ARTICLE |
Correspondence to: Junji Sagara, Dept. of Molecular Oncology and Angiology, Research Center on Aging and Adaptation, Shinshu U. School of Medicine, Asahi 3-1-1, Matsumoto 390-8621, Nagano, Japan.
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is a pyrin N-terminal homology domain (PYD)- and caspase recruitment domain (CARD)-containing a proapoptotic molecule. This molecule has also been identified as a target of methylation-induced silencing (TMS)-1. We cloned the ASC cDNA by immunoscreening using an anti-ASC monoclonal antibody. In this study, we determined the binding site of the anti-ASC monoclonal antibody on ASC and analyzed the expression of ASC in normal human tissues. ASC expression was observed in anterior horn cells of the spinal cord, trophoblasts of the placental villi, tubule epithelium of the kidney, seminiferous tubules and Leydig cells of the testis, hepatocytes and interlobular bile ducts of the liver, squamous epithelial cells of the tonsil and skin, hair follicle, sebaceous and eccrine glands of the skin, and peripheral blood leukocytes. In the colon, ASC was detected in mature epithelial cells facing the luminal side rather than immature cells located deeper in the crypts. These observations indicate that high levels of ASC are abundantly expressed in epithelial cells and leukocytes, which are involved in host defense against external pathogens and in well-differentiated cells, the proliferation of which is regulated.
(J Histochem Cytochem 49:12691275, 2001)
Key Words: ASC, TMS-1, tissue distribution, PYD, CARD, apoptosis, differentiation
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
APOPTOSIS plays important roles in regulating the growth and development of organisms and also mediates normal and neoplastic tissue growth by the removal of excess cells (
ASC, composed of an N-terminal pyrin N-terminal homology domain (PYD) and a C-terminal CARD, oligomerizes and enhances anti-cancer drug-induced apoptosis (
Although we initially cloned ASC cDNA by immunoscreening using an anti-ASC monoclonal antibody, which domain of ASC is recognized by the antibody has not been determined. In this study we determined the site on ASC recognized by the anti-ASC monoclonal antibody and analyzed the cellular distributions of ASC in various tissues to obtain information about the cells in which ASC functions. Here we report that ASC is differentially expressed in human tissues in a manner dependent on both maturation and cell type.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Construction of Expression Plasmids
The entire open reading frame of ASC was inserted into the EcoRI and SalI sites of pEGFP-C2 (CLONTECH; Palo Alto, CA) to produce pEGFP-ASC. Deletion mutants of pEGFP-ASC-1-37, pEGFP-ASC-
1-58, pEGFP-ASC-
1-100 (CARD), and pEGFP-ASC-
101-195 (PYD) were constructed by polymerase chain reaction (PCR) using including a
gt11 clone of the entire open reading frame of ASC (
Transfection, Expression of GFP-fused Proteins and Determination of the Binding Site of the Anti-ASC Monoclonal Antibody
Approximately 1 x 106 COS-7 cells were transfected with expression plasmids using LipofectAMINE-PLUS reagent (Life Technologies; Rockville, MD) according to the manufacturer's instructions. Localizations of GFP-ASC-WT, GFP-ASC-1-37, GFP-ASC-
1-58, GFP-ASC-
1-100 (CARD), and GFP-ASC-
101-195 (PYD) in transfected COS-7 cells were analyzed by immunofluorescence microscopy (Zeiss; Oberkochen, Germany). Transfected COS-7 cells were lysed with SDS sample buffer (
In Situ Hybridization of ASC Transcripts and Immunohistochemistry of ASC
To detect ASC transcripts in normal human placenta, in situ hybridization was carried out using a non-radioactively labeled RNA probe. Paraffin-embedded blocks of normal human placenta fixed for 48 hr in 20% buffered formalin (pH 7.4) were selected from the pathology files of the Central Clinical Laboratories, (Shinshu University Hospital, Matsumoto, Japan). An ASC-specific nucleotide sequence (nucleotides 101250) was amplified by PCR using upstream primer (5'-GCTCTAGACGACGCCATCCTGGATGCGC-3') and 3'-primers (5'-GGGGTACCTTGTCGGTGAGGTCCAAGGC-3'), where the Xba I and Kpn I sites are underlined. This amplified DNA fragment was cloned into the Xba I and Kpn I sites of pGEM-3Zf (+) (Promega; Madison, WI), and the resultant vector was used for construction of the RNA probe. A digoxigenin-labeled antisense RNA probe was obtained using Xba I-digested template and T7 RNA polymerase with DIG RNA labeling mixture (Roche Molecular Biochemicals; Mannheim, Germany). Similarly, a sense probe was prepared for negative control experiments using a Kpn I-digested template and SP6 RNA polymerase with the DIG RNA labeling mixture, and ISH was performed as described previously (
Isolation of Polymorphonuclear Leukocytes, Monocytes, T-lymphocytes, and B-lymphocytes from Human Peripheral Blood
Human peripheral leukocytes obtained from ourselves and our colleagues with their informed consent were isolated by FicollPaque (Amersham Pharmacia Biotech; Uppsala, Sweden) centrifugation of heparinized peripheral blood under conditions described previously (
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Determination of the Epitope for the Anti-ASC Monoclonal Antibody
Several deletion mutants of ASC or intact ASC fused with GFP (Fig 1A) were overexpressed in COS-7 cells, and the GFP-fusion proteins were detected by Western blotting using an anti-ASC MAb or an anti-GFP MAb (Fig 1B). Then, both GFP-ASC-WT and GFP-ASC-101-195 (PYD) were detected using the anti-ASC MAb, whereas GFP-ASC-
1-37, GFP-ASC-
1-58, and GFP-ASC-
1-100 (CARD) were not detected (Fig 1B, left panel). Expression of GFP constructs was also confirmed by Western blotting using the anti-GFP MAb (Fig 1B, right panel).
|
Subcellular Localizations of the Deletion Mutants of ASC Fused to GFP Were Analyzed by Fluorescence Microscopy
The subcellular localizations of deletion mutants of ASC presented in Fig 1A were examined. GFP-ASC-WT appeared as perinuclear speck-like aggregates in the transfected COS-7 cells (Fig 2Aa2Ac). GFP-ASC-1-37 (data not shown), GFP-ASC-
1-58 (data not shown), GFP-ASC-
1-100 (CARD) (Fig 2Ba2Bc), and GFP-ASC-
101-195 (PYD) (Fig 2Ca2Cc) appeared as filament-like aggregates in the transfected COS-7 cells. GFP, used as a control, was diffusely localized in the cytoplasm in transfected COS-7 cells (Fig 2Da2Dc).
ASC Expression in the Human Placenta Was Identified by ISH Analysis and IHC
Because we had no data about the distribution of ASC in individual cells, we chose the placenta because it includes a mixture of various types of cells. We analyzed ASC expression in the placenta by two independent experimental methods. We examined whether the data regarding ASC expression obtained by histochemical analysis using the anti-ASC MAb were the same as those for ASC gene expression determined by ISH analysis with an ASC-specific DNA probe (Fig 3A). By ISH, definite signals for ASC mRNA were demonstrated in syncytiotrophoblasts and cytotrophoblasts (Fig 3Ba3Bc), in which ASC protein was actually detected by IHC with the anti-ASC MAb (Fig 3Ca3Cc). These results established that the placental trophoblasts lining chorionic villi synthesize ASC and that the anti-ASC MAb specifically recognizes this particular protein.
ASC Expression in Normal Human Tissues Was Demonstrated by IHC
We examined the tissue distribution of ASC in normal human tissues using the anti-ASC MAb in paraffin-embedded sections from a variety of tissues. ASC was detected in a variety of cells, including those of the placenta, anterior horn cells of the spinal cord (Fig 4Aa4Ac), renal tubules of the kidney (Fig 4Ba4Bc), seminiferous tubules and Leydig cells of the testis (Fig 4Ca4Cc), hepatocytes and interlobular bile ducts of the liver (Fig 4Da4Dd), squamous epithelium of the tonsil (Fig 5Aa5Ac) and skin (Fig 5Ba5Bc), hair follicles, sebaceous and eccrine glands of the skin (Fig 5Ba5Bc), epithelial cells in the colon (Fig 6Aa6Ac), and peripheral blood leukocytes (data not shown). No significant ASC expression was detected in the ciliated epithelium of the trachea (data not shown), glomeruli of the kidney (Fig 4Ba), cardiac muscle, alveolar epithelium of the lung, or lymphocytes (the lymphatic follicle of the tonsil is shown in Fig 5Ab). The results are summarized in Table 1. We also observed that ASC appeared as speck-like aggregates in peripheral blood leukocytes separated and incubated for 24 hr (data not shown) similar to those observed in apoptotic cells (
|
|
ASC Expression in Normal Human Tissues Was Analyzed by Western Blotting
We also examined the expression of ASC using the anti-ASC MAb in normal human tissues from frozen samples and in CD14-positive monocytes, CD3-positive T-lymphocytes, CD20-positive B-lymphocytes, and polymorphonuclear leukocytes (PMNs) separated by cell sorting. ASC expression was detected in the spleen, small intestine, and colon. A trace amount of ASC expression was detected in the kidney (Fig 7A). In peripheral blood, a high level of ASC expression was detected in CD14-positive monocytes, and low levels were detected in PMNs and CD3-positive T-lymphocytes (Fig 7B).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Anti-ASC MAb Bound to PYD of ASC
The anti-ASC MAb utilized in this study was used for the initial immunoscreening to identify ASC (
Is ASC a Possible Key Adaptor Molecule Between PYD-containing Proteins and CARD-containing Proteins?
The speck-like aggregation of GFP-ASC-WT was different from the filament-like aggregation of GFP-ASC-1-100 (CARD) or GFP-ASC-
101-195 (PYD). This was believed to be due to an intramolecular heterophilic interaction between PYD and CARD, which is involved in the oligomerization of ASC (
Protein Expression of ASC Was Detected in the Epithelial Surface and in Some Differentiated Functional Cells for Which Proliferation is Regulated
The expression patterns of ASC associated with differentiation in squamous epithelium in the skin (Fig 5Ba5Bc), tonsil (Fig 5Aa5Ac), and intestinal mucosal epithelium (Fig 6Aa6Ac), were very similar (
We previously reported high levels of ASC mRNA expression in the spleen, which contains large numbers of lymphocytes (
Recently, ASC was independently identified as TMS-1 and was shown to confer lack of ASC expression through methylation-mediated silencing, a survival advantage (
![]() |
Acknowledgments |
---|
Supported by a Grant-in-Aid 12670109 from the Ministry of Education, Science and Culture, Japan.
We thank Drs Hiroshi Zenda and Shin Ohta (Department of Pharmacy, Shinshu University Hospital) for encouragement during this work and Masanobu Momose (Central Clinical Laboratory, Shinshu University Hospital) for technical assistance.
Received for publication January 29, 2001; accepted May 16, 2001.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arends MJ, Wyllie AH (1991) Apoptosis: mechanisms and roles in pathology. Int Rev Exp Pathol 32:223-254[Medline]
Berkow RL, Tzeng DY, Williams LV, Baehner RL (1983) The comparative responses of human polymorphonuclear leukocytes obtained by counterflow centrifugal elutriation and Ficoll-Hypaque density centrifugation. I. Resting volume, stimulus-induced superoxide production, and primary and specific granule release. J Lab Clin Med 102:732-742[Medline]
Bertin J, DiStefano PS (2000) The PYRIN domain: a novel motif found in apoptosis and inflammation proteins. Cell Death Differ 7:1273-1274[Medline]
Chinnaiyan AM, O'Rourke K, Tewari M, Dixit VM (1995) FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis. Cell 81:505-512[Medline]
Chu ZL, Pio F, Xie Z, Welsh K, Krajewska M, Krajewski S, Godzik A, Reed JC (2001) A novel enhancer of the Apaf1 apoptosome involved in cytochrome c-dependent caspase activation and apoptosis. J Biol Chem 276:9239-9245
Conway KE, McConnell BB, Bowring CE, Donald CD, Warren ST, Vertino PM (2000) TMS1, a novel proapoptotic caspase recruitment domain protein, is a target of methylation-induced gene silencing in human breast cancers. Cancer Res 60:6236-6242
Feinstein E, Kimchi A, Wallach D, Boldin M, Varfolomeev E (1995) The death domain: a module shared by proteins with diverse cellular functions. Trends Biochem Sci 20:342-344[Medline]
Hall PA, Coates PJ, Ansari B, Hopwood D (1994) Regulation of cell number in the mammalian gastrointestinal tract: the importance of apoptosis. J Cell Sci 107:3569-3577
Hlaing T, Guo RF, Dilley KA, Loussia JM, Morrish TA, Shi MM, Vincenz C, Ward PA (2001) Molecular cloning and characterization of DEFCAP-L and -S, two isoforms of a novel member of the mammalian Ced-4 family of apoptosis proteins. J Biol Chem 276:9230-9238
Hofmann K (1999) The modular nature of apoptotic signaling proteins. Cell Mol Life Sci 55:1113-1128[Medline]
Hofmann K, Bucher P, Tschopp J (1997) The CARD domain: a new apoptotic signalling motif. Trends Biochem Sci 22:155-156[Medline]
Inohara N, Ogura Y, Chen FF, Muto A, Núñez G (2001) Human Nod1 confers responsiveness to bacterial lipopolysaccharides. J Biol Chem 276:2551-2554
Inohara N, Núñez G (2000) Genes with homology to mammalian apoptosis regulators identified in zebrafish. Cell Death Differ 7:509-510[Medline]
Kawakami M, Nakayama J (1997) Enhanced expression of prostate-specific membrane antigen gene in prostate cancer as revealed by in situ hybridization. Cancer Res 57:2321-2324[Abstract]
Kerr JF, Wyllie AH, Currie AR (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26:239-257[Medline]
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685[Medline]
Martinon F, Hofmann K, Tschopp J (2001) The pyrin domain: a possible member of the death domain-fold family implicated in apoptosis and inflammation. Curr Biol 11:R118-120[Medline]
Masumoto J, Taniguchi S, Ayukawa K, Sarvotham H, Kishino T, Niikawa N, Hidaka E, Katsuyama T, Higuchi T, Sagara J (1999) ASC, a novel 22-kDa protein, aggregates during apoptosis of human promyelocytic leukemia HL-60 cells. J Biol Chem 274:33835-33838
Masumoto J, Taniguchi S, Nakayama K, Ayukawa K, Sagara J (2001a) Murine ortholog of ASC, a CARD-containing protein, self-associates and exhibits restricted distribution in developing mouse embryos. Exp Cell Res 262:128-133[Medline]
Masumoto J, Taniguchi S, Sagara J (2001b) Pyrin N-terminal homology domain- and caspase recruitment domain-dependent oligomerization of ASC. Biochem Biophys Res Commun 280:652-655[Medline]
McConnell BB, Vertino PM (2000) Activation of a caspase-9-mediated apoptotic pathway by subcellular redistribution of the novel caspase recruitment domain protein TMS1. Cancer Res 60:6243-6247
Núñez G, Benedict MA, Hu Y, Inohara N (1998) Caspases: the proteases of the apoptotic pathway. Oncogene 17:3237-3245[Medline]
Ogura Y, Inohara N, Benito A, Chen FF, Yamaoka S, Nunez G (2001) Nod2, a Nod1/Apaf-1 family member that is restricted to monocytes and activates NFB. J Biol Chem 276:4812-4818
Pawlowski K, Pio F, Chu Z, Reed JC, Godzik A (2001) PAADa new protein domain associated with apoptosis, cancer and autoimmune diseases. Trends Biochem Sci 26:85-87[Medline]
Staub E, Dahl E, Rosenthal A (2001) The DAPIN family: a novel domain links apoptotic and interferon response proteins. Trends Biochem Sci 26:83-85[Medline]