Identification of Novel Regulatory Regions Induced by Intrauterine Growth Restriction in Rat Islets.

Intrauterine growth restriction (IUGR) leads to the development of type 2 diabetes in adulthood, and the permanent alterations in gene expression implicate an epigenetic mechanism. Using a rat model of IUGR, we performed TrueSeq-HELP Tagging to assess the association of DNA methylation changes and gene dysregulation in islets. We identified 511 differentially methylated regions (DMRs) and 4377 significantly altered single CpG sites. Integrating the methylome and our published transcriptome data sets resulted in the identification of pathways critical for islet function. The identified DMRs were enriched with transcription factor binding motifs, such as Elk1, Etv1, Foxa1, Foxa2, Pax7, Stat3, Hnf1, and AR. In silico analysis of 3-dimensional chromosomal interactions using human pancreas and islet Hi-C data sets identified interactions between 14 highly conserved DMRs and 35 genes with significant expression changes at an early age, many of which persisted in adult islets. In adult islets, there were far more interactions between DMRs and genes with significant expression changes identified with Hi-C, and most of them were critical to islet metabolism and insulin secretion. The methylome was integrated with our published genome-wide histone modification data sets from IUGR islets, resulting in further characterization of important regulatory regions of the genome altered by IUGR containing both significant changes in DNA methylation and specific histone marks. We identified novel regulatory regions in islets after exposure to IUGR, suggesting that epigenetic changes at key transcription factor binding motifs and other gene regulatory regions may contribute to gene dysregulation and an abnormal islet phenotype in IUGR rats.

The Transcriptome and Epigenome Reveal Novel Changes in Transcription Regulation During Pancreatic Rat Islet Maturation.

Islet function is critical for normal glucose homeostasis. Unlike adult ß cells, fetal and neonatal islets are more proliferative and have decreased insulin secretion in response to stimuli. However, the underlying mechanisms governing functional maturity of islets have not been completely elucidated. Pancreatic islets comprise different cell types. The microenvironment of islets and interactions between these cell types are critical for ß-cell development and maturation. Thus, the study of intact islets is optimal to identify novel molecular mechanisms controlling islet functional development. Transcriptomes and genome-wide histone landscapes of H3K4me3, H3K27me3, and H3K27Ac from intact islets isolated from 2- and 10-week-old Sprague-Dawley rats were integrated to elucidate genes and pathways modulating islet development, as well as the contribution of epigenetic regulation. A total of 4489 differentially expressed genes were identified; 2289 and 2200 of them were up- and down-regulated in 10-week islets, respectively. Ingenuity Pathway Analysis revealed critical pathways regulating functional maturation of islets, including nutrient sensing, neuronal function, immune function, cell replication, and extracellular matrix. Furthermore, we identified significant changes in enrichment of H3K4me3, H3K27me3, and H3K27Ac marks, which correlated with expression changes of genes critical for islet function. These histone marks were enriched at critical transcription factor-binding motifs, such as Hoxa9, C/EBP-ß, Gata1, Foxo1, E2f1, E2f3, and Mafb. In addition, our chromatin immunoprecipitation sequencing data revealed multiple potential bivalent genes whose poised states changed with maturation. Collectively, our current study identified critical novel pathways for mature islet function and suggested a role for histone modifications in regulating islet development and maturation.

Human Placental Transcriptome Reveals Critical Alterations in Inflammation and Energy Metabolism with Fetal Sex Differences in Spontaneous Preterm Birth.

A well-functioning placenta is crucial for normal gestation and regulates the nutrient, gas, and waste exchanges between the maternal and fetal circulations and is an important endocrine organ producing hormones that regulate both the maternal and fetal physiologies during pregnancy. Placental insufficiency is implicated in spontaneous preterm birth (SPTB). We proposed that deficits in the capacity of the placenta to maintain bioenergetic and metabolic stability during pregnancy may ultimately result in SPTB. To explore our hypothesis, we performed a RNA-seq study in male and female placentas from women with SPTB (<36 weeks gestation) compared to normal pregnancies (≥38 weeks gestation) to assess the alterations in the gene expression profiles. We focused exclusively on Black women (cases and controls), who are at the highest risk of SPTB. Six hundred and seventy differentially expressed genes were identified in male SPTB placentas. Among them, 313 and 357 transcripts were increased and decreased, respectively. In contrast, only 61 differentially expressed genes were identified in female SPTB placenta. The ingenuity pathway analysis showed alterations in the genes and canonical pathways critical for regulating inflammation, oxidative stress, detoxification, mitochondrial function, energy metabolism, and the extracellular matrix. Many upstream regulators and master regulators important for nutrient-sensing and metabolism were also altered in SPTB placentas, including the PI3K complex, TGFB1/SMADs, SMARCA4, TP63, CDKN2A, BRCA1, and NFAT. The transcriptome was integrated with published human placental metabolome to assess the interactions of altered genes and metabolites. Collectively, significant and biologically relevant alterations in the transcriptome were identified in SPTB placentas with fetal sex disparities. Altered energy metabolism, mitochondrial function, inflammation, and detoxification may underly the mechanisms of placental dysfunction in SPTB.

Intrauterine Inflammation Alters the Transcriptome and Metabolome in Placenta.

Placental insufficiency is implicated in spontaneous preterm birth (SPTB) associated with intrauterine inflammation. We hypothesized that intrauterine inflammation leads to deficits in the capacity of the placenta to maintain bioenergetic and metabolic stability during pregnancy ultimately resulting in SPTB. Using a mouse model of intrauterine inflammation that leads to preterm delivery, we performed RNA-seq and metabolomics studies to assess how intrauterine inflammation alters gene expression and/or modulates metabolite production and abundance in the placenta. 1871 differentially expressed genes were identified in LPS-exposed placenta. Among them, 1,149 and 722 transcripts were increased and decreased, respectively. Ingenuity pathway analysis showed alterations in genes and canonical pathways critical for regulating oxidative stress, mitochondrial function, metabolisms of glucose and lipids, and vascular reactivity in LPS-exposed placenta. Many upstream regulators and master regulators important for nutrient-sensing and mitochondrial function were also altered in inflammation exposed placentae, including STAT1, HIF1α, mTOR, AMPK, and PPARα. Comprehensive quantification of metabolites demonstrated significant alterations in the glucose utilization, metabolisms of branched-chain amino acids, lipids, purine and pyrimidine, as well as carbon flow in TCA cycle in LPS-exposed placenta compared to control placenta. The transcriptome and metabolome were also integrated to assess the interactions of altered genes and metabolites. Collectively, significant and biologically relevant alterations in the placenta transcriptome and metabolome were identified in placentae exposed to intrauterine inflammation. Altered mitochondrial function and energy metabolism may underline the mechanisms of inflammation-induced placental dysfunction.

Transcriptomic and Quantitative Proteomic Profiling Reveals Signaling Pathways Critical for Pancreatic Islet Maturation.

Pancreatic ß-cell dysfunction and reduced insulin secretion play a key role in the pathogenesis of diabetes. Fetal and neonatal islets are functionally immature and have blunted glucose responsiveness and decreased insulin secretion in response to stimuli and are far more proliferative. However, the mechanisms underlying functional immaturity are not well understood. Pancreatic islets are composed of a mixture of different cell types, and the microenvironment of islets and interactions between these cell types are critical for ß-cell development and maturation. RNA sequencing and quantitative proteomic data from intact islets isolated from fetal (embryonic day 19) and 2-week-old Sprague-Dawley rats were integrated to compare their gene and protein expression profiles. Ingenuity Pathway Analysis (IPA) was also applied to elucidate pathways and upstream regulators modulating functional maturation of islets. By integrating transcriptome and proteomic data, 917 differentially expressed genes/proteins were identified with a false discovery rate of less than 0.05. A total of 411 and 506 of them were upregulated and downregulated in the 2-week-old islets, respectively. IPA revealed novel critical pathways associated with functional maturation of islets, such as AMPK (adenosine monophosphate-activated protein kinase) and aryl hydrocarbon receptor signaling, as well as the importance of lipid homeostasis/signaling and neuronal function. Furthermore, we also identified many proteins enriched either in fetal or 2-week-old islets related to extracellular matrix and cell communication, suggesting that these pathways play critical roles in islet maturation. Our present study identified novel pathways for mature islet function in addition to confirming previously reported mechanisms, and provided new mechanistic insights for future research on diabetes prevention and treatment.

Altered Transcription Factor Binding and Gene Bivalency in Islets of Intrauterine Growth Retarded Rats.

Intrauterine growth retardation (IUGR), which induces epigenetic modifications and permanent changes in gene expression, has been associated with the development of type 2 diabetes. Using a rat model of IUGR, we performed ChIP-Seq to identify and map genome-wide histone modifications and gene dysregulation in islets from 2- and 10-week rats. IUGR induced significant changes in the enrichment of H3K4me3, H3K27me3, and H3K27Ac marks in both 2-wk and 10-wk islets, which were correlated with expression changes of multiple genes critical for islet function in IUGR islets. ChIP-Seq analysis showed that IUGR-induced histone mark changes were enriched at critical transcription factor binding motifs, such as C/EBPs, Ets1, Bcl6, Thrb, Ebf1, Sox9, and Mitf. These transcription factors were also identified as top upstream regulators in our previously published transcriptome study. In addition, our ChIP-seq data revealed more than 1000 potential bivalent genes as identified by enrichment of both H3K4me3 and H3K27me3. The poised state of many potential bivalent genes was altered by IUGR, particularly Acod1, Fgf21, Serpina11, Cdh16, Lrrc27, and Lrrc66, key islet genes. Collectively, our findings suggest alterations of histone modification in key transcription factors and genes that may contribute to long-term gene dysregulation and an abnormal islet phenotype in IUGR rats.

Dysregulation of Neuronal Genes by Fetal-Neonatal Iron Deficiency Anemia Is Associated with Altered DNA Methylation in the Rat Hippocampus.

Early-life iron deficiency results in long-term abnormalities in cognitive function and affective behavior in adulthood. In preclinical models, these effects have been associated with long-term dysregulation of key neuronal genes. While limited evidence suggests histone methylation as an epigenetic mechanism underlying gene dysregulation, the role of DNA methylation remains unknown. To determine whether DNA methylation is a potential mechanism by which early-life iron deficiency induces gene dysregulation, we performed whole genome bisulfite sequencing to identify loci with altered DNA methylation in the postnatal day (P) 15 iron-deficient (ID) rat hippocampus, a time point at which the highest level of hippocampal iron deficiency is concurrent with peak iron demand for axonal and dendritic growth. We identified 229 differentially methylated loci and they were mapped within 108 genes. Among them, 63 and 45 genes showed significantly increased and decreased DNA methylation in the P15 ID hippocampus, respectively. To establish a correlation between differentially methylated loci and gene dysregulation, the methylome data were compared to our published P15 hippocampal transcriptome. Both datasets showed alteration of similar functional networks regulating nervous system development and cell-to-cell signaling that are critical for learning and behavior. Collectively, the present findings support a role for DNA methylation in neural gene dysregulation following early-life iron deficiency.

Defiant: (DMRs: easy, fast, identification and ANnoTation) identifies differentially Methylated regions from iron-deficient rat hippocampus.

BACKGROUND: Identification of differentially methylated regions (DMRs) is the initial step towards the study of DNA methylation-mediated gene regulation. Previous approaches to call DMRs suffer from false prediction, use extreme resources, and/or require library installation and input conversion. RESULTS: We developed a new approach called Defiant to identify DMRs. Employing Weighted Welch Expansion (WWE), Defiant showed superior performance to other predictors in the series of benchmarking tests on artificial and real data. Defiant was subsequently used to investigate DNA methylation changes in iron-deficient rat hippocampus. Defiant identified DMRs close to genes associated with neuronal development and plasticity, which were not identified by its competitor. Importantly, Defiant runs between 5 to 479 times faster than currently available software packages. Also, Defiant accepts 10 different input formats widely used for DNA methylation data. CONCLUSIONS: Defiant effectively identifies DMRs for whole-genome bisulfite sequencing (WGBS), reduced-representation bisulfite sequencing (RRBS), Tet-assisted bisulfite sequencing (TAB-seq), and HpaII tiny fragment enrichment by ligation-mediated PCR-tag (HELP) assays.

Transcriptomic Analysis Reveals Novel Mechanisms Mediating Islet Dysfunction in the Intrauterine Growth-Restricted Rat.

Intrauterine growth restriction (IUGR) increases the risk of type 2 diabetes developing in adulthood. In previous studies that used bilateral uterine artery ligation in a rat model of IUGR, age-associated decline in glucose homeostasis and islet function was revealed. To elucidate mechanisms contributing to IUGR pathogenesis, the islet transcriptome was sequenced from 2-week-old rats, when in vivo glucose tolerance is mildly impaired, and at 10 weeks of age, when rats are hyperglycemic and have reduced ß-cell mass. RNA sequencing and functional annotation with Ingenuity Pathway Analysis revealed temporal changes in IUGR islets. For instance, gene expression involving amino acid metabolism was significantly reduced primarily at 2 weeks of age, but ion channel expression, specifically that involved in cell-volume regulation, was more disrupted in adult IUGR islets. Additionally, we observed alterations in the microenvironment of IUGR islets with extracellular matrix genes being significantly increased at 2 weeks of age and significantly decreased at 10 weeks. Specifically, hyaluronan synthase 2 expression and hyaluronan staining were increased in IUGR islets at 2 weeks of age (P < 0.05). Mesenchymal stromal cell-derived factors that have been shown to preserve islet allograft function, such as Anxa1, Cxcl12, and others, also were increased at 2 weeks and decreased in adult islets. Finally, comparisons of differentially expressed genes with those of type 2 diabetic human islets support a role for these pathways in human patients with diabetes. Together, these data point to new mechanisms in the pathogenesis of IUGR-mediated islet dysfunction in type 2 diabetes.

Late-gestation maternal dietary methyl donor and cofactor supplementation in sheep partially reverses protection against allergic sensitization by IUGR.

Perinatal exposures are associated with altered risks of childhood allergy. Human studies and our previous work suggest that restricted growth in utero (IUGR) is protective against allergic disease. The mechanisms are not clearly defined, but reduced fetal abundance and altered metabolism of methyl donors are hypothesized as possible underlying mechanisms. Therefore, we examined whether late-gestation maternal dietary methyl donor and cofactor supplementation of the placentally restricted (PR) sheep pregnancy would reverse allergic protection in progeny. Allergic outcomes were compared between progeny from control pregnancies (CON; n = 49), from PR pregnancies without intervention (PR; n = 28), and from PR pregnancies where the dam was fed a methyl donor plus cofactor supplement from day 120 of pregnancy until delivery (PR + Methyl; n = 25). Both PR and PR + Methyl progeny were smaller than CON; supplementation did not alter birth size. PR was protective against cutaneous hypersensitivity responses to ovalbumin (OVA; P < 0.01 in singletons). Cutaneous hypersensitivity responses to OVA in PR + Methyl progeny were intermediate to and not different from the responses of CON and PR sheep. Cutaneous hypersensitivity responses to house dust mites did not differ between treatments. In singleton progeny, upper dermal mast cell density was greater in PR + Methyl than in PR or CON (each P < 0.05). The differences in the cutaneous allergic response were not explained by treatment effects on circulating immune cells or antibodies. Our results suggest that mechanisms underlying in utero programming of allergic susceptibility by IUGR and methyl donor availability may differ and imply that late-gestation methyl donor supplementation may increase allergy risk.

Fetal iron deficiency induces chromatin remodeling at the Bdnf locus in adult rat hippocampus.

Fetal and subsequent early postnatal iron deficiency causes persistent impairments in cognitive and affective behaviors despite prompt postnatal iron repletion. The long-term cognitive impacts are accompanied by persistent downregulation of brain-derived neurotrophic factor (BDNF), a factor critical for hippocampal plasticity across the life span. This study determined whether early-life iron deficiency epigenetically modifies the Bdnf locus and whether dietary choline supplementation during late gestation reverses these modifications. DNA methylation and histone modifications were assessed at the Bdnf-IV promoter in the hippocampus of rats [at postnatal day (PND) 65] that were iron-deficient (ID) during the fetal-neonatal period. Iron deficiency was induced in rat pups by providing pregnant and nursing dams an ID diet (4 mg/kg Fe) from gestational day (G) 2 through PND7, after which iron deficiency was treated with an iron-sufficient (IS) diet (200 mg/kg Fe). This paradigm resulted in about 60% hippocampal iron loss on PND15 with complete recovery by PND65. For choline supplementation, pregnant rat dams were given dietary choline (5 g/kg) from G11 through G18. DNA methylation was determined by quantitative sequencing of bisulfite-treated DNA, revealing a small alteration at the Bdnf-IV promoter. Chromatin immunoprecipitation analysis showed increased HDAC1 binding accompanied by reduced binding of RNA polymerase II and USF1 at the Bdnf-IV promoter in formerly ID rats. These changes were correlated with altered histone methylations. Prenatal choline supplementation reverses these epigenetic modifications. Collectively, the findings identify epigenetic modifications as a potential mechanism to explicate the long-term repression of Bdnf following fetal and early postnatal iron deficiency.

Keratinocyte growth factor and glucocorticoid induction of human peroxiredoxin 6 gene expression occur by independent mechanisms that are synergistic.

AIMS: Peroxiredoxin 6 (Prdx6), a 1-cys Prdx has both peroxidase and phospholipase A2 activities, protecting against oxidative stress and regulating pulmonary surfactant phospholipid metabolism. This study determined the mechanism by which keratinocyte growth factor (KGF) and the glucocorticoid analogue, dexamethasone (Dex), induce increased Prdx6 expression. RESULTS: Transcriptional activation by KGF in both A549 lung adenocarcinoma cells and rat lung alveolar epithelial type II (ATII) cells utilizes an antioxidant response element (ARE), located between 357 and 349 nucleotides before the PRDX6 translational start, that is also necessary for upregulation of the human PRDX6 promoter in response to oxidative stress. Activation is mediated by binding of the transcription factor, Nrf2, to the ARE as shown by experiments using siRNA against Nrf2 and by transfecting ATII cells isolated from lungs of Nrf2 null mice. KGF triggers the migration of Nrf2 from cytoplasm to nucleus where it binds to the PRDX6 promoter as shown by chromatin immunoprecipitation assays. Activation of transcription by Dex occurs through a glucocorticoid response element located about 750 nucleotides upstream of the PRDX6 translational start. INNOVATION: This study demonstrates that KGF can activate an ARE in a promoter without reactive oxygen species involvement and that KGF and Dex can synergistically activate the PRDX6 promoter and protect cells from oxidative stress. CONCLUSION: These two different activators work through different DNA elements. Their combined effect on transcription of the reporter gene is synergistic; however, at the protein level, the combined effect is additive and protects cells from oxidative damage.

Functional interaction of glutathione S-transferase pi and peroxiredoxin 6 in intact cells.

Peroxiredoxin 6 (Prdx6) is a 1-Cys member of the peroxiredoxin superfamily that plays an important role in antioxidant defense. Glutathionylation of recombinant Prdx6 mediated by π glutathione S-transferase (GST) is required for reduction of the oxidized Cys and completion of the peroxidatic catalytic cycle in vitro. This study investigated the requirement for πGST in intact cells. Transfection with a plasmid construct expressing πGST into MCF7, a cell line that lacks endogenous πGST, significantly increased phospholipid peroxidase activity as measured in cell lysates and protected intact cells against a peroxidative stress. siRNA knockdown indicated that this increased peroxidase activity was Prdx6 dependent. Interaction between πGST and Prdx6, evaluated by the Duolink Proximity Ligation Assay, was minimal under basal conditions but increased dramatically following treatment of cells with the oxidant, tert-butyl hydroperoxide. Interaction was abolished by mutation of C47, the active site for Prdx6 peroxidase activity. Depletion of cellular GSH by treatment of cells with buthionine sulfoximine had no effect on the interaction of Prdx6 and πGST. These data are consistent with the hypothesis that oxidation of the catalytic cysteine in Prdx6 is required for its interaction with πGST and that the interaction plays an important role in regenerating the peroxidase activity of Prdx6.

The roles of peroxidase and phospholipase A2 activities of peroxiredoxin 6 in protecting pulmonary microvascular endothelial cells against peroxidative stress.

AIMS: Peroxiredoxin 6 (Prdx6), a bifunctional enzyme with glutathione peroxidase and phospholipase A(2) (PLA(2)) activities, has been demonstrated as playing a critical role in antioxidant defense of the lung. Our aim was to evaluate the relative role of each activity in Prdx6-mediated protection of mouse pulmonary microvascular endothelial cells (PMVECs) against the peroxidative stress of treatment with tert-butyl hydroperoxide (tBOOH). RESULTS: PMVEC from Prdx6 null mice showed increased lethality on tBOOH exposure (50-200 µM) compared with wild-type (WT) controls. Treatment with 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33), a Prdx6 PLA(2) activity inhibitor, increased the sensitivity of WT cells to peroxidative stress, but did not further sensitize Prdx6 null cells. Lethality in Prdx6 null PMVEC was "rescued" by transfection with a construct leading to the expression of WT rat Prdx6. Expression of mutant Prdx6 with either peroxidase activity or PLA(2) activity alone each partially rescued the survival of Prdx6 null cells, while constructs with both active sites mutated failed to rescue. Co-transfection with two different constructs, each expressing one activity, rescued cells as well as the WT construct. INNOVATION AND CONCLUSION: Contrary to the general assumption that the peroxidase activity is the main mechanism for Prdx6 antioxidant function, these results indicate that the PLA(2) activity also plays a substantial role in protecting cells against oxidant stress caused by an exogenous hydroperoxide.

Peroxiredoxin 6 phosphorylation and subsequent phospholipase A2 activity are required for agonist-mediated activation of NADPH oxidase in mouse pulmonary microvascular endothelium and alveolar macrophages.

Peroxiredoxin 6 (Prdx6), a bifunctional enzyme with glutathione peroxidase and phospholipase A2 (PLA(2)) activities, participates in the activation of NADPH oxidase 2 (NOX2) in neutrophils, but the mechanism for this effect is not known. We now demonstrate that Prdx6 is required for agonist-induced NOX2 activation in pulmonary microvascular endothelial cells (PMVEC) and that the effect requires the PLA(2) activity of Prdx6. Generation of reactive oxygen species (ROS) in response to angiotensin II (Ang II) or phorbol 12-myristate 13-acetate was markedly reduced in perfused lungs and isolated PMVEC from Prdx6 null mice. Rac1 and p47(phox), cytosolic components of NOX2, translocated to the endothelial cell membrane after Ang II treatment in wild-type but not Prdx6 null PMVEC. MJ33, an inhibitor of Prdx6 PLA(2) activity, blocked agonist-induced PLA(2) activity and ROS generation in PMVEC by >80%, whereas inhibitors of other PLA(2)s were ineffective. Transfection of Prx6 null cells with wild-type and C47S mutant Prdx6, but not with mutants of the PLA(2) active site (S32A, H26A, and D140A), "rescued" Ang II-induced PLA(2) activity and ROS generation. Ang II treatment of wild-type cells resulted in phosphorylation of Prdx6 and its subsequent translocation from the cytosol to the cell membrane. Phosphorylation as well as PLA(2) activity and ROS generation were markedly reduced by the MAPK inhibitor, U0126. Thus, agonist-induced MAPK activation leads to Prdx6 phosphorylation and translocation to the cell membrane, where its PLA(2) activity facilitates assembly of the NOX2 complex and activation of the oxidase.

S-glutathionylation activates STIM1 and alters mitochondrial homeostasis.

Oxidant stress influences many cellular processes, including cell growth, differentiation, and cell death. A well-recognized link between these processes and oxidant stress is via alterations in Ca(2+) signaling. However, precisely how oxidants influence Ca(2+) signaling remains unclear. Oxidant stress led to a phenotypic shift in Ca(2+) mobilization from an oscillatory to a sustained elevated pattern via calcium release-activated calcium (CRAC)-mediated capacitive Ca(2+) entry, and stromal interaction molecule 1 (STIM1)- and Orai1-deficient cells are resistant to oxidant stress. Functionally, oxidant-induced Ca(2+) entry alters mitochondrial Ca(2+) handling and bioenergetics and triggers cell death. STIM1 is S-glutathionylated at cysteine 56 in response to oxidant stress and evokes constitutive Ca(2+) entry independent of intracellular Ca(2+) stores. These experiments reveal that cysteine 56 is a sensor for oxidant-dependent activation of STIM1 and demonstrate a molecular link between oxidant stress and Ca(2+) signaling via the CRAC channel.

Mitochondrial function and nuclear factor-kappaB-mediated signaling in radiation-induced bystander effects.

Although radiation-induced bystander effects have been well described over the past decade, the mechanisms of the signaling processes involved in the bystander phenomenon remain unclear. In the present study, using the Columbia University charged particle microbeam, we found that mitochondrial DNA-depleted human skin fibroblasts (rho(o)) showed a higher bystander mutagenic response in confluent monolayers when a fraction of the same population were irradiated with lethal doses compared with their parental mitochondrial-functional cells (rho(+)). However, using mixed cultures of rho(o) and rho(+) cells and targeting only one population of cells with a lethal dose of alpha-particles, a decreased bystander mutagenesis was uniformly found in nonirradiated bystander cells of both cell types, indicating that signals from one cell type can modulate expression of bystander response in another cell type. In addition, we found that Bay 11-7082, a pharmacologic inhibitor of nuclear factor-kappaB (NF-kappaB) activation, and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a scavenger of nitric oxide (NO), significantly decreased the mutation frequency in both bystander rho(o) and rho(+) cells. Furthermore, we found that NF-kappaB activity and its dependent proteins, cyclooxygenase-2 (COX-2) and inducible NO synthase (iNOS), were lower in bystander rho(o) cells when compared with their rho(+) counterparts. Our results indicated that mitochondria play an important role in the regulation of radiation-induced bystander effects and that mitochondria-dependent NF-kappaB/iNOS/NO and NF-kappaB/COX-2/prostaglandin E2 signaling pathways are important to the process.

Genotoxic mechanisms of asbestos fibers: role of extranuclear targets.

Asbestos fibers are carcinogenic to both humans and experimental animals. The continued discoveries of exposure routes whereby the general public is exposed to asbestos suggest a long-term, low-dose exposure for a large number of people. However, the mechanisms by which asbestos induces malignancy are not entirely understood. In previous studies, we have shown that asbestos is an effective gene and chromosomal mutagen when assayed using the highly sensitive AL mutation assay and that the mutagenicity is mediated by reactive oxygen species. The objective of the present study is to determine the origin of these radical species, particularly reactive nitrogen species, in fiber mutagenesis. Using the radical probe 5',6'-chloromethyl-2',7'-dihydroxyphenoxazine diacetate to trap reactive radical species, we showed that crocidolite increased the levels of oxyradicals in cytoplasts, in the absence of the nucleus, in a dose-dependent manner, which was reduced significantly by cotreatment with the radical scavenger dimethyl sulfoxide. Treatment of enucleated cells with crocidolite asbestos followed by rescue fusion using karyoplasts from control cells resulted in significant mutant induction, indicating that the nuclear-cytoplasmic interaction is necessary for fiber mutagenesis. Using the fluorescent probe 2,3-diaminonaphthotriazole, crocidolite fibers were shown to induce a dose-dependent increase of nitric oxide production, which was suppressed significantly by concurrent treatment with the nitric oxide synthase inhibitor, NG-methyl-L-arginine (L-NMMA). Similarly, there was a dose-dependent decrease in the mutation yield induced by crocidolites in the presence of graded doses of L-NMMA. These data showed that extranuclear targets play an essential role in the initiation of oxidative damage that mediates fiber mutagenesis in mammalian cells.

Phospholipase C-delta1 is a critical target for tumor necrosis factor receptor-mediated protection against adriamycin-induced cardiac injury.

The clinical application of adriamycin, an exceptionally good chemotherapeutic agent, is limited by its dose-related cardiomyopathy. Our recent study showed that tumor necrosis factor-alpha (TNF-alpha) receptors mediated cytoprotective signaling against adriamycin-induced mitochondrial injury and cardiomyocyte apoptosis. In the present study, we investigated the potential targets of TNF receptor-mediated cytoprotective signaling by global genome microarray analysis using wild-type and TNF receptor-deficient mice. Microarray analysis revealed that adriamycin treatment induced the down-regulation of several mitochondrial functions and energy production-related genes in double TNF receptor-deficient mice, notably, phospholipase C-delta1, a protein involved in fatty acid metabolism and calcium regulation. The role of phospholipase C-delta1 in TNF receptor-mediated cardioprotection against adriamycin-induced injury was evaluated by measuring changes in cardiac function using high-frequency ultrasound biomicroscopy. Selective inhibition of phospholipase C activity in wild-type mice by its inhibitor, U73122, exacerbated adriamycin-induced cardiac dysfunction. Inhibition of phospholipase C-delta1 resulted in the significant decrease of left ventricular ejection fraction and fractional shortening, and the decreased levels were similar to those observed in adriamycin-treated double TNF receptor-deficient mice. The data derived from the global genome analysis identified phospholipase C-delta1 as an important target for TNF receptors and revealed the critical role of TNF receptor signaling in the protection against adriamycin-induced cardiotoxicity.

TNF receptor deficiency reveals a translational control mechanism for adriamycin-induced Fas expression in cardiac tissues.

Adriamycin, ADR, a potent chemotherapeutic agent, has been demonstrated to cause cardiomyocyte apoptosis, in part, via the Fas/Fas ligand-mediated cell death pathway. Our previous studies suggested that TNF-alpha receptors may mediate cardioprotection against ADR toxicity by the suppression of the Fas-mediated pathway. However, the role of TNF-alpha receptors in this process is unclear. In the present study, we extended our initial observation to determine the molecular mechanisms by which ADR induced Fas expression in the presence and absence of TNF receptors. Our results demonstrated that ADR-mediated p53 and AP-1 interaction and increased Fas mRNA levels independent of TNF receptors. However, the levels of Fas proteins only increased in the cardiac tissues of TNF receptor-deficient mice. These results demonstrated that the suppression of ADR-induced Fas expression by TNF receptors was not regulated at transcriptional levels, but may be regulated at a translational level.