RESUMEN
WNT signaling pathways play an important role in both development and disease. By analyzing the signaling capabilities of commercially available WNT3a preparations towards the PI3K/AKT/GSK3 signaling pathway, we discovered unexpected inconsistencies from lot to lot of recombinant WNT3a. We provide evidence that: (1) The ability to trigger AKT/GSK3 signaling varies dramatically between different lots of WNT3a, without any variation in their ability to activate the canonical WNT/ß-catenin signaling. (2) sFRP1, a WNT signaling inhibitor, is unable to interfere with the activation of AKT/GSK3 signaling induced by some of the WNT3a lots. (3) Pharmacological inhibition of AKT/GSK3 phosphorylation by PI3K inhibitors fails to affect the stabilization of ß-catenin, the central effector of the canonical WNT/ß-catenin signaling pathway. In summary, while all tested lots of recombinant WNT3a activated WNT/ß-catenin pathway, our results suggest that individual lots of recombinant WNT3a activate the PI3K/AKT/GSK3 pathway in a WNT-independent manner, hampering thus the analysis of regulation of PI3K/AKT/GSK3 by WNT ligand.
Asunto(s)
Transducción de Señal/efectos de los fármacos , Proteínas Wnt/farmacología , Composición de Medicamentos , Glucógeno Sintasa Quinasa 3 , Humanos , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Proteínas Recombinantes , Proteína Wnt3 , Proteína Wnt3ARESUMEN
alpha-Secretase cleavage of the amyloid precursor protein (APP) is of great interest because it prevents the formation of the Alzheimer-linked amyloid-beta peptide. APP belongs to a conserved gene family including the two paralogues APP-like protein (APLP) 1 and 2. Insulin-like growth factor-1 (IGF-1) stimulates the shedding of all three proteins. IGF-1-induced shedding of both APP and APLP1 is dependent on phosphatidylinositol 3-kinase (PI3-K), whereas APLP2 shedding is independent of this signaling pathway. Here, we used human neuroblastoma SH-SY5Y cells to investigate the involvement of protein kinase C (PKC) in the proteolytic processing of endogenously expressed members of the APP family. Processing was induced by IGF-1 or retinoic acid, another known stimulator of APP alpha-secretase shedding. Our results show that stimulation of APP and APLP1 processing involves multiple signaling pathways, whereas APLP2 processing is mainly dependent on PKC. Next, we wanted to investigate whether the difference in the regulation of APLP2 shedding compared with APP shedding could be due to involvement of different processing enzymes. We focused on the two major alpha-secretase candidates ADAM10 and TACE, which both are members of the ADAM (a disintegrin and metalloprotease) family. Shedding was analyzed in the presence of the ADAM10 inhibitor GI254023X, or after transfection with small interfering RNAs targeted against TACE. The results clearly demonstrate that different alpha-secretases are involved in IGF-1-induced processing. APP is mainly cleaved by ADAM10, whereas APLP2 processing is mediated by TACE. Finally, we also show that IGF-1 induces PKC-dependent phosphorylation of TACE.
Asunto(s)
Proteínas ADAM/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas ADAM/antagonistas & inhibidores , Proteína ADAM10 , Proteína ADAM17 , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Antineoplásicos/farmacología , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Neuroblastoma/metabolismo , Fosforilación , Proteína Quinasa C/metabolismo , ARN Interferente Pequeño/farmacología , Tretinoina/farmacología , Células Tumorales CultivadasRESUMEN
Retinoic acid stimulates alpha-secretase processing of amyloid precursor protein (APP) and decreases beta-secretase cleavage that leads to amyloid-beta formation. Here, we investigated the effect of retinoic acid on the two putative alpha-secretases, the disintegrin metalloproteinases ADAM10 and TACE, and the beta-site cleaving enzyme BACE1, in human neuroblastoma SH-SY5Y cells. Western blot analysis showed that exposure to retinoic acid resulted in significantly increased levels of ADAM10 and TACE, suggesting that regulation of alpha-secretases causes the effects on APP processing. The presence of the phosphatidylinositol 3-kinase inhibitor LY 294002 selectively reduced the effect on ADAM10 protein levels but not on ADAM10 mRNA levels as determined by RT-PCR. On the other hand, the effect on TACE was shown to be dependent on protein kinase C, since it was completely blocked in the presence of the inhibitor bisindolylmaleimide XI. Our data indicate that different signalling pathways are involved in retinoic acid-induced up-regulation of the secretases.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Neuroblastoma/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Quinasa C/metabolismo , Receptores de Superficie Celular/metabolismo , Transducción de Señal/efectos de los fármacos , Tretinoina/administración & dosificación , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Nexinas de Proteasas , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The mammalian amyloid precursor protein (APP) protein family consists of the APP and the amyloid precursor-like proteins 1 and 2 (APLP1 and APLP2). The neurotoxic amyloid beta-peptide (Abeta) originates from APP, which is the only member of this protein family implicated in Alzheimer disease. However, the three homologous proteins have been proposed to be processed in similar ways and to have essential and overlapping functions. Therefore, it is also important to take into account the effects on the processing and function of the APP-like proteins in the development of therapeutic drugs aimed at decreasing the production of Abeta. Insulin and insulin-like growth factor-1 (IGF-1) have been shown to regulate APP processing and the levels of Abeta in the brain. In the present study, we show that IGF-1 increases alpha-secretase processing of endogenous APP and also increases ectodomain shedding of APLP1 and APLP2 in human SH-SY5Y neuroblastoma cells. We also investigated the role of different IGF-1-induced signaling pathways, using specific inhibitors for phosphatidylinositol 3-kinase and mitogen-activated protein kinase (MAPK). Our results indicate that phosphatidylinositol 3-kinase is involved in ectodomain shedding of APP and APLP1, but not APLP2, and that MAPK is involved only in the ectodomain shedding of APLP1.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Proteínas del Tejido Nervioso/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Línea Celular Tumoral , Humanos , Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Sistema de Señalización de MAP Quinasas , Fosfatidilinositol 3-Quinasas/metabolismo , Estructura Terciaria de ProteínaRESUMEN
The amyloid precursor protein (APP) belongs to a conserved gene family, also including the amyloid precursor-like proteins, APLP1 and APLP2. We have previously shown that all members of the APP protein family are up-regulated upon retinoic acid (RA)-induced neuronal differentiation of SH-SY5Y neuroblastoma cells. Here, we demonstrate that RA also affects the processing of APLP2 and APP, as shown by increased shedding of both sAPLP2 and sAPPalpha, as well as elevated levels of the APP intracellular domains (AICDs). Brain-derived neurotrophic factor (BDNF) has been reported to induce APP promoter activity and RA induces expression of the tyrosine kinase receptor B (TrkB) in neuroblastoma cells. We show that the increase in shedding of both APLP2 and APP in response to RA is not mediated through the TrkB receptor. However, BDNF concomitant with RA increased the expression of APP even further. In addition, the secretion of sAPLP2 and sAPPalpha as well as the levels of AICDs were increased in response to BDNF. In contrast, the levels of membrane-bound APP C-terminal fragment C99 significantly decreased. Our results suggest that RA and BDNF shifts APP processing towards the alpha-secretase pathway. In addition, we show that RA and BDNF regulate N-linked glycosylation of APLP1.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Factor Neurotrófico Derivado del Encéfalo/farmacología , Diferenciación Celular/efectos de los fármacos , Espacio Extracelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Tretinoina/farmacología , Western Blotting/métodos , Línea Celular Tumoral , Interacciones Farmacológicas , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Neuroblastoma , Estructura Terciaria de Proteína/fisiología , Factores de TiempoRESUMEN
Amyloid precursor protein (APP) belongs to a conserved gene family, also including the amyloid precursor-like proteins, APLP1 and APLP2. The function of these three proteins is not yet fully understood. One of the proposed roles of APP is to promote neurite outgrowth. The aim of this study was to investigate the regulation of the expression levels of APP family members during neurite outgrowth. We observed that retinoic acid (RA)-induced neuronal differentiation of human SH-SY5Y cells resulted in increased expression of APP, APLP1 and APLP2. We also examined the effect of the NFkappaB, AP-1 and c-Jun N-terminal kinase inhibitor curcumin (diferuloylmethane) on the RA-induced expression levels of these proteins. We found that treatment with curcumin counteracted the RA-induced mRNA expression of all APP family members. In addition, we observed that curcumin treatment resulted in neurite retraction without any effect on cell viability. Surprisingly, curcumin had differential effects on the APLP protein levels in RA-differentiated cells. RA-induced APLP1 protein expression was blocked by curcumin, while the APLP2 protein levels were further increased. APP protein levels were not affected by curcumin treatment. We propose that the sustained levels of APP and the elevated levels of APLP2, in spite of the reduced mRNA expression, are due to altered proteolytic processing of these proteins. Furthermore, our results suggest that APLP1 does not undergo the same type of regulated processing as APP and APLP2.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Diferenciación Celular/fisiología , Sistema Nervioso Central/embriología , Proteínas del Tejido Nervioso/metabolismo , Neuritas/metabolismo , Neuroblastoma/metabolismo , Tretinoina/metabolismo , Precursor de Proteína beta-Amiloide/genética , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Sistema Nervioso Central/citología , Sistema Nervioso Central/metabolismo , Curcumina/farmacología , Inhibidores Enzimáticos/farmacología , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Proteínas del Tejido Nervioso/genética , Neuritas/efectos de los fármacos , Neuroblastoma/tratamiento farmacológico , ARN Mensajero/metabolismo , Factor de Transcripción AP-1/antagonistas & inhibidores , Factor de Transcripción AP-1/metabolismo , Tretinoina/farmacologíaRESUMEN
The amyloid precursor protein (APP) and its proteolytic cleavage products, the amyloid beta peptides, have been implicated as a cause of Alzheimer's disease. Peptide nucleic acids (PNA), the DNA mimics, have been shown to block the expression of specific proteins at both transcriptional and translational levels. Generally, the cellular uptake of PNA is low. However, recent studies have indicated that the effect of unmodified antisense PNA uptake is more pronounced in nervous tissue. In this study we have shown that biotinylated PNA directed to the initiator codon region of the APP mRNA (-4 - +11) was taken up into the cytoplasm of primary rat cerebellar granule cells and cortical astrocytes, using fluorescence and confocal microscopy studies. Uptake of PNA was faster in neurons than in astrocytes. Western blotting analysis showed that APP was strongly down-regulated in both neurons and astrocytes. Thus, unmodified PNA can be used for studies on the function of APP in neurons and astrocytes.