RESUMO
We reported previously that prostaglandin E2 (PGE2) up-regulates IL-23 in vitro in bone marrow-derived dendritic cells and in vivo in models of collagen-induced arthritis and inflammatory bowel disease, leading to preferential Th17 development and activity. There is very little information on the molecular mechanisms involved in the PGE2-induced up-regulation of Il23a gene expression. In this study we investigated the signaling pathways and transcription factors involved in the stimulatory effect of PGE2. Although PGE2 does not induce IL-23p19 expression by itself, it synergizes with both extra- and intracellular Toll-like receptor ligands and with inflammatory cytokines such as TNFα. We established that the effect of PGE2 in conjunction with either LPS or TNFα is mediated through the EP4 receptor and the cAMP-dependent activation of both protein kinase A (PKA) and exchange protein activated by cAMP (EPAC). Using the EP4 agonist PGE(1)OH in conjunction with TNFα, we found that PKA-induced phosphorylation of cAMP-response element-binding protein ((P)CREB) and EPAC-induced phosphorylation of C/AATT enhancer-binding protein ß ((P)C/EBPß) mediate the stimulatory effect of PGE2 on IL-23p19 expression. This is the first report of CREB and C/EBPß involvement in Il23a promoter activation. Mutation within the putative CREB and C/EBP sites combined with in vivo DNA binding (ChIP) assays identified the distal CREB site (-1125) and the two proximal C/EBP sites (-274 and -232) as essential for PKA-activated CREB and EPAC-activated C/EBPß-induced IL-23p19 expression.
Assuntos
Células da Medula Óssea/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Células Dendríticas/metabolismo , Dinoprostona/fisiologia , Subunidade p19 da Interleucina-23/metabolismo , Adenilil Ciclases/metabolismo , Alprostadil/análogos & derivados , Alprostadil/farmacologia , Animais , Sítios de Ligação , Células da Medula Óssea/imunologia , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Dendríticas/imunologia , Regulação da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina , Interleucina-12/genética , Interleucina-12/metabolismo , Subunidade p19 da Interleucina-23/genética , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Ligação Proteica , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Sistemas do Segundo Mensageiro , Receptores Toll-Like/metabolismo , Transcrição Gênica , Fator de Necrose Tumoral alfa/fisiologia , Regulação para CimaRESUMO
MS is an autoimmune disease characterized by immune cell infiltration in the CNS, leading to cumulative disability. IFN-ß, used clinically in RR-MS reduces lesion formation and rates of relapse. Although the molecular mechanisms are not entirely elucidated, myeloid cells appear to be a major target for the therapeutic effects of IFN-ß. DCs have a critical role in experimental models of MS through their effect on encephalitogenic Th1/Th17 cell differentiation and expansion. Here we focused on the effects of IFN-ß on DC expression of cytokines involved in the control of Th1/Th17 differentiation and expansion. Administration of IFN-ß to mice immunized with MOG35-55 inhibited IL-12 and IL-23 expression in splenic DC and reduced in vivo differentiation of Th1/Th17 cells. IFN-ß affected cytokine expression in TLR-stimulated DC in a similar manner in vitro, inhibiting IL-12 and IL-23 and stimulating IL-10 at both mRNA and protein levels, by signaling through IFNAR. We investigated the role of the signaling molecules STAT1/STAT2, IRF-1 and IRF-7, and of the PI3KâGSK3 pathway. IFN-ß inhibition of the IL-12 subunits p40 and p35 was mediated through STAT1/STAT2, whereas inhibition of IL-23 was STAT1 dependent, and the stimulatory effect on IL-10 expression was mediated through STAT2. IFN-ß induces IRF-7 and, to a lesser degree, IRF-1. However, neither IRF mediated the effects of IFN-ß on IL-12, IL-23, or IL-10. We found that the PI3K pathway mediated IL-12 inhibition but did not interfere with the inhibition of IL-23 or stimulation of IL-10.
Assuntos
Células Dendríticas/imunologia , Interferon beta/imunologia , Interleucina-10/imunologia , Subunidade p35 da Interleucina-12/imunologia , Subunidade p40 da Interleucina-12/imunologia , Interleucina-23/imunologia , Transdução de Sinais/imunologia , Receptores Toll-Like/imunologia , Animais , Células Dendríticas/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Fator Regulador 1 de Interferon/genética , Fator Regulador 1 de Interferon/imunologia , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/imunologia , Interferon beta/genética , Interleucina-10/genética , Subunidade p35 da Interleucina-12/genética , Subunidade p40 da Interleucina-12/genética , Interleucina-23/genética , Camundongos , Camundongos Knockout , Glicoproteína Mielina-Oligodendrócito/farmacologia , Fragmentos de Peptídeos/farmacologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/imunologia , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/imunologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Baço/citologia , Baço/imunologia , Células Th17/citologia , Células Th17/imunologia , Receptores Toll-Like/genéticaRESUMO
Although Crohn's disease has been traditionally considered to be Th1-mediated, the newly identified Th17 cells emerged recently as crucial participants. Th1/Th17 differentiation is controlled primarily by the IL-12 family of cytokines secreted by activated dendritic cells (DCs) and macrophages. IL-23 and IL-12/IL-27 have opposite effects, supporting the Th17 and Th1 phenotypes, respectively. We found that PGE(2), a major lipid mediator released in inflammatory conditions, shifts the IL-12/IL-23 balance in DCs in favor of IL-23, and propose that high levels of PGE(2) exacerbate the inflammatory process in inflammatory bowel disease through the IL-23-->IL-17 axis. We assessed the effects of PGE(2) on IL-12, IL-27, and IL-23 and found that PGE(2) promotes IL-23, inhibits IL-12 and IL-27 expression and release from stimulated DCs, and subsequently induces IL-17 production in activated T cells. The effects of PGE(2) are mediated through the EP2/EP4 receptors on DCs. In vivo, we assessed the effects of PGE analogs in an experimental model for inflammatory bowel disease and found that the exacerbation of clinical symptoms and histopathology correlated with an increase in IL-23 and IL-17, a decrease in IL-12p35 expression in colon and mesenteric lymph nodes, and a substantial increase in the number of infiltrating neutrophils and of CD4(+)IL-17(+) T cells in the colonic tissue. These studies suggest that high levels of PGE(2) exacerbate the inflammatory process through the preferential expression and release of DC-derived IL-23 and the subsequent support of the autoreactive/inflammatory Th17 phenotype.
Assuntos
Colite/imunologia , Colo/imunologia , Dinoprostona/metabolismo , Doenças Inflamatórias Intestinais/imunologia , Interleucina-17/metabolismo , Interleucina-23/metabolismo , Animais , Antiulcerosos/farmacologia , Colite/induzido quimicamente , Colo/efeitos dos fármacos , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Dinoprostona/farmacologia , Modelos Animais de Doenças , Doenças Inflamatórias Intestinais/induzido quimicamente , Interleucina-12/antagonistas & inibidores , Interleucina-17/análise , Interleucina-23/análise , Interleucinas/antagonistas & inibidores , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Misoprostol/farmacologia , Neutrófilos/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Ácido Trinitrobenzenossulfônico/toxicidadeRESUMO
Rat liver arginase (arginase I) is potently inactivated by diethyl pyrocarbonate, with a second-order rate constant of 113M(-1)s(-1) for the inactivation process at pH 7.0, 25 degrees C. Partial protection from inactivation is provided by the product of the reaction, l-ornithine, while nearly complete protection is afforded by the inhibitor pair, l-ornithine and borate. The role of H141 has been probed by mutagenesis, chemical modulation, and X-ray diffraction. The hyper-reactivity of H141 towards diethyl pyrocarbonate can be explained by its proximity to E277. A proton shuttling role for H141 is supported by its conformational mobility observed among the known arginase structures. H141 is proposed to serve as an acid/base catalyst, deprotonating the metal-bridging water molecule to generate the metal-bridging hydroxide nucleophile, and by protonating the amino group of the product to facilitate its departure.
Assuntos
Arginase/química , Histidina/química , Animais , Arginase/antagonistas & inibidores , Boratos/química , Cristalografia por Raios X , Dietil Pirocarbonato/química , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ornitina/química , Conformação Proteica , RatosRESUMO
Hyperargininemia is a rare autosomal disorder that results from a deficiency in hepatic type I arginase. This deficiency is the consequence of random point mutations that occur throughout the gene. The G235R patient mutation has been proposed to affect the catalytic activity and structural integrity of the protein [D. E. Ash, L. R. Scolnick, Z. F. Kanyo, J. G. Vockley, S. D. Cederbaum, and D. W. Christianson (1998) Mol. Genet. Metab. 64, 243-249]. The G235R (patient) and G235A (control) arginase mutants of rat liver arginase have been generated to probe the effects of these point mutations on the structure and function of hepatic type I arginase. Both mutant arginases were trimeric by gel filtration, but the control G235A mutant had 56% of wild-type activity and the G235R mutant had less than 0.03% activity compared to the wild-type enzyme. The G235R mutant contained undetectable levels of tightly bound manganese as determined by electron paramagnetic resonance, while the G235A mutant had a Mn(II) stoichiometry of 2 Mn/subunit. Molecular modeling indicates that the introduction of an arginine residue at position 235 results in a major rearrangement of the metal ligands that compromise Mn(II) binding.
Assuntos
Arginase/genética , Arginase/fisiologia , Fígado/metabolismo , Mutação Puntual , Animais , Arginase/química , Sítios de Ligação , Cromatografia em Gel , Espectroscopia de Ressonância de Spin Eletrônica , Estabilidade Enzimática , Hiperargininemia/enzimologia , Hiperargininemia/etiologia , Cinética , Manganês/química , Metais/metabolismo , Modelos Químicos , Modelos Moleculares , Peso Molecular , Mutagênese Sítio-Dirigida , Ratos , Relação Estrutura-Atividade , TemperaturaRESUMO
Arginase is a binuclear manganese metalloenzyme that hydrolyzes l-arginine to form l-ornithine and urea. The three-dimensional structures of D128E, D128N, D232A, D232C, D234E, H101N, and H101E arginases I have been determined by X-ray crystallographic methods to elucidate the roles of the first-shell metal ligands in the stability and catalytic activity of the enzyme. This work represents the first structure-based dissection of the binuclear manganese cluster using site-directed mutagenesis and X-ray crystallography. Substitution of the metal ligands compromises the catalytic activity of the enzyme, either by the loss or disruption of the metal cluster or the nucleophilic metal-bridging hydroxide ion. However, the substitution of the metal ligands or the reduction of Mn(2+)(A) or Mn(2+)(B) occupancy does not compromise enzyme-substrate affinity as reflected by K(M), which remains relatively invariant across this series of arginase variants. This implicates a nonmetal binding site for substrate l-arginine in the precatalytic Michaelis complex, as proposed based on analysis of the native enzyme structure (Kanyo, Z. F., Scolnick, L. R., Ash, D. E., and Christianson, D. W. (1996) Nature 383, 554-557).
Assuntos
Arginase/metabolismo , Manganês/metabolismo , Substituição de Aminoácidos , Animais , Arginase/genética , Cristalografia por Raios X , Ligantes , Ratos , Relação Estrutura-AtividadeRESUMO
Arginase is a manganese metalloenzyme that catalyzes the hydrolysis of L-arginine to form L-ornithine and urea. The structure and stability of the binuclear manganese cluster are critical for catalytic activity as it activates the catalytic nucleophile, metal-bridging hydroxide ion, and stabilizes the tetrahedral intermediate and its flanking states. Here, we report X-ray structures of a series of inhibitors bound to the active site of arginase, and each inhibitor exploits a different mode of coordination with the Mn(2+)(2) cluster. Specifically, we have studied the binding of fluoride ion (F(-); an uncompetitive inhibitor) and L-arginine, L-valine, dinor-N(omega)-hydroxy-L-arginine, descarboxy-nor-N(omega)-hydroxy-L-arginine, and dehydro-2(S)-amino-6-boronohexanoic acid. Some inhibitors, such as fluoride ion, dinor-N(omega)-hydroxy-L-arginine, and dehydro-2(S)-amino-6-boronohexanoic acid, cause the net addition of one ligand to the Mn(2+)(2) cluster. Other inhibitors, such as descarboxy-nor-N(omega)-hydroxy-L-arginine, simply displace the metal-bridging hydroxide ion of the native enzyme and do not cause any net change in the metal coordination polyhedra. The highest affinity inhibitors displace the metal-bridging hydroxide ion (and sometimes occupy a Mn(2+)(A) site found vacant in the native enzyme) and maintain a conserved array of hydrogen bonds with their alpha-amino and -carboxylate groups.
Assuntos
Arginase/química , Inibidores Enzimáticos/química , Manganês , Animais , Arginase/antagonistas & inibidores , Arginina/análogos & derivados , Arginina/química , Sítios de Ligação , Cristalografia por Raios X , Flúor/química , Ligação de Hidrogênio , Metaloproteínas/antagonistas & inibidores , Metaloproteínas/química , Estrutura Molecular , Ligação Proteica , Ratos , Proteínas Recombinantes , Valina/químicaRESUMO
Arginase is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of l-arginine to form l-ornithine and urea. The X-ray crystal structure of a fully active, truncated form of human arginase II complexed with a boronic acid transition state analogue inhibitor has been determined at 2.7 A resolution. This structure is consistent with the hydrolysis of l-arginine through a metal-activated hydroxide mechanism. Given that human arginase II appears to play a role in regulating l-arginine bioavailability to NO synthase in human penile corpus cavernosum smooth muscle, the inhibition of human arginase II is a potential new strategy for the treatment of erectile dysfunction [Kim, N. N., Cox, J. D., Baggio, R. F., Emig, F. A., Mistry, S., Harper, S. L., Speicher, D. W., Morris, S. M., Ash, D. E., Traish, A. M., and Christianson, D. W. (2001) Biochemistry 40, 2678-2688]. Since NO synthase is found in human clitoral corpus cavernosum and vagina, we hypothesized that human arginase II is similarly present in these tissues and functions to regulate l-arginine bioavailability to NO synthase. Accordingly, hemodynamic studies conducted with a boronic acid arginase inhibitor in vivo are summarized, suggesting that the extrahepatic arginase plays a role in both male and female sexual arousal. Therefore, arginase II is a potential target for the treatment of male and female sexual arousal disorders.