Kaempferol attenuates streptozotocin-induced diabetic nephropathy by downregulating TRAF6 expression: The role of TRAF6 in diabetic nephropathy.

ETHNOPHARMACOLOGICAL RELEVANCE: Kaempferia rhizome is a famous traditional herbal medical in tropical and subtropical areas. Kaempferol (KPF) is one of the main bioactive compounds in Kaempferia rhizome, with anti-oxidant/anti-inflammatory effects demonstrated in various disease models, including cancers, obesity and diabetes. AIM OF THE STUDY: Inflammation plays an important role in the pathogenesis of diabetic nephropathy (DN). TRAF6 functions as a signal transducer in toll-like receptor 4 and NF-κB pro-inflammatory signaling pathway. We aimed at investigate whether KPF is able to mitigate inflammatory responses by regulating TRAF6 in DN. MATERIAL AND METHODS: C57BL/6 mice were injected with streptozotocin to induce type 1 DN. NRK-52E, a tubular epithelial cell line, was used for in vitro analysis. TRAF6 was knockdown using siRNA in vitro and AAV2/2-shRNA in vivo. The anti-DN and inflammatory effects of KPF or knockdown of TRAF6 were evaluated by investigating renal filtration index, pathological changes of kidney tissue. Proinflammatory cytokine levels were detected using ELISA. NF-κB pathway and protein levels of related pathways were detected through Western blot. RESULTS: KPF significantly reduced renal inflammation, fibrosis, and kidney dysfunction in diabetic mice. These effects were associated with a downregulation of TRAF6 in diabetic mouse kidneys, indicating the potential role of TRAF6. Knockdown of TRAF6 in mice through AAV2-shTRAF6 confirmed the importance of TRAF6 in DN. In vitro, treatment of KPF in NRK-52E cells attenuated high glucose (HG)-induced inflammatory and fibrogenic responses, associated with downregulated TRAF6 expression. The conclusion was further confirmed in NRK-52E cells by knocking down the expression and by overexpression of TRAF6. CONCLUSION: Our findings provide direct evidence that TRAF6 mediates diabetes-induced inflammation leading to renal dysfunction. We also show that KPF is a potential therapeutic agent to reduce inflammatory responses in DN. Also, TRAF6 may represent an interesting target to combat DN.

Downregulation of lncRNA NEAT1 Ameliorates LPS-Induced Inflammatory Responses by Promoting Macrophage M2 Polarization via miR-125a-5p/TRAF6/TAK1 Axis.

The lncRNA nuclear enriched abundant transcript 1 (NEAT1) promotes sepsis-inflammatory responses and acute kidney injury (AKI), but little known about the underlying mechanisms. This study aims to investigate the roles of NEAT1 in regulating macrophage polarization and its potential for alleviating inflammatory responses during sepsis pathogenesis. Mouse RAW264.7 macrophages were treated with lipopolysaccharide (LPS) as a cellular inflammatory model. NEAT1 shRNA, miR-125a-5p mimics, and TRAF6-overexpressing vector were used to transfect RAW264.7 cells. NEAT1, miR-125a-5p, and mRNA levels of functional genes were detected by quantitative RT-PCR. Protein abundances were analyzed by western blotting. Macrophage polarization was evaluated by flow cytometry. The bindings of miR-125a-5p with NEAT1 or TRAF6 gene were validated by dual luciferase reporter assay. LPS treatment promoted NEAT1 and suppressed miR-125a-5p expression in mouse macrophage cells. NEAT1 silencing by shRNAs promoted macrophage M2 polarization under LPS treatment, which upregulated miR-125a-5p expression, repressed TRAF6 expression and TAK1 protein phosphorylation in macrophages. These cellular and molecular changes induced by NEAT1 shRNAs were abrogated by miR-125a-5p inhibitors. Moreover, miR-125a-5p mimics suppressed TRAF6 expression and TAK1 protein phosphorylation in LPS-treated macrophages, thus causing macrophage M2 polarization under LPS treatment. TRAF6 overexpression abrogated the miR-125a-5p mimics-induced macrophage M2 polarization. miR-125a-5p could directly bind to NEAT1 or TRAF6 gene in macrophages. lncRNA NEAT1 knockdown ameliorates LPS-induced inflammation by promoting macrophage M2 polarization via miR-125a-5p/TRAF6/TAK1 axis.

MiR-143 Inhibits Osteoclastogenesis by Targeting RANK and NF-κB and MAPK Signaling Pathways.

OBJECTIVE: To explore the effect of miRNA-143 on osteoclast formation and provide new ideas for the treatment of osteoporosis. METHODS: Mice macrophage lines RAW264.7 cells after transfection were divided into four groups: control group, RANKL group, RANKL combined with miR-143 mimics group and RANKL combined with miR-NC group. TARCP staining was used to observe the effect of miR-143 on osteoclast formation. The expression of RANK, TRAF6 and NFATc-1 in the upstream of RANKL pathway was detected by real-time quantitative PCR (RT qPCR) and Western blotting (WB). The binding of miR-143 to TNFRSF11A was detected by double Luciferase Reporter Analysis. The effect of miR-143 on the expression of NF-κB (p65, I-κB-α) signal pathway in osteoclasts was detected. The effects of I-BET151 on the expression of osteoclast-specific genes TRACP, MMP 9, CtsK and c-Src were detected. RESULTS: The positive level of osteoclasts in RANKL group and RANKL combined with miR-NC group was significantly higher than that of RANKL combined with miR-143 mimics group and control group (P < 0.05). The expression levels of RANK, TRAF6, NFATc-1, TRACP, MMP-9, CtsK and c-Src in RANKL group and RANKL combined with miR-NC group were significantly higher than those of RANKL combined with miR-143 mimics group and control group (P < 0.05). The expression levels of I-κB-α were significantly lower than that of RANKL combined with miR-143 mimics group and control group (P<0.05). CONCLUSION: MiR-143 can inhibit the expression of RANK, TRAF6 and downstream NFATc-1 in the RANKL pathway, thereby inhibiting the RANK/RANKL pathway. MiR-143 can inhibit the signal pathway of NF-κB (p65, I-κB-α). MiR-143 inhibits the expression of osteoclast-specific genes TRACP, MMP 9, CtsK and c-Src. That is to say, miR-143 inhibits osteoclast formation by targeting RANK, NF- κB and MAPK signaling pathways.

Swimming Impacts on Pancreatic Inflammatory Cytokines, miR-146a and NF-кB Expression Levels in Type-2 Diabetic Rats.

BACKGROUND: Obesity-induced chronic inflammation is a key component in the pathogenesis of insulin resistance and type-2 diabetes Objective: This study aimed to evaluate the effect of swimming exercise on pancreatic expression levels of inflammatory cytokines, miR-146a and NF-кB in type-2 diabetic male rats. METHODS: Twenty- eight male Wistar rats were divided into four groups: control (Con), exercise, diabetes and diabetic exercise (n = 7). Diabetes induction performed by the combination of high-fat diet (HFD, 4 weeks) and streptozotocin (35 mg/kg. ip). After induction of diabetes, the rats swam in the exercise groups for 12 weeks. Then, blood and tissue samples were collected. RESULTS: Our results indicated a significant increase in expression levels of miR-146, NF-κB and inflammatory cytokines (IL-6, TNF-α, and IL-1ß) while a significant decrease in pancreatic expression levels of TRAF6 and IRAK1 in diabetic group as compared to the control group. In contrast, swimming exercise resulted in a significant decrease in expression levels of miR-146a, NF-кB and inflammatory cytokines and a significant increase in expression levels of TRAF6 and IRAK1 in the exercise-diabetic group compared to the diabetic group. CONCLUSION: Our results indicated a significant increase in expression levels of miR-146, NF-κB and inflammatory cytokines (IL-6, TNF-α, and IL-1ß) while a significant decrease in pancreatic expression levels of TRAF6 and IRAK1 in diabetic group as compared to the control group. In contrast, swimming exercise resulted in a significant decrease in expression levels of miR-146a, NF-кB and inflammatory cytokines and a significant increase in expression levels of TRAF6 and IRAK1 in the exercise-diabetic group compared to the diabetic group.

TRAF6 correlated to invasion and poor prognosis of glioblastoma via elevating MMP9 expression.

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.

Gender difference in the effect of progesterone on neonatal hypoxic/ischemic brain injury in mouse.

This study investigated the effects of progesterone (PROG) on neonatal hypoxic/ischemic (NHI) brain injury, the differences in effects between genders, and the underlying mechanisms. NHI brain injury was established in both male and female neonatal mice induced by occlusion of the left common carotid artery followed by hypoxia. The mice were treated with PROG or vehicle. Fluoro-Jade B staining (F-JB), long term behavior testing, and brain magnetic resonance image (MRI) were applied to evaluate neuronal death, neurological function, and brain damage. The underlying molecular mechanisms were also investigated by Western blots. The results showed that, in the male mice, administration of PROG significantly reduced neuronal death, improved the learning and memory function impaired by cerebral HI, decreased infarct size, and maintained the thickness of the cortex after cerebral HI. PROG treatment, however, did not show significant neuroprotective effects on female mice subjected to HI. In addition, the data demonstrated a gender difference in the expression of tumor necrosis factor receptor 1 (TNFR1), TNF receptor associated factor 6 (TRAF6), Fas associated protein with death domain (FADD), and TIR-domain-containing adapter-inducing interferon-ß (TRIF) between males and females. Our results indicated that treatment with PROG had beneficial effects on NHI injured brain in acute stage and improved the long term cognitive function impaired by cerebral HI in male mice. In addition, the activation of TNF and TRIF mediated signaling in response to cerebral HI and the treatment of PROG varied between genders, which highly suggested that gender differences should be emphasized in evaluating neonatal HI brain injury and PROG effects, as well as the underlying mechanisms.

Mice with miR-146a deficiency develop severe gouty arthritis via dysregulation of TRAF 6, IRAK 1 and NALP3 inflammasome.

BACKGROUND: MicroRNAs (miRNAs) serve as important regulators of inflammatory and immune responses and are implicated in several immune disorders including gouty arthritis. The expression of miR-146a is upregulated in the peripheral blood mononuclear cells of patients with inter-critical gout when compared to normouricemic and hyperuricemic controls and those patients with acute gout flares. However, the role of miR-146a in the development of gout remains unknown. Here, we used miR-146a knockout (KO) mice to test miR-146a function in a monosodium urate (MSU)-induced gouty arthritis model. METHODS: The footpad or ankle joint of miR-146a KO and wild-type (WT) mice were injected with an MSU suspension to induce acute gouty arthritis. Bone marrow-derived macrophages (BMDMs) were stimulated with MSU and the gene expression of miR-146a; interleukin 1 beta (IL-1ß); tumor necrosis factor-α (TNF-α); and the NACHT, LRR and PYD domains-containing protein 3 (NALP3) inflammasome was evaluated. TNF-α and IL-1ß protein levels in BMDMs were assessed by fluorescence-activated cell sorting and western blot analyses. Gene and protein levels of TNF receptor-associated factor 6 (TRAF6) and IL-1 receptor-associated kinase (IRAK1), the targets of miR-146a, were also measured. RESULTS: Significantly increased paw swelling and index and ankle joint swelling were observed in miR-146a KO mice compared to WT controls after MSU treatment. MiR-146a expression in BMDMs from WT mice was dramatically upregulated at 4 h following MSU stimulation. Additionally, the expression of IL-1ß, TNF-α, and NALP3 was higher in BMDMs from miR-146a KO mice after exposure to MSU crystals compared to those from WT mice. Consistent with the observed gene expression, the IL-1ß and TNF-α proteins were upregulated in miR-146a KO mice. Additionally quantitative RT-PCR and western blot demonstrated that TRAF6 and IRAK1 were dramatically upregulated in BMDMs from miR-146 KO mice compared to those from WT mice. CONCLUSIONS: Collectively, these observations suggest that miR-146a provides negative feedback regulation of gouty arthritis development and lack of miR-146a enhances gouty arthritis via upregulation of TRAK6, IRAK-1, and the NALP3 inflammasome function.

Tumor necrosis factor receptor-associated factor 6 mediated the promotion of salivary adenoid cystic carcinoma progression through Smad-p38-JNK signaling pathway induced by TGF-ß.

BACKGROUND: Tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) has been proved to play an important role in tumorigenesis, invasion, and metastasis. However, its precise role salivary adenoid cystic carcinoma (SACC) has not been determined. The aim of this study was to explore the role of TRAF6 in SACC including invasion and metastasis of SACC cells. MATERIALS AND METHODS: Immunohistochemistry and quantitative real-time PCR were performed in SACC tissues paired with their adjacent normal tissues to analyze the expression of TRAF6. Downstream proteins expression was explored when TRAF6 was knockdown by siRNA. RESULTS: The results show that TRAF6 is upregulated in SACC samples, especially in SACC with metastasis, which is closely correlated with an aggressive phenotype (P = .0073) and shorter life survival span (P = .0061) in SACC patients. Knockdown of TRAF6 can attenuate the promotion effect of SACC cell invasion induced by TGF-ß. Western blot results also showed that silencing TRAF6 expression can inhibit the activation of SMAD2, SMAD3, ERK, p38, and JNK induced by TGF-ß in SACC cells. CONCLUSION: These data suggested that TRAF6 regulates TGF-ß-mediated SACC progression through SMAD2/3-ERK-p38-JNK cascades.

Effect of Tumor Necrosis Factor Inhibitor Therapy on Osteoclasts Precursors in Rheumatoid Arthritis.

Objective. Tumor necrosis factor (TNF) increases circulating osteoclast (OC) precursors numbers by promoting their proliferation and differentiation. The aim of this study was to assess the effect of TNF inhibitors (TNFi) on the differentiation and activity of OC in rheumatoid arthritis (RA) patients. Methods. Seventeen RA patients treated with TNFi were analyzed at baseline and after a minimum follow-up period of 6 months. Blood samples were collected to assess receptor activator of nuclear factor kappa-B ligand (RANKL) surface expression on circulating leukocytes and frequency and phenotype of monocyte subpopulations. Quantification of serum levels of bone turnover markers, in vitro OC differentiation assays, and qRT-PCR for OC specific genes was performed. Results. After TNFi therapy, patients had reduced RANKL surface expression in B-lymphocytes and the frequency of circulating classical CD14brightCD16- monocytes was decreased. Serum levels of sRANKL, sRANKL/OPG ratio, and CTX-I were reduced in RA patients after TNFi treatment. Moreover, after exposure to TNFi, osteoclast differentiation and activity were decreased, as well as the expression of TRAF6 and cathepsin K. Conclusion. We propose that TNFi arrests bone loss and erosion, through two pathways: direct reduction of osteoclast precursor numbers and inhibition of intracellular signaling pathways acting through TRAF6.

TLR4-MyD88-TRAF6-TAK1 Complex-Mediated NF-κB Activation Contribute to the Anti-Inflammatory Effect of V8 in LPS-Induced Human Cervical Cancer SiHa Cells.

The synthetic compound 7-4-[Bis-(2-hydroxyethyl)-amino]-butoxy-5-hydroxy-8-methoxy-2-phenylchromen-4-one (V8) is a novel flavonoid-derived compound. In this study, we investigated the effects of V8 on Toll-like receptor 4 (TLR4)-mediated inflammatory reaction in human cervical cancer SiHa cells and lipopolysaccharide (LPS)-induced TLR4 activity in cervical cancer SiHa (HPV16+) cells, but not in HeLa (HPV18+) and C33A (HPV-) cells. In addition, V8 inhibited LPS-induced expression of TLR4, MyD88, TRAF6 and phosphorylation of TAK1, and their interaction with TLR4 in SiHa cells, resulting in an inhibition of TLR4-MyD88-TRAF6-TAK1 complex. Moreover, V8 blocked LPS-induced phosphorylation of IκB and IKK, resulting in inhibition of the nuclear translocation of P65-NF-κB in SiHa cells. We also found that V8 reduced the expression of NF-κB target genes, such as those for COX-2, iNOS, IL-6, IL-8, CCL-2, and TNF-α in LPS-stimulated SiHa cells. These results suggested that V8 exerted an anti-inflammatory effect on SiHa cells by inhibiting the TLR4-MyD88-TRAF6-TAK1 complex-mediated NF-κB activation.

Synergistic antitumor effects of radiation and proteasome inhibitor treatment in pancreatic cancer through the induction of autophagy and the downregulation of TRAF6.

Ninety percent of human pancreatic cancer is characterized by activating K-RAS mutations. TRAF6 is an oncogene that plays a vital role in K-RAS-mediated oncogenesis. We investigated the synergistic effect of combining ionizing radiation (IR) and proteasome inhibitor (MG132). Furthermore, following combined treatment with IR and MG132, we analyzed the expression of TRAF6 and the mechanism of human pancreatic cancer cell death in vitro and in an orthotopic pancreatic cancer mouse model. The combined treatment groups displayed synergistic cell killing effects and induced endoplasmic reticulum stress in human pancreatic cancer cells. The combined treatment groups were characterized by enhanced cytotoxicity, which resulted from increased autophagy induction through the inhibition of TRAF6. Significantly reduced cytotoxicity was observed following MG132 and IR treatment of MIA PaCa-2 cells pre-treated with 3-MA (an autophagy inhibitor). Down-regulation of TRAF6 led to a significant increase in apoptosis and autophagy. In an orthotopic xenograft model of SCID mice, combination MG132 and IR therapy resulted in a significant increase in the tumor growth delay time and a decreased tumor tissue expression of TRAF6. IR combined with a proteasome inhibitor or TRAF6 inhibition could represent a new therapeutic strategy for human pancreatic cancer.

Using a novel microRNA delivery system to inhibit osteoclastogenesis.

Previously, we developed a novel microRNA (miRNA) delivery system based on bacteriophage MS2 virus-like particles (MS2 VLPs). In this current study, we used this system to transport miR-146a into human peripheral blood mononuclear cells (PBMCs), and demonstrated the inhibition of osteoclastogenesis in precursors. Two cytokines, receptor activator of NF-κB ligand (RANKL), and macrophage-colony stimulating factor (M-CSF) were used to induce osteoclastogenesis. MS2 VLPs were transfected into PBMCs. qRT-PCR was applied to measure expression levels of miR-146a and osteoclast (OC)-specific genes. Western blot (WB) was conducted to evaluate miR-146a downstream target proteins: epidermal growth factor receptor (EGFR) and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6). The formation and activity of OCs were assessed by cytochemical staining and bone resorption assay, respectively. In PBMCs treated with MS2-miR146a VLPs, qRT-PCR assays showed increased expression of miR-146a (p < 0.01) and decreased expression of all four OC-specific genes (p < 0.05). WB results indicated decreased expression of EGFR (p < 0.01) and TRAF6 (p < 0.05). The number of OCs decreased markedly and bone resorption assay demonstrated inhibited activity. This miR-146a delivery system could be applied to induce overexpression of miR-146a and to inhibit the differentiation and function of OCs.

Corilagin Protects Against HSV1 Encephalitis Through Inhibiting the TLR2 Signaling Pathways In Vivo and In Vitro.

In this study, we tried to explore the molecular mechanism that Corilagin protected against herpes simplex virus-1 encephalitis through inhibiting the TLR2 signaling pathways in vivo and in vitro. As a result, Corilagin significantly prevented increase in the levels of TLR2 and its downstream mediators following Malp2 or HSV-1 challenge. On the other hand, in spite of TLR2 knockdown, Corilagin could still significantly suppress the expression of P38 and NEMO, phosphor-P38, and nuclear factor kappa B. The mRNA and protein expression of TLR2 and its downstream mediators in the brain tissue were also significantly lowered in mice treated with Corilagin. In addition, Corilagin inhibited expression of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 protein. In conclusion, Corilagin shows the potential to protect against HSV-1-induced encephalitis, and the beneficial effects may be mediated by inhibiting TLR2 signaling pathways.

TRAF6 is over-expressed in pancreatic cancer and promotes the tumorigenicity of pancreatic cancer cells.

Pancreatic cancer is one of the most lethal malignancies, with a poor response to chemotherapy and therefore it is important to identify novel therapeutic targets. TNF receptor-associated factor 6 (TRAF6) , a regulator of NF-κB signaling, has been found recently to be involved in tumorigenesis. However, its function in pancreatic cancer remains poorly understood. Here, we found that the expression of TRAF6 was up-regulated in pancreatic cancer tissues. Moreover, over-expression of TRAF6 in pancreatic cancer cells promoted cell proliferation and migration, whereas down-regulation of TRAF6 impaired the tumorigenicity of pancreatic cancer cells in vitro and in vivo. Mechanistically, TRAF6 regulated the expression of multiple genes involved in cell growth, apoptosis and migration. Our results suggested several important roles of TRAF6 in the pathogenesis of pancreatic cancer. TRAF6 might therefore represent a potential therapeutic target.

MicroRNA-146a provides feedback regulation of lyme arthritis but not carditis during infection with Borrelia burgdorferi.

MicroRNAs have been shown to be important regulators of inflammatory and immune responses and are implicated in several immune disorders including systemic lupus erythematosus and rheumatoid arthritis, but their role in Lyme borreliosis remains unknown. We performed a microarray screen for expression of miRNAs in joint tissue from three mouse strains infected with Borrelia burgdorferi. This screen identified upregulation of miR-146a, a key negative regulator of NF-κB signaling, in all three strains, suggesting it plays an important role in the in vivo response to B. burgdorferi. Infection of B6 miR-146a-/- mice with B. burgdorferi revealed a critical nonredundant role of miR-146a in modulating Lyme arthritis without compromising host immune response or heart inflammation. The impact of miR-146a was specifically localized to the joint, and did not impact lesion development or inflammation in the heart. Furthermore, B6 miR-146a-/- mice had elevated levels of NF-κB-regulated products in joint tissue and serum late in infection. Flow cytometry analysis of various lineages isolated from infected joint tissue of mice showed that myeloid cell infiltration was significantly greater in B6 miR-146a-/- mice, compared to B6, during B. burgdorferi infection. Using bone marrow-derived macrophages, we found that TRAF6, a known target of miR-146a involved in NF-κB activation, was dysregulated in resting and B. burgdorferi-stimulated B6 miR-146a-/- macrophages, and corresponded to elevated IL-1ß, IL-6 and CXCL1 production. This dysregulated protein production was also observed in macrophages treated with IL-10 prior to B. burgdorferi stimulation. Peritoneal macrophages from B6 miR-146a-/- mice also showed enhanced phagocytosis of B. burgdorferi. Together, these data show that miR-146a-mediated regulation of TRAF6 and NF-κB, and downstream targets such as IL-1ß, IL-6 and CXCL1, are critical for modulation of Lyme arthritis during chronic infection with B. burgdorferi.

Potential anti-osteoporotic activity of low-molecular weight hyaluronan by attenuation of osteoclast cell differentiation and function in vitro.

Due to some severe side effects or lack of efficacy of currently used synthetic drugs, such as bisphosphonates (BPs), the search for new therapeutic agents that can more effectively prevent and treat osteoporosis (OP) has been an increasingly important topic of research. In this study, the low-molecular weight hyaluronan (LMW-HA, 50 kDa) produced by enzymatic degradation of high-molecular weight hyaluronan (HMW-HA, 1922 kDa) from Streptococcus zooepidemicus was evaluated in vitro for its anti-osteoclastogenic potentials using RAW 264.7 murine macrophage cells. LMW-HA (25-200 µg/ml) dose-dependently inhibited the receptor activator of NF-κB ligand (RANKL)-induced tartrate-resistance acid phosphatase (TRAP) activity and the formation of multinucleated osteoclasts. Western blot analysis showed that LMW-HA reduced the RANKL-induced expression of tumor necrosis factor receptor-associated factor 6 (TRAF6), gelsolin and c-Src-proline-rich tyrosine kinase 2 suggesting that it could inhibit actin ring formation of osteoclast cells. In addition, LMW-HA inhibited the bone resorption activity of osteoclastic cells by dose-dependently attenuating the RANKL-induced expression of carbonic anhydrase II and integrin ß3. RT-PCR analysis showed that LMW-HA dose-dependently decreased the expression of osteoclast-specific genes, such as matrix metalloproteinase 9 (MMP-9) and cathepsin K, suggesting that it has potential to inhibit the differentiation of osteoclastic cells. Taken collectively, these results suggested that LMW-HA (50 kDa) has significant anti-osteoporotic activity in vitro and may be used as a potent functional ingredient in health beneficial foods or as a therapeutic agent to prevent or treat OP.

Effect of TRAF6 on the biological behavior of human lung adenocarcinoma cell.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a unique adaptor protein of the tumor necrosis factor receptor-associated factor family that mediates both tumor necrosis factor receptor and interleukin-1 receptor/Toll-like receptor signaling. A recent study showed that TRAF6 played an important role in tumorigenesis and invasion through activation of nuclear factor kappa B (NF-κB). However, the biological role of TRAF6 remains unknown in lung cancer up to now. To address the expression of TRAF6 in lung cancer cells, four lung cancer cell lines (A549, HCC827, NCI-H292, and 95-D) and human bronchial epithelial cells were used to detect the expression of TRAF6 protein by western blotting. Results indicated that TRAF6 displayed an upregulation in human lung cancer cell lines. To investigate the effects of TRAF6 on the biological behavior of human lung adenocarcinoma cell, we generated human lung adenocarcinoma A549 cell line in which TRAF6 was depleted. The results showed that downregulation of TRAF6 could decrease cell viability, suppress cell proliferation and invasion, and promote cell apoptosis. At the same time, we explored the effects of TRAF6 on the expression of the following proteins: phosphor-NF-κB (p-p65), cyclin D1, caspase-3, and matrix metalloproteinase 9 (MMP9). Downregulation of TRAF6 could decrease the expression of p-p65, cyclin D1, and MMP9 and increase the expression of caspase-3. All these results suggested that TRAF6 might be involved in the potentiation of growth, proliferation, and invasion of A549 cell line, as well as the inhibition of A549 cell apoptosis by the activation of NF-κB. To make a long story short, the overexpression of TRAF6 might be related to the tumorigenesis and invasion of lung cancer.

Up-regulated tumor necrosis factor-associated factor 6 level is correlated with apoptosis in the rat cerebral ischemia and reperfusion.

Tumor necrosis-associated factor 6 (TRAF6) performs critical roles in mediating apoptosis-associated inflammatory processes in multiple cell types, but its role in cerebral ischemia-reperfusion (I/R) injury is still unclear. In the present study, we established a middle cerebral artery occlusion (MCAO) reperfusion model in rat, and evaluated both the cerebral inflammatory damage and the cell apoptosis by TTC staining and TUNEL method, respectively. The expression of TRAF6 and the neural cell apoptosis was examined during the I/R pathophysiological process. Cerebral ischemia injury induced significant neuronal cell apoptosis, but after the onset of reperfusion, cell apoptosis was gradually alleviated. In accord with the trend of I/R injury and cell apoptosis, up-regulated TRAF6 mRNA expression and caspase-3 cleavage level were observed in the ischemia stage and the early stage of reperfusion accordingly, which indicated that the activation of TRAF6 correlated positively with the cell apoptosis. Immunohistochemistry staining further showed that the TRAF6 was mainly localized in the neuronal cells. Thus, our study suggested that TRAF6 is involved in the inflammatory process induced by cerebral ischemia-reperfusion, and functions partially as a pro-inflammatory adaptor to mediate cell apoptosis.

Association of self-DNA mediated TLR9-related gene, DNA methyltransferase, and cytokeratin protein expression alterations in HT29-cells to DNA fragment length and methylation status.

To understand the biologic role of self-DNA bound to Toll-like Receptor 9 (TLR9), we assayed its effect on gene and methyltransferase expressions and cell differentiation in HT29 cells. HT29 cells were incubated separately with type-1 (normally methylated/nonfragmented), type-2 (normally methylated/fragmented), type-3 (hypermethylated/nonfragmented), or type-4 (hypermethylated/fragmented) self-DNAs. Expression levels of TLR9-signaling and proinflammatory cytokine-related genes were assayed by qRT-PCR. Methyltransferase activity and cell differentiation were examined by using DNA methyltransferase (DNMT1, -3A, -3B) and cytokeratin (CK) antibodies. Treatment with type-1 DNA resulted in significant increase in TLR9 expression. Type-2 treatment resulted in the overexpression of TLR9-related signaling molecules (MYD88A, TRAF6) and the IL8 gene. In the case of type-3 treatment, significant overexpression of NFkB, IRAK2, and IL8 as well as downregulation of TRAF6 was detected. Using type-4 DNA, TRAF6 and MYD88A gene expression was upregulated, while MYD88B, IRAK2, IL8, and TNFSF10 were all underexpressed. CK expression was significantly higher only after type-1 DNA treatment. DNMT3A expression could also be induced by type-1 DNA treatment. DNA structure may play a significant role in activation of the TLR9-dependent and even independent proinflammatory pathways. There may be a molecular link between TLR9 signaling and DNMT3A. The mode of self-DNA treatment may influence HT29 cell differentiation.

TRAF6 and p62 inhibit amyloid ß-induced neuronal death through p75 neurotrophin receptor.

Amyloid ß (Aß) aggregates are the primary component of senile plaques in Alzheimer disease (AD) patient's brain. Aß is known to bind p75 neurotrophin receptor (p75(NTR)) and mediates Aß-induced neuronal death. Recently, we showed that NGF leads to p75(NTR) polyubiquitination, which promotes neuronal cell survival. Here, we demonstrate that Aß stimulation impaired the p75(NTR) polyubiquitination. TRAF6 and p62 are required for polyubiquitination of p75(NTR) on NGF stimulation. Interestingly, we found that overexpression of TRAF6/p62 restored p75(NTR) polyubiquitination upon Aß/NGF treatment. Aß significantly reduced NF-κB activity by attenuating the interaction of p75(NTR) with IKKß. p75(NTR) increased NF-κB activity by recruiting TRAF6/p62, which thereby mediated cell survival. These findings indicate that TRAF6/p62 abrogated the Aß-mediated inhibition of p75(NTR) polyubiquitination and restored neuronal cell survival.