Resveratrol attenuates CXCL11 expression induced by proinflammatory cytokines in retinal pigment epithelial cells

Dysfunction of the retinal pigment epithelium (RPE) resulting from chronic inflammation is implicated in the pathogenesis of age-related macular degeneration (AMD). RPE cells adjacent to drusen deposits in the AMD eye are known to contain CXCL11, a chemokine involved in inflammatory cell recruitment. We investigated the CXCL11 production by the human RPE (ARPE-19) cells under inflammatory conditions and tested its response to resveratrol, a naturally occurring anti-inflammatory antioxidant. A proinflammatory cytokine mixture consisting of IFN-γ, IL-1β and TNF-α highly increased CXCL11 mRNA expression and CXCL11 protein secretion by ARPE-19 cells. Resveratrol substantially inhibited the proinflammatory cytokines-induced CXCL11 production while partially blocking nuclear factor-κB activation. This inhibitory action of resveratrol was also observed for the cytokines-induced expression of chemokines CXCL9, CCL2 and CCL5. Our results indicate that resveratrol could potentially attenuate RPE inflammatory response implicated in the pathogenesis of AMD.     

TRAF2 Suppresses Basal IKK Activity in Resting Cells and TNFα Can Activate IKK in TRAF2 and TRAF5 Double Knockout Cells

Tumor necrosis factor receptor (TNFR)-associated factor 2 (TRAF2) and TRAF5 are adapter proteins involved in TNFα-induced activation of the c-Jun N-terminal kinase and nuclear factor κB (NF-κB) pathways. Currently, TNFα-induced NF-κB activation is believed to be impaired in TRAF2 and TRAF5 double knockout (T2/5 DKO) cells. Here, we report instead that T2/5 DKO cells exhibit high basal IκB kinase (IKK) activity and elevated expression of NF-κB-dependent genes in unstimulated conditions. Although TNFα-induced receptor-interacting protein 1 ubiquitination is indeed impaired in T2/5 DKO cells, TNFα stimulation further increases IKK activity in these cells, resulting in significantly elevated expression of NF-κB target genes to a level higher than that in wild-type cells. Inhibition of NIK in T2/5 DKO cells attenuates basal IKK activity and restores robust TNFα-induced IKK activation to a level comparable with that seen in wild-type cells. This suggests that TNFα can activate IKK in the absence of TRAF2 and TRAF5 expression and receptor-interacting protein 1 ubiquitination. In addition, both the basal and TNFα-induced expression of anti-apoptotic proteins are normal in T2/5 DKO cells, yet these DKO cells remain sensitive to TNFα-induced cell death, due to the impaired recruitment of anti-apoptotic proteins to the TNFR1 complex in the absence of TRAF2. Thus, our data demonstrate that TRAF2 negatively regulates basal IKK activity in resting cells and inhibits TNFα-induced cell death by recruiting anti-apoptotic proteins to the TNFR1 complex rather than by activating the NF-κB pathway.     

EB病毒潜伏膜蛋白1通过结合TRAFs调控NF-κB

为了探讨EB病毒潜伏膜蛋白1(LMP1)的致瘤机制,对鼻咽癌中LMP1激活重要的核转录因子NF-κB机制进行了研究.首先,采用免疫共沉淀-蛋白质印迹在稳定表达LMP1的鼻咽癌细胞系HNE2-LMP1中证实LMP1与TRAF1,2,3结合形成免疫共沉淀复合物,进一步以野生型LMP1及其三种突变体的鼻咽癌细胞系LMP1（野生型,wt）、HNE2-LMP1 del187～351(CTAR1缺失型)、HNE2-LMP1(1～231)(CTAR2缺失型)、HNE2-LMP1(1～187)(羧基端胞浆区缺失型)、HNE2-pSG5(空白载体型)为材料,结合NF-κB报道基因质粒（pGL2-NF-κB-luc）的荧光素酶活性表达分析NF-κB的活性,证实:较之母细胞, 野生型LMP1活化NF-κB达13.8倍, LMP1(1～187)几乎不活化NF-κB,LMP1(1～231)活化NF-κB达4.9倍, LMP1(del187～351)活化NF-κB达9.1倍;TRAF1过表达升高LMP1(wt)及LMP1(1～231)介导的NF-κB活性,而对LMP1(del 187～351)活化NF-κB无影响;TRAF3过表达或TRAF3负显性突变体抑制LMP1(wt)及LMP1(1～231)介导的NF-κB活性,而不影响LMP1(del 187～351)活化NF-κB; TRAF2过表达升高LMP1(wt)、LMP1 (1～231)及LMP1(del 187～351)介导的NF-κB活性.这些结果表明：鼻咽癌中LMP1通过TRAF1、TRAF2或TRAF3调控NF-κB,TRAF1和TRAF3主要通过CTAR1发挥作用,TRAF2的作用主要是通过CTAR1和CTAR2介导的.     

Neutral sphingomyelinase inhibition decreases ER stress-mediated apoptosis and inducible nitric oxide synthase in retinal pigment epithelial cells

Endoplasmic reticulum (ER) stress and excessive nitric oxide production via the induction of inducible nitric oxide synthase (NOS2) have been implicated in the pathogenesis of ocular diseases characterized by retinal degeneration. Previous studies have revealed the sphingomyelinase/ceramide pathway in the regulation of NOS2 induction. Thus, the objective of this study was to determine the activity of the sphingomyelinase/ceramide pathway, assess nitric oxide production, and examine apoptosis in human retinal pigment epithelial (RPE) cells undergoing ER stress. Sphingomyelinase (SMase) activity; nuclear factor κB (NF-κB) activation; NOS2, nitrite/nitrate, and nitrotyrosine levels; and apoptosis were determined in cultured human RPE cell lines subjected to ER stress via exposure to tunicamycin. Induction of ER stress was confirmed by increased intracellular levels of ER stress markers including phosphorylated PKR-like ER kinase, C/EBP-homologous protein, and 78-kDa glucose-regulated protein. ER stress increased nuclear translocation of NF-κB, NOS2 expression, nitrite/nitrate levels, and nitrotyrosine formation and caused apoptosis in RPE cell lines. Inhibition of neutral SMase (N-SMase) activity via GW 4869 treatment caused a significant reduction in nuclear translocation of NF-κB, NOS2 expression, nitrite/nitrate levels, nitrotyrosine formation, and apoptosis in ER-stressed RPE cells. In conclusion, N-SMase inhibition reduced nitrative stress and apoptosis in RPE cells undergoing ER stress. Obtained data suggest that NOS2 can be regulated by N-SMase in RPE cells experiencing ER stress.     

Molecular cloning of rock bream's (Oplegnathus fasciatus) tumor necrosis factor receptor-associated factor 2 and its role in NF-κB activiation

Tumor necrosis factor receptor-associated factor 2 (TRAF2) acts as a transducer of tumor necrosis factor-α (TNF-α)-triggered cell signals which results in inflammation, cell proliferation and antiapoptotic response. In this study, we have cloned cDNA of rock bream (Oplegnathus fasciatus) TRAF2, and analyzed its function in activation of NF-κB. The full length cDNA of rock bream TRAF2 consisted of 95 bp 5' UTR, 335 bp 3' UTR, and 1563 bp ORF encoding 520 amino acids that contained N-terminal RING-type and TRAF-type zinc finger domains and a C-terminal TRAF domain. The deduced amino acid sequence of rock bream TRAF2 showed more than 75% identity with other fish TRAF2s, and even as high as 56% identity with mouse and human TRAF2 proteins. To know whether the rock bream TRAF2 involves in NF-κB activation, Epithelioma papulosum cyprini (EPC) cells harboring an NF-κB reporting vector were transfected with a vector expressing rock bream TRAF2 or a control empty vector. NF-κB activity of EPC cells was significantly increased by exposure to the rock bream recombinant TNF-α. EPC cells transfected with the vector expressing rock bream TRAF2 showed significantly higher NF-κB activity by stimulation with the recombinant TNF-α than cells transfected with a control empty vector, suggesting the present rock bream TRAF2 acts as a transducer of TNF-α-mediated cell signals that enhance NF-κB activation.     

Identification and characterization of receptor-interacting protein 2 as a TNFR-associated factor 3 binding partner

Tumor necrosis factor receptor-associated factor 3 (TRAF3) is a highly versatile immune regulator that positively controls type I interferon production, but negatively regulates the activation of mitogen-activated protein kinase and alternative nuclear factor-κB signaling. The precise function of TRAF3 in different signaling pathways remains unclear. Thus, in a yeast two-hybrid assay, TRAF3 was used as the bait to screen a human spleen cDNA library for TRAF3 interactors that may potentially mediate TRAF3-regulated functions. Receptor-interacting protein 2 (RIP2) was identified as a TRAF3 binding partner. The interaction between TRAF3 and RIP2 was further confirmed by mammalian two-hybrid, co-immunoprecipitation and GST pull-down assays, and this interaction was also verified by immunoprecipitation of endogenous proteins in Ramos cells, a human B lymphoma cell line. RIP2 is an activator of NF-κB. We therefore examined the effect of TRAF3 in RIP2-induced NF-κB activation. The result showed that TRAF3 could inhibit RIP2-induced NF-κB activation. Given the high expression of RIP2 in the B lymphoma cell line and endogenous interaction between TRAF3 and RIP2 in Ramos cells, the role of RIP2 was further studied. The result demonstrated that RIP2 knockdown was capable of increasing the expression of TRAF3 and suppressing the activation of alternative NF-кB pathway in Ramos cells. These findings suggest that functional interactions between RIP2 and TRAF3 may provide some clues to the mechanisms of TRAF3-involvement in both positive and negative regulatory functions.     

Mutational analysis of TRAF6 reveals a conserved functional role of the RING dimerization interface and a potentially necessary but insufficient role of RING-dependent TRAF6 polyubiquitination towards NF-κB activation

TRAF6 is an E3 ubiquitin ligase that plays a pivotal role in the activation of NF-κB by innate and adaptive immunity stimuli. TRAF6 consists of a highly conserved carboxyl terminal TRAF-C domain which is preceded by a coiled coil domain and an amino terminal region that contains a RING domain and a series of putative zinc-finger motifs. The TRAF-C domain contributes to TRAF6 oligomerization and mediates the interaction of TRAF6 with upstream signaling molecules whereas the RING domain comprises the core of the ubiquitin ligase catalytic domain. In order to identify structural elements that are important for TRAF6-induced NF-κB activation, mutational analysis of the TRAF-C and RING domains was performed. Alterations of highly conserved residues of the TRAF-C domain of TRAF6 did not affect significantly the ability of the protein to activate NF-κB. On the other hand a number of functionally important residues (L77, Q82, R88, F118, N121 and E126) for the activation of NF-κB were identified within the RING domain of TRAF6. Interestingly, several homologues of these residues in TRAF2 were shown to have a conserved functional role in TRAF2-induced NF-κB activation and lie at the dimerization interface of the RING domain. Finally, whereas alteration of Q82, R88 and F118 compromised both the K63-linked polyubiquitination of TRAF6 and its ability to activate NF-κB, alteration of L77, N121 and E126 diminished the NF-κB activating function of TRAF6 without affecting TRAF6 K63-linked polyubiquitination. Our results support a conserved functional role of the TRAF RING domain dimerization interface and a potentially necessary but insufficient role for RING-dependent TRAF6 K63-linked polyubiquitination towards NF-κB activation in cells.     

Diosmetin inhibits the growth and invasion of gastric cancer by interfering with M2 phenotype macrophage polarization

Overturning M2 phenotype macrophage polarization is a promising therapeutic strategy for gastric cancer (GC). Diosmetin (DIO) is a natural flavonoid with antitumor effect. The aim of this study was to investigate the effect of DIO on polarization of M2 phenotype macrophages in GC. THP-1 cells were induced to M2 phenotype macrophages and co-cultured with AGS cells. The effects of DIO were determined by flow cytometry, qRT-PCR, CCK-8, Transwell, and western blot. To explore the mechanisms, THP-1 cells were transfected with adenoviral vectors containing tumor necrosis factor receptor-associated factor 2 (TRAF2) or si-TRAF2. DIO (0, 5, 10, and 20 μM) restrained the M2 phenotype macrophage polarization. In addition, DIO (20 μM) reversed the increased viability and invasion of AGS cells induced by the co-culture of M2 macrophages. Mechanistically, TRAF2 knockdown inhibited the effect of M2 phenotype macrophages on AGS cells' growth and invasion. Furthermore, DIO (20 μM) was found to decrease TRAF2/NF-κB activity in GC cells. However, TRAF2 overexpressed reversed the inhibitory effect of DIO on the co-culture system. The in vivo study confirmed that DIO treatment (50 mg/kg) could repress the growth of GC. DIO treatment markedly reduced the expressions of Ki-67 and N-cadherin, and decreased the protein levels of TRAF2 and p-NF-κB/NF-κB. In conclusion, DIO inhibited the growth and invasion of GC cells by interfering with M2 phenotype macrophage polarization through repression of the TRAF2/NF-κB signaling pathway.     

Interleukin-2 induces extracellular matrix synthesis and TGF-β2 expression in retinal pigment epithelial cells

Macular fibrosis is a vital obstacle of vision acuity improvement of age-related macular degeneration patients. This study was to investigate the effects of interleukin 2 (IL-2) on epithelial-mesenchymal transition (EMT), extracellular matrix (ECM) synthesis and transforming growth factor β2 (TGF-β2) expression in retinal pigment epithelial (RPE) cells. 10 μg/L IL-2 was used to induce fibrosis in RPE cells for various times. Western blot was used to detect the EMT marker α-smooth muscle actin (α-SMA), ECM markers fibronectin (Fn) and type 1 collagen (COL-1), TGF-β2, and the activation of the JAK/STAT3 and NF-κB signaling pathway. Furthermore, JAK/STAT3 and NF-κB signaling pathways were specifically blocked by WP1066 or BAY11-7082, respectively, and the expression of α-SMA, COL-1, Fn and TGF-β2 protein were detected. Wound healing and Transwell assays were used to measure cell migration ability of IL-2 with or without WP1066 or BAY11-7082. After induction of IL-2, the expressions of Fn, COL-1, TGF-β2 protein were significantly increased, and this effect was correlated with IL-2 treatment duration, while α-SMA protein expression did not change significantly. Both WP1066 and BAY11-7082 could effectively downregulate the expression of Fn, COL-1 and TGF-β2 induced by IL-2. What's more, both NF-κB and JAK/STAT3 inhibitors could suppress the activation of the other signaling pathway. Additionally, JAK/STAT3 inhibitor WP1066 and NF-κB inhibitor BAY 11-7082 could obviously decrease RPE cells migration capability induced by IL-2. IL-2 promotes cell migration, ECM synthesis and TGF-β2 expression in RPE cells via JAK/STAT3 and NF-κB signaling pathways, which may play an important role in proliferative vitreoretinopathy.     

CD44 aptamer mediated cargo delivery to lysosomes of retinal pigment epithelial cells to prevent age-related macular degeneration

Age related macular degeneration (AMD) is a progressive, neurodegenerative disorder that leads to the severe loss of central vision in elderlies. The health of retinal pigment epithelial (RPE) cells is critical for the onset of AMD. Chronic oxidative stress along with loss of lysosomal activity is a major cause for RPE cell death during AMD. Hence, development of a molecule for targeted lysosomal delivery of therapeutic protein/drugs in RPE cells is important to prevent RPE cell death during AMD. Using human RPE cell line (ARPE-19 cells) as a study model, we confirmed that hydrogen peroxide (H₂O₂) induced oxidative stress results in CD44 cell surface receptor overexpression in RPE cells; hence, an important target for specific delivery to RPE cells during oxidative stress. We also demonstrate that the known nucleic acid CD44 aptamer - conjugated with a fluorescent probe (FITC) - is delivered into the lysosomes of CD44 expressing ARPE-19 cells. Hence, as a proof of concept, we demonstrate that CD44 aptamer may be used for lysosomal delivery of cargo to RPE cells under oxidative stress, similar to AMD condition. Since oxidative stress may induce wet and dry AMD, both, along with proliferative vitreoretinopathy, CD44 aptamer may be applicable as a carrier for targeted lysosomal delivery of therapeutic cargoes in ocular diseases showing oxidative stress in RPE cells.     

泛素化在TRAF2介导的NF-κB信号通路中的作用

NF-κB（核因子κ增强子结合蛋白）是核转录因子家族成员,具有调节免疫、炎症和细胞存活的功能.它可被TRAF2（tumor necrosis factor receptor associated factor 2,肿瘤坏死因子受体相关因子2）等相关因子活化.TRAF2包含了N-端的环指结构域和C-端的高度保守结构域.它通过与肿瘤坏死因子受体超家族成员相互作用,介导了下游信号通路.而TRAF2的泛素化在过程中是关键的,鞘磷脂作为TRAF2的泛素化连接酶辅助因子,在TRAF2介导的NF-κB信号通路中发挥重要作用.     

Tannic acid suppresses ultraviolet B-induced inflammatory signaling and complement factor B on human retinal pigment epithelial cells

Ultraviolet B (UVB) radiation may cause the inflammation of retinal pigment epithelium (RPE) cells and play a role in development of age-related macular degeneration (AMD). The activation of the complement factor B (CFB) gene has been shown to be involved in formation of AMD. Here our results revealed that UVB induces IL-6/STAT3 signaling activation and the UVB-induced STAT3 is able to regulate the CFB expression in ARPE-19 cells. Tannic acid (TA) is a kind of water-soluble polyphenol and may have anti-inflammation effects. We also found that TA attenuates the UVB-induced IL-6 protein production, the STAT3 phosphorylation and the CFB expression. Taken together, these findings suggest UVB-induced inflammation of RPE can be mediated through the IL-6/STAT3/CFB pathway, and TA has a protected effect via the inhibition to the inflammatory response.     

Opposing roles for TRAF1 in the alternative versus classical NF-κB pathway in T cells

T cells lacking TRAF1 hyperproliferate in response to T cell receptor signaling but have impaired signaling downstream of specific TNFR family members such as 4-1BB. Here we resolve this paradox by showing that while TRAF1 is required for maximal activation of the classical NF-κB pathway downstream of 4-1BB in primary T cells, TRAF1 also restricts the constitutive activation of NIK in anti-CD3-activated T cells. Activation of the alternative NF-κB pathway is restricted in unstimulated cells by a cIAP1/2:TRAF2:TRAF3:NIK complex. Using knockdown of NIK by siRNA we show that in activated CD8 T cells TRAF1 is also involved in this process and that constitutive activation of the alternative NF-κB pathway is responsible for costimulation independent hyperproliferation and excess cytokine production in TRAF1-deficient CD8 T cells compared with WT CD8 T cells. The T cell costimulatory molecule 4-1BB critically regulates the survival of activated and memory CD8 T cells. We demonstrate that stimulation through 4-1BB induces cIAP1-dependent TRAF3 degradation and activation of the alternative NF-κB pathway. We also show that while both TRAF1 and cIAP1 have non-redundant roles in suppressing the alternative NF-κB pathway in T cells activated in the absence of costimulation, activation of the classical NF-κB pathway downstream of 4-1BB requires TRAF1, whereas cIAP1 plays a redundant role with cIAP2. Collectively these results demonstrate that TRAF1 plays a critical role in regulating T cell activation both through restricting the costimulation independent activation of NIK in activated T cells and by promoting the 4-1BB-induced classical NF-κB pathway.     

Numbl inhibits glioma cell migration and invasion by suppressing TRAF5-mediated NF-κB activation

The Notch signaling regulator Numblike (Numbl) is expressed in the brain, but little is known regarding its role in the pathophysiology of glial cells. In this paper, we report that Numbl expression was down-regulated in high-grade human glioma tissue samples and glioblastoma cell lines. To investigate the role of Numbl in glioma migration and invasion, we generated human glioma cell lines in which Numbl was either overexpressed or depleted. Overexpression of Numbl suppressed, while elimination of Numbl promoted, the migration and invasion of glioma cells. Numbl inhibited glioma migration and invasion by dampening NF-κB activity. Furthermore, Numbl interacted directly with tumor necrosis factor receptor-associated factor 5 (TRAF5), which signals upstream and is required for the activation of NF-κB, and committed it to proteasomal degradation by promoting K48-linked polyubiquitination of TRAF5. In conclusion, our data suggest that Numbl negative regulates glioma cell migration and invasion by abrogating TRAF5-induced activation of NF-κB.     

The TRAF3 adaptor protein drives proliferation of anaplastic large cell lymphoma cells by regulating multiple signaling pathways

T cells devoid of tumor necrosis factor receptor associated factor-3 (Traf3) exhibit decreased proliferation, sensitivity to apoptosis, and an improper response to antigen challenge. We therefore hypothesized that TRAF3 is critical to the growth of malignant T cells. By suppressing TRAF3 protein in different cancerous T cells, we found that anaplastic large cell lymphoma (ALCL) cells require TRAF3 for proliferation. Since reducing TRAF3 results in aberrant activation of the noncanonical nuclear factor-κB (NF-κB) pathway, we prevented noncanonical NF-κB signaling by suppressing RelB together with TRAF3. This revealed that TRAF3 regulates proliferation independent of the noncanonical NF-κB pathway. However, suppression of NF-κB-inducing kinase (NIK) along with TRAF3 showed that high levels of NIK have a partial role in blocking cell cycle progression. Further investigation into the mechanism by which TRAF3 regulates cell division demonstrated that TRAF3 is essential for continued PI3K/AKT and JAK/STAT signaling. In addition, we found that while NIK is dispensable for controlling JAK/STAT activity, NIK is critical to regulating the PI3K/AKT pathway. Analysis of the phosphatase and tensin homolog (PTEN) showed that NIK modulates PI3K/AKT signaling by altering the localization of PTEN. Together our findings implicate TRAF3 as a positive regulator of the PI3K/AKT and JAK/STAT pathways and reveal a novel function for NIK in controlling PI3K/AKT activity. These results provide further insight into the role of TRAF3 and NIK in T cell malignancies and indicate that TRAF3 differentially governs the growth of B and T cell cancers.