Astrocytic Yin Yang 1 is critical for murine brain development and protection against apoptosis, oxidative stress, and inflammation.

The transcription factor Yin Yang 1 (YY1) is ubiquitously expressed in mammalian cells, regulating the expression of a variety of genes involved in proliferation, differentiation, and apoptosis in a context-dependent manner. While it is well-established that global YY1 knockout (KO) leads to embryonic death in mice and that YY1 deletion in neurons or oligodendrocytes induces impaired brain function, the role of astrocytic YY1 in the brain remains unknown. We investigated the role of astrocytic YY1 in the brain using a glial fibrillary acidic protein (GFAP)-specific YY1 conditional KO (YY1 cKO) mouse model to delete astrocytic YY1. Astrocytic YY1 cKO mice were tested for behavioral phenotypes, such as locomotor activity, coordination, and cognition, followed by an assessment of relevant biological pathways using RNA-sequencing analysis, immunoblotting, and immunohistochemistry in the cortex, midbrain, and cerebellum. YY1 cKO mice showed abnormal phenotypes, movement deficits, and cognitive dysfunction. At the molecular level, astrocytic YY1 deletion altered the expression of genes associated with proliferation and differentiation, p53/caspase apoptotic pathways, oxidative stress response, and inflammatory signaling including NF-κB, STAT, and IRF in all regions. Astrocytic YY1 deletion significantly increased the expression of GFAP as astrocytic activation and Iba1 as microglial activation, indicating astrocytic YY1 deletion activated microglia as well. Accordingly, multiple inflammatory cytokines and chemokines including TNF-α and CXCL10 were elevated. Combined, these novel findings suggest that astrocytic YY1 is a critical transcription factor for normal brain development and locomotor activity, motor coordination, and cognition. Astrocytic YY1 is also essential in preventing pathological oxidative stress, apoptosis, and inflammation.

Mechanisms of manganese-induced neurotoxicity and the pursuit of neurotherapeutic strategies.

Chronic exposure to elevated levels of manganese via occupational or environmental settings causes a neurological disorder known as manganism, resembling the symptoms of Parkinson's disease, such as motor deficits and cognitive impairment. Numerous studies have been conducted to characterize manganese's neurotoxicity mechanisms in search of effective therapeutics, including natural and synthetic compounds to treat manganese toxicity. Several potential molecular targets of manganese toxicity at the epigenetic and transcriptional levels have been identified recently, which may contribute to develop more precise and effective gene therapies. This review updates findings on manganese-induced neurotoxicity mechanisms on intracellular insults such as oxidative stress, inflammation, excitotoxicity, and mitophagy, as well as transcriptional dysregulations involving Yin Yang 1, RE1-silencing transcription factor, transcription factor EB, and nuclear factor erythroid 2-related factor 2 that could be targets of manganese neurotoxicity therapies. This review also features intracellular proteins such as PTEN-inducible kinase 1, parkin, sirtuins, leucine-rich repeat kinase 2, and α-synuclein, which are associated with manganese-induced dysregulation of autophagy/mitophagy. In addition, newer therapeutic approaches to treat manganese's neurotoxicity including natural and synthetic compounds modulating excitotoxicity, autophagy, and mitophagy, were reviewed. Taken together, in-depth mechanistic knowledge accompanied by advances in gene and drug delivery strategies will make significant progress in the development of reliable therapeutic interventions against manganese-induced neurotoxicity.

Manganese phosphorylates Yin Yang 1 at serine residues to repress EAAT2 in human H4 astrocytes.

Impairment of the astrocytic glutamate transporter excitatory amino acid transporter 2 (EAAT2) is associated with neurological disorders such as Parkinson's disease (PD), Alzheimer's disease (AD), and manganism, a neurological disorder caused by overexposure to manganese (Mn) which shares the features of sporadic PD. Mechanisms of Mn-induced neurotoxicity include dysregulation of EAAT2 following activation of the transcription factor Yin Yang 1 (YY1) by transcriptional upregulation, but the posttranslational mechanisms by which YY1 is activated to repress EAAT2 remain to be elucidated. In the present study, we tested if Mn activates YY1 through posttranslational phosphorylation in cultured H4 human astrocytes, leading to EAAT2 repression. The results demonstrate that Mn exposure induced phosphorylation of YY1 at serine residues via kinases Aurora B kinase (AurkB) and Casein kinase II (CK2), leading to YY1 nuclear translocation, YY1/HDAC interactions, binding to the EAAT2 promoter, and consequent decreases in EAAT2 promoter activity and mRNA/protein levels. Although further studies are warranted to fully elucidate the mechanisms of Mn-induced YY1 phosphorylation and resultant EAAT2 impairment, our findings indicate that serine phosphorylation of YY1 via AurkB and CK2 is critical, at least in part, to its activation and transcriptional repression of EAAT2.

Manganese-induced reactive oxygen species activate IκB kinase to upregulate YY1 and impair glutamate transporter EAAT2 function in human astrocytes in vitro.

Dysregulation of the astrocytic glutamate transporter excitatory amino acid transporter 2 (EAAT2) is associated with several neurological disorders, including Parkinson's disease, Alzheimer's disease, and manganism, the latter induced by chronic exposure to high levels of manganese (Mn). Mechanisms of Mn-induced neurotoxicity include impairment of EAAT2 function secondary to the activation of the transcription factor Yin Yang 1 (YY1) by nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). However, the upstream mechanisms by which Mn-induced NF-κB activates YY1 remain to be elucidated. In the present study, we used the H4 human astrocyte cell line to test if Mn activates YY1 through the canonical NF-κB signaling pathway, leading to EAAT2 repression. The results demonstrate that Mn exposure induced phosphorylation of the upstream kinase IκB kinase (IKK-ß), leading to NF-κB p65 translocation, increased YY1 promoter activity, mRNA/protein levels, and consequently repressed EAAT2. Results also demonstrated that Mn-induced oxidative stress and subsequent TNF-α production were upstream of IKK-ß activation, as antioxidants attenuated Mn-induced TNF-α production and IKK-ß activation. Moreover, TNF-α inhibition attenuated the Mn-induced activation of IKK-ß and YY1. Taken together, Mn-induced oxidative stress and TNF-α mediates activation of NF-κB signaling and YY1 upregulation, leading to repression of EAAT2. Thus, targeting reactive oxygen species (ROS), TNF-α and IKK-ß may attenuate Mn-induced YY1 activation and consequent EAAT2 repression.

Nutrient intakes from supplement and factors associated with supplement use among breast cancer survivors: A cross-sectional study.

OBJECTIVE: We investigated the contribution of supplement use to total nutrient intake, the prevalence of inadequate nutrient intake and the factors associated with supplement use among breast cancer survivors. METHODS: A total of 701 Korean breast cancer survivors were included. We calculated the contribution of dietary supplements to total nutrient intake and the proportion of the population below the estimated average requirements (EARs) or exceeding the tolerable upper intake levels (ULs). Stepwise logistic regression was used to identify factors associated with dietary supplement use. RESULTS: A total of 66.5% of the survivors used dietary supplements, with multivitamins and minerals being the most commonly consumed ones. The per cent contribution of supplement to the total intake was the highest for vitamin C. 28.2%-55.4% of the non-users consumed below the EAR of riboflavin, folate and calcium; 6.1%, 4.9% and 6.5% of the supplement users consumed above the UL of vitamins A and C, and iron, respectively. Supplement users had higher education levels or longer survival time. CONCLUSION: 66.5% of Korean breast cancer survivors used dietary supplements. A higher education level or prolonged survival time was associated with higher use of dietary supplements.

Astrocyte-specific deletion of the transcription factor Yin Yang 1 in murine substantia nigra mitigates manganese-induced dopaminergic neurotoxicity.

Manganese (Mn)-induced neurotoxicity resembles Parkinson's disease (PD), but the mechanisms underpinning its effects remain unknown. Mn dysregulates astrocytic glutamate transporters, GLT-1 and GLAST, and dopaminergic function, including tyrosine hydroxylase (TH). Our previous in vitro studies have shown that Mn repressed GLAST and GLT-1 via activation of transcription factor Yin Yang 1 (YY1). Here, we investigated if in vivo astrocytic YY1 deletion mitigates Mn-induced dopaminergic neurotoxicity, attenuating Mn-induced reduction in GLAST/GLT-1 expression in murine substantia nigra (SN). AAV5-GFAP-Cre-GFP particles were infused into the SN of 8-week-old YY1 flox/flox mice to generate a region-specific astrocytic YY1 conditional knockout (cKO) mouse model. 3 weeks after adeno-associated viral (AAV) infusion, mice were exposed to 330 µg of Mn (MnCl2 30 mg/kg, intranasal instillation, daily) for 3 weeks. After Mn exposure, motor functions were determined in open-field and rotarod tests, followed by Western blotting, quantitative PCR, and immunohistochemistry to assess YY1, TH, GLAST, and GLT-1 levels. Infusion of AAV5-GFAP-Cre-GFP vectors into the SN resulted in region-specific astrocytic YY1 deletion and attenuation of Mn-induced impairment of motor functions, reduction of TH-expressing cells in SN, and TH mRNA/protein levels in midbrain/striatum. Astrocytic YY1 deletion also attenuated the Mn-induced decrease in GLAST/GLT-1 mRNA/protein levels in midbrain. Moreover, YY1 deletion abrogated its interaction with histone deacetylases in astrocytes. These results indicate that astrocytic YY1 plays a critical role in Mn-induced neurotoxicity in vivo, at least in part, by reducing astrocytic GLAST/GLT-1. Thus, YY1 might be a potential target for treatment of Mn toxicity and other neurological disorders associated with dysregulation of GLAST/GLT-1.

Manganese-induced neurodegenerative diseases and possible therapeutic approaches.

INTRODUCTION: Neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis and prion disease represent important public health concerns. Exposure to high levels of  heavy metals such as manganese (Mn) may contribute to their development. AREAS COVERED: In this critical review, we address the role of Mn in the etiology of neurodegenerative diseases and discuss emerging treatments of Mn overload, such as chelation therapy. In addition, we discuss natural and synthetic compounds under development as prospective therapeutics. Moreover, bioinformatic approaches to identify new potential targets and therapeutic substances to reverse the neurodegenerative diseases are discussed. EXPERT OPINION: Here, the authors highlight the importance of better understanding the molecular mechanisms of toxicity associated with neurodegenerative diseases, and the role of Mn in these diseases. Additional emphasis should be directed to the discovery of new agents to treat Mn-induced diseases, since present day chelator therapies have limited bioavailability. Furthermore, the authors encourage the scientific community to develop research using libraries of compounds to screen those compounds that show efficacy in regulating brain Mn levels. In addition, bioinformatics may provide novel insight for pathways and clinical treatments associated with Mn-induced neurodegeneration, leading to a new direction in Mn toxicological research.

Efficacy of parenteral glutamine supplementation in adult hematopoietic stem cell transplantation patients.

BACKGROUND: Hematopoietic stem cell transplantation (HSCT) patients need parenteral nutrition because of nausea, vomiting, and mucositis caused by conditioning regimens. The demand for glutamine increases during the HSCT period. We evaluated the effects of glutamine-containing parenteral nutrition on the clinical outcomes of HSCT patients. METHODS: In this retrospective analysis, we reviewed HSCT patients from Seoul National University from August 2013 to July 2017. Depending on their glutamine supplementation status, 91 patients were divided into 2 groups: glutamine group (N=44) and non-glutamine group (N=47). We analyzed the rate of weight change, infection (clinically/microbiologically documented), complications (duration of mucositis and neutropenia, acute graft versus host disease), and 100-days mortality in each group. RESULTS: Regarding the clinical characteristics of the patients, there were no significant differences between the 2 groups except that there was a larger proportion of myeloablative conditioning regimen in the glutamine group (P=0.005). In the glutamine group, the average number of days of glutamine use, parenteral nutrition, and mucositis was 7.6±1.4, 14.6±9.9, and 13.3±9.5, respectively. Furthermore, multivariate analysis revealed odds ratios of 0.37 (95% CI, 0.14-0.96; P=0.042) and 0.08 (95% CI, 0.01-0.98; P=0.048) for clinically documented infection and 100-days mortality, respectively, in the glutamine group. CONCLUSION: Results showed that the glutamine group had less clinically documented infection and 100-days mortality than the non-glutamine group, but the other outcomes did not show significant differences. The extended duration of glutamine supplementation according to the period of total parenteral nutrition and mucositis should be considered.

Effect of raw potato starch on the gut microbiome and metabolome in mice.

The gut microbiome plays a pivotal role in human health and is affected by various factors. To investigate the association between phenotypic and microbiota-related changes in the gut and a raw starch-based diet, we fed mice with different starch substitutes (corn, wheat, rice, and potato) for 16 weeks. The potato starch-fed group showed the lowest weight gain and fat tissue accumulation of all the groups, as well as the highest insulin sensitivity. Taxonomic analysis indicated that the proportions of Akkermansia, Rikenellaceae, and Sutterella showed the greatest increase in the ceca of mice fed raw potato starch. In addition, the gut microbiota of the raw potato starch group showed the highest carbohydrate and energy metabolism of all the groups, as confirmed by cecal metabolite analysis. The raw potato starch group also produced the highest propionic acid content. Our results showed that the differences in the digestibility of each starch, differences in the phenotype in terms of digestibility, and changes in intestinal microbiota were connected, and it was confirmed that potato starch, which had the lowest digestibility, caused the greatest difference in intestinal microbe composition and metabolism.

Effects of raw potato starch on body weight with controlled glucose delivery.

Starch digestion in the gastrointestinal tract has different properties depending on its botanical source. In this study, corn, wheat, rice, and potato starches were used to test the digestion properties of mammalian mucosal α-glucosidase in an in vitro assay, and their physiological effects were investigated in male C57BL/6 mice. The results clearly demonstrated that potato starch caused the lowest rates of glucose generation, suggesting that it can attenuate the postprandial glucose spike. Interestingly, a potato starch-based diet caused significantly (P < 0.05) lower weight gain and fat accumulation compared to diets based on other starches, through increased insulin sensitivity. This result suggests that potato starch-based products can be used to regulate postprandial blood glucose levels, aiding in the control of metabolic diseases.

Effects of bentonite Bgp35b-p on the gut microbiota of mice fed a high-fat diet.

BACKGROUND: Bentonite is a natural clay mineral with health-promoting effects due to its high adsorption abilities with high cation-exchange capacity. Previously, we found an anti-obesity effect for Bgp35b-p bentonite produced in South Korea, where its high adsorbent ability of dietary lipids possibly partially removed the lipidic environment in the gut (unpublished). It is hypothesized that Bgp35b-p affects the intestinal microbial community, and thus the microbial changes were investigated via next-generation sequencing targeting the bacterial 16S rRNA gene and bioinformatics using QIIME (Quantitative Insights Into Microbial Ecology) were performed on feces of C57BL/6 male mice fed a high-fat diet (HFD) with the Bgp35b-p. RESULTS: The HFD caused microbial dysbiosis, characterized by a decrease in the relative abundance of Bacteroidetes and an increase in abundance of Firmicutes and Proteobacteria. It was found that HFD + Bgp35b-p led to significant changes in the microbial compositions of family-level bacteria known as short-chain fatty-acid-producing bacteria. The relative abundance of Ruminococcaceae was significantly increased, and the abundances of Clostridiaceae and Lachnospiraceae were decreased by HFD + Bgp35b-p, shifting close to that in mice fed a normal diet. CONCLUSION: Bgp35b-p induced compositional changes in intestinal microbiota, which can be considered as a prebiotic effect, thus suggesting that bentonite may be a potential prebiotic functional supplement. © 2018 Society of Chemical Industry.

Manganese Control of Glutamate Transporters' Gene Expression.

Manganese (Mn) is an essential trace element, serving as a cofactor for several enzymes involved in various cellular and biochemical reactions in human body. However, chronic overexposure to Mn from occupational or environmental sources induces a neurological disorder, characterized by psychiatric, cognitive, and motor abnormalities, referred to as manganism. Mn-induced neurotoxicity is known to target astrocytes since these cells preferentially accumulate Mn. Astrocytes are the most abundant non-neuronal glial cells in the brain, and they play a critical role in maintaining the optimal glutamate levels to prevent excitotoxic death. The fine regulation of glutamate in the brain is accomplished by two major glutamate transporters - glutamate transporter-1 (GLT-1) and glutamate aspartate transporter (GLAST) that are predominantly expressed in astrocytes. Excitotoxic neuronal injury has been demonstrated as a critical mechanism involved in Mn neurotoxicity and implicated in the pathological signs of multiple neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Recent evidences also establish that Mn directly deregulates the expression and function of both astrocytic glutamate transporters by decreasing mRNA and protein levels of GLT-1 and GLAST. Herein, we will review the mechanisms of Mn-induced gene regulation of glutamate transporters at the transcriptional level and their role in Mn toxicity.

Transcriptional Regulation of Human Transforming Growth Factor-α in Astrocytes.

Transforming growth factor-alpha (TGF-α) is known to play multifunctional roles in the central nervous system (CNS), including the provision of neurotropic properties that protect neurons against various neurotoxic insults. Previously, we reported that TGF-α mediates estrogen-induced enhancement of glutamate transporter GLT-1 function in astrocytes. However, the regulatory mechanism of TGF-α at the transcriptional level remains to be established. Our findings revealed that the human TGF-α promoter contains consensus sites for several transcription factors, such as NF-κB and yin yang 1 (YY1). NF-κB served as a positive regulator of TGF-α promoter activity, corroborated by observations that overexpression of NF-κB p65 increased, while mutation in the NF-κB binding sites in the TGF-α promoter reduced the promoter activity in rat primary astrocytes. Pharmacological inhibition of NF-κB with pyrrolidine dithiocarbamate (PDTC; 50 µM) or quinazoline (QNZ; 10 µM) also abolished TGF-α promoter activity, and NF-κB directly bound to its consensus site in the TGF-α promoter as evidenced by electrophoretic mobility shift assay (EMSA). Dexamethasone (DX) increased TGF-α promoter activity by activation of NF-κB. Treatment of astrocytes with 100 nM of DX for 24 h activated its glucocorticoid receptor and signaling proteins, including MAPK, PI3K/Akt, and PKA, via non-genomic pathways, to enhance TGF-α promoter activity and expression. YY1 served as a critical negative regulator of the TGF-α promoter as overexpression of YY1 decreased, while mutation of YY1 binding site in the promoter increased TGF-α promoter activity. Treatment for 3 h with 250 µM of manganese (Mn), an environmental neurotoxin, decreased astrocytic TGF-α expression by activation of YY1. Taken together, our results suggest that NF-κB is a critical positive regulator, whereas YY1 is a negative regulator of the TGF-α promoter. These findings identify potential molecular targets for neurotherapeutics that may modulate TGF-α regulation and afford neuroprotection.

Transcriptional Regulation of the Astrocytic Excitatory Amino Acid Transporter 1 (EAAT1) via NF-κB and Yin Yang 1 (YY1).

Astrocytic glutamate transporter excitatory amino acid transporter (EAAT) 1, also known as glutamate aspartate transporter (GLAST) in rodents, is one of two glial glutamate transporters that are responsible for removing excess glutamate from synaptic clefts to prevent excitotoxic neuronal death. Despite its important role in neurophysiological functions, the molecular mechanisms of EAAT1 regulation at the transcriptional level remain to be established. Here, we report that NF-κB is a main positive transcription factor for EAAT1, supported by the following: 1) EAAT1 contains two consensus sites for NF-κB, 2) mutation of NF-κB binding sites decreased EAAT1 promoter activity, and 3) activation of NF-κB increased, whereas inhibition of NF-κB decreased EAAT1 promoter activity and mRNA/protein levels. EGF increased EAAT1 mRNA/protein levels and glutamate uptake via NF-κB. The transcription factor yin yang 1 (YY1) plays a role as a critical negative regulator of EAAT1, supported by the following: 1) the EAAT1 promoter contains multiple consensus sites for YY1, 2) overexpression of YY1 decreased EAAT1 promoter activity and mRNA/protein levels, and 3) knockdown of YY1 increased EAAT1 promoter activity and mRNA/protein levels. Manganese decreased EAAT1 expression via YY1. Epigenetic modifiers histone deacetylases (HDACs) served as co-repressors of YY1 to further decrease EAAT1 promoter activity, whereas inhibition of HDACs reversed manganese-induced decrease of EAAT1 expression. Taken together, our findings suggest that NF-κB is a critical positive regulator of EAAT1, mediating the stimulatory effects of EGF, whereas YY1 is a negative regulator of EAAT1 with HDACs as co-repressors, mediating the inhibitory effects of manganese on EAAT1 regulation.

Genetic dys-regulation of astrocytic glutamate transporter EAAT2 and its implications in neurological disorders and manganese toxicity.

Astrocytic glutamate transporters, the excitatory amino acid transporter (EAAT) 2 and EAAT1 (glutamate transporter 1 and glutamate aspartate transporter in rodents, respectively), are the main transporters for maintaining optimal glutamate levels in the synaptic clefts by taking up more than 90% of glutamate from extracellular space thus preventing excitotoxic neuronal death. Reduced expression and function of these transporters, especially EAAT2, has been reported in numerous neurological disorders, including amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, schizophrenia and epilepsy. The mechanism of down-regulation of EAAT2 in these diseases has yet to be fully established. Genetic as well as transcriptional dys-regulation of these transporters by various modes, such as single nucleotide polymorphisms and epigenetics, resulting in impairment of their functions, might play an important role in the etiology of neurological diseases. Consequently, there has been an extensive effort to identify molecular targets for enhancement of EAAT2 expression as a potential therapeutic approach. Several pharmacological agents increase expression of EAAT2 via nuclear factor κB and cAMP response element binding protein at the transcriptional level. However, the negative regulatory mechanisms of EAAT2 have yet to be identified. Recent studies, including those from our laboratory, suggest that the transcriptional factor yin yang 1 plays a critical role in the repressive effects of various neurotoxins, such as manganese (Mn), on EAAT2 expression. In this review, we will focus on transcriptional epigenetics and translational regulation of EAAT2.

Role of transcription factor yin yang 1 in manganese-induced reduction of astrocytic glutamate transporters: Putative mechanism for manganese-induced neurotoxicity.

Astrocytes are the most abundant non-neuronal glial cells in the brain. Once relegated to a mere supportive role for neurons, contemporary dogmas ascribe multiple active roles for these cells in central nervous system (CNS) function, including maintenance of optimal glutamate levels in synapses. Regulation of glutamate levels in the synaptic cleft is crucial for preventing excitotoxic neuronal injury. Glutamate levels are regulated predominantly by two astrocytic glutamate transporters, glutamate transporter 1 (GLT-1) and glutamate aspartate transporter (GLAST). Indeed, the dysregulation of these transporters has been linked to several neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD) and Parkinson's disease (PD), as well as manganism, which is caused by overexposure to the trace metal, manganese (Mn). Although Mn is an essential trace element, its excessive accumulation in the brain as a result of chronic occupational or environmental exposures induces a neurological disorder referred to as manganism, which shares common pathological features with Parkinsonism. Mn decreases the expression and function of both GLAST and GLT-1. Astrocytes are commonly targeted by Mn, and thus reduction in astrocytic glutamate transporter function represents a critical mechanism of Mn-induced neurotoxicity. In this review, we will discuss the role of astrocytic glutamate transporters in neurodegenerative diseases and Mn-induced neurotoxicity.

Yin Yang 1 is a repressor of glutamate transporter EAAT2, and it mediates manganese-induced decrease of EAAT2 expression in astrocytes.

Impairment of astrocytic glutamate transporter (GLT-1; EAAT2) function is associated with multiple neurodegenerative diseases, including Parkinson's disease (PD) and manganism, the latter being induced by chronic exposure to high levels of manganese (Mn). Mn decreases EAAT2 promoter activity and mRNA and protein levels, but the molecular mechanism of Mn-induced EAAT2 repression at the transcriptional level has yet to be elucidated. We reveal that transcription factor Yin Yang 1 (YY1) is critical in repressing EAAT2 and mediates the effects of negative regulators, such as Mn and tumor necrosis factor alpha (TNF-α), on EAAT2. YY1 overexpression in astrocytes reduced EAAT2 promoter activity, while YY1 knockdown or mutation of the YY1 consensus site of the EAAT2 promoter increased its promoter activity and attenuated the Mn-induced repression of EAAT2. Mn increased YY1 promoter activity and mRNA and protein levels via NF-κB activation. This led to increased YY1 binding to the EAAT2 promoter region. Epigenetically, histone deacetylase (HDAC) classes I and II served as corepressors of YY1, and, accordingly, HDAC inhibitors increased EAAT2 promoter activity and reversed the Mn-induced repression of EAAT2 promoter activity. Taken together, our findings suggest that YY1, with HDACs as corepressors, is a critical negative transcriptional regulator of EAAT2 and mediates Mn-induced EAAT2 repression.

Osteoblastogenesis and osteoprotection enhanced by flavonolignan silibinin in osteoblasts and osteoclasts.

Bone-remodeling imbalance induced by decreased osteoblastogenesis and increased bone resorption is known to cause skeletal diseases such as osteoporosis. Silibinin is the major active constituent of silymarin, the mixture of flavonolignans extracted from blessed milk thistle (Silybum marianum). Numerous studies suggest that silibinin is a powerful antioxidant and has anti-hepatotoxic properties and anti-cancer effects against carcinoma cells. This study investigated that silibinin had bone-forming and osteoprotective effects in in vitro cell systems of murine osteoblastic MC3T3-E1 cells and RAW 264.7 murine macrophages. MC3T3-E1 cells were incubated in osteogenic media in the presence of 1-20 µM silibinin up to 15 days. Silibinin accelerated cell proliferation and promoted matrix mineralization by enhancing bone nodule formation by calcium deposits. In addition, silibinin furthered the induction of osteoblastogenic biomarkers of alkaline phosphatase, collagen type 1, connective tissue growth factor, and bone morphogenetic protein-2. Differentiated MC3T3-E1 cells enhanced secretion of receptor activator of nuclear factor-κB ligand (RANKL) essential for osteoclastogenesis, which was reversed by silibinin. On the other hand, RAW 264.7 cells were pre-incubated with 1-20 µM silibinin for 5 days in the presence of RANKL. Non-toxic silibinin markedly attenuated RANK transcription and intracellular adhesion molecule-1 expression elevated by RANKL, thereby suppressing the differentiation of macrophages to multi-nucleated osteoclasts. It was also found that silibinin retarded tartrate-resistant acid phosphatase and cathepsin K induction and matrix metalloproteinase-9 activity elevated by RANKL through disturbing TRAF6-c-Src signaling pathways. These results demonstrate that silibinin was a potential therapeutic agent promoting bone-forming osteoblastogenesis and encumbering osteoclastic bone resorption.

Oleanolic acid reduces markers of differentiation in 3T3-L1 adipocytes.

Oleanolic acid is a triterpenoid compound that is widely present in vegetables, medicinal herbs, and other plants and has potent antioxidant and antiinflammatory properties. However, the potential of oleanolic acid to offset obesity is not clear. This study tested the hypothesis that oleanolic acid suppresses the differentiation of 3T3-L1 adipocytes by downregulating cellular induction of peroxisome proliferators-activated receptor γ (PPARγ) and cytidine-cytidine-adenosine-adenosine-thymidine (CCAAT) enhancer binding protein α (C/EBPα). The 3T3-L1 adipocytes were cultured and differentiated in Dulbecco modified Eagle medium containing 10% fetal bovine serum for 6 to 8 days in the absence and presence of 1 to 25 µmol/L oleanolic acid according to differentiating protocols. Nontoxic oleanolic acid, at 25 µmol/L or less, dose-dependently attenuated lipid accumulation in differentiated adipocytes as evidenced by Oil Red O staining. Western blot analysis showed that the induction of PPARγ and C/EBPα was markedly attenuated in differentiated and oleanolic acid-treated adipocytes at their transcriptional messenger RNA levels. Furthermore, this study examined whether oleanolic acid dampened the induction of visfatin, a proinflammatory and visceral fat-specific adipokine expressed in adipocytes. Visfatin expression was inhibited in differentiated adipocytes exposed to a PPARγ inhibitor GW9662. In addition, the visfatin production was significantly repressed in 25 µmol/L oleanolic acid-treated adipocytes, possibly through blocking PPARγ activation. These results demonstrate that oleanolic acid may be a promising agent to disturb adipocyte differentiation and suppress obesity-associated inflammation.

Fatty fish and fish omega-3 fatty acid intakes decrease the breast cancer risk: a case-control study.

BACKGROUND: Although it is believed that fish omega-3 fatty acids may decrease breast cancer risk, epidemiological evidence has been inconclusive. This study examined the association between fish and fish omega-3 fatty acids intake with the risk of breast cancer in a case-control study of Korean women. METHODS: We recruited 358 incident breast cancer patients and 360 controls with no history of malignant neoplasm from the National Cancer Center Hospital between July 2007 and April 2008. The study participants were given a 103-item food intake frequency questionnaire to determine their dietary consumption of fish (fatty and lean fish) and omega-3 fatty acids derived from fish (eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA)). RESULTS: Using a multivariate logistic regression model, high intake of fatty fish was associated with a reduced risk for breast cancer in both pre- and postmenopausal women (OR [95% CI] for highest vs. lowest intake quartiles, p for trend: 0.19 [0.08 to 0.45], p < 0.001 for premenopausal women, 0.27 [0.11 to 0.66], p = 0.005 for postmenopausal women). Similarly, reductions in breast cancer risk were observed among postmenopausal subjects who consumed more than 0.101 g of EPA (OR [95% CI]: 0.38 [0.15 to 0.96]) and 0.213 g of DHA (OR [95% CI]: 0.32 [0.13 to 0.82]) from fish per day compared to the reference group who consumed less than 0.014 g of EPA and 0.037 g of DHA per day. Among premenopausal women, there was a significant reduction in breast cancer risk for the highest intake quartiles of omega-3 fatty acids (ORs [95% CI]: 0.46 [0.22 to 0.96]), compared to the reference group who consumed the lowest quartile of intake. CONCLUSION: These results suggest that high consumption of fatty fish is associated with a reduced risk for breast cancer, and that the intake of omega-3 fatty acids from fish is inversely associated with postmenopausal breast cancer risk.