RESUMO
Balancing cellular demise and survival constitutes a key feature of resilience mechanisms that underlie the control of epithelial tissue damage. These resilience mechanisms often limit the burden of adaptive cellular stress responses to internal or external threats. We recently identified Diedel, a secreted protein/cytokine, as a potent antagonist of apoptosis-induced regulated cell death in the Drosophila intestinal midgut epithelium during aging. Here, we show that Diedel is a ligand for RGD-binding Integrins and is thus required for maintaining midgut epithelial cell attachment to the extracellular matrix (ECM)-derived basement membrane. Exploiting this function of Diedel, we uncovered a resilience mechanism of epithelial tissues, mediated by Integrin-ECM interactions, which shapes cell death spreading through the regulation of cell detachment and thus cell survival. Moreover, we found that resilient epithelial cells, enriched for Diedel-Integrin-ECM interactions, are characterized by membrane association of Catalase, thus preserving extracellular reactive oxygen species (ROS) balance to maintain epithelial integrity. Intracellular Catalase can relocalize to the extracellular membrane to limit cell death spreading and repair Integrin-ECM interactions induced by the amplification of extracellular ROS, which is a critical adaptive stress response. Membrane-associated Catalase, synergized with Integrin-ECM interactions, likely constitutes a resilience mechanism that helps balance cellular demise and survival within epithelial tissues.
Assuntos
Drosophila , Matriz Extracelular , Animais , Catalase/metabolismo , Adesão Celular , Drosophila/metabolismo , Epitélio/metabolismo , Matriz Extracelular/metabolismo , Integrinas/metabolismo , Mucosa Intestinal/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Epidermal growth factor receptor (EGFR) signaling drives the formation of many types of cancer, including colon cancer. Docosahexaenoic acid (DHA, 22â¶6Δ4,7,10,13,16,19), a chemoprotective long-chain n-3 polyunsaturated fatty acid suppresses EGFR signaling. However, the mechanism underlying this phenotype remains unclear. Therefore, we used super-resolution microscopy techniques to investigate the mechanistic link between EGFR function and DHA-induced alterations to plasma membrane nanodomains. Using isogenic in vitro (YAMC and IMCE mouse colonic cell lines) and in vivo (Drosophila, wild type and Fat-1 mice) models, cellular DHA enrichment via therapeutic nanoparticle delivery, endogenous synthesis, or dietary supplementation reduced EGFR-mediated cell proliferation and downstream Ras/ERK signaling. Phospholipid incorporation of DHA reduced membrane rigidity and the size of EGFR nanoclusters. Similarly, pharmacological reduction of plasma membrane phosphatidic acid (PA), phosphatidylinositol-4,5-bisphosphate (PIP2) or cholesterol was associated with a decrease in EGFR nanocluster size. Furthermore, in DHA-treated cells only the addition of cholesterol, unlike PA or PIP2, restored EGFR nanoscale clustering. These findings reveal that DHA reduces EGFR signaling in part by reshaping EGFR proteolipid nanodomains, supporting the feasibility of using membrane therapy, i.e., dietary/drug-related strategies to target plasma membrane organization, to reduce EGFR signaling and cancer risk.
Assuntos
Ácidos Docosa-HexaenoicosRESUMO
Aging is characterized by degeneration of unique tissues. However, dissecting the interconnectedness of tissue aging remains a challenge. Here, we employ a muscle-specific DNA damage model in Drosophila to reveal secreted factors that influence systemic aging in distal tissues. Utilizing this model, we uncovered a cytokine-Diedel-that, when secreted from muscle or adipose, can attenuate age-related intestinal tissue degeneration by promoting proliferative homeostasis of stem cells. Diedel is both necessary and sufficient to limit tissue degeneration and regulate lifespan. Secreted homologs of Diedel are also found in viruses, having been acquired from host genomes. Focusing on potential mechanistic overlap between cellular aging and viral-host cell interactions, we found that Diedel is an inhibitor of apoptosis and can act as a systemic rheostat to modulate cell death during aging. These results highlight a key role for secreted antagonists of apoptosis in the systemic coordination of tissue aging.
Assuntos
Envelhecimento/fisiologia , Apoptose , Citocinas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/fisiologia , Vírus/metabolismo , Animais , Dano ao DNA , Hormese , Intestinos , Longevidade , Camundongos , Músculos/metabolismoRESUMO
The low density lipoprotein receptor-related protein 1 (LRP1) is a ubiquitously expressed cell surface receptor that protects from intracellular cholesterol accumulation. However, the underlying mechanisms are unknown. Here we show that the extracellular (α) chain of LRP1 mediates TGFß-induced enhancement of Wnt5a, which limits intracellular cholesterol accumulation by inhibiting cholesterol biosynthesis and by promoting cholesterol export. Moreover, we demonstrate that the cytoplasmic (ß) chain of LRP1 suffices to limit cholesterol accumulation in LRP1(-/-) cells. Through binding of Erk2 to the second of its carboxyl-terminal NPXY motifs, LRP1 ß-chain positively regulates the expression of ATP binding cassette transporter A1 (ABCA1) and of neutral cholesterol ester hydrolase (NCEH1). These results highlight the unexpected functions of LRP1 and the canonical Wnt5a pathway and new therapeutic potential in cholesterol-associated disorders including cardiovascular diseases.
Assuntos
Colesterol/metabolismo , Receptores de LDL/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Via de Sinalização Wnt , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Animais , Células HEK293 , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Receptores de LDL/química , Receptores de LDL/genética , Esterol Esterase/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética , Proteínas Wnt/metabolismo , Proteína Wnt-5aRESUMO
Src homology and collagen A (ShcA) is an adaptor protein that binds to tyrosine kinase receptors. Its germ line deletion is embryonic lethal with abnormal cardiovascular system formation, and its role in cardiovascular development is unknown. To investigate its functional role in cardiovascular development in mice, ShcA was deleted in cardiomyocytes and vascular smooth muscle cells by crossing ShcA flox mice with SM22a-Cre transgenic mice. Conditional mutant mice developed signs of severe dilated cardiomyopathy, myocardial infarctions, and premature death. No evidence of a vascular contribution to the phenotype was observed. Histological analysis of the heart revealed aberrant sarcomeric Z-disk and M-band structures, and misalignments of T-tubules with Z-disks. We find that not only the ErbB3/Neuregulin signaling pathway but also the baroreceptor reflex response, which have been functionally associated, are altered in the mutant mice. We further demonstrate that ShcA interacts with Caveolin-1 and the costameric protein plasma membrane Ca(2+)/calmodulin-dependent ATPase (PMCA), and that its deletion leads to abnormal dystrophin signaling. Collectively, these results demonstrate that ShcA interacts with crucial proteins and pathways that link Z-disk and costamere.
Assuntos
Costâmeros/metabolismo , Coração/embriologia , Miócitos Cardíacos/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Alelos , Animais , Aorta Torácica/metabolismo , Pressão Sanguínea , Sobrevivência Celular , Distrofina/metabolismo , Ecocardiografia , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Imageamento por Ressonância Magnética , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Fenótipo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , RNA Interferente Pequeno/metabolismo , Ratos , Receptor ErbB-3/metabolismo , Proteínas Adaptadoras da Sinalização Shc/genética , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de SrcRESUMO
Osteopontin (OPN), a multifunctional glycophosphoprotein, has been reported to contribute to the development and progression of cardiac remodeling and hypertrophy. Cardiac-specific OPN knockout mice were protected against hypertrophy and fibrosis mediated by Ang II. Recently, transgenic mice expressing the active form of the Na(+)/H(+) exchanger isoform 1 (NHE1) developed spontaneous hypertrophy in association with elevated levels of OPN. The mechanism by which active NHE1 induces OPN expression and contributes to the hypertrophic response remains unclear. To validate whether expression of the active form of NHE1 induces OPN, cardiomyocytes were stimulated with Ang II, a known inducer of both OPN and NHE1. Ang II induced hypertrophy and increased OPN protein expression (151.6 ± 28.19 %, P < 0.01) and NHE1 activity in H9c2 cardiomyoblasts. Ang II-induced hypertrophy and OPN protein expression were regressed in the presence of an NHE1 inhibitor, EMD 87580, or a calcineurin inhibitor, FK506. In addition, our results indicated that activation of NHE1-induced NFAT3 translocation into the nucleus and a significant activation of the transcription factor Gata4 (NHE1: 149 ± 28 % of control, P < 0.05). NHE1-induced activation of Gata4 was inhibited by FK506. In summary, our results suggest that activation of NHE1 induces hypertrophy through the activation of NFAT3/Gata4 and OPN expression.
Assuntos
Proteínas de Transporte de Cátions/genética , Fator de Transcrição GATA4/genética , Hipertrofia/genética , Fatores de Transcrição NFATC/metabolismo , Osteopontina/biossíntese , Trocadores de Sódio-Hidrogênio/genética , Animais , Proteínas de Transporte de Cátions/metabolismo , Regulação da Expressão Gênica , Hipertrofia/patologia , Camundongos , Camundongos Knockout , Mioblastos/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Osteopontina/genética , Trocador 1 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/metabolismo , Tacrolimo/administração & dosagemRESUMO
Although the role of the Wnt pathway in colon carcinogenesis has been described previously, it has been recently demonstrated that Wnt signaling originates from highly dynamic nano-assemblies at the plasma membrane. However, little is known regarding the role of oncogenic APC in reshaping Wnt nanodomains. This is noteworthy, because oncogenic APC does not act autonomously and requires activation of Wnt effectors upstream of APC to drive aberrant Wnt signaling. Here, we demonstrate the role of oncogenic APC in increasing plasma membrane free cholesterol and rigidity, thereby modulating Wnt signaling hubs. This results in an overactivation of Wnt signaling in the colon. Finally, using the Drosophila sterol auxotroph model, we demonstrate the unique ability of exogenous free cholesterol to disrupt plasma membrane homeostasis and drive Wnt signaling in a wildtype APC background. Collectively, these findings provide a link between oncogenic APC, loss of plasma membrane homeostasis and CRC development.
Assuntos
Via de Sinalização Wnt , beta Catenina , Animais , beta Catenina/genética , beta Catenina/metabolismo , Carcinogênese/genética , Membrana Celular/metabolismo , Colo/metabolismo , Drosophila/metabolismo , Via de Sinalização Wnt/genéticaRESUMO
The low density lipoprotein receptor-related protein (LRP1) is a transmembrane receptor that integrates multiple signaling pathways. Its cytoplasmic domain serves as docking sites for several adaptor proteins such as the Src homology 2/α-collagen (ShcA), which also binds to several tyrosine kinase receptors such as the insulin-like growth factor 1 (IGF-1) receptor. However, the physiological significance of the physical interaction between LRP1 and ShcA, and whether this interaction modifies tyrosine kinase receptor signaling, are still unknown. Here we report that LRP1 forms a complex with the IGF-1 receptor, and that LRP1 is required for ShcA to become sensitive to IGF-1 stimulation. Upon IGF-1 treatment, ShcA is tyrosine phosphorylated and translocates to the plasma membrane only in the presence of LRP1. This leads to the recruitment of the growth factor receptor-bound protein 2 (Grb2) to ShcA, and activation of the Ras/MAP kinase pathway. Conversely, in the absence of ShcA, IGF-1 signaling bifurcates toward the Akt/mammalian target of rapamycin pathway and accelerates adipocyte differentiation when cells are stimulated for adipogenesis. These results establish the LRP1-ShcA complex as an essential component in the IGF-1-regulated pathway for MAP kinase and Akt/mammalian target of rapamycin activation, and may help to understand the IGF-1 signaling shift from clonal expansion to growth-arrested cells and differentiation during adipogenesis.
Assuntos
Regulação da Expressão Gênica , Receptor IGF Tipo 1/metabolismo , Receptores de LDL/metabolismo , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Células 3T3-L1 , Adipócitos/citologia , Animais , Diferenciação Celular , Fibroblastos/metabolismo , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Camundongos , Camundongos Transgênicos , Fosforilação , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Serina-Treonina Quinases TOR/metabolismo , Tirosina/química , Proteínas ras/metabolismoRESUMO
Ras signaling originates from transient nanoscale compartmentalized regions of the plasma membrane composed of specific proteins and lipids. The highly specific lipid composition of these nanodomains, termed nanoclusters, facilitates effector recruitment and therefore influences signal transduction. This suggests that Ras nanocluster proteolipid composition could represent a novel target for future chemoprevention interventions. There is evidence that consumption of fish oil containing long-chain n-3 polyunsaturated fatty acids (n-3 PUFA) such as eicosapentaenoic acid (EPA, 20:5Δ5,8,11,14,17) and docosahexaenoic acid (DHA, 22:6Δ4,7,10,13,16,19) may reduce colon cancer risk in humans, yet the mechanism underlying this effect is unknown. Here, we demonstrate that dietary n-3 PUFA reduce the lateral segregation of cholesterol-dependent and -independent nanoclusters, suppressing phosphatidic acid-dependent oncogenic KRas effector interactions, via their physical incorporation into plasma membrane phospholipids. This results in attenuation of oncogenic Ras-driven colonic hyperproliferation in both Drosophila and murine models. These findings demonstrate the unique properties of dietary n-3 PUFA in the shaping of Ras nanoscale proteolipid complexes and support the emerging role of plasma membrane-targeted therapies.Significance: The influence of dietary long chain n-3 polyunsaturated fatty acids on plasma membrane protein nanoscale organization and KRas signaling supports development of plasma membrane-targeted therapies in colon cancer.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/14/3899/F1.large.jpg Cancer Res; 78(14); 3899-912. ©2018 AACR.
Assuntos
Membrana Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ácidos Graxos Ômega-3/farmacologia , Proteolipídeos/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Membrana Celular/metabolismo , Células Cultivadas , Colesterol/metabolismo , Dieta , Ácidos Docosa-Hexaenoicos/farmacologia , Drosophila/metabolismo , Ácido Eicosapentaenoico/análogos & derivados , Ácido Eicosapentaenoico/farmacologia , Óleos de Peixe , Camundongos , Fosfolipídeos/metabolismoRESUMO
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
RESUMO
ShcA is an adaptor protein that binds to the cytoplasmic tail of receptor tyrosine kinases and of the Low Density Lipoprotein-related receptor 1 (LRP1), a trans-membrane receptor that protects against atherosclerosis. Here, we examined the role of endothelial ShcA in atherosclerotic lesion formation. We found that atherosclerosis progression was markedly attenuated in mice deleted for ShcA in endothelial cells, that macrophage content was reduced at the sites of lesions, and that adhesion molecules such as the intercellular adhesion molecule-1 (ICAM-1) were severely reduced. Our data indicate that transcriptional regulation of ShcA by the zinc-finger E-box-binding homeobox 1 (ZEB1) and the Hippo pathway effector YAP, promotes ICAM-1 expression independently of p-NF-κB, the primary driver of adhesion molecules expressions. In addition, ShcA suppresses endothelial Akt and nitric oxide synthase (eNOS) expressions. Thus, through down regulation of eNOS and ZEB1-mediated ICAM-1 up regulation, endothelial ShcA promotes monocyte-macrophage adhesion and atherosclerotic lesion formation. Reducing ShcA expression in endothelial cells may represent an obvious therapeutic approach to prevent atherosclerosis.
RESUMO
Studies using pharmacological and genetic approaches have shown that increased activity/expression of the Na+/H+ exchanger isoform 1 (NHE1) play a critical role in the pathogenesis of cardiac hypertrophy. Despite the importance of NHE1 in cardiac hypertrophy, severe cerebrovascular side effects were associated with the use of NHE1 inhibitors when administered to patients with myocardial infarctions. p90 ribosomal S6 Kinase (RSK), a downstream regulator of the mitogen-activated protein kinase pathway, has also been implicated in cardiac hypertrophy. We hypothesized that RSK plays a role in the NHE1 induced cardiomyocyte hypertrophic response. Infection of H9c2 cardiomyoblasts with the active form of the NHE1 adenovirus induced hypertrophy and was associated with an increase in the phosphorylation of RSK (P<0.05). Parameters of hypertrophy such as cell area, protein content and atrial natriuretic mRNA expression were significantly reduced in H9c2 cardiomyoblasts infected with active NHE1 in the presence of dominant negative RSK (DN-RSK) (P<0.05). These results confirm that NHE1 lies upstream of RSK. Increased phosphorylation and activation of GATA4 at Ser261 was correlated with increased RSK phosphorylation. This increase was reversed upon inhibition of RSK or NHE1. These findings demonstrate for the first time that the NHE1 mediated hypertrophy is accounted for by increased activation and phosphorylation of RSK, which subsequently increased the phosphorylation of GATA4; eventually activating fetal gene transcriptional machinery.
Assuntos
Miócitos Cardíacos/enzimologia , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Trocadores de Sódio-Hidrogênio/fisiologia , Animais , Linhagem Celular , Ativação Enzimática , Fator de Transcrição GATA4/metabolismo , Regulação da Expressão Gênica , Hipertrofia/enzimologia , Miócitos Cardíacos/fisiologia , Fosforilação , Processamento de Proteína Pós-Traducional , Ratos , Trocador 1 de Sódio-HidrogênioRESUMO
Enhanced expression and activity of the Na+/H+ exchanger isoform 1 (NHE1) has been implicated in cardiomyocyte hypertrophy in various experimental models. The upregulation of NHE1 was correlated with an increase in osteopontin (OPN) expression in models of cardiac hypertrophy (CH), and the mechanism for this remains to be delineated. To determine whether the expression of active NHE1-induces OPN and contributes to the hypertrophic response in vitro, cardiomyocytes were infected with the active form of the NHE1 adenovirus or transfected with OPN silencing RNA (siRNA-OPN) and characterized for cardiomyocyte hypertrophy. Expression of NHE1 in cardiomyocytes resulted in a significant increase in cardiomyocyte hypertrophy markers: cell surface area, protein content, ANP mRNA and expression of phosphorylated-GATA4. NHE1 activity was also significantly increased in cardiomyocytes expressing active NHE1. Interestingly, transfection of cardiomyocytes with siRNA-OPN significantly abolished the NHE1-induced cardiomyocyte hypertrophy. siRNA-OPN also significantly reduced the activity of NHE1 in cardiomyocytes expressing NHE1 (68.5±0.24%; P<0.05), confirming the role of OPN in the NHE1-induced hypertrophic response. The hypertrophic response facilitated by NHE1-induced OPN occurred independent of the extracellular-signal-regulated kinases and Akt, but required p90-ribosomal S6 kinase (RSK). The ability of OPN to facilitate the NHE1-induced hypertrophic response identifies OPN as a potential therapeutic target to reverse the hypertrophic effect induced by the expression of active NHE1.
Assuntos
Cardiomegalia/metabolismo , Miócitos Cardíacos/metabolismo , Osteopontina/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Cardiomegalia/genética , Cardiomegalia/patologia , Crescimento Celular , Células Cultivadas , Miócitos Cardíacos/patologia , Osteopontina/genética , Fosforilação , RNA Interferente Pequeno , Ratos , Transdução de Sinais/fisiologia , Trocador 1 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/genética , TransfecçãoRESUMO
Vascular calcification is a hallmark of advanced atherosclerosis. Here we show that deletion of the nuclear receptor PPARγ in vascular smooth muscle cells of low density lipoprotein receptor (LDLr)-deficient mice fed an atherogenic diet high in cholesterol, accelerates vascular calcification with chondrogenic metaplasia within the lesions. Vascular calcification in the absence of PPARγ requires expression of the transmembrane receptor LDLr-related protein-1 in vascular smooth muscle cells. LDLr-related protein-1 promotes a previously unknown Wnt5a-dependent prochondrogenic pathway. We show that PPARγ protects against vascular calcification by inducing the expression of secreted frizzled-related protein-2, which functions as a Wnt5a antagonist. Targeting this signalling pathway may have clinical implications in the context of common complications of atherosclerosis, including coronary artery calcification and valvular sclerosis.