RESUMO
SARS-CoV-2 infections are rapidly spreading around the globe. The rapid development of therapies is of major importance. However, our lack of understanding of the molecular processes and host cell signaling events underlying SARS-CoV-2 infection hinders therapy development. We use a SARS-CoV-2 infection system in permissible human cells to study signaling changes by phosphoproteomics. We identify viral protein phosphorylation and define phosphorylation-driven host cell signaling changes upon infection. Growth factor receptor (GFR) signaling and downstream pathways are activated. Drug-protein network analyses revealed GFR signaling as key pathways targetable by approved drugs. The inhibition of GFR downstream signaling by five compounds prevents SARS-CoV-2 replication in cells, assessed by cytopathic effect, viral dsRNA production, and viral RNA release into the supernatant. This study describes host cell signaling events upon SARS-CoV-2 infection and reveals GFR signaling as a central pathway essential for SARS-CoV-2 replication. It provides novel strategies for COVID-19 treatment.
Assuntos
Antivirais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/genética , Fosfatidilinositol 3-Quinase/genética , Receptores de Fatores de Crescimento/genética , Proteínas Virais/genética , Corticosteroides/uso terapêutico , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Betacoronavirus/imunologia , Betacoronavirus/patogenicidade , Células CACO-2 , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Humanos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Receptores de Fatores de Crescimento/antagonistas & inibidores , Receptores de Fatores de Crescimento/metabolismo , SARS-CoV-2 , Transdução de Sinais , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
All multicellular animals express receptors for growth factors (GFs) and extracellular matrix (ECM) molecules. Integrin-type ECM receptors anchor cells to their surroundings and concomitantly activate intracellular signal transduction pathways. The same signaling mechanisms are regulated by GF receptors (GFRs). Recently, intensive research efforts have revealed novel mechanisms describing how the two receptor systems collaborate at many different levels. Integrins can directly bind to GFs and promote their activation. Adhesion receptors also organize signaling platforms and assist GFRs or even activate them via ligand-independent mechanisms. Furthermore, integrins can orchestrate endocytosis and recycling of GFRs. Here, we review the present knowledge about the interplay between integrins and GFRs and discuss recent ideas of how this collaboration may explain some previous controversies in integrin research.
Assuntos
Endocitose/fisiologia , Integrinas/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Transdução de Sinais/fisiologia , Proteínas Angiogênicas/metabolismo , Animais , Membrana Celular/metabolismo , Matriz Extracelular/metabolismo , Humanos , Integrinas/química , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Modelos Moleculares , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Receptores de Fatores de Crescimento/químicaRESUMO
Several different signaling pathways that regulate cell proliferation and differentiation are initiated by binding of ligands to cell-surface and membrane-bound enzyme-linked receptors, such as receptor tyrosine kinases and serine-threonine kinases. They prompt phosphorylation of tyrosine and serine-threonine residues and initiate downstream signaling pathways and priming of intracellular molecules that convey the signal in the cytoplasm and nucleus, with transcriptional activation of specific genes enriching cell growth and survival-related cascades. These cell processes are rhythmically driven by molecular clockworks endowed in every cell type and when deregulated play a crucial role in cancer onset and progression. Growth factors and their matching receptor-dependent signaling are frequently overexpressed and/or dysregulated in many cancer types. In this review we focus on the interplay between biological clocks and Growth Factor Receptor-dependent signaling in the context of carcinogenesis.
Assuntos
Carcinogênese , Transdução de Sinais , Humanos , Carcinogênese/metabolismo , Carcinogênese/genética , Carcinogênese/patologia , Animais , Receptores de Fatores de Crescimento/metabolismo , Ritmo Circadiano/genética , Ritmo Circadiano/fisiologia , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/genéticaRESUMO
Endosomal trafficking of receptors and associated proteins plays a critical role in signal processing. Until recently, it was thought that trafficking was shut down during cell division. Thus, remarkably, the regulation of trafficking during division remains poorly characterized. Here we delineate the role of mitotic kinases in receptor trafficking during asymmetric division. Targeted perturbations reveal that Cyclin-dependent Kinase 1 (CDK1) and Aurora Kinase promote storage of Fibroblast Growth Factor Receptors (FGFRs) by suppressing endosomal degradation and recycling pathways. As cells progress through metaphase, loss of CDK1 activity permits differential degradation and targeted recycling of stored receptors, leading to asymmetric induction. Mitotic receptor storage, as delineated in this study, may facilitate rapid reestablishment of signaling competence in nascent daughter cells. However, mutations that limit or enhance the release of stored signaling components could alter daughter cell fate or behavior thereby promoting oncogenesis.
Assuntos
Aurora Quinases/fisiologia , Proteína Quinase CDC2/fisiologia , Mitose/fisiologia , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Animais , Animais Geneticamente Modificados , Aurora Quinases/genética , Proteína Quinase CDC2/genética , Proteínas de Ciclo Celular/metabolismo , Ciona intestinalis/embriologia , Ciona intestinalis/genética , Embrião não Mamífero , Mitose/genética , Transporte Proteico/genética , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento/genética , Receptores de Fatores de Crescimento/metabolismo , Transdução de Sinais/genética , Distribuição Tecidual/genéticaRESUMO
Many small molecule kinase inhibitors (SMKIs), used predominantly in cancer therapy, have been implicated in serious clinical cardiac adverse events, which means that traditional preclinical drug development assays were not sufficient for identifying these cardiac liabilities. To improve clinical cardiac safety predictions, the effects of SMKIs targeting many different signaling pathways were studied using human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) in combined assays designed for the detection of both electrophysiological (proarrhythmic) and non-electrophysiological (non-proarrhythmic) drug-induced cardiotoxicity. Several microplate-based assays were used to quantitate cell death, apoptosis, mitochondrial damage, energy depletion, and oxidative stress as mechanism-based non-electrophysiological cardiomyocyte toxicities. Microelectrode arrays (MEA) were used to quantitate in vitro arrhythmic events (iAEs), field potential duration (FPD) prolongation, and spike amplitude suppression (SAS) as electrophysiological effects. To enhance the clinical relevance, SMKI-induced cardiotoxicities were compared by converting drug concentrations into multiples of reported clinical maximum therapeutic plasma concentration, "FoldCmax", for each assay. The results support the conclusion that the combination of the hPSC-CM based electrophysiological and non-electrophysiological assays have significantly more predictive value than either assay alone and significantly more than the current FDA-recommended hERG assay. In addition, the combination of these assays provided mechanistic information relevant to cardiomyocyte toxicities, thus providing valuable information on potential drug-induced cardiotoxicities early in drug development prior to animal and clinical testing. We believe that this early information will be helpful to guide the development of safer and more cost-effective drugs.
Assuntos
Miócitos Cardíacos/efeitos dos fármacos , Células-Tronco Pluripotentes/fisiologia , Inibidores de Proteínas Quinases/farmacologia , Diferenciação Celular , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Inibidores de Proteínas Quinases/química , Receptores de Fatores de Crescimento/genética , Receptores de Fatores de Crescimento/metabolismoRESUMO
BACKGROUND: Angiogenesis is primarily attributed to the excessive proliferation and migration of endothelial cells. Targeting the vascular endothelial growth factor (VEGF) is therefore significant in anti-angiogenic therapy. Although these treatments have not reached clinical expectations, the upregulation of alternative angiogenic pathways (endoglin/Smad1) may play a critical role in drug (VEGF-neutralizing agents) resistance. Enhanced endoglin expression following a VEGF-neutralizing therapy (semaxanib®) was noted in patients. Treatment with an endoglin-targeting antibody augmented VEGF expression in human umbilical vein endothelial cells (HUVECs). Therefore, approaches that inhibit both the androgen and VEGF pathways enhance the HUVECs cytotoxicity and reverse semaxanib resistance. The purpose of this study was to find natural-occurring compounds that inhibited the endoglin-targeting pathway. METHODS: Curcuminoids targeting endoglin were recognized from two thousand compounds in the Traditional Chinese Medicine Database@Taiwan (TCM Database@Taiwan) using Discovery Studio 4.5. RESULTS: Our results, obtained using cytotoxicity, migration/invasion, and flow cytometry assays, showed that curcumin (Cur) and demethoxycurcumin (DMC) reduced angiogenesis. In addition, Cur and DMC downregulated endoglin/pSmad1 phosphorylation. CONCLUSIONS: The study first showed that Cur and DMC demonstrated antiangiogenic activity via the inhibition of endoglin/Smad1 signaling. Synergistic effects of curcuminoids (i.e., curcumin and DMC) and semaxanib on HUVECs were found. This might be attributed to endoglin/pSmad1 downregulation in HUVECs. Combination treatment with curcuminoids and a semaxanib is therefore expected to reverse semaxanib resistance.
Assuntos
Curcumina , Fator A de Crescimento do Endotélio Vascular , Movimento Celular , Proliferação de Células , Curcumina/farmacologia , Diarileptanoides/farmacologia , Endoglina/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Neovascularização Patológica/metabolismo , Fosforilação , Receptores de Fatores de Crescimento/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Chronic kidney disease (CKD) is a slow-developing, progressive deterioration of renal function. The final common pathway in the pathophysiology of CKD involves glomerular sclerosis, tubular atrophy and interstitial fibrosis. Transforming growth factor-beta (TGF-ß) stimulates the differentiation of fibroblasts towards myofibroblasts and the production of extracellular matrix (ECM) molecules, and thereby interstitial fibrosis. It has been shown that endoglin (ENG, CD105), primarily expressed in endothelial cells and fibroblasts, can function as a co-receptor of TGF signaling. In several human organs, endoglin tends to be upregulated when chronic damage and fibrosis is present. We hypothesize that endoglin is upregulated in renal interstitial fibrosis and plays a role in the progression of CKD. We first measured renal endoglin expression in biopsy samples obtained from patients with different types of CKD, i.e., IgA nephropathy, focal segmental glomerulosclerosis (FSGS), diabetic nephropathy (DN) and patients with chronic allograft dysfunction (CAD). We showed that endoglin is upregulated in CAD patients (p < 0.001) and patients with DN (p < 0.05), compared to control kidneys. Furthermore, the amount of interstitial endoglin expression correlated with eGFR (p < 0.001) and the amount of interstitial fibrosis (p < 0.001), independent of the diagnosis of the biopsies. Finally, we investigated in vitro the effect of endoglin overexpression in TGF-ß stimulated human kidney fibroblasts. Overexpression of endoglin resulted in an enhanced ACTA2, CCN2 and SERPINE1 mRNA response (p < 0.05). It also increased the mRNA and protein upregulation of the ECM components collagen type I (COL1A1) and fibronectin (FN1) (p < 0.05). Our results suggest that endoglin is an important mediator in the final common pathway of CKD and could be used as a possible new therapeutic target to counteract the progression towards end-stage renal disease (ESRD).
Assuntos
Nefropatias Diabéticas , Endoglina , Falência Renal Crônica , Insuficiência Renal Crônica , Humanos , Nefropatias Diabéticas/metabolismo , Endoglina/genética , Endoglina/metabolismo , Células Endoteliais/metabolismo , Fibrose , Rim/metabolismo , Falência Renal Crônica/patologia , Receptores de Fatores de Crescimento/metabolismo , Insuficiência Renal Crônica/metabolismo , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Osteosarcoma is a primary malignant bone tumor arising from bone-forming mesenchymal cells in children and adolescents. Despite efforts to understand the biology of the disease and identify novel therapeutics, the survival of osteosarcoma patients remains dismal. We have concurrently profiled the copy number and gene expression of 226 osteosarcoma samples as part of the Strategic Partnering to Evaluate Cancer Signatures (SPECS) initiative. Our results demonstrate the heterogeneous landscape of osteosarcoma in younger populations by showing the presence of genome-wide copy number abnormalities occurring both recurrently among samples and in a high frequency. Insulin growth factor receptor 1 (IGF1R) is a receptor tyrosine kinase which binds IGF1 and IGF2 to activate downstream pathways involved in cell apoptosis and proliferation. We identify prevalent amplification of IGF1R corresponding with increased gene expression in patients with poor survival outcomes. Our results substantiate previously tenuously associated copy number abnormalities identified in smaller datasets (13q34+, 20p13+, 4q35-, 20q13.33-), and indicate the significance of high fibroblast growth factor receptor 2 (FGFR2) expression in distinguishing patients with poor prognosis. FGFR2 is involved in cellular proliferation processes such as division, growth and angiogenesis. In summary, our findings demonstrate the prognostic significance of several genes associated with osteosarcoma pathogenesis.
Assuntos
Neoplasias Ósseas , Osteossarcoma , Adolescente , Biomarcadores , Neoplasias Ósseas/diagnóstico , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Criança , DNA , Variações do Número de Cópias de DNA , Regulação Neoplásica da Expressão Gênica , Humanos , Insulina/metabolismo , Osteossarcoma/diagnóstico , Osteossarcoma/genética , Osteossarcoma/metabolismo , Prognóstico , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Receptores de Fatores de Crescimento/metabolismoRESUMO
The neurotrophin growth factors bind and activate two types of cell surface receptors: the tropomyosin receptor kinase (Trk) family and p75. TrkA, TrkB, and TrkC are bound preferentially by nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3 (NT3), respectively, to activate neuroprotective signals. The p75 receptors are activated by all neurotrophins, and paradoxically in neurodegenerative disease p75 is upregulated and mediates neurotoxic signals. To test neuroprotection strategies, we engineered NT3 to broadly activate Trk receptors (mutant D) or to reduce p75 binding (mutant RK). We also combined these features in a molecule that activates TrkA, TrkB, and TrkC but has reduced p75 binding (mutant DRK). In neurodegenerative disease mouse models in vivo, the DRK protein is a superior therapeutic agent compared with mutant D, mutant RK, and wild-type neurotrophins and protects a broader range of stressed neurons. This work rationalizes a therapeutic strategy based on the biology of each type of receptor, avoiding activation of p75 toxicity while broadly activating neuroprotection in stressed neuronal populations expressing different Trk receptors. SIGNIFICANCE STATEMENT: The neurotrophins nerve growth factor, brain-derived neurotrophic factor, and neurotrophin 3 each can activate a tropomyosin receptor kinase (Trk) A, TrkB, or TrkC receptor, respectively, and all can activate a p75 receptor. Trks and p75 mediate opposite signals. We report the engineering of a protein that activates all Trks, combined with low p75 binding, as an effective therapeutic agent in vivo.
Assuntos
Fatores de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuroproteção/fisiologia , Engenharia de Proteínas/métodos , Receptor trkA/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Animais , Axotomia/efeitos adversos , Neuropatias Diabéticas/tratamento farmacológico , Neuropatias Diabéticas/genética , Neuropatias Diabéticas/metabolismo , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Fatores de Crescimento Neural/administração & dosagem , Fatores de Crescimento Neural/genética , Proteínas do Tecido Nervoso/genética , Neuroproteção/efeitos dos fármacos , Nervo Óptico/efeitos dos fármacos , Nervo Óptico/metabolismo , Receptor trkA/genética , Receptores de Fatores de Crescimento/genéticaRESUMO
The toll-like receptor (TLR) family consists of vital receptors responsible for pattern recognition in innate immunity, making them the core proteins involved in pathogen detection and eliciting immune responses. The most studied member of this family, TLR4, has been the center of attention regarding its contributory role in many inflammatory diseases including sepsis shock and asthma. Notably, mounting pieces of evidence have proved that this receptor is aberrantly expressed on the tumor cells and the tumor microenvironment in a wide range of cancer types and it is highly associated with the initiation of tumorigenesis as well as tumor progression and drug resistance. Cancer therapy using TLR4 inhibitors has recently drawn scientists' attention, and the promising results of such studies may pave the way for more investigation in the foreseeable future. This review will introduce the key proteins of the TLR4 pathway and how they interact with major growth factors in the tumor microenvironment. Moreover, we will discuss the many aspects of tumor progression affected by the activation of this receptor and provide an overview of the recent therapeutic approaches using various TLR4 antagonists.
Assuntos
Neoplasias/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Antineoplásicos/uso terapêutico , Progressão da Doença , Humanos , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Neoplasias/patologia , Receptor Cross-Talk , Receptores de Fatores de Crescimento/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/antagonistas & inibidores , Microambiente TumoralRESUMO
BACKGROUND: Streptococcus pneumoniae meningitis is a destructive central nervous system (CNS) infection with acute and long-term neurological disorders. Previous studies suggest that p75NTR signaling influences cell survival, apoptosis, and proliferation in brain-injured conditions. However, the role of p75NTR signaling in regulating pneumococcal meningitis (PM)-induced neuroinflammation and altered neurogenesis remains largely to be elucidated. METHODS: p75NTR signaling activation in the pathological process of PM was assessed. During acute PM, a small-molecule p75NTR modulator LM11A-31 or vehicle was intranasally administered for 3 days prior to S. pneumoniae exposure. At 24 h post-infection, clinical severity, histopathology, astrocytes/microglia activation, neuronal apoptosis and necrosis, inflammation-related transcription factors and proinflammatory cytokines/mediators were evaluated. Additionally, p75NTR was knocked down by the adenovirus-mediated short-hairpin RNA (shRNA) to ascertain the role of p75NTR in PM. During long-term PM, the intranasal administration of LM11A-31 or vehicle was continued for 7 days after successfully establishing the PM model. Dynamic changes in inflammation and hippocampal neurogenesis were assessed. RESULTS: Our results revealed that both 24 h (acute) and 7, 14, 28 day (long-term) groups of infected rats showed increased p75NTR expression in the brain. During acute PM, modulation of p75NTR through pretreatment of PM model with LM11A-31 significantly alleviated S. pneumoniae-induced clinical severity, histopathological injury and the activation of astrocytes and microglia. LM11A-31 pretreatment also significantly ameliorated neuronal apoptosis and necrosis. Moreover, we found that blocking p75NTR with LM11A-31 decreased the expression of inflammation-related transcription factors (NF-κBp65, C/EBPß) and proinflammatory cytokines/mediators (IL-1ß, TNF-α, IL-6 and iNOS). Furthermore, p75NTR knockdown induced significant changes in histopathology and inflammation-related transcription factors expression. Importantly, long-term LM11A-31 treatment accelerated the resolution of PM-induced inflammation and significantly improved hippocampal neurogenesis. CONCLUSION: Our findings suggest that the p75NTR signaling plays an essential role in the pathogenesis of PM. Targeting p75NTR has beneficial effects on PM rats by alleviating neuroinflammation and promoting hippocampal neurogenesis. Thus, the p75NTR signaling may be a potential therapeutic target to improve the outcome of PM.
Assuntos
Hipocampo/patologia , Meningite Pneumocócica/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/fisiologia , Doenças Neuroinflamatórias/patologia , Receptores de Fatores de Crescimento/metabolismo , Animais , Modelos Animais de Doenças , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Isoleucina/análogos & derivados , Isoleucina/farmacologia , Morfolinas/farmacologia , Neurogênese/efeitos dos fármacos , Doenças Neuroinflamatórias/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Epithelioid sarcomas and rhabdoid tumors are rare, aggressive malignancies with poor prognosis. Both are characterized by INI1 alterations and deregulation of growth factor receptors albeit their interaction has not been elucidated. METHODS: In this study, we investigated the activity of a panel of epigenetic modulators and receptor tyrosine kinase inhibitors in vitro on respective cell lines as well as on primary patient-derived epithelioid sarcoma cells, and in vivo on xenografted mice. Focusing on histone deacetylase (HDAC) inhibitors, we studied the mechanism of action of this class of agents, its effect on growth factor receptor regulation, and changes in epithelial-to-mesenchymal transition by using cell- and RT-qPCR-based assays. RESULTS: Pan-HDAC inhibitor panobinostat exhibited potent anti-proliferative activity at low nanomolar concentrations in A204 rhabdoid tumor, and VAESBJ/GRU1 epithelioid sarcoma cell lines, strongly induced apoptosis, and resulted in significant tumor growth inhibition in VAESBJ xenografts. It differentially regulated EGFR, FGFR1 and FGFR2, leading to downregulation of EGFR in epithelioid sarcoma and to mesenchymal-to-epithelial transition whereas in rhabdoid tumor cells, EGFR was strongly upregulated and reinforced the mesenchymal phenotype. All three cell lines were rendered more susceptible towards combination with EGFF inhibitor erlotinib, further enhancing apoptosis. CONCLUSIONS: HDAC inhibitors exhibit significant anticancer activity due to their multifaceted actions on cytotoxicity, differentiation and drug sensitization. Our data suggest that the tailored, tissue-specific combination of HDAC inhibitors with therapeutics which target cellular salvage mechanisms might increase their therapeutic relevance.
Assuntos
Transição Epitelial-Mesenquimal/efeitos dos fármacos , Inibidores de Histona Desacetilases/uso terapêutico , Panobinostat/uso terapêutico , Receptores de Fatores de Crescimento/metabolismo , Tumor Rabdoide/tratamento farmacológico , Sarcoma/tratamento farmacológico , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Inibidores de Histona Desacetilases/farmacologia , Humanos , Camundongos , Camundongos Nus , Panobinostat/farmacologia , Tumor Rabdoide/patologia , Sarcoma/patologiaRESUMO
The tropomyosin-related kinase (Trk) family consists of three receptor tyrosine kinases (RTKs) called TrkA, TrkB, and TrkC. These RTKs are regulated by the neurotrophins, a class of secreted growth factors responsible for the development and function of neurons. The Trks share a high degree of homology and utilize overlapping signaling pathways, yet their signaling is associated with starkly different outcomes in certain cancers. For example, in neuroblastoma, TrkA expression and signaling correlates with a favorable prognosis, whereas TrkB is associated with poor prognoses. To begin to understand how activation of the different Trks can lead to such distinct cellular outcomes, we investigated differences in kinase activity and duration of autophosphorylation for the TrkA and TrkB tyrosine kinase domains (TKDs). We find that the TrkA TKD has a catalytic efficiency that is â¼2-fold higher than that of TrkB, and becomes autophosphorylated in vitro more rapidly than the TrkB TKD. Studies with mutated TKD variants suggest that a crystallographic dimer seen in many TrkA (but not TrkB) TKD crystal structures, which involves the kinase-insert domain, may contribute to this enhanced TrkA autophosphorylation. Consistent with previous studies showing that cellular context determines whether TrkB signaling is sustained (promoting differentiation) or transient (promoting proliferation), we also find that TrkB signaling can be made more transient in PC12 cells by suppressing levels of p75NTR. Our findings shed new light on potential differences between TrkA and TrkB signaling, and suggest that subtle differences in signaling dynamics can lead to substantial shifts in the cellular outcome.
Assuntos
Neuroblastoma/metabolismo , Receptor trkA/metabolismo , Receptor trkB/metabolismo , Transdução de Sinais/genética , Animais , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Domínio Catalítico , Diferenciação Celular/genética , Proliferação de Células/genética , Técnicas de Silenciamento de Genes , Cinética , Mutação , Fatores de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/farmacologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neuroblastoma/enzimologia , Neuroblastoma/genética , Células PC12 , Fosforilação , Domínios Proteicos , RNA Interferente Pequeno , Ratos , Receptor trkA/química , Receptor trkA/genética , Receptor trkB/química , Receptor trkB/genética , Receptores de Fatores de Crescimento/genética , Receptores de Fatores de Crescimento/metabolismo , Proteínas Recombinantes , Transdução de Sinais/efeitos dos fármacosRESUMO
Ovarian response of collared peccaries (Pecari tajacu), after hormonal stimulation with gonadotropin association (eCG/hCG), was accessed by both gene expression and follicular development. Thus, collared peccaries (n = 8) were treated with the dose used for sows (swine dose, SWD) or with dose adjusted for peccary's weight (allometric dose, ALD). The gene expression of receptors was evaluated for both gonadotropins (FSHR and LHCGR) and growth factors (proteins codified by TGFßR-1, BMPR1-A and BMPR2 genes) in antral follicles, cortex and corpora haemorrhagica (CH). Five days after gonadotropin injection, all females presented CH. The ovulation rate was similar (p > .05) between SWD (4.00 ± 1.17) and ALD (2.50 ± 0.43) group. The total number of follicles per animal and amounts of small (<3 mm), medium (3-5 mm) and large (>5 mm) follicles was similar among groups. However, SWD produced large follicles heavier than ALD group, as accessed by weight of follicular wall biopsies. Ovarian follicles expressed both gonadotropin and growth factor receptors at levels which are independent from gonadotropin dose. In conclusion, the two gonadotropin doses (SWD and ALD) can be used for ovarian stimulation of collared peccary. Additionally, FSH and growth factors (TGFßR-1, BMPR1-A and BMPR2) receptors are more expressed in the early follicle development, while LH receptor seems to be more important in the final of follicular growth.
Assuntos
Artiodáctilos/fisiologia , Gonadotropina Coriônica/farmacologia , Ovário/efeitos dos fármacos , Animais , Peso Corporal , Gonadotropina Coriônica/administração & dosagem , Feminino , Folículo Ovariano/efeitos dos fármacos , Ovulação/efeitos dos fármacos , Receptores da Gonadotropina/genética , Receptores da Gonadotropina/metabolismo , Receptores de Fatores de Crescimento/genética , Receptores de Fatores de Crescimento/metabolismoRESUMO
Glioblastoma is characterized by the extensive vascularization with poor prognosis. Targeting both tumor cell and angiogenesis may present an effective therapeutic strategy for glioblastoma. Monensin, a polyether ionophore antibiotic, has been recently recognized as promising anticancer drug candidate due to its potent and selective anti-tumor activities. However, little is known on the effects of monensin on tumor angiogenesis. In this work, we investigated the effects and underlying mechanisms of monensin on glioblastoma angiogenesis and growth. We show that monensin at nanomolar concentrations inhibits early stages of capillary network formation of glioblastoma endothelial cell. Monensin inhibited multiple endothelial cellular events, including migration, growth and survival, without affecting adhesion to Matrigel. We further demonstrate that monensin acts on endothelial cells via suppressing VEGFR- and EGFR-mediated signaling pathways. Monensin also inhibits proliferation and induces apoptosis in a panel of glioblastoma cells. However, monensin is more effective in targeting endothelial cells than tumor cells. Using glioblastoma growth xenograft mice model, we show that monensin at tolerable dose effectively inhibits glioblastoma growth. Of note, there is a significant decreased tumor vascularization from monensin-treated mice. Our work clearly demonstrates the anti-angiogenic activity of monensin and its ability in suppressing multiple tyrosine kinase receptor-mediated pathways. Our findings suggest that is a useful addition to the treatment armamentarium for glioblastoma.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Monensin/uso terapêutico , Neovascularização Patológica/tratamento farmacológico , Receptores de Fatores de Crescimento/metabolismo , Inibidores da Angiogênese/farmacologia , Animais , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Glioblastoma/irrigação sanguínea , Glioblastoma/metabolismo , Humanos , Camundongos Nus , Monensin/farmacologia , Neovascularização Patológica/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Despite commonly used for coronary artery bypass surgery, saphenous vein (SV) grafts have significantly lower patency rates in comparison to internal thoracic artery (ITA) grafts, which might be due to the structural characteristics of the vessel wall but also due to differences in oxidative stress adaptation and molecular signaling and regulation. This human post mortem study included a total of 150 human bypass grafts (75 SV grafts and 75 ITA grafts) obtained from 60 patients divided into five groups due to the time period of implantation: group 1: baseline group without grafting; group 2: 1 day; group 3: > 1 day-1 week; group 4: > 1 week-1 month; group 5: > 1 month-1 year. Pieces of 3 mm length were fixed with formaldehyde, dehydrated, wax embedded, cut into sections of 3 µm thickness, and histologically and immunohistochemically examined. Over the whole time period, we observed a lower neointima formation and a better preserved media in ITA grafts with a higher percentage of TNF-α, PDGFR-α, and VEGF-A in nearly all vessel wall layers, a higher amount of MMP-7, MMP-9, EGFR, and bFGF positive cells in SV grafts and a timely different peak not only between ITA and SV grafts but also within the various vessel wall layers of both graft types. Since most of the examined growth factors, growth factor receptors and cytokines are regulated by MAPKs, our results suggest an activation of different pathways in both vessel graft types immediately after bypass grafting.
Assuntos
Ponte de Artéria Coronária , Citocinas/análise , Peptídeos e Proteínas de Sinalização Intercelular/análise , Receptores de Fatores de Crescimento/análise , Veia Safena/metabolismo , Artérias Torácicas/metabolismo , Citocinas/metabolismo , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Receptores de Fatores de Crescimento/metabolismo , Veia Safena/cirurgia , Artérias Torácicas/cirurgia , Fatores de TempoRESUMO
Keloid is a benign skin tumor characterized by its cell hyperproliferative activity, invasion into normal skin, uncontrolled growth, overproduction and deposition of extracellular matrices and high recurrence rate after various therapies. Nintedanib is a receptor tyrosine kinase inhibitor targeting VEGF, PDGF, FGF, and TGF-ß receptors with proved efficacy in anti-angiogenesis and in treating various types of cancers. In this study, we investigated the effects of nintedanib on keloid fibroblasts in both in vitro and ex vivo models. Keloid fibroblasts were prepared from 54 keloid scar samples in active stages collected from 49 patients. We found that nintedanib (1-4 µM) dose-dependently suppressed cell proliferation, induced G0/G1 cell cycle arrest, and inhibited migration and invasion of keloid fibroblasts. The drug also significantly inhibited the gene and protein expression of collagen I (COL-1) and III (COL-3), fibronectin (FN), and connective growth factor (CTGF), as well as the gene expression of other pathological factors, such as alpha smooth muscle actin (α-SMA), plasminogen activator inhibitor-1 (PAI-1), FK506-binding protein 10 (FKBP10), and heat shock protein 47 (HSP47) in keloid fibroblasts. Furthermore, nintedanib treatment significantly suppressed the phosphorylation of p38, JNK, ERK, STAT3, and Smad, enhanced endocytosis of various growth factor receptors. Using an ex vivo tissue explant model, we showed that nintedanib significantly suppressed cell proliferation, migration, and collagen production. The drug also significantly disrupted microvessel structure ex vivo. In summary, our results demonstrate that nintedanib is likely to become a potential targeted drug for keloid systemic therapy.
Assuntos
Fibroblastos/efeitos dos fármacos , Indóis/farmacologia , Queloide/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Adolescente , Adulto , Idoso , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Criança , Colágeno/metabolismo , Relação Dose-Resposta a Droga , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Fosforilação/efeitos dos fármacos , Adulto JovemRESUMO
We recently showed that sodium nitroprusside (SNP), a NO donor, attenuated hypertension in spontaneously hypertensive rats (SHR). Since hypertension is associated with enhanced proliferation and hypertrophy of vascular smooth muscle cells (VSMC), the present study examines whether in vivo treatment of SHR with SNP could also inhibit the augmented proliferation of VSMC and explore the signaling mechanisms. Treatment of 8 week old SHR and Wistar Kyoto rats with SNP twice a week for 2 weeks inhibited the enhanced proliferation of VSMC from SHR, the enhanced expression of angiotensin II type 1 (AT1) receptor, and enhanced activation of c-Src and growth factor receptors and ERK1/2 signaling pathways. In addition, SNP also inhibited the overexpression of cell cycle proteins including cyclins D1, Cdk4, and phosphorylated pRB and restored the downregulated Cdk inhibitors p21Cip1 and p27Kip1 expression towards control levels. Furthermore, SNP-induced inhibition of enhanced levels of the AT1 receptor and enhanced proliferation was reversed by L-NAME, an inhibitor of nitric oxide synthase. These results suggest that the SNP-induced antiproliferative effect may be mediated through the inhibition of enhanced expression of the AT1 receptor, cell cycle proteins and activation of c-Src, growth factor receptors, and MAP kinase signaling.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Nitroprussiato/farmacologia , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Células Cultivadas , Hipertensão/tratamento farmacológico , Hipertensão/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Espécies Reativas de Oxigênio/metabolismoRESUMO
To investigate the mechanism dexmedetomidine in relieving the neurotoxicity of a developing brain induced by sevoflurane. Sprague-Dawley rats, 6 days old, were randomly divided into three groups. Rats in the control group were inhaled with air after injection of normal saline; rats in the sevoflurane group were injected with normal saline and inhaled with 3% sevoflurane for 2 h in three consecutive day; rats in the dexmedetomidine group were inhaled with 3% sevoflurane after intraperitoneal injection of dexmedetomidine 25 µg/kg. WB results showed that mBDNF, pTrkB/TrkB, and CREB were significantly decreased in the hippocampus of the sevoflurane group, which are significantly upregulated in the dexmedetomidine group. In the sevoflurane group, proBDNF, P75NRT, and RhoA were significantly increased, which were significantly lower than those in the dexmedetomidine group than those in the sevoflurane group. The expression BDNF was downregulated in the sevoflurane group, while the proBDNF was upregulated in the sevoflurane group. In the Morris water maze test, the escape latency of the sevoflurane group was significantly prolonged. In sevoflurane groups, the number of crossing platform was significantly reduced, the synaptic protein decreased significantly, and this effect was reversed in rats of the dexmedetomidine group. Dexmedetomidine could reduce synaptic plasticity decline in developing rats induced by sevoflurane, through downregulating the proBDNF-p75NTR-RhoA pathway and upregulating BDNF-TrkB-CREB.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Dexmedetomidina/farmacologia , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptor trkB/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Sevoflurano/toxicidade , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Feminino , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Fármacos Neuroprotetores/farmacologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacosRESUMO
The carotid body may undergo plasticity changes during development/ageing and in response to environmental (hypoxia and hyperoxia), metabolic, and inflammatory stimuli. The different cell types of the carotid body express a wide series of growth factors and corresponding receptors, which play a role in the modulation of carotid body function and plasticity. In particular, type I cells express nerve growth factor, brain-derived neurotrophic factor, neurotrophin 3, glial cell line-derived neurotrophic factor, ciliary neurotrophic factor, insulin-like-growth factor-I and -II, basic fibroblast growth factor, epidermal growth factor, transforming growth factor-α and -ß, interleukin-1ß and -6, tumor necrosis factor-α, vascular endothelial growth factor, and endothelin-1. Many specific growth factor receptors have been identified in type I cells, indicating autocrine/paracrine effects. Type II cells may also produce growth factors and express corresponding receptors. Future research will have to consider growth factors in further experimental models of cardiovascular, metabolic, and inflammatory diseases and in human (normal and pathologic) samples. From a methodological point of view, microarray and/or proteomic approaches would permit contemporary analyses of large groups of growth factors. The eventual identification of physical interactions between receptors of different growth factors and/or neuromodulators could also add insights regarding functional interactions between different trophic mechanisms.