RESUMEN
Bladder cancer (BC) is a common and malignant tumor of the urinary tract, and its treatment options are limited. Tectoridin (TEC) has antitumor activity against prostate and colon cancer, but its effects on BC are poorly understood. BC cells were treated with increasing concentrations of TEC, and its effects on cell proliferation, migration, invasiveness, and apoptosis were assessed. Xenograft mouse model was used to evaluate the influences of TEC on BC tumor growth. Western blot analysis was conducted to explore the downstream pathways affected by TEC. TEC treatment decreased BC cell viability in a dose-dependent manner (IC50 ≈ 25 µM), and inhibited cell proliferation, migration, and invasiveness while promoting apoptosis. Clinical analysis revealed high expression of RAB27B in BC tumor tissues, particularly in advanced stages, correlating with an unfavorable prognosis. In vitro experiments demonstrated that TEC suppressed the PI3K/MAPK pathway by targeting RAB27B, and overexpression of RAB27B counteracted the antitumor effects of TEC. In xenograft models, TEC administration suppressed tumor growth, reduced tumor volume, inhibited cell proliferation, and suppressed the PI3K/MAPK pathway, highlighting its potential as an inhibitor of tumor growth. TEC suppresses BC tumor growth by targeting RAB27B and inactivating the PI3K/MAPK signaling and may provide a promising therapeutic target for BC treatment.
Asunto(s)
Proliferación Celular , Isoflavonas , Fosfatidilinositol 3-Quinasas , Neoplasias de la Vejiga Urinaria , Proteínas de Unión al GTP rab , Animales , Femenino , Humanos , Ratones , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones Endogámicos BALB C , Ratones Desnudos , Fosfatidilinositol 3-Quinasas/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas de Unión al GTP rab/efectos de los fármacos , Proteínas de Unión al GTP rab/metabolismo , Transducción de Señal/efectos de los fármacos , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Isoflavonas/farmacologíaRESUMEN
Leucine-rich repeat kinase 2 (LRRK2), the major causative gene product of autosomal-dominant Parkinson's disease, is a protein kinase that phosphorylates a subset of Rab GTPases. Since pathogenic LRRK2 mutations increase its ability to phosphorylate Rab GTPases, elucidating the mechanisms of how Rab phosphorylation is regulated by LRRK2 is of great importance. We have previously reported that chloroquine-induced lysosomal stress facilitates LRRK2 phosphorylation of Rab10 to maintain lysosomal homeostasis. Here we reveal that Rab10 phosphorylation by LRRK2 is potently stimulated by treatment of cells with a set of lysosome stressors and clinically used lysosomotropic drugs. These agents commonly promoted the formation of LRRK2-coated enlarged lysosomes and extracellular release of lysosomal enzyme cathepsin B, the latter being dependent on LRRK2 kinase activity. In contrast to the increase in Rab10 phosphorylation, treatment with lysosomotropic drugs did not increase the enzymatic activity of LRRK2, as monitored by its autophosphorylation at Ser1292 residue, but rather enhanced the molecular proximity between LRRK2 and its substrate Rab GTPases on the cytosolic surface of lysosomes. Lysosomotropic drug-induced upregulation of Rab10 phosphorylation was likely a downstream event of Rab29 (Rab7L1)-mediated enzymatic activation of LRRK2. These results suggest a regulated process of Rab10 phosphorylation by LRRK2 that is associated with lysosomal overload stress, and provide insights into the novel strategies to halt the aberrant upregulation of LRRK2 kinase activity.
Asunto(s)
Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Lisosomas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/efectos de los fármacos , Lisosomas/efectos de los fármacos , Ratones , Fosforilación/efectos de los fármacos , Proteínas de Unión al GTP rab/efectos de los fármacosRESUMEN
In a cell line, stably expressing α1A-adrenoceptors fused to the mCherry red fluorescent protein, noradrenaline, methoxamine, and oxymetazoline induced concentration-dependent increases in intracellular calcium. All of these agents increase α1A-adrenoceptor phosphorylation and internalization. Transient co-expression of these receptors with Rab proteins tagged with the enhanced Green Fluorescent Protein was employed to estimate α1A-adrenoceptor-Rab interaction using Förster Resonance Energy Transfer. Noradrenaline and methoxamine increased α1A-adrenoceptor interaction with Rab5 and Rab7 but did not modify it with Rab9. Oxymetazoline induced adrenoceptor interaction with Rab5 and Rab9 and only an insignificant increase in Rab7 signal. Phorbol myristate acetate increased α1A-adrenoceptor interaction with Rab5 and Rab9 but did not modify it with Rab7. The agonists and the active phorbol ester, all of which induce receptor phosphorylation and internalization, favor receptor interaction with Rab5, i.e., association with early endosomes. Cell stimulation with phorbol myristate acetate induced the α1A-adrenoceptors to interact with the late endosomal marker, Rab9, suggesting that the receptors are directed to slow recycling endosomes once they have transited to the Trans-Golgi network to be retrieved to the plasma membrane. The agonists noradrenaline and methoxamine likely induce a faster recycling and might direct some of the adrenoceptors toward degradation and/or very slow recycling to the plasma membrane. Oxymetazoline produced a mixed pattern of interaction with the Rab proteins. These data indicate that α1A-adrenoceptor agonists can trigger different vesicular traffic and receptor fates within the cells.
Asunto(s)
Agonistas de Receptores Adrenérgicos alfa 1/farmacología , Ésteres del Forbol/farmacología , Receptores Adrenérgicos alfa 1/efectos de los fármacos , Proteínas de Unión al GTP rab/efectos de los fármacos , Calcio/metabolismo , Línea Celular , Endosomas/efectos de los fármacos , Humanos , Proteínas Luminiscentes , Metoxamina/farmacología , Norepinefrina/farmacología , Oximetazolina/farmacología , Fosforilación , Acetato de Tetradecanoilforbol/farmacología , Proteínas de Unión al GTP rab5/efectos de los fármacos , Red trans-Golgi/efectos de los fármacos , Proteína Fluorescente RojaRESUMEN
OBJECTIVE: To explore the specific molecular mechanisms of Danshensu (DSS) in the treatment of ischemia reperfusion injury (IRI). METHODS: IRI model was established with isolated rat hearts by performing global ischaemia for 30 min, and then followed by 60 min reperfusion. Also, H9C2 cells were subjected to 4-h hypoxia followed by 3-h reoxygenation. Then 10 µmol/L DSS were added in the reperfusion/reoxygenation step to intervene IRI. Cardiac function, structural change and apoptosis were respectively tested by Langendorff System, hematoxylin and eosin (HE) and terminal-deoxynucleotidyl transferase mediated nick endabeling (TUNEL) stainings. Then lactate dehydrogenase (LDH), reactive oxygen species (ROS), superoxide gasification enzyme (SOD) and glutathione peroxidase (GSH-PX) were detected by enzyme-linked immunosorbent assay (ELISA). Sirt1/FoxO1/Rab7 Signal Pathway was monitored at both protein and mRNA levels. RESULTS: The results showed that IRI not only greatly attenuated cardiac function (LVDP and ±dp/dtmax, P<0.01, P<0.05) and increased the level of the marker enzymes (cardiac troponin T, LDH, P<0.01) from the coronary effluents, but also markedly induced changes in the structure of cardiomyocytes and contributed to apoptosis, which were mediated by boosted endogenous ROS. However, after treatment with DSS all above indexes were improved, which was related to activating Sirt1/FoxO1/Rab7 signal pathway accompanied with the enhancement of antioxidant defense system, such as superoxide gasification enzyme and glutathione peroxidase. CONCLUSION: DSS is able to protect hearts from IRI, which may be attributable to inhibiting excessive ROS through Sirt1/FoxO1/Rab7 signaling.
Asunto(s)
Lactatos/farmacología , Miocitos Cardíacos/efectos de los fármacos , Daño por Reperfusión , Transducción de Señal/efectos de los fármacos , Animales , Antioxidantes/farmacología , Modelos Animales de Enfermedad , Isquemia , L-Lactato Deshidrogenasa/metabolismo , Miocitos Cardíacos/metabolismo , Proteínas del Tejido Nervioso , Estrés Oxidativo/efectos de los fármacos , Ratas , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/metabolismo , Sirtuina 1/efectos de los fármacos , Proteínas de Unión al GTP rab/efectos de los fármacos , Proteínas de Unión a GTP rab7RESUMEN
Autophagy is an evolutionarily conserved lysosomal degradation pathway, yet the underlying mechanisms remain poorly understood. Nicotinic acid adenine dinucleotide phosphate (NAADP), one of the most potent Ca(2+) mobilizing messengers, elicits Ca(2+) release from lysosomes via the two pore channel 2 (TPC2) in many cell types. Here we found that overexpression of TPC2 in HeLa or mouse embryonic stem cells inhibited autophagosomal-lysosomal fusion, thereby resulting in the accumulation of autophagosomes. Treatment of TPC2 expressing cells with a cell permeant-NAADP agonist, NAADP-AM, further induced autophagosome accumulation. On the other hand, TPC2 knockdown or treatment of cells with Ned-19, a NAADP antagonist, markedly decreased the accumulation of autophagosomes. TPC2-induced accumulation of autophagosomes was also markedly blocked by ATG5 knockdown. Interestingly, inhibiting mTOR activity failed to increase TPC2-induced autophagosome accumulation. Instead, we found that overexpression of TPC2 alkalinized lysosomal pH, and lysosomal re-acidification abolished TPC2-induced autophagosome accumulation. In addition, TPC2 overexpression had no effect on general endosomal-lysosomal degradation but prevented the recruitment of Rab-7 to autophagosomes. Taken together, our data demonstrate that TPC2/NAADP/Ca(2+) signaling alkalinizes lysosomal pH to specifically inhibit the later stage of basal autophagy progression.
Asunto(s)
Álcalis/metabolismo , Autofagia , Canales de Calcio/metabolismo , Lisosomas/metabolismo , Fusión de Membrana , Fagosomas/metabolismo , Animales , Autofagia/efectos de los fármacos , Calcio/farmacología , Diferenciación Celular/efectos de los fármacos , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Endosomas/ultraestructura , Células HeLa , Humanos , Concentración de Iones de Hidrógeno/efectos de los fármacos , Lisosomas/efectos de los fármacos , Lisosomas/ultraestructura , Fusión de Membrana/efectos de los fármacos , Ratones , NADP/análogos & derivados , NADP/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fagosomas/efectos de los fármacos , Fagosomas/ultraestructura , Unión Proteica/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Proteínas de Unión al GTP rab/efectos de los fármacos , Proteínas de Unión a GTP rab7RESUMEN
X-linked nephrogenic diabetes insipidus (XNDI), a severe pathological condition characterized by greatly impaired urine-concentrating ability of the kidney, is caused by inactivating mutations in the V2 vasopressin receptor (V2R) gene. The lack of functional V2Rs prevents vasopressin-induced shuttling of aquaporin-2 (AQP2) water channels to the apical plasma membrane of kidney collecting duct principal cells, thus promoting water reabsorption from urine to the interstitium. At present, no specific pharmacological therapy exists for the treatment of XNDI. We have previously reported that the cholesterol-lowering drug lovastatin increases AQP2 membrane expression in renal cells in vitro. Here we report the novel finding that fluvastatin, another member of the statins family, greatly increases kidney water reabsorption in vivo in mice in a vasopressin-independent fashion. Consistent with this observation, fluvastatin is able to increase AQP2 membrane expression in the collecting duct of treated mice. Additional in vivo and in vitro experiments indicate that these effects of fluvastatin are most likely caused by fluvastatin-dependent changes in the prenylation status of key proteins regulating AQP2 trafficking in collecting duct cells. We identified members of the Rho and Rab families of proteins as possible candidates whose reduced prenylation might result in the accumulation of AQP2 at the plasma membrane. In conclusion, these results strongly suggest that fluvastatin, or other drugs of the statin family, may prove useful in the therapy of XNDI.
Asunto(s)
Acuaporina 2/genética , Diabetes Insípida Nefrogénica/fisiopatología , Ácidos Grasos Monoinsaturados/farmacología , Indoles/farmacología , Túbulos Renales Colectores/metabolismo , Animales , Acuaporina 2/biosíntesis , Membrana Celular/metabolismo , Diabetes Insípida Nefrogénica/tratamiento farmacológico , Diabetes Insípida Nefrogénica/genética , Ácidos Grasos Monoinsaturados/uso terapéutico , Fluvastatina , Indoles/uso terapéutico , Túbulos Renales Colectores/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Prenilación/efectos de los fármacos , Proteínas de Unión al GTP rab/efectos de los fármacos , Proteínas de Unión al GTP rab/metabolismo , Quinasas Asociadas a rho/efectos de los fármacos , Quinasas Asociadas a rho/metabolismoRESUMEN
In plants, Rab and Arf GTPases are key regulators of vesicle trafficking. To investigate whether these small GTPases (SG) play a role in the regulation of the regeneration of latex (the cytoplasm of the rubber-producing laticifer cell) in Hevea brasiliensis (Hevea hereafter), full-length cDNAs that encode four HbRab and two HbArf GTPases were cloned. The four HbRab proteins showed specific GTP-binding activity when expressed in Escherichia coli. Transcript expression of the six SG genes was investigated by real-time RT-PCR. All genes revealed to be expressed in each of the six Hevea tissues examined, but the expression patterns were different. Four genes, HbArf1, HbRab2, HbRab3 and HbRab4, displayed a preferential expression in latex. The expression of all genes was upregulated by the act of latex exploitation (tapping), and HbRab1 had the highest level of upregulation. Wounding markedly upregulated the expression of two SG genes (HbRab1 and HbArf2), and exogenous methyl jasmonate upregulated all six SG genes. Wounding might upregulate the expression of HbRab1 and HbArf2 through a jasmonic acid-mediated signaling pathway. None of the genes were markedly upregulated by Ethrel (an ethylene releaser and latex stimulator); instead, HbArf2 and HbRab4 were downregulated significantly after a 24 h treatment with Ethrel. This paper gives the first description of Rab and Arf GTPases in Hevea and provides clues for their involvement, HbRab1 in particular, in latex regeneration.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Hevea/enzimología , Hevea/genética , Reguladores del Crecimiento de las Plantas/farmacología , Proteínas de Unión al GTP rab/metabolismo , Factores de Ribosilacion-ADP/efectos de los fármacos , Factores de Ribosilacion-ADP/genética , Acetatos/farmacología , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Secuencia de Consenso , Ciclopentanos/farmacología , ADN Complementario/química , ADN Complementario/genética , Flores/efectos de los fármacos , Flores/enzimología , Flores/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas/genética , Hevea/efectos de los fármacos , Látex/química , Látex/metabolismo , Datos de Secuencia Molecular , Compuestos Organofosforados/farmacología , Oxilipinas/farmacología , Filogenia , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Proteínas de Plantas/efectos de los fármacos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tallos de la Planta/efectos de los fármacos , Tallos de la Planta/enzimología , Tallos de la Planta/genética , Estructura Terciaria de Proteína , ARN de Planta/genética , Semillas/efectos de los fármacos , Semillas/enzimología , Semillas/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Proteínas de Unión al GTP rab/efectos de los fármacos , Proteínas de Unión al GTP rab/genéticaRESUMEN
UNLABELLED: C5L2, a G-protein-coupled receptor (GPCR), has been identified as an ASP (C3adesArg) and C5a receptor. Controversy exists regarding both ligand binding and functionality. ASP activation of C5L2 is proposed to regulate fat storage. C5L2 is also proposed as a decoy receptor for C5a, an inflammatory mediator, based on absence of Ca(2+) or chemotaxis changes. AIMS: (i) to evaluate C5L2 receptor activation and recycling using recombinant ASP (rASP) and rC5a and (ii) assess receptor trafficking of S323I-C5L2 mutation previously identified in a family and demonstrated to have altered functionality. RESULTS: stably transfected C5L2-HEK cells were sorted using fluorescent-ASP (Fluos-ASP) binding. Following 2-h serum-free pretreatment, C5L2 was typically localized to the cell-surface. beta-Arrestin-2-GFP transiently transfected C5L2-HEK cells demonstrated rASP and rC5a-dependent beta-arrestin-2-GFP translocation, which showed time-dependent intracellular colocalization with C5L2. Without ligand or C5L2 transfection, no translocation was identified at any time point. Ligand-dependent (rASP and rC5a) C5L2 endocytosis was time-dependent with a 1-h nadir, and was clathrin- and cholesterol-dependent. Transiently transfected Rab-GFP proteins (Rabs 5, 7 and 11) demonstrated time-dependent colocalization of Rab5, Rab7, and Rab11 with C5L2. In contrast to C5L2, a large proportion of stably transfected S323I-C5L2 did not localize to the cell-surface. While S323I-C5L2 was competent for Fluos-ASP and (125)I-ASP binding, although at a reduced level, there was no ligand-mediated receptor phosphorylation. Further, there was no ligand-mediated activation of beta-arrestin-2-GFP translocation, and no downstream functional activation of glucose transport or triglyceride synthesis. CONCLUSION: C5L2 is a functional metabolic receptor, and serine 323 is important for ASP induced functionality.
Asunto(s)
Arrestinas/metabolismo , Complemento C3a/inmunología , Endocitosis/inmunología , Receptores de Quimiocina/inmunología , Proteínas de Unión al GTP rab/metabolismo , Acilación , Arrestinas/efectos de los fármacos , Línea Celular , Complemento C3a/farmacología , Complemento C5a/farmacología , Endocitosis/efectos de los fármacos , Humanos , Mutación , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Receptor de Anafilatoxina C5a , Receptores de Quimiocina/agonistas , Receptores de Quimiocina/genética , Proteínas Recombinantes/farmacología , Serina/genética , Transfección , Arrestina beta 2 , beta-Arrestinas , Proteínas de Unión al GTP rab/efectos de los fármacosRESUMEN
Small GTP-binding protein, Rab27, has been implicated in the regulation of different types of membrane trafficking, including melanosome transport in melanocytes and regulated secretion events in a wide variety of secretory cells. We have previously shown that Rab27 is involved in the control of isoproterenol (IPR)-induced amylase release from rat parotid acinar cells. Although Rab27 is predominantly localized on secretory granules under resting conditions, changes to its intracellular localization after beta-stimulation have never been elucidated. The present study investigated IPR-induced redistribution of Rab27B in the parotid acinar cells, revealing translocation from secretory granules to the subapical region after 5 min of IPR treatment and then diffusion into the cytosol after 30 min of IPR treatment. Dissociation of Rab27B from the apical plasma membrane is probably mediated through the Rab GDP dissociation inhibitor (GDI) in the cytosol extracting GDP-bound Rab protein from membranes, as a dramatic increase in the amount of the Rab27B-GDI complex in the cytosol was observed 30 min after stimulation with IPR. These results indicate that, in parotid acinar cells, Rab27B is translocated, in a time-dependent manner, from secretory granules into the apical plasma membrane as a result of exposure to IPR, and then into the cytosol through binding with the GDI.
Asunto(s)
Agonistas Adrenérgicos beta/farmacología , Isoproterenol/farmacología , Glándula Parótida/efectos de los fármacos , Proteínas de Unión al GTP rab/efectos de los fármacos , Amilasas/análisis , Animales , Biomarcadores/análisis , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Células Cultivadas , Citosol/efectos de los fármacos , Citosol/enzimología , Inhibidores de Disociación de Guanina Nucleótido/farmacología , Glándula Parótida/citología , Glándula Parótida/enzimología , Ratas , Vesículas Secretoras/efectos de los fármacos , Vesículas Secretoras/enzimología , Factores de Tiempo , Proteína 2 de Membrana Asociada a Vesículas/análisis , Proteínas de Unión al GTP rab/farmacocinéticaRESUMEN
AIM: To elucidate the role of Rab23 in hepatocellular carcinoma (HCC) by assessing the expression of Rab23 in HCC tissue and in HCC cell lines. METHODS: Primary tumors (n = 100) were stained with Rab23 antibodies using immunohistochemistry and in situ hybridization in tissue microarrays. Relationships between gene expression and pathology parameters were analysed. The biological significance of Rab23 in Hep-3B cells was examined by knocking down Rab23 gene expression. We designed a pair of double-stranded RNAs against human rab23 and transfected siRNA into Hep-3B cells. Rab23 expression in these cells was examined using RT-PCR and Western blots. We investigated cell growth by MTT assays and fluorescence-activated cell sorting. RESULTS: High cytoplasmic and nuclear expression of Rab23 was found in 38 of 71 (53.5%) and in 49 of 68 HCC patients (72%) respectively, which correlated with tumor size. HCC cell lines expressed Rab23. In Hep3B cells, siRNA for Rab23 decreased Rab23 mRNA by 4.5-fold and protein expression by 2-fold. Survival rates at 24 and 48 h for Hep-3B cells transfected with siRNA were lower and about 30% Hep-3B cells were apoptotic. Knocking down rab23 suppressed Hep3B cell growth, suggesting that rab23 could play an important role in Hep3B cell growth. CONCLUSION: Rab23 is overexpressed and/or activated in HCC. Rab23 may be both a HCC predictor and a target for treating HCC.
Asunto(s)
Antineoplásicos/uso terapéutico , Carcinoma Hepatocelular/etiología , Neoplasias Hepáticas/etiología , Proteínas de Unión al GTP rab/efectos de los fármacos , Proteínas de Unión al GTP rab/fisiología , Apoptosis/efectos de los fármacos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/fisiopatología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas Hedgehog/fisiología , Humanos , Hígado/citología , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/fisiopatología , ARN Interferente Pequeño/farmacología , Transducción de Señal/fisiología , Transfección , Proteínas de Unión al GTP rab/genéticaRESUMEN
Cells require growth factors to support glucose metabolism for survival and growth. It is unclear, however, how noninsulin growth factors may regulate glucose uptake and glucose transporters. We show that the hematopoietic growth factor interleukin (IL)3, maintained the glucose transporter Glut1 on the cell surface and promoted Rab11a-dependent recycling of intracellular Glut1. IL3 required phosphatidylinositol-3 kinase activity to regulate Glut1 trafficking, and activated Akt was sufficient to maintain glucose uptake and surface Glut1 in the absence of IL3. To determine how Akt may regulate Glut1, we analyzed the role of Akt activation of mammalian target of rapamycin (mTOR)/regulatory associated protein of mTOR (RAPTOR) and inhibition of glycogen synthase kinase (GSK)3. Although Akt did not require mTOR/RAPTOR to maintain surface Glut1 levels, inhibition of mTOR/RAPTOR by rapamycin greatly diminished glucose uptake, suggesting Akt-stimulated mTOR/RAPTOR may promote Glut1 transporter activity. In contrast, inhibition of GSK3 did not affect Glut1 internalization but nevertheless maintained surface Glut1 levels in IL3-deprived cells, possibly via enhanced recycling of internalized Glut1. In addition, Akt attenuated Glut1 internalization through a GSK3-independent mechanism. These data demonstrate that intracellular trafficking of Glut1 is a regulated component of growth factor-stimulated glucose uptake and that Akt can promote Glut1 activity and recycling as well as prevent Glut1 internalization.
Asunto(s)
Citocinas/farmacología , Transportador de Glucosa de Tipo 1/metabolismo , Glucosa/farmacocinética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Línea Celular , Membrana Celular/metabolismo , Citocinas/metabolismo , Transportador de Glucosa de Tipo 1/efectos de los fármacos , Glucógeno Sintasa Quinasa 3/metabolismo , Células Madre Hematopoyéticas/citología , Interleucina-3/metabolismo , Interleucina-3/farmacología , Ratones , Proteínas Quinasas/efectos de los fármacos , Proteínas Quinasas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/genética , Sirolimus/farmacología , Serina-Treonina Quinasas TOR , Proteínas de Unión al GTP rab/efectos de los fármacos , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Retinoic acid (RA), used as first-line therapy for acute promyelocytic leukemia (APL), exerts its antileukemic activity by inducing blast differentiation and activating tumor-selective TNF-related apoptosis-inducing ligand (TRAIL) signaling. To identify downstream mediators of RA signaling, we used retrovirus-mediated insertion mutagenesis in PLB985 leukemia cells and established the RA-resistant cell line WY-1. In PLB985, but not WY-1 cells, RA induced TRAIL and its DR4 and DR5 receptors. Knocking down TRAIL expression by RNA interference blocked RA-induced apoptosis. WY-1 cells are defective for RA-induced differentiation, G1 arrest and exhibit co-resistance to TRAIL. In WY-1 cells, a single virus copy is integrated into a novel RA-regulated gene termed RAM (retinoic acid modulator). RAM is expressed in the myelomonocytic lineage and extinguished by RA in PLB985, but not WY-1 cells. Whereas knocking down RAM expression by RNA interference promoted RA-induced differentiation and TRAIL-triggered apoptosis of PLB985 and WY-1 cells, overexpression of the predicted 109 amino-acid RAM open reading frame did not alter RA signaling in PLB985 cells. This indicates that, apart from encoding the putative RAM protein, RAM RNA may exert additional functions that are impaired by the retrovirus insertion. Our study demonstrates that RA induction of the TRAIL pathway is also operative in leukemia cells lacking an RARalpha oncofusion protein and identifies RAM as a novel RA-dependent modulator of myeloid differentiation and death.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Resistencia a Antineoplásicos/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Glicoproteínas de Membrana/metabolismo , Tretinoina/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas de Unión al GTP rab/genética , Secuencia de Aminoácidos , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/farmacología , Secuencia de Bases , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/genética , Leucemia Mieloide/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/farmacología , Datos de Secuencia Molecular , Mutagénesis Insercional , ARN Largo no Codificante , Receptores de Ácido Retinoico/efectos de los fármacos , Receptores de Ácido Retinoico/genética , Receptores de Ácido Retinoico/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF , Receptores del Factor de Necrosis Tumoral/efectos de los fármacos , Receptores del Factor de Necrosis Tumoral/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Receptor alfa de Ácido Retinoico , Receptores X Retinoide/efectos de los fármacos , Receptores X Retinoide/metabolismo , Transducción de Señal , Ligando Inductor de Apoptosis Relacionado con TNF , Tretinoina/metabolismo , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/farmacología , Proteínas de Unión al GTP rab/efectos de los fármacos , Proteínas de Unión al GTP rab/metabolismo , Proteínas rab27 de Unión a GTPRESUMEN
Chronic exposure of pubertal male rats to ethanol results in a decline in serum testosterone, increased gonadotropins, pituitary luteinizing hormone (LH) and follicle stimulating hormone (FSH) content, and decreased or inappropriately normal serum LH and FSH levels, suggesting impaired secretory release of gonadotropins. The molecular mechanisms behind this disorder are undefined, but a disruption of vesicle-mediated secretory processes is possible because intracellular protein trafficking pathways are involved in secretion of glycoproteins such as FSH and LH. Because small GTP-binding proteins of Rab family have been implicated as key regulators of membrane and protein trafficking in mammalian cells, this study was designed to test if ethanol-impaired pituitary FSH and LH secretion is associated with changes in Rab proteins, particularly Rab1B, Rab3B, Rab6, and Rab11. Male Sprague-Dawley rats 35 days old were pair-fed a Lieber-DeCarli diet with ethanol or without ethanol for 5 to 60 days. After ethanol exposure, serum testosterone levels decreased while LH and FSH were inappropriately unchanged. Immunohistochemical staining showed decreased Rab1B, Rab3B, and Rab11 protein levels in ethanol-treated pituitaries. Immunoblotting showed that ethanol induced a transient reduction in Rab6 after 5 days of ethanol exposure, whereas Rab3B decreased after 20 days, Rab11 after 30 days, and Rab1B after 60 days. Despite these changes in Rab proteins, mRNA levels were unaffected by ethanol exposure. We concluded that reductions in key Rab proteins may lead to altered vesicle trafficking and may play a role in disruption of pituitary FSH and LH secretion caused by ethanol.
Asunto(s)
Depresores del Sistema Nervioso Central/toxicidad , Etanol/toxicidad , Enfermedades de la Hipófisis/inducido químicamente , Proteínas de Unión al GTP rab/metabolismo , Animales , Western Blotting , Peso Corporal/efectos de los fármacos , Hormona Folículo Estimulante/sangre , Inmunohistoquímica , Hormona Luteinizante/sangre , Masculino , Enfermedades de la Hipófisis/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Testosterona/sangre , Factores de Tiempo , Proteínas de Unión al GTP rab/biosíntesis , Proteínas de Unión al GTP rab/efectos de los fármacosRESUMEN
Ethanol exposure induces retention of glycoproteins in growing astrocytes. We examined the intracellular sites at which this retention occurs and investigated whether this effect is accompanied by alterations in the Golgi complex and microtubular system. We studied the effects of ethanol on the Golgi complex structure, as well as on the secretory pathway functionality by monitoring both the transport of the VSV-G protein and the protein levels of several molecules involved in the regulation of this pathway. Ethanol was found to delay VSV-G transport, modify Golgi complex morphology, and reduce the number of secretory vesicles. Moreover, ethanol affected the levels of mannosidase II, p58, betaCOP, rbet1, and several Rab GTPases. It also affected microtubule organization and polymerization and the levels of the motor proteins kinesin and dynein. Most of these effects were dose-dependent. These alterations, together with those previously reported concerning biosynthesis of glycoconjugates, provide novel insights into how ethanol impairs brain development.
Asunto(s)
Astrocitos/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/crecimiento & desarrollo , Etanol/toxicidad , Aparato de Golgi/efectos de los fármacos , Trastornos del Sistema Nervioso Inducidos por Alcohol/metabolismo , Trastornos del Sistema Nervioso Inducidos por Alcohol/fisiopatología , Animales , Astrocitos/metabolismo , Encéfalo/fisiopatología , Células Cultivadas , Proteína Coatómero/efectos de los fármacos , Proteína Coatómero/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Trastornos del Espectro Alcohólico Fetal/metabolismo , Trastornos del Espectro Alcohólico Fetal/fisiopatología , Aparato de Golgi/metabolismo , Lectinas de Unión a Manosa/efectos de los fármacos , Lectinas de Unión a Manosa/metabolismo , Manosidasas/efectos de los fármacos , Manosidasas/metabolismo , Proteínas de la Membrana/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Proteínas Motoras Moleculares/efectos de los fármacos , Proteínas Motoras Moleculares/metabolismo , Embarazo , Efectos Tardíos de la Exposición Prenatal/metabolismo , Efectos Tardíos de la Exposición Prenatal/fisiopatología , Proteínas Qc-SNARE/efectos de los fármacos , Proteínas Qc-SNARE/metabolismo , Ratas , Vesículas Secretoras/efectos de los fármacos , Vesículas Secretoras/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Proteínas de Transporte Vesicular/efectos de los fármacos , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión al GTP rab/efectos de los fármacos , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Mammalian cells cultured in the presence of high concentrations of sucrose demonstrate large, phase-lucent, osmotically swollen vacuoles. Three normal human fibroblast cell lines exposed to 100 mM of sucrose for 24 h demonstrated increased expression of lysosomal, intracellular vesicle trafficking, cholesterol biosynthesis, and fatty acid metabolism genes. Most steps of the cholesterol biosynthesis pathway were upregulated including HMG CoA reductase, which catalyzes the rate-limiting step of cholesterol biosynthesis. The lysosomal genes neuraminidase, CLN3, and CLCN5 and the small GTP-binding proteins Rab7L1 and Arl7 were also increased. A Rab7L1-GFP fusion protein was overexpressed in human fibroblasts and was demonstrated to localize primarily to the Golgi apparatus, and in some cells to the membranes bounding vesicles in the perinuclear region. Increased levels of the transcription factor C/EBP were found in nuclear extracts from cells exposed to sucrose for 12 h, relative to matched controls suggesting regulation of gene expression following sucrose-induced vacuolation may be coordinated, at least in part, by the transcription factor C/EBP. Sucrose-induced vacuolation is a useful model in which to study the regulation of lysosomal gene expression and biogenesis.
Asunto(s)
Colesterol/genética , Fibroblastos/metabolismo , Lisosomas/genética , Glicoproteínas de Membrana , Chaperonas Moleculares , Sacarosa/farmacología , Vacuolas/metabolismo , Extractos Celulares/química , Línea Celular , Núcleo Celular/química , Canales de Cloruro/efectos de los fármacos , Canales de Cloruro/metabolismo , Colesterol/biosíntesis , Colesterol/metabolismo , Ácidos Grasos/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Regulación de la Expresión Génica , Aparato de Golgi/metabolismo , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/metabolismo , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Neuraminidasa/efectos de los fármacos , Neuraminidasa/metabolismo , Proteínas/efectos de los fármacos , Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Vacuolas/efectos de los fármacos , Proteínas de Unión al GTP rab/efectos de los fármacos , Proteínas de Unión al GTP rab/metabolismoAsunto(s)
Proteínas de Unión al GTP/efectos de los fármacos , Proteínas de Unión al GTP/metabolismo , Prenilación de Proteína , Transferasas Alquil y Aril/metabolismo , Farnesiltransferasa , Proteínas de Unión al GTP/química , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Prenilación de Proteína/efectos de los fármacos , Proteínas de Unión al GTP rab/efectos de los fármacos , Proteínas de Unión al GTP rab/metabolismo , Proteínas ras/efectos de los fármacos , Proteínas ras/metabolismo , Proteínas de Unión al GTP rho/efectos de los fármacos , Proteínas de Unión al GTP rho/metabolismoAsunto(s)
Leishmania mexicana/fisiología , Leucina/análogos & derivados , Leucina/toxicidad , Proteínas de Unión al GTP rab/fisiología , Animales , Cinética , Leishmania mexicana/efectos de los fármacos , Mutagénesis , Proteínas Protozoarias/efectos de los fármacos , Proteínas Protozoarias/genética , Proteínas Protozoarias/fisiología , Proteínas de Unión al GTP rab/efectos de los fármacos , Proteínas de Unión al GTP rab/genética , Proteínas de Unión a GTP rab7RESUMEN
Previously, we showed that live Salmonella-containing phagosomes (LSP) recruit early acting Rab5 and promote fusion with early endosomes, thus avoiding transport to the lysosomes. Therefore, live Salmonella survive in a specialized compartment. Here we show that scavenger-receptor-mediated intracellular delivery of muramyl dipeptide (MDP) to macrophages leads to efficient killing of Salmonella both in vitro and in vivo. To understand the intracellular trafficking modulation of Salmonella by delivery of MDP, we investigated the levels of endocytic Rab proteins, which are the major regulators of vesicular transport. Western blot analysis reveals reduced Rab5 and enhanced Rab7 content in the maleylated bovine serum albumin-MDP (MBSA-MDP)-treated cells. The reduced content of Rab5 in the treated cells and on phagosomes inhibits the fusion of Salmonella-containing phagosomes with early endosomes, and the enhanced Rab7 content in these cells facilitated targeting of LSP to lysosomes, which contain cathepsin D and vacuolar ATPase, for killing. In vitro reconstitution of lysosomal transport demonstrated that a reduced content of Rab5 and an enhanced level of Rab7 in MBSA-MDP-treated cells is primarily responsible for targeting Salmonella to lysosomes. Intracellular delivery of MDP thus offers a general strategy against macrophage-associated infections caused by intracellular pathogens that survive in the host cell by resisting transport to lysosomes.
Asunto(s)
Acetilmuramil-Alanil-Isoglutamina/farmacología , Lisosomas/metabolismo , Macrófagos/metabolismo , Fagocitosis/fisiología , Infecciones por Salmonella/metabolismo , Salmonella typhimurium/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Células Cultivadas , Interacciones Huésped-Parásitos/efectos de los fármacos , Interacciones Huésped-Parásitos/fisiología , Lisosomas/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Fagocitosis/efectos de los fármacos , Fagosomas/efectos de los fármacos , Fagosomas/metabolismo , Infecciones por Salmonella/tratamiento farmacológico , Infecciones por Salmonella/inmunología , Salmonella typhimurium/inmunología , Proteínas de Unión al GTP rab/efectos de los fármacos , Proteínas de Unión al GTP rab5/efectos de los fármacos , Proteínas de Unión al GTP rab5/metabolismo , Proteínas de Unión a GTP rab7RESUMEN
An in vitro model of GH-responsive cells was subjected to microarray analysis to identify a novel gene regulated by GH. This 258 amino acid protein, we term GH Regulated TBC Protein-1 (GRTP1), contains the TBC signature motif of GTPase activator proteins of Rab-like small GTPases. Northern blot analysis revealed a 1.3 kb major mRNA species, most abundant in testes. TaqMan assay confirmed that in the mouse, Grtp1 is expressed at highest levels in testes, with lesser abundance in intestine, kidney, lung, and liver. In the testis, expression of Grtp1 significantly increases post-pubertally. Administration of GH to mice increased levels of GRTP1 mRNA in testes (140%), but decreased GRTP1 mRNA abundance in kidney (50%) and liver (25%). Grtp1 was localized to mouse proximal chromosome 8. Orthologs of this protein are present in human, mouse, rat, and drosophila suggesting that GRTP1 has an important biological role(s).
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Hormona del Crecimiento/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , Humanos , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , Ratas , Proteínas de Unión al GTP rab/efectos de los fármacos , Proteínas de Unión al GTP rab/genéticaRESUMEN
The Rab/Ypt small G proteins are essential for intracellular vesicle trafficking in mammals and yeast. The vesicle-docking process requires that Ypt proteins are located in the vesicle membrane. C-terminal geranylgeranyl anchors mediate the membrane attachment of these proteins. The Rab escort protein (REP) is essential for the recognition of Rab/Ypt small G proteins by geranylgeranyltransferase II (GGTase II) and for their delivery to acceptor membranes. What effect an alteration in the levels of prenylated Rab/Ypt proteins has on vesicle transport or other cellular processes is so far unknown. Here, we report the characterization of a yeast REP mutant, mrs6-2, in which reduced prenylation of Ypt proteins occurs even at the permissive temperature. A shift to the restrictive temperature does not alter exponential growth during the first 3 h. The amount of Sec4p, but not Ypt1p, bound to vesicle membranes is reduced 2.5 h after the shift compared with wild-type or mrs6-2 cells incubated at 25 degrees C. In addition, vesicles fail to be polarized towards the bud and small budded binucleate cells accumulate at this time point. Growth in 1 M sorbitol or overexpression of MLC1, encoding a myosin light chain able to bind the unconventional type V myosin Myo2, or of genes involved in cell wall maintenance, such as SLG1, GFA1 and LRE1, suppresses mrs6-2 thermosensitivity. Our data suggest that, at least at high temperature, a critical minimal level of Ypt protein prenylation is required for maintaining vesicle polarization.