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

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.

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

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-ß-activated kinase 1 (TAK1)-IκB kinase ß (IKKß)-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.

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

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.

Genome-wide RNAi screen reveals a new role of a WNT/CTNNB1 signaling pathway as negative regulator of virus-induced innate immune responses.

To identify new regulators of antiviral innate immunity, we completed the first genome-wide gene silencing screen assessing the transcriptional response at the interferon-ß (IFNB1) promoter following Sendai virus (SeV) infection. We now report a novel link between WNT signaling pathway and the modulation of retinoic acid-inducible gene I (RIG-I)-like receptor (RLR)-dependent innate immune responses. Here we show that secretion of WNT2B and WNT9B and stabilization of ß-catenin (CTNNB1) upon virus infection negatively regulate expression of representative inducible genes IFNB1, IFIT1 and TNF in a CTNNB1-dependent effector mechanism. The antiviral response is drastically reduced by glycogen synthase kinase 3 (GSK3) inhibitors but restored in CTNNB1 knockdown cells. The findings confirm a novel regulation of antiviral innate immunity by a canonical-like WNT/CTNNB1 signaling pathway. The study identifies novel avenues for broad-spectrum antiviral targets and preventing immune-mediated diseases upon viral infection.

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

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.

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

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 IFNß, 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.

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

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-ß (IKKß) in Ang II-treated cells, a kinase involved in inflammatory reactions. In light of a study suggesting a role of IKKß 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 IKKß 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 IKKß. Consistent with this, the use of different short hairpin RNA constructs targeting IKKß 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 IKKß inhibitor N-(6-chloro-7-methoxy-9H-ß-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 IKKß inhibition for the treatment of cardiovascular diseases.

Deep Tissue Penetration of Bottle-Brush Polymers via Cell Capture Evasion and Fast Diffusion.

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.

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.

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 IKKß 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, IKKß subunit, tumor growth factor ß-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 IKKß and the activation of the IKK complex by Ang II. Rapid phosphorylation of IKKß requires a second messenger-dependent pathway composed of PKCα-TRAF6-TAK1, whereas sustained phosphorylation and activation of IKKß requires the MEK1/2-ERK1/2-RSK pathway. Importantly, simultaneously targeting components of these two pathways completely blunts the phosphorylation of IKKß 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 IKKß, which is an essential step in the proinflammatory activity of Ang II.

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

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.

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

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 ß (IKKß) 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 IKKß nuclear factor-κB transcription factors inflammatory pathway. Importantly, this constitutive pro-inflammatory IKKß 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 IKKß inhibitor or IKKß knockdown (KD) (clustered regularly interspaced short palindromic repeats/Cas9-mediated IKKß 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 IKKß reduces the immunopotency of atherosclerosis+T2DM MSC by changing their secretome composition. Modulation of IKKß in atherosclerosis+T2DM MSCs enhances their myocardial repair ability.

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

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.

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

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 ß isoforms are active in unstimulated resting cells and are involved in the constitutive turnover of ß-catenin, a transcriptional coactivator involved in cell proliferation, differentiation, and lineage commitment. Interestingly, studies have demonstrated the regulatory roles of both GSK-3 and ß-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 ß-catenin control the antiviral innate immune response to RNA and DNA virus infections.

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

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.

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

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.

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

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.

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

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.

In vivo interferon regulatory factor 3 tumor suppressor activity in B16 melanoma tumors.

Delivery of transcription factors to cancer cells to reprogram gene expression may represent a novel strategy to augment the production of immune stimulatory cytokines and trigger a more potent antitumor response. In the present study, a bicistronic retroviral vector (AP2) was used to transduce B16-F0 melanoma cells with IFN regulatory factor (IRF)-3, which has been shown to activate type I IFN genes (IFN-beta and IFN-alpha) as well as other cytokines. Gene-modified B16 melanoma cells were inoculated s.c. into C57BL/6 syngeneic mice. In animals receiving IRF-3 B16 melanoma cells, tumors grew at a 4- to 5-fold reduced rate, and tumors that developed from these mice had a moderate-to-dense infiltration of inflammatory cells, whereas only low levels of lymphocyte infiltration were observed in mock-transduced B16 tumors. Furthermore, tumor growth was not inhibited in severe-combined immunodeficient mice after inoculation of IRF-3-expressing B16 cells, which suggested that IRF-3-mediated antitumor responses were dependent on a functional adaptive lymphocyte response. Interestingly, these in vivo effects on tumor growth correlated with higher mRNA expression of chemokines such as MIP-1beta, RANTES, and IP-10, as well as dramatic increases in vitro in the inducibility of cytokine mRNA such as IFN-beta, TNF-alpha and interleukin 6. Our results demonstrate that with weakly antigenic tumors such as B16 melanoma, IRF-3 gene transfer can mediate important antitumor responses. These findings suggest a novel role for IRF-3 as a potential molecular target for gene therapy of cancer.

Disruption of the B-cell specific transcriptional program in HHV-8 associated primary effusion lymphoma cell lines.

Primary effusion lymphoma (PEL) is a lymphoproliferative disease of B-cell origin that is associated with HHV-8 infection. PEL cells harbor a non-B, non-T phenotype and lack significant surface immunoglobulin (Ig) expression, a characteristic that has not been fully explained. In the present study, we demonstrate that PEL cells constitutively express interferon regulatory factor (IRF)-4, a transcription factor that regulates the activity of the immunoglobulin light-chain enhancer elements lambdaB and kappaE3' through binding to a composite Ets-IRF site. IRF-4 activity requires its physical interaction with PU.1, an Ets family member involved in the activation of genes essential for B-cell development. However, in PEL-derived B-cell lines, PU.1 expression was completely abrogated; expression of the B cell specific transcription factor Oct-2, which is known to regulate PU.1 expression, was also abolished. Moreover, the B-cell-specific coactivator of octamer factors, BOB-1/OcaB, was expressed at very decreased levels in PEL cells. Ectopic expression of Oct-2 was able to fully restore PU.1 promoter activity in the PEL cell line BCBL-1, while PU.1 expression also reconstituted the activity of the lambdaB Ets-IRF site. In addition, protein levels of BSAP/Pax-5 and IRF-8/ICSBP were undetectable in PEL cells. The pattern of transcription factor ablation observed in PEL was found to be comparable to that observed in classical Hodgkin's disease-derived cell lines, which also lack B-cell-specific surface markers. These observations indicate that disruption of the B-cell-specific transcriptional program is likely to contribute to the incomplete B-cell phenotype characteristic of PEL cells.

Position of Src tyrosine kinases in the interaction between angiotensin II and endothelin in in vivo vascular protein synthesis.

OBJECTIVES: Endothelin is a necessary intermediate in the trophic action of angiotensin II during hypertension-induced resistance artery remodeling in vivo. Since Src tyrosine kinases can be activated by both agonists, we studied their role in the trophic action of angiotensin II, endothelin and their interaction in rat small mesenteric arteries. METHODS AND RESULTS: Twenty-six hour infusion of high-dose angiotensin II (400 ng/kg per min) or endothelin (5 pmol/kg per min) via osmotic pumps significantly enhanced vascular protein synthesis in vivo. When angiotensin II was used as the trophic stimulus, treatment with a Src tyrosine kinase inhibitor (PP2, 0.5 mg/kg, starting at 21 h of the 26-h stimulation) produced a significant attenuation of extracellular regulated kinase 1 (ERK 1) phosphorylation and of protein synthesis. However, PP2 administered at 21 h or throughout the 26-h infusion did not abrogate the elevation of protein synthesis induced by endothelin. Moreover, endothelin did not enhance the phosphorylation of ERK 1/2 in small mesenteric arteries. We confirmed that angiotensin II stimulated the expression of prepro-endothelin mRNA in small mesenteric arteries in a Src-dependent manner, as the response was inhibited by PP2. To support the specific inhibitory activity of PP2 on Src tyrosine kinases in vivo, angiotensin II-induced phosphorylation of cortactin, a Src-specific substrate, was inhibited by PP2. CONCLUSION: Src tyrosine kinases represent an important signaling element in angiotensin II-induced endothelin production in small arteries in vivo. However, Src tyrosine kinases did not appear to contribute to the trophic signaling of endothelin, suggesting that they lie upstream of endothelin in the angiotensin II-endothelin-protein synthesis cascade.