RESUMEN
Cyclopentenone prostaglandins (cyPG) with antiinflammatory and antiproliferative properties have been envisaged as leads for the development of therapeutic agents. Because cyPG effects are mediated in part by the formation of covalent adducts with critical signaling proteins, it is important to assess the specificity of this interaction. By using biotinylated derivatives of 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)-B) and PGA(1) (PGA(1)-B) we herein provide novel evidence for the differential selectivity of protein modification by distinct cyPG. The marked quantitative and qualitative differences in the binding of 15d-PGJ(2)-B and PGA(1)-B to cellular proteins were related to a differential reactivity in the presence of glutathione (GSH), both in vitro and in intact cells. Therefore GSH levels may influence not only the intensity but also the specificity of cyPG action.
Asunto(s)
Glutatión/fisiología , Prostaglandina D2/análogos & derivados , Prostaglandinas A/farmacología , Proteínas/efectos de los fármacos , Animales , Ciclopentanos/farmacología , Glutatión/farmacología , Ratones , Células 3T3 NIH , Prostaglandina D2/farmacología , Proteínas/metabolismoRESUMEN
Cyclopentenone prostanoids (cyP) arise as important modulators of inflammation and cell proliferation. Although their physiological significance has not been fully elucidated, their potent biological effects have spurred their study as leads for the development of therapeutic agents. A key determinant of cyP action is their ability to bind to thiol groups in proteins or in glutathione through Michael addition. Even though several protein targets for cyP addition have been identified, little is known about the structural determinants from the protein or the cyP that drive this modification. The results herein presented provide the first evidence that cyP with different structures target distinct thiol sites in a protein molecule, namely, H-Ras. Whereas 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) and Delta12-PGJ2 preferentially target the C-terminal region containing cysteines 181 and 184, PGA1 and 8-iso-PGA1 bind mainly to cysteine 118, located in the GTP-binding motif. The biological counterparts of this specificity are the site-selective modification and activation of H-Ras in cells and the differential interaction of cyP with H, N, and K-Ras proteins. Cysteine 184 is unique to H-Ras, whereas cysteine 118 is present in the three Ras homologues. Consistent with this, PGA1 binds to and activates H-, N-, and K-Ras, thus differing from the preferential interaction of 15d-PGJ2 with H-Ras. These results put forward the possibility of influencing the selectivity of cyP-protein addition by modifying cyP structure. Furthermore, they may open new avenues for the development of cyP-based drugs.
Asunto(s)
Prostaglandinas/farmacología , Proteínas ras/efectos de los fármacos , Proteínas ras/metabolismo , Animales , Antiinflamatorios/metabolismo , Antiinflamatorios/farmacología , Sitios de Unión/efectos de los fármacos , Cisteína/química , Cisteína/efectos de los fármacos , Regulación de la Expresión Génica , Células HeLa , Humanos , Ratones , Prostaglandina D2/análogos & derivados , Prostaglandina D2/metabolismo , Prostaglandina D2/farmacología , Prostaglandinas/química , Prostaglandinas/metabolismo , Prostaglandinas A/metabolismo , Prostaglandinas A/farmacología , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/clasificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masas en Tándem , Células Tumorales Cultivadas , Proteínas ras/química , Proteínas ras/clasificaciónRESUMEN
The induction of cyclooxygenase-2 (COX-2) and the production of PGE(2) in response to pathogen-associated molecular patterns decorated with mannose moieties were studied in human monocytes and monocyte-derived macrophages (MDM). Saccharomyces cerevisiae mannan was a robust agonist, suggesting the involvement of the mannose receptor (MR). MR expression increased along the macrophage differentiation route, as judged from both its surface display assessed by flow cytometry and the ability of MDM to ingest mannosylated BSA. Treatment with mannose-BSA, a weak agonist of the MR containing a lower ratio of attached sugar compared with pure polysaccharides, before the addition of mannan inhibited COX-2 expression, whereas this was not observed when agonists other than mannan and zymosan were used. HeLa cells, which were found to express MR mRNA, showed a significant induction of COX-2 expression upon mannan challenge. Conversely, mannan did not induce COX-2 expression in HEK293 cells, which express the mRNA encoding Endo180, a parent receptor pertaining to the MR family, but not the MR itself. These data indicate that mannan is a strong inducer of COX-2 expression in human MDM, most likely by acting through the MR route. Because COX-2 products can be both proinflammatory and immunomodulatory, these results disclose a signaling route triggered by mannose-decorated pathogen-associated molecular patterns, which can be involved in both the response to pathogens and the maintenance of homeostasis.
Asunto(s)
Dinoprostona/biosíntesis , Lectinas Tipo C/fisiología , Macrófagos/metabolismo , Lectinas de Unión a Manosa/fisiología , Manosa/metabolismo , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Receptores de Superficie Celular/fisiología , Complejo Antígeno-Anticuerpo/farmacología , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Ciclooxigenasa 2 , Contaminación de Medicamentos , Glucanos , Células HeLa , Humanos , Lipopolisacáridos/farmacología , Macrófagos/citología , Macrófagos/enzimología , Mananos/farmacología , Manosa/fisiología , Receptor de Manosa , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/fisiología , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Monocitos/citología , Monocitos/enzimología , Monocitos/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Polisacáridos/farmacología , ARN Mensajero/biosíntesis , Receptores de Superficie Celular/metabolismo , Receptores Toll-Like , Zimosan/farmacologíaRESUMEN
THP-1 monocytic cells were stimulated with IgG-ovalbumin equivalence immune complexes (IC) and mAb reacting with both FcgammaRI and FcgammaRIIA. All of these stimuli were capable of activating the cells; however, different patterns of response were observed as regards activation of the p42-MAP/ERK kinase, triggering of the NF-kappaB/Rel system, production of chemotactic cytokines, and induction of the expression of cyclooxygenase-2 (COX-2). Activation of p42-MAP/ERK kinase was a constant finding, which occurred regardless of the type of stimulus applied, for instance, homotypic stimulation of a single type of receptor by cross-linking with specific mAb or heterotypic stimulation with both types of antibodies and IC. However, the activation of the MAP/ERK kinase cascade was not connected to the triggering of cytosolic phospholipase A(2) (cPLA(2)) and arachidonic acid (AA) release. The heterotypic stimulation of FcgammaR induced the expression of COX-2 in a time and dose-dependent manner and activated the NF-kappaB system as judged from the degradation of IkappaB-alpha protein. In summary, the present data indicate that activation of the p42-MAP/ERK pathway occurs after cross-linking FcgammaRI and FcgammaRIIA receptors in monocytic cells; however, this is not coupled to the cPLA(2) route, which leads to the release of AA. Noteworthy,heterotypic activation involving combined cross-linking of both FcgammaRI and FcgammaRIIA has a robust effect on the oxidative metabolism of AA by a mechanism involving kappaB-dependent trans-activation of COX-2.
Asunto(s)
Ácido Araquidónico/metabolismo , Proteínas I-kappa B , Isoenzimas/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Prostaglandina-Endoperóxido Sintasas/genética , Receptores de IgG/metabolismo , Secuencia de Bases , Línea Celular , Quimiocina CCL2/biosíntesis , Reactivos de Enlaces Cruzados , Ciclooxigenasa 2 , Proteínas de Unión al ADN/metabolismo , Activación Enzimática , Humanos , Mediadores de Inflamación/metabolismo , Proteínas de la Membrana , Inhibidor NF-kappaB alfa , FN-kappa B/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regulación hacia ArribaRESUMEN
The role of the opsonic receptors FcgammaR and CR3 on the release of arachidonic acid (AA) by human monocytes was studied using IgG-ovalbumin (OVA) equivalence immune complexes (IC), anti-OVA IgG bound to OVA-coupled latex beads, and C3bi-bound IC. Release of AA was produced by IC and latex-OVA beads bound to IgG, whereas binding of C3bi to IC inhibited the ability of IC to release AA. In contrast, coating of zymosan particles with C3bi enhanced AA release as compared with that produced by non-coated particles. Masking of C3bi on C3bi-bound IC by incubation with anti-C3 IgG resulted in the recovery of their ability to release AA, thereby suggesting that binding of C3b by IC reduces their flogogenic effects, whereas opsonization of microbial walls by complement may enhance their proinflammatory potential. The binding/uptake of opsonized zymosan particles was inhibited by anti-CR3 Ab and C3bi-bound IC, but not by beta-glucan, mannan, and anti-Toll-like receptor 2 Ab. These findings show that cooperative engagement of CR3 on both the lectin-like site involved in beta-glucan binding and the I-domain involved in C3bi binding, as it can be observed in the innate immune response, produces AA release, whereas the unique interaction of C3bi-bound IC with the I-domain of CR3, as it may occur in the adaptive immune response, diverts the IC lattice from a productive interaction with FcgammaR linked to AA release.
Asunto(s)
Ácido Araquidónico/metabolismo , Antígeno de Macrófago-1/metabolismo , Monocitos/metabolismo , Receptores de IgG/metabolismo , Receptores Inmunológicos/metabolismo , Antígeno CD11b/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Citometría de Flujo , Glucanos/metabolismo , Humanos , Immunoblotting , Lectinas/metabolismo , Leucocitos Mononucleares/metabolismo , Macrófagos/metabolismo , Mananos/química , Ovalbúmina/metabolismo , Fagocitosis , Polisacáridos/química , Unión Proteica , Estructura Terciaria de Proteína , Factores de Tiempo , Zimosan/químicaRESUMEN
Monocytic cells were stimulated with IgG-OVA equivalence immune complexes, mAb reacting with FcgammaRI, FcgammaRIIA, and FcgammaRIII, LPS, TNF-alpha, and the combination of ionomycin and phorbol ester, to address their effects on the expression of the mRNAs encoding for chemokines. Stimulation of monocytes with immune complexes induced a rapid expression of macrophage-inflammatory protein (MIP)-1alpha, MIP-1beta, and IL-8 mRNAs. In contrast, RANTES mRNA was already detectable in resting cells and only increased after 16 h of stimulation. A similar pattern was observed following homotypic stimulation of FcgammaR with mAb reacting with FcgammaRI and FcgammaRIIA, but not with a mAb reacting with FcgammaRIII, a subtype of receptor not expressed in THP-1 cells, thus indicating that both FcgammaRI and FcgammaRIIA are involved in the response. The pattern of chemokine induction elicited by LPS and the combination of ionomycin and PMA showed some similarities to those produced by FcgammaR cross-linking, although expression of IFN-gamma-inducible protein 10 mRNA was also observed in response to those agonists. The production of MIP-1alpha, MIP-1beta, and RANTES proteins encompassing the induction of their mRNAs was confirmed by specific ELISA. Experiments to address the transcription factors involved in the regulation of MIP-1alpha using pharmacological agents and EMSA showed the possible involvement of CCAAT/enhancer-binding protein beta sites and ruled out the functional significance of both NF-AT and AP-1 sites.