TAK1-binding proteins (TAB)2 and TAB3 are redundantly required for TLR-induced cytokine production in macrophages.

Transforming growth factor-ß-activated kinase 1 (TAK1) plays a pivotal role in innate and adaptive immunity. TAK1 is essential for the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB pathways downstream of diverse immune receptors, including Toll-like receptors (TLRs). Upon stimulation with TLR ligands, TAK1 is activated via recruitment to lysine 63-linked polyubiquitin chain through TAK1-binding proteins (TAB) 2 and TAB3. However, the physiological importance of TAB2 and TAB3 in macrophages is still controversial. A previous study has shown that mouse bone marrow-derived macrophages (BMDMs) isolated from mice double deficient for TAB2 and TAB3 produced tumor necrosis factor (TNF)-α and interleukin (IL)-6 to the similar levels as control wild-type BMDMs in response to TLR ligands such as lipopolysaccharide (LPS) or Pam3CSK4, indicating that TAB2 and TAB3 are dispensable for TLR signaling. In this study, we revisited the role of TAB2 and TAB3 using an improved mouse model. We observed a significant impairment in the production of pro-inflammatory cytokines and chemokine in LPS- or Pam3CSK4-treated BMDMs deficient for both TAB2 and TAB3. Double deficiency of TAB2 and TAB3 resulted in the decreased activation of NF-κB and MAPK pathways as well as the slight decrease in TAK1 activation in response to LPS or Pam3CSK4. Notably, the TLR-mediated expression of inhibitor of NF-κB (IκB)ζ was severely compromised at the protein and mRNA levels in the TAB2/TAB3 double-deficient BMDMs, thereby impeding IL-6 production. Our results suggest that TAB2 and TAB3 play a redundant and indispensable role in TLR signaling pathway.

GRIM-19 is a target of mycobacterial Zn2+ metalloprotease 1 and indispensable for NLRP3 inflammasome activation.

Tuberculosis is a communicable disease caused by Mycobacterium tuberculosis which primarily infects macrophages and establishes intracellular parasitism. A mycobacterial virulence factor Zn2+ metalloprotease 1 (Zmp1) is known to suppress interleukin (IL)-1ß production by inhibiting caspase-1 resulting in phagosome maturation arrest. However, the molecular mechanism of caspase-1 inhibition by Zmp1 is still elusive. Here, we identified GRIM-19 (also known as NDUFA13), an essential subunit of mitochondrial respiratory chain complex I, as a novel Zmp1-binding protein. Using the CRISPR/Cas9 system, we generated GRIM-19 knockout murine macrophage cell line J774.1 and found that GRIM-19 is essential for IL-1ß production during mycobacterial infection as well as in response to NLRP3 inflammasome-activating stimuli such as extracellular ATP or nigericin. We also found that GRIM-19 is required for the generation of mitochondrial reactive oxygen species and NLRP3-dependent activation of caspase-1. Loss of GRIM-19 or forced expression of Zmp1 resulted in a decrease in mitochondrial membrane potential. Our study revealed a previously unrecognized role of GRIM-19 as an essential regulator of NLRP3 inflammasome and a molecular mechanism underlying Zmp1-mediated suppression of IL-1ß production during mycobacterial infection.

miR-935 Inhibits Oral Squamous Cell Carcinoma and Targets Inositol Polyphosphate-4-phosphatase Type IA (INPP4A).

BACKGROUND/AIM: Oral squamous cell carcinoma (OSCC) is a common malignancy with poor prognosis. Therefore, novel therapeutic options are needed to improve prognosis of OSCC. Recently, microRNAs (miRs) have received increasing attention as a potential therapeutic tool for carcinomas. However, no definitive miR-based drugs for patients with OSCC have been reported to date. The aim of this study was to identify new miRs potentially involved in cellular processes associated with OSCC malignancy, which could lead to novel therapeutic strategies. MATERIALS AND METHODS: We identified miRs that are modulated in OSCC and possibly regulate OSCC malignancy, using miR microarray on OSCC cell lines. RESULTS: miR-935 and miR-509-3p were down-regulated in OSCC cell lines and patient tissues. When miR-935 was overexpressed in HSC-3-M3 cells, proliferation, migration, and invasion of the cell line was suppressed, whereas apoptosis was increased. Moreover, we showed that the gene inositol polyphosphate-4-phosphatase type I A (INPP4A) is a potential target whose expression is positively regulated by miR-935. CONCLUSION: miR-935 may function as a tumor suppressor by inhibiting OSCC malignancy via INPP4A induction. Therefore, miR-935 can be a new therapeutic candidate for OSCC treatment.

Effects of Psidium guajava leaf extract on secretion systems of Gram-negative enteropathogenic bacteria.

We screened a total of 672 plant-tissue extracts to search for phytochemicals that inhibit the function of the type III secretion system (T3SS) of enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC). Among candidates examined, we found that an extract from the leaves of Psidium guajava (guava) inhibited the secretion of the EspB protein from EPEC and EHEC without affecting bacterial growth. The guava extract (GE) also inhibited EPEC and EHEC from adhering to and injecting EspB protein into HEp-2 cells. GE seemed to block the translocation of EspB from the bacterial cells to the culture medium. In addition to EPEC and EHEC, GE also inhibited the T3SS of Yersinia pseudotuberculosis and Salmonella enterica serovar Typhimurium. After exposure to GE, Y. pseudotuberculosis stopped the secretion of Yop proteins and lost its ability to induce the apoptosis of mouse bone marrow-derived macrophages. S. Typhimurium exposed to GE ceased the secretion of Sip proteins and lost its ability to invade HEp-2 cells. GE inhibited EspC secretion, the type V secretion protein of EPEC, but not Shiga toxin2 from EHEC. Thus, our results suggest that guava leaves contain a novel type of antimicrobial compound that could be used for the therapeutic treatment and prevention of gram-negative enteropathogenic bacterial infections.

TAK1 regulates resident macrophages by protecting lysosomal integrity.

Hematopoietic cell survival and death is critical for development of a functional immune system. Here, we report that a protein kinase, TAK1, is selectively required for resident macrophage integrity during embryogenesis. Hematopoietic lineage-specific deletion of Tak1 gene (Tak1HKO) caused accumulation of cellular debris in the thymus in perinatal mice. Although no overt alteration in thymocytes and blood myeloid populations was observed in Tak1HKO mice, we found that thymic and lung macrophages were diminished. In the in vitro setting, Tak1 deficiency caused profound disruption of lysosomes and killed bone marrow-derived macrophages (BMDMs) without any exogenous stressors. Inhibition of the lysosomal protease, cathepsin B, partially blocked Tak1-deficient BMDM death, suggesting that leakage of the lysosomal contents is in part the cause of cell death. To identify the trigger of this cell death, we examined involvement of TNF and Toll-like receptor pathways. Among them, we found that deletion of Tnfr1 partially rescued cell death. Finally, we show that Tnfr1 deletion partially restored thymic and lung macrophages in vivo. These results suggest that autocrine and potentially paracrine TNF kills Tak1-deficient macrophages during development. Our results reveal that TAK1 signaling maintains proper macrophage populations through protecting lysosomal integrity.

TAK1 determines susceptibility to endoplasmic reticulum stress and leptin resistance in the hypothalamus.

Sustained endoplasmic reticulum (ER) stress disrupts normal cellular homeostasis and leads to the development of many types of human diseases, including metabolic disorders. TAK1 (also known as MAP3K7) is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family and is activated by a diverse set of inflammatory stimuli. Here, we demonstrate that TAK1 regulates ER stress and metabolic signaling through modulation of lipid biogenesis. We found that deletion of Tak1 increased ER volume and facilitated ER-stress tolerance in cultured cells, which was mediated by upregulation of sterol-regulatory-element-binding protein (SREBP)-dependent lipogenesis. In the in vivo setting, central nervous system (CNS)-specific Tak1 deletion upregulated SREBP-target lipogenic genes and blocked ER stress in the hypothalamus. Furthermore, CNS-specific Tak1 deletion prevented ER-stress-induced hypothalamic leptin resistance and hyperphagic obesity under a high-fat diet (HFD). Thus, TAK1 is a crucial regulator of ER stress in vivo, which could be a target for alleviation of ER stress and its associated disease conditions.

Activated macrophage survival is coordinated by TAK1 binding proteins.

Macrophages play diverse roles in tissue homeostasis and immunity, and canonically activated macrophages are critically associated with acute inflammatory responses. It is known that activated macrophages undergo cell death after transient activation in some settings, and the viability of macrophages impacts on inflammatory status. Here we report that TGFß- activated kinase (TAK1) activators, TAK1-binding protein 1 (TAB1) and TAK1-binding protein 2 (TAB2), are critical molecules in the regulation of activated macrophage survival. While deletion of Tak1 induced cell death in bone marrow derived macrophages even without activation, Tab1 or Tab2 deletion alone did not profoundly affect survival of naïve macrophages. However, in lipopolysaccharide (LPS)-activated macrophages, even single deletion of Tab1 or Tab2 resulted in macrophage death with both necrotic and apoptotic features. We show that TAB1 and TAB2 were redundantly involved in LPS-induced TAK1 activation in macrophages. These results demonstrate that TAK1 activity is the key to activated macrophage survival. Finally, in an in vivo setting, Tab1 deficiency impaired increase of peritoneal macrophages upon LPS challenge, suggesting that TAK1 complex regulation of macrophages may participate in in vivo macrophage homeostasis. Our results demonstrate that TAB1 and TAB2 are required for activated macrophages, making TAB1 and TAB2 effective targets to control inflammation by modulating macrophage survival.

TAK1 kinase switches cell fate from apoptosis to necrosis following TNF stimulation.

TNF activates three distinct intracellular signaling cascades leading to cell survival, caspase-8-mediated apoptosis, or receptor interacting protein kinase 3 (RIPK3)-dependent necrosis, also called necroptosis. Depending on the cellular context, one of these pathways is activated upon TNF challenge. When caspase-8 is activated, it drives the apoptosis cascade and blocks RIPK3-dependent necrosis. Here we report the biological event switching to activate necrosis over apoptosis. TAK1 kinase is normally transiently activated upon TNF stimulation. We found that prolonged and hyperactivation of TAK1 induced phosphorylation and activation of RIPK3, leading to necrosis without caspase activation. In addition, we also demonstrated that activation of RIPK1 and RIPK3 promoted TAK1 activation, suggesting a positive feedforward loop of RIPK1, RIPK3, and TAK1. Conversely, ablation of TAK1 caused caspase-dependent apoptosis, in which Ripk3 deletion did not block cell death either in vivo or in vitro. Our results reveal that TAK1 activation drives RIPK3-dependent necrosis and inhibits apoptosis. TAK1 acts as a switch between apoptosis and necrosis.

Structural insights into E2-E3 interaction for LC3 lipidation.

The members of the LC3/Atg8 family of proteins are covalently attached to phagophore and autophagosomal membranes. At the last step of the LC3 lipidation cascade, LC3 is transferred from the E2 enzyme ATG3 to phosphatidylethanolamine (PE). This transfer is stimulated by the ATG12-ATG5-ATG16L1 E3 complex, but the mechanism is not fully understood. We recently found that ATG12 of the E3 binds to a short sequence in the flexible region (FR) of ATG3 with high affinity, and that this interaction is critical for E2-E3 complex formation. These findings, together with detailed structural analyses of this interaction, define the properties of ATG12 and provide new insights of how LC3 transfer begins with ATG3 recruitment by ATG12.

Structural basis of ATG3 recognition by the autophagic ubiquitin-like protein ATG12.

The autophagic ubiquitin-like protein (ublp) autophagy-related (ATG)12 is a component of the ATG12∼ATG5-ATG16L1 E3 complex that promotes lipid conjugation of members of the LC3 ublp family. A role of ATG12 in the E3 complex is to recruit the E2 enzyme ATG3. Here we report the identification of the ATG12 binding sequence in the flexible region of human ATG3 and the crystal structure of the minimal E3 complexed with the identified binding fragment of ATG3. The structure shows that 13 residues of the ATG3 fragment form a short ß-strand followed by an α-helix on a surface area that is exclusive to ATG12. Mutational analyses of ATG3 confirm that four residues whose side chains make contacts with ATG12 are important for E3 interaction as well as LC3 lipidation. Conservation of these four critical residues is high in metazoan organisms and plants but lower in fungi. A structural comparison reveals that the ATG3 binding surface on ATG12 contains a hydrophobic pocket corresponding to the binding pocket of LC3 that accommodates the leucine of the LC3-interacting region motif. These findings establish the mechanism of ATG3 recruitment by ATG12 in higher eukaryotes and place ATG12 among the members of signaling ublps that bind liner sequences.

Lose the battle to win the war: bacterial strategies for evading host inflammasome activation.

The inflammasome is composed of nucleotide-binding, oligomerization domain (NOD)-like receptor (NLR) proteins, and leads to caspase-1 activation and subsequent secretion of the proinflammatory cytokines interleukin 1ß (IL-1ß) and interleukin-18 (IL-18). After certain pathogenic bacteria infect host cells, such as macrophages, NLR-mediated inflammasome activation is triggered to form part of the host defenses against the invading pathogens. However, recent evidence has shown that bacteria have strategies for evading inflammasome activation in host cells. In this review, we focus on NLR-mediated inflammasome activation and bacterial evasion of the inflammasome as part of the battle between the host defenses and pathogens.

Structure of the human ATG12~ATG5 conjugate required for LC3 lipidation in autophagy.

The autophagy factor ATG12~ATG5 conjugate exhibits E3 ligase-like activity which facilitates the lipidation of members of the LC3 family. The crystal structure of the human ATG12~ATG5 conjugate bound to the N-terminal region of ATG16L1, the factor that recruits the conjugate to autophagosomal membranes, reveals an integrated architecture in which ATG12 docks onto ATG5 through conserved residues. ATG12 and ATG5 are oriented such that other conserved residues on each molecule, including the conjugation junction, form a continuous surface patch. Mutagenesis data support the importance of both the interface between ATG12 and ATG5 and the continuous patch for E3 activity. The ATG12~ATG5 conjugate interacts with the E2 enzyme ATG3 with high affinity through another surface location that is exclusive to ATG12, suggesting a different role of the continuous patch in E3 activity. These findings provide a foundation for understanding the mechanism of LC3 lipidation.

TAK1 (MAP3K7) signaling regulates hematopoietic stem cells through TNF-dependent and -independent mechanisms.

A cytokine/stress signaling kinase Tak1 (Map3k7) deficiency is known to impair hematopoietic progenitor cells. However, the role of TAK1 signaling in the stem cell function of the hematopoietic system is not yet well defined. Here we characterized hematopoietic stem cells (HSCs) harboring deletion of Tak1 and its activators, Tak1 binding proteins 1 and 2 (Tab1 and Tab2) using a competitive transplantation assay in a mouse model. Tak1 single or Tab1/Tab2 double deletions completely eliminated the reconstitution activity of HSCs, whereas Tab1 or Tab2 single deletion did not cause any abnormality. Tak1 single or Tab1/Tab2 double deficient lineage-negative, Sca-1(+), c-Kit(+) (LSK) cells did not proliferate and underwent cell death. We found that Tnfr1 deficiency restored the reconstitution activity of Tak1 deficient bone marrow cells for 6-18 weeks. However, the reconstitution activity of Tak1- and Tnfr1-double deficient bone marrow cells declined over the long term, and the number of phenotypically identified long-term hematopoietic stem cells were diminished. Our results indicate that TAB1- or TAB2-dependent activation of TAK1 is required for maintenance of the hematopoietic system through two mechanisms: one is prevention of TNF-dependent cell death and the other is TNF-independent maintenance of long-term HSC.

TGFß-activated kinase 1 (TAK1)-binding proteins (TAB) 2 and 3 negatively regulate autophagy.

Transforming growth factor ß-activated protein kinase 1 (TAK1)-binding protein 2 (TAB2) and its close homolog TAB3 are initially characterized as adapter proteins essential for TAK1 activation in response to interleukin-1ß and tumour necrosis factor-α. However, the physiological roles of TAB2 and TAB3 are still not fully understood. Here we report that TAB2 and TAB3 bind to Beclin1 and colocalize in the cytoplasm. TAB2 also interacts with ATG13 and is phosphorylated by ULK1. Overexpression of TAB2 or TAB3 induces punctate localization of ATG5 under the normal culture condition. Knockdown of TAB2 and TAB3 results in the decrease in endogenous protein level of p62/SQSTM1 under the normal culture condition, while overexpression of TAB2 results in the accumulation of p62/SQSTM1 independently of TAK1. The decrease of p62/SQSTM1 induced by the knockdown of TAB2 and TAB3 is largely dependent on ATG5. These results suggest that TAB2 and TAB3 negatively regulate autophagy independently of TAK1 activity.

A major lipid raft protein raftlin modulates T cell receptor signaling and enhances th17-mediated autoimmune responses.

The membrane microdomains known as lipid rafts have been shown to act as platforms for the initiation of various receptor signals. Through proteomic analysis, we have identified a novel protein termed Raftlin (raft-linking protein) as a major protein in lipid rafts. To determine the physiological and immunological functions of Raftlin in mammals, we generated Raftlin-deficient mice, as well as Raftlin-transgenic (Tg) mice. Although Raftlin was originally identified in B cells, we observe no severe abnormalities in the B cells of these mice, presumably due to a high expression of Raftlin-homologue (Raftlin-2). T cells, in contrast, expressed a substantial amount of Raftlin but no Raftlin-2. In Raftlin-deficient mice, T cell-dependent Ab production was reduced, and experimental autoimmune encephalomyelitis, a Th17-dependent autoimmune disease model, was ameliorated. In Raftlin-Tg mice, in contrast, Ab production was enhanced and experimental autoimmune encephalomyelitis was more severe. Cytokine production, especially that of IL-17, was reduced in Raftlin-deficient T cells, while it was enhanced in Raftlin-Tg T cells. We found that these changes were associated with the strength of the TCR-mediated signals. Importantly, localization of Lck protein in the lipid rafts was enhanced by Raftlin overexpression and reduced by Raftlin deficiency. These data indicate that Raftlin modulates TCR signals and is necessary for the fine-tuning of T cell-mediated immune responses.

Cyclic adenosine monophosphate suppresses the transcription of proinflammatory cytokines via the phosphorylated c-Fos protein.

Intracellular cyclic adenosine monophosphate (cAMP) suppresses innate immunity by inhibiting proinflammatory cytokine production from monocytic cells. Enhanced expression of interleukin-10 (IL-10) has been suggested to be the mechanism of suppression. However, cAMP is still capable of suppressing production of the cytokines TNF-alpha and IL-12 in IL-10-deficient dendritic cells (DCs). Here, we demonstrated that the transcription factor c-Fos was responsible for the cAMP-mediated suppression of inflammatory cytokine production. c-Fos accumulated at high amounts in response to cAMP and lipopolysaccharide (LPS). Overexpression of c-Fos suppressed LPS-induced cytokine production, whereas cAMP-mediated suppression of TNF-alpha and IL-12 was impaired in Fos(-/-) DCs or in RAW264.7 cells treated with c-Fos siRNA. c-Fos physically interacted with p65 protein and reduced the recruitment of p65 to the Tnf promoter. Multiple sites of c-Fos were phosphorylated by the IKKbeta protein. Thus, we propose that c-Fos is a substrate of IKKbeta and is responsible for the immunosuppressive effect of cAMP.

An F-box protein, FBXW5, negatively regulates TAK1 MAP3K in the IL-1beta signaling pathway.

TAK1, a member of the MAP3K family, plays an essential role in activation of JNK/p38 MAPKs and IKK in the IL-1beta and TNFalpha signaling pathway. Upon stimulation, TAK1 is rapidly and transiently activated. While the activation mechanism of TAK1 in these signaling pathways is well characterized, how its activity is terminated still remains unclear. To identify the molecule(s) involved in TAK1 regulation, we performed tandem affinity purification (TAP) in HeLa cells stably expressing TAP-tagged TAK1. FBXW5, an F-box family protein, was identified as a previously unknown component of the IL-1beta-induced TAK1 complex. FBXW5 associated with endogenous TAK1 in an IL-1beta-dependent manner. Overexpression of FBXW5 inhibited IL-1beta-induced activation of JNK/p38 MAPKs and NF-kappaB as well as phosphorylation of TAK1 on Thr187. Conversely, knockdown of FBXW5 resulted in the prolonged activation of TAK1 upon IL-1beta stimulation. These results suggest that FBXW5 negatively regulates TAK1 in the IL-1beta signaling pathway.

TRAF6 and MEKK1 play a pivotal role in the RIG-I-like helicase antiviral pathway.

Type I interferons (IFN-alpha/beta) are essential for immune defense against viruses and induced through the actions of the cytoplasmic helicases, RIG-I and MDA5, and their downstream adaptor molecule IPS-1. TRAF6 and the downstream kinase TAK1 have been shown to be essential for the production of proinflammatory cytokines through the TLR/MyD88/TRIF pathway. Although binding of TRAF6 with IPS-1 has been demonstrated, the role of the TRAF6 pathway in IFN-alpha/beta production has not been fully understood. Here, we demonstrate that TRAF6 is critical for IFN-alpha/beta induction in response to viral infection and intracellular double-stranded RNA, poly(I:C). Activation of NF-kappaB, JNK, and p38, but not IRF3, was impaired in TRAF6-deficient mouse embryo fibroblasts in response to vesicular stomatitis virus and poly(I:C). However, TAK1 was not required for IFN-beta induction in this process, since normal IFN-alpha/beta production was observed in TAK1-deficient mouse embryo fibroblasts. Instead, another MAP3K, MEKK1, was important for the activation of the IFN-beta promoter in response to poly(I:C). Forced expression of MEKK1 in combination with IRF3 was sufficient for the induction of IFN-beta, whereas suppression of MEKK1 expression by small interfering RNA inhibited the induction of IFN-beta by poly(I:C). These data suggest that IPS-1 requires TRAF6 and MEKK1 to activate NF-kappaB and mitogen-activated protein kinases that are critical for the optimal induction of type I interferons.

FLN29 deficiency reveals its negative regulatory role in the Toll-like receptor (TLR) and retinoic acid-inducible gene I (RIG-I)-like helicase signaling pathway.

FLN29 was identified as an interferon (IFN)-inducible gene, and it has been shown to suppress Toll-like receptor 4-mediated NF-kappaB activation by binding to TRAF6. To elucidate the physiological roles of FLN29, we generated FLN29-deficient mice. FLN29 deficiency resulted in hyper-response to LPS both in vivo and in vitro, demonstrating the negative regulatory role of FLN29 in TLR4 signaling. Furthermore, we found that FLN29(-/-) mice exhibited increased susceptibility to poly(I:C)-induced septic shock compared with WT mice. FLN29(-/-) fibroblasts were highly resistant to vesicular stomatitis virus infection, and these cells produced more IFN-beta than WT cells did in response to not only intracellular poly(I:C) but also overexpression of IPS-1. Forced expression of FLN29 inhibited the IPS-1-dependent activation of both NF-kappaB and IRF3. We also found that FLN29 could interact with TRIF, IPS-1, TRAF3, and TRAF6. Together, these results suggest that FLN29, in addition to playing a negative regulatory role in the TLR4 signaling pathway, negatively regulates the RIG-I-like helicase signaling pathway at the level of IPS-1/TRAF6 and IPS-1/TRAF3 complexes.

SOCS3 regulates p21 expression and cell cycle arrest in response to DNA damage.

Genotoxic agents such as ionizing radiation trigger cell cycle arrest at the G1/S and G2/M checkpoints, allowing cells to repair damaged DNA before entry into mitosis. DNA damage-induced G1 arrest involves p53-dependent expression of p21 (Cip1/Waf-1), which inhibits cyclin-dependent kinases and blocks S phase entry. While much of the core DNA damage response has been well-studied, other signaling proteins that intersect with and modulate this response remain uncharacterized. In this study, we identify Suppressor of Cytokine Signaling (SOCS)-3 as an important regulator of radiation-induced G1 arrest. SOCS3-deficient fibroblasts fail to undergo G1 arrest and accumulate in the G2/M phase of the cell cycle. SOCS3 knockout cells phosphorylate p53 and H2AX normally in response to radiation, but fail to upregulate p21 expression. In addition, STAT3 phosphorylation is elevated in SOCS3-deficient cells compared to WT cells. Normal G1 arrest can be restored in SOCS3 KO cells by retroviral transduction of WT SOCS3 or a dominant-negative mutant of STAT3. Our results suggest a novel function for SOCS3 in the control of genome stability by negatively regulating STAT3-dependent radioresistant DNA synthesis, and promoting p53-dependent p21 expression.