An NF-κB–Dependent Role for JunB in the Induction of Proinflammatory Cytokines in LPS-Activated Bone Marrow–Derived Dendritic Cells

BACKGROUND: Dendritic cells (DCs) play a key role in the induction of adaptive and memory immune responses. Upon encounter with pathogens, they undergo a complex maturation process and migrate toward lymphoid organs where they stimulate immune effector cells. This process is associated with dramatic transcriptome changes, pointing to a paramount role for transcription factors in DC activation and function. The regulation and the role of these transcription factors are however ill-defined and require characterization. Among those, AP-1 is a family of dimeric transcription complexes with an acknowledged role in the control of immunity. However, it has not been studied in detail in DCs yet. METHODOLOGY/PRINCIPAL FINDINGS: Here, we have investigated the regulation and function of one of its essential components, JunB, in primary bone marrow-derived DCs induced to maturate upon stimulation by Escherichia coli lipopolysaccharide (LPS). Our data show fast and transient NF-kappaB-dependent transcriptional induction of the junb gene correlating with the induction of the TNFalpha, IL-6, and IL-12 proinflammatory cytokines. Inhibition of JunB protein induction by RNA interference hampered the transcriptional activation of the TNF-alpha, IL-6, and IL-12p40 genes. Consistently, chromatin immunoprecipitation experiments showed LPS-inducible binding of JunB at AP-1-responsive sites found in promoter regions of these genes. Concomitant LPS-inducible NF-kappaB/p65 binding to these promoters was also observed. CONCLUSIONS/SIGNIFICANCE: We identified a novel role for JunB--that is, induction of proinflammatory cytokines in LPS-activated primary DCs with NF-kappaB acting not only as an inducer of JunB, but also as its transcriptional partner

Stable transmission of targeted gene modification using single-stranded oligonucleotides with flanking LNAs

Targeted mutagenesis directed by oligonucleotides (ONs) is a promising method for manipulating the genome in higher eukaryotes. In this study, we have compared gene editing by different ONs on two new target sequences, the eBFP and the rd1 mutant photoreceptor βPDE cDNAs, which were integrated as single copy transgenes at the same genomic site in 293T cells. Interestingly, antisense ONs were superior to sense ONs for one target only, showing that target sequence can by itself impart strand-bias in gene editing. The most efficient ONs were short 25 nt ONs with flanking locked nucleic acids (LNAs), a chemistry that had only been tested for targeted nucleotide mutagenesis in yeast, and 25 nt ONs with phosphorothioate linkages. We showed that LNA-modified ONs mediate dose-dependent target modification and analyzed the importance of LNA position and content. Importantly, when using ONs with flanking LNAs, targeted gene modification was stably transmitted during cell division, which allowed reliable cloning of modified cells, a feature essential for further applications in functional genomics and gene therapy. Finally, we showed that ONs with flanking LNAs aimed at correcting the rd1 stop mutation could promote survival of photoreceptors in retinas of rd1 mutant mice, suggesting that they are also active in vivo

SUMOylation inhibitor TAK-981 (subasumstat) synergizes with 5-azacytidine in preclinical models of acute myeloid leukemia

Acute myeloid leukemias (AML) are severe hematomalignancies with dismal prognosis. The post-translational modification SUMOylation plays key roles in leukemogenesis and AML response to therapies. Here, we show that TAK-981 (subasumstat), a first-in-class SUMOylation inhibitor, is endowed with potent anti-leukemic activity in various preclinical models of AML. TAK-981 targets AML cell lines and patient blast cells in vitro and in vivo in xenografted mice with minimal toxicity on normal hematopoietic cells. Moreover, it synergizes with 5-azacytidine (AZA), a DNA-hypomethylating agent now used in combination with the BCL-2 inhibitor venetoclax to treat AML patients unfit for standard chemotherapies. Interestingly, TAK-981+AZA combination shows higher anti-leukemic activity than AZA+venetoclax combination both in vitro and in vivo, at least in the models tested. Mechanistically, TAK-981 potentiates the transcriptional reprogramming induced by AZA, promoting apoptosis, alteration of the cell cycle and differentiation of the leukemic cells. In addition, TAK-981+AZA treatment induces many genes linked to inflammation and immune response pathways. In particular, this leads to the secretion of type-I interferon by AML cells. Finally, TAK-981+AZA induces the expression of natural killer-activating ligands (MICA/B) and adhesion proteins (ICAM-1) at the surface of AML cells. Consistently, TAK-981+AZA-treated AML cells activate natural killer cells and increase their cytotoxic activity. Targeting SUMOylation with TAK-981 may thus be a promising strategy to both sensitize AML cells to AZA and reduce their immune-escape capacities

The Alpha Helix of Ubiquitin Interacts with Yeast Cyclin-Dependent Kinase Subunit CKS1 †

International audienc

Multisite Protein Kinase A and Glycogen Synthase Kinase 3β Phosphorylation Leads to Gli3 Ubiquitination by SCF(βTrCP)

Gli3 is a zinc finger transcription factor proteolytically processed into a truncated repressor lacking C-terminal activation domains. Gli3 processing is stimulated by protein kinase A (PKA) and inhibited by Hedgehog signaling, a major signaling pathway in vertebrate development and disease. We show here that multisite glycogen synthase kinase 3β (GSK3β) phosphorylation and ubiquitination by SCF(βTrCP) are required for Gli3 processing. We identified multiple βTrCP-binding sites related to the DSGX(2)(-)(4)S motif in Gli3, which are intertwined with PKA and GSK3β sites, and SCF(βTrCP) target lysines that are essential for processing. Our results support a simple model whereby PKA triggers a cascade of Gli3 phosphorylation by GSK3β and CK1 that leads to direct βTrCP binding and ubiquitination by SCF(βTrCP). Binding of βTrCP to Gli3 N- and C-terminal domains lacking DSGX(2)(-)(4)S-related motifs was also observed, which could reflect indirect interaction via other components of Hedgehog signaling, such as the tumor suppressor Sufu. Gli3 therefore joins a small set of transcription factors whose processing is regulated by the ubiquitin-proteasome pathway. Our study sheds light on the role of PKA phosphorylation in Gli3 processing and will help to analyze how dose-dependent tuning of Gli3 processing is achieved by Hedgehog signaling

The NF-κB member p65 controls glutamine metabolism through miR-23a.

International audienceCancer cells have elevated aerobic glycolysis that is termed the Warburg effect. But several tumor cells, including leukemic cells, also increase glutamine metabolism, which is initiated by glutaminase (GLS). The microRNA (miRNA) miR-23 targets GLS mRNA and inhibits expression of GLS protein. Here we show that in human leukemic Jurkat cells the NF-κB p65 subunit binds to miR-23a promoter and inhibits miR-23a expression. Histone deacetylase (HDAC) inhibitors release p65-induced inhibition. Jurkat cells growing in glutamine decrease proliferation due to cell accumulation in G0/G1 phase. Nevertheless, cells get used to this new source of energy by increasing GLS expression, which correlates with an increase in p65 expression and its translocation to the nucleus, leading to a higher basal NF-κB activity. Jurkat cells overexpressing p65 show increase basal GLS expression and proliferate faster than control cells in glutamine medium. Overexpressing miR-23a in leukemic cells impaired glutamine use and induces mitochondrial dysfunction leading to cell death. Therefore, p65 activation decreases miR-23a expression, which facilitates glutamine consumption allowing leukemic cells to use this alternative source of carbon and favoring their adaptation to the metabolic environment

Characterization of <i>E. coli</i> LPS-induced BMDC maturation.

<p>(A) <i>Purity of BMDCs</i>. Bone marrow cells were cultured in the presence of GM-CSF and IL-4 for 7 days and analyzed by flow cytometry for the presence of CD11c and MHC II. (B) <i>Induction of CD40 and CD80 by LPS</i>. BMDCs were LPS-stimulated for 24 hours and analyzed for the induction of CD40 and CD80 by flow cytometry. Error bars correspond to standard deviation from 5 independent experiments. (C) <i>Cytokine induction</i>. BMDCs were LPS-stimulated for the indicated periods of time and cytokines were assayed from culture supernatant by ELISA. The presented data correspond to a representative experiment.</p

Binding of JunB and NF-κB/p65 to the promoters of TNFα, IL-6 and IL-12p40 genes in LPS-stimulated BMDCs.

<p>(A) <i>Binding sites for AP-1 and NF-κB in the promoter regions of TNFα, IL-6 and IL-12p40</i>. The grey bars with the inverted arrows (enh) indicate the enhancer-containing fragment which is amplified in ChIP experiments to visualize JunB and NF-κB binding. The black bars with the inverted arrows (cont) located downstream of the various genes indicate the amplified negative control fragments used in the ChIP experiments to exclude non-specific JunB and NF-κB binding. (B, C and D) <i>Binding of JunB to the promoter regions of the TNFα, IL-6 and IL-12p40 genes</i>. BMDCs were LPS-stimulated for various periods of time and ChIP experiments were carried out for assessing the presence of JunB in the cytokine gene promoter regions. PI corresponds to negative control immunoprecipitations with preimmune sera. Non-specific binding of JunB was excluded by qPCR analysis of a DNA fragment devoid of any AP-1 site and located downstream of each gene (not shown). (E, F and G) <i>Binding of NF-κB/p65 to promoter regions of the TNFα, IL-6 and IL-12p40 genes</i>. The experiments were carried as in B, C and D, except that an anti-NF-κB/p65 antiserum was used instead of the anti-JunB one. JunB and NF-κB bindings in the enhancer region are presented in arbitrary units as well as the parallel negative control ChIPs carried out with a preimmune serum (PI). Calculations were made with respect to the amplification of the “cont” negative control fragment for each gene.</p