Among the members of the inhibitor of apoptosis (IAP) protein family, only Livin and survivin have been reported to have variant forms. We have found a variant form of c-IAP2 through the interaction with the X protein of HBV using the yeast two-hybrid system. In contrast to the wild-type c-IAP2, the variant form has two stretches of sequence in the RING domain that are repeated in the C-terminus that would disrupt the RING domain. We demonstrate that the variant form has an inhibitory effect on TNF-mediated NF-kappaB activation unlike the wild-type c-IAP2, which increases TNF- mediated NF-kappaB activation. These results suggest that this variant form has different activities from the wild-type and the RING domain may be involved in the regulation of TNF-induced NF-kappaB activation.
Apoptosis ; Humans ; Inhibitor of Apoptosis Proteins* ; NF-kappa B ; TNF Receptor-Associated Factor 2 ; TNF Receptor-Associated Factor 6 ; Two-Hybrid System Techniques

Among the members of the inhibitor of apoptosis (IAP) protein family, only Livin and survivin have been reported to have variant forms. We have found a variant form of c-IAP2 through the interaction with the X protein of HBV using the yeast two-hybrid system. In contrast to the wild-type c-IAP2, the variant form has two stretches of sequence in the RING domain that are repeated in the C-terminus that would disrupt the RING domain. We demonstrate that the variant form has an inhibitory effect on TNF-mediated NF-kappaB activation unlike the wild-type c-IAP2, which increases TNF- mediated NF-kappaB activation. These results suggest that this variant form has different activities from the wild-type and the RING domain may be involved in the regulation of TNF-induced NF-kappaB activation.
Apoptosis ; Humans ; Inhibitor of Apoptosis Proteins* ; NF-kappa B ; TNF Receptor-Associated Factor 2 ; TNF Receptor-Associated Factor 6 ; Two-Hybrid System Techniques

Sequestosome 1 (SQSTM1, p62), a ubiquitin binding protein, plays a role in cell signaling, oxidative stress, and autophagy. However, its functional role in inflammatory signaling is controversial. Recent studies have shown that p62 is negatively implicated in inflammatory responses. But, the precise molecular mechanisms by which p62 regulates inflammatory responses remain unclear. In this study, we report on a new regulatory role for p62 in TLR4-mediated signaling. p62 overexpression led to the suppression of NF-κB activation and the production of pro-inflammatory cytokines, TNF-α, IL-6, and IL-1β in response to TLR4 stimulation. In contrast, p62(−/−) mouse embryonic fibroblast (MEF) cells exhibited marked enhancement of NF-κB activation and production of pro-inflammatory cytokines by TLR4 stimulation, compared to p62(+/+) MEF cells. Additionally, the TLR4-induced activation of signal transduction was significantly augmented in p62(−/−) MEF cells, indicating that p62 was negatively implicated in TLR4-mediated signaling. Biochemical studies revealed that p62 interacted with the internal domain of evolutionarily conserved signaling intermediate in Toll pathways (ECSIT), which is critical for associating with the TNF receptor associated factor 6 (TRAF6)-ECSIT complex to activate NF-κB in TLR4 signaling. Interestingly, p62-ECSIT interaction inhibited the interaction between TRAF6 and ECSIT and attenuated the ubiquitination of ECSIT. Furthermore, upon LPS challenge, the mortality of p62(−/−) (p62-knockout) mice was markedly enhanced compared to p62(+/+) (p62 wild-type) mice. Taken together, our data demonstrate that p62 negatively regulated TLR4 signaling via functional regulation of the TRAF6-ECSIT complex.
Animals ; Autophagy ; Carrier Proteins ; Cytokines ; Fibroblasts ; Interleukin-6 ; Mice ; Mortality ; Oxidative Stress ; Signal Transduction ; TNF Receptor-Associated Factor 6 ; Toll-Like Receptor 4 ; Ubiquitin ; Ubiquitination

OBJECTIVETo explore the effect of MiR-146a regulator function on the inflammatory response in neuroglia cell (microglia).

METHODSBV2 cells were transfected by MiR-146a mimics,and then stimulated by lipopolysaccharide (LPS). MiR-146a expression was measured by real-time polymerase chain reaction (real-time PCR). Interleukin (IL)-6 and tumor necrosis factor α (TNFα) were measured by enzyme-linked immunosorbent assay (ELISA). Furthermore, IL-1 receptor-associated kinase 1 (IRAK1) and TNF receptor-associated factor 6 (TRAF6) were detected by PCR and Western blotting.

RESULTSCompared to the normal control group, MiR-146a expression was significantly elevated by transfection with MiR-146a mimics (t=5.846, P=0.0021). The expression levels of IRAK1, TRAF6, TNFα, and IL-6 significantly increased in the LPS-stimulated BV2 cells compared to the non-stimulated BV2. The enhancement of MiR-146a resulted in significantly decreased IL-6 (t=5.200, P=0.0003) and TNFα (t=9.812, P<0.0001) secretion. The mRNA (t=5.353, P=0.0007) and protein (t=6.980, P=0.0009) levels of TRAF6, but not IRAK1, also significantly decreased.

CONCLUSIONMiR-146a may negatively suppress the inflammatory response of BV2 cells by regulating the expression of IRAF6 molecules in the TLR4 signaling pathway.

Blotting, Western ; Cell Line ; Enzyme-Linked Immunosorbent Assay ; Humans ; Inflammation ; Interleukin-1 Receptor-Associated Kinases ; Interleukin-6 ; Lipopolysaccharides ; MicroRNAs ; RNA, Messenger ; Real-Time Polymerase Chain Reaction ; Signal Transduction ; TNF Receptor-Associated Factor 6 ; Transfection ; Tumor Necrosis Factor-alpha

BACKGROUND/OBJECTIVES: Carnosic acid (CA), found in rosemary (Rosemarinus officinalis) leaves, is known to exhibit anti-obesity and anti-inflammatory activities. However, whether its anti-inflammatory potency can contribute to the amelioration of obesity has not been elucidated. The aim of the current study was to investigate the effect of CA on Toll-like receptor 4 (TLR4) pathways in the presence of lipopolysaccharide (LPS) in 3T3-L1 adipocytes. MATERIALS/METHODS: 3T3-L1 adipocytes were treated with CA (0-20 microM) for 1 h, followed by treatment with LPS for 30 min; mRNA expression of adipokines and protein expression of TLR4-related molecules were then measured. RESULTS: LPS-stimulated 3T3-L1 adipocytes showed elevated mRNA expression of tumor necrosis factor (TNF)-alpha, interleukin-6, and monocyte chemoattractant protein-1, and CA significantly inhibited the expression of these adipokine genes. LPS-induced up regulation of TLR4, myeloid differentiation factor 88, TNF receptor-associated factor 6, and nuclear factor-kappaB, as well as phosphorylated extracellular receptor-activated kinase were also suppressed by pre-treatment of 3T3-L1 adipocytes with CA. CONCLUSIONS: Results of this study suggest that CA directly inhibits TLR4-MyD88-dependent signaling pathways and decreases the inflammatory response in adipocytes.
Adipocytes* ; Adipokines ; Chemokine CCL2 ; Inflammation ; Interleukin-6 ; Myeloid Differentiation Factor 88 ; Obesity ; Phosphotransferases ; RNA, Messenger ; TNF Receptor-Associated Factor 6 ; Toll-Like Receptor 4 ; Tumor Necrosis Factor-alpha ; Up-Regulation

MicroRNAs (miRNAs) are small, single-stranded, non-coding RNA molecules of 20~22 nucleotides, which are involved in many biologic functions such as development, cell proliferation, differentiation, and apoptosis. In addition to these biologic functions, recent reports have demonstrated that miRNAs play important roles in the development of the immune system and the regulation of immune responses. Dysregulation of miRNAs might be involved in the pathogenesis of autoimmune diseases such as rheumatoid arthritis (RA). Recent studies have shown that miR-146a, miR-155, and miR-203 are overexpressed in RA and that miR-124a is under expressed in RA. miR-146 downregulates the expression of IL-1 receptor associated kinase 1 and TNF receptor-associated factor 6 involved in IL-1beta signaling, and miR-155 suppresses the expression of matrix metalloproteinases 1 and 3, suggesting that these miRNAs act as negative feedback regulators of inflammation and tissue damage in RA. In this report, we review the current knowledge about miRNAs and summarize the involvement of miRNAs in RA.
Apoptosis ; Arthritis, Rheumatoid ; Autoimmune Diseases ; Cell Proliferation ; Immune System ; Inflammation ; Interleukin-1 ; Matrix Metalloproteinases ; MicroRNAs ; Nucleotides ; Phosphotransferases ; RNA, Untranslated ; TNF Receptor-Associated Factor 6

MicroRNAs (miRNAs) are small, single-stranded, non-coding RNA molecules of 20~22 nucleotides, which are involved in many biologic functions such as development, cell proliferation, differentiation, and apoptosis. In addition to these biologic functions, recent reports have demonstrated that miRNAs play important roles in the development of the immune system and the regulation of immune responses. Dysregulation of miRNAs might be involved in the pathogenesis of autoimmune diseases such as rheumatoid arthritis (RA). Recent studies have shown that miR-146a, miR-155, and miR-203 are overexpressed in RA and that miR-124a is under expressed in RA. miR-146 downregulates the expression of IL-1 receptor associated kinase 1 and TNF receptor-associated factor 6 involved in IL-1beta signaling, and miR-155 suppresses the expression of matrix metalloproteinases 1 and 3, suggesting that these miRNAs act as negative feedback regulators of inflammation and tissue damage in RA. In this report, we review the current knowledge about miRNAs and summarize the involvement of miRNAs in RA.
Apoptosis ; Arthritis, Rheumatoid ; Autoimmune Diseases ; Cell Proliferation ; Immune System ; Inflammation ; Interleukin-1 ; Matrix Metalloproteinases ; MicroRNAs ; Nucleotides ; Phosphotransferases ; RNA, Untranslated ; TNF Receptor-Associated Factor 6

OBJECTIVETo investigate the effect of small interfering RNA (siRNA)-mediated inhibition of tumor necrosis factor receptor associated factor 6(TRAF6) gene on murine odontoblast-like cell line, MDPC-23 cell and the effect of TRAF6 on MDPC-23 cell proliferation.

METHODSThe vectors expressing siRNA against TRAF6 were constructed and introduced into MDPC-23 cell with lipofectin , and the cell line with stable expression of siRNA of TRAF6 was obtained by G418 screening and colony culture. Reverse transcription-PCR (RT-PCR) and Western blotting were performed to detect the expression of TRAF6. The proliferation of transfected MDPC-23 cell was investigated through methabenzthiazuron (MTT) and flow cytometry (FCM) assay.

RESULTSThe positive single colony was screened out, and was found to express siRNA against TRAF6 effectively because both TRAF6 mRNA and protein relative expression were significantly decreased in the experimental group (pSUPER-TRAF6siRNA: mRNA 0.163 ± 0.008, protein 0.215 ± 0.006) compared with controls (pSUPER: mRNA 0.778 ± 0.017, protein 0.964 ± 0.007 (P < 0.001). The A value of treated pSUPER-TRAF6siRNA cells (3 d: 0.46 ± 0.03, 5 d: 1.35 ± 0.06) was increased compared with controls (P<0.01). The result of proliferation index (PrI) was also increased compared with controls [pSUPER- TRAF6siRNA: (24.1 ± 2.2)%; pSUPER (11.2 ± 1.0)%; control (10.5 ± 0.7)%, P < 0.01].

CONCLUSIONSThe transcription and expression of TRAF6 gene were inhibited. The proliferation ability was increased in MDPC-23 cells by the constructed pSUPER-TRAF6siRNA vector. It may further influence the formation and repair of dentin, and may be involved in the regulation of normal tooth eruption and process of dentin repair after injury.

Animals ; Cell Line, Tumor ; Cell Proliferation ; Flow Cytometry ; Gene Silencing ; Genetic Vectors ; Mice ; Odontoblasts ; cytology ; RNA, Messenger ; RNA, Small Interfering ; TNF Receptor-Associated Factor 6 ; genetics ; Transfection

OBJECTIVETo investigate the down-regulated TRAF6 gene expression and its effects on proliferation and apoptosis in multiple myeloma (MM) cells.

METHODSDetection of TRAF6 expression were conducted by RT-PCR and Western blot in MM cell lines of KM3, U266, RPMI8226 and primary cells from patients. RPMI8226 cell lines were transfected with siRNA of TRAF6. The efficiency of transfection was identified by using of fluorescence microscope, RT-PCR, and Western blot. The levels of proliferation were analyzed by CCK-8 method under the different concentrations of siRNA. Apoptosis rate were detected with Hoechst33258/PI double staining by flow cytometry. Apoptosis related proteins Bcl-2, BAX, and NF-κB signal pathway were observed before and after siRNA transfection by Western blot.

RESULTSThe levels of TRAF6 mRNA and protein in MM cell lines, especially in primary myeloma cells, were significantly higher than those in controls. After transfected with 50 nmol/L siRNA in RPMI8226 cells, the relative level of TRAF6 mRNA (0.49±0.24) was significantly lower than that in non-transfected group (1.87±0.23) and idling group (1.74±0.35). The proliferation rate of siRNA transfected cells decreased with dose dependence (P<0.01). The apoptosis rates increased from 11.20% (before transfection) to 51.82% (after transfection), accompanied by down-regulated Bcl-2 protein, NF-κB signal pathway (p-p65 and p52), and up-regulated BAX protein.

CONCLUSIONTRAF6 expression was high in myeloma cells. TRAF6 siRNA could inhibit proliferation of myeloma cells and induce apoptosis mediated by NF-κB classical and alternative pathway in myeloma cells.

Case-Control Studies ; Cell Proliferation ; Down-Regulation ; Female ; Gene Expression ; Humans ; Male ; Multiple Myeloma ; metabolism ; pathology ; TNF Receptor-Associated Factor 6 ; genetics ; metabolism ; Tumor Cells, Cultured

To investigate the effects of interleukin (IL)-17-mediated autophagy on the TNF receptor associated factor (TRAF6)/extracellular signal-regulated kinase (ERK)/p38 pathway and osteoclast differentiation. Mouse bone marrow-derived macrophages (BMM) were cultured with a medium containing 30 ng/mL macrophage colony stimulating factor and 50 ng/mL receptor activator of nuclear factor-kappa B ligard (RANKL), and IL-17 (0.01, 0.1, 1.0, 10 ng/mL) was added for intervention (IL-17 group). Tartrate-resistant acid phosphatase (TRAP) staining was used to observe TRAP positive multinucleated cells; phalloidin fluorescent staining was used to detect actin ring circumference; toluidine blue staining was used to analyze bone resorption lacuna formation. To further examine the mechanism of the effect of IL-17-mediated autophagy on the differentiation of osteoclasts, the control group used RANKL medium to culture mouse macrophage RAW264.7 cells, while the IL-17 group was treated with IL-17 (0.01, 0.1, 1.0, /mL). Western blot was used to detect the expression of autophagy-related proteins Beclin-1, microtubule-associated protein 1 light chain 3 (LC3) and osteoclast-related proteins c-fos and nuclear factor of activated T cell 1 (NFATc1) after treatment with different concentrations of IL-17. The expression of LC3, NFATc1, TRAF6/ERK/p38 signaling pathway related proteins were detected in IL-17 and autophagy inhibitor 3-MA group. The number of TRAP positive multinucleated cells, the circumference of the actin ring and the area of bone resorption lacuna in IL-17 group treated with IL-17 (0.01, 0.1, were significantly higher than those in the control group. In IL-17 treated RAW264.7 cells, the expression of c-fos, NFATc1, Beclin-1, LC3, TRAF6, p-ERK, and p-p38 was all significantly up-regulated (all 0.05). After treatment with the autophagy inhibitor 3-MA, the expression levels of LC3, NFATc1, TRAF6, p-ERK, and p-p38 all decreased significantly (all 0.05). IL-17 can promote the expression of autophagy proteins and enhance the differentiation ability of osteoclast precursor cells, and the TRAF6/ERK/p38 signaling pathway may be involved in this process.
Animals ; Autophagy ; Bone Resorption ; Cell Differentiation ; Extracellular Signal-Regulated MAP Kinases ; Interleukin-17 ; Mice ; NFATC Transcription Factors/metabolism* ; Osteoclasts/metabolism* ; RANK Ligand/metabolism* ; TNF Receptor-Associated Factor 6