Transforming Growth Factor Β-activated Kinase Research Articles

αTAT1-induced tubulin acetylation promotes ameloblastoma migration and invasion

AbstractAmeloblastoma (AB) is the most common benign epithelial odontogenic tumor occurring in the jawbone. AB is a slowly growing tumor but sometimes shows a locally invasive and an aggressive growth pattern with a marked bone resorption. In addition, the local recurrence and distant metastasis of AB also sometimes occurs, which resembles one of the typical malignant potentials. From these points of view, to understand better the mechanisms of AB cell migration or invasion is necessary for the better clinical therapy and improvements of the patients' quality of life. Microtubules in eukaryotic cells reveal the shape of hollow cylinders made up of polymerized alpha (α)- and beta (β)-tubulin dimers and form the cytoskeleton together with microfilaments and intermediate filaments. Microtubules play important roles in cell migration by undergoing assembly and disassembly with post-translational modifications. Stability of microtubules caused by their acetylation is involved in cell migration. In this study, we investigated the expression and distribution of acetylated α-tubulin and alpha-tubulin N-acetyltransferase 1 (αTAT1), an enzyme which acetylates Lys-40 in α-tubulin, in AB specimens, and analyzed how tubulin was acetylated by αTAT1 activation in a human AB cell line, AM-1. Finally, we clarified that TGF-β-activated kinase1 (TAK1) was phosphorylated by TGF-β stimulation, then, induced tubulin acetylation via αTAT1 activation, which subsequently activated the migration and invasion of AB cells.

TGF-β-activated kinase-1 inhibitor LL-Z1640-2 reduces joint inflammation and bone destruction in mouse models of rheumatoid arthritis by inhibiting NLRP3 inflammasome, TACE, TNF-α and RANKL expression.

ObjectivesAberrant NLRP3 inflammasome activation has been demonstrated in rheumatoid arthritis (RA), which may contribute to debilitating inflammation and bone destruction. Here, we explored the efficacy of the potent TGF‐β‐activated kinase‐1 (TAK1) inhibitor LL‐Z1640‐2 (LLZ) on joint inflammation and bone destruction in collagen‐induced arthritis (CIA).MethodsLL‐Z1640‐2 was administered every other day in CIA mice. Clinical and histological evaluation was performed. Priming and activation of NLRP3 inflammasome and osteoclastogenic activity were assessed.ResultsNLRP3 inflammasome formation was observed in synovial macrophages and osteoclasts (OCs) in CIA mice. TACE and RANKL were also overexpressed in synovial macrophages and fibroblasts, respectively, in the CIA joints. Treatment with LLZ mitigated all the above changes. As a result, LLZ markedly suppressed synovial hypertrophy and pannus formation to alleviate pain and inflammation in CIA mice. LLZ could block the priming and activation of NLRP3 inflammasome in RAW264.7 macrophage cell line, primary bone marrow macrophages and OCs upon treatment with LPS followed by ATP, thereby suppressing their IL‐1β production. LLZ also suppressed LPS‐induced production of TACE and TNF‐α in bone marrow macrophages and abolished IL‐1β‐induced production of MMP‐3, IL‐6 and RANKL in synovial fibroblasts. In addition, LLZ directly inhibits RANKL‐mediated OC formation and activation.ConclusionTAK1 inhibition with LLZ may become a novel treatment strategy to effectively alleviate inflammasome‐mediated inflammation and RANKL‐induced osteoclastic bone destruction in joints alongside its potent suppression of TNF‐α and IL‐6 production and proteinase‐mediated pathological processes in RA.

TAB1 binding induced p38α conformation change: an accelerated molecular dynamics simulation study.

p38α mitogen-activated protein kinase (MAPK) undergoes autophosphorylation induced by the binding of TGFβ-activated kinase 1 binding protein 1 (TAB1) in myocardial ischemia. Investigation of the conformational transformations in p38α triggered by TAB1 binding is motivated by the need to find selective p38α activation inhibitors to treat myocardial ischemia. Herein, the conformational transformations of p38α were studied via all-atom accelerated molecular dynamics simulations and principal component analysis. With the binding of TAB1, the conformational changes of p38α auto-activation were characterized by the movement of the activation loop (A-loop) away from the αG helix toward the αF, αE helixes and L16-loop. In addition, a diverse intermediate state with an extensional and phosphorylated A-loop different from the transition intermediate state was explored. The conformational changes, including the A-loop alpha-structure breaking and the stronger hydrogen bond network formation, are accompanied by the extension of the A-loop and more intramolecular interactions in p38α. TAB1 correlates with other regions of p38α that are distal from the TAB1-binding site, including the A-loop, αC helix, and L16-loop, which regulates the intramolecular correlation of p38α. And, the phosphorylation further enhances the correlations between the A-loop and the other regions of p38α. The correlation results imply the regulation process of p38α conformational transformations. These findings will improve our understanding of the autophosphorylation of kinase and facilitate the development of selective inhibitors for the treatment of ischemic injury.

Caffeic Acid Inhibits RANKL and TNFa-induced Osteoclastogenesis by Targeting TAK1-p44/42 MAPK

BACKGROUND: The potential of the caffeic acid in other important Receptor Activator Nuclear Factor kB Ligand (RANKL)-Tumor Necrosis Factor (TNF)a-induced osteoclastogenic signaling pathways has not been known. Therefore, the current study was conducted to explore as well as to understand the inhibition potential of caffeic acid.METHODS: RAW264.7 cells were cultured, treated with caffeic acid, RANKL and TNFa. Tartrate Resistant Acid Phosphatase (TRAP) staining was performed to detect TRAP+ osteoclast-like polynuclear cells. To detect the activity of p44/42 Mitogen Activated Protein Kinase (MAPK), Akt, and Transforming Growth Factor-β-activated Kinase (TAK)1, the phosphorylated forms of the proteins were investigated with the immunoblotting assay.RESULTS: Pre-treatment of caffeic acid inhibited the RANKL and TNFa-induced differentiation of RAW264.7 cells into TRAP+ osteoclast-like polynuclear cells. RANKL and TNFa induced phosphorylation of p44/42 MAPK at Thr202/Tyr204, phosphorylation of Akt at both Ser473 and Thr308 and phosphorylation of TAK1 at Ser412. Pre-treatment with caffeic acid prior to the RANKL and TNFa induction, inhibited the phosphorylation of MAPK, and TAK1, but not Akt.CONCLUSION: Caffeic acid might regulate the RANKL-TNFa-induced osteoclastogenic pathway in RAW264.7 by targeting TAK1, which later activation of p44/42 MAPK was abolished.KEYWORDS: caffeic acid, osteoclastogenesis, p44/42, Erk1/2, Akt, TAK1, RAW264.7

Benzoyl-xanthone derivative induces apoptosis in MCF-7 cells by binding TRAF6.

TNF receptor-associated factor 6 (TRAF6) has been reported to be associated with the development of cancer. Nevertheless, the exact role of TRAF6 in cancer remains unclear. The purpose of the present study was to explore the mechanism of 2-benzoyl-3-hydroxy-4-methyl-9H-xanthen-9-one leading to the inhibition of the activation of AKT and TGF-β-activated kinase 1 (TAK1), and to the apoptosis of MCF-7 cells. Using a computational docking program and examination of AKT and TAK1 level changes, a new small molecule was identified, 2-benzoyl-3-hydroxy-4-methyl-9H-xanthen-9-one, which competitively bound to TRAF6. Next, the effect of this new compound on MCF-7 cells' biological behavior was studied in vitro. MTT assays were used to investigate cell viability; flow cytometry and invasion assays were performed to detect early apoptosis and invasion in MCF-7 cells, respectively. Immunoprecipitation, western blotting and caspase-3/9 activity assays were carried out to explore changes in protein expression. Briefly, the present data indicated that 2-benzoyl-3-hydroxy-4-methyl-9H-xanthen-9-one could suppress proliferation, induce early apoptosis and inhibit invasion in MCF-7 cells by suppressing the expression of Bcl-2 and promoting the expression of Bax, caspase-9, and caspase-3. These findings indicated that 2-benzoyl-3-hydroxy-4-methyl-9H-xanthen-9-one could induce apoptosis by inhibiting the activation of AKT and TAK1, and affecting the Bcl-2/Bax-caspase-9-caspase-3 pathway by competitively binding with TRAF6.

Breviscapine alleviates NASH by inhibiting TGF-β-activated kinase 1-dependent signaling.

Background and AimsNAFLD is a key component of metabolic syndrome, ranging from nonalcoholic fatty liver to NASH, and is now becoming the leading cause of cirrhosis and HCC worldwide. However, due to the complex and unclear pathophysiological mechanism, there are no specific approved agents for treating NASH. Breviscapine, a natural flavonoid prescription drug isolated from the traditional Chinese herb Erigeron breviscapus, exhibits a wide range of pharmacological properties, including effects on metabolism. However, the anti‐NASH efficacy and mechanisms of breviscapine have not yet been characterized.Approach and ResultsWe evaluated the effects of breviscapine on the development of hepatic steatosis, inflammation, and fibrosis in vivo and in vitro under metabolic stress. Breviscapine treatment significantly reduced lipid accumulation, inflammatory cell infiltration, liver injury, and fibrosis in mice fed a high‐fat diet, a high‐fat/high‐cholesterol diet, or a methionine‐ and choline‐deficient diet. In addition, breviscapine attenuated lipid accumulation, inflammation, and lipotoxicity in hepatocytes undergoing metabolic stress. RNA‐sequencing and multiomics analyses further indicated that the key mechanism linking the anti‐NASH effects of breviscapine was inhibition of TGF‐β‐activated kinase 1 (TAK1) phosphorylation and the subsequent mitogen‐activated protein kinase signaling cascade. Treatment with the TAK1 inhibitor 5Z‐7‐oxozeaenol abrogated breviscapine‐mediated hepatoprotection under metabolic stress. Molecular docking illustrated that breviscapine directly bound to TAK1.ConclusionBreviscapine prevents metabolic stress–induced NASH progression through direct inhibition of TAK1 signaling. Breviscapine might be a therapeutic candidate for the treatment of NASH.

Intestinal ischemia-reperfusion induces the release of IL-17A to regulate cell inflammation, apoptosis and barrier damage.

Intestinal ischemia-reperfusion (I/R) injury promotes the release of IL-17A, and previous studies have indicated that TGF-β activated kinase 1 (TAK1) is an important signaling molecule in the regulatory function of IL-17A. The present study aimed to explore the potential effects of IL-17A release in intestinal I/R injury, and to investigate the underlying regulatory mechanisms. Initially, the expression levels of TAK1 and JNK in a hypoxia/reoxygenation model were determined, and the effects of TAK1-knockdown on JNK phosphorylation and the viability, inflammation, apoptosis and barrier function of Caco-2 cells were assessed using Cell Counting Kit-8, reverse transcription-quantitative PCR, TUNEL and transepithelial electrical resistance assays, respectively. Subsequently, an antibody targeting IL-17A was used, and the effects of the IL-17A antibody on the expression levels of TAK1 as well as cell viability, inflammation, apoptosis and barrier function were determined. The results of the present study demonstrated that TAK1-knockdown markedly reduced JNK phosphorylation and improved the levels of cell viability, inflammation, apoptosis and barrier function via the MAPK signaling pathway. In addition, treatment with the IL-17A antibody inhibited the expression of TAK1, and reversed the aforementioned effects of TAK1 on Caco-2 cells. In conclusion, intestinal I/R induces the release of IL-17A to regulate cell viability, inflammation, apoptosis and barrier damage via the TAK1/MAPK signaling pathway.

An SNP at the target site of cid-miR-nov-1043 in the TOLLIP 3′ UTR decreases mortality rate in grass carp subjected to ENU-induced mutagenesis following grass carp reovirus infection

N-ethyl-N-nitrosourea (ENU) selection is a useful technique to generate new mutations that may cause some functional changes in the gene. Through our previous genomic bulked segregant analysis (BSA), one single nucleotide polymorphism (SNP) at the 3′ UTR of Toll interacting protein gene (TOLLIP982T>C) was identified in grass carp (Ctenopharyngodon idella) subjected to ENU-induced mutagenesis. We found that the overexpression of cid-miR-nov-1043 mimics significantly suppressed the luciferase activity of the TOLLIP 3′ UTR, but TOLLIP982T>C mutation at the target site can decrease the binding affinity between the miRNA cid-miR-nov-1043 and TOLLIP 3′ UTR, reducing the inhibition of TOLLIP mRNA transcription in grass carp subjected to ENU-induced mutagenesis. More importantly, we demonstrated that TOLLIP mRNA transcription levels in the gills, liver, kidney and the isolate white cells of the mutant grass carp were significantly (p < 0.01) higher than those in the corresponding tissues from the wild-type grass carp following infection with Grass Carp Reovirus (GCRV) for seven days, while the downstream gene of TOLLIP transforming growth factor β-activated kinase 1 (TAK1) and TAK1-binding protein 1 (TAB1), were higher expressed in wild-type grass carp. As a negative regulator in the pro-inflammatory pathway of NF-κB, TOLLIP inhibits the excessive inflammation in ENU grass carp after GCRV infection. Consistent with the TOLLIP expression, histopathological results demonstrated more severe inflammation in wild-type grass carp, compared to the TOLLIP982T>C mutant grass carp on the seventh day. Severe inflammation will lead to thoroughly infiltration of chloride and inflammatory cells in the gill filaments. This seriously hindered the exchange of oxygen, which ultimately disrupted blood circulation. Meanwhile, the survival rate of the mutant grass carp was significantly (p < 0.01) higher than that of the wild-type grass carp, indicating that the TOLLIP982T>C mutants showed strong anti-viral abilities. Our results revealed that an SNP in the TOLLIP 3′ UTR may contribute to the suppression of serve inflammation subjected to ENU-induced mutagenesis following GCRV infection, which may be helpful for future resistant breeding development of grass carp.

Regulator of G-protein signaling 14 protects the liver from ischemia-reperfusion injury by suppressing TGF-β-activated kinase 1 activation.

Background and AimsHepatic ischemia–reperfusion injury (IRI) is a common complication of hepatectomy and liver transplantation. However, the mechanisms underlying hepatic IRI have not been fully elucidated. Regulator of G‐protein signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates the G‐protein and mitogen‐activated protein kinase (MAPK) signaling pathways. However, the role of RGS14 in hepatic IRI remains unclear.Approach and ResultsWe found that RGS14 expression increased in mice subjected to hepatic ischemia–reperfusion (IR) surgery and during hypoxia reoxygenation in hepatocytes. We constructed global RGS14 knockout (RGS14‐KO) and hepatocyte‐specific RGS14 transgenic (RGS14‐TG) mice to establish 70% hepatic IRI models. Histological hematoxylin and eosin staining, levels of alanine aminotransferase and aspartate aminotransferase, expression of inflammatory factors, and apoptosis were used to assess liver damage and function in these models. We found that RGS14 deficiency significantly aggravated IR‐induced liver injury and activated hepatic inflammatory responses and apoptosis in vivo and in vitro. Conversely, RGS14 overexpression exerted the opposite effect of the RGS14‐deficient models. Phosphorylation of TGF‐β‐activated kinase 1 (TAK1) and its downstream effectors c‐Jun N‐terminal kinase (JNK) and p38 increased in the liver tissues of RGS14‐KO mice but was repressed in those of RGS14‐TG mice. Furthermore, inhibition of TAK1 phosphorylation rescued the effect of RGS14 deficiency on JNK and p38 activation, thus blocking the inflammatory responses and apoptosis.ConclusionsRGS14 plays a protective role in hepatic IR by inhibiting activation of the TAK1–JNK/p38 signaling pathway. This may be a potential therapeutic strategy for reducing incidences of hepatic IRI in the future.

Takinib Inhibits Inflammation in Human Rheumatoid Arthritis Synovial Fibroblasts by Targeting the Janus Kinase-Signal Transducer and Activator of Transcription 3 (JAK/STAT3) Pathway.

TGF β-activated kinase 1 (TAK1) is an important participant in inflammatory pathogenesis for diseases such as rheumatoid arthritis (RA) and gouty arthritis. The central position it occupies between the mitogen activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) pathways makes it an attractive therapeutic target. As this field has developed in recent years, several novel inhibitors have been presented as having specific activity that reduces the TAK1 function either covalently as in the case of 5Z-7-oxozeanol (5Z7O) or reversibly (NG-25). However, the mechanism through which takinib elicits its anti-inflammatory activity remains elusive. While this inhibitor shows great promise, a thorough analysis of its inhibitor function and its potential off-target effects is necessary before addressing its clinical potential or its use in inflammatory conditions. An analysis through Western blot showed an unexpected increase in IL-1β-induced TAK1 phosphorylation—a prerequisite for and indicator of its functional potential—by takinib while simultaneously demonstrating the inhibition of the JAK/STAT pathway in human rheumatoid arthritis synovial fibroblasts (RASFs) in vitro. In THP-1 monocyte-derived macrophages, takinib again led to the lipopolysaccharide-induced phosphorylation of TAK1 without a marked inhibition of the TAK1 downstream effectors, namely, of c-Jun N-terminal kinase (JNK), phospho-c-Jun, NF-κB phospho-p65 or phospho-IκBα. Taken together, these findings indicate that takinib inhibits inflammation in these cells by targeting multiple signaling pathways, most notably the JAK/STAT pathway in human RASFs.

Phosphocreatine attenuates doxorubicin-induced cardiotoxicity by inhibiting oxidative stress and activating TAK1 to promote myocardial survival in vivo and in vitro

Myocardial apoptosis and necroptosis are the major etiological factor during doxorubicin (DOX) induced cardiotoxicity, and one of the important reasons that limit the drug’s clinical application. Up to date, its mechanism has not been fully elucidated. The protective role of phosphocreatine (PCr) in heart surgery and medical cardiology has been observed in numerous clinical trials. This study aimed to evaluate cardioprotective actions of PCr against DOX-induced cardiotoxicity and investigate the underlying mechanism involving in transforming growth factor β-activated kinase 1 (TAK1) mediated myocardial survive signaling pathway. Male Sprague-Dawleyrats were intraperitoneally (ip) injected with normal saline (NS) or DOX (2 mg/kg) alone or DOX with PCr (200 mg/kg) used as animal model. The data showed that DOX significantly impaired cardiac function and structure, induced oxidative stress, myocardial apoptosis and necroptosis, and dramatically down-regulated the expression level of TAK1, while the intervention of PCr obviously attenuated cardiac dysfunction, oxidative stress, myocardial apoptosis and necroptosis, especially alleviated the decrease of TAK1 expression. In vitro analysis, after H9c2 cells were pretreated with or without PCr (0.5 mM) or N-Acetyl-L-cysteine (NAC, 0.5 mM) or 5Z-7-oxozeaenol (5z-7-Ox, 1 μM) for 1 h, subsequently treated with DOX (1 μM) for 24 h. The results revealed that inhibition of TAK1 further deteriorated apoptotic and necroptotic cell death induced by DOX in H9c2 cells, but didn’t affect oxidative stress. While the pretreatment of PCr or NAC enhanced antioxidant activity to reduce oxidative stress, significantly alleviated apoptotic and necroptotic cell death induced by DOX in H9c2 cells. Consistent with the results in vivo, PCr or NAC significantly inhibited the decrease of TAK1 expression induced by DOX. In conclusion, oxidative stress induced by DOX inhibits the expression of TAK1, and leads to myocardial apoptotic and necroptotic death, while the intervention of PCr increases antioxidant activity to alleviate oxidative stress, which in turn activates TAK1 signaling pathway to promote myocardial survival, and finally attenuate DOX-induced cardiotoxicity.

Oxidative stress-induced senescence mediates inflammatory and fibrotic phenotypes in fibroblasts from systemic sclerosis patients.

SSc is an autoimmune connective tissue disorder characterized by inflammation and fibrosis. Although constitutive activation of fibroblasts is proposed to be responsible for the fibrotic and inflammatory features of the disease, the underlying mechanism remains elusive, and effective therapeutic targets are still lacking. The aim of this study was to evaluate the role of oxidative stress-induced senescence and its contribution to the pro-fibrotic and pro-inflammatory phenotypes of fibroblasts from SSc patients. Dermal fibroblasts were isolated from SSc (n = 13) and healthy (n = 10) donors. Fibroblasts' intracellular and mitochondrial reactive oxygen species (ROS) were determined by flow cytometry. Mitochondrial function was measured by Seahorse XF24 analyser. Fibrotic and inflammatory gene expressions were assessed by qPCR and key pro-inflammatory components of the fibroblasts' secretome (IL-6 and IL-8) were quantified by ELISA. Compared with healthy fibroblasts, SSc fibroblasts displayed higher levels of both intracellular and mitochondrial ROS. Oxidative stress in SSc fibroblasts induced the expression of fibrotic genes and activated the TGF-β-activated kinase 1 (TAK1)-IκB kinase β (IKKβ)-IFN regulatory factor 5 (IRF5) inflammatory signalling cascade. These cellular responses paralleled the presence of a DNA damage response, a senescence-associated secretory phenotype and a fibrotic response. Treatment of SSc fibroblasts with ROS scavengers reduced their pro-inflammatory secretome production and fibrotic gene expression. Oxidative stress-induced cellular senescence in SSc fibroblasts underlies their pro-inflammatory and pro-fibrotic phenotypes. Targeting redox imbalance of SSc fibroblasts enhances their in vitro functions and could be of relevance for SSc therapy.

Celastrol Attenuates RANKL-Induced Osteoclastogenesis in vitro and Reduces Titanium Particle-Induced Osteolysis and Ovariectomy-Induced Bone Loss in vivo.

Excessive bone resorption by osteoclasts contributes significantly to osteoclast-related diseases such as periprosthetic osteolysis and osteoporosis. Osteolysis in a titanium particle-induced calvarial model and bone loss in an ovariectomized mice model occurred similarly to those in humans; thus, these models can be used to evaluate potential therapies for aseptic prosthetic loosening and osteoporosis. Celastrol, which is extracted from the seeds of the genus Tripterygium, has been thoroughly investigated for its anti-inflammatory and anti-cancer pharmacological effects. However, the mechanisms involving bone metabolism by which celastrol inhibits osteoclastogenesis are not yet fully understood. We demonstrated that celastrol inhibited the receptor activator of nuclear factor κB ligand-induced osteoclastogenesis and the bone resorptive function of osteoclasts in vitro by inhibiting the activation of transforming growth factor β-activated kinase 1-mediated NF-κB and mitogen-activated protein kinase signaling pathways and downregulating osteoclastogenesis marker-related genes. Furthermore, celastrol was also shown to be beneficial in both the titanium particle-induced osteolysis calvarial and the murine ovariectomy-induced bone loss. Collectively, our results suggested that celastrol is promising for the prevention of aseptic prosthetic loosening and osteoporosis in the treatment of osteolytic diseases induced by disrupted osteoclast formation and function.

Inducible ATP1B1 Upregulates Antiviral Innate Immune Responses by the Ubiquitination of TRAF3 and TRAF6.

The antiviral innate immune responses are crucial steps during host defense and must be strictly regulated, but the molecular mechanisms of control remain unclear. In this study, we report increased expression of human ATPase Na+/K+ transporting subunit β 1(ATP1B1) after DNA and RNA virus infections. We found that the expression of ATP1B1 can inhibit viral replication and increase the levels of IFNs, IFN-stimulated genes, and inflammatory cytokines. Knockdown of ATP1B1 by specific short hairpin RNA had the opposite effects. Upon viral infection, ATP1B1 was induced, interacted with TRAF3 and TRAF6, and potentiated the ubiquitination of these proteins, leading to increased phosphorylation of downstream molecules, including TGF-β-activated kinase 1 (TAK1) and TANK-binding kinase 1 (TBK1). These results reveal a previously unrecognized role of ATP1B1 in antiviral innate immunity and suggest a novel mechanism for the induction of IFNs and proinflammatory cytokines during viral infection.

The stabilization of yes-associated protein by TGFβ-activated kinase 1 regulates the self-renewal and oncogenesis of gastric cancer stem cells.

Gastric cancer (GC) is the most frequent digestive system malignant tumour and the second most common cause of cancer death globally. Cancer stem cell (CSC) is a small percentage of cancer cells in solid tumours that have differentiation, self‐renewal and tumorigenic capabilities. They have an active participation in the initiation, development, metastasis, recurrence and resistance of tumours to chemotherapy and radiotherapy. Gastric cancer stem cells (GCSCs) have been shown to be correlated with GC initiation and metastasis. In this study, we found that TAK1 expression level in GC tissues was significantly increased compared to the adjacent non‐cancerous tissues by RT‐qPCR, Western blot and immunohistochemistry. TAK1 has been identified as a critical molecule that promoted a variety of malignant GC phenotypes both in vivo and in vitro and promoted the self‐renewal of GCSCs. Mechanistically, TAK1 was up‐regulated by IL‐6 and prevented the degradation of yes‐associated protein (YAP) in the cytoplasm by binding to YAP. Thus, TAK1 promoted the SOX2 and SOX9 transcription and the self‐renewal and oncogenesis of GCSCs. Our findings provide insights into the mechanism of self‐renewal and tumorigenesis of TAK1 in GCSCs and have broad implications for clinical therapies.

Nasturtium officinale Extract Suppresses Osteoclastogenesis in RAW 264 Cells by Inhibiting IκB-Kinase β

Osteoclasts are large, multinucleated, bone-absorbing cells and play a crucial role in osteolytic bone diseases such as osteopetrosis and rheumatoid arthritis. Therefore, controlling osteoclast differentiation and activation has been considered a promising strategy to prevent and treat osteolytic diseases. In this study, we demonstrate, using the mouse monocyte-derived macrophage-like cell line RAW 264, that extract from Nasturtium officinale or watercress, an herb of European origin, suppresses receptor activator of nuclear factor-κB ligand-induced osteoclast differentiation in vitro . N. officinale extract decreased the emergence of tartrate-resistant acid phosphatase-positive differentiated multinuclear cells and inhibited their bone-absorbing activity. The extract decreased expression of genes associated with osteoclast differentiation and function. Induction of nuclear factor of activated T cells c1 (NFATc1), the master transcriptional regulator of osteoclastogenesis, was blunted by N. officinale extract. Activation of nuclear factor-κB and mitogen-activated protein kinases pathways, both of which are necessary for NFATc1 induction and osteoclast differentiation, was also suppressed by the extract. Among upstream kinases, activity of IκB-kinase β (IKKβ), but not that of TGFβ-activated kinase 1, was inhibited by N. officinale extract in vitro. Pharmacological inhibition of IKKβ by a specific inhibitor PS1145 in RAW 264 cells mostly recaptured the inhibitory action of N. officinale extract. These findings provide a novel pharmacological action of N. officinale and its potential usefulness for the prevention of osteoporosis.

The E3 Ubiquitin-Protein Ligase RNF4 Promotes TNF-α-Induced Cell Death Triggered by RIPK1.

Receptor-interacting protein kinase 1 (RIPK1) is a key component of the tumor necrosis factor (TNF) receptor signaling complex that regulates both pro- and anti-apoptotic signaling. The reciprocal functions of RIPK1 in TNF signaling are determined by the state of the posttranslational modifications (PTMs) of RIPK1. However, the underlying mechanisms associated with the PTMs of RIPK1 are unclear. In this study, we found that RING finger protein 4 (RNF4), a RING finger E3 ubiquitin ligase, is required for the RIPK1 autophosphorylation and subsequent cell death. It has been reported that RNF4 negatively regulates TNF-α-induced activation of the nuclear factor-κB (NF-κB) through downregulation of transforming growth factor β-activated kinase 1 (TAK1) activity, indicating the possibility that RNF4-mediated TAK1 suppression results in enhanced sensitivity to cell death. However, interestingly, RNF4 was needed to induce RIPK1-mediated cell death even in the absence of TAK1, suggesting that RNF4 can promote RIPK1-mediated cell death without suppressing the TAK1 activity. Thus, these observations reveal the existence of a novel mechanism whereby RNF4 promotes the autophosphorylation of RIPK1, which provides a novel insight into the molecular basis for the PTMs of RIPK1.

TRIM26 positively regulates the inflammatory immune response through K11-linked ubiquitination of TAB1.

Protein ubiquitination plays an important role in the regulation of TGF-β-activated kinase 1 (TAK1)-mediated NF-κB activation. It is well established that TAK1 activation is tightly regulated with its binding partners, TAK1-binding proteins (TAB1-3). However, the tight regulation of TAK1 activation remains elusive. Here, using Trim26-knockout mice and Trim26-transgenic mice, we found that TRIM26 acts as a positive regulator of TAK1 activation by ubiquitinating its binding partner TAB1. Knockout of Trim26 inhibited TAK1 activation and downstream kinases activation, thus decreasing the induction of proinflammatory cytokines following LPS, TNF-α, and IL-1β stimulation. Mechanistically, TRIM26 catalyzes the K11-linked polyubiquitination of TAB1 at Lys294, Lys319, and Lys335 to enhance the activation of TAK1 and subsequent NF-κB and MAPK signaling. Consequently, Trim26 deficiency protects mice from LPS-induced septic shock in vivo. Moreover, Trim26 deficiency attenuates the severity of dextran sodium sulfate (DSS)-induced colitis. Thus, these finding provides a novel insight into how TAK1 activation is regulated through TRIM26-mediated ubiquitination of TAB1 and reveals the new function of TRIM26 in the regulation of the inflammatory innate immune response.

Sensing of mycobacterial arabinogalactan by galectin-9 exacerbates mycobacterial infection.

Mycobacterial arabinogalactan (AG) is an essential cell wall component of mycobacteria and a frequent structural and bio‐synthetical target for anti‐tuberculosis (TB) drug development. Here, we report that mycobacterial AG is recognized by galectin‐9 and exacerbates mycobacterial infection. Administration of AG‐specific aptamers inhibits cellular infiltration caused by Mycobacterium tuberculosis (Mtb) or Mycobacterium bovis BCG, and moderately increases survival of Mtb‐infected mice or Mycobacterium marinum‐infected zebrafish. AG interacts with carbohydrate recognition domain (CRD) 2 of galectin‐9 with high affinity, and galectin‐9 associates with transforming growth factor β‐activated kinase 1 (TAK1) via CRD2 to trigger subsequent activation of extracellular signal‐regulated kinase (ERK) as well as induction of the expression of matrix metalloproteinases (MMPs). Moreover, deletion of galectin‐9 or inhibition of MMPs blocks AG‐induced pathological impairments in the lung, and the AG‐galectin‐9 axis aggravates the process of Mtb infection in mice. These results demonstrate that AG is an important virulence factor of mycobacteria and galectin‐9 is a novel receptor for Mtb and other mycobacteria, paving the way for the development of novel effective TB immune modulators.

Mycobacterium tuberculosis protein kinase G acts as an unusual ubiquitinating enzyme to impair host immunity.

Upon Mycobacterium tuberculosis (Mtb) infection, protein kinase G (PknG), a eukaryotic-type serine-threonine protein kinase (STPK), is secreted into host macrophages to promote intracellular survival of the pathogen. However, the mechanisms underlying this PknG-host interaction remainunclear. Here, we demonstrate that PknG serves both as a ubiquitin-activating enzyme (E1) and a ubiquitin ligase (E3) to trigger the ubiquitination and degradation of tumor necrosis factor receptor-associated factor 2 (TRAF2) and TGF-β-activated kinase 1 (TAK1), thereby inhibiting the activation of NF-κB signaling and host innate responses. PknG promotes the attachment of ubiquitin (Ub) to the ubiquitin-conjugating enzyme (E2) UbcH7 via an isopeptide bond (UbcH7 K82-Ub), rather than the usual C86-Ub thiol-ester bond. PknG induces the discharge of Ub from UbcH7 by acting as an isopeptidase, before attaching Ub to its substrates. These results demonstrate that PknG acts as an unusual ubiquitinating enzyme to remove key components of the innate immunity system, thus providing a potential target for tuberculosis treatment.