RESUMO
We recently described morgana as an essential protein able to regulate centrosome duplication and genomic stability, by inhibiting ROCK. Here we show that morgana (+/-) mice spontaneously develop a lethal myeloproliferative disease resembling human atypical chronic myeloid leukemia (aCML), preceded by ROCK hyperactivation, centrosome amplification, and cytogenetic abnormalities in the bone marrow (BM). Moreover, we found that morgana is underexpressed in the BM of patients affected by atypical CML, a disorder of poorly understood molecular basis, characterized by nonrecurrent cytogenetic abnormalities. Morgana is also underexpressed in the BM of a portion of patients affected by Philadelphia-positive CML (Ph(+) CML) caused by the BCR-ABL oncogene, and in this condition, morgana underexpression predicts a worse response to imatinib, the standard treatment for Ph(+) CML. Thus, morgana acts as an oncosuppressor with different modalities: (1) Morgana underexpression induces centrosome amplification and cytogenetic abnormalities, and (2) in Ph(+) CML, it synergizes with BCR-ABL signaling, reducing the efficacy of imatinib treatment. Importantly, ROCK inhibition in the BM of patients underexpressing morgana restored the efficacy of imatinib to induce apoptosis, suggesting that ROCK inhibitors, combined with imatinib treatment, can overcome suboptimal responses in patients in which morgana is underexpressed.
Assuntos
Benzamidas/farmacologia , Proteínas de Transporte/fisiologia , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Fusão bcr-abl/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Cromossomo Filadélfia , Piperazinas/farmacologia , Pirimidinas/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Animais , Apoptose , Western Blotting , Medula Óssea/metabolismo , Medula Óssea/patologia , Proliferação de Células , Citometria de Fluxo , Proteínas de Fusão bcr-abl/genética , Humanos , Mesilato de Imatinib , Técnicas Imunoenzimáticas , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Chaperonas Moleculares , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismoRESUMO
Melusin is a protein selectively expressed in skeletal muscles and heart and highly conserved in vertebrates. Melusin is part of the heat shock protein 90 machinery and acts as molecular chaperone in controlling cardiomyocyte survival and adaptive hypertrophy signaling pathways in the heart in response to different stress conditions. The role of melusin has been extensively investigated in genetically modified mice over the past years disclosing an important cardioprotective function of this unique muscle-specific chaperone protein in different pathological conditions. This review highlights the findings in animal models and the molecular mechanisms underlying melusin cardioprotective function.
Assuntos
Cardiomiopatias/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas Musculares/metabolismo , Miocárdio/metabolismo , Animais , Humanos , CamundongosRESUMO
Morgana/CHP-1 is a ubiquitously expressed protein able to inhibit ROCK II kinase activity. We have previously demonstrated that morgana haploinsufficiency leads to multiple centrosomes, genomic instability, and higher susceptibility to tumour development. While a large fraction of human cancers has shown morgana down-regulation, a small subset of tumours was shown to express high morgana levels. Here we demonstrate that high morgana expression in different breast cancer subtypes correlates with high tumour grade, mitosis number, and lymph node positivity. Moreover, morgana overexpression induces transformation in NIH-3T3 cells and strongly protects them from various apoptotic stimuli. From a mechanistic point of view, we demonstrate that morgana causes PTEN destabilization, by inhibiting ROCK activity, hence triggering the PI3K/AKT survival pathway. In turn, morgana down-regulation in breast cancer cells that express high morgana levels increases PTEN expression and leads to sensitization of cells to chemotherapy.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Transdução de Sinais/fisiologia , Quinases Associadas a rho/metabolismo , Animais , Neoplasias da Mama/patologia , Centrossomo/patologia , Regulação para Baixo/fisiologia , Feminino , Humanos , Camundongos , Chaperonas Moleculares , Fosfatidilinositol 3-Quinases/metabolismo , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Melusin is a muscle-specific protein which interacts with ß1 integrin cytoplasmic domain and acts as chaperone protein. Its overexpression induces improved resistance to cardiac overload delaying left ventricle dilation and reducing the occurrence of heart failure. Here, we investigated possible protective effect of melusin overexpression against acute ischemia/reperfusion (I/R) injury with or without Postconditioning cardioprotective maneuvers. Melusin transgenic (Mel-TG) mice hearts were subjected to 30-min global ischemia followed by 60-min reperfusion. Interestingly, infarct size was reduced in Mel-TG mice hearts compared to wild-type (WT) hearts (40.3 ± 3.5 % Mel-TG vs. 59.5 ± 3.8 % WT hearts; n = 11 animals/group; P < 0.05). The melusin protective effect was also demonstrated by measuring LDH release, which was 50 % lower in Mel-TG compared to WT. Mel-TG hearts had a higher baseline level of AKT, ERK1/2 and GSK3ß phosphorylation, and displayed increased phospho-kinases level after I/R compared to WT mice. Post-ischemic Mel-TG hearts displayed also increased levels of the anti-apoptotic factor phospho-BAD. Importantly, pharmacological inhibition of PI3K/AKT (Wortmannin) and ERK1/2 (U0126) pathways abrogated the melusin protective effect. Notably, HSP90, a chaperone known to protect heart from I/R injury, showed high levels of expression in the heart of Mel-TG mice suggesting a possible collaboration of this molecule with AKT/ERK/GSK3ß pathways in the melusin-induced protection. Postconditioning, known to activate AKT/ERK/GSK3ß pathways, significantly reduced IS and LDH release in WT hearts, but had no additive protective effects in Mel-TG hearts. These findings implicate melusin as an enhancer of AKT and ERK pathways and as a novel player in cardioprotection from I/R injury.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas Musculares/metabolismo , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/metabolismo , Animais , Proteínas do Citoesqueleto/genética , Modelos Animais de Doenças , Ativação Enzimática , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Proteínas de Choque Térmico HSP90/metabolismo , Masculino , Camundongos Transgênicos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Musculares/genética , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/patologia , Fosfatidilinositol 3-Quinase/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Fatores de Tempo , Regulação para CimaRESUMO
The ability of cardiomyocytes to detect mechanical and humoral stimuli is critical for adaptation of the myocardium in response to new conditions and for sustaining the increased workload during stress. While certain stimuli mediate a beneficial adaptation to stress conditions, others result in maladaptive remodelling, ultimately leading to heart failure. Specific signalling pathways activating either adaptive or maladaptive cardiac remodelling have been identified. Paradoxically, however, in a number of cases, the transduction pathways involved in such opposing responses engage the same signalling proteins. A notable example is the Raf-MEK1/2-ERK1/2 signalling pathway that can control both adaptive and maladaptive remodelling. ERK1/2 signalling requires a signalosome complex where a scaffold protein drives the assembly of these three kinases into a linear pathway to facilitate their sequential phosphorylation, ultimately targeting specific effector molecules. Interestingly, a number of different Raf-MEK1/2-ERK1/2 scaffold proteins have been identified, and their role in determining the adaptive or maladaptive cardiac remodelling is a promising field of investigation for the development of therapeutic strategies capable of selectively potentiating the adaptive response.
Assuntos
Insuficiência Cardíaca/fisiopatologia , Coração/fisiopatologia , Sistema de Sinalização das MAP Quinases/fisiologia , Miocárdio/patologia , Animais , Arrestinas/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Humanos , Camundongos , Miócitos Cardíacos/citologia , Fosforilação , Estrutura Terciária de Proteína , Transdução de Sinais , beta-Arrestinas , Quinases raf/metabolismoRESUMO
Extracellular signal-regulated kinase 1/2 (ERK1/2) signalling is a key pathway in cardiomyocyte hypertrophy and survival in response to many different stress stimuli. We have previously characterized melusin as a muscle-specific chaperone protein capable of ERK1/2 signalling activation in the heart. Here, we show that in the heart, melusin forms a supramolecular complex with the proto-oncogene c-Raf, MEK1/2 (also known as MAPKK1/2) and ERK1/2 and that melusin-bound mitogen-activated protein kinases (MAPKs) are activated by pressure overload. Moreover, we demonstrate that both focal adhesion kinase (FAK) and IQ motif-containing GTPase activating protein 1 (IQGAP1), a scaffold protein for the ERK1/2 signalling cascade, are part of the melusin complex and are required for ERK1/2 activation in response to pressure overload. Finally, analysis of isolated neonatal cardiomyocytes indicates that both FAK and IQGAP1 regulate melusin-dependent cardiomyocyte hypertrophy and survival through ERK1/2 activation.
Assuntos
Cardiomiopatia Hipertrófica/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Musculares/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Regulação Alostérica , Animais , Cardiomiopatia Hipertrófica/tratamento farmacológico , Cardiomiopatia Hipertrófica/patologia , Cardiomiopatia Hipertrófica/fisiopatologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Proteínas do Citoesqueleto/genética , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Coração/efeitos dos fármacos , Coração/fisiologia , Coração/fisiopatologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Chaperonas Moleculares/genética , Complexos Multienzimáticos/metabolismo , Proteínas Musculares/genética , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Estresse Fisiológico , Proteínas Ativadoras de ras GTPase/genéticaRESUMO
Morgana/CHP-1 (CHORD containing protein-1) has been recently shown to be necessary for proper cell divisions. However, the presence of the protein in postmitotic tissues such as brain and striated muscle suggests that morgana/CHP-1 has additional cellular functions. Here we show that morgana/CHP-1 behaves like an HSP90 co-chaperone and possesses an independent molecular chaperone activity towards denatured proteins. The expression time profile of morgana/Chp-1 in NIH3T3 cells in response to heat stress is similar to that of Hsp70, a classical effector of Heat Shock Factor-1 mediated stress response. Moreover, overexpression of morgana/CHP-1 in NIH3T3 cells leads to the increased stress resistance of the cells. Interestingly, morgana/Chp-1 upregulation in response to transient global brain ischemia lasts longer in ischemia-resistant regions of the gerbil hippocampus than in vulnerable ones, suggesting the involvement of morgana/CHP-1 in natural protective mechanisms in vivo.
Assuntos
Proteínas de Transporte/fisiologia , Células/metabolismo , Citoproteção/genética , Estresse Fisiológico/genética , Animais , Isquemia Encefálica/genética , Isquemia Encefálica/patologia , Proteínas de Transporte/genética , Células Cultivadas , Gerbillinae , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Choque Térmico HSP90/fisiologia , Resposta ao Choque Térmico/genética , Resposta ao Choque Térmico/fisiologia , Temperatura Alta , Camundongos , Chaperonas Moleculares/genética , Chaperonas Moleculares/fisiologia , Células NIH 3T3RESUMO
Hypertension affects nearly 20% of the population in Western countries and strongly increases the risk for cardiovascular diseases. In the pathogenesis of hypertension, the vasoactive peptide of the renin-angiotensin system, angiotensin II and its G protein-coupled receptors (GPCRs), play a crucial role by eliciting reactive oxygen species (ROS) and mediating vessel contractility. Here we show that mice lacking the GPCR-activated phosphoinositide 3-kinase (PI3K)gamma are protected from hypertension that is induced by administration of angiotensin II in vivo. PI3Kgamma was found to play a role in angiotensin II-evoked smooth muscle contraction in two crucial, distinct signaling pathways. In response to angiotensin II, PI3Kgamma was required for the activation of Rac and the subsequent triggering of ROS production. Conversely, PI3Kgamma was necessary to activate protein kinase B/Akt, which, in turn, enhanced L-type Ca(2+) channel-mediated extracellular Ca(2+) entry. These data indicate that PI3Kgamma is a key transducer of the intracellular signals that are evoked by angiotensin II and suggest that blocking PI3Kgamma function might be exploited to improve therapeutic intervention on hypertension.
Assuntos
Angiotensina II/farmacologia , Hipertensão/prevenção & controle , Músculo Liso Vascular/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/deficiência , Vasoconstritores/farmacologia , Animais , Aorta , Cálcio/metabolismo , Células Cultivadas , Hipertensão/induzido quimicamente , Isoenzimas/antagonistas & inibidores , Isoenzimas/deficiência , Masculino , Artérias Mesentéricas , Camundongos , Camundongos Knockout , Músculo Liso Vascular/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Espécies Reativas de Oxigênio/metabolismo , VasoconstriçãoRESUMO
The ErbB2 oncogene is often overexpressed in breast tumors and associated with poor clinical outcome. p130Cas represents a nodal scaffold protein regulating cell survival, migration, and proliferation in normal and pathological cells. The functional role of p130Cas in ErbB2-dependent breast tumorigenesis was assessed by its silencing in breast cancer cells derived from mouse mammary tumors overexpressing ErbB2 (N202-1A cells), and by its reexpression in ErbB2-transformed p130Cas-null mouse embryonic fibroblasts. We demonstrate that p130Cas is necessary for ErbB2-dependent foci formation, anchorage-independent growth, and in vivo growth of orthotopic N202-1A tumors. Moreover, intranipple injection of p130Cas-stabilized siRNAs in the mammary gland of Balbc-NeuT mice decreases the growth of spontaneous tumors. In ErbB2-transformed cells, p130Cas is a crucial component of a functional molecular complex consisting of ErbB2, c-Src, and Fak. In human mammary cells, MCF10A.B2, the concomitant activation of ErbB2, and p130Cas overexpression sustain and strengthen signaling, leading to Rac1 activation and MMP9 secretion, thus providing invasive properties. Consistently, p130Cas drives N202-1A cell in vivo lung metastases colonization. These results demonstrate that p130Cas is an essential transducer in ErbB2 transformation and highlight its potential use as a novel therapeutic target in ErbB2 positive human breast cancers.
Assuntos
Transformação Celular Neoplásica , Proteína Substrato Associada a Crk/fisiologia , Genes erbB-2 , Neoplasias Mamárias Experimentais/patologia , Animais , Linhagem Celular Tumoral , Proteína Substrato Associada a Crk/genética , Feminino , Inativação Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Células NIH 3T3 , RNA Interferente Pequeno , Transdução de SinaisRESUMO
Cardiac hypertrophy is an adaptive response to a variety of mechanical and hormonal stimuli, and represents an early event in the clinical course leading to heart failure. By gene inactivation, we demonstrate here a crucial role of melusin, a muscle-specific protein that interacts with the integrin beta1 cytoplasmic domain, in the hypertrophic response to mechanical overload. Melusin-null mice showed normal cardiac structure and function in physiological conditions, but when subjected to pressure overload--a condition that induces a hypertrophic response in wild-type controls--they developed an abnormal cardiac remodeling that evolved into dilated cardiomyopathy and contractile dysfunction. In contrast, the hypertrophic response was identical in wild-type and melusin-null mice after chronic administration of angiotensin II or phenylephrine at doses that do not increase blood pressure--that is, in the absence of cardiac biomechanical stress. Analysis of intracellular signaling events induced by pressure overload indicated that phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) was specifically blunted in melusin-null hearts. Thus, melusin prevents cardiac dilation during chronic pressure overload by specifically sensing mechanical stress.
Assuntos
Baixo Débito Cardíaco , Cardiomegalia , Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto , Integrina beta1/metabolismo , Proteínas Musculares/metabolismo , Angiotensina II/farmacologia , Animais , Coartação Aórtica , Fenômenos Biomecânicos , Proteínas de Transporte/genética , Ecocardiografia , Feminino , Inativação Gênica , Ventrículos do Coração/anatomia & histologia , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/patologia , Hemodinâmica , Masculino , Camundongos , Camundongos Knockout , Proteínas Musculares/genética , Músculo Esquelético/citologia , Músculo Esquelético/fisiologia , Miocárdio/citologia , Miocárdio/metabolismo , Fenilefrina/farmacologia , Transdução de Sinais/fisiologia , Estresse Mecânico , Vasoconstritores/farmacologia , Função VentricularRESUMO
We previously demonstrated that integrin-dependent adhesion activates STAT5A, a well known target of IL-3-mediated signaling. Here, we show that in endothelial cells the active beta1 integrin constitutively associates with the unphosphorylated IL-3 receptor (IL-3R) beta common subunit. This association is not sufficient for activating downstream signals. Indeed, only upon fibronectin adhesion is Janus Kinase 2 (JAK2) recruited to the beta1 integrin-IL-3R complex and triggers IL-3R beta common phosphorylation, leading to the formation of docking sites for activated STAT5A. These events are IL-3 independent but require the integrity of the IL-3R beta common. IL-3 treatment increases JAK2 activation and STAT5A and STAT5B tyrosine and serine phosphorylation and leads to cell cycle progression in adherent cells. Expression of an inactive STAT5A inhibits cell cycle progression upon IL-3 treatment, identifying integrin-dependent STAT5A activation as a priming event for IL-3-mediated S phase entry. Consistently, overexpression of a constitutive active STAT5A leads to anchorage-independent cell cycle progression. Therefore, these data provide strong evidence that integrin-dependent STAT5A activation controls IL-3-mediated proliferation.
Assuntos
Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Células Endoteliais/metabolismo , Matriz Extracelular/metabolismo , Integrina beta1/metabolismo , Proteínas do Leite/metabolismo , Receptores de Interleucina-3/metabolismo , Transativadores/metabolismo , Adesão Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Células Cultivadas , Proteínas de Ligação a DNA/genética , Fibronectinas/metabolismo , Humanos , Interleucina-3/metabolismo , Interleucina-3/farmacologia , Janus Quinase 2 , Proteínas do Leite/genética , Fosforilação/efeitos dos fármacos , Subunidades Proteicas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fase S/fisiologia , Fator de Transcrição STAT5 , Transdução de Sinais/fisiologia , Transativadores/genética , Ativação Transcricional/fisiologia , Proteínas Supressoras de TumorRESUMO
BACKGROUND: Unloading/disuse induces skeletal muscle atrophy in bedridden patients and aged people, who cannot prevent it by means of exercise. Because interventions against known atrophy initiators, such as oxidative stress and neuronal NO synthase (nNOS) redistribution, are only partially effective, we investigated the involvement of melusin, a muscle-specific integrin-associated protein and a recognized regulator of protein kinases and mechanotransduction in cardiomyocytes. METHODS: Muscle atrophy was induced in the rat soleus by tail suspension and in the human vastus lateralis by bed rest. Melusin expression was investigated at the protein and transcript level and after treatment of tail-suspended rats with atrophy initiator inhibitors. Myofiber size, sarcolemmal nNOS activity, FoxO3 myonuclear localization, and myofiber carbonylation of the unloaded rat soleus were studied after in vivo melusin replacement by cDNA electroporation, and muscle force, myofiber size, and atrogene expression after adeno-associated virus infection. In vivo interference of exogenous melusin with dominant-negative kinases and other atrophy attenuators (Grp94 cDNA; 7-nitroindazole) on size of unloaded rat myofibers was also explored. RESULTS: Unloading/disuse reduced muscle melusin protein levels to about 50%, already after 6 h in the tail-suspended rat (P < 0.001), and to about 35% after 8 day bed rest in humans (P < 0.05). In the unloaded rat, melusin loss occurred despite of the maintenance of ß1D integrin levels and was not abolished by treatments inhibiting mitochondrial oxidative stress, or nNOS activity and redistribution. Expression of exogenous melusin by cDNA transfection attenuated atrophy of 7 day unloaded rat myofibers (-31%), compared with controls (-48%, P = 0.001), without hampering the decrease in sarcolemmal nNOS activity and the increase in myonuclear FoxO3 and carbonylated myofibers. Infection with melusin-expressing adeno-associated virus ameliorated contractile properties of 7 day unloaded muscles (P ≤ 0.05) and relieved myofiber atrophy (-33%) by reducing Atrogin-1 and MurF-1 transcripts (P ≤ 0.002), despite of a two-fold increase in FoxO3 protein levels (P = 0.03). Atrophy attenuation by exogenous melusin did not result from rescue of Akt, ERK, or focal adhesion kinase activity, because it persisted after co-transfection with dominant-negative kinase forms (P < 0.01). Conversely, melusin cDNA transfection, combined with 7-nitroindazole treatment or with cDNA transfection of the nNOS-interacting chaperone Grp94, abolished 7 day unloaded myofiber atrophy. CONCLUSIONS: Disuse/unloading-induced loss of melusin is an early event in muscle atrophy which occurs independently from mitochondrial oxidative stress, nNOS redistribution, and FoxO3 activation. Only preservation of melusin levels and sarcolemmal nNOS localization fully prevented muscle mass loss, demonstrating that both of them act as independent, but complementary, master switches of muscle disuse atrophy.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteína Forkhead Box O3/metabolismo , Elevação dos Membros Posteriores/fisiologia , Proteínas Musculares/metabolismo , Atrofia Muscular/genética , Óxido Nítrico Sintase Tipo I/metabolismo , Animais , Feminino , Humanos , Ratos , Ratos Wistar , TransfecçãoRESUMO
The early gene early growth response (Egr-1), a broadly expressed member of the zing-finger family of transcription factors, is induced in many cell types by a variety of growth and differentiation stimuli, including epidermal growth factor (EGF). Here we demonstrate that Egr-1 expression is mainly regulated by integrin-mediated adhesion. Integrin-dependent adhesion plays a dual role in Egr-1 regulation, either being sufficient "per se" to induce Egr-1, or required for EGF-dependent expression of Egr-1, which occurs only in adherent cells and not in cells in suspension. To dissect the molecular basis of integrin-dependent Egr-1 regulation, we show by FLIM-based FRET that in living cells beta1-integrin associates with the EGF receptor (EGFR) and that EGF further increases the extent complex formation. Interestingly, Egr-1 induction depends on integrin-dependent PI3K/Akt activation, as indicated by the decrease in Egr-1 levels in presence of the pharmacological inhibitor LY294002, the kinase-defective Akt mutant and Akt1/2 shRNAs. Indeed, upon adhesion activated Akt translocates into the nucleus and phosphorylates FoxO1, a Forkhead transcription factors. Consistently, FoxO1silencing results in Egr-1-increased levels, indicating that FoxO1 behaves as a negative regulator of Egr-1 expression. These data demonstrate that integrin/EGFR cross-talk is required for expression of Egr-1 through a novel regulatory cascade involving the activation of the PI3K/Akt/Forkhead pathway.
Assuntos
Proteína 1 de Resposta de Crescimento Precoce/genética , Receptores ErbB/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Integrina beta1/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/enzimologia , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Proteína Forkhead Box O1 , Humanos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Receptor Cross-Talk/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
BACKGROUND: Melusin is a muscle specific signaling protein, required for compensatory hypertrophy response in pressure-overloaded heart. The role of Melusin in heart function has been established both by loss and gain of function experiments in murine models. With the aim of verifying the hypothesis of a potential role of the Melusin encoding gene, ITGB1BP2, in the modification of the clinical phenotype of human cardiomyopathies, we screened the ITGB1BP2 gene looking for genetic variations possibly associated to the pathological phenotype in three selected groups of patients affected by hypertension and dilated or hypertrophic cardiomyopathy METHODS: We analyzed ITGB1BP2 by direct sequencing of the 11 coding exons and intron flanking sequences in 928 subjects, including 656 hypertensive or cardiopathic patients and 272 healthy individuals. RESULTS: Only three nucleotide variations were found in patients of three distinct families: a C>T missense substitution at position 37 of exon 1 causing an amino acid change from His-13 to Tyr in the protein primary sequence, a duplication (IVS6+12_18dupTTTTGAG) near the 5'donor splice site of intron 6, and a silent 843C>T substitution in exon 11. CONCLUSIONS: The three variations of the ITGB1BP2 gene have been detected in families of patients affected either by hypertension or primary hypertrophic cardiomyopathy; however, a clear genotype/phenotype correlation was not evident. Preliminary functional results and bioinformatic analysis seem to exclude a role for IVS6+12_18dupTTTTGAG and 843C>T in affecting splicing mechanism.Our analysis revealed an extremely low number of variations in the ITGB1BP2 gene in nearly 1000 hypertensive/cardiopathic and healthy individuals, thus suggesting a high degree of conservation of the melusin gene within the populations analyzed.
Assuntos
Cardiomiopatia Dilatada/genética , Cardiomiopatia Hipertrófica/genética , Proteínas do Citoesqueleto/genética , Variação Genética , Hipertensão/genética , Proteínas Musculares/genética , Idoso , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Análise de Sequência de DNARESUMO
Using two-hybrid screening, we isolated the integrin cytoplasmic domain-associated protein (ICAP-1), an interactor for the COOH terminal region of the beta1A integrin cytoplasmic domain. To investigate the role of ICAP-1 in integrin-mediated adhesive function, we expressed the full-length molecule in NIH3T3 cells. ICAP-1 expression strongly prevents NIH3T3 cell spreading on extracellular matrix. This inhibition is transient and can be counteracted by coexpression of a constitutively activated mutant of Cdc42, suggesting that ICAP-1 acts upstream of this GTPase. In addition, we found that ICAP-1 binds both to Cdc42 and Rac1 in vitro, and its expression markedly inhibits activation of these GTPases during integrin-mediated cell adhesion to fibronectin as detected by PAK binding assay. In the attempt to define the molecular mechanism of this inhibition, we show that ICAP-1 reduces both the intrinsic and the exchange factor-induced dissociation of GDP from Cdc42; moreover, purified ICAP-1 displaces this GTPase from cellular membranes. Together, these data show for the first time that ICAP-1 regulates Rho family GTPases during integrin-mediated cell matrix adhesion, acting as guanine dissociation inhibitor.
Assuntos
Proteínas de Transporte/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , Proteínas rho de Ligação ao GTP/metabolismo , Células 3T3 , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Células COS , Divisão Celular , Tamanho Celular , Guanosina Difosfato/metabolismo , Integrinas/metabolismo , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Ratos , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Especificidade por Substrato , Fatores de Tempo , Técnicas do Sistema de Duplo-Híbrido , Proteína cdc42 de Ligação ao GTP/antagonistas & inibidores , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Cell matrix adhesion is required for cell proliferation and survival. Here we report that mutation by gene targeting of the cytoplasmic tail of beta1 integrin leads to defective proliferation and survival both in vivo and in vitro. Primary murine embryonic fibroblasts (MEFs) derived from mutant homozygotes display defective cell cycle coupled to impaired activation of the FAK-PI3K-Akt and Rac-JNK signaling pathways. Expression in homozygous MEFs of a constitutively active form of Rac is able to rescue proliferation, survival, and JNK activation. Moreover, although showing normal Erk phosphorylation, mutant cells fail to display Erk nuclear translocation upon fibronectin adhesion. However, expression of the constitutively activated form of Rac restores Erk nuclear localization, suggesting that adhesion-dependent Rac activation is necessary to integrate signals directed to promote MAPK activity. Altogether, our data provide the evidence for an epistatic interaction between the beta1 integrin cytoplasmic domain and Rac, and indicate that this anchorage-dependent signaling pathway is crucial for cell growth control.
Assuntos
Integrina beta1/fisiologia , Proteínas rac de Ligação ao GTP/fisiologia , Alelos , Animais , Adesão Celular , Ciclo Celular , Divisão Celular , Sobrevivência Celular , Morte Fetal , Fibroblastos , Marcação de Genes , Homozigoto , Integrina beta1/genética , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Isoformas de Proteínas , Estrutura Terciária de ProteínaRESUMO
Imatinib currently represents the standard treatment in the early chronic phase of chronic myelogenous leukemia (CML), thanks to the high percentage of cytogenetic complete remission achieved, but it is yet unclear to what extent it can eradicate leukemia. Therefore, different vaccination strategies have been suggested, mainly based on the exploitment of the junctional peptides spanning the fusion region of the Bcr/Abl proteins. To identify new potential immunologic targets, 63 Philadelphia chromosome-positive patients and 6 BCR/ABL-positive cell lines were tested in nested reverse transcriptase PCR to detect the presence of BCR/ABL transcripts arising from the alternative splicing of the main BCR/ABL transcripts. We could detect BCR/ABL transcripts with junctions between BCR exon 1, 13, or 14 and ABL exon 4 in approximately 80% of patients and 84% of cell lines, beside the main fusion transcripts. Translation products of these transcripts were characterized at their COOH terminus by a large amino acid portion derived from the out of frame (OOF) reading of ABL gene. These proteins were detected in BCR/ABL-positive cell lines by immunoprecipitation and immunohistochemistry. Finally, we determined whether OOF-specific CD8+ T cells could be found in the peripheral blood of CML patients and whether they could acquire effector function following in vitro sensitization with OOF-derived peptides predicted to bind to human leucocyte antigen (HLA)-A2 and HLA-A3 molecules. We detected the presence of OOF-specific CD8+ T cells in four of four patients studied, and in one case, these T cells exhibited specific cytotoxic activity against both peptide-pulsed targets and autologous primary CML cells.
Assuntos
Proteínas de Fusão bcr-abl/imunologia , Imunoterapia/métodos , Leucemia Mielogênica Crônica BCR-ABL Positiva/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Éxons , Proteínas de Fusão bcr-abl/genética , Antígeno HLA-A2/imunologia , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Isoformas de Proteínas , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T Citotóxicos/imunologiaRESUMO
Melusin is a mammalian muscle specific CHORD containing protein capable of activating signal transduction pathways leading to cardiomyocytes hypertrophy in response to mechanical stress. To define melusin function we searched for molecular partners possibly involved in melusin dependent signal transduction. Here we show that melusin and heat shock proteins are co-regulated. Moreover, melusin directly binds to Hsp90, a ubiquitous chaperone involved in regulating several signaling pathways. In addition, melusin interacts with Sgt1, an Hsp90 binding molecule. Melusin does not behave as an Hsp90 substrate but rather as a chaperone capable to protect citrate synthase from heat induced aggregation. These results describe melusin as a new component of the Hsp90 chaperone machinery.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Musculares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Ciclo Celular/genética , Proteínas do Citoesqueleto/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP90/genética , Imunoprecipitação , Camundongos , Chaperonas Moleculares/genética , Proteínas Musculares/genética , Estrutura Terciária de ProteínaRESUMO
Diabetes mellitus is a main risk factor for vascular diseases. Vascular injury induced by diabetes mellitus is characterized by endothelial dysfunction attributable to an increased oxidative stress. So far, the molecular mechanisms involved in the vasculotoxic effects of diabetes are only partially known. We examined the effect of diabetes mellitus on oxidative stress and Rac-1 activation, a small G-protein involved in the activation of NADPH oxidase. Our results show that oxidative stress in vessels of different murine models of diabetes mellitus and in endothelial cells treated with high glucose is associated with an increased Rac-1/PAK binding and Rac-1 translocation from cytosol to plasma membrane, thus demonstrating an enhanced Rac-1 activity. More important, selective Rac-1 inhibition by an adenoviral vector carrying a dominant negative mutant of Rac-1 protected from oxidative stress and vascular dysfunction induced by diabetes mellitus. Our study demonstrates that Rac-1 plays a crucial role in diabetes-induced vascular injury, and it could be a target of novel therapeutic approaches to reduce vascular risk in diabetes mellitus.
Assuntos
Angiopatias Diabéticas/prevenção & controle , Neuropeptídeos/antagonistas & inibidores , Proteínas rac de Ligação ao GTP/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Animais , Células Cultivadas , Endotélio Vascular/fisiologia , Glucose/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , NADPH Oxidases/metabolismo , Neuropeptídeos/fisiologia , Estresse Oxidativo , Proteína Quinase C/fisiologia , Proteína Quinase C beta , Proteínas rac de Ligação ao GTP/fisiologia , Proteínas rac1 de Ligação ao GTP/fisiologiaRESUMO
To investigate the mechanisms through which p130Cas adaptor protein is linked to tumorigenesis, we generated mouse mammary tumor virus (MMTV)-p130Cas mice overexpressing p130Cas in the mammary gland. MMTVp130Cas transgenic mice are characterized by extensive mammary epithelial hyperplasia during development and pregnancy and by delayed involution at the end of lactation. These phenotypes are associated with activation of Src kinase, extracellular signal-regulated kinase 1/2, mitogen-activated protein kinase, and Akt pathways, leading to an increased rate of proliferation and a decreased apoptosis. A double-transgenic line derived from crossing MMTV-p130Cas with MMTV-HER2-Neu mice expressing the activated form of the HER2-Neu oncogene develops multifocal mammary tumors with a significantly shorter latency than the HER2-Neu parental strain alone. Mammary epithelial cells isolated from tumors of double-transgenic mice display increased tyrosine phosphorylation, c-Src, and Akt activation compared with cells derived from HER2-Neu tumors. In addition, p130Cas down-regulation by RNA interference increases apoptosis in HER2-Neu-expressing cells, indicating that p130Cas regulates cell survival. Consistently with the double-transgenic mice model, p130Cas is overexpressed in a significant subset of human breast cancers and high levels of p130Cas in association with HER2 expression correlate with elevated proliferation. These findings provide evidences for a role of p130Cas as a positive regulator of both proliferation and survival in normal and transformed mammary epithelial cells. Its overexpression contributes to HER2-Neu-induced breast tumorigenesis, thus identifying this protein as a putative target for clinical therapy.