Transforming Growth Factor-β-activated Kinase 1 Research Articles

TAK1 as the mediator in the protective effect of propofol on renal interstitial fibrosis induced by ischemia/reperfusion injury

Ischemia-reperfusion injury (IRI), which is a major cause of acute and chronic renal dysfunction, induces both apoptosis and fibrotic processes. The mitogen-activated protein kinase kinase kinase transforming growth factor-β-activated kinase 1 (TAK1) was implicated in the processes of inflammation and fibrosis. The protective effect of propofol on renal functionality after acute kidney injury (AKI) in mice has been identified, whereas the mechanisms underlying fibrosis induced by kidney injury remain obscure. Herein, we investigated whether the protective effect of propofol on renal interstitial fibrosis induced by ischemia/reperfusion injury was modulated by TAK1 in renal ischemia /reperfusion (I/R) mouse models. The results of immunohistochemistry and western blotting revealed that TAK1 was significantly upregulated in IR group versus the control group, which was reversed by propofol administration. In addition, fibronectin (FN), α-smooth muscle actin (α-SMA) and type I collagen (COL1) were significantly downregulated and Tunnel staining revealed the number of tubular apoptotic cells was markedly reduced in IRP group versus IR group. Collectively, our results validated that propofol could ameliorate the IRI-induced renal interstitial fibrosis in mice by downregulation of TAK1 and inhibition of apoptosis at the early stage.

Abstract 240: TGFβ-Activated Kinase 1 is Required for Arteriovenous Fistula Maturation

Objectives: Arteriovenous fistulae (AVF) remain the optimal conduit for hemodialysis access but continue to have high rates of primary failure to mature. Transforming growth factor-β-activated kinase 1 (TAK1) plays important roles in regulation of extracellular matrix (ECM) production and deposition as well as inflammatory signaling and prevention of apoptosis; however the function of TAK1 in mechanotransduction in response to hemodynamic changes such as occur during AVF maturation is not well understood. Since deposition of ECM is critical to AVF maturation, we hypothesized that TAK1 is a critical regulator of AVF maturation. Methods: Aortocaval fistulae were created via needle puncture in wild-type (WT) C57BL/6J mice. AVF diameter was serially assessed weekly by duplex ultrasound; AVF were harvested at days 7 or 21 for histological analysis using computerized morphometry, as well as qPCR and Western blot. Some mice were treated with either the TAK1 inhibitor 5Z-7-oxozeaenol (OZ, 0.5mg/kg/day, 7 days) or Lentiviral TAK1 ShRNA (1x 10 8 pfu/ml; adventitial transduction). Mouse lung endothelial cells (MLECs) were exposed to laminar shear stress (3 or 20 dyne/cm 2 , 1h); TAK1 signaling was detected by Western blot and immunofluorescence. Results: TAK1 mRNA expression was increased at days 7 and 21 in AVF (2.0-fold; day 21; p<0.05) compared with sham controls. In AVF treated with OZ there was reduced fistula diameter (p=0.0059) and wall thickness (p=0.031) at day 21, as well as reduced collagen and fibronectin deposition (p<0.05), compared to controls. TAK1 knockdown with ShRNA also showed reduced fistula diameter at day 21 (p<0.05), compared to controls. MLECs exposed to arterial magnitudes of laminar shear stress showed increased TAK1 phosphorylation and downstream signaling (p<0.05) compared to MLECs exposed to venous shear stress or static conditions, which was reduced in cells either pretreated with OZ (p<0.0001) or transfected with TAK1 ShRNA (p<0.05). Conclusions: TAK1 regulates ECM production during AVF maturation. Strategies to alter TAK1 function in vivo may be a novel therapeutic approach to improve AVF maturation.

Effect of RNA Interference-silenced TAK1 on Kasumi-1 cell Proliferation Inhibition Induced by As2O3 and Its Mechanism

To explore the effect of transforming growth factor-β activated kinase-1(TAK1) gene silenced by RNA interference on proliferation inhibition of Kasumi-1 cells induced by As2O3 and its mechanism. The experiments were divided into 4 groups, including control group(Kasumi-1 cells treated with non-specific siRNA), TAK1 specific siRNA treated group (Kasumi 1 treated with TAK specific siRNA), As2O3 treated group (Kasumi 1 cells treated with As2O3) and combined treated group (Kasumi 1 cells treated with TAK1 specific siRNA plus As2O3). The proliferation inhibition rate of Kasami 1 cells was detected by CCK-8 method, the apoptotic rate of cells was detected by flow eytometry, the expressions of TAK1, phosphorylated c-Jun N-terminal kinase(p-JNK) and apoptosis-related proteins were detected by Western blot. As2O3 could inhibit Kasumi-1 cell proliferation in a dose-dependent manner between 0.5 to 20 µmol/L with IC50 of (3.79±0.36) µmol/L at 24 h, and also inhibit Kasumi-1 cell proliferation in a dose-dependent manner between 0.5 to 10 µmol/L with IC50 of (2.38±0.17) µmol/L at 48 h, but then the inhibitory effect reached plateau. After treating Kasumi-1 cells with TAK1 siRNA and 3.5 µmol/L As2O3 for 24 h, the proliferation inhibition rate was (10.86±1.64)% and (49.80±2.19)%, meanwhile the apoptosis rate was (8.47±0.75)% and (24.78±2.14)%, all significantly higher than those in control group (P<0.05, P<0.01). The proliferation inhibition rate and apoptosis rate of the combined treated group were significantly higher than that in control and single treated groups (P<0.05, P<0.01), TAK1 silencing and 3.5 µmol/L of As2O3 could decrease the expression of TAK1, p-JNK, c-Fos, c-Jun and BCL-2 in different degrees, and increase the expression levels of BAX and the activated (cleaved) caspase-3, 9 with statistically significant differences as compared with control group (P< 0.05). When Kasumi-1 cells were treated with TAK1 specific siRNA plus As2O3 for 24 h, protein expression levels were all significantly greater than that in the single-treated groups (P< 0.05). TAK1 silencing and As2O3 can separately and synergistically inhibit Kasumi-1 cell proliferation which probably relates with the inducing apoptosis via the JNK and mitochondrial pathway. Meanwhile, TAK1 silencing enhances the inhibitory effect of As2O3 on Kasumi-1 cell proliferation.

Silencing TAK1 alters gene expression signatures in bladder cancer cells.

The aim of the present study was to identify the differentially expressed genes (DEGs) that are induced by the silencing of transforming growth factor-β-activated kinase 1 (TAK1) in bladder cancer cells and to analyze the potential biological effects. Dataset GSE52452 from mutant fibroblast growth factor receptor 3 (FGFR3) bladder cancer cells transfected with control siRNA or TAK1-specific siRNA was downloaded from Gene Expression Omnibus. The DEGs between the two groups were identified using Limma package following data pre-processing by Affy in Bioconductor. Enrichment analysis of DEGs was performed using the Database for Annotation, Visualization and Integrated Discovery, followed by functional annotation using TRANSFAC, TSGene and TAG databases. Integrated networks were constructed by Cytoscape and sub-networks were extracted employing BioNet, followed by enrichment analysis of DEGs in the sub-network. A total of 43 downregulated and 21 upregulated genes were obtained. The downregulated genes were enriched in five pathways, including NOD-like receptor signaling pathway and functions related to cellular response. The upregulated genes were associated with cellular developmental processes. Transcription factor EGR1 and 9 tumor-associated genes were screened from the DEGs. Among the DEGs, 10 hub nodes may represent important roles in the complex metabolic network, including EGFR, CYP3A5, MAP3K7, GSTA1, PTHLH, ALDH1A1, KCND2, EGR1, ARRB1 and ITPR1. Additionally, EGFR was correlated with ERBB2, GRB2 and PIK3R1, and these were enriched in ErbB signaling pathway and various cancer-associated pathways. Silencing TAK1 may decrease cellular response to chemical stimulus via downregulating CYP3A5, MAP3K7, GSTA1, ALDH1A1, ARRB1 and ITPR1; increase cancer cell development via upregulating EGFR, EGR1 and PTHLH; and regulate cancer metastasis through EGFR, ERBB2, GRB2 and PIK3R1.

Adenovirus-Mediated Small Interfering RNA Targeting TAK1 Ameliorates Joint Inflammation with Collagen-Induced Arthritis in Mice.

Transforming growth factor β-activated kinase-1 (TAK1) is a key upstream kinase in cell signaling during inflammation, which regulates the expression of inflammatory mediators. Small interfering RNA (siRNA) against TAK1 offers promise as a potential therapeutic strategy in immune-mediated inflammatory disorder including rheumatoid arthritis. Here, we are to evaluate the therapeutic effects of intra-articular administration of adenoviral-mediated siRNA against TAK1 (ad-siRNA-TAK1) on collagen-induced arthritis (CIA) in mice. Ad-siRNA-TAK1 was constructed. The murine RAW 264.7 macrophages were infected with ad-siRNA-TAK1, and the silencing specificity of TAK1 was assessed by quantitative polymerase chain reaction (PCR) and western blot. DBA/1 mice were injected intra-articularly with ad-siRNA-TAK1. Development and severity of arthritis was assessed histologically. Synovial inflammation and bone destruction were determined by hematoxylin and eosin (HE) staining. Articular and serum concentrations of tumor necrosis factor-α, interleukin-1, and interleukin-6 were determined using enzyme-linked immunosorbent assay. Levels of phosphorylated p38, c-Jun N-terminal kinase (JNK), and extracellular-signal-regulated kinase (ERK) were detected by western blot. In vitro, ad--siRNA-TAK1 efficiently inhibited the expression of TAK1 at both mRNA and protein levels. In vivo, intra-articular injection of ad-siRNA-TAK1 efficiently alleviated joint inflammation, decreased the expression of pro-inflammatory mediators, and suppressed JNK pathways. Our results demonstrate the efficiency of ad--siRNA-TAK1 in controlling joint inflammation of CIA, which is associated with the suppression of the expression of pro-inflammatory cytokines and JNK activation.

Enhancement of hyperthermia-induced apoptosis by 5Z-7-oxozeaenol, a TAK1 inhibitor, in Molt-4 cells

Purpose: Transforming growth factor-β-activated kinase1 (TAK1) plays an anti-apoptotic role in response to multiple stresses. TAK1 inhibitor, 5Z-7-oxozeaenol (OZ) has been studied for its apoptotic effects. However, the combined effect of OZ with physical stresses remains to be elusive. Therefore, in this study we focussed to determine the combined effects of OZ with hyperthermia (HT) using Molt-4 cell line.Materials and methods: Molt-4 cells were pre-treated with OZ for 1 h followed by heat exposure (44 °C, 10 min) and harvested 24 h after incubation at 37 °C, apoptosis was measured by Annexin V-FITC/PI double staining assay using flow cytometry and cell growth was observed by cell counting assay. Further mechanism involved in the combination was investigated by measuring mitochondrial membrane potential (MMP), intracellular ROS generation, expression of apoptosis related protein, intracellular calcium ion level and Fas activity.Results: Combination of OZ with HT significantly enhances MMP loss and superoxide generation. Furthermore, OZ pre-treatment promotes caspase-8 cleavage, Fas externalisation, caspase 3 activity and intracellular calcium ion levels. OZ pre-treatment decreased the expression of HT-induced Bcl-2 and increased the expression of pro-apoptotic Bax, while markedly suppressed the phosphorylation of JNK and p38. In addition, increased expression of CHOP following combined treatment indicates that ER stress may also involve in the enhancement of HT-induced apoptosis.Conclusion: Our data showed for the first time that OZ sensitizes Molt-4 cells to HT-induced apoptosis via extrinsic and intrinsic apoptotic pathways. Furthermore, ROS and ER stress may also play role in the enhancement of HT-induced apoptosis by OZ.

Effect of curcumin on phosphorylation of TAK1 during endotoxin-induced lung injury in rats

Objective To evaluate the effect of curcumin on the phosphorylation of transforming growth factor-β-activated kinase 1 (TAK1) during endotoxin-induced lung injury in rats. Methods Eighteen pathogen-free healthy male Sprague-Dawley rats, weighing 180-220 g, were divided into 3 groups (n=6 each) using a random number table: control group (group C), lung injury group (group LI) and curcumin group (group CUR). Lung injury was induced by intra-tracheal instillation of lipopolysaccharide 5 mg/kg in 1 ml of normal saline in LI and CUR groups.Curcumin 200 mg/kg was injected intraperitoneally at 30 min before establishment of the model in group CUR, while the equal volume of dimethyl sulfoxide was given instead in group LI.The rats were sacrificed at 12 h after administration of lipopolysaccharide or normal saline, and lungs were removed for determination of the expression of TAK1 and phosphorylated TAK1 (p-TAK1) in lung tissues.The right upper lobe of the lung was removed for determination of wet/dry weight ratio (W/D ratio). Results Compared with group C, the W/D ratio was significantly increased, and the expression of TAK1 and p-TAK1 was up-regulated in group LI (P<0.05). Compared with group LI, the W/D ratio was significantly decreased, and the expression of TAK1 and p-TAK1 was down-regulated in group CUR (P<0.05). Conclusion The mechanism by which curcumin attenuates endotoxin-induced lung injury is related to inhibition of the phosphorylation of TAK1 in lung tissues of rats. Key words: Curcumin; Endotoxemia; Respiratory distress syndrome, adult; Transforming growth factor beta

Structure-guided development of covalent TAK1 inhibitors

TAK1 (transforming growth factor-β-activated kinase 1) is an essential intracellular mediator of cytokine and growth factor signaling and a potential therapeutic target for the treatment of immune diseases and cancer. Herein we report development of a series of 2,4-disubstituted pyrimidine covalent TAK1 inhibitors that target Cys174, a residue immediately adjacent to the ‘DFG-motif’ of the kinase activation loop. Co-crystal structures of TAK1 with candidate compounds enabled iterative rounds of structure-based design and biological testing to arrive at optimized compounds. Lead compounds such as 2 and 10 showed greater than 10-fold biochemical selectivity for TAK1 over the closely related kinases MEK1 and ERK1 which possess an equivalently positioned cysteine residue. These compounds are smaller, more easily synthesized, and exhibit a different spectrum of kinase selectivity relative to previously reported macrocyclic natural product TAK1 inhibitors such as 5Z-7-oxozeanol.

The GABAB positive allosteric modulators CGP7930 and GS39783 stimulate ERK1/2 signalling in cells lacking functional GABAB receptors

The present study shows that the GABAB positive allosteric modulators (PAMs) CGP7930 and GS39783 stimulate extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) signalling in cells that do not express functional GABAB receptors. In human SH-SY5Y neuroblastoma cells, CGP7930 and GS39783 induced a time- and concentration-dependent increase in ERK1/2 phosphorylation with potencies similar to those displayed as GABAB PAMs. Conversely, γ-aminobutyric acid and the GABAB receptor agonists (-)baclofen and SKF97541 were completely inactive. CGP7930 and GS39783 enhanced the nuclear localization of phospho-ERK1/2 and CGP7930 promoted the phosphorylation of the transcription factors Elk-1 and CREB. CGP7930-induced ERK1/2 stimulation was insensitive to pertussis toxin, the Gq/11 antagonist YM254890 and the phospholipase C-β inhibitor U-73122, but was completely blocked by the MEK1/2 inhibitor PD98059. Inhibition of insulin-like growth factor-1, platelet--derived growth factor, phosphoinositide 3-kinase and Akt activities potentiated CGP7930-induced ERK1/2 phosphorylation. CGP7930 enhanced the phosphorylation of myristoylated alanine-rich protein kinase C (PKC) substrate and inhibition of PKC attenuated the ERK1/2 stimulation. Over-expression of N17Ras, a dominant negative mutant of c-Ras, or inhibition of c-Raf by GW5074 partially antagonized CGP7930-induced ERK1/2 activation. CGP7930 enhanced the phosphorylation of transforming growth factor-β-activated kinase 1 (TAK-1) and TAK-1 inhibition by 5Z-7-oxozeaenol reduced CGP7930-induced ERK1/2 phosphorylation. CGP7930 activated ERK1/2 in CHO-K1 fibroblasts, which lack endogenous GABAB receptors, but not in HEK-293 cells, indicating that the response displayed cell type specificity. These data demonstrate that CGP7930 and GS39783 can trigger ERK1/2 signalling, a critical modulator of mood and drug addiction, independently of an action on GABAB receptors.

Inactivation of transforming growth factor-β-activated kinase 1 promotes taxol efficacy in ovarian cancer cells.

Resistance to taxol represents a major obstacle for long-term remission in ovarian cancer. Transforming Growth Factor-β-Activated Kinase 1 (TAK1) is a critical component in immune response pathway. However, the role of TAK1 in the development of chemoresistance in ovarian cancer remains unknown. Here, we showed that in vitro, taxol-resistant cells expressed higher TAK1, and the ratio of p-TAK1/TAK1 positively associated with taxol resistance in ovarian cancer cells. Inactivation of TAK1 by inhibitor 5Z-7-oxozeaenol or gene knockdown sensitized taxol cytotoxicity in vitro, promoting cell apoptosis and mitosis arrest. Moreover, resistant cells were much more sensitive to the combined TAK1 inhibitor and taxol treatment than their parental counterparts. Using xenograft mouse model, we found that 5Z-7-oxozeaenol significantly enhanced taxol efficacy in vivo. Thus, targeting TAK1 pathway is a promising strategy to enhance taxol response in ovarian cancer treatment.

The role of extracellular vesicles in mediating progression, metastasis and potential treatment of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a major cause of cancer-related death worldwide. As vectors for intercellular information exchange, the potential role of extracellular vesicles (EVs) in HCC formation, progression and therapy has been widely investigated. In this review, we explore the current status of the researches in this field. Altogether there is undeniable evidence that EVs play a crucial role in HCC development, metastasis. Moreover, EVs have shown great potential as drug delivery systems (DDSs) for the treatment of HCC. Exosomal miRNAs derived from HCC cells can enhance transformed cell growth in recipient cells by modulating the expression of transforming growth factor-β activated kinase-1(TAK1) and downstream signaling molecules. Furthermore, vacuolar protein sortin 4 homolog A(VPS4A) and insulin-like growth factor(IGF)-1 regulate exosome-mediated miRNAs transfer. Immune cells- derived EVs containing integrin αMβ2 or CD147 may facilitate HCC metastasis. In addition, EVs-mediated shuttle of long non-coding RNAs (lncRNAs), specifically linc- VLDLR and linc-ROR promote chemoresistance of malignant cells. Heat shock proteins (HSPs)-harboring exosomes derived from HCC tumor cells increase the antitumor effect of natural killer (NK) cells, thus enhancing HCC immunotherapy. Indeed, inhibition of HCC tumor growth has been associated with tumor cell-derived exosomes (TEX)-pulsed dentritic cells (DCs). Exosomes are also essential in liver metastasis during colorectal carcinoma (CRC) and pancreatic ductal adenocarcinomas (PDAC). Therefore, as nucleic acid and drug delivery vehicles, EVs show a tremendous potential for effective treatment against HCC.

TLR and TNF-R1 activation of the MKK3/MKK6-p38α axis in macrophages is mediated by TPL-2 kinase.

Previous studies suggested that Toll-like receptor (TLR) stimulation of the p38α MAP kinase (MAPK) is mediated by transforming growth factor-β-activated kinase 1 (TAK1) activation of MAPK kinases, MKK3, MKK4 and MKK6. We used quantitative mass spectrometry to monitor tumour progression locus 2 (TPL-2)-dependent protein phosphorylation following TLR4 stimulation with lipopolysaccharide, comparing macrophages from wild-type mice and Map3k8D270A/D270A mice expressing catalytically inactive TPL-2 (MAP3K8). In addition to the established TPL-2 substrates MKK1/2, TPL-2 kinase activity was required to phosphorylate the activation loops of MKK3/6, but not of MKK4. MKK3/6 activation required IκB kinase (IKK) phosphorylation of the TPL-2 binding partner nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB1) p105, similar to MKK1/2 activation. Tumour necrosis factor (TNF) stimulation of MKK3/6 phosphorylation was similarly dependent on TPL-2 catalytic activity and IKK phosphorylation of NF-κB1 p105. Owing to redundancy of MKK3/6 with MKK4, Map3k8D270A mutation only fractionally decreased lipopolysaccharide activation of p38α. TNF activation of p38α, which is mediated predominantly via MKK3/6, was substantially reduced. TPL-2 catalytic activity was also required for MKK3/6 and p38α activation following macrophage stimulation with Mycobacterium tuberculosis and Listeria monocytogenes. Our experiments demonstrate that the IKK/NF-κB1 p105/TPL-2 signalling pathway, downstream of TAK1, regulates MKK3/6 and p38α activation in macrophages in inflammation.

The long non-coding RNA, SNHG6-003, functions as a competing endogenous RNA to promote the progression of hepatocellular carcinoma.

The expression of long non-coding RNAs (lncRNAs) is dysregulated in hepatocellular carcinoma (HCC). However, the functions and contributions of lncRNAs remain largely unknown. Here, we identified a critical role of SNHG6-003 in HCC. We found that five SNHG6 transcripts were differentially expressed in HCC tissues while only the SNHG6-003 had an oncogenic function. Ectopic expression of SNHG6-003 in HCC cells promoted cell proliferation and induced drug resistance, whereas SNHG6-003 knockdown promoted apoptosis. Moreover, SNHG6-003 functioned as a competitive endogenous RNA (ceRNA), effectively becoming sponge for miR-26a/b and thereby modulating the expression of transforming growth factor-β-activated kinase 1 (TAK1). Importantly, expression analysis revealed that both SNHG6-003 and TAK1 were upregulated in human cancers, exhibiting a co-expression pattern. In HCC patients, high expression of SNHG6-003 closely correlated with tumor progression and shorter survival. Thus, targeting the ceRNA network involving SNHG6-003 may be used as a treatment strategy against HCC.

Lack of TAK1 in dendritic cells inhibits the contact hypersensitivity response induced by trichloroethylene in local lymph node assay

Trichloroethylene (TCE) is a ubiquitous environmental contaminant. Occupational TCE exposure has been associated with severe, generalized contact hypersensitivity (CHS) skin disorder. The development of CHS depends on innate and adaptive immune functions. Transforming growth factor-β activated kinase-1 (TAK1) controls the survival of dendritic cells (DCs) that affect the immune system homeostasis. We aimed to investigate the role of TAK1 activity in DC on TCE-induced CHS response. Control mice and DC-specific TAK1 deletion mice were treated with 80% (v/v) TCE using local lymph node assay (LLNA) to establish a TCE-induced CHS model. The draining lymph nodes (DLNs) were excised and the lymphocytes were measure for proliferation by BrdU-ELISA, T-cell phenotype analysis by flow cytometry and signaling pathway activation by western blot. The ears were harvested for histopathological analysis. Control mice in the 80% TCE group displayed an inflammatory response in the ears, increased lymphocyte proliferation, elevated regulatory T-cell and activated T-cell percentages, and more IFN-γ producing CD8+ T cells in DLNs. In contrast to control mice, DC-specific TAK1 deletion mice in the 80% TCE group showed an abolished CHS response and this was associated with defective T-cell expansion, activation and IFN-γ production. This effect may occur through Jnk and NF-κB signaling pathways. Overall, this study demonstrates a pivotal role of TAK1 in DCs in controlling TCE-induced CHS response and suggests that targeting TAK1 function in DCs may be a viable approach to preventing and treating TCE-related occupational health hazards.

Enhancement of hyperthermia-induced apoptosis by 5Z-7-oxozeaenol, a TAK1 inhibitor, in A549 cells.

KRAS mutant lung cancers have long been considered as untreatable with drugs. Transforming growth factor-β-activated kinase 1 (TAK1) appears to play an anti-apoptotic role in response to multiple stresses and has been reported to be a responsive kinase that regulates cell survival in KRAS-dependent cells. In this study, in order to find a useful approach to treat KRAS mutant lung cancer, we focused on the combined effects of 5Z-7-oxozeaenol, a TAK1 inhibitor, with hyperthermia (HT) in KRAS mutant lung cancer cell line A549. Annexin V-FITC/PI assay, cell cycle analysis, and colony formation assay revealed a significant enhancement in apoptosis induced by HT treatment, when the cells were pre-incubated with 5Z-7-oxozeaenol in a dose-dependent manner. The enhanced apoptosis by 5Z-7-oxozeaenol was accompanied by a significant increase in reactive oxygen species (ROS) generation and loss of mitochondrial membrane potential (MMP). In addition, western blot showed that 5Z-7-oxozeaenol enhanced HT-induced expressions of cleaved caspase-3, cleaved caspase-8, and HSP70 and decreased HT-induced expressions of Bcl-2, p-p38, p-JNK, and LC3. Moreover, 5Z-7-oxozeaenol pre-treatment resulted in a marked elevation of intracellular calcium level which might be associated with endoplasmic reticulum (ER) stress-related pathway. Taken together, our data provides further insights of the mechanism of action of 5Z-7-oxozeaenol and HT treatment, and their potential application as a novel approache to treat patients with KRAS mutant lung cancer.

Transforming Growth Factor-β-Activated Kinase 1 Is Required for Human FcγRIIIb-Induced Neutrophil Extracellular Trap Formation.

Neutrophils (PMNs) are the most abundant leukocytes in the blood. PMN migrates from the circulation to sites of infection where they are responsible for antimicrobial functions. PMN uses phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes. Several stimuli, including bacteria, fungi, and parasites, and some pharmacological compounds, such as Phorbol 12-myristate 13-acetate (PMA), are efficient inducers of NETs. Antigen–antibody complexes are also capable of inducing NET formation. Recently, it was reported that FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. Direct cross-linking of FcγRIIA or integrins did not promote NET formation. FcγRIIIb-induced NET formation presented different kinetics from PMA-induced NET formation, suggesting differences in signaling. Because FcγRIIIb also induces a strong activation of extracellular signal-regulated kinase (ERK) and nuclear factor Elk-1, and the transforming growth factor-β-activated kinase 1 (TAK1) has recently been implicated in ERK signaling, in the present report, we explored the role of TAK1 in the signaling pathway activated by FcγRIIIb leading to NET formation. FcγRIIIb was stimulated by specific monoclonal antibodies, and NET formation was evaluated in the presence or absence of pharmacological inhibitors. The antibiotic LL Z1640-2, a selective inhibitor of TAK1 prevented FcγRIIIb-induced, but not PMA-induced NET formation. Both PMA and FcγRIIIb cross-linking induced phosphorylation of ERK. But, LL Z1640-2 only inhibited the FcγRIIIb-mediated activation of ERK. Also, only FcγRIIIb, similarly to transforming growth factor-β-induced TAK1 phosphorylation. A MEK (ERK kinase)-specific inhibitor was able to prevent ERK phosphorylation induced by both PMA and FcγRIIIb. These data show for the first time that FcγRIIIb cross-linking activates TAK1, and that this kinase is required for triggering the MEK/ERK signaling pathway to NETosis.

Rifaximin Improves Clostridium difficile Toxin A-Induced Toxicity in Caco-2 Cells by the PXR-Dependent TLR4/MyD88/NF-κB Pathway.

Background: Clostridium difficile infections (CDIs) caused by Clostridium difficile toxin A (TcdA) lead to severe ulceration, inflammation and bleeding of the colon, and are difficult to treat.Aim: The study aimed to evaluate the effect of rifaximin on TcdA-induced apoptosis in intestinal epithelial cells and investigate the role of PXR in its mechanism of action.Methods: Caco-2 cells were incubated with TcdA and treated with rifaximin (0.1-10 μM) with or without ketoconazole (10 μM). The transepithelial electrical resistance (TEER) and viability of the treated cells was determined. Also, the expression of zona occludens-1 (ZO-1), toll-like receptor 4 (TLR4), Bcl-2-associated X protein (Bax), transforming growth factor-β-activated kinase-1 (TAK1), myeloid differentiation factor 88 (MyD88), and nuclear factor-kappaB (NF-κB) was determined.Results: Rifaximin treatment (0.1, 1.0, and 10 μM) caused a significant and concentration-dependent increase in the TEER of Caco-2 cells (360, 480, and 680% vs. TcdA treatment) 24 h after the treatment and improved their viability (61, 79, and 105%). Treatment also concentration-dependently decreased the expression of Bax protein (-29, -65, and -77%) and increased the expression of ZO-1 (25, 54, and 87%) and occludin (71, 114, and 262%) versus TcdA treatment. The expression of TLR4 (-33, -50, and -75%), MyD88 (-29, -60, and -81%) and TAK1 (-37, -63, and -79%) were also reduced with rifaximin versus TcdA treatment. Ketoconazole treatment inhibited these effects.Conclusion: Rifaximin improved TcdA-induced toxicity in Caco-2 cells by the PXR-dependent TLR4/MyD88/NF-κB pathway mechanism, and may be useful in the treatment of CDIs.

High glucose induced-macrophage activation through TGF-β-activated kinase 1 signaling pathway.

Transforming growth factor-β-activated kinase 1 (TAK1) plays a pivotal role in innate immune responses and kidney disease, and is critically involved in macrophage activation. However, there is a paucity of data to explore the role of high glucose (HG) in the regulation of TAK1 signaling and its functional role in macrophage activation. We assume that TAK1 signaling in hyperglycemic condition could be a key factor leading to macrophage activation and inflammation response. Mice macrophages were seeded on a 96-well cell culture plate; cell viability was tested after treatment with different concentration of TAK1 inhibitors. Cells were divided into groups (OZ300; MC; NC; HG; HG+OZ30, 100, 300nM) and treated for given time course. Monocyte chemotactic protein1(MCP-1) and tumor necrosis factor-α (TNF-α) mRNA levels were evaluated by qRT-PCR. Flow cytometry and confocal microscopy are used to analyse the activated macrophage induced by HG. Expression levels of p-TAK1, TAB 1, p-JNK, p-p38MAPK, NF-κBpp65 were detected by western blot. Nuclear translocation of NF-κBp65 was assessed by confocal microscopy. Our data revealed that high glucose not only significantly increased macrophage activation and subsequently abnormal high-expression of MCP-1 and TNF-α, but likewise remarkably enhanced TAK1 activation, MAPK phosphorylation, NF-κB expression in macrophages. Furthermore, pharmacological inhibition of TAK1 attenuated high glucose-triggered signal pathways, macrophage activation and inflammatory cytokines in a simulated diabetic environment. Our findings suggested that high glucose activated macrophages mainly in TAK1/MAPKs and TAK1/NF-κB-dependent manners, which lead to the polarization of macrophages towards a pro-inflammatory phenotype, and finally lead to diabetic nephropathy. In sum, the study raises novel data about the molecular mechanisms involved in the high glucose-mediated inflammatory response in macrophages.

TAK1 promotes epithelial-mesenchymal transition of lens epithelial cells

Transforming growth factor-β-activated kinase-1 (TAK1) is thought to play a key role in the initiation of Smad-independent TGF-β signaling. This study investigated the role of TAK1 in the epithelial-mesenchymal transition (EMT) lens epithelial cells. TAK1 was overexpressed in the HLE B-3 cell line by transfecting TAK1-pcDNA3 and TAK1-binding protein 1 (TAB1)-pcDNA3 plasmids. The expression levels of TAK1, phospho-TAK1, E-cadherin, and fibronectin were detected by Western blot analysis and immunocytofluorescence to analyze the effects of overexpression. The levels of α-SMA and type I collagen were analyzed by real-time PCR. Quantitative data were analyzed by Student's t test or one-way analysis of variance (ANOVA) (multiple comparisons using LSD test). Western blot analysis showed in the TAK1-pcDNA3 plasmids group, expression of TAK1 proteins (1.00±0.03) with a maximum upregulation of approximately 80% at 24 h than it was in the control group (0.19±0.09)(t=8.02, P< 0.01); Western blot analysis showed in the TAB1-pcDNA3 plasmids group, expression of TAB1 proteins (1.00±0.02) with a maximum upregulation of approximately 78% at 24 h than it was in the control group (0.22±0.08)(t=7.63, P<0.01). The levels of E-cadherin/Beta-actin had significant differences among control, overexpression of TAK1 together with TAB1, overexpression of TAK1, and overexpression of TAB1 (1.00±0.02, 0.12±0.03, 0.98±0.09, 0.92±0.08;F=31.03, P<0.01). The levels of fibronectin/Beta-actin had significant differences among control, overexpression of TAK1 together with TAB1, overexpression of TAK1, and overexpression of TAB1 (0.11±0.03, 1.00±0.05, 0.16±0.04, 0.21±0.05;F=35.12, P<0.01). Overexpression of TAK1 with TAB1 resulted in upregulated expression of fibronectin, and downregulated expression of E-cadherin. The expression of E-cadherin was increased and the expression of fibronectin was decreased by TAK1 siRNA and TAK1 chemical inhibitors in the presence of TGF-β2. These data reveal that TAK1 can induce the EMT of HLE cells, and the inhibition of TAK1 phosphorylation may be a potential novel therapeutic target for the prevention and treatment of posterior capsule opacification. (Chin J Ophthalmol, 2016, 52: 278-284).

Novel epidermal growth factor receptor pathway mediates release of human β-defensin 3 from Helicobacter pylori-infected gastric epithelial cells.

Persistent Helicobacter pylori (H. pylori) infection in hostile gastric mucosa can result in gastric diseases. Helicobacter pylori induces to express antimicrobial peptides from gastric epithelial cells, especially human β-defensin 3 (hBD3), as an innate immune response, and this expression of hBD3 is mediated by epidermal growth factor receptor (EGFR) activation. In this study, we found that phosphorylation of a serine residue of EGFR via transforming growth factor β-activated kinase-1 (TAK1), and subsequent p38α activation is essential for H. pylori-induced hBD3 release from gastric epithelial cells. We showed that this pathway was dependent on H. pylori type IV secretion system and was independent of H. pylori-derived CagA or peptidoglycan. H. pylori infection induced phosphorylation of serine residue of EGFR, and this phosphorylation was followed by internalization of EGFR; consequently, hBD3 was released at an early phase of the infection. In the presence of TAK1 or p38α inhibitors, synthesis of hBD3 was completely inhibited. Similar results were observed in EGFR-, TAK1- or p38α-knockdown cells. However, NOD1 knockdown in gastric epithelial cells did not inhibit hBD3 induction. Our study has firstly demonstrated that this novel EGFR activating pathway functioned to induce hBD3 at an early phase of H. pylori infection.