Electromobility Gel Shift Assays Research Articles

Abstract Tu013: ETV2 Transcriptionally Activates Rig1 Gene Expression and Promotes Reprogramming of the Endothelial Lineage

ETV2 is a master regulator and pioneer factor for the endothelial lineage. Previous studies have established that this transcription factor was essential for embryogenesis and direct reprogramming of fibroblasts to endothelial cells. ETV2-mediated direct reprogramming holds therapeutic potential in treating ischemic tissues including ischemic heart disease, yet the underlying molecular mechanisms remain to be completely defined. In the current studies, we focused on deciphering the mechanisms that governed ETV2-mediated reprogramming, including the role of RIG1 inflammatory responses. We hypothesized that ETV2 transcriptionally activated Rig1 gene expression, increased expression of its downstream network [RIG1-like receptor (RLR) pathway] and modulated the reprogramming of fibroblasts to endothelial cells. Mouse embryonic fibroblasts (MEFs) with inducible ETV2 expression system were used to overexpress ETV2, and single-cell RNA-seq defined transcript expression involved in RLR signaling pathways. ChIP-seq, electromobility gel shift assays (EMSA), and transcriptional assays were used to verify that ETV2 was a direct upstream activator of Rig1 gene expression. Both RIG1 and RLR pathway proteins were significantly upregulated in ETV2-overexpressing MEFs. ETV2 mediated the Rig1 upregulation through direct binding to the Rig1 promoter and transcriptional activation of Rig1 expression. Knockdown of Rig1 using shRNA significantly (p < 0.05) reduced the efficiency of fibroblast to endothelial cell reprogramming more than 70%. These results highlight that 1) ETV2 activates inflammatory pathways and 2) RIG1 is direct downstream target of ETV2 and together they have an essential role in the reprogramming process. These findings extend our current understanding of the molecular mechanisms underlying ETV2-mediated reprogramming and will be important in the design of a revascularization strategy for the treatment of ischemic heart disease.

Neuroprotective Capability of Narcissoside in 6-OHDA-Exposed Parkinson's Disease Models through Enhancing the MiR200a/Nrf-2/GSH Axis and Mediating MAPK/Akt Associated Signaling Pathway.

We assessed the antioxidant potential of narcissoside from Sambucus nigra flowers (elderflowers) in Parkinson's disease models in vitro and in vivo. The results showed that narcissoside lessened the 6-hydroxydopamine (6-OHDA)-induced increase in reactive oxygen species (ROS) and apoptosis in SH-SY5Y cells. In the 6-OHDA-exposed Caenorhabditis elegans model, narcissoside reduced degeneration of dopaminergic neurons and ROS generation, and also improved dopamine-related food-sensitive behavior and shortened lifespan. Moreover, NCS increased total glutathione (GSH) by increasing the expression of the catalytic subunit and modifier subunit of γ-glutamylcysteine ligase in cells and nematodes. Treatment with a GSH inhibitor partially abolished the anti-apoptotic ability of narcissoside. Furthermore, narcissoside diminished the 6-OHDA-induced phosphorylation of JNK and p38, while rising activities of ERK and Akt in resisting apoptosis. The antioxidant response element (ARE)-luciferase reporter activity analysis and electromobility gel shift assay showed that narcissoside promotes the transcriptional activity mediated by Nrf2. Finally, we found that narcissoside augmented the expression of miR200a, a translational inhibitor of the Nrf2 repressor protein Keap1. Downregulation of Nrf2 and miR200a by RNAi and anti-miR200a, respectively, reversed the neuroprotective ability of narcissoside. In summary, narcissoside can enhance the miR200a/Nrf2/GSH antioxidant pathway, alleviate 6-OHDA-induced apoptosis, and has the neuroprotective potential.

P130 And pRb in the Maintenance of Transient Quiescence of Mesenchymal Stem Cells.

An effective regulation of quiescence plays a key role in the differentiation, plasticity, and prevention of stem cells from becoming malignant. The state of quiescence is being controlled by the pRb family proteins which show overlapping functions in cell cycle regulation; however, their roles in controlling the proliferation of mesenchymal stem cells (MSCs) remain to be understood. This study investigated the regulation of transient quiescence using growth curves, proliferation assay, the cytometric evaluation of cell cycle, Western blotting, and the electromobility gel shift assay (EMSA) on synchronized MSCs of the C3H10Т1/2 and control cells with different statuses of pRb proteins. It has been found that functional steady-state level of p130 but not pRb plays a critical role for entering, exiting, and maintenance of transient quiescence in multipotent mesenchymal stem cells.

Exploring inflammatory and apoptotic signatures in distinct Crohn's disease phenotypes: Way towards molecular stratification of patients and targeted therapy

BackgroundCrohn's disease (CD) is chronic inflammatory bowel disease with different phenotypic characteristics influencing disease prognosis and therapeutic strategies. The aim of this pilot study was to analyze selected inflammatory and apoptotic markers in non-inflamed and inflamed samples of ileal mucosa of non-stricturing/non-penetrating (NS/NP) and stricturing (S) CD mucosal phenotypes in order to characterize their distinct profiles. MethodsFrom twenty CD patients (9 NS/NP, 11 S) paired non-inflamed and inflamed ileal biopsies were collected and used for analysis of cytokine (TNF and IL6) and apoptotic (Bcl2, Bax, Fas and FasL) genes' expression levels by real-time PCR, while NFκB transcriptional potency was assessed by electromobility gel shift assay. ResultsOur results demonstrated significant upregulation of TNF and IL6 in inflamed area of both NS/NP (p = 0.03, p = 0.01) and S phenotypes (p = 0.04, p = 0.04), respectively. However, TNF increase was more prominent in NS/NP compared to S inflamed mucosa (p = 0.02). Also, level of proapoptotic Bax was significantly higher in NS/NP compared to S inflamed mucosa (p = 0.01). Opposing transcription potency of NFκB has been detected between two phenotypes: being decreased in NS/NP (p = 0.07) and increased in S (p = 0.1) inflamed compared to non-inflamed mucosa, demonstrating trend towards statistical significance. ConclusionsWe found that two distinct CD phenotypes have specific molecular signatures. Obtained results could direct improvement of current and development of new therapeutic strategies based on more specific molecular stratification of CD patients.

Solution Structure of an Archaeal DNA Binding Protein with an Eukaryotic Zinc Finger Fold

While the basal transcription machinery in archaea is eukaryal-like, transcription factors in archaea and their viruses are usually related to bacterial transcription factors. Nevertheless, some of these organisms show predicted classical zinc fingers motifs of the C2H2 type, which are almost exclusively found in proteins of eukaryotes and most often associated with transcription regulators. In this work, we focused on the protein AFV1p06 from the hyperthermophilic archaeal virus AFV1. The sequence of the protein consists of the classical eukaryotic C2H2 motif with the fourth histidine coordinating zinc missing, as well as of N- and C-terminal extensions. We showed that the protein AFV1p06 binds zinc and solved its solution structure by NMR. AFV1p06 displays a zinc finger fold with a novel structure extension and disordered N- and C-termini. Structure calculations show that a glutamic acid residue that coordinates zinc replaces the fourth histidine of the C2H2 motif. Electromobility gel shift assays indicate that the protein binds to DNA with different affinities depending on the DNA sequence. AFV1p06 is the first experimentally characterised archaeal zinc finger protein with a DNA binding activity. The AFV1p06 protein family has homologues in diverse viruses of hyperthermophilic archaea. A phylogenetic analysis points out a common origin of archaeal and eukaryotic C2H2 zinc fingers.

Equilibrium binding and kinetic characterization of putative tetracycline repressor family transcription regulator Fad35R from Mycobacterium tuberculosis

Fatty acids play critical role in the survival and virulence of Mycobacterium tuberculosis (Mtb). Activation of fatty acids by acyl-CoA synthetases (Fad) into fatty acyl-CoA is the first and one of the crucial steps in fatty acid metabolism. Mtb possesses 36 fatty acyl-CoA synthetases, unlike Escherichia coli, which has single enzyme. However, the mechanisms by which the expression of these multiple Fad genes is regulated remain uncharacterized. We characterized the DNA- and ligand-binding properties of a putative tetracycline repressor family regulator, named Fad35R, located upstream of the Fad35 gene and ScoA-citE operon. We identified a palindromic regulatory motif upstream of Fad35 and characterized the binding of Fad35R to this motif. Equilibrium binding studies show that Fad35R binds to this motif with high affinity (K(d) ∼ 0.033 μm) and the specificity of binding was confirmed by an electromobility gel shift assay. Kinetic studies indicate that faster association (k(a,avg) ∼ 5.4 × 10(4) m(-1) · s(-1)) and slower dissociation rates (k(d,avg) ∼ 5.84 × 10(-4) s(-1)) confer higher affinity. The affinity for the promoter is maximum at 300 mm NaCl but decreases rapidly beyond this range. Ligand-binding studies indicate that Fad35R binds specifically to tetracycline and also binds to fatty acid derivatives. The promoter-binding affinity is decreased significantly in the presence of palmityl-CoA, suggesting that Fad35R can sense the levels of activated fatty acids and alter its DNA-binding activity. Our results suggest that Fad35R may be the functional homologue of FadR and controls the expression of genes in a metabolite-dependent manner.

Structure and Function Relationship Study of Allium Organosulfur Compounds on Upregulating the Pi Class of Glutathione S-Transferase Expression

Allium organosulfides are potential chemopreventive compounds due to their effectiveness on the induction of phase II detoxification enzyme expression. In this study, we examined the structure and function relationship among various alk(en)yl sulfides on the expression of the pi class of glutathione S-transferase (GSTP) in rat Clone 9 cells, and what mechanism is involved. Cells were treated with 300 μM dipropyl sulfide (DPS), dipropyl disulfide (DPDS), propyl methyl sulfide (PMS), and propyl methyl disulfide (PMDS) for 48 h. DPDS and PMDS displayed more potency on GSTP protein and mRNA induction than that of DPS and PMS. Next, we compared the effectiveness of DPDS, PMDS, and diallyl disulfide (DADS), which have the same number of sulfur atoms but differ in the side alk(en)yl groups. The maximum increases on protein expression, mRNA level, and enzyme activity were noted in cells treated with DADS, followed by DPDS and PMDS. A reporter assay showed that three disulfides increased GSTP enhancer I (GPE I) activity (P < 0.05) in the order DADS > DPDS ≥ PMDS. Electromobility gel shift assays showed that the DNA binding of GPE I to nuclear proteins reached a maximum at 1 to 3 h after alk(en)yl disulfide treatment. Supershift assay revealed that c-jun bound to GPE I. Silencing of extracellular signal-regulated kinase (ERK) 2 expression inhibited c-jun activation and GSTP induction. Results suggest that both the type of alk(en)yl groups and number of sulfur atoms are determining factors of allium organosulfides on inducing GSTP expression, and it is likely related to the ERK-c-Jun-GPE I pathway.

Critical role of transcription factor PU.1 in the expression of CD80 and CD86 on dendritic cells

AbstractIn this study, we investigated the role of a transcription factor, PU.1, in the regulation of CD80 and CD86 expression in dendritic cells (DCs). A chromatin immunoprecipitation assay revealed that PU.1 is constitutively bound to the CD80 and CD86 promoters in bone marrow–derived DCs. In addition, co-expression of PU.1 resulted in the transactivation of the CD80 and CD86 promoters in a reporter assay. The binding of PU.1 to cis-enhancing regions was confirmed by electromobility gel-shift assay. As expected, inhibition of PU.1 expression by short interfering RNA (siRNA) in bone marrow–derived DCs resulted in marked down-regulation of CD80 and CD86 expression. Moreover, overexpression of PU.1 in murine bone marrow–derived lineage-negative cells induced the expression of CD80 and CD86 in the absence of monocyte/DC-related growth factors and/or cytokines. Based on these results, we conclude that PU.1 is a critical factor for the expression of CD80 and CD86. We also found that subcutaneous injection of PU.1 siRNA or topical application of a cream-emulsified PU.1 siRNA efficiently inhibited murine contact hypersensitivity. Our results suggest that PU.1 is a potential target for the treatment of immune-related diseases.

GATA Depletion Impacts Insulin-like Growth Factor 1 mRNA and Protein Levels in Luteinizing Porcine Granulosa Cells1

GATA4 and GATA6 are zinc-finger transcription factors that regulate specific genes involved in steroidogenesis. Using RNA interference (RNAi)-mediated reduction of GATA4 and/or GATA6 with microarray analysis, we aimed to identify novel GATA target genes in luteinizing porcine granulosa cells under vehicle- and cAMP-treated conditions. Microarray analysis identified IGF1 mRNA to be cAMP- and GATA-responsive, and real-time PCR demonstrated that the cAMP-induced increase in IGF1 mRNA was reduced under conditions of GATA6 depletion and GATA4 plus GATA6 depletion, but not GATA4 depletion. Insulin-like growth factor 1 protein levels in media were also decreased by GATA6 or GATA4 plus GATA6 reduction. IGFBP2 and IGFBP4 mRNAs were increased and IGFBP5 mRNA decreased with vehicle and cAMP treatment under GATA4 plus GATA6 RNAi conditions. GATA6 reduction alone increased basal IGFBP4 and decreased IGFBP5 with both vehicle and cAMP, and GATA4 reduction alone lowered cAMP IGFBP5 levels with cAMP. No changes in IGFBP3 mRNA were observed with GATA reduction relative to the control RNAi condition. Levels of insulin-like growth factor binding proteins 2-5 in media as assessed by Western ligand blotting were not altered by GATA reduction. Electromobility gel shift assays with two GATA-containing oligonucleotides of the IGF1 5'-regulatory region showed GATA4 and GATA6 could bind the more proximal GATA-B site. These studies indicate that although GATA4 and GATA6 can bind the porcine IGF1 5'-region, GATA6 is functionally most important for cAMP-stimulated mRNA levels. Using microarray analysis, we identified other mRNAs that were altered by GATA-reduced conditions, including ALDH1, DIO2, and EDNRB. Our findings further support GATA as a coordinator of endocrine/paracrine/autocrine signals in the ovary.

Methionine restriction up‐regulates the expression of the pi class of glutathione S‐transferase partially via the extracellular signal‐regulated kinase‐activator protein‐1 signaling pathway initiated by glutathione depletion

Understanding the molecular events underlying gene regulation by amino acids has attracted increasing attention. Here, we explored whether the mechanism by which methionine restriction affects the expression of the pi class of glutathione S-transferase (GSTP) is related to oxidative stress initiated by glutathione (GSH) depletion. Rat primary hepatocytes were cultured in an L-15-based medium in the absence or presence of 200 muM L-buthionine sulfoximine (BSO) or in a methionine-restricted L-15 medium supplemented with 20 muM L-methionine up to 72 h. BSO and methionine restriction time-dependently induced GSTP mRNA and protein expression in a similar pattern accompanied by a decrease in the cellular GSH level. The phosphorylation of extracellular signal-regulated kinase (ERK), but not of c-Jun NH(2)-terminal kinase and p38, was stimulated by methionine restriction and BSO. Electromobility gel shift assay showed that the DNA-binding activity of nuclear activator protein-1 (AP-1) increased in cells exposed to methionine restriction or BSO. With the ERK inhibitor FR180204, AP-1 activation and GSTP expression were abolished. Moreover, the induction of GSTP by methionine restriction and BSO was reversed by GSH monoethyl ester and N-acetylcysteine. Our results suggest that methionine restriction up-regulates GSTP gene expression, which appears to be initiated by the ERK-AP-1 signaling pathway through GSH depletion in rat hepatocytes.

Sulforaphane and α-Lipoic Acid Upregulate the Expression of the π Class of Glutathione S-Transferase through c-Jun and Nrf2 Activation

The anticarcinogenic effect of dietary organosulfur compounds has been partly attributed to their modulation of the activity and expression of phase II detoxification enzymes. Our previous studies indicated that garlic allyl sulfides upregulate the expression of the π class of glutathione S-transferase (GSTP) through the activator protein-1 pathway. Here, we examined the modulatory effect of sulforaphane (SFN) and α-lipoic acid (LA) or dihydrolipoic acid (DHLA) on GSTP expression in rat Clone 9 liver cells. Cells were treated with LA or DHLA (50–600 μmol/L) or SFN (0.2–5 μmol/L) for 24 h. Immunoblots and real-time PCR showed that SFN, LA, and DHLA dose dependently induced GSTP protein and mRNA expression. Compared with the induction by the garlic organosulfur compound diallyl trisulfide (DATS), the effectiveness was in the order of SFN > DATS > LA = DHLA. The increase in GSTP enzyme activity in cells treated with 5 μmol/L SFN, 50 μmol/L DATS, and 600 μmol/L LA and DHLA was 172, 75, 122, and 117%, respectively (P < 0.05). A reporter assay showed that the GSTP enhancer I (GPEI) was required for GSTP induction by the organosulfur compounds. Electromobility gel shift assays showed that the DNA binding of GPEI to nuclear proteins reached a maximum at 0.5–1 h after SFN, LA, and DHLA treatment. Super-shift assay revealed that the transcription factors c-jun and nuclear factor erythroid-2 related factor 2 (Nrf2) were bound to GPEI. These results suggest that SFN and LA in either its oxidized or reduced form upregulate the transcription of the GSTP gene by activating c-jun and Nrf2 binding to the enhancer element GPEI.

Diverse poly(A) binding proteins mediate internal translational initiation by a plant viral IRES

During 5'-cap-dependent translation, methylated 5'-cap and 3'-poly(A) tail work synergistically in a poly(A) binding protein (PABP)-dependent manner to facilitate translation via promoting the formation of a closed mRNA loop. On the other hand, during internal translation initiation, the requirement for and the roles of 3'-poly(A) tail and PABP vary depending on specific characteristics of each internal ribosomal entry site (IRES). In this study, we analyzed the effect of 3'-poly(A) tail and phylogenetically divergent PABPs on a polypurine tract-containing IRES element derived from the coat protein gene of crucifer-infecting tobamovirus (CrTMV IRESCP). We find that mutations in the internal polypurine tract decrease IRES activity in a heterologous (mammalian) system in vivo. Moreover, these mutations decrease the high-affinity binding of all phylogenetically divergent PABPs derived from Arabidopsis and yeast in electro mobility gel shift assays in vitro. Partial PABP depletion and reconstitution assays using Arabidopsis-derived PABP2, 3, 5, 8 and yeast Pab1p provide further evidence that CrTMV IRESCP requires PABP for maximal activity. Furthermore, stronger enhancement in the presence of 3'-poly(A) and the absence of 5'-methylated cap suggests a potential joint interaction between PABP, the CrTMV IRESCP and the 3'-poly(A).

Analysis of a Promoter Polymorphism in the SMDF Neuregulin 1 Isoform in Schizophrenia

Background/Aims: Neuregulin 1 (NRG1) is a positional candidate gene in schizophrenia (SZ). Two major susceptibility loci in the NRG1 gene approximately one million nucleotides apart have been identified in genetic studies. Several candidate functional allelic variants have been described that might be involved in disease susceptibility. However, the findings are still preliminary. We recently mapped active promoters and other regulatory domains in several SZ and bipolar disorder (BD) candidate genes using ChIP-chip (chromatin immunoprecipitation hybridized to microarrays). One was the promoter for the NRG1 isoform, SMDF, which maps to the 3′ end of the gene complex. Analysis of the SNP database revealed several polymorphisms within the approximate borders of the region immunoprecipitated in our ChIP-chip experiments, one of which is rs7825588. Methods: This SNP was analyzed in patients with SZ and BD and its effect on promoter function was assessed by electromobility gel shift assays and luciferase reporter constructs. Results: A significant increase in homozygosity for the minor allele was found in patients with SZ (genotype distribution χ² = 7.32, p = 0.03) but not in BD (genotype distribution χ² = 0.52, p = 0.77). Molecular studies demonstrated modest, but statistically significant allele-specific differences in protein binding and promoter function. Conclusion: The findings suggest that homozygosity for rs725588 could be a risk genotype for SZ.

Variations in the promoter of CYP21A2 gene identified in a Chinese patient with simple virilizing form of 21‐hydroxylase deficiency

The variations in the transcriptional regulatory regions of CYP21A2 were rarely investigated in patients with 21-hydroxylase deficiency (21-OHD). The present study aims to verify that the variations in the promoter of CYP21A2 relate to the classical form of 21OHD. CYP21A2 was screened for mutations in 20 patients with the simple virilizing form of 21OHD, including the promoter region. The transcriptional activities of the variants in the promoter were investigated using a dual-reporter luciferase assay system and electromobility gel shift assays. The heterozygous variants -447 A > G, -443InsA, -306G > C, -295T > C, -294 A > C, -283 A > G, -281T > G, -210T > C, -199C > T, -196 A > T, -126C > T, -113G > A, -110T > C, -103 A > G and -4C > T in the promoter of CYP21A2 gene were identified in a patient with simple virilizing form of 21OHD. The transcriptional activities of the promoter with the variants were reduced to 50% of the wild type. We speculated that the 15 variants in the promoter of CYP21A2 combined with a compound heterozygous mutation Q318X lead to a simple virilizing form of 21OHD.

Mucosal NOD2 expression and NF-κB activation in pediatric Crohnʼs disease

Recent advances in the pathogenesis of Crohn's disease (CD) have suggested that an aberrant innate immune response initiates the cascade of events leading to T-cell activation and to disease development. NOD2 protein, which is mainly expressed by innate immunity cells, appears to play a key role against bacteria by triggering a host defense response through the activation of the transcriptor factor NF-kappaB and a consequent proinflammatory cytokine production. The present study was aimed at investigating the expression and activity of NOD2, NF-kappaB, and of 2 proinflammatory cytokines, TNFalpha and IL-1beta, in mucosal biopsies of CD affected children compared to healthy controls. In all, 22 children with active CD and 10 matched controls were entered in the study. mRNA and protein expressions were detected using reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blot; NF-kappaB binding activity was assessed by electromobility gel shift assay (EMSA). NOD2 and IL-1beta mRNAs were upregulated in CD children. Protein levels of NOD2, TNFalpha, and nuclear NF-kappaB, as well as the binding activity of NF-kappaB to a consensus DNA sequence, were significantly increased in inflamed mucosa of patients as compared to controls. Moreover, NF-kappaB activity was strongly upregulated in patients also when bound to the NOD2 promoter site. No difference was seen between patients and controls when NF-kappaB binding activity was determined in the uninflamed tissue. This study suggests that altered mechanisms regulating NOD2 induction, NF-kappaB activation and cytokine production may contribute to dysregulate the innate immune response underlying pediatric CD.

Interferons Downregulate the Expression of WT1 in Myeloid Leukemia through the JAK/STAT Pathway and This Potentiates Chemotherapy Induced Apoptosis.

Introduction: Expression of the WT1 gene is tightly regulated during normal hematopoietic development, and dysregulation of WT1 expression can lead to leukemia. However, relatively little is known about how the WT1 gene is regulated. Whereas WT1 overexpression blocks myeloid progenitor differentiation, the interferons (IFN, both type I and type II) have been shown to promote this differentiation, which underlies their clinical use in the myeloproliferative disorders. We hypothesized that the effect of IFN on myelopoeisis was via regulation of WT1 expression.Materials and Methods: We examined the effect of interferon-(both α and γ) treatment on WT1 mRNA expression on the CML cell line K562 and the AML cell line KG1. We studied the gene expression by RT-PCR and the protein expression by western blotting and flow cytometry by methods previously described. WT1 gene was knocked down using shRNA transfected by standard lipofectamine method. Cell viability was measured by annexin V staining by FACS.Results: Interferon (α and γ) treatment resulted in downregulation of WT1 mRNA expression indicating a common IFN-induced mechanism(s). Importantly, downregulation of WT1 was also associated with downregulation of downstream WT1 targets, such as the bcl-2 and c-myc. Blocking STAT1 phosphorylation by epigallocatechin (EGCG) completely abrogated this effect of IFNg on WT1 protein expression. These findings also suggested that this regulation was primarily at the transcription level, which lead us to initially examine the WT1 promoter. Analysis of the promoter region revealed no canonical STAT1 binding sites; however IFN-induced STAT1 signaling transcriptionally regulates the expression of the transcription factor IRF-1. We first analyzed the WT1 promoter (Transcription Element Search Software, www.cbil.upenn.edu/tess) for possible IRF-1 binding sites and identified a putative site at -222 to - 240 of transcription start site. The actual interaction was confirmed by Electromobility gel shift assay (EMSA) using oligonucleotides derived from the putative sequence. Treatment with IFNg substantially increased the amount of shifted probe, consistent with increased oligonucleotide binding by IRF-1 protein. IFNg treatment alone had little effect on cell viability. However, treatment with IFNg or INFa resulted in significant sensitization of both K562 and KG1 to chemotherapy and significantly increases apoptosis in both cell lines. Since it is possible that IFN-mediated sensitization is due to one of the myriad of interferon responses we specifically down regulated WT1 expression by shRNA in K 562 cells. The chosen shRNA clone alone induced some apoptosis but the combination of WT1 silencing plus etoposide markedly increased cell death.Conclusions: In summary our data strongly support the hypothesis that downregulation of WT1 expression impairs the survival of leukemic blasts, and sensitizes these cells to chemotherapy-induced apoptosis. The data also demonstrate that WT1 expression can be modulated by clinically available agents (INFa), and point to potentially new strategies to target AML. Ongoing studies in primary AML cells will not only confirm the findings above but also lay the foundation for therapeutic targeting of a key protein necessary for leukemia cell survival in the face of cytotoxic therapy.

Environmental Toxicants May Modulate Osteoblast Differentiation by a Mechanism Involving the Aryl Hydrocarbon Receptor

The AHR mediates many of the toxicological effects of aromatic hydrocarbons. We show that AHR expression in osteoblasts parallels the induction of early bone-specific genes involved in maturation. The AHR may not only mediate the effects of toxicants, but with an as yet unidentified ligand, be involved in the differentiation pathways of osteoblasts. Metabolic bone diseases arise as a result of an imbalance in bone cell activities. Recent evidence suggests that environmental toxicants may be contributing factors altering these activities. One candidate molecule implicated in mediating the toxic effects of exogenous compounds is the aryl hydrocarbon receptor (AHR). Osteoblasts isolated from neonatal rat calvaria were analyzed for AHR expression by quantitative PCR, Western blot, and immunohistochemistry. In addition, AHR activation was evaluated by electromobility gel shift assay and fluorescence microscopy. Our findings showed AHR expression in mature osteoblasts in vivo. The pattern of AHR expression peaks after alkaline phosphatase and before induction of osteocalcin. We first show that AHR functions as a transactivating receptor in osteoblasts, as evidenced by its ligand-dependent migration to the nucleus and its association with known dioxin response elements. AHR activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) mediated the induction of cytochrome p450 1A1 and cycloxygenase-2 protein levels. This effect could be inhibited by the potent AHR antagonist, 3'4 methoxynitroflavone. Furthermore, lead treatment of osteoblasts upregulates the expression of AHR mRNA and protein levels, supporting a novel mechanism whereby lead in the skeleton may increase the sensitivity of bone cells to toxicant exposure. These data imply that the AHR mediates the effects of aromatic toxicants on bone and that AHR expression is regulated during osteoblast differentiation.

Diallyl Disulfide and Diallyl Trisulfide Up-Regulate the Expression of the π Class of Glutathione S-Transferase via an AP-1-Dependent Pathway

Garlic organosulfur compounds are recognized as potential chemopreventive compounds. This protection is related to the induction of phase II detoxification enzymes. We previously reported that diallyl disulfide (DADS) and diallyl trisulfide (DATS) up-regulate the gene expression of the pi class of glutathione S-transferase (GSTP) and that an enhancer element named GPE I is required for this induction. In the present study, we further investigated the signal pathway involved in DADS and DATS up-regulation of this detoxification enzyme in Clone 9 cells. Cells were cultured with 25-200 micromol/L of DADS or DATS for 24 h. Western and Northern blots showed that both garlic allyl sulfides concentration dependently induced GSTP protein and mRNA expression, respectively. Changes in GST activity toward ethacrynic acid were consistent with the increase in GSTP expression (P < 0.05). Electromobility gel shift assay showed that the DNA binding activity of nuclear activator protein-1 (AP-1) is concentration-dependently increased in the presence of DADS and DATS as compared with that of the control cells. The phosphorylation of c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), but not of p38, was stimulated in the presence of both garlic allyl sulfides. Pretreatment with SP600125 and PD98059, which are JNK and ERK inhibitors, respectively, abolished the increase in AP-1-DNA binding activity and also the induction of GSTP protein by either allyl sulfide. Our results indicate that the effectiveness of DADS and DATS on GSTP expression is likely related to the JNK-AP-1 and ERK-AP-1 signaling pathways and, thus, that DADS and DATS enhance the binding of AP-1 to GPE I.

Zebrafish klf4 plays a global role in primitive erythropoiesis

Mammalian Kruppel-like factors (KLFs) play essential roles during primitive and definitive erythropoiesis. In zebrafish myeloid and erythroid cell populations develop in spatially distinct regions of the developing embryo. Erythrocytes arise from the posterior LPM and express genes such as embryonic globins and gata1, whereas myeloid cells form in the anterior LPM and express genes such as spi1 and myeloperoxidase. Klf4 is the zebrafish homologue of neptune, a Xenopus KLF which acts early in the haematopoietic program [1,2]. Early expression of klf4 throughout the anterior and posterior LPM from the 1-somite stage of development suggested an early role in blood development. Knockdown of klf4 by two non-overlapping morpholinos (MOs) resulted in anaemia and pericardial oedema at 24 hpf. Expression profiling of klf4-MO versus control MO-injected embryos at 18 hpf using Compugen 16K oligo arrays suggested a role for klf4 in determining various cell fates which are derived from the LPM, particularly primitive erythropoiesis. Klf4 morphants showed a dramatic down-regulation of embryonic globin gene expression by both WISH and qRT–PCR (> 1000-fold) at 24 hpf, but no down-regulation of gata1 gene expression. There was a slight up-regulation (> 5-fold) in the level of definitive haematopoietic specific transcripts, runx1 and cmyb, at 24 hpf and adult globin (> 5-fold) at 36 hpf. This may reflect an expanded secondary/larval haematopoietic wave. These results suggest a restricted role for klf4 in primitive/ICM erythropoiesis in a manner analogous to the role of klf2 in mammalian primitive/yolk sac erythropoiesis. We reinvestigated organisation of the zebrafish globin locus and found a conserved extended CACC box element within the embryonic β-like globin gene promoters which bind Klf4 well in electromobility gel shift assays. On the other hand, Klf4 does not bind well to a divergent CACC box element in the adult β-like globin gene promoter.

NOD2/CARD15 and NF‐κB Expression in Mucosal Biopsies in Children with Active Crohn's Disease

Background: The chronically relapsing inflammatory bowel disease (IBD), including Crohn's Disease (CD) and ulcerative colitis (UC), appear to be the consequence of overly aggressive immune responses to enteric bacterial components in genetically predisposed individuals. A significant advance in the understanding of CD was achieved by the identification of NOD2/CARD15 as the first susceptibility gene for this disease in the Caucasian populations. NOD2/CARD15 is a specific pattern recognition receptor protein which plays a main role in the regulation of inflammatory processes in mammals. It detects a specific microbial cell wall component (peptidoglycan) and induces the activation of the NF-κB signalling pathway, affecting the downstream cytokines production. Aims: To investigate the mRNA expression of NOD2/CARD15 in fresh intestinal mucosal specimens of children with CD in comparison with normal controls in order to validate the hypothesis of a NOD2 up-regulation in CD, as previously experimentally suggested. To assess changes of the nuclear expression and activity of transcription factor NF-κB between patient and control tissues in order to establish a possible relationship between the induction of NOD2 and that of NF-κB. Finally, to measure the mRNA expression level of IL-1beta following NOD2 induction and NF-κB activation. Methods: 20 children (mean age:12) with active CD plus 10 age matched controls entered into the study; mRNA was extracted from unfixed, frozen biopsy specimens and expression levels of NOD2/CARD15 and IL-1beta were detected by using RT-PCR reactions. NF-κB p65 protein expression was detected by Western Blot analysis, while NF-κB activity was assessed by electromobility gel shift assay (EMSA). Image analysis and quantifications of the bands were performed by a densitometer GS-700 model using the Software Quantity One. Results: densitometrical analyses showed that CD patients expressed markedly higher levels of NOD2/CARD15 and IL-1beta mRNA as compared to controls. Moreover, significantly enhanced NF-κB protein expression was found in inflamed tissues of CD patients as compared to normal controls. Other analyses are under investigation. Conclusions: We have shown for the first time that under inflammatory conditions NOD2/CARD15 is up-regulated in intestinal mucosal biopsies from children with active CD. This over-expression is accompanied by a marked nuclear protein expression of the transcription factor NF-κB and a strong secretion of IL-1beta as compared to controls. Our data strengthen the view that NOD2/CARD15 has a crucial role in the etiopathogenesis of CD.