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1.
Biochem Biophys Rep ; 27: 101041, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34189278

RESUMO

Coronin-1, a hematopoietic cell-specific actin-binding protein, is thought to be involved in the phagocytic process through its interaction with actin filaments. The dissociation of coronin-1 from phagosomes after its transient accumulation on the phagosome surface is associated with lysosomal fusion. We previously reported that 1) coronin-1 is phosphorylated by protein kinase C (PKC), 2) coronin-1 has two phosphorylation sites, Ser-2 and Thr-412, and 3) Thr-412 of coronin-1 is phosphorylated during phagocytosis. In this study, we examined which PKC isoform is responsible for the phosphorylation of coronin-1 at Thr-412 by using isotype-specific PKC inhibitors and small interfering RNAs (siRNAs). Thr-412 phosphorylation of coronin-1 was suppressed by Gö6976, an inhibitor of PKCα and PKCßI. This phosphorylation was attenuated by siRNA for PKCα, but not by siRNA for PKCß. Furthermore, Thr-412 of coronin-1 was phosphorylated by recombinant PKCα in vitro, but not by recombinant PKCß. We next examined the effects of Gö6976 on the intracellular distribution of coronin-1 in HL60 cells during phagocytosis. The confocal fluorescence microscopic observation showed that coronin-1 was not dissociated from phagosomes in Gö6976-treated cells. These results indicate that phosphorylation of coronin-1 at Thr-412 by PKCα regulates intracellular distribution during phagocytosis.

2.
Yakugaku Zasshi ; 141(4): 579-589, 2021.
Artigo em Japonês | MEDLINE | ID: mdl-33790123

RESUMO

Staphylococcus aureus is a common pathogen causing a wide range of infectious diseases in humans and animals. This bacterium secretes a variety of exoproteins, including toxins known as superantigens, such as toxic shock syndrome toxin-1 (TSST-1) and enterotoxins. Staphylococcal superantigen-like (SSL) proteins are a family of exoproteins showing structural similarities with superantigens but no superantigenic activity. This family is composed of 14 members (SSL1-SSL14), and recent studies have revealed that these members exhibit various immunomodulatory activities: e.g., inhibition of antibody and complement functions, impairment of leukocyte trafficking, modulation of receptor functions, inappropriate activation of immunocytes, and inhibition of blood coagulation. These activities have been proposed to contribute to immune evasion of the bacteria. The interactions between SSL proteins and their target molecules in the host immune system and the pathophysiological roles of SSL proteins in the bacterial infections are reviewed in this article.


Assuntos
Toxinas Bacterianas/imunologia , Enterotoxinas/imunologia , Exotoxinas/imunologia , Sistema Imunitário/imunologia , Imunomodulação , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologia , Staphylococcus aureus/metabolismo , Superantígenos/imunologia , Animais , Coagulação Sanguínea , Humanos , Leucócitos/imunologia , Camundongos
3.
Biol Pharm Bull ; 42(6): 937-943, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31155590

RESUMO

Hydroxyoctadecadienoic acids (HODEs) are generated by oxidation of linoleic acid in vivo and thought to mediate various pathophysiological responses. In this study, we examined the effects of HODEs on EL4 mouse lymphoma cell growth and found that 9-(E,Z)-HODE inhibited EL4 cell growth in a dose-dependent manner, whereas no such growth inhibition was observed with other isomers (9-(E,E)-, 13-(Z,E)-, or 13-(E,E)-HODE), suggesting that the growth-inhibitory effect of HODEs was stereospecific. Analysis by flow cytometry (FACS) with annexin V and propidium iodide (PI) staining showed that 9-(E,Z)-HODE induced apoptosis with G2/M phase arrest. We next examined the growth inhibition profile of 9-(E,Z)-HODE against a panel of 39 human cancer cell lines (JFCR39). The fingerprint of growth inhibition by 9-(E,Z)-HODE exhibited a high degree of similarity to that by MLN4924, an inhibitor of NEDD8-activating enzyme. The intracellular NEDD8 (ubiquitin-like protein) expression in EL4 cells was decreased by the treatment with 9-(E,Z)-HODE as assessed by immunoblotting and flow cytometry. In conclusion, 9-(E,Z)-HODE specifically induced G2/M phase arrest and apoptosis, and the decrease of NEDD8 expression might be involved in this effect.


Assuntos
Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Ácidos Graxos Insaturados/farmacologia , Linfoma/metabolismo , Animais , Linhagem Celular Tumoral , Ácidos Graxos Insaturados/química , Linfoma/patologia , Camundongos , Proteína NEDD8/metabolismo , Estereoisomerismo
4.
Biol Pharm Bull ; 42(6): 982-988, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31155595

RESUMO

Staphylococcus aureus produces a variety of exoproteins that interfere with host immune systems. We attempted to purify cytotoxins against human leukocytic cells from the culture supernatant of S. aureus by a combination of ammonium sulfate precipitation, ion-exchange chromatography on a CM-cellulose column and HPLC on a Mono S 5/50 column. A major protein possessing cytotoxicity to HL60 human promyelocytic leukemia cells was purified, and the protein was identified as α-hemolysin (Hla, α-toxin) based on its molecular weight (34 kDa) and N-terminal amino acid sequence. Flow cytometric analysis suggested differential cytotoxicity of Hla against different human peripheral blood leukocyte populations. After cell fractionation with density-gradient centrifugation, we found that peripheral blood mononuclear cells (PBMCs) were more susceptible to Hla than polymorphonuclear leukocytes. Moreover, cell surface marker analysis suggested that Hla exhibited slightly higher cytotoxicity against CD14-positive PBMCs (mainly monocytes) than CD3- or CD19-positive cells (T or B lymphocytes). From these results, we conclude that human leukocytes have different susceptibility to Hla depending on their cell lineages, and thereby the toxin may modulate the host immune response.


Assuntos
Toxinas Bacterianas/farmacologia , Proteínas Hemolisinas/farmacologia , Leucócitos/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Leucócitos/imunologia
5.
Biochem Biophys Res Commun ; 508(1): 263-269, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30497774

RESUMO

Staphylococcal α-hemolysin (Hla) is a principal small ß-barrel pore forming toxin. It targets a variety of mammalian cells including immune cells; however little is known about its effects on mast cells. In this study, we examined whether Hla affects the degranulation of mast cells. Although Hla bound to the surface of bone marrow-derived mast cells (BMMCs) and formed SDS-stable oligomers on the cells, Hla alone induced neither cytotoxicity nor obvious release of a granule enzyme, ß-hexosaminidase. However, Hla more than doubled the releases of ß-hexosaminidase from BMMCs induced by FcεRI cross-linking or treatment with ionomycin. The augmentation of the enzyme release by rHla was impaired in the presence of 130 mM of extracellular KCl. The mutants of Hla that lacked pore-formation did not augment the release of the enzyme. These findings demonstrate that Hla is able to enhance the degranulation of mast cells induced by FcεRI cross-linking and ionomycin, although it alone does not induce the degranulation, and the pore-formation of Hla followed by potassium efflux is involved in the augmentation. These findings propose a previously unrecognized role for Hla in S. aureus-associated allergic and inflammatory processes via augmentation of mast cell responses.


Assuntos
Toxinas Bacterianas/farmacologia , Degranulação Celular/efeitos dos fármacos , Reagentes de Ligações Cruzadas/farmacologia , Proteínas Hemolisinas/farmacologia , Ionomicina/farmacologia , Receptores de IgE/metabolismo , Staphylococcus aureus/química , Animais , Toxinas Bacterianas/química , Sobrevivência Celular/efeitos dos fármacos , Proteínas Hemolisinas/química , Mastócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
6.
Biochem Biophys Res Commun ; 508(4): 1162-1167, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30554660

RESUMO

The family of staphylococcal superantigen-like proteins (SSLs) have a structure similar to bacterial superantigens but exhibit no superantigenic activity. These exoproteins have recently been shown to disturb the host immune defense system. One family member, SSL5, was reported to bind to human leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) and matrix metalloproteinase-9 (MMP-9) and to interfere with leukocyte trafficking. In the present study, we explored human plasma proteins bound by glutathione S-transferase (GST)-tagged recombinant SSL5 (GST-SSL5) and identified plasma protease C1 inhibitor (C1Inh) as a major SSL5-binding protein based on the results of peptide mass fingerprinting analysis with MALDI-TOFMS. GST-SSL5 was found to attenuate the inhibitory activity of recombinant histidine-tagged C1Inh (C1Inh-His) toward complement C1s. We also observed that the treatment of C1Inh-His with neuraminidase markedly decreased its binding to GST-SSL5. Moreover, C1Inh-His produced by Lec2 mutant cells (deficient in sialic acid biosynthesis) showed much lower binding affinity for SSL5 than that produced by the wild-type CHO-K1 cells, as assessed by pull-down assay. These results suggest that SSL5 binds to C1Inh in a sialic acid-dependent fashion and modulates the host immune defense through perturbation of the complement system in association with S. aureus infection.


Assuntos
Proteínas de Bactérias/metabolismo , Proteína Inibidora do Complemento C1/metabolismo , Staphylococcus aureus/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Células HEK293 , Humanos , Ácido N-Acetilneuramínico/metabolismo , Polissacarídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes/metabolismo
7.
Int J Mol Sci ; 19(12)2018 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-30544870

RESUMO

It has recently been recognized that inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), upregulate the secretion of matrix metalloproteinase-9 (MMP-9) from cancer cells and thereby promote peritoneal dissemination. In this study, we found that TNF-α also stimulated peritoneal mesothelial cells to secrete MMP-9 as assessed by zymography. MMP-9 gene expression in mesothelial cells induced by TNF-α was confirmed by quantitative RT-PCR analysis. We then utilized the reconstituted artificial mesothelium, which was composed of a monolayer of mesothelial cells cultured on a Matrigel layer in a Boyden chamber system, to examine the effects of TNF-α on carcinoma cell invasion. The transmigration of MKN1 human gastric carcinoma cells through the reconstituted mesothelium was promoted by TNF-α in a dose-dependent manner. The increased MKN1 cell migration was partially inhibited by the anti-α3 integrin antibody, indicating that the invasion process involves an integrin-dependent mechanism. Finally, we observed that the invasion of MMP-9-knockdown MKN1 cells into Matrigel membranes was potentiated by the exogenous addition of purified proMMP-9. These results suggest that TNF-α-induced MMP-9 secretion from mesothelial cells plays an important role in the metastatic dissemination of gastric cancer.


Assuntos
Epitélio/patologia , Metaloproteinase 9 da Matriz/metabolismo , Peritônio/patologia , Neoplasias Gástricas/enzimologia , Neoplasias Gástricas/patologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Meios de Cultivo Condicionados/farmacologia , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Camundongos , Invasividade Neoplásica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
8.
Monoclon Antib Immunodiagn Immunother ; 37(5): 212-217, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30362929

RESUMO

Staphylococcus aureus secretes a family of exoproteins structurally homologous to bacterial superantigens, such as toxic shock syndrome toxin-1 (TSST-1), and those exoproteins are thus called staphylococcal superantigen-like proteins (SSLs). Recent studies have revealed that SSLs play roles in evasion of the host defense by disturbing host immune responses. We previously reported that staphylococcal superantigen-like protein 5 (SSL5; a member of the SSL family) inhibited matrix metalloproteinase-9 (MMP-9), which is crucial for leukocyte recruitment to sites of infection. In this study, we established a mouse hybridoma clone (30G5C) producing a monoclonal antibody specific for SSL5. In immunoblotting analysis using recombinant His-tagged SSL1 to SSL14 (His-SSLs), the antibody was found to specifically recognize SSL5 without crossreactivity with other His-SSLs. The antibody bound to the C-terminal region of SSL5 (ß-grasp domain), but did not interfere with the binding of SSL5 to MMP-9, suggesting that this antibody is useful for identification of SSL5-producing S. aureus and screening for inhibitors of the SSL5/MMP-9 complex formation.


Assuntos
Anticorpos Monoclonais/imunologia , Proteínas de Bactérias/imunologia , Staphylococcus aureus/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Toxinas Bacterianas/imunologia , Enterotoxinas/imunologia , Humanos , Camundongos , Ligação Proteica , Staphylococcus aureus/patogenicidade , Superantígenos/imunologia
9.
Biochem Biophys Res Commun ; 503(4): 3235-3241, 2018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-30149916

RESUMO

We investigated the fate of proheparanase added to the culture media of mast cells. A recombinant protein mimicking proheparanase was continuously internalized into mastocytoma cells as well as bone marrow- and peritoneal cell-derived mast cells. Internalized heparanase molecules were accumulated in granules and a significant portion was released by stimulation with ionomycin, indicating that the internalized heparanase was sorted into secretory granules. The pro-form heparanase was processed into a mature and an active form inside the cells, in which intracellular heparin was fragmented by the mature enzyme. The internalization was substantially inhibited by addition of heparin and heparan sulfate to the culture medium, suggesting that glycosaminoglycan is involved in the uptake pathway. Out of four syndecans, expression of syndecan-3 and syndecan-4, especially cell surface syndecan-4, was detected in the mastocytoma cells. Two knockdown clones transfected with a shRNA expression vector targeting the syndecan-4 gene took up significantly lower amounts of heparanase than mock cells. We propose that some exogenous substances like proheparanase can be incorporated into mast cell granules via a glycosaminoglycan-mediated, especially syndecan-4-dependent, uptake pathway.


Assuntos
Glucuronidase/metabolismo , Mastócitos/fisiologia , Sindecana-4/metabolismo , Animais , Degranulação Celular , Células Cultivadas , Endocitose , Glicosaminoglicanos/metabolismo , Heparina/metabolismo , Heparitina Sulfato/metabolismo , Mastócitos/citologia , Mastócitos/metabolismo , Camundongos , Transporte Proteico , Proteínas Recombinantes/metabolismo , Transdução de Sinais
10.
Biochem Biophys Res Commun ; 497(2): 713-718, 2018 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-29462623

RESUMO

Staphylococcal superantigen like 5 (SSL5) is an exotoxin produced by S. aureus and has a strong inhibitory effect on MMP-9 enzymatic activity. However, the mechanism of inhibition remains unclear. We sought to identify the responsible regions of SSL5 for the interaction with MMP-9 by comparing a series of domain swap and deletion mutants of SSL5. Binding analyses revealed that SSL5 had two regions for binding to MMP-9 catalytic domain, ß1-3 region (25SKELKNVTGY RYSKGGKHYL IFDKNRKFTR VQIFGK60) in N-terminal half and α4ß9 region (138KELDFKLRQY LIQNFDLYKK FPKDSKIKVI MKD170) in C-terminal half. The collagen binding domain and zinc-chelating histidine residues of MMP-9 were not essential for the specific binding to SSL5. The domain swap mutants of SSL5 that conserved ß1-3 but not α4ß9 region inhibited the gelatinolysis by MMP-9, and the mutant of SSL7 that substituted ß1-3 region to that of SSL5 acquired the binding and inhibitory activity. Furthermore, the polypeptide that harbored ß1-3 region of SSL5 inhibited gelatinolysis by MMP-9. Taken together, SSL5 inhibits the MMP9 activity through binding to the catalytic domain, and the ß1-3 region is responsible for the inhibition of proteolytic activity of MMP-9.


Assuntos
Proteínas de Bactérias/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Linhagem Celular , Humanos , Metaloproteinase 9 da Matriz/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/química
11.
Microbiol Immunol ; 62(3): 168-175, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29328525

RESUMO

Staphylococcal superantigen-like proteins (SSL) show no superantigenic activity but have recently been considered to act as immune suppressors. It was previously reported that SSL5 bound to P-selectin glycoprotein ligand-1 (PSGL-1) and matrix metalloproteinase (MMP)-9, leading to inhibition of leukocyte adhesion and invasion. These interactions were suggested to depend on sialic acid-containing glycans of MMP-9, but the roles of sialic acids in the interaction between SSL5 and MMP-9 are still controversial. In the present study, we prepared recombinant glutathione S-transferase-tagged SSL5 (GST-SSL5) and analyzed its binding capacity to MMP-9 by pull-down assay after various modifications of its carbohydrate moieties. We observed that GST-SSL5 specifically bound to MMP-9 from a human monocytic leukemia cell line (THP-1 cells) and inhibited its enzymatic activity in a concentration-dependent manner. After MMP-9 was treated with neuraminidase, its binding activity towards GST-SSL5 was markedly decreased. Furthermore, recombinant MMP-9 produced by sialic acid-deficient Lec2 mutant cells showed much lower affinity for SSL5 than that produced by wild-type CHO-K1 cells. Treatment of MMP-9 with PNGase F to remove N-glycan resulted in no significant change in the GST-SSL5/MMP-9 interaction. In contrast, the binding of GST-SSL5 to MMP-9 secreted from THP-1 cells cultured in the presence of an inhibitor for the biosynthesis of O-glycan (benzyl-GalNAc) was weaker than the binding of GST-SSL5 to MMP-9 secreted from untreated cells. These results strongly suggest the importance of the sialic acid-containing O-glycans of MMP-9 for the interaction of MMP-9 with GST-SSL5.


Assuntos
Proteínas de Bactérias/metabolismo , Ligação Competitiva , Metaloproteinase 9 da Matriz/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Polissacarídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Clonagem Molecular , DNA Bacteriano/genética , Ativação Enzimática , Ensaios Enzimáticos , Humanos , Evasão da Resposta Imune , Metaloproteinase 9 da Matriz/química , Glicoproteínas de Membrana/metabolismo , Ácido N-Acetilneuramínico/química , Neuraminidase , Polissacarídeos/química , Ligação Proteica , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo , Proteínas Recombinantes/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/imunologia , Superantígenos/genética , Superantígenos/metabolismo , Células THP-1
12.
Biochem Biophys Res Commun ; 485(1): 201-208, 2017 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-28193526

RESUMO

Staphylococcal superantigen-like proteins (SSLs) are a family of exoproteins of Staphylococcus aureus. We have shown that SSL10 binds to vitamin K-dependent coagulation factors and inhibits blood coagulation induced by recalcification of citrated plasma. SSL10 was revealed to bind to coagulation factors via their γ-carboxyglutamic acid (Gla) domain. In this study we attempted to identify the responsible sequence of SSL10 for the interaction with coagulation factors. We prepared a series of domain swap mutants between SSL10 and its paralog SSL7 that does not interact with coagulation factors, and examined their binding activity to immobilized prothrombin using ELISA-like binding assay. The domain swap mutants that contained SSL10ß1-ß3 (23MEMKN ISALK HGKNN LRFKF RGIKI QVL60) bound to immobilized prothrombin, and mutants that contained SSL10ß10-ß12 (174SFYNL DLRSK LKFKY MGEVI ESKQI KDIEV NLK207) also retained the binding activity. On the other hand, mutants that lacked these two regions did not bind to prothrombin. These sequences, each alone, bound to prothrombin as 33 amino acid length polypeptides. These results suggest that SSL10 has two responsible sequences for the binding to prothrombin. These prothrombin-binding peptides would contribute to the development of new anticoagulants.


Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Protrombina/metabolismo , Staphylococcus aureus/metabolismo , Sequência de Aminoácidos , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Humanos , Mutação , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/química , Staphylococcus aureus/genética
13.
PLoS One ; 11(9): e0162208, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27588757

RESUMO

We previously reported that mouse bone marrow-derived macrophages (BMDMs) that had been co-cultured with platelets exhibited lower susceptibility to bacterial lipopolysaccharide (LPS) and produced lower levels of nitric oxide (NO) and inflammatory cytokines including TNF-α and IL-6. The suppression of macrophage responses was mediated, at least in part, by platelet supernatant. In the present study, we assessed phenotypic changes of BMDMs induced by incubation with the supernatant from thrombin-activated platelets (PLT-sup) and found that BMDMs cultured with PLT-sup (PLT-BMDMs) expressed a lower level of inducible NO synthase (iNOS) and a higher level of arginase-1, both of which are involved in the L-arginine metabolism, upon stimulation with LPS or zymosan. We also examined possible modulation of the NF-κB signaling pathway and observed suppression of IκBα phosphorylation and a decrease of NF-κB p65 expression in LPS-stimulated PLT-BMDMs. These results suggest that PLT-sup suppresses inflammatory responses of BMDMs via negative regulation of NF-κB signaling leading to lowered expression of iNOS and enhanced L-arginine catabolism by arginase-1.


Assuntos
Arginase/metabolismo , Plaquetas/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Técnicas de Cocultura , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C
14.
World J Gastroenterol ; 22(8): 2503-11, 2016 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-26937138

RESUMO

AIM: To examine the expression of SphK1, an oncogenic kinase that produces sphingosine 1-phosphate (S1P), and its correlation with the expression of LPAR2, a major lysophosphatidic acid (LPA) receptor overexpressed in various cancers, in human colorectal cancer. METHODS: Real-time reverse-transcription polymerase chain reaction was used to measure the mRNA expression of SphK1, LPAR2, and the three major S1P receptors in 27 colorectal cancer samples and corresponding normal tissue samples. We also examined the correlation between the expression of SphK1 and LPAR2. RESULTS: Colorectal cancer tissue in 22 of 27 patients had higher levels of SphK1 mRNA than in normal tissue. In two-thirds of the samples, SphK1 mRNA expression was more than two-fold higher than in normal tissue. Consistent with previous reports, LPAR2 mRNA expression in 20 of 27 colorectal cancer tissue samples was higher compared to normal tissue samples. Expression profiles of all three major S1P receptors, S1PR1, S1PR2, and S1PR3, varied without any trend, with no significant difference in expression between cancer and normal tissues. A highly significant positive correlation was found between SphK1 and LPAR2 expression [Pearson's correlation coefficient (r) = 0.784 and P < 0.01]. The mRNA levels of SphK1 and LPAR2 did not correlate with TNM stage. CONCLUSION: Our findings suggest that S1P and LPA may play important roles in the development of colorectal cancer via the upregulation of SphK1 and LPAR2, both of which could serve as new therapeutic targets in the treatment of colorectal cancer.


Assuntos
Adenocarcinoma/genética , Neoplasias Colorretais/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Receptores de Ácidos Lisofosfatídicos/genética , Adenocarcinoma/enzimologia , Adenocarcinoma/patologia , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Lisoesfingolipídeo/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptores de Esfingosina-1-Fosfato , Regulação para Cima
15.
Artigo em Inglês | MEDLINE | ID: mdl-26788840

RESUMO

Integrins are a major family of adhesion molecules, consisting of heterodimers (α and ß subunits). Several reports have suggested the presence of splice variants in the cytoplasmic domain of certain integrin subunits. In the present study, we detected mRNA of integrin α3 splice variants (α3A and α3B) by RT-PCR using total RNA from the human brain as a template. The α3B variant lacks the sequence coded by exon 25 and appears to be generated by alternative splicing. We established mouse hybridomas producing monoclonal antibodies (both of which are of IgG1 class) specific for each variant. Each antibody exhibited specific reactivity towards the corresponding integrin α3 variant in Western blotting and immunoprecipitation experiments, suggesting it to be a useful tool for detection of the respective integrin variant.


Assuntos
Anticorpos Monoclonais/genética , Encéfalo/fisiologia , Integrina alfa3/imunologia , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Humanos , Hibridomas , Integrina alfa3/genética , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Subunidades Proteicas/genética , Subunidades Proteicas/imunologia
16.
Platelets ; 27(4): 344-50, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26588084

RESUMO

Considerable evidence has been accumulated concerning the roles of platelets in immune responses. In the present study, we examined the functional modulation of macrophages by platelets. When mouse bone marrow-derived macrophages (BMDMs) were co-cultured with platelets, BMDMs produced lower levels of nitric oxide (NO), tumor necrosis factor-α (TNF)-α, and interleukin (IL)-6 in response to a bacterial endotoxin (LPS) and zymosan. The attenuation in the macrophage susceptibility to LPS appeared to be mediated by soluble factors secreted from platelets. The mRNA levels of NOS2 (iNOS), TNF-α, and IL-6 in LPS-stimulated BMDMs that had been cultured with a conditioned medium of platelets were also decreased as analyzed by RT-qPCR. The ability of the platelet-conditioned medium to suppress macrophage NO production was recovered in a high-molecular-weight fraction (>670 kDa) after gel-filtration chromatography on a Superose 6 column. These results suggest that platelets control the susceptibility of macrophages to prevent excessive responses to LPS and provide mechanistic insight into a previous report that experimental thrombocytopenia aggravated organ failure in LPS-induced endotoxemia.


Assuntos
Plaquetas/metabolismo , Citocinas/biossíntese , Endotoxinas/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Óxido Nítrico/biossíntese , Animais , Linhagem Celular , Meios de Cultivo Condicionados/farmacologia , Humanos , Lipopolissacarídeos/imunologia , Ativação de Macrófagos/imunologia , Macrófagos/efeitos dos fármacos , Camundongos , Trombina/farmacologia , Zimosan/imunologia
17.
Pharm Res ; 31(10): 2868-75, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24792831

RESUMO

PURPOSE: NMSO3, a sulfated derivative of sialic acid, is a specific inhibitor for P-selectin (CD62P)-mediated cell adhesion. We attempted to apply liposomes modified with NMSO3 for selective targeting of activated platelets. METHODS: The binding of fluorescently labeled NMSO3-containing liposomes (NMSO3-liposomes) to CHO cells expressing P-selectin (CHO-P cells) and activated platelets were examined. The distribution of NMSO3-liposomes incorporated into the cells was observed by fluorescence microscopy. RESULTS: The binding assay revealed that NMSO3-liposomes specifically bound to immobilized P-selectin and CHO-P cells in a dose-dependent manner. The binding of NMSO3-liposomes to CHO-P cells was much stronger than that to the parental CHO-K1 cells. Fluorescence microscopic observation showed that NMSO3-liposomes were incorporated into CHO-P cells after the binding and distributed throughout the cytoplasm of the cell. NMSO3-liposomes bound more strongly to thrombin-activated platelets than to resting platelets, as assessed by flow cytometry. CONCLUSIONS: These results suggest that NMSO3-liposomes can be applied for selective drug delivery to activated platelets.


Assuntos
Plaquetas/efeitos dos fármacos , Portadores de Fármacos/química , Lipídeos/administração & dosagem , Ácido N-Acetilneuramínico/análogos & derivados , Nanoestruturas/química , Selectina-P/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Animais , Plaquetas/metabolismo , Células CHO , Adesão Celular/efeitos dos fármacos , Cricetulus , Citometria de Fluxo , Humanos , Lipídeos/farmacologia , Lipossomos , Ácido N-Acetilneuramínico/administração & dosagem , Ácido N-Acetilneuramínico/farmacologia , Selectina-P/genética , Ativação Plaquetária/efeitos dos fármacos , Transfecção
18.
Clin Exp Metastasis ; 31(3): 285-91, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24292405

RESUMO

Macrophages infiltrating tumor tissues (tumor-associated macrophages, TAM) affect the malignant behaviors of tumor cells. We previously reported that monocytes were differentiated into TAM-like cells secreting matrix metalloproteinase (MMP)-9 by co-culture with tumor cells, and that cell adhesion to extracellular matrix (ECM) proteins played a critical role in the differentiation. In this study, we found that the monocyte differentiation was promoted by laminin-332 (laminin-5), a major epithelial ECM component. We also demonstrated that the proteolytic processing of the γ2 chain of laminin-332 was essential for its activity but that the N-terminal short arm of the γ2 chain inhibited MMP-9 secretion. These results indicate that the activity of laminin-332 for monocyte differentiation is dynamically regulated by the proteolytic processing of the γ2 chain.


Assuntos
Moléculas de Adesão Celular/metabolismo , Diferenciação Celular/genética , Metaloproteinase 9 da Matriz/metabolismo , Monócitos/citologia , Adesão Celular , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Técnicas de Cocultura , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Humanos , Macrófagos/metabolismo , Metaloproteinase 9 da Matriz/genética , Neoplasias/genética , Neoplasias/patologia , Calinina
19.
J Biol Chem ; 288(30): 21569-80, 2013 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-23754290

RESUMO

The staphylococcal superantigen-like protein (SSL) family is composed of 14 exoproteins sharing structural similarity with superantigens but no superantigenic activity. Target proteins of four SSLs have been identified to be involved in host immune responses. However, the counterparts of other SSLs have been functionally uncharacterized. In this study, we have identified porcine plasma prothrombin as SSL10-binding protein by affinity purification using SSL10-conjugated Sepharose. The resin recovered the prodomain of prothrombin (fragment 1 + 2) as well as factor Xa in pull-down analysis. The equilibrium dissociation constant between SSL10 and prothrombin was 1.36 × 10(-7) M in surface plasmon resonance analysis. On the other hand, the resin failed to recover γ-carboxyglutamic acid (Gla) domain-less coagulation factors and prothrombin from warfarin-treated mice, suggesting that the Gla domain of the coagulation factors is essential for the interaction. SSL10 prolonged plasma clotting induced by the addition of Ca(2+) and factor Xa. SSL10 did not affect the protease activity of thrombin but inhibited the generation of thrombin activity in recalcified plasma. S. aureus produces coagulase that non-enzymatically activates prothrombin. SSL10 attenuated clotting induced by coagulase, but the inhibitory effect was weaker than that on physiological clotting, and SSL10 did not inhibit protease activity of staphylothrombin, the complex of prothrombin with coagulase. These results indicate that SSL10 inhibits blood coagulation by interfering with activation of coagulation cascade via binding to the Gla domain of coagulation factor but not by directly inhibiting thrombin activity. This is the first finding that the bacterial protein inhibits blood coagulation via targeting the Gla domain of coagulation factors.


Assuntos
Ácido 1-Carboxiglutâmico/imunologia , Proteínas de Bactérias/imunologia , Fator Xa/imunologia , Protrombina/imunologia , Staphylococcus aureus/imunologia , Superantígenos/imunologia , Ácido 1-Carboxiglutâmico/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/metabolismo , Sítios de Ligação/imunologia , Ligação Competitiva/imunologia , Coagulação Sanguínea/imunologia , Cálcio/imunologia , Cálcio/metabolismo , Coagulase/imunologia , Coagulase/metabolismo , Eletroforese em Gel de Poliacrilamida , Fator Xa/metabolismo , Humanos , Soros Imunes/imunologia , Soros Imunes/metabolismo , Camundongos , Dados de Sequência Molecular , Ligação Proteica/imunologia , Protrombina/metabolismo , Staphylococcus aureus/metabolismo , Superantígenos/metabolismo , Ressonância de Plasmônio de Superfície , Suínos , Trombina/imunologia , Trombina/metabolismo
20.
Biochem Biophys Res Commun ; 433(1): 127-32, 2013 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-23485472

RESUMO

Staphylococcal superantigen-like protein (SSL), a family of exotoxins composed of 14 SSLs, exhibits no superantigenic activity despite of its structural similarity with superantigens. Several SSLs have been revealed to bind to host immune molecules such as IgA, IgG, complement and cell surface molecules expressed on immune cells, but the physiological function of SSL family has not been fully identified. In this study we attempted to isolate host target proteins of SSLs from human breast milk using SSLs-conjugated Sepharose. SSL8-conjugated Sepharose specifically recovered tenascin C (TNC), a multimodular and multifunctional extracellular matrix protein. Pull down analysis using SSL8-conjugated Sepharose and recombinant truncated fragments of TNC revealed that SSL8 interacts with fibronectin (FN) type III repeats 1-5 of TNC. The interaction of TNC with immobilized FN was attenuated, the scratch wound closure by HaCaT human keratinocytes was delayed and the inhibition of cell spreading on FN by TNC was recovered in the presence of SSL8. These findings suggest that SSL8 binds to TNC, thereby inhibits the TNC-FN interaction and motility of keratinocytes. The present study added a novel role of SSL family protein as an interrupting molecule against the function of extracellular matrix.


Assuntos
Exotoxinas/metabolismo , Fibronectinas/metabolismo , Queratinócitos/fisiologia , Staphylococcus aureus/imunologia , Staphylococcus aureus/metabolismo , Superantígenos/metabolismo , Tenascina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular , Movimento Celular , Feminino , Fibronectinas/genética , Humanos , Queratinócitos/imunologia , Leite Humano/imunologia , Leite Humano/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Staphylococcus aureus/patogenicidade , Tenascina/genética , Cicatrização
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