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The nuclear factor-κB (NFκB) family of transcription factors has been implicated in inflammatory disorders, viral infections, and cancer. Most of the drugs that inhibit NFκB show significant side effects, possibly due to sustained NFκB suppression. Drugs affecting induced, but not basal, NFκB activity may have the potential to provide therapeutic benefit without associated toxicity. NFκB activation by stress-inducible cell cycle inhibitor p21 was shown to be mediated by a p21-stimulated transcription-regulating kinase CDK8. CDK8 and its paralog CDK19, associated with the transcriptional Mediator complex, act as coregulators of several transcription factors implicated in cancer; CDK8/19 inhibitors are entering clinical development. Here we show that CDK8/19 inhibition by different small-molecule kinase inhibitors or shRNAs suppresses the elongation of NFκB-induced transcription when such transcription is activated by p21-independent canonical inducers, such as TNFα. On NFκB activation, CDK8/19 are corecruited with NFκB to the promoters of the responsive genes. Inhibition of CDK8/19 kinase activity suppresses the RNA polymerase II C-terminal domain phosphorylation required for transcriptional elongation, in a gene-specific manner. Genes coregulated by CDK8/19 and NFκB include IL8, CXCL1, and CXCL2, which encode tumor-promoting proinflammatory cytokines. Although it suppressed newly induced NFκB-driven transcription, CDK8/19 inhibition in most cases had no effect on the basal expression of NFκB-regulated genes or promoters; the same selective regulation of newly induced transcription was observed with other transcription signals potentiated by CDK8/19. This selective role of CDK8/19 identifies these kinases as mediators of transcriptional reprogramming, a key aspect of development and differentiation as well as pathological processes. [ABSTRACT FROM AUTHOR]

Lysine methylation of the p65 subunit of nuclear factor κB (NF-κB) on K218 and K221 together or K37 alone strongly enhances gene expression in response to cytokines. We analyzed the effects of K-to-Q mutations in the REL homology domain of p65 on the response to IL-1β in 293 cells with low levels of p65. The K218/221Q mutation greatly reduced the expression of 39 of 82 genes, whereas the K37Q mutation reduced the expression of 23 different genes. Enhanced expression of the lysine demethylase FBXL11, which catalyzes the demethylation of K218 and K221 specifically, inhibited the expression of most of the genes that were inhibited by the DKQ mutation. CHIP-Seq analysis showed that the K218/221Q mutation greatly reduces the affinity of p65 for many promoters and that the K37Q mutation does not. Structural modeling showed that the newly introduced methyl groups of K218 and K221 interact directly with DNA to increase the affinity of p65 for specific κB sites. Thus, the K218/221Q and K37Q mutations have dramatically different effects because methylations of these residues affect different genes by distinct mechanisms. [ABSTRACT FROM AUTHOR]

The ubiquitous inducible transcription factor NF-κB plays central roles in immune and inflammatory responses and in tumorigenesis. Complex posttranslational modifications of the p65 subunit (RelA) are a major aspect of the extremely flexible regulation of NF-κB activity. Although phosphorylation, acetylation, ubiquitination, and lysine methylation of NF-κB have been well described, arginine methylation has not yet been found. We now report that, in response to IL-1β, the p65 subunit of NF-κB is dimethylated on arginine 30 (R30) by protein-arginine methyltransferase 5 (PRMT5). Expression of the R30A and R30K mutants of p65 substantially decreased the ability of NF-κB to bind to κB elements and to drive gene expression. A model in which dimethyl R30 is placed into the crystal structure of p65 predicts new van der Waals contacts that stabilize intraprotein interactions and indirectly increase the affinity of p65 for DNA. PRMT5 was the only arginine methyltransferase that coprecipitated with p65, and its overexpression increased NF-κB activity, whereas PRMT5 knockdown had the opposite effect. Microarray analysis revealed that ~85% of the NF-κB-inducible genes that are down-regulated by the R30A mutation are similarly down-regulated by knocking PRMT5 down. Many cytokine and chemokine genes are among these, and conditioned media from cells expressing the R30A mutant of p65 had much less NF-κB-inducing activity than media from cells expressing the wild-type protein. PRMT5 is overexpressed in many types of cancer, often to a striking degree, indicating that high levels of this enzyme may promote tumorigenesis, at least in part by facilitating NF-κB-induced gene expression. [ABSTRACT FROM AUTHOR]

Following its tyrosine phosphorylation. STAT3 is methylated on k140 by the histone methyl transferase SET9 and demethylated by LSD1 when it is bound to a subset of the promoters that it activates. Methylation of K140 is a negative regulatory event, because its blockade greatly increases the steady-state amount of activated STAT3 and the expression of many (i.e., SOCS3) but not all (i.e., CD 14) STAT3 target genes. Biological relevance is shown by the observation that overexpression of SOCS3 when k140 cannot be methylatod blocks the ability of cells to activate STAT3 in response to IL-6. 1(140 methylation does not occur with mutants of STAT3 that do not enter nuclei or bind to DNA. Following treatment with lL-6, events at the SOCS3 promoter occur in an ordered sequence, as shown by chromatin immunoprecipitations. Y705-phosphoryl- STAT3 binds first and 5727 is then phosphorylated, followed by the coincident binding of SET9 and dimethylation of K140, and lastly by the binding of LSD1. We conclude that the lysine methylation of promoter-bound STAT3 leads to biologically important down-regulation of the dependent responses and that SET9. which is known to help provide an activating methylation mark to H3K4, is recruited to the newly activated SOCS3 promoter by STAT3. [ABSTRACT FROM AUTHOR]

Dysregulation of autophagy, a cellular catabolic mechanism essential for degradation of misfolded proteins, has been implicated in multiple neurodegenerative diseases. However, the mechanisms that lead to the autophagy dysfunction are still not clear. Based on the results of a genome-wide screen, we show that reactive oxygen species (ROS) serve as common mediators upstream of the activation of the type III PI3 kinase, which is critical for the initiation of autophagy. Furthermore, ROS play an essential function in the induction of the type III PI3 kinase and autophagy in response to amyloid β peptide, the main pathogenic mediator of Alzheimer's disease (AD). However, lysosomal blockage also caused by Aβ is independent of ROS. In addition, we demonstrate that autophagy is transcriptionally down-regulated during normal aging in the human brain. Strikingly, in contrast to normal aging, we observe transcriptional upregulation of autophagy in the brains of AD patients, suggesting that there might be a compensatory regulation of autophagy. Interestingly, we show that an AD drug and an AD drug candidate have inhibitory effects on autophagy, raising the possibility that decreasing input into the lysosomal system may help to reduce cellular stress in AD. Finally, we provide a list of candidate drug targets that can be used to safely modulate levels of autophagy without causing cell death. [ABSTRACT FROM AUTHOR]

NF-KB, a central coordinator of immune and inflammatory responses, must be tightly regulated. We describe a NF-KB regulatory pathway that is driven by reversible lysine methylation of the p65 subunit, carried out by a lysine methylase, the nuclear receptor- binding SET domain-containing protein 1 (NSD1), and a lysine demethylase, F-box and leucine-rich repeat protein 11 (FBXL11). Overexpression of FBXL11 inhibits NF-KB activity, and a high level of NSD1 activates NF-KB and reverses the inhibitory effect of FBXL1 1, whereas reduced expression of NSD1 decreases NF-KB acti- vation. The targets are K21 Sand K221 of p65, which are methylated in cells with activated NF-KB. Overexpression of FBXL11 slowed the growth of HT29 cancer cells, whereas shRNA-mediated knockdown had the opposite effect, and these phenotypes were dependent on K218/K221 methylation. In mouse embryo fibroblasts, the activation of most p65-dependent genes relied on K218/K221 methylation. Importantly, expression of the FBXL 11 gene is driven by NF-KB, revealing a negative regulatory feedback loop. We conclude that reversible lysine methylation of NF-KB is an important element in the complex regulation of this key transcription factor. [ABSTRACT FROM AUTHOR]

We describe a highly efficient use of lentiviral validation-based insertional mutagenesis (VBIM) to generate large populations of mammalian cells in which a strong promoter is inserted into many different genomic loci, causing greatly increased expression of downstream sequences. Many different selections or screens can follow, to isolate dominant mutant clones with a desired phenotypic change. The inserted promoter can be excised or silenced at will, to prove that the insertion caused the mutation. Cloning DNA flanking the insertion site identifies the locus precisely. VBIM virus particles are pseudotyped with VSV G protein, allowing efficient infection of most mammalian cell types, including non-dividing cells, and features are included that give high yields of stable virus stocks. In several different selections, useful mutants have been obtained at frequencies of approximately 10-6 or higher. We used the VBIM technique to isolate mutant human cells in which the F-box leucine-rich protein 11 (FBXL11), a histone H3K36 demethylase, is shown to be a negative regulator of NFκB. High levels of FBXL11 block the ability of NFicB to bind to DNA or activate gene expression, and siRNA-mediated reduction of FBXL11 expression has the opposite effects. The H212A mutation of FBXL11 abolishes both its histone H3K36 demethylase activity and its ability to inhibit NFκB. Thus, we have used a powerful tool for mutagenesis of mammalian cells to reveal an aspect of the complex regulation of NFκB-dependent signaling. [ABSTRACT FROM AUTHOR]

Some tumor cell lines secrete high concentrations of TGFβ or IL-1. Similarly high concentrations of each of these cytokines cross-activate the other pathway: TGFβ activates NFκB, and IL-1β activates Smads. The IL-1 signaling components IRAK, MyD88, TRAF6, and TAK1 are all required for cross-activation of NFKB by TGF/3. Knockdown experiments revealed that both TGFβ receptor subunits are required for IL-1β to activate Smads, and the IL-1 receptor is required for TGFβ to activate NFκB. Coimmunoprecipitations showed that either TGFβ or IL-1β stimulate ligand-dependent association of all three receptor subunits. Furthermore, cross-talk between the TGFβ and IL-1 signaling pathways leads to dose-dependent cross-control of gene expression. These interactions provide new insight into biological responses to IL-1 and TGFβ in the proximity of tumors that secrete high concentrations of these factors and probably also at sites of inflammation, where the local concentrations of these cytokines are likely to be high. [ABSTRACT FROM AUTHOR]

Genetic dissection of signaling pathways in mammalian cells involves screening or selecting phenotypic mutants obtained by a variety of techniques. Limitations in current methods include inadequate genome coverage and difficulty in validating the link between mutation and phenotype. We describe an improved method for insertional mutagenesis with retroviral vectors and show that the ability to induce mutations increases greatly if a randomly inserted promoter directs transcription into the host DNA. The mutant phenotype is due to the expression of a hybrid transcript derived from the vector and the insertion site. Because other alleles of the affected gene remain intact, the phenotype is dominant, but is reversible by inactivating the promoter. for example, by site-specific recombination. Importantly, in mutant clones with multiple inserts, limited excision yields progeny with different patterns of inserts remaining. Characterizing these progeny allows the mutant phenotype to be associated with a specific target gene. Relative simplicity and robust target validation make the method suitable for a broad range of applications. We have used this technique to search for proteins that regulate NE-κB- dependent signaling in human cells. Two validated targets are the relA gene, which codes for the NF-κR p65 subunit, and the NF-κB regulator act1. Overexpression of the corresponding proteins, caused by insertion of a promoter into the first intron of each gene, leads to NF-κB-dependent secretion of factors that activate NF-κB through cell-surface receptors, establishing an autocrine loop. [ABSTRACT FROM AUTHOR]

The basis of constitutive activation of NF-κB, essential for survival and resistance to apoptosis in many tumors, is not well understood. We find that transforming growth factor β2 (TGFβ2), predominantly in its latent form, is secreted by several different types of tumor cell lines that exhibit constitutively active NF-κB and that TGFβ2 potently stimulates the activation of NF-κB in reporter cells. Suppression of TGFβ2 expression by small interfering RNA kills prostate cancer PC3 cells, indicating that the TGFβ2-NF-κB pathway is important for their viability. These findings identify TGFβ2 as a potential target for therapeutic strategies to inhibit the growth of tumor cells that depend on constitutively active NE-κB, or to sensitize them to treatment with cytotoxic drugs. [ABSTRACT FROM AUTHOR]

Investigates the effect of targeted disruption of the MKK4 gene, a member of the mitogen-activated protein kinase kinase group of dual specificity protein kinases that function as an activator of the c-Jun NH2-terminal kinase (JNK) in vitro. Inhibition of c-Jun NH2-terminal kinase activation; Embryonic death; Defects in AP-1 transcriptional activity.

Catalysts with a refined electronic structure are highly desirable for promoting the oxygen evolution reaction (OER) kinetics and reduce the charge overpotentials for lithium-oxygen (Li-O 2 ) batteries. However, bridging the orbital interactions inside the catalyst with external orbital coupling between catalysts and intermediates for reinforcing OER catalytic activities remains a grand challenge. Herein, we report a cascaded orbital-oriented hybridization, namely alloying hybridization in intermetallic Pd 3 Pb followed by intermolecular orbital hybridization between low-energy Pd atom and reaction intermediates, for greatly enhancing the OER electrocatalytic activity in Li-O 2 battery. The oriented orbital hybridization in two axes between Pb and Pd first lowers the d band energy level of Pd atoms in the intermetallic Pd 3 Pb; during the charging process, the low-lying 4d xz/yz and 4d z 2 orbital of the Pd further hybridizes with 2π* and 5σ orbitals of lithium superoxide (LiO 2 ) (key reaction intermediate), eventually leading to lower energy levels of antibonding and, thus, weakened orbital interaction toward LiO 2 . As a consequence, the cascaded orbital-oriented hybridization in intermetallic Pd 3 Pb considerably decreases the activation energy and accelerates the OER kinetics. The Pd 3 Pb-based Li-O 2 batteries exhibit a low OER overpotential of 0.45 V and superior cycle stability of 175 cycles at a fixed capacity of 1,000 mAh g -1 , which is among the best in the reported catalysts. The present work opens up a way for designing sophisticated Li-O 2 batteries at the orbital level.

Vγ9Vδ2 T cells play an important role in the development and progression of psoriasis vulgaris (PV), but how they promote skin inflammation and the molecular mechanisms underlying Vγ9Vδ2 T cell dysfunction are poorly understood. Here, we show that circulating Vγ9Vδ2 T cells are decreased and exhibit enhanced proliferation and increased production of IFN-γ and TNF-α in PV patients. Monocytes from PV patients express higher levels of the phosphoantigen sensor butyrophilin 3A1 (BTN3A1) than monocytes from healthy controls. Blockade of BTN3A1 suppresses Vγ9Vδ2 T cell activation and abolishes the difference in Vγ9Vδ2 T cell activation between PV patients and healthy controls. The CD14 + cells in PV skin lesions highly express BTN3A1 and juxtapose to Vδ2 T cells. In addition, IFN-γ induces the up-regulation of BTN3A1 on monocytes. Collectively, our results demonstrate a crucial role of BTN3A1 on monocytes in regulating Vγ9Vδ2 T cell activation and highlight BTN3A1 as a potential therapeutic target for psoriasis.