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1.
Oncogene ; 38(26): 5142-5157, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30894682

RESUMO

The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a transcriptional target of the lineage-survival oncogene NKX2-1/TTF-1 in lung adenocarcinomas. In addition to its kinase-dependent role, ROR1 functions as a scaffold protein to facilitate interaction between caveolin-1 (CAV1) and CAVIN1, and consequently maintains caveolae formation, which in turn sustains pro-survival signaling toward AKT from multiple receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR), MET (proto-oncogene, receptor tyrosine kinase), and IGF-IR (insulin-like growth factor receptor 1). Therefore, ROR1 is an attractive target for overcoming EGFR-TKI resistance due to various mechanisms such as EGFR T790M double mutation and bypass signaling from other RTKs. Here, we report that ROR1 possesses a novel scaffold function indispensable for efficient caveolae-dependent endocytosis. CAVIN3 was found to bind with ROR1 at a site distinct from sites for CAV1 and CAVIN1, a novel function required for proper CAVIN3 subcellular localization and caveolae-dependent endocytosis, but not caveolae formation itself. Furthermore, evidence of a mechanistic link between ROR1-CAVIN3 interaction and consequential caveolae trafficking, which was found to utilize a binding site distinct from those for ROR1 interactions with CAV1 and CAVIN1, with RTK-mediated pro-survival signaling towards AKT in early endosomes in lung adenocarcinoma cells was also obtained. The present findings warrant future study to enable development of novel therapeutic strategies for inhibiting the multifaceted scaffold functions of ROR1 in order to reduce the intolerable death toll from this devastating cancer.


Assuntos
Adenocarcinoma de Pulmão/patologia , Cavéolas/fisiologia , Endocitose , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pulmonares/patologia , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Animais , Células COS , Cavéolas/metabolismo , Sobrevivência Celular/genética , Células Cultivadas , Endocitose/genética , Células HEK293 , Células HeLa , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ligação Proteica/fisiologia , Células Sf9 , Transdução de Sinais/genética , Spodoptera
2.
Virology ; 529: 160-168, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30710800

RESUMO

To comprehensively understand the endocytosis of Sapelovirus A (PSV) entry into PK-15 cells, we studied PSV infection in the context of cell perturbations through drug inhibition, siRNA silencing and overexpression of dominant negative (DN) mutants. We showed here that PSV infection of PK-15 cells was unaffected by pretreated with chlorpromazine, EIPA, knockdown of the clathrin heavy chain or overexpression of Eps15 DN mutant. Conversely, PSV infection was sensitive to NH4Cl, chloroquine, dynasore, nystatin, MßCD and wortmannin with reduced PSV VP1 expression levels and virus titer. Additionally, PSV invasion leaded to rapid actin rearrangement and disruption of the cellular actin network enhanced PSV infection. After internalization the virus was transported to late endosomes and/or cycling endosomes that requires the participation of Rab7 and Rab11. Our findings demonstrate that PSV uses caveolae-dependent endocytosis as the predominant entry portal into PK-15 cells which requires low pH, dynamin, Rab7 and Rab11.


Assuntos
Cavéolas/fisiologia , Endocitose/fisiologia , Picornaviridae/fisiologia , Internalização do Vírus , Proteínas rab de Ligação ao GTP/fisiologia , Animais , Linhagem Celular , Sobrevivência Celular , Concentração de Íons de Hidrogênio , Suínos
3.
BMB Rep ; 52(2): 111-112, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30760383

RESUMO

Although many studies have reported that the breakdown of the blood-brain barrier (BBB) represents one of the major pathological changes in aging, the mechanism underlying this process remains relatively unexplored. In this study, we described that acid sphingomyelinase (ASM) derived from endothelial cells plays a critical role in BBB disruption in aging. ASM levels were elevated in the brain endothelium and plasma of aged humans and mice, resulting in BBB leakage through an increase in caveolae-mediated transcytosis. Moreover, ASM caused damage to the caveolae-cytoskeleton via protein phosphatase 1-mediated ezrin/radixin/moesin dephosphorylation in primary mouse brain endothelial cells. Mice overexpressing brain endothelial cell-specific ASM exhibited acceleration of BBB impairment and neuronal dysfunction. However, genetic inhibition and endothelial specific knock-down of ASM in mice improved BBB disruption and neurocognitive impairment during aging. Results of this study revealed a novel role of ASM in the regulation of BBB integrity and neuronal function in aging, thus highlighting the potential of ASM as a new therapeutic target for anti-aging. [BMB Reports 2019; 52(2): 111-112].


Assuntos
Envelhecimento/fisiologia , Barreira Hematoencefálica/fisiologia , Esfingomielina Fosfodiesterase/fisiologia , Animais , Encéfalo/metabolismo , Cavéolas/fisiologia , Células Endoteliais/metabolismo , Humanos , Camundongos , Esfingomielina Fosfodiesterase/genética , Junções Íntimas/metabolismo , Transcitose/fisiologia
4.
Nanoscale ; 10(26): 12386-12397, 2018 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-29926047

RESUMO

Transcytosis of nanoparticles (NPs) is emerging as an attractive alternative to the paracellular route in cancer drug delivery with studies suggesting targeting caveolae-mediated endocytosis to maximize NP transcytosis. However, there are limited studies on transcytosis of NPs, especially for corona-coated NPs. Most studies focused on cellular uptake as an indirect measure of the NP's transcellular permeability (Pd). Here, we probed the effect of protein corona on the uptake and transcytosis of 20, 40, 100, and 200 nm polystyrene nanoparticles (pNP-PC) across HUVECs in a microfluidic channel that modelled the microvasculature. We observed increased cell uptake with size of pNP-PC although it was the smallest 20 nm pNP-PC that exhibited the highest transcellular Pd. In the absence of corona however, cell uptake decreased with size, and the largest 200 nm pNP-PEG exhibited the lowest transcellular Pd. By inhibiting caveolae-mediated endocytosis in HUVECs, smaller pNPs had a larger drop in cell uptake than larger pNPs, regardless of surface coating. However, only the smallest (20 nm) and largest (200 nm) pNP-PC had a decrease in Pd following inhibition with MßCD. Our findings showed that the protein corona affected the transcytosis of NPs, and their uptake by caveolae-mediated endocytosis did not necessarily lead to transcytosis.


Assuntos
Cavéolas/fisiologia , Endocitose , Nanopartículas/metabolismo , Coroa de Proteína/química , Transcitose , Células Endoteliais da Veia Umbilical Humana , Humanos , Dispositivos Lab-On-A-Chip , Poliestirenos
5.
Curr Biol ; 28(8): R402-R405, 2018 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-29689223

RESUMO

Caveolae are one of the most abundant and striking features of the plasma membrane of many mammalian cell types. These surface pits have fascinated biologists since their discovery by the pioneers of electron microscopy in the middle of the last century, but we are only just starting to understand their multiple functions. Molecular understanding of caveolar formation is advancing rapidly and we now know that sculpting the membrane to generate the characteristic bulb-shaped caveolar pit involves the coordinated action of integral membrane proteins and peripheral membrane coat proteins in a process dependent on their multiple interactions with membrane lipids. The resulting structure is further stabilised by protein complexes at the caveolar neck. Caveolae can bud to generate an endocytic carrier but can also be disassembled in response to specific stimuli to function as a mechanoprotective device. These structures have also been linked to numerous signalling pathways. Here, we will briefly summarise the current molecular and structural understanding of caveolar formation and dynamics, discuss how the crucial structural components of caveolae work together to generate a dynamic sensing domain, and discuss the implications of recent studies on the diverse roles proposed for caveolae in different cells and tissues.


Assuntos
Cavéolas/metabolismo , Cavéolas/fisiologia , Membrana Celular/fisiologia , Animais , Caveolinas/metabolismo , Estruturas da Membrana Celular/fisiologia , Humanos , Mamíferos , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Transdução de Sinais/fisiologia
6.
Vet Res ; 49(1): 16, 2018 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-29439726

RESUMO

Cell-penetrating peptide (CPP) is a promising cargo for delivering bioactive molecules. In this study, the N terminus of VP1 from chicken anemia virus, designated as CVP1, was found to carry enriched arginine residues with α-helix. By confocal imaging, flow cytometry and MTT assay, we identified CVP1 as a novel, safe and efficient CPP. CVP1-FITC peptide could entry different types of cells tested with dose dependence, but without cytotoxic effects. Compared with TAT-FITC peptide, the CVP1-FITC peptide showed much higher cell-penetrating activity. Moreover, CVP1 could successfully deliver ß-glycosidase, poly (I:C) and plasmid into HCT116 cells. Inhibitors and temperature sensitivity analysis further indicated that the cell-penetrating activity of CVP1 was based on ATP-dependent and caveolae-mediated endocytosis. All these data demonstrate that CVP1 has efficient cell-penetrating activity and great potential for developing a novel delivery vector.


Assuntos
Cavéolas/fisiologia , Peptídeos Penetradores de Células/administração & dosagem , Vírus da Anemia da Galinha/fisiologia , Animais , Cavéolas/virologia , Linhagem Celular , Galinhas , Cães , Sistemas de Liberação de Medicamentos/veterinária , Endocitose/fisiologia , Células HCT116 , Células HEK293 , Humanos , Células Madin Darby de Rim Canino
7.
Nat Commun ; 9(1): 168, 2018 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-29330478

RESUMO

Effective suppression of JAK-STAT signalling by the inducible inhibitor "suppressor of cytokine signalling 3" (SOCS3) is essential for limiting signalling from cytokine receptors. Here we show that cavin-1, a component of caveolae, is a functionally significant SOCS3-interacting protein. Biochemical and confocal imaging demonstrate that SOCS3 localisation to the plasma membrane requires cavin-1. SOCS3 is also critical for cavin-1 stabilisation, such that deletion of SOCS3 reduces the expression of cavin-1 and caveolin-1 proteins, thereby reducing caveola abundance in endothelial cells. Moreover, the interaction of cavin-1 and SOCS3 is essential for SOCS3 function, as loss of cavin-1 enhances cytokine-stimulated STAT3 phosphorylation and abolishes SOCS3-dependent inhibition of IL-6 signalling by cyclic AMP. Together, these findings reveal a new functionally important mechanism linking SOCS3-mediated inhibition of cytokine signalling to localisation at the plasma membrane via interaction with and stabilisation of cavin-1.


Assuntos
Proteínas de Membrana/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Animais , Cavéolas/fisiologia , Deleção de Genes , Regulação da Expressão Gênica , Células HEK293 , Humanos , Janus Quinases/genética , Janus Quinases/metabolismo , Proteínas de Membrana/genética , Camundongos , Ligação Proteica , Proteínas de Ligação a RNA/genética , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas/genética
8.
Reprod Sci ; 25(8): 1231-1242, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29113580

RESUMO

During early pregnancy, uterine epithelial cells (UECs) become less adherent to the underlying basal lamina and are subsequently removed so the blastocyst can invade the underlying stroma. This process involves the removal of focal adhesions from the basal plasma membrane of UECs. These focal adhesions are thought to be internalized by caveolae, which significantly increase in abundance at the time of blastocyst implantation. A recent in vitro study indicated that prominin-2 prevents the formation of caveolae by sequestering membrane cholesterol. The present study examines whether prominin-2 affects the formation of caveolae and loss of focal adhesions in UECs during normal and ovarian hyperstimulation (OH) pregnancy in the rat. At the time of fertilization during normal pregnancy, prominin-2 is distributed throughout the basolateral plasma membrane. However, at the time of implantation and coincident with an increase in caveolae, prominin-2 is lost from the basal plasma membrane. In contrast, prominin-2 remains in the basolateral plasma membrane throughout OH pregnancy. Transmission electron microscopy showed that this membrane contained few caveolae throughout OH pregnancy. Our results indicate that prominin-2 prevents the formation of caveolae. We suggest the retention of prominin-2 in the basal plasma membrane during OH pregnancy prevents the formation of caveolae and is responsible for the retention of focal adhesions in this membrane, thereby contributing to the reduced implantation rate observed after such treatments.


Assuntos
Cavéolas/fisiologia , Glicoproteínas de Membrana/fisiologia , Útero/fisiologia , Animais , Cavéolas/metabolismo , Cavéolas/ultraestrutura , Caveolina 1/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Estrogênios/administração & dosagem , Estrogênios/fisiologia , Feminino , Adesões Focais/fisiologia , Glicoproteínas de Membrana/metabolismo , Ovariectomia , Gravidez , Progesterona/administração & dosagem , Progesterona/fisiologia , Ratos Wistar , Útero/metabolismo , Útero/ultraestrutura
9.
Placenta ; 57: 137-143, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28864003

RESUMO

INTRODUCTION: Studies in animal models have shown that unidirectional vesicular transport of amniotic fluid across the amnion plays a primary role in regulating amniotic fluid volume. Our objective was to explore vesicle type, vesicular uptake and intracellular distribution of vesicles in human amnion cells using high- and super-resolution fluorescence microscopy. METHODS: Placental amnion was obtained at cesarean section and amnion cells were prepared and cultured. At 20%-50% confluence, the cells were incubated with fluorophore conjugated macromolecules for 1-30 min at 22 °C or 37 °C. Fluorophore labeled macromolecules were selected as markers of receptor-mediated caveolar and clathrin-coated vesicular uptake as well as non-specific endocytosis. After fluorophore treatment, the cells were fixed, imaged and vesicles counted using Imaris® software. RESULTS: Vesicular uptake displayed first order saturation kinetics with half saturation times averaging 1.3 min at 37 °C compared to 4.9 min at 22 °C, with non-specific endocytotic uptake being more rapid at both temperatures. There was extensive cell-to-cell variability in uptake rate. Under super-resolution microscopy, the pattern of intracellular spatial distribution was distinct for each macromolecule. Co-localization of fluorescently labeled macromolecules was very low at vesicular dimensions. CONCLUSIONS: In human placental amnion cells, 1) vesicular uptake of macromolecules is rapid, consistent with the concept that vesicular transcytosis across the amnion plays a role in the regulation of amniotic fluid volume; 2) uptake is temperature dependent and variable among individual cells; 3) the unique intracellular distributions suggest distinct functions for each vesicle type; 4) non-receptor mediated vesicular uptake may be a primary vesicular uptake mechanism.


Assuntos
Âmnio/citologia , Cavéolas/fisiologia , Vesículas Revestidas por Clatrina/fisiologia , Endocitose , Células Epiteliais/fisiologia , Feminino , Humanos , Substâncias Macromoleculares , Gravidez
10.
Biophys J ; 113(5): 1047-1059, 2017 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-28877488

RESUMO

Caveolae are signal transduction centers, yet their subcellular distribution and preservation in cardiac myocytes after cell isolation are not well documented. Here, we quantify caveolae located within 100 nm of the outer cell surface membrane in rabbit single-ventricular cardiomyocytes over 8 h post-isolation and relate this to the presence of caveolae in intact tissue. Hearts from New Zealand white rabbits were either chemically fixed by coronary perfusion or enzymatically digested to isolate ventricular myocytes, which were subsequently fixed at 0, 3, and 8 h post-isolation. In live cells, the patch-clamp technique was used to measure whole-cell plasma membrane capacitance, and in fixed cells, caveolae were quantified by transmission electron microscopy. Changes in cell-surface topology were assessed using scanning electron microscopy. In fixed ventricular myocardium, dual-axis electron tomography was used for three-dimensional reconstruction and analysis of caveolae in situ. The presence and distribution of surface-sarcolemmal caveolae in freshly isolated cells matches that of intact myocardium. With time, the number of surface-sarcolemmal caveolae decreases in isolated cardiomyocytes. This is associated with a gradual increase in whole-cell membrane capacitance. Concurrently, there is a significant increase in area, diameter, and circularity of sub-sarcolemmal mitochondria, indicative of swelling. In addition, electron tomography data from intact heart illustrate the regular presence of caveolae not only at the surface sarcolemma, but also on transverse-tubular membranes in ventricular myocardium. Thus, caveolae are dynamic structures, present both at surface-sarcolemmal and transverse-tubular membranes. After cell isolation, the number of surface-sarcolemmal caveolae decreases significantly within a time frame relevant for single-cell research. The concurrent increase in cell capacitance suggests that membrane incorporation of surface-sarcolemmal caveolae underlies this, but internalization and/or micro-vesicle loss to the extracellular space may also contribute. Given that much of the research into cardiac caveolae-dependent signaling utilizes isolated cells, and since caveolae-dependent pathways matter for a wide range of other study targets, analysis of isolated cell data should take the time post-isolation into account.


Assuntos
Cavéolas , Ventrículos do Coração/citologia , Miócitos Cardíacos/citologia , Animais , Cavéolas/fisiologia , Separação Celular , Células Cultivadas , Capacitância Elétrica , Tomografia com Microscopia Eletrônica , Imagem Tridimensional , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Mitocôndrias/fisiologia , Modelos Biológicos , Miócitos Cardíacos/fisiologia , Técnicas de Patch-Clamp , Coelhos , Sarcolema/fisiologia , Propriedades de Superfície , Fixação de Tecidos
11.
Mol Biol Cell ; 28(22): 3095-3111, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-28904206

RESUMO

Caveolin-1 (CAV1) is an essential component of caveolae and is implicated in numerous physiological processes. Recent studies have identified heterozygous mutations in the CAV1 gene in patients with pulmonary arterial hypertension (PAH), but the mechanisms by which these mutations impact caveolae assembly and contribute to disease remain unclear. To address this question, we examined the consequences of a familial PAH-associated frameshift mutation in CAV1, P158PfsX22, on caveolae assembly and function. We show that C-terminus of the CAV1 P158 protein contains a functional ER-retention signal that inhibits ER exit and caveolae formation and accelerates CAV1 turnover in Cav1-/- MEFs. Moreover, when coexpressed with wild-type (WT) CAV1 in Cav1-/- MEFs, CAV1-P158 functions as a dominant negative by partially disrupting WT CAV1 trafficking. In patient skin fibroblasts, CAV1 and caveolar accessory protein levels are reduced, fewer caveolae are observed, and CAV1 complexes exhibit biochemical abnormalities. Patient fibroblasts also exhibit decreased resistance to a hypo-osmotic challenge, suggesting the function of caveolae as membrane reservoir is compromised. We conclude that the P158PfsX22 frameshift introduces a gain of function that gives rise to a dominant negative form of CAV1, defining a new mechanism by which disease-associated mutations in CAV1 impair caveolae assembly.


Assuntos
Caveolina 1/genética , Caveolina 1/metabolismo , Cavéolas/fisiologia , Endocitose , Retículo Endoplasmático , Fibroblastos/metabolismo , Mutação da Fase de Leitura/genética , Humanos , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/metabolismo , Mutação , Transporte Proteico
12.
Curr Biol ; 27(19): 2951-2962.e5, 2017 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-28943089

RESUMO

Caveolae introduce flask-shaped convolutions into the plasma membrane and help to protect the plasma membrane from damage under stretch forces. The protein components that form the bulb of caveolae are increasingly well characterized, but less is known about the contribution of proteins that localize to the constricted neck. Here we make extensive use of multiple CRISPR/Cas9-generated gene knockout and knockin cell lines to investigate the role of Eps15 Homology Domain (EHD) proteins at the neck of caveolae. We show that EHD1, EHD2, and EHD4 are recruited to caveolae. Recruitment of the other EHDs increases markedly when EHD2, which has been previously detected at caveolae, is absent. Construction of knockout cell lines lacking EHDs 1, 2, and 4 confirms this apparent functional redundancy. Two striking sets of phenotypes are observed in EHD1,2,4 knockout cells: (1) the characteristic clustering of caveolae into higher-order assemblies is absent; and (2) when the EHD1,2,4 knockout cells are subjected to prolonged cycles of stretch forces, caveolae are destabilized and the plasma membrane is prone to rupture. Our data identify the first molecular components that act to cluster caveolae into a membrane ultrastructure with the potential to extend stretch-buffering capacity and support a revised model for the function of EHDs at the caveolar neck.


Assuntos
Proteínas de Transporte/genética , Cavéolas/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas Nucleares/genética , Proteínas de Transporte Vesicular/genética , Animais , Fenômenos Biomecânicos , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Camundongos , Células NIH 3T3 , Proteínas Nucleares/metabolismo , Estresse Mecânico , Proteínas de Transporte Vesicular/metabolismo
13.
Proc Natl Acad Sci U S A ; 114(20): E4010-E4019, 2017 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-28461495

RESUMO

The fibroblast growth factor (FGF) homologous factor FGF13, a noncanonical FGF, has been best characterized as a voltage-gated Na+ channel auxiliary subunit. Other cellular functions have been suggested, but not explored. In inducible, cardiac-specific Fgf13 knockout mice, we found-even in the context of the expected reduction in Na+ channel current-an unanticipated protection from the maladaptive hypertrophic response to pressure overload. To uncover the underlying mechanisms, we searched for components of the FGF13 interactome in cardiomyocytes and discovered the complete set of the cavin family of caveolar coat proteins. Detailed biochemical investigations showed that FGF13 acts as a negative regulator of caveolae abundance in cardiomyocytes by controlling the relative distribution of cavin 1 between the sarcolemma and cytosol. In cardiac-specific Fgf13 knockout mice, cavin 1 redistribution to the sarcolemma stabilized the caveolar structural protein caveolin 3. The consequent increase in caveolae density afforded protection against pressure overload-induced cardiac dysfunction by two mechanisms: (i) enhancing cardioprotective signaling pathways enriched in caveolae, and (ii) increasing the caveolar membrane reserve available to buffer membrane tension. Thus, our results uncover unexpected roles for a FGF homologous factor and establish FGF13 as a regulator of caveolae-mediated mechanoprotection and adaptive hypertrophic signaling.


Assuntos
Cardiomegalia/metabolismo , Cavéolas/fisiologia , Caveolinas/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Miócitos Cardíacos/fisiologia , Animais , Cardiomegalia/etiologia , Cardiomegalia/patologia , Modelos Animais de Doenças , Feminino , Fatores de Crescimento de Fibroblastos/genética , Fibrose , Masculino , Microdomínios da Membrana/metabolismo , Camundongos Knockout , Miocárdio/patologia , Miócitos Cardíacos/ultraestrutura , Pressão , Sarcolema/fisiologia , Sarcolema/ultraestrutura
14.
Curr Diab Rep ; 17(3): 19, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28283950

RESUMO

PURPOSE OF REVIEW: Diabetic nephropathy, a major microvascular complication of diabetes and the most common cause of end-stage renal disease, is characterized by prominent accumulation of extracellular matrix. The membrane microdomains caveolae, and their integral protein caveolin-1, play critical roles in the regulation of signal transduction. In this review we discuss current knowledge of the contribution of caveolin-1/caveolae to profibrotic signaling and the pathogenesis of diabetic kidney disease, and assess its potential as a therapeutic target. RECENT FINDINGS: Caveolin (cav)-1 is key to facilitating profibrotic signal transduction induced by several stimuli known to be pathogenic in diabetic nephropathy, including the most prominent factors hyperglycemia and angiotensin II. Phosphorylation of cav-1 on Y14 is an important regulator of these responses. In vivo studies support a pathogenic role for caveolae in the progression of diabetic nephropathy. Targeting caveolin-1/caveolae would enable inhibition of multiple profibrotic pathways, representing a novel and potentially potent therapeutic option for diabetic nephropathy.


Assuntos
Caveolina 1/fisiologia , Nefropatias Diabéticas/etiologia , Animais , Cavéolas/fisiologia , Caveolina 1/antagonistas & inibidores , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/fisiopatologia , Matriz Extracelular/metabolismo , Humanos , Estresse Oxidativo , Transdução de Sinais/fisiologia
15.
Am J Physiol Cell Physiol ; 312(4): C459-C477, 2017 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-28122734

RESUMO

Caveolins (Cavs) are ~20 kDa scaffolding proteins that assemble as oligomeric complexes in lipid raft domains to form caveolae, flask-shaped plasma membrane (PM) invaginations. Caveolae ("little caves") require lipid-lipid, protein-lipid, and protein-protein interactions that can modulate the localization, conformational stability, ligand affinity, effector specificity, and other functions of proteins that are partners of Cavs. Cavs are assembled into small oligomers in the endoplasmic reticulum (ER), transported to the Golgi for assembly with cholesterol and other oligomers, and then exported to the PM as an intact coat complex. At the PM, cavins, ~50 kDa adapter proteins, oligomerize into an outer coat complex that remodels the membrane into caveolae. The structure of caveolae protects their contents (i.e., lipids and proteins) from degradation. Cellular changes, including signal transduction effects, can destabilize caveolae and produce cavin dissociation, restructuring of Cav oligomers, ubiquitination, internalization, and degradation. In this review, we provide a perspective of the life cycle (biogenesis, degradation), composition, and physiologic roles of Cavs and caveolae and identify unanswered questions regarding the roles of Cavs and cavins in caveolae and in regulating cell physiology.1.


Assuntos
Cavéolas/fisiologia , Caveolinas/metabolismo , Fenômenos Fisiológicos Celulares/fisiologia , Proteínas de Membrana/metabolismo , Transporte Proteico/fisiologia , Proteínas de Ligação a RNA/metabolismo , Animais , Humanos , Modelos Biológicos
16.
Proc Natl Acad Sci U S A ; 113(28): 7834-9, 2016 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-27342861

RESUMO

Lipid membrane curvature plays important roles in various physiological phenomena. Curvature-regulated dynamic membrane remodeling is achieved by the interaction between lipids and proteins. So far, several membrane sensing/sculpting proteins, such as Bin/amphiphysin/Rvs (BAR) proteins, are reported, but there remains the possibility of the existence of unidentified membrane-deforming proteins that have not been uncovered by sequence homology. To identify new lipid membrane deformation proteins, we applied liposome-based microscopic screening, using unbiased-darkfield microscopy. Using this method, we identified phospholipase Cß1 (PLCß1) as a new candidate. PLCß1 is well characterized as an enzyme catalyzing the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2). In addition to lipase activity, our results indicate that PLCß1 possessed the ability of membrane tubulation. Lipase domains and inositol phospholipids binding the pleckstrin homology (PH) domain of PLCß1 were not involved, but the C-terminal sequence was responsible for this tubulation activity. Computational modeling revealed that the C terminus displays the structural homology to the BAR domains, which is well known as a membrane sensing/sculpting domain. Overexpression of PLCß1 caused plasma membrane tubulation, whereas knockdown of the protein reduced the number of caveolae and induced the evagination of caveolin-rich membrane domains. Taken together, our results suggest a new function of PLCß1: plasma membrane remodeling, and in particular, caveolae formation.


Assuntos
Cavéolas/fisiologia , Fosfolipase C beta/metabolismo , Animais , Lipossomos , Camundongos , Camundongos Endogâmicos C57BL , Células Swiss 3T3
17.
J Physiol ; 594(20): 5941-5957, 2016 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-27121987

RESUMO

KEY POINTS: Hyperfibrinogenaemia (HFg) results in vascular remodelling, and fibrinogen (Fg) and amyloid ß (Aß) complex formation is a hallmark of Alzheimer's disease. However, the interconnection of these effects, their mechanisms and implications in cerebrovascular diseases are not known. Using a mouse model of HFg, we showed that at an elevated blood level, Fg increases cerebrovascular permeability via mainly caveolar protein transcytosis. This enhances deposition of Fg in subendothelial matrix and interstitium making the immobilized Fg a readily accessible substrate for binding Aß and cellular prion protein (PrPC ), the protein that is thought to have a greater effect on memory than Aß. We showed that enhanced formation of Fg-Aß and Fg-PrPC complexes are associated with reduction in short-term memory. The present study delineates a new mechanistic pathway for vasculo-neuronal dysfunctions found in inflammatory cardiovascular and cerebrovascular diseases associated with an elevated blood level of Fg. ABSTRACT: Many cardiovascular diseases are associated with inflammation and as such are accompanied by an increased blood level of fibrinogen (Fg). Besides its well-known prothrombotic effects Fg seems to have other destructive roles in developing microvascular dysfunction that include changes in vascular reactivity and permeability. Increased permeability of brain microvessels has the most profound effects as it may lead to cerebrovascular remodelling and result in memory reduction. The goal of the present study was to define mechanisms of cerebrovascular permeability and associated reduction in memory induced by elevated blood content of Fg. Genetically modified, transgenic hyperfibrinogenic (HFg) mice were used to study cerebrovascular transcellular and paracellular permeability in vivo. The extent of caveolar formation and the role of caveolin-1 signalling were evaluated by immunohistochemistry (IHC) and Western blot (WB) analysis in brain samples from experimental animals. Formation of Fg complexes with amyloid ß (Aß) and with cellular prion protein (PrPC ) were also assessed with IHC and WB analysis. Short-term memory of mice was assessed by novel object recognition and Y-maze tests. Caveolar protein transcytosis was found to have a prevailing role in overall increased cerebrovascular permeability in HFg mice. These results were associated with enhanced formation of caveolae. Increased formation of Fg-PrPC and Fg-Aß complexes were correlated with reduction in short-term memory in HFg mice. Using the model of hyperfibrinogenaemia, the present study shows a novel mechanistic pathway of inflammation-induced and Fg-mediated reduction in short-term memory.


Assuntos
Circulação Cerebrovascular/fisiologia , Transtornos Cerebrovasculares/metabolismo , Transtornos Cerebrovasculares/patologia , Fibrinogênio/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Cavéolas/metabolismo , Cavéolas/fisiologia , Caveolina 1/metabolismo , Memória de Curto Prazo/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Permeabilidade
18.
PLoS One ; 11(3): e0151556, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26977592

RESUMO

Endothelial cells respond to a large range of stimuli including circulating lipoproteins, growth factors and changes in haemodynamic mechanical forces to regulate the activity of endothelial nitric oxide synthase (eNOS) and maintain blood pressure. While many signalling pathways have been mapped, the identities of membrane domains through which these signals are transmitted are less well characterized. Here, we manipulated bovine aortic endothelial cells (BAEC) with cholesterol and the oxysterol 7-ketocholesterol (7KC). Using a range of microscopy techniques including confocal, 2-photon, super-resolution and electron microscopy, we found that sterol enrichment had differential effects on eNOS and caveolin-1 (Cav1) colocalisation, membrane order of the plasma membrane, caveolae numbers and Cav1 clustering. We found a correlation between cholesterol-induced condensation of the plasma membrane and enhanced high density lipoprotein (HDL)-induced eNOS activity and phosphorylation suggesting that cholesterol domains, but not individual caveolae, mediate HDL stimulation of eNOS. Vascular endothelial growth factor (VEGF)-induced and shear stress-induced eNOS activity was relatively independent of membrane order and may be predominantly controlled by the number of caveolae on the cell surface. Taken together, our data suggest that signals that activate and phosphorylate eNOS are transmitted through distinct membrane domains in endothelial cells.


Assuntos
Células Endoteliais/metabolismo , Microdomínios da Membrana/fisiologia , Óxido Nítrico Sintase Tipo III/metabolismo , Animais , Aorta/citologia , Bovinos , Cavéolas/fisiologia , Caveolina 1/análise , Linhagem Celular , Colesterol/farmacologia , Cromatografia Líquida de Alta Pressão , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/ultraestrutura , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Humanos , Processamento de Imagem Assistida por Computador , Cetocolesteróis/farmacologia , Lipoproteínas HDL/farmacologia , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/ultraestrutura , Microscopia Eletrônica , Reologia , Estresse Mecânico , Fator A de Crescimento do Endotélio Vascular/farmacologia , beta-Ciclodextrinas/farmacologia
19.
Am J Physiol Lung Cell Mol Physiol ; 310(11): L1078-87, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-27016585

RESUMO

Caveolae are stiff plasma membrane microdomains implicated in various cell response mechanisms like Ca(2+) signaling and mechanotransduction. Pulmonary arterial smooth muscle cells (PASMC) transduce mechanical stimuli into Ca(2+) increase via plasma membrane stretch-activated channels (SAC). This mechanotransduction process is modified in pulmonary hypertension (PH) during which stretch forces are increased by the increase in arterial blood pressure. We propose to investigate how caveolae are involved in the pathophysiology of PH and particularly in mechanotransduction. PASMC were freshly isolated from control rats (Ctrl rats) and rats suffering from PH induced by 3 wk of chronic hypoxia (CH rats). Using a caveolae disrupter (methyl-ß-cyclodextrin), we showed that SAC activity measured by patch-clamp, stretch-induced Ca(2+) increase measured with indo-1 probe and pulmonary arterial ring contraction to osmotic shock are enhanced in Ctrl rats when caveolae are disrupted. In CH rats, SAC activity, Ca(2+), and contraction responses to stretch are all higher compared with Ctrl rats. However, in contrast to Ctrl rats, caveolae disruption in CH-PASMC, reduces SAC activity, Ca(2+) responses to stretch and arterial contractions. Furthermore, by means of immunostainings and transmission electron microscopy, we observed that caveolae and caveolin-1 are expressed in PASMC from both Ctrl and CH rats and localize close to subplasmalemmal sarcoplasmic reticulum (ryanodine receptors) and mitochondria, thus facilitating Ca(2+) exchanges, particularly in CH. In conclusion, caveolae are implicated in mechanotransduction in Ctrl PASMC by buffering mechanical forces. In PH-PASMC, caveolae form a distinct Ca(2+) store facilitating Ca(2+) coupling between SAC and sarcoplasmic reticulum.


Assuntos
Cavéolas/fisiologia , Hipertensão Pulmonar/metabolismo , Mecanotransdução Celular , Animais , Sinalização do Cálcio , Caveolina 1/metabolismo , Células Cultivadas , Hipertensão Pulmonar/patologia , Masculino , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Ratos Wistar
20.
Birth Defects Res C Embryo Today ; 108(1): 45-64, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26991990

RESUMO

Caveolae, an almost ubiquitous, structural component of the plasma membrane, play a critical role in many functions essential for proper cell function, including membrane trafficking, signal transduction, extracellular matrix remodeling, and tissue regeneration. Three main types of caveolin proteins have been identified from caveolae since the discovery of caveolin-1 in the early 1990s. All three (Cav-1, Cav-2, and Cav-3) play crucial roles in mammalian physiology, and can effect pathogenesis in a wide range of human diseases. While many biological activities of caveolins have been uncovered since its discovery, their role and regulation in embryonic develop remain largely poorly understood, although there is increasing evidence that caveolins may be linked to lung and brain birth defects. Further investigations are clearly needed to decipher how caveolae/caveolins mediate cellular functions and activities of normal embryogenesis and how their perturbations contribute to developmental disorders.


Assuntos
Cavéolas/patologia , Cavéolas/fisiologia , Caveolina 1/metabolismo , Animais , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Desenvolvimento Embrionário/fisiologia , Humanos , Transdução de Sinais/fisiologia
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