Element Consensus Sequence Research Articles

Type I Interferon Activates PD-1 Expression through Activation of the STAT1-IRF2 Pathway in Myeloid Cells.

PD-1 (Programmed cell death protein 1) regulates the metabolic reprogramming of myeloid-derived suppressor cells and myeloid cell differentiation, as well as the type I interferon (IFN-I) signaling pathway in myeloid cells in the tumor microenvironment. PD-1, therefore, is a key inhibitory receptor in myeloid cells. However, the regulation of PD-1 expression in myeloid cells is unknown. We report that the expression level of PDCD1, the gene that encodes the PD-1 protein, is positively correlated with the levels of IFNB1 and IFNAR1 in myeloid cells in human colorectal cancer. Treatment of mouse myeloid cell lines with recombinant IFNβ protein elevated PD-1 expression in myeloid cells in vitro. Knocking out IFNAR1, the gene that encodes the IFN-I-specific receptor, diminished the inductive effect of IFNβ on PD-1 expression in myeloid cells in vitro. Treatment of tumor-bearing mice with a lipid nanoparticle-encapsulated IFNβ-encoding plasmid (IFNBCOL01) increased IFNβ expression, resulting in elevated PD-1 expression in tumor-infiltrating myeloid cells. At the molecular level, we determined that IFNβ activates STAT1 (signal transducer and activator of transcription 1) and IRFs (interferon regulatory factors) in myeloid cells. Analysis of the cd279 promoter identified IRF2-binding consensus sequence elements. ChIP (chromatin immunoprecipitation) analysis determined that the pSTAT1 directly binds to the irf2 promoter and that IRF2 directly binds to the cd279 promoter in myeloid cells in vitro and in vivo. In colon cancer patients, the expression levels of STAT1, IRF2 and PDCD1 are positively correlated in tumor-infiltrating myeloid cells. Our findings determine that IFNβ activates PD-1 expression at least in part by an autocrine mechanism via the stimulation of the pSTAT1-IRF2 axis in myeloid cells.

GSK3β/NF-κB -dependent transcriptional regulation of homeostatic hepatocyte Tnf production.

Maintenance of hepatocyte homeostasis plays an important role in mediating the pathogenesis of many diseases. A growing body of literature has established a critical role played by tumor necrosis factor-α (TNFα) in maintaining hepatocyte homeostasis; however, the transcriptional mechanisms underlying constitutive Tnf expression are unknown. Whole liver fractions and primary hepatocytes from adult control C57BL/6 mice and the murine hepatocyte cell line AML12 were assessed for constitutive Tnf expression. Impacts of glycogen synthase kinase-3 β (GSK3β) and nuclear factor κB (NF-κB) inhibition on constitutive Tnf expression were assessed in AML12 cells. Finally, AML12 cell proliferation following GSK3β and NF-κB inhibition was evaluated. Constitutive Tnf gene expression is present in whole liver, primary hepatocytes, and cultured AML12 hepatocytes. Cytokine-induced Tnf gene expression is regulated by NF-κB activation. Pharmacological inhibition of GSK3β resulted in a time- and dose-dependent inhibition of Tnf gene expression. GSK3β inhibition decreased nuclear levels of the NF-κB subunits p65 and p50. We determined that NF-κB transcription factor subunit p65 binds to consensus sequence elements present in the murine TNFα promoter and inhibition of GSK3β decreases binding and subsequent Tnf expression. Finally, AML12 cell growth was significantly reduced following GSK3β and NF-κB inhibition. These results demonstrate that GSK3β and NF-κB are essential for mediating Tnf expression and constitutive hepatocyte cell growth. These findings add to a growing body of literature on TNFα mediated hepatocyte homeostasis and identify novel molecular mechanisms involved in mediating response to various disease states in the liver.NEW & NOTEWORTHY Maintenance of hepatocyte homeostasis plays an important role in controlling the pathogenesis of many diseases. Our findings add to a growing body of literature on tumor necrosis factor-α (TNFα)-mediated hepatocyte homeostasis and identify novel molecular mechanisms involved in regulating this response.

Innate Immune Zonation in the Liver: NF-κB (p50) Activation and C-Reactive Protein Expression in Response to Endotoxemia Are Zone Specific.

Hepatic innate immune function plays an important role in the pathogenesis of many diseases. Importantly, a growing body of literature has firmly established the spatial heterogeneity of hepatocyte metabolic function; however, whether innate immune function is zonated remains unknown. To test this question, we exposed adult C57BL/6 mice to endotoxemia, and hepatic tissue was assessed for the acute phase response (APR). The zone-specific APR was evaluated in periportal and pericentral/centrilobular hepatocytes isolated using digitonin perfusion and on hepatic tissue using RNAscope and immunohistochemistry. Western blot, EMSA, chromatin immunoprecipitation, and immunohistochemistry were used to determine the role of the transcription factor NF-κB in mediating hepatic C-reactive protein (CRP) expression. Finally, the ability of mice lacking the NF-κB subunit p50 (p50-/-) to raise a hepatic APR was evaluated. We found that endotoxemia induces a hepatocyte transcriptional APR in both male and female mice, with Crp, Apcs, Fga, Hp, and Lbp expression being enriched in pericentral/centrilobular hepatocytes. Focusing our work on CRP expression, we determined that NF-κB transcription factor subunit p50 binds to consensus sequence elements present in the murine CRP promoter. Furthermore, pericentral/centrilobular hepatocyte p50 nuclear translocation is temporally associated with zone-specific APR during endotoxemia. Lastly, the APR and CRP expression is blunted in endotoxemic p50-/- mice. These results demonstrate that the murine hepatocyte innate immune response to endotoxemia includes zone-specific activation of transcription factors and target gene expression. These results support further study of zone-specific hepatocyte innate immunity and its role in the development of various disease states.

The Role of the NRF2 Pathway in Maintaining and Improving Cognitive Function.

Nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a redox-sensitive transcription factor that binds to the antioxidant response element consensus sequence, decreasing reactive oxygen species and regulating the transcription of a wide array of genes, including antioxidant and detoxifying enzymes, regulating genes involved in mitochondrial function and biogenesis. Moreover, NRF2 has been shown to directly regulate the expression of anti-inflammatory mediators reducing the expression of pro-inflammatory cytokines. In recent years, attention has turned to the role NRF2 plays in the brain in different diseases such Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and others. This review focused on the evidence, derived in vitro, in vivo and from clinical trials, supporting a role for NRF2 activation in maintaining and improving cognitive function and how its activation can be used to elicit neuroprotection and lead to cognitive enhancement. The review also brings a critical discussion concerning the possible prophylactic and/or therapeutic use of NRF2 activators in treating cognitive impairment-related conditions.

Repetitive DNA symmetry elements negatively regulate gene expression in embryonic stem cells

Transcription factor (TF) binding to genomic DNA elements constitutes one of the key mechanisms that regulates gene expression program in cells. Both consensus and nonconsensus DNA sequence elements influence the recognition specificity of TFs. Based on the analysis of experimentally determined c-Myc binding preferences to genomic DNA, here we statistically predict that certain repetitive, nonconsensus DNA symmetry elements can relatively reduce TF-DNA binding preferences. This is in contrast to a different set of repetitive, nonconsensus symmetry elements that can increase the strength of TF-DNA binding. Using c-Myc enhancer reporter system containing consensus motif flanked by nonconsensus sequences in embryonic stem cells, we directly demonstrate that the enrichment in such negatively regulating repetitive symmetry elements is sufficient to reduce the gene expression level compared with native genomic sequences. Negatively regulating repetitive symmetry elements around consensus c-Myc motif and DNA sequences containing consensus c-Myc motif flanked by entirely randomized sequences show similar expression baseline. A possible explanation for this observation is that rather than complete repression, negatively regulating repetitive symmetry elements play a regulatory role in fine-tuning the reduction of gene expression, most probably by binding TFs other than c-Myc.

Anatomy and evolution of a DNA replication origin.

DNA amplification occurs at the DNA puff II/9A locus in the fungus fly Sciara coprophila. As a foundation to study the molecular mechanism for the initiating events of II/9A DNA re-replication, we have sequenced 14kb spanning a DNase hypersensitive site (DHS) upstream of the 1kb amplification origin and through transcription units II/9-1 and II/9-2 downstream of the origin. These elements are annotated as well as the ORC binding site at the origin and the transition point (TP) between continuous and discontinuous DNA syntheses that marks the origin of bidirectional replication at the nucleotide level. A 9bp motif found at the TP is repeated near the other end of the 1 kb ORI and may identify a putative second TP. The steroid hormone ecdysone induces DNA amplification as well as transcription and puffing at locus II/9A. Within the 14 kb, several matches to the ecdysone response element (EcRE) consensus sequence were identified, including some in the amplification origin region. EcRE O-P is at a central axis of a remarkable symmetry, equidistant to the TPs that are themselves equidistant to EcRE O-1 and EcRE O-2. DNA sequence alterations have occurred throughout the II/9A region in a newly discovered polymorphism (#2). Polymorphism #2 is not specific to developmental stage, sex, or tissue, and it does not impair DNA amplification. The DHS, both 9bp TP sequences, and EcREs O-1, O-P, and O-2 are conserved between the polymorphism #1 and #2 sequences, suggesting their functional importance and retention during evolutionary selection. Moreover, a 72 bp sequence in the Sciara DHS at DNA puff II/9A is conserved in DNA puff C-3 of Rhynchosciara americana. Comparisons are discussed between the Sciara II/9A amplicon and the chorion locus amplicon on the third chromosome of Drosophila.

The Role of Cytoplasmic Polyadenylation Element Binding Protein 2 Alternative Splicing in Chronic Hypoxia

Cytosolic polyadenylation element binding (CPEB) proteins are established as mRNA binding proteins that, upon interacting with a 3 ‘UTR consensus sequence, regulate the translation of their mRNA targets. Moreover, the stress-induced CPEB family member, CPEB2, has been shown to interact with the oxygen-dependent subunit of the HIF1 (hypoxia-inducible factor 1) transcription factor, HIF1a, in order to repress its translation. However, our lab has previously demonstrated in cancer cells that alternatively spliced isoforms of CPEB2 regulate the translation of HIF1a in opposing fashions: the CPEB2A isoform represses its translation, while the CPEB2B isoform activates its translation. Although it is well established that during hypoxia, HIF1α levels are initially upregulated due to a decrease in proteasomal degradation, we hypothesize that during chronic hypoxia, the expression of HIF1α is maintained through a stress-induced translational mechanism, likely alongside a decrease in proteolytic activity. In this study we demonstrate that targeting the CPEB2B splice isoform via antisense oligonucleotides has an inhibitory effect on the translation of nascent HIF1α protein during chronic hypoxia, but not during acute hypoxia exposure, and that this this regulatory pathway is dependent on the initiation factor eIF3H. We further demonstrate that CPEB2A and CPEB2B bind differentially to cytoplasmic polyadenylation element consensus sequences, which may depend on surrounding sequence context that can be detected through RNA foot-printing assays. Finally, we characterize how CPEB2A and B expression impacts new blood vessel growth in a 3D vasculature cell model. These findings are important, since they provide evidence that this splicing event could potentially act as a therapeutic target for treating chronic hypoxia-related pulmonary diseases.

Ori-Finder 3: a web server for genome-wide prediction of replication origins in Saccharomyces cerevisiae.

DNA replication is a fundamental process in all organisms; this event initiates at sites termed origins of replication. The characteristics of eukaryotic replication origins are best understood in Saccharomyces cerevisiae. For this species, origin prediction algorithms or web servers have been developed based on the sequence features of autonomously replicating sequences (ARSs). However, their performances are far from satisfactory. By utilizing the Z-curve methodology, we present a novel pipeline, Ori-Finder 3, for the computational prediction of replication origins in S. cerevisiae at the genome-wide level based solely on DNA sequences. The ARS exhibiting both an AT-rich stretch and ARS consensus sequence element can be predicted at the single-nucleotide level. For the identified ARSs in the S. cerevisiae reference genome, 83 and 60% of the top 100 and top 300 predictions matched the known ARS records, respectively. Based on Ori-Finder 3, we subsequently built a database of the predicted ARSs identified in more than a hundred S. cerevisiae genomes. Consequently, we developed a user-friendly web server including the ARS prediction pipeline and the predicted ARSs database, which can be freely accessed at http://tubic.tju.edu.cn/Ori-Finder3.

XACT-Seq Comprehensively Defines the Promoter-Position and Promoter-Sequence Determinants for Initial-Transcription Pausing

Pausing by RNA polymerase (RNAP) during transcription elongation, in which a translocating RNAP uses a "stepping" mechanism, has been studied extensively, but pausing by RNAP during initial transcription, in which a promoter-anchored RNAP uses a "scrunching" mechanism, has not. We report a method that directly defines the RNAP-active-center position relative to DNA with single-nucleotide resolution (XACT-seq; "crosslink-between-active-center-and-template sequencing"). We apply this method to detect and quantify pausing in initial transcription at 411 (∼4,000,000) promoter sequences invivo in Escherichia coli. The results show initial-transcription pausing can occur in each nucleotide addition during initial transcription, particularly the first 4 to 5 nucleotide additions. The results further show initial-transcription pausing occurs at sequences that resemble the consensus sequence element for transcription-elongation pausing. Our findings define the positional and sequence determinants for initial-transcription pausing and establish initial-transcription pausing is hard coded by sequence elements similar to those for transcription-elongation pausing.

Ajoene, a Major Organosulfide Found in Crushed Garlic, Induces NAD(P)H:quinone Oxidoreductase Expression Through Nuclear Factor E2-related Factor-2 Activation in Human Breast Epithelial Cells

BackgroundNAD(P)H:quinone oxidoreductase-1 (NQO1) is a widely-distributed flavin adenine dinucleotide-dependent flavoprotein that promotes obligatory 2-electron reductions of quinones, quinoneimines, nitroaromatics, and azo dyes. This reduces quinone levels and thereby minimizes generation of excess reactive oxygen species (ROS) formed by redox cycling, and concurrent depletion of intracellular thiol pools. Ajoene is derived from crushed garlic. It is formed by a reaction involving two allicin molecules, and is composed of allyl sulfide and vinyl disulfide. Ajoene is present in two isomers, E- and Z-form.MethodsExpression of antioxidant enzymes and nuclear factor E2-related factor-2 (Nrf2) was measured by Western blot analysis. NQO1 promoter activity was assessed by the luciferase reporter gene assay. ROS accumulation was monitored by using the fluorescence-generating probe 2′,7′-dichlorofluorescein diacetate. The intracellular glutathione levels were measured by using a commercially available kit.ResultsZ-ajoene significantly up-regulated the expression of representative antioxidant enzyme NQO1 in non-tumorigenic breast epithelial MCF-10A cells at non-toxic concentrations. Z-ajoene enhanced up-regulation and nuclear translocation of Nrf2, which plays a pivotal role in the induction of many genes encoding antioxidant enzymes and other cytoprotective proteins. Z-ajoene treatment also increased the activity of nqo1-promoter harboring antioxidant response element consensus sequences in MCF-10A cells. Silencing of Nrf2 by small interfering RNA abrogated ajoene-induced expression of NQO1. Z-ajoene activated extracellular signal-regulated kinase (ERK). Inhibition of ERK activation by U0126 abrogated ability of Z-ajoene to activate Nrf2 and to induce NQO1 expression. Intracellular ROS accumulation was observed after treatment with Z-ajoene, whereas the E-isoform was not effective. The inhibition of ROS by treatment with N-acetylcysteine, a radical scavenger, abrogated Z-ajoene-induced expression of NQO1 as well as activation of ERK and Nrf2, suggesting that Z-ajoene augments the Nrf2-dependent antioxidant defense via ROS generation and ERK activation.ConclusionsZ-ajoene induces NQO1 expression in MCF-10A cells through ROS-mediated activation of Nrf2.

Programmable mutually exclusive alternative splicing for generating RNA and protein diversity

Alternative splicing performs a central role in expanding genomic coding capacity and proteomic diversity. However, programming of splicing patterns in engineered biological systems remains underused. Synthetic approaches thus far have predominantly focused on controlling expression of a single protein through alternative splicing. Here, we describe a modular and extensible platform for regulating four programmable exons that undergo a mutually exclusive alternative splicing event to generate multiple functionally-distinct proteins. We present an intron framework that enforces the mutual exclusivity of two internal exons and demonstrate a graded series of consensus sequence elements of varying strengths that set the ratio of two mutually exclusive isoforms. We apply this framework to program the DNA-binding domains of modular transcription factors to differentially control downstream gene activation. This splicing platform advances an approach for generating diverse isoforms and can ultimately be applied to program modular proteins and increase coding capacity of synthetic biological systems.

Ferulic acid improves self-renewal and differentiation of human tendon-derived stem cells by upregulating early growth response 1 through hypoxia.

We aimed to investigate the potential beneficial effect of ferulic acid (FA) on stemness of human tendon-derived stem cells (hTSCs) in vitro and to elucidate the underlying molecular mechanism. The self-renewal ability of hTSCs was evaluated by colony formation and cell proliferation was determined by CCK-8 kit. Adipogenesis, osteogenesis, and chondrogenesis were determined by Oil Red O, Alizarin Red, and Alcian Blue stainings, respectively. Relative mRNA levels of PPARγ, Col2A1, Acan, Runx2, HIF1α, and EGR1 were measured with real-time PCR. Protein levels of HIF1α and EGR1 were detected by western blot. Direct binding of HIF1α with EGR1 promoter was analyzed by ChIP assay. Hypoxia-induced expression of EGR1 was interrogated by luciferase reporter assay. We demonstrated that FA treatment improved both self-renewal ability and multi-differentiation potential of hTSCs. FA induced hypoxia which in turn upregulated EGR1 expression via direct association with its hypoxia response element consensus sequence. Furthermore, we showed that both HIF1α and EGR1 were required for the enhancing effects of FA on hTSC self-renewal and differentiation. We hereby characterize the beneficial effect of FA on the stemness of hTSCs and highlight the critical role of HIF1α-EGR1 axis in this process.

Significant antitumor effect of an antibody against TMEM180, a new colorectal cancer-specific molecule.

The present state of therapy for colorectal cancer (CRC) is far from satisfactory, highlighting the need for new targets for this disease. We identified a new CRC‐specific molecule, TMEM180, a predicted 11‐pass transmembrane protein that apparently functions as a cation symporter. We developed an anti‐TMEM180 mAb and then succeeded in humanizing the mAb. Immunohistochemistry (IHC) in CRC with the mAb showed a similar positivity rate as compared with anti‐epidermal growth factor receptor mAb, and IHC with anti‐TMEM180 mAb did not show staining in major organs used in this study. Immune electron microscopy clearly indicated that TMEM180 was present on the tumor exosome. The TMEM180 promoter region contains 10 hypoxia‐responsive element consensus sequences; accordingly, SW480 cells upregulated TMEM180 under low‐oxygen conditions. Anti‐TMEM180 mAb has in vitro antibody‐dependent cell‐mediated cytotoxicity and complement‐dependent cytotoxicity activity, and SW480 CRC xenografts were eradicated by the mAb. These data indicate that TMEM180 may be a new CRC marker and that a mAb against this protein could be used as antibody‐based therapy against CRC.

Development of an Inducible Secretory Expression System in Bacillus licheniformis Based on an Engineered Xylose Operon.

The xylose operon can be an efficient biological component for regulatory expression uses in Bacillus licheniformis. However, its characteristic susceptibility to carbon catabolite repression (CCR) makes its application inconvenient. In this study, plasmids harboring the wild-type operons from three Bacillus species were constructed and introduced into B. licheniformis. These plasmids ensured secretory expression of maltogenic α-amylase (BLMA) in B. licheniformis under strict regulation. The glucose-mediated CCR was then alleviated by engineering the xylose operon of the expression system. Evidence showed that mutations in the highly conserved nucleotides of the identified catabolite responsive element (cre) consensus sequence prevented association of the regulator CcpA with DNA, thus resulting in an increase in BLMA activity of up to 12-fold. Furthermore, features of this engineered system for inducible expression were investigated. Induction in mid-log phase using 10 g/L xylose at 37 °C was found to be beneficial for promoting the accumulation of recombinant product, and the maximum yield of BlmMA reached 715.4 U/mL. This study contributes to the industrial application of the generally recognized as safe (GRAS) workhorse B. licheniformis.

Abstract 1716: The PARP inhibitor rucaparib activates the STING pathway and enhances antitumor responses of immune checkpoint inhibitors in BRCA deficient syngeneic models

Abstract Background: The poly(ADP ribose) polymerase (PARP) inhibitor rucaparib effectively kills homologous recombination (HR) deficient cells through impeding DNA repair that leads to DNA damage, apoptosis, and cell death. Detection of cytosolic DNA by the stimulator of interferon genes (STING) pathway mediates type I interferon (IFN) production and activates the immune system. Following rucaparib treatment, the accumulation of damaged DNA in HR impaired tumors may elicit an immune response through STING signaling, and enhance rucaparib activity as a single agent or in combination with immune checkpoint blockade. To test this hypothesis, rucaparib efficacy and mechanism of action were evaluated using BRCA deficient syngeneic ovarian tumor models. Results: Single agent rucaparib showed potent antitumor activity in the BRCAmut BrKras and ID8B3.15 models. In the BrKras model, rucaparib treatment resulted in complete regression and prevented tumor formation upon re-challenge. However, this efficacy was abolished with anti-CD8 but not anti-CD4 depletion. CD8 tumor infiltrating lymphocytes (TILs) but not CD4 TILs increased with rucaparib exposure, and expression profiling of rucaparib treated tumors showed activation of the type I IFN pathway. In vitro PCR assays identified several targets that were upregulated upon rucaparib treatment including Ccl5 and Cxcl10. Notably, increased Ccl5 and Cxcl10 levels were observed in BRCAmut cells but not in BRCAwt cells, and knockdown of the STING pathway genes MB21D1, IRF3, TBK1, and STING (TMEM173) abrogated rucaparib induction of Ccl5 and Cxcl10. Furthermore, rucaparib showed transcriptional activation of IFN type I signaling in BRCAmut reporter cells expressing the IFN stimulated response element consensus sequence driving luciferase expression, but required 6-fold higher concentrations in BRCAwt reporter cells. Similarly, a 10-fold higher rucaparib dose was needed to inhibit proliferation of BRCAwt cells compared to BRCAmut cells in a cell viability assay. Consistent with the in vitro results, rucaparib combined with anti-programmed death 1 or anti-programmed death ligand 1 therapy improved the survival and augmented antitumor responses in BRCA deficient syngeneic tumor models. Conclusions: Rucaparib treatment in HR deficient cells, and at a higher concentration in HR proficient cells, triggers type I IFN signaling through the STING pathway, which participates in the single agent efficacy of rucaparib and enhances the combination of rucaparib and checkpoint inhibitors in syngeneic models. These findings provide further evidence supporting the rationale for combining rucaparib with checkpoint therapy for the treatment of patients with HR defective cancers. Citation Format: Minh Nguyen, Liliane Robillard, Kevin K. Lin, Thomas C. Harding, Andrew D. Simmons. The PARP inhibitor rucaparib activates the STING pathway and enhances antitumor responses of immune checkpoint inhibitors in BRCA deficient syngeneic models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1716.

Targeting transcription factors in acute myeloid leukemia.

Transcription factors recognize and bind to consensus sequence elements that are specific for each transcription factor, and the transcription factors then regulate downstream gene expression. In the bone marrow, transcription factors, such as C/EBPα, PU.1, and RUNX1, control essential genes to maintain the normal hematopoietic system. Dysregulation of transcription factors caused by gene mutations, chromosomal aberrations, or aberrant expression can lead to cancer, including acute myeloid leukemia. In the past, transcription factors were not considered "druggable" targets. However, a better understanding of the pathology of malignant tumors and mechanisms of transcriptional regulation has enabled us to develop novel therapeutic strategies that target transcription factors. In this review, we focus on transcription factors that play important roles in leukemogenesis and current efforts and prospects in the development of transcriptional therapy. We believe that such a therapeutic approach will benefit patients with cancers that involve acute myeloid leukemia in the near future.

Commentary: A Possible Mechanism of Zika Virus Associated Microcephaly: Imperative Role of Retinoic Acid Response Element (RARE) Consensus Sequence Repeats in the Viral Genome.

Commentary: A Possible Mechanism of Zika Virus Associated Microcephaly: Imperative Role of Retinoic Acid Response Element (RARE) Consensus Sequence Repeats in the Viral Genome.

Celastrol improves self-renewal and differentiation of human tendon-derived stem cells by suppressing Smad7 through hypoxia

BackgroundWe aimed to evaluate the potential enhancing effect of celastrol on the stemness of human tendon-derived stem cells (hTSCs) in vitro and the underlying molecular mechanisms.MethodsThe capability of hTSC self-renewal was assessed by cell proliferation and colony formation as determined with the CCK-8 kit. Adipogenesis, chondrogenesis, and osteogenesis were determined by Oil Red O, Alcian Blue, and Alizarin Red staining, respectively. The relative mRNA levels of Sox9, PPARγ, Runx2, Smad7, and HIF1α were determined by real-time polymerase chain reaction (PCR). The levels of Smad7 and HIF1α protein were measured by immunoblotting. The chromatin immunoprecipitation (ChIP) assay was used to assess the direct binding of HIF1α to the Smad7 promoter. Suppression of Smad7 induced by hypoxia was examined using the luciferase reporter assay.ResultsWe found that treatment with celastrol resulted in improvement in both the multi-differentiation potential and self-renewal capability of hTSCs. Celastrol elicited hypoxia and subsequently suppressed the expression of Smad7 through direct association with the hypoxia response element consensus sequence. Further, we demonstrated that both Smad7 and HIF1α were involved in the beneficial effects of celastrol on the differentiation and self-renewal of hTSCs.ConclusionsWe demonstrated the positive effect of celastrol on the stemness of hTSCs and elucidated the essential role of the HIF1α-Smad7 pathway in this process.

Hepatic Lipid Accumulation and Nrf2 Expression following Perinatal and Peripubertal Exposure to Bisphenol A in a Mouse Model of Nonalcoholic Liver Disease.

Background:Exposure to chemicals during critical windows of development may re-program liver for increased risk of nonalcoholic fatty liver disease (NAFLD). Bisphenol A (BPA), a plastics component, has been described to impart adverse effects during gestational and lactational exposure. Our work has pointed to nuclear factor E2-related factor 2 (Nrf2) being a modulator of hepatic lipid accumulation in models of NAFLD.Objectives:To determine if chemical exposure can prime liver for steatosis via modulation of NRF2 and epigenetic mechanisms.Methods:Utilizing BPA as a model exposure, pregnant CD-1 mice were administered BPA via osmotic minipumps from gestational day 8 through postnatal day (PND)16. The offspring were weaned on PND21 and exposed to same dose of BPA via their drinking water through PND35. Tissues were collected from pups at week 5 (W5), and their littermates at week 39 (W39).Results:BPA increased hepatic lipid content concomitant with increased Nrf2 and pro-lipogenic enzyme expression at W5 and W39 in female offspring. BPA exposure increased Nrf2 binding to a putative antioxidant response element consensus sequence in the sterol regulatory-element binding protein-1c (Srebp-1c) promoter. Known Nrf2 activators increased SREBP-1C promoter reporter activity in HepG2 cells. Methylated DNA immunoprecipitation-PCR and pyrosequencing revealed that developmental BPA exposure induced hypomethylation of the Nrf2 and Srebp-1c promoters in livers of W5 mice, which was more prominent in W39 mice than in others.Conclusion:Exposure to a xenobiotic during early development induced persistent fat accumulation via hypomethylation of lipogenic genes. Moreover, increased Nrf2 recruitment to the Srebp-1c promoter in livers of BPA-exposed mice was observed. Overall, the underlying mechanisms described a broader impact beyond BPA exposure and can be applied to understand other models of NAFLD. https://doi.org/10.1289/EHP664

SG2-Type R2R3-MYB Transcription Factor MYB15 Controls Defense-Induced Lignification and Basal Immunity in Arabidopsis.

Lignification of cell wall appositions is a conserved basal defense mechanism in the plant innate immune response. However, the genetic pathway controlling defense-induced lignification remains unknown. Here, we demonstrate the Arabidopsis thaliana SG2-type R2R3-MYB transcription factor MYB15 as a regulator of defense-induced lignification and basal immunity. Loss of MYB15 reduces the content but not the composition of defense-induced lignin, whereas constitutive expression of MYB15 increases lignin content independently of immune activation. Comparative transcriptional and metabolomics analyses implicate MYB15 as necessary for the defense-induced synthesis of guaiacyl lignin and the basal synthesis of the coumarin metabolite scopoletin. MYB15 directly binds to the secondary wall MYB-responsive element consensus sequence, which encompasses the AC elements, to drive lignification. The myb15 and lignin biosynthetic mutants show increased susceptibility to the bacterial pathogen Pseudomonas syringae, consistent with defense-induced lignin having a major role in basal immunity. A scopoletin biosynthetic mutant also shows increased susceptibility independently of immune activation, consistent with a role in preformed defense. Our results support a role for phenylalanine-derived small molecules in preformed and inducible Arabidopsis defense, a role previously dominated by tryptophan-derived small molecules. Understanding the regulatory network linking lignin biosynthesis to plant growth and defense will help lignin engineering efforts to improve the production of biofuels and aromatic industrial products as well as increase disease resistance in energy and agricultural crops.