RESUMO
Herpes simplex virus 1 (HSV-1) causes a number of clinical manifestations including cold sores, keratitis, meningitis and encephalitis. Although current drugs are available to treat HSV-1 infection, they can cause side effects such as nephrotoxicity. Moreover, owing to the emergence of drug-resistant HSV-1 strains, new anti-HSV-1 compounds are needed. Because many viruses exploit cellular host proteases and encode their own viral proteases for survival, we investigated the inhibitory effects of a panel of protease inhibitors (TLCK, TPCK, E64, bortezomib, or MG132) on HSV-1 replication and several host cell signaling pathways. We found that HSV-1 infection suppressed c-Raf-MEK1/2-ERK1/2-p90RSK signaling in host cells, which facilitated viral replication. The mechanism by which HSV-1 inhibited ERK signaling was mediated through the polyubiquitination and proteasomal degradation of Ras-guanine nucleotide-releasing factor 2 (Ras-GRF2). Importantly, the proteasome inhibitor MG132 inhibited HSV-1 replication by reversing ERK suppression in infected cells, inhibiting lytic genes (ICP5, ICP27 and UL42) expression, and overcoming the downregulation of Ras-GRF2. These results indicate that the suppression of ERK signaling via proteasomal degradation of Ras-GRF2 is necessary for HSV-1 infection and replication. Given that ERK activation by MG132 exhibits anti-HSV-1 activity, these results suggest that the proteasome inhibitor could serve as a novel therapeutic agent against HSV-1 infection.
Assuntos
Antivirais/farmacologia , Herpesvirus Humano 1/efeitos dos fármacos , Leupeptinas/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Animais , Bortezomib/farmacologia , Caspases/metabolismo , Chlorocebus aethiops , Replicação do DNA/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Leucina/análogos & derivados , Leucina/farmacologia , Modelos Biológicos , Inibidor de NF-kappaB alfa/metabolismo , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Poliubiquitina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Estabilidade Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Tosilina Clorometil Cetona/farmacologia , Tosilfenilalanil Clorometil Cetona/farmacologia , Células Vero , Replicação Viral/efeitos dos fármacosRESUMO
Candida parapsilosis sensu stricto (C. parapsilosis) has emerged as the second/third commonest Candida species isolated from hospitals worldwide. Candida spp. possess numerous virulence attributes, including peptidases that play multiple roles in both physiological and pathological events. So, fungal peptidases are valid targets for new drugs development. With this premise in mind, we have evaluated the effect of serine peptidase inhibitors (SPIs) on both cell biology and virulence aspects of C. parapsilosis. First, five different SPIs, phenylmethylsulfonyl fluoride, benzamidine, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, N-α-tosyl-L-lysine chloromethyl ketone hydrochloride, and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) were tested, and TPCK showed the best efficacy to arrest fungal growth. Subsequently, the ability of TPCK to modulate physiopathological processes was investigated. Overall, TPCK was able to (i) inhibit the cell-associated serine peptidase activities, (ii) promote morphometric and ultrastructural alterations, (iii) induce an increase in the intracellular oxidation level, which culminates in a vigorous lipid peroxidation and accumulation of neutral lipids in cytoplasmic inclusions, (iv) modulate the expression/exposition of surface structures, such as mannose/glucose-rich glycoconjugates, N-acetylglucosamine-containing molecules, chitin, polypeptides and surface aspartic peptidases, (v) reduce the adhesion to either polystyrene or glass surfaces as well as to partially disarticulate the mature biofilm, (vi) block the fungal interaction with macrophages, and (vii) protect Galleria mellonella from fungal infection, enhancing larvae survivability. Altogether, these results demonstrated that TPCK induced several changes over fungal biology besides the interference with aspects associated to C. parapsilosis virulence and pathogenesis, which indicates that SPIs could be novel promising therapeutic agents in dealing with candidiasis.
Assuntos
Antifúngicos/farmacologia , Candida parapsilosis/efeitos dos fármacos , Candidíase/prevenção & controle , Inibidores de Serina Proteinase/farmacologia , Tosilfenilalanil Clorometil Cetona/farmacologia , Animais , Antifúngicos/administração & dosagem , Candida parapsilosis/citologia , Candida parapsilosis/crescimento & desenvolvimento , Adesão Celular/efeitos dos fármacos , Modelos Animais de Doenças , Larva/microbiologia , Lepidópteros/microbiologia , Estresse Oxidativo , Inibidores de Serina Proteinase/administração & dosagem , Análise de Sobrevida , Tosilfenilalanil Clorometil Cetona/administração & dosagem , Resultado do Tratamento , Virulência/efeitos dos fármacosRESUMO
Laquinimod is an immunomodulatory compound that has shown neuroprotective benefits in clinical trials for multiple sclerosis. Laquinimod ameliorates both white and gray matter damage in human patients, and prevents axonal degeneration in animal models of multiple sclerosis. Axonal damage and white matter loss are a common feature shared between different neurodegenerative diseases. Caspase-6 activation plays an important role in axonal degeneration on the molecular level. Increased activity of caspase-6 has been demonstrated in brain tissue from presymptomatic Huntington disease mutation carriers, and it is an early marker of axonal dysfunction. Since laquinimod is currently undergoing a clinical trial in Huntington disease (LEGATO-HD, clinicaltrials.gov ID: NCT02215616), we set out to evaluate its impact on neuronal caspase-6 activation. We find that laquinimod ameliorates DNA-damage induced activation of caspase-6 in primary neuronal cultures. This is an indirect effect that is not mediated by direct inhibition of the enzyme. The investigation of potential caspase-6 activating mechanisms revealed that laquinimod reduces the expression of Bax, a pro-apoptotic molecule that causes mitochondrial cytochrome c release and caspase activation. Bax expression is furthermore increased in striatal tissues from the YAC128 mouse model of HD in an age-dependent manner. Our results demonstrate that laquinimod can directly downregulate neuronal apoptosis pathways relevant for axonal degeneration in addition to its known effects on astrocytes and microglia in the CNS. It targets a pathway that is relevant for the pathogenesis of HD, supporting the hypothesis that laquinimod may provide clinical benefit.
Assuntos
Caspase 6/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Quinolonas/farmacologia , Proteína X Associada a bcl-2/metabolismo , Animais , Antineoplásicos Fitogênicos/farmacologia , Células COS , Camptotecina/farmacologia , Córtex Cerebral/citologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Síndrome de Down/genética , Síndrome de Down/patologia , Humanos , Proteína Huntingtina/genética , Camundongos , Camundongos Transgênicos , Mutação/genética , Inibidores da Síntese de Proteínas/farmacologia , Fatores de Tempo , Tosilfenilalanil Clorometil Cetona/análogos & derivados , Tosilfenilalanil Clorometil Cetona/farmacologia , Proteína X Associada a bcl-2/genéticaRESUMO
Cigarette smoking is one of the most important risk factors for the development of various diseases. Nicotine is the most extensively investigated component of cigarette smoke, and a comprehensive analysis of the genes induced by nicotine stimulation revealed that interleukin-8 (IL-8) was induced in oral squamous cell carcinoma cell (OSCC). Based on this background, the signaling mechanisms of nicotine-mediated IL-8 induction in OSCC was investigated. Augmented IL-8 secretion by Ca9-22 cells was blocked by the NF-κB inhibitor L-1-4'-tosylamino-phenylethyl-chloromethyl ketone (TPCK) and the nicotinic acetylcholine receptor (nAChR)-specific inhibitor α-bungarotoxin (αBtx). The downstream signaling pathway was further examined by pre-incubating the cells with inhibitors against mitogen-activated protein kinase (MEK), protein kinase C (PKC), and Ca(2+)/calmodulin-dependent kinase II (CaMK II). Only the CaMK II inhibitor was found to exert an inhibitory effect on nicotine-mediated IL-8 secretion. Pre-treatment of the Ca9-22 cells with the Ca(2+) chelator BAPTA-AM drastically inhibited IL-8 secretion. Although nicotine stimulation induced the phosphorylation of the NF-κB p65 subunit, pre-treatment with BAPTA-AM was found to inhibit this activity significantly. CaMK II-dependent p65 phosphorylation was confirmed by pre-incubation of the cells with CaMK II inhibitor. The results from this study indicate that the binding of nicotine to nAChR induces Ca(2+) influx, which results in the activation and phosphorylation of CaMK II and NF-κB p65, respectively. Nicotine-mediated IL-8 induction should be a trigger for the initiation of various diseases.
Assuntos
Cálcio/metabolismo , Células Epiteliais/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Interleucina-8/antagonistas & inibidores , Regiões 5' não Traduzidas , Bungarotoxinas/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Genes Reporter , Células HT29 , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Luciferases/genética , Luciferases/metabolismo , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mucosa Bucal/efeitos dos fármacos , Mucosa Bucal/metabolismo , Mucosa Bucal/patologia , Nicotina/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Transdução de Sinais , Tosilfenilalanil Clorometil Cetona/farmacologia , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismoRESUMO
In Crohn's disease (CD), hierarchical architecture of the inflammatory network, including subordination of IL-18, an IFN-γ-inducing cytokine, to the inflammasome, have remained undeciphered. Heterogeneity among patients of such a subordination cannot be evaluated by animal models, monofactorial in their etiology and homogenous in disease progression. To address these issues, we set up an ex vivo model of inflamed mucosa explant cultures from patients with active long-standing CD. Th1 cytokine production, especially IFN-γ and IL-18, was assessed in relation with inflammation intensity. Subordination of the Th1 response to caspase-1, effector of the inflammasome, was determined in explant cultures subjected to pharmacological inhibition of caspase-1 by YVAD. We showed a correlation between secreted IFN-γ/IL-18 levels, and caspase-1 activation, with inflammation intensity of intestinal CD mucosa explants. Inhibition of caspase-1 activation using the specific inhibitor YVAD identified a homogenous non responder group featuring a caspase-1-independent IL-18/IFN-γ response, and a heterogenous responder group, in which both IL-18 and IFN-γ responses were caspase-1-dependent, with a 40-70% range of inhibition by YVAD. These findings bring out the concept of heterogeneity of subordination of the Th1 response to inflammasome activation among CD patients. This ex vivo model should have therapeutic relevance in allowing to determine eligibility of CD patients for new targeted therapies.
Assuntos
Caspase 1/metabolismo , Colo/metabolismo , Doença de Crohn/metabolismo , Íleo/metabolismo , Interferon gama/metabolismo , Interleucina-18/metabolismo , Mucosa Intestinal/metabolismo , Adulto , Idoso , Biomarcadores/metabolismo , Caspase 1/química , Inibidores de Caspase/farmacologia , Colo/efeitos dos fármacos , Colo/enzimologia , Colo/patologia , Doença de Crohn/imunologia , Doença de Crohn/patologia , Doença de Crohn/cirurgia , Resistência a Medicamentos , Ativação Enzimática , Feminino , Humanos , Íleo/efeitos dos fármacos , Íleo/enzimologia , Íleo/patologia , Inflamassomos/efeitos dos fármacos , Inflamassomos/imunologia , Inflamassomos/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/enzimologia , Mucosa Intestinal/patologia , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Técnicas de Cultura de Tecidos , Tosilfenilalanil Clorometil Cetona/análogos & derivados , Tosilfenilalanil Clorometil Cetona/farmacologia , Adulto JovemRESUMO
Nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) is regulated by a myriad of signaling cascades including glycogen synthase kinase (GSK) 3ß and plays a Janus role in podocyte injury. In vitro, lipopolysaccharide (LPS) or adriamycin (ADR) elicited podocyte injury and cytoskeletal disruption, associated with NFκB activation and induced expression of NFκB target molecules, including pro-survival Bcl-xL and podocytopathic mediators like MCP-1, cathepsin L, and B7-1. Broad-range inhibition of NFκB diminished the expression of all NFκB target genes, restored cytoskeleton integrity, but potentiated apoptosis. In contrast, blockade of GSK3ß by lithium or 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8) mitigated the expression of podocytopathic mediators, ameliorated podocyte injury, but barely affected Bcl-xL expression or sensitized apoptosis. Mechanistically, GSK3ß was sufficient and essential for RelA/p65 phosphorylation, specifically at serine 467, which specifies the expression of selective NFκB target molecules, including podocytopathic mediators, but not Bcl-xL. In vivo, lithium or TDZD-8 therapy improved podocyte injury and proteinuria in mice treated with LPS or ADR, concomitant with the suppression of podocytopathic mediators, but retained Bcl-xL in glomerulus. Broad-range inhibition of NFκB conferred similar but much weakened antiproteinuric and podoprotective effects accompanied with a blunted glomerular expression of Bcl-xL and marked podocyte apoptosis. Thus, the GSK3ß-dictated fine-tuning of NFκB may serve as a novel therapeutic target for podocytopathy.
Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , NF-kappa B/metabolismo , Podócitos/metabolismo , Fator de Transcrição RelA/metabolismo , Animais , Apoptose/efeitos dos fármacos , Antígeno B7-1/metabolismo , Catepsina L/metabolismo , Movimento Celular , Células Cultivadas , Quimiocina CCL2/metabolismo , Doxorrubicina , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta , Glomérulos Renais , Lipopolissacarídeos , Lítio/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/antagonistas & inibidores , Fosforilação , Podócitos/efeitos dos fármacos , Podócitos/patologia , Proteinúria/induzido quimicamente , Proteinúria/metabolismo , Pirrolidinas/farmacologia , Inibidores de Serina Proteinase/farmacologia , Tiadiazóis/farmacologia , Tiocarbamatos/farmacologia , Tosilfenilalanil Clorometil Cetona/farmacologia , Proteína bcl-X/metabolismoRESUMO
Among the many immunomodulatory and anti-tumor activities, IFN-γ up-regulates tumor cell death mediated by Fas receptor (FasR). Our and several other studies have demonstrated the involvement of trypsin-like proteases (TLPs) in the mode of action of IFN-γ. In the present study, we tried to unravel the role of serine proteases in IFN-γ induced Fas-mediated cell death. Our present results show that both tosyl-l-Lysine chloromethylketone (TLCK), a trypsin like protease inhibitor and tosyl-l-phenylalanine chloromethylketone (TPCK) - a chymotrypsin like protease (CLP) inhibitor, sensitize HeLa cells to Fas-mediated cell death. The combined effect of these protease inhibitors with anti-Fas was stronger than additive. In contrast, elastase inhibitor III (EI), which also contains the chloromethyl ketone moiety, was not active. Furthermore, co-addition of TLCK or TPCK with IFN-γ markedly enhanced Fas-induced cell death. IFN-γ led to up-regulation of FasR on its own, which was further enhanced by the co-addition of TLCK or TPCK. This was evident both by increased expression of Fas receptor on cell surface and by elevated Fas mRNA level. This study may provide the basis for the design of a novel combinatory therapeutic strategy that could enhance the eradication of tumors.
Assuntos
Apoptose/efeitos dos fármacos , Interferon gama/farmacologia , Neoplasias/tratamento farmacológico , Inibidores de Serina Proteinase/farmacologia , Receptor fas/biossíntese , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Proteína Ligante Fas/metabolismo , Células HT29 , Células HeLa , Humanos , Neoplasias/patologia , RNA Mensageiro/biossíntese , Serina Endopeptidases/metabolismo , Tosilina Clorometil Cetona/farmacologia , Tosilfenilalanil Clorometil Cetona/farmacologia , Regulação para Cima , Receptor fas/genéticaRESUMO
BACKGROUND/AIMS: Renalase is a recently discovered, kidney-specific monoamine oxidase that metabolizes circulating catecholamines. These findings present new insights into hypertension and chronic kidney diseases. Previous data demonstrated that renalase was mainly secreted from proximal tubules which could be evoked by catecholamines. The purpose of this study is to investigate whether renalase expression is induced by epinephrine via α-adrenoceptor/NFκB pathways. METHODS: HK2 cells were utilized to explore renalase expression in response to epinephrine in vitro. Phentolamine, an α-adrenoceptor antagonist, and Tosyl Phenylalanyl Chloromethyl Ketone (TPCK) were used to block α-adrenoceptor and to knock down the transcription factor NFκB, respectively. Renalase expression was analyzed using Western blot and quantitative PCR. RESULTS: Both protein and mRNA levels of renalase in HK2 cells increased in response to epinephrine (P<0.05). Epinephrine-evoked renalase expression was attenuated by phentolamine and TPCK separately (P<0.05). CONCLUSION: Epinephrine evokes renalase secretion via α-adrenoceptor/NF-κB pathways in renal proximal tubular epithelial cells.
Assuntos
Agonistas alfa-Adrenérgicos/farmacologia , Epinefrina/farmacologia , Células Epiteliais/metabolismo , Túbulos Renais Proximais/metabolismo , Monoaminoxidase/metabolismo , NF-kappa B/efeitos dos fármacos , Receptores Adrenérgicos alfa/efeitos dos fármacos , Antagonistas Adrenérgicos alfa/farmacologia , Linhagem Celular , Epinefrina/antagonistas & inibidores , Células Epiteliais/efeitos dos fármacos , Humanos , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Fentolamina/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Tosilfenilalanil Clorometil Cetona/farmacologiaRESUMO
Autophagy is involved in both the cell protective and the cell death process but its mechanism is largely unknown. The present work unravels a novel intracellular mechanism by which the serpin α1-antitrypsin (AAT) acts as a novel negative regulator of autophagic cell death. For the first time, the role of intracellularly synthesized AAT, other than in liver cells, is demonstrated. Autophagic cell death was induced by N-α-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and tamoxifen. By utilizing a fluorescently tagged TPCK analog, AAT was "fished out" (pulled out) as a TPCK intracellular protein target. The interaction was further verified by competition binding experiments. Both inducers caused downregulation of AAT expression associated with activation of trypsin-like proteases. Furthermore, silencing AAT by siRNA induced autophagic cell death. Moreover, AAT administration to cultured cells prevented autophagic cell death. This new mechanism could have implications in the treatment of diseases by the regulation of AAT levels in which autophagy has a detrimental function. Furthermore, the results imply that the high synthesis of endogenous AAT by cancer cells could provide a novel resistance mechanism of cancer against autophagic cell death.
Assuntos
Autofagia/fisiologia , alfa 1-Antitripsina/metabolismo , Autofagia/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Células HT29 , Humanos , Células MCF-7 , Inibidores da Síntese de Proteínas/farmacologia , RNA Interferente Pequeno/genética , Tamoxifeno/farmacologia , Tosilfenilalanil Clorometil Cetona/farmacologia , Tripsina/metabolismo , alfa 1-Antitripsina/genéticaRESUMO
PROBLEM: How is the tumor necrosis factor (TNF) α-induced inhibitor of apoptosis (IAP) protein expression in endometriotic stromal cells (ESCs) involved in cell viability and signaling pathways? METHOD OF STUDY: Endometriotic stromal cells were isolated from ovarian chocolate cysts in 20 patients who underwent laparoscopic surgery. IAP protein expression and IκB phosphorylation were evaluated by Western blot analysis. Interleukin (IL)-8 protein expression and cell proliferation were assessed by ELISA. RESULTS: Cellular IAP (cIAP)-2 protein expression in endometriotic tissue was higher than that of endometrium. TNFα markedly enhanced cIAP-2 protein expression in ESCs. Pretreatment with a nuclear factor (NF)-κB inhibitor attenuated TNFα-induced cIAP-2 expression. An antagonist of IAPs abrogated TNFα-induced cIAP-2 protein expression and showed a decrease in TNFα-induced IL-8 protein expression and BrdU incorporation in ESCs. CONCLUSIONS: TNFα and its downstream NFκB pathway have proven to be critical regulators of highly expressed cIAP-2 in ESCs. cIAP-2 may be a novel therapeutic target for endometriosis.
Assuntos
Endometriose/imunologia , Endométrio/patologia , Proteínas Inibidoras de Apoptose/metabolismo , Células Estromais/fisiologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Endometriose/tratamento farmacológico , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas I-kappa B/metabolismo , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Proteínas Inibidoras de Apoptose/genética , Interleucina-8/metabolismo , Terapia de Alvo Molecular , NF-kappa B/antagonistas & inibidores , Oligopeptídeos/farmacologia , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Tosilfenilalanil Clorometil Cetona/farmacologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
The zebrafish is a powerful genetic model that has only recently been used to dissect developmental pathways involved in oncogenesis. We hypothesized that operative pathways during embryogenesis would also be used for oncogenesis. In an effort to define RAS target genes during embryogenesis, gene expression was evaluated in Tg(hsp70-HRAS(G12V)) zebrafish embryos subjected to heat shock. dusp6 was activated by RAS, and this was used as the basis for a chemical genetic screen to identify small molecules that interfere with RAS signaling during embryogenesis. A KRAS(G12D)-induced zebrafish embryonal rhabdomyosarcoma was then used to assess the therapeutic effects of the small molecules. Two of these inhibitors, PD98059 and TPCK, had anti-tumor activity as single agents in both zebrafish embryonal rhabdomyosarcoma and a human cell line of rhabdomyosarcoma that harbored activated mutations in NRAS. PD98059 inhibited MEK1 whereas TPCK suppressed S6K1 activity; however, the combined treatment completely suppressed eIF4B phosphorylation and decreased translation initiation. Our work demonstrates that the activated pathways in RAS induction during embryogenesis are also important in oncogenesis and that inhibition of these pathways suppresses tumor growth.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Rabdomiossarcoma/patologia , Transdução de Sinais , Peixe-Zebra/embriologia , Proteínas ras/metabolismo , Animais , Animais Geneticamente Modificados , Linhagem Celular Tumoral , Fatores de Iniciação em Eucariotos/metabolismo , Flavonoides/farmacologia , Humanos , MAP Quinase Quinase 1/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Biossíntese de Proteínas , Rabdomiossarcoma/genética , Rabdomiossarcoma/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Tosilfenilalanil Clorometil Cetona/farmacologia , Transgenes , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
The aim of the present study was to determine the correlation between angiogenesis and the differential expression of growth factors and their receptors when myocardial microvascular endothelial cells (MMVECs) were co-cultured with mast cell granules (MCGs) under hyperglycemic conditions. MMVECs and mast cells (MCs) were isolated from Wistar rats. An in vitro angiogenesis assay was used to observe any differences when MMVECs were co-cultured with MCGs in normal or hyperglycemic medium. The mRNA and protein expression of growth factors and their receptors were analyzed by real-time reverse transcription (RT)-PCR and western blot analysis. Real-time RT-PCR analysis demonstrated the upregulated mRNA and protein expression of vascular endothelial growth factor (VEGF) in the MMVECs; however, the expression of its receptor, fms-like tyrosine kinase-1 (Flt-1) and fetal liver kinase-1 (Flk1), decreased significantly, and the angiogenic ability of the MMVECs decreased under hyperglycemic conditions. The angiogenic ability of the MMVECs cultured under hyperglycemic conditions (even after the addition of MCGs) was inferior to that of the MMVECs cultured under normal glucose conditions. The specific inhibitor of tryptase, N-tosyl-L-lysine chloromethyl ketone (TLCK), suppressed angiogenesis regardless of the glucose concentration, and the specific inhibitor of chymase, N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK), was not as effective as TLCK, which was mainly detected under hyperglycemic conditions. High glucose levels have a profound effect on angiogenesis; this effect may be more pronounced than the effects of MCGs on angiogenesis.
Assuntos
Células Endoteliais/metabolismo , Glucose/farmacologia , Hiperglicemia/patologia , Mastócitos/metabolismo , Microvasos/citologia , Miocárdio/citologia , Neovascularização Fisiológica/efeitos dos fármacos , Animais , Técnicas de Cocultura , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Hiperglicemia/metabolismo , Masculino , Mastócitos/citologia , Mastócitos/efeitos dos fármacos , Neovascularização Fisiológica/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tosilina Clorometil Cetona/farmacologia , Tosilfenilalanil Clorometil Cetona/farmacologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Cicatrização/efeitos dos fármacosRESUMO
AIM: To investigate the effects of CpG ODN (CpG oligodeoxynucleotides) to model the action of bacterial challenge on pulpal matrix metalloproteinase-13 (MMP-13) expression and elucidate the associated intracellular signalling pathways. METHODOLOGY: Real-time PCR was used to detect the effects of CpG ODN on MMP-13 mRNA expression levels in a murine odontoblast-lineage cell line (OLCs). The possible involvement of TLR9/MyD88, NF-κB or MAPK pathways involved in the CpG ODN-induced MMP-13 expression was examined by real-time PCR, transient transfection, luciferase activity assay and ELISA. Western blotting was performed to assay the phosphorylation of ERK at a range of time points. RESULTS: MMP-13 was constitutively expressed in OLCs, and their exposure to CpG ODN significantly increased MMP-13 expression. Pre-treatment of OLCs with the inhibitory peptide MyD88, or chloroquine, attenuated the CpG ODN-induced expression of MMP-13. Treatment of the OLCs with CpG ODN increased NF-κB-luciferase activity. This activity was decreased by the over-expression of a nondegrading mutant of IκBα (IκBαSR), although enhanced by the over-expression of NF-κB p65. MMP-13 expression induced by CpG ODN was markedly suppressed by NF-κB inhibitors (pyrrolidine dithiocarbamate, PDTC), IκBα phosphorylation inhibitors (Bay 117082) or IκB protease inhibitor (L-1-tosylamido-2-phenylethyl chloromethyl ketone, TPCK). The inhibitor of ERK1/2, U0126, but not inhibitors of p38 MAPK and JNK, SB203580 and SP600125, decreased CpG ODN-mediated MMP-13 expression. CONCLUSION: The CpG ODN-induced MMP-13 expression in OLCs is mediated through TLR9, NF-κB and the ERK pathway indicating that potentially the recognition of CpG ODN by TLR9 on odontoblasts may regulate the remodelling of injured dental pulp and hard tissues by inducing MMP-13 expression.
Assuntos
Ilhas de CpG , Polpa Dentária/enzimologia , Metaloproteinase 13 da Matriz/efeitos dos fármacos , Oligodesoxirribonucleotídeos/farmacologia , Animais , Antracenos/farmacologia , Linhagem Celular , Cloroquina/farmacologia , Polpa Dentária/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , Proteínas I-kappa B/farmacologia , Imidazóis/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Fator 88 de Diferenciação Mieloide/farmacologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/efeitos dos fármacos , Nitrilas/farmacologia , Odontoblastos/efeitos dos fármacos , Odontoblastos/enzimologia , Fosforilação , Piridinas/farmacologia , Pirrolidinas/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacos , Sulfonas/farmacologia , Tiocarbamatos/farmacologia , Receptor Toll-Like 9/efeitos dos fármacos , Tosilfenilalanil Clorometil Cetona/farmacologia , Fator de Transcrição RelA/farmacologiaRESUMO
N-alpha-tosyl-L-phenylalanyl chloromethyl ketone (TPCK) has anti-tumorigenic properties, but its direct cellular targets are unknown. Previously, we showed TPCK inhibited the PDKl-dependent AGC kinases RSK, Akt and S6K1 without inhibiting PKA, ERK1/2, PI3K, and PDK1 itself. Here we show TPCK-inhibition of the RSK-related kinases MSK1 and 2, which can be activated independently of PDK1. Mass spectrometry analysis of RSK1, Aktl, S6K1 and MSK1 immunopurified from TPCK-treated cells identified TPCK adducts on cysteines located in conserved activation loop Phenylalanine-Cysteine (Phe-Cys) motifs. Mutational analysis of the Phe-Cys residues conferred partial TPCK resistance. These studies elucidate a primary mechanism by which TPCK inhibits several AGC kinases, inviting consideration of TPCK-like compounds in chemotherapy given their potential for broad control of cellular growth, proliferation and survival.
Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/química , Tosilfenilalanil Clorometil Cetona/farmacologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência de Bases , Sequência Conservada , Cisteína/química , Primers do DNA/genética , Células HEK293 , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fenilalanina/química , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Proto-Oncogênicas c-akt/genética , Piruvato Desidrogenase Quinase de Transferência de Acetil , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 90-kDa/química , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Homologia de Sequência de AminoácidosRESUMO
Proteolysis carried out by different proteases control cellular processes during development and regeneration. Here we investigated the function of the proteasome and other proteases in the process of intestinal regeneration using as a model the sea cucumber Holothuria glaberrima. This echinoderm possesses the ability to regenerate its viscera after a process of evisceration. Enzymatic activity assays showed that intestinal extracts at different stages of regeneration possessed chymotrypsin-like activity. This activity was inhibited by i) MG132, a reversible inhibitor of chymotrypsin and peptidylglutamyl peptidase hydrolase (PGPH) activities of the proteasome, ii) E64d, a permeable inhibitor of cysteine proteases and iii) TPCK, a serine chymotrypsin inhibitor, but not by epoxomicin, an irreversible and potent inhibitor of all enzymatic activities of the proteasome. To elucidate the role which these proteases might play during intestinal regeneration, we carried out in vivo experiments injecting MG132, E64d and TPCK into regenerating animals. The results showed effects on the size of the regenerating intestine, cell proliferation and collagen degradation. These findings suggest that proteolysis by several proteases is important in the regulation of intestinal regeneration in H. glaberrima.
Assuntos
Holothuria/fisiologia , Intestinos/fisiologia , Organogênese/fisiologia , Proteólise/efeitos dos fármacos , Regeneração/fisiologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proliferação de Células/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , Holothuria/efeitos dos fármacos , Intestinos/efeitos dos fármacos , Leucina/análogos & derivados , Leucina/farmacologia , Leupeptinas/farmacologia , Organogênese/efeitos dos fármacos , Regeneração/efeitos dos fármacos , Inibidores de Serina Proteinase/farmacologia , Tosilfenilalanil Clorometil Cetona/farmacologiaRESUMO
High mobility group box chromosomal protein 1 (HMGB-1) is a widely studied, ubiquitous nuclear protein that is present in eukaryotic cells, and plays a crucial role in inflammatory response. However, the effects of HMGB-1 on human synovial fibroblasts are largely unknown. In this study, we investigated the intracellular signaling pathway involved in HMGB-1-induced IL-6 production in human synovial fibroblast cells. HMGB-1 caused concentration- and time-dependent increases in IL-6 production. HMGB-1-mediated IL-6 production was attenuated by receptor for advanced glycation end products (RAGE) monoclonal antibody (Ab) or siRNA. Pretreatment with c-Src inhibitor (PP2), Akt inhibitor and NF-κB inhibitor (pyrrolidine dithiocarbamate and L-1-tosylamido-2-phenylenylethyl chloromethyl ketone) also inhibited the potentiating action of HMGB-1. Stimulation of cells with HMGB-1 increased the c-Src and Akt phosphorylation. HMGB-1 increased the accumulation of p-p65 in the nucleus, as well as NF-κB luciferase activity. HMGB-1-mediated increase of NF-κB luciferase activity was inhibited by RAGE Ab, PP2 and Akt inhibitor or RAGE siRNA, or c-Src and Akt mutant. Our results suggest that HMGB-1-increased IL-6 production in human synovial fibroblasts via the RAGE receptor, c-Src, Akt, p65, and NF-κB signaling pathways.
Assuntos
Proteína HMGB1/metabolismo , Interleucina-6/biossíntese , NF-kappa B/metabolismo , Proteína Oncogênica v-akt/metabolismo , Quinases da Família src/metabolismo , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Redes e Vias Metabólicas/efeitos dos fármacos , NF-kappa B/antagonistas & inibidores , Proteína Oncogênica v-akt/antagonistas & inibidores , Pirimidinas/farmacologia , Pirrolidinas/farmacologia , Inibidores de Serina Proteinase/farmacologia , Transdução de Sinais , Membrana Sinovial/efeitos dos fármacos , Tiocarbamatos/farmacologia , Tosilfenilalanil Clorometil Cetona/farmacologia , Quinases da Família src/antagonistas & inibidoresRESUMO
OBJECTIVE: The purpose of the present study was to characterize the collagenolytic activity in a sonicated extract of Tannerella forsythia and to investigate the activation of proMMMP-2 and -9 by the T. forsythia extract. METHODS: The T. forsythia extract was incubated with type I collagen. The cleaved products were then analyzed by SDS-PAGE using the method of Laemmli. We studied the effects of cysteine, DTT, CaCl(2), and various proteinase inhibitors on collagenolytic activity. A HT1080 cell culture supernatant containing proMMP-2 and -9 was incubated with the T. forsythia extract and analyzed for the activation of proMMP-2 and -9 by gelatin zymography. RESULTS: The T. forsythia extract degraded type I collagen. Cysteine increased the collagenolytic activity of the extract, and 5mM CaCl(2) was required for this activity. The collagenolytic activity of T. forsythia was inhibited by N-ethylmaleimide, iodoacetamide, iodoacetic acid, EDTA, and leupeptin, but not by PMSF, E-64, TLCK, or TPCK. When proMMP-2 and -9 were incubated with the T. forsythia extract, gelatinases with the relative molecular masses of MMP-2 and -9 were produced. CONCLUSION: The present study suggests that T. forsythia extract is able to degrade type I collagen and activate proMMP-2 and -9.
Assuntos
Bacteroides/metabolismo , Colágeno Tipo I/metabolismo , Cloreto de Cálcio/farmacologia , Catepsinas/antagonistas & inibidores , Linhagem Celular Tumoral , Quelantes/farmacologia , Cisteína/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Ácido Edético/farmacologia , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Precursores Enzimáticos/metabolismo , Etilmaleimida/farmacologia , Gelatinases/metabolismo , Humanos , Iodoacetamida/farmacologia , Ácido Iodoacético/farmacologia , Leucina/análogos & derivados , Leucina/farmacologia , Leupeptinas/farmacologia , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Metaloproteinase 9 da Matriz/efeitos dos fármacos , Metaloproteinase 9 da Matriz/metabolismo , Fluoreto de Fenilmetilsulfonil/farmacologia , Inibidores de Proteases/farmacologia , Inibidores de Serina Proteinase/farmacologia , Tosilina Clorometil Cetona/farmacologia , Tosilfenilalanil Clorometil Cetona/farmacologiaRESUMO
The protein Puma (p53-upregulated modulator of apoptosis) belongs to the BH3-only group of the Bcl-2 family and is a major regulator of apoptosis. Although the transcriptional regulation of Puma is clearly established, little is known about the regulation of its expression at the protein levels. We show here that various signals--including the cytokine TGFß, the death effector TRAIL or chemical drugs such as anisomycin--downregulate Puma protein levels via a novel pathway based on the sequential activation of caspase-3 and a protease inhibited by the serpase inhibitor N-tosyl-L-phenylalanine chloromethyl ketone. This pathway is specific for Puma because (1) the levels of other BH3-only proteins, such as Bim and Noxa were not modified by these stimuli and (2) this caspase-mediated degradation was dependent on both the BH3 and C-terminal domains of Puma. Our data also show that Puma is regulated during the caspase-3-dependent differentiation of murine embryonic stem cells and suggest that this pathway may be relevant and important during caspase-mediated cell differentiation not associated with apoptosis.
Assuntos
Proteínas Reguladoras de Apoptose , Caspase 3 , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Fragmentos de Peptídeos/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Serina Proteases , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Tosilfenilalanil Clorometil Cetona , Fator de Crescimento Transformador beta/farmacologia , Animais , Anisomicina/farmacologia , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Inativação Gênica/fisiologia , Humanos , Camundongos , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Serina Proteases/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/genética , Tosilfenilalanil Clorometil Cetona/farmacologia , Fator de Crescimento Transformador beta/genéticaRESUMO
This study is focused on the links between the major products of inflammation and cell damage induced by the administration of lipopolysaccharide (LPS) from Salmonella typhimurium in embryonal cardiomyocytes. LPS treatment for 72 hours induced transcription factor NF-kappaB activation, inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 expression, nitric oxide (NO) and tumor necrosis factor (TNF)-alpha release. Moreover, LPS administration induced a significant cell loss, reversed by the NO-synthases inhibitor, suggesting a relationship between cell damage and iNOS-dependent NO overproduction. Cell death was reversed by the specific NF-kappaB inhibitor, TPCK, whereas COX-2 specific inhibitor determined an increase of cell damage in terms of apoptosis, as observed by YO-PRO immunostaining, DNA laddering analysis and caspase-3 activation. Overall these findings evidenced a selective role for NF-kappaB in mediating NO-induced cell damage and a protective action by COX-2 in LPS-treated embryonal cardiomyocytes. The reflection of these experiments on human cardiac pathology will be discussed.
Assuntos
Coração/embriologia , Inflamação/imunologia , Lipopolissacarídeos/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Células Cultivadas , Embrião de Galinha , Ciclo-Oxigenase 2/química , Ciclo-Oxigenase 2/metabolismo , Inibidores Enzimáticos/farmacologia , Técnicas In Vitro , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Transdução de Sinais , Tosilfenilalanil Clorometil Cetona/farmacologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Phloroglucinol (PG), a polyphenolic compound, has been proposed to show free radical scavenging, anti-tumor, and immunomodulation effects on immune cells. In this study, we investigated PG for its immunological activity and their molecular mechanisms, specifically focusing on the functional activation of nuclear factor-kappaB (NFkappaB), an important transcription factor involved in the activation and differentiation of lymphocytes, and subsequent recruitment of murine lymphocytes. METHODS: We tested whether PG may contribute to the activation of immune response through the NFkappaB pathway by (3)H-thymidine incorporation assay, Western blot, intracellular cytokine assays and Electrophoretic mobility shift assay (EMSA) in murine lymphocytes. RESULTS: PG markedly enhanced the proliferation of lymphocytes by inducing the degradation and phosphorylation of IkappaB, which leads to the activation of NFkappaB p65. Also, PG induced the activation of mitogen-activated protein kinases (MAPKs) such as ERK1/2, JNK, and p38, upstream molecules of NFkappaB pathway. In addition, PG augmented the secretion of interleukin (IL)-2 in mature T lymphocytes and their production of IL-2. Furthermore, the application of TPCK significantly reduced the activation of lymphocytes by PG via inhibiting the NFkappaB activation. CONCLUSION: These results suggest that PG induces the activation of lymphocytes by inducing the proliferative activity and secretion of IL-2 through the classical NFkappaB. GENERAL SIGNIFICANCE: The effect of PG on lymphocyte activation was assayed for the first time. These results would also elucidate its underlying mechanism.