMPK inhibitor Compound C. Indeed, salicylate treatment induced the phosphorylation of AMPK, which was involved in the induction of PGC-1α, NRF1, and TFAM. Importantly, inhibition of PGC-1α expression using PGC-1α small RNA interference abolished the effects of salicylate on mitochondrial biogenesis. These results suggest that salicylate has a potential therapeutic capacity against mitochondrial dysfunction in diabetes.

Saccharomyces cerevisiae Red1 protein exhibits nonhomologous DNA end-joining activity and potentiates Hop1-promoted pairing of double-stranded DNA.

Elucidation of the function of synaptonemal complex (SC) in Saccharomyces cerevisiae has mainly focused on in vivo analysis of recombination-defective meiotic mutants. Consequently, significant gaps remain in the mechanistic understanding of the activities of various SC proteins and the functional relationships among them. S. cerevisiae Hop1 and Red1 are essential structural components of the SC axial/lateral elements. Previous studies have demonstrated that Hop1 is a structure-selective DNA-binding protein exhibiting high affinity for the Holliday junction and promoting DNA bridging, condensation, and pairing between double-stranded DNA molecules. However, the exact mode of action of Red1 remains unclear, although it is known to interact with Hop1 and to suppress the spore viability defects of hop1 mutant alleles. Here, we report the purification and functional characterization of the full-length Red1 protein. Our results revealed that Red1 forms a stable complex with Hop1 in vitro and provided quantitative insights into their physical interactions. Mechanistically, Red1 preferentially associated with the Holliday junction and 3-way junction rather than with single- or double-stranded DNA with overhangs. Although Hop1 and Red1 exhibited similar binding affinities toward several DNA substrates, the two proteins displayed some significant differences. Notably, Red1, by itself, lacked DNA-pairing ability; however, it potentiated Hop1-promoted intermolecular pairing between double-stranded DNA molecules. Moreover, Red1 exhibited nonhomologous DNA end-joining activity, thus revealing an unexpected role for Red1 in recombination-based DNA repair. Collectively, this study presents the first direct insights into Red1's mode of action and into the mechanism underlying its role in chromosome synapsis and recombination.

Recombinant human erythropoietin stimulates melanoma tumor growth through activation of initiation factor eIF4E.

Recombinant human erythropoietin (EPO) is standard treatment for anemia in cancer patients. Recent clinical trials suggest that EPO may accelerate tumor progression and increase mortality. However, the evidence supporting a growth-promoting effect of EPO has remained controversial. Employing an in vivo model of B16 murine melanoma, we observed that administration of EPO to tumor bearing C57BL/6 mice resulted in pronounced acceleration of melanoma growth. Our in vitro studies demonstrate that B16 murine melanoma cells express EPOR, both at the protein and mRNA levels. Interestingly, expression of EPOR was retained in the established tumors. EPO stimulation of B16 cells enhanced proliferation and protein synthesis rates, and correlated with activation of the receptor associated Janus kinase 2 (Jak2) as well as phosphorylation of extracellular signal-regulated kinase (Erk) 1/2 and Akt kinases. Treatment with EPO and Jak-2 antagonists significantly inhibited EPO-mediated B16 cell proliferation. Moreover, EPO dose-dependently induced the phosphorylation and activation of the translation initiation factor eIF4E as well as the phosphorylation of its repressor, the eIF4E binding protein 4E-BP1. Finally, using eIF4E small interfering RNA (siRNA), we observed that EPO-mediated stimulation of B16 cell proliferation is eIF4E-dependent. Our results indicate that EPO exerts a powerful stimulatory effect on cell proliferation and de novo protein synthesis in melanoma cells through activation of the initiation factor eIF4E.

Glucose, amino acids and fatty acids directly regulate ghrelin and NUCB2/nesfatin-1 in the intestine and hepatopancreas of goldfish (Carassius auratus) in vitro.

Ghrelin and nesfatin-1 are two peptidyl hormones primarily involved in food intake regulation. We previously reported that the amount of dietary carbohydrates, protein and lipids modulates the expression of these peptides in goldfish in vivo. In the present work, we aimed to characterize the effects of single nutrients on ghrelin and nesfatin-1 in the intestine and hepatopancreas. First, immunolocalization of ghrelin and NUCB2/nesfatin-1 in goldfish hepatopancreas cells was studied by immunohistochemistry. Second, the effects of 2 and 4hour-long exposures of cultured intestine and hepatopancreas sections to glucose, l-tryptophan, oleic acid, linolenic acid (LNA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on ghrelin and nesfatin-1 gene and protein expression were studied. Co-localization of ghrelin and NUCB2/nesfatin-1 in the cytoplasm of goldfish hepatocytes was found. Exposure to glucose led to an upregulation of preproghrelin and a downregulation of nucb2/nesfatin-1 in the intestine. l-Tryptophan mainly decreased the expression of both peptides in the intestine and hepatopancreas. Fatty acids, in general, downregulated NUCB2/nesfatin-1 in the intestine, but only the longer and highly unsaturated fatty acids inhibited preproghrelin. EPA exposure led to a decrease in preproghrelin, and an increase in nucb2/nesfatin-1 expression in hepatopancreas after 2h. These results show that macronutrients exert a dose- and time-dependent, direct regulation of ghrelin and nesfatin-1 in the intestine and hepatopancreas, and suggest a role for these hormones in the digestive process and nutrient metabolism.

Regulation of the mammalian heat shock factor 1.

Living organisms are endowed with the capability to tackle various forms of cellular stress due to the presence of molecular chaperone machinery complexes that are ubiquitous throughout the cell. During conditions of proteotoxic stress, the transcription factor heat shock factor 1 (HSF1) mediates the elevation of heat shock proteins, which are crucial components of the chaperone complex machinery and function to ameliorate protein misfolding and aggregation and restore protein homeostasis. In addition, HSF1 orchestrates a versatile transcriptional programme that includes genes involved in repair and clearance of damaged macromolecules and maintenance of cell structure and metabolism, and provides protection against a broad range of cellular stress mediators, beyond heat shock. Here, we discuss the structure and function of the mammalian HSF1 and its regulation by post-translational modifications (phosphorylation, sumoylation and acetylation), proteasomal degradation, and small-molecule activators and inhibitors.

The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange.

Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA.

Rodent Models of Amyotrophic Lateral Sclerosis.

Amyotrophic Lateral Sclerosis (ALS) is a motor neuron disease affecting upper and lower motor neurons in the central nervous system. Patients with ALS develop extensive muscle wasting and atrophy leading to paralysis and death 3 to 5 years after disease onset. The condition may be familial (fALS 10%) or sporadic ALS (sALS, 90%). The large majority of fALS cases are due to genetic mutations in the Superoxide dismutase 1 gene (SOD1, 15% of fALS) and repeat nucleotide expansions in the gene encoding C9ORF72 (∼ 40% to 50% of fALS and ∼ 10% of sALS). Studies suggest that ALS is mediated through aberrant protein homeostasis (i.e., ER stress and autophagy) and/or changes in RNA processing (as in all non-SOD1-mediated ALS). In all of these cases, animal models suggest that the disorder is mediated non-cell autonomously, i.e., not only motor neurons are involved, but glial cells including microglia, astrocytes, and oligodendrocytes, and other neuronal subpopulations are also implicated in the pathogenesis. Provided in this unit is a review of ALS rodent models, including discussion of their relative advantages and disadvantages. Emphasis is placed on correlating the model phenotype with the human condition and the utility of the model for defining the disease process. Information is also presented on RNA processing studies in ALS research, with particular emphasis on the newest ALS rodent models.

Synthesis and Structure-Activity Relationships of Quaternary Coptisine Derivatives as Potential Anti-ulcerative Colitis Agents.

Thirty quaternary coptisine derivatives from a synthesized library were found to activate the in vitro transcription of x-box-binding protein 1 (XBP1). Among these, the dihydrocoptisines were demonstrated by in vitro XBP1 transcriptional activity assays and animal experiments to be much more active anti-ulcerative colitis (UC) agents than quaternary coptisines, tetrahydrocoptisines, and the positive control. Unsubstituted dihydrocoptisine exhibited more significant anti-UC efficacy than dihydrocoptisines substituted at the C-8 or C-13 position. The EC50 value of dihydrocoptisine for XBP1 transcriptional activation was 2.25 nM. Dihydrocoptisine exhibited a significant dose-effect relationship, as indicated by biomarkers in in vitro and in vivo experiments. According to this study, the starting material's reductive states and the substitution patterns of the dihydrocoptisines were determined to be the critical parameters for modulating their anti-UC efficacy, and the dihydrocoptisine skeleton was designated as the key pharmacophore. The synthesized dihydrocoptisine is a promising lead for developing anti-UC drugs.

Shikonin causes apoptosis by up-regulating p73 and down-regulating ICBP90 in human cancer cells.

Shikonin, a natural naphthoquinone isolated from the Chinese traditional medicine Zi Cao (purple gromwell), is known to suppress the growth of several cancer cell types. In this study, we evaluated the pro-apoptotic effects of shikonin on MCF-7 and HeLa cells, and investigated the underlying mechanism. Shikonin-induced apoptosis was associated with activation of caspase-3, poly(ADP-ribose) polymerase (PARP) cleavage, up-regulation of p73, and down-regulation of BCL-2. Shikonin also induced up-regulation of the tumor suppressor gene, p16(INK4A). Increasing transcriptional activity of p16(INK4A) by shikonin treatment, we observed in luciferase promoter assay, reflects reduced promoter binding by down-regulation of ICBP90 (inverted CCAAT box binding protein, 90 kDa), which are involved in down-regulation of its partner, DNMT1 (DNA methyltransferase 1). On the basis of these results, we conclude that shikonin causes apoptosis via a p73-related, caspase-3-dependent pathway.

PPAR gamma activation is neuroprotective in a Drosophila model of ALS based on TDP-43.

Amyotrophic Lateral Sclerosis (ALS) is a progressive neuromuscular disease for which there is no cure. We have previously developed a Drosophila model of ALS based on TDP-43 that recapitulates several aspects of disease pathophysiology. Using this model, we designed a drug screening strategy based on the pupal lethality phenotype induced by TDP-43 when expressed in motor neurons. In screening 1200 FDA-approved compounds, we identified the PPARγ agonist pioglitazone as neuroprotective in Drosophila. Here, we show that pioglitazone can rescue TDP-43-dependent locomotor dysfunction in motor neurons and glia but not in muscles. Testing additional models of ALS, we find that pioglitazone is also neuroprotective when FUS, but not SOD1, is expressed in motor neurons. Interestingly, survival analyses of TDP or FUS models show no increase in lifespan, which is consistent with recent clinical trials. Using a pharmacogenetic approach, we show that the predicted Drosophila PPARγ homologs, E75 and E78, are in vivo targets of pioglitazone. Finally, using a global metabolomic approach, we identify a set of metabolites that pioglitazone can restore in the context of TDP-43 expression in motor neurons. Taken together, our data provide evidence that modulating PPARγ activity, although not effective at improving lifespan, provides a molecular target for mitigating locomotor dysfunction in TDP-43 and FUS but not SOD1 models of ALS in Drosophila. Furthermore, our data also identify several 'biomarkers' of the disease that may be useful in developing therapeutics and in future clinical trials.

TXNL1 induces apoptosis in cisplatin resistant human gastric cancer cell lines.

Cisplatin is one of the most commonly used drugs in the treatment of gastric cancer. However, drug resistance is a major obstacle for effective treatment and originates in multiple mechanisms such as enhanced DNA repair and anti-apoptosis. Our previous results demonstrated that XRCC1 was a key regulator of cisplatin induced DNA damage and apoptosis. TXNL1, a member of the thioredoxin family, negatively regulated the expression of XRCC1 via the ubiquitin-proteasome pathway. Here, we investigated the role of TXNL1 in the apoptosis induced by cisplatin. Our data showed that the expression of TXNL1 in the cisplatin resistant gastric cancer cell lines BGC823/DDP and SGC7901/DDP cells was significantly lower compared with the cisplatin sensitive cell lines BGC823 and SGC7901. Inhibition of the expression of TXNL1 in BGC823 and SGC7901 cells led to increased resistance to cisplatin induced apoptosis and cell death detected by Tunel and clonogenic assay, respectively. In contrast, over expression of TXNL1 in BGC823/DDP and SGC7901/DDP cells lead to higher cisplatin induced apoptosis and cell death. Moreover, our results demonstrated that the mechanism of TXNL1 regulating cisplatin-induced apoptosis was closely associated with Bcl-2 mediated mitochondria apoptosis pathway. In conclusion, these findings suggest that TXNL1 was a feasible modulator and potential chemotherapeutic target for the cisplatin resistant phenotype of human gastric cancer cells.

p53-Dependent apoptosis induced in human bronchial epithelial (16-HBE) cells by PM(2.5) sampled from air in Guangzhou, China.

Epidemiological studies have shown that air pollution particulate matter (PM) is associated with increased respiratory morbidity and mortality. However, the mechanisms are not fully understood. Oxidative stress-mediated apoptosis plays an important role in the occurrence of respiratory diseases. In this study, human bronchial epithelial (16-HBE) cells were exposed to different concentrations (16-128 µg/ml) of PM(2.5) for 24 h to investigate the apoptosis induced by PM(2.5). The results showed that PM(2.5) exposure significantly induced apoptosis, DNA strand breaks, and oxidative damage in a dose-dependent manner in 16-HBE cells. The expression of p53 and p73 increased significantly along with the dose of PM(2.5) in 16-HBE cells, whereas the expression of p21(Cip1/WAF1) decreased; the expression of mdm2 increased and then decreased, but not significantly. Taken together, these observations indicate that PM(2.5) may lead to oxidative damage and induce apoptosis through the p53-dependent pathway in 16-HBE cells. p53-Dependent apoptosis mediated by DNA strand breaks may be an important mechanism of PM(2.5)-induced apoptosis in 16-HBE cells.

Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide.

In the 1950s, the drug thalidomide, administered as a sedative to pregnant women, led to the birth of thousands of children with multiple defects. Despite the teratogenicity of thalidomide and its derivatives lenalidomide and pomalidomide, these immunomodulatory drugs (IMiDs) recently emerged as effective treatments for multiple myeloma and 5q-deletion-associated dysplasia. IMiDs target the E3 ubiquitin ligase CUL4-RBX1-DDB1-CRBN (known as CRL4(CRBN)) and promote the ubiquitination of the IKAROS family transcription factors IKZF1 and IKZF3 by CRL4(CRBN). Here we present crystal structures of the DDB1-CRBN complex bound to thalidomide, lenalidomide and pomalidomide. The structure establishes that CRBN is a substrate receptor within CRL4(CRBN) and enantioselectively binds IMiDs. Using an unbiased screen, we identified the homeobox transcription factor MEIS2 as an endogenous substrate of CRL4(CRBN). Our studies suggest that IMiDs block endogenous substrates (MEIS2) from binding to CRL4(CRBN) while the ligase complex is recruiting IKZF1 or IKZF3 for degradation. This dual activity implies that small molecules can modulate an E3 ubiquitin ligase and thereby upregulate or downregulate the ubiquitination of proteins.

Telmisartan exerts anti-tumor effects by activating peroxisome proliferator-activated receptor-γ in human lung adenocarcinoma A549 cells.

Telmisartan, a member of the angiotensin II type 1 receptor blockers, is usually used for cardiovascular diseases. Recent studies have showed that telmisartan has the property of PPARγ activation. Meanwhile, PPARγ is essential for tumor proliferation, invasion and metastasis. In this work we explore whether telmisartan could exert anti-tumor effects through PPARγ activation in A549 cells. MTT and trypan blue exclusion assays were included to determine the survival rates and cell viabilities. RT-PCR and western blotting were used to analyze the expression of ICAM-1, MMP-9 and PPARγ. DNA binding activity of PPARγ was evaluated by EMSA. Our data showed that the survival rates and cell viabilities of A549 cells were all reduced by telmisartan in a time- and concentration-dependent manner. Meanwhile, our results also demonstrated that telmisartan dose-dependently inhibited the expression of ICAM-1 and MMP-9. Moreover, the cytotoxic and anti-proliferative effects, ICAM-1 and MMP-9 inhibitive properties of telmisartan were totally blunted by the PPARγ antagonist GW9662. Our findings also showed that the expression of PPARγ was up-regulated by telmisartan in a dose dependent manner. And, the EMSA results also figured out that DNA binding activity of PPARγ was dose-dependently increased by telmisartan. Additionally, our data also revealed that telmisartan-induced PPARγ activation was abrogated by GW9662. Taken together, our results indicated that telmisartan inhibited the expression of ICAM-1 and MMP-9 in A549 cells, very likely through the up-regulation of PPARγ synthesis.

Chronic exposure to low doses of pharmaceuticals disturbs the hepatic expression of circadian genes in lean and obese mice.

Drinking water can be contaminated with pharmaceuticals. However, it is uncertain whether this contamination can be harmful for the liver, especially during obesity. Hence, the goal of our study was to determine whether chronic exposure to low doses of pharmaceuticals could have deleterious effects on livers of lean and obese mice. To this end, lean and ob/ob male mice were treated for 4 months with a mixture of 11 drugs provided in drinking water at concentrations ranging from 10 to 106 ng/l. At the end of the treatment, some liver and plasma abnormalities were observed in ob/ob mice treated with the cocktail containing 106 ng/l of each drug. For this dosage, a gene expression analysis by microarray showed altered expression of circadian genes (e.g. Bmal1, Dbp, Cry1) in lean and obese mice. RT-qPCR analyses carried out in all groups of animals confirmed that expression of 8 different circadian genes was modified in a dose-dependent manner. For some genes, a significant modification was observed for dosages as low as 10²-10³ ng/l. Drug mixture and obesity presented an additive effect on circadian gene expression. These data were validated in an independent study performed in female mice. Thus, our study showed that chronic exposure to trace pharmaceuticals disturbed hepatic expression of circadian genes, particularly in obese mice. Because some of the 11 drugs can be found in drinking water at such concentrations (e.g. acetaminophen, carbamazepine, ibuprofen) our data could be relevant in environmental toxicology, especially for obese individuals exposed to these contaminants.

Characterization of the metabolic effects of irisin on skeletal muscle in vitro.

AIMS: This work explored the effects of irisin on metabolism, gene expression and mitochondrial content in cultured myocytes. METHODS: C2C12 myocytes were treated with various concentrations of irisin for various durations. Glycolysis and oxidative metabolism were quantified by measurement of extracellular acidification and oxygen consumption, respectively. Metabolic gene expression was measured by quantitative real-time polymerase chain reaction (qRT-PCR) and mitochondrial content was assessed by flow cytometry and confocal microscopy. RESULTS: Cells treated with irisin exhibited significantly increased oxidative metabolism. Irisin treatment also significantly increased mitochondrial uncoupling at various doses and durations. Lastly, treatment with irisin also significantly elevated metabolic gene expression including peroxisome proliferator-activated receptor γ coactivator-1 alpha (PGC-1α), nuclear respiratory factor 1 (NRF1), mitochondrial transcription factor A (TFAM), irisin, glucose transporter 4 (GLUT4) and mitochondrial uncoupling protein 3 (UCP3) leading to increased mitochondrial biogenesis. CONCLUSIONS: Our observations are the first to document increased metabolism in myocytes through irisin-mediated induction of mitochondrial biogenesis and uncoupling with corresponding gene expression. These observations support the need for further investigation into the therapeutic and pharmacological effects of irisin, as well as development of irisin-based therapy.

TLR dependent XBP-1 activation induces an autocrine loop in rheumatoid arthritis synoviocytes.

X-box binding protein 1 (XBP1) is a central regulator of the endoplasmic reticulum (ER) stress response. It is induced via activation of the IRE1 stress sensor as part of the unfolded protein response (UPR) and has been implicated in several diseases processes. XBP1 can also be activated in direct response to Toll-like receptor (TLR) ligation independently of the UPR but the pathogenic significance of this mode of XBP1 activation is not well understood. Here we show that TLR-dependent XBP1 activation is operative in the synovial fibroblasts (SF) of patients with active rheumatoid arthritis (RA). We investigated the expression of ER stress response genes in patients with active RA and also in patients in remission. The active (spliced) form of (s)XBP1 was significantly overexpressed in the active RA group compared to healthy controls and patients in remission. Paradoxically, expression of nine other ER stress response genes was reduced in active RA compared to patients in remission, suggestive of a UPR-independent process. However, sXBP1 was induced in SF by TLR4 and TLR2 stimulation, resulting in sXBP1-dependent interleukin-6 and tumour necrosis factor (TNF) production. We also show that TNF itself induces sXBP1 in SF, thus generating a potential feedback loop for sustained SF activation. These data confirm the first link between TLR-dependent XBP1 activation and human inflammatory disease. sXBP1 appears to play a central role in this process by providing a convergence point for two different stimuli to maintain activation of SF.