NFκB signaling is of paramount importance in the regulation of apoptosis, proliferation, and inflammatory responses during human development and homeostasis, as well as in many human cancers. Receptor Tyrosine Kinases (RTKs), including the Fibroblast Growth Factor Receptors (FGFRs) are also important in development and disease. However, a direct relationship between growth factor signaling pathways and NFκB activation has not been previously described, although FGFs have been known to antagonize TNFα-induced apoptosis.
Here, we demonstrate an interaction between FGFR4 and IKKβ (Inhibitor of NFκB Kinase β subunit), an essential component in the NFκB pathway. This novel interaction was identified utilizing a yeast two-hybrid screen  and confirmed by coimmunoprecipitation and mass spectrometry analysis. We demonstrate tyrosine phosphorylation of IKKβ in the presence of activated FGFR4, but not kinase-dead FGFR4. Following stimulation by TNFα (Tumor Necrosis Factor α) to activate NFκB pathways, FGFR4 activation results in significant inhibition of NFκB signaling as measured by decreased nuclear NFκB localization, by reduced NFκB transcriptional activation in electophoretic mobility shift assays, and by inhibition of IKKβ kinase activity towards the substrate GST-IκBα in in vitro assays. FGF19 stimulation of endogenous FGFR4 in TNFα-treated DU145 prostate cancer cells also leads to a decrease in IKKβ activity, concomitant reduction in NFκB nuclear localization, and reduced apoptosis. Microarray analysis demonstrates that FGF19 + TNFα treatment of DU145 cells, in comparison with TNFα alone, favors proliferative genes while downregulating genes involved in apoptotic responses and NFκB signaling.
These results identify a compelling link between FGFR4 signaling and the NFκB pathway, and reveal that FGFR4 activation leads to a negative effect on NFκB signaling including an inhibitory effect on proapoptotic signaling. We anticipate that this interaction between an RTK and a component of NFκB signaling will not be limited to FGFR4 alone.
Corneal inflammation associated with ocular adenoviral infection is caused by leukocytic infiltration of the subepithelial stroma in response to expression of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) by infected corneal cells. We have shown that these two chemokines are activated by the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase (ERK) and p38 for IL-8, and Jun-terminal kinase (JNK) for MCP-1. It is also well established that transcription of each of these chemokines is tightly controlled by the nuclear factor kappa B (NFκB) transcription factor family. Therefore, we sought to better understand the differential regulation of chemokine expression by NFκB in adenoviral infection of the cornea.
Primary keratocytes derived from human donor corneas were treated with signaling inhibitors and small interfering RNA specific to MAPKs, and infected with adenovirus for different time periods before analysis. Activation of specific NFκB subunits was analyzed by western blot, confocal microscopy, electromobility shift assay, and chromatin immunoprecipitation, and chemokine expression was quantified by enzyme-linked immunosorbent assay.
Upon adenoviral infection, NFκB p65, p50, and cREL subunits translocate to the nucleus. This translocation is blocked by inhibitors of specific MAPK signaling pathways. Confocal microscopy showed that inhibitors of the p38, JNK, and ERK pathways differentially inhibited NFκB nuclear translocation, while PP2, an inhibitor of Src family kinases, completely inhibited NFκB nuclear translocation. Western blot analysis revealed that activation of specific NFκB subunits was time dependent following infection. Chromatin immunoprecipitation experiments indicated that binding of NFκB p65 and p50 subunits to the IL-8 promoter upon viral infection was differentially reduced by chemical inhibitors of MAPKs. Electromobility shift assay and luciferase assay analysis revealed that transactivation of IL-8 occurred with binding by the NFκB p65 homodimer or NFκB p65/p50 heterodimer as early as 1 h post infection, whereas MCP-1 expression was dependent upon the NFκB cREL but not the p65 subunit, and occurred 4 h after IL-8 induction. Finally, knockdown of NFκB p65 by short interfering RNA abrogated IL-8 but not MCP-1 expression after adenoviral infection.
The kinetics of NFκB subunit activation are partly responsible for the observed pattern of acute inflammation in the adenoviral-infected cornea. MAPKs differentially regulate chemokine expression in adenoviral keratitis by differential and time-dependent activation of specific NFκB subunits.
The protein kinase regulated by double-stranded RNA (dsRNA), PKR, is implicated in a range of biologic processes, including apoptotic death and interferon antiviral responses, based in part on studies with mouse cells genetically deficient in Pkr. To test the role of the PKR protein in human cells, an RNA interference silencing strategy was used to generate stable HeLa cell lines with less than 2% of the PKR protein (PKR deficient) compared to either parental or control knockdown HeLa lines. Phosphorylation of the α subunit of eukaryotic initiation factor 2 on serine 51 was not detectably increased in response to dsRNA in PKR-deficient HeLa cells but was elevated severalfold in PKR-sufficient cells. PKR-deficient cells displayed reduced dsRNA-induced apoptosis compared to PKR-sufficient cell lines, whereas tumor necrosis factor alpha (TNF-α)-induced apoptosis was comparable between the HeLa lines. NF-κB was activated to a comparable extent in PKR-deficient and PKR-sufficient HeLa cells upon treatment with either dsRNA or TNF-α. The antiviral response against vesicular stomatitis virus was reduced in interferon-treated PKR-deficient compared to PKR-sufficient HeLa cells. However, the growth of two human viruses, adenovirus and reovirus, was unaffected by the PKR knockdown. Surprisingly, the yield of mutant adenovirus that fails to encode VAI RNA was not enhanced in PKR-deficient cells, indicating the importance of host factors in addition to PKR in conferring the VAI RNA phenotype.
DNA microarray assay performance is commonly compromised by spot-spot probe and signal variations as well as heterogeneity within printed microspots. Accurate metrics for captured DNA target signal rely upon uniform spot distribution of both probe and target DNA to yield reliable hybridized signal. While often presumed, this is neither easily achieved nor often proven experimentally. High resolution imaging techniques were used to determine spot heterogeneity in identical DNA array microspots comprising varied ratios of unlabeled and dye-labeled DNA probes contact printed onto commercial arraying surfaces. Epifluorescence imaging data for individual array microspots were correlated with time-of-flight secondary ion mass spectrometry (TOF-SIMS) chemical state imaging of the same spots. Epifluorescence imaging intensity distinguished varying DNA density distributed both within a given spot and from spot-to-spot. TOF-SIMS chemical analysis confirmed these heterogeneous printed DNA distributions by tracking bound Cy3 dye-, DNA base, and phosphate- specific ion fragments often correlating to fluorescence patterns within identical spots. TOF-SIMS ion fragments originating from probe DNA and Cy3 dye are enriched in microspot centers, correlating with high fluorescence intensity regions. Both TOF-SIMS and epifluorescence supports Marangoni flow effects on spot drying, with high density DNA-Cy3 located in spot centers and non-homogeneous DNA distribution within printed spots. Microspot image dimensional analysis results for DNA droplet spreading show differing DNA densities across printed spots. The study directly supports different DNA probe chemical and spatial microenvironments within spots that yield spot-spot signal variations known to affect DNA target hybridization efficiencies and kinetics. These variations critically affect probe-target duplex formation and DNA array signal generation.
microarray; chemical imaging; fluorescence; mass spectrometry; surface analysis; nucleic acid; hybridization
Summary: ChIP-based technology is becoming the leading technology to globally profile thousands of transcription factors and elucidate the transcriptional regulation mechanisms in living cells. It has evolved rapidly in recent years, from hybridization with spotted or tiling microarray (ChIP-chip), to pair-end tag sequencing (ChIP-PET), to current massively parallel sequencing (ChIP-seq). Although there are many tools available for identifying binding sites (peaks) for ChIP-chip and ChIP-seq, few of them are available as easy-accessible online web tools for processing both ChIP-chip and ChIP-seq data for the ChIP-based user community. As such, we have developed a comprehensive web application tool for processing ChIP-chip and ChIP-seq data. Our web tool W-ChIPeaks employed a probe-based (or bin-based) enrichment threshold to define peaks and applied statistical methods to control false discovery rate for identified peaks. The web tool includes two different web interfaces: PELT for ChIP-chip, BELT for ChIP-seq, where both were tested on previously published experimental data. The novel features of our tool include a comprehensive output for identified peaks with GFF, BED, bedGraph and .wig formats, annotated genes to which these peaks are related, a graphical interpretation and visualization of the results via a user-friendly web interface.
Supplementary information: Supplementary data are available at Bioinformatics online.
Transcription factor (TF)-DNA binding loci are explored by analyzing massive datasets generated with application of Chromatin Immuno-Precipitation (ChIP)-based high-throughput sequencing technologies. These datasets suffer from a bias in the information about binding loci availability, sample incompleteness and diverse sources of technical and biological noises. Therefore adequate mathematical models of ChIP-based high-throughput assay(s) and statistical tools are required for a robust identification of specific and reliable TF binding sites (TFBS), a precise characterization of TFBS avidity distribution and a plausible estimation the total number of specific TFBS for a given TF in the genome for a given cell type.
We developed an exploratory mixture probabilistic model for a specific and non-specific transcription factor-DNA (TF-DNA) binding. Within ChiP-seq data sets, the statistics of specific and non-specific DNA-protein binding is defined by a mixture of sample size-dependent skewed functions described by Kolmogorov-Waring (K-W) function (Kuznetsov, 2003) and exponential function, respectively. Using available Chip-seq data for eleven TFs, essential for self-maintenance and differentiation of mouse embryonic stem cells (SC) (Nanog, Oct4, sox2, KLf4, STAT3, E2F1, Tcfcp211, ZFX, n-Myc, c-Myc and Essrb) reported in Chen et al (2008), we estimated (i) the specificity and the sensitivity of the ChiP-seq binding assays and (ii) the number of specific but not identified in the current experiments binding sites (BSs) in the genome of mouse embryonic stem cells. Motif finding analysis applied to the identified c-Myc TFBSs supports our results and allowed us to predict many novel c-Myc target genes.
We provide a novel methodology of estimating the specificity and the sensitivity of TF-DNA binding in massively paralleled ChIP sequencing (ChIP-seq) binding assay. Goodness-of fit analysis of K-W functions suggests that a large fraction of low- and moderate- avidity TFBSs cannot be identified by the ChIP-based methods. Thus the task to identify the binding sensitivity of a TF cannot be technically resolved yet by current ChIP-seq, compared to former experimental techniques. Considering our improvement in measuring the sensitivity and the specificity of the TFs obtained from the ChIP-seq data, the models of transcriptional regulatory networks in embryonic cells and other cell types derived from the given ChIp-seq data should be carefully revised.
Transcription is a sophisticated multi-step process in which RNA polymerase II (Pol II) transcribes a DNA template into RNA in concert with a broad array of transcription initiation, elongation, capping, termination, and histone modifying factors. Recent global analyses of Pol II distribution have indicated that many genes are regulated during the elongation phase, shedding light on a previously underappreciated mechanism for controlling gene expression. Understanding how various factors regulate transcription elongation in living cells has been greatly aided by chromatin immunoprecipitation (ChIP) studies, which can provide spatial and temporal resolution of protein-DNA binding events. The coupling of ChIP with DNA microarray and high-throughput sequencing technologies (ChIP-chip and ChIP-seq) has significantly increased the scope of ChIP studies and genome-wide maps of Pol II or elongation factor binding sites can now be readily produced. However, while ChIP-chip/ChIP-seq data allow for high-resolution localization of protein-DNA binding sites, they are not sufficient to dissect protein function. Here we describe techniques for coupling ChIP-chip/ChIP-seq with genetic, chemical, and experimental manipulation to obtain mechanistic insight from genome-wide protein-DNA binding studies. We have employed these techniques to discern immature promoter-proximal Pol II from productively elongating Pol II, and infer a critical role for the transition between initiation and full elongation competence in regulating development and gene induction in response to environmental signals.
transcription elongation; gene expression; ChIP-chip; ChIP-seq
We have developed a high-density microarray platform consisting of nano-biofilms of Candida albicans. A robotic microarrayer was used to print yeast cells of C. albicans encapsulated in a collagen matrix at a volume as low as 50 nL onto surface-modified microscope slides. Upon incubation, the cells grow into fully formed “nano-biofilms”. The morphological and architectural complexity of these biofilms were evaluated by scanning electron and confocal scanning laser microscopy. The extent of biofilm formation was determined using a microarray scanner from changes in fluorescence intensities due to FUN 1 metabolic processing. This staining technique was also adapted for antifungal susceptibility testing, which demonstrated that, similar to regular biofilms, cells within the on-chip biofilms displayed elevated levels of resistance against antifungal agents (fluconazole and amphotericin B). Thus, results from structural analyses and antifungal susceptibility testing indicated that despite miniaturization, these biofilms display the typical phenotypic properties associated with the biofilm mode of growth. In its final format, the C. albicans biofilm chip (CaBChip) is composed of 768 equivalent and spatially distinct nano-biofilms on a single slide; multiple chips can be printed and processed simultaneously. Compared to current methods for the formation of microbial biofilms, namely the 96-well microtiter plate model, this fungal biofilm chip has advantages in terms of miniaturization and automation, which combine to cut reagent use and analysis time, minimize labor intensive steps, and dramatically reduce assay costs. Such a chip should accelerate the antifungal drug discovery process by enabling rapid, convenient and inexpensive screening of hundreds-to-thousands of compounds simultaneously.
Viral hepatitis due to hepatitis B virus and hepatitis C virus are major public health problems all over the world. Traditional detection methods including polymerase chain reaction (PCR)-based assays and enzyme-linked immunosorbent assays (ELISA) are expensive and time-consuming. In our assay, a protein chip assay using Nano-gold Immunological Amplification and Silver Staining (NIASS) method was applied to detect HBV and HCV antibodies rapidly and simultaneously.
Chemically modified glass slides were used as solid supports (named chip), on which several antigens, including HBsAg, HBeAg, HBcAg and HCVAg (a mixture of NS3, NS5 and core antigens) were immobilized respectively. Colloidal nano-gold labelled staphylococcal protein A (SPA) was used as an indicator and immunogold silver staining enhancement technique was applied to amplify the detection signals, producing black image on array spots, which were visible with naked eyes. To determine the detection limit of the protein chip assay, a set of model arrays in which human IgG was spotted were structured and the model arrays were incubated with different concentrations of anti-IgG. A total of 305 serum samples previously characterized with commercial ELISA were divided into 4 groups and tested in this assay.
We prepared mono-dispersed, spherical nano-gold particles with an average diameter of 15 ± 2 nm. Colloidal nano-gold-SPA particles observed by TEM were well-distributed, maintaining uniform and stable. The optimum silver enhancement time ranged from 8 to 12 minutes. In our assay, the protein chips could detect serum antibodies against HBsAg, HBeAg, HBcAg and HCVAg with the absence of the cross reaction. In the model arrays, the anti-IgG as low as 3 ng/ml could be detected. The data for comparing the protein chip assay with ELISA indicated that no distinct difference (P > 0.05) existed between the results determined by our assay and ELISA respectively.
Results showed that our assay can be applied with serology for the detection of HBV and HCV antibodies rapidly and simultaneously in clinical detection.
Double-stranded RNA (dsRNA)-activated protein kinase (PKR) is a critical upstream mediator of the ribotoxic stress response (RSR) to the trichothecene deoxynivalenol (DON) and other translational inhibitors. Here, we employed HeLa cell lysates to: (1) characterize PKR’s interactions with the ribosome and ribosomal RNA (rRNA); (2) demonstrate cell-free activation of ribosomal-associated PKR and (3) integrate these findings in a unified model for RSR. Robust PKR-dependent RSR was initially confirmed in intact cells. PKR basally associated with 40S, 60S, 80S and polysome fractions at molar ratios of 7, 2, 23 and 3, respectively. Treatment of ATP-containing HeLa lysates with DON or the ribotoxins anisomycin and ricin concentration-dependently elicited phosphorylation of PKR and its substrate eIF2α. These phosphorylations could be blocked by PKR inhibitors. rRNA immunoprecipitation (RNA-IP) of HeLa lysates with PKR-specific antibody and sequencing revealed that in the presence of DON or not, the kinase associated with numerous discrete sites on both the 18S and 28S rRNA molecules, a number of which contained double-stranded hairpins. These findings are consistent with a sentinel model whereby multiple PKR molecules basally associate with the ribosome positioning them to respond to ribotoxin-induced alterations in rRNA structure by dimerizing, autoactivating and, ultimately, evoking RSR.
PKR; ribotoxic stress; rRNA; deoxynivalenol; anisomycin; ricin; translational inhibitor; quantitiative Western analysis; RNA immunoprecipitation
NFκB signaling is critical for expression of genes involved in the vascular injury response. We have shown that estrogen (17β-estradiol, E2) inhibits expression of these genes in an estrogen receptor (ER)-dependent manner in injured rat carotid arteries and in tumor necrosis factor (TNF)-α treated rat aortic smooth muscle cells (RASMCs). This study tested whether E2 inhibits NFκB signaling in RASMCs and defined the mechanisms.
TNF-α treated RASMCs demonstrated rapid degradation of IκBα (10–30 min), followed by dramatic increases in IκBα mRNA and protein synthesis (40–60 min). E2 enhanced TNF-α induced IκBα synthesis without affecting IκBα degradation. Chromatin immunoprecipitation (ChIP) assays revealed that E2 pretreatment both enhanced TNF-α induced binding of NFκB p65 to the IκBα promoter and suppressed TNF-α induced binding of NFκB p65 to and reduced the levels of acetylated histone 3 at promoters of monocyte chemotactic protein (MCP)-1 and cytokine-induced neutrophil chemoattractant (CINC)-2β genes. ChIP analyses also demonstrated that ERβ can be recruited to the promoters of MCP-1 and CINC-2β during co-treatment with TNF-α and E2.
These data demonstrate that E2 inhibits inflammation in RASMCs by two distinct mechanisms: promoting new synthesis of IκBα, thus accelerating a negative feedback loop in NFκB signaling, and directly inhibiting binding of NFκB to the promoters of inflammatory genes. This first demonstration of multifaceted modulation of NFκB signaling by E2 may represent a novel mechanism by which E2 protects the vasculature against inflammatory injury.
Systematic chromatin immunoprecipitation (chIP-chip) experiments have become a central technique for mapping transcriptional interactions in model organisms and humans. However, measurement of chromatin binding does not necessarily imply regulation, and binding may be difficult to detect if it is condition or cofactor dependent. To address these challenges, we present an approach for reliably assigning transcription factors (TFs) to target genes that integrates many lines of direct and indirect evidence into a single probabilistic model. Using this approach, we analyze publicly available chIP-chip binding profiles measured for yeast TFs in standard conditions, showing that our model interprets these data with significantly higher accuracy than previous methods. Pooling the high-confidence interactions reveals a large network containing 363 significant sets of factors (TF modules) that cooperate to regulate common target genes. In addition, the method predicts 980 novel binding interactions with high confidence that are likely to occur in so-far untested conditions. Indeed, using new chIP-chip experiments we show that predicted interactions for the factors Rpn4p and Pdr1p are observed only after treatment of cells with methyl-methanesulfonate, a DNA-damaging agent. We outline the first approach for consistently integrating all available evidences for TF–target interactions and we comprehensively identify the resulting TF module hierarchy. Prioritizing experimental conditions for each factor will be especially important as increasing numbers of chIP-chip assays are performed in complex organisms such as humans, for which “standard conditions” are ill defined.
Transcription factors (TFs) bind close to their target genes for regulating transcript levels depending on cellular conditions. Each gene may be regulated differently from others through the binding of specific groups of TFs (TF modules). Recently, a wide variety of large-scale measurements about transcriptional networks has become available. Here the authors present a framework for consistently integrating all of this evidence to systematically determine the precise set of genes directly regulated by each TF (i.e., TF–target interactions). The framework is applied to the yeast Saccharomyces cerevisiae using seven distinct sources of evidences to score all possible TF–target interactions in this organism. Subsequently, the authors employ another newly developed algorithm to reveal TF modules based on the top 5,000 TF–target interactions, yielding more than 300 TF modules. The new scoring scheme for TF–target interactions allows predicting the binding of TFs under so-far untested conditions, which is demonstrated by experimentally verifying interactions for two TFs (Pdr1p, Rpn4p). Importantly, the new methods (scoring of TF–target interactions and TF module identification) are scalable to much larger datasets, making them applicable to future studies in humans, which are thought to have substantially larger numbers of TF–target interactions.
Heart failure, a progressive, fatal disease of the heart muscle, is a state of chronic inflammation and injury. Heat shock protein (HSP) 72, a ubiquitous protective protein that is well-established as cardioprotective, is not increased in heart failure. In contrast, HSP60 levels are doubled in the failing heart. We hypothesized that HSF-1 is not activated in heart failure and that the increased expression of HSP60 was driven by NFκB activation. To test this hypothesis, we measured levels of heat shock factor (HSF) −1 and −2, the transcription factors controlling HSP expression, which were increased in heart failure. There was no increased phosphorylation of serine 230 or serine 303/307 in HSF-1, which are thought to regulate its activity; EMSA showed no increase in HSF binding activity with heart failure. Nonetheless, mRNA was increased for HSP60, but not HSP72. In contrast to HSF, NFκB activity was increased in heart failure. HSP60, but not HSP72, contained NFκB binding elements. ChIP assay demonstrated increased binding of NFκB to both of the NFκB binding elements in the heart failure HSP60 gene. TNFα treatment was used to test the role of NFκB activation in HSP60 expression in a cardiac cell line. TNFα increased HSP60 expression, and this could be prevented by pretreatment with siRNA inhibiting p65 expression. In conclusion, HSP72 is not increased in heart failure because HSF activity is not changed; increased expression of HSP60 may be driven by NFκB activation.
HSP60; HSP72; Heart failure; NFκB; ERK 1/2; GSK
Host response to viral RNA genomes and replication products represents an effective strategy to combat viral invasion. PKR is a Ser/Thr protein kinase that binds to dsRNA, autophosphorylates its kinase domain, and subsequently phosphorylates eukaryotic initiation factor 2α (eIF2α). This results in attenuation of protein translation, preventing synthesis of necessary viral proteins. In certain DNA viruses, PKR function can be evaded by transcription of highly structured virus-encoded dsRNA inhibitors that bind to and inactivate PKR. We probe here the mechanism of PKR inhibition by two viral inhibitor RNAs, EBERI (from Epstein-Barr) and VAI (from human adenovirus). Native gel shift mobility assays and isothermal titration calorimetry experiments confirmed that the RNA-binding domains of PKR are sufficient and necessary for the interaction with dsRNA inhibitors. Both EBERI and VAI are effective inhibitors of PKR activation by preventing trans-autophosphorylation between two PKR molecules. The RNA inhibitors prevent self-association of PKR molecules, providing a mechanistic basis for kinase inhibition. A variety of approaches indicated that dsRNA inhibitors remain associated with PKR under activating conditions, as opposed to activator dsRNA molecules that dissociate due to reduced affinity for the phosphorylated form of PKR. Finally, we show using a HeLa cell extract system that inhibitors of PKR result in translational recovery by the protein synthesis machinery. These data indicate that inhibitory dsRNAs bind preferentially to the latent, dephosphorylated form of PKR and prevent dimerization that is required for trans-autophosphorylation.
RNA; PKR; kinase; inhibition; virus
The IL-6/STAT3 and TNFα/NFκB pathways are emerging as critical mediators of inflammation-associated colon cancer. TNFR2 expression is increased in inflammatory bowel diseases, the azoxymethane/dextran sodium sulfate (AOM/DSS) model of colitis-associated cancer, and by combined IL-6 and TNFα. The molecular mechanisms that regulate TNFR2 remain undefined. This study used colon cancer cell lines to test the hypothesis that IL-6 and TNFα induce TNFR2 via STAT3 and/or NFκB. Basal and IL-6 + TNFα-induced TNFR2 were decreased by pharmacological STAT3 inhibition. NFκB inhibition had little effect on IL-6 + TNFα-induced TNFR2, but did inhibit induction of endogenous IL-6 and TNFR2 in cells treated with TNFα alone. Chromatin immunoprecipitation (ChIP) revealed cooperative effects of IL-6 + TNFα to induce STAT3 binding to a -1578 STAT response element in the TNFR2 promoter, but no effect on NFκB binding to consensus sites. Constitutively active STAT3 was sufficient to induce TNFR2 expression. Over-expression of SOCS3, a cytokine-inducible STAT3 inhibitor, which reduces tumorigenesis in preclinical models of colitis-associated cancer, decreased cytokine-induced TNFR2 expression and STAT3 binding to the -1578 STAT response element. SOCS3 over-expression also decreased proliferation of colon cancer cells and dramatically decreased anchorage-independent growth of colon cancer cells, even cells over-expressing TNFR2. Collectively, these studies demonstrate that IL-6 and TNFα-induced TNFR2 expression in colon cancer cells is mediated primarily by STAT3, and provide evidence that TNFR2 may contribute to the tumor-promoting roles of STAT3.
TNFR2; STAT3; Colon Cancer; Inflammation; SOCS3
Extravasation of circulating cancer cells is a key event of metastatic dissemination that is initiated by the adhesion of cancer cells to endothelial cells. It requires interactions between adhesion receptors on endothelial cells and their counter-receptors on cancer cells. Notably, E-selectin, a major endothelial adhesion receptor, interacts with Death receptor-3 present on metastatic colon carcinoma cells. This interaction confers metastatic properties to colon cancer cells by promoting the adhesion of cancer cells to endothelial cells and triggering the activation of the pro-migratory p38 and pro-survival ERK pathways in the cancer cells. In the present study, we investigated further the mechanisms by which the E-selectin-activated pathways downstream of DR3 confer a survival advantage to colon cancer cells.
Cell survival has been ascertained by using the WST-1 assay and by evaluating the activation of the PI3 kinase/NFκB survival axis. Apoptosis has been assayed by determining DNA fragmentation by Hoechst staining and by measuring cleavage of caspases-8 and -3. DR3 isoforms have been identified by PCR. For more precise quantification, targeted PCR reactions were carried out, and the amplified products were analyzed by automated chip-based microcapillary electrophoresis on an Agilent 2100 Bioanalyzer instrument.
Interaction between DR3-expressing HT29 colon carcinoma cells and E-selectin induces the activation of the PI3K/Akt pathway. Moreover, p65/RelA, the anti-apoptotic subunit of NFκB, is rapidly translocated to the nucleus in response to E-selectin. This translocation is impaired by the PI3K inhibitor LY294002. Furthermore, inhibition of the PI3K/Akt pathway increases the cleavage of caspase 8 in colon cancer cells treated with E-selectin and this effect is still further increased when both ERK and PI3K pathways are concomitantly inhibited. Intriguingly, metastatic colon cancer cell lines such as HT29 and SW620 express higher levels of a splice variant of DR3 that has no trans-membrane domain and no death domain.
Colon cancer cells acquire an increased capacity to survive via the activation of the PI3K/NFκB pathway following the stimulation of DR3 by E-selectin. Generation of a DR3 splice variant devoid of death domain can further contribute to protect against apoptosis.
Death receptor-3; E-selectin; colon cancer; PI3 kinase; splice variant
The long terminal repeat (LTR) region of leukemia viruses plays a critical role in tissue tropism and pathogenic potential of the viruses. We have previously reported that U3-LTR from Moloney murine and feline leukemia viruses (Mo-MuLV and FeLV) upregulate specific cellular genes in trans in an integration-independent way. The U3-LTR region necessary for this action does not encode a protein but instead makes a specific RNA transcript. Because several cellular genes transactivated by the U3-LTR can also be activated by NFκB, and because the antiapoptotic and growth promoting activities of NFκB have been implicated in leukemogenesis, we investigated whether FeLV U3-LTR can activate NFκB signaling. Here we demonstrate that FeLV U3-LTR indeed upregulates NFκB signaling pathway via activation of Ras-Raf-IκB kinase (IKK) and degradation of IκB. LTR-mediated transcriptional activation of genes did not require new protein synthesis suggesting an active role of the LTR transcript in the process. Using Toll-like receptor (TLR) deficient HEK293 cells and PKR−/− mouse embryo fibroblasts we further demonstrate that although dsRNA activated protein kinase R (PKR) is not necessary, TLR3 is required for the activation of NFκB by the LTR. Our study thus demonstrates involvement of a TLR3 dependent but PKR independent dsRNA mediated signaling pathway for NFκB activation and thus provides a new mechanistic explanation of LTR-mediated cellular gene transactivation.
Leukemia virus; LTR; transactivation; NFκB; TLR3
Ultraviolet (UV) radiation activates cell signaling pathways in melanocytes. As a result of altered signaling pathways and UV-induced cellular damage, melanocytes can undergo oncogenesis and develop into melanomas. In this study, we investigated the effect of UV-radiation on p38 MAPK (mitogen-activated protein kinase), JNK and NFκB pathways to determine which plays a major role in stimulating TNFα secretion in human HEM (melanocytes) and MM96L (melanoma) cells. MM96L cells exhibited 3.5-fold higher p38 activity than HEM cells at 5 min following UVA + B radiation and 1.6-fold higher JNK activity at 15–30 min following UVB+A radiation, while NFκB was minimally activated in both cells. Irradiated HEM cells had the greatest fold of TNFα secretion (UVB: 109-fold, UVA + B: 103-fold & UVB+A: 130-fold) when co-exposed to IL1α. The p38 inhibitor, SB202190, inhibited TNFα release by 93% from UVB-irradiated HEM cells. In the UVB-irradiated MM96L cells, both SB202190 and sulfasalazine (NFκB inhibitor) inhibited TNFα release by 52%. Although, anisomycin was a p38 MAPK activator, it inhibited TNFα release in UV-irradiated cells. This suggests that UV-mediated TNFα release may occur via different p38 pathway intermediates compared to those stimulated by anisomycin. As such, further studies into the functional role p38 MAPK plays in regulating TNFα release in UV-irradiated melanocyte-derived cells are warranted.
UV; melanocytes; melanoma; TNFα; p38; JNK; NFκB; anisomycin
Near equal rates of incidence and mortality emphasize the need for novel targeted approaches for better management of pancreatic cancer patients. Inflammatory molecules NFκB and Stat3 are overexpressed in pancreatic tumors. Inhibition of one protein allows cancer cells to survive using the other. The goal of the present study is to determine whether targeting Stat3/NFκB cross talk with a natural product Nexrutine (Nx) can inhibit inflammatory signaling in pancreatic cancer.
HPNE, HPNE-Ras, BxPC3, Capan-2, MIA PaCa-2 and AsPC-1 cells were tested for growth, apoptosis, Cox-2, NFκB and Stat3 level in response to Nx treatment. Transient expression, gel shift, ChIP was used to examine transcriptional regulation of Cox-2. Stat3 knockdown was used to decipher Stat3/NFκB cross talk. Histopathological and immunoblotting evaluation was performed on BK5-Cox2 transgenic mice treated with Nx. In vivo expression of prostaglandin receptor EP4 was analyzed in a retrospective cohort of pancreatic tumors using a TMA.
Nx treatment inhibited growth of pancreatic cancer cells through induction of apoptosis. Reduced levels and activity of Stat3, NFκB and their cross talk led to transcriptional suppression of Cox-2 and subsequent decreased levels of PGE2 and PGF2. Stat3 knockdown studies suggest Stat3 as negative regulator of NFκB activation. Nx intervention reduced the levels of NFκB, Stat3 and fibrosis in vivo. Expression of prostaglandin receptor EP4 that is known to play a role in fibrosis was significantly elevated in human pancreatic tumors.
Dual inhibition of Stat3-NFκB by Nx may overcome problems associated with inhibition of either pathway.
Pancreatic cancer; Nexrutine; Stat3; NFκB; Prostaglandin receptor EP4; Cox-2
Tumor necrosis factor α (TNFα)-stimulated nuclear factor (NF) κB activation plays a key role in the pathogenesis of inflammatory bowel disease (IBD). Phosphorylation of NFκB inhibitory protein (IκB) leading to its degradation and NFκB activation, is regulated by the multimeric IκB kinase complex, including IKKα and IKKβ. We recently reported that 5-aminosalicylic acid (5-ASA) inhibits TNFα-regulated IκB degradation and NFκB activation. To determine the mechanism of 5-ASA inhibition of IκB degradation, we studied young adult mouse colon (YAMC) cells by immunodetection and in vitro kinase assays. We show 5-ASA inhibits TNFα-stimulated phosphorylation of IκBα in intact YAMC cells. Phosphorylation of a glutathione S-transferase-IκBα fusion protein by cellular extracts or immunoprecipitated IKKα isolated from cells treated with TNFα is inhibited by 5-ASA. Recombinant IKKα and IKKβ autophosphorylation and their phosphorylation of glutathione S-transferase-IκBα are inhibited by 5-ASA. However, IKKα serine phosphorylation by its upstream kinase in either intact cells or cellular extracts is not blocked by 5-ASA. Surprisingly, immunodepletion of cellular extracts suggests IKKα is predominantly responsible for IκBα phosphorylation in intestinal epithelial cells. In summary, 5-ASA inhibits TNFα-stimulated IKKα kinase activity toward IκBα in intestinal epithelial cells. These findings suggest a novel role for 5-ASA in the management of IBD by disrupting TNFα activation of NFκB.
The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. For instance, on the single cell level, distinct microenvironments can impose specific differentiation, migration, and proliferation of phenotypes, and on the tissue level the microenvironment may control processes as complex as morphogenesis and tumorigenesis1. Not only do the cell and molecular contents of microenvironments impact the cells within them, but also does the elasticity2 and geometry3 of the tissue. Defined as the sum total of cell-cell, -ECM, and –soluble factor interactions, in addition to physical characteristics, the microenvironment is highly complex. The phenotypes of cells within a tissue are partially due to their genomic content and partially due to the combinatorial interactions with the molecular and physical components of the microenviroment. A major challenge is to link specific combinations of microenvironmental components with distinctive behaviours.
Here, we present the microenvironment microarray (MEArray) platform for cell-based functional screening of interactions with combinatorial microenvironments4. The method allows for simultaneous control of the molecular composition and the elastic modulus, and combines the use of widely available microarray and micropatterning technologies. MEArray screens require as few as 10,000 cells per array, which facilitates functional studies of rare cell types such as adult progenitor cells. A limitation of the technology is that entire tissue microenvironments cannot be completely recapitulated on MEArrays. However, comparison of responses in the same cell type to numerous related microenvironments, for instance pairwise combinations of ECM proteins that characterize a given tissue, will provide insights into how microenvironmental components elicit tissue-specific functional phenotypes.
MEArrays can be printed using a wide variety of recombinant growth factors, cytokines, and purified ECM proteins, and combinations thereof. The platform is limited only by the availability of specific reagents. MEArrays are amenable to time-lapsed analysis, but most often are used for end point analyses of cellular functions that are measureable with fluorescent probes. For instance, DNA synthesis, apoptosis, acquisition of differentiated states, or production of specific gene products are commonly measured. Briefly, the basic flow of an MEArray experiment is to prepare slides coated with the printing substrata and to prepare the master plate of proteins that are to be printed. Then the arrays are printed with a microarray robot, cells are allowed to attach, grow in culture, and then are chemically fixed upon reaching the experimental endpoint. Fluorescent or colorimetric assays, imaged with traditional microscopes or microarray scanners, are used to reveal relevant molecular and cellular phenotypes (Figure 1).
Microenvironment; microarray; MEArray; functional cell-based assay
The inducible IκB kinase (IKKi/IKKɛ) is a recently described serine-threonine kinase that activates the transcription factors NFκB, interferon regulatory factor-3 (IRF3) and CCAAA/enhancer-binding protein (C/EBPδ). Several inflammatory agents have been shown to induce the expression of the IKKi gene in macrophages and other cell types but the mechanism is unknown. We have found that the IKKi expression was constitutive in human chondrocytes from OA cartilage and a human chondrocytic cell line C28/I2 but was up-regulated by the inflammatory cytokines TNFα or IL-1β in an NFκB dependent manner. To understand the constitutive and inducible expression of the IKKi gene we localized the transcription start site (TSS), cloned and sequenced a 2 kb genomic DNA fragment 5’ of the TSS and characterized the putative promoter region (PPR), and identified the motifs therein that are required for basal and cytokine-induced IKKi gene promoter activity. We found that IKKi core promoter was TATA-less and by using PCR generated deletion mutants of the PPR we found that the cis-elements responsible for basal transcriptional activity were located between −51 to −100 bp upstream of the TSS while the cytokine response elements were located distally between −501 and −1000 bp upstream of the TSS. The DNA region containing the cytokine response elements had two κB sites as the most relevant regulatory motifs. The results of site-directed mutagenesis revealed that the κB site located between −833 and −847 bp upstream of the TSS was biologically functional and required for cytokine-induced IKKi promoter activity in human chondrocytes and HeLa cells. The silencing of the other κB site (−816/−802) was positional, rather than sequence-specific. Over-expression of NFκB p65 mimics the TNFα-induced activation of the IKKi promoter. In addition, the gel shift assay suggested that NFκB p65 is responsible for activation of the IKKi promoter. These data for the first time characterize the promoter region of IKKi and provide further insights into its transcriptional regulation in human chondrocytes and other cell types.
IKKi/IKKɛ Promoter; cis-acting element; Transcriptional regulation; NFκB, Chondrocyte; 5’-RACE, 5’-rapid amplification of cDNA ends; ActD, actinomycin D; bp, base pair(s); BRE, TFIIB responsive element; CHX, cycloheximide; DPE, downstream promoter element; EMSA, electrophoretic mobility shift assay; EMSSA, electrophoretic mobility supershift assay; IκB, inhibitor of NFκB; IKKi/IKKɛ, inducible IκB kinase; IL, interleukin; Inr, initiator; NFκB, nuclear factor kappa B; nt, nucleotide(s); PPR, putative promoter region; RT-PCR, reverse transcriptase-polymerase chain reaction; SEAP, secreted form of human placental alkaline phosphatase; TBP, TATA-binding protein; TNF, tumor necrosis factor; TSS, transcription start site
The NFκB pathway is a major source of pro-inflammatory cytokines, which may contribute to cancer chemoresistance. We showed that constitutive NFκB activity is characteristic of the ovarian cancer stem cells (OCSCs). The aim of this study is to determine if the inhibition of NFκB by Eriocalyxin B (EriB) in the OCSCs may induce cell death in otherwise chemoresistant cells.
OCSCs and mature ovarian cancer cells (mOCCs) were treated with increasing concentrations of EriB. Cell viability was measured using the Celltiter 96 assay and caspase activity was quantified using Caspase-Glo™ assay. Cytokine levels were quantified using xMAP technology.
EriB decreased the percent of viable cells in all cultures tested with GI50 of 0.5 −1µM after 48h of treatment. The intracellular changes associated with EriB-induced cell death are: 1) inhibition of NF-κB activity; 2) decreased cytokine production; 3) activation of caspases; and 4) down-regulation of XIAP. In addition, EriB is able to sensitize OCSCs to TNFα and FasL-mediated cell death.
Inhibition of the NFκB pathway induces cell death in the OCSCs. Since the OCSCs may represent the source of recurrence and chemoresistance, the use of NFκB inhibitors like EriB may prevent recurrence in ovarian cancer patients.
inflammation; nuclear factor kappa B; TNF-a; cancer stem cells; ovarian cancer; ovarian cancer stem cells
Chemokine (C-C motif) ligand 2, also known as monocyte chemoattractant protein 1 (MCP-1) is an important factor for the pathogenesis of HIV-associated neurocognitive disorders (HAND). The mechanisms of MCP-1-mediated neuropathogenesis, in part, revolve around its neuroinflammatory role and the recruitment of monocytes into the central nervous system (CNS) via the disrupted blood-brain barrier (BBB). We have previously demonstrated that HIV-1/HIV-1 Tat upregulate platelet-derived growth factor (PDGF)-BB, a known cerebrovascular permeant; subsequently, the present study was aimed at exploring the regulation of MCP-1 by PDGF-BB in astrocytes with implications in HAND. Specifically, the data herein demonstrate that exposure of human astrocytes to HIV-1 LAI elevated PDGF-B and MCP-1 levels. Furthermore, treating astrocytes with the human recombinant PDGF-BB protein significantly increased the production and release of MCP-1 at both the RNA and protein levels. MCP-1 induction was regulated by activation of extracellular-signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinases and phosphatidylinositol 3-kinase (PI3K)/Akt pathways and the downstream transcription factor, nuclear factor κB (NFκB). Chromatin immunoprecipitation (ChIP) assays demonstrated increased binding of NFκB to the human MCP-1 promoter following PDGF-BB exposure. Conditioned media from PDGF-BB-treated astrocytes increased monocyte transmigration through human brain microvascular endothelial cells (HBMECs), an effect that was blocked by STI-571, a tyrosine kinase inhibitor (PDGF receptor (PDGF-R) blocker). PDGF-BB-mediated release of MCP-1 was critical for increased permeability in an in vitro BBB model as evidenced by blocking antibody assays. Since MCP-1 is linked to disease severity, understanding its modulation by PDGF-BB could aid in understanding the proinflammatory responses in HAND. These results suggest that astrocyte activation by PDGF-BB exaggerates monocyte recruitment into the brain via MCP-1 and underscores the critical role astrocytes play in HAND.
We have developed DNA microarray techniques for studying Corynebacterium glutamicum. A set of 52 C. glutamicum genes encoding enzymes from primary metabolism was amplified by PCR and printed in triplicate onto glass slides. Total RNA was extracted from cells harvested during the exponential-growth and lysine production phases of a C. glutamicum fermentation. Fluorescently labeled cDNAs were prepared by reverse transcription using random hexamer primers and hybridized to the microarrays. To establish a set of benchmark metrics for this technique, we compared the variability between replicate spots on the same slide, between slides hybridized with cDNAs from the same labeling reaction, and between slides hybridized with cDNAs prepared in separate labeling reactions. We found that the results were both robust and statistically reproducible. Spot-to-spot variability was 3.8% between replicate spots on a given slide, 5.0% between spots on separate slides (though hybridized with identical, labeled cDNA), and 8.1% between spots from separate slides hybridized with samples from separate reverse transcription reactions yielding an average spot to spot variability of 7.1% across all conditions. Furthermore, when we examined the changes in gene expression that occurred between the two phases of the fermentation, we found that results for the majority of the genes agreed with observations made using other methods. These procedures will be a valuable addition to the metabolic engineering toolbox for the improvement of C. glutamicum amino acid-producing strains.