Takahiro Doi

NF-κB inhibits TNF-induced accumulation of ROS that mediate prolonged MAPK activation and necrotic cell death

NF‐κB downregulates tumor necrosis factor (TNF)‐induced c‐Jun N‐terminal kinase (JNK) activation that promotes cell death, but the mechanism is not yet fully understood. By using murine embryonic fibroblasts (MEFs) that are deficient in TNF receptor‐associated factor (TRAF) 2 and TRAF5 (DKO) or p65 NF‐κB subunit (p65KO), we demonstrate here that TNF stimulation leads to accumulation of reactive oxygen species (ROS), which is essential for prolonged mitogen‐activated protein kinase (MAPK) activation and cell death. Interestingly, dying cells show necrotic as well as apoptotic morphological changes as assessed by electron microscopy and flow cytometry, and necrotic, but not apoptotic, cell death is substantially inhibited by antioxidant. Importantly, TNF does not induce ROS accumulation or prolonged MAPK activation in wild‐type MEFs, indicating that TRAF‐mediated NF‐κB activation normally suppresses the TNF‐induced ROS accumulation that subsequently induces prolonged MAPK activation and necrotic cell death

Transient and Selective NF-κB p65 Serine 536 Phosphorylation Induced by T Cell Costimulation Is Mediated by IκB Kinase β and Controls the Kinetics of p65 Nuclear Import

Full transcriptional activity of the nuclear, DNA-bound form of NF-κB requires additional posttranslational modifications. In this study, we systematically mapped the T cell costimulation-induced phosphorylation sites within the C-terminal half of the strongly trans-activating NF-κB p65 subunit and identified serine 536 as the main phosphorylation site. The transient kinetics of serine 536 phosphorylation paralleled the kinetics of IκBα and IκB kinase (IKK) phosphorylation and also mirrored the principle of T cell costimulation. The TCR-induced pathway leading to serine 536 phosphorylation is regulated by the kinases Cot (Tpl2), receptor interacting protein, protein kinase Cθ, and NF-κB-inducing kinase, but is independent from the phosphatidylinositol 3-kinase/Akt signaling pathway. Loss-of-function and gain-of-function experiments showed phosphorylation of p65 serine 536 by IKKβ, but not by IKKα. Phosphorylation occurs within the cytoplasmic and intact NF-κB/IκBα complex and requires prior phosphorylation of IκBα at serines 32 and 36. Reconstitution of p65−/− cells either with wild-type p65 or a p65 mutant containing a serine to alanine mutation revealed the importance of this phosphorylation site for cytosolic IκBα localization and the kinetics of p65 nuclear import.

Phosphorylation of serine 276 is essential for p65 NF-κB subunit-dependent cellular responses

Phosphorylation of several serine residues especially in the transactivation (TA) domain of p65 NF-kappaB subunit has been suggested to be important for its transcriptional activity. However, the responsible phosphorylation site of p65 remains controversial. To investigate the biological significance of phosphorylation and to determine the critical phosphorylation sites of p65, we reconstituted murine embryonic fibroblasts (MEFs) from p65(-/-) mice with various serine to alanine (SA)-substituted mutants of p65. Unexpectedly, mutants in the TA domain, including S529A, S536A, and S529A/S536A, completely rescued the defect of p65(-/-) MEFs as assessed by tumor necrosis factor (TNF)- or interleukin-1 (IL-1)-induced IL-6 production and protection from TNF-induced cell death. On the other hand, S276A mutant had an impaired ability to rescue these responses. Moreover, TNF-induced phosphorylation of p65 was severely impaired in S276A mutant, indicating that S276 is the major phosphorylation site of p65 and its phosphorylation is essential for p65-dependent cellular responses.

A Proximal κB Site in the IL-23 p19 Promoter Is Responsible for RelA- and c-Rel-Dependent Transcription

IL-23 is a heterodimeric cytokine composed of a unique p19 subunit and a common p40 subunit is shared with IL-12. IL-23 promotes the inflammatory response by inducing the expansion of CD4+ cells producing IL-17. The regulation of p19 gene expression has been less studied than that of p40 subunit expression, which in macrophages is well known to be dependent on NF-κB. To clarify the role of NF-κB in expression of the p19 gene, we analyzed mRNA levels in NF-κB-deficient macrophages. As reported to occur in dendritic cells, p19 expression was dramatically reduced in c-rel-deficient macrophages. Moreover, we found that p19 expression was halved in rela-deficient macrophages, but it was enhanced in p52-deficient macrophages. The p19 promoter contains three putative κB sites, located at nt −642 to −632 (κB–642), nt −513 to −503 (κB–513), and nt −105 to −96 (κB–105), between the transcription start site and −937 bp upstream in the p19 promoter region. Although EMSA analysis indicated that both κB–105 and κB–642, but not κB–513, bound to NF-κB in vitro, luciferase-based reporter assays showed that the most proximal κB site, κB–105, was uniquely indispensable to the induction of p19 transcription. Chromatin immunoprecipitation demonstrated in vivo association of RelA, c-Rel, and p50 with κB–105 of the p19 promoter. These results provide the evidence that the association of RelA and c-Rel with the proximal κB site in the p19 promoter is required to induce of p19 expression.

Tumor Necrosis Factor α Represses Bone Morphogenetic Protein (BMP) Signaling by Interfering with the DNA Binding of Smads through the Activation of NF-κB

Bone morphogenetic proteins (BMPs) induce not only bone formation in vivo but also osteoblast differentiation of mesenchymal cells in vitro. Tumor necrosis factor α (TNFα) inhibits both osteoblast differentiation and bone formation induced by BMPs. However, the molecular mechanisms of these inhibitions remain unknown. In this study, we found that TNFα inhibited the alkaline phosphatase activity and markedly reduced BMP2- and Smad-induced reporter activity in MC3T3-E1 cells. TNFα had no effect on the phosphorylation of Smad1, Smad5, and Smad8 or on the nuclear translocation of the Smad1-Smad4 complex. In p65-deficient mouse embryonic fibroblasts, overexpression of p65, a subunit of NF-κB, inhibited BMP2- and Smad-induced reporter activity in a dose-dependent manner. Furthermore, this p65-mediated inhibition of BMP2- and Smad-responsive promoter activity was restored after inhibition of NF-κB by the overexpression of the dominant negative IκBα. Although TNFα failed to affect receptor-dependent formation of the Smad1-Smad4 complex, p65 associated with the complex. Chromatin immunoprecipitation and electrophoresis mobility shift assays revealed that TNFα suppressed the DNA binding of Smad proteins to the target gene. Importantly, the specific NF-κB inhibitor, BAY11-7082, abolished these phenomena. These results suggest that TNFα inhibits BMP signaling by interfering with the DNA binding of Smads through the activation of NF-κB.

Interleukin-11 Links Oxidative Stress and Compensatory Proliferation

In dying cells, reactive oxygen species stimulate the production of a cytokine that triggers the healthy neighboring cells to proliferate. Dying Cells Protect the Neighborhood In addition to releasing a number of factors that induce the production of proinflammatory cytokines, dying cells can promote wound healing and tissue homeostasis by inducing the proliferation of surrounding cells in a process known as compensatory proliferation. Nishina et al. found that in dying hepatocytes reactive oxygen species triggered the production of the cytokine interleukin-11 (IL-11), which induced the proliferation of surrounding cells by activating the transcription factor STAT3. Consistent with these in vitro findings, IL-11 signaling protected hepatocytes in a mouse model of acetaminophen-induced liver injury, and mice deficient in a component of the IL-11 receptor had exacerbated disease. Together, these findings suggest that IL-11 stimulates compensatory proliferation in response to oxidative stress. Apoptotic cells can stimulate the compensatory proliferation of surrounding cells to maintain tissue homeostasis. Although oxidative stress is associated with apoptosis and necrosis, whether it contributes to compensatory proliferation is unknown. Here, we showed that interleukin-11 (IL-11), a member of the IL-6 family of proinflammatory cytokines, was produced by cells in an oxidative stress–dependent manner. IL-11 production depended on the activation in dying cells of extracellular signal–regulated kinase 2, which in turn caused the phosphorylation and accumulation of the transcription factor Fra-1 by preventing its proteasome-dependent degradation. Fra-1 was subsequently recruited to the Il11 promoter and activated gene transcription. Upon acute liver injury in mice, IL-11 was mainly produced by hepatocytes in response to reactive oxygen species that were presumably released from dying hepatocytes. IL-11 that was secreted by the dying cells then induced the phosphorylation of the transcription factor STAT3 in adjacent healthy hepatocytes, which resulted in their compensatory proliferation. Furthermore, an IL-11 receptor (IL-11R) agonist enhanced the proliferation of hepatocytes and ameliorated oxidative stress upon acetaminophen-induced liver injury. Conversely, the effects of acetaminophen were exacerbated in mice deficient in the IL-11R α subunit. Together, these results suggest that IL-11 provides a functional link between oxidative stress and compensatory proliferation.

Neural Activity in Cortical Area V4 Underlies Fine Disparity Discrimination

Primates are capable of discriminating depth with remarkable precision using binocular disparity. Neurons in area V4 are selective for relative disparity, which is the crucial visual cue for discrimination of fine disparity. Here, we investigated the contribution of V4 neurons to fine disparity discrimination. Monkeys discriminated whether the center disk of a dynamic random-dot stereogram was in front of or behind its surrounding annulus. We first behaviorally tested the reference frame of the disparity representation used for performing this task. After learning the task with a set of surround disparities, the monkey generalized its responses to untrained surround disparities, indicating that the perceptual decisions were generated from a disparity representation in a relative frame of reference. We then recorded single-unit responses from V4 while the monkeys performed the task. On average, neuronal thresholds were higher than the behavioral thresholds. The most sensitive neurons reached thresholds as low as the psychophysical thresholds. For subthreshold disparities, the monkeys made frequent errors. The variable decisions were predictable from the fluctuation in the neuronal responses. The predictions were based on a decision model in which each V4 neuron transmits the evidence for the disparity it prefers. We finally altered the disparity representation artificially by means of microstimulation to V4. The decisions were systematically biased when microstimulation boosted the V4 responses. The bias was toward the direction predicted from the decision model. We suggest that disparity signals carried by V4 neurons underlie precise discrimination of fine stereoscopic depth.

Processing of the NF-κB2 precursor p100 to p52 is critical for RANKL-induced osteoclast differentiation

Gene targeting of the p50 and p52 subunits of NF‐κB has shown that NF‐κB plays a critical role in osteoclast differentiation. However, the molecular mechanism by which NF‐κB regulates osteoclast differentiation is still unclear. To address this issue, we analyzed alymphoplasia (aly/aly) mice in which the processing of p100 to p52 does not occur owing to an inactive form of NF‐κB‐inducing kinase (NIK). Aly/aly mice showed a mild osteopetrosis with significantly reduced osteoclast numbers. RANKL‐induced osteoclastogenesis from bone marrow cells of aly/aly mice also was suppressed. RANKL still induced the degradation of IκBα and activated classical NF‐κB, whereas processing of p100 to p52 was abolished by the aly/aly mutation. Moreover, RANKL‐induced expression of NFATc1 was impaired in aly/aly bone marrow. Overexpression of constitutively active IKKα or p52 restored osteoclastogenesis in aly/aly cells. Finally, transfection of either wild‐type p100, p100ΔGRR that cannot be processed to p52, or p52 into NF‐κB2‐deficient cells followed by RANKL treatment revealed a strong correlation between the number of osteoclasts induced by RANKL and the ratio of p52 to p100 expression. Our data provide a new finding for a previously unappreciated role for NF‐κB in osteoclast differentiation.

Matching and correlation computations in stereoscopic depth perception

A fundamental task of the visual system is to infer depth by using binocular disparity. To encode binocular disparity, the visual cortex performs two distinct computations: one detects matched patterns in paired images (matching computation); the other constructs the cross-correlation between the images (correlation computation). How the two computations are used in stereoscopic perception is unclear. We dissociated their contributions in near/far discrimination by varying the magnitude of the disparity across separate sessions. For small disparity (0.03°), subjects performed at chance level to a binocularly opposite-contrast (anti-correlated) random-dot stereogram (RDS) but improved their performance with the proportion of contrast-matched (correlated) dots. For large disparity (0.48°), the direction of perceived depth reversed with an anti-correlated RDS relative to that for a correlated one. Neither reversed nor normal depth was perceived when anti-correlation was applied to half of the dots. We explain the decision process as a weighted average of the two computations, with the relative weight of the correlation computation increasing with the disparity magnitude. We conclude that matching computation dominates fine depth perception, while both computations contribute to coarser depth perception. Thus, stereoscopic depth perception recruits different computations depending on the disparity magnitude.

Agonistic Antibody to TLR4/MD-2 Protects Mice from Acute Lethal Hepatitis Induced by TNF-α

LPS is recognized by a heterodimer consisting of TLR4 and its coreceptor MD-2. LPS signal causes excessive inflammation and tissue damage. In this study, we show that a mAb to TLR4/MD-2 protected mice from acute lethal hepatitis caused by LPS/d-galactosamine. The protective effect of the mAb was not due to inhibition of LPS response, because serum TNF-α, which was induced by LPS and caused lethal hepatitis, was 10 times up-regulated by the mAb pretreatment. Moreover, this mAb induced antiapoptotic genes in liver in a TLR4/MD-2-dependent manner. These results demonstrated that an agonistic mAb to TLR4/MD-2 protected mice from LPS/d-galactosamine-induced acute lethal hepatitis by delivering a protective signal activating NF-κB through TLR4/MD-2.