Abstract 584: Allograft Inflammatory Factor-1 is Required for NfκB Pathway Activity in Macrophages and Atherosclerosis

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Lander Egaña-Gorroño ◽  
Prameladevi Chinnasamy ◽  
Nicholas Sibinga
Keyword(s):  
Oxidized Ldl ◽  
Critical Role ◽  
Immunohistochemical Analysis ◽  
Nuclear Factor Κb ◽  
Raw 264.7 Cells ◽  
Luciferase Reporter ◽  
Inflammatory Factor ◽  
Atherosclerotic Lesions ◽  
Nfκb Pathway

Introduction: In preliminary studies, we found that Allograft inflammatory factor-1 (AIF1) supports MΦ migration, phagocytosis, survival and pro-inflammatory cytokine secretion. Moreover, AIF1 limits necrotic core formation in atherosclerotic lesions in vivo . Nuclear Factor-κB (NFκB)-mediated signal transduction has been established at different stages of atherosclerosis. We hypothesize that AIF1-regulated processes in atherosclerosis may be mediated through effects on NFκB signaling. Methods: Bone marrow (BM) derived MΦs were isolated and immortalized from wt and Aif1 -/- mice and stimulated with oxidized-LDL (50 ug/ul; oxLDL). Lysates were immunoblotted for total and phosphorylated (active) p65 NFκB, and for total and phosphorylated forms of the IκBα repressor. Aif1 expression in mouse Raw 264.7 cells was knocked down (KD) using siRNA, and NFκB reporter activity, measured by a luciferase reporter, was assessed after adding LPS+IFN-γ. Immunohistochemical analysis for phospho-p65 NFκB was performed in atherosclerotic lesions (aortic roots) from Apoe -/- (SKO) and Apoe -/- ;Aif1 -/- (DKO) mice maintained on high fat diet for 16 weeks. Results: In AIF1-deficient BMMΦs stimulated with oxLDL, we found no differences in the levels of total p65 NFκB and IκBα, but interestingly, phospho-p65 NFκB levels were significantly reduced and phospho-IκBα levels increased compared to wt cells (P<0.05). AIF1 KD using siRNA significantly reduced NFκB activity compared to scrambled control (scrambled control vs. AIF1 KD; 40% vs. 22% luciferase activity, P<0.05), and this impairment was rescued with the addition of AIF1 cDNA. In vivo , NFκB phospho-p65 staining showed that in comparison to SKO samples, DKO aortic roots had decreased phospho-p65 NFκB (SKO vs. DKO; 7.0 vs. 4.0 +nuclei/aortic root, P<0.05). Conclusions: AIF1 is required for NFκB activation in MΦs and moreover, AIF1 enhances NFκB activity in atherosclerotic lesions in vivo . Because NFkB has been closely linked to both cytokine expression and cell survival signaling, these results point to a critical role for AIF1 in pro-inflammatory MΦ functions. Future studies involve identifying the precise steps of the pathway controlled by AIF1, and the mechanisms by which AIF1 affects NFκB signaling.

scholarly journals RNF141 interacts with KRAS to promote colorectal cancer progression

Oncogene ◽  
2021 ◽  
Author(s):  
Jiuna Zhang ◽  
Xiaoyu Jiang ◽  
Jie Yin ◽  
Shiying Dou ◽  
Xiaoli Xie ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Plasma Membrane ◽  
Tumor Growth ◽  
Cancer Progression ◽  
Critical Role ◽  
Ring Finger ◽  
Immunohistochemical Analysis ◽  
Bimolecular Fluorescence Complementation

AbstractRING finger proteins (RNFs) play a critical role in cancer initiation and progression. RNF141 is a member of RNFs family; however, its clinical significance, roles, and mechanism in colorectal cancer (CRC) remain poorly understood. Here, we examined the expression of RNF141 in 64 pairs of CRC and adjacent normal tissues by real-time PCR, Western blot, and immunohistochemical analysis. We found that there was more expression of RNF141 in CRC tissue compared with its adjacent normal tissue and high RNF141 expression associated with T stage. In vivo and in vitro functional experiments were conducted and revealed the oncogenic role of RNF141 in CRC. RNF141 knockdown suppressed proliferation, arrested the cell cycle in the G1 phase, inhibited migration, invasion and HUVEC tube formation but promoted apoptosis, whereas RNF141 overexpression exerted the opposite effects in CRC cells. The subcutaneous xenograft models showed that RNF141 knockdown reduced tumor growth, but its overexpression promoted tumor growth. Mechanistically, liquid chromatography-tandem mass spectrometry indicated RNF141 interacted with KRAS, which was confirmed by Co-immunoprecipitation, Immunofluorescence assay. Further analysis with bimolecular fluorescence complementation (BiFC) and Glutathione-S-transferase (GST) pull-down assays showed that RNF141 could directly bind to KRAS. Importantly, the upregulation of RNF141 increased GTP-bound KRAS, but its knockdown resulted in a reduction accordingly. Next, we demonstrated that RNF141 induced KRAS activation via increasing its enrichment on the plasma membrane not altering total KRAS expression, which was facilitated by the interaction with LYPLA1. Moreover, KRAS silencing partially abolished the effect of RNF141 on cell proliferation and apoptosis. In addition, our findings presented that RNF141 functioned as an oncogene by upregulating KRAS activity in a manner of promoting KRAS enrichment on the plasma membrane in CRC.

scholarly journals Essential role of IPS-1 in innate immune responses against RNA viruses

2006 ◽  
Vol 203 (7) ◽  
pp. 1795-1803 ◽  
Author(s):  
Himanshu Kumar ◽  
Taro Kawai ◽  
Hiroki Kato ◽  
Shintaro Sato ◽  
Ken Takahashi ◽  
...  
Keyword(s):  
Rna Viruses ◽  
Rna Virus ◽  
Critical Role ◽  
Nuclear Factor Κb ◽  
Type I ◽  
Innate Immune Responses ◽  
Antiviral Responses ◽  
Deficient Mice

IFN-β promoter stimulator (IPS)-1 was recently identified as an adapter for retinoic acid–inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (Mda5), which recognize distinct RNA viruses. Here we show the critical role of IPS-1 in antiviral responses in vivo. IPS-1–deficient mice showed severe defects in both RIG-I– and Mda5-mediated induction of type I interferon and inflammatory cytokines and were susceptible to RNA virus infection. RNA virus–induced interferon regulatory factor-3 and nuclear factor κB activation was also impaired in IPS-1–deficient cells. IPS-1, however, was not essential for the responses to either DNA virus or double-stranded B-DNA. Thus, IPS-1 is the sole adapter in both RIG-I and Mda5 signaling that mediates effective responses against a variety of RNA viruses.

scholarly journals Bovine Foamy Virus Transactivator BTas Interacts with Cellular RelB To Enhance Viral Transcription

2010 ◽  
Vol 84 (22) ◽  
pp. 11888-11897 ◽  
Author(s):  
Jian Wang ◽  
Juan Tan ◽  
Hongyan Guo ◽  
Qicheng Zhang ◽  
Rui Jia ◽  
...  
Keyword(s):  
Foamy Virus ◽  
Nuclear Factor Κb ◽  
Luciferase Reporter ◽  
Viral Transcription ◽  
Bovine Foamy Virus ◽  
Intracellular Parasites ◽  
Positive Feedback Circuit ◽  
Cellular Machinery

ABSTRACT Viruses are obligate intracellular parasites that depend on cellular machinery for their efficient transcription and replication. In a previous study we reported that bovine foamy virus (BFV) is able to activate the nuclear factor κB (NF-κB) pathway through the action of its transactivator BTas to enhance viral transcription. However, the mechanism used by NF-κB to enhance BFV transcription remains elusive. To address this question, we employed a yeast two-hybrid assay to screen for BTas-interacting proteins. We found that RelB, a member of NF-κB protein family, interacts with BTas. We confirmed the putative RelB-BTas interaction in vitro and in vivo and identified the protein regions responsible for the RelB-BTas interaction. Using a luciferase reporter assay, we next showed that RelB enhances BFV transcription (BTas-induced long terminal repeat [LTR] transactivation) and that this process requires both the localization of the RelB-BTas interaction in the nucleus and the Rel homology domain of RelB. The knockdown of the cellular endogenous RelB protein using small interfering RNA (siRNA) significantly attenuated BTas-induced LTR transcription. The results of chromatin immunoprecipitation (ChIP) analysis showed that endogenous RelB binds to the viral LTR in BFV-infected cells. Together, these results suggest that BFV engages the RelB protein as a cotransactivator of BTas to enhance viral transcription. In addition, our findings indicate that BFV infection upregulates cellular RelB expression through BTas-induced NF-κB activation. Thus, this study demonstrates the existence of a positive-feedback circuit in which BFV utilizes the host's NF-κB pathway through the RelB protein for efficient viral transcription.

scholarly journals Long non-coding RNA TUG1 promotes cell progression in hepatocellular carcinoma via regulating miR-216b-5p/DLX2 axis

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Qun Dai ◽  
Jingyi Deng ◽  
Jinrong Zhou ◽  
Zhuhong Wang ◽  
Xiao-feng Yuan ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cancer Progression ◽  
Noncoding Rna ◽  
Critical Role ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Marked Rise ◽  
Hcc Cells

Abstract Background Accumulating evidence indicates that the long noncoding RNA taurine upregulated gene 1(TUG1) plays a critical role in cancer progression and metastasis. However, the overall biological role and clinical significance of TUG1 in hepatocellular carcinoma (HCC) remain largely unknown. Methods The expressions of TUG1, microRNA-216b-5p and distal-less homeobox 2 (DLX2) were detected by Quantitative real-time polymerase chain reaction (qRT-PCR). The target relationships were predicted by StarBase v.2.0 or TargetScan and confirmed by dual-luciferase reporter assay. The cell growth, apoptosis, migration and invasion were detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Flow cytometry and Transwell assays, respectively. All protein expression levels were detected by western blot. Tumor xenografts were implemented to explore the role of TUG1 in vivo. Results We found that there was a marked rise in TUG1 expression in HCC tissues and cells, and knockdown of TUG1 repressed the growth and metastasis and promoted apoptosis of HCC cells. In particular, TUG1 could act as a ceRNA, effectively becoming a sink for miR-216b-5p to fortify the expression of DLX2. Additionally, repression of TUG1 impared the progression of HCC cells by inhibiting DLX2 expression via sponging miR-216b-5p in vitro. More importantly, TUG1 knockdown inhibited HCC tumor growth in vivo through upregulating miR-216b-5p via inactivation of the DLX2. Conclusion TUG1 interacting with miR-216b-5p contributed to proliferation, metastasis, tumorigenesis and retarded apoptosis by activation of DLX2 in HCC.

scholarly journals Function of Native OmtA In Vivo and Expression and Distribution of This Protein in Colonies of Aspergillus parasiticus

2002 ◽  
Vol 68 (11) ◽  
pp. 5718-5727 ◽  
Author(s):  
Li-Wei Lee ◽  
Ching-Hsun Chiou ◽  
John E. Linz
Keyword(s):  
Fusion Protein ◽  
Aspergillus Parasiticus ◽  
Polyclonal Antibodies ◽  
Critical Role ◽  
Immunohistochemical Analysis ◽  
Cell Types ◽  
Temporal Association ◽  
Thin Sections

ABSTRACT The activities of two enzymes, a 168-kDa protein and a 40-kDa protein, OmtA, purified from the filamentous fungus Aspergillus parasiticus were reported to convert the aflatoxin pathway intermediate sterigmatocystin to O-methylsterigmatocystin in vitro. Our initial goal was to determine if OmtA is necessary and sufficient to catalyze this reaction in vivo and if this reaction is necessary for aflatoxin synthesis. We generated A. parasiticus omtA-null mutant LW1432 and a maltose binding protein-OmtA fusion protein expressed in Escherichia coli. Enzyme activity analysis of OmtA fusion protein in vitro confirmed the reported catalytic function of OmtA. Feeding studies conducted with LW1432 demonstrated a critical role for OmtA, and the reaction catalyzed by this enzyme in aflatoxin synthesis in vivo. Because of a close regulatory link between aflatoxin synthesis and asexual sporulation (conidiation), we hypothesized a spatial and temporal association between OmtA expression and conidiospore development. We developed a novel time-dependent colony fractionation protocol to analyze the accumulation and distribution of OmtA in fungal colonies grown on a solid medium that supports both toxin synthesis and conidiation. OmtA-specific polyclonal antibodies were purified by affinity chromatography using an LW1432 protein extract. OmtA was not detected in 24-h-old colonies but was detected in 48-h-old colonies using Western blot analysis; the protein accumulated in all fractions of a 72-h-old colony, including cells (0 to 24 h) in which little conidiophore development was observed. OmtA in older fractions of the colony (24 to 72 h) was partly degraded. Fluorescence-based immunohistochemical analysis conducted on thin sections of paraffin-embedded fungal cells from time-fractionated fungal colonies demonstrated that OmtA is evenly distributed among different cell types and is not concentrated in conidiophores. These data suggest that OmtA is present in newly formed fungal tissue and then is proteolytically cleaved as cells in that section of the colony age.

scholarly journals MicroRNA-218 Negatively Regulates Osteoclastogenic Differentiation by Repressing the Nuclear Factor-κB Signaling Pathway and Targeting Tumor Necrosis Factor Receptor 1

2018 ◽  
Vol 48 (1) ◽  
pp. 339-347 ◽  
Author(s):  
Weiwei Wang ◽  
Lei Yang ◽  
Dan Zhang ◽  
Chao Gao ◽  
Jie Wu ◽  
...  
Keyword(s):  
Bioinformatics Analysis ◽  
Tumor Necrosis Factor Receptor ◽  
Nuclear Factor Κb ◽  
Raw 264.7 Cells ◽  
Luciferase Reporter ◽  
Raw 264.7 ◽  
Nuclear Factor ◽  
Reporter Assay ◽  
Trap Staining ◽  
Necrosis Factor

Background/Aims: Postmenopausal osteoporosis is a common disease associated with estrogen deficiency leading to bone loss and bone tissue changes. The resultant bone fragility and increased risk of fracture has serious adverse effects on health and quality of life of the elderly, making it an important health issue. MicroRNA-218 (miR-218) is closely related to the development of osteoporosis. In this study, we investigated the regulatory mechanisms of miR-218 in osteoclastogenesis. Methods: We investigated miR-218 levels on differentiation of RAW 264.7 cells into osteoclasts compared with normal cells. Next, RAW 264.7 cells were transfected with miR-218 mimics or inhibitors to study the role of miR-218 in osteoclastogenic differentiation. Tartrate-resistant acid phosphatase (TRAP) staining was performed to determine osteoclastogenic differentiation. Bioinformatics analysis and luciferase reporter assay were used to identify and validate miR-218 target genes. Results: miR-218 was downregulated following RAW 264.7 cell differentiation into osteoclasts. miR-218 overexpression attenuated osteoclast differentiation, whereas low miR-218 expression promoted it as demonstrated by increased expression of osteoclast-specific genes and TRAP staining. Bioinformatics analysis and the luciferase reporter assay showed that tumor necrosis factor receptor 1 (TNFR1), a cell membrane receptor of TNF (TNF is an activator of nuclear factor-κB [NF-κB]), is a direct target of miR-218. Conclusions: Our findings indicate that miR-218 regulates osteoclastogenic differentiation negatively by repressing NF-κB signaling by targeting TNFR1, suggesting that targeting miR-218 may be a therapeutic approach in postmenopausal osteoporosis.

scholarly journals Transcriptional co-operativity between distant retinoic acid response elements in regulation of Cyp26A1 inducibility

2005 ◽  
Vol 392 (1) ◽  
pp. 241-248 ◽  
Author(s):  
Olivier Loudig ◽  
Glenn A. Maclean ◽  
Naomi L. Dore ◽  
Luong Luu ◽  
Martin Petkovich
Keyword(s):  
Retinoic Acid ◽  
Mutational Analysis ◽  
Transcriptional Start Site ◽  
Critical Role ◽  
Transcriptional Response ◽  
Luciferase Reporter ◽  
Maximal Response ◽  
Fold Induction

Cyp26A1 encodes an RA (retinoic acid)-catabolizing CYP (cytochrome P450) protein that plays a critical role in regulating RA distribution in vivo. Cyp26A1 expression is inducible by RA, and the locus has previously been shown to contain a RARE (RA response element), R1, within the minimal promoter [Loudig, Babichuk, White, Abu-Abed, Mueller and Petkovich (2000) Mol. Endocrinol. 14, 1483–1497]. In the present study, we report the identification of a second functional RARE (R2) located 2.0 kb upstream of the Cyp26A1 transcriptional start site. Constructs containing murine sequences encompassing both R1 and R2 showed that these elements work together to generate higher transcriptional activity upon treatment with RA than those containing R1 alone. Inclusion of R2 also dramatically enhanced the sensitivity of reporter constructs to RA, as even treatment with 10−8 M RA resulted in a 5-fold induction of reporter activity. Mutational analysis identified R2 as the functional element responsible for the increased RA inducibility of promoter constructs. The element was shown to bind RARγ (RA receptor γ)/RXRα (retinoid X receptor α) heterodimers in vitro, and inclusion of nuclear receptors in transfections boosted the transcriptional response. A construct containing both R1 and R2 was used to generate a stable luciferase reporter cell line that can be used as a tool to identify factors regulating Cyp26A1 expression. The analysis of R1 and R2 has led to the proposal that the two elements work synergistically to provide a maximal response to RA and that R2 is an upstream enhancer.

Mechanism by which H-2g, a glucose analog of blood group H antigen, mediates angiogenesis

Blood ◽  
2005 ◽  
Vol 105 (6) ◽  
pp. 2343-2349 ◽  
Author(s):  
Kui Zhu ◽  
Mohammed Asif Amin ◽  
Yuanyuan Zha ◽  
Lisa A. Harlow ◽  
Alisa E. Koch
Keyword(s):  
Growth Factor ◽  
Critical Role ◽  
Aortic Ring ◽  
Nuclear Factor Κb ◽  
Janus Kinase ◽  
Intercellular Adhesion Molecule ◽  
Soluble Form ◽  
Intercellular Adhesion Molecule 1

AbstractThe 4A11 antigen is a unique cytokine-inducible antigen up-regulated on rheumatoid arthritis (RA) synovial endothelial cells (ECs) compared with normal ECs. Previously, we showed that in soluble form, this antigen, Lewisy-6/H-5-2 (Ley/H) or its glucose analog, 2-fucosyl lactose (H-2g), induced the expression of EC intercellular adhesion molecule-1 (ICAM-1) and leukocyte-endothelial adhesion through the Janus kinase 2 (JAK2)–signal transducer and activator of transcription 3 (STAT3) pathway. Currently, we show that H-2g induces release of EC angiogenic basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), an effect inhibited by decoy nuclear factor κB (NFκB) oligodeoxynucleotide (ODN). JAK2 and phosphoinositide-3 kinase (PI3K) are 2 upstream kinases of NFκB activated by H-2g, as confirmed by an inhibitor of kappa B kinase (IKKβ) assay. In vitro, H-2g induces vascular sprouting in the rat aortic ring model, whereas blockade of JAK2, PI3K, or NFκB inhibits sprouting. Likewise, in the in vivo mouse Matrigel plug angiogenesis assay, chemical inhibitors and antisense or decoy ODNs of JAK2, PI3K, or NFκB decrease angiogenesis, confirming the importance of these pathways in H-2g–induced EC signaling. The critical role of Ley/H involvement in angiogenesis and its signaling pathways may provide new targets for therapy of diseases characterized by pathologic neovascularization.

scholarly journals SAG/ROC2 E3 ligase regulates skin carcinogenesis by stage-dependent targeting of c-Jun/AP1 and IκB-α/NF-κB

2007 ◽  
Vol 178 (6) ◽  
pp. 1009-1023 ◽  
Author(s):  
Qingyang Gu ◽  
G. Tim Bowden ◽  
Daniel Normolle ◽  
Yi Sun
Keyword(s):  
Early Stage ◽  
Nuclear Factor Κb ◽  
Tumor Formation ◽  
Luciferase Reporter ◽  
Activator Protein 1 ◽  
Cellular Functions ◽  
Accelerated Proliferation ◽  
F Box Protein

Sensitive to apoptosis gene (SAG)/regulator of cullins-2–Skp1-cullin–F-box protein (SCF) E3 ubiquitin ligase regulates cellular functions through ubiquitination and degradation of protein substrates. We report that, when expressed in mouse epidermis driven by the K14 promoter, SAG inhibited TPA-induced c-Jun levels and activator protein-1 (AP-1) activity in both in vitro primary culture, in vivo transgenic mice, and an AP-1– luciferase reporter mouse model. After AP-1 inactivation, epidermal proliferation induced by 7,12-dimethylbenz(a)-anthracene/12-O-tetradecanoylphorbol-13-acetate at the early stage of carcinogenesis was substantially inhibited. Later stage tumor formation was also substantially inhibited with prolonged latency and reduced frequency of tumor formation. Interestingly, SAG expression increased tumor size, not because of accelerated proliferation, but caused by reduced apoptosis resulting, at least in part, from nuclear factor κB (NF-κB) activation. Thus, SAG, in a manner depending on the availability of F-box proteins, demonstrated early-stage suppression of tumor formation by promoting c-Jun degradation, thereby inhibiting AP-1, and later-stage enhancement of tumor growth, by promoting inhibitor of κBα degradation to activate NF-κB and inhibit apoptosis.

scholarly journals Flow-dependent YAP/TAZ activities regulate endothelial phenotypes and atherosclerosis

2016 ◽  
Vol 113 (41) ◽  
pp. 11525-11530 ◽  
Author(s):  
Kuei-Chun Wang ◽  
Yi-Ting Yeh ◽  
Phu Nguyen ◽  
Elaine Limqueco ◽  
Jocelyn Lopez ◽  
...  
Keyword(s):  
Target Genes ◽  
Critical Role ◽  
Atherosclerotic Lesion ◽  
Tissue Growth ◽  
Binding Motif ◽  
Disturbed Flow ◽  
Atherosclerotic Lesions ◽  
En Face

The focal nature of atherosclerotic lesions suggests an important role of local hemodynamic environment. Recent studies have demonstrated significant roles of Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in mediating mechanotransduction and vascular homeostasis. The objective of this study is to investigate the functional role of YAP/TAZ in the flow regulation of atheroprone endothelial phenotypes and the consequential development of atherosclerotic lesions. We found that exposure of cultured endothelial cells (ECs) to the atheroprone disturbed flow resulted in YAP/TAZ activation and translocation into EC nucleus to up-regulate the target genes, including cysteine-rich angiogenic inducer 61 (CYR61), connective tissue growth factor (CTGF), and ankyrin repeat domain 1 (ANKRD1). In contrast, the athero-protective laminar flow suppressed YAP/TAZ activities. En face analysis of mouse arteries demonstrated an increased nuclear localization of YAP/TAZ and elevated levels of the target genes in the endothelium in atheroprone areas compared with athero-protective areas. YAP/TAZ knockdown significantly attenuated the disturbed flow induction of EC proliferative and proinflammatory phenotypes, whereas overexpression of constitutively active YAP was sufficient to promote EC proliferation and inflammation. In addition, treatment with statin, an antiatherosclerotic drug, inhibited YAP/TAZ activities to diminish the disturbed flow-induced proliferation and inflammation. In vivo blockade of YAP/TAZ translation by morpholino oligos significantly reduced endothelial inflammation and the size of atherosclerotic lesions. Our results demonstrate a critical role of the activation of YAP/TAZ by disturbed flow in promoting atheroprone phenotypes and atherosclerotic lesion development. Therefore, inhibition of YAP/TAZ activation is a promising athero-protective therapeutic strategy.

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