PYPAF3, a PYRIN-containing APAF-1-like Protein, Is a Feedback Regulator of Caspase-1-dependent Interleukin-1β Secretion

Abstract

PYPAF3 is a member of the PYRIN-containing apoptotic protease-activating factor-1-like proteins (PYPAFs, also called NALPs). Among the members of this family, PYPAF1, PYPAF5, PYPAF7, and NALP1 have been shown to induce caspase-1-dependent interleukin-1β secretion and NF-κB activation in the presence of the adaptor molecule ASC. On the other hand, we recently discovered that PYNOD, another member of this family, is a suppressor of these responses. Here, we show that PYPAF3 is the second member that inhibits caspase-1-dependent interleukin-1β secretion. In contrast, PYPAF2/NALP2 does not inhibit this response but rather inhibits the NF-κB activation that is induced by the combined expression of PYPAF1 and ASC. 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 hematopoietic and non-hematopoietic lineages. Stimulation of monocytic THP-1 cells with lipopolysaccharide or interleukin-1β induced PYPAF3 mRNA expression. Furthermore, the stable expression of PYPAF3 in THP-1 cells abrogated the ability of the cells to produce interleukin-1β in response to lipopolysaccharide. These results suggest that PYPAF3 is a feedback regulator of interleukin-1β secretion. Thus, PYPAF2 and PYPAF3, together with PYNOD, constitute an anti-inflammatory subgroup of PYPAFs. PYPAF3 is a member of the PYRIN-containing apoptotic protease-activating factor-1-like proteins (PYPAFs, also called NALPs). Among the members of this family, PYPAF1, PYPAF5, PYPAF7, and NALP1 have been shown to induce caspase-1-dependent interleukin-1β secretion and NF-κB activation in the presence of the adaptor molecule ASC. On the other hand, we recently discovered that PYNOD, another member of this family, is a suppressor of these responses. Here, we show that PYPAF3 is the second member that inhibits caspase-1-dependent interleukin-1β secretion. In contrast, PYPAF2/NALP2 does not inhibit this response but rather inhibits the NF-κB activation that is induced by the combined expression of PYPAF1 and ASC. 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 hematopoietic and non-hematopoietic lineages. Stimulation of monocytic THP-1 cells with lipopolysaccharide or interleukin-1β induced PYPAF3 mRNA expression. Furthermore, the stable expression of PYPAF3 in THP-1 cells abrogated the ability of the cells to produce interleukin-1β in response to lipopolysaccharide. These results suggest that PYPAF3 is a feedback regulator of interleukin-1β secretion. Thus, PYPAF2 and PYPAF3, together with PYNOD, constitute an anti-inflammatory subgroup of PYPAFs. Interleukin (IL) 1The abbreviations used are: IL-1β, interleukin-1β; Ipaf, IL-1-converting enzyme-protease-activating factor; CARD, caspase recruitment domain; Apaf, apoptotic protease-activating factor; PYPAF, PYRIN-containing Apaf-1-like protein; TNF, tumor necrosis factor; LRR, leucine-rich repeat; RIP-DD, receptor-interacting protein death domain; HA, human influenza virus hemagglutinin epitope; LPS, lipopolysaccharide; RT, reverse transcription; ELISA, enzyme-linked immunosorbent assay. -1β is produced upon microbial infection mainly from monocytes/macrophages and neutrophils. IL-1β plays a fundamental role in protecting animals against infectious agents by activating immune cells, inducing a variety of cytokines and acute phase proteins and acting as a pyrogen (1Dinarello C.A. N. Engl. J. Med. 1984; 311: 1413-1418Crossref PubMed Scopus (923) Google Scholar, 2Dinarello C.A. Int. J. Tissue React. 1992; 14: 65-75PubMed Google Scholar). On the other hand, excessive production of IL-1β induces septic shock and is fatal for animals. Therefore, the regulation of IL-1β production is an important issue in infectious diseases from both the pathophysiological and pharmacological points of view. However, the regulatory mechanisms of IL-1β production are poorly understood. IL-1β is produced as an inactive proprotein and is converted to the active mature protein through proteolytic cleavage by caspase-1. Recently, IL-1β-converting enzyme-protease-activating factor (Ipaf, also called caspase recruitment domain (CARD) 12), which has homology with apoptotic protease-activating factor (Apaf)-1 and promotes caspase-1-dependent IL-1β maturation, was discovered (3Poyet J.L. Srinivasula S.M. Tnani M. Razmara M. Fernandes-Alnemri T. Alnemri E.S. J. Biol. Chem. 2001; 276: 28309-28313Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar). More recently, several members of another subfamily of Apaf-1-like proteins having an N-terminal PYRIN domain (called PYRIN-containing Apaf-1-like proteins (PYPAFs) or NALPs), namely PYPAF1/cryopyrin, PYPAF5, PYPAF7, and NALP1, have been found to promote caspase-1-dependent IL-1β maturation in the presence of an adaptor molecule, ASC (4Wang L. Manji G.A. Grenier J.M. Al-Garawi A. Merriam S. Lora J.M. Geddes B.J. Briskin M. DiStefano P.S. Bertin J. J. Biol. Chem. 2002; 277: 29874-29880Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar, 5Grenier J.M. Wang L. Manji G.A. Huang W.J. Al-Garawi A. Kelly R. Carlson A. Merriam S. Lora J.M. Briskin M. DiStefano P.S. Bertin J. FEBS Lett. 2002; 530: 73-78Crossref PubMed Scopus (222) Google Scholar, 6Martinon F. Burns K. Tschopp J. Mol. Cell. 2002; 10: 417-426Abstract Full Text Full Text PDF PubMed Scopus (4307) Google Scholar). On the other hand, two other Apaf-1-like proteins, Nod1 and Nod2, were discovered to be cytoplasmic sensors for cell-invading microbes (7Inohara N. Nunez G. Nat. Rev. Immunol. 2003; 3: 371-382Crossref PubMed Scopus (870) Google Scholar). These findings shed light on how microbial infection induces the activation of caspase-1 and the secretion of mature IL-1β (7Inohara N. Nunez G. Nat. Rev. Immunol. 2003; 3: 371-382Crossref PubMed Scopus (870) Google Scholar, 8Tschopp J. Martinon F. Burns K. Nat. Rev. Mol. Cell. Biol. 2003; 4: 95-104Crossref PubMed Scopus (601) Google Scholar). On the other hand, little is known about the functions of other Apaf-1-like proteins in caspase-1 activation and/or IL-1β maturation. PYPAF2 has been contradictorily reported both to and not to promote caspase-1-dependent IL-1β secretion (5Grenier J.M. Wang L. Manji G.A. Huang W.J. Al-Garawi A. Kelly R. Carlson A. Merriam S. Lora J.M. Briskin M. DiStefano P.S. Bertin J. FEBS Lett. 2002; 530: 73-78Crossref PubMed Scopus (222) Google Scholar, 9Agostini L. Martinon F. Burns K. McDermott M.F. Hawkins P.N. Tschopp J. Immunity. 2004; 20: 319-325Abstract Full Text Full Text PDF PubMed Scopus (1402) Google Scholar). Recently, we discovered that PYNOD, a novel member of the PYPAFs, has the potential to inhibit caspase-1-dependent IL-1β secretion as well as ASC-mediated NF-κB activation (10Wang Y. Hasegawa M. Imamura R. Kinoshita T. Kondo C. Konaka K. Suda T. Int. Immunol. 2004; 16: 777-786Crossref PubMed Scopus (103) Google Scholar). This was the first example of a PYPAF member playing a negative regulatory role in IL-1β secretion. This finding prompted us to investigate whether other ill-characterized members of this family have activities similar to PYNOD. Here we investigated the effects of PYPAF2 and PYPAF3 expression on NF-κB activation and IL-1β secretion. These two proteins are highly homologous and likely to be products of a very recent gene duplication event. We found that PYPAF3 inhibits caspase-1-dependent IL-1β secretion but not ASC-mediated NF-κB, while PYPAF2 inhibits the latter but not the former. Further study suggested that PYPAF3 is a feedback regulator of caspase-1-dependent IL-1β secretion. Reagents—Recombinant mouse IL-1β and tumor necrosis factor (TNF)-α was purchased from PeproTech (London, UK) and Genzyme (Cambridge, MA), respectively. Lipopolysaccharide from Escherichia coli 055:B5 was purchased from Sigma. Plasmids—A cDNA encoding human PYPAF2 (IMAGE:2821428) was purchased from Open Biosystems (Huntsville, AL). A cDNA encoding human PYPAF3 was amplified by RT-PCR from human peripheral blood lymphocyte poly(A)(+) RNA. The cDNAs encoding PYPAF2, PYPAF3, and their leucine-rich repeat (LRR)-truncated mutants (PYPAF2dLRR (amino acids 1–683) and PYPAF3dLRR (amino acids 1–685), respectively) were cloned into the pEF-Bos mammalian expression vector (11Mizushima S. Nagata S. Nucleic Acids Res. 1990; 18: 5322Crossref PubMed Scopus (1499) Google Scholar). Plasmids expressing human PYPAF1, PYPAF1dLRR (amino acids 1–739), a dominant negative mutant of TRADD (amino acids 196–312 with alanine substitution at amino acids 296–299), procaspase-1, pro-IL-1β, ASC, MyD88, and receptor-interacting protein death domain (RIP-DD, amino acids 558–671) with or without an N-terminal FLAG-tag were described previously (10Wang Y. Hasegawa M. Imamura R. Kinoshita T. Kondo C. Konaka K. Suda T. Int. Immunol. 2004; 16: 777-786Crossref PubMed Scopus (103) Google Scholar). The human influenza virus hemagglutinin epitope (HA) was introduced as needed using pHA-EAK and pEAK-HA, which are pEAK8-derived mammalian expression vectors in which the sequence for HA is inserted at the N- and C-terminal side of the multicloning site, respectively. pEAK-HA was kindly provided by Dr. Hisashi Miyamori (Kanazawa University, Cancer Research Institute). Human Bcl-XL DNA was a kind gift from Dr. Shigeomi Shimizu (Osaka University Medical School), and it was subcloned into pHA-EAK. Cell Lines and Transfection—The HEK293, 293T, HepG2, ECV304, PK-1, SW480, K562, Jurkat, U937, and THP-1 cell lines were described previously (10Wang Y. Hasegawa M. Imamura R. Kinoshita T. Kondo C. Konaka K. Suda T. Int. Immunol. 2004; 16: 777-786Crossref PubMed Scopus (103) Google Scholar). The HeLa (epitheloid carcinoma) cell line was obtained from Dr. Nobuhiro Nakamura (Kanazawa University, Faculty of Pharmaceutical Science). The HL-60 (promyelocytic leukemia) cell line was obtained from Dr. Shinji Nakao (Kanazawa University Graduate School of Medical Science). For luciferase reporter assays and IL-1β secretion assays, HEK293 cells were plated onto 48-well plates at a density of 4 × 104 cells/well, and transfection was carried out 18 h later using linear polyethyleneimine (Mr about 25,000, Polysciences Inc., Warrington, PA) as described previously (12Boussif O. Lezoualc'h F. Zanta M.A. Mergny M.D. Scherman D. Demeneix B. Behr J.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7297-7301Crossref PubMed Scopus (5654) Google Scholar). For immunoprecipitation assays, 293T cells were plated onto 6-well plates at density of 5 × 105 cells/well, and transfection was carried out 18 h later using Lipofectamine Plus (Invitrogen). To generate stable transfectants, THP-1 cells were transfected with pHA-EAK-PYPAF3 by electroporation. After selection with puromycin for 4 weeks, stable transfectants expressing HA-tagged PYPAF3 were selected by Western blot analysis. Luciferase Reporter Assays for NF-κB Activity—HEK293 cells in 48-well plates were transfected with plasmids including 15 ng/well of pNF-κB-Luc (carrying a firefly luciferase cDNA driven by 5× NF-κB-binding sites; Stratagene, La Jolla, CA) and 3 ng/well of pRL-TK (carrying Renilla luciferase cDNA driven by the HSV-TK promoter; Promega, Madison, WI). The total amount of DNA in each transfection was kept constant (300 ng/well) by the addition of empty vector (pEF-BOS). After 24 h, cell lysates were prepared and the firefly, and Renilla luciferase activities were measured using the dual-luciferase reporter assay system (Promega). The relative luciferase activity was calculated as follows: relative luciferase activity = firefly luciferase activity/renilla luciferase activity. IL-1β Secretion Assays—HEK293 cells in 48-well plates were transfected with plasmids including those expressing pro-IL-1β (80 ng/well) and procaspase-1 (2 ng/well, unless otherwise specified). The total amount of DNA in each transfection was kept constant (300 ng/well) by the addition of empty vector (pEF-BOS). After 26 h, the culture supernatants were collected and subjected to ELISA for IL-1β using the Human IL-1β OptEIA ELISA Set (Pharmingen). Immunoprecipitation and Western Blot Analysis—Immunoprecipitation and Western blotting were carried out as previously described (10Wang Y. Hasegawa M. Imamura R. Kinoshita T. Kondo C. Konaka K. Suda T. Int. Immunol. 2004; 16: 777-786Crossref PubMed Scopus (103) Google Scholar), except that transfection and cell culture were performed in 6-well plates and that rabbit polyclonal and mouse monoclonal anti-HA antibodies (Sigma) were used for immunoprecipitation and Western blots, respectively. PYPAF-3-specific antisera were prepared from mice immunized with purified glutathione S-transferase-fused PYPAF-3 PYRIN domain (amino acids 1–93). Assays for mRNA Expression—Panels of first-strand cDNA from multiple human tissues (Human I and Human II) were purchased from Clontech (Palo Alto, CA). Total RNA from cultured cell lines was prepared using the RNeasy mini kit (Qiagen, Hilden, Germany). RT-PCR analysis was performed as described previously (13Fukui M. Imamura R. Umemura M. Kawabe T. Suda T. J. Immunol. 2003; 171: 1868-1874Crossref PubMed Scopus (45) Google Scholar). The primers used to detect human PYPAF2 mRNA were as follows: sense, 5′-gcaaaggatgaagtcagaga-3′; antisense, 5′-ccctccacccttatgtagat-3′, while those for PYPAF3 mRNA were as follows: sense, 5′-gcagtgggctgaattcttct-3′; antisense, 5′-gcctgacagagaatccacaa-3′. The amount of template cDNA was adjusted so that a similar amount of a PCR fragment of β-actin was generated within the linear range of the PCR. PYPAF2 Inhibits the NF-κB Activation Induced by the Combined Expression of PYPAF1 and ASC—We first investigated the effect of PYPAF2 and PYPAF3 expression on ASC-mediated NF-κB activation in HEK293 cells using luciferase reporter assays. Consistent with a previous report (14Manji G.A. Wang L. Geddes B.J. Brown M. Merriam S. Al-Garawi A. Mak S. Lora J.M. Briskin M. Jurman M. Cao J. DiStefano P.S. Bertin J. J. Biol. Chem. 2002; 277: 11570-11575Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar), PYPAF1 promoted this response, and truncation of the LRRs (PYPAF1dLRR) enhanced this activity (Fig. 1A). In contrast, PYPAF2 and PYPAF3 and even their LRR-truncated mutants failed to show such an activity (Fig. 1A). Instead, PYPAF2 dose-dependently inhibited the NF-κB activation induced by PYPAF1dLRR plus ASC (Fig. 1B). PYPAF3 showed little or no such activity. It was previously shown that PYPAF4 inhibits NF-κB activation induced by TNF-α or IL-1β (15Fiorentino L. Stehlik C. Oliveira A. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). In contrast, PYPAF2 not inhibit NF-κB while a dominant negative mutant of TRADD inhibited this response, as a (Fig. In this PYPAF2 is similar to (10Wang Y. Hasegawa M. Imamura R. Kinoshita T. Kondo C. Konaka K. Suda T. Int. Immunol. 2004; 16: 777-786Crossref PubMed Scopus (103) Google PYPAF3 Inhibits IL-1β we investigated whether PYPAF2 and PYPAF3 promotes caspase-1-dependent IL-1β secretion in the presence of ASC in HEK293 on whether or not PYPAF2 promotes this response (5Grenier J.M. Wang L. Manji G.A. Huang W.J. Al-Garawi A. Kelly R. Carlson A. Merriam S. Lora J.M. Briskin M. DiStefano P.S. Bertin J. FEBS Lett. 2002; 530: 73-78Crossref PubMed Scopus (222) Google Scholar, 9Agostini L. Martinon F. Burns K. McDermott M.F. Hawkins P.N. Tschopp J. Immunity. 2004; 20: 319-325Abstract Full Text Full Text PDF PubMed Scopus (1402) Google Scholar). In IL-1β secretion was not induced by PYPAF2, PYPAF3, or their of the presence or of ASC (Fig. and A and while it was induced by PYPAF1 and PYPAF1dLRR in the presence of ASC as reported previously (4Wang L. Manji G.A. Grenier J.M. Al-Garawi A. Merriam S. Lora J.M. Geddes B.J. Briskin M. DiStefano P.S. Bertin J. J. Biol. Chem. 2002; 277: 29874-29880Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar, Y. Hasegawa M. Imamura R. Kinoshita T. Kondo C. Konaka K. Suda T. Int. Immunol. 2004; 16: 777-786Crossref PubMed Scopus (103) Google Scholar). We investigated the effect of PYPAF2 or PYPAF3 expression on the caspase-1-dependent IL-1β secretion induced by PYPAF1dLRR plus ASC. In to of activity against ASC-mediated NF-κB activation described PYPAF3 and dose-dependently inhibited the IL-1β while PYPAF2, which inhibited the ASC-mediated NF-κB not (Fig. To the by which PYPAF3 inhibits caspase-1-dependent IL-1β we investigated the effect of PYPAF3 on the IL-1β secretion induced by caspase-1 In the of ASC and PYPAF1, caspase-1 induced IL-1β secretion dose-dependently (Fig. The expression of PYPAF3 but not PYPAF2 inhibited this response that PYPAF3 caspase-1 activation or PYPAF3 Inhibits of and how PYPAF3 inhibited the caspase-1-dependent IL-1β secretion in we investigated the of pro-IL-1β and procaspase-1 by Western blot analysis. C-terminal HA-tagged pro-IL-1β was with procaspase-1 in 293T these an anti-HA the mature of IL-1β (Fig. The expression of PYPAF3 (Fig. but not PYPAF2 (Fig. dose-dependently inhibited the of N-terminal procaspase-1 was in 293T cells to induce or these which to the N-terminal fragment the was by Western blotting using an (Fig. PYPAF3 and dose-dependently inhibited the of (Fig. that PYPAF3 inhibits procaspase-1 PYPAF2 inhibited this at a but PYPAF3 (Fig. PYPAF3 with Both and investigated whether are PYPAF3 and caspase-1 or These proteins were expressed in 293T cells, and the cell lysates were subjected to immunoprecipitation analysis. Immunoprecipitation of caspase-1 or IL-1β but not in the of PYPAF3 (Fig. A and In the immunoprecipitation of PYPAF3 but not Bcl-XL in the of caspase-1 and IL-1β (Fig. A and These results that PYPAF3 with both caspase-1 and caspase-1 or IL-1β was expressed in 293T cells, it was that caspase-1 or IL-1β with PYPAF3 through the other Therefore, We the expression of caspase-1 and IL-1β mRNA in 293T RT-PCR to caspase-1 cDNA was that caspase-1 is not expressed in this cell On the other hand, we a to IL-1β cDNA we investigated whether a amount of IL-1β is in 293T We not detect IL-1β protein in the culture supernatants or cell lysates from 293T cells using ELISA of the presence or of unless IL-1β was expressed by transfection Thus, the expression of IL-1β protein in 293T cells was very These results suggest that caspase-1 and IL-1β with PYPAF3 of each Expression of PYPAF2 and PYPAF3 expression of PYPAF2 and PYPAF3 mRNA in human tissues and cell lines was investigated by Both PYPAF2 and PYPAF3 mRNA were in tissues (Fig. However, skeletal expressed neither PYPAF2 PYPAF3 and heart and expressed of PYPAF2 and no PYPAF3 PYPAF2 and PYPAF3 mRNA were in many but not cell including hematopoietic and non-hematopoietic cell lines (Fig. However, the expression of these mRNAs in the cell lines were not well with each Lipopolysaccharide and IL-1β PYPAF3 Expression in THP-1 and investigated whether induce PYPAF3 expression in hematopoietic cell using HL-60 acute cell and THP-1 acute monocytic enhanced the expression of PYPAF3 but not PYPAF2 mRNA in THP-1 cells (Fig. was not in Jurkat, or IL-1β also induced PYPAF3 mRNA expression in THP-1 cells in a and (Fig. Furthermore, and IL-1β enhanced PYPAF3 mRNA expression in peripheral blood cells as by RT-PCR (Fig. These results suggest that of the IL-1β results in the of PYPAF3, which has the potential to inhibit IL-1β in PYPAF-3 Inhibits IL-1β Secretion from THP-1 to into the of the ability of PYPAF3 to inhibit IL-1β we investigated whether PYPAF3 inhibits the IL-1β production in THP-1 To this we stable transfectants expressing N-terminal HA-tagged Western blot analysis using anti-HA or antibodies failed to detect a protein to however, we a with anti-HA or antibodies in but not in transfectants (Fig. and not We of PYPAF3 transfectants, and of produced little or no IL-1β upon (Fig. two while transfectants empty vector produced a amount of IL-1β these These results that PYPAF3 inhibits the IL-1β production in THP-1 In this we that PYPAF3 inhibits caspase-1-dependent IL-1β while PYPAF2 inhibits ASC-mediated NF-κB We recently found that a novel PYPAF which we PYNOD, inhibits both of these activities (10Wang Y. Hasegawa M. Imamura R. Kinoshita T. Kondo C. Konaka K. Suda T. Int. Immunol. 2004; 16: 777-786Crossref PubMed Scopus (103) Google Scholar). In PYPAF4 was previously reported to inhibit the NF-κB activation induced by TNF-α or IL-1β (15Fiorentino L. Stehlik C. Oliveira A. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, these members to constitute an anti-inflammatory subgroup of PYPAFs, in to the which PYPAF1, PYPAF5, PYPAF7, and PYPAF3 with both caspase-1 and IL-1β and inhibited the of it is not whether PYPAF3 inhibit the of IL-1β by caspase-1. On the other hand, PYPAF2, PYNOD, inhibited the ASC-mediated but not NF-κB These results suggest that the mechanisms by which PYPAF2 and inhibit NF-κB activation are from that of which to and inhibits NF-κB activation induced by cytokines (15Fiorentino L. Stehlik C. Oliveira A. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). PYPAF2 and PYPAF3 are products of highly homologous that to each other on with a (7Inohara N. Nunez G. Nat. Rev. Immunol. 2003; 3: 371-382Crossref PubMed Scopus (870) Google Scholar, 8Tschopp J. Martinon F. Burns K. Nat. Rev. Mol. Cell. Biol. 2003; 4: 95-104Crossref PubMed Scopus (601) Google Scholar). In only to these two human has been found K. A. J.M. Stehlik C. L. J. S. R. S. F. G. A. Res. 2003; PubMed Scopus Google Scholar). Thus, it is likely that these two were generated as the of a very recent gene duplication event. inhibits both caspase-1-dependent IL-1β and ASC-mediated NF-κB and are for each of these Wang and T. it is that the gene for PYPAF2 and PYPAF3 a protein that both of these which have been by PYPAF2 and In this it be to whether the mouse protein these two human proteins has both This and PYPAF2 and PYPAF3 be for the of these PYPAF2 and PYPAF3 mRNAs were broadly expressed in with a expressed neither PYPAF2 PYPAF3, while heart and expressed PYPAF2 but not is mainly expressed in skeletal muscle, and (10Wang Y. Hasegawa M. Imamura R. Kinoshita T. Kondo C. Konaka K. Suda T. Int. Immunol. 2004; 16: 777-786Crossref PubMed Scopus (103) Google Scholar). Thus, it is likely that and similar functions in tissues and/or the expression of PYPAF2 and PYPAF3 their expression in a or cell line were not well with each Furthermore, induced the expression of PYPAF3 but not PYPAF2 mRNA in THP-1 These results suggest that the expression of these mRNAs is their on the we the N-terminal HA-tagged PYPAF3 protein expressed in stable transfectants of we detect only a which to the N-terminal fragment of PYPAF3 of the PYRIN domain and the that PYPAF3 is in these In we transfected HEK293 cells with a expressing the N-terminal of PYPAF3 to the fragment together with expression plasmids for caspase-1 and pro-IL-1β, it inhibited the IL-1β secretion but a expressing PYPAF3 not We are to the of PYPAF3 for activity. Stimulation by or IL-1β induced the expression of PYPAF3 mRNA in monocytic THP-1 Furthermore, stable transfectants of this cell line expressing PYPAF3 were in the IL-1β Therefore, it is likely that the PYPAF3 is a feedback regulator for IL-1β secretion. that an production of IL-1β is highly to such a feedback regulation be very important pathophysiological and are proteins that have been reported to with caspase-1 and inhibit caspase-1-dependent IL-1β secretion M.A. W.J. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, Stehlik C. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, M. Srinivasula S.M. Wang L. J.L. Geddes B.J. DiStefano P.S. Bertin J. Alnemri E.S. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). However, the for these proteins have been found in but not in mice K. A. J.M. Stehlik C. L. J. S. R. S. F. G. A. Res. 2003; PubMed Scopus Google Scholar). In contrast, PYPAF3 and have in mice (10Wang Y. Hasegawa M. Imamura R. Kinoshita T. Kondo C. Konaka K. Suda T. Int. Immunol. 2004; 16: 777-786Crossref PubMed Scopus (103) Google Scholar, K. A. J.M. Stehlik C. L. J. S. R. S. F. G. A. Res. 2003; PubMed Scopus Google Scholar). Therefore, these proteins fundamental as caspase-1 in This be investigated by in the We for and with