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Drug nanocarriers (NCs) capable of crossing the vascular endothelium and deeply penetrating into dense tissues of the CNS could potentially transform the management of neurological diseases. In the present study, we investigated the interaction of bottle-brush (BB) polymers with different biological barriers in vitro and in vivo and compared it to nanospheres of similar composition. In vitro internalization and permeability assays revealed that BB polymers are not internalized by brain-associated cell lines and translocate much faster across a blood-brain barrier model compared to nanospheres of similar hydrodynamic diameter. These observations performed under static, no-flow conditions were complemented by dynamic assays performed in microvessel arrays on chip and confirmed that BB polymers can escape the vasculature compartment via a paracellular route. BB polymers injected in mice and zebrafish larvae exhibit higher penetration in brain tissues and faster extravasation of microvessels located in the brain compared to nanospheres of similar sizes. The superior diffusivity of BBs in extracellular matrix-like gels combined with their ability to efficiently cross endothelial barriers via a paracellular route position them as promising drug carriers to translocate across the blood-brain barrier and penetrate dense tissue such as the brain, two unmet challenges and ultimate frontiers in nanomedicine.

Objective: 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. Fibroblasts' intracellular and mitochondrial reactive oxygen species (ROS) were determined by flow cytometry. Mitochondrial function was measured by Seahorse XF24 analyser. Fibrotic and inflammatory gene expressions were assessed by qPCR and key pro-inflammatory components of the fibroblasts' secretome (IL-6 and IL-8) were quantified by ELISA., Results: Compared with 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 TGF-β-activated kinase 1 (TAK1)-IκB kinase β (IKKβ)-IFN regulatory factor 5 (IRF5) inflammatory signalling 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., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: journals.permissions@oup.com.)

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., Competing Interests: The authors have declared that no competing interests exist.

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.

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.

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:CD4 + T-cell suppression was assessed in allogeneic coculture systems. Compared with nonatherosclerotic-MSCs, atherosclerotic-MSCs displayed higher levels of both intracellular ( P =0.006) and mitochondrial ( P =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 P <0.0001, interleukin-8/C-X-C motif chemokine ligand 8 P =0.04, and monocyte chemoattractant protein-1/chemokine ligand 2 P =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 ( P =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., (© 2017 American Heart Association, Inc.)

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., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

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., (© FASEB.)

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Ubiquitously expressed interferon regulatory factor 3 (IRF-3) is directly activated after virus infection and functions as a key activator of the immediate-early alpha/beta interferon (IFN) genes, as well as the RANTES chemokine gene. In the present study, a tetracycline-inducible expression system expressing a constitutively active form of IRF-3 (IRF-3 5D) was combined with DNA microarray analysis to identify target genes regulated by IRF-3. Changes in mRNA expression profiles of 8,556 genes were monitored after Tet-inducible expression of IRF-3 5D. Among the genes upregulated by IRF-3 were transcripts for several known IFN-stimulated genes (ISGs). Subsequent analysis revealed that IRF-3 directly induced the expression of ISG56 in an IFN-independent manner through the IFN-stimulated responsive elements (ISREs) of the ISG56 promoter. These results demonstrate that, in addition to its role in the formation of a functional immediate-early IFN-beta enhanceosome, IRF-3 is able to discriminate among ISRE-containing genes involved in the establishment of the antiviral state as a direct response to virus infection.

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.

The mechanisms of cellular recognition for virus infection remain poorly understood despite the wealth of information regarding the signaling events and transcriptional responses that ensue. Host cells respond to viral infection through the activation of multiple signaling cascades, including the activation of NF-kappaB, c-Jun/ATF-2 (AP-1), and the interferon regulatory factors (IRFs). Although viral products such as double-stranded RNA (dsRNA) and the processes of viral binding and fusion have been implicated in the activation of NF-kappaB and AP-1, the mechanism(s) of IRF-1, IRF-3, and IRF-7 activation has yet to be fully elucidated. Using recombinant measles virus (MeV) constructs, we now demonstrate that phosphorylation-dependent IRF-3 activation represents a novel cellular detection system that recognizes the MeV nucleocapsid structure. At low multiplicities of infection, IRF-3 activation is dependent on viral transcription, since UV cross-linking and a deficient MeV containing a truncated polymerase L gene failed to induce IRF-3 phosphorylation. Expression of the MeV nucleocapsid (N) protein, without the requirement for any additional viral proteins or the generation of dsRNA, was sufficient for IRF-3 activation. In addition, the nucleocapsid protein was found to associate with both IRF-3 and the IRF-3 virus-activated kinase, suggesting that it may aid in the colocalization of the kinase and the substrate. Altogether, this study suggests that IRF-3 recognizes nucleocapsid structures during the course of an MeV infection and triggers the induction of interferon production.

Recent molecular, biochemical, and gene disruption studies have demonstrated the essential role of interferon (IFN) regulatory factor-3, (IRF-3) and IRF-7 in the activation of type I IFN gene expression and the induction of the antiviral state. Both transcription factors share structural and functional properties, as well as a common mechanism of activation through C-terminal phosphorylation. The purpose of this review is to summarize recent investigations indicating that similar signalling pathways are likely involved in the activation of IRF-3 and IRF-7. Moreover, unique biochemical events, such as coactivator association and differential recognition of cis-acting elements, also illustrate the capacity of IRF-3 and IRF-7 to selectively regulate type I IFN and IFN-stimulated gene (ISG) expression.

Infection of host cells by viruses leads to the activation of multiple signaling pathways, resulting in the expression of host genes involved in the establishment of the antiviral state. Among the transcription factors mediating the immediate response to virus is interferon regulatory factor-3 (IRF-3) which is post-translationally modified as a result of virus infection. Phosphorylation of latent cytoplasmic IRF-3 on serine and threonine residues in the C-terminal region leads to dimerization, cytoplasmic to nuclear translocation, association with the p300/CBP coactivator, and stimulation of DNA binding and transcriptional activities. We now demonstrate that IRF-3 is a phosphoprotein that is uniquely activated via virus-dependent C-terminal phosphorylation. Paramyxoviridae including measles virus and rhabdoviridae, vesicular stomatitis virus, are potent inducers of a unique virus-activated kinase activity. In contrast, stress inducers, growth factors, DNA-damaging agents, and cytokines do not induce C-terminal IRF-3 phosphorylation, translocation or transactivation, but rather activate a MAPKKK-related signaling pathway that results in N-terminal IRF-3 phosphorylation. The failure of numerous well characterized pharmacological inhibitors to abrogate virus-induced IRF-3 phosphorylation suggests the involvement of a novel kinase activity in IRF-3 regulation by viruses.

Angiotensin II (Ang II), the primary effector of the renin-angiotensin system, is a multifunctional hormone that plays an important role in vascular function. In addition to its classical vasoconstrictor action, more recent studies demonstrated that Ang II stimulates the growth of a number of cell types, including vascular smooth muscle cells (SMC) (reviewed in [1-3]). In vivo studies have shown that chronic infusion of Ang II leads to the development of vascular hypertrophy in rats, whereas administration of angiotensin-converting enzyme (ACE) inhibitors or Ang II receptor antagonists prevents or regresses vascular hypertrophy in models of genetic and experimental hypertension [4]. Consistent with in vivo data, several laboratories have shown that Ang II stimulates protein synthesis and induces cellular hypertrophy, but not cell proliferation, in cultured aortic SMC [5-9]. Ang II also induces directed migration (chemotaxis) of vascular SMC [10, 11], although its effect is less prominent than that of platelet-derived growth factor (PDGF). The cellular mechanisms underlying these diverse actions of Ang II are not clearly understood but are likely to involve the activation of distinct signaling pathways.

Virus infection of target cells can result in different biological outcomes: lytic infection, cellular transformation, or cell death by apoptosis. Cells respond to virus infection by the activation of specific transcription factors involved in cytokine gene regulation and cell growth control. The ubiquitously expressed interferon regulatory factor 3 (IRF-3) transcription factor is directly activated following virus infection through posttranslational modification. Phosphorylation of specific C-terminal serine residues results in IRF-3 dimerization, nuclear translocation, and activation of DNA-binding and transactivation potential. Once activated, IRF-3 transcriptionally up regulates alpha/beta interferon genes, the chemokine RANTES, and potentially other genes that inhibit viral infection. We previously generated constitutively active [IRF-3(5D)] and dominant negative (IRF-3 DeltaN) forms of IRF-3 that control target gene expression. In an effort to characterize the growth regulatory properties of IRF-3, we observed that IRF-3 is a mediator of paramyxovirus-induced apoptosis. Expression of the constitutively active form of IRF-3 is toxic, preventing the establishment of stably transfected cells. By using a tetracycline-inducible system, we show that induction of IRF-3(5D) alone is sufficient to induce apoptosis in human embryonic kidney 293 and human Jurkat T cells as measured by DNA laddering, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay, and analysis of DNA content by flow cytometry. Wild-type IRF-3 expression augments paramyxovirus-induced apoptosis, while expression of IRF-3 DeltaN blocks virus-induced apoptosis. In addition, we demonstrate an important role of caspases 8, 9, and 3 in IRF-3-induced apoptosis. These results suggest that IRF-3, in addition to potently activating cytokine genes, regulates apoptotic signalling following virus infection.

Platelet-derived growth factor-BB (PDGF-BB) acts as a full mitogen for cultured aortic smooth muscle cells (SMC), promoting DNA synthesis and cell proliferation. In contrast, angiotensin II (Ang II) induces cellular hypertrophy as a result of increased protein synthesis, but is unable to drive cells into S phase. In an effort to understand the molecular basis for this differential growth response, we have examined the downstream effects of PDGF-BB and Ang II on regulators of the cell cycle machinery in rat aortic SMC. Both PDGF-BB and Ang II were found to stimulate the accumulation of G(1) cyclins with similar kinetics. In addition, little difference was observed in the expression level of their catalytic partners, Cdk4 and Cdk2. However, while both factors increased the enzymatic activity of Cdk4, only PDGF-BB stimulated Cdk2 activity in late G(1) phase. The lack of activation of Cdk2 in Ang II-treated cells was causally related to the failure of Ang II to stimulate phosphorylation of the enzyme on threonine and to downregulate p27(Kip1) expression. By contrast, exposure to PDGF-BB resulted in a progressive and dramatic reduction in the level of p27(Kip1) protein. The time course of p27(Kip1) decline was correlated with a reduced rate of synthesis and an increased rate of degradation of the protein. Importantly, the repression of p27(Kip1) synthesis by PDGF-BB was associated with a marked attenuation of Kip1 gene transcription and a corresponding decrease in Kip1 mRNA accumulation. We also show that the failure of Ang II to promote S phase entry is not related to the autocrine production of transforming growth factor-beta1 by aortic SMC. These results identify p27(Kip1) as an important regulator of the phenotypic response of vascular SMC to mitogenic and hypertrophic stimuli.

Interferons are a large family of multifunctional secreted proteins involved in antiviral defense, cell growth regulation and immune activation. Viral infection induces transcription of multiple IFN genes, a response that is in part mediated by the interferon regulatory factors (IRFs). The initially characterized members IRF-1 and IRF-2 are now part of a growing family of transcriptional regulators that has expanded to nine members. The functions of the IRFs have also expanded to include distinct roles in biological processes such as pathogen response, cytokine signaling, cell growth regulation and hematopoietic development. The aim of this review is to provide an update on the novel discoveries in the area of IRF transcription factors and the important roles of the new generation of IRFs--particularly IRF-3, IRF-4 and IRF-7.

We monitored cardiac angiotensin II concentration and AT1-receptor density after long-term blockade of the renin-angiotensin system in inbred control hamsters treated with placebo or losartan (100 mg/kg/day) and cardiomyopathic hamsters treated with placebo, low-(30 mg/kg/day), or high-dose (100 mg/kg/day) losartan or quinapril (100 mg/kg/day). All treatments were started at age 50 days. Angiotensin II-receptor density and affinity were measured by radioligand-binding assays, and ventricular angiotensin II concentration was determined by radioimmunoassay. After 125 and 275 days of treatment, both doses of losartan significantly reduced AT1-receptor density, whereas quinapril had no effect. The administration of both drugs resulted in significant reductions in ventricular angiotensin II concentration. The prolonged administration of losartan was associated with an increase in cardiac hypertrophy, suggesting that angiotensin II signaling is not directly involved or at least does not play a major role in the remodeling process observed in cardiomyopathic hamsters.

In the present study, we have examined the effect of increased cyclic AMP (cAMP) levels on the stimulatory action of angiotensin II (Ang II) on protein synthesis. Treatment with cAMP-elevating agents potently inhibited Ang II-induced protein synthesis in rat aortic smooth muscle cells and in rat fibroblasts expressing the human AT1 receptor. The inhibition was dose-dependent and was observed at all concentrations of the peptide. To explore the mechanism of cAMP action, we have analyzed the effects of forskolin and 3-isobutyl-1-methylxanthine on various receptor-mediated responses. Elevation of cAMP did not alter the binding properties of the AT1 receptor and did not interfere with the activation of phospholipase C or the induction of early growth response genes by Ang II. Likewise, Ang II-dependent activation of the mitogen-activated protein kinases ERK1/ERK2 and p70 S6 kinase was unaffected by cAMP. In contrast, we found that increased concentration of cAMP strongly inhibited the stimulatory effect of Ang II on protein tyrosine phosphorylation. Specifically, cAMP abolished Ang II-induced tyrosine phosphorylation of the focal adhesion-associated protein paxillin and of the tyrosine kinase Tyk2. These results identify a novel mechanism by which the cAMP signaling system may exert growth-inhibitory effects in specific cell types.

Mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) is a dual-specificity protein phosphatase encoded by an immediate-early gene responsive to growth factors and stress. The MKP-1 protein selectively inactivates MAP kinases in vitro by dephosphorylation of the regulatory Thr and Tyr residues. Little is known on the mechanisms that regulate MKP-1 gene expression. Here, we demonstrate that Ca2+ is both necessary and sufficient for the induction of MKP-1 gene expression. Treatment of Rat1 fibroblasts with the Ca2+ chelating agent BAPTA completely suppressed serum-induced MKP-1 expression in a dose- and time-dependent manner. The inhibitory effect of BAPTA was observed at the level of the protein and the mRNA. Importantly, Ca2+ chelation blocked the induction of MKP-1 expression in response to all stimuli tested and in different cell types. Increasing the intracellular concentration of Ca2+ with the ionophore A23187 was sufficient to induce MKP-1 mRNA and protein expression in rat fibroblasts. We also provide evidence that activation of MAP kinases is not an absolute requirement for induction of the MKP-1 gene. Exposure of rat fibroblasts to A23187 induced MKP-1 expression without activating the JNK and p38 MAP kinase pathways. Also, inhibition of the ERK pathway with the selective MEK inhibitor PD98059 did not interfere with serum-stimulated MKP-1 mRNA expression. These results will help define the regulatory mechanisms that govern MKP-1 gene transcription in target cells.

A common response of cells to mitogenic and hypertrophic factors is the activation of high rates of protein synthesis. To investigate the molecular basis of this action, we have used the recently developed MAP kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor PD 98059 to examine the involvement of the ERK pathway in the regulation of global protein synthesis by growth factors in rat aortic smooth muscle cells (SMC). Incubation with PD 98059 blocked angiotensin II (AII)-dependent phosphorylation and enzymatic activity of both MEK1 and MEK2 isoforms, leading to inhibition of the phosphorylation and activation of p44(mapk) and p42(mapk). The compound was found to selectively inhibit activation of the ERK pathway by AII, but not the stimulation of p70 S6 kinase, phospholipase C, or tyrosine phosphorylation. Most importantly, treatment of aortic SMC with PD 98059 potently inhibited AII-stimulated protein synthesis with a half-maximal inhibitory concentration of 4.3 microM. The effect of PD 98059 was not restricted to AII, since the compound also blocked to various extent the induction of protein synthesis by growth factors acting through tyrosine kinase receptors, G protein-coupled receptors, or protein kinase C. These results provide strong evidence that activation of ERK isoforms is an obligatory step for growth factor-induced protein synthesis in aortic SMC.