PTBP1-TRAF6 Axis Aggravates Chronic Obstructive Pulmonary Disease by Promoting M1 Macrophage Polarization and Impairing Clearance of Neutrophil Extracellular Traps

Little is known about the combined effect of fluoride (F)and arsenic (As) on bone metabolism. This study aims to explore the effect of co-exposure to F and As on the expressions of TNF receptor-associated factor 6 (TRAF-6), nuclear factor-kappa B (NF-κB), and the related factors in cell and animal experiments. With the rats exposed to different doses of F, As, and combined F-As, we found that F exposure doses were positively correlated with the protein expression of receptor activator of nuclear factor-kappa B ligand (RANKL), receptor activator of nuclear factor-kappa B (RANK), TRAF-6, NF-κB, and nuclear factor of activated T cells (NFAT-c1) (P < 0.001). As exposure doses were negatively correlated with RANK, TRAF-6, NF-κB, and NFAT-c1 (P < 0.001). The effect of F and As interaction on the protein expression of RANKL, TRAF-6, NF-κB, and NFAT-c1 was significant in bone tissue (P < 0.05). In the cellular experiment, F could promote the mRNA expression of RANK, TRAF-6, and NFAT-c1. A higher concentration of As could inhibit the mRNA expression of Tartrate-resistant acid phosphatase (TRAP), RANK, TRAF-6, and NFAT-c1. The effect of F and As interaction on the mRNA expression of TRAP, RANK, TRAF-6, and NFATc1 in osteoclasts was significant (P < 0.001). In conclusion, the expression of TRAF-6 and NF-κB pathway was affected by F and As co-exposure in osteogenic differentiation, and As could antagonize the promoting effect of F on the expression of TRAF-6, TRAP, RANKL, RANK, NF-κB, and NFAT-c1 in these exposure levels. These results could provide a scientific basis for understanding the interaction of F and As in bone formation.

Huntington disease (HD) is a neurodegenerative disorder caused by an expansion of polyglutamines in the first exon of huntingtin (HTT), which confers aggregation-promoting properties to amino-terminal fragments of the protein (N-HTT). Mutant N-HTT aggregates are enriched for ubiquitin and contain ubiquitin E3 ligases, thus suggesting a role for ubiquitination in aggregate formation. Here, we report that tumor necrosis factor receptor-associated factor 6 (TRAF6) binds to WT and polyQ-expanded N-HTT in vitro as well as to endogenous full-length proteins in mouse and human brain in vivo. Endogenous TRAF6 is recruited to cellular inclusions formed by mutant N-HTT. Transient overexpression of TRAF6 promotes WT and mutant N-HTT atypical ubiquitination with Lys6, Lys27, and Lys29 linkage formation. Both interaction and ubiquitination seem to be independent from polyQ length. In cultured cells, TRAF6 enhances mutant N-HTT aggregate formation, whereas it has no effect on WT N-HTT protein localization. Mutant N-HTT inclusions are enriched for ubiquitin staining only when TRAF6 and Lys6, Lys27, and Lys29 ubiquitin mutants are expressed. Finally, we show that TRAF6 is up-regulated in post-mortem brains from HD patients where it is found in the insoluble fraction. These results suggest that TRAF6 atypical ubiquitination warrants investigation in HD pathogenesis.

Regulation of Metabolic Homeostasis By TRAF6 Contributes to the Leukemia Progression

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an adaptor/scaffold protein that mediates several important signaling pathways, including the tumor necrosis factor-R:NF-kappaB pathway, involved in immune surveillance, inflammation, etc. Because most studies of TRAF6 function have focused primarily on its role as an adaptor molecule in signaling pathways in the cytoplasm, the potential functions of TRAF6 in other cellular compartments has not been previously investigated. Here, we demonstrate that TRAF6 resides not only in the cellular cytoplasm but is also found in the nuclei of both normal and malignant B lymphocytes. TRAF6 does not possess a nuclear localization signal but enters the nucleus through the nuclear pore complex containing RanGap1. Chromatin immunoprecipitation cloning experiments demonstrated that nuclear TRAF6 associates with c-Myb within the 5'-end of the c-Myb promoter. Further analysis showed that nuclear TRAF6 is modified by small ubiquitin-related modifier-1, interacts with histone deacetylase 1, and represses c-Myb-mediated transactivation. Thus, TRAF6 negatively regulates c-Myb through a novel repressor function in the nuclei of both normal and malignant B-lymphocytes that could represent a novel control mechanism that maintains cell homeostasis and immune surveillance.

Myocardial infarction arises from an excessive or prolonged inflammatory response, leading to ventricular remodeling or impaired cardiac function. Macrophages exhibit different polarization types associated with inflammation both at steady state and after myocardial infarction. Exosomal miR-146a-5p has been identified as an important molecule in the cardiovascular field in recent years. However, the effect of cardiomyocyte-derived exosomal miR-146a-5p on macrophages has not yet been elucidated. Initially, we found that exosomes with low expression of miR-146a-5p derived from myocardial infarction tissues modulated macrophage polarization. To determine whether cardiomyocyte-derived exosomal miR-146a-5p mediated macrophage polarization, we treated macrophages with exosomes rich in miR-146a-5p collected from neonatal mouse cardiomyocytes. The effects of exosomal miR-146a-5p on macrophage polarization were measured using RT-qPCR, transwell assays, and western blotting. The results showed that the increased expression of miR-146a-5p promoted M1 macrophage polarization, inhibited M2 macrophage polarization, and increased the expression of VEGFA. However, the decreased expression of exosomalmiR-146a-5p showed the opposite trends. Interestingly, in contrast to treatment with the solitary miR-146a-5p mimic, exosomal miR-146a-5p derived from neonatal mouse cardiomyocytes reduced TNFα and iNOS expression. In addition, when macrophages were activated by the miR-146a-5p mimic or exosomal miR-146a-5p, the expression of TNF receptor-associated factor 6 (TRAF6), a target gene of miR-146a-5p, was reduced significantly. Taken together, these findings indicate that exosomal miR-146a-5p derived from cardiomyocytes could stimulate M1 macrophage polarization to induce an inflammatory reaction, while targeting TRAF6, exerting an anti-inflammatory effect. Exosomal miR-146a-5p plays important roles in macrophages, illuminating a novel potential therapeutic target in myocardial infarction.

Background: Considerable data have shown that circular RNAs (circRNAs) mediate the pathogenesis of chronic obstructive pulmonary disease (COPD). The study aims to analyze the function and mechanism of circ_0026466 in COPD. Methods: Human bronchial epithelial cells (16HBE) were treated with cigarette smoke extract (CSE) to establish a COPD cell model. Quantitative real-time polymerase chain reaction and Western blot were used to detect the expression of circ_0026466, microRNA-153-3p (miR-153-3p), TNF receptor associated factor 6 (TRAF6), cell apoptosis-related proteins, and NF-κB pathway-related proteins. Cell viability, proliferation, apoptosis, and inflammation were investigated by cell counting kit-8, EdU assay, flow cytometry, and enzyme-linked immunosorbent assay, respectively. Oxidative stress was evaluated by lipid peroxidation malondialdehyde assay kit and superoxide dismutase activity assay kit. The interaction between miR-153-3p and circ_0026466 or TRAF6 was confirmed by dual-luciferase reporter assay and RNA pull-down assay. Results: Circ_0026466 and TRAF6 expression were significantly increased, but miR-153-3p was decreased in the blood samples of smokers with COPD and CSE-induced 16HBE cells when compared with controls. CSE treatment inhibited the viability and proliferation of 16HBE cells but induced cell apoptosis, inflammation, and oxidative stress, but these effects were attenuated after circ_0026466 knockdown. Circ_0026466 interacted with miR-153-3p and regulated CSE-caused 16HBE cell damage by targeting miR-153-3p. Additionally, TRAF6, a target gene of miR-153-3p, regulated CSE-induced 16HBE cell injury by combining with miR-153-3p. Importantly, circ_0026466 activated NF-κB pathway by targeting the miR-153-3p/TRAF6 axis. Conclusion: Circ_0026466 absence protected against CSE-triggered 16HBE cell injury by activating the miR-153-3p/TRAF6/NF-κB pathway, providing a potential therapeutic target for COPD.

Objective To research the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6) in rectus abdominis of gastric cancer patients and abdominal benign disease patients, and discuss the relationship between nutritional status and prognosis of gastric cancer patients with TRAF6. Methods Biopsies of the rectus abdominis were obtained intraoperatively from 64 gastric cancer patients and 53 subjects undergoing surgery for benign abdominal diseases. The expression levels of TRAF6 mRNA and protein were assessed through quantitative real time reverse transcriptase PCR (RT-QPCR) and Western blotting, and the relationships between nutritional status and prognosis of gastric cancer patients and the expression status of TRAF6 were analyzed. Results TRAF6 mRNA was significantly upregulated in muscle of stage Ⅰ (2.080±0.611), stage Ⅱ (2.126±0.640), stage Ⅲ (3.127±0.953), stage Ⅳ (3.289±0.734) gastric cancer compared with the control group (1.249±0.723), t=4.107, P=0.001; t=4.128, P=0.000; t=8.741, P=0.000; t=9.208, P=0.000. TRAF6 protein was significantly upregulated in muscle of stage Ⅰ (0.198±0.042), stage Ⅱ (0.209±0.051), stage Ⅲ (0.295±0.072), stage Ⅳ (0.345±0.084) gastric cancer compared with the control group (0.088±0.013), t=5.203, P=0.000; t= 5.642, P=0.000; t=9.351, P=0.000; t=9.854, P=0.000. TRAF6 was upregulated in 67.2% (43/64) of gastric cancer patients. The expression of TRAF6 in muscles of gastric cancer was associated with the percent of weight loss (χ2=12.231, P=0.000), the level of serum albumin (χ2=22.808, P=0.000) and the survival rate (χ2=3.933, P=0.047). Conclusion TRAF6 is significantly upregulated in rectus abdominis of gastric cancer, suggesting that TRAF6 may play an important role in progression and prognosis of cancer cachexia, and its overexpression suggests unfavorable prognosis. Key words: Stomach neoplasms; Rectus abdominis; Cachexia; Tumor necrosis factor receptor-asso-ciated factor 6

Tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) has been reported to be associated with the development of various cancers. However, the role of TRAF6 in lung cancer remains unclear. To explore the expression and clinicopathological significance of TRAF6 protein in lung cancer tissues. Three hundred and sixty-five lung cancer samples and thirty normal lung tissues were constructed into 3 microarrays. The expression of TRAF6 protein was determined using immunohistochemistry (IHC). Furthermore, correlations between the expression of TRAF6 and clinicopathological parameters were investigated. The expression of TRAF6 in total lung cancer tissues (365 cases), as well as in small cell lung cancer (SCLC, 26 cases) and non-small cell lung cancer (NSCLC, 339 cases) was significantly higher compared with that in normal lung tissues. The ROC curve showed that the area under curve of TRAF6 was 0.663 (95%CI 0.570~0.756) for lung cancer. The diagnostic sensitivity and specificity of TRAF6 were 52.6% and 80%, respectively. In addition, the expression of TRAF6 was correlated with clinical TNM stage, tumor size and lymph node metastasis in all lung cancers. Consistent correlations were also observed for NSCLCs. TRAF6 might be an oncogene and the expression of TRAF6 protein is related to the progression of lung cancer. Thus, TRAF6 might become a target for diagnosis and gene therapy for lung cancer patients.

The tumor necrosis factor receptor (TNFR)-associated factor 4 (TRAF4) is a member of TRAF family proteins that act as major signal transducers of the TNF receptor and the interleukin-1 receptor/Toll-like receptor (IL-1R/TLR) superfamily. TRAF4 has been reported to be overexpressed in various human cancers. However, the exact mechanisms that regulate the expression of TRAF4 still remain elusive. The objective of the present study was to investigate the regulatory mechanism of TRAF4 expression in prostate cancer. We initially identified microRNA-29a (miR‑29a) as a possible candidate to bind TRAF4 3' untranslated region (3'UTR) by the algorithm, TargetScan. The expression of TRAF4 mRNA and protein was inversely associated with miR-29a expression in prostate cancer cell lines (LNCaP, DU145 and PC3). TRAF4 expression was reduced by the introduction of mimic miR-29a in LNCaP cells. Luciferase activity from the construct harboring wild-type TRAF4 3'UTR was reduced by the mimic miR-29a and this reduction was diminished by introducing mutations at the predicted miR-29a binding site. On the other hand, TRAF4 was upregulated when transfected with the inhibitor of miR-29a in DU145 and PC3 cells. TRAF4 was significantly upregulated in patients with metastatic prostate cancer compared to those with localized prostate cancer. Furthermore, there was a significant inverse correlation between TRAF4 and miR-29a expression in tumor tissues from radical prostatectomy. Considered together, our results suggest that the tumor suppressor microRNA, miR-29a, is one of the regulators of TRAF4 expression in metastatic prostate cancer.

Objective: To explore the role of tumor necrosis factor receptor-associated factor 4 (TRAF4) in promoting the abnormal activation of epidermal growth factor receptor (EGFR) and its effect on lung cancer cell proliferation, migration and invasion. Methods: Tumor tissues from patients who underwent lung adenocarcinoma resection at The First Affiliated Hospital of Second Military Medical University, from January 2015 to May 2017 were collected, and the expressions of TRAF4 and Ki-67 in lung cancer tissues were detected by immunohistochemistry, the mRNA levels of Cyclin D and Vimentin were detected by real-time fluorescence quantitative PCR (qRT-PCR). The effect of TRAF4 on the tumor growth ability of lung cancer A549 cells was investigated by the xenograft model, the effect of TRAF4 or EGFR on the tumor proliferation ability was detected by using cell counting kit 8 (CCK8) and BrdU assay, and the migration and invasion abilities of tumor cells were detected by Transwell assay. Different structural domain deletion expression vectors of TRAF4 and EGFR were constructed to transfect cells, and the interaction mode of TRAF4 and EGFR was investigated by immunoprecipitation assay. Results: The expression of TRAF4 in non-small cell lung cancer (NSCLC) tissues was positively correlated with the expressions of Ki-67, cyclin D, and vimentin (r2: 0.438, 0.695, and 0.736, respectively, all P＜0.01). Immunohistochemical assay of tumor tissues from NSCLC patients showed that tissues with high expression of TRAF4 were also high in Ki-67. Patients with high TRAF4 expression (TRAF4 positivity >30%) had a shorter progression-free survival (PFS) time than that of patients with low TRAF4 expression (TRAF4 positivity ≤30%) (median PFS of 12 and 19 months, respectively; P=0.034). Traf4-/- cells had a weakened proliferative capacity than traf4+/+ cells and formed tumors with smaller size (P＜0.05). The expression level of Ki-67 in the tumor tissues formed by traf4-/- cells [(45.6±8.7)%] was lower than that in the tumor tissues formed by traf4+/+ cells [(62.3±10.3)%, P=0.015], the mRNA levels of cyclin D (1.01±0.15) and vimentin (1.01±0.12) in the traf4-/- cells were lower than those of the traf4+/+ cells (3.41±0.32 and 3.12±0.18, respectively, both P＜0.05).The western blot results showed that, with the elevated intracellular expression level of TRAF4, phosphorylation level of EGFR was significantly increased in both wild-type EGFR and activation mutant EGFR-expression cells. The capacities of proliferation, migration and invasion of A549 cells was weakened after EGFR knockdown (all P＜0.01). Immunoprecipitation experiments showed that TRAF4 binds to the peptide segment of the near-membrane region of EGFR through the TRAF structural domain, and the mutual binding between EGFR molecules was enhanced under TRAF4 overexpression conditions. Increasing TRAF4 expression promoted EGFR molecular phosphorylation and activation of downstream signaling. Conclusions: TRAF4 expression is elevated in NSCLC tissues and tumor cells, which promotes tumor proliferation, migration and invasion. TRAF4 directly binds to EGFR molecules, enhances its own phosphorylation and activates the downstream signaling pathway by promoting the interaction between EGFR molecules.

Tumor necrosis factor (TNF) receptor-associated factor 4 (TRAF4) was initially identified as a gene amplified and overexpressed in breast carcinoma. The present study investigated the expression and anti-apoptotic function of TRAF4 in human breast cancer MCF-7 cells. TRAF4 was found to be localized in the cytoplasm and nuclei of MCF-7 cells by immunofluorescence staining and western blotting. The expression of TRAF4 in normal MCF-10A breast cells was found to be lower than in MCF-7 and MDA-MB-231 breast cancer cells. Following TNF-α treatment, TRAF4 depletion by siRNA in the MCF-7 cells was observed to suppress cell proliferation and the nuclear expression of nuclear factor κB was significantly reduced. The percentage of early apoptotic cells in the MCF-7 cells was augmented upon TRAF4-knockdown, and an increase in G1 phase cells and a decrease in S phase cells was detected. These results indicate that TRAF4 has anti-apoptotic effects on apoptosis induced by TNF-α in MCF-7 cells.

Aberrant expression of tumor necrosis factor receptor-associated factor 6 (TRAF6) was reported in several types of cancers and it was demonstrated to promote tumor progression. In glioblastoma multiforme (GBM), TRAF6 depression by miRNA could decrease GBM cell resistance to temozolomide, but the prognostic values of TRAF6 and its functions in GBM progression have not been elucidated. In our study, the expression of TRAF6 and matrix metalloprotein 9 (MMP9) in 101 cases of GBM were investigated using immunohistochemistry. Twelve pairs of GBM frozen tissues and their corresponding adjacent tissues were collected during operation prospectively, and TRAF6 and MMP9 mRNA levels in them were detected using qRT-PCR. The correlations between TRAF6, MMP9, and clinicopathological factors were analyzed using the Chi-square test, and the prognostic value of TRAF6 and MMP9 were evaluated using univariate and multivariate analysis. The effect of TRAF6 and MMP9 on GBM cell invasion and proliferation was detected with experiments in vitro, and the correlation between TRAF6 and MMP9 expression was explored by regulating their expression with overexpression or knockdown. The expression of TRAF6 and MMP9 in GBM tissues was significantly higher than that in adjacent tissues. The expression of TRAF6 and MMP9 was significantly associated in GBM tissues. Both TRAF6 and MMP9 correlated with poor prognosis of GBM, and TRAF6 was identified as an independent prognostic factor of GBM. TRAF6 could promote invasion instead of proliferation of GBM cells via elevating expression of MMP9. TRAF6 was identified as an independent prognostic factor of GBM, with ability to promote invasion of GBM cells via elevating expression of MMP9.

The present study aimed to verify tumor necrosis factor receptor‑associated factor6 (TRAF6) as the target gene of microRNA-124 (miR-124). In addition, the expression of miR‑124 was investigated in osteosarcoma tissues and cells, and its effects on the biological characteristics of osteosarcoma cells were determined, in order to provide an experimental and theoretical basis for the application of TRAF6 in the treatment of osteosarcoma. A fluorescence reporter enzyme system was used to verify TRAF6 as a target gene of miR‑124, and western blotting was used to detect the effects of miR‑124 on the protein expression levels of TRAF6 in cells. The expression levels of miR‑124 were detected in osteosarcoma tissues and an osteosarcoma cell line (MG‑63) by quantitative polymerase chain reaction (qPCR). In addition, a total of 48h post‑transfection of MG‑63 cells with a miR‑124 mimic, qPCR was used to detect the expression levels of miR‑124, and the effects of miR‑124 on the viability of MG‑63 human osteosarcoma cells was determined using the MTT method. The effects of miR‑124 on the cell cycle progression and apoptosis of MG‑63 cells were analyzed by flow cytometry, whereas the effects of miR‑124 on the migration of MG‑63 cells was detected using the Transwell invasion chamber analysis method. A TRAF6 recombinant expression plasmid (pcDNA3.1‑TRAF6) was also constructed, and MG‑63 cells were transfected with the recombinant plasmid and a miR‑124 mimic, in order to further validate the biological role of miR‑124 via the regulation of TRAF6. The results of the present study indicated that, compared with in the normal control group, the expression levels of miR‑124 were significantly increased in MG‑63 cells transfected with a miR‑124 mimic (P<0.01). In addition, the luciferase reporter gene system demonstrated that, compared with in the control group, relative luciferase activity was significantly reduced in the miR‑124 mimic group (P<0.01). The results of MTT analysis indicated that cell viability was also significantly reduced in response to the overexpression of miR‑124 in MG‑63 cells (P<0.01). Flow cytometric analysis demonstrated that the proportion of cells in Sphase and G2/Mphase was significantly decreased (P<0.01) in cells overexpressing miR‑124, and the number of apoptotic cells was significantly increased (P<0.01). Furthermore, the results of the Transwell invasion assay suggested that the number of invasive cells was significantly decreased following enhanced expression of miR‑124 (P<0.01). In MG‑63 cells overexpressing miR‑124 and TRAF6, the results of MTT, flow cytometric and Transwell assay analyses demonstrated that the overexpression of TRAF6 had the opposite biological effects compared to miR‑124 overexpression. In conclusion, the present study indicated that the expression levels of miR‑124 were downregulated in human osteosarcoma tissues and cells, and that miR‑124 is associated with negative regulation of TRAF6 expression; therefore, the role of TRAF6 in primary osteosarcoma may be regulated by miR‑124. Therapeutic strategies that enhance miR‑124 expression or inhibit TRAF6 expression may be beneficial for the treatment of patients with osteosarcoma.

p63, a member of the p53 family, is overexpressed in squamous cell carcinoma of the head and neck (SCCHN) and some other tumors of epithelial origin. As a transcription factor, p63 can bind to p53-type response elements and there is some overlap between p53 family transcriptional targets. Tumor necrosis factor receptor associated factor 4 (TRAF4) is a p53 regulated gene which is overexpressed in many human carcinomas. We investigated the involvement of p63 in regulation of TRAF4 and the expression of the TRAF4 protein in SCCHN. Disrupting endogenous p63 expression resulted in downregulation of TRAF4 mRNA and protein in an SCCHN cell line. Endogenous p63 bound to the TRAF4 promoter in vivo and reporter assays showed that p63, p73 and p53 can all transactivate TRAF4, with TAp63 isoforms being the most potent activators. The level of TRAF4 activation by TAp63 was two-fold higher than by p53, and TRAF4 was 10-fold more responsive to TAp63 than another p63-target, IGFBP3. Nuclear expression of TRAF4 was seen in normal oral epithelium and highly/moderately differentiated SCCHN, whereas cytoplasmic expression of TRAF4 was seen in poorly differentiated SCCHN. These results indicate that TRAF4 is a common target of p53 family members and that localization of TRAF4 is associated with differentiation of SCCHN cells.

To investigate the role of tumor necrosis factor receptor-associated factor 6 (TRAF6) in regulating Bacillus Calmette-Guérin (BCG)-induced macrophage apoptosis. The expression of TRAF6 in peripheral blood samples of 50 patients with active tuberculosis (TB) and 50 healthy individuals were detected using quantitative real-time PCR (qPCR). RAW264.7 macrophages were infected with BCG at different MOI and for different lengths of time, and the changes in expressions of Caspase 3 and TRAF6 were detected with Western blotting and qPCR. In a RAW264.7 cell model of BCG infection with TRAF6 knockdown established using RNA interference technique, the bacterial load was measured and cell apoptotic rate and mitochondrial membrane potential (MMP) were determined with flow cytometry. The expression levels of TRAF6, Caspase 3, PARP, BAX and Bcl-2 in the cells were detected using Western blotting, and the expressions of TRAF6 and Caspase 3 were also examined with immunofluorescence assay. The expression of TRAF6 was significantly upregulated in the peripheral blood of patients with active TB as compared with healthy subjects (P < 0.001). In RAW264.7 cells, BCG infection significantly increased the expressions of Caspase 3 and TRAF6, which were the highest in cells infected for 18 h and at the MOI of 15. TRAF6 knockdown caused a significant increase of bacterial load in BCG-infected macrophages (P=0.05), lowered the cell apoptotic rate (P < 0.001) and reduced the expressions of Caspase 3 (P=0.002) and PARP (P < 0.001). BCG-infected RAW264.7 cells showed a significantly increased MMP (P < 0.001), which was lowered by TRAF6 knockdown (P < 0.001); the cells with both TRAF6 knockdown and BCG infection showed a lowered BAX expression (P=0.005) and an increased expression of Bcl-2 (P=0.04). TRAF6 promotes BCG-induced macrophage apoptosis by regulating the intrinsic apoptosis pathway.