Jun Ninomiya-Tsuji - US grants

DESCRIPTION (provided by applicant): Bone integrity is maintained through the coordinated action of bone resorption by osteoclasts and bone formation by osteoblasts. RANKL (receptor activator of nuclear factor kappaB (NF-kappaB) ligand) is a key factor for differentiation and activation of osteoclasts. Chronic inflammation and cancer metastasis to bone induce excessive bone resorption primarily by enhancing production of RANKL. RANKL binds to its receptor RANK (receptor activator of NF-kappaB) and initiates intercellular signaling by recruiting intercellular adaptor TRAF6 (tumor necrosis factor receptor-associated factor 6). RANKL/RANK/TRAF6-mediated signaling leads to activation of transcription factor NF-kappaB, mitogen-activated kinases JNK and p38, oncogene product c-Src and transcription factor c-Fos expression, which are important for osteoclastogenesis. However, the link between RANK/TRAF6 and the downstream effectors has not been identified. We have previously demonstrated that TAK1 (transforming growth factor beta activated kinase), a member of MAPKKK (mitogen-activated kinase kinase kinase), plays essential roles in proinflammatory signaling and cell differentiation. Recently, we have found that RANKL induces endogenous association of TAK1 and TAB2 (TAK1 binding protein 2) with RANK/TRAF6. Therefore, we hypothesize that TAK1 and TAB2 also play essential roles in RANKL signaling pathway. In this exploratory/developmental proposal, we will determine the roles of TAK1/TAB2 in RANKL-dependent signaling pathways and obtain data to develop in vivo model to further verify the specific roles of TAK1/TAB2 in osteoclastogenesis. Our specific Aims are: 1) To determine the roles of TAK1/TAB2 in RANKL-induced NF-kappaB, JNK, p38 and c-Src activation, c-Fos induction and osteoclastogenesis: 2) To design and create mutant TAK1 and TAB2 proteins that lack the ability to transmit the RANKL-dependent signaling but are intact in mediating other signaling pathways. We will plan to use those mutants to demonstrate functional roles of the RANK/TRAF6/TAB2/TAK1 signaling in osteoclastogenesis in the future study. These studies will advance the understanding of the molecular mechanisms underlying the osteoclastogenesis and may offer novel therapeutic targets for bone diseases caused by excessive osteoclastogenesis.

DESCRIPTION (provided by applicant): Cytokines play important roles in human development and disease. Specificity and cross-talk of cytokine signaling pathways appear to be important for fine-tuning stress responses and cell fate decision during development. Transforming growth factor beta (TGF-beta) is involved in cell growth, differentiation, tissue remodeling, immune response and angiogenesis. Interleukin 1 (IL-1) pathway plays a central role in the generation of inflammatory responses. We have found that both TGF-beta and IL-1 activate TGF-beta activated kinase 1 (TAK1) MAPKKK. Active form of TAK1 can enhance both TGF-beta- and IL-l-dependent transcription. In response to IL-1 stimulation, TAK1 activates transcription factors AP-1 and NF-KappaB. While TGF-6 stimulation does activate TAK1, the role of TAK1 in TGF-beta signaling pathway is not known. Recently, we found that TAK1 associates with a transcriptional repressor SnoN, a negative regulator of TGF-beta signaling. TAK1 induces degradation of SnoN. We hypothesize that TGF-beta activates TAK1 to induce phosphorylation of SnoN and targets SnoN for proteasomal degradation, thereby up-regulating TGF-b signal transduction. In addition, we hypothesize that TGF-beta and IL-1 activate TAK1 in distinct manner via specific scaffold/regulatory proteins to induce their unique cellular responses. Thus, the overall objectives of this proposal are; to delineate the pathway and functional role of TAK1 in TGF-beta signaling and to elucidate the mechanisms through which TAK1 regulates signal pathway specificity. To accomplish these objectives and to test our hypotheses we will: i) determine the mechanism and role of TAK1-induced SnoN degradation in TGF-b signaling pathway; ii) isolate and characterize molecules associated with TAK1 and iii) generate a skin specific knockout of TAK1 to characterize the in vivo role of TAK1 in a tissue in which TGF-beta play important roles. These studies will address unsolved questions regarding the mechanisms of TGF-beta and IL-1 family signaling and will provide an understanding of the physiological function of TAK 1 in vivo.

TAK1 kinase is an indispensable intermediate in the intracellular signaling of innate immune responses. TAK1 is activated by many of distinct factors including Toll-like receptor ligands, intracellular microorganism sensor (NOD like receptor) ligands, IL-1 and TNF. TAK1 upregulates proinflammatory responses through activation of NF-B and mitogen activated protein kinase pathways. Thus, TAK1 is generally considered to be a positive regulator of inflammation. However, we have recently found that the targeted deletion of TAK1 in the epithelium of skin and intestine results in severe inflammation. These inflammatory conditions in the TAK1 mutant mice resemble chronic inflammatory diseases such as psoriasis in the skin and Crohn's disease in the intestine. We have found that TAK1 deletion causes accumulation of reactive oxygen species (ROS), and that inhibition of ROS can completely rescue cell death in cultured epithelial cells. Importantly, we found that treatment of the antioxidants in the epithelial-specific TAK1 deletion mice could prevent the epithelial cell death and diminishes inflammation. Therefore, we hypothesize that ablation of TAK1 signaling in epithelial cells causes dysregulation of ROS that is involved in epithelial cell death and inflammation. The objectives of this proposal are;1) to determine the mechanism by which ROS regulates epithelial cell death and inflammation;2) to identify the cause of ROS accumulation in TAK1-deficient epithelium. Outcomes from this project will delineate the relationship between ROS regulation and chronic inflammation, which could result in new approaches to regulate inflammation.

DESCRIPTION (provided by applicant): TAK1 kinase is an essential signaling intermediate involving multiple signaling pathways including TNF, IL-1, and stress pathways. We have recently demonstrated that the targeted deletion of TAK1 in multiple epithelial tissues causes cell death and inflammatory conditions. Thus, TAK1 is critically involved in tissue homeostasis by regulating cell death. Although TAK1 regulation of pro-inflammatory signaling leading to cytokine production has been well studied, the TAK1 pathways regulating cell death remain elusive. We have identified that TAK1 regulates the level of reactive oxygen species (ROS). TAK1 binding proteins, TAB1 and TAB2, differentially participate in TAK1 signaling;TAB2 mediates cytokine-induced TAK1 activation, whereas TAB1 mediates activation of TAK1 specifically in response to stress. We hypothesize that TAK1 regulates cell survival and inflammation in vivo by modulating ROS, and that TAB1 and TAB2 regulate TAK1-cell survival signaling in response to stimulus unique to each protein. The long-term objective is to delineate the TAK1 signaling network regulating tissue homeostasis. In short- term, we aim to determine the roles of TAK1, TAB1 and TAB2 in ROS-dependent cell death pathway. Outcomes from this project will enhance our understanding of tissue homeostasis specifically regulation of ROS, cell death and inflammation, which could lead to new approaches to improve many inflammatory conditions that are associated with ROS. PUBLIC HEALTH RELEVANCE: To maintain tissue integrity, cells need to prevent unscheduled cell death, which could induce tissue damages and inflammation. In many tissues, potential cell death inducers such as cytokines and stressors constantly present even in normal conditions;however cells are resistant to those inducers. We have found that mice having deletion of TAK1 kinase in the epithelial tissues spontaneously develop tissue damages associated with cell death. This suggests that TAK1 kinase activity is important to prevent cell death in normal tissues. In this project, we will determine the mechanism by which TAK1 controls cell death and define how TAK1 kinase activity is regulated in normal tissues. The outcomes enhance our understanding of the regulatory mechanism of tissue integrity, which could lead new approaches to prevent tissue damage-associated pathogenic conditions.

Energy metabolism is tightly regulated, and disruptions in the metabolic homeostasis could lead to metabolic syndromes such as obesity and type II diabetes. Inflammation is a prominent disrupter of energy metabolism. Thus, understanding molecular pathways of inflammation-induced metabolic disorders is critical to combat metabolic diseases. Inflammatory signaling molecules such as c-Jun N-terminal kinase (JNK) and NF-?B have been identified as mediators of metabolic diseases. JNK and NF-?B inhibit insulin receptor substrate 1, which is responsible for insulin resistance in several tissues including liver and adipose tissues. In addition to the JNK- and NF-?B-dependent mechanisms, emerging evidence indicates that endoplasmic reticulum (ER) stress is the major pathway linking inflammation and metabolic disorders. However, the molecular mechanism by which inflammation induces ER stress is not yet clear. We found that deletion of a protein kinase TAK1 protects cells from ER stress, and that neuron-specific TAK1 deletion blocks inflammation- associated metabolic disorders including excess weight gain. TAK1 belongs to the mitogen-activated protein kinase kinase kinase (MAP3K) family, and is an intermediate of inflammatory signaling pathways. TAK1 can activate JNK and NF-?B; however, activity of JNK and NF-?B is unaltered in the deletion of Tak1 in neurons. Thus, TAK1 modulates ER stress and energy metabolism through a previously uncharacterized pathway. In an effort to determine a new downstream pathway of TAK1, we have found that TAK1 inhibits a transcription factor SREBP, which is the key regulator of lipogenesis. Lipogenesis is important for membrane biogenesis and its alteration impacts the ER mass and function. We hypothesize that inflammation-induced TAK1 activation downregulates membrane biogenesis through inhibiting SREBP-dependent lipogenesis, which is causally associated with ER stress and the disorder in neuronal regulation of systemic energy metabolism. In this project, we will delineate: 1) the molecular mechanism by which TAK1 modulates lipogenesis, 2) the role of TAK1-lipogenesis pathway in ER stress, and 3) the link between neuronal TAK1-lipogenesis-ER stress pathway and systemic metabolic disorders. Outcomes of this project will reveal a new mechanistic link between inflammation and energy metabolism.

Abstract Innate immune signaling pathways are activated in response to exposure to microorganisms, and generally are effective in preventing pathogen invasion through inducing inflammation and host cell death. However, its aberrant activation is known to be causally associated with many inflammatory diseases e.g. cancers and neurodegeneration, as it could cause tissue damage through inflammation and cell death. The innate immune signaling pathways are highly complex as they have evolved in response to evolving microorganisms trying to evade the host immunity. Thus, the regulatory mechanisms of innate immunity particularly their signaling connections/networks are incompletely understood. Understanding the complexities of the innate immune signaling network is highly anticipated to impact our ability to develop strategies to fight pathogen infection and to treat inflammatory diseases. We have been studying mitogen-activated protein kinase kinase kinase 7 (MAP3K7), known as TAK1, since its discovery. Initially we identified that TAK1 mediates transcriptional activation of inflammatory responses by activating both MAPK cascades and NF-?B pathways. More recently, through our characterization of numerous tissue-specific Tak1-deficient mouse models we have revealed that TAK1 also participates in cell death. However, there remain unanswered fundamental questions; why and how do the inflammatory and cell death pathways converge through TAK1? The R35 stable funding mechanism is highly suitable for this challenging project. We have all the materials, e.g. genetically engineered mouse models and pharmacological modulators, and experience for answering the above central question. For the next 5 years, we propose to determine the molecular mechanisms of how inflammatory and cell death pathways are connected at TAK1 and of how aberrant activation of TAK1 leads to inflammatory diseases.