RESUMEN
Mitochondrial disorders are a group of pathologies characterized by impairment of mitochondrial function mainly due to defects of the respiratory chain and consequent organellar energetics. This affects organs and tissues that require an efficient energy supply, such as brain and skeletal muscle. They are caused by mutations in both nuclear- and mitochondrial DNA (mtDNA)-encoded genes and their clinical manifestations show a great heterogeneity in terms of age of onset and severity, suggesting that patient-specific features are key determinants of the pathogenic process. In order to correlate the genetic defect to the clinical phenotype, we used a cell culture model consisting of fibroblasts derived from patients with different mutations in the mtDNA-encoded ND5 complex I subunit and with different severities of the illness. Interestingly, we found that cells from patients with the 13514A>G mutation, who manifested a relatively late onset and slower progression of the disease, display an increased autophagic flux when compared with fibroblasts from other patients or healthy donors. We characterized their mitochondrial phenotype by investigating organelle turnover, morphology, membrane potential and Ca(2+) homeostasis, demonstrating that mitochondrial quality control through mitophagy is upregulated in 13514A>G cells. This is due to a specific downregulation of mitochondrial Ca(2+) uptake that causes the stimulation of the autophagic machinery through the AMPK signaling axis. Genetic and pharmacological manipulation of mitochondrial Ca(2+) homeostasis can revert this phenotype, but concurrently decreases cell viability. This indicates that the higher mitochondrial turnover in complex I deficient cells with this specific mutation is a pro-survival compensatory mechanism that could contribute to the mild clinical phenotype of this patient.
Asunto(s)
Autofagia , Señalización del Calcio , Complejo I de Transporte de Electrón/genética , Fibroblastos/fisiología , Proteínas Mitocondriales/genética , Calcio/metabolismo , Canales de Calcio/metabolismo , Células Cultivadas , Complejo I de Transporte de Electrón/metabolismo , Homeostasis , Humanos , Potencial de la Membrana Mitocondrial , Mitocondrias/metabolismo , Enfermedades Mitocondriales/enzimología , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/patología , Dinámicas Mitocondriales , Proteínas Mitocondriales/metabolismo , Mutación Puntual , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismoRESUMEN
During ageing skeletal muscles undergo a process of structural and functional remodelling that leads to sarcopenia, a syndrome characterized by loss of muscle mass and force and a major cause of physical frailty. To determine the causes of sarcopenia and identify potential targets for interventions aimed at mitigating ageing-dependent muscle wasting, we focussed on the main signalling pathway known to control protein turnover in skeletal muscle, consisting of the insulin-like growth factor 1 (IGF1), the kinase Akt and its downstream effectors, the mammalian target of rapamycin (mTOR) and the transcription factor FoxO. Expression analyses at the transcript and protein level, carried out on well-characterized cohorts of young, old sedentary and old active individuals and on mice aged 200, 500 and 800 days, revealed only modest age-related differences in this pathway. Our findings suggest that during ageing there is no downregulation of IGF1/Akt pathway and that sarcopenia is not due to FoxO activation and upregulation of the proteolytic systems. A potentially interesting result was the increased phosphorylation of the ribosomal protein S6, indicative of increased activation of mTOR complex1 (mTORC1), in aged mice. This result may provide the rationale why rapamycin treatment and caloric restriction promote longevity, since both interventions blunt activation of mTORC1; however, this change was not statistically significant in humans. Finally, genetic perturbation of these pathways in old mice aimed at promoting muscle hypertrophy via Akt overexpression or preventing muscle loss through inactivation of the ubiquitin ligase atrogin1 were found to paradoxically cause muscle pathology and reduce lifespan, suggesting that drastic activation of the IGF1-Akt pathway may be counterproductive, and that sarcopenia is accelerated, not delayed, when protein degradation pathways are impaired.
Asunto(s)
Envejecimiento/fisiología , Factores de Transcripción Forkhead/fisiología , Factor I del Crecimiento Similar a la Insulina/fisiología , Músculo Esquelético/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Transducción de Señal/fisiología , Serina-Treonina Quinasas TOR/fisiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Proteína 7 Relacionada con la Autofagia , Femenino , Proteína Forkhead Box O1 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/fisiología , Modelos Animales , Proteínas Musculares/genética , Proteínas Musculares/fisiología , Proteínas Ligasas SKP Cullina F-box/genética , Proteínas Ligasas SKP Cullina F-box/fisiología , Sarcopenia/fisiopatología , Serpina E2/genética , Serpina E2/fisiología , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/fisiología , Adulto JovenRESUMEN
Endoplasmic reticulum stress is emerging as an important modulator of different pathologies and as a mechanism contributing to cancer cell death in response to therapeutic agents. In several instances, oxidative stress and the onset of endoplasmic reticulum (ER) stress occur together; yet, the molecular events linking reactive oxygen species (ROS) to ER stress-mediated apoptosis are currently unknown. Here, we show that PERK (RNA-dependent protein kinase (PKR)-like ER kinase), a key ER stress sensor of the unfolded protein response, is uniquely enriched at the mitochondria-associated ER membranes (MAMs). PERK(-/-) cells display disturbed ER morphology and Ca(2+) signaling as well as significantly weaker ER-mitochondria contact sites. Re-expression of a kinase-dead PERK mutant but not the cytoplasmic deletion mutant of PERK in PERK(-/-) cells re-establishes ER-mitochondria juxtapositions and mitochondrial sensitization to ROS-mediated stress. In contrast to the canonical ER stressor thapsigargin, during ROS-mediated ER stress, PERK contributes to apoptosis twofold by sustaining the levels of pro-apoptotic C/EBP homologous protein (CHOP) and by facilitating the propagation of ROS signals between the ER and mitochondria through its tethering function. Hence, this study reveals an unprecedented role of PERK as a MAMs component required to maintain the ER-mitochondria juxtapositions and propel ROS-mediated mitochondrial apoptosis. Furthermore, it suggests that loss of PERK may cause defects in cell death sensitivity in pathological conditions linked to ROS-mediated ER stress.
Asunto(s)
Apoptosis/efectos de los fármacos , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , eIF-2 Quinasa/metabolismo , Animales , Señalización del Calcio , Línea Celular , Estrés del Retículo Endoplásmico/efectos de los fármacos , Células HCT116 , Humanos , Luz , Ratones , Membranas Mitocondriales/metabolismo , Tapsigargina/farmacología , Factor de Transcripción CHOP/metabolismo , Respuesta de Proteína Desplegada , eIF-2 Quinasa/antagonistas & inhibidores , eIF-2 Quinasa/genéticaRESUMEN
Transmembrane BAX inhibitor motif-containing (TMBIM)-6, also known as BAX-inhibitor 1 (BI-1), is an anti-apoptotic protein that belongs to a putative family of highly conserved and poorly characterized genes. Here we report the function of TMBIM3/GRINA in the control of cell death by endoplasmic reticulum (ER) stress. Tmbim3 mRNA levels are strongly upregulated in cellular and animal models of ER stress, controlled by the PERK signaling branch of the unfolded protein response. TMBIM3/GRINA synergies with TMBIM6/BI-1 in the modulation of ER calcium homeostasis and apoptosis, associated with physical interactions with inositol trisphosphate receptors. Loss-of-function studies in D. melanogaster demonstrated that TMBIM3/GRINA and TMBIM6/BI-1 have synergistic activities against ER stress in vivo. Similarly, manipulation of TMBIM3/GRINA levels in zebrafish embryos revealed an essential role in the control of apoptosis during neuronal development and in experimental models of ER stress. These findings suggest the existence of a conserved group of functionally related cell death regulators across species beyond the BCL-2 family of proteins operating at the ER membrane.
Asunto(s)
Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Respuesta de Proteína Desplegada/genética , Factor de Transcripción Activador 4/metabolismo , Animales , Apoptosis , Drosophila melanogaster , Estrés del Retículo Endoplásmico , Fibroblastos/metabolismo , Células HEK293 , Células HeLa , Homeostasis , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Ratones , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Pez Cebra , eIF-2 Quinasa/metabolismoRESUMEN
Voltage-dependent anion channels (VDACs) are expressed in three isoforms, with common channeling properties and different roles in cell survival. We show that VDAC1 silencing potentiates apoptotic challenges, whereas VDAC2 has the opposite effect. Although all three VDAC isoforms are equivalent in allowing mitochondrial Ca(2+) loading upon agonist stimulation, VDAC1 silencing selectively impairs the transfer of the low-amplitude apoptotic Ca(2+) signals. Co-immunoprecipitation experiments show that VDAC1, but not VDAC2 and VDAC3, forms complexes with IP(3) receptors, an interaction that is further strengthened by apoptotic stimuli. These data highlight a non-redundant molecular route for transferring Ca(2+) signals to mitochondria in apoptosis.
Asunto(s)
Apoptosis , Señalización del Calcio , Calcio/metabolismo , Mitocondrias/metabolismo , Canal Aniónico 1 Dependiente del Voltaje/metabolismo , Apoptosis/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Células HeLa , Humanos , Peróxido de Hidrógeno/farmacología , Inmunoprecipitación , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Mitocondrias/efectos de los fármacos , Isoformas de Proteínas/metabolismoRESUMEN
Despite advanced knowledge on the genetic basis of oxidative phosphorylation-related diseases, the molecular and/or cellular determinants for tissue-specific dysfunction are not completely understood. Here, we report the cellular events associated with mitochondrial respiratory Complex II deficiency occurring before cell death. Mutation or chronic inhibition of Complex II determined a large increase of basal and agonist-evoked Ca(2+) signals in the cytosol and the mitochondria, in parallel with mitochondrial dysfunction characterized by membrane potential (Δψ(mit)) loss, [ATP] reduction and increased reactive oxygen species production. Cytosolic and mitochondrial Ca(2+) overload are linked to increased endoplasmic reticulum (ER) Ca(2+) leakage, and to SERCA2b and PMCA proteasome-dependent degradation. Increased [Ca(2+)](mit) is also contributed by decreased mitochondrial motility and increased ER-mitochondria contact sites. Interestingly, increased intracellular [Ca(2+)] activated on the one hand a compensatory Ca(2+)-dependent glycolytic ATP production and determined on the second hand mitochondrial pathology. These results revealed the primary function for Ca(2+) signalling in the control of mitochondrial dysfunction and cellular bioenergetics outcomes linked to respiratory chain Complex II deficiency.
Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Complejo II de Transporte de Electrones/metabolismo , Metabolismo Energético , Mitocondrias/metabolismo , Adenosina Trifosfato/metabolismo , Apoptosis , Células Cultivadas , Regulación hacia Abajo , Complejo II de Transporte de Electrones/deficiencia , Complejo II de Transporte de Electrones/genética , Retículo Endoplásmico/metabolismo , Fibroblastos/metabolismo , Humanos , Potencial de la Membrana Mitocondrial/fisiología , Nitrocompuestos/farmacología , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Propionatos/farmacología , Piridonas/farmacología , Especies Reactivas de Oxígeno/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismoRESUMEN
Mammalian life span can be controlled by p66Shc protein through regulation of cellular response to oxidative stress. We investigated age-related changes in the amount of p66Shc and its Ser36-phosphorylated form in various mouse organs and tissues and correlated it with the level of antioxidant enzymes. Comparing to the newborn, in adult 6-month-old mice, the level of p66Shc was increased particularly in liver, lungs, skin and diaphragm. In older animals the level of p66Shc decreased while signaling pathway responsible for Ser36 phosphorylation of p66Shc protein seemed to be continually enhanced. The amount of p66Shc phosphorylated at Ser36, significantly increased with age, resulted in higher free radical production and, in consequence accumulation of damages caused by free radicals. The increased amount of Ser36-phosphorylated p66Shc in livers of 12- and 23-month-old mice was correlated with the decreased level of antioxidant enzymes. Moreover, we found that p66Shc is a resident of mitochondria- and plasma membrane-associated membranes and that its level there depends on the age of animal.
Asunto(s)
Envejecimiento/metabolismo , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Animales , Animales Recién Nacidos , Antioxidantes/metabolismo , Células Cultivadas , Femenino , Radicales Libres/metabolismo , Hígado/metabolismo , Ratones , Modelos Biológicos , Fosforilación , Serina/química , Proteínas Adaptadoras de la Señalización Shc/química , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Fracciones Subcelulares/metabolismo , Distribución TisularRESUMEN
There is a growing consensus that the various forms of cell death (necrosis, apoptosis and autophagy) are not separated by strict boundaries, but rather share molecular effectors and signaling routes. Among the latter, a clear role is played by calcium (Ca(2+)), the ubiquitous second messenger involved in the control of a broad variety of physiological events. Fine tuning of intracellular Ca(2+) homeostasis by anti- and proapoptotic proteins shapes the Ca(2+) signal to which mitochondria and other cellular effectors are exposed, and hence the efficiency of various cell death inducers. Here, we will review: (i) the evidence linking calcium homeostasis to the regulation of apoptotic, and more recently autophagic cell death, (ii) the discussion of mitochondria as a critical, although not unique checkpoint and (iii) the molecular and functional elucidation of ER/mitochondria contacts, corresponding to the mitochondria-associated membrane (MAM) subfraction and proposed to be a specialized signaling microdomain.
Asunto(s)
Apoptosis/fisiología , Calcio/fisiología , Retículo Endoplásmico/metabolismo , Animales , Calcio/metabolismo , Citosol/metabolismo , HumanosRESUMEN
OBJECTIVES: Polycystin-1 (PC1), a signalling receptor regulating Ca(2+)-permeable cation channels, is mutated in autosomal dominant polycystic kidney disease, which is typically characterized by increased cell proliferation. However, the precise mechanisms by which PC1 functions on Ca(2+) homeostasis, signalling and cell proliferation remain unclear. Here, we investigated the possible role of PC1 as a modulator of non-capacitative Ca(2+) entry (NCCE) and Ca(2+) oscillations, with downstream effects on cell proliferation. RESULTS AND DISCUSSION: By employing RNA interference, we show that depletion of endogenous PC1 in HEK293 cells leads to an increase in serum-induced Ca(2+) oscillations, triggering nuclear factor of activated T cell activation and leading to cell cycle progression. Consistently, Ca(2+) oscillations and cell proliferation are increased in PC1-mutated kidney cystic cell lines, but both abnormal features are reduced in cells that exogenously express PC1. Notably, blockers of the NCCE pathway, but not of the CCE, blunt abnormal oscillation and cell proliferation. Our study therefore provides the first demonstration that PC1 modulates Ca(2+) oscillations and a molecular mechanism to explain the association between abnormal Ca(2+) homeostasis and cell proliferation in autosomal dominant polycystic kidney disease.
Asunto(s)
Señalización del Calcio , Riñón/patología , Canales Catiónicos TRPP/metabolismo , Línea Celular , Línea Celular Transformada , Proliferación Celular , Codón sin Sentido/genética , Citoplasma/metabolismo , Activación Enzimática , Humanos , Riñón/enzimología , Modelos Biológicos , Factores de Transcripción NFATC/metabolismo , Riñón Poliquístico Autosómico Dominante/enzimología , Riñón Poliquístico Autosómico Dominante/patología , Proteína Quinasa C-alfa/metabolismo , Interferencia de ARNRESUMEN
The reduction of intracellular 1,4,5-inositol trisphosphate (IP(3)) levels stimulates autophagy, whereas the enhancement of IP(3) levels inhibits autophagy induced by nutrient depletion. Here, we show that knockdown of the IP(3) receptor (IP(3)R) with small interfering RNAs and pharmacological IP(3)R blockade is a strong stimulus for the induction of autophagy. The IP(3)R is known to reside in the membranes of the endoplasmic reticulum (ER) as well as within ER-mitochondrial contact sites, and IP(3)R blockade triggered the autophagy of both ER and mitochondria, as exactly observed in starvation-induced autophagy. ER stressors such as tunicamycin and thapsigargin also induced autophagy of ER and, to less extent, of mitochondria. Autophagy triggered by starvation or IP(3)R blockade was inhibited by Bcl-2 and Bcl-X(L) specifically targeted to ER but not Bcl-2 or Bcl-X(L) proteins targeted to mitochondria. In contrast, ER stress-induced autophagy was not inhibited by Bcl-2 and Bcl-X(L). Autophagy promoted by IP(3)R inhibition could not be attributed to a modulation of steady-state Ca(2+) levels in the ER or in the cytosol, yet involved the obligate contribution of Beclin-1, autophagy-related gene (Atg)5, Atg10, Atg12 and hVps34. Altogether, these results strongly suggest that IP(3)R exerts a major role in the physiological control of autophagy.
Asunto(s)
Autofagia , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Animales , Autofagia/genética , Calcio/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Privación de Alimentos , Células HeLa , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inhibidores , Compuestos Macrocíclicos/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Oxazoles/farmacología , Isoformas de Proteínas/metabolismo , Ratas , Proteína bcl-X/metabolismoRESUMEN
Recent data shed light on two novel aspects of the mitochondria-Ca2+ liaison. First, it was extensively investigated how Ca2+ handling is controlled by mitochondrial shape, and positioning; a playground also of cell death and survival regulation. On the other hand, significant progress has been made to explore how intra- and near-mitochondrial Ca2+ signals modify mitochondrial morphology and cellular distribution. Here, we shortly summarize these advances and provide a model of Ca2+-mitochondria interactions.
Asunto(s)
Señalización del Calcio , Mitocondrias/metabolismo , Animales , Evolución Biológica , Retículo Endoplásmico/metabolismo , HumanosRESUMEN
Recent data have revealed an unexpected role of Bcl-2 in modulating the steady-state levels and agonist-dependent fluxes of Ca(2+) ions. Direct monitoring of endoplasmic reticulum (ER) Ca(2+) concentration with recombinant probes reveals a lower state of filling in Bcl-2-overexpressing cells and a higher leak rate from the organelle. The broader set of indirect data using cytosolic probes reveals a more complex scenario, as in many cases no difference was detected in the Ca(2+) content of the intracellular pools. At the same time, Ca(2+) signals have been shown to affect important checkpoints of the apoptotic process, such as mitochondria, thus tuning the sensitivity of cells to various challenges. In this contribution, we will review (i) the data on the effect of Bcl-2 on [Ca(2+)](er), (ii) the functional significance of the Ca(2+)-signalling alteration and (iii) the current insight into the possible mechanisms of this effect.
Asunto(s)
Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Homeostasis , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Apoptosis , Señalización del Calcio , Humanos , Proteínas Proto-Oncogénicas c-bcl-2/clasificación , Proteínas Proto-Oncogénicas c-bcl-2/genéticaRESUMEN
BACKGROUND: Nonsense mutations in coagulation factor (F) VII potentially cause a lethal hemorrhagic diathesis. Readthrough of nonsense mutations by aminoglycosides has been studied in a few human disease models with variable results. OBJECTIVES: We investigated the K316X and W364X FVII mutations, associated with intracranial hemorrhage, and their correction by aminoglycosides. The rare nonsense mutations in FVII represent favorite models to test this strategy, because even tiny increases in the amount of functional full-length protein in patients could ameliorate hemorrhagic phenotypes. RESULTS: A FVII-green fluorescent protein (GFP) chimaera provided us with a fluorescent model of FVII expression in living cells. Appreciable fluorescence in cells transfected with nonsense FVII-GFP mutants was detected upon geneticin treatment, thus demonstrating suppression of premature translation termination. To investigate the rescue of FVII function, nonsense variants of the native FVII without GFP (p316X-FVII and p364X-FVII) were transfected and found to secrete low amounts of FVII (approximately 1% of Wt-FVII activity), thus suggesting a spontaneous stop codon readthrough. Geneticin treatment of cells resulted in a significant and dose-dependent increase of secreted FVII molecules (p316X-FVII, 24 +/- 12 ng mL(-1), 3.6 +/- 0.8% of Wt-FVII activity; p364X-FVII, 26 +/- 10 ng mL(-1), 3.7+/-0.6%) characterized by reduced specific activity, thus indicating the synthesis of dysfunctional proteins. Similar results were observed with gentamicin, a commonly used aminoglycoside of potential interest for patient treatment. CONCLUSIONS: Our approach, extendable to other coagulation factors, represents an effective tool for a systematic study of the effects of aminoglycosides and neighboring sequences on nonsense codon readthrough. These results provide the rationale for a mutation-specific therapeutic approach in FVII deficiency.
Asunto(s)
Antibacterianos/farmacología , Codón sin Sentido , Factor VII/genética , Regulación de la Expresión Génica/efectos de los fármacos , Gentamicinas/farmacología , Adolescente , Animales , Coagulación Sanguínea/efectos de los fármacos , Línea Celular , Preescolar , Cricetinae , Relación Dosis-Respuesta a Droga , Factor VII/metabolismo , Deficiencia del Factor VII/sangre , Deficiencia del Factor VII/genética , Proteínas Fluorescentes Verdes/genética , Humanos , Masculino , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónAsunto(s)
Adenosina Trifosfato/biosíntesis , Proteínas de Unión al GTP/metabolismo , Daño por Reperfusión Miocárdica/prevención & control , Miocitos Cardíacos/metabolismo , Transglutaminasas/metabolismo , Animales , Proteínas de Unión al GTP/deficiencia , Proteínas de Unión al GTP/genética , Técnicas In Vitro , Masculino , Potenciales de la Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias Cardíacas/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Miocitos Cardíacos/citología , Proteína Glutamina Gamma Glutamiltransferasa 2 , Receptores Adrenérgicos alfa 1/deficiencia , Receptores Adrenérgicos alfa 1/genética , Receptores Adrenérgicos alfa 1/metabolismo , Transducción de Señal , Transglutaminasas/deficiencia , Transglutaminasas/genéticaAsunto(s)
Apoptosis/efectos de los fármacos , Inhibidores de la Proteasa del VIH/farmacología , Proteínas Inmediatas-Precoces/farmacología , Proteínas Inhibidoras de la Apoptosis/farmacología , Proteínas Inflamatorias de Macrófagos/farmacología , Mitocondrias/efectos de los fármacos , Proteínas Virales/farmacología , Apoptosis/fisiología , Calcio/metabolismo , Ceramidas/farmacología , Citomegalovirus , Células Gigantes/efectos de los fármacos , Células Gigantes/patología , Células Gigantes/ultraestructura , Células HeLa , Humanos , Peróxido de Hidrógeno/farmacología , Mitocondrias/fisiología , Nelfinavir/farmacología , Ritonavir/farmacología , Saquinavir/farmacología , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
Mitochondrial Ca2+ uptake controls cellular functions as diverse as aerobic metabolism, cytosolic Ca2+signalling and mitochondrial participation in apoptosis. Modulatory inputs converging on the organelle can regulate this process, determining the final outcome of Ca2+-mediated cell stimulation. We investigated in HeLa cells and primary skeletal myotubes the effect on Ca2+ signalling of the transcriptional peroxisome-proliferator-activated-receptor-gamma-coactivator-1alpha (PGC-1alpha), which triggers organelle biogenesis and modifies the mitochondrial proteome. PGC-1alpha selectively reduced mitochondrial Ca2+ responses to cell stimulation by reducing the efficacy of mitochondrial Ca2+ uptake sites and increasing organelle volume. In turn, this affected ER Ca2+ release and cytosolic responses in HeLa cells. Most importantly, the modulation of mitochondrial Ca2+ uptake significantly reduced cellular sensitivity to the Ca2+-mediated proapoptotic effect of C2 ceramide. These results reveal a primary role of PGC-1alpha in shaping mitochondrial participation in calcium signalling, that underlies its protective role against stress and proapoptotic stimuli in pathophysiological conditions.
Asunto(s)
Apoptosis , Señalización del Calcio , Calcio/metabolismo , Proteínas de Choque Térmico/metabolismo , Mitocondrias Musculares/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Factores de Transcripción/metabolismo , Agonistas Adrenérgicos beta/farmacología , Albuterol/farmacología , Animales , Canales de Calcio/metabolismo , Inhibidores Enzimáticos/farmacología , Células HeLa , Proteínas de Choque Térmico/genética , Histamina/farmacología , Homeostasis , Humanos , Receptores de Inositol 1,4,5-Trifosfato , Canales Iónicos , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Mitocondrias Musculares/efectos de los fármacos , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Dilatación Mitocondrial , Fibras Musculares Esqueléticas/patología , Factor Nuclear 1 de Respiración/genética , Factor Nuclear 1 de Respiración/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Ratas , Receptores Citoplasmáticos y Nucleares/metabolismo , Esfingosina/análogos & derivados , Esfingosina/farmacología , Factores de Transcripción/genética , Transfección , Proteína Desacopladora 2RESUMEN
The ability of cadmium to disrupt calcium homeostasis has been known since a long time, but the precise cellular targets of its toxic action are still debated. A great problem in the interpretation of data has been associated with the ability of cadmium to strongly bind traditional calcium probes. Aequorin, the well-characterized calcium-sensitive photoprotein, was used as intracellular calcium indicator during cadmium injury in NIH 3T3 murine fibroblasts. NIH 3T3 cells were transfected with a cDNA construct containing aequorin fused to a truncated glutamate receptor, which directs the probe to the outer surface of intracellular membranes. At first, we tested if different cadmium concentrations were able to modify the rate of light emission by aequorin showing that cadmium concentrations <15 microM were ineffective on aequorin luminescence. Hence, aequorin chimeras revealed as a useful tool in the analyses of Cd2+/Ca2+ interference. To directly investigate the role of Cd2+ in Ca2+ homeostasis, we have started to selectively measure the free Ca2+ concentration in different cell compartments. Here, we report that cadmium reduces the transient free calcium signal after stimulation of cells with bradykinin. Further studies are in progress to clarify the role of mitochondria and endoplasmic reticulum in cadmium-induced alterations of Ca2+ homeostasis in order to link signal transduction modifications with the onset of apoptosis induced by cadmium exposure.
Asunto(s)
Aequorina/metabolismo , Cadmio/toxicidad , Calcio/análisis , Sustancias Luminiscentes/farmacología , Proteínas Recombinantes de Fusión/metabolismo , Aequorina/genética , Animales , Apoptosis/efectos de los fármacos , Cadmio/metabolismo , Calcio/metabolismo , Relación Dosis-Respuesta a Droga , Sustancias Luminiscentes/química , Ratones , Microscopía de Contraste de Fase , Células 3T3 NIH , Proteínas Recombinantes de Fusión/genética , Espectrofotometría Atómica , Factores de TiempoRESUMEN
Extracellular agonists mobilize Ca2+ from SERCA-comprising intracellular Ca2+ stores located in both the Golgi apparatus and the endoplasmic reticulum. Ca2+ release from both these compartments was studied in HeLa cells stably expressing the luminescent Ca2+ indicator aequorin specifically targeted to these compartments. Changes in lumenal [Ca2+] as detected by the aequorin measurements were correlated with parallel changes in total Ca2+ content of the stores. The latencies and initial rates of Ca2+ release from the Golgi apparatus and the endoplasmic reticulum were quite similar. However, maximal Ca2+ release measured with Golgi-targeted aequorin terminated faster than that from the endoplasmic reticulum. The rate and extent of Ca2+ depletion from both compartments correlated well with the peak amplitude of the cytosolic [Ca2+] rise. Time-course experiments further revealed that the peak of the cytosolic Ca2+ response occurred before the lumenal [Ca2+] reached its lowest level. We conclude that both the Golgi apparatus and the endoplasmic reticulum contribute to the rise in cytosolic [Ca2+] upon agonist stimulation, but the kinetics of the Ca2+ release are different.
Asunto(s)
Aequorina/biosíntesis , Aequorina/genética , Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Aequorina/metabolismo , Relación Dosis-Respuesta a Droga , Retículo Endoplásmico/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Aparato de Golgi/efectos de los fármacos , Células HeLa , Humanos , Inositol 1,4,5-Trifosfato/farmacologíaRESUMEN
Mutations in the ubiquitously expressed secretory-pathway Ca(2+)-ATPase (SPCA1) Ca(2+) pump result in Hailey-Hailey disease, which almost exclusively affects the epidermal part of the skin. We have studied Ca(2+) signaling in human keratinocytes by measuring the free Ca(2+) concentration in the cytoplasm and in the lumen of both the Golgi apparatus and the endoplasmic reticulum. These signals were compared with those recorded in SPCA1-overexpressing and control COS-1 cells. Both the sarco(endo)plasmic-reticulum Ca(2+)-ATPase (SERCA) and SPCA1 can mediate Ca(2+) uptake into the Golgi stacks. Our results indicate that keratinocytes mainly used the SPCA1 Ca(2+) pump to load the Golgi complex with Ca(2+) whereas the SERCA Ca(2+) pump was mainly used in control COS-1 cells. Cytosolic Ca(2+) signals in keratinocytes induced by extracellular ATP or capacitative Ca(2+) entry were characterized by an unusually long latency reflecting extra Ca(2+) buffering by an SPCA1-containing Ca(2+) store, similarly as in SPCA1-overexpressing COS-1 cells. Removal of extracellular Ca(2+) elicited spontaneous cytosolic Ca(2+) transients in keratinocytes, similarly as in SPCA1-overexpressing COS-1 cells. With respect to Ca(2+) signaling keratinocytes and SPCA1-overexpressing COS-1 cells therefore behaved similarly but differed from control COS-1 cells. The relatively large contribution of the SPCA1 pumps for loading the Golgi stores with Ca(2+) in keratinocytes may, at least partially, explain why mutations in the SPCA1 gene preferentially affect the skin in Hailey-Hailey patients.
Asunto(s)
Señalización del Calcio , ATPasas Transportadoras de Calcio/metabolismo , Calcio/metabolismo , Queratinocitos/metabolismo , Animales , Células COS , ATPasas Transportadoras de Calcio/genética , Células Cultivadas , Citoplasma/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Humanos , TransfecciónRESUMEN
Studies on pancreatic acinar cells provided the original evidence for the Ca(2+) releasing action of inositol 1,4,5-trisphosphate (IP(3)). Ironically, this system has presented problems for the general theory that IP(3) acts primarily on the endoplasmic reticulum (ER), because the IP(3)-elicited Ca(2+) release occurs in the apical pole, which is dominated by zymogen granules (ZGs) and apparently contains very little ER. Using confocal and two-photon microscopy and a number of different ER-specific fluorescent probes, we have now investigated in detail the distribution of the ER in living pancreatic acinar cells. It turns out that although the bulk of the ER, as expected, is clearly located in the baso-lateral part of the cell, there is significant invasion of ER into the granular pole and each ZG is in fact surrounded by strands of ER. This structural evidence from living cells, in conjunction with recent functional studies demonstrating the high Ca(2+) mobility in the ER lumen, provides the framework for a coherent and internally consistent theory for cytosolic Ca(2+) signal generation in the apical secretory pole, in which the primary Ca(2+) release occurs from ER extensions in the granular pole supplied with Ca(2+) from the main store at the base of the cell by the tunnel function of the ER.