Sachiko Komazawa-Sakon

An antiapoptotic protein, c‐FLIPL, directly binds to MKK7 and inhibits the JNK pathway

Inhibition of NF‐κB activation increases susceptibility to tumor necrosis factor (TNF)α‐induced cell death, concurrent with caspases and prolonged c‐Jun N‐terminal kinase (JNK) activation, and reactive oxygen species (ROS) accumulation. However, the detailed mechanisms are unclear. Here we show that cellular FLICE‐inhibitory protein (c‐FLIP) is rapidly lost in NF‐κB activation‐deficient, but not wild‐type fibroblasts upon TNFα stimulation, indicating that NF‐κB normally maintains the cellular levels of c‐FLIP. The ectopic expression of the long form of c‐FLIP (c‐FLIPL) inhibits TNFα‐induced prolonged JNK activation and ROS accumulation in NF‐κB activation‐deficient fibroblasts. Conversely, TNFα induces prolonged JNK activation and ROS accumulation in c‐Flip−/− fibroblasts. Moreover, c‐FLIPL directly interacts with a JNK activator, MAP kinase kinase (MKK)7, in a TNFα‐dependent manner and inhibits the interactions of MKK7 with MAP/ERK kinase kinase 1, apoptosis‐signal‐regulating kinase 1, and TGFβ‐activated kinase 1. This stimuli‐dependent interaction of c‐FLIPL with MKK7 might selectively suppress the prolonged phase of JNK activation. Taken that ROS promote JNK activation and activation of the JNK pathway may promote ROS accumulation, c‐FLIPL might block this positive feedback loop, thereby suppressing ROS accumulation.

Crucial role for autophagy in degranulation of mast cells

BACKGROUND Autophagy plays a crucial role in controlling various biological responses including starvation, homeostatic turnover of long-lived proteins, and invasion of bacteria. However, a role for autophagy in development and/or function of mast cells is unknown. OBJECTIVE To investigate a role for autophagy in mast cells, we generated bone marrow-derived mast cells (BMMCs) from mice lacking autophagy related gene (Atg) 7, an essential enzyme for autophagy induction. METHODS Bone marrow-derived mast cells were generated from bone marrow cells of control and IFN-inducible Atg7-deficient mice, and morphologic and functional analyses were performed. RESULTS We found that conversion of type I to type II light chain (LC3)-II, a hallmark of autophagy, was constitutively induced in mast cells under full nutrient conditions, and LC3-II localized in secretory granules of mast cells. Although deletion of Atg7 did not impair the development of BMMCs, Atg7(-/-) BMMCs showed severe impairment of degranulation, but not cytokine production on FcεRI cross-linking. Intriguingly, LC3-II but not LC3-I was co-localized with CD63, a secretory lysosomal marker, and was released extracellularly along with degranulation in Atg7(+/+) but not Atg7(-/-) BMMCs. Moreover, passive cutaneous anaphylaxis reactions were severely impaired in mast cell-deficient WBB6F1-W/W(V) mice reconstituted with Atg7(-/-) BMMCs compared with Atg7(+/+) BMMCs. CONCLUSION These results suggest that autophagy is not essential for the development but plays a crucial role in degranulation of mast cells. Thus, autophagy might be a potential target to treat allergic diseases in which mast cells are critically involved.

c-FLIP Maintains Tissue Homeostasis by Preventing Apoptosis and Programmed Necrosis

The antiapoptotic protein c-FLIP blocks multiple cell death pathways in mice. FLIPping Multiple Death Signals Off The gene c-Flip, which encodes the antiapoptotic protein c-FLIP, is expressed in response to nuclear factor κB (NF-κB) activation. NF-κB–mediated protection of the intestine and liver from proapoptotic signaling is important for tissue maintenance (homeostasis). Avoiding the embryonic lethality caused by complete knockout of c-Flip in mice, Piao et al. selectively deleted c-Flip in intestinal epithelial cells (IECs) or hepatocytes. Whereas c-FLIP–deficient IECs exhibited tumor necrosis factor (TNF)–dependent apoptosis and programmed necrosis, a cell death process morphologically and mechanistically distinct from that of apoptosis, leading to perinatal death of the mice, c-FLIP–deficient hepatocytes exhibited apoptosis and programmed necrosis, and mice died in a TNF-independent manner. Induced loss of c-FLIP in hepatocytes in adult mice led to lethal hepatitis, which was prevented by blocking multiple proinflammatory factors that trigger apoptosis. Together, these data show that c-FLIP blocks both apoptosis and programmed necrosis to maintain tissue homeostasis and suggest that targeting both cell death pathways may be effective in treating certain viral infections in which c-FLIP abundance is reduced. As a catalytically inactive homolog of caspase-8, a proapoptotic initiator caspase, c-FLIP blocks apoptosis by binding to and inhibiting caspase-8. The transcription factor nuclear factor κB (NF-κB) plays a pivotal role in maintaining the homeostasis of the intestine and the liver by preventing death receptor–induced apoptosis, and c-FLIP plays a role in the NF-κB–dependent protection of cells from death receptor signaling. Because c-Flip–deficient mice die in utero, we generated conditional c-Flip–deficient mice to investigate the contribution of c-FLIP to homeostasis of the intestine and the liver at developmental and postnatal stages. Intestinal epithelial cell (IEC)– or hepatocyte-specific deletion of c-Flip resulted in perinatal lethality as a result of the enhanced apoptosis and programmed necrosis of the IECs and the hepatocytes. Deficiency in the gene encoding tumor necrosis factor–α (TNF-α) receptor 1 (Tnfr1) partially rescued perinatal lethality and the development of colitis in IEC-specific c-Flip–deficient mice but did not rescue perinatal lethality in hepatocyte-specific c-Flip–deficient mice. Moreover, adult mice with interferon (IFN)–inducible deficiency in c-Flip died from hepatitis soon after depletion of c-FLIP. Pretreatment of IFN-inducible c-Flip–deficient mice with a mixture of neutralizing antibodies against TNF-α, Fas ligand (FasL), and TNF-related apoptosis-inducing ligand (TRAIL) prevented hepatitis. Together, these results suggest that c-FLIP controls the homeostasis of IECs and hepatocytes by preventing cell death induced by TNF-α, FasL, and TRAIL.

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.

Fusion of OTT to BSAC Results in Aberrant Up-regulation of Transcriptional Activity

OTT/RBM15-BSAC/MAL/MKL1/MRTF-A was identified as a fusion transcript generated by t(1;22)(p13;q13) in acute megakaryoblastic leukemia. Previous studies have shown that BSAC (basic, SAP, and coiled-coil domain) activates the promoters containing CArG boxes via interaction with serum response factor, and OTT (one twenty-two) negatively regulates the development of megakaryocytes and myeloid cells. However, the mechanism by which OTT-BSAC promotes leukemia is largely unknown. Here we show that OTT-BSAC, but not BSAC or OTT strongly activates several promoters containing a transcription factor Yin Yang 1-binding sequence. In addition, although BSAC predominantly localizes in the cytoplasm and its nuclear translocation is considered to be regulated by the Rho-actin signaling pathway, OTT-BSAC exclusively localizes in the nucleus. Moreover, OTT interacts with histone deacetylase 3, but this interaction is abolished in OTT-BSAC. Collectively, these functional and spatial changes of OTT and BSAC caused by the fusion might perturb their functions, culminating in the development of acute megakaryoblastic leukemia.

Cellular FLICE-Inhibitory Protein Regulates Tissue Homeostasis

Cellular FLICE-inhibitory protein (cFLIP) is structurally related to caspase-8, but lacks its protease activity. Cflip gene encodes several splicing variants including short form (cFLIPs) and long form (cFLIPL). cFLIPL is composed of two death effector domains at the N terminus and a C-terminal caspase-like domain, and cFLIPs lacks the caspase-like domain. Our studies reveal that cFLIP plays a central role in NF-κB-dependent survival signals that control apoptosis and programmed necrosis. Germline deletion of Cflip results in embryonic lethality due to enhanced apoptosis and programmed necrosis; however, the combined deletion of the death-signaling regulators, Fadd and Ripk3, prevents embryonic lethality in Cflip-deficient mice. Moreover, tissue-specific deletion of Cflip reveals cFLIP as a crucial regulator that maintains tissue homeostasis of immune cells, hepatocytes, intestinal epithelial cells, and epidermal cells by preventing apoptosis and programmed necrosis.

Depletion of myeloid cells exacerbates hepatitis and induces an aberrant increase in histone H3 in mouse serum

Tissue‐resident macrophages and bone marrow (BM)‐derived monocytes play a crucial role in the maintenance of tissue homeostasis; however, their contribution to recovery from acute tissue injury is not fully understood. To address this issue, we generated an acute murine liver injury model using hepatocyte‐specific Cflar‐deficient (CflarHep‐low) mice. Cellular FLICE‐inhibitory protein expression was down‐regulated in Cflar‐deficient hepatocytes, which thereby increased susceptibility of hepatocytes to death receptor–induced apoptosis. CflarHep‐low mice developed acute hepatitis and recovered with clearance of apoptotic hepatocytes at 24 hours after injection of low doses of tumor necrosis factor α (TNFα), which could not induce hepatitis in wild‐type (WT) mice. Depletion of Kupffer cells (KCs) by clodronate liposomes did not impair clearance of dying hepatocytes or exacerbate hepatitis in CflarHep‐low mice. To elucidate the roles of BM‐derived monocytes and neutrophils in clearance of apoptotic hepatocytes, we examined the effect of depletion of these cells on TNFα‐induced hepatitis in CflarHep‐low mice. We reconstituted CflarHep‐low mice with BM cells from transgenic mice in which human diphtheria toxin receptor (DTR) was expressed under control of the lysozyme M (LysM) promoter. TNFα‐induced infiltration of myeloid cells, including monocytes and neutrophils, was completely ablated in LysM‐DTR BM‐reconstituted CflarHep‐low mice pretreated with diphtheria toxin, whereas KCs remained present in the livers. Under these experimental conditions, LysM‐DTR BM‐reconstituted CflarHep‐low mice rapidly developed severe hepatitis and succumbed within several hours of TNFα injection. We found that serum interleukin‐6 (IL‐6), TNFα, and histone H3 were aberrantly increased in LysM‐DTR BM‐reconstituted, but not in WT BM‐reconstituted, CflarHep‐low mice following TNFα injection. Conclusion: These findings indicate an unexpected role of myeloid cells in decreasing serum IL‐6, TNFα, and histone H3 levels via the suppression of TNFα‐induced hepatocyte apoptosis. (Hepatology 2017;65:237‐252).

Blockade of TNF receptor superfamily 1 (TNFR1)–dependent and TNFR1-independent cell death is crucial for normal epidermal differentiation

Background: A delicate balance between cell death and keratinocyte proliferation is crucial for normal skin development. Previous studies have reported that cellular FLICE (FADD‐like ICE)‐inhibitory protein plays a crucial role in prevention of keratinocytes from TNF‐&agr;–dependent apoptosis and blocking of dermatitis. However, a role for cellular FLICE‐inhibitory protein in TNF‐&agr;–independent cell death remains unclear. Objective: We investigated contribution of TNF‐&agr;–dependent and TNF‐&agr;–independent signals to the development of dermatitis in epidermis‐specific Cflar‐deficient (CflarE‐KO) mice. Methods: We examined the histology and expression of epidermal differentiation markers and inflammatory cytokines in the skin of CflarE‐KO;Tnfrsf1a+/− and CflarE‐KO;Tnfrsf1a−/− mice. Mice were treated with neutralizing antibodies against Fas ligand and TNF‐related apoptosis‐inducing ligand to block TNF‐&agr;–independent cell death of CflarE‐KO;Tnfrsf1a−/− mice. Results: CflarE‐KO;Tnfrsf1a−/− mice were born but experienced severe dermatitis and succumbed soon after birth. CflarE‐KO;Tnfrsf1a+/− mice exhibited embryonic lethality caused by massive keratinocyte apoptosis. Although keratinocytes from CflarE‐KO;Tnfrsf1a−/− mice still died of apoptosis, neutralizing antibodies against Fas ligand and TNF‐related apoptosis‐inducing ligand substantially prolonged survival of CflarE‐KO;Tnfrsf1a−/− mice. Expression of inflammatory cytokines, such as Il6 and Il17a was increased; conversely, expression of epidermal differentiation markers was severely downregulated in the skin of CflarE‐KO;Tnfrsf1a−/− mice. Treatment of primary keratinocytes with IL‐6 and, to a lesser extent, IL‐17A suppressed expression of epidermal differentiation markers. Conclusion: TNF receptor superfamily 1 (TNFR1)–dependent or TNFR1‐independent apoptosis of keratinocytes promotes inflammatory cytokine production, which subsequently blocks epidermal differentiation. Thus blockade of both TNFR1‐dependent and TNFR1‐independent cell death might be an alternative strategy to treat skin diseases when treatment with anti–TNF‐&agr; antibody alone is not sufficient. GRAPHICAL ABSTRACT Figure. No caption available.

Novel method to rescue a lethal phenotype through integration of target gene onto the X-chromosome

The loss-of-function mutations of serine protease inhibitor, Kazal type 1 (SPINK1) gene are associated with human chronic pancreatitis, but the underlying mechanisms remain unknown. We previously reported that mice lacking Spink3, the murine homologue of human SPINK1, die perinatally due to massive pancreatic acinar cell death, precluding investigation of the effects of SPINK1 deficiency. To circumvent perinatal lethality, we have developed a novel method to integrate human SPINK1 gene on the X chromosome using Cre-loxP technology and thus generated transgenic mice termed “X-SPINK1“. Consistent with the fact that one of the two X chromosomes is randomly inactivated, X-SPINK1 mice exhibit mosaic pattern of SPINK1 expression. Crossing of X-SPINK1 mice with Spink3+/− mice rescued perinatal lethality, but the resulting Spink3−/−;XXSPINK1 mice developed spontaneous pancreatitis characterized by chronic inflammation and fibrosis. The results show that mice lacking a gene essential for cell survival can be rescued by expressing this gene on the X chromosome. The Spink3−/−;XXSPINK1 mice, in which this method has been applied to partially restore SPINK1 function, present a novel genetic model of chronic pancreatitis.