Tae-Bong Kang

RIG-I RNA Helicase Activation of IRF3 Transcription Factor Is Negatively Regulated by Caspase-8-Mediated Cleavage of the RIP1 Protein

Excessive responses to pattern-recognition receptors are prevented by regulatory mechanisms that affect the amounts and activities of the downstream signaling proteins. We report that activation of the transcription factor IRF3 by the ribonucleic acid sensor RIG-I was restricted by caspase-8-mediated cleavage of the RIP1 protein, which resulted in conversion of RIP1 from a signaling enhancer to a signaling inhibitor. The proteins RIP1 and caspase-8 were recruited to the RIG-I complex after viral infection and served antagonistic regulatory roles. Conjugation of ubiquitin chains to RIP1 facilitated assembly of the RIG-I complex, resulting in enhanced phosphorylation of IRF3. However, the ubiquitination of RIP1 also rendered it susceptible to caspase-8-mediated cleavage that yielded an inhibitory RIP1 fragment. The dependence of RIP1 cleavage on the same molecular change as that facilitating RIG-I signaling allows for RIG-I signaling to be restricted in its duration without compromising its initial activation.

Programmed necrosis in inflammation: Toward identification of the effector molecules

Apoptosis, necrosis, and pyroptosis The routes to cell death are many, and distinguishing which path a particular cell may have taken remains a challenge. Wallach et al. review current understanding of how programmed necrotic cell death contributes to inflammation. Science, this issue p. 10.1126/science.aaf2154 BACKGROUND Inflammatory lesions often contain dead cells. Cell death in developmental processes characteristically occurs by apoptosis, a form of programmed cell death in which dying cells are phagocytized before undergoing membrane damage. In inflammation, however, cell death is often necrotic, where cellular constituents are released after membrane rupture. The death of cells in inflammation was until recently thought to result from other changes in the inflamed tissue. This view has changed, however, owing to several findings: that necrotic cell death can be induced by biomolecules known to initiate inflammation [such as the cytokine tumor necrosis factor (TNF) or the pathogen component lipopolysaccharide (LPS)]; that it can be dictated in a programmed manner by distinct sets of signaling mechanisms; and that it yields release of some cellular components capable of facilitating inflammation. It now seems probable that necrotic cell death is not always a consequence of inflammation, but is sometimes rather its trigger. To confirm this notion, we need reliable tools for detection of programmed necrosis in vivo. Because programmed necrosis cannot be distinguished morphologically from accidental cell death, its identification in inflamed tissues must be based on its distinctive molecular details. ADVANCES We now have quite detailed knowledge of the mechanisms initiating apoptotic cell death and those initiating two forms of programmed necrosis—necroptosis and pyroptosis. Apoptosis is triggered by proteases of the caspase family. Necroptosis is triggered by specific protein kinases, most crucially receptor-interacting protein kinase–3 (RIPK3). Pyroptosis is triggered by caspases distinct from those mediating apoptosis, and whose activation yields proteolytic activation of the inflammatory cytokines interleukin-1β (IL-1β) and IL-18. All of these molecular initiators of death programs, however, also contribute to the initiation of nondeadly cell functions and are thus not specific markers for death. Two proteins were recently found to act further downstream in the signaling pathways leading to programmed necrosis. One, the pseudokinase mixed lineage kinase domain–like protein (MLKL), is crucial for necroptosis. The other, gasdermin-D (GSDMD), after cleavage by the pyroptosis-mediating caspases, is a major player in their induction of death. Mere expression of activated MLKL, or of the N-terminal proteolytic fragment of GSDMD, can trigger necrotic death. The finding that MLKL and GSDMD play roles in necroptosis and pyroptosis raises hopes that we are approaching the identification of molecules that exclusively serve these forms of death. OUTLOOK What are the parameters that can reliably allow us to define a cause-effect relationship between necrotic death and inflammation—and more generally, to define the causal relationships between any disease and its co-occurring pathogenic events? In the case of cell death, this question boils down to the need to identify molecular events that contribute specifically enough to allow their use as definitive molecular probes. The effectors of death—proteins found to mediate deadly changes in the cell—are likely to have that absolute specificity. Whether MLKL and GSDMD are such death effectors has yet to be established. Some studies suggest that MLKL directly permeabilizes membranes, whereas others suggest that it does not. Regarding GSDMD, there is still no knowledge of the mechanisms by which its N-terminal fragment triggers death. Identifying death-effector molecules will be of immense importance to medicine. Such molecules will not only be the best markers for detecting programmed necrosis, but will also serve as the optimal targets for its pharmaceutical arrest in disease. Effector mechanisms in lytic and nonlytic cell death (A) In apoptosis, caspases cleave substrate proteins that orchestrate the cell-death process. (B) In necroptosis, protein kinases phosphorylate MLKL, thereby activating it. Phospho-MLKL then causes cell lysis

Mutation of a Self-Processing Site in Caspase-8 Compromises Its Apoptotic but Not Its Nonapoptotic Functions in Bacterial Artificial Chromosome-Transgenic Mice

Caspase-8, the proximal enzyme in the death-induction pathway of the TNF/nerve growth factor receptor family, is activated upon juxtaposition of its molecules within the receptor complexes and is then self-processed. Caspase-8 also contributes to the regulation of cell survival and growth, but little is known about the similarities or the differences between the mechanisms of these nonapoptotic functions and of the enzyme’s apoptotic activity. In this study, we report that in bacterial artificial chromosome-transgenic mice, in which the aspartate residue upstream of the initial self-processing site in caspase-8 (D387) was replaced by alanine, induction of cell death by Fas is compromised. However, in contrast to caspase-8-deficient mice, which die in utero at mid-gestation, the mice mutated at D387 were born alive and seemed to develop normally. Moreover, mice with the D387A mutation showed normal in vitro growth responses of T lymphocytes to stimulation of their Ag receptor as well as of B lymphocytes to stimulation by LPS, normal differentiation of bone marrow macrophage precursors in response to M-CSF, and normal generation of myeloid colonies by the bone marrow hematopoietic progenitors, all of which are compromised in cells deficient in caspase-8. These finding indicated that self-processing of activated caspase-8 is differentially involved in the different functions of this enzyme: it is needed for the induction of cell death through the extrinsic cell death pathway but not for nonapoptotic functions of caspase-8.

Role of caspase-8 in hepatocyte response to infection and injury in mice†

Caspase‐8 has been implicated in signaling for apoptotic cell death and for certain nonapoptotic functions. However, knowledge of actual physiological or pathophysiological processes to which this enzyme contributes is lacking. Using a mouse model and employing the conditional knockout approach to delete the caspase‐8 gene specifically in the liver, we found that caspase‐8 deficiency in hepatocytes facilitates infection of the liver by Listeria monocytogenes, attenuates the hepatocyte proliferation wave during the first 48 hours after partial hepatectomy and, depending on the genetic background of the mice, prompts a chronic inflammatory response to the hepatectomy, as a result of which the proliferation of hepatocytes, although initially suppressed, might later be persistently enhanced, resulting in significant hepatomegaly. Conclusion: These findings indicate that caspase‐8 participates in regulation of the cellular response to infection and injury and that it does so by affecting various cellular functions, including cell death, cell proliferation, and induction of inflammation. (HEPATOLOGY 2007.)

Concepts of tissue injury and cell death in inflammation: a historical perspective

Emerging evidence indicates that the molecular mechanisms of cell death have regulatory roles in inflammation and that the molecular changes that are associated with different forms of cell death affect the course of inflammation in different ways. In this Timeline article, we discuss how our understanding of the mechanisms and functional roles of tissue injury and cell death in inflammation has evolved on the basis of almost two centuries of study. We describe how such ideas have led to our current models of cell death and inflammation, and we highlight the remaining gaps in our knowledge of the subject.

Caspase-8 deficiency facilitates cellular transformation in vitro

Caspase-8 is frequently deficient in several kinds of human tumors, suggesting that certain effects of this enzyme restrict tumor development. To examine the nature of the cellular function whose regulation by caspase-8 contributes to its antitumor effect, we assessed the impact of caspase-8 deficiency on cell transformation in vitro. Caspase-8-deficient mouse embryonic fibroblasts immortalized with the SV40 T antigen did not survive when cultured in soft agar, and were nontumorogenic in nude mice. However, the rate of transformation of these cells during their continuous growth in culture, as reflected in the observed emergence of cells that do grow in soft agar and are able to form tumors in nude mice, was far higher than that of cells expressing caspase-8. These findings indicate that caspase-8 deficiency can contribute to cancer development in a way that does not depend on the enzymes participation in killing of the tumor cells by host immune cytotoxic mechanisms, or on its involvement in the cell-death process triggered upon detachment of the cells from their substrate, but rather concerns cell-autonomous mechanisms that affect the rate of cell transformation.

The extrinsic cell death pathway and the élan mortel.

Early in the exploration of the chemical nature of life, it was widely believed that the molecules of living organisms, by their very nature, differ from those of inorganic material molecules and possess a vital force (‘élan vital’). Similarly, early scientific thinking on the subject of cell death and its induction by cytotoxic cells of the immune system was pervaded by a sense that the molecules mediating these functions possess intrinsic deadly activity and are dedicated exclusively to death-related tasks. This impression was also reflected in the initial notions of the mode of action of intracellular proteins that signal for death. It is now gradually becoming clear, however, that proteins participating in death induction also have functions unrelated to death. Nevertheless, as exemplified by studies of the function of caspase-8 (an enzyme that signals both for activation of the extrinsic cell-death pathway and for non-death-related effects), analysis of the mechanistic basis for such heterogeneity might allow identification of distinct structural determinants in the proteins participating in death induction that do bear death specificity.

Anti-inflammatory and anti-allergic effects of Agrimonia pilosa Ledeb extract on murine cell lines and OVA-induced airway inflammation

ETHNOPHARMACOLOGICAL EVIDENCE Agrimonia pilosa Ledeb (Rosaceae, AP) has long been used as a traditional medicine in Korea and other Asian countries to treat various diseases. AIM OF THE STUDY In the present study, the anti-inflammatory and anti-allergic effects of AP extract in in vitro cell lines and in vivo mouse model of inflammation and the molecular mechanisms involved were reported. MATERIALS AND METHODS Using Raw 264.7 murine macrophages the effects of methanol extract of AP in lipopolysaccharide (LPS)-induced production of inflammatory mediators were measured. Further IgE-DNP-induced interleukin (IL)-4 production and degranulation in RBL-2H3 rat basophilic cell lines was also estimated. To investigate the anti-asthmatic effect of AP in vivo, airway inflammation in ovalbumin (OVA)-induced mouse model was used. RESULTS AP attenuated the production of inflammatory mediators such as NO, PGE(2) and pro-inflammatory cytokines in LPS-induced Raw 264.7 cells. Further, AP inhibited IL-4 production and degranulation in IgE-DNP-induced RBL-2H3 cells. Furthermore, AP attenuated the infiltration of immune cells into lung, cytokines production in broncho-alveolar lavage fluid (BALF) and airway-hyperresponsiveness (AHR) on OVA-induced mouse model of inflammation. CONCLUSION Our results showed that AP attenuated the activation of macrophages, basophils, and inhibited the OVA-induced airway inflammation. The molecular mechanisms leading to APs potent anti-inflammatory and anti-allergic effects might be through regulation of TRIF-dependent and Syk-PLCγ/AKT signaling pathways, suggesting that AP may provide a valuable therapeutic strategy in treating various inflammatory diseases including asthma.

Anti-Inflammatory Effect of Emodin via Attenuation of NLRP3 Inflammasome Activation

Emodin, an active constituent of oriental herbs, is widely used to treat allergy, inflammation, and other symptoms. This study provides the scientific basis for the anti-inflammasome effects of emodin on both in vitro and in vivo experimental models. Bone marrow-derived macrophages were used to study the effects of emodin on inflammasome activation by using inflammasome inducers such as ATP, nigericin, and silica crystals. The lipopolysaccharide (LPS)-induced endotoxin shock model was employed to study the effect of emodin on in vivo efficacy. Emodin treatment attenuated interleukin (IL)-1β secretion via the inhibition of NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation induced by ATP, nigericin, and silica crystals. Further, emodin ameliorated the severity of NLRP3 inflammasome-mediated symptoms in LPS-induced endotoxin mouse models. This study is the first to reveal mechanism-based evidence, especially with respect to regulation of inflammasome activation, substantiating traditional claims of emodin in the treatment of inflammation-related disorders.

Caveolin-1-dependent apoptosis induced by fibrin degradation products.

In mice lacking the blood coagulation regulator thrombomodulin, fibrinolytic degradation products (FDP) of fibrin induce apoptotic cell death of a specialized cell type in the placenta, polyploid trophoblast giant cells. Here, we document that this bioactivity of FDP is conserved in human FDP, is not limited to trophoblast cells, and is associated with an Aalpha-chain segment of fibrin fragment E (FnE). The majority of proapoptotic activity is arginine-glycine-aspartic acid (RGD)-independent and requires caveolin-1-dependent cellular internalization of FnE. Internalization through caveoli is mediated by an epitope contained within Aalpha52-81 that is necessary and sufficient for cellular uptake of FnE. Aalpha52-81 does not cause apoptosis itself, and competitively inhibits FnE internalization and apoptosis induction. Apoptotic activity per se resides within Aalpha17-37 and requires the N-terminal neoepitope generated by release of fibrinopeptide A. Cellular internalization of FnE elicits depression of mitochondrial function and consequent apoptosis that is strictly dependent on the activity of caspases 9 and 3. These findings describe the molecular details of a novel mechanism linking fibrin degradation to cell death in the placenta, which may also contribute to pathologic alterations in nonplacental vascular beds that are associated with fibrinolysis.