RESUMEN
BACKGROUND: Faster off-rate competitive enzyme inhibitors are generally more sensitive than slower off-rate ones to binding inhibition by enzyme substrates. We previously reported that the cyclic adenosine monophosphate concentration in dopamine D1 receptor-expressing medium spiny neurons (D1-MSNs) may be higher than that in D2-MSNs. Consequently, compared with slower off-rate phosphodiesterase 10A inhibitors, faster off-rate ones comparably activated D2-MSNs but partially activated D1-MSNs. We further investigated the pharmacological profiles of phosphodiesterase 10A inhibitors with different off-rates. METHODS: Phosphodiesterase 10A inhibitors with slower (T-609) and faster (T-773) off-rates were used. D1- and D2-MSN activation was assessed by substance P and enkephalin mRNA induction, respectively, in rodents. Antipsychotic-like effects were evaluated by MK-801- and methamphetamine-induced hyperactivity and prepulse inhibition in rodents. Cognition was assessed by novel object recognition task and radial arm maze in rats. Prefrontal cortex activation was evaluated by c-Fos immunohistochemistry in rats. Gene translations in D1- and D2-MSNs were evaluated by translating ribosome affinity purification and RNA sequencing in mice. RESULTS: Compared with T-609, T-773 comparably activated D2-MSNs but partially activated D1-MSNs. Haloperidol (a D2 antagonist) and T-773, but not T-609, produced antipsychotic-like effects in all paradigms. T-773, but not T-609 or haloperidol, activated the prefrontal cortex and improved cognition. Overall gene translation patterns in D2-MSNs by all drugs and those in D1-MSNs by T-773 and T-609 were qualitatively similar. CONCLUSIONS: Differential pharmacological profiles among those drugs could be attributable to activation balance of D1- and D2-MSNs. The "balanced activation" of MSNs by faster off-rate phosphodiesterase 10A inhibitors may be favorable to treat schizophrenia.
Asunto(s)
Antipsicóticos/farmacología , Cuerpo Estriado/efectos de los fármacos , Neuronas GABAérgicas/efectos de los fármacos , Aprendizaje por Laberinto/efectos de los fármacos , Nootrópicos/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Hidrolasas Diéster Fosfóricas , Corteza Prefrontal/efectos de los fármacos , Receptores de Dopamina D1/efectos de los fármacos , Receptores de Dopamina D2/efectos de los fármacos , Reconocimiento en Psicología/efectos de los fármacos , Animales , Conducta Animal/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Ratas , Ratas Long-Evans , Ratas Sprague-DawleyRESUMEN
The endoplasmic reticulum (ER) stress protein mesencephalic astrocyte-derived neurotrophic factor (MANF) has been reported to protect cells from stress-induced cell death before and after its secretion; however, the conditions under which it is secreted are not known. Accordingly, we examined the mechanism of MANF release from cultured ventricular myocytes and HeLa cells, both of which secrete proteins via the constitutive pathway. Although the secretion of proteins via the constitutive pathway is not known to increase upon changes in intracellular calcium, MANF secretion was increased within 30 min of treating cells with compounds that deplete sarcoplasmic reticulum (SR)/ER calcium. In contrast, secretion of atrial natriuretic factor from ventricular myocytes was not increased by SR/ER calcium depletion, suggesting that not all secreted proteins exhibit the same characteristics as MANF. We postulated that SR/ER calcium depletion triggered MANF secretion by decreasing its retention. Consistent with this were co-immunoprecipitation and live cell, zero distance, photo affinity cross-linking, demonstrating that, in part, MANF was retained in the SR/ER via its calcium-dependent interaction with the SR/ER-resident protein, GRP78 (glucose-regulated protein 78 kDa). This unusual mechanism of regulating secretion from the constitutive secretory pathway provides a potentially missing link in the mechanism by which extracellular MANF protects cells from stresses that deplete SR/ER calcium. Consistent with this was our finding that administration of recombinant MANF to mice decreased tissue damage in an in vivo model of myocardial infarction, a condition during which ER calcium is known to be dysregulated, and MANF expression is induced.
Asunto(s)
Calcio/metabolismo , Isquemia Miocárdica/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Proteínas/metabolismo , Retículo Sarcoplasmático/metabolismo , Animales , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico , Células HeLa , Proteínas de Choque Térmico/metabolismo , Humanos , Masculino , Glicoproteínas de Membrana/metabolismo , Ratones , Isquemia Miocárdica/patología , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/patología , Miocitos Cardíacos/metabolismo , Factores de Crecimiento Nervioso/fisiología , Unión Proteica , Señales de Clasificación de Proteína , Estructura Terciaria de Proteína , Proteínas/fisiología , Ratas , Retículo Sarcoplasmático/efectos de los fármacos , Tapsigargina/farmacología , Tunicamicina/farmacologíaRESUMEN
Proper folding of secreted and transmembrane proteins made in the rough endoplasmic reticulum (ER) requires oxygen for disulfide bond formation. Accordingly, ischemia can impair ER protein folding and initiate the ER stress response, which we previously showed is activated in the ischemic heart and in culture cardiac myocytes subjected to simulated ischemia. ER stress and ischemia activate the transcription factor, activating transcription factor 6 (ATF6), which induces numerous genes, many of which have not been identified, or examined in the heart. Using an ATF6 transgenic mouse model, we previously showed that ATF6 protected the heart from ischemic damage; however, the mechanism of this protection remains to be determined. In this study, we showed that, in the mouse heart, and in cultured cardiac myocytes, ATF6 induced the protein disulfide isomerase associated 6 (PDIA6) gene, which encodes an ER enzyme that catalyzes protein disulfide bond formation. Moreover, in cultured cardiac myocytes, ER stress-mediated PDIA6 promoter activation was ATF6-dependent, and required an ER stress response element (ERSE) and a nearby CCAAT box element. Electromobility shift assays and chromatin immunoprecipitation showed that ATF6 bound to the ERSE in the PDIA6 promoter, in vitro, and in the mouse heart, in vivo. Gain- and loss-of-function studies showed that PDIA6 protected cardiac myocytes against simulated ischemia/reperfusion-induced death in a manner that was dependent on the catalytic activity of PDIA6. Thus, by facilitating disulfide bond formation, and enhanced ER protein folding, PDIA6 may contribute to the protective effects of ATF6 in the ischemic mouse heart.
Asunto(s)
Factor de Transcripción Activador 6/metabolismo , Muerte Celular/fisiología , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Daño por Reperfusión/fisiopatología , Factor de Transcripción Activador 6/genética , Animales , Muerte Celular/genética , Inmunoprecipitación de Cromatina , Cromatografía Liquida , Ensayo de Cambio de Movilidad Electroforética , Estrés del Retículo Endoplásmico/genética , Estrés del Retículo Endoplásmico/fisiología , Immunoblotting , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , MicroARNs/genética , Proteína Disulfuro Isomerasas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Espectrometría de Masas en TándemRESUMEN
Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive lipid involved in multiple physiological processes. Importantly, dysregulated S1P levels are associated with several pathologies, including cardiovascular and inflammatory diseases and cancer. This report describes the successful production and characterization of a murine monoclonal antibody, LT1002, directed against S1P, using novel immunization and screening methods applied to bioactive lipids. We also report the successful generation of LT1009, the humanized variant of LT1002, for potential clinical use. Both LT1002 and LT1009 have high affinity and specificity for S1P and do not cross-react with structurally related lipids. Using an in vitro bioassay, LT1002 and LT1009 were effective in blocking S1P-mediated release of the pro-angiogenic and prometastatic cytokine, interleukin-8, from human ovarian carcinoma cells, showing that both antibodies can out-compete S1P receptors in binding to S1P. In vivo anti-angiogenic activity of all antibody variants was demonstrated using the murine choroidal neovascularization model. Importantly, intravenous administration of the antibodies showed a marked effect on lymphocyte trafficking. The resulting lead candidate, LT1009, has been formulated for Phase 1 clinical trials in cancer and age-related macular degeneration. The anti-S1P antibody shows promise as a novel, first-in-class therapeutic acting as a "molecular sponge" to selectively deplete S1P from blood and other compartments where pathological S1P levels have been implicated in disease progression or in disorders where immune modulation may be beneficial.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Lisofosfolípidos/inmunología , Esfingosina/análogos & derivados , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/farmacología , Especificidad de Anticuerpos/inmunología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neovascularización Coroidal/prevención & control , Reacciones Cruzadas/inmunología , Femenino , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Pesadas de Inmunoglobulina/metabolismo , Interleucinas/metabolismo , Cinética , Lisofosfolípidos/metabolismo , Degeneración Macular/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones SCID , Mutagénesis , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Unión Proteica , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Esfingosina/inmunología , Esfingosina/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
S1P has been proposed to contribute to cancer progression by regulating tumor proliferation, invasion, and angiogenesis. We developed a biospecific monoclonal antibody to S1P to investigate its role in tumorigenesis. The anti-S1P mAb substantially reduced tumor progression and in some cases eliminated measurable tumors in murine xenograft and allograft models. Tumor growth inhibition was attributed to antiangiogenic and antitumorigenic effects of the antibody. The anti-S1P mAb blocked EC migration and resulting capillary formation, inhibited blood vessel formation induced by VEGF and bFGF, and arrested tumor-associated angiogenesis. The anti-S1P mAb also neutralized S1P-induced proliferation, release of proangiogenic cytokines, and the ability of S1P to protect tumor cells from apoptosis in several tumor cell lines, validating S1P as a target for therapy.