RESUMO
mTORC1 is known to activate sterol regulatory element-binding proteins (SREBPs) including SREBP-2, a master regulator of cholesterol synthesis. Through incompletely understood mechanisms, activated mTORC1 triggers translocation of SREBP-2, an endoplasmic reticulum (ER) resident protein, to the Golgi where SREBP-2 is cleaved to translocate to the nucleus and activate gene expression for cholesterol synthesis. Low ER cholesterol is a well-established trigger for SREBP-2 activation. We thus investigated whether mTORC1 activates SREBP-2 by reducing cholesterol delivery to the ER. We report here that mTORC1 activation is accompanied by low ER cholesterol and an increase of SREBP-2 activation. Conversely, a decrease in mTORC1 activity coincides with a rise in ER cholesterol and a decrease in SERBP-2 activity. This rise in ER cholesterol is of lysosomal origin: blocking the exit of cholesterol from lysosomes by U18666A or NPC1 siRNA prevents ER cholesterol from increasing and, consequently, SREBP-2 is activated without mTORC1 activation. Furthermore, when mTORC1 activity is low, cholesterol is delivered to lysosomes through two membrane trafficking pathways: autophagy and rerouting of endosomes to lysosomes. Indeed, with dual blockade of both pathways by Atg5-/- and dominant-negative rab5, ER cholesterol fails to increase when mTORC1 activity is low, and SREBP-2 is activated. Conversely, overexpressing constitutively active Atg7, which forces autophagy and raises ER cholesterol even when mTORC1 activity is high, suppresses SREBP-2 activation. We conclude that mTORC1 actively suppresses autophagy and maintains endosomal recycling, thereby preventing endosomes and autophagosomes from reaching lysosomes. This results in a reduction of cholesterol in the ER and activation of SREBP-2.
Assuntos
Autofagossomos/fisiologia , Colesterol/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Células HEK293 , HumanosRESUMO
The plasma membrane of mammalian cells undergoes constitutive endocytosis, endocytic sorting, and recycling, which delivers nutrients to the lysosomes. The receptors, along with membrane lipids, are normally returned to the plasma membrane to sustain this action. It is not known, however, whether this process is influenced by metabolic conditions. Here we report that endocytic recycling requires active mechanistic target of rapamycin (aka mammalian target of rapamycin) (mTORC1), a master metabolic sensor. Upon mTORC1 inactivation, either by starvation or by inhibitor, recycling receptors and plasma membrane lipids, such as transferrin receptors and sphingomyelin, are delivered to the lysosomes. This lysosomal targeting is independent of canonical autophagy: both WT and Atg5-/- mouse embryonic fibroblasts responded similarly. Furthermore, we identify hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), an endosomal sorting complexes required for transport (ESCORT-0) component, as a downstream target of mTORC1. Hrs requires mTORC1 activity to maintain its protein expression level. Silencing Hrs without decreasing mTORC1 activity is sufficient to target transferrin and sphingomyelin to the lysosomes. It is thus evident that the canonical recycling pathway is under the regulation of mTORC1 and likely most predominant in proliferating cells where mTORC1 is highly active.
Assuntos
Embrião de Mamíferos/metabolismo , Endocitose/fisiologia , Fibroblastos/metabolismo , Lisossomos/metabolismo , Complexos Multiproteicos/metabolismo , Esfingomielinas/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Transferrina/metabolismo , Animais , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Transporte Biológico Ativo/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Embrião de Mamíferos/citologia , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Fibroblastos/citologia , Lisossomos/genética , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Knockout , Complexos Multiproteicos/genética , Esfingomielinas/genética , Serina-Treonina Quinases TOR/genética , Transferrina/genéticaRESUMO
Stains are known to be anti-inflammatory, but the mechanism remains poorly understood. Here we show that macrophages, either treated with statin in vitro or from statin-treated mice, have reduced cholesterol levels and higher expression of Jmjd3, a H3K27me3 demethylase. We provide evidence that lowering cholesterol levels in macrophages suppresses the ATP synthase in the inner mitochondrial membrane (IMM) and changes the proton gradient in the mitochondria. This activates NFkB and Jmjd3 expression to remove the repressive marker H3K27me3. Accordingly, the epigenome is altered by the cholesterol reduction. When subsequently challenged by the inflammatory stimulus LPS (M1), both macrophages treated with statins in vitro or isolated from statin-treated mice in vivo, express lower levels pro-inflammatory cytokines than controls, while augmenting anti-inflammatory Il10 expression. On the other hand, when macrophages are alternatively activated by IL4 (M2), statins promote the expression of Arg1, Ym1, and Mrc1. The enhanced expression is correlated with the statin-induced removal of H3K27me3 from these genes prior to activation. In addition, Jmjd3 and its demethylase activity are necessary for cholesterol to modulate both M1 and M2 activation. We conclude that upregulation of Jmjd3 is a key event for the anti-inflammatory function of statins on macrophages.
RESUMO
Statins are known to be anti-inflammatory, but the mechanism remains poorly understood. Here, we show that macrophages, either treated with statin in vitro or from statin-treated mice, have reduced cholesterol levels and higher expression of Jmjd3, a H3K27me3 demethylase. We provide evidence that lowering cholesterol levels in macrophages suppresses the adenosine triphosphate (ATP) synthase in the inner mitochondrial membrane and changes the proton gradient in the mitochondria. This activates nuclear factor kappa-B (NF-κB) and Jmjd3 expression, which removes the repressive marker H3K27me3. Accordingly, the epigenome is altered by the cholesterol reduction. When subsequently challenged by the inflammatory stimulus lipopolysaccharide (M1), macrophages, either treated with statins in vitro or isolated from statin-fed mice, express lower levels proinflammatory cytokines than controls, while augmenting anti-inflammatory Il10 expression. On the other hand, when macrophages are alternatively activated by IL-4 (M2), statins promote the expression of Arg1, Ym1, and Mrc1. The enhanced expression is correlated with the statin-induced removal of H3K27me3 from these genes prior to activation. In addition, Jmjd3 and its demethylase activity are necessary for cholesterol to modulate both M1 and M2 activation. We conclude that upregulation of Jmjd3 is a key event for the anti-inflammatory function of statins on macrophages.
Assuntos
Colesterol , Inibidores de Hidroximetilglutaril-CoA Redutases , Histona Desmetilases com o Domínio Jumonji , Macrófagos , Mitocôndrias , Regulação para Cima , Histona Desmetilases com o Domínio Jumonji/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Animais , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Colesterol/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Anti-Inflamatórios/farmacologia , Camundongos Endogâmicos C57BL , MasculinoRESUMO
The mammalian olfactory system detects an unlimited variety of odorants with a limited set of odorant receptors. To cope with the complexity of the odor world, each odorant receptor must detect many different odorants. The demand for low odor selectivity creates problems for the transduction process: the initial transduction step, the synthesis of the second messenger cAMP, operates with low efficiency, mainly because odorants bind only briefly to their receptors. Sensory cilia of olfactory receptor neurons have developed an unusual solution to this problem. They accumulate chloride ions at rest and discharge a chloride current upon odor detection. This chloride current amplifies the receptor potential and promotes electrical excitation. We have studied this amplification process by examining identity, subcellular localization, and regulation of its molecular components. We found that the Na(+)/K(+)/2Cl(-) cotransporter NKCC1 is expressed in the ciliary membrane, where it mediates chloride accumulation into the ciliary lumen. Gene silencing experiments revealed that the activity of this transporter depends on the kinases SPAK and OSR1, which are enriched in the cilia together with their own activating kinases, WNK1 and WNK4. A second Cl(-) transporter, the Cl(-)/HCO(3)(-) exchanger SLC4A1, is expressed in the cilia and may support Cl(-) accumulation. The calcium-dependent chloride channel TMEM16B (ANO2) provides a ciliary pathway for the excitatory chloride current. These findings describe a specific set of ciliary proteins involved in anion-based signal amplification. They provide a molecular concept for the unique strategy that allows olfactory sensory neurons to operate as efficient transducers of weak sensory stimuli.
Assuntos
Neurônios Receptores Olfatórios/fisiologia , Animais , Proteína 1 de Troca de Ânion do Eritrócito/genética , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Sequência de Bases , Cloretos/metabolismo , Cílios/fisiologia , Primers do DNA/genética , Retroalimentação Fisiológica , Inativação Gênica , Transporte de Íons , Camundongos , Camundongos Transgênicos , Antígenos de Histocompatibilidade Menor , Modelos Neurológicos , Fosforilação , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Ratos , Ratos Wistar , Receptores Odorantes/fisiologia , Transdução de Sinais/fisiologia , Olfato/fisiologia , Simportadores de Cloreto de Sódio-Potássio/genética , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Membro 2 da Família 12 de Carreador de Soluto , Proteína Quinase 1 Deficiente de Lisina WNKRESUMO
Calcium-activated chloride channels are expressed in chemosensory neurons of the nose and contribute to secretory processes and sensory signal transduction. These channels are thought to be members of the family of anoctamins (alternative name: TMEM16 proteins), which are opened by micromolar concentrations of intracellular Ca(2+). Two family members,ANO 1 (TMEM16A) and ANO 2 (TMEM16B), are expressed in the various sensory and respiratory tissues of the nose.We have examined the tissue specificity and sub-cellular localization of these channels in the nasal respiratory epithelium and in the five chemosensory organs of the nose: the main olfactory epithelium, the septal organ of Masera, the vomeronasal organ, the Grueneberg ganglion and the trigeminal system. We have found that the two channels show mutually exclusive expression patterns. ANO 1 is present in the apical membranes of various secretory epithelia in which it is co-localized with the water channel aquaporin 5. It has also been detected in acinar cells and duct cells of subepithelial glands and in the supporting cells of sensory epithelia. In contrast, ANO 2 expression is restricted to chemosensory neurons in which it has been detected in microvillar and ciliary surface structures. The different expression patterns of ANO 1 and ANO 2 have been observed in the olfactory, vomeronasal and respiratory epithelia. No expression has been detected in the Grueneberg ganglion or trigeminal sensory fibers. On the basis of this differential expression, we derive the main functional features of ANO 1 and ANO 2 chloride channels in the nose and suggest their significance for nasal physiology.
Assuntos
Canais de Cloreto/metabolismo , Mucosa Nasal/metabolismo , Animais , Anoctamina-1 , Anoctaminas , Gânglios Sensitivos/metabolismo , Camundongos , Mucosa Olfatória/citologia , Mucosa Olfatória/metabolismo , RatosRESUMO
PURPOSE: In the vertebrate retina, calcium-activated chloride channels are expressed in photoreceptor synaptic terminals. These channels are involved in the control of transmitter release in the dark. The search for their molecular identity has recently lead to the localization of the protein anoctamin 2 (also TMEM16B) in the outer plexiform layer of the rodent retina. Since both rod and cone photoreceptors have their terminals in this layer, it was not clear which of these express anoctamin 2. Here, we examine rod spherules and cone pedicles for expression of anoctamin 2. METHODS: Expression of anoctamin genes was studied in the rat eye using RT-PCR. Immunohistochemical experiments were used to localize anoctamins and chloride transporters with their regulatory kinases. Photoreceptor synaptic proteins, as well as the lectins Peanut agglutinin and Griffonia simplicifolia agglutinin, were used to distinguish retinal structures. RESULTS: Anoctamin 1, 2, and 10 were found to be expressed in the eye. Anoctamin 2 was expressed as a splice variant that includes exon 15 of the genomic structure. The protein is exclusively expressed in rod terminals and is not present in cone pedicles. Expression is not clustered at the ribbon complex, but spread across the presynaptic membrane where it colocalizes with the plasma membrane calcium pump. The electroneutral chloride transporter NKCC1 is expressed in photoreceptor terminals, together with its regulatory kinases SPAK and OSR1. CONCLUSIONS: Rod photoreceptor terminals possess the molecular machinery for chloride accumulation and for the generation of calcium-dependent chloride currents conducted through anoctamin 2 channels. We discuss this finding in the framework of the established hypothesis that calcium-activated chloride channels are part of a feedback inhibition mechanism that limits transmitter release in the dark.
Assuntos
Canais de Cloreto/genética , Regulação da Expressão Gênica/fisiologia , Terminações Pré-Sinápticas/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Animais , Anoctamina-1 , Anoctaminas , Western Blotting , Canais de Cloreto/metabolismo , Primers do DNA/química , Técnica Indireta de Fluorescência para Anticorpo , Cobaias , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Retina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Simportadores de Cloreto de Sódio-Potássio/genética , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Membro 2 da Família 12 de Carreador de SolutoRESUMO
Calcium-dependent chloride channels serve critical functions in diverse biological systems. Driven by cellular calcium signals, the channels codetermine excitatory processes and promote solute transport. The anoctamin (ANO) family of membrane proteins encodes three calcium-activated chloride channels, named ANO 1 (also TMEM16A), ANO 2 (also TMEM16B), and ANO 6 (also TMEM16F). Here we examined how ANO 1 and ANO 2 interact with Ca(2+)/calmodulin using nonstationary current analysis during channel activation. We identified a putative calmodulin-binding domain in the N-terminal region of the channel proteins that is involved in channel activation. Binding studies with peptides indicated that this domain, a regulatory calmodulin-binding motif (RCBM), provides two distinct modes of interaction with Ca(2+)/calmodulin, one at submicromolar Ca(2+) concentrations and one in the micromolar Ca(2+) range. Functional, structural, and pharmacological data support the concept that calmodulin serves as a calcium sensor that is stably associated with the RCBM domain and regulates the activation of ANO 1 and ANO 2 channels. Moreover, the predominant splice variant of ANO 2 in the brain exhibits Ca(2+)/calmodulin-dependent inactivation, a loss of channel activity within 30 s. This property may curtail ANO 2 activity during persistent Ca(2+) signals in neurons. Mutagenesis data indicated that the RCBM domain is also involved in ANO 2 inactivation, and that inactivation is suppressed in the retinal ANO 2 splice variant. These results advance the understanding of Ca(2+) regulation in anoctamin Cl(-) channels and its significance for the physiological function that anoctamin channels subserve in neurons and other cell types.