RESUMO
Eph receptor and ephrin signaling has a major role in segregating distinct cell populations to form sharp borders. Expression of interacting Ephs and ephrins typically occurs in complementary regions, such that polarised activation of both components occurs at the interface. Forward signaling through Eph receptors can drive cell segregation, but it is unclear whether reverse signaling through ephrins can also contribute. We have tested the role of reverse signaling, and of polarised versus non-polarised activation, in assays in which contact repulsion drives cell segregation and border sharpening. We find that polarised forward signaling drives stronger segregation than polarised reverse signaling. Nevertheless, reverse signaling contributes since bidirectional Eph and ephrin activation drives stronger segregation than unidirectional forward signaling alone. In contrast, non-polarised Eph activation drives little segregation. We propose that although polarised forward signaling is the principal driver of segregation, reverse signaling enables bidirectional repulsion which prevents mingling of each population into the other.
Assuntos
Efrinas/fisiologia , Receptores da Família Eph/fisiologia , Transdução de Sinais , Movimento Celular , Polaridade Celular , Efrinas/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Transdução de Sinais/genéticaRESUMO
CDP diacylglycerol synthase (CDS) catalyses the conversion of phosphatidic acid (PA) to CDP-diacylglycerol, an essential intermediate in the synthesis of phosphatidylglycerol, cardiolipin and phosphatidylinositol (PI). CDS activity has been identified in mitochondria and endoplasmic reticulum of mammalian cells apparently encoded by two highly-related genes, CDS1 and CDS2. Cardiolipin is exclusively synthesised in mitochondria and recent studies in cardiomyocytes suggest that the peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1α and ß) serve as transcriptional regulators of mitochondrial biogenesis and up-regulate the transcription of the CDS1 gene. Here we have examined whether CDS1 is responsible for the mitochondrial CDS activity. We report that differentiation of H9c2 cells with retinoic acid towards cardiomyocytes is accompanied by increased expression of mitochondrial proteins, oxygen consumption, and expression of the PA/PI binding protein, PITPNC1, and CDS1 immunoreactivity. Both CDS1 immunoreactivity and CDS activity were found in mitochondria of H9c2 cells as well as in rat heart, liver and brain mitochondria. However, the CDS1 immunoreactivity was traced to a peripheral p55 cross-reactive mitochondrial protein and the mitochondrial CDS activity was due to a peripheral mitochondrial protein, TAMM41, not an integral membrane protein as expected for CDS1. TAMM41 is the mammalian equivalent of the recently identified yeast protein, Tam41. Knockdown of TAMM41 resulted in decreased mitochondrial CDS activity, decreased cardiolipin levels and a decrease in oxygen consumption. We conclude that the CDS activity present in mitochondria is mainly due to TAMM41, which is required for normal mitochondrial function.
Assuntos
Cardiolipinas/biossíntese , Diacilglicerol Colinofosfotransferase/metabolismo , Mitocôndrias Cardíacas/metabolismo , Proteínas Mitocondriais/metabolismo , Miócitos Cardíacos/metabolismo , Consumo de Oxigênio/fisiologia , Animais , Cardiolipinas/genética , Linhagem Celular , Diacilglicerol Colinofosfotransferase/genética , Mitocôndrias Cardíacas/genética , Proteínas Mitocondriais/genética , Miócitos Cardíacos/citologia , RatosRESUMO
Atherosclerosis is an inflammatory disease of the vasculature regulated by cytokines. Macrophages play a crucial role at all stages of this disease, including regulation of foam cell formation, the inflammatory response and stability of atherosclerotic plaques. For example, matrix metalloproteinases produced by macrophages play an important role in modulating plaque stability. More recently, the ADAMTS proteases, which are known to play a key role in the control of cartilage degradation during arthritis, have been found to be expressed in atherosclerotic lesions and suggested to have potentially important functions in the control of plaque stability. Unfortunately, the action of cytokines on the expression of ADAMTS family in macrophages is poorly understood. We have investigated the effect of classical cytokines (IFN-γ and TGF-ß) and those that have been recently identified (TL1A and IL-17) on the expression of ADAMTS-1, -4 and -5 in human macrophages. The expression of all three ADAMTS members was induced during differentiation of monocytes into macrophages. TGF-ß had a differential action with induction of ADAMTS-1 and -5 expression and attenuation in the levels of ADAMTS-4. In contrast, IFN-γ suppressed the expression of ADAMTS-1 without having an effect on ADAMTS-4 and -5. Although TL-1A or IL-17A alone had little effect on the expression of all the members, they induced their expression synergistically when present together. These studies provide new insight into the regulation of key ADAMTS family members in human macrophages by major cytokines in relation to atherosclerosis.
Assuntos
Proteínas ADAM/metabolismo , Aterosclerose/metabolismo , Macrófagos/metabolismo , Pró-Colágeno N-Endopeptidase/metabolismo , Proteínas ADAM/biossíntese , Proteína ADAMTS1 , Proteína ADAMTS4 , Proteína ADAMTS5 , Diferenciação Celular , Linhagem Celular Tumoral , Humanos , Interferon gama/metabolismo , Interleucina-17/metabolismo , Macrófagos/imunologia , Monócitos/metabolismo , Placa Aterosclerótica/imunologia , Pró-Colágeno N-Endopeptidase/biossíntese , Fator de Crescimento Transformador beta/metabolismo , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismoRESUMO
A key event during the formation of lipid-rich foam cells during the progression of atherosclerosis is the uptake of modified low-density lipoproteins (LDL) by macrophages in response to atherogenic mediators in the arterial intima. In addition to scavenger receptor-dependent uptake of LDL, macropinocytosis is known to facilitate the uptake of LDL through the constitutive and passive internalization of large quantities of extracellular solute. In this study we confirm the ability of macropinocytosis to facilitate the uptake of modified LDL by human macrophages and show its modulation by TGF-ß, IFN-γ, IL-17A and IL-33. Furthermore we show that the TGF-ß-mediated inhibition of macropinocytosis is a Smad-2/-3-independent process.
Assuntos
Aterosclerose/patologia , Células Espumosas/patologia , Interferon gama/metabolismo , Interleucina-17/metabolismo , Interleucinas/metabolismo , Pinocitose/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo , Aterosclerose/imunologia , Transporte Biológico/efeitos dos fármacos , Diferenciação Celular , Células Cultivadas , Citocalasina D/farmacologia , Células Espumosas/imunologia , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Humanos , Inflamação/imunologia , Interleucina-33 , Lipoproteínas LDL/metabolismo , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , Inibidores da Síntese de Ácido Nucleico/farmacologia , Interferência de RNA , RNA Interferente Pequeno , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad3/genética , Proteína Smad3/metabolismoRESUMO
Liver X receptors (LXRs) belong to the nuclear receptor superfamily of ligand-dependent transcription factors. LXRs are activated by oxysterols, metabolites of cholesterol, and therefore act as intracellular sensors of this lipid. There are two LXR genes (α and ß) that display distinct tissue/cell expression profiles. LXRs interact with regulatory sequences in target genes as heterodimers with retinoid X receptor. Such direct targets of LXR actions include important genes implicated in the control of lipid homeostasis, particularly reverse cholesterol transport. In addition, LXRs attenuate the transcription of genes associated with the inflammatory response indirectly by transrepression. In this review, we describe recent evidence that both highlights the key roles of LXRs in atherosclerosis and inflammation and provides novel insights into the mechanisms underlying their actions. In addition, we discuss the major limitations of LXRs as therapeutic targets for the treatment of atherosclerosis and how these are being addressed.
Assuntos
Aterosclerose/metabolismo , Inflamação/metabolismo , Metabolismo dos Lipídeos/fisiologia , Fígado/metabolismo , Receptores Nucleares Órfãos/fisiologia , Animais , Humanos , Receptores X do FígadoRESUMO
The development of atherosclerosis, a chronic inflammatory disease characterized by the formation of arterial fibrotic plaques, has been shown to be reduced by IL-33 in vivo. However, whether IL-33 can directly affect macrophage foam cell formation, a key feature of atherosclerotic plaques, has not been determined. In this study, we investigated whether IL-33 reduces macrophage foam cell accumulation in vivo and if IL-33 reduces their formation in vitro using THP-1 and primary human monocyte-derived macrophages. In Apolipoprotein E(-/-) mice fed on a high fat diet, IL-33 treatment significantly reduced the accumulation of macrophage-derived foam cells in atherosclerotic plaques. IL-33 also reduced macrophage foam cell formation in vitro by decreasing acetylated and oxidized low-density lipoprotein uptake, reducing intracellular total and esterified cholesterol content and enhancing cholesterol efflux. These changes were associated with IL-33-mediated reduction in the expression of genes involved in modified low-density lipoprotein uptake, such as CD36, and simultaneous increase in genes involved in cholesterol efflux, including Apolipoprotein E, thereby providing a mechanism for such an action for this cytokine. IL-33 also decreased the expression of key genes implicated in cholesterol esterification and triglyceride storage, including Acyl-CoA:cholesterol acyltransferase 1 and Adipocyte differentiation-related protein. Furthermore, using bone marrow-derived macrophages from ST2(-/-) mice, we demonstrate that the IL-33 receptor, ST2, is integral to the action of IL-33 on macrophage foam cell formation. In conclusion, IL-33 has a protective role in atherosclerosis by reducing macrophage foam cell formation suggesting that IL-33 maybe a potential therapeutic agent against atherosclerosis.
Assuntos
Aterosclerose/prevenção & controle , Células Espumosas/efeitos dos fármacos , Interleucinas/farmacologia , Macrófagos/efeitos dos fármacos , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerose/genética , Aterosclerose/metabolismo , Western Blotting , Linhagem Celular , Células Cultivadas , Colesterol/metabolismo , Ésteres do Colesterol/metabolismo , Feminino , Células Espumosas/metabolismo , Células Espumosas/patologia , Expressão Gênica/efeitos dos fármacos , Humanos , Proteína 1 Semelhante a Receptor de Interleucina-1 , Interleucina-33 , Lipoproteínas LDL/metabolismo , Lipoproteínas LDL/farmacocinética , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Phosphatidylinositol is the parent lipid for the synthesis of seven phosphorylated inositol lipids and each of them play specific roles in numerous processes including receptor-mediated signalling, actin cytoskeleton dynamics and membrane trafficking. PI synthesis is localised to the endoplasmic reticulum (ER) whilst its phosphorylated derivatives are found in other organelles where the lipid kinases also reside. Phosphorylation of PI to phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) at the plasma membrane and to phosphatidylinositol 4-phosphate (PI4P) at the Golgi are key events in lipid signalling and Golgi function respectively. Here we review a family of proteins, phosphatidylinositol transfer proteins (PITPs), that can mobilise PI from the ER to provide the substrate to the resident kinases for phosphorylation. Recent studies identify specific and overlapping functions for the three soluble PITPs (PITPα, PITPß and PITPNC1) in phospholipase C signalling, neuronal function, membrane trafficking, viral replication and in cancer metastases.
Assuntos
Fosfatidilinositóis/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Transdução de SinaisRESUMO
Chronic stimulation (24â¯h) with vasopressin leads to hypertrophy in H9c2 cardiomyoblasts and this is accompanied by continuous activation of phospholipase C. Consequently, vasopressin stimulation leads to a depletion of phosphatidylinositol levels. The substrate for phospholipase C is phosphatidylinositol (4, 5) bisphosphate (PIP2) and resynthesis of phosphatidylinositol and its subsequent phosphorylation maintains the supply of PIP2. The resynthesis of PI requires the conversion of phosphatidic acid to CDP-diacylglycerol catalysed by CDP-diacylglycerol synthase (CDS) enzymes. To examine whether the resynthesis of PI is regulated by vasopressin stimulation, we focussed on the CDS enzymes. Three CDS enzymes are present in mammalian cells: CDS1 and CDS2 are integral membrane proteins localised at the endoplasmic reticulum and TAMM41 is a peripheral protein localised in the mitochondria. Vasopressin selectively stimulates an increase CDS1 mRNA that is dependent on protein kinase C, and can be inhibited by the AP-1 inhibitor, T-5224. Vasopressin also stimulates an increase in cFos protein which is inhibited by a protein kinase C inhibitor. We conclude that vasopressin stimulates CDS1 mRNA through phospholipase C, protein kinase C and cFos and provides a potential mechanism for maintenance of phosphatidylinositol levels during long-term phospholipase C signalling.
Assuntos
Diacilglicerol Colinofosfotransferase/metabolismo , Miócitos Cardíacos/citologia , Fosfolipases Tipo C/metabolismo , Vasopressinas/farmacologia , Animais , Linhagem Celular , Hipertrofia/etiologia , Miócitos Cardíacos/efeitos dos fármacos , Fosfatidilinositóis/metabolismo , Proteína Quinase C , Proteínas Proto-Oncogênicas c-fos , RatosRESUMO
The lipid transporters of the phosphatidylinositol transfer protein (PITP) family dictate phosphoinositide compartmentalization, and specific phosphoinositides play crucial roles in signaling cascades, membrane traffic, ion channel regulation, and actin dynamics. Although PITPs are enriched in the brain, their physiological functions in neuronal signaling pathways in vivo remain ill defined. We describe a CRISPR/Cas9-generated zebrafish mutant in a brain-specific, conserved class II PITP member, pitpnc1a. Zebrafish pitpnc1a mutants are healthy but display widespread aberrant neuronal activity and increased wakefulness across the day-night cycle. The loss of Pitpnc1a increases insulin-like growth factor (IGF) signaling in the brain, and inhibition of IGF pathways is sufficient to rescue both neuronal and behavioral hyperactivity in pitpnc1a mutants. We propose that Pitpnc1a-expressing neurons alter behavior via modification of neuro-modulatory IGF that acts on downstream wake-promoting circuits.
Assuntos
Fator de Crescimento Insulin-Like I/metabolismo , Proteínas de Membrana Transportadoras/uso terapêutico , Vigília/fisiologia , Animais , Proteínas de Membrana Transportadoras/farmacologia , Transdução de Sinais , Peixe-ZebraRESUMO
The anti-atherogenic cytokine TGF-ß inhibits macrophage foam cell formation by suppressing the expression of key genes implicated in the uptake of modified lipoproteins. We have previously shown a critical role for p38 MAPK and JNK in the TGF-ß-mediated regulation of apolipoprotein E expression in human monocytes. However, the roles of these two MAPK pathways in the control of expression of key genes involved in the uptake of modified lipoproteins in human macrophages is poorly understood and formed the focus of this study. TGF-ß activated both p38 MAPK and JNK, and knockdown of p38 MAPK or c-Jun, a key downstream target of JNK action, demonstrated their requirement in the TGF-ß-inhibited expression of several key genes implicated in macrophage lipoprotein uptake. The potential role of c-Jun and specific co-activators in the action of TGF-ß was investigated further by studies on the lipoprotein lipase gene. c-Jun did not directly interact with the minimal promoter region containing the TGF-ß response elements and a combination of transient transfection and knock down assays revealed an important role for SRC-1. These studies provide novel insights into the mechanisms underlying the TGF-ß-mediated inhibition of macrophage gene expression associated with the control of cholesterol homeostasis.
RESUMO
Atherosclerosis is an inflammatory disorder of the vasculature regulated by cytokines. Amongst the cytokines, IL-33 attenuates the development of atherosclerosis in mouse model systems via several mechanisms, including inhibition of macrophage foam cell formation and promotion of a Th1 to Th2 shift. Proteases produced by macrophages, such as matrix metalloproteinases and members of ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family, play potential roles in regulating atherosclerotic plaque stability. Despite such importance, the action of IL-33 on the expression of such proteases has not been analyzed. We have therefore investigated the effect of IL-33 on the expression of ADAMTS-1, -4 and -5 in human macrophages. Immunohistochemical analysis showed that these three proteases were expressed in human atherosclerotic lesions, particularly by macrophages and, to a lesser extent, by smooth muscle cells and endothelial cells. The expression of ADAMTS-1, -4 and -5 in human macrophages was specifically inhibited by IL-33. The action of IL-33 on the expression of these ADAMTS members was mediated through its receptor ST2. IL-33 activated ERK1/2, JNK1/2 and c-Jun, but not p38 MAPK or Akt, in human macrophages. RNA interference assays using a combination of adenoviral encoding small hairpin RNA and small interfering RNA showed a requirement of ERK1/2, JNK1/2, c-Jun, PI3Kγ and PI3Kδ, but not p38α, in the IL-33-inhibited expression of these ADAMTS isoforms. These studies provide novel insights into the expression of ADAMTS-1, -4 and -5 in human atherosclerotic lesions and the regulation of their expression in human macrophages by the key anti-atherogenic cytokine IL-33.
Assuntos
Citocinas/metabolismo , Desintegrinas/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Interleucinas/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Metaloproteases/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Trombospondinas/metabolismo , Animais , Aterosclerose/metabolismo , Humanos , Macrófagos/metabolismo , Camundongos , Transdução de SinaisRESUMO
Cardiovascular disease is the biggest killer globally and the principal contributing factor to the pathology is atherosclerosis; a chronic, inflammatory disorder characterized by lipid and cholesterol accumulation and the development of fibrotic plaques within the walls of large and medium arteries. Macrophages are fundamental to the immune response directed to the site of inflammation and their normal, protective function is harnessed, detrimentally, in atherosclerosis. Macrophages contribute to plaque development by internalizing native and modified lipoproteins to convert them into cholesterol-rich foam cells. Foam cells not only help to bridge the innate and adaptive immune response to atherosclerosis but also accumulate to create fatty streaks, which help shape the architecture of advanced plaques. Foam cell formation involves the disruption of normal macrophage cholesterol metabolism, which is governed by a homeostatic mechanism that controls the uptake, intracellular metabolism, and efflux of cholesterol. It has emerged over the last 20 years that an array of cytokines, including interferon-γ, transforming growth factor-ß1, interleukin-1ß, and interleukin-10, are able to manipulate these processes. Foam cell targeting, anti-inflammatory therapies, such as agonists of nuclear receptors and statins, are known to regulate the actions of pro- and anti-atherogenic cytokines indirectly of their primary pharmacological function. A clear understanding of macrophage foam cell biology will hopefully enable novel foam cell targeting therapies to be developed for use in the clinical intervention of atherosclerosis.
Assuntos
Artérias/patologia , Aterosclerose/terapia , Células Espumosas , Inflamação/terapia , Macrófagos/metabolismo , Terapia de Alvo Molecular/métodos , Placa Aterosclerótica/terapia , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/uso terapêutico , Artérias/efeitos dos fármacos , Artérias/imunologia , Artérias/metabolismo , Aterosclerose/complicações , Aterosclerose/imunologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Transporte Biológico/efeitos dos fármacos , Colesterol/imunologia , Células Espumosas/efeitos dos fármacos , Células Espumosas/imunologia , Células Espumosas/metabolismo , Células Espumosas/patologia , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Inflamação/complicações , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Interferon gama/imunologia , Interleucina-10/imunologia , Interleucina-1beta/imunologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipoproteínas/imunologia , Macrófagos/imunologia , Macrófagos/patologia , Placa Aterosclerótica/complicações , Placa Aterosclerótica/imunologia , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/imunologia , Fator de Crescimento Transformador beta/imunologiaRESUMO
Atherosclerosis is an inflammatory disorder of the vasculature that is orchestrated by the action of cytokines. Macrophages play a prominent role in all stages of this disease, including foam cell formation, production of reactive oxygen species, modulation of the inflammatory response and the regulation of the stability of atherosclerotic plaques. The role of the matrix metalloproteinase family in the control of plaque stability is well established. A disintegrin and metalloproteinase with thrombospondin motif (ADAMTS) family has been implicated in several diseases and the expression of ADAMTS-4 in macrophages of atherosclerotic lesions has suggested a potential role for this protease in atherosclerosis. However, the action of cytokines on the expression of ADAMTS-4 in macrophages is poorly understood. We have investigated here the effect of transforming growth factor-ß (TGF-ß) on ADAMTS-4 expression in macrophages along with the regulatory mechanisms underlying its actions. Consistent with the anti-atherogenic role of TGF-ß, this cytokine decreased the expression of ADAMTS-4 mRNA and protein in human macrophages. Transient transfection assays showed that the -100 to +10 promoter region contained the minimal TGF-ß response elements. Small-interfering RNA-mediated knockdown revealed a critical role for Smads, p38 mitogen-activated protein kinase and c-Jun in the action of TGF-ß on ADAMTS-4 mRNA expression. These studies show for the first time that TGF-ß inhibits the expression of ADAMTS-4 in human macrophages and identifies the signalling pathways underlying this response. The inhibition of macrophage ADAMTS-4 expression is likely to contribute to the anti-atherogenic, plaque stabilisation action of TGF-ß.
Assuntos
Proteínas ADAM/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Macrófagos/efeitos dos fármacos , Pró-Colágeno N-Endopeptidase/metabolismo , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas ADAM/genética , Proteína ADAMTS4 , Aterosclerose/tratamento farmacológico , Aterosclerose/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Humanos , Macrófagos/metabolismo , Pró-Colágeno N-Endopeptidase/genética , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta1/uso terapêuticoRESUMO
The ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) proteases are secreted enzymes that regulate extracellular matrix turnover by degrading specific matrix components. Roles for the proteases in inflammation and atherosclerosis have been suggested by a number of recent studies, and the role of ADAMTS-4 and -5 in the breakdown of aggrecan and subsequent degradation of cartilage during osteoarthritis has also been established. The ability of the ADAMTS proteases to degrade versican, the primary proteoglycan in the vasculature, is thought to be central to any hypothesized role for the proteases in atherosclerosis. In this review, we introduce the structure and function of the ADAMTS family of proteases and review the literature that links them with inflammation and atherosclerosis.