Publications by authors named "Marc J Servant"

33 Publications

Oxidative stress-induced senescence mediates inflammatory and fibrotic phenotypes in fibroblasts from systemic sclerosis patients.

Rheumatology (Oxford) 2021 Jun 11. Epub 2021 Jun 11.

Faculty of Pharmacy, Université de Montréal, Québec, Canada.

Objective: Systemic sclerosis (SSc) is an autoimmune connective tissue disorder characterized by inflammation and fibrosis. Although constitutive activation of fibroblasts is proposed to be responsible for the fibrotic and inflammatory features of the disease, the underlying mechanism remains elusive and, effective therapeutic targets are still lacking. The aim of this study was to evaluate the role of oxidative stress-induced senescence and its contribution to the pro-fibrotic and pro-inflammatory phenotypes of fibroblasts from SSc patients.

Methods: Dermal fibroblasts were isolated from SSc (n = 13) and healthy (n = 10) donors. Fibroblast's intracellular and mitochondrial reactive oxygen species were determined by flow cytometry. Mitochondrial function measured by Seahorse XF24 analyzer. Fibrotic and inflammatory gene expressions were assessed by qPCR and key pro-inflammatory components of the fibroblasts' secretome (interleukin (IL) 6 and IL8) were quantified by ELISA.

Results: Compared to healthy fibroblasts, SSc fibroblasts displayed higher levels of both intracellular and mitochondrial ROS. Oxidative stress in SSc fibroblasts induced the expression of fibrotic genes and activated the transforming growth factor-β-activated kinase 1 (TAK1) -IκB kinase β (IKKβ)- interferon regulatory factor 5 (IRF5) inflammatory signaling cascade. These cellular responses paralleled the presence of a DNA damage response, a senescence-associated secretory phenotype and a fibrotic response. Treatment of SSc fibroblasts with ROS scavengers reduced their pro-inflammatory secretome production and fibrotic gene expression.

Conclusions: Oxidative stress-induced cellular senescence in SSc fibroblasts underlies their pro-inflammatory and pro-fibrotic phenotypes. Targeting redox imbalance of SSc fibroblasts enhances their in vitro functions and could be of relevance for SSc therapy.
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http://dx.doi.org/10.1093/rheumatology/keab477DOI Listing
June 2021

TRK-Fused Gene (TFG), a protein involved in protein secretion pathways, is an essential component of the antiviral innate immune response.

PLoS Pathog 2021 01 7;17(1):e1009111. Epub 2021 Jan 7.

Faculty of Pharmacy, Université de Montréal, Montréal, Canada.

Antiviral innate immune response to RNA virus infection is supported by Pattern-Recognition Receptors (PRR) including RIG-I-Like Receptors (RLR), which lead to type I interferons (IFNs) and IFN-stimulated genes (ISG) production. Upon sensing of viral RNA, the E3 ubiquitin ligase TNF Receptor-Associated Factor-3 (TRAF3) is recruited along with its substrate TANK-Binding Kinase (TBK1), to MAVS-containing subcellular compartments, including mitochondria, peroxisomes, and the mitochondria-associated endoplasmic reticulum membrane (MAM). However, the regulation of such events remains largely unresolved. Here, we identify TRK-Fused Gene (TFG), a protein involved in the transport of newly synthesized proteins to the endomembrane system via the Coat Protein complex II (COPII) transport vesicles, as a new TRAF3-interacting protein allowing the efficient recruitment of TRAF3 to MAVS and TBK1 following Sendai virus (SeV) infection. Using siRNA and shRNA approaches, we show that TFG is required for virus-induced TBK1 activation resulting in C-terminal IRF3 phosphorylation and dimerization. We further show that the ability of the TRAF3-TFG complex to engage mTOR following SeV infection allows TBK1 to phosphorylate mTOR on serine 2159, a post-translational modification shown to promote mTORC1 signaling. We demonstrate that the activation of mTORC1 signaling during SeV infection plays a positive role in the expression of Viperin, IRF7 and IFN-induced proteins with tetratricopeptide repeats (IFITs) proteins, and that depleting TFG resulted in a compromised antiviral state. Our study, therefore, identifies TFG as an essential component of the RLR-dependent type I IFN antiviral response.
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http://dx.doi.org/10.1371/journal.ppat.1009111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790228PMC
January 2021

Loss of interleukin-17 receptor D promotes chronic inflammation-associated tumorigenesis.

Oncogene 2021 01 11;40(2):452-464. Epub 2020 Nov 11.

Institute for Research in Immunology and Cancer, Montreal, Quebec, Canada.

Interleukin-17 receptor D (IL-17RD), also known as similar expression to Fgf genes (SEF), is proposed to act as a signaling hub that negatively regulates mitogenic signaling pathways, like the ERK1/2 MAP kinase pathway, and innate immune signaling. The expression of IL-17RD is downregulated in certain solid tumors, which has led to the hypothesis that it may exert tumor suppressor functions. However, the role of IL-17RD in tumor biology remains to be studied in vivo. Here, we show that genetic disruption of Il17rd leads to the increased formation of spontaneous tumors in multiple tissues of aging mice. Loss of IL-17RD also promotes tumor development in a model of colitis-associated colorectal cancer, associated with an exacerbated inflammatory response. Colon tumors from IL-17RD-deficient mice are characterized by a strong enrichment in inflammation-related gene signatures, elevated expression of pro-inflammatory tumorigenic cytokines, such as IL-17A and IL-6, and increased STAT3 tyrosine phosphorylation. We further show that RNAi depletion of IL-17RD enhances Toll-like receptor and IL-17A signaling in colon adenocarcinoma cells. No change in the proliferation of normal or tumor intestinal epithelial cells was observed upon genetic inactivation of IL-17RD. Our findings establish IL-17RD as a tumor suppressor in mice and suggest that the protein exerts its function mainly by limiting the extent and duration of inflammation.
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http://dx.doi.org/10.1038/s41388-020-01540-4DOI Listing
January 2021

Ex vivo Ikkβ ablation rescues the immunopotency of mesenchymal stromal cells from diabetics with advanced atherosclerosis.

Cardiovasc Res 2021 Feb;117(3):756-766

Faculty of Pharmacy, University of Montreal, C.P. 6128, Succursale Centre-Ville, Montréal, QC H3C 3J7, Canada.

Aims: Diabetes is a conventional risk factor for atherosclerotic cardiovascular disease and myocardial infarction (MI) is the most common cause of death among these patients. Mesenchymal stromal cells (MSCs) in patients with type 2 diabetes mellitus (T2DM) and atherosclerosis have impaired ability to suppress activated T-cells (i.e. reduced immunopotency). This is mediated by an inflammatory shift in MSC-secreted soluble factors (i.e. pro-inflammatory secretome) and can contribute to the reduced therapeutic effects of autologous T2DM and atherosclerosis-MSC post-MI. The signalling pathways driving the altered secretome of atherosclerosis- and T2DM-MSC are unknown. Specifically, the effect of IκB kinase β (IKKβ) modulation, a key regulator of inflammatory responses, on the immunopotency of MSCs from T2DM patients with advanced atherosclerosis has not been studied.

Methods And Results: MSCs were isolated from adipose tissue obtained from patients with (i) atherosclerosis and T2DM (atherosclerosis+T2DM MSCs, n = 17) and (ii) atherosclerosis without T2DM (atherosclerosis MSCs, n = 17). MSCs from atherosclerosis+T2DM individuals displayed an inflammatory senescent phenotype and constitutively expressed active forms of effectors of the canonical IKKβ nuclear factor-κB transcription factors inflammatory pathway. Importantly, this constitutive pro-inflammatory IKKβ signature resulted in an altered secretome and impaired in vitro immunopotency and in vivo healing capacity in an acute MI model. Notably, treatment with a selective IKKβ inhibitor or IKKβ knockdown (KD) (clustered regularly interspaced short palindromic repeats/Cas9-mediated IKKβ KD) in atherosclerosis+T2DM MSCs reduced the production of pro-inflammatory secretome, increased survival, and rescued their immunopotency both in vitro and in vivo.

Conclusions: Constitutively active IKKβ reduces the immunopotency of atherosclerosis+T2DM MSC by changing their secretome composition. Modulation of IKKβ in atherosclerosis+T2DM MSCs enhances their myocardial repair ability.
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http://dx.doi.org/10.1093/cvr/cvaa118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7898947PMC
February 2021

Roles of GSK-3 and β-Catenin in Antiviral Innate Immune Sensing of Nucleic Acids.

Cells 2020 04 7;9(4). Epub 2020 Apr 7.

Faculty of Pharmacy, Université de Montréal, Montréal, QC H3C3J7, Canada.

The rapid activation of the type I interferon (IFN) antiviral innate immune response relies on ubiquitously expressed RNA and DNA sensors. Once engaged, these nucleotide-sensing receptors use distinct signaling modules for the rapid and robust activation of mitogen-activated protein kinases (MAPKs), the IκB kinase (IKK) complex, and the IKK-related kinases IKKε and TANK-binding kinase 1 (TBK1), leading to the subsequent activation of the activator protein 1 (AP1), nuclear factor-kappa B (NF-κB), and IFN regulatory factor 3 (IRF3) transcription factors, respectively. They, in turn, induce immunomodulatory genes, allowing for a rapid antiviral cellular response. Unlike the MAPKs, the IKK complex and the IKK-related kinases, ubiquitously expressed glycogen synthase kinase 3 (GSK-3) α and β isoforms are active in unstimulated resting cells and are involved in the constitutive turnover of β-catenin, a transcriptional coactivator involved in cell proliferation, differentiation, and lineage commitment. Interestingly, studies have demonstrated the regulatory roles of both GSK-3 and β-catenin in type I IFN antiviral innate immune response, particularly affecting the activation of IRF3. In this review, we summarize current knowledge on the mechanisms by which GSK-3 and β-catenin control the antiviral innate immune response to RNA and DNA virus infections.
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http://dx.doi.org/10.3390/cells9040897DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226782PMC
April 2020

Mitochondrial Oxidative Stress Reduces the Immunopotency of Mesenchymal Stromal Cells in Adults With Coronary Artery Disease.

Circ Res 2018 01 7;122(2):255-266. Epub 2017 Nov 7.

From the Department of Anatomy and Cell Biology (O.K.M.), Department of Physiology (U.S.), Divisions of Cardiac Surgery and Surgical Research, Department of Surgery (D.S.T.), Division of Rheumatology, Department of Medicine (I.C., M.L.) McGill University, Montreal, Quebec, Canada; Shriners Hospital for Children (R.H.); Department of Cellular and Molecular Medicine, Faculty of Medicine (A.C., Y.B.), University of Ottawa, Ontario, Canada; and Faculty of Pharmacy (M.J.S.), University of Montreal, Quebec, Canada.

Rationale: Mesenchymal stromal cells (MSCs) are promising therapeutic strategies for coronary artery disease; however, donor-related variability in cell quality is a main cause of discrepancies in preclinical studies. In vitro, MSCs from individuals with coronary artery disease have reduced ability to suppress activated T-cells. The mechanisms underlying the altered immunomodulatory capacity of MSCs in the context of atherosclerosis remain elusive.

Objective: The aim of this study was to assess the role of mitochondrial dysfunction in the impaired immunomodulatory properties of MSCs from patients with atherosclerosis.

Methods And Results: Adipose tissue-derived MSCs were isolated from atherosclerotic (n=38) and nonatherosclerotic (n=42) donors. MSCs:CD4T-cell suppression was assessed in allogeneic coculture systems. Compared with nonatherosclerotic-MSCs, atherosclerotic-MSCs displayed higher levels of both intracellular (=0.006) and mitochondrial (=0.03) reactive oxygen species reflecting altered mitochondrial function. The increased mitochondrial reactive oxygen species levels of atherosclerotic-MSCs promoted a phenotypic switch characterized by enhanced glycolysis and an altered cytokine secretion (interleukin-6 <0.0001, interleukin-8/C-X-C motif chemokine ligand 8 =0.04, and monocyte chemoattractant protein-1/chemokine ligand 2 =0.01). Furthermore, treatment of atherosclerotic-MSCs with the reactive oxygen species scavenger N-acetyl-l-cysteine reduced the levels of interleukin-6, interleukin-8/C-X-C motif chemokine ligand 8, and monocyte chemoattractant protein-1/chemokine ligand 2 in the MSC secretome and improved MSCs immunosuppressive capacity (=0.03).

Conclusions: An impaired mitochondrial function of atherosclerotic-MSCs underlies their altered secretome and reduced immunopotency. Interventions aimed at restoring the mitochondrial function of atherosclerotic-MSCs improve their in vitro immunosuppressive ability and may translate into enhanced therapeutic efficiency.
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http://dx.doi.org/10.1161/CIRCRESAHA.117.311400DOI Listing
January 2018

Fine-Tuning of the RIG-I-Like Receptor/Interferon Regulatory Factor 3-Dependent Antiviral Innate Immune Response by the Glycogen Synthase Kinase 3/β-Catenin Pathway.

Mol Cell Biol 2015 Sep 22;35(17):3029-43. Epub 2015 Jun 22.

Faculty of Pharmacy, Université de Montréal, Montréal, Québec, Canada

Induction of an antiviral innate immune response relies on pattern recognition receptors, including retinoic acid-inducible gene 1-like receptors (RLR), to detect invading pathogens, resulting in the activation of multiple latent transcription factors, including interferon regulatory factor 3 (IRF3). Upon sensing of viral RNA and DNA, IRF3 is phosphorylated and recruits coactivators to induce type I interferons (IFNs) and selected sets of IRF3-regulated IFN-stimulated genes (ISGs) such as those for ISG54 (Ifit2), ISG56 (Ifit1), and viperin (Rsad2). Here, we used wild-type, glycogen synthase kinase 3α knockout (GSK-3α(-/-)), GSK-3β(-/-), and GSK-3α/β double-knockout (DKO) embryonic stem (ES) cells, as well as GSK-3β(-/-) mouse embryonic fibroblast cells in which GSK-3α was knocked down to demonstrate that both isoforms of GSK-3, GSK-3α and GSK-3β, are required for this antiviral immune response. Moreover, the use of two selective small-molecule GSK-3 inhibitors (CHIR99021 and BIO-acetoxime) or ES cells reconstituted with the catalytically inactive versions of GSK-3 isoforms showed that GSK-3 activity is required for optimal induction of antiviral innate immunity. Mechanistically, GSK-3 isoform activation following Sendai virus infection results in phosphorylation of β-catenin at S33/S37/T41, promoting IRF3 DNA binding and activation of IRF3-regulated ISGs. This study identifies the role of a GSK-3/β-catenin axis in antiviral innate immunity.
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http://dx.doi.org/10.1128/MCB.00344-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4525315PMC
September 2015

Systemic sclerosis immunoglobulin induces growth and a pro-fibrotic state in vascular smooth muscle cells through the epidermal growth factor receptor.

PLoS One 2014 13;9(6):e100035. Epub 2014 Jun 13.

Université de Montréal, Faculté de Pharmacie, Montreal, Quebec, Canada.

Objective: It has been suggested that autoantibodies in systemic sclerosis (SSc) may induce the differentiation of cultured fibroblasts into myofibroblasts through platelet-derived growth factor receptor (PDGFR) activation. The present study aims to characterize the effects of SSc IgG on vascular smooth muscle cells (VSMCs) and to determine if stimulatory autoantibodies directed to the PDGFR can be detected, and whether they induce a profibrotic response in primary cultured VSMCs.

Methods: Cultured VSMCs were exposed to IgG fractions purified from SSc-patient or control sera. VSMC responses were then analyzed for ERK1/2 and Akt phosphorylation, PDGFR immunoprecipitation, cellular proliferation, protein synthesis, and pro-fibrotic changes in mRNA expression.

Results: Stimulatory activity in IgG fractions was more prevalent and intense in the SSc samples. SSc IgG immunoprecipitated the PDGFR with greater avidity than control IgG. Interestingly, activation of downstream signaling events (e.g. Akt, ERK1/2) was independent of PDGFR activity, but required functional EGFR. We also detected increased protein synthesis in response to SSc IgG (p<0.001) and pro-fibrotic changes in gene expression (Tgfb1 +200%; Tgfb2 -23%; p<0.001)) in VSMCs treated with SSc IgG.

Conclusion: When compared to control IgG, SSc IgG have a higher stimulation index in VSMCs. Although SSc IgG interact with the PDGFR, the observed remodeling signaling events occur through the EGFR in VSMC. Our data thus favour a model of transactivation of the EGFR by SSc-derived PDGFR autoantibodies and suggest the use of EGFR inhibitors in future target identification studies in the field of SSc.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0100035PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4057313PMC
December 2015

TNF-α expression in neutrophils and its regulation by glycogen synthase kinase-3: a potentiating role for lithium.

FASEB J 2014 Aug 6;28(8):3679-90. Epub 2014 May 6.

Centre de Recherche du Centre Hospitalier Universitaire de Québec and Faculty of Medicine, Université Laval, Quebec City, Quebec, Canada; and

Glycogen synthase kinase 3 (GSK-3) is associated with several cellular systems, including immune response. Lithium, a widely used pharmacological treatment for bipolar disorder, is a GSK-3 inhibitor. GSK-3α is the predominant isoform in human neutrophils. In this study, we examined the effect of GSK-3 inhibition on the production of TNF-α by neutrophils. In the murine air pouch model of inflammation, lithium chloride (LiCl) amplified TNF-α release. In lipopolysaccharide-stimulated human neutrophils, GSK-3 inhibitors mimicked the effect of LiCl, each potentiating TNF-α release after 4 h, in a concentration-dependent fashion, by up to a 3-fold increase (ED50 of 1 mM for lithium). LiCl had no significant effect on cell viability. A positive association was revealed between GSK-3 inhibition and prolonged activation of the p38/MNK1/eIF4E pathway of mRNA translation. Using lysine and arginine labeled with stable heavy isotopes followed by quantitative mass spectrometry, we determined that GSK-3 inhibition markedly increases (by more than 3-fold) de novo TNF-α protein synthesis. Our findings shed light on a novel mechanism of control of TNF-α expression in neutrophils with GSK-3 regulating mRNA translation and raise the possibility that lithium could be having a hitherto unforeseen effect on inflammatory diseases.
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http://dx.doi.org/10.1096/fj.14-251900DOI Listing
August 2014

Sustained activation of interferon regulatory factor 3 during infection by paramyxoviruses requires MDA5.

J Innate Immun 2014 30;6(5):650-62. Epub 2014 Apr 30.

CRCHUM - Centre Hospitalier de l'Université de Montréal, Montreal, Que., Canada.

Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are the main cytosolic sensors of single-stranded RNA viruses, including paramyxoviruses, and are required to initiate a quick and robust innate antiviral response. Despite different ligand-binding properties, the consensus view is that RIG-I and MDA5 trigger common signal(s) to activate interferon regulatory factor 3 (IRF-3) and NF-κB, and downstream antiviral and proinflammatory cytokine expression. Here, we performed a thorough analysis of the temporal involvement of RIG-I and MDA5 in the regulation of IRF-3 during respiratory syncytial virus (RSV) infection. Based on specific RNA interference-mediated knockdown of RIG-I and MDA5 in A549 cells, we confirmed that RIG-I is critical for the initiation of IRF-3 phosphorylation, dimerization and downstream gene expression. On the other hand, our experiments yielded the first evidence that knockdown of MDA5 leads to early ubiquitination and proteasomal degradation of active IRF-3. Conversely, ectopic expression of MDA5 prolonged RIG-I-induced IRF-3 activation. Altogether, we provide novel mechanistic insight into the temporal involvement of RIG-I and MDA5 in the innate antiviral response. While RIG-I is essential for initial IRF-3 activation, engagement of induced MDA5 is essential to prevent early degradation of IRF-3, thereby sustaining IRF-3-dependent antiviral gene expression. MDA5 plays a similar role during Sendai virus infection suggesting that this model is not restricted to RSV amongst paramyxoviruses.
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http://dx.doi.org/10.1159/000360764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4846353PMC
May 2015

Pyroglutamylated RF-amide peptide (QRFP) gene is regulated by metabolic endotoxemia.

Mol Endocrinol 2014 Jan 1;28(1):65-79. Epub 2013 Jan 1.

Faculty of Pharmacy (C.J., M.M., S.M., M.J.S., H.O.), Université de Montréal C.P. 6128, Succursale Centre-Ville, Québec, Canada, H3C 3J7; and Laboratory of Molecular and Cellular Endocrinology (R.G., D.G., E.G.), Department of Internal Medicine, University of Turin, Corso Dogliotti 14, 10126 Turin, Italy.

Pyroglutamylated RF-amide peptide (QRFP) is involved in the regulation of food intake, thermogenesis, adipogenesis, and lipolysis. The expression of QRFP in adipose tissue is reduced in diet-induced obesity, a mouse model in which plasma concentrations of endotoxins are slightly elevated. The present study investigated the role of metabolic endotoxemia (ME) on QRFP gene regulation. Our results uncovered the expression of QRFP in murine macrophages and cell lines. This expression has been found to be decreased in mice with ME. Low doses of lipopolysaccharide (LPS) transiently down-regulated QRFP by 59% in RAW264.7 macrophages but not in 3T3-L1 adipocytes. The effect of LPS on QRFP expression in macrophages was dependent on the inhibitor of kB kinase and TIR-domain-containing adapter-inducing interferon (IFN)-β (TRIF) but not myeloid differentiation primary response gene 88. IFN-β was induced by ME in macrophages. IFN-β sustainably reduced QRFP expression in macrophages (64%) and adipocytes (49%). IFN-γ down-regulated QRFP (74%) in macrophages only. Both IFNs inhibited QRFP secretion from macrophages. LPS-stimulated macrophage-conditioned medium reduced QRFP expression in adipocytes, an effect blocked by IFN-β neutralizing antibody. The effect of IFN-β on QRFP expression was dependent on phosphoinositide 3-kinase, p38 MAPK, and histone deacetylases. The effect of IFN-γ was dependent on MAPK/ERK kinase 1/2 and histone deacetylases. Macrophage-conditioned medium containing increased amounts of QRFP preserved adipogenesis in adipocytes. In conclusion, LPS induces IFN-β release from macrophages, which reduces QRFP expression in both macrophages and adipocytes in an autocrine/paracrine-dependent manner, suggesting QRFP as a potential biomarker in ME.
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http://dx.doi.org/10.1210/me.2013-1027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5426650PMC
January 2014

Role of IκB kinase-β in the growth-promoting effects of angiotensin II in vitro and in vivo.

Arterioscler Thromb Vasc Biol 2013 Dec 17;33(12):2850-7. Epub 2013 Oct 17.

From the Faculty of Pharmacy, Université de Montréal, Montréal, Canada.

Objective: Angiotensin II (Ang II) is implicated in processes underlying the development of arterial wall remodeling events, including cellular hypertrophy and inflammation. We previously documented the activation of IκB kinase-β (IKKβ) in Ang II-treated cells, a kinase involved in inflammatory reactions. In light of a study suggesting a role of IKKβ in angiogenesis through its effect on the tuberous sclerosis (TSC)1/2-mammalian target of rapamycin complex 1 pathway in cancer cells, we hypothesized that targeting IKKβ could reduce arterial remodeling events by affecting both the inflammatory and the growth-promoting response of Ang II.

Approach And Results: Treatment of aortic vascular smooth muscle cells with Ang II induced the rapid and sustained phosphorylation of TSC1 on Ser511, which paralleled the activation of effectors of the mammalian target of rapamycin complex 1 pathway. Furthermore, we show that Ser511 of TSC1 acted as a phosphoacceptor site for Ang II-activated IKKβ. Consistent with this, the use of different short hairpin RNA constructs targeting IKKβ reduced Ang II-induced TSC1, S6 kinase, and eukaryotic translation initiation factor 4E-binding protein 1 phosphorylation and the rate of protein synthesis. Overexpression of TSC1 lacking Ser511 in vascular smooth muscle cells also exerted detrimental effects on the hypertrophic effect of Ang II. Furthermore, the selective IKKβ inhibitor N-(6-chloro-7-methoxy-9H-β-carbolin-8-yl)-2 methylnicotinamide reduced the inflammatory response and dose-dependently diminished Ang II-induced TSC1 phosphorylation and effectors of the mammalian target of rapamycin complex 1 pathway, leading to inhibition of protein synthesis in vitro and in rat arteries in vivo.

Conclusions: Our findings provide new insights into the molecular understanding of the pathological role of Ang II and assist in identifying the beneficial effects of IKKβ inhibition for the treatment of cardiovascular diseases.
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http://dx.doi.org/10.1161/ATVBAHA.113.302487DOI Listing
December 2013

Proteomic profiling of the TRAF3 interactome network reveals a new role for the ER-to-Golgi transport compartments in innate immunity.

PLoS Pathog 2012 5;8(7):e1002747. Epub 2012 Jul 5.

Faculty of Pharmacy, Université de Montréal, Montréal, Québec Canada.

Tumor Necrosis Factor receptor-associated factor-3 (TRAF3) is a central mediator important for inducing type I interferon (IFN) production in response to intracellular double-stranded RNA (dsRNA). Here, we report the identification of Sec16A and p115, two proteins of the ER-to-Golgi vesicular transport system, as novel components of the TRAF3 interactome network. Notably, in non-infected cells, TRAF3 was found associated with markers of the ER-Exit-Sites (ERES), ER-to-Golgi intermediate compartment (ERGIC) and the cis-Golgi apparatus. Upon dsRNA and dsDNA sensing however, the Golgi apparatus fragmented into cytoplasmic punctated structures containing TRAF3 allowing its colocalization and interaction with Mitochondrial AntiViral Signaling (MAVS), the essential mitochondria-bound RIG-I-like Helicase (RLH) adaptor. In contrast, retention of TRAF3 at the ER-to-Golgi vesicular transport system blunted the ability of TRAF3 to interact with MAVS upon viral infection and consequently decreased type I IFN response. Moreover, depletion of Sec16A and p115 led to a drastic disorganization of the Golgi paralleled by the relocalization of TRAF3, which under these conditions was unable to associate with MAVS. Consequently, upon dsRNA and dsDNA sensing, ablation of Sec16A and p115 was found to inhibit IRF3 activation and anti-viral gene expression. Reciprocally, mild overexpression of Sec16A or p115 in Hec1B cells increased the activation of IFNβ, ISG56 and NF-κB -dependent promoters following viral infection and ectopic expression of MAVS and Tank-binding kinase-1 (TBK1). In line with these results, TRAF3 was found enriched in immunocomplexes composed of p115, Sec16A and TBK1 upon infection. Hence, we propose a model where dsDNA and dsRNA sensing induces the formation of membrane-bound compartments originating from the Golgi, which mediate the dynamic association of TRAF3 with MAVS leading to an optimal induction of innate immune responses.
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http://dx.doi.org/10.1371/journal.ppat.1002747DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3390413PMC
January 2013

Tumor necrosis factor receptor-associated factor-6 and ribosomal S6 kinase intracellular pathways link the angiotensin II AT1 receptor to the phosphorylation and activation of the IkappaB kinase complex in vascular smooth muscle cells.

J Biol Chem 2010 Oct 21;285(40):30708-18. Epub 2010 Jul 21.

Faculty of Pharmacy and Groupe de Recherche Universitaire sur le Médicament, Université de Montréal, Montreal H3C 3J7, Canada.

Activation of NF-κB transcription factors by locally produced angiotensin II (Ang II) is proposed to be involved in chronic inflammatory reactions leading to atherosclerosis development. However, a clear understanding of the signaling cascades coupling the Ang II AT1 receptors to the activation of NF-κB transcription factors is still lacking. Using primary cultured aortic vascular smooth muscle cells, we show that activation of the IKK complex and NF-κB transcription factors by Ang II is regulated by phosphorylation of the catalytic subunit IKKβ on serine residues 177 and 181 in the activation T-loop. The use of pharmacological inhibitors against conventional protein kinases C (PKCs), mitogen-activated/extracellular signal-regulated kinase (MEK) 1/2, ribosomal S6 kinase (RSK), and silencing RNA technology targeting PKCα, IKKβ subunit, tumor growth factor β-activating kinase-1 (TAK1), the E3 ubiquitin ligase tumor necrosis factor receptor-associated factor-6 (TRAF6), and RSK isoforms, demonstrates the requirement of two distinct signaling pathway for the phosphorylation of IKKβ and the activation of the IKK complex by Ang II. Rapid phosphorylation of IKKβ requires a second messenger-dependent pathway composed of PKCα-TRAF6-TAK1, whereas sustained phosphorylation and activation of IKKβ requires the MEK1/2-ERK1/2-RSK pathway. Importantly, simultaneously targeting components of these two pathways completely blunts the phosphorylation of IKKβ and the proinflammatory effect of the octapeptide. This is the first report demonstrating activation of TAK1 by the AT1R. We propose a model whereby TRAF6-TAK1 and ERK-RSK intracellular pathways independently and sequentially converge to the T-loop phosphorylation for full activation of IKKβ, which is an essential step in the proinflammatory activity of Ang II.
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http://dx.doi.org/10.1074/jbc.M110.126433DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2945565PMC
October 2010

GPR103b functions in the peripheral regulation of adipogenesis.

Mol Endocrinol 2010 Aug 9;24(8):1615-25. Epub 2010 Jun 9.

Faculty of Pharmacy, Université de Montréal Case Postale 6128, Succursale Centre-Ville, Montréal, Québec, Canada H3C 3J7.

The activation of G protein-coupled receptor 103 (GPR103) by its endogenous peptidic ligands, QRFPs, is involved in the central regulation of feeding by increasing food intake, body weight, and fat mass after intracerebroventricular injection in mice. However, the role of GPR103 in regulating peripheral metabolic pathways has not yet been explored. The present study aimed to investigate the role of GPR103 in adipogenesis and lipid metabolism using 3T3-L1 adipocyte cells. Our results show that differentiated 3T3-L1 cells expressed the GPR103b subtype mRNA and protein, as well as QRFP mRNA. QRFP-43 and -26 induced an increase in triglyceride accumulation of 50 and 41%, respectively, and elicited a dose-dependent increase in fatty acid uptake, by up to approximately 60% at the highest concentration, in 3T3-L1-differentiated cells. QRFP-43 and -26 inhibited isoproterenol (ISO)-induced lipolysis in a dose-dependent manner, with IC(50)s of 2.3 +/- 1.2 and 1.1 +/- 1.0 nm, respectively. The expression of genes involved in lipid uptake (FATP1, CD36, LPL, ACSL1, PPAR-gamma, and C/EBP-alpha), was increased by 2- to 3-fold after treatment with QRFP. The effects of QRFP on ISO-induced lipolysis and fatty acid uptake were abolished when GPR103b was silenced. In a mouse model of diet-induced obesity, the expression of GPR103b in epididymal fat pads was elevated by 16-fold whereas that of QRFP was reduced by 46% compared to lean mice. Furthermore, QRFP was bioactive in omental adipocytes from obese individuals, inhibiting ISO-induced lipolysis in these cells. Our results suggest that GPR103b and QRFP work in an autocrine/paracrine manner to regulate adipogenesis.
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http://dx.doi.org/10.1210/me.2010-0010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5417454PMC
August 2010

Direct and indirect induction by 1,25-dihydroxyvitamin D3 of the NOD2/CARD15-defensin beta2 innate immune pathway defective in Crohn disease.

J Biol Chem 2010 Jan 30;285(4):2227-31. Epub 2009 Nov 30.

Department of Physiology, Montreal General Hospital, Canada.

Vitamin D signaling through its nuclear vitamin D receptor has emerged as a key regulator of innate immunity in humans. Here we show that hormonal vitamin D, 1,25-dihydroxyvitamin D(3), robustly stimulates expression of pattern recognition receptor NOD2/CARD15/IBD1 gene and protein in primary human monocytic and epithelial cells. The vitamin D receptor signals through distal enhancers in the NOD2 gene, whose function was validated by chromatin immunoprecipitation and chromatin conformation capture assays. A key downstream signaling consequence of NOD2 activation by agonist muramyl dipeptide is stimulation of NF-kappaB transcription factor function, which induces expression of the gene encoding antimicrobial peptide defensin beta2 (DEFB2/HBD2). Pretreatment with 1,25-dihydroxyvitamin D(3) synergistically induced NF-kappaB function and expression of genes encoding DEFB2/HBD2 and antimicrobial peptide cathelicidin in the presence of muramyl dipeptide. Importantly, this synergistic response was also seen in macrophages from a donor wild type for NOD2 but was absent in macrophages from patients with Crohn disease homozygous for non-functional NOD2 variants. These studies provide strong molecular links between vitamin D deficiency and the genetics of Crohn disease, a chronic incurable inflammatory bowel condition, as Crohn's pathogenesis is associated with attenuated NOD2 or DEFB2/HBD2 function.
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http://dx.doi.org/10.1074/jbc.C109.071225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2807280PMC
January 2010

Hereditary inclusion body myopathy-linked p97/VCP mutations in the NH2 domain and the D1 ring modulate p97/VCP ATPase activity and D2 ring conformation.

Mol Cell Biol 2009 Aug 8;29(16):4484-94. Epub 2009 Jun 8.

The Nicholas Conor Institute for Pediatric Cancer Research, 9710 Scranton Road, Suite 170, San Diego, CA 92121, USA.

Hereditary inclusion body myopathy associated with early-onset Paget disease of bone and frontotemporal dementia (hIBMPFTD) is a degenerative disorder caused by single substitutions in highly conserved residues of p97/VCP. All mutations identified thus far cluster within the NH(2) domain or the D1 ring, which are both required for communicating conformational changes to adaptor protein complexes. In this study, biochemical approaches were used to identify the consequences of the mutations R155P and A232E on p97/VCP structure. Assessment of p97/VCP oligomerization revealed that p97(R155P) and p97(A232E) formed hexameric ring-shaped structures of approximately 600 kDa. p97(R155P) and p97(A232E) exhibited an approximately 3-fold increase in ATPase activity compared to wild-type p97 (p97(WT)) and displayed increased sensitivity to heat-induced upregulation of ATPase activity. Protein fluorescence analysis provided evidence for conformational differences in the D2 rings of both hIBMPFTD mutants. Furthermore, both mutations increased the proteolytic susceptibility of the D2 ring. The solution structures of all p97/VCP proteins revealed a didispersed distribution of a predominant hexameric population and a minor population of large-diameter complexes. ATP binding significantly increased the abundance of large-diameter complexes for p97(R155P) and p97(A232E), but not p97(WT) or the ATP-binding mutant p97(K524A). Therefore, we propose that hIBMPFTD p97/VCP mutants p97(R155P) and p97(A232E) possess structural defects that may compromise the mechanism of p97/VCP activity within large multiprotein complexes.
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http://dx.doi.org/10.1128/MCB.00252-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2725746PMC
August 2009

Ubiquitin-regulated recruitment of IkappaB kinase epsilon to the MAVS interferon signaling adapter.

Mol Cell Biol 2009 Jun 20;29(12):3401-12. Epub 2009 Apr 20.

Lady Davis Institute for Medical Research, Jewish General Hospital, 3755 Cote Ste. Catherine, Montreal, Quebec, Canada H3T 1E2.

Induction of the antiviral interferon response is initiated upon recognition of viral RNA structures by the RIG-I or Mda-5 DEX(D/H) helicases. A complex signaling cascade then converges at the mitochondrial adapter MAVS, culminating in the activation of the IRF and NF-kappaB transcription factors and the induction of interferon gene expression. We have previously shown that MAVS recruits IkappaB kinase epsilon (IKKepsilon) but not TBK-1 to the mitochondria following viral infection. Here we map the interaction of MAVS and IKKepsilon to the C-terminal region of MAVS and demonstrate that this interaction is ubiquitin dependent. MAVS is ubiquitinated following Sendai virus infection, and K63-linked ubiquitination of lysine 500 (K500) of MAVS mediates recruitment of IKKepsilon to the mitochondria. Real-time PCR analysis reveals that a K500R mutant of MAVS increases the mRNA level of several interferon-stimulated genes and correlates with increased NF-kappaB activation. Thus, recruitment of IKKepsilon to the mitochondria upon MAVS K500 ubiquitination plays a modulatory role in the cascade leading to NF-kappaB activation and expression of inflammatory and antiviral genes. These results provide further support for the differential role of IKKepsilon and TBK-1 in the RIG-I/Mda5 pathway.
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http://dx.doi.org/10.1128/MCB.00880-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2698723PMC
June 2009

The IKK-related kinases: from innate immunity to oncogenesis.

Cell Res 2008 Sep;18(9):889-99

Faculté de Pharmacie, Université de Montréal, C.P. 6128, succursale Centre-Ville, Montréal, Québec, Canada H3C 3J7.

Over the past four years, the field of the innate immune response has been highly influenced by the discovery of the IkappaB kinase (IKK)-related kinases, TANK Binding Kinase 1 (TBK1) and IKKi, which regulate the activity of interferon regulatory factor (IRF)-3/IRF-7 and NF-kappaB transcription factors. More recently, additional essential components of the signaling pathways that activate these IKK homologues have been discovered. These include the RNA helicases RIGi and MDA5, and the downstream mitochondrial effector known as CARDIF/MAVS/VISA/IPS-1. In addition to their essential functions in controlling the innate immune response, recent studies have highlighted a role of these kinases in cell proliferation and oncogenesis. The canonical IKKs are well recognized to be a bridge linking chronic inflammation to cancer. New findings now suggest that the IKK-related kinases TBK1 and IKKi also participate in signaling pathways that impact on cell transformation and tumor progression. This review will therefore summarize and discuss the role of TBK1 and IKKi in cellular transformation and oncogenesis by focusing on their regulation and substrate specificity.
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http://dx.doi.org/10.1038/cr.2008.273DOI Listing
September 2008

Roles of ubiquitination in pattern-recognition receptors and type I interferon receptor signaling.

Cytokine 2008 Sep 15;43(3):359-67. Epub 2008 Aug 15.

Faculté de Pharmacie, Université de Montréal, CP 6128, succursale Centre-Ville, Montréal, Québec, Canada.

Post-translational protein modifications are involved in all functions of living cells. This includes the ability of cells to recognize pathogens and regulate genes involved in their clearance, a concept known as innate immunity. While phosphorylation mechanisms play essential roles in regulating different aspects of the innate immune response, ubiquitination is now recognized as another post-translational modification that works in parallel with phosphorylation to orchestrate the final proper innate immune response against invading pathogens. More precisely, this review will discuss the most recent advances that address the role of ubiquitination in pattern-recognition receptors and type I interferon receptor signaling.
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http://dx.doi.org/10.1016/j.cyto.2008.07.012DOI Listing
September 2008

Phosphorylation of IRF-3 on Ser 339 generates a hyperactive form of IRF-3 through regulation of dimerization and CBP association.

J Virol 2008 Apr 13;82(8):3984-96. Epub 2008 Feb 13.

Faculté de Pharmacie, Université de Montréal, C.P. 6128, succursale Centre-Ville, Montréal, Québec, Canada.

The IkappaB kinase-related kinases, TBK1 and IKKi, were recently shown to be responsible for the C-terminal phosphorylation of IRF-3. However, the identity of the phosphoacceptor site(s) targeted by these two kinases remains unclear. Using a biological assay based on the IRF-3-mediated production of antiviral cytokines, we demonstrate here that all Ser/Thr clusters of IRF-3 are required for its optimal transactivation capacity. In vitro kinase assays using full-length His-IRF-3 as a substrate combined with mass spectrometry analysis revealed that serine 402 and serine 396 are directly targeted by TBK1. Analysis of Ser/Thr-to-Ala mutants revealed that the S396A mutation, located in cluster II, abolished IRF-3 homodimerization, CBP association, and nuclear accumulation. However, production of antiviral cytokines was still present in IRF-3 S396A-expressing cells. Interestingly, mutation of serine 339, which is involved in IRF-3 stability, also abrogated CBP association and dimerization without affecting gene transactivation as long as serine 396 remained available for phosphorylation. Complementation of IRF-3-knockout mouse embryonic fibroblasts also revealed a compensatory mechanism of serine 339 and serine 396 in the ability of IRF-3 to induce expression of the interferon-stimulated genes ISG56 and ISG54. These data lead us to reconsider the current model of IRF-3 activation. We propose that conventional biochemical assays used to measure IRF-3 activation are not sensitive enough to detect the small fraction of IRF-3 needed to elicit a biological response. Importantly, our study establishes a molecular link between the role of serine 339 in IRF-3 homodimerization, CBP association, and its destabilization.
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http://dx.doi.org/10.1128/JVI.02526-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2292984PMC
April 2008

Signaling pathways involved in the cardioprotective effects of cannabinoids.

J Pharmacol Sci 2006 Oct 7;102(2):155-66. Epub 2006 Oct 7.

Faculty of Pharmacy, Université de Montréal, Canada.

The aim of the present article is to review the cardioprotective properties of cannabinoids, with an emphasis on the signaling pathways involved. Cannabinoids have been reported to protect against ischemia in rat isolated hearts, as well as in rats and mice in vivo. Although these effects have been observed mostly with a pre-treatment of a cannabinoid, we report that the selective CB(2)-receptor agonist JWH133 is able to reduce infarct size when administered either before ischemia, during the entire ischemic period, or just upon reperfusion. Little is known about the signaling pathways involved in these cardioprotective effects. Likely candidates include protein kinase C (PKC) and mitogen-activated protein kinases (MAPK) since they are activated during ischemia-reperfusion and contribute to the protective effect ischemic preconditioning. The use of pharmacological inhibitors suggests that PKC, p38 MAPK, and p42/p44 MAPK (ERK1/2) contribute to the protective effect of cannabinoids. In addition, perfusion with JWH133 in healthy hearts caused an increase in both p38 MAPK phosphorylation level and activity, whereas the CB(1)-receptor agonist ACEA was associated with an increase in the phosphorylation status of both ERK1 and ERK2 without any change in activity. During ischemia, both agonists doubled p38 MAPK activity, whereas ERK1/2 phosphorylation level and activity during reperfusion were enhanced only by the CB(1)-receptor agonist. Finally, although nitric oxide (NO) was shown to exert both pro and anti-apoptotic effects on cardiomyocytes, with an apparently controversial effect on myocardial survival, our data suggest that NO may contribute to the cardioprotective effect of some cannabinoids.
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http://dx.doi.org/10.1254/jphs.crj06011xDOI Listing
October 2006

Involvement of the IkappaB kinase (IKK)-related kinases tank-binding kinase 1/IKKi and cullin-based ubiquitin ligases in IFN regulatory factor-3 degradation.

J Immunol 2006 Oct;177(8):5059-67

Faculty of Pharmacy, University of Montreal, Montreal, Canada.

Activation of the innate arm of the immune system following pathogen infection relies on the recruitment of latent transcription factors involved in the induction of a subset of genes responsible for viral clearance. One of these transcription factors, IFN regulatory factor 3 (IRF-3), is targeted for proteosomal degradation following virus infection. However, the molecular mechanisms involved in this process are still unknown. In this study, we show that polyubiquitination of IRF-3 increases in response to Sendai virus infection. Using an E1 temperature-sensitive cell line, we demonstrate that polyubiquitination is required for the observed degradation of IRF-3. Inactivation of NEDD8-activating E1 enzyme also results in stabilization of IRF-3 suggesting the NEDDylation also plays a role in IRF-3 degradation following Sendai virus infection. In agreement with this observation, IRF-3 is recruited to Cullin1 following virus infection and overexpression of a dominant-negative mutant of Cullin1 significantly inhibits the degradation of IRF-3 observed in infected cells. We also asked whether the C-terminal cluster of phosphoacceptor sites of IRF-3 could serve as a destabilization signal and we therefore measured the half-life of C-terminal phosphomimetic IRF-3 mutants. Interestingly, we found them to be short-lived in contrast to wild-type IRF-3. In addition, no degradation of IRF-3 was observed in TBK1(-/-) mouse embryonic fibroblasts. All together, these data demonstrate that virus infection stimulates a host cell signaling pathway that modulates the expression level of IRF-3 through its C-terminal phosphorylation by the IkappaB kinase-related kinases followed by its polyubiquitination, which is mediated in part by a Cullin-based ubiquitin ligase.
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http://dx.doi.org/10.4049/jimmunol.177.8.5059DOI Listing
October 2006

Endothelin is a dose-dependent trophic factor and a mitogen in small arteries in vivo.

Cardiovasc Res 2006 Jul 3;71(1):61-8. Epub 2006 Mar 3.

Faculty of Pharmacy, Université de Montréal, 2900 Edouard-Montpetit, Pavillon Jean-Coutu, room 3197, P.O. Box 6128, Station Centre-Ville Montréal, Québec, Canada H3C 3J7.

Objective: Endothelin (ET) modulates cellular processes relevant to vascular remodeling, but there is still some debate as to the potential of ET to be a trophic factor or a mitogen. Moreover, the signaling of ET in vivo to produce these effects is largely unknown.

Methods: 3H-leucine and 3H-thymidine incorporation in rat small mesenteric arteries was studied with several doses of ET-1 (0.1-10 pmol/kg/min) administered for 26 h in vivo.

Results: The EC50 for protein synthesis was four times lower than that of DNA synthesis, with maximal effects around 1 and 3 pmol/kg/min, respectively. At 5 pmol/kg/min, ET enhanced CDK2 activity by reducing the binding of its inhibitor p27(Kip1). In contrast, the binding was enhanced at 0.5 pmol/kg/min. The reduced binding observed at 5 pmol/kg/min could not be explained by changes of p27(Kip1) or CDK2 content. Phosphorylation of p27(Kip1) on serine 10 was significantly reduced at 5 pmol/kg/min ET. Although the phosphoinositide 3-kinase pathway was activated, it did not contribute to the protein or DNA synthesis responses. Administration of 1 or 5 pmol/kg/min ET-1 for 28 days increased the thickness and cross-sectional area of the small mesenteric artery due to hypertrophy and hyperplasia, respectively, thus confirming the results obtained in acute conditions.

Conclusion: ET modulates p27(Kip1) binding to CDK2, producing hypertrophy at low and hyperplasia at higher concentrations. Taken together, these results suggest that ET can act both as a trophic factor and as a mitogen in an in vivo environment, depending on its local concentration.
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http://dx.doi.org/10.1016/j.cardiores.2006.02.029DOI Listing
July 2006

The proinflammatory actions of angiotensin II are dependent on p65 phosphorylation by the IkappaB kinase complex.

J Biol Chem 2006 May 2;281(19):13275-13284. Epub 2006 Mar 2.

Faculty of Pharmacy, University of Montreal, Montreal, Quebec H3C 3J7, Canada. Electronic address:

The vasoactive hormone angiotensin II (Ang II) probably triggers inflammatory cardiovascular diseases by activating transcription factors such as NF-kappaB. We describe here a novel mode of NF-kappaB activation in cultured vascular smooth muscle cells exposed to Ang II. Ang II treatment resulted in an increase in the phosphotransferase activity of the IKK complex, which was mediated through the AT1 receptor subtype. The typical phosphorylation and proteasome-dependent degradation of the NF-kappaB inhibitor IkappaBalpha were not observed. Rather, Ang II treatment of vascular smooth muscle cells led to the phosphorylation of p65 on serine 536, a signal detected in both the cytoplasm and the nuclear compartments. The use of pharmacological inhibitors that inhibit the activation of MEK by Ang II revealed that phosphorylation of p65 on serine 536 did not require the MEK-ERK-RSK signaling pathway. On the other hand, specifically targeting the IKKbeta subunit of the IKK complex by overexpression of a dominant negative version of IKKbeta (IKKbeta K44A) or silencing RNA technology demonstrated that the IKKbeta subunit of the IKK complex was responsible for the detected phosphoserine 536 signal in Ang II-treated cells. Characterization of the signaling pathway leading to activation of the IKK complex by Ang II revealed that neither epidermal growth factor receptor transactivation nor the phosphatidylinositol 3-kinase-AKT signaling cascade were involved. Collectively, our data demonstrate that the proinflammatory activity of Ang II is independent of the classical pathway leading to IkappaBalpha phosphorylation and degradation but clearly depends on the recruitment of an IKK complex signaling cascade leading to phosphorylation of p65 on serine 536.
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http://dx.doi.org/10.1074/jbc.M512815200DOI Listing
May 2006

Roles of an IkappaB kinase-related pathway in human cytomegalovirus-infected vascular smooth muscle cells: a molecular link in pathogen-induced proatherosclerotic conditions.

J Biol Chem 2005 Mar 24;280(9):7477-86. Epub 2004 Dec 24.

Faculty of Pharmacy, University of Montreal, Montreal, Quebec H3C 3J7, Canada.

Viral and bacterial pathogens have long been suspected to affect atherogenesis directly. However, mechanisms linking innate immunity to chronic inflammatory diseases such as atherosclerosis are still poorly defined. Here we show that infection of primary human aortic smooth muscle cells (HAOSMC) with human cytomegalovirus (HCMV) leads to activation of the novel IkappaB kinase (IKK)-related kinase, Tank-binding kinase-1 (TBK1), a major effector of the cellular innate immune response. We demonstrate that part of the HCMV inflammatory response is most likely mediated via this novel kinase because the canonical IKK complex was only poorly activated upon infection of HAOSMC. An increase in TBK1 phosphotransferase activity led to a strong activation of the interferon regulatory factor (IRF)-3 transcription factor as measured by its C-terminal phosphorylation, dimerization, and DNA binding activity. In addition to TBK1, HAOSMC also express another IKK-related kinase isoform, IKKepsilon, albeit at a lower level. Nevertheless, both isoforms were required for full activation of IRF-3 by HCMV. The transcripts of proatherosclerotic genes Ccl5 (encoding for the chemokine RANTES (regulated upon activation, normal T cell expressed and secreted)) and Cxcl10 (encoding for the chemokine IP-10 (interferon-gamma-inducible protein 10)) were induced in an IRF-3-dependent manner after HCMV infection of smooth muscle cells. In addition, cytokine arrays analysis showed that RANTES and IP-10 were the predominant chemokines present in the supernatant of HCMV-infected HAOSMC. Activation of the TBK1/IRF-3 pathway was independent of epidermal growth factor receptor and pertussis toxin-sensitive G protein-coupled receptor activation. Our results thus add additional molecular clues to a possible role of HCMV as a modulator of atherogenesis through the induction of a proinflammatory response that is, in part, dependent of an IKK-related kinase pathway.
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http://dx.doi.org/10.1074/jbc.M410392200DOI Listing
March 2005

Convergence of the NF-kappaB and interferon signaling pathways in the regulation of antiviral defense and apoptosis.

Ann N Y Acad Sci 2003 Dec;1010:237-48

Lady Davis Institute for Medical Research, Jewish General Hospital, Departments of Microbiology and Immunology and Medicine, McGill University, Montreal, Canada H3T 1E2.

The ubiquitously expressed interferon regulatory factor 3 (IRF-3) is directly activated following virus infection and functions as a key activator of the immediate-early Type 1 interferon (IFN) genes. Using DNA microarray analysis (8,556 genes) in Jurkat T cells inducibly expressing constitutively active IRF-3, several target genes directly regulated by IRF-3 were identified. Among the genes upregulated by IRF-3 were transcripts for a subset of known IFN-stimulated genes (ISGs), including ISG56, which functions as an inhibitor of translation initiation. Phosphorylation of C-terminal Ser/Thr residues--(382)GGASSLENTVDLHISNSHPLSLTSDQY(408)-is required for IRF-3 activation. Using C-terminal point mutations and a novel phosphospecific antibody, Ser396 was characterized as the minimal phosphoacceptor site required in vivo for IRF-3 activation following Sendai virus (SeV) infection, expression of viral nucleocapsid, or double-stranded RNA (dsRNA) treatment. The identity of the virus-activated kinase (VAK) activity that targets and activates IRF-3 and IRF-7 has remained a critical missing link in the understanding of interferon signaling. We report that the IKK-related kinases-IKKepsilon/TBK-1-are components of VAK that mediate IRF-3 and IRF-7 phosphorylation and thus functionally link the NF-kappaB and IRF pathways in the development of the antiviral response.
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http://dx.doi.org/10.1196/annals.1299.042DOI Listing
December 2003

Identification of the minimal phosphoacceptor site required for in vivo activation of interferon regulatory factor 3 in response to virus and double-stranded RNA.

J Biol Chem 2003 Mar 10;278(11):9441-7. Epub 2003 Jan 10.

Terry Fox Molecular Oncology Group, Lady Davis Institute for Medical Research and the Departments of Microbiology & Immunology and Medicine, McGill University, Montreal, Quebec H3T 1E2, Canada.

The ubiquitously expressed latent interferon regulatory factor (IRF) 3 transcription factor is activated in response to virus infection by phosphorylation events that target a cluster of Ser/Thr residues, (382)GGASSLENTVDLHISNSHPLSLTSDQY(408) at the C-terminal end of the protein. To delineate the minimal phosphoacceptor sites required for IRF-3 activation, several point mutations were generated and tested for transactivation potential and cAMP-response element-binding protein-binding protein/p300 coactivator association. Expression of the IRF-3 S396D mutant alone was sufficient to induce type I IFN beta, IFNalpha1, RANTES, and the interferon-stimulated gene 561 promoters. Using SDS-PAGE and immunoblotting with a novel phosphospecific antibody, we show for the first time that, in vivo, IRF-3 is phosphorylated on Ser(396) following Sendai virus infection, expression of viral nucleocapsid, and double-stranded RNA treatment. These results demonstrate that Ser(396) within the C-terminal Ser/Thr cluster is targeted in vivo for phosphorylation following virus infection and plays an essential role in IRF-3 activation. The inability of the phosphospecific antibody to detect Ser(396) phosphorylation in lipopolysaccharide-treated cells suggests that other major pathways may be involved in IRF-3 activation following Toll-like receptor 4 stimulation.
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http://dx.doi.org/10.1074/jbc.M209851200DOI Listing
March 2003

Multiple signaling pathways leading to the activation of interferon regulatory factor 3.

Biochem Pharmacol 2002 Sep;64(5-6):985-92

Terry Fox Molecular Oncology Group, Lady Davis Institute-Jewish General Hospital, McGill University, 3755 Cote Ste., Catherine Montreal, Que., Canada.

Virus infection of susceptible cells activates multiple signaling pathways that orchestrate the activation of genes, such as cytokines, involved in the antiviral and innate immune response. Among the kinases induced are the mitogen-activated protein (MAP) kinases, Jun-amino terminal kinases (JNK) and p38, the IkappaB kinase (IKK) and DNA-PK. In addition, virus infection also activates an uncharacterized VAK responsible for the C-terminal phosphorylation and subsequent activation of interferon regulatory factor 3 (IRF-3). Virus-mediated activation of IRF-3 through VAK is dependent on viral entry and transcription, since replication deficient virus failed to induce IRF-3 activity. The pathways leading to VAK activation are not well characterized, but IRF-3 appears to represent a novel cellular detection pathway that recognizes viral nucleocapsid (N) structure. Recently, the range of inducers responsible for IRF-3 activation has increased. In addition to virus infection, recognition of bacterial infection mediated through lipopolysaccharide by Toll-like receptor 4 has also been reported. Furthermore, MAP kinase kinase kinase (MAP KKK)-related pathways and DNA-PK induce N-terminal phosphorylation of IRF-3. This review summarizes recent observations in the identification of novel signaling pathways leading to IRF-3 activation.
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http://dx.doi.org/10.1016/s0006-2952(02)01165-6DOI Listing
September 2002

The interferon antiviral response: from viral invasion to evasion.

Curr Opin Infect Dis 2002 Jun;15(3):259-67

Terry Fox Molecular Oncology Group, Lady Davis Institute for Medical Research and Department of Medicine, McGill University, Montreal, Quebec, Canada.

One of the initial responses of an organism to infection by pathogenic viruses is the synthesis of antiviral cytokines such as the type I interferons (interferon-alpha/beta), interleukins, and other proinflammatory cytokines and chemokines. Interferons provide a first line of defence against virus infections by generating an intracellular environment that restricts virus replication and signals the presence of a viral pathogen to the adaptive arm of the immune response. Interferons stimulate cells in the local environment to activate a network of interferon-stimulated genes, which encode proteins that have antiviral, antiproliferative and immunomodulatory activities. The present review focuses on recent reports that describe the activation of multiple signalling pathways following virus infection, new candidate genes that are implicated in the establishment of the antiviral state, and the strategies used by viruses and their specific viral products to antagonize and evade the host antiviral response.
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http://dx.doi.org/10.1097/00001432-200206000-00008DOI Listing
June 2002
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