RESUMO
Lichen planopilaris (LPP) and frontal fibrosing alopecia (FFA) are primary cicatricial alopecia that cause a major impact on quality of life due to irreversible hair loss and symptoms as itching, burning and pain. They are characterized by permanent loss of hair follicle stem cells (HFSCs) by pathomechanisms still poorly understood, resulting in poor efficacy of currently available treatments. Caveolae are flask-shaped lipid rafts invaginated within the plasma membrane of multiple cell types. Although their role in the HF physiology and pathophysiology is relatively unknown, we have previously demonstrated that the primary structural component of caveolae (caveolin-1 or Cav1) is upregulated in FFA. Thus, we propose to investigate the expression and localization of caveolae-associated structural proteins (Cav1, Cav2, and Cavin-1) and HFSCs (identified by K15) in both LPP and FFA. We analyzed 4 patients with LPP biopsied in affected and non-affected (NA) scalp, 4 patients with FFA biopsied in affected scalp and 4 healthy controls. Affected scalp of LPP and FFA demonstrated increased levels of Cav1 and Cavin-1 compared with HC and LPP-NA. Moreover, Cav1, Cav2 and Cavin1 all exhibit high colocalization with K15 and their expression appears to be negatively correlated, supporting the hypothesis that these proteins are important players in LPP/FFA and may serve as therapeutic targets in future treatments.
Assuntos
Alopecia , Cavéolas , Caveolina 1 , Folículo Piloso , Líquen Plano , Regulação para Cima , Humanos , Alopecia/patologia , Alopecia/metabolismo , Folículo Piloso/patologia , Folículo Piloso/metabolismo , Líquen Plano/metabolismo , Líquen Plano/patologia , Pessoa de Meia-Idade , Feminino , Caveolina 1/metabolismo , Masculino , Cavéolas/metabolismo , Couro Cabeludo/patologia , Adulto , Queratina-15/metabolismo , Idoso , Biópsia , Fibrose , Células-Tronco/metabolismo , Células-Tronco/patologia , Proteínas de Ligação a RNA/metabolismoRESUMO
Quiescin/sulfhydryl oxidase (QSOX1) is a secreted flavoprotein that modulates cellular proliferation, migration and adhesion, roles attributed to its ability to organize the extracellular matrix. We previously showed that exogenously added QSOX1b induces smooth muscle cells migration in a process that depends on its enzymatic activity and that is mediated by hydrogen peroxide derived from Nox1, a catalytic subunit of NAD(P)H oxidases. Here, we report that exogenous QSOX1b also stimulates the migration of L929 fibroblasts and that this effect is regulated by its endocytosis. The use of endocytosis inhibitors and caveolin 1-knockdown demonstrated that this endocytic pathway is caveola-mediated. QSOX1b colocalized with Nox1 in intracellular vesicles, as detected by confocal fluorescence, suggesting that extracellular QSOX1b is endocytosed with the transmembrane Nox1. These results reveal that endosomal QSOX1b is a novel intracellular redox regulator of cell migration.
Assuntos
Cavéolas , NADPH Oxidases , Fibroblastos , Endocitose , Proliferação de CélulasRESUMO
Caveolae-associated signaling toward mitochondria contributes to the cardioprotective mechanisms against ischemia-reperfusion (I/R) injury induced by ischemic postconditioning. In this work, we evaluated the role that the actin-cytoskeleton network exerts on caveolae-mitochondria communication during postconditioning. Isolated rat hearts subjected to I/R and to postconditioning were treated with latrunculin A, a cytoskeleton disruptor. Cardiac function was compared between these hearts and those exposed only to I/R and to the cardioprotective maneuver. Caveolae and mitochondria structures were determined by electron microscopy and maintenance of the actin-cytoskeleton was evaluated by phalloidin staining. Caveolin-3 and other putative caveolae-conforming proteins were detected by immunoblot analysis. Co-expression of caveolin-3 and actin was evaluated both in lipid raft fractions and in heart tissue from the different groups. Mitochondrial function was assessed by respirometry and correlated with cholesterol levels. Treatment with latrunculin A abolishes the cardioprotective postconditioning effect, inducing morphological and structural changes in cardiac tissue, reducing F-actin staining and diminishing caveolae formation. Latrunculin A administration to post-conditioned hearts decreases the interaction between caveolae-forming proteins, the co-localization of caveolin with actin and inhibits oxygen consumption rates in both subsarcolemmal and interfibrillar mitochondria. We conclude that actin-cytoskeleton drives caveolae signaling to mitochondria during postconditioning, supporting their functional integrity and contributing to cardiac adaption against reperfusion injury.
Assuntos
Cavéolas , Traumatismo por Reperfusão , Ratos , Animais , Cavéolas/metabolismo , Actinas/metabolismo , Caveolina 3/metabolismo , Citoesqueleto/metabolismo , Caveolina 1/metabolismo , Traumatismo por Reperfusão/metabolismo , Mitocôndrias/metabolismoRESUMO
Caveolae are small plasma membrane invaginations constituted for membrane proteins namely caveolins and cytosolic proteins termed cavins, which can occupy up to 50% of the surface of mammalian cells. The caveolae have been involved with a variety of cellular processes including regulation of cellular signaling. Insulin is a hormone that mediates a variety of physiological processes through activation of insulin receptor (IR), which is a tyrosine kinase receptor expressed in all mammalian tissues. Insulin induces activation of signal transducers and activators of transcription (STAT) family members including STAT5. In this study, we demonstrate, for the first time, that insulin induces phosphorylation of STAT5 at tyrosine-694 (STAT5-Tyr(P)694), STAT5 nuclear accumulation and an increase in STAT5-DNA complex formation in MCF-7 breast cancer cells. Insulin also induces nuclear accumulation of STAT5-Tyr(P)694, caveolin-1, and IR in MCF-7 cells. STAT5 nuclear accumulation and the increase of STAT5-DNA complex formation require the integrity of caveolae and microtubule network. Moreover, insulin induces an increase and nuclear accumulation of STAT5-Tyr(P)694 in MDA-MB-231 breast cancer cells. In conclusion, results demonstrate that caveolae and microtubule network play an important role in STAT5-Tyr(P)694, STAT5 nuclear accumulation and STAT5-DNA complex formation induced by insulin in breast cancer cells.
Assuntos
Neoplasias da Mama , Cavéolas , Animais , Humanos , Feminino , Cavéolas/metabolismo , Insulina/farmacologia , Insulina/metabolismo , Células MCF-7 , Fator de Transcrição STAT5/metabolismo , Neoplasias da Mama/metabolismo , Caveolina 1/genética , Caveolina 1/metabolismo , Fosforilação , Tirosina/metabolismo , DNA/metabolismo , Mamíferos/metabolismoRESUMO
BACKGROUND: Adipocytes from lipodystrophic Agpat2-/- mice have impaired adipogenesis and fewer caveolae. Herein, we examined whether these defects are associated with changes in lipid composition or abnormal levels of caveolae-associated proteins. Lipidome changes were quantified in differentiated Agpat2-/- adipocytes to identify lipids with potential adipogenic roles. METHODS: Agpat2-/- and wild type brown preadipocytes were differentiated in vitro. Plasma membrane was purified by ultracentrifugation. Number of caveolae and caveolae-associated proteins, as well as sterol, sphingolipid, and phospholipid lipidome were determined across differentiation. RESULTS: Differentiated Agpat2-/- adipocytes had decreased caveolae number but conserved insulin signaling. Caveolin-1 and cavin-1 levels were equivalent between Agpat2-/- and wild type adipocytes. No differences in PM cholesterol and sphingolipids abundance were detected between genotypes. Levels of phosphatidylserine at day 10 of differentiation were increased in Agpat2-/- adipocytes. Wild type adipocytes had increased whole cell triglyceride, diacylglycerol, phosphatidylglycerol, phosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, and trihexosyl ceramide, and decreased 24,25-dihydrolanosterol and sitosterol, as a result of adipogenic differentiation. By contrast, adipogenesis did not modify whole cell neutral lipids but increased lysophosphatidylcholine, sphingomyelin, and trihexosyl ceramide levels in Agpat2-/- adipocytes. Unexpectedly, adipogenesis decreased PM levels of main phospholipids in both genotypes. CONCLUSION: In Agpat2-/- adipocytes, decreased caveolae is not associated with changes in PM cholesterol nor sphingolipid levels; however, increased PM phosphatidylserine content may be implicated. Abnormal lipid composition is associated with the adipogenic abnormalities of Agpat2 -/- adipocytes but does not prevent insulin signaling.
Assuntos
Aciltransferases/metabolismo , Adipócitos/metabolismo , Adipogenia/fisiologia , Cavéolas/metabolismo , Membrana Celular/metabolismo , Colesterol/metabolismo , Esfingolipídeos/metabolismo , Animais , Insulina/metabolismo , Metabolismo dos Lipídeos/fisiologia , Lipidômica/métodos , Lipídeos/fisiologia , Camundongos , Transdução de Sinais/fisiologiaRESUMO
Caveolae constitute membrane domains critical for the organization and synchronization of different signaling molecules related to numerous cell processes such as cell migration, invasion, and differentiation. Caveolin-1 (Cav-1) is the main integral membrane protein of these domains. Recently, it was found that a normal expression of aquaporin-3 (AQP3) is required for extravillous trophoblast (EVT) cell migration. Our aim was to investigate the role of caveolae in the migration, invasion, and endovascular differentiation of human EVT cells during placentation and its interaction with AQP3. EVT cells (Swan 71 cell line) were cultured in complete Dulbecco's modified Eagle's medium-nutrient mixture F12 and treated with 5 mM methyl-ß-cyclodextrin (MßCD) to disrupt caveolae. We found that after MßCD treatment, Cav-1 protein was undetectable. In this condition, the ability of the cells to migrate was significantly decreased compared with the control cells, while no differences were observed in the number of invading cells and the metalloproteinases activity between control and MßCD-treated cells. Surprisingly, the disruption of caveolae significantly enhanced EVT endovascular differentiation. On the contrary, the silencing of AQP3, negatively affected tube-like formation. The theoretical analysis of the primary sequence of AQP3 protein revealed a putative Cav-1-binding site. In addition, immunoprecipitation and double immunofluorescence assays showed that AQP3 colocalized with Cav-1. These results showed that during placentation an intact caveola in EVT cells may be necessary for AQP3 and Cav-1 interaction and any perturbations might result in serious pregnancy disorders.
Assuntos
Aquaporina 3/genética , Cavéolas/metabolismo , Caveolina 1/genética , Trofoblastos/metabolismo , Sítios de Ligação , Diferenciação Celular/genética , Movimento Celular/genética , Feminino , Humanos , Placentação/genética , Gravidez , Ligação Proteica , Mapas de Interação de Proteínas/genética , Transdução de Sinais , beta-CiclodextrinasRESUMO
Caveolae are plasma membrane invaginations enriched with high cholesterol and sphingolipid content; they also contain caveolin proteins in their structure. Endothelial nitric oxide synthase (eNOS), an enzyme that synthesizes nitric oxide (NO) by converting L-arginine to L-citrulline, is highly concentrated in plasma membrane caveolae. Hypertension is associated with decreased NO production and impaired endothelium-dependent relaxation. Understanding the molecular mechanisms that follow hypertension is important. For this study, we hypothesized that spontaneously hypertensive rat (SHR) vessels should have a smaller number of caveolae, and that the caveolae structure should be disrupted in these vessels. This should impair the eNOS function and diminish NO bioavailability. Therefore, we aimed to investigate caveolae integrity and density in SHR aortas and mesenteric arteries and the role played by caveolae in endothelium-dependent relaxation. We have been able to show the presence of caveolae-like structures in SHR aortas and mesenteric arteries. Increased phenylephrine-induced contractile response after treatment with dextrin was related to lower NO release. In addition, impaired acetylcholine-induced endothelium-dependent relaxation could be related to decreased caveolae density in SHR vessels. The most important finding of this study was that cholesterol depletion with dextrin induced eNOS phosphorylation at Serine1177 (Ser1177) and boosted reactive oxygen species (ROS) production in normotensive rat and SHR vessels, which suggested eNOS uncoupling. Dextrin plus L-NAME or BH4 decreased ROS production in aorta and mesenteric arteries supernatant's of both SHR and normotensive groups. Human umbilical vein endothelial cells (HUVECs) treated with dextrin confirmed eNOS uncoupling, as verified by the reduced eNOS dimer/monomer ratio. BH4, L-arginine, or BH4 plus L-arginine inhibited eNOS monomerization. All these results showed that caveolae structure and integrity are essential for endothelium-dependent relaxation. Additionally, a smaller number of caveolae is associated with hypertension. Finally, caveolae disruption promotes eNOS uncoupling in normotensive and hypertensive rat vessels and in HUVECs.
Assuntos
Cavéolas/patologia , Endotélio Vascular/fisiopatologia , Hipertensão/fisiopatologia , Artérias Mesentéricas/patologia , Espécies Reativas de Oxigênio/metabolismo , Acetilcolina/farmacologia , Animais , Aorta/metabolismo , Aorta/patologia , Cavéolas/metabolismo , Cavéolas/ultraestrutura , Células Endoteliais da Veia Umbilical Humana , Humanos , Hipertensão/metabolismo , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Fenilefrina/farmacologia , Ratos Endogâmicos SHR , Ratos Wistar , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologiaRESUMO
BACKGROUND: Poly-lactic acid nanoparticles (PLA-NP) are a type of polymeric NP, frequently used as nanomedicines, which have advantages over metallic NP such as the ability to maintain therapeutic drug levels for sustained periods of time. Despite PLA-NP being considered biocompatible, data concerning alterations in cellular physiology are scarce. METHODS: We conducted an extensive evaluation of PLA-NP biocompatibility in human lung epithelial A549 cells using high throughput screening and more complex methodologies. These included measurements of cytotoxicity, cell viability, immunomodulatory potential, and effects upon the cells' proteome. We used non- and green-fluorescent PLA-NP with 63 and 66 nm diameters, respectively. Cells were exposed with concentrations of 2, 20, 100 and 200 µg/mL, for 24, 48 and 72 h, in most experiments. Moreover, possible endocytic mechanisms of internalization of PLA-NP were investigated, such as those involving caveolae, lipid rafts, macropinocytosis and clathrin-coated pits. RESULTS: Cell viability and proliferation were not altered in response to PLA-NP. Multiplex analysis of secreted mediators revealed a low-level reduction of IL-12p70 and vascular epidermal growth factor (VEGF) in response to PLA-NP, while all other mediators assessed were unaffected. However, changes to the cells' proteome were observed in response to PLA-NP, and, additionally, the cellular stress marker miR155 was found to reduce. In dual exposures of staurosporine (STS) with PLA-NP, PLA-NP enhanced susceptibility to STS-induced cell death. Finally, PLA-NP were rapidly internalized in association with clathrin-coated pits, and, to a lesser extent, with lipid rafts. CONCLUSIONS: These data demonstrate that PLA-NP are internalized and, in general, tolerated by A549 cells, with no cytotoxicity and no secretion of pro-inflammatory mediators. However, PLA-NP exposure may induce modification of biological functions of A549 cells, which should be considered when designing drug delivery systems. Moreover, the pathways of PLA-NP internalization we detected could contribute to the improvement of selective uptake strategies.
Assuntos
Materiais Biocompatíveis/química , Cavéolas/metabolismo , Células Epiteliais/efeitos dos fármacos , Microdomínios da Membrana , Nanopartículas/química , Poliésteres/química , Células A549 , Sobrevivência Celular , Clatrina/química , Sistemas de Liberação de Medicamentos , Células Epiteliais/citologia , Humanos , Interleucina-12/metabolismo , MicroRNAs/metabolismo , Tamanho da Partícula , Pinocitose , Proteoma , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
An important transcellular transport mechanism in the blood-brain barrier (BBB) involves caveolae, which are specialized delta-shaped domains of the endothelial plasma membrane that are rich in cholesterol, glycosphingolipids and the scaffolding protein Caveolina-1 (Cav-1). In this work, we investigated whether the increase in endocytosis and transendothelial vesicular trafficking in rat cerebellum after blood-brain barrier breakdown (BBBb) induced by Phoneutria nigriventer spider venom (PNV) was mediated by caveolae. The expression of Cav-1, phosphorylated Cav-1 (pCav-1), dynamin-2 (Dyn2), Src kinase family (SKF) and matrix-metalloproteinase-9 (MMP9), proteins involved in caveolar dynamics and BBB opening, was investigated. Immunofluorescence, western blotting (WB) and transmission electron microscopy were used to assess changes at 1, 2, 5, 24 and 72h post-venom. WB showed upregulation of Cav-1, Dyn2 and MMP9 at 1, 5 and 72h (corresponding, respectively, to intervals when intoxication was most evident, when signs of recovery were present, and when no intoxication was detectable). In contrast, pCav-1 and SKF, which are essential for internalization and transport, decreased when Cav-1 and Dyn2, proteins essential for caveolar formation, were increased. Overall, these changes indicated that vesicular trafficking across the endothelium (high pCav/SKF levels) coincided with lower numbers of caveolae (Cav-1/Dyn2 downregulation) and lower expression of MMP9. Thus, the internalization (disassembly) of caveolae alternates with caveolar neoformation (assembly), resulting in changes in caveolar density in the endothelium membrane. These caveolar dynamics imply tensional mechanical stress that is important in triggering key signaling mechanisms. We conclude that PNV-induced breakdown of transcellular transport in the BBB is caused by an increase in caveolae-mediated endocytosis; this effect was correlated with the progression of temporal signs of envenoming. Caveolar dynamics are probably involved in shear stress and BBBb regulatory mechanisms in this experimental model.