Role Of TGF-β-activated Kinase 1 Research Articles

Induction of pro-inflammatory genes by fibronectin DAMPs in three fibroblast cell lines: Role of TAK1 and MAP kinases.

Changes in the organization and structure of the fibronectin matrix are believed to contribute to dysregulated wound healing and subsequent tissue inflammation and tissue fibrosis. These changes include an increase in the EDA isoform of fibronectin as well as the mechanical unfolding of fibronectin type III domains. In previous studies using embryonic foreskin fibroblasts, we have shown that fibronectin's EDA domain (FnEDA) and the partially unfolded first Type III domain (FnIII-1c) function as Damage Associated Molecular Pattern (DAMP) molecules to stimulate the induction of inflammatory cytokines by serving as agonists for Toll-Like Receptor-4 (TLR4). However, the role of signaling molecules downstream of TLR-4 such as TGF-β Activated Kinase 1 (TAK1) and Mitogen activated protein kinases (MAPK) in regulating the expression of fibronectin DAMP induced inflammatory genes in specific cell types is not known. In the current study, we evaluate the molecular steps regulating the fibronectin driven induction of inflammatory genes in three human fibroblast cell lines: embryonic foreskin, adult dermal, and adult kidney. The fibronectin derived DAMPs each induce the phosphorylation and activation of TAK1 which results in the activation of two downstream signaling arms, IKK/NF-κB and MAPK. Using the specific inhibitor 5Z-(7)-Oxozeanol as well as siRNA, we show TAK1 to be a crucial signaling mediator in the release of cytokines in response to fibronectin DAMPs in all three cell types. Finally, we show that FnEDA and FnIII-1c induce several pro-inflammatory cytokines whose expression is dependent on both TAK1 and JNK MAPK and highlight cell-type specific differences in the gene-expression profiles of the fibroblast cell-lines.

Targeted regulation of TAK1 counteracts dystrophinopathy in a DMD mouse model.

Muscular dystrophies make up a group of genetic neuromuscular disorders that involve severe muscle wasting. TGF-β-activated kinase 1 (TAK1) is an important signaling protein that regulates cell survival, growth, and inflammation. TAK1 has been recently found to promote myofiber growth in the skeletal muscle of adult mice. However, the role of TAK1 in muscle diseases remains poorly understood. In the present study, we have investigated how TAK1 affects the progression of dystrophic phenotype in the mdx mouse model of Duchenne muscular dystrophy (DMD). TAK1 is highly activated in the dystrophic muscle of mdx mice during the peak necrotic phase. While targeted inducible inactivation of TAK1 inhibits myofiber injury in young mdx mice, it results in reduced muscle mass and contractile function. TAK1 inactivation also causes loss of muscle mass in adult mdx mice. By contrast, forced activation of TAK1 through overexpression of TAK1 and TAB1 induces myofiber growth without having any deleterious effect on muscle histopathology. Collectively, our results suggest that TAK1 is a positive regulator of skeletal muscle mass and that targeted regulation of TAK1 can suppress myonecrosis and ameliorate disease progression in DMD.

TAK1 regulates skeletal muscle mass and mitochondrial function.

Skeletal muscle mass is regulated by a complex array of signaling pathways. TGF-β-activated kinase 1 (TAK1) is an important signaling protein, which regulates context-dependent activation of multiple intracellular pathways. However, the role of TAK1 in the regulation of skeletal muscle mass remains unknown. Here, we report that inducible inactivation of TAK1 causes severe muscle wasting, leading to kyphosis, in both young and adult mice.. Inactivation of TAK1 inhibits protein synthesis and induces proteolysis, potentially through upregulating the activity of the ubiquitin-proteasome system and autophagy. Phosphorylation and enzymatic activity of AMPK are increased, whereas levels of phosphorylated mTOR and p38 MAPK are diminished upon inducible inactivation of TAK1 in skeletal muscle. In addition, targeted inactivation of TAK1 leads to the accumulation of dysfunctional mitochondria and oxidative stress in skeletal muscle of adult mice. Inhibition of TAK1 does not attenuate denervation-induced muscle wasting in adult mice. Finally, TAK1 activity is highly upregulated during overload-induced skeletal muscle growth, and inactivation of TAK1 prevents myofiber hypertrophy in response to functional overload. Overall, our study demonstrates that TAK1 is a key regulator of skeletal muscle mass and oxidative metabolism.

Activation of TGF-β activated kinase 1 promotes colon mucosal pathogenesis in inflammatory bowel disease.

The etiology and mechanisms for inflammatory bowel disease (IBD) are incompletely known. Determination of new, clinically important mechanisms for intestinal inflammation is imperative for developing effective therapies to treat IBD. We sought to define a widespread mechanism for colon mucosal inflammation via the activation of TGF‐β activated Kinase 1 (TAK1), a central regulator of cellular inflammatory actions. Activation of TAK1 and the downstream inflammatory signaling mediators was determined in pediatric patients with ulcerative colitis (UC) or Crohn's disease (CD) as well as in DSS‐induced and spontaneous IBD in mice. The role of TAK1 in facilitating intestinal inflammation in murine models of IBD was investigated by using (5Z)‐7‐Oxozeaenol, a highly selective pharmacological inhibitor of TAK1. We found hyper‐activation of TAK1 in patients with UC or CD and in murine models of IBD. Pharmacological inhibition of TAK1 prevented loss in body weight, disease activity, microscopic histopathology, infiltration of inflammatory cells in the colon mucosa, and elevated proinflammatory cytokine production in two murine models of IBD. We demonstrated that at the early phase of the disease activation of TAK1 is restricted in the epithelial cells. However, at a more advanced stage of the disease, TAK1 activation predominantly occurs in nonepithelial cells, especially in macrophages. These findings elucidate the activation of TAK1 as crucial in promoting intestinal inflammation. Thus, the TAK1 activation pathway may represent a suitable target to design new therapies for treating IBD in humans.

Abstract 44: TAK1 Regulates Caspase 8 Activation and Necroptotic Signaling via Multiple Cell Death Checkpoints

Necroptosis has emerged as a new form of programmed cell death implicated in a number of pathological conditions such as ischemic injury, neurodegenerative disease, and viral infection. Recent studies indicate that TGFβ-activated kinase 1 (TAK1) is nodal regulator of necroptotic cell death, but the underlying molecular regulatory mechanisms remain elusive. Here we reported that TAK1 regulates necroptotic signaling as well as caspase 8 activation through both NFκB-dependent and -independent mechanisms. Inhibition of TAK1 promoted TNFα-induced necroptosis through the induction of RIP1 phosphorylation/activation and necrosome formation, in the presence of ongoing caspase activation. Further, inhibition of TAK1 triggered two caspase 8 activation pathways through the induction of RIP1-FADD-caspase 8 complex as well as FLIP cleavage/degradation. Mechanistically, our data uncovered an essential role of the adaptor protein TRADD in caspase 8 activation and necrosome formation triggered by TAK1 inhibition. Moreover, ablation of the deubiqutinase CYLD prevented both apoptotic and necroptotic signaling induced by TAK1 inhibition, whereas deletion of the E3 ubiquitin ligase TRAF2 had the opposite effect. Finally, blocking the ubiquitin-proteasome pathway prevented the degradation of key necroptotic signaling proteins and necrosome formation. Thus we identified novel regulatory mechanisms underling the critical role of TAK1 in necroptotic signaling through regulation of multiple cell death checkpoints. Targeting key components of the necroptotic pathway (e.g., TRADD and CYLD) and the ubiquitin-proteasome pathway may represent novel therapeutic strategies for pathological conditions driven by necroptosis.

TAK1 Is a Key Regulator of Skeletal Muscle Maintenance in Mice

TGFβ-activated kinase 1 (TAK1), a member of the MEK kinase (MAP3K) family, has been demonstrated to be a component of signaling pathways leading to the activation of NF-kB and activator protein-1 (AP-1) in response to cytokines and growth factors. Our previous study has shown that TAK1 is a critical regulator for the proliferation and differentiation of cultured C2C12 myoblasts. However, the role of TAK1 in regulation of skeletal muscle mass has not yet been investigated. PURPOSE: To determine if skeletal muscle-specific deletion of TAK1 alters the skeletal muscle mass and function in adult mice. METHODS: Skeletal muscle-specific TAK1 knockout (TAK1mko, n=5) mice were generated by crossing HSA-cre mice with floxed TAK1 (i.e. TAK1f/f) mice. Tamoxifen was injected intraperitoneally to inactivate TAK1 and the mice were fed with tamoxifen-containing standard chow for the entire duration of the experiment. TAK1f/f mice (n=6) were used as control. All mice were sacrificed at approximately 3 months of age and the tibialis anterior (TA) and soleus (SOL) muscles were excised for further analysis. RESULTS: TAK1mko mice were found to have reduced body weight (14%) and TA muscle weight (10%) compared to TAK1f/f mice. Mean fiber-cross sectional area (CSA) was decreased in TAK1mko mice by 42% and 39% in TA and SOL muscles, respectively. TAK1mko mice showed lower grip strength compared to control mice by 39%. Genetic ablation of TAK1 in skeletal muscle significantly reduced the levels of specific muscle proteins whereas the levels of atrogin-1, poly-ubiquitinated proteins, and proteasome activity were increased. CONCLUSION: These data demonstrate that TAK1 is essential for maintenance of skeletal muscle in adult animals. Supported by NIH Grant AR059810 and AG029623 to AK.

TAK1 inhibition accelerates cellular senescence of retinal pigment epithelial cells.

Oxidative stress and cellular senescence are known to contribute to the development of AMD; however, the mechanism is not fully understood. This study investigated the role of TGF-β-activated kinase 1 (TAK1) in the senescence of RPE cells as a model for the development of dry AMD. Cultured human RPE cells were treated with the TAK1 inhibitor 5Z-7-oxozeaenol for 1 hour, and then treated with 200 μM hydrogen peroxide for 1 hour. Human RPE cells that were not pretreated with TAK1 inhibitor for 1 hour served as controls. Senescence-associated β-galactosidase (SA-β-gal) activity was detected by histochemistry, and p53 expression by immunoblotting. Cell-cycle and apoptosis rate in RPE cells were determined by flow cytometry. The TAK1 expression in human RPE cells was high and was altered on oxidative stress. Transforming growth factor-β-activated kinase 1 inhibition led to reduction in cell proliferation, cell-cycle arrest at G0/G1, and increased SA-β-gal expression, all known to be features of cell senescence. Exposure of cells to oxidative stress combined with inhibition of TAK1 activity decreased the expression of apoptotic proteins, such as p53, and promoted cellular senescence. Aberrant TAK1 activity in RPE cells triggered their secretion of factors that induced hypertrophy and fibrotic changes in neighboring cells. The in vitro evidence indicated a role for TAK1 in the onset of senescence in RPE cells. The data shown hereby demonstrated that TAK1 activity is essential for maintaining normal function of RPE cells. Elucidation of its role in mechanisms underlying RPE cellular senescence induction may potentiate development of powerful tools for halting the development of dry AMD.

Inhibition of TAK1 (MAP3K7) upregulates unfolded protein response and prevents high‐fat diet‐induced obesity (765.4)

High‐fat diet (HFD) ingestion induces endoplasmic reticulum (ER) stress in hypothalamic neurons leading to dysregulation of food intake and eventually obesity. Therefore, alleviating ER stress in the hypothalamus would be beneficial for treating obesity. Unfolded protein response (UPR) signaling pathways are activated to protect cells from ER stress. Several different pathways initiate induction of chaperones, attenuation of translation and protein degradation in UPR. However, it’s still elusive how each UPR pathway is regulated upon ER stress. TGF‐β‐activated kinase 1 (TAK1) is a member of MAP3K and known to be involved in various stress response signaling. In the present study, we investigated the role of TAK1 in ER stress response. We found that deletion of Tak1 protected cells from ER stress‐induced cell death. Intriguingly, induction of a molecular chaperon, BiP/GRP78, which is one of the major targets of UPR, was highly upregulated in Tak1‐deficient cells. These data suggest that TAK1 negatively regulates UPR and deletion of Tak1 alleviates ER stress and cell damage by upregulating BiP/GRP78. To examine the physiological relevance for the TAK1 regulation of ER stress, we generated mice harboring neuron‐specific deletion of Tak1 and tested the effect of Tak1 deletion in HFD‐induced obesity. We found that neuron‐specific deletion of Tak1 significantly reduced food intake and weight gain upon HFD feeding compared to wild type mice, suggesting that TAK1 may promote hypothalamic neuronal damage through ER stress. Our results revealed a new regulatory mechanism of ER stress which is involved in neuronal homeostasis.

The MAP3K TAK1: A Chock Block to Liver Cancer Formation

Abstract TGF-β-activated kinase 1 (TAK1) is a MAP3K family member that activates NF-κB and JNK via Toll-like receptors and the receptors for IL-1, TNF-α, and TGF-β. Because the TAK1 downstream molecules NF-κB and JNK have opposite effects on cell death and carcinogenesis, the role of TAK1 in the liver is unpredictable. To address this issue, we generated hepatocyte-specific Tak1 -deficient ( Tak1ΔHEP ) mice. The Tak1ΔHEP mice displayed spontaneous hepatocyte death, compensatory proliferation, inflammatory cell infiltration, and perisinusoidal fibrosis at age 1 month. Older Tak1ΔHEP mice developed multiple cancer nodules characterized by increased expression of fetal liver genes including α-fetoprotein. Cultures of primary hepatocytes deficient in Tak1 exhibited spontaneous cell death that was further increased in response to TNF-α. TNF-α increased caspase-3 activity but activated neither NF-κB nor JNK in Tak1 -deficient hepatocytes. Genetic abrogation of TNF receptor type I (TNFRI) in Tak1ΔHEP mice reduced liver damage, inflammation, and fibrosis compared with unmodified Tak1ΔHEP mice. In conclusion, hepatocyte-specific deletion of TAK1 in mice resulted in spontaneous hepatocyte death, inflammation, fibrosis, and carcinogenesis that was partially mediated by TNFR signaling, indicating that TAK1 is an essential component for cellular homeostasis in the liver. Abstract The MAP3-kinase TGF-β-activated kinase 1 (TAK1) critically modulates innate and adaptive immune responses and connects cytokine stimulation with activation of inflammatory signaling pathways. Here, we report that conditional ablation of TAK1 in liver parenchymal cells (hepatocytes and cholangiocytes) causes hepatocyte dysplasia and early-onset hepatocarcinogenesis, coinciding with biliary ductopenia and cholestasis. TAK1-mediated cancer suppression is exerted through activating NF-κB in response to tumor necrosis factor (TNF) and through preventing Caspase-3-dependent hepatocyte and cholangiocyte apoptosis. Moreover, TAK1 suppresses a procarcinogenic and pronecrotic pathway, which depends on NF-κB-independent functions of the IκB-kinase (IKK)-subunit NF-κB essential modulator (NEMO). Therefore, TAK1 serves as a gatekeeper for a protumorigenic, NF-κB-independent function of NEMO in parenchymal liver cells.

Disruption of TAK1 in hepatocytes causes hepatic injury, inflammation, fibrosis, and carcinogenesis

TGF-beta-activated kinase 1 (TAK1) is a MAP3K family member that activates NF-kappaB and JNK via Toll-like receptors and the receptors for IL-1, TNF-alpha, and TGF-beta. Because the TAK1 downstream molecules NF-kappaB and JNK have opposite effects on cell death and carcinogenesis, the role of TAK1 in the liver is unpredictable. To address this issue, we generated hepatocyte-specific Tak1-deficient (Tak1DeltaHEP) mice. The Tak1DeltaHEP mice displayed spontaneous hepatocyte death, compensatory proliferation, inflammatory cell infiltration, and perisinusoidal fibrosis at age 1 month. Older Tak1DeltaHEP mice developed multiple cancer nodules characterized by increased expression of fetal liver genes including alpha-fetoprotein. Cultures of primary hepatocytes deficient in Tak1 exhibited spontaneous cell death that was further increased in response to TNF-alpha. TNF-alpha increased caspase-3 activity but activated neither NF-kappaB nor JNK in Tak1-deficient hepatocytes. Genetic abrogation of TNF receptor type I (TNFRI) in Tak1DeltaHEP mice reduced liver damage, inflammation, and fibrosis compared with unmodified Tak1DeltaHEP mice. In conclusion, hepatocyte-specific deletion of TAK1 in mice resulted in spontaneous hepatocyte death, inflammation, fibrosis, and carcinogenesis that was partially mediated by TNFR signaling, indicating that TAK1 is an essential component for cellular homeostasis in the liver.