Leukotriene A: stereochemistry and enzymatic conversion to leukotriene B

Leukotriene A was assigned the structure 5(S)-$\text{trans}$-5,6-oxido-7,9-$\text{trans}\text{-11,14-}\text{cis}$-eicosatetraenoic acid by the enzymatic conversion of a synthetic product of known stereochemistry into the naturally occurring isomer of 5(S),12(R)-dihydroxy-6,8,10,14-eicosatetraenoic acid in human polymorphonuclear leukocytes.     

11-Trans leukotriene C: A naturally occurring slow reacting substance

A slow reacting substance, produced by murine mastocytoma cells, has been shown to have the structure 5(S)-hydroxy-6(R)-$\text{S}$-glutathionyl-7,9,11-$\text{trans}\text{-14-}\text{cis}\text{-eicosatetraenoic acid (11-}\text{trans}$ leukotriene C, previously referred to as leukotriene C-2) by ultraviolet spectroscopy, amino acid analyses, lipoxygenase conversion and comparisions with a synthetic compound of known structure and stereochemistry.     

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.     

Foxp3 inhibits RORgammat-mediated IL-17A mRNA transcription through direct interaction with RORgammat

The cytokine, transforming growth factor-beta1 (TGF-beta1), converts naive T cells into regulatory T cells that prevent autoimmunity. However, in the presence of interleukin (IL)-6, TGF-beta1 has also been found to promote differentiation into IL-17-producing helper T (Th17) cells that are deeply involved in autoimmunity and inflammation. However, it has not been clarified how TGF-beta1 and IL-6 determine such a distinct fate. Here we found that a master regulator for Th17, retinoic acid-related orphan receptor gammat (RORgammat), was rapidly induced by TGF-beta1 regardless of the presence of IL-6. IL-6 reduced Foxp3 expression, and overexpression of Foxp3 in a T cell line resulted in a strong reduction of IL-17A expression. We have characterized the IL-17A promoter and found that RORgammat binding is sufficient for activation of the minimum promoter in the HEK 293T cells. RORgammat-mediated IL-17A promoter activation was suppressed by forced expression of Foxp3. Foxp3 directly interacted with RORgammat through exon 2 region of Foxp3. The exon 2 region and forkhead (FKH) domain of Foxp3 were necessary for the suppression of RORgammat-mediated IL-17A promoter activation. We propose that induction of Foxp3 is the mechanism for the suppression of Th17 and polarization into inducible Treg.     

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.     

Stereochemistry of leukotriene C-1

Leukotriene C-1, a “Slow Reacting Substance” (SRS), has been shown to possess the molecular Structure depicted by V (5(S)-hydroxy-6(R)-$\text{S}$-glutathionyl-7,9-$\text{trans}$-11,14-$\text{cis}$-eicosatetraenoic acid) by its identity with a totally synthetic product of known structure and stereochemistry.     

TAK1 is critical for IkappaB kinase-mediated activation of the NF-kappaB pathway

Cytokine treatment stimulates the IkappaB kinases, IKKalpha and IKKbeta, which phosphorylate the IkappaB proteins, leading to their degradation and activation of NF-kappaB regulated genes. A clear definition of the specific roles of IKKalpha and IKKbeta in activating the NF-kappaB pathway and the upstream kinases that regulate IKK activity remain to be elucidated. Here, we utilized small interfering RNAs (siRNAs) directed against IKKalpha, IKKbeta and the upstream regulatory kinase TAK1 in order to better define their roles in cytokine-induced activation of the NF-kappaB pathway. In contrast to previous results with mouse embryo fibroblasts lacking either IKKalpha or IKKbeta, which indicated that only IKKbeta is involved in cytokine-induced NF-kappaB activation, we found that both IKKalpha and IKKbeta were important in activating the NF-kappaB pathway. Furthermore, we found that the MAP3K TAK1, which has been implicated in IL-1-induced activation of the NF-kappaB pathway, was also critical for TNFalpha-induced activation of the NF-kappaB pathway. TNFalpha activation of the NF-kappaB pathway is associated with the inducible binding of TAK1 to TRAF2 and both IKKalpha and IKKbeta. This analysis further defines the distinct in vivo roles of IKKalpha, IKKbeta and TAK1 in cytokine-induced activation of the NF-kappaB pathway.     

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.     

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

Transforming growth factor β-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-1β 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.