General Summary of Division of Immunology and Molecular Biology (2006-2008)

In the last three years, our study has been aiming to clarify the tripartite relationship among apoptosis, inflammation and cancer. It is generally believed that apoptosis is an important process to prevent oncogenesis, and that inflammation is a causative factor for cancer development. In terms of the relationship between apoptosis and inflammation, it has been said that apoptosis does not induce inflammation. However, recent studies demonstrated that apoptosis and inflammation uses many common protein molecules for signal transduction. For example, Fas ligand, an apoptosis inducing factor, was found to be a potent inducer of inflammation in vivo. Furthermore, we previously discovered that in a mouse model of chronic

hepatitis that eventually develops hepatic cancer, administration of a neutralizing antibody against Fas ligand　suppressed the hepatic inflammation and cancer development. Based on these results, we are investigating the functions of proteins (including Fas ligand) that play a role at the crossroad of apoptosis and inflammation, aiming to discover new findings useful for cancer therapy.

A) Study for the molecular mechanism of the inflammatory activity of Fas ligand.

Fas ligand is famous as a death factor; however, transplantation of Fas ligand-expressing cells into the peritoneal cavity of a mouse induces peritonitis associated with massive neutrophil infiltration. In addition, Fas ligand induces production of IL-8, a chemokine for neutrophils, in the human embryonic kidney (HEK)-293 cell line. Our previous study revealed that NF-kB plays an important role in the Fas ligand-induced IL-8 production and that caspase-8 is involved in this response. This time, we further demonstrated that AP-1 is also required for optimal IL-8 production, and that caspase-8 and JNK play essential roles in this response.

B) Study for the molecular mechanism of ASC-mediated apoptosis

ASC was originally discovered as a protein that forms a large aggregate in an apoptotic HL-60 human leukemia cell treated with a chemotherapeutic drug. This protein was independently discovered as a product of the gene called TMS1 whose expression in various human cancer tissues were suppressed by DNA methylation. It has been also reported that the expression of ASC is controlled by p53 tumor suppressor, and that ASC is involved in etoposide-induced apoptosis of tumor cells. These results suggest that ASC is a novel tumor suppressor. In addition, ASC was recently identified as an adaptor protein that mediates inflammatory and apoptotic signals from some members of the NLR family (such as cryopyrin and CARD12) that function as cytoplasmic sensors for pathogens and activate cellular innate immune responses. The molecular mechanism of ASC-mediated apoptosis has been controversially reported. Initially, it was reported to be caspase-9 dependent; however, this notion was recently challenged. Therefore, we have investigated that molecular mechanism of ASC-mediated apoptosis and clearly demonstrated that caspase-8 plays an important role for this response. Just like the extrinsic pathway of apoptosis that is initiated by a death factor, ASC-mediated apoptosis in type-2 cells depends on proteolytic activation of Bid by caspase-8.

Recently, we also discovered that ASC mediates necrotic cell death under some conditions. Our current study is aiming to clarify the molecular mechanism of ASC-mediated necrosis. We also investigate possible use of ASC as a molecular target for cancer therapy.

C) The regulatory mechanisms of NLR proteins

Several members of NLR proteins functions (including cryopyrin) as cytoplasmic sensors for pathogens and activator for apoptosis and inflammation as described above. However, we previously discovered other members of NLRs such as PYNOD, PYPAF2 and PYPAF3 function as negative regulator for ASC and caspase-1. This time, we have searched for cryopyrin-binding proteins using the yeast-two hybrid system. As a result, we have identified FAF1 as a novel cryopyrin-binding protein, and found that FAF1 inhibits cryopyrin-mediated NF-kB activation. In addition, we have established PYNOD-transgenic mice, and we are now investigating the in vivo functions of PYNOD.

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

Norihiko Matsumoto, Ryu Imamura, and Takashi Suda

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 kB (NF-kB) 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-kB 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 (siRNAs), or using inhibitors for pan-caspase and caspase-8 but not caspase-1 or caspase-3. Furthermore, wild-type, 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.

Figure. Fas ligand is well known death factor that induces apoptosis in a caspase-8 dependent manner. We previously demonstrated that stimulation by Fas ligand induces IL-1b secretion in LPS-primed mouse macrophages and IL-8 secretion in human embryonic kidney (HEK)-293 cells. Interestingly, it was found that both of these inflammatory responses are caspase-8 dependent. In this study, we further discovered that activation of Fas by Fas ligand induces AP-1 activation in a caspase-8- and JNK-dependent manner. This AP-1 activation is required for the Fas ligand-induced IL-8 production in HEK293 cells.

Fas-Associated Factor 1 is a negative regulator of PYRIN-containing Apaf-1-like protein 1

Takeshi Kinoshita, Chiaki Kondoh, Mizuho Hasegawa, Ryu Imamura, and Takashi Suda

PYRIN-containing apoptotic protease-activating factor-1-like proteins (PYPAFs, also called NALPs) participate in inflammatory signaling by regulating NF-kB activation and cytokine processing, and have been implicated in autoimmune and inflammatory disorders. However, the precise mechanisms that regulate the signal pathway leading to NF-kB activation are not completely understood. Here, we used yeast-two hybrid assays to identify Fas associated factor 1 (FAF1) as a protein interacting with the pyrin domains of several PYPAFs. In these assays, FAF1 interacted strongly with PYPAF1, PYPAF3, and PYPAF7, moderately with PYPAF2 and PYNOD, but not at all with the pyrin domains of pyrin or the adaptor molecule ASC. The interaction between FAF1 and PYPAF1 in mammalian cells was confirmed by immunoprecipitation assays, and the Fas-interacting domain of FAF1 was critical for this interaction. When coexpressed in HEK293 cells, FAF1 interfere with NF-B activation induced by PYPAF1 and ASC. A FAF1 mutant lacking the Fas-interacting domain showed significantly reduced ability to inhibit NF-kB activation. Furthermore, down-regulation of endogenous FAF1 protein augmented LPS-induced IL-8 production, a biological marker for NF-kB activation, in monocytic cells. Finally, the level of FAF1 expression in THP-1 cells increased in response to NF-kB stimulation. These findings suggest that FAF1 functions as a negative regulator of an NF-kB signal pathway that involves PYPAF1 and ASC.

Figure 1. FAF1 interacts with the pyrin domains of PYPAF1, PYPAF2, PYPAF3, PYNOD, and PYPAF7, but not with those of pyrin, ASC and PYPAF6 in Yeast-two hybrid assays.	Figure 2. Cotransfection of a constitutive active mutant of PYPAF1 (PYPAF1-DLRR) and ASC induces NF-kB activation as revealed by a luciferase reporter assay in HEK293 cells. This NF-kB activation was inhibited by cotransfection of FAF1.

Expression of NLRP7 (PYPAF3) protein in endometrial cancer tissues

Satoshi Ohno*, Takeshi Kinoshita*, Yumiko Ohno, Toshinari Minamoto, Nobutaka Suzuki, Masaki Inoue and Takashi Suda (*Both authors equally contributed to this work)

Nucleotide-binding domain and leucine-rich repeat-containing family, pyrin domain-containing 7 (NLRP7) (pyrin-containing apoptotic protease activating factor-1-like protein 3; PYPAF3, NACHT domain-, leucine-rich repeat, and pyrin domain-containing 7; NALP7) has been thought to contribute to innate immunity and inflammation. Although expression of NLRP7 in human seminoma tissues and several cancer cell lines has been demonstrated, the pathophysiological and prognostic importance in cancer tissues has not been defined. In this study, a series of 70 endometrial cancer cases that had undergone curative resection was studied to determine the correlation between NLRP7 expression and clinico-pathological characteristics in human endometrial cancer tissue. Tissue specimens were evaluated for NLRP7 by immunohistochemistry. NLRP7 expression was positive in cancer cells in 7 cases (10%). There was a statistical relationship between the depth of tumor invasion and NLRP7 expression (p=0.0326). NLRP7 expression showed a trend for being associated with poor prognosis. Conclusion: Tumor-produced NLRP7, associated with myometrial invasion, might provide additional prognostic information in endometrial cancer patients.

Figure 1. Representative sections of endometrial cancer with immunohistochemical staining of NLRP7. Strong cytoplasmic staining is observed in the invasion front of the tumor (×40; inset, ×200).	Figure 2. The Kaplan-Meier survival curves of 70 patients with endometrial carcinoma in relation to NLRP7 expression are shown.

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

M. Hasegawa, K. Kawase, N. Inohara, R. Imamura, W-C.　Yeh, T. Kinoshita, and T. Suda

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 CARD12-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, while a requirement for caspase-9 depended on the cell type. In addition, CLARP/FLIP (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.

	c
Figure. a, b) MDP induces apoptosis in MAIL8 cells expressing CARD12-NOD2 chimera protein and ASC. c) ASC, like death receptors, mediates two types of apoptosis depending on the cell type, in a manner involving caspase-8

Publications

1. Kinoshita, T., Kondoh, C., Hasegawa, M., Imamura, R., and Suda, T. (2006) Fas-associated Factor 1 is a negative regulator of PYRIN-containing Apaf-1-like protein 1, Int. Immunol., 18:1701-1706.

2. El Kasmi, K.C., Holst J, Coffre, M., Mielke, L., de Pauw, A., Lhocine, N., Smith, A.M., Rutschman, R., Kaushal, D., Shen, Y., Suda, T., Donnelly, R.P., Myers, M.G. Jr., Alexander, W., Vignali, D.A., Watowich, S.S., Ernst, M., Hilton, D.J., Murray, P.J. (2006) General nature of the STAT3-activated anti-inflammatory response. J. Immunol., 177:7880-7888.

3. Hasegawa, M., Kawase, K., Inohara, N., Imamura, R., Yeh, W-C.,Kinoshita, T., and Suda, T. (2007) Mechanism of ASC-mediated apoptosis: Bid-dependent apoptosis in type II cells, Oncogene, 26:1748-1756.

4. Fujisawa, A., Kambe, N., Saito, M., Nishikomori, R., Tanizaki, H., Kanazawa, N., Adachi, S., Heike, T., Sagara, J., Suda, T., Nakahata, T., Miyachi, Y. (2007) Disease-associated mutations in CIAS1 induce cathepsin B-dependent rapid cell death of human THP-1 monocytic cells, Blood, 109:2903-2911.

5. Umemura, M., Yahagi, A., Hamada, S., Begum, M. D., Watanabe, H., Kawakami, K., Suda, T., Sudo, K., Nakae, S., Iwakura, Y., and Matsuzaki, G. (2007) IL-17-mediated regulation of innate and acquired immune response against pulmonary Mycobacterium bovis BCG infection, J. Immunol., 178:3786-3796.

6. Matsumoto, N., Imamura, R., Suda, T. (2007) Caspase-8- and JNK-dependent AP-1 activation is required for Fas ligand-induced IL-8 production. FEBS J. 274:2376-2384

7. Ohno, S.*,　Kinoshita, T.*, Ohno, Y., Minamoto, T., Suzuki N., Inoue M., and Suda, T. (2008) Expression of NLRP7 (PYPAF3, NALP7) Protein in Endometrial Cancer Tissues. Anticancer Res. 28:2493-2497. (* Both authors equally contributed to this work.)