Immediate inhibition of spinal secretory phospholipase A2 prevents the pain and elevated spinal neuronal hyperexcitability and neuroimmune regulatory genes that develop with nerve root compression.

Cervical nerve root injury induces a host of inflammatory mediators in the spinal cord that initiate and maintain neuronal hyperexcitability and pain. Secretory phospholipase A2 (sPLA2) is an enzyme that has been implicated as a mediator of pain onset and maintenance in inflammation and neural injury. Although sPLA2 modulates nociception and excitatory neuronal signaling in vitro, its effects on neuronal activity and central sensitization early after painful nerve root injury are unknown. This study investigated whether inhibiting spinal sPLA2 at the time of nerve root compression (NRC) modulates the pain, dorsal horn hyperexcitability, and spinal genes involved in glutamate signaling, nociception, and inflammation that are seen early after injury. Rats underwent a painful C7 NRC injury with immediate intrathecal administration of the sPLA2 inhibitor thioetheramide-phosphorlycholine. Additional groups underwent either injury alone or sham surgery. One day after injury, behavioral sensitivity, spinal neuronal excitability, and spinal cord gene expression for glutamate receptors (mGluR5 and NR1) and transporters (GLT1 and EAAC1), the neuropeptide substance P, and pro-inflammatory cytokines (TNFα, IL1α, and IL1ß) were assessed. Treatment with the sPLA2 inhibitor prevented mechanical allodynia, attenuated neuronal hyperexcitability in the spinal dorsal horn, restored the proportion of spinal neurons classified as wide dynamic range, and reduced genes for mGluR5, substance P, IL1α, and IL1ß to sham levels. These findings indicate spinal regulation of central sensitization after painful neuropathy and suggest that spinal sPLA2 is implicated in those early spinal mechanisms of neuronal excitability, perhaps via glutamate signaling, neurotransmitters, or inflammatory cascades.

Effect of short-term sterigmatocystin exposure on lipid peroxidation and glutathione redox system and expression of glutathione redox system regulatory genes in common carp liver.

Sterigmatocystin (STC) is structurally close to the mycotoxin aflatoxin B1 as it shares its biosynthetic pathway with aflatoxins. The purpose of the present study was to investigate the short-term (24 h) effects of STC contaminated diet at different doses (1 mg, 2 mg and 4 mg STC kg-1 feed) in one year old common carp juveniles. Liver samples were taken in 8-h intervals. The markers of the lipid peroxidation showed moderate changes after the application of sterigmatocystin-contaminated diet, significant elevations were only observed in the lowest applied dose group of sterigmatocystin after 16 h of exposure. Reduced glutathione content showed higher levels than control group after 16 h of exposure as effect of low dose of sterigmatocystin. Glutathione peroxidase (GPX4) activity was lower than control in the group treated with 2 mg STC kg-1 feed after 24 h of exposure. Gene expression measurements of keap1, nrf2, gpx4a, gpx4b and gss genes revealed a dual response. Down-regulation or near control values were observed 8 h after exposure which was followed by an induction 16 and 24 h after exposure. In case of gsr, gene expression values returned to control levels by the 24th hour. In summary, these results suggest that lower doses of STC caused oxidative stress earlier than higher doses, which efficiently activated the Keap1-Nrf2 pathway, while higher doses revealed long-drawn activation of this pathway.

Differential genotoxic and epigenotoxic effects of graphene family nanomaterials (GFNs) in human bronchial epithelial cells.

The widespread applications of graphene family nanomaterials (GFNs) raised the considerable concern over human health and environment. The cyto-genotoxic potentiality of GFNs has attracted much more attention, albeit the potential effects on the cellular epigenome remain largely unknown. The effects of GFNs on cellular genome were evaluated with single and double stranded DNA damage and DNA repair gene expressions while the effects on epigenome was accomplished by addressing the global DNA methylation and expression of DNA methylation machineries at non-cytotoxic to moderately cytotoxic doses in in vitro system. We used five different representatives of GFNs-pristine (GNP-Prist), carboxylated (GNP-COOH) and aminated (GNP-NH2) graphene nanoplatelets as well as single layer (SLGO) and few layer (FLGO) graphene oxide. The order of single stranded DNA damage was observed as GNP-Prist ≥ GNP-COOH>GNP-NH2≥FLGO>SLGO at 10mg/L and marked dose dependency was found in SLGO. The GFNs possibly caused genotoxicity by affecting nucleotide excision repair and non-homologus end joining repair systems. Besides, dose dependent increase in global DNA methylation (hypermethylation) were observed in SLGO/FLGO exposure and conversely, GNPs treatment caused hypomethylation following the order as GNP-COOH>GNP-NH2 ≥ GNP-Prist. The decrements of DNA methyltransferase (DNMT3B gene) and methyl-CpG binding domain protein (MBD1) genes were probably the cause of global hypomethylation induced by GNPs. Conversely, the de novo methylation through the up-regulation of DNMT3B and MBD1 genes gave rise to the global DNA hypermethylation in SLGO/FLGO treated cells. In general, the GFNs induced genotoxicity and alterations of global DNA methylation exhibited compounds type specificity with differential physico-chemical properties. Taken together, our study suggests that the GFNs could cause more subtle changes in gene expression programming by modulating DNA methylation status and this information would be helpful for their prospective use in biomedical field.

Morphology-based mammalian stem cell tests reveal potential developmental toxicity of donepezil.

Various compounds, including therapeutic drugs, can adversely impact the survival and development of embryos in the uterus. Identification of such development-interfering agents is a challenging task, although multi-angle approaches--including the use of in vitro toxicology studies involving embryonic stem cells--should alleviate some of the current difficulties. In the present study, we utilized the in vitro elongation of embryoid bodies (EBs) derived from mouse embryonal carcinoma stem cell line P19C5 as a model of early embryological events, specifically that of gastrulation and axial patterning. From our study, we identified donepezil, a medication indicated for the management of Alzheimer's disease, as a potential developmental toxicant. The extent of P19C5 EB axial elongation was diminished by donepezil in a dose-dependent manner. Although donepezil is a known inhibitor of acetylcholinesterase, interference of elongation was not mediated through this enzyme. Quantitative reverse-transcriptase PCR revealed that donepezil altered the expression pattern of a specific set of developmental regulator genes involved in patterning along the anterior-posterior body axis. When tested in mouse whole embryo culture, donepezil caused morphological abnormalities including impaired somitogenesis. Donepezil also diminished elongation morphogenesis of EBs generated from human embryonic stem cells. These results suggest that donepezil interferes with axial elongation morphogenesis of early embryos by altering the expression pattern of regulators of axial development.

Regulated genes in psoriatic skin during treatment with fumaric acid esters.

BACKGROUND: Fumaric acid esters (FAEs) are widely used in Europe for the treatment of psoriasis because of their clinical efficacy and favourable safety profile. However, the mechanisms of action by which FAEs improve psoriasis remain largely unknown. OBJECTIVES: To identify pathways and mechanisms affected by FAE treatment and to compare these with pathways affected by treatment with the antitumour necrosis factor (anti-TNF)-α biologic etanercept. METHODS: In a prospective cohort study, 50 patients with plaque psoriasis were treated with FAEs for 20 weeks. Nine patients were randomly selected for gene expression profiling of plaque biopsies from week 0 and week 12. The groups consisted of FAE responders [> Psoriasis Area and Severity Index (PASI)-75 improvement] and nonresponders (< PASI-50 improvement). Changes in gene expression profiles were analysed using Ingenuity Pathway Analysis (IPA) and the outcome was compared with gene expression affected by etanercept. RESULTS: Response to FAE treatment was associated with a ≥ 2-fold change (P < 0.05) in the expression of 458 genes. In FAE responders the role of interleukin-17A in the psoriasis pathway was most significantly activated. Glutathione and Nrf2 pathway molecules were specifically induced by FAE treatment and not by etanercept treatment, representing an FAE-specific effect in psoriatic skin. In addition, FAE treatment specifically induced the transcription factors PTTG1, NR3C1, GATA3 and NFκBIZ in responding patients. CONCLUSIONS: FAE treatment induces glutathione and Nrf2 pathway genes in lesional skin of patients with psoriasis. In responders, FAEs specifically regulate the transcription factors PTTG1, NR3C1, GATA3 and NFκBIZ, which are important in normal cutaneous development, and the T-helper (Th)2 and Th17 pathways, respectively.

Polymyxin resistance of Pseudomonas aeruginosa phoQ mutants is dependent on additional two-component regulatory systems.

Pseudomonas aeruginosa can develop resistance to polymyxin as a consequence of mutations in the PhoPQ regulatory system, mediated by covalent lipid A modification. Transposon mutagenesis of a polymyxin-resistant phoQ mutant defined 41 novel loci required for resistance, including two regulatory systems, ColRS and CprRS. Deletion of the colRS genes, individually or in tandem, abrogated the polymyxin resistance of a ΔphoQ mutant, as did individual or tandem deletion of cprRS. Individual deletion of colR or colS in a ΔphoQ mutant also suppressed 4-amino-L-arabinose addition to lipid A, consistent with the known role of this modification in polymyxin resistance. Surprisingly, tandem deletion of colRS or cprRS in the ΔphoQ mutant or individual deletion of cprR or cprS failed to suppress 4-amino-L-arabinose addition to lipid A, indicating that this modification alone is not sufficient for PhoPQ-mediated polymyxin resistance in P. aeruginosa. Episomal expression of colRS or cprRS in tandem or of cprR individually complemented the Pm resistance phenotype in the ΔphoQ mutant, while episomal expression of colR, colS, or cprS individually did not. Highly polymyxin-resistant phoQ mutants of P. aeruginosa isolated from polymyxin-treated cystic fibrosis patients harbored mutant alleles of colRS and cprS; when expressed in a ΔphoQ background, these mutant alleles enhanced polymyxin resistance. These results define ColRS and CprRS as two-component systems regulating polymyxin resistance in P. aeruginosa, indicate that addition of 4-amino-L-arabinose to lipid A is not the only PhoPQ-regulated biochemical mechanism required for resistance, and demonstrate that colRS and cprS mutations can contribute to high-level clinical resistance.

Azacytidine inhibits the proliferation of human promyelocytic leukemia cells (HL60) by demethylation of MGMT, DAPK and p16 genes.

BACKGROUND: Azacytidine (Aza) was the first demethylation agent identified that may inhibit DNA methyltransferases and reverse DNA hypermethylation, restoring the expression of silenced tumor suppressor genes in patients with myelodysplastic syndromes (MDS). It is unclear whether azacytidine can alter the proliferative and apoptotic changes in myeloid leukemia cells, and methylation changes induced by this drug have remained poorly characterized in therapy-related models. METHODS: The proliferation rate of azacytidine on HL60 cells was determined by the MTT protocol. Methylation-specific PCR (MSP) and RT-PCR were used respectively to detect gene methylation status changes and expression levels of p16, Death associated protein kinase (DAPK) and O(6)-methylguanine-DNA methyltransferase (MGMT) before and after treatment with azacytidine. RESULTS: Azacytidine inhibited HL60 cell proliferation and showed a time- and dose-dependent effect. MSP showed hypermethylated p16, DAPK, and MGMT genes before azacytidine treatment. Complete demethylation was seen in p16 and DAPK genes and partial demethylation in the MGMT gene after co-culture with azacytidine. The expression level of p16, DAPK and MGMT genes in HL60 cells was up-regulated after treatment with azacytidine. CONCLUSIONS: The CpG islands of p16, DAPK and MGMT genes are hypermethylated in HL60 cells. Azacytidine inhibits proliferation of leukemic cells by hypomethylation of p16, DAPK and MGMT genes.

Valproic acid reversed pathologic endothelial cell gene expression profile associated with ischemia-reperfusion injury in a swine hemorrhagic shock model.

BACKGROUND: Vascular endothelial cells serve as the first line of defense for end organs after ischemia and reperfusion injuries. The full etiology of this dysfunction is poorly understood, and valproic acid (VPA) has proven to be beneficial after traumatic injury. The purpose of this study was to determine the mechanism of action through which VPA exerts its beneficial effects. METHODS: Sixteen Yorkshire swine underwent a standardized protocol for an ischemia-reperfusion injury through hemorrhage and a supraceliac cross-clamp with ensuing 6-hour resuscitation. The experimental swine (n = 6), received VPA at cross-clamp application and were compared with a sham (n = 5) and injury-control models (n = 5). Aortic endothelium was harvested, and microarray analysis was performed along with a functional clustering analysis with gene transcript validation using relative quantitative polymerase chain reaction. RESULTS: Clinical comparison of experimental swine matched for sex, weight, and length demonstrated that VPA significantly decreased resuscitative requirements, with improved hemodynamics and physiologic laboratory measurements. Six transcript profiles from the VPA treatment were compared with the 1536 gene transcripts (529 up and 1007 down) from sham and injury-control swine. Microarray analysis and a Database for Annotation, Visualization and Integrated Discovery functional pathway analysis approach identified biologic processes associated with pathologic vascular endothelial function, specifically through functional cluster pathways involving apoptosis/cell death and angiogenesis/vascular development, with five specific genes (THBS1, TNFRSF12A, ANGPTL4, RHOB, and RTN4) identified as members of both functional clusters. This study also examined gene expression of transforming growth factor (TGF)-ß (TGF-ß1, TGF-ß2, and TGF-ß-releasing thrombospondin 1 [THBS1]) and genes expressing vascular endothelial growth factor (VEGF) C, VEGFD, and VEGFR1 and found that these genes were involved in the endothelial functional preservation associated with VPA administration. CONCLUSIONS: VPA minimized pathologic endothelial cell function through the TGF-ß and VEGF functional pathways. This study also implicates that integrated functional modeling and analysis will enable advancements in endothelial dysfunction using a systems biology approach.

Black tea polyphenol (theaflavin) downregulates MMP-2 in human melanoma cell line A375 by involving multiple regulatory molecules.

The tumor-inhibiting property of black tea polyphenol, theaflavin, is well documented. Matrix metalloproteinases (MMPs) play a pivotal role in tumor invasion through degradation of extracellular matrix (ECM). In the present study, we observed the effect of theaflavin on MMP-2, which is upregulated in most tumor types, and its regulatory molecules, in human melanoma cell line, A375. The treatment of theaflavin downregulated the gelatinolytic activity, mRNA and protein expression of MMP-2. It reduced the mRNA and protein expression of membrane type-1 MMP (MT1-MMP) and induced mRNA and protein expression of tissue inhibitor of MMP-2 (TIMP-2), suggesting theaflavin's inhibitory effect on MMP-2 activation. Theaflavin reduced the binding of A375 cell to ECM ligands demonstrating that theaflavin treatment hinders cell-ECM adhesion, cell motility, and integrin-mediated MMP-2 activation. Theaflavin treatment inhibited the protein expression FAK EGFR and ERK, suggesting that, theaflavin treatment downregulates the molecules participating in MMP-2 secretion and regulation. The downregulation of NFchiB suggests downregulation of MMP-2 transactivation. Theaflavin also reduced the tumor volume in syngenic black mice. Thus, we report that theaflavin causes an inhibition of the expression and activity of pro-MMP-2 by a process involving multiple regulatory molecules in human melanoma cells, A375.

[Regulation mechanism for rig-g gene expression induced by all-trans retinoic acid].

To investigate the molecular mechanisms of all-trans retinoic acid (ATRA)-induced rig-g gene expression and to better understand the signal transduction of ATRA during acute promyelocytic leukemia (APL) cell differentiation, the luciferase reporter assay, co-immunoprecipitation and chromatin immunoprecipitation were used to clarify the basic transcriptional factors, which directly initiated the expression of rig-g gene. The results showed that the expression of STAT2, IRF-9 and IRF-1 could be upregulated by ATRA with different kinetics in NB4 cells. IRF-9 was able to interact with STAT2 to form a complex, which could bind the rig-g gene promoter and trigger the rig-g expression. IRF-1 alone could also activate the reporter gene containing rig-g gene promoter, but C/EBPalpha could strongly inhibit this transcription activity of IRF-1. It is concluded that during ATRA-induced APL cell differentiation, IRF-1 is first upregulated by ATRA, and then IRF-1 increases the protein levels of IRF-9 and STAT2 with the downregulation of C/EBPalpha. The complex of IRF-9 and STAT2 is the primary transcriptional factor for rig-g gene induction. This study will be helpful for better understanding the signal transduction networks of ATRA during the course of APL cell differentiation.

Evaluation of high-throughput genotoxicity assays used in profiling the US EPA ToxCast chemicals.

Three high-throughput screening (HTS) genotoxicity assays-GreenScreen HC GADD45a-GFP (Gentronix Ltd.), CellCiphr p53 (Cellumen Inc.) and CellSensor p53RE-bla (Invitrogen Corp.)-were used to analyze the collection of 320 predominantly pesticide active compounds being tested in Phase I of US. Environmental Protection Agency's ToxCast research project. Between 9% and 12% of compounds were positive for genotoxicity in the assays. However, results of the varied tests only partially overlapped, suggesting a strategy of combining data from a battery of assays. The HTS results were compared to mutagenicity (Ames) and animal tumorigenicity data. Overall, the HTS assays demonstrated low sensitivity for rodent tumorigens, likely due to: screening at a low concentration, coverage of selected genotoxic mechanisms, lack of metabolic activation and difficulty detecting non-genotoxic carcinogens. Conversely, HTS results demonstrated high specificity, >88%. Overall concordance of the HTS assays with tumorigenicity data was low, around 50% for all tumorigens, but increased to 74-78% (vs. 60% for Ames) for those compounds producing tumors in rodents at multiple sites and, thus, more likely genotoxic carcinogens. The aim of the present study was to evaluate the utility of HTS assays to identify potential genotoxicity hazard in the larger context of the ToxCast project, to aid prioritization of environmentally relevant chemicals for further testing and assessment of carcinogenicity risk to humans.

Spx mediates oxidative stress regulation of the methionine sulfoxide reductases operon in Bacillus subtilis.

BACKGROUND: All aerobically grown living cells are exposed to oxidative damage by reactive oxygen species (ROS). A major damage by ROS to proteins is caused by covalent modifications of methionine residues giving methionine sulfoxide (Met-SO). Methionine sulfoxide reductases are enzymes able to regenerate methionine and restore protein function after oxidative damage. RESULTS: We characterized the methionine sulfoxide reductase genes msrA and msrB in Bacillus subtilis, forming an operon transcribed from a single sigma A-dependent promoter. The msrAB operon was specifically induced by oxidative stress caused by paraquat (PQ) but not by H2O2. Spx, a global oxidative stress regulator in B. subtilis, is primarily responsible for this PQ-specific induction of msrAB expression. In support of this finding, an spx deletion mutant is extremely sensitive to PQ, and increased expression of msrA was identified in a clpX mutant in which Spx accumulated. However, the Spx effect was also visible under conditions where the protein did not accumulate (PQ treatment), suggesting a specific molecular effect at the level of the Spx protein. Indeed, the CXXC motif of Spx was found essential for its function in the PQ-specific induction of msrAB expression. PQ caused a modification of Spx requiring at least one of the cysteines of the CXXC motif of Spx. The PQ modified form of Spx showed a dynamic change in vivo. CONCLUSION: The Spx mediated PQ-specific regulation pathway of the msrAB operon in B. subtilis is reported. Our results suggest that PQ induced the expression of msrAB partially through an oxidation on Spx via modification of its CXXC motif.

High-specificity and high-sensitivity genotoxicity assessment in a human cell line: validation of the GreenScreen HC GADD45a-GFP genotoxicity assay.

The battery of genetic toxicity tests required by most regulatory authorities includes both bacterial and mammalian cell assays and identifies practically all genotoxic carcinogens. However, the relatively high specificity of the Salmonella mutagenicity assay (Ames test) is offset by the low specificity of the established mammalian cell assays, which leads to difficulties in the interpretation of the biological relevance of results. This paper describes a new high-throughput assay that links the regulation of the human GADD45a gene to the production of Green Fluorescent Protein (GFP). A study of 75 well-characterised genotoxic and non-genotoxic compounds with diverse mechanisms of DNA-damage induction (including aneugens) reveals that the assay responds positively to all classes of genotoxic damage with both high specificity and high sensitivity. The current micro-well assay format does not include metabolic activation, but a separate low-throughput protocol demonstrates a successful proof-of-principle for an S9 metabolic activation assay with the model pro-mutagen cyclophosphamide. The test should be of value both as a tool in the selection of candidate compounds for further development, where additional data may be required because of conflicting information from the in vitro test battery, or in product development areas where the use of animals is to be discontinued. As a microplate assay however, it has the qualities of high throughput and low compound use that will facilitate its application in early screening for genotoxic liability.

Hypermethylation patterns in the Fhit regulatory region are tissue specific.

DNA hypermethylation is associated with decreased expression of tumor suppressor genes. We previously observed decreased Fhit expression and Fhit promoter region hypermethylation in rodent tumors induced by various carcinogens, and noted that the 5' regulatory regions in the promoter, exon 1, and intron 1 were differentially methylated, depending on the tissue of origin. Because different carcinogens were used for induction of tumors of the different organs, we could not conclude that the methylation patterns were tissue-specific. To determine if in rat tissues: (1) Fhit methylation status is related to expression levels and (2) Fhit methylation patterns were tissue- or carcinogen-specific, we examined Fhit methylation status and expression levels in DMBA- and MNU-induced benign and malignant mammary tumors. Fhit intron 1 was methylated in 3/9 DMBA and all of MNU-induced benign mammary tumors, in association with reduced Fhit expression levels; Fhit promoter and intron 1 were methylated in all DMBA and MNU-induced carcinomas in association with highly reduced Fhit expression levels. Treatment of rat cancer cells in vitro with the DNA methyltransferase inhibitor, 5'-Aza-2'deoxycytidine, for 4 d, increased Fhit expression and altered the methylation status. Before treatment, both promoter and intron 1 regions were methylated; after treatment, only intron 1 remained methylated. Thus, in carcinogen-exposed rat tissues there is an overall association of Fhit expression with regulatory region methylation, and hypermethylation patterns did not vary with carcinogen. The specific patterns of hypermethylated CpGs in the Fhit regulatory regions thus appear to be tissue-specific.

Transcriptional regulation of tyrosine hydroxylase by estrogen: opposite effects with estrogen receptors alpha and beta and interactions with cyclic AMP.

Reported effects of estrogen administration on tyrosine hydroxylase (TH) gene expression are confusing. Therefore, we studied the mechanism of regulation of TH transcription by estrogen with different estradiol receptor (ER) subtypes. PC12 cells, transiently co-transfected with expression vector for ERalpha or ERbeta, and luciferase gene under control of the TH promoter, were treated with 17 beta-estradiol (E2). E2 doubled luciferase activity with ERalpha; however, it was decreased with ERbeta. Mapping the TH promoter showed that the putative half estrogen response element (ERE) motif at - 675, as well as the activation protein 1 motif at - 205, were not required for response to E2 with either ER. The specificity protein 1/early growth response gene 1 (Egr 1) motif was required for the E2-elicited response with ERbeta, but not with ERalpha. Deletion of the cyclic AMP/Ca2+ response element (CRE/CaRE) nearly abolished E2-triggered responses with either ER. Further analysis revealed an imperfect canonical putative ERE overlapping with CRE/CaRE and Nurr1 response element. Oligonucleotides spanning this ERE displayed binding to ER, Cyclic AMP Response Element Binding Protein (CREB) and other proteins. Moreover, E2 attenuated the increase in TH transcription seen with cyclic AMP analogs. Thus, TH is transcriptionally regulated by estradiol in opposite directions depending on ER subtype. The overlapping ERE and CRE/CaRE may integrate interactions elicited by various regulators of TH transcription including cAMP and estrogens.

Molecular cloning and analysis of the mouse gicerin gene.

Gicerin is a cell adhesion molecule, which has five immunoglobulin-like loop structures in an extracellular domain followed by a single transmembrane domain and a short cytoplasmic tail. We have reported that gicerin participates in neurite extension and structural organization of the nervous system, and its expression in the nervous system is high during the development and dramatically decreased after birth. To elucidate the mechanism how the expression of gicerin is regulated, we performed a genomic cloning of a mouse gicerin. A fragment of 16 kbp genomic clone contained 8 kbp gicerin gene composed of 16 exons with 6 kbp upstream region. Genomic cloning revealed that two isoforms of gicerin were generated by an alternative splicing of exon 15 results in cytoplasmic domains composed of either 63 or 21 amino acids. As for an expressional regulation of gicerin, we found that the mRNA content of gicerin in PC12 cells was regulated by cAMP. Quantitative-PCR analysis revealed that forskolin induced four-fold increase of gicerin mRNA. To characterize the involvement of its promoter region, we examined the promoter activity in PC12 cells by a luciferase-reporter assay. We found that a CRE site located at 60 bp upstream of gicerin gene was responsible for the increase of its mRNA induced by forskolin.

Transcriptional regulation of thioredoxin reductase 1 expression by cadmium in vascular endothelial cells: role of NF-E2-related factor-2.

Thioredoxin reductase (TrxR) is a selenoprotein that catalyzes the reduction of the active site disulfide of thioredoxin (Trx), which regulates the redox status of the cells. In the present study, we found that TrxR1, one of the three TrxR isozymes, was induced by cadmium as well as tumor necrosis factor alpha (TNFalpha) in bovine arterial endothelial cells (BAEC), and investigated the mechanism of cadmium-induced TrxR1 expression. We here showed that cadmium, differently from TNFalpha, enhanced the promoter activity of the 5'-flanking region of human TrxR1 gene (nucleotides -1692 to +49). Deletion and site-directed mutation of antioxidant responsive element (ARE) (nucleotides -62 to -48) in this region abolished the response to cadmium. Overexpression of NF-E2-related factor-2 (Nrf2) augmented the TrxR1 promoter activity. In contrast, overexpression of the dominant negative mutant of Nrf2 suppressed cadmium-induced activation of TrxR1 promoter through the ARE. Chromatin immunoprecipitation (ChIP) assays showed that anti-Nrf2 antibody precipitated ARE from the chromatin of the cadmium-treated cells. These results indicated that cadmium-induced TrxR1 gene expression is mediated by the activation of Nrf2 transcription factor and its binding to ARE in the TrxR1 gene promoter. We further found that in addition to cadmium, the activators of Nrf2, such as diethyl maleate (DEM) and arsenite, induced both TrxR1 and Trx gene expression in BAEC. Nrf2 might play an important role in the regulation of the cellular Trx system consisting of Trx and TrxR.

Intronic hormone response elements mediate regulation of FKBP5 by progestins and glucocorticoids.

Expression of FKBP51, a large molecular weight immunophilin, is strongly enhanced by glucocorticoids, progestins, and androgens. However, the activity of a 3.4-kb fragment of the FKBP51 gene (FKBP5) promoter was only weakly increased by progestin and we show here that it is unresponsive to glucocorticoids and androgens. The entire FKBP5 was scanned for consensus hormone response elements (HREs) using MatInspector. We found that 2 regions of intron E, which are conserved in rat and mouse FKBP5, contain HRE-like sequences with high match scores. Deoxyribonucleic acid fragments (approximately 1 kb in length) containing these regions were amplified and tested in reporter gene assays for steroid responsiveness. One region of intron E of FKBP5 (pIE2) conferred both glucocorticoid and progestin responsiveness to 2 heterologous reporter genes, whereas the other, less-conserved region of intron E (pIE1) was responsive only to progestins. The inclusion of pIE1 upstream of pIE2 (pIE1IE2) enhanced progestin but not glucocorticoid responsiveness. None of the constructs containing intronic sequences was responsive to androgens. Mutation of the putative HREs within pIE1 and pIE2 eliminated hormone responsiveness. Electrophoretic mobility shift assays demonstrated that progesterone receptors (PR) bound to the HRE in pIE1, whereas both PR and glucocorticoid receptors interacted with the HRE in pIE2. These data suggest that distal intronic elements significantly contribute to transcriptional regulation of FKBP5 by glucocorticoids and progestins.

Ras inhibition leads to transcriptional activation of p53 and down-regulation of Mdm2: two mechanisms that cooperatively increase p53 function in colon cancer cells.

Activated Ras, operating through the Raf/MEK/ERK pathway, is known to regulate transcription of both Mdm2 and its inhibitor p19ARF, resulting in opposing effects on the tumor suppressor protein p53. We show here that a decrease in Ras in SW480 cells induced either by the Ras inhibitor farnesylthiosalicylic acid (FTS) or by K-Ras antisense oligonucleotides, resulted in a similar increase in p53 protein. The increase in p53 was accompanied by an increase in p21(waf1/cip1) mRNA transcripts and protein. Consistent with the Ras/Raf/MEK/ERK-mediated control of Mdm2, treatment of SW480 cells with the Ras inhibitor FTS caused a marked (80%) decrease in Mdm2, which itself would account for the increase in p53. However, FTS also caused a 1.6-fold increase in p53 mRNA, indicative of a Ras-dependent mechanism that regulates p53 transcription. Thus, oncogenic Ras appears to attenuate p53 in SW480 cells by two independent regulatory mechanisms, the one leading to increased Mdm2-dependent p53 degradation and the other leading to a decrease in p53 transcription.

The heat-shock-induced suppression of the IkappaB/NF-kappaB cascade is due to inactivation of upstream regulators of IkappaBalpha through insolubilization.

Heat shock (HS) was found to suppress the IkappaB/NF-kappaB cascade via the inhibition of IkappaB kinase (IKK) activity; however, the mechanism has not been clear. This study was undertaken to elucidate the detail of the mechanism involved. TNF-alpha-induced activation of IKK was suppressed by HS in human bronchial epithelial cells, and this was associated with the absence of IKK in the immunoprecipitates. It was not due to a degradation of IKK, but due to a conversion of IKK from a soluble to an insoluble form. IKK lost its activity rapidly upon exposure to HS in vitro. The time course of the insolubilization of IKK coincided with the decrease in IKK activity. However, inhibition of IKK insolubilization by the induction of thermotolerance did not reverse the HS-induced suppression of IKK activation and IkappaBalpha degradation. Upstream activators of IKK, such as NF-kappaB-inducing kinase (NIK) and IL-1R-associated kinase (IRAK) were also insolubilized by HS. The HS-induced insolubilization of NIK was not blocked by the induction of thermotolerance. Overexpression of NIK resumed TNF-alpha-induced activation of IKK in thermotolerant cells. These results indicate that the loss of activity of NIK, IRAK, and IKK through insolubilization is responsible for the HS-induced suppression of IkappaB/NF-kappaB pathway.