RESUMO
OBJECTIVES: For many years the p38 MAP kinase (MAPK) has been a major anti-inflammatory target for the development of an oral therapy for rheumatoid arthritis (RA). However, disappointing results from Phase II clinical studies suggest that adaptations may occur, which allow escape from blockade of the p38 pathway. In this study we investigated whether p38 inhibition mediated JNK activation represents such an escape mechanism. METHODS: Interaction between the JNK and p38 pathways was studied in TNF-α stimulated THP-1 monocytes, primary macrophages and fibroblast-like synoviocytes from OA and RA patients using pharmacological inhibitors and siRNAs. RESULTS: TNF-α induced phosphorylation of JNK and c-Jun was sustained by p38 inhibitors in monocytes, primary macrophages and FLS. Upregulation of Mip1α, Mip1ß and IL-8 mRNAs and protein were observed upon p38 inhibition. More importantly, inhibition of MK2, the substrate of p38 did not sustain JNK activation upon TNF-α activation and did not elevate Mip1α, Mip1ß and IL-8 chemokines as compared to TNF-α alone. In this study, TNF-α or IL-1ß induced JNK activation is sustained by p38 inhibition, resulting in enhanced chemokine secretion. CONCLUSIONS: Based on the suggested role of these chemokines in RA pathogenesis, the upregulation of these chemokines may provide an explanation for the lack of efficacy of p38 inhibitors in Phase II. The absence of any effect of MK2 inhibition in our models on this mechanism, while coming with similar efficacy on blocking p38, provides support for further investigations to reveal the potential of MK2 inhibition as a novel treatment of RA.
Assuntos
Artrite Reumatoide/tratamento farmacológico , Quimiocinas/metabolismo , Inibidores Enzimáticos/farmacologia , Fibroblastos/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Artrite Reumatoide/enzimologia , Artrite Reumatoide/imunologia , Linhagem Celular , Quimiocinas/imunologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Imidazóis/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Monócitos/citologia , Naftalenos/farmacologia , Cultura Primária de Células , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Pirazóis/farmacologia , Piridinas/farmacologia , Membrana Sinovial/citologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Mycophenolic acid is the active ingredient of the immunosuppressant mycophenolate mofetil that is widely used in transplantation medicine and autoimmunity. Mycophenolic acid inhibits inosine monophosphate dehydrogenase, an enzyme involved in biosynthesis of guanine nucleotides required for lymphocyte clonal expansion. Here, we present novel insights into the mechanisms underlying mycophenolic acid-mediated suppression of human CD4+ T cells. Upon CD3/CD28 stimulation, mycophenolic acid inhibited T cell IL-17, IFN-γ and TNF-α production but not IL-2 production. Phenotypic analysis showed that drug treatment enhanced the expression of negative co-stimulators PD-1, CTLA-4 and the transcription factor FoxP3 and decreased the expression of positive co-stimulators CD27 and CD28, whereas CD25 was unaffected. Mycophenolic acid-treated cells were anergic, but not suppressive, and at the same time proved hyperblastoid with high metabolic activity. Moreover, a reduced Akt/mTOR and STAT5 signaling was observed. Interestingly, the co-stimulatory molecule CD70 was uniquely and dose-dependently upregulated on mycophenolic acid-treated T cells and found to be directly linked to target enzyme inhibition. CD70 on mycophenolic acid-treated cells proved functional: an anti-CD70 agonist was found to restore both STAT5 and Akt/mTOR signaling and may thereby prevent apoptosis and promote survival. These novel insights may contribute to optimization of protocols for MPA-based immunosuppressive regimens.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Ligante CD27/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Nucleotídeos de Guanina/metabolismo , Ácido Micofenólico/farmacologia , Transdução de Sinais/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Western Blotting , Antígenos CD28/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Citometria de Fluxo , Humanos , IMP Desidrogenase/metabolismo , Interferon gama/metabolismo , Interleucina-17/metabolismo , Interleucina-2/metabolismo , Transdução de Sinais/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND AND PURPOSE: The p38 kinase regulates the release of proinflammatory cytokines including tumour-necrosis factor-alpha (TNFalpha) and is regarded as a potential therapeutic target in rheumatoid arthritis (RA). Using the novel p38 inhibitor Org 48762-0, we investigated the therapeutic potential of p38 inhibition and compared this to anti-mouse (m)TNFalpha antibody treatment in murine collagen-induced arthritis (CIA). EXPERIMENTAL APPROACH: Pharmacological profiles of Org 48762-0 were characterized in kinase assays, cellular assays and in lipopolysaccharide (LPS)-induced inflammation in mice. The effects of Org 48762-0 and of mTNFalpha-neutralization on established arthritis were examined in murine CIA. KEY RESULTS: Org 48762-0 potently inhibited p38alpha kinase with a high degree of kinase selectivity. In cellular assays, Org 48762-0 reduced LPS-induced TNFalpha release. Oral administration of Org 48762-0 in mice showed drug-like pharmacokinetic properties and inhibited LPS-induced cytokine production. These pharmacological characteristics of Org 48762-0 prompted a comparison of therapeutic efficacy with mTNFalpha-neutralization in CIA. Org 48762-0 and anti-mTNFalpha antibody treatment equally inhibited development of arthritis when evaluated macroscopically. Radiological analyses revealed protection against bone damage for both treatments, although statistical difference was reached with Org 48762-0 treatment only. Further, micro-computed tomographical and histopathological analyses confirmed the protective effects of Org 48762-0 on joint damage. CONCLUSIONS AND IMPLICATIONS: Pharmacological targeting of p38 kinase provided good protection against joint tissue damage in CIA. In our experiments, neutralization of mTNFalpha produced less prominent suppression of bone damage. Our data suggest a therapeutic potential for selective and potent p38 inhibitors in RA.
Assuntos
Anticorpos Bloqueadores/uso terapêutico , Artrite Experimental/tratamento farmacológico , Inibidores Enzimáticos/uso terapêutico , Piridinas/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Animais , Anti-Inflamatórios/uso terapêutico , Artrite Experimental/patologia , Western Blotting , Cartilagem/patologia , Endotoxemia/tratamento farmacológico , Inibidores Enzimáticos/farmacocinética , Feminino , Inflamação/induzido quimicamente , Lipopolissacarídeos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Prednisolona/uso terapêutico , Especificidade por Substrato , Tomografia Computadorizada por Raios XRESUMO
Aberrant expression of platelet-derived growth factor and its receptor (PDGFR) has been implicated in various human disorders, including cardiovascular disease and certain types of cancer. Inhibitors of the tyrosine kinase activity of PDGFR are leads in the development of novel agents to combat these diseases. We describe here a novel, potent inhibitor of PDGFR tyrosine kinase, 3-(4-dimethylamino-benzylidenyl)-2-indolinone (DMBI). The compound also inhibits signal transduction through fibroblast growth factor receptor 1 (FGFR1), but is not active towards epidermal growth factor receptor (EGFR) or c-Src tyrosine kinase. The activity of DMBI and other tyrosine kinase inhibitors was compared in a cell-based assay as well as in an assay based on purified recombinant platelet-derived growth factor beta-receptor (beta-PDGFR) lacking the transmembrane and ligand-binding domain. We showed that this truncated beta-PDGFR could dimerize, and that dimerization was required for tyrosine kinase activity. Tyrosine kinase activity was modulated by inhibitors of beta-PDGFR autophosphorylation in cells, but not by specific inhibitors of EGFR or c-Src tyrosine kinase. We conclude that beta-PDGFR lacking the transmembrane and ligand-binding domain retains the essential properties of the full-length receptor tyrosine kinase.
Assuntos
Inibidores Enzimáticos/farmacologia , Substâncias de Crescimento/farmacologia , Indóis/farmacologia , Receptores Proteína Tirosina Quinases/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Células 3T3 , Animais , Becaplermina , Carcinoma de Células Escamosas , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Citoplasma/enzimologia , Fator de Crescimento Epidérmico/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Cinética , Camundongos , Músculo Liso Vascular , Fosforilação , Fosfotirosina/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Proto-Oncogênicas c-sis , Artéria Pulmonar , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/isolamento & purificação , Receptor beta de Fator de Crescimento Derivado de Plaquetas , Receptores do Fator de Crescimento Derivado de Plaquetas/genética , Receptores do Fator de Crescimento Derivado de Plaquetas/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
Antistasin is a Factor Xa inhibitor that is present in the salivary glands of the Mexican leech Haementeria officinalis. The antistasin protein consists of 119 amino acids, of which residues 1-55 (domain I) are 56% similar to residues 56-110 (domain II). Of the nine C-terminal amino acids (residues 111-119; domain III), four are positively charged. The reactive site for Factor Xa is located in domain I. In this study we assessed the role of separate domains and of individual amino acids in the reactive site for the inhibition of Factor Xa. A series of mutants was constructed and expressed in Chinese hamster ovary (CHO) cells. In vitro chromogenic assays for Factor Xa show that domain I is sufficient for inhibition of Factor Xa. Domains II and III neither contain any intrinsic Factor Xa inhibitory activity, nor contribute to the activity of domain I. Furthermore, domain II does not become a Factor Xa inhibitor by partially adaptating its sequence towards that of the reactive site in domain I. Mutation of the cysteine at position 33 is not crucial for Factor Xa inhibition, suggesting a relatively rigid reactive site loop structure.