Tumor Necrosis Factor-alpha Treatment Research Articles

Herpes Simplex Virus 1-Encoded Tegument Protein VP16 Abrogates the Production of Beta Interferon (IFN) by Inhibiting NF-κB Activation and Blocking IFN Regulatory Factor 3 To Recruit Its Coactivator CBP

Host cells activate innate immune signaling pathways to defend against invading pathogens. To survive within an infected host, viruses have evolved intricate strategies to counteract host immune responses. Herpesviruses, including herpes simplex virus type 1 (HSV-1), have large genomes and therefore have the capacity to encode numerous proteins that modulate host innate immune responses. Here we define the contribution of HSV-1 tegument protein VP16 in the inhibition of beta interferon (IFN-β) production. VP16 was demonstrated to significantly inhibit Sendai virus (SeV)-induced IFN-β production, and its transcriptional activation domain was not responsible for this inhibition activity. Additionally, VP16 blocked the activation of the NF-κB promoter induced by SeV or tumor necrosis factor alpha treatment and expression of NF-κB-dependent genes through interaction with p65. Coexpression analysis revealed that VP16 selectively blocked IFN regulatory factor 3 (IRF-3)-mediated but not IRF-7-mediated transactivation. VP16 was able to bind to IRF-3 but not IRF-7 in vivo, based on coimmunoprecipitation analysis, but it did not affect IRF-3 dimerization, nuclear translocation, or DNA binding activity. Rather, VP16 interacted with the CREB binding protein (CBP) coactivator and efficiently inhibited the formation of the transcriptional complexes IRF-3-CBP in the context of HSV-1 infection. These results illustrate that VP16 is able to block the production of IFN-β by inhibiting NF-κB activation and interfering with IRF-3 to recruit its coactivator CBP, which may be important to the early events leading to HSV-1 infection.

TNF-alpha and melphalan modulate a specific group of early expressed genes in a murine melanoma model

BackgroundCutaneous melanoma displays high morbidity and mortality rates. Isolated limb perfusion with melphalan (Mel) is used for the treatment of non-resectable, locally advanced extremity melanomas. When combined with tumor necrosis factor alpha (TNF-alpha) treatment, the complete response varies between 70% and 90%. The mechanisms underlying the effects of Mel and TNF-alpha are not completely understood. We evaluated the impact of systemic Mel and TNF-alpha administration on tumor growth, analyzed the morphological changes promoted by each treatment, and identified early expressed genes in response to Mel and TNF-alpha treatment, either alone or in combination, in a murine melanoma model. MethodsSix- to eight-week-old male mice were subcutaneously inoculated with B16F10 melanoma cells and then intravenously injected with TNF-alpha, melphalan or a combination of both drugs when the tumors reached 1.0cm2. Tumor growth was monitored every other day, and histological analysis was performed when the tumors reached 3.0cm2. Total RNA was extracted from the resected tumors and submitted to amplification, labeling and hybridization on an oligonucleotide microarray (Fox Chase Cancer Center). Tumor growth and histological parameters were compared using ANOVA. Survival curves were calculated using the Kaplan–Meier method. Two-way ANOVA was used to identify differentially expressed genes among the various treatments, and Dunn’s test was used for pair-wise comparisons. ResultsSystemic administration of Mel impaired tumor growth (p<0.001), improved animal survival (p<0.001), and decreased mitotic rate (p=0.049). Treatment with TNF-alpha alone had no impact, neither on tumor growth, nor on survival, but it increased necrosis (p<0.024) and decreased mitotic rates (p=0.001) in the tumors. Combined treatment with Mel and TNF-alpha had similar effects in tumor growth, survival, necrosis and mitotic rate as observed with individual treatments. Moreover, 118 genes were found differentially expressed by microarray analysis and 10% of them were validated by RT- real time PCR. In our model we found that the treatments regulate genes that play important roles in tumorigenesis such as cell adhesion (Pard3, Pecam1, Ilk, and Dlg5), proliferation (Tcfe3 and Polr1e), cell motility (Kifap3, Palld, and Arhgef6), apoptosis (Bcl2l11), and angiogenesis (Flt1and Ptprj). ConclusionsOur data reproduces, in mice, some of the features observed in melanoma patients treated with the combination of Mel and TNF-alpha. The identification of genes with altered expression by these drugs both individually and in combination might help in the understanding of their mechanism of action and, as a consequence, improved strategies that could impact their clinical application.

Critical Roles of Glucocorticoid-Induced Leucine Zipper in Infectious Bursal Disease Virus (IBDV)-Induced Suppression of Type I Interferon Expression and Enhancement of IBDV Growth in Host Cells via Interaction with VP4

Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive avian disease caused by IBD virus (IBDV). Although IBDV-induced immunosuppression has been well established, the underlying exact molecular mechanism for such induction is not very clear. We report here the identification of IBDV VP4 as an interferon suppressor by interaction with the glucocorticoid-induced leucine zipper (GILZ) in host cells. We found that VP4 suppressed the expression of type I interferon in HEK293T cells after tumor necrosis factor alpha (TNF-α) treatment or Sendai virus (SeV) infection and in DF-1 cells after poly(I·C) stimulation. In addition, the VP4-induced suppression of type I interferon could be completely abolished by knockdown of GILZ by small interfering RNA (siRNA). Furthermore, knockdown of GILZ significantly inhibited IBDV growth in host cells, and this inhibition could be markedly mitigated by anti-alpha/beta interferon antibodies in the cell cultures (P < 0.001). Thus, VP4-induced suppression of type I interferon is mediated by interaction with GILZ, a protein that appears to inhibit cell responses to viral infection.

Annexin A2 Modulates Radiation-Sensitive Transcriptional Programming and Cell Fate

We previously established annexin A2 as a radioresponsive protein associated with anchorage independent growth in murine epidermal cells. In this study, we demonstrate annexin A2 nuclear translocation in human skin organotypic culture and murine epidermal cells after exposure to X radiation (10-200 cGy), supporting a conserved nuclear function for annexin A2. Whole genome expression profiling in the presence and absence of annexin A2 [shRNA] identified fundamentally altered transcriptional programming that changes the radioresponsive transcriptome. Bioinformatics predicted that silencing AnxA2 may enhance cell death responses to stress in association with reduced activation of pro-survival signals such as nuclear factor kappa B. This prediction was validated by demonstrating a significant increase in sensitivity toward tumor necrosis factor alpha-induced cell death in annexin A2 silenced cells, relative to vector controls, associated with reduced nuclear translocation of RelA (p65) following tumor necrosis factor alpha treatment. These observations implicate an annexin A2 niche in cell fate regulation such that AnxA2 protects cells from radiation-induced apoptosis to maintain cellular homeostasis at low-dose radiation.

TNF-α influences the lateral dynamics of TNF receptor I in living cells

In mammalian cells, inflammation is mainly mediated by the binding of tumor necrosis factor alpha to tumor necrosis factor receptor 1. In this study, we investigated lateral dynamics of TNF-R1 before and after ligand binding using high-density single-particle tracking in combination with photoactivated localization microscopy. Our single-molecule data indicates the presence of tumor necrosis factor receptor 1 with different mobilities in the plasma membrane, suggesting different molecular organizations. Cholesterol depletion led to a decrease of slow receptor species and a strong increase in the average diffusion coefficient. Moreover, as a consequence of tumor necrosis factor-alpha treatment, the mean diffusion coefficient moderately increased while its distribution narrowed. Based on our observation, we propose a refined mechanism on the structural arrangement and activation of tumor necrosis factor receptor 1 in the plasma membrane.

Profound Effect of Profiling Platform and Normalization Strategy on Detection of Differentially Expressed MicroRNAs – A Comparative Study

BackgroundAdequate normalization minimizes the effects of systematic technical variations and is a prerequisite for getting meaningful biological changes. However, there is inconsistency about miRNA normalization performances and recommendations. Thus, we investigated the impact of seven different normalization methods (reference gene index, global geometric mean, quantile, invariant selection, loess, loessM, and generalized procrustes analysis) on intra- and inter-platform performance of two distinct and commonly used miRNA profiling platforms.Methodology/Principal FindingsWe included data from miRNA profiling analyses derived from a hybridization-based platform (Agilent Technologies) and an RT-qPCR platform (Applied Biosystems). Furthermore, we validated a subset of miRNAs by individual RT-qPCR assays. Our analyses incorporated data from the effect of differentiation and tumor necrosis factor alpha treatment on primary human skeletal muscle cells and a murine skeletal muscle cell line. Distinct normalization methods differed in their impact on (i) standard deviations, (ii) the area under the receiver operating characteristic (ROC) curve, (iii) the similarity of differential expression. Loess, loessM, and quantile analysis were most effective in minimizing standard deviations on the Agilent and TLDA platform. Moreover, loess, loessM, invariant selection and generalized procrustes analysis increased the area under the ROC curve, a measure for the statistical performance of a test. The Jaccard index revealed that inter-platform concordance of differential expression tended to be increased by loess, loessM, quantile, and GPA normalization of AGL and TLDA data as well as RGI normalization of TLDA data.Conclusions/SignificanceWe recommend the application of loess, or loessM, and GPA normalization for miRNA Agilent arrays and qPCR cards as these normalization approaches showed to (i) effectively reduce standard deviations, (ii) increase sensitivity and accuracy of differential miRNA expression detection as well as (iii) increase inter-platform concordance. Results showed the successful adoption of loessM and generalized procrustes analysis to one-color miRNA profiling experiments.

Profound effect of profiling platform and normalization strategy on detection of differentially expressed microRNAs

Adequate normalization minimizes the effects of systematic technical variations and is a prerequisite for getting meaningful biological changes. However, there is inconsistency about miRNA normalization performances and recommendations. Thus, we investigated the impact of seven different normalization methods (reference gene index, global geometric mean, quantile, invariant selection, loess, loessM, and generalized procrustes analysis) on intra- and inter-platform performance of two distinct and commonly used miRNA profiling platforms. We included data from miRNA profiling analyses derived from a hybridization-based platform (Agilent Technologies) and an RT-qPCR platform (Applied Biosystems). Furthermore, we validated a subset of miRNAs by individual RT-qPCR assays. Our analyses incorporated data from the effect of differentiation and tumor necrosis factor alpha treatment on primary human skeletal muscle cells and a murine skeletal muscle cell line. Distinct normalization methods differed in their impact on (i) standard deviations, (ii) the area under the receiver operating characteristic (ROC) curve, (iii) the similarity of differential expression. Loess, loessM, and quantile analysis were most effective in minimizing standard deviations on the Agilent and TLDA platform. Moreover, loess, loessM, invariant selection and generalized procrustes analysis increased the area under the ROC curve, a measure for the statistical performance of a test. The Jaccard index revealed that inter-platform concordance of differential expression tended to be increased by loess, loessM, quantile, and GPA normalization of AGL and TLDA data as well as RGI normalization of TLDA data. We recommend the application of loess, or loessM, and GPA normalization for miRNA Agilent arrays and qPCR cards as these normalization approaches showed to (i) effectively reduce standard deviations, (ii) increase sensitivity and accuracy of differential miRNA expression detection as well as (iii) increase inter-platform concordance. Results showed the successful adoption of loessM and generalized procrustes analysis to one-color miRNA profiling experiments.

Fulminant liver failure models with subsequent encephalopathy in the mouse

A reliable model of fulminant liver failure (FLF) is urgently required in this research field. This study aimed to develop a murine FLF model. We used three groups of male C57BL/6 mice: control, with azoxymethane treatment (AOM group), and with galactosamine and tumor necrosis factor-alpha treatment (Gal+TNF-alpha group). The effects of body temperature (BT) control on survival in all three groups were investigated. Using BT control, we compared the survival, histopathological findings and biochemical/coagulation profiles between the two experimental groups. The effects of hydration on international normalized ratios of prothrombin time (PT-INRs) were also checked. Dose-dependent survival curves were constructed for both experimental groups. Neurological behavior was assessed using a coma scale. No unexpected BT effects were seen in the control group. The AOM group, but not the Gal+TNF-alpha group, showed a significant difference in survival curves between those with and without BT care. Histopathological assessment showed consistent FLF findings in both experimental groups with BT care. There were significant differences between the experimental groups in aspartate aminotransferase levels and PT-INRs, and significant differences in PT-INRs between the sufficiently and insufficiently hydrated groups. There were significant differences between FLF models in the duration of each coma stage, with significant differences in stages 1 and 3 as percentages of the disease state (stages 1-4). The two FLF models with BT care showed different survival curves in the dose-dependent survival study. AOM provides a good FLF model, but requires a specialized environment and careful BT control. Other FLF models may also be useful, depending on the research purpose. Thoughtful attention to caregiving and close observation are indispensable for successful FLF models.

Hepatotoxicity mediated by pyrazole (cytochrome P450 2E1) plus tumor necrosis factor alpha treatment occurs in c-Jun N-terminal kinase 2−/− but not in c-Jun N-terminal kinase 1 −/− mice

Cytochrome P450 2E1 (CYP2E1) induction and tumor necrosis factor alpha (TNF-α) production are key risk factors in alcoholic liver injury. Increased oxidative stress from CYP2E1 induction by pyrazole in vivo sensitizes the liver to TNF-α-induced hepatotoxicity by a mechanism involving the activation of c-jun N-terminal kinase (JNK) and mitochondrial damage. The aim of this study was to evaluate whether JNK1 or JNK2 plays a role in this potentiated hepatotoxicity. Wild-type (WT), jnk1(-/-) , and jnk2(-/-) mice were used to identify changes of hepatotoxicity, damage to mitochondria, and production of oxidative stress after pyrazole plus TNF-α treatment. Increased serum alanine aminotransferase, inflammatory infiltration, and central necrosis were observed in the jnk2(-/-) and WT mice treated with pyrazole plus TNF-α, but not in the jnk1(-/-) mice. Pyrazole elevated the activity and protein level of CYP2E1 in all mice. There was a significant increase of malondialdehyde, 4-hydroxynonenal adducts, 3-nitrotyrosine, and inducible nitric oxide synthase in the jnk2(-/-) and WT mice, compared to the jnk1(-/-) mice, upon pyrazole plus TNF-α treatment, or compared to mice treated with either pyrazole alone or TNF-α alone. The antioxidants, catalase, phospholipid hydroperoxide glutathione peroxidase, thioredoxin, and glutathione were lowered, and cytochrome c was released from the mitochondria in the jnk2(-/-) and WT mice. Mitochondrial production of superoxide was increased in the jnk2(-/-) and WT mice, compared to the jnk1(-/-) mice, upon pyrazole plus TNF-α treatment. Electron microscopy showed altered mitochondrial structure in the jnk2(-/-) and WT mice, but not the jnk1(-/-) mice. JNK1 plays a role in the hepatotoxicity, mitochondrial dysfunction, and oxidative stress mediated by pyrazole plus TNF-α treatment. These findings raise the question as to the potential mechanisms of JNK1 activation related to alcoholic liver injury.

Dietary apigenin regulates high glucose and hypoxic reoxygenation-induced reductions in apelin expression in human endothelial cells

The early stages of vascular endothelial dysfunction enhance angiogenic stimulation and strongly influence vascular rearrangement. The aim of this study was to determine whether a short period of high glucose (HG, 30 mM glucose) plus tumor necrosis factor alpha (TNFα) treatment or reoxygenation after hypoxia (H/R) alters the expression levels of apelin in human endothelial cells. In addition, we also examined the effects of the dietary flavonoid apigenin on apelin expression. Human endothelial cell lines were treated with HG plus TNFα or subjected to H/R. The expression levels of genes and proteins were then assessed by the reverse transcriptase polymerase chain reaction, Western blotting and immunofluorescence analyses. The expression level of apelin was significantly higher in the HG group following exposure to reoxygenation or TNFα. Reoxygenation after hypoxia decreased the expression levels of apelin and fatty acid transport protein (FATP) 1 compared with those observed during hypoxia alone and normoxia in a normal glucose concentration. Inversely, apigenin augmented H/R-reduced apelin and FATP1 expression in endothelial cells. Based on our findings, we propose that the early stages of endothelial disorder subtly influence angiogenesis and that HG and H/R stimulate vascular rearrangement and are involved in fatty acid uptake. Furthermore, dietary apigenin might improve the expression of angiogenic genes and fatty acid uptake.

Dexamethasone treatment of tumor necrosis factor-alpha challenged organ of Corti explants activates nuclear factor kappa B signaling that induces changes in gene expression that favor hair cell survival

The objective was to determine the role of nuclear factor kappa B (NFκB) in dexamethasone base (DXMb) protection of auditory hair cells from tumor necrosis factor-alpha (TNFα)-induced loss on gene expression and cell signaling levels. Organ of Corti (OC) explants from 3-day-old rats were cultured under one of the following conditions: (1) media only--no treatment; (2) media+TNFα; (3) media+TNFα+DXMb; (4) media+TNFα+DXMb+NFκB-Inhibitor (NFκB-I); or (5) media+TNFα+DXMb+NFκBI-Scrambled control (NFκBI-C). A total of 60 organ of Corti explants (OC) were stained with FITC-Phalloidin after 96 h in culture (conditions 1-5) for hair cell counts and imaging of surface characteristics. A total of 108 OC were used for gene expression studies (i.e. B-actin, Bax, Bcl-2, Bcl-xl, and TNFR1) after 0, 24, or 48 h in vitro (conditions 1-4). A total of 86 OC were cultured (conditions 1-3) for 48 h, 36 of which were used for phosphorylated NFκB (p-NFκB) ELISA studies and 50 for whole mount anti-p-NFκB immunostain experiments. TNFα+DXMb exposed cultures demonstrated significant upregulation in anti-apoptotic Bcl-2 and Bcl-xl genes and downregulation in pro-apoptotic Bax gene expression; DXMb treatment of TNFα explants also lowered the Bax/Bcl-2 ratio and inhibited TNFR1 upregulation. After inhibiting NFκB activity with NFκB-I, the gene expression profile following TNFα+DXMb treatment now mimics that of TNFα-challenged OC explants. The levels of p-NFκB and the degree of nuclear translocation are significantly greater in TNFα+DXMb exposed OC explants than observed in the TNFα and control groups in the middle+basal turns of OC explants. These findings were supported by the results of the hair cell counts and the imaging results obtained from the whole mount OC specimens. DXMb protects against TNFα-induced apoptosis of auditory hair cells in vitro via activation of NFκB signaling in hair cell nuclei, and regulation of the expression levels of anti- and pro-apoptotic genes and a pro-inflammatory gene.

Activating transcription factor‐2 in survival mechanisms in head and neck carcinoma cells

Activating transcription factor-2 (ATF2) is associated with tumor progression but is not well studied in head and neck squamous cell carcinoma (HNSCC). Its effects in stress and its importance in other survival mechanisms were studied. ATF2 expression and nuclear activation were confirmed in HNSCC. After modulation of ATF2, in vitro effects on proliferation and chemosensitivity were studied. Effects on in vivo tumor growth and interleukin 8 (IL-8) expression were determined. Tumor necrosis factor-alpha (TNF-α) treatment was used to further evaluate cytokine production and chemosensitivity. Reductions of ATF2 resulted in significant nuclear p-ATF2 activation, cisplatin resistance, and augmented IL-8 expression without affecting in vivo tumor growth. In this setting, TNF increases p-p38 phosphorylation and chemosensitivity while further enhancing IL-8 production. Our data suggest regulatory roles for ATF2 in TNF-related mechanisms of HNSCC. Its perturbation and nuclear activation are associated with significant effects on survival and cytokine production.

Markers Distinguishing Mesenchymal Stem Cells from Fibroblasts Are Downregulated with Passaging

Expansion of plastic-adherent bone marrow-derived mesenchymal stem cells (MSCs) results in gradual loss of osteogenic potential after passage 5-6. One explanation is contamination of MSC cultures with mature cells including fibroblasts. Identification and elimination of fibroblasts from MSC cultures could improve MSC yield and differentiation potential and also prevent tumor formation after MSC transplantation. However, no specific markers currently exist that can reliably discriminate between MSCs and fibroblasts. Flow cytometry analysis demonstrated that markers currently used to define MSCs, such as CD105, CD166, CD90, CD44, CD29, CD73, and CD9, are also expressed on human skin or lung fibroblasts. However, the level of expression of CD166 was significantly higher and that of CD9 was significantly lower in MSCs than in fibroblasts. CD146 was expressed only in MSCs. Using small focused microarrays, new markers differentially expressed in MSCs and fibroblasts were identified. Real-time polymerase chain reaction confirmed that expression of CD106, integrin alpha 11, and insulin-like growth factor-2 in MSCs was at least 10-fold higher than in fibroblasts; whereas expression of matrix metalloproteinase 1 and matrix metalloproteinase 3 was almost 100-fold lower. Flow cytometry and immunostaining demonstrated that CD106 protein expression on cell surface could be upregulated in MSCs but not in fibroblasts by the treatment with tumor necrosis factor-alpha. Comparison of surface expression of commonly used and newly identified MSC markers in MSCs cultures of passage 2 and passage 6 demonstrated that CD106 (with and without tumor necrosis factor-alpha treatment), integrin alpha 11, and CD146 were downregulated in MSCs of passage 6, and CD9 was upregulated; whereas all other markers did not change. Newly identified markers that have robust differences of expression in MSCs and fibroblasts on gene and protein level could be used for quality control of MSC cultures after expansion, cryopreservation, gene transfection, and other manipulations.

P65 Negatively Regulates Transcription of the Cyclin E Gene

NF-kappaB family members play a pivotal role in many cellular and organismal functions, including the cell cycle. As an activator of cyclin D1 and p21(Waf1) genes, NF-kappaB has been regarded as a critical modulator of cell cycle. To study the involvement of NF-kappaB in G(1)/S phase regulation, the levels of selected transcriptional regulators were monitored following overexpression of NF-kappaB or its physiological induction by tumor necrosis factor-alpha. Cyclin E gene was identified as a major transcriptional target of NF-kappaB. Recruitment of NF-kappaB to the cyclin E promoter was correlated with the transrepression of cyclin E gene. Ligation-mediated PCR and micrococcal nuclease-Southern assays suggested the nucleosomal nature of this region while chromatin immunoprecipitation analysis confirmed the exchange of cofactors following tumor necrosis factor-alpha treatment or release from serum starvation. There was a progressive reduction in cyclin E transcription along with the accumulation of catalytically inactive cyclin E-cdk2 complexes and arrest of cells in G(1)/S-phase. Thus, our study clearly establishes NF-kappaB as a negative regulator of cell cycle through transcriptional repression of cyclin E.

Growth Inhibition and Apoptosis Induction by Tumor Necrosis Factor-α in Human Urethral Rhabdosphincter Satellite Cells

A decrease in the human urethral rhabdosphincter is reported with aging due to apoptosis, which may be a cause of urinary incontinence in the elderly population. To explore this mechanism we investigated the effects of tumor necrosis factor-alpha (Upstate, Temecula, California) on human urethral rhabdosphincter satellite cells. Human urethral rhabdosphincter satellite cells were cultured and selected by magnetic affinity cell sorting, extended their life span. Apoptosis induction was examined by flow cytometry and immunocytochemistry. Caspase cascade activation was determined by Western blot analysis. After tumor necrosis factor receptor expression was confirmed we determined the tumor necrosis factor signaling pathway. Tumor necrosis factor-alpha inhibited human urethral rhabdosphincter satellite cell proliferation. It caused some cells to stain positive for annexin V-fluorescein isothiocyanate but not for propidium iodide, suggesting the induction of early phase apoptosis. Flow cytometry revealed an increased sub-G1 fraction. Western blot analysis showed activation of caspase-8 and 3, and cleavage of poly (adenosine diphosphate-ribose) polymerase. Tumor necrosis factor receptor expression at the mRNA and protein levels was confirmed by reverse transcriptase-polymerase chain reaction and Western blot analysis, respectively. IkappaBalpha phosphorylation was noted within 2 to 5 minutes after tumor necrosis factor-alpha treatment. The tumor necrosis factor-alpha antagonist etanercept (Wyeth, Collegeville, Pennsylvania) inhibited IkappaBalpha activation and reversed tumor necrosis factor-alpha effects on human urethral rhabdosphincter satellite cells. Since tumor necrosis factor-alpha induces growth inhibition and apoptosis of human urethral rhabdosphincter satellite cells via tumor necrosis factor receptor activation, it may be involved in age related decreases in the number of human urethral rhabdosphincter cells and be a causative factor for urinary incontinence in the elderly population.

Human plasminogen kringle 1-5 reduces atherosclerosis and neointima formation in mice by suppressing the inflammatory signaling pathway.

Activation of vascular endothelial cells plays an important role in atherogenesis and plaque instability. Recent research has demonstrated that late-stage inhibition of plaque angiogenesis by angiostatin (kringle 1-4) reduces macrophage accumulation and slows the progression of advanced atherosclerosis. Kringle 1-5 (K(1-5)) is a variant of angiostatin that contains the first five kringle domains of plasminogen. To investigate whether K(1-5) has an inhibitory effect on early-stage atherosclerosis, using the apolipoprotein E (ApoE)-deficient mouse model and a carotid artery ligation model. ApoE-deficient mice received K(1-5) treatment for 4 weeks, and the severity of aortic atherosclerosis was measured. In the ligation model, the left common carotid arteries of C57BL/6 mice were ligated near the carotid bifurcation, and the mice received K(1-5) for 4 weeks. Human umbilical vein endothelial cells were pretreated with K(1-5) before tumor necrosis factor-alpha (TNF-alpha) treatment to explore the anti-inflammatory effect of K(1-5). The areas of the lesion in the aortas of ApoE-deficient mice that received K(1-5) treatment were notably decreased, and the formation of carotid neointima in the C57BL/6 mice was decreased by treatment with K(1-5). Expression of TNF-alpha-induced intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 was inhibited by K(1-5) treatment, possibly via downregulation of translocation of nuclear factor-kappaB and expression of reactive oxygen species. K(1-5) reduced atherosclerosis and neointima formation in mice, possibly through inhibition of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression in endothelial cells.

Redox Regulation of Tumor Necrosis Factor Signaling

Tumor necrosis factor-alpha (TNF) is a key cytokine that has been shown to play important physiologic (e.g., inflammation) and pathophysiologic (e.g., various liver pathologies) roles. In liver and other tissues, TNF treatment results in the simultaneous activation of an apoptotic pathway (i.e., TRADD, RIP, JNK) and a survival pathway mediated by NF-kappaB transcription of survival genes (i.e., GADD45beta, Mn-SOD, cFLIP). The cellular response (e.g., proliferation versus apoptosis) to TNF is determined by the balance between the apoptotic signaling pathway and the NF-kappaB survival pathway stimulated by TNF. Reactive oxygen species (ROS) are important modulators of signaling pathways and can regulate both apoptotic signaling and NF-kappaB transcription triggered by TNF. ROS are important in mediating the sustained activation of JNK, to help mediate apoptosis after TNF treatment. In some cells, ROS are second messengers that mediate apoptosis after TNF stimulation. Conversely, ROS can cause redox modifications that inhibit NF-kappaB activation, which can lead to cell death triggered by TNF. Consequently, the redox status of cells can determine the biologic response that TNF will induce in cells. In many liver pathologies, ROS generated extrinsically (e.g., inflammation) or intrinsically (i.e., drugs, toxins) may act in concert with TNF to promote hepatocyte death and liver injury through redox inhibition of NF-kappaB.

C Terminus of Hsc70-interacting Protein Promotes Smooth Muscle Cell Proliferation and Survival through Ubiquitin-mediated Degradation of FoxO1

Forkhead transcription factors (FoxOs) play a pivotal role in controlling cellular proliferation and survival. The cellular level of these factors is tightly regulated through the phosphoinositide 3-kinase/Akt and ubiquitin-mediated degradation. However, the ubiquitin ligases responsible for the degradation of FoxO1 and the relevance of this regulation to smooth muscle cell (SMC) proliferation and survival have not been fully identified. Here we showed that overexpression of C terminus of Hsc70-interacting protein (CHIP) promoted ubiquitination and degradation of FoxO1 in SMCs in response to tumor necrosis factor-alpha. Both the U-box (containing ubiquitin ligase activity) and the charged (essential for FoxO1 binding) domains within CHIP were required for CHIP-mediated FoxO1 down-regulation. Moreover, interaction and ubiquitination of FoxO1 by CHIP depended on phosphorylation of FoxO1 at Ser-256. Furthermore, overexpression of CHIP repressed FoxO1-mediated transactivation and its proapoptotic function following tumor necrosis factor-alpha treatment. In contrast, knockdown of CHIP by small interfering RNA enhanced FoxO1-mediated transactivation and its effect on SMC proliferation and survival. Taken together, our data indicate that CHIP is a negative regulator of FoxO1 activity through ubiquitin-mediated degradation, and inhibition of CHIP may serve as a potential therapeutic target for reducing proliferative arterial diseases.

Inhibitor of nuclear factor‐kappaB alpha derepresses hypoxia‐inducible factor‐1 during moderate hypoxia by sequestering factor inhibiting hypoxia‐inducible factor from hypoxia‐inducible factor 1α

Hypoxia and inflammation often develop concurrently in numerous diseases, and both hypoxia-inducible factor (HIF)-1alpha and nuclear factor-kappaB (NF-kappaB) are key transcription factors of stress response genes. An NF-kappaB inhibitor, inhibitor of NF-kappaB alpha (IkappaB alpha), was found to interact with factor inhibiting HIF (FIH) and to be hydroxylated by FIH. However, FIH did not functionally regulate IkappaB alpha, and the consequence of the FIH-IkappaB alpha interaction thus remains uncertain. In the present study, we tested the possibility that IkappaB alpha regulates FIH. FIH-IkappaB alpha binding was confirmed by yeast two-hybrid and coimmunoprecipitation analyses. Functionally, IkappaB alpha expression further enhanced the transcriptional activity of HIF-1alpha under hypoxic conditions. Furthermore, IkappaB alpha knockdown repressed HIF-1alpha activity. Mechanistically, IkappaB alpha derepressed HIF-1alpha activity by inhibiting the FIH-mediated Asn803 hydroxylation of HIF-1alpha. It was also found that IkappaB alpha activated HIF-1alpha by sequestering FIH from HIF-1alpha. However, the effect of IkappaB alpha on HIF-1alpha activity was only observed in atmospheres containing 1% or more of oxygen. After tumor necrosis factor-alpha treatment, IkappaB alpha downregulation, Asn803 hydroxylation and HIF-1alpha inactivation all occurred up to 8 h, but subsided later. On the basis of these results, we propose that IkappaB alpha plays a positive regulatory role during HIF-1-mediated gene expression. Therefore, IkappaB alpha, owing to its interactions with NF-kappaB and HIF-1alpha, may play a pivotal role in the crosstalk between the molecular events that underlie inflammatory and hypoxic responses.

Influence of F protein of hepatitis C virus subtype 1b inhibits on human hepatocellular carcinoma HepG2 cell apoptosis

To investigate the effects of F protein of hepatitis C virus subtype 1b on the apoptosis of human hepatocellular carcinoma HepG2 cells. HepG2 cells were transfected with recombinant plasmid pcDNA3.0-F-EGFP and pcDNA3.0-F-EGFP-HepG2 strain was exposed to Act-D and tumor necrosis factor alpha (TNF alpha) treatment in order to induce cell apoptosis with positive control pcDNA3.0-C-EGFP-HepG2, negative control pcDNA3.0-C-EGFP-HepG2 and blank control HepG2. Annexin V-FITC/PI of flow cytometry was performed to determine the number of apoptotic cells. DNA Ladder was used to observe the isolation of apoptotic DNA fragments in the apoptotic cells. pcDNA3.0-F-EGFP- HepG2 cell strain showed a much delayed apoptosis as well as obviously lowering the apoptotic rate when compared with the pcDNA3.0-HepG2 strain and HepG2 strain (P < 0.001). The introduction and expression of extraneous gene (the F gene of hepatitis C virus subtype 1b) could significantly inhibit the apoptosis of HepG2 cells.