Cryopyrin and pyrin activate caspase-1, but not NF-kappaB, via ASC oligomerization.

Mutations in cryopyrin and pyrin proteins are responsible for several autoinflammatory disorders in humans, suggesting that these proteins play important roles in regulating inflammation. Using a HEK293 cell-based reconstitution system that stably expresses ASC and procaspase-1 we demonstrated that neither cryopyrin nor pyrin or their corresponding disease-associated mutants could significantly activate NF-kappaB in this system. However, both cryopyrin and two disease-associated cryopyrin mutants induced ASC oligomerization and ASC-dependent caspase-1 activation, with the disease-associated mutants being more potent than the wild-type (WT) cryopyrin, because of increased self-oligomerization. Contrary to the proposed anti-inflammatory activity of WT pyrin, our results demonstrated that pyrin, like cryopyrin, can also assemble an inflammasome complex with ASC and procaspase-1 leading to ASC oligomerization, caspase-1 activation and interleukin-1beta processing. Thus, we propose that pyrin could function as a proinflammatory molecule.

Expression of lamins depends on epidermal differentiation and transformation.

BACKGROUND: It has been suggested that A- and B-type lamins, proteins of the nuclear lamina, play important roles in the morphogenesis of the nucleus and cellular differentiation. OBJECTIVE: To investigate the expression of these nuclear proteins in normal skin and some keratinocytic tumours of the skin. METHODS: We examined by means of immunohistochemistry the expression of lamins in normal skin and some keratinocytic tumours of the skin, such as squamous cell carcinoma (SCC), basal cell carcinoma (BCC), Bowen's disease, solar keratosis, keratoacanthoma and seborrhoeic keratosis. RESULTS: In normal skin, A-type lamin was expressed in all epidermal cells, but the expression level of B-type lamins diminished from basal cells to granular cells. In keratinocytic tumours, the expression of A-type lamin was reduced, especially in BCCs, Bowen's disease and poorly differentiated SCCs. B-type lamins were reduced and exhibited heterogeneous expression patterns in most well-differentiated SCCs and keratoacanthomas. Antibodies against B-type lamins stained only peripheral cells of the lobules in keratoacanthomas, while no regular staining patterns were seen in well-differentiated SCCs. CONCLUSIONS: Lamin expression depends on the differentiation and transformation of the human skin. This finding should be useful for the diagnosis of keratinocytic tumours.

Calponin h1 induced a flattened morphology and suppressed the growth of human fibrosarcoma HT1080 cells.

Calponin h1 (CNh1) is an actin-binding protein that is expressed mainly in smooth muscle cells and is known to regulate smooth muscle contraction. Recently, re-expression of CNh1 in leiomyosarcoma cell lines is reported to suppress cell proliferation and tumorigenicity. However, little is known about the associated cellular structural and functional changes. Since CNh1 is also detected in normal fibroblasts, we hypothesised that CNh1 would also inhibit cell proliferation of the fibrosarcoma cells, HT1080, in which CNh1 is suppressed. An expression vector of human CNh1 complementary DNA was transfected into human HT1080 cells by a calcium-phosphate precipitation method. CNh1-transfected cells exhibited a flattened morphology with organised actin filaments, a significant decrease in cell motility and enhancement in adhesion to fibronectin in association with an increase in integrin alpha5beta1 expression. Anchorage-independent growth and tumorigenicity in nude mice were suppressed in the CNh1-transfected cells. Our results suggest that CNh1 may have a role as a tumour suppressor in human fibrosarcoma by influencing cytoskeletal activities.

Expression of apoptosis-associated speck-like protein containing a caspase recruitment domain, a pyrin N-terminal homology domain-containing protein, in normal human tissues.

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.

Characterization of cytosolic glutathione S-transferase in cultured astrocytes.

To elucidate the contribution of glutathione S-transferase (GST) and glutathione peroxidase (GPx) to the protection against oxidative stress in rat brain, we prepared GST and GPx from newborn rat liver, brain and cultured astrocytes, and investigated the characteristics and kinetics of the enzymes. The activity of cytosolic GST of the cultured astrocytes toward 1-chloro-2,4-dinitrobenzene (CDNB) was much higher than that of GPx toward peroxides. The GST activity toward 4-hydroxy-2-nonenal (4HNE) was almost the same as the GPx activity. GST isozymes were purified from the cytosolic fraction of the liver and astrocytes. In the case of the astrocytes, a major GST isozyme with an isoelectric point (pI) of 9.02 accounted for approximately 40% of total GST activity toward CDNB, while hepatic GST isozymes showed seven peaks in the basic region. Each of astrocytes and liver showed a single GST peak with high activity toward 4HNE, namely AVIII and LVIII, respectively, and both of them had a similar pI value of about 6.7. The kinetic parameters of AVIII and LVIII were found to be similar to each other. These data suggest that the same types of GST isozymes are expressed in the astrocytes and liver, and take part mainly in the detoxification of 4HNE.

Pyrin N-terminal homology domain- and caspase recruitment domain-dependent oligomerization of ASC.

ASC was first identified as a caspase recruitment domain (CARD)-containing proapoptotic molecule that forms insoluble aggregates during apoptosis. Here, we report both the pyrin N-terminal homology domain (PYD) and CARD domains are involved in the aggregation of ASC. Preliminary experiments indicated that overexpression of ASC formed filament-like aggregates in COS-7 cells. Expression experiments using green fluorescent protein (GFP) constructs showed that not only the GFP-ASC-CARD but also the GFP-ASC-PYD formed filament-like aggregates in COS-7 cells. We confirmed these filament-like aggregates of both the ASC-PYD and the ASC-CARD due to homophilic interaction by immunoprecipitation method. We also demonstrated that the ASC-PYD associated with the ASC-CARD by heterophilic interaction. These observations suggest that the dimerization of the PYD as well as the CARD plays an important role in the oligomerization of ASC as an adaptor molecule.

Murine ortholog of ASC, a CARD-containing protein, self-associates and exhibits restricted distribution in developing mouse embryos.

ASC (apoptosis-associated speck-like protein containing a CARD) was first identified as a cytosolic soluble protein that forms insoluble aggregates and enhances etoposide-induced apoptosis. We have cloned a murine ortholog of ASC (mASC) comprising 193 amino acids with a well-conserved pyrin N-terminal homology domain and caspase recruitment domain (CARD). mASC fused with green fluorescent protein appeared as a speck in transfected COS-7 cells and showed self-association. We analyzed mASC gene expression in developing embryos by in situ hybridization and found it to have a restricted distribution in mouse embryos. At E9.5, mASC was strongly expressed in the telencephalon, thalamic areas of the diencephalon, heart, and liver. Northern blotting analysis revealed that the mASC gene was expressed ubiquitously in multiple organs in adult mice. These findings indicate that mASC shows conservation of not only the primary structure of human ASC but also the ability to aggregate and has some similarity in its distribution to other CARD-containing molecules, including the apoptosis regulator Apaf-1.

The rabies virion-associated 100-kDa polypeptide (VAP100) is a host-derived minor component of the viral envelope.

We investigated a minor polypeptide component of 100-kDa detected in the rabies virion (referred to as VAP100) by using a monoclonal antibody (mAb), #16743, which was shown to recognize the SDS-denatured VAP100 antigen by immunoblot analyses. Although the VAP100 antigen was hardly detectable in the cell by usual immunoblot methods with this mAb, we could detect the antigen by a luminescent immunoblot method as well as by immunoprecipitation from the metabolically radiolabeled cell lysates and virions. Fluorescent antibody (FA) staining with mAb #16743 detected the uniformly distributed antigen on the formalin-fixed normal BHK-21 cells, while slight accumulation of the antigen was also seen in the Golgi area when the cells were permeabilized by treatment with Triton X-100 after fixation. Rabies virus infection induced alteration of the behavior of VAP100 to show a spotted distribution pattern in virus-infected cells. Double FA staining with mAb #16743 and rabbit antibody against the rabies virus envelope antigen demonstrated colocalized distribution of the viral envelope antigens and VAP100 in the cell. From these results, we think that VAP100 is a membrane-associated component of the cell, and its colocalized distribution with the viral envelope antigens in the cell implicates an intimate association of the VAP100 with viral envelope protein(s) and a reflection of possible involvement in the efficient incorporation of VAP100 into the virion.

La autoantigen is cleaved in the COOH terminus and loses the nuclear localization signal during apoptosis.

La autoantigen is a 47-kDa nuclear protein that binds to nascent polymerase III transcripts and a number of viral RNAs. We show that La protein was cleaved to generate a 43-kDa fragment during apoptosis of human leukemic HL-60 cells treated with camptothecin or etoposide. Immunofluorescence microscopy showed that the La protein level was increased in the cytoplasm during apoptosis of HL-60 cells. In addition, UV irradiation of HeLa cells led to the cleavage and redistribution of La protein upon apoptosis. Several lines of evidence show that La protein is cleaved by caspase-3 or closely related proteases at Asp-374 in the COOH terminus. When the full-length (La) and COOH-terminally truncated (La delta C374) forms of La protein were expressed as fusion proteins with green fluorescence protein (GFP), GFP-La delta C374 was predominantly cytoplasmic, whereas GFP-La was localized in the nucleus. These results suggest that La protein loses the nuclear localization signal residing in the COOH terminus upon cleavage and is thus redistributed to the cytoplasm during apoptosis.

ASC, a novel 22-kDa protein, aggregates during apoptosis of human promyelocytic leukemia HL-60 cells.

The cytoskeletal and/or nuclear matrix molecules responsible for morphological changes associated with apoptosis were identified using monoclonal antibodies (mAbs). We developed mAbs against Triton X-100-insoluble components of HL-60 cells pretreated with all-trans retinoic acid. In particular, one mAb recognized a 22-kDa protein that exhibited intriguing behavior by forming an aggregate and appearing as a speck during apoptosis induced by retinoic acid and other anti-tumor drugs. Cloning and sequencing of its cDNA revealed that this protein comprises 195 amino acids and that its C-terminal half has a caspase recruitment domain (CARD) motif, characteristic of numerous proteins involved in apoptotic signaling. We referred to this protein as ASC (apoptosis-associated speck-like protein containing a CARD). The ASC gene was mapped on chromosome 16p11.2-12. The antisense oligonucleotides of ASC were found to reduce the expression of ASC, and consequently, etoposide-mediated apoptosis of HL-60 cells was suppressed. Our results indicate that ASC is a novel member of the CARD-containing adaptor protein family.

Expression of moesin and its associated molecule CD44 in epithelial skin tumors.

Moesin, one of the ERM (ezrin; radixin; moesin) family members, is directly associated with the cytoplasmic domain of CD44, which is now thought to be related to the metastatic potential of tumor cells. Using immunohistochemistry we investigated the expression of moesin in normal epidermis and various kinds of epithelial skin tumors: squamous cell carcinoma, verrucous carcinoma, Bowen's disease, solar keratosis, keratoacanthoma, basal cell carcinoma, and extramammary Paget's disease. Normal skin showed positive epidermal staining for moesin with the exception of the stratum corneum. The expression of moesin varied with the type of skin tumor. In basal cell carcinoma, Bowen's disease, and extramammary Paget's disease, moesin expression was either faint or negative. In contrast to Bowen's disease, invasive squamous cell carcinoma showed more intense and heterogeneous staining of the cytoplasm and the cell membrane. Verrucous carcinoma was weakly positive, with a tendency for the moesin to be distributed in the cell membrane. The staining pattern of moesin varied among the different kinds of epithelial skin tumors, and its expression was generally similar to that of the standard form of CD44. These results suggest that moesin is closely inter-related with CD44 in epithelial skin cells as seen in other cellular systems, and that the variable pattern of moesin staining among the skin tumor cells could reflect expression disorders associated with the transformation.

Differential expression of moesin in cells of hematopoietic lineage and lymphatic systems.

Moesin is a member of the ERM family consisting of ezrin, radixin, and moesin. The protein is located in the plasma membrane similarly to ezrin and radixin, and is thought to regulate cellular movements and morphological changes. Using monoclonal antibody CR-22, the specificity of which against human moesin was confirmed by immunoprecipitation and western blotting analysis, we immunohistochemically stained various formalin-fixed and paraffin-embedded human tissues, in particular, clots of bone marrow and lymphatic tissues, to examine moesin expression in cells of hematopoietic lineage and lymphatic systems. In the bone marrow, moesin was expressed in myeloid cells, while little staining was detected in erythroid cells. Moesin was highly expressed in both the center and the periphery of mature megakaryocytes. In the lymphatic tissues, moesin was strongly expressed by T-lymphocytes in the paracortex. In the mantle zone, the periphery of the germinal center, moesin was expressed by small lymphocytes which were identified as B-lymphocytes. Furthermore, in areas of inflammation, moesin was expressed in both the center and the periphery of neutrophils, whereas in some neutrophils in distant areas, moesin was localized at the cellular periphery. These results suggest that differential expression of moesin in these cells is involved in their morphology and specialized functions.

Moesin and CD44 expression in cutaneous melanocytic tumours.

The ERM (ezrin, radixin and moesin) family members, located just beneath the plasma membranes, are thought to be involved in the association of action filaments with the plasma membrane. One of the family members, moesin, is reported to bind to CD44. Splice variants of CD44 are thought to be associated with tumour progression or differentiation. Our aim was to investigate immunohistochemically the expression of moesin together with CD44 on paraffin tissue sections of a series of melanocytic tumours. The material included 12 ordinary melanocytic naevi, six Spitz naevi, eight dysplastic naevi, six blue naevi, seven malignant melanomas in situ, 15 primary malignant melanomas, five metastatic melanomas to the skin and five lymph node metastases. In the normal skin and the melanocytic tumours the expression of moesin was largely similar to that of CD44 standard. Strong moesin staining was observed in benign melanocytic lesions and melanomas in situ. However, the expression was decreased in advanced malignant melanomas. The moesin labelling in melanoma cells was downregulated with the depth of dermal invasion. The immunoreactivity was also diminished in the skin metastases and the lymph node metastases of melanoma. These results suggest that in melanocytic tumours, the alternation in the expression of moesin may be involved in the progression of malignancy.

The use of sequence comparison to detect 'identities' in tRNA genes.

We have developed a computational method that detects 'identities' in tRNA genes by using principal component analysis to classify the sequences of bases in tRNA genes into groups of similar sequences and then comparing the distribution of sequences of bases, in order to extract characteristic bases that are conserved within a group but differ between groups. These classification and comparison procedures are applied recursively to classify the sequences into hierarchical groups, so that multiple levels of characteristic sites can be detected. By using this computational method, we were able to detect many characteristic sites in the T and D domains of tRNAs, as well as the characteristic sites that had already been detected experimentally. This suggests that bases not only in the contact regions but also in the elbow regions, which determine the structure and dynamics of the whole tRNA molecule, are important to the tRNA-aminoacyl tRNA synthetase recognition.

The 21-kDa polypeptide (VAP21) in the rabies virion is a CD99-related host cell protein.

In our monoclonal antibody (MAb) stocks prepared against the BHK-21 cell antigens, two (#11875 and 28276) recognized a 21-kDa polypeptide (referred to as VAP21) which is efficiently incorporated into the rabies virion. By using these MAbs, we isolated the cDNA clones that encoded a polypeptide of 144 amino acids from our BHK-21 cell cDNA library. Based on the following evidence, the cDNA was assumed to encode a full-length sequence of VAP21 antigen: i) expression of the cDNA in animal cells resulted in the production of a polypeptide recognized by the two MAbs, and its electrophoretic mobility was the same as that of authentic VAP21 antigen; and ii) immunization with the products from the cDNA-transformed E. coli cells raised specific antibodies in rabbits that recognized a 21-kDa polypeptide in the virion. From the deduced amino acid sequence, it is suggested that the VAP21 antigen has a molecular structure of type-I transmembrane protein containing characteristic proline-rich and glycine-rich regions in its ectodomain. Homology searches resulted in finding homologous sequences (totally about 40% homology) in the human MIC2 gene product (CD99; 32-kDa) of T lymphocytes. These results suggest that the VAP21 antigen in the rabies virion is a cellular CD99-related transmembrane protein.

Immunological studies of a 21 kDa cellular component efficiently incorporated into rabies virion grown in a BHK-21 cell culture.

To investigate cellular components incorporated into the rabies virion, monoclonal antibodies (MAbs) were screened based on their reactivity with additional virion components. Two of the MAbs we prepared recognized a virion-associated 21 kDa polypeptide (referred to as VAP21) from a BHK-21 cell. Since the MAbs precipitated the rabies virion and trypsin digestion eliminated the VAP21 antigen from the virion but alkaline treatment (pH 11) did not, VAP21 seems to be anchored into the viral envelope and exposed on the virion surface. Although quantitative immunoblot analyses indicated an apparently increased concentration of VAP21 in the virion, the ratio of the content of VAP21 to that of viral glycoprotein (G) was several times decreased as compared to the ratio of those in the cell. These data suggest that sorting of VAP21 occurs during the viral budding process on the cell but that it might be inefficient, probably due to a more intimate association of VAP21 with the viral envelope proteins. This assumption seems to be consistent with the results of immunofluorescence studies; that is, VAP21 displayed colocalized distribution with viral envelope antigens in the cell. From these results, it is suggested that VAP21 closely associates with the viral envelope proteins in the cell, and this association might cause passive but relatively efficient incorporation of VAP21 into the virion.

Glutathione efflux from cultured astrocytes.

The characteristics and kinetics of GSH efflux from the monolayer culture of rat astrocytes were investigated. GSH efflux was dependent on temperature, with a Q10 value of 2.0 between 37 and 25 degrees C. The GSH efflux rate showed a hyperbolic dependency on the intracellular GSH concentration. The data were fitted well to the Michaelis-Menten model, giving the following kinetic parameter values: Km = 127 nmol/mg of protein; Vmax = 0.39 nmol/min/mg of protein. p-Chloromercuribenzenesulfonic acid, a thiol-reactive agent impermeable to the cell membrane, lowered the GSH efflux rate by 25% without affecting the intracellular GSH content. These results suggest that a carrier is involved in the efflux of GSH. The GSH content of cultured astrocytes showed a marked increase when the cells were exposed to insults, such as sodium arsenite, cadmium chloride, and glucose/glucose oxidase that lead to the generation of hydrogen peroxide. The increase in GSH content was attributed to the induction of the cystine transport activity by the agents. Although the intracellular GSH concentration and GSH efflux were increased, the kinetics of GSH efflux were not affected by those agents that imposed the oxidative stress. Because the Km value is very large, it is suggested that astrocytes release GSH depending on their GSH concentration in a wide range.

Transgene expression in mammary glands of newborn rats.

Transgene expression in the mammary glands of newborn rats was studied to establish an early selection system for transgenic animals producing exogenous proteins in their milk during lactation. A fusion gene composed of the bovine alpha S1 casein gene promoter and the human growth hormone gene was microinjected into rat embryos. Transgenic lines that produced human growth hormone in their milk were established and used in this study. Immediately after birth, and without any hormone treatment, human growth hormone was found in the extracts of mammary glands from both male and female rats derived from the line secreting human growth hormone in their milk. The expression of the transgene in mammary glands of newborn rats was also detected by the presence of human growth hormone mRNA. Nontransgenic newborn rats did not express the human growth hormone gene in their mammary glands, while the mRNA for rat alpha casein, an endogenous milk protein, was found in all mammary glands from both transgenic and nontransgenic neonates. These results show that analyzing the expression of transgenes in the mammary glands of neonates is a valuable tool to select the desired transgenic animals and to shorten the selection schedules establishing the transgenic animals.

Induction of cystine transport activity in mouse peritoneal macrophages by bacterial lipopolysaccharide.

The transport of cystine has been investigated in mouse peritoneal macrophages cultured in vitro. The transport activity for cystine was very low in freshly isolated macrophages but was potently induced during culture in the presence of bacterial lipopolysaccharide (LPS) at concentrations as low as 0.1 ng/ml. The transport activity for cystine was enhanced when the cells were incubated with tumour necrosis factor-alpha (TNF-alpha), but not with interferon-gamma (IFN-gamma) or interleukin-1. IFN-gamma was rather repressive in the induction of the activity by LPS or TNF-alpha. The transport activity for cystine induced by LPS has been characterized. Cystine was transported mainly by Na(+)-independent system and the uptake of cystine was inhibited by extracellular glutamate and homocysteate, but not by aspartate, indicating that the transport of cystine in macrophages treated with LPS is mediated by System xc-. Glutathione content of the macrophages increased when they were exposed to LPS, and this increase was, at least in part, attributable to the induced activity of the cystine transport.

Studies on the antiviral activity of protein kinase inhibitors against the replication of vesicular stomatitis virus.

Several derivatives of K-252a, a protein kinase inhibitor isolated from Nocardiopsis sp., were investigated for their effects on the replication of vesicular stomatitis virus (VSV) in BHK-21 cell cultures. Among those we tested, KT5926, which preferentially inhibits the myosin light chain kinase (MLCK), suppressed the viral replication by 95-99% at 15 microM. K-252a, which inhibits a broad spectrum of cellular protein kinase, similarly affected the viral replication. Other derivatives, KT5720 and KT5823, that are known to inhibit the cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG), respectively, did not suppress VSV replication even at a high concentration as 15 microM. None of these inhibitors affected the Sindbis virus replication in BHK-21 cells under similar assay conditions as used for VSV. KT5926 and K-252a seemed to affect the VSV replication at the step(s) after the viral invasion, resulting in decreased viral RNA synthesis. Neither substance inhibited cellular casein kinase (CK) II which is known to be involved in phosphorylation of the nonstructural (NS) protein, a non-catalytic subunit of the viral RNA polymerase. These results suggest that the inhibition of VSV replication by KT5926 and K-252a is not a secondary effect due to generalized suppression of host cell activities, and that the VSV replication requires the KT5926-sensitive function(s) in the cell which would be performed by an enzyme(s) other than CK II.