Ryu Imamura

Signalling into the T-Cell Nucleus: NFAT Regulation

The nuclear factor of activated T cells (NFAT) plays an important role in T-cell biology. Activation of T cells results in the rapid calcineurin-dependent translocation of NFAT transcription factors from the cytoplasm to the nucleus. This translocation process coupled to the subsequent active maintenance of NFAT in the nucleus compartment is critical for the induction of expression of several genes encoding cytokines and membrane proteins that modulate immune responses. The molecular cloning of the NFAT family of transcription factors has facilitated rapid progress in the understanding of the signalling mechanisms that control the activity of NFAT.

The membrane-bound but not the soluble form of human Fas ligand is responsible for its inflammatory activity

The ectopic expression of Fas ligand (FasL/CD95L) in tissues or tumors induces neutrophil infiltration and the destruction of the tissues or the rejection of tumors. It has been suggested thatthe infiltrated neutrophils are responsible for the latter phenomena. FasL is synthesized as a type II transmembrane protein, and soluble FasL is produced by a proteolytic mechanism from the membrane‐bound form. We previously demonstrated that uncleavable membrane‐bound FasL of mice induces IL‐1β release from inflammatory cells, and suggested that the IL‐1β enhances neutrophil infiltration. However, recent papers reported that human soluble FasL is directly chemoattractive to neutrophils in vitro and proposed that the soluble form of FasL is responsible for its inflammatory activity. Therefore, in this report, we investigated which form is responsible for the inflammatory activities of human FasL. We produced tumor cell lines expressing one or both forms of human FasL. Cells expressing both forms or only the membrane‐bound form of FasL induced neutrophil infiltration when transplanted into the peritoneal cavity of syngeneic mice, while cells expressing only the soluble form did not. Purified soluble FasL failed to induce neutrophil infiltration in vivo. IL‐1β release from inflammatory peritoneal exudate and acceleration of tumor rejection were also mediated by membrane‐bound but not soluble FasL. These results indicate that the membrane‐bound form of FasL is primarily responsible for its inflammatory activity.

Anti-Inflammatory Activity of PYNOD and Its Mechanism in Humans and Mice

Many members of the nucleotide-binding and oligomerization domain (NOD)- and leucine-rich-repeat–containing protein (NLR) family play important roles in pathogen recognition and inflammation. However, we previously reported that human PYNOD/NLRP10, an NLR-like protein consisting of a pyrin domain and a NOD, inhibits inflammatory signal mediated by caspase-1 and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) in reconstitution experiments using HEK293 cells. In this study, we investigated the molecular mechanism of PYNOD’s anti-inflammatory activity in vitro and its expression and function in mice. Human PYNOD inhibited the autoprocessing of caspase-1 and caspase-1–mediated IL-1β processing and suppressed the aggregation of ASC, a hallmark of ASC activation. Interestingly, the NOD of human PYNOD was sufficient to inhibit caspase-1–mediated IL-1β secretion, whereas its pyrin domain was sufficient to inhibit ASC-mediated NF-κB activation and apoptosis and to reduce ASC’s ability to promote caspase-1–mediated IL-1β production. Mouse PYNOD protein was detected in the skin, tongue, heart, colon, peritoneal macrophages, and several cell lines of hematopoietic and myocytic lineages. Mouse PYNOD colocalized with ASC aggregates in LPS + R837-stimulated macrophages; however, unlike human PYNOD, mouse PYNOD failed to inhibit ASC aggregation. Macrophages and neutrophils from PYNOD-transgenic mice exhibited reduced IL-1β processing and secretion upon microbial infection, although mouse PYNOD failed to inhibit caspase-1 processing, which was inhibited by caspase-4 inhibitor z-LEED-fluoromethylketone. These results suggest that mouse PYNOD colocalizes with ASC and inhibits caspase-1–mediated IL-1β processing without inhibiting caspase-4 (mouse caspase-11)–mediated caspase-1 processing. Furthermore, PYNOD-transgenic mice were resistant to lethal endotoxic shock. Thus, PYNOD is the first example of an NLR that possesses an anti-inflammatory function in vivo.

Roles of the PI3K/Akt pathway and autophagy in TLR3 signaling-induced apoptosis and growth arrest of human prostate cancer cells

Toll-like receptors (TLRs) are widely expressed in immune cells and play a crucial role in many aspects of the immune response. Although some types of TLRs are also expressed in cancer cells, the effects and mechanisms of TLR signaling in cancer cells have not yet been fully elucidated. In the present study, we analyzed the effects of polyinosinic-polycytidylic acid [poly(I:C)], a TLR3 ligand, on three TLR3-expressing human prostate cancer cell lines (LNCaP, PC3, and DU145). We then further characterized the underlying mechanisms, focusing on the poly(I:C)-sensitive LNCaP cell line. Poly(I:C) significantly reduced the viability of LNCaP cells TLR3 and endosome dependently. One mechanism for the antitumor effect was caspase-dependent apoptosis, and another mechanism was poly(I:C)-induced growth arrest. Cell survival and proliferation of LNCaP cells depended on the PI3K/Akt pathway, and PI3K/Akt inhibitors induced apoptosis and growth arrest similar to poly(I:C) treatment. Additionally, poly(I:C) treatment caused dephosphorylation of Akt in LNCaP cells, but transduction of the constitutively active form of Akt rendered LNCaP cells resistant to poly(I:C). Immunoblot analysis of proliferation- and apoptosis-related molecules in poly(I:C)-treated LNCaP cells revealed participation of cyclinD1, c-Myc, p53, and NOXA. Interestingly, poly(I:C) treatment of LNCaP cells was accompanied by autophagy, which was cytoprotective toward poly(I:C)-induced apoptosis. Together, these findings indicate that TLR3 signaling triggers apoptosis and growth arrest of LNCaP cells partially through inactivation of the PI3K/Akt pathway and that treatment-associated autophagy plays a cytoprotective role.

Pathogen-Associated Molecular Patterns Sensitize Macrophages to Fas Ligand-Induced Apoptosis and IL-1β Release

Antigenic stimulation activates T cells and simultaneously destines them to die by Fas-mediated apoptosis. In this study, we demonstrated that various pathogen-associated molecular patterns up-regulated Fas expression in macrophages and sensitized them specifically to Fas ligand (FasL), but not to other apoptosis-inducing agents such as TNF-α, etoposide (VP-16), and staurosporine. Toll-like receptor, NF-κB, and p38 mitogen-activated protein kinase mediated these responses. LPS stimulation induced the expression of Fas, caspase 8, cellular FLIP Bfl-1/A1, and Bcl-x, but not FasL, TNFR p55, Bak, Bax, and Bad at the transcriptional level. Thus, LPS selectively induced the expression of apoptotic molecules of the Fas death pathway (except for cellular FLIP) and antiapoptotic molecules of the mitochondrial death pathway. However, the kinetics of macrophage disappearance following Escherichia coli-induced peritonitis was similar between wild-type and Fas-deficient mice, suggesting that Fas is not essential for the turnover of activated macrophages in T cell-independent inflammation. In contrast, LPS-activated macrophages produced a large amount of IL-1β upon FasL stimulation. Thus, unlike the activation-induced cell death of T cells, the sensitization of macrophages to FasL by pathogen-associated molecular patterns seems to be a proinflammatory rather than an anti-inflammatory event.

Mechanism of ASC-mediated apoptosis: Bid-dependent apoptosis in type II cells

Apoptosis-associated speck-like protein containing a CARD (ASC) is an adaptor molecule that mediates apoptotic and inflammatory signals, and implicated in tumor suppression. However, the mechanism of ASC-mediated apoptosis has not been well elucidated. Here, we investigated the molecular mechanisms of ASC-mediated apoptosis in several cell lines using a caspase recruitment domain 12-Nod2 chimeric protein that transduces the signal from muramyl dipeptide into ASC-mediated apoptosis. Experiments using dominant-negative mutants, small-interfering RNAs and peptide inhibitors for caspases indicated that caspase-8 was generally required for ASC-mediated apoptosis, whereas a requirement for caspase-9 depended on the cell type. In addition, caspase-like apoptosis-regulatory protein (CLARP)/Fas-like inhibitor protein, a natural caspase-8 inhibitor, suppressed ASC-mediated apoptosis, and Clarp−/− mouse embryonic fibroblasts were highly sensitive to ASC-mediated apoptosis. Bax-deficient HCT116 cells were resistant to ASC-mediated apoptosis as reported previously, although we failed to observe colocalization of ASC and Bax in cells. Like Fas-ligand-induced apoptosis, the ASC-mediated apoptosis was inhibited by Bcl-2 and/or Bcl-XL in type-II but not type-I cell lines. Bid was cleaved upon ASC activation, and suppression of endogenous Bid expression using small-interfering RNAs in type-II cells reduced the ASC-mediated apoptosis. These results indicate that ASC, like death receptors, mediates two types of apoptosis depending on the cell type, in a manner involving caspase-8.

Caspase‐8‐ and JNK‐dependent AP‐1 activation is required for Fas ligand‐induced IL‐8 production

Despite a dogma that apoptosis does not induce inflammation, Fas ligand (FasL), a well‐known death factor, possesses pro‐inflammatory activity. For example, FasL induces nuclear factor κB (NF‐κB) activity and interleukin 8 (IL‐8) production by engagement of Fas in human cells. Here, we found that a dominant negative mutant of c‐Jun, a component of the activator protein‐1 (AP‐1) transcription factor, inhibits FasL‐induced AP‐1 activity and IL‐8 production in HEK293 cells. Selective inhibition of AP‐1 did not affect NF‐κB activation and vice versa, indicating that their activations were not sequential events. The FasL‐induced AP‐1 activation could be inhibited by deleting or introducing the lymphoproliferation (lpr)‐type point mutation into the Fas death domain (DD), knocking down the Fas‐associated DD protein (FADD), abrogating caspase‐8 expression with small interfering RNAs, or using inhibitors for pan‐caspase and caspase‐8 but not caspase‐1 or caspase‐3. Furthermore, wildtype, but not a catalytically inactive mutant, of caspase‐8 reconstituted the FasL‐induced AP‐1 activation in caspase‐8‐deficient cells. Fas ligand induced the phosphorylation of two of the three major mitogen‐activated protein kinases (MAPKs): extracellular signal‐regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK) but not p38 MAPK. Unexpectedly, an inhibitor for JNK but not for MAPK/ERK kinase inhibited the FasL‐induced AP‐1 activation and IL‐8 production. These results demonstrate that FasL‐induced AP‐1 activation is required for optimal IL‐8 production, and this process is mediated by FADD, caspase‐8, and JNK.

Caspase-1 Protein Induces Apoptosis-associated Speck-like Protein Containing a Caspase Recruitment Domain (ASC)-mediated Necrosis Independently of Its Catalytic Activity

Background: ASC mediates apoptosis and necrosis of tumor cells and necrosis of microbe-infected macrophages. Results: ASC mediates necrosis only when cells express caspase-1; however, inhibition of caspase-1 proteolytic activity did not suppress the necrosis. Conclusion: Caspase-1 but not its proteolytic activity is essential for ASC-mediated necrosis. Significance: This study explains why ASC induces apoptosis or necrosis depending on the cell type. The adaptor protein, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), connects pathogen/danger sensors such as NLRP3 and NLRC4 with caspases and is involved in inflammation and cell death. We have found that ASC activation induced caspase-8-dependent apoptosis or CA-074Me (cathepsin B inhibitor)-inhibitable necrosis depending on the cell type. Unlike necroptosis, another necrotic cell death, ASC-mediated necrosis, was neither RIP3-dependent nor necrostatin-1-inhibitable. Although acetyl–YVAD–chloromethylketone (Ac-YVAD-CMK) (caspase-1 inhibitor) did not inhibit ASC-mediated necrosis, comprehensive gene expression analyses indicated that caspase-1 expression coincided with the necrosis type. Furthermore, caspase-1 knockdown converted necrosis-type cells to apoptosis-type cells, whereas exogenous expression of either wild-type or catalytically inactive caspase-1 did the opposite. Knockdown of caspase-1, but not Ac-YVAD-CMK, suppressed the monocyte necrosis induced by Staphylococcus and Pseudomonas infection. Thus, the catalytic activity of caspase-1 is dispensable for necrosis induction. Intriguingly, a short period of caspase-1 knockdown inhibited IL-1β production but not necrosis, although longer knockdown suppressed both responses. Possible explanations of this phenomenon are discussed.

Mechanism and repertoire of ASC-mediated gene expression

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an adaptor molecule that mediates inflammatory and apoptotic signals. Although the role of ASC in caspase-1-mediated IL-1β and IL-18 maturation is well known, ASC also induces NF-κB activation and cytokine gene expression in human cells. In this study, we investigated the molecular mechanism and repertoire of ASC-induced gene expression in human cells. We found that the specific activation of ASC induced AP-1 activity, which was required for optimal IL8 promoter activity. ASC activation also induced STAT3-, but not STAT1-, IFN-stimulated gene factor 3- or NF-AT-dependent reporter gene expression. The ASC-mediated AP-1 activation was NF-κB-independent and primarily cell-autonomous response, whereas the STAT3 activation required NF-κB activation and was mediated by a factor that can act in a paracrine manner. ASC-mediated AP-1 activation was inhibited by chemical or protein inhibitors for caspase-8, caspase-8-targeting small-interfering RNA, and p38 and JNK inhibitors, but not by a caspase-1 inhibitor, caspase-9 or Fas-associated death domain protein (FADD) dominant-negative mutants, FADD- or RICK-targeting small-interfering RNAs, or a MEK inhibitor, indicating that the ASC-induced AP-1 activation is mediated by caspase-8, p38, and JNK, but does not require caspase-1, caspase-9, FADD, RICK, or ERK. DNA microarray analyses identified 75 genes that were induced by ASC activation. A large proportion of them was related to transcription (23%), inflammation (21%), or cell death (16%), indicating that ASC is a potent inducer of inflammatory and cell death-related genes. This is the first report of ASC-mediated AP-1 activation and the repertoire of genes induced downstream of ASC activation.

Fas ligand induces cell-autonomous IL-23 production in dendritic cells, a mechanism for fas ligand-induced IL-17 production

Fas ligand (FasL) has the potential to induce inflammation accompanied by massive neutrophil infiltration. We previously reported that FasL rapidly induces the production of various inflammatory cytokines including IL-1β and IL-17. In this study, we investigated the mechanism of the FasL-induced IL-17 production. We found that the culture supernatant of mouse resident peritoneal exudate cells (PEC) cocultured with FasL-expressing tumor (FFL) cells induced IL-17 production in freshly isolated resident PEC. Anti-IL-1β Ab strongly inhibited the IL-17-inducing activity. However, rIL-1β by itself induced only weak IL-17 production. Intriguingly, anti-IL-12 Ab but not an IL-15-neutralizing agent, IL15R-Fc, strongly inhibited the FasL-induced IL-17-inducing activity. IL-23, which shares the p40 subunit with IL-12, but not IL-12 itself, induced IL-17 production synergistically with IL-1β in resident PEC. FasL induced the production of IL-23 in PEC in vivo and in vitro, and IL-17 production following the i.p. injection of FFL cells was severely impaired in p40−/− mice, indicating that IL-23 plays an important role in the FasL-induced IL-17 production. FFL also induced the production of IL-23 in bone marrow- or PEC-derived dendritic cells (DCs). Finally, FasL induced only weak p40 production in a mixture of p40−/− and Fas−/− DC, indicating that FasL induces IL-23 production in DC mainly in a cell-autonomous manner.