Jun Ninomiya-Tsuji

Innate immune signaling pathways are activated in response to exposure to microorganisms, and effectively prevent pathogen invasion through inducing inflammation and host cell death. As we are living under pathogen-rich environment, the innate intracellular signaling pathways have long been contributing to human survival. However, having myriad hygienic and medical evolutions, humans are now having less chances of exposure to many pathogens and live longer. As a result, the innate immune signaling pathways, which may be aberrantly activated over the time, are not always beneficial to human survival but are also causally associated with many age-related inflammatory diseases. Thus, controlling innate immunity emerges as an important goal in current biomedical research. However, its complexity has been preventing our complete understanding. We have been working on one of the key innate immune signaling molecules, a protein kinase MAP3K7, commonly known as TAK1. TAK1 is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family and activates MAPK cascades, but is also a prominent activator of NF-��������B pathway, which transcriptionally activates inflammatory responses. TAK1 also physically interacts with other innate immune signaling molecules such as caspase 8, receptor interacting protein kinase 1 and 3 (RIPK1 and RIPK3), and participates in their cell death pathways. Our series of in vivo TAK1 studies in the mouse model have unexpectedly revealed that both inhibition and activation of TAK1 elicits inflammation and cell death, which is seemingly contradictory. Interestingly, other innate immune signaling molecules, caspase 8 and RIPK1, also exhibit contradicting consequences by activation and inhibition. It seems that the innate immune signaling pathways are designed to induce inflammation regardless of whether they are activated or inhibited. We hypothesize that these puzzling responses are host counter attack mechanisms to deal with evolving pathogens that have gained the abilities to inhibit the host innate immunity. For the next five years, our research aims at determining the roles and mechanisms of such seemingly unreasonable regulation of the innate immune signaling pathways by defining the TAK1 signaling pathways. We will further determine how aberrant activation of TAK1 occurs and how it contributes to inflammatory diseases.

Clear cell sarcoma (CCS) is an aggressive bone and soft tissue cancer of young adults. It is caused by gene fusions between EWS (Ewing������������������s sarcoma oncogene) and ATF1 (activating transcription factor) or CREB (cAMP-responsive element binding protein), giving rise to chimeric oncoproteins consisting of the N-terminal EWS domain fused to a C-terminal half of truncated ATF1 and CREB domains. The chimeric EWS/ATF1 and EWS/CREB oncoproteins are potent transcription factors that bind to CRE (cAMP-responsive element) via the ATF1 and CREB domains. Normal human ATF1 and CREB are activated by stimulus-induced phosphorylation, in which ATF1 at Ser63 and CREB at Ser133 are phosphorylated by protein kinase A (PKA) or related kinases in response to various growth factors and hormones. In contrast, the molecular mechanism through which the transcription function of EWS/ATF1 and EWS/CREB is regulated remains unknown. The primary reason for this problem is the fact that truncated ATF1 and CREB domains fused to EWS do not contain these canonical Ser63 and Ser133 PKA-mediated phosphorylation sites. However, we recently identified the conserved new phosphorylation sites in the chimeric oncoproteins of ATF1 at Ser198 and CREB at Ser271. Intriguingly, we found that HIPK2 (homeodomain interacting protein kinase 2), but not PKA, phosphorylates EWS/ATF1 at Ser198 of the ATF1 domain and suppresses the transcription of c-FOS, one of the key EWS/ATF1 target genes driving aggressive cell proliferation of CCS. Indeed, EWS/ATF1 can be phosphorylated at Ser198 of the ATF1 domain in human CCS cells. Furthermore, we preliminary identified candidates of ATF1 Ser198 phosphatases. Thus, we have found two new potential reversible regulators of EWS/ATF1 oncoprotein through Ser198 phosphorylation. We will test our hypothesis that the phosphorylation status of EWS/ATF1 at Ser198 determines the EWS/ATF1 transcription activity on key target genes responsible for aggressive proliferation of CCS cancer cells. We will characterize the phosphorylation and dephosphorylation of EWS/ATF1 at Ser198 associated with its transcription function and CCS malignant phenotype. As ATF1 and CREB contain highly conserved phosphorylation sites, we anticipate the same regulatory mechanism of the EWS/CREB fusion oncoprotein via Ser271 phosphorylation in the truncated CREB domain. HIPK2 as well as Ser198 ATF1 phosphatases characterized in this proposal can be a new molecular target for more selective and effective inactivation of these fusion oncoproteins in CCS.

Inflammation is one of the key biological processes in Alzheimer������������������s disease (AD). Glial cells activate neuronal inflammatory signaling leading to beta-amyloid deposition and tauopathy. Reciprocally, neuron-derived pathology makes glial cells inflamed. Disrupting this inflammatory loop is emerging in blocking AD progression. We are investigating a protein kinase TAK1, which plays a vital role in intracellular inflammatory signaling in most types of cells. Inhibition of TAK1 not only blocks inflammatory responses but also elicits apoptosis. We recently found that hippocampal TAK1 is activated by aging or by mutant beta-amyloid expression, and that deletion of Tak1 alleviates AD pathology in mouse models. Thus, TAK1 activation is causally associated with AD, and inhibition of TAK1 could be effective in AD treatment. We propose to define the mechanism and effectiveness of inhibition of TAK1 as a mean of AD treatment using AD mouse models.

Macrophages are essential cells for tissue maintenance and mediating inflammation, and are found ubiquitously throughout the body. They are primary phagocytic cells that engulf and destroy pathogens, parasites and any unwanted material. However, some pathogens have been able to subvert and thwart their attempts at being eliminating by macrophages. The pathogen-derived mechanisms to evade macrophage defense include disrupting the immune response. Mitogen-activated protein kinase kinase kinase 7 (MAP3K7), also known as TAK1, is a key intermediate molecule of the intracellular inflammatory signaling pathways, and is, therefore, one of the major targets of immune disruption by pathogens. However, disruption of TAK1 not only blocks inflammatory signaling but also triggers cell death, which is believed to be a host counter defense mechanism to limit pathogen survival and colonization. This project aims to determine the TAK1 ablation-derived host defense mechanism, which has not yet been fully characterized. Our preliminary studies have thus far revealed that ablation of Tak1 limits intracellular bacterial growth. Tak1 deficiency is associated with activation of both apoptosis and necroptosis, but is most notably associated with increases of mitochondrial reactive oxygen species (ROS). We aim to; i) gain mechanistic insight of how TAK1 ablation limits intracellular bacterial growth by defining the roles and relationship of apoptosis, necroptosis and ROS in macrophages; ii) understand the mechanisms by which TAK1 regulates mitochondrial reactive oxygen species. Outcomes of this project would yield fruitful understanding of the host defense mechanisms present within macrophages, and could serve as a platform to develop better approaches in infectious diseases.

Metabolism is tightly regulated and disruptions in metabolic homeostasis could lead to metabolic diseases such as type 2 diabetes. Inflammation is the prominent disrupter of metabolic homeostasis, which is associated with hyperglycemia, hypercholesterolemia, hypertriglyceridemia and obesity. Thus, understanding of 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 disruption. JNK and NF-kB modulate and inhibit insulin receptor substrate 1 (IRS1), which is clearly responsible for insulin resistance in several tissues including liver and adipose tissues. However, inflammation is associated with many other metabolic disorders including impairment of hypothalamic neurons, which regulate appetite and glucose homeostasis. Many of the mechanisms by which inflammation regulates metabolism still remain to be identified. We found that neuron-specific deletion of a protein kinase TAK1 blocks inflammation-associated obesity. TAK1 belongs to the mitogen-activated protein kinase kinase kinase (MAP3K) family., and a intermediate of inflammatory signaling pathway. TAK1 can activate JNK or NF-��������B; however, we found that activity of JNK and NF-��������B is unchanged in Tak1 deletion in neurons. Thus, TAK1 modulate neurons and systemic metabolism through a mechanism independently of JNK or NF-��������B. In the effort to determine new downstream pathways of TAK1, we have identified lipogenesis is increased by Tak1 deletion. TAK1 modulates lipid metabolism, which may alter overall metabolic state in the cells. We hypothesize that inflammation-induced TAK1 activation changes cellular metabolism through modulating lipogenesis, which is one of the pathway of inflammation-induced metabolic disorder. In this project: 1) we will delineate the TAK1-dependent mechanism for metabolic regulation at the cellular level: 2) we will determine how the TAK1-mediated cellular level metabolic change modulates systemic metabolic homeostasis such as weight control. Outcomes of this project will reveal a new mechanistic link between inflammation and metabolism, and could provide therapeutic targets in inflammation-induced metabolic diseases.

Oxidative stress is a continual threat to cells during exposure of environmental toxicants such as arsenic. To cope with oxidative stress, cells have evolved the Nrf2-ARE (antioxidant responsive element) pathway that activates transcription of an array of detoxification genes. Nrf2 is trapped in the cytoplasm by the inhibitor protein Keap1, but Keap1 oxidized or adducted by xenobiotics releases Nrf2 to translocate into nucleus. Like other transcription factors, Nrf2 should be tightly regulated in the nucleus; however, it remains largely uncharacterized. We have recently reported that p66Shc, a prooxidant and anti-longevity gene, is transcriptionally activated by various xenobiotics through the Nrf2-ARE pathway in some but not all cell types we tested. We hypothesized that a nuclear inhibitory protein may suppress Nrf2 transcription function in these non-responsive cells. According to our preliminary results, the inhibitory protein appears to be Aiolos (IKZF3), an Ikaros family transcription factor. We will characterize expression of p66Shc and major Nrf2-regulated antioxidant genes and Nrf2 binding to AREs in our tet-inducible Aiolos cells. Another issue is that p66Shc has been understood as a mitochondrial prooxidant protein, whereas we demonstrated that p66Shc is a cytoplasmic antioxidant protein. We have found that a cleaved form of p66Shc is accumulated in mitochondria after xenobiotic treatment. Thus, p66Shc may play an antioxidant role in the cytoplasm but become a prooxidant protein after translocation to mitochondria and subsequent proteolytic cleavage following xenobiotic exposure. We will assess whether a mitochondrial protease is involved in the formation of the cleaved p66Shc isoform.

The CREB (cAMP-response element binding) transcription factor is a stimulus-induced phospho-protein that is involved in numerous cell signaling pathways. Dysfunction and deregulation of CREB and CREB-interacting proteins cause human diseases such as cancer and neuronal cell damage. CREB appears to play a key role in cell defense and survival in various tissues; however, the mechanisms through which CREB is involved in cell survival and the reason why deregulation of CREB function causes these human diseases remain largely unknown. CREB phosphorylation at Ser-133 is the major posttranslational modification that enhances CREB activity in response to various receptor-coupled stimuli. However, the status of CREB Ser-133 phosphorylation was not always correlated with CREB transcription function, suggesting that another event along with CREB Ser-133 phosphorylation seems to be involved in CREB regulation in a stimulus-specific manner. This research project may provide evidence and a critical answer to theses unsolved problems because we recently found that HIPK2 (homeodomain interacting protein kinase 2), a genotoxic stress responsive kinase, activates CREB via phosphorylation of a new serine site (Ser-271) but not Ser-133, resulting in activation of CREB transcription function. We will test our hypothesis that HIPK2 is a new regulator of the CREB transcription factor in genotoxic and oxidative stress condition via phosphorylation of this new serine site. The proposed experiments will focus on characterization of molecular mechanism through which CREB phosphorylation by HIPK2 activates its transcription function as well as downstream events including expression of target genes and cellular susceptibility to genotoxic stress in in vitro and in vivo models. The scientific impact of this research will be broad and significant in many research areas because CREB regulates numerous cellular functions in many types of tissues, therefore the unveiled new CREB regulation from successful completion of this proposal will be evaluated in various physiological and disease conditions closely associated with CREB activity.

The gestation period is uniquely susceptible to environmental exposures. Highly active epigenetic reprogramming is likely to contribute to such high susceptibility. Aberrant epigenetic modulations during embryogenesis could persist and lead to adult diseases such as type II diabetes (T2D) and obesity. This proposal seeks to collect essential preliminary data to identify the molecular mechanism through which environmental exposure induces epigenetic modulations in the fetal brain that can cause T2D in adulthood. Leptin resistance in the hypothalamus is known to impair glucose homeostasis as well as eating behavior, and is caused by disruption of normal hypothalamic leptin signaling. A protein kinase, TAK1, is commonly activated under stress conditions induced by exposure to toxic metals, including arsenic. We recently found that activation of TAK1 is associated with leptin resistance in the hypothalamus. Furthermore, we found that TAK1 regulates energy metabolism, which could alter the cellular concentrations of key donor molecules in epigenetic modifications. Interestingly, alteration of metabolites has been reported in the arsenic exposed human cohort study. Our main hypothesis is Hypothalamic TAK1 activation by toxic metal exposure causes leptin resistance through epigenetic modification. Such changes occur at a high efficiency during embryogenesis and persist to adulthood.

Iron is an essential element by serving as a constituent of vital cellular proteins involved in a variety of cellular functions; however, excess iron is detrimental because it catalyzes formation of reactive oxygen species (ROS). Disorder of iron homeostasis involving iron deficiency or overload is associated with various human health problems such as neurodegenerative disease, cancer and aging. Fine-tuning of intracellular iron levels is therefore essential for maintaining normal cellular function and physiological metabolic balance. Ferritin is the major iron-storage protein in eukaryotic cells and it plays a crucial role in regulation of iron metabolism by detoxifying and storing intracellular excess iron in a non-toxic but bioavailable form. Ferritin synthesis is regulated at both transcriptional and translational levels. Translational regulatory mechanism of ferritin by iron has been extensively studied and well characterized. In contrast, iron-independent transcriptional regulation of the ferritin gene under such conditions as cells need to limit iron availability remains incompletely understood. In particular, little is known about ferritin transcriptional regulation through chromatin remodeling mechanism under oxidative stress conditions. Transcription of ferritin and a battery of antioxidant genes are regulated by a conserved enhancer, termed the ARE (antioxidant responsive element). We hypothesize that chromatin remodeling and associated factors we have recently identified on the human ferritin ARE can serve as crucial proteins that regulate ferritin transcription and iron homeostasis. The proposed experiments will focus on characterization of these new ARE-interacting proteins and their roles in chromatin modifications adjacent to ARE-regulated ferritin and antioxidant genes. The scientific impact of this research will be broad and significant because it will not only provide new insight into the basic transcriptional mechanism of a group of antioxidant genes via coordinated regulation of transcription factors and chromatin-remodeling factors, but also define new regulatory proteins responsible for cellular antioxidant response and iron homeostasis under oxidative stress conditions that are associated with various iron- and ROS-involving human diseases.

Hematopoietic TAK1 is Required for Maintaining Lysosomal Integrity and Cell Survival in Resident Macrophages Sakamachi, Y., Mihaly, S. M., Morioka, S., and Ninomiya-Tsuji, J. Dept. of Biological Sciences, North Carolina State University, Raleigh, NC, 27695 USA Transforming growth factor-β activated kinase 1 (TAK1) is an intracellular signaling molecule regulating apoptosis and necrosis (necroptosis) in several cell types, including epithelial and endothelial cells. Aberrant cell death of hematopoietic cells, both in excess and in deficiency, is known to cause a number of diseased conditions, but the role of TAK1 in hematopoietic cells is not yet fully determined. This study aimed to determine the role of TAK1 in the hematopoietic system, specifically during development, where hematopoietic cells are involved not only in immunity but also in morphogenesis. Here, we report that hematopoietic TAK1 plays a critical role predominantly in tissue-resident macrophage maintenance and survival. Hematopoietic lineage-specific deletion of Tak1 gene (Tak1HKO) showed diminished thymic and lung resident macrophages, accumulation of cellular debris in the embryonic thymus, and impaired alveolar expansion in perinatal mice, which were accompanied by animal mortality. However, thymocytes, splenic lymphocytes, and myeloid cells developed normally in Tak1HKO mice, suggesting that TAK1 is required selectively for the maintenance of macrophages in the embryonic hematopoietic system. Resident macrophages are known to play a vital role during embryonic development, and depletion or alterations of macrophage function during organogenesis can lead to developmental defects or embryonic lethality. Thus, loss of macrophage in Tak1HKO mice is likely the cause of accumulation of dead cell debris and impaired lung development. To investigate the role of TAK1 in macrophages, we utilized bone marrow-derived macrophages (BMDMs). We found that Tak1 gene deletion induced profound cell death in BMDMs without exogenous stimulation. Since macrophages express cell death-inducing receptors (e.g. TNF receptor (TNFR) and pattern recognition receptors), autocrine signaling may be involved in the cell death. However, the ablations of toll-like receptor (TLR) signaling intermediates Trif or Myd88 did not block cell death, while Tnfr1-deletion only partially restored cell viability in Tak1-deficient BMDMs. This suggests the involvement of TNF/TLR-independent mechanisms of cell death, potentially associated with cell intrinsic mechanisms. Tak1-deficient BMDMs exhibited marginal activation of caspase-3, suggesting non-apoptotic types of cell death. We found that Tak1-deficient BMDMs exhibited aberrant lysosomal structures and potential leakage of lysosomal proteins. Consistently, inhibition of a lysosomal protease, cathepsin B, reduced Tak1-deficient BMDM death. Furthermore, we also found that ROS was highly upregulated in Tak1-deficient BMDMs, and that ROS scavenger effectively prevented cell death. Since active phagocytosis in macrophages is associated with lysosomal ROS production, Tak1 deficiency- induced failure in elimination of ROS may be the cause of the lysosome rupture-induced cell death in macrophages. Our results collectively suggest a novel function of TAK1 in maintaining lysosome integrity in macrophages, which is essential for resident macrophage survival and organ development.