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Research Progress of the Center for the Development of Molecular Target Drugs (2003-2005)

 

Sudafs group

A) Molecular mechanism of Fas ligand-induced inflammation

Fas ligand (FasL) is a prototype of death factor that induces apoptosis by binding to its receptor, Fas. We previously demonstrated that anti-Fas ligand antibody administration ameliorates various inflammatory diseases, and prevent chronic-hepatitis-induced hepatic cancer development using animal models. We also demonstrated that Fas ligand-induced caspase activation in neutrophils causes not only apoptosis but also proteolytic maturation and release of IL-1b, which in turn induces massive neutrophil infiltration in vivo. In the last three years, we further demonstrated that Fas ligand induces production of various cytokines including IL-1b, IL-6, IL-17, IL-18, IL-23, KC, and Mip2 in mice. Fas ligand induces IL-23 production in dendritic cells at the mRNA level in a cell-autonomous manner. The IL-23 then induces IL-17 production in T cells synergistically with IL-1b. We also found that Fas ligand induces IL-8 production in human embryonic kidney (HEK) 293 cells in a manner dependent on the NF-kB activation. The Fas ligand-induced NF-kB activation is a cell-autonomous response and requires FADD and caspase-8.

 

B) Molecular mechanisms of activation and regulation of ASC-mediated inflammation.

ASC is an adaptor molecule that mediates apoptotic and inflammatory signals from several Apaf-1-like molecules, including CARD12/Ipaf, PYPAF1/cryopyrin, PYPAF5, PYPAF7, and NALP1. ASC is also implicated in tumor suppression, because the ASC gene expression is suppressed in various cancer cells. We have established an experimental system in which muramyl dipeptide, the bacterial component recognized by another Apaf-1-like molecule, Nod2, induced an interaction between a CARD12-Nod2 chimeric protein and ASC, and elicited cell-autonomous NF-kB activation leading to IL-8 production. Using this system, we demonstrated that caspase-8 plays an essential role in ASC-mediated NF-kB activation. On the other hand, we have found that some members of Apaf-1-like proteins such as PYNOD, PYPAF2 and PYPAF3 inhibit ASC-mediated NF-kB activation and or caspase-1-mediated proteolytic maturation of IL-1b.

 

 

Molecular mechanisms of Fas ligand-induced inflammation.

 

M. Umemura, T. Kawabe, H. Kidoya, K. Shudo, M. Fukui, M. Asano, Y. Iwakura, G. Matsuzaki,A. Yahagi, R. Imamura, and T. Suda

 

      Fas ligand (FasL) has been well characterized as a death factor. However, recent studies revealed that ectopic expression of FasL induces inflammation associated with massive neutrophil infiltration. We previously demonstrated that the neutrophil infiltration-inducing activity of FasL is partly dependent on but partly independent of IL-1b. Therefore, we investigated the cytokine profile of peritoneal lavage fluid obtained from mice that received intraperitoneal injections of FFL, a FasL-expressing tumor cell line. We found that FFL injection caused a marked increase of not only IL-1b but also IL-6, IL-17, IL-18, KC/chemokine CXC ligand 1, and macrophage inflammatory protein (MIP)-2, but not of IL-1a, IFN-g, TGF-b, or TNF-a. Among cells transfected to express individually IL-1b, IL-6, IL-17, or IL-18, only those expressing IL-1b and IL-17 induced neutrophil infiltration. Co-administration of the anti-IL-17 antibody with FFL diminished the peritoneal KC levels and neutrophil infiltration in IL-1-deficient mice. In addition, the expression of IL-17 by the tumor cells inhibited tumor growth in wild-type and nude mice. These results suggest that IL-17 is involved in FasL-induced inflammation and tumor rejection in the absence of IL-1b.

      Then, 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 FFL cells induced IL-17 production in freshly isolated resident PEC. Anti-IL-1b Ab strongly inhibited the IL-17-inducing activity. However, recombinant IL-1b 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-1b 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. Finally, FasL induced only weak p40 production in a mixture of p40-/- and Fas-/- dendritic cells, indicating that FasL induces IL-23 production in dendritic cells mainly in a cell-autonomous manner.

1.           Umemura, M., et al., Int. Immunol., 16:1099-108, 2004

2.           Kidoya, H., et al., J. Immunol., 175:8024-31, 2005

 

 

Molecular Mechanism of Fas ligand-induced IL-8 Production

 

R. Imamura, N. Matsumoto, M. Hasegawa, K. Konaka, and T. Suda

 

It has been believed that apoptosis does not induce inflammation. However, there are remarkable similarities between the molecular mechanisms of apoptosis and inflammation. Fas (CD95) is not an exception and recent studies revealed that Fas ligand (FasL)-Fas system possesses inflammatory activity. We recently found that FasL induces production of the inflammatory chemokine IL-8 in human cell lines and FasL-induced NF-kB and AP-1 activation is required for this IL-8 production. Our further analyses revealed that the death domain of Fas, FADD, and caspase-8, which are essential for the apoptosis signaling, are required for both NF-kB and AP-1 activation by FasL. However, rsponses of NF-kB and AP-1 activation are independent of each other. In the NF-kB signaling pathway, we also showed that TRADD and RIP, which are essential for the TNF-a-induced NF-kB activation, were not involved in the FasL-induced NF-kB activation and CLARP/FLIP inhibited the FasL- but not the TNF-a-induced NF-kB activation. More interestingly, our results revealed that enzymatic activity of caspase-8 is required for both NF-kB and AP-1 activation induced by FasL. Further characterization of these pathways will help us to understand and, hopefully, to control the FasL-induced inflammation. (Imamura, R. et al., J. Biol. Chem., 279:46415-46423, 2004)

 

Figure

In the case of FasL-Fas system, contrary to TNF-a-TNFR system, signaling pathway of apoptosis and transcription factor activation separates downstream of caspase-8 and enzymatic activity of caspase-8 is required for both pathways. Current our goal is identification of targets (substrates) of caspase-8, which are important for FasL-induced NFkB and/or AP-1 activation.


 

 

PYNOD, a Novel Apaf-1/CED4-like Protein is an Inhibitor of ASC and Caspase-1

 

Y. Wang, M. Hasegawa, R. Imamura, T. Kinoshita, C. Kondo, K. Konaka, and T. Suda

 

      Recently, a large subfamily of nucleotide-binding and oligomerization domain-containing proteins that have an N-terminal pyrin-like domain and C-terminal leucine-rich repeats has been described. In this study, we identified PYNOD, a novel member of this family that lacks the leucine-rich repeats. We found that human PYNOD mRNA is expressed in various tissues and at high levels in heart, skeletal muscle, and brain. It is also expressed in various cell lines, including haematopoietic cell lines. PYNOD oligomerizes and binds to ASC, an adaptor protein that plays a role in apoptotic and inflammatory signal transduction, and to caspase-1 and IL-1b. PYNOD inhibits ASC-mediated NF-kB activation and apoptosis, and caspase-1-mediated IL-1b maturation, and it does so in the presence and absence of constitutively active mutants of CARD12 and PYPAF1, which are enhancers of these processes. Thus, PYNOD is a novel regulator of apoptosis and inflammation. (Wang, Y. et al., Int. Immunol. 16:777-86, 2004)

 

 

Figure

PYNOD is a member of Apaf-1-like proteins and inhibits ASC-mediated NF-kB activation and apoptosis, and caspase-1-mediated IL-1b activation.

 

 

PYPAF3, a PYRIN-containing Apaf-1-like protein, is a feedback regulator of caspase-1-dependent interleukin-1b secretion

 

T. Kinoshita, Y. Wang, M. Hasegawa, R. Imamura, T. Suda

 

      PYPAF3 is a member of the PYRIN-containing apoptotic protease-activating factor-1-like proteins (PYPAFs) that are thought to function in inflammatory signaling pathways. Among the members of this family, PYPAF1, PYPAF5, PYPAF7, and NALP1 have been shown to induce caspase-1-dependent interleukin-1b secretion and NF-kB activation in the presence of the adaptor molecule ASC. On the other hand, we recently identified PYNOD, another member of this family, as a suppressor of these responses. In this study, we showed that PYPAF3 is the second member that inhibits caspase-1-dependent interleukin-1b secretion (Fig. 1) and that PYPAF2 does not inhibit this response, but rather inhibits the ASC-mediated NF-kB activation (Fig. 2). Both PYPAF2 and PYPAF3 mRNAs are broadly expressed in a variety of tissues; however, neither is expressed in skeletal muscle, and only PYPAF2 mRNA is expressed in heart and brain. They are also expressed in many cell lines of both haematopoietic and non-haematopoietic lineages. Stimulation of monocytic THP-1 cells with lipopolysaccharide or interleukin-1b induced PYPAF3 mRNA expression (Fig. 3). Furthermore, the stable expression of PYPAF3 in THP-1 cells abrogated the cellsf ability to produce interleukin-1b in response to lipopolysaccharide. These results suggest that PYPAF3 is a feedback regulator of interleukin-1b secretion. Thus, PYPAF2 and PYPAF3, together with PYNOD, may constitute an anti-inflammatory subgroup of PYPAFs. (Kinoshita, T. et al., J. Biol. Chem. 280:21720-5, 2005)

 

 

 

 

 

 

 

ASC-mediated NF-kB Activation Leading to IL-8 Production Requires Caspase-8 and Is Inhibited by CLARP

 

M. Hasegawa, R. Imamura, T. Kinoshita, N. Matsumoto, J. Masumoto, N. Inohara,

and T. Suda

 

      ASC is an adaptor molecule that mediates apoptotic and inflammatory signals from several Apaf-1-like molecules, including CARD12/Ipaf. ASC is also implicated in tumor suppression, because the ASC gene expression is suppressed in various cancer cells. To characterize the signaling pathway mediated by ASC, we established cell lines in which muramyl dipeptide, the bacterial component recognized by another Apaf-1-like molecule, Nod2, induced an interaction between a CARD12-Nod2 chimeric protein and ASC, and elicited cell-autonomous NF-kB activation. This response required caspase-8, and was suppressed by CLARP/FLIP, an inhibitor of caspase-8. The catalytic activity of caspase-8 was required for the ASC-mediated NF-kB activation when caspase-8 was expressed at an endogenous level, although it was not essential when caspase-8 was overexpressed. In contrast, FADD, the adaptor protein linking Fas and caspase-8, was not required for this response. Consistently, ASC recruited Caspase-8 and CLARP but not FADD and Nod2 to its speck-like aggregates in cells. Finally, muramyl dipeptide induced IL-8 production in MAIL8 cells. These results are the first to indicate that caspase-8 plays an important role in the ASC-mediated NF-kB activation, and that the ASC-mediated NF-kB activation actually induces physiologically relevant gene expression. (Hasegawa M, et al., J. Biol. Chem. 280: 15122-30, 2005.)

 

Figure

DREDD, the drosophila homolog of caspase-8 has been shown to play an important role in NF-kB activation. In mammals, caspase-8 also plays an important role in Fas ligand-induced and ASC-mediated NF-kB activation.

 


Publications

1.  Kidoya, H., Umemura, U., Kawabe, T., Matsuzaki, G., Yahagi, A., Imamura, R., and Suda, T. (2005) Fas ligand induces cell-autonomous IL-23 production in dendritic cells, a mechanism for Fas ligand-induced IL-17 production. J. Immunol. 175:8024-31.

2.  Kinoshita, T., Wang, Y., Hasegawa, M., Imamura, R., and Suda, T. (2005) PYPAF3, a PYRIN-containing APAF-1-like protein, is a feedback regulator of caspase-1-dependent interleukin-1b secretion. J. Biol. Chem. 280:21720-5

3.  Hasegawa M, Imamura R, Kinoshita T, Matsumoto N, Masumoto J, Inohara N, and Suda T. (2005) ASC-mediated NF-kB activation leading to interleukin-8 production requires caspase-8 and is inhibited by CLARP. J. Biol. Chem. 280: 15122-30.

4.  Suda, T. (2005) Physiological and pathological roles of apoptosis. Nippon Rinsho. Suppl 4:395-400. Review. (Japanese)

5.  Imamura, R., Konaka, K., Matsumoto, N., Hasegawa, M., Fukui, M., Mukaida, N., Kinoshita, T., and Suda, T. (2004) Fas ligand induces cell-autonomous NF-kB activation and IL-8 production by a mechanism distinct from that of TNF-a. J. Biol. Chem. 279:46415-23.

6.  Umemura, M., Kawabe, T., Shudo, K., Kidoya, H., Fukui, M., Asano, M., Iwakura, Y., Matsuzaki, G., Imamura, R., and Suda, T. (2004) Involvement of IL-17 in Fas ligand-induced inflammation. Int. Immunol. 16:1099-108.

7.  Wang, Y., Hasegawa, M., Imamura, R., Kinoshita, T., Kondo, C., Konaka, K., Suda, T. (2004) PYNOD, a novel Apaf-1/CED4-like protein is an inhibitor of ASC and caspase-1. Int. Immunol. 16:777-86.

8.  Nakamoto Y, Suda T, Momoi T, and Kaneko S. (2004) Different procarcinogenic potentials of lymphocyte subsets in a transgenic mouse model of chronic hepatitis B. Cancer Res. 64:3326-3333

9.  Uekita, T., Gotoh, I., Kinoshita, T., Itoh, Y., Sato, H., Shiomi, T., Okada, Y., and Seiki, M. (2004). Membrane-type 1 matrix metalloproteinase cytoplasmic tail-binding protein-1 is a new member of the Cupin superfamily. A possible multifunctional protein acting as an invasion suppressor down-regulated in tumors. J Biol Chem 279, 12734-43.

10. Fukui, M., Imamura, R., Umemura, M., Kawabe, T., Suda, T. (2003) Pathogen-associated molecular patterns sensitize macrophages to Fas ligand-induced apoptosis and IL-1beta release. J. Immunol.. 171:1868-74.

11. Tachiiri, A., Imamura, R., Wang, Y., Fukui, M., Umemura, M., and Suda, T. (2003) Genomic structure and inducible expression of the IL-22 receptor a chain in mice. Gene. Immun., 4:153-159