RESUMO
We recently identified elevated annexin A6 (AnxA6) protein levels in Niemann-Pick-type C1 (NPC1) mutant cells. In these cells, AnxA6 depletion rescued the cholesterol accumulation associated with NPC1 deficiency. Here, we demonstrate that elevated AnxA6 protein levels in NPC1 mutants or upon pharmacological NPC1 inhibition, using U18666A, were not due to upregulated AnxA6 mRNA expression, but caused by defects in AnxA6 protein degradation. Two KFERQ-motifs are believed to target AnxA6 to lysosomes for chaperone-mediated autophagy (CMA), and we hypothesized that the cholesterol accumulation in endolysosomes (LE/Lys) triggered by the NPC1 inhibition could interfere with the CMA pathway. Therefore, AnxA6 protein amounts and cholesterol levels in the LE/Lys (LE-Chol) compartment were analyzed in NPC1 mutant cells ectopically expressing lysosome-associated membrane protein 2A (Lamp2A), which is well known to induce the CMA pathway. Strikingly, AnxA6 protein amounts were strongly decreased and coincided with significantly reduced LE-Chol levels in NPC1 mutant cells upon Lamp2A overexpression. Therefore, these findings suggest Lamp2A-mediated restoration of CMA in NPC1 mutant cells to lower LE-Chol levels with concomitant lysosomal AnxA6 degradation. Collectively, we propose CMA to permit a feedback loop between AnxA6 and cholesterol levels in LE/Lys, encompassing a novel mechanism for regulating cholesterol homeostasis in NPC1 disease.
Assuntos
Anexina A6/metabolismo , Colesterol/metabolismo , Endossomos/metabolismo , Retroalimentação Fisiológica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lisossomos/metabolismo , Mutação/genética , Proteólise , Animais , Células CHO , Cricetulus , Endossomos/ultraestrutura , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/ultraestrutura , Modelos Biológicos , Proteína C1 de Niemann-PickRESUMO
Cholesterol accumulation in late endosomes is a prevailing phenotype of Niemann-Pick type C1 (NPC1) mutant cells. Likewise, annexin A6 (AnxA6) overexpression induces a phenotype reminiscent of NPC1 mutant cells. Here, we demonstrate that this cellular cholesterol imbalance is due to AnxA6 promoting Rab7 inactivation via TBC1D15, a Rab7-GAP. In NPC1 mutant cells, AnxA6 depletion and eventual Rab7 activation was associated with peripheral distribution and increased mobility of late endosomes. This was accompanied by an enhanced lipid accumulation in lipid droplets in an acyl-CoA:cholesterol acyltransferase (ACAT)-dependent manner. Moreover, in AnxA6-deficient NPC1 mutant cells, Rab7-mediated rescue of late endosome-cholesterol export required the StAR-related lipid transfer domain-3 (StARD3) protein. Electron microscopy revealed a significant increase of membrane contact sites (MCS) between late endosomes and ER in NPC1 mutant cells lacking AnxA6, suggesting late endosome-cholesterol transfer to the ER via Rab7 and StARD3-dependent MCS formation. This study identifies AnxA6 as a novel gatekeeper that controls cellular distribution of late endosome-cholesterol via regulation of a Rab7-GAP and MCS formation.
Assuntos
Anexina A6/genética , Colesterol/genética , Proteínas Ativadoras de GTPase/genética , Doença de Niemann-Pick Tipo C/genética , Proteínas rab de Ligação ao GTP/genética , Animais , Células CHO , Proteínas de Transporte/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Colesterol/metabolismo , Cricetulus , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Endossomos/genética , Endossomos/metabolismo , Humanos , Proteínas de Membrana/genética , Doença de Niemann-Pick Tipo C/metabolismo , Doença de Niemann-Pick Tipo C/patologia , Domínios Proteicos/genética , Transporte Proteico/genética , RNA Interferente Pequeno/genética , proteínas de unión al GTP Rab7RESUMO
The epicardium plays a crucial role in embryonic heart development and adult heart repair; however, the molecular events underlying its maturation remain unknown. Wt1, one of the main markers of the embryonic epicardium, is essential for epicardial development and function. Here, we analyse the transcriptomic profile of epicardial-enriched cells at different stages of development and from control and epicardial-specific Wt1 knockout (Wt1KO) mice. Transcriptomic and cell morphology analyses of epicardial cells from epicardial-specific Wt1KO mice revealed a defect in the maturation process of the mutant epicardium, including sustained upregulation of Bmp4 expression and the inability of mutant epicardial cells to transition into a mature squamous phenotype. We identified Bmp4 as a transcriptional target of Wt1, thus providing a molecular basis for the retention of the cuboidal cell shape observed in the Wt1KO epicardium. Accordingly, inhibition of the Bmp4 signalling pathway both ex vivo and in vivo rescued the cuboidal phenotype of the mutant epicardium. Our findings indicate the importance of the cuboidal-to-squamous transition in epicardial maturation, a process regulated by Wt1.
Assuntos
Proteína Morfogenética Óssea 4/metabolismo , Proteína Morfogenética Óssea 4/farmacologia , Pericárdio/citologia , Pericárdio/metabolismo , Proteínas WT1/metabolismo , Animais , Forma Celular/efeitos dos fármacos , Forma Celular/genética , Células Cultivadas , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Coração/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Miocárdio/metabolismo , Miocárdio/ultraestrutura , Pericárdio/efeitos dos fármacos , Pericárdio/ultraestrutura , Proteínas WT1/genéticaRESUMO
Annexins are a family of proteins that bind to phospholipids in a calcium-dependent manner. Earlier studies implicated annexin A6 (AnxA6) to inhibit secretion and participate in the organization of the extracellular matrix. We recently showed that elevated AnxA6 levels significantly reduced secretion of the extracellular matrix protein fibronectin (FN). Because FN is directly linked to the ability of cells to migrate, this prompted us to investigate the role of AnxA6 in cell migration. Up-regulation of AnxA6 in several cell models was associated with reduced cell migration in wound healing, individual cell tracking and three-dimensional migration/invasion assays. The reduced ability of AnxA6-expressing cells to migrate was associated with decreased cell surface expression of αVß3 and α5ß1 integrins, both FN receptors. Mechanistically, we found that elevated AnxA6 levels interfered with syntaxin-6 (Stx6)-dependent recycling of integrins to the cell surface. AnxA6 overexpression caused mislocalization and accumulation of Stx6 and integrins in recycling endosomes, whereas siRNA-mediated AnxA6 knockdown did not modify the trafficking of integrins. Given our recent findings that inhibition of cholesterol export from late endosomes (LEs) inhibits Stx6-dependent integrin recycling and that elevated AnxA6 levels cause LE cholesterol accumulation, we propose that AnxA6 and blockage of LE cholesterol transport are critical for endosomal function required for Stx6-mediated recycling of integrins in cell migration.
Assuntos
Anexina A6/metabolismo , Colesterol/metabolismo , Endossomos/metabolismo , Integrina alfa5beta1/metabolismo , Integrina alfaVbeta3/metabolismo , Proteínas Qa-SNARE/metabolismo , Animais , Anexina A6/antagonistas & inibidores , Anexina A6/genética , Células CHO , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Movimento Celular , Células Cultivadas , Cricetulus , Endossomos/ultraestrutura , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Humanos , Integrina alfa5beta1/antagonistas & inibidores , Integrina alfaVbeta3/antagonistas & inibidores , Camundongos , Microscopia Confocal , Microscopia de Vídeo , Proteínas Qa-SNARE/antagonistas & inibidores , Proteínas Qa-SNARE/genética , Interferência de RNA , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Imagem com Lapso de TempoRESUMO
BACKGROUND AND PURPOSE: Annexin A6 (AnxA6) is a calcium-dependent phospholipid-binding protein that can be recruited to the plasma membrane to function as a scaffolding protein to regulate signal complex formation, endo- and exocytic pathways as well as distribution of cellular cholesterol. Here, we have investigated how AnxA6 influences the membrane order. EXPERIMENTAL APPROACH: We used Laurdan and di-4-ANEPPDHQ staining in (i) artificial membranes; (ii) live cells to investigate membrane packing and ordered lipid phases; and (iii) a super-resolution imaging (photoactivated localization microscopy, PALM) and Ripley's K second-order point pattern analysis approach to assess how AnxA6 regulates plasma membrane order domains and protein clustering. KEY RESULTS: In artificial membranes, purified AnxA6 induced a global increase in membrane order. However, confocal microscopy using di-4-ANEPPDHQ in live cells showed that cells expressing AnxA6, which reduces plasma membrane cholesterol levels and modifies the actin cytoskeleton meshwork, displayed a decrease in membrane order (â¼15 and 30% in A431 and MEF cells respectively). PALM data from Lck10 and Src15 membrane raft/non-raft markers revealed that AnxA6 expression induced clustering of both raft and non-raft markers. Altered clustering of Lck10 and Src15 in cells expressing AnxA6 was also observed after cholesterol extraction with methyl-ß-cyclodextrin or actin cytoskeleton disruption with latrunculin B. CONCLUSIONS AND IMPLICATIONS: AnxA6-induced plasma membrane remodelling indicated that elevated AnxA6 expression decreased membrane order through the regulation of cellular cholesterol homeostasis and the actin cytoskeleton. This study provides the first evidence from live cells that support current models of annexins as membrane organizers.
Assuntos
Anexina A6/metabolismo , Membrana Celular/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/química , Humanos , Lipídeos/química , Camundongos Knockout , Microscopia de FluorescênciaRESUMO
Inhibition of cholesterol export from late endosomes causes cellular cholesterol imbalance, including cholesterol depletion in the trans-Golgi network (TGN). Here, using Chinese hamster ovary (CHO) Niemann-Pick type C1 (NPC1) mutant cell lines and human NPC1 mutant fibroblasts, we show that altered cholesterol levels at the TGN/endosome boundaries trigger Syntaxin 6 (Stx6) accumulation into VAMP3, transferrin, and Rab11-positive recycling endosomes (REs). This increases Stx6/VAMP3 interaction and interferes with the recycling of αVß3 and α5ß1 integrins and cell migration, possibly in a Stx6-dependent manner. In NPC1 mutant cells, restoration of cholesterol levels in the TGN, but not inhibition of VAMP3, restores the steady-state localization of Stx6 in the TGN. Furthermore, elevation of RE cholesterol is associated with increased amounts of Stx6 in RE. Hence, the fine-tuning of cholesterol levels at the TGN-RE boundaries together with a subset of cholesterol-sensitive SNARE proteins may play a regulatory role in cell migration and invasion.
Assuntos
Colesterol/metabolismo , Endossomos/metabolismo , Proteínas Qa-SNARE/metabolismo , Rede trans-Golgi/metabolismo , Animais , Células CHO , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Movimento Celular , Cricetinae , Cricetulus , Humanos , Integrina alfa5beta1/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteína C1 de Niemann-Pick , Ligação Proteica , Transporte Proteico , Proteínas Qa-SNARE/química , Receptores de Vitronectina/metabolismo , Proteínas SNARE/metabolismo , Proteína 3 Associada à Membrana da Vesícula/química , Proteína 3 Associada à Membrana da Vesícula/metabolismo , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Adult regenerative myogenesis is central for restoring normal tissue structure and function after muscle damage. In muscle repair after injury, as in severe myopathies, damaged and necrotic fibers are removed by infiltrating inflammatory cells and then replaced by muscle stem cells or satellite cells, which will fuse to form new myofibers. Extracellular proteolysis mediated by uPA-generated plasmin plays a critical role in controlling inflammation and satellite-cell-dependent myogenesis. α-enolase has been described as plasminogen receptor in several cell types, where it acts concentrating plasmin proteolytic activity on the cell surface. In this study, we investigated whether α-enolase plasminogen receptor plays a regulatory role during the muscular repair process. Inhibitors of α-enolase/plasminogen binding: MAb11G1 (a monoclonal antibody against α-enolase) and ε-aminocaproic acid, EACA (a lysine analogue) inhibited the myogenic abilities of satellite cells-derived myoblasts. Furthermore, knockdown of α-enolase decreased myogenic fusion of myoblasts. Injured wild-type mice and dystrophic mdx mice were also treated with MAb11G1 and EACA. These treatments had negative impacts on muscle repair impairing satellite cell functions in vitro in agreement with blunted growth of new myofibers in vivo. Furthermore, both MAb11G1 and EACA treatments impaired adequate inflammatory cell infiltration and promoted extracellular matrix deposition in vivo, which resulted in persistent degeneration. These results demonstrate the novel requirement of α-enolase for restoring homeostasis of injured muscle tissue, by controlling the pericellular localization of plasmin activity.
Assuntos
Desenvolvimento Muscular , Músculo Esquelético/fisiologia , Fosfopiruvato Hidratase/metabolismo , Plasminogênio/metabolismo , Aminocaproatos/farmacologia , Animais , Fibrinolisina/metabolismo , Camundongos , Mioblastos/enzimologia , Mioblastos/fisiologia , Fosfopiruvato Hidratase/antagonistas & inibidores , Ligação Proteica , Regeneração/efeitos dos fármacosRESUMO
α-Enolase is a key glycolytic enzyme in the cytoplasm of prokaryotic and eukaryotic cells and is considered a multifunctional protein. α-enolase is expressed on the surface of several cell types, where it acts as a plasminogen receptor, concentrating proteolytic plasmin activity on the cell surface. In addition to glycolytic enzyme and plasminogen receptor functions, α-Enolase appears to have other cellular functions and subcellular localizations that are distinct from its well-established function in glycolysis. Furthermore, differential expression of α-enolase has been related to several pathologies, such as cancer, Alzheimer's disease, and rheumatoid arthritis, among others. We have identified α-enolase as a plasminogen receptor in several cell types. In particular, we have analyzed its role in myogenesis, as an example of extracellular remodelling process. We have shown that α-enolase is expressed on the cell surface of differentiating myocytes, and that inhibitors of α-enolase/plasminogen binding block myogenic fusion in vitro and skeletal muscle regeneration in mice. α-Enolase could be considered as a marker of pathological stress in a high number of diseases, performing several of its multiple functions, mainly as plasminogen receptor. This paper is focused on the multiple roles of the α-enolase/plasminogen axis, related to several pathologies.