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Multiple myeloma (MM) is a malignancy of plasma cells whose antibody secretion creates proteotoxic stress relieved by the N-end rule pathway, a proteolytic system that degrades Narginylated proteins in the proteasome. When the proteasome is inhibited, protein cargo is alternatively targeted for autophagic degradation by binding to the ZZ-domain of p62/sequestosome-1. Here, we demonstrate that XRK3F2, a selective ligand for the ZZ-domain, dramatically improved two major responses to the proteasome inhibitor bortezomib by increasing: 1) killing of human MM cells by stimulating both bortezomib mediated apoptosis and necroptosis, a process regulated by p62; and 2) preservation of bone mass by stimulating osteoblasts differentiation and inhibiting osteoclastic bone destruction. Co-administration of bortezomib and XRK3F2 inhibited both branches of the bimodal N-end rule pathway exhibited synergistic anti-MM effects on MM cell lines and CD138+ cells from MM patients, and prevented stromal-mediated MM cell survival. In mice with established human MM, coadministration of bortezomib and XRK3F2 decreased tumor burden and prevented the progression of MM-induced osteolytic disease by inducing new bone formation more effectively than either single agent alone. The results suggest that p62-ZZ ligands enhance the anti-MM efficacy of proteasome inhibitors and can reduce MM morbidity and mortality by improving bone health.
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Paget's disease (PD) is characterized by increased numbers of abnormal osteoclasts (OCLs) that drive exuberant bone formation, but the mechanisms responsible for the increased bone formation remain unclear. We previously reported that OCLs from 70% of PD patients express measles virus nucleocapsid protein (MVNP), and that transgenic mice with targeted expression of MVNP in OCLs (MVNP mice) develop bone lesions and abnormal OCLs characteristic of PD. In this report, we examined if OCL-derived sphingosine-1-phosphate (S1P) contributed to the abnormal bone formation in PD, since OCL-derived S1P can act as a coupling factor to increase normal bone formation via binding S1P-receptor-3 (S1PR3) on osteoblasts (OBs). We report that OCLs from MVNP mice and PD patients expressed high levels of sphingosine kinase-1 (SphK-1) compared with wild-type (WT) mouse and normal donor OCLs. SphK-1 production by MVNP-OCLs was interleukin-6 (IL-6)-dependent since OCLs from MVNP/IL-6-/- mice expressed lower levels of SphK-1. Immunohistochemistry of bone biopsies from a normal donor, a PD patient, WT and MVNP mice confirmed increased expression levels of SphK-1 in OCLs and S1PR3 in OBs of the PD patient and MVNP mice compared with normal donor and WT mice. Further, MVNP-OCLs cocultured with OBs from MVNP or WT mice increased OB-S1PR3 expression and enhanced expression of OB differentiation markers in MVNP-OBs precursors compared with WT-OBs, which was mediated by IL-6 and insulin-like growth factor 1 secreted by MVNP-OCLs. Finally, the addition of an S1PR3 antagonist (VPC23019) to WT or MVNP-OBs treated with WT and MVNP-OCL-conditioned media (CM) blocked enhanced OB differentiation of MVNP-OBs treated with MVNP-OCL-CM. In contrast, the addition of the SIPR3 agonist, VPC24191, to the cultures enhanced osterix and Col-1A expression in MVNP-OBs treated with MVNP-OCL-CM compared with WT-OBs treated with WT-OCL-CM. These results suggest that IL-6 produced by PD-OCLs increases S1P in OCLs and S1PR3 on OBs, to increase bone formation in PD.
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Lisofosfolipídeos/metabolismo , Osteíte Deformante/metabolismo , Osteoclastos/metabolismo , Esfingosina/análogos & derivados , Animais , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Immunoblotting , Imuno-Histoquímica , Interleucina-6/metabolismo , Masculino , Camundongos , Osteoclastos/citologia , Osteogênese/fisiologia , Fosforilação/fisiologia , Esfingosina/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismoRESUMO
In addition to exerting a potent anti-elastase function, α-1 antitrypsin (A1AT) maintains the structural integrity of the lung by inhibiting endothelial inflammation and apoptosis. A main serpin secreted in circulation by hepatocytes, A1AT requires uptake by the endothelium to achieve vasculoprotective effects. This active uptake mechanism, which is inhibited by cigarette smoking (CS), involves primarily clathrin- but also caveola-mediated endocytosis and may require active binding to a receptor. Because circulating A1AT binds to high-density lipoprotein (HDL), we hypothesized that scavenging receptors are candidates for endothelial uptake of the serpin. Although the low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) internalizes only elastase-bound A1AT, the scavenger receptor B type I (SR-BI), which binds and internalizes HDL and is modulated by CS, may be involved in A1AT uptake. Transmission electron microscopy imaging of colloidal gold-labeled A1AT confirmed A1AT endocytosis in both clathrin-coated vesicles and caveolae in endothelial cells. SR-BI immunoprecipitation identified binding to A1AT at the plasma membrane. Pretreatment of human lung microvascular endothelial cells with SR-B ligands (HDL or LDL), knockdown of SCARB1 expression, or neutralizing SR-BI antibodies significantly reduced A1AT uptake by 30-50%. Scarb1 null mice exhibited decreased A1AT lung content following systemic A1AT administration and reduced lung anti-inflammatory effects of A1AT supplementation during short-term CS exposure. In turn, A1AT supplementation increased lung SR-BI expression and modulated circulating lipoprotein levels in wild-type animals. These studies indicate that SR-BI is an important mediator of A1AT endocytosis in pulmonary endothelium and suggest a cross talk between A1AT and lipoprotein regulation of vascular functions.
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Células Endoteliais/metabolismo , Receptores Depuradores Classe B/fisiologia , Fumar/metabolismo , alfa 1-Antitripsina/metabolismo , Animais , Ligação Competitiva , Células Cultivadas , Endocitose , Endotélio Vascular/patologia , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
The engulfment function of macrophages relies on complex molecular interactions involving both lipids and proteins. In particular, the clearance of apoptotic bodies (efferocytosis) is enabled by externalization on the cell target of phosphatidylserine lipids, which activate receptors on macrophages, suggesting that (local) specific lipid-protein interactions are required at least for the initiation of efferocytosis. However, in addition to apoptotic cells, macrophages can engulf foreign bodies that vary substantially in size from a few nanometers to microns, suggesting that nonspecific interactions over a wide range of length scales could be relevant. Here, we use model lipid membranes (made of phosphatidylcholine, phosphatidylserine, and ceramide) and rat alveolar macrophages to show how lipid bilayer properties probed by small-angle x-ray scattering and solid-state (2)H NMR correlate with engulfment rates measured by flow cytometry. We find that engulfment of protein-free model lipid vesicles is promoted by the presence of phosphatidylserine lipids but inhibited by ceramide, in accord with a previous study of apoptotic cells. We conclude that the roles of phosphatidylserine and ceramide in phagocytosis is based, at least in part, on lipid-mediated modification of membrane physical properties, including interactions at large length scales as well as local lipid ordering and possible domain formation.
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Lipossomos/metabolismo , Macrófagos/metabolismo , Fagocitose , Animais , Linhagem Celular , Ceramidas/química , Ceramidas/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lipossomos/química , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Ligação Proteica , Alvéolos Pulmonares/citologia , RatosRESUMO
Prolonged exposure to cigarette smoking is the main risk factor for emphysema, a component of chronic obstructive pulmonary diseases (COPDs) characterized by destruction of alveolar walls. Moreover, smoking is associated with pulmonary artery remodeling and pulmonary hypertension, even in the absence of COPD, through as yet unexplained mechanisms. In murine models, elevations of intra- and paracellular ceramides in response to smoking have been implicated in the induction of lung endothelial cell apoptosis, but the role of ceramides in human cell counterparts is yet unknown. We modeled paracrine increases (outside-in) of palmitoyl ceramide (Cer16) in primary human lung microvascular cells. In naive cells, isolated from nonsmokers, Cer16 significantly reduced cellular proliferation and induced caspase-independent apoptosis via mitochondrial membrane depolarization, apoptosis-inducing factor translocation, and poly(ADP-ribose) polymerase cleavage. In these cells, caspase-3 was inhibited by ceramide-induced Akt phosphorylation, and by the induction of autophagic microtubule-associated protein-1 light-chain 3 lipidation. In contrast, cells isolated from smokers exhibited increased baseline proliferative features associated with lack of p16(INK4a) expression and Akt hyperphosphorylation. These cells were resistant to Cer16-induced apoptosis, despite presence of both endoplasmic reticulum stress response and mitochondrial membrane depolarization. In cells from smokers, the prominent up-regulation of Akt pathways inhibited ceramide-triggered apoptosis, and was associated with elevated sphingosine and high-mobility group box 1, skewing the cell's response toward autophagy and survival. In conclusion, the cell responses to ceramide are modulated by an intricate cross-talk between Akt signaling and sphingolipid metabolites, and profoundly modified by previous cigarette smoke exposure, which selects for an apoptosis-resistant phenotype.
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Apoptose/efeitos dos fármacos , Ceramidas/toxicidade , Células Endoteliais/efeitos dos fármacos , Pulmão/irrigação sanguínea , Ácidos Palmíticos/toxicidade , Fumaça/efeitos adversos , Fumar/efeitos adversos , Estresse Fisiológico/efeitos dos fármacos , Adaptação Fisiológica , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Proteína HMGB1/metabolismo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Comunicação Parácrina/efeitos dos fármacos , Fenótipo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
Chronic obstructive pulmonary disease (COPD) includes a spectrum of conditions that have in common varying degrees of airflow obstruction, such as chronic bronchitis and emphysema. There is an increasing evidence of involvement of sphingolipids as key molecular mediators or biomarkers of disease in emphysema, chronic bronchitis, and more recently in asthma, another disease characterized by (reversible) airflow obstruction. Given the recognized central role of oxidative stress and inflammatory stimuli along with involvement of immune responses, apoptosis, and tissue remodeling in the development of chronic obstructive lung diseases, it is not surprising that sphingolipids have been shown to play important role in their pathobiology. In particular the pro-apoptotic effects of ceramide were suspected as events in the lung destruction that occurs as a result of apoptotic loss of structural cells comprising the alveolar walls, such as microvascular endothelial cells and alveolar epithelial cells. In addition, the role of ceramide was investigated in models of larger airway epithelial cell stress responses to cigarette smoke, in the context of ensuing airway remodeling and inflammation. This chapter discusses current evidence of sphingolipid perturbations in experimental models of COPD and relevant links to human disease based on translational and epidemiological data.
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Pulmão/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Transdução de Sinais , Esfingolipídeos/metabolismo , Animais , Asma/metabolismo , Asma/fisiopatologia , Bronquite Crônica/metabolismo , Bronquite Crônica/fisiopatologia , Modelos Animais de Doenças , Humanos , Pulmão/fisiopatologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologiaRESUMO
Multiple myeloma (MM) is an incapacitating hematological malignancy characterized by accumulation of cancerous plasma cells in the bone marrow (BM) and production of an abnormal monoclonal protein (M-protein). The BM microenvironment has a key role in myeloma development by facilitating the growth of the aberrant plasma cells, which eventually interfere with the homeostasis of the bone cells, exacerbating osteolysis and inhibiting osteoblast differentiation. Recent recognition that metabolic reprograming has a major role in tumor growth and adaptation to specific changes in the microenvironmental niche have led to consideration of the role of sphingolipids and the enzymes that control their biosynthesis and degradation as critical mediators of cancer since these bioactive lipids have been directly linked to the control of cell growth, proliferation, and apoptosis, among other cellular functions. In this review, we present the recent progress of the research investigating the biological implications of sphingolipid metabolism alterations in the regulation of myeloma development and its progression from the pre-malignant stage and discuss the roles of sphingolipids in in MM migration and adhesion, survival and proliferation, as well as angiogenesis and invasion. We introduce the current knowledge regarding the role of sphingolipids as mediators of the immune response and drug-resistance in MM and tackle the new developments suggesting the manipulation of the sphingolipid network as a novel therapeutic direction for MM.
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Multiple myeloma (MM) remains incurable for most patients due to the emergence of drug resistant clones. Here we report a p53-independent mechanism responsible for Growth Factor Independence-1 (GFI1) support of MM cell survival by its modulation of sphingolipid metabolism to increase the sphingosine-1-phosphate (S1P) level regardless of the p53 status. We found that expression of enzymes that control S1P biosynthesis, SphK1, dephosphorylation, and SGPP1 were differentially correlated with GFI1 levels in MM cells. We detected GFI1 occupancy on the SGGP1 gene in MM cells in a predicted enhancer region at the 5' end of intron 1, which correlated with decreased SGGP1 expression and increased S1P levels in GFI1 overexpressing cells, regardless of their p53 status. The high S1P:Ceramide intracellular ratio in MM cells protected c-Myc protein stability in a PP2A-dependent manner. The decreased MM viability by SphK1 inhibition was dependent on the induction of autophagy in both p53WT and p53mut MM. An autophagic blockade prevented GFI1 support for viability only in p53mut MM, demonstrating that GFI1 increases MM cell survival via both p53WT inhibition and upregulation of S1P independently. Therefore, GFI1 may be a key therapeutic target for all types of MM that may significantly benefit patients that are highly resistant to current therapies.
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A decreased clearance of apoptotic cells (efferocytosis) by alveolar macrophages (AM) may contribute to inflammation in emphysema. The up-regulation of ceramides in response to cigarette smoking (CS) has been linked to AM accumulation and increased detection of apoptotic alveolar epithelial and endothelial cells in lung parenchyma. We hypothesized that ceramides inhibit the AM phagocytosis of apoptotic cells. Release of endogenous ceramides via sphingomyelinase or exogenous ceramide treatments dose-dependently impaired apoptotic Jurkat cell phagocytosis by primary rat or human AM, irrespective of the molecular species of ceramide. Similarly, in vivo augmentation of lung ceramides via intratracheal instillation in rats significantly decreased the engulfment of instilled target apoptotic thymocytes by resident AM. The mechanism of ceramide-induced efferocytosis impairment was dependent on generation of sphingosine via ceramidase. Sphingosine treatment recapitulated the effects of ceramide, dose-dependently inhibiting apoptotic cell clearance. The effect of ceramide on efferocytosis was associated with decreased membrane ruffle formation and attenuated Rac1 plasma membrane recruitment. Constitutively active Rac1 overexpression rescued AM efferocytosis against the effects of ceramide. CS exposure significantly increased AM ceramides and recapitulated the effect of ceramides on Rac1 membrane recruitment in a sphingosine-dependent manner. Importantly, CS profoundly inhibited AM efferocytosis via ceramide-dependent sphingosine production. These results suggest that excessive lung ceramides may amplify lung injury in emphysema by causing both apoptosis of structural cells and inhibition of their clearance by AM.
Assuntos
Apoptose/efeitos dos fármacos , Ceramidas/farmacologia , Macrófagos Alveolares/metabolismo , Fumar/efeitos adversos , Animais , Membrana Celular/metabolismo , Membrana Celular/patologia , Ceramidases/metabolismo , Ceramidas/metabolismo , Relação Dose-Resposta a Droga , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Células Jurkat , Macrófagos Alveolares/patologia , Masculino , Enfisema Pulmonar/metabolismo , Enfisema Pulmonar/patologia , Ratos , Ratos Sprague-Dawley , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Multiple myeloma (MM) is the second most common haematological malignancy and is characterized by a clonal proliferation of neoplastic plasma cells within the bone marrow. MM is the most frequent cancer involving the skeleton, causing osteolytic lesions, bone pain and pathological fractures that dramatically decrease MM patients' quality of life and survival. MM bone disease (MBD) results from uncoupling of bone remodelling in which excessive bone resorption is not compensated by new bone formation, due to a persistent suppression of osteoblast activity. Current management of MBD includes antiresorptive agents, bisphosphonates and denosumab, that are only partially effective due to their inability to repair the existing lesions. Thus, research into agents that prevent bone destruction and more importantly repair existing lesions by inducing new bone formation is essential. This review discusses the mechanisms regulating the uncoupled bone remodelling in MM and summarizes current advances in the treatment of MBD. LINKED ARTICLES: This article is part of a themed issue on The molecular pharmacology of bone and cancer-related bone diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.9/issuetoc.
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Conservadores da Densidade Óssea , Mieloma Múltiplo , Osteólise , Conservadores da Densidade Óssea/uso terapêutico , Humanos , Mieloma Múltiplo/tratamento farmacológico , Osteoblastos , Qualidade de VidaRESUMO
The integrity of lung alveoli is maintained by proper circulating levels of alpha-1 antitrypsin (A1AT). Next to cigarette smoking, A1AT deficiency is a major risk factor for lung emphysema development. We recently reported that in addition to neutralizing neutrophil elastases in the extracellular compartment, A1AT is internalized by lung endothelial cells and inhibits apoptosis. We hypothesized that the intracellular uptake of A1AT by endothelial cells may be required for its protective function; therefore, we studied the mechanisms of A1AT internalization by primary rat lung microvascular endothelial cells and the effect of cigarette smoke on this process both in vitro and in vivo (in mice). Purified A1AT was taken up intracellularly by endothelial cells in a time-dependent, dose-dependent, and conformer-specific manner and was detected in the cytoplasm of endothelial cells of nondiseased human lung sections. Despite a critical role for caveoli in endothelial cell endocytosis in general, specific inhibition of clathrin-mediated, but not caveoli-mediated, endocytosis profoundly decreased A1AT internalization and reversed the A1AT's antiapoptotic action. Further more, A1AT associated with clathrin heavy chains, but not with caveolin-1 in the plasma membrane fraction of endothelial cells. Interestingly, cigarette smoke exposure significantly inhibited A1AT uptake both in endothelial cells and in the mouse lung and altered the intracellular distribution of clathrin heavy chains. Our results suggest that clathrin-mediated endocytosis regulates A1AT intracellular function in the lung endothelium and may be an important determinant of the serpin's protection against developing cigarette smoke-induced emphysema.
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Endocitose , Endotélio/fisiologia , Pulmão/citologia , Fumaça/efeitos adversos , alfa 1-Antitripsina/metabolismo , Animais , Clatrina/metabolismo , Enfisema/etiologia , Endotélio/metabolismo , Humanos , Camundongos , Ratos , Ratos Sprague-Dawley , Nicotiana , alfa 1-Antitripsina/sangueRESUMO
Multiple Myeloma (MM) induces bone destruction, decreases bone formation, and increases marrow angiogenesis in patients. We reported that osteocytes (Ocys) directly interact with MM cells to increase tumor growth and expression of Ocy-derived factors that promote bone resorption and suppress bone formation. However, the contribution of Ocys to enhanced marrow vascularization in MM is unclear. Since the MM microenvironment is hypoxic, we assessed if hypoxia and/or interactions with MM cells increases pro-angiogenic signaling in Ocys. Hypoxia and/or co-culture with MM cells significantly increased Vegf-a expression in MLOA5-Ocys, and conditioned media (CM) from MLOA5s or MM-MLOA5 co-cultured in hypoxia, significantly increased endothelial tube length compared to normoxic CM. Further, Vegf-a knockdown in MLOA5s or primary Ocys co-cultured with MM cells or neutralizing Vegf-a in MM-Ocy co-culture CM completely blocked the increased endothelial activity. Importantly, Vegf-a-expressing Ocy numbers were significantly increased in MM-injected mouse bones, positively correlating with tumor vessel area. Finally, we demonstrate that direct contact with MM cells increases Ocy Fgf23, which enhanced Vegf-a expression in Ocys. Fgf23 deletion in Ocys blocked these changes. These results suggest hypoxia and MM cells induce a pro-angiogenic phenotype in Ocys via Fgf23 and Vegf-a signaling, which can promote MM-induced marrow vascularization.
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Medula Óssea/irrigação sanguínea , Fatores de Crescimento de Fibroblastos/fisiologia , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Neovascularização Patológica/genética , Osteócitos/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Reabsorção Óssea/etiologia , Linhagem Celular , Feminino , Fator de Crescimento de Fibroblastos 23 , Expressão Gênica/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Osteócitos/metabolismo , Osteogênese , Microambiente TumoralRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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We hereby provide the initial portrait of lincNORS, a spliced lincRNA generated by the MIR193BHG locus, entirely distinct from the previously described miR-193b-365a tandem. While inducible by low O2 in a variety of cells and associated with hypoxia in vivo, our studies show that lincNORS is subject to multiple regulatory inputs, including estrogen signals. Biochemically, this lincRNA fine-tunes cellular sterol/steroid biosynthesis by repressing the expression of multiple pathway components. Mechanistically, the function of lincNORS requires the presence of RALY, an RNA-binding protein recently found to be implicated in cholesterol homeostasis. We also noticed the proximity between this locus and naturally occurring genetic variations highly significant for sterol/steroid-related phenotypes, in particular the age of sexual maturation. An integrative analysis of these variants provided a more formal link between these phenotypes and lincNORS, further strengthening the case for its biological relevance.
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Homeostase , Oxigênio/metabolismo , RNA Longo não Codificante/fisiologia , Esteróis/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Colesterol/metabolismo , Estrogênios/metabolismo , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/metabolismo , Humanos , Células MCF-7 , Fenótipo , Polimorfismo de Nucleotídeo Único , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
The de novo pathway of ceramide synthesis has been implicated in the pathogenesis of excessive lung apoptosis and murine emphysema. Intracellular and paracellular-generated ceramides may trigger apoptosis and propagate the death signals to neighboring cells, respectively. In this study we compared the sphingolipid signaling pathways triggered by the paracellular- versus intracellular-generated ceramides as they induce lung endothelial cell apoptosis, a process important in emphysema development. Intermediate-chain length (C(8:0)) extracellular ceramides, used as a surrogate of paracellular ceramides, triggered caspase-3 activation in primary mouse lung endothelial cells, similar to TNF-alpha-generated endogenous ceramides. Inhibitory siRNA against serine palmitoyl transferase subunit 1 but not acid sphingomyelinase inhibited both C(8:0) ceramide- and TNF-alpha (plus cycloheximide)-induced apoptosis, consistent with the requirement for activation of the de novo pathway of sphingolipid synthesis. Tandem mass spectrometry analysis detected increases in both relative and absolute levels of C(16:0) ceramide in response to C(8:0) and TNF-alpha treatments. These results implicate the de novo pathway of ceramide synthesis in the apoptotic effects of both paracellular ceramides and TNF-alpha-stimulated intracellular ceramides in primary lung endothelial cells. The serine palmitoyl synthase-regulated ceramides synthesis may contribute to the amplification of pulmonary vascular injury induced by excessive ceramides.
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Apoptose/fisiologia , Ceramidas/metabolismo , Células Endoteliais/metabolismo , Pulmão/citologia , Transdução de Sinais/fisiologia , Esfingolipídeos/metabolismo , Animais , Caspase 3/metabolismo , Células Cultivadas , Ceramidas/química , Células Endoteliais/citologia , Ativação Enzimática , Humanos , Camundongos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Tissue transglutaminase (TG2), an enzyme involved in protein cross-linking and overexpressed in ovarian tumors, has antiapoptotic effects in cancer cells and may play a role in response to chemotherapy. In this study, we investigated the role of TG2 in the sensitivity of ovarian cancer cells to cisplatin. By using stable knockdown and overexpression strategies, we demonstrate that the level of expression of TG2 regulates apoptosis induced by cisplatin in SKOV3 and OV-90 ovarian cancer cells. Interestingly, not only TG2 knockdown but also a TG2 enzymatic inhibitor (KCC009) sensitized SKOV3 cells to cisplatin. To understand the mechanism by which TG2 exerts its antiapoptotic role, we examined the effects of protein kinase B (Akt) and nuclear factor-kappa B (NF-kappaB), two survival pathways commonly involved in development of drug resistance. Overexpression of the constitutively active p65 subunit of NF-kappaB, but not constitutively active Akt, rescued cells with diminished TG2 expression from cisplatin-induced apoptosis. This implicates activation of NF-kappaB as the main cisplatin resistance mechanism downstream of TG2. Indeed, NF-kappaB activity is decreased and the level of the inhibitory subunit I kappaB alpha is increased in ovarian cancer cells engineered to express diminished levels of TG2 or treated with the enzymatic inhibitor, KCC009. Our data show that TG2 prevents apoptosis induced by cisplatin by activating the NF-kappaB survival pathway in ovarian cancer cells.
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Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Cisplatino/farmacologia , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Transglutaminases/fisiologia , Caspase 9/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Proteínas de Ligação ao GTP , Humanos , Isoxazóis/farmacologia , NF-kappa B/metabolismo , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/patologia , Proteína 2 Glutamina gama-Glutamiltransferase , Transdução de Sinais , Transglutaminases/análise , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismoRESUMO
BACKGROUND: In spite of major advances in treatment, multiple myeloma (MM) is currently an incurable malignancy due to the emergence of drug-resistant clones. We previously showed that MM cells upregulate the transcriptional repressor, growth factor independence 1 (Gfi1), in bone marrow stromal cells (BMSCs) that induces prolonged inhibition of osteoblast differentiation. However, the role of Gfi1 in MM cells is unknown. METHODS: Human primary CD138+ and BMSC were purified from normal donors and MM patients' bone marrow aspirates. Gfi1 knockdown and overexpressing cells were generated by lentiviral-mediated shRNA. Proliferation/apoptosis studies were done by flow cytometry, and protein levels were determined by Western blot and/or immunohistochemistry. An experimental MM mouse model was generated to investigate the effects of MM cells overexpressing Gfi1 on tumor burden and osteolysis in vivo. RESULTS: We found that Gfi1 expression is increased in patient's MM cells and MM cell lines and was further increased by co-culture with BMSC, IL-6, and sphingosine-1-phosphate. Modulation of Gfi1 in MM cells had major effects on their survival and growth. Knockdown of Gfi1 induced apoptosis in p53-wt, p53-mutant, and p53-deficient MM cells, while Gfi1 overexpression enhanced MM cell growth and protected MM cells from bortezomib-induced cell death. Gfi1 enhanced cell survival of p53-wt MM cells by binding to p53, thereby blocking binding to the promoters of the pro-apoptotic BAX and NOXA genes. Further, Gfi1-p53 binding could be blocked by HDAC inhibitors. Importantly, inoculation of MM cells overexpressing Gfi1 in mice induced increased bone destruction, increased osteoclast number and size, and enhanced tumor growth. CONCLUSIONS: These results support that Gfi1 plays a key role in MM tumor growth, survival, and bone destruction and contributes to bortezomib resistance, suggesting that Gfi1 may be a novel therapeutic target for MM.
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Proteínas de Ligação a DNA/biossíntese , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Osteogênese/fisiologia , Fatores de Transcrição/biossíntese , Animais , Apoptose/fisiologia , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Feminino , Humanos , CamundongosRESUMO
Changes in systemic redox balance can alter platelet activation and aggregation. Acute pulmonary embolism (PE) is a systematic inflammatory disease associated with mechanical shear stress, increased thrombin, catecholamines, serotonin and hemolysis, which cumulatively can hyperactivate platelets and accelerate their turnover. We tested the hypothesis that platelets from patients with moderately severe PE will show hyperstimulation and a pre-apoptotic phenotype associated with microparticles (MPs) in plasma. Blood for platelet respiration and thromboelastography (TEG) was obtained at diagnosis and 24h later from patients (n=76) with image-proven PE, SBP>90mmHg and right ventricular dysfunction demonstrated by echocardiogram or elevated biomarkers. Controls (n=12) were healthy volunteers. At diagnosis, platelets from PE patients had significantly elevated baseline oxygen consumption compared with controls, explained primarily by accelerated electron transport and oxygen wasting with no measurable extramitochondrial oxygen consumption. On thromboelastography, unstimulated thrombin-independent maximum amplitude was increased with PE, 19±14.1 vs.10.5±7.8mm in controls (p=0.002). Compared with controls, platelets from PE patients showed elevated mitochondrial reactive oxygen species with decreased mitochondrial Bcl-2 protein content and increased cytosolic cytochrome C, coincident with strong annexin V binding, P selectin release from lysed platelets and in plasma MPs compared to controls (p<0.05). These results show evidence of platelet hyperactivation and apoptosis in patients with acute PE, and provide preliminary theoretical basis for further exploration of platelet inhibition in patients with more severe PE.
Assuntos
Apoptose , Ativação Plaquetária , Embolia Pulmonar/sangue , Embolia Pulmonar/complicações , Trombofilia/sangue , Trombofilia/complicações , Doença Aguda , Adulto , Idoso , Coagulação Sanguínea , Plaquetas/metabolismo , Plaquetas/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Estresse Oxidativo , Embolia Pulmonar/metabolismo , Embolia Pulmonar/patologia , Espécies Reativas de Oxigênio/metabolismo , Trombofilia/metabolismo , Trombofilia/patologiaRESUMO
Cigarette smoking (CS), the main risk factor for COPD (chronic obstructive pulmonary disease) in developed countries, decreases alveolar macrophages (AM) clearance of both apoptotic cells and bacterial pathogens. This global deficit of AM engulfment may explain why active smokers have worse outcomes of COPD exacerbations, episodes characterized by airway infection and inflammation that carry high morbidity and healthcare cost. When administered as intravenous supplementation, the acute phase-reactant alpha-1 antitrypsin (A1AT) reduces the severity of COPD exacerbations in A1AT deficient (AATD) individuals and of bacterial pneumonia in murine models, but the effect of A1AT on AM scavenging functions has not been reported. Apoptotic cell clearance (efferocytosis) was measured in human AM isolated from patients with COPD, in primary rat AM or differentiated monocytes exposed to CS ex vivo, and in AM recovered from mice exposed to CS. A1AT (100 µg/mL, 16 h) significantly ameliorated efferocytosis (by ~50%) in AM of active smokers or AM exposed ex vivo to CS. A1AT significantly improved AM global engulfment, including phagocytosis, even when cells were simultaneously challenged with apoptotic and Fc-coated (bacteria-like) targets. The improved efferocytosis in A1AT-treated macrophages was associated with inhibition of tumor necrosis factor-α converting enzyme (TACE) activity, decreased mannose receptor shedding, and markedly increased abundance of efferocytosis receptors (mannose- and phosphatidyl serine receptors and the scavenger receptor B2) on AM plasma membrane. Directed airway A1AT treatment (via inhalation of a nebulized solution) restored in situ airway AM efferocytosis after CS exposure in mice. The amelioration of CS-exposed AM global engulfment may render A1AT as a potential therapy for COPD exacerbations.
Assuntos
Macrófagos Alveolares/imunologia , Nicotiana/química , Fagocitose/efeitos dos fármacos , Fumaça/efeitos adversos , alfa 1-Antitripsina/farmacologia , Proteína ADAM17/metabolismo , Adulto , Animais , Líquido da Lavagem Broncoalveolar/citologia , Estudos de Casos e Controles , Células Cultivadas , Feminino , Humanos , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/etiologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/patologia , Ratos , Ratos Sprague-Dawley , Nicotiana/metabolismo , alfa 1-Antitripsina/análise , alfa 1-Antitripsina/metabolismoRESUMO
Circulating endothelial microparticles (EMPs) are emerging as biomarkers of chronic obstructive pulmonary disease (COPD) in individuals exposed to cigarette smoke (CS), but their mechanism of release and function remain unknown. We assessed biochemical and functional characteristics of EMPs and circulating microparticles (cMPs) released by CS. CS exposure was sufficient to increase microparticle levels in plasma of humans and mice, and in supernatants of primary human lung microvascular endothelial cells. CS-released EMPs contained predominantly exosomes that were significantly enriched in let-7d, miR-191; miR-126; and miR125a, microRNAs that reciprocally decreased intracellular in CS-exposed endothelium. CS-released EMPs and cMPs were ceramide-rich and required the ceramide-synthesis enzyme acid sphingomyelinase (aSMase) for their release, an enzyme which was found to exhibit significantly higher activity in plasma of COPD patients or of CS-exposed mice. The ex vivo or in vivo engulfment of EMPs or cMPs by peripheral blood monocytes-derived macrophages was associated with significant inhibition of efferocytosis. Our results indicate that CS, via aSMase, releases circulating EMPs with distinct microRNA cargo and that EMPs affect the clearance of apoptotic cells by specialized macrophages. These targetable effects may be important in the pathogenesis of diseases linked to endothelial injury and inflammation in smokers.