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1.
Genes (Basel) ; 14(7)2023 07 16.
Article in English | MEDLINE | ID: mdl-37510359

ABSTRACT

Bisphenols are environmental toxins with endocrine disruptor activity, yet bisphenol A (BPA) and its analogs are still widely used in manufacturing plastic products. There is evidence showing that BPA elicits inflammation in humans and animals, but the target cell types of BPA are not well understood. In this study, we sought to determine BPA's direct effect on macrophages and BPA immunotoxicity in mouse intestine. Ghrelin is an important nutrient-sensing hormone, acting through its receptor growth hormone secretagogue receptor (GHSR) to regulate metabolism and inflammation. We found that BPA promotes intestinal inflammation, showing increased infiltrating immune cells in colons and enhanced expression of Ghsr and pro-inflammatory cytokines and chemokines, such as Il6 and Ccl2, in colonic mucosa. Moreover, we found that both long- and short-term BPA exposure elevated pro-inflammatory monocytes and macrophages in mouse peripheral blood mononuclear cells (PBMC) and peritoneal macrophages (PM), respectively. To determine the role of GHSR in BPA-mediated inflammation, we generated Ghsr deletion mutation in murine macrophage RAW264.7 using CRISPR gene editing. In wild-type RAW264.7 cells, the BPA exposure promotes macrophage pro-inflammatory polarization and increases Ghsr and cytokine/chemokine Il6 and Ccl2 expression. Interestingly, Ghsr deletion mutants showed a marked reduction in pro-inflammatory cytokine/chemokine expression in response to BPA, suggesting that GHSR is required for the BPA-induced pro-inflammatory response. Further understanding how nutrient-sensing GHSR signaling regulates BPA intestinal immunotoxicity will help design new strategies to mitigate BPA immunotoxicity and provide policy guidance for BPA biosafety.


Subject(s)
Leukocytes, Mononuclear , Receptors, Ghrelin , Animals , Mice , Chemokines , Cytokines/genetics , Cytokines/metabolism , Inflammation/chemically induced , Interleukin-6/genetics , Leukocytes, Mononuclear/metabolism , Macrophages/metabolism , Nutrients , Receptors, Ghrelin/genetics , Receptors, Ghrelin/metabolism
2.
Redox Biol ; 54: 102377, 2022 08.
Article in English | MEDLINE | ID: mdl-35763934

ABSTRACT

The metastasis-associated lung adenocarcinoma transcript1 (MALAT1) is a long noncoding RNA (lncRNA) and is known for its role in cancer development and prognosis. In this study, we report that MALAT1 plays an important role in regulating acute inflammatory responses in sepsis. In patient samples, MALAT1 expression was positively correlated with severity of sepsis. In cultured macrophages, LPS treatment significantly induced MALAT1 expression, while genetic ablation of MALAT1 greatly reduced proinflammatory cytokine levels. Furthermore, MALAT1-ablated mice had significantly increased survival rates in cecal ligation and puncture (CLP)-induced sepsis and LPS-induced endotoxemia. One novel and salient feature of MALAT1-ablated mice is greatly reduced ROS level in macrophages and other cell types and increased glutathione/oxidized glutathione (GSH/GSSG) ratio in macrophages, suggesting an increased antioxidant capacity. We showed a mechanism for MALAT1 ablation leading to enhanced antioxidant capacity is through activation of methionine cycle by epitranscriptomical regulation of methionine adenosyltransferase 2A (MAT2A). MAT2A 3'UTR can be methylated by METTL16 which was known to directly bind to MALAT1. MALAT1 ablation was found to reduce methylation in MAT2A hairpin1 and increase MAT2A protein levels. Our results suggest a MALAT1-METTL16-MAT2A interactive axis which may be targeted for treatments of sepsis.


Subject(s)
Adenocarcinoma , MicroRNAs , RNA, Long Noncoding/genetics , Sepsis , Animals , Antioxidants , Lipopolysaccharides , Mice , MicroRNAs/genetics , Sepsis/metabolism
3.
J Vis Exp ; (136)2018 06 25.
Article in English | MEDLINE | ID: mdl-29985354

ABSTRACT

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic ß cells which will lead to the development of type 2 diabetes mellitus (T2DM). Although the mechanisms for the chemical-induced ß cell damage were unclear and likely to be complex, one recurring finding is that these chemicals induce oxidative stress leading to the generation of excessive reactive oxygen species (ROS) which induce damage to the ß cell. To identify potential diabetogenic environmental chemicals, we isolated pancreatic islet cells from C57BL/6 mice and cultured islet cells in 96-well cell culture plates; then, the islet cells were dosed with chemicals and the ROS generation was detected by 2',7'-dichlorofluorescein (DCFH-DA) fluorescent dye. Using this method, we found that bisphenol A (BPA), Benzo[a]pyrene (BaP), and polychlorinated biphenyls (PCBs), could induce high levels of ROS, suggesting that they may potentially induce damage in islet cells. This method should be useful for screening diabetogenic xenobiotics. In addition, the cultured islet cells may also be adapted for in vitro analysis of chemical-induced toxicity in pancreatic cells.


Subject(s)
Diabetes Mellitus, Type 2/diagnosis , Environmental Exposure/adverse effects , Environmental Pollutants/toxicity , Insulin-Secreting Cells/metabolism , Animals , Humans , Insulin-Secreting Cells/pathology , Mice , Mice, Inbred C57BL , Reactive Oxygen Species , Xenobiotics
4.
Biochem Pharmacol ; 152: 94-103, 2018 06.
Article in English | MEDLINE | ID: mdl-29577871

ABSTRACT

The metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a long noncoding RNA and its overexpression is associated with the development of many types of malignancy. MALAT1 null mice show no overt phenotype. However, in transcriptome analysis of MALAT1 null mice we found significant upregulation of nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) regulated antioxidant genes including Nqo1 and Cat with significant reduction in reactive oxygen species (ROS) and greatly reduced ROS-generated protein carbonylation in hepatocyte and islets. We performed lncRNA pulldown assay using biotinylated antisense oligonucleotides against MALAT1 and found MALAT1 interacted with Nrf2, suggesting Nrf2 is transcriptionally regulated by MALAT1. Exposure to excessive ROS has been shown to cause insulin resistance through activation of c-Jun N-terminal kinase (JNK) which leads to inhibition of insulin receptor substrate 1 (IRS-1) and insulin-induced phosphorylation of serine/threonine kinase Akt. We found MALAT1 ablation suppressed JNK activity with concomitant insulin-induced activation of IRS-1 and phosphorylation of Akt suggesting MALAT1 regulated insulin responses. MALAT1 null mice exhibited sensitized insulin-signaling response to fast-refeeding and glucose/insulin challenges and significantly increased insulin secretion in response to glucose challenge in isolated MALAT1 null islets, suggesting an increased insulin sensitivity. In summary, we demonstrate that MALAT1 plays an important role in regulating insulin sensitivity and has the potential as a therapeutic target for the treatment of diabetes as well as other diseases caused by excessive exposure to ROS.


Subject(s)
Insulin/pharmacology , RNA, Long Noncoding/metabolism , Reactive Oxygen Species/metabolism , Animals , Gene Expression Regulation/drug effects , Male , Mice , Mice, Knockout , Oxidative Stress , Protein Carbonylation , RNA, Long Noncoding/genetics , Signal Transduction
5.
PLoS One ; 13(2): e0192264, 2018.
Article in English | MEDLINE | ID: mdl-29389953

ABSTRACT

Metastasis-associated lung adenocarcinoma transcript-1 (MALAT-1) is a long non-coding RNA (lncRNA) that is a negative prognostic factor for patients with pancreatic cancer and several other tumors. In this study, we show that knockdown of MALAT-1 in Panc1 and other pancreatic cancer cell lines decreases cell proliferation, survival and migration. We previously observed similar results for the lncRNAs HOTTIP and HOTAIR in Panc1 cells; however, RNAseq comparison of genes regulated by MALAT-1 shows minimal overlap with HOTTIP/HOTAIR-regulated genes. Analysis of changes in gene expression after MALAT-1 knockdown shows that this lncRNA represses several tumor suppressor-like genes including N-myc downregulated gene-1 (NDRG-1), a tumor suppressor in pancreatic cancer that is also corepressed by EZH2 (a PRC2 complex member). We also observed that Specificity proteins Sp1, Sp3 and Sp4 are overexpressed in Panc1 cells and Sp knockdown or treatment with small molecules that decrease Sp proteins expression also decrease MALAT-1 expression. We also generated Kras-overexpressing p53L/L;LSL-KrasG12DL/+;p48Cre+/- (p53L/L/KrasG12D) and p53L/+;LSLKrasG12DL/+;p48Cre+/- (p53L/+/KrasG12D) mice which are p53 homo- and heterozygous, respectively. These mice rapidly develop pancreatic ductal adenocarcinoma-like tumors and were crossed with MALAT-1-/- mice. We observed that the loss of one or two MALAT-1 alleles in these Ras overexpressing mice does not significantly affect the time to death; however, the loss of MALAT-1 in the p53-/+ (heterozygote) mice slightly increases their lifespan.


Subject(s)
Biomarkers, Tumor/genetics , Pancreatic Neoplasms/pathology , RNA, Long Noncoding/genetics , Animals , Cell Line, Tumor , Down-Regulation , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Humans , Mice , Mice, Transgenic , Pancreatic Neoplasms/genetics
6.
Toxicol Lett ; 275: 67-76, 2017 Jun 05.
Article in English | MEDLINE | ID: mdl-28428138

ABSTRACT

Pregnane X receptor (PXR) plays an important role in protecting cells from mutagenic DNA damages induced by endogenous and exogenous toxicants. This protective function is often attributed to the PXR-regulated metabolic detoxification. Here we report a novel potential mechanism that PXR reduces benzo-[α]-pyrene(BaP)-induced DNA damage through inhibiting the transcriptional activity of aryl hydrocarbon receptor (AhR) which plays a pivotal role in the bioactivation of BaP. We have utilized three well-characterized cell lines, i.e. Hepa1c1c7, AhR +/+; Bpr lacks AhR obligatory partner ARNT; Tao, lacks AhR, to analyze pivotal role of AhR/ARNT complex in mediating the BaP-induced DNA damages using comet assay (single-cell gel electrophoresis). We found that PXR activation could significantly inhibit BaP-induced DNA damage in the HepG2 cells as well as mouse hepatocytes. Using PXR-null and wild type mouse hepatocytes we showed that PXR activation by pregnenolone 16α-carbonitrile (PCN) significantly inhibited BaP-induced DNA damage and this protective effect was abolished in PXR-null hepatocytes. Mechanistically, PXR activation inhibited expression of AhR-target genes for CYP1A1, CYP1B1 and CYP1A2 that are required for BaP biotransformation in cultured liver cells, or in the livers of C57BL/6J mice. Using an AhR-responsive reporter assay as well as chromatin immunoprecipitation assay we found that PXR activation transcriptionally represses AhR-regulated gene expression. Furthermore, we found that PXR directly bound AhR at its DNA-binding domain, and this association may play a role in preventing of the AhR from binding to its target genes as shown in the ChIP assay. Taken together, our study has revealed a novel mechanism by which PXR protects liver cells from BaP-induced DNA damage through inhibiting the BaP biotransformation.


Subject(s)
Benzo(a)pyrene/toxicity , Cytochrome P-450 CYP1A1/metabolism , DNA Damage , Liver/drug effects , Receptors, Aryl Hydrocarbon/metabolism , Receptors, Steroid/metabolism , Animals , Cell Culture Techniques , Comet Assay , Cytochrome P-450 CYP1A1/genetics , Hep G2 Cells , Hepatocytes/drug effects , Hepatocytes/metabolism , Humans , Liver/metabolism , Male , Mice, Inbred C57BL , Mice, Knockout , Pregnane X Receptor , Receptors, Aryl Hydrocarbon/genetics , Receptors, Steroid/genetics , Signal Transduction
7.
Oncol Rep ; 37(4): 2153-2160, 2017 Apr.
Article in English | MEDLINE | ID: mdl-28260109

ABSTRACT

Long non-coding RNAs (lncRNA) are key regulators of gene expression both at the transcriptional and post-transcriptional levels. The lncRNA metastasis associated lung adenocarcinoma transcript 1 (Malat1) is overexpressed in many types of cancer, including hepatocarcinoma, and induces cell proliferation in several cell lines in vitro. However, the direct causal effects of Malat1 on hepatocyte proliferation and liver carcinogenesis in vivo are not fully understood. To better determine the contribution of Malat1 to hepatocarcinoma oncogenesis, this study was aimed at testing the hypothesis that its absence confers resistance to the development of liver tumors. Male Malat1-/- mice and their wild-type (WT) littermates were studied one year after treatment with the genotoxic agent diethylnitrosamine (DEN), a potent inducer of liver cancer. As expected, in WT mice, Malat1 expression was significantly higher in hepatic tumors than in healthy liver regions. Altered hepatic mRNA levels of Ki67, HDAC3, NFκB and p27 were observed in DEN-treated Malat1-/- mice. Despite this, these mice were characterized by similar liver weight, prevalence of tumors, and histological features compared to those of their WT littermates. In parallel, plasma lipids and glucose homeostasis did not significantly differ between DEN-treated groups. These findings support a role for Malat1 as a marker of liver carcinogenesis, but also suggest that its role in the regulation of hepatocyte hyperproliferation in mice is either minimal or masked by redundant and/or overwhelming mechanisms.


Subject(s)
Carcinoma, Hepatocellular/pathology , Diethylnitrosamine/adverse effects , Liver Neoplasms/pathology , RNA, Long Noncoding/genetics , Animals , Blood Glucose/metabolism , Carcinoma, Hepatocellular/chemically induced , Carcinoma, Hepatocellular/genetics , Gene Deletion , Gene Expression Regulation, Neoplastic , Lipids/blood , Liver Neoplasms/chemically induced , Liver Neoplasms/genetics , Male , Mice , Organ Size , Up-Regulation
8.
Cell Cycle ; 15(22): 3115-3120, 2016 Nov 16.
Article in English | MEDLINE | ID: mdl-27686860

ABSTRACT

Eukaryotic initiation factor 2A (eIF2A) is a 65-kDa protein that was first identified in the early 1970s as a factor capable of stimulating initiator methionyl-tRNAi (Met-tRNAMeti) binding to 40S ribosomal subunits in vitro. However, in contrast to the eIF2, which stimulates Met-tRNAMeti binding to 40S ribosomal subunits in a GTP-dependent manner, eIF2A didn't reveal any GTP-dependence, but instead was found to direct binding of the Met-tRNAMeti to 40S ribosomal subunits in a codon-dependent manner. eIF2A appears to be highly conserved across eukaryotic species, suggesting conservation of function in evolution. The yeast Saccharomyces cerevisae eIF2A null mutant revealed no apparent phenotype, however, it was found that in yeast eIF2A functions as a suppressor of internal ribosome entry site (IRES)-mediated translation. It was thus suggested that eIF2A my act by impinging on the expression of specific mRNAs. Subsequent studies in mammalian cell systems implicated eIF2A in non-canonical (non-AUG-dependent) translation initiation events involving near cognate UUG and CUG codons. Yet, the role of eIF2A in cellular functions remains largely enigmatic. As a first step toward characterization of the eIF2A function in mammalian systems in vivo, we have obtained homozygous eIF2A-total knockout (KO) mice, in which a gene trap cassette was inserted between eIF2A exons 1 and 2 disrupting expression of all exons downstream of the insertion. The KO mice strain is viable and to date displays no apparent phenotype. We believe that the eIF2A KO mice strain will serve as a valuable tool for researchers studying non-canonical initiation of translation in vivo.


Subject(s)
Eukaryotic Initiation Factor-2/deficiency , Animals , Base Sequence , Eukaryotic Initiation Factor-2/genetics , Eukaryotic Initiation Factor-2/metabolism , Gene Expression Regulation , Mice, Inbred C57BL , Mice, Knockout
9.
J Steroid Biochem Mol Biol ; 157: 48-60, 2016 Mar.
Article in English | MEDLINE | ID: mdl-25917081

ABSTRACT

The orphan nuclear receptors NR4A1, NR4A2 and NR4A3 are immediate early genes induced by multiple stressors, and the NR4A receptors play an important role in maintaining cellular homeostasis and disease. There is increasing evidence for the role of these receptors in metabolic, cardiovascular and neurological functions and also in inflammation and inflammatory diseases and in immune functions and cancer. Despite the similarities of NR4A1, NR4A2 and NR4A3 and their interactions with common cis-genomic elements, they exhibit unique activities and cell-/tissue-specific functions. Although endogenous ligands for NR4A receptors have not been identified, there is increasing evidence that structurally-diverse synthetic molecules can directly interact with the ligand binding domain of NR4A1 and act as agonists or antagonists, and ligands for NR4A2 and NR4A3 have also been identified. Since NR4A receptors are key factors in multiple diseases, there are opportunities for the future development of NR4A ligands for clinical applications in treating multiple health problems including metabolic, neurologic and cardiovascular diseases, other inflammatory conditions, and cancer.


Subject(s)
DNA-Binding Proteins/metabolism , Nuclear Receptor Subfamily 4, Group A, Member 1/metabolism , Nuclear Receptor Subfamily 4, Group A, Member 2/metabolism , Receptors, Steroid/metabolism , Receptors, Thyroid Hormone/metabolism , Arthritis/metabolism , Cardiovascular Diseases/metabolism , Homeostasis , Humans , Immunity, Cellular , Inflammation/metabolism , Ligands , Metabolic Diseases/genetics , Metabolic Diseases/metabolism , Neoplasms/metabolism , Nuclear Receptor Subfamily 4, Group A, Member 1/genetics , Nuclear Receptor Subfamily 4, Group A, Member 2/genetics
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