RESUMO
Pulmonary arterial hypertension (PAH) is a sex-biased disease with female sex as a significant risk factor. Increased expression of the long noncoding RNA X-inactive-specific transcript (Xist), as induced by an intersectin-1s protein fragment with proliferative potential (EHITSN), may explain the sexual dimorphism of female pulmonary artery endothelial cells (ECs) and at least in part, the imbalance sex/ratio of PAH. Xist is essential for X-chromosome inactivation and dosage compensation of X-linked genes. Herein, increased Xist expression was detected in a subset of ECs and lung tissue samples of male patients with PAH. The role of different Xist expression levels in ECs of male patients with PAH (ECPAH) was studied in several lines of male ECPAH in conjunction with molecular, biochemical, morphologic, and functional approaches. Male ECPAH showed on average 10.3-fold increase in high Xist versus low Xist, a significant association between Xist levels and their proliferative potential, and a heterogeneous methylation of the Xist/XIST antisense RNA (Tsix) locus. Interestingly, Xist up-regulation in male ECPAH decreased the expression of Krueppel-like factor 2 (Klf2), via EHITSN interaction with enhancer of zeste polycomb repressive complex 2 subunit (EZH2), the catalytic subunit of the polycomb repressive complex 2. Moreover, the studies demonstrate that EHITSN-triggered p38/ETS domain-containing protein Elk1/AP-1 transcription factor subunit (c-Fos) signaling is a pathologic mechanism central to ECPAH proliferation and the dynamic crosstalk with cell cycle regulatory proteins cyclin A1/cyclin D2 and Xist-EZH2-Klf2 interaction participate directly and differentially in establishing the proliferative profile of male ECPAH.
Assuntos
Hipertensão Arterial Pulmonar , RNA Longo não Codificante , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Masculino , Humanos , Hipertensão Arterial Pulmonar/genética , Hipertensão Arterial Pulmonar/metabolismo , Hipertensão Arterial Pulmonar/patologia , Feminino , Células Endoteliais/metabolismo , Proliferação de Células/genética , Pessoa de Meia-Idade , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Elk-1 do Domínio ets/metabolismo , Proteínas Elk-1 do Domínio ets/genética , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Regulação da Expressão Gênica , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , AdultoRESUMO
Pulmonary arterial hypertension (PAH) is a sex-biased disease with a poorly understood female prevalence. Emerging research suggests that nonhormonal factors, such as the XX or XY sex chromosome complement and sex bias in gene expression, may also lead to sex-based differences in PAH incidence, penetrance, and progression. Typically, one of females' two X chromosomes is epigenetically silenced to offer a gender-balanced gene expression. Recent data demonstrate that the long noncoding RNA X-inactive specific transcript, essential for X chromosome inactivation and dosage compensation of X-linked gene expression, shows elevated levels in female PAH lung specimens compared with controls. This molecular event leads to incomplete inactivation of the females' second X chromosome, abnormal expression of X-linked gene(s) involved in PAH pathophysiology, and a pulmonary artery endothelial cell (PAEC) proliferative phenotype. Moreover, the pathogenic proliferative p38 mitogen-activated protein kinase/ETS transcription factor ELK1 (Elk1)/cFos signaling is mechanistically linked to the sexually dimorphic proliferative response of PAECs in PAH. Apprehending the complicated relationship between long noncoding RNA X-inactive specific transcript and X-linked genes and how this relationship integrates into a sexually dimorphic proliferation of PAECs and PAH sex paradox remain challenging. We highlight herein new findings related to how the sex chromosome complement and sex-differentiated epigenetic mechanisms to control gene expression are decisive players in the sexual dimorphism of PAH. Pharmacologic interventions in the light of the newly elucidated mechanisms are discussed.
Assuntos
Hipertensão Arterial Pulmonar , RNA Longo não Codificante , Feminino , Humanos , Hipertensão Arterial Pulmonar/genética , Artéria Pulmonar , Caracteres Sexuais , Cromossomos Sexuais/genéticaRESUMO
The sex-biased disease pulmonary arterial hypertension (PAH) is characterized by the proliferation and overgrowth of dysfunctional pulmonary artery endothelial cells (PAECs). During inflammation associated with PAH, granzyme B cleaves intersectin-1 to produce N-terminal (EHITSN) and C-terminal (SH3A-EITSN) protein fragments. In a murine model of PAH, EHITSN triggers plexiform arteriopathy via p38-ELK1-c-Fos signaling. The SH3A-EITSN fragment also influences signaling, having dominant-negative effects on ERK1 and ERK2 (also known as MAPK3 and MAPK1, respectively). Using PAECs engineered to express tagged versions of EHITSN and SH3A-EITSN, we demonstrate that the two ITSN fragments increase both p38-ELK1 activation and the ratio of p38 to ERK1 and ERK2 activity, leading to PAEC proliferation, with female cells being more responsive than male cells. Furthermore, expression of EHITSN substantially upregulates the expression and activity of the long non-coding RNA Xist in female PAECs, which in turn upregulates the X-linked gene ELK1 and represses expression of krüppel-like factor 2 (KLF2). These events are recapitulated by the PAECs of female idiopathic PAH patients, and may account for their proliferative phenotype. Thus, upregulation of Xist could be an important factor in explaining sexual dimorphism in the proliferative response of PAECs and the imbalanced sex ratio of PAH.
Assuntos
Hipertensão Pulmonar , Artéria Pulmonar , Animais , Proliferação de Células , Células Cultivadas , Células Endoteliais , Feminino , Humanos , Masculino , Camundongos , Caracteres SexuaisRESUMO
As time progresses, our understanding of disease pathology is propelled forward by technological advancements. Much of the advancements that aid in understanding disease mechanics are based on animal studies. Unfortunately, animal models often fail to recapitulate the entirety of the human disease. This is especially true with animal models used to study pulmonary arterial hypertension (PAH), a disease with two distinct phases. The first phase is defined by nonspecific medial and adventitial thickening of the pulmonary artery and is commonly reproduced in animal models, including the classic models (ie, hypoxia-induced pulmonary hypertension and monocrotaline lung injury model). However, many animal models, including the classic models, fail to capture the progressive, or second, phase of PAH. This is a stage defined by plexogenic arteriopathy, resulting in obliteration and occlusion of the small- to mid-sized pulmonary vessels. Each of these two phases results in severe pulmonary hypertension that directly leads to right ventricular hypertrophy, decompensated right-sided heart failure, and death. Fortunately, newly developed animal models have begun to address the second, more severe, side of PAH and aid in our ability to develop new therapeutics. Moreover, p38 mitogen-activated protein kinase activation emerges as a central molecular mediator of plexiform lesions in both experimental models and human disease. Therefore, this review will focus on plexiform arteriopathy in experimental animal models of PAH.
Assuntos
Modelos Animais de Doenças , Hipertensão Arterial Pulmonar/patologia , Artéria Pulmonar/patologia , Animais , Progressão da Doença , Humanos , Hipóxia/complicações , Hipóxia/patologia , Indóis , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Knockout , Hipertensão Arterial Pulmonar/etiologia , Hipertensão Arterial Pulmonar/metabolismo , Pirróis , RatosRESUMO
Recently, we demonstrated in cultured endothelial cells and in vivo that deficiency of an isoform of intersectin-1, ITSN-1s, impairs caveolae and clathrin-mediated endocytosis and functionally upregulates compensatory pathways and their morphological carriers (i.e. enlarged endocytic structures, membranous rings or tubules) that are normally underrepresented. We now show that these endocytic structures internalize the broadly expressed transforming growth factor ß receptor I (TGFß-RI or TGFBR1), also known as Alk5, leading to its ubiquitylation and degradation. Moreover, the apoptotic or activated vascular cells of the ITSN-1s-knockdown mice release Alk5-bearing microparticles to the systemic circulation. These interact with and transfer Alk5 to endocytosis-deficient endothelial cells, resulting in lung endothelial cell survival and phenotypic alteration towards proliferation through activation of Erk1 and Erk2 (also known as MAPK3 and MAPK1, respectively). We also show that non-productive assembly of the Alk5-Smad-SARA (Smad anchor for receptor activation, also known as ZFYVE9) signaling complex and preferential formation of the Alk5-mSos-Grb2 complex account for Erk1/2 activation downstream of Alk5 and proliferation of pulmonary endothelial cells. Taken together, our studies demonstrate a functional relationship between the intercellular transfer of Alk5 by microparticles and endothelial cell survival and proliferation, and define a novel molecular mechanism for TGFß and Alk5-dependent Erk1/2(MAPK) signaling that is significant for proliferative signaling and abnormal growth.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteína Smad2/metabolismo , Animais , Apoptose , Cavéolas/metabolismo , Linhagem Celular , Sobrevivência Celular , Endocitose , Células Endoteliais/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Receptor do Fator de Crescimento Transformador beta Tipo I , Transdução de SinaisRESUMO
Here we addressed the role of intersectin-2L (ITSN-2L), a guanine nucleotide exchange factor for the Rho GTPase Cdc42, in the mechanism of caveola endocytosis in endothelial cells (ECs). Immunoprecipitation and co-localization studies showed that ITSN-2L associates with members of the Cdc42-WASp-Arp2/3 actin polymerization pathway. Expression of Dbl homology-pleckstrin homology (DH-PH) region of ITSN-2L (DH-PH(ITSN-2L)) induced specific activation of Cdc42, resulting in formation of extensive filopodia, enhanced cortical actin, as well as a shift from G-actin to F-actin. The "catalytically dead" DH-PH domain reversed these effects and induced significant stress fiber formation, without a detectable shift in actin pools. A biotin assay for caveola internalization indicated a significant decrease in the uptake of biotinylated proteins in DH-PH(ITSN-2L)-transfected cells compared with control and 1 microM jasplakinolide-treated cells. ECs depleted of ITSN-2L by small interfering RNA, however, showed decreased Cdc42 activation and actin remodeling similar to the defective DH-PH, resulting in 62% increase in caveola-mediated uptake compared with controls. Thus, ITSN-2L, a guanine nucleotide exchange factor for Cdc42, regulates different steps of caveola endocytosis in ECs by controlling the temporal and spatial actin polymerization and remodeling sub-adjacent to the plasma membrane.
Assuntos
Actinas/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Cavéolas/metabolismo , Endocitose/fisiologia , Células Endoteliais/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/genética , Proteínas Adaptadoras de Transporte Vesicular/antagonistas & inibidores , Proteínas Adaptadoras de Transporte Vesicular/genética , Células Endoteliais/citologia , Ativação Enzimática/fisiologia , Humanos , Estrutura Terciária de Proteína/fisiologia , Pseudópodes/genética , Pseudópodes/metabolismo , RNA Interferente Pequeno/genética , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Proteína cdc42 de Ligação ao GTP/genéticaRESUMO
Pirfenidone is an anti-inflammatory and anti-fibrotic drug that has shown efficacy in lung and kidney fibrosis. Because inflammation and fibrosis have been linked to the progression of osteoarthritis, we investigated the effects of oral Pirfenidone in a mouse model of cartilage injury, which results in chronic inflammation and joint-wide fibrosis in mice that lack hyaluronan synthase 1 (Has1-/- ) in comparison to wild-type. Femoral cartilage was surgically injured in wild-type and Has1-/- mice, and Pirfenidone was administered in food starting after 3 days. At 4 weeks, Pirfenidone reduced the appearance, on micro-computed tomography, of pitting in subchondral bone at, and cortical bone surrounding, the site of cartilage injury. This corresponded with a reduction in fibrotic tissue deposits as observed with gross joint surface photography. Pirfenidone resulted in significant recovery of trabecular bone parameters affected by joint injury in Has1-/- mice, although the effect in wild-type was less pronounced. Pirfenidone also increased Safranin-O staining of growth plate cartilage after cartilage injury and sham operation in both genotypes. Taken together with the expression of selected extracellular matrix, inflammation, and fibrosis genes, these results indicate that Pirfenidone may confer chondrogenic and bone-protective effects, although the well-known anti-fibrotic effects of Pirfenidone may occur earlier in the wound-healing response than the time point examined in this study. Further investigations to identify the specific cell populations in the joint and signaling pathways that are responsive to Pirfenidone are warranted, as Pirfenidone and other anti-fibrotic drugs may encourage tissue repair and prevent progression of post-traumatic osteoarthritis. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:365-376, 2018.
Assuntos
Osso e Ossos/efeitos dos fármacos , Cartilagem Articular/lesões , Traumatismos do Joelho/patologia , Osteoartrite do Joelho/prevenção & controle , Piridonas/uso terapêutico , Animais , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/patologia , Fibrose/prevenção & controle , Glicosaminoglicanos/análise , Hialuronan Sintases/fisiologia , Ácido Hialurônico/análise , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Pulmonary endothelial cells' (ECs) injury and apoptotic death are necessary and sufficient for the pathogenesis of the acute respiratory distress syndrome (ARDS), regardless of epithelial damage. Interaction of dysfunctional ECs with circulatory extracellular vesicles (EVs) holds therapeutic promise in ARDS. However, the presence in the blood of long-term ARDS survivors of EVs with a distinct phenotype compared to the EVs of non-surviving patients is not reported. With a multidisciplinary translational approach, we studied EVs from the blood of 33 patients with moderate-to-severe ARDS. RESULTS: The EVs were isolated from the blood of ARDS and control subjects. Immunoblotting and magnetic beads immunoisolation complemented by standardized flow cytometry and nanoparticles tracking analyses identified in the ARDS patients a subset of EVs with mesenchymal stem cell (MSC) origin (CD73+CD105+Cd34-CD45-). These EVs have 4.7-fold greater counts compared to controls and comprise the transforming growth factor-beta receptor I (TßRI)/Alk5 and the Runx1 transcription factor. Time course analyses showed that the expression pattern of two Runx1 isoforms is critical for ARDS outcome: the p52 isoform shows a continuous expression, while the p66 is short-lived. A high ratio Runx1p66/p52 provided a survival advantage, regardless of age, sex, disease severity or length of stay in the intensive care unit. Moreover, the Runx1p66 isoform is transiently expressed by cultured human bone marrow-derived MSCs, it is released in the EVs recoverable from the conditioned media and stimulates the proliferation of lipopolysaccharide (LPS)-treated ECs. The findings are consistent with a causal effect of Runx1p66 expression on EC proliferation. Furthermore, morphological and functional assays showed that the EVs bearing the Runx1p66 enhanced junctional integrity of LPS-injured ECs and decreased lung histological severity in the LPS-treated mice. CONCLUSIONS: The expression pattern of Runx1 isoforms might be a reliable circulatory biomarker of ARDS activity and a novel determinant of the molecular mechanism for lung vascular/tissue repair and recovery after severe injury.
RESUMO
Intersectin, a multiple Eps15 homology and Src homology 3 (SH3) domain-containing protein, is a component of the endocytic machinery in neurons and nonneuronal cells. However, its role in endocytosis via caveolae in endothelial cells (ECs) is unclear. We demonstrate herein by coimmunoprecipitation, velocity sedimentation on glycerol gradients, and cross-linking that intersectin is present in ECs in a membrane-associated protein complex containing dynamin and SNAP-23. Electron microscopy (EM) immunogold labeling studies indicated that intersectin associated preferentially with the caveolar necks, and it remained associated with caveolae after their fission from the plasmalemma. A cell-free system depleted of intersectin failed to support caveolae fission from the plasma membrane. A biotin assay used to quantify caveolae internalization and extensive EM morphological analysis of ECs overexpressing wt-intersectin indicated a wide range of morphological changes (i.e., large caveolae clusters marginated at cell periphery and pleiomorphic caveolar necks) as well as impaired caveolae internalization. Biochemical evaluation of caveolae-mediated uptake by ELISA showed a 68.4% inhibition by reference to control. We also showed that intersectin interaction with dynamin was important in regulating the fission and internalization of caveolae. Taken together, the results indicate the crucial role of intersectin in the mechanism of caveolae fission in endothelial cells.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular , Proteínas de Transporte/metabolismo , Cavéolas/metabolismo , Membrana Celular/metabolismo , Endocitose/fisiologia , Células Endoteliais/metabolismo , Animais , Proteínas de Transporte/fisiologia , Cavéolas/fisiologia , Compartimento Celular/fisiologia , Membrana Celular/fisiologia , Células Cultivadas , Clonagem Molecular , Dinaminas/metabolismo , Dinaminas/fisiologia , Células Endoteliais/fisiologia , Ensaio de Imunoadsorção Enzimática , Humanos , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Modelos Moleculares , Proteínas Qb-SNARE , Proteínas Qc-SNARE , Ratos , Ratos Sprague-DawleyRESUMO
A characteristic feature of idiopathic pulmonary fibrosis (IPF) is accumulation of apoptotic resistant fibroblasts/myofibroblasts in the fibroblastic foci. As caveolin (Cav)-null mice develop pulmonary fibrosis (PF), we hypothesized that the participating fibroblasts display an apoptosis-resistant phenotype. To test this hypothesis and identify the molecular mechanisms involved we isolated lung fibroblasts from Cav-null mice and examined the expression of several inhibitors of apoptosis (IAPs), of c-FLIP, of Bcl-2 proteins and of the death receptor CD95/Fas. We found significant increase in XIAP and c-FLIP constitutive protein expression with no alteration of Bcl-2 and lower levels of CD95/Fas. The isolated fibroblasts were then treated with the CD95/Fas ligand (FasL) to induce apoptosis. While the morphological and biochemical alterations induced by FasL were similar in wild-type (wt) and Cav-null mouse lung fibroblasts, the time course and the extent of the alterations were greater in the Cav-null fibroblasts. Several salient features of Cav-null fibroblasts response such as loss of membrane potential, fragmentation of the mitochondrial continuum concurrent with caspase-8 activation, and subsequent Bid cleavage, prior to caspase-3 activation were detected. Furthermore, M30 antigen formation, phosphatidylserine expression and DNA fragmentation were caspase-3 dependent. SiRNA-mediated silencing of XIAP and c-FLIP, individually or combined, enhanced the sensitivity of lung fibroblasts to FasL-induced apoptosis. Pharmacological inhibition of Bcl-2 had no effect. Together our findings support a mechanism in which CD95/Fas engagement activates caspase-8, inducing mitochondrial apoptosis through Bid cleavage. XIAP and c-FLIP fine tune this process in a cell-type specific manner.
RESUMO
Abstract Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are severe syndromes resulting from the diffuse damage of the pulmonary parenchyma. ALI and ARDS are induced by a plethora of local or systemic insults, leading to the activation of multiple pathways responsible for injury, resolution, and repair or scarring of the lungs. Despite the large efforts aimed at exploring the roles of different pathways in humans and animal models and the great strides made in understanding the pathogenesis of ALI/ARDS, the only viable treatment options are still dependent on ventilator and cardiovascular support. Investigation of the pathophysiological mechanisms responsible for initiation and resolution or advancement toward lung scarring in ALI/ARDS animal models led to a better understanding of the disease's complexity and helped in elucidating the links between ALI and systemic multiorgan failure. Although animal models of ALI/ARDS have pointed out a variety of new ideas for study, there are still limited data regarding the initiating factors, the critical steps in the progression of the disease, and the central mechanisms dictating its resolution or progression to lung scarring. Recent studies link deficiency of intersectin-1s (ITSN-1s), a prosurvival protein of lung endothelial cells, to endothelial barrier dysfunction and pulmonary edema as well as to the repair/recovery from ALI. This review discusses the effects of ITSN-1s deficiency on pulmonary endothelium and its significance in the pathology of ALI/ARDS.
RESUMO
Intersectin-1s (ITSN-1s), a protein containing five SH3 (A-E) domains, regulates via the SH3A the function of dynamin-2 (dyn2) at the endocytic site. ITSN-1s expression was modulated in mouse lung endothelium by liposome delivery of either a plasmid cDNA encoding myc-SH3A or a specific siRNA targeting ITSN-1 gene. The lung vasculature of SH3A-transduced and ITSN-1s- deficient mice was perfused with gold albumin (Au-BSA) to analyze by electron microscopy the morphological intermediates and pathways involved in transendothelial transport or with dinitrophenylated (DNP)-BSA to quantify by ELISA its transport. Acute modulation of ITSN-1s expression decreased the number of caveolae, impaired their transport, and opened the interendothelial junctions, while upregulating compensatory nonconventional endocytic/transcytotic structures. Chronic inhibition of ITSN-1s further increased the occurrence of nonconventional intermediates and partially restored the junctional integrity. These findings indicate that ITSN-1s expression is required for caveolae function and efficient transendothelial transport. Moreover, our results demonstrate that ECs are highly adapted to perform their transport function while maintaining lung homeostasis.
RESUMO
It is known that platelet-activating factor (PAF) induces severe endothelial barrier leakiness, but the signaling mechanisms remain unclear. Here, using a wide range of biochemical and morphological approaches applied in both mouse models and cultured endothelial cells, we addressed the mechanisms of PAF-induced disruption of interendothelial junctions (IEJs) and of increased endothelial permeability. The formation of interendothelial gaps filled with filopodia and lamellipodia is the cellular event responsible for the disruption of endothelial barrier. We observed that PAF ligation of its receptor induced the activation of the Rho GTPase Rac1. Following PAF exposure, both Rac1 and its guanine nucleotide exchange factor Tiam1 were found associated with a membrane fraction from which they co-immunoprecipitated with PAF receptor. In the same time frame with Tiam1-Rac1 translocation, the junctional proteins ZO-1 and VE-cadherin were relocated from the IEJs, and formation of numerous interendothelial gaps was recorded. Notably, the response was independent of myosin light chain phosphorylation and thus distinct from other mediators, such as histamine and thrombin. The changes in actin status are driven by the PAF-induced localized actin polymerization as a consequence of Rac1 translocation and activation. Tiam1 was required for the activation of Rac1, actin polymerization, relocation of junctional associated proteins, and disruption of IEJs. Thus, PAF-induced IEJ disruption and increased endothelial permeability requires the activation of a Tiam1-Rac1 signaling module, suggesting a novel therapeutic target against increased vascular permeability associated with inflammatory diseases.
Assuntos
Permeabilidade Capilar/fisiologia , Células Endoteliais/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Junções Intercelulares/metabolismo , Neuropeptídeos/metabolismo , Fator de Ativação de Plaquetas/farmacologia , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Actinas/metabolismo , Animais , Antígenos CD/metabolismo , Caderinas/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/citologia , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Cadeias Leves de Miosina/metabolismo , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Fator de Ativação de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/agonistas , Glicoproteínas da Membrana de Plaquetas/metabolismo , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T , Proteína da Zônula de Oclusão-1RESUMO
Caveolae transcytosis with its diverse mechanisms-fluid phase, adsorptive, and receptor-mediated-plays an important role in the continuous exchange of molecules across the endothelium. We will discuss key features of endothelial transcytosis and caveolae that have been studied recently and have increased our understanding of caveolae function in transcytosis at the molecular level. During transcytosis, caveolae "pinch off" from the plasma membrane to form discrete vesicular carriers that shuttle to the opposite front of endothelial cells, fuse with the plasma membrane, and discharge their cargo into the perivascular space. Endothelial transcytosis exhibits distinct properties, the most important being rapid and efficient coupling of endocytosis to exocytosis on opposite plasma membrane. We address herein the membrane fusion-fission reactions that underlie transcytosis. Caveolae move across the endothelial cells with their cargo predominantly in the fluid phase through an active process that bypasses the lysosomes. Endothelial transcytosis is a constitutive process of vesicular transport. Recent studies show that transcytosis can be upregulated in response to pathological stimuli. Transcytosis via caveolae is an important route for the regulation of endothelial barrier function and may participate in different vascular diseases.
Assuntos
Permeabilidade Capilar/fisiologia , Endocitose/fisiologia , Células Endoteliais/metabolismo , Exocitose/fisiologia , Animais , Transporte Biológico/fisiologia , Cavéolas/metabolismo , Cavéolas/fisiologia , HumanosRESUMO
Intersectins (ITSNs) are multidomain adaptor proteins implicated in endocytosis, regulation of actin polymerization, and Ras/MAPK signaling. We have previously shown that ITSN-1s is required for caveolae fission and internalization in endothelial cells (ECs). In the present study, using small interfering RNA to knock down ITSN-1s protein expression, we demonstrate a novel role of ITSN-1s as a key antiapoptotic protein. Knockdown of ITSN-1s in ECs activated the mitochondrial pathway of apoptosis as determined by genomic DNA fragmentation, extensive mitochondrial fission, activation of the proapoptotic proteins BAK and BAX, and cytochrome c efflux from mitochondria. ITSN-1 knockdown acts as a proapoptotic signal that causes mitochondrial outer membrane permeabilization, dissipation of the mitochondrial membrane potential, and generation of reactive oxygen species. These effects were secondary to decreased activation of Erk1/2 and its direct activator MEK. Bcl-X(L) overexpression prevented BAX activation and the apoptotic ECs death induced by suppression of ITSN-1s. Our findings demonstrate a novel role of ITSN-1s as a negative regulator of the mitochondrial pathway-dependent apoptosis secondary to activation of the Erk1/2 survival signaling pathway.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Apoptose/fisiologia , Endotélio Vascular/fisiologia , Mitocôndrias/fisiologia , Sequência de Bases , Células Cultivadas , Citocromos c/metabolismo , Endotélio Vascular/citologia , Humanos , Marcação In Situ das Extremidades Cortadas , Microscopia Eletrônica , Microscopia de Fluorescência , Mitocôndrias/enzimologia , RNA Interferente Pequeno , Transdução de SinaisRESUMO
We determined the organization of target (t) SNARE proteins on the basolateral endothelial plasma membrane (PM) and their role in the mechanism of caveolar fusion. Studies were performed in a cell-free system involving endothelial PM sheets and isolated biotin-labeled caveolae. We monitored the fusion of caveolae with the PM by the detection of biotin-streptavidin complexes using correlative high resolution fluorescence microscopy and gold labeling electron microscopy on ultrathin sections of PM sheets. Imaging of PM sheets demonstrated and biochemical findings showed that the t-SNARE proteins present in endothelial cells (SNAP-23 and syntaxin-4) formed cholesterol-dependent clusters in discrete areas of the PM. Upon fusion of caveolae with the target PM, 50% of the caveolae co-localized with the t-SNARE clusters, indicating that these caveolae were at the peak of the fusion reaction. Fluorescent streptavidin staining of PM sheets correlated with the ultrastructure in the same area. These findings demonstrate that t-SNARE clusters in the endothelial target PM serve as the fusion sites for caveolae during exocytosis.