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1.
Cell ; 162(1): 33-44, 2015 Jul 02.
Article in English | MEDLINE | ID: mdl-26140591

ABSTRACT

SNPs affecting disease risk often reside in non-coding genomic regions. Here, we show that SNPs are highly enriched at mouse strain-selective adipose tissue binding sites for PPARγ, a nuclear receptor for anti-diabetic drugs. Many such SNPs alter binding motifs for PPARγ or cooperating factors and functionally regulate nearby genes whose expression is strain selective and imbalanced in heterozygous F1 mice. Moreover, genetically determined binding of PPARγ accounts for mouse strain-specific transcriptional effects of TZD drugs, providing proof of concept for personalized medicine related to nuclear receptor genomic occupancy. In human fat, motif-altering SNPs cause differential PPARγ binding, provide a molecular mechanism for some expression quantitative trait loci, and are risk factors for dysmetabolic traits in genome-wide association studies. One PPARγ motif-altering SNP is associated with HDL levels and other metabolic syndrome parameters. Thus, natural genetic variation in PPARγ genomic occupancy determines individual disease risk and drug response.


Subject(s)
Hypoglycemic Agents/metabolism , PPAR gamma/genetics , PPAR gamma/metabolism , Polymorphism, Single Nucleotide , Adipose Tissue , Animals , Gene Expression , Humans , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Transcription Factors/metabolism
2.
Genes Dev ; 32(15-16): 1035-1044, 2018 08 01.
Article in English | MEDLINE | ID: mdl-30006480

ABSTRACT

The nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) is known to regulate lipid metabolism in many tissues, including macrophages. Here we report that peritoneal macrophage respiration is enhanced by rosiglitazone, an activating PPARγ ligand, in a PPARγ-dependent manner. Moreover, PPARγ is required for macrophage respiration even in the absence of exogenous ligand. Unexpectedly, the absence of PPARγ dramatically affects the oxidation of glutamine. Both glutamine and PPARγ have been implicated in alternative activation (AA) of macrophages, and PPARγ was required for interleukin 4 (IL4)-dependent gene expression and stimulation of macrophage respiration. Indeed, unstimulated macrophages lacking PPARγ contained elevated levels of the inflammation-associated metabolite itaconate and express a proinflammatory transcriptome that, remarkably, phenocopied that of macrophages depleted of glutamine. Thus, PPARγ functions as a checkpoint, guarding against inflammation, and is permissive for AA by facilitating glutamine metabolism. However, PPARγ expression is itself markedly increased by IL4. This suggests that PPARγ functions at the center of a feed-forward loop that is central to AA of macrophages.


Subject(s)
Glutamine/metabolism , Macrophage Activation , Macrophages/metabolism , PPAR gamma/physiology , Animals , Cell Respiration , Cells, Cultured , Fatty Acids/metabolism , Gene Expression/drug effects , Glucose/metabolism , Interleukin-4/physiology , Macrophages/drug effects , Macrophages/immunology , Mice, Inbred C57BL , Mice, Knockout , PPAR gamma/genetics , Rosiglitazone , Thiazolidinediones/pharmacology
3.
Mol Cell ; 56(1): 128-139, 2014 Oct 02.
Article in English | MEDLINE | ID: mdl-25201415

ABSTRACT

In unicellular organisms, initiation is the rate-limiting step in transcription; in metazoan organisms, the transition from initiation to productive elongation is also important. Here, we show that the RNA polymerase II (RNAPII)-associated multiprotein complex, Integrator, plays a critical role in both initiation and the release of paused RNAPII at immediate early genes (IEGs) following transcriptional activation by epidermal growth factor (EGF) in human cells. Integrator is recruited to the IEGs in a signal-dependent manner and is required to engage and recruit the super elongation complex (SEC) to EGF-responsive genes to allow release of paused RNAPII and productive transcription elongation.


Subject(s)
RNA Polymerase II/metabolism , Transcription Initiation, Genetic , Transcriptional Activation , Epidermal Growth Factor/metabolism , Epidermal Growth Factor/physiology , HeLa Cells , Humans
4.
Genes Dev ; 28(9): 1018-28, 2014 May 01.
Article in English | MEDLINE | ID: mdl-24788520

ABSTRACT

Rosiglitazone (rosi) is a powerful insulin sensitizer, but serious toxicities have curtailed its widespread clinical use. Rosi functions as a high-affinity ligand for peroxisome proliferator-activated receptor γ (PPARγ), the adipocyte-predominant nuclear receptor (NR). The classic model, involving binding of ligand to the NR on DNA, explains positive regulation of gene expression, but ligand-dependent repression is not well understood. We addressed this issue by studying the direct effects of rosi on gene transcription using global run-on sequencing (GRO-seq). Rosi-induced changes in gene body transcription were pronounced after 10 min and correlated with steady-state mRNA levels as well as with transcription at nearby enhancers (enhancer RNAs [eRNAs]). Up-regulated eRNAs occurred almost exclusively at PPARγ-binding sites, to which rosi treatment recruited coactivators, including MED1, p300, and CBP. In contrast, transcriptional repression by rosi involved a loss of coactivators from eRNA sites devoid of PPARγ and enriched for other transcription factors, including AP-1 factors and C/EBPs. Thus, rosi activates and represses transcription by fundamentally different mechanisms that could inform the future development of anti-diabetic drugs.


Subject(s)
Adipocytes/drug effects , Gene Expression Regulation/drug effects , Hypoglycemic Agents/pharmacology , PPAR gamma/metabolism , Thiazolidinediones/pharmacology , 3T3-L1 Cells , Animals , Humans , Mediator Complex Subunit 1/metabolism , Mice , Protein Binding , Rosiglitazone , Transcriptome
5.
J Biol Chem ; 286(3): 1938-50, 2011 Jan 21.
Article in English | MEDLINE | ID: mdl-21097508

ABSTRACT

The Crohn's disease and early onset sarcoidosis susceptibility protein, NOD2, coordinates innate immune signaling pathways. Because dysregulation of this coordination can lead to inflammatory disease, maintaining appropriate activation of the NOD2 signaling pathway is paramount in immunologic homeostasis. In this work, we identify the atypical tumor necrosis factor-associated factor (TRAF) family member, TRAF4, as a key negative regulator of NOD2 signaling. TRAF4 inhibits NOD2-induced NF-κB activation and directly binds to NOD2 to inhibit NOD2-induced bacterial killing. We find that two consecutive glutamate residues in NOD2 are required for interaction with TRAF4 and inhibition of NOD2 signaling because mutation of these residues abrogated both TRAF4 binding and inhibition of NOD2. This work identifies a novel negative regulator of NOD2 signaling. Additionally, it defines a TRAF4 binding motif within NOD2 involved in termination of innate immune signaling responses.


Subject(s)
Crohn Disease/metabolism , Down-Regulation , Nod2 Signaling Adaptor Protein/metabolism , TNF Receptor-Associated Factor 4/metabolism , Amino Acid Motifs , Animals , Crohn Disease/genetics , Crohn Disease/immunology , Genetic Predisposition to Disease/genetics , HEK293 Cells , Humans , Immunity, Innate , Mice , NF-kappa B/genetics , NF-kappa B/immunology , NF-kappa B/metabolism , Nod2 Signaling Adaptor Protein/genetics , Nod2 Signaling Adaptor Protein/immunology , Protein Binding , Salmonella Infections/immunology , Salmonella Infections/metabolism , Salmonella typhimurium/immunology , Salmonella typhimurium/metabolism , Signal Transduction/genetics , Signal Transduction/immunology , TNF Receptor-Associated Factor 4/genetics , TNF Receptor-Associated Factor 4/immunology
6.
Curr Biol ; 18(18): 1402-8, 2008 Sep 23.
Article in English | MEDLINE | ID: mdl-18775659

ABSTRACT

The Crohn's-disease-susceptibility protein, NOD2, coordinates signaling responses upon intracellular exposure to bacteria. Although NOD2 is known to activate NFkappaB, little is known about the molecular mechanisms by which NOD2 coordinates functionally separate signaling pathways such as NFkappaB, JNK, and p38 to regulate cytokine responses. Given that one of the characteristics of Crohn's disease is an altered cytokine response to normal bacterial flora, the coupling of signaling pathways could be important for Crohn's-disease pathophysiology. We find that a MAP3K, MEKK4, binds to RIP2 to sequester RIP2 from the NOD2 signaling pathway. This MEKK4:RIP2 complex dissociates upon exposure to the NOD2 agonist, MDP, allowing NOD2 to bind to RIP2 and activate NFkappaB. MEKK4 thus sequesters RIP2 to inhibit the NOD2:RIP2 complex from activating NFkappaB signaling pathways, and Crohn's-disease-associated NOD2 polymorphisms cannot compete with MEKK4 for RIP2 binding. Lastly, we find that MEKK4 helps dictate signal specificity downstream of NOD2 activation as knockdown of MEKK4 in macrophages exposed to MDP causes increased NFkappaB activity, absent p38 activity, and hyporesponsiveness to TLR2 and TLR4 agonists. These biochemical findings suggest that basal inhibition of the NOD2-driven NFkappaB pathway by MEKK4 could be important in the pathogenesis of Crohn's disease.


Subject(s)
MAP Kinase Kinase Kinase 4/metabolism , Nod2 Signaling Adaptor Protein/physiology , Receptor-Interacting Protein Serine-Threonine Kinase 2/metabolism , Cell Line , Homeostasis , Humans , MAP Kinase Kinase Kinase 1/metabolism , MAP Kinase Kinase Kinase 2/metabolism , MAP Kinase Kinase Kinase 3/metabolism , MAP Kinase Kinase Kinase 4/genetics , Signal Transduction , Transfection
7.
Nat Med ; 25(3): 507-516, 2019 03.
Article in English | MEDLINE | ID: mdl-30842678

ABSTRACT

Quantitative changes in leptin concentration lead to alterations in food intake and body weight, but the regulatory mechanisms that control leptin gene expression are poorly understood. Here we report that fat-specific and quantitative leptin expression is controlled by redundant cis elements and trans factors interacting with the proximal promoter together with a long noncoding RNA (lncOb). Diet-induced obese mice lacking lncOb show increased fat mass with reduced plasma leptin levels and lose weight after leptin treatment, whereas control mice do not. Consistent with this finding, large-scale genetic studies of humans reveal a significant association of single-nucleotide polymorphisms (SNPs) in the region of human lncOb with lower plasma leptin levels and obesity. These results show that reduced leptin gene expression can lead to a hypoleptinemic, leptin-responsive form of obesity and provide a framework for elucidating the pathogenic mechanism in the subset of obese patients with low endogenous leptin levels.


Subject(s)
Leptin/genetics , Obesity/genetics , RNA, Long Noncoding/genetics , Animals , Body Weight/drug effects , Body Weight/genetics , Diet, High-Fat , Eating/drug effects , Eating/genetics , Enhancer Elements, Genetic/genetics , Female , Gene Expression Regulation , Humans , Leptin/metabolism , Leptin/pharmacology , Male , Mice , Mice, Knockout , Mice, Transgenic , Obesity/metabolism , Polymorphism, Single Nucleotide
8.
ACS Med Chem Lett ; 10(6): 857-862, 2019 Jun 13.
Article in English | MEDLINE | ID: mdl-31223438

ABSTRACT

RIP1 regulates cell death and inflammation and is believed to play an important role in contributing to a variety of human pathologies, including immune-mediated inflammatory diseases and cancer. While small-molecule inhibitors of RIP1 kinase have been advanced to the clinic for inflammatory diseases and CNS indications, RIP1 inhibitors for oncology indications have yet to be described. Herein we report on the discovery and profile of GSK3145095 (compound 6). Compound 6 potently binds to RIP1 with exquisite kinase specificity and has excellent activity in blocking RIP1 kinase-dependent cellular responses. Highlighting its potential as a novel cancer therapy, the inhibitor was also able to promote a tumor suppressive T cell phenotype in pancreatic adenocarcinoma organ cultures. Compound 6 is currently in phase 1 clinical studies for pancreatic adenocarcinoma and other selected solid tumors.

9.
Cancer Cell ; 34(5): 757-774.e7, 2018 11 12.
Article in English | MEDLINE | ID: mdl-30423296

ABSTRACT

Pancreatic ductal adenocarcinoma (PDA) is characterized by immune tolerance and immunotherapeutic resistance. We discovered upregulation of receptor-interacting serine/threonine protein kinase 1 (RIP1) in tumor-associated macrophages (TAMs) in PDA. To study its role in oncogenic progression, we developed a selective small-molecule RIP1 inhibitor with high in vivo exposure. Targeting RIP1 reprogrammed TAMs toward an MHCIIhiTNFα+IFNγ+ immunogenic phenotype in a STAT1-dependent manner. RIP1 inhibition in TAMs resulted in cytotoxic T cell activation and T helper cell differentiation toward a mixed Th1/Th17 phenotype, leading to tumor immunity in mice and in organotypic models of human PDA. Targeting RIP1 synergized with PD1-and inducible co-stimulator-based immunotherapies. Tumor-promoting effects of RIP1 were independent of its co-association with RIP3. Collectively, our work describes RIP1 as a checkpoint kinase governing tumor immunity.


Subject(s)
Carcinoma, Pancreatic Ductal/immunology , Immune Tolerance/immunology , Macrophages/immunology , Pancreatic Neoplasms/immunology , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , T-Lymphocytes, Cytotoxic/immunology , Th1 Cells/immunology , Th17 Cells/immunology , Animals , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/pathology , Cell Line, Tumor , Humans , Immune Tolerance/genetics , L Cells , Lymphocyte Activation/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Receptor-Interacting Protein Serine-Threonine Kinases/antagonists & inhibitors , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , STAT1 Transcription Factor/metabolism , Th1 Cells/cytology , Th17 Cells/cytology
10.
J Exp Med ; 214(12): 3687-3705, 2017 Dec 04.
Article in English | MEDLINE | ID: mdl-29089374

ABSTRACT

A variant of the autophagy gene ATG16L1 is associated with Crohn's disease, an inflammatory bowel disease (IBD), and poor survival in allogeneic hematopoietic stem cell transplant recipients. We demonstrate that ATG16L1 in the intestinal epithelium is essential for preventing loss of Paneth cells and exaggerated cell death in animal models of virally triggered IBD and allogeneic hematopoietic stem cell transplantation. Intestinal organoids lacking ATG16L1 reproduced this loss in Paneth cells and displayed TNFα-mediated necroptosis, a form of programmed necrosis. This cytoprotective function of ATG16L1 was associated with the role of autophagy in promoting mitochondrial homeostasis. Finally, therapeutic blockade of necroptosis through TNFα or RIPK1 inhibition ameliorated disease in the virally triggered IBD model. These findings indicate that, in contrast to tumor cells in which autophagy promotes caspase-independent cell death, ATG16L1 maintains the intestinal barrier by inhibiting necroptosis in the epithelium.


Subject(s)
Apoptosis , Autophagy , Carrier Proteins/metabolism , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Animals , Autophagy-Related Proteins , Caliciviridae Infections/pathology , Caliciviridae Infections/virology , Cell Survival , Cytoprotection , Epithelial Cells/metabolism , Epithelial Cells/pathology , Gene Deletion , Graft vs Host Disease/pathology , Graft vs Host Disease/therapy , Hematopoietic Stem Cell Transplantation , Homeostasis , Mice , Mice, Inbred C57BL , Mitochondria/metabolism , Mitochondria/ultrastructure , Mutation/genetics , Necrosis , Norovirus/physiology , Organoids/pathology , Paneth Cells/metabolism , Paneth Cells/pathology , Receptor-Interacting Protein Serine-Threonine Kinases/antagonists & inhibitors , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Tumor Necrosis Factor-alpha/metabolism
12.
Cell Rep ; 4(2): 352-61, 2013 Jul 25.
Article in English | MEDLINE | ID: mdl-23871670

ABSTRACT

While the I kappa kinase (IKK) scaffolding protein NF-κB essential modulator (NEMO) binds to polyubiquitin chains to transmit inflammatory signals, NEMO itself is also ubiquitinated in response to a variety of inflammatory agonists. Although there have been hints that polyubiquitination of NEMO is essential for avoiding inflammatory disorders, the in vivo physiologic role of NEMO ubiquitination is unknown. In this work, we knock in a NEMO allele in which two major inflammatory agonist-induced ubiquitination sites cannot be ubiquitinated. We show that mice with a nonubiquitinatable NEMO allele display embryonic lethality. Heterozygous females develop inflammatory skin lesions, decreased B cell numbers, and hypercellular spleens. Embryonic lethality can be complemented by mating onto a TNFR1(-/-) background, at the cost of severe steatohepatitis and early mortality, and we also show that NEMO ubiquitination is required for optimal innate immune signaling responses. These findings suggest that NEMO ubiquitination is crucial for NF-κB activity in response to innate immune agonists.


Subject(s)
Intracellular Signaling Peptides and Proteins/genetics , NF-kappa B/immunology , Animals , Female , Gene Knock-In Techniques , Intracellular Signaling Peptides and Proteins/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , NF-kappa B/genetics , NF-kappa B/metabolism , Signal Transduction/immunology , Ubiquitination/immunology
13.
Mol Cell Biol ; 32(13): 2479-89, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22547678

ABSTRACT

Despite their homology, IκB kinase α (IKKα) and IKKß have divergent roles in NF-κB signaling. IKKß strongly activates NF-κB while IKKα can downregulate NF-κB under certain circumstances. Given this, identifying independent substrates for these kinases could help delineate their divergent roles. Peptide substrate array technology followed by bioinformatic screening identified TRAF4 as a substrate for IKKα. Like IKKα, TRAF4 is atypical within its family because it is the only TRAF family member to negatively regulate innate immune signaling. IKKα's phosphorylation of serine-426 on TRAF4 was required for this negative regulation. Binding to the Crohn's disease susceptibility protein, NOD2, is required for TRAF4 phosphorylation and subsequent inhibition of NOD2 signaling. Structurally, serine-426 resides within an exaggerated ß-bulge in TRAF4 that is not present in the other TRAF proteins, and phosphorylation of this site provides a structural basis for the atypical function of TRAF4 and its atypical role in NOD2 signaling.


Subject(s)
I-kappa B Kinase/metabolism , Immunity, Innate , TNF Receptor-Associated Factor 4/metabolism , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Cell Line , Crohn Disease/genetics , Crohn Disease/immunology , Crohn Disease/metabolism , DNA Primers/genetics , Down-Regulation , HCT116 Cells , HEK293 Cells , Humans , I-kappa B Kinase/genetics , I-kappa B Kinase/immunology , Mice , Models, Molecular , Molecular Sequence Data , NF-kappa B/metabolism , Nod2 Signaling Adaptor Protein/genetics , Nod2 Signaling Adaptor Protein/metabolism , Peptide Library , Phosphorylation , Protein Stability , Protein Structure, Tertiary , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Serine/chemistry , Signal Transduction/immunology , Substrate Specificity , TNF Receptor-Associated Factor 4/chemistry , TNF Receptor-Associated Factor 4/immunology
14.
Curr Biol ; 19(15): 1255-63, 2009 Aug 11.
Article in English | MEDLINE | ID: mdl-19592251

ABSTRACT

BACKGROUND: The inability to coordinate the signaling pathways that lead to proper cytokine responses characterizes the pathogenesis of inflammatory diseases such as Crohn's disease. The Crohn's disease susceptibility protein, NOD2, helps coordinate cytokine responses upon intracellular exposure to bacteria, and this signal coordination by NOD2 is accomplished, in part, through K63-linked polyubiquitin chains that create binding surfaces for the scaffolding of signaling complexes. RESULTS: In this work, we show that the NOD2 signaling partner, RIP2, is directly K63-polyubiquitinated by ITCH, an E3 ubiquitin ligase that when lost genetically causes widespread inflammatory disease at mucosal surfaces. We show that ITCH is responsible for RIP2 polyubiquitination in response to infection with listeria monocytogenes. We also show that NOD2 can bind polyubiquitinated RIP2 and that whereas ITCH E3 ligase activity is required for optimal NOD2:RIP2-induced p38 and JNK activation, ITCH inhibits NOD2:RIP2-induced nuclear factor kappa B (NFkappaB) activation. This effect can be seen independently at the whole-genome level by microarray analysis of muramyl dipeptide (MDP)-treated Itch(-/-) primary macrophages. CONCLUSIONS: These findings suggest that ITCH helps regulate NOD2-dependent signal transduction pathways and, as such, may be involved in the pathogenesis of NOD2-mediated inflammatory disease.


Subject(s)
Crohn Disease/metabolism , Nod2 Signaling Adaptor Protein/metabolism , Receptor-Interacting Protein Serine-Threonine Kinase 2/metabolism , Repressor Proteins/metabolism , Signal Transduction/physiology , Ubiquitin-Protein Ligases/metabolism , Acetylmuramyl-Alanyl-Isoglutamine , Cell Line , Humans , Immunoblotting , Immunoprecipitation , Listeria monocytogenes , NF-kappa B/metabolism , Oligonucleotide Array Sequence Analysis , RNA, Small Interfering/genetics , Ubiquitination
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