The p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs) play important roles in the host innate immune response. The protein kinase regulated by RNA (PKR) is implicated in p38 MAPK activation in response to proinflammatory signals in mouse embryonic fibroblasts. To test the role of PKR in the activation of p38 and JNK MAPKs in human cells following viral infection, HeLa cells made stably deficient in PKR by using an RNA interference strategy were compared to cells with sufficient PKR. The phosphorylation of both p38 and JNK in cells with sufficient PKR was activated following either infection with an E3L deletion (ΔE3L) mutant of vaccinia virus or transfection with double-stranded RNA (dsRNA) in the absence of infection with wild-type vaccinia virus. The depletion of PKR by stable knockdown impaired the phosphorylation of both p38 and JNK induced by either the ΔE3L mutant virus or dsRNA but not that induced by tumor necrosis factor alpha. The PKR-dependent activation of MAPKs in ΔE3L mutant-infected cells was abolished by treatment with cytosine β-d-arabinoside. The complementation of PKR-deficient cells with the human PKR wild-type protein, but not with the PKR catalytic mutant (K296R) protein, restored p38 and JNK phosphorylation following ΔE3L mutant virus infection. Transient small interfering RNA knockdown established that the p38 and JNK kinase activation following ΔE3L infection was dependent upon RIG-I-like receptor signal transduction pathway components, including the mitochondrial adapter IPS-1 protein.
Interferons (IFNs) are antiviral cytokines that selectively regulate gene expression through several signaling pathways including nuclear factor κB (NFκB). To investigate the specific role of NFκB in IFN signaling, we performed gene expression profiling after IFN treatment of embryonic fibroblasts derived from normal mice or mice with targeted deletion in NFκB p50 and p65 genes. Interestingly, several antiviral and immunomodulatory genes were induced higher by IFN in NFκB knockout cells. Chromatin immunoprecipitation (ChIP) experiments demonstrated that NFκB was basally bound to the promoters of these genes, while IFN treatment resulted in the recruitment of STAT1 and STAT2 to these promoters. However, in NFκB knockout cells IFN induced STAT binding as well as the binding of the IFN regulatory factor-1 (IRF1) to the ISG promoters. IRF1 binding closely correlated with enhanced gene induction. Moreover, NFκB suppressed both antiviral and immunomodulatory actions of IFN against influenza virus. Our results identify a novel negative regulatory role of NFκB in IFN-induced gene expression and biological activities, and suggest that modulating NFκB activity may provide a new avenue for enhancing the IFN's therapeutic effectiveness.
The protein kinase regulated by RNA (PKR) enhances both activation of mitogen-activated protein kinases and the induction of interferon beta (IFN-β) by measles virus defective in C protein expression (Cko). Here we used complementation of human cell lines stably deficient in PKR (PKRkd) to probe the basis of these PKR-mediated responses. We found that PKRkd HeLa and amnion U cell lines were defective for virus-mediated activation of IFN induction signaling components compared to PKR-sufficient control cells. Complementation of PKRkd cells with wildtype PKR, but not with PKR mutants defective in either catalytic activity or dsRNA binding activity, restored JNK, p38 and ATF-2 phosphorylation and enhanced IFN-β induction following infection. By contrast to mammalian PKR, the Z-DNA binding domain-containing fish homologue of PKR, PKZ, lacked the capacity to enhance Cko virus-mediated IFN-β induction. Furthermore, inhibition of virus growth was observed with Cko-infected PKRkd cells complemented with PKR but not with PKZ.
PKR; protein kinase; interferon; innate immunity
The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.
Bioengineering; Issue 59; Materials discovery; Surface characterization; Polymer library; High throughput; Cell attachment
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 immunosuppressive and antiinflammatory actions of glucocorticoid hormones are mediated by their transrepression of activating protein-1 (AP-1) and nuclear factor-kappa B (NFκB) transcription factors. Inhibition of the c-Jun NH2-terminal kinase (JNK) signaling pathway, the main mediator of AP-1 activation, has been described in extracts of hormone-treated cells. Here, we show by confocal laser microscopy, enzymatic assays, and immunoblotting that the synthetic glucocorticoid dexamethasone inhibited tumor necrosis factor α (TNF-α)–induced phosphorylation and activation of JNK in the cytoplasm and nucleus of intact HeLa cells. As a result, c-Jun NH2-terminal domain phosphorylation and induction were impaired. Dexamethasone did not block the TNF-α–induced JNK nuclear translocation, but rather induced, per se, nuclear accumulation of the enzyme. Consistently with previous findings, a glucocorticoid receptor mutant (GRdim), which is deficient in dimerization, DNA binding, and transactivation, but retains AP-1 transrepressing activity, was as efficient as wild-type GR in mediating the same effects of dexamethasone on JNK in transfected Cos-7 cells. Our results show that glucocorticoids antagonize the TNF-α–induced activation of AP-1 by causing the accumulation of inactive JNK without affecting its subcellular distribution.
dexamethasone; activating protein-1; tumor necrosis factor α; c-Jun NH2-terminal kinase; nuclear translocation
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.
The inhibition of apoptosis by Toxoplasma gondii is governed by its modulation of several signaling cascades including the NFκappaB and JNK pathways. This is evident in the dysregulation of JNK activation following treatment with UV and TNFα, both apoptogenic stimuli. Infection-mediated interference with the JNK cascade was found to be highly reproducible in HeLa cells. In light of emerging evidence regarding cross talk between the JNK and NFκB cascades, we examined the impact of infection in wild type and RelA/p65−/− mouse embryonic fibroblasts (MEF). Remarkably, parasite infection failed to significantly impact both UV and TNFα-mediated JNK phosphorylation in both cell lines suggesting a cell type specific effect. Furthermore siRNA-mediated knockdown of RelA/p65 failed to impact the parasite mediated effects on stimulus dependent activation of JNK in HeLa cells. Finally, the infection mediated suppression of JNK phosphorylation in HeLa cells did not result in decreased JNK kinase activity. Rather, the reduced levels of phospho-JNK in infected cells correlated with increased phosphatase activity noted by the partial rescue of the phenotype following treatment with okadaic acid. Taken together the results indicate that manipulation of the JNK-pathway does not involve NFκB and is furthermore not a central component of the parasite enforced block of apoptosis. It further highlights the complexity of these systems and the danger of extrapolating results both within and across pathogen-host cell systems based on limited studies.
Toxoplasma gondii; apoptosis; JNK; NFκB
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
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
The goal of the present study was to investigate hepato-protective effects of growth factor (GF) arrays during alcohol injury. Hepatocyte growth factor (HGF) and bone morphogenetic protein (BMP)7 were mixed with collagen (I) and robotically printed onto standard glass slides to create arrays of 500 μm diameter spots. Primary rat hepatocytes were seeded on top of the arrays forming clusters corresponding in size to the underlying protein spots. Cell arrays were then injured in culture by exposure to 100 mM ethanol for 48h. Hepatocytes residing on GF spots were found to have less apoptosis then cells cultured on collagen-only spots. Least apoptosis (0.3 % as estimated by TUNEL assay) was observed on HGF/BMP7/collagen spots whereas most apoptosis (17.3%) was seen on collagen-only arrays. Interestingly, the extent of alcohol-induced apoptosis in hepatocytes varied based on the concentration of printed GF. In addition to preventing apoptosis, printed GFs contributed to maintenance of epithelial phenotype during alcohol injury as evidenced by higher levels of E-cadherin expression in HGF-protected hepatocytes. Importantly, GF microarrays could be used to investigate heterotypic interactions in the context of liver injury. To highlight this, stellate cells - nonparenchymal liver cells involved in fibrosis - were added to hepatocytes residing on arrays of either HGF/collagen or collagen-only spots. Exposure of these cocultures to ethanol followed by RT-PCR analysis revealed that stellate cells residing alongside HGF-protected hepatocytes were significantly less activated (less fibrotic) compared to controls. Overall, our results demonstrate that GF microarray format can be used to screen anti-fibrotic and anti-apoptotic effects of growth factors as well as to investigate how signals delivered to a specific cell type modulate heterotypic cellular interactions.
Protein microarrays; Micropatterned cocultures; Hepatocytes; Growth factor microarrays; Apoptosis; Fibrosis; Ethanol injury
In the assembly of microarrays and microarray-based chemical assays and enzymatic bioassays, most approaches use pins for contact spotting. Acoustic dispensing is a technology capable of nanoliter transfers by using acoustic energy to eject liquid sample from an open source well. Although typically used for well plate transfers, when applied to microarraying it avoids drawbacks of undesired physical contact with sample, difficulty in assembling multicomponent reactions on a chip by readdressing, a rigid mode of printing that lacks patterning capabilities, and time-consuming wash steps. We demonstrated the utility of acoustic dispensing by delivering human cathepsin L in a drop-on-drop fashion into individual 50-nanoliter, pre-spotted reaction volumes to activate enzyme reactions at targeted positions on a microarray. We generated variable-sized spots ranging from 200 to 750 μm (and higher), and handled the transfer of fluorescent bead suspensions with increasing source well concentrations of 0.1 to 10 ×108 beads/mL in a linear fashion. There are no tips that can clog and liquid dispensing CVs are generally below 5%. This platform expands the toolbox for generating analytical arrays and meets needs associated with spatially-addressed assembly of multicomponent microarrays on the nanoliter scale.
Microarray; acoustic dispensing; pin spotting; no-contact dispensing; assay development; lab on a chip; screening
Cellular stresses, including growth factor deprivation, inflammatory cytokines or viral infection promote RAX/PACT-dependent activation of the double-stranded RNA-dependent protein kinase, PKR, to phosphorylate eIF2α, resulting in translation inhibition and apoptosis. In addition, PKR has been reported to regulate p53, STAT1 and NFκB. Here, we report that RAX/PACT interacts with the SUMO E2 ligase Ubc9 to stimulate p53-Ubc9 association and reversible p53 sumoylation on lysine 386. In addition, expression of RAX/PACT in a variety of cell lines promotes p53 stability and activity to increase p53 target gene expression. Significantly, while the expression of RAX/PACT, PKR or p53 alone has little effect on the cell cycle of p53-null H1299 cells, co-expression of p53 with either RAX/PACT or PKR promotes a 25–35% increase of cells in G1. In contrast, co-expression of RAX/PACT with the sumoylation-deficient p53(K386R) mutant or with the desumoylase SENP1 fails to induce such a G1 arrest. Furthermore, co-expression of p53, RAX/PACT and the dominant-negative PKR(K296R) mutant inhibits RAX/PACT-induced, p53-dependent G1 growth arrest and expression of RAX/PACT in pkr+/+ but not pkr−/− MEF cells promotes p53 and p21 expression following gamma irradiation. Significantly, p53 stability is decreased in cells with reduced RAX/PACT or PKR following doxorubicin treatment, and expression of exogenous RAX/PACT promotes phosphorylation of wild-type but not p53(K386R) on serine 392. Collectively, results indicate that, in response to stress, the RAX/PACT-PKR signaling pathway may inhibit p53 protein turnover by a sumoylation-dependent mechanism with promotion of p53 phosphorylation and translational activation leading to G1 cell cycle arrest.
p53; PKR; RAX; PACT; Ubc9; sumoylation
Background & Aims
Chronic liver disease is associated with endotoxemia, oxidative stress, increased endocannabinoids and decreased cardiac responsiveness. Endocannabinoids activate the tumor necrosis factor-alpha (TNFα)–nuclear factor κB (NFκB) pathway. However, how they interact with each other remains obscure. We therefore aimed to clarify the relationship between the TNFα–NFκB pathway and endocannabinoids in the pathogenesis of cardiodepression of cholestatic bile duct ligated (BDL) mice.
BDL mice with TNFα knockout (TNFα−/−) and infusion of anti-TNFα antibody were used. Cardiac mRNA and protein expression of NFκBp65, c-Jun-N-terminal kinases (JNK), p38 mitogen-activated protein kinase (p38MAPK), extracelullar-signal-regulated kinase (ERK), inducible nitric oxide synthase (iNOS), Copper/Zinc and Magnesium-superoxide dismutase (Cu/Zn- and Mn-SOD), cardiac anandamide, 2-arachidonoylglycerol (2-AG), nitric oxide (NOx) and glutathione, and plasma TNFα were measured. The effects of TNFα, cannabinoid receptor (CB1) antagonist AM251 and the endocannabinoid reuptake inhibitor UCM707, on the contractility of isolated cardiomyocytes, were assessed.
In BDL mice, cardiac mRNA and protein expression of NFκBp65, p38MAPK, iNOS, NOx, anandamide, and plasma TNFα were increased, whereas glutathione, Cu/Zn-SOD, and Mn-SOD were decreased. Cardiac contractility was blunted in BDL mice. Anti-TNFα treatment in BDL mice decreased cardiac anandamide and NOx, reduced expression of NFκBp65, p38MAPK, and iNOS, enhanced expression of Cu/Zn-SOD and Mn-SOD, increased reductive glutathione and restored cardiomyocyte contractility. TNFα-depressed contractility was worsened by UCM707, whereas AM251 improved contractility.
Increased TNFα, acting via NFκB–iNOS and p38MAPK signaling pathways, plays an important role in the pathogenesis of cardiodepression in BDL mice. TNFα also suppressed contractility by increasing oxidative stress and endocannabinoid activity.
Cardiac dysfunction; Endocannabinoids; Tumor necrosis factor-alpha (TNFα); Nuclear factor-B (NFκB); Inducible nitric oxide synthase (iNOS) 3
Dimethylaminoparthenolide (DMAPT) is a water soluble parthenolide analogue with preclinical activity in hematologic malignancies. Using NSCLC cell lines (A549, H522) and an immortalized human bronchial epithelial cell line (BEAS2B) and TCC cell lines (UMUC-3, HT-1197, HT-1376) and a bladder papilloma (RT-4), we aimed to characterize DMAPT's anti-cancer activity in tobacco associated neoplasms. Flow cytometric, electrophorectic mobility gel shift assays (EMSA), and western blot studies measured generation of reactive oxygen species (ROS), inhibition of NFκB DNA binding, and changes in cell cycle distribution and apoptotic proteins. DMAPT generated ROS with subsequent JNK activation and also decreased NFκB DNA binding and anti-apoptotic proteins, TRAF-2 and XIAP. DMAPT induced apoptotic cell death and altered cell cycle distribution with upregulation of p21 and p73 levels in a cell type dependent manner. DMAPT suppressed cyclin D1 in BEAS2B. DMAPT retained NFκB and cell cycle inhibitory activity in the presence of the tobacco carcinogen nitrosamine ketone, 4(methylnitrosamino)-1-(3–pyridyl)-1-butanone (NNK). Using a BrdU accumulation assay, 5 to 20μM of DMAPT was shown to inhibit cellular proliferation of all cell lines by more than 95%. Oral dosing of DMAPT suppressed in vivo A549 and UMUC-3 subcutaneous xenograft growth by 54% (p=0.015) and 63% (p<0.01) respectively and A549 lung metastatic volume by 28% (p=0.043). In total this data demonstrates DMAPT's novel anti-cancer properties in both early and late stage tobacco associated neoplasms as well as its significant in vivo activity. The data provides support for the conduct of clinical trials in TCC and NSCLC.
Dimethylaminoparthenolide; cell cycle; apoptosis; cancer
Vascular dysfunction contributes to the pathogenesis of various cardiovascular diseases. Dietary supplements, including fish oil, dietary fibers, and various natural products, and exercise training exert vasoprotective effects. However, the mechanisms underlying the vasoprotective benefits of dietary supplements and physical activity demand extensive investigation. Accumulating evidence suggests that inflammatory cytokine tumor necrosis factor-alpha (TNFα) plays a pivotal role in the dysregulation of macrovascular and microvascular function. TNFα induces vascular inflammation, monocyte adhesion to endothelial cells, vascular oxidative stress, apoptosis, and atherogenic response and participates in the regulation of thrombosis and coagulation through multiple signaling pathways involving NFκB, Sp1, activator protein 1, JNK, p38, STAT3, and so forth. Dietary supplements and exercise training decrease TNFα production and ameliorate TNFα-mediated pathological changes in vasculature. Thus, the inhibitory effects of dietary supplements and physical exercise on TNFα production and TNFα signaling may contribute to their vasoprotective properties.
Many cancers arise at sites of infection and inflammation. Cellular senescence, a permanent state of cell cycle arrest that provides a barrier against tumorigenesis, is accompanied by elevated proinflammatory cytokines such as IL1, IL6, IL8 and TNFα. Here we demonstrate that media conditioned by cells undergoing any of the three main forms of senescence, i.e. replicative, oncogene- and drug-induced, contain high levels of IL1, IL6, and TGFb capable of inducing reactive oxygen species (ROS)-mediated DNA damage response (DDR). Persistent cytokine signaling and activated DDR evoke senescence in normal bystander cells, accompanied by activation of the JAK/STAT, TGFβ/SMAD and IL1/NFκB signaling pathways. Whereas inhibition of IL6/STAT signaling had no effect on DDR induction in bystander cells, inhibition of either TGFβ/SMAD or IL1/NFκB pathway resulted in decreased ROS production and reduced DDR in bystander cells. Simultaneous inhibition of both TGFβ/SMAD and IL1/NFκB pathways completely suppressed DDR indicating that IL1 and TGFβ cooperate to induce and/or maintain bystander senescence. Furthermore, the observed IL1- and TGFβ-induced expression of NAPDH oxidase Nox4 indicates a mechanistic link between the senescence-associated secretory phenotype (SASP) and DNA damage signaling as a feature shared by development of all major forms of paracrine bystander senescence.
senescence-associated secretome; DNA damage response; cytokines; JAK/STAT3; TGFβ; NFκB; IL6; IL1β; Nox4; autocrine and paracrine signaling; tumor microenvironment
Microarray analysis is a critically important technology for genome-enabled biology, therefore it is essential that the data obtained be reliable. Current software and normalization techniques for microarray analysis rely on the assumption that fluorescent background within spots is essentially the same throughout the glass slide and can be measured by fluorescence surrounding the spots. This assumption is not valid if background fluorescence is spot-localized. Inaccurate estimates of background fluorescence under the spot create a source of error, especially for low expressed genes. We have identified spot-localized, contaminating fluorescence in the Cy3 channel on several commercial and in-house printed microarray slides. We determined through mock hybridizations (without labeled target) that pre-hybridization scans could not be used to predict the contribution of this contaminating fluorescence after hybridization because the change in spot-to-spot fluorescence after hybridization was too variable. Two solutions to this problem were identified. First, allowing 4 h of exposure to air prior to printing on to Corning UltraGAPS slides significantly reduced contaminating fluorescence intensities to approximately the value of the surrounding glass. Alternatively, application of a novel, hyperspectral imaging scanner and multivariate curve resolution algorithms, allowed the spectral contributions of Cy3 signal, glass, and contaminating fluorescence to be distinguished and quantified after hybridization.
We describe a novel application of microarray technology for comparative genomics of bacteria in which libraries of entire genomes rather than the sequence of a single genome or sets of genes are arrayed on the slide and then probed for the presence or absence of specific genes and/or gene alleles.
We first adopted a 96-well high throughput working protocol to efficiently isolate high quality genomic DNA. We then optimized conditions to print genomic DNA onto a glass slide with high density (up to 15000 spots) and to sensitively detect gene targets in each genome spot using fluorescently labeled DNA probe. Finally, we created an E. coli reference collection array and probed it for the presence or absence of the hemolysin (hly) gene using a dual channel non-competing hybridization strategy. Results from the array hybridization matched perfectly with previous tests.
This new form of microarray technology, Library on a Slide, is an efficient way for sharing and utilizing large strain collections in comparative genomic analyses.
The dsRNA-dependent protein kinase (PKR) is a key mediator of the anti-viral and anti-proliferative effects of interferon. Unphosphorylated PKR is characterized by inhibitory interactions between the kinase and RNA binding domains (RBDs), but the structural details of the latent state and its unraveling during activation are not well understood. To study PKR regulation by NMR we assigned a large portion of the backbone resonances of the catalytically inactive K296R kinase domain, and performed 15N-HSQC titrations of this kinase domain with the RBDs. Chemical shift perturbations in the kinase indicate that RBD2 binds to the substrate eIF2α docking site in the kinase C-lobe. Consistent with these results, a mutation in the eIF2α docking site, F495A displays weaker interactions with the RBD. The full-length RBD1+2 binds more strongly to the kinase domain than RBD2 alone. The observed chemical shift changes extend from the eIF2α binding site into the kinase N-lobe and inside the active site, consistent with weak interactions between the N-terminal part of the RBD and the kinase.
Despite the fact that many genomes have been decoded, proteome chips comprising individually purified proteins have been reported only for budding yeast, mainly because of the complexity and difficulty of high-throughput protein purification. To facilitate proteomics studies in prokaryotes, we have developed a high-throughput protein purification protocol that allowed us to purify 4,256 proteins encoded by the Escherichia coli K12 strain within 10 h. The purified proteins were then spotted onto glass slides to create E. coli proteome chips. We used these chips to develop assays for identifying proteins involved in the recognition of potential base damage in DNA. By using a group of DNA probes, each containing a mismatched base pair or an abasic site, we found a small number of proteins that could recognize each type of probe with high affinity and specificity. We further evaluated two of these proteins, YbaZ and YbcN, by biochemical analyses. The assembly of libraries containing DNA probes with specific modifications and the availability of E. coli proteome chips have the potential to reveal important interactions between proteins and nucleic acids that are time-consuming and difficult to detect using other techniques.
The gingival epithelium is becoming known as a regulator of the oral innate immune responses to a variety of insults such as bacteria and chemicals, including those chemicals found in cigarette smoke. We investigated the effects of whole cigarette smoke on cell-surface-expressed Toll-like receptors (TLR)-2, −4 and −6, human β-defensin (HBD) and proinflammatory cytokine expression and production in primary human gingival epithelial cells. Whole cigarette smoke was shown to increase TLR2, TLR4 and TLR6 expression. Cigarette smoke led to ERK1/2, p38 and JNK phosphorylation in conjunction with nuclear factor-κB (NFκB) translocation into the nucleus. TLR expression following cigarette smoke exposure was down regulated by the use of ERK1/2, p38, JNK MAP kinases, and NFκB inhibitors, suggesting the involvement of these signaling pathways in the cellular response against cigarette smoke. Cigarette smoke also promoted HBD2, HBD3, IL-1β, and IL-6 expression through the ERK1/2 and NFκB pathways. Interestingly, the modulation of TLR, HBD, and cytokine expression was maintained long after the gingival epithelial cells were exposed to smoke. By promoting TLR, HBDs, and proinflammatory cytokine expression and production, cigarette smoke may contribute to innate immunity dysregulation, which may have a negative effect on human health.
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.
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.
The regulation of apoptosis under basal (non-stress) conditions is crucial for normal mammalian development and also for normal cellular turnover in different tissues throughout life. Deficient regulation of basal apoptosis, or its perturbation, can result in impaired development and/or disease states including cancer. In contrast to stress-induced apoptosis the regulation of apoptosis under basal conditions is poorly understood. To address this issue we have compared basal- and stress-induced apoptosis in human epithelial cells of normal and cancerous origins. For this purpose we focussed our study on the opposing pro-apoptotic JNK/anti-apoptotic NFκB pathways.
Combinatorial RNAi plus gene knockout were employed to access and map basal regulatory pathways of apoptosis. Follow-on, in-depth analyses included exogenous expression of phosphorylation mutants and chromatin immunoprecipitation. We demonstrate that basal apoptosis is constitutively suppressed by JNK2 in a range of human cancer cell lines. This effect was not observed in non-cancer cells. Silencing JNK2 by RNAi resulted in JNK1-dependent apoptosis of cancer cells via up-regulation of the AP-1 factor c-Jun. Unexpectedly we discovered that JNK1 and c-Jun promote basal apoptosis in the absence of “activating phosphorylations” typically induced by stress. Hypo-phosphorylated c-Jun accumulated to high levels following JNK2 silencing, auto-regulated its own expression and suppressed expression of Bcl-3, an unusual IκB protein and regulator of NFκB. Basal apoptosis was mediated by components of the TNFα response pathway but was mechanistically distinct from TNFα-induced apoptosis.
Our results demonstrate that mechanistically distinct pathways operate to regulate apoptosis in mammalian cells under basal (physiological) versus stress-induced conditions. We also describe a novel apoptotic network which governs the basal survival of cancer cells. Such information is crucial for understanding normal cellular turnover during mammalian development and subsequently throughout life. This information also opens new avenues for therapeutic intervention in human proliferative disease states including cancer.