RESUMO
Many pathological conditions lead to altered intracellular pH (pHi) disrupting normal cellular functions. The chemical exchange saturation transfer (CEST) method, known as Amine and Amide Concentration Independent Detection (AACID), can produce image contrast that is predominantly dependent on tissue intracellular pHi. The AACID value is linearly related to the ratio of the 3.5 ppm amide CEST effect and the 2.75 ppm amine CEST effect in the physiological range. However, the amine CEST effect at 2 ppm is often more clearly defined in vivo, and may provide greater sensitivity to pH changes. The purpose of the current study was to compare AACID measurement precision utilizing the 2.0 and 2.75 ppm amine CEST effects. We hypothesized that the 2.0 ppm amine CEST resonance would produce measurements with greater sensitivity to pH changes. In the current study, we compare the range of the AACID values obtained in 24 mice with brain tumors and in normal tissue using the 2 ppm and 2.75 ppm amine resonances. All CEST data were acquired on a 9.4T MRI scanner. The AACID measurement range increased by 39% when using the 2 ppm amine resonance compared to the 2.75 ppm resonance, with decreased measurement variability across the brain. These data indicate that in vivo pH measurements made using AACID CEST can be enhanced by incorporating the 2 ppm amine resonance. This approach should be considered for pH measurements made over short intervals when no changes are expected in the concentration of metabolites that contribute to the 2 ppm amine resonance.
Assuntos
Amidas , Aminas , Animais , Encéfalo/diagnóstico por imagem , Concentração de Íons de Hidrogênio , Imageamento por Ressonância Magnética/métodos , CamundongosRESUMO
INTRODUCTION: Non-invasively distinguishing aggressive from non-aggressive brain tumors is an important clinical challenge. Intracellular pH (pHi) regulation is essential for normal cell function and is normally maintained within a narrow range. Cancer cells are characterized by a reversed intracellular to extracellular pH gradient, compared to healthy cells, that is maintained by several distinct mechanisms. Previous studies have demonstrated acute pH modulation in glioblastoma detectable by chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) after blocking individual pH regulatory mechanisms. The purpose of the current study was to simultaneously block five pH regulatory mechanisms while also providing glucose as an energy substrate. We hypothesized that this approach would increase the acute pH modulation effect allowing the identification of aggressive cancer. METHODS: Using a 9.4 T MRI scanner, CEST spectra were acquired sensitive to pHi using amine/amide concentration independent detection (AACID). Twelve mice were scanned approximately 11 ± 1 days after implanting 105 U87 human glioblastoma multiforme cells in the brain, before and after intraperitoneal injection of a combination of five drugs (quercetin, cariporide, dichloroacetate, acetazolamide, and pantoprazole) with and without glucose. RESULTS: Two hours after combination drug injection there was a significant 0.1 ± 0.03 increase in tumor AACID value corresponding to a 0.4 decrease in pHi. After injecting the drug combination with glucose the AACID value increased by 0.18 ± 0.03 corresponding to a 0.72 decrease in pHi. AACID values were also slightly increased in contralateral tissue. CONCLUSIONS: The combined drug treatment with glucose produced a large acute CEST MRI contrast indicating tumor acidification, which could be used to help localize brain cancer and monitor tumor response to chemotherapy.
Assuntos
Neoplasias Encefálicas/patologia , Encéfalo/patologia , Ácido Dicloroacético/farmacologia , Glioblastoma/patologia , Quercetina/farmacologia , Topiramato/farmacologia , Animais , Antioxidantes/farmacologia , Apoptose , Encéfalo/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Proliferação de Células , Feminino , Glioblastoma/tratamento farmacológico , Humanos , Concentração de Íons de Hidrogênio , Hipoglicemiantes/farmacologia , Camundongos , Camundongos Nus , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Brain-derived neurotrophic factor (BDNF) facilitates multiple aspects of neuronal differentiation and cellular physiology by activating the high-affinity receptor tyrosine kinase, TrkB. While it is known that both BDNF and TrkB modulate cellular processes involved in learning and memory, exactly how TrkB cross-talks and modulates signaling downstream of excitatory ionotropic receptors, such as the NMDA receptor (NMDAR), are not well understood. A model that we have investigated involves the signaling molecule RasGrf1, a guanine nucleotide exchange factor for both Ras and Rac. We previously identified RasGrf1 as a novel Trk binding partner that facilitates neurite outgrowth in response to both nerve growth factor (NGF) (Robinson et al. in J Biol Chem 280:225-235, 2005) and BDNF (Talebian et al. in J Mol Neurosci 49:38-51, 2013); however, RasGrf1 can also bind the NR2B subunit of the NMDAR (Krapivinsky et al. in Neuron 40:775-784, 2003) and stimulate long-term depression (LTD) (Li et al. in J Neurosci 26:1721-1729, 2006). We have addressed a model that TrkB facilitates learning and memory via two processes. First, TrkB uncouples RasGrf1 from NR2B and facilitates a decrease in NMDA signaling associated with LTD (p38-MAPK). Second, the recruitment of RasGrf1 to TrkB enhances neurite outgrowth and pERK activation and signaling associated with learning and memory. We demonstrate that NMDA recruits RasGrf1 to NR2B; however, co-stimulation with BDNF uncouples this association and recruits RasGrf1 to TrkB. In addition, activation of TrkB stimulates the tyrosine phosphorylation of RasGrf1 which increases neurite outgrowth (Talebian et al. in J Mol Neurosci 49:38-51, 2013), and the tyrosine phosphorylation of NR2B (Tyr1472) (Nakazawa et al. in J Biol Chem 276:693-699, 2001) which facilitates NMDAR cell surface retention (Zhang et al. in J Neurosci 28:415-24, 2008). Collectively, these data demonstrate that TrkB alters NMDA signaling by a dual mechanism that uncouples LTD and, in turn, stimulates neuronal growth and the signaling pathways associated with learning and memory.
Assuntos
Encéfalo/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Encéfalo/fisiologia , Células HEK293 , Humanos , Depressão Sináptica de Longo Prazo , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Ligação Proteica , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , ras-GRF1/metabolismoRESUMO
The response of tumor intracellular pH to a pharmacological challenge could help identify aggressive cancer. Chemical exchange saturation transfer (CEST) is an MRI contrast mechanism that is dependent on intracellular pH (pHi). pHi is important in the maintenance of normal cell function and is normally maintained within a narrow range by the activity of transporters located at the plasma membrane. In cancer, changes in pHi have been correlated with both cell proliferation and cell death. Quercetin is a bioflavonoid and monocarboxylate transporter (MCT) inhibitor. Since MCTs plays a significant role in maintaining pH balance in the tumor microenvironment, we hypothesized that systemically administered quercetin could selectively acidify brain tumors. The goals of the current study were to determine whether CEST MRI measurements sensitive to tumor pH could detect acidification after quercetin injection and to measure the magnitude of the pH change (ΔpH). Using a 9.4 T MRI, amine and amide concentration independent detection (AACID) CEST spectra were acquired in six mice approximately 15 ± 1 days after implanting 105 U87 human glioblastoma multiforme cells in the brain, before and after administration of quercetin (dose: 200 mg/kg) by intraperitoneal injection. Three additional mice were studied as controls and received only vehicle dimethyl sulfoxide (DMSO) injection. Repeated measures t-test was used to compare AACID changes in tumor and contralateral tissue regions of interest. Two hours after quercetin injection there was a significant increase in tumor AACID by 0.07 ± 0.03 corresponding to a 0.27 decrease in pHi, and no change in AACID in contralateral tissue. There was also a small average increase in AACID in tumors within the three mice injected with DMSO only. The use of the natural compound quercetin in combination with pH weighted MRI represents a unique approach to cancer detection that does not require injection of an imaging contrast agent.
Assuntos
Neoplasias Encefálicas/química , Glioblastoma/química , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Quercetina/farmacologia , Animais , Neoplasias Encefálicas/diagnóstico por imagem , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Glioblastoma/diagnóstico por imagem , Humanos , Concentração de Íons de Hidrogênio , Imageamento por Ressonância Magnética , CamundongosRESUMO
Glioblastoma is an aggressive brain cancer that is very difficult to treat. Clinically, it is important to be able to distinguish aggressive from non-aggressive brain tumors. Previous studies have shown that some drugs can induce a rapid change in intracellular pH that could help to identify aggressive cancer. The sodium proton exchanger (NHE1) plays a significant role in maintaining pH balance in the tumor microenvironment. Cariporide is a sodium proton exchange inhibitor that is well tolerated by humans in cardiac applications. We hypothesized that cariporide could selectively acidify brain tumors. The purpose of this study was to determine whether amine/amide concentration-independent detection (AACID) chemical exchange saturation transfer (CEST) MRI measurement of tumor pHi could detect acidification after cariporide injection. Using a 9.4T MRI scanner, CEST spectra were acquired in six mice approximately 14 days after implanting 105 U87 human glioblastoma multiforme cells in the brain, before and after administration of cariporide (dose: 6 mg/kg) by intraperitoneal injection. Three additional mice were studied as controls and received only vehicle injection (DMSO + PBS). Repeated measures t test was used to examine changes in tumor and contralateral tissue regions of interest. Two hours after cariporide injection, there was a significant 0.12 ± 0.03 increase in tumor AACID value corresponding to a 0.48 decrease in pHi and no change in AACID value in contralateral tissue. A small but significant increase of 0.04 ± 0.017 in tumor AACID value was also observed following vehicle injection. This study demonstrates that acute CEST MRI contrast changes, indicative of intracellular acidification, after administration of cariporide could help localize glioblastoma.
Assuntos
Acidose/patologia , Antiarrítmicos/toxicidade , Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Guanidinas/toxicidade , Sulfonas/toxicidade , Acidose/induzido quimicamente , Acidose/diagnóstico por imagem , Animais , Feminino , Concentração de Íons de Hidrogênio , Imageamento por Ressonância Magnética , Camundongos , Microambiente TumoralRESUMO
Intracellular pH (pHi) plays an important role in the maintenance of normal cell function, and is maintained within a narrow range by the activity of transporters located at the plasma membrane. Modulation of tumor pHi may influence proliferation, apoptosis, chemotherapy resistance, and thermosensitivity. Chemical exchange saturation transfer (CEST) is a novel MRI contrast mechanism that is dependent on cellular pH. Amine and amide concentration-independent detection (AACID) is a recently developed CEST contrast method that is intracellular pH (pHi) weighted. Dichloroacetate (DCA) can alter tumor pHi by inhibiting the enzyme pyruvate dehydrogenase kinase causing reduced lactate (increasing pHi), or by decreasing the expression of monocarboxylate transporters and vacuolar ATPase leading to reduced pHi. Since the net in vivo effect of DCA on pHi is difficult to predict, the purpose of this study was to quantify the magnitude of acute pHi change in glioblastoma after a single DCA injection using AACID CEST MRI. Using a 9.4T MRI scanner, CEST spectra were acquired in six mice approximately 14 days after implanting 105 U87 human glioblastoma multiforme (GBM) cells in the brain, before and after intravenous injection of DCA (dose: 200 mg/kg). Three additional mice received only phosphate buffered saline (PBS) injection and were studied as controls. Repeated measures t test was used to compare AACID changes in tumor and contralateral tissue regions of interest. One hour after DCA injection there was a significant increase in tumor AACID level by 0.04 ± 0.01 corresponding to a 0.16 decrease in pHi, and no change in AACID in contralateral tissue. Inspection of AACID maps following PBS injection showed no differences. The use of DCA to induce a tumor specific pH change detectable by AACID CEST MRI is consistent with previous studies that have shown similar effects for lonidamine and topiramate. This study demonstrates that a single dose of DCA can be used as a pharmacological challenge to induced rapid tumor intracellular acidification.
Assuntos
Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/metabolismo , Ácido Dicloroacético/administração & dosagem , Glioblastoma/diagnóstico por imagem , Glioblastoma/metabolismo , Imageamento por Ressonância Magnética/métodos , Animais , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Meios de Contraste , Ácido Dicloroacético/metabolismo , Concentração de Íons de Hidrogênio , CamundongosRESUMO
Proteins of the Src homology and collagen (Shc) family are typically involved in signal transduction events involving Ras/MAPK and PI3K/Akt pathways. In the nervous system, they function proximal to the neurotrophic factors that regulate cell survival, differentiation, and neuron-specific characteristics. The least characterized homolog, ShcD, is robustly expressed in the developing and mature nervous system, but its contributions to neural cell circuitry are largely uncharted. We now report that ShcD binds to active Ret, TrkA, and TrkB neurotrophic factor receptors predominantly via its phosphotyrosine-binding (PTB) domain. However, in contrast to the conventional Shc adaptors, ShcD suppresses distal phosphorylation of the Erk MAPK. Accordingly, genetic knock-out of mouse ShcD enhances Erk phosphorylation in the brain. In cultured cells, this capacity is tightly aligned to phosphorylation of ShcD CH1 region tyrosine motifs, which serve as docking platforms for signal transducers, such as Grb2. Erk suppression is relieved through independent mutagenesis of the PTB domain and the CH1 tyrosine residues, and successive substitution of these tyrosines breaks the interaction between ShcD and Grb2, thereby promoting TrkB-Grb2 association. Erk phosphorylation can also be restored in the presence of wild type ShcD through Grb2 overexpression. Conversely, mutation of the ShcD SH2 domain results in enhanced repression of Erk. Although the SH2 domain is a less common binding interface in Shc proteins, we demonstrate that it associates with the Ptpn11 (Shp2) phosphatase, which in turn regulates ShcD tyrosine phosphorylation. We therefore propose a model whereby ShcD competes with neurotrophic receptors for Grb2 binding and opposes activation of the MAPK cascade.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Glicoproteínas de Membrana/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-ret/metabolismo , Receptor trkA/metabolismo , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Motivos de Aminoácidos , Linhagem Celular , MAP Quinases Reguladas por Sinal Extracelular/genética , Proteína Adaptadora GRB2/genética , Proteína Adaptadora GRB2/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Fosforilação/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas c-ret/genética , Receptor trkA/genética , Receptor trkB , Proteínas Adaptadoras da Sinalização Shc/genéticaRESUMO
Reversal of the intracellular/extracellular pH gradient is a hallmark of malignant tumors and is an important consideration in evaluating tumor growth potential and the effectiveness of anticancer therapies. Glioblastoma multiforme (GBM) brain tumors have increased expression of the carbonic anhydrase (CA) isozymes CAII, CAIX and CAXII that contribute to the altered regulation of intracellular pH (pHi). The anti-epileptic drug topiramate (TPM) inhibits CA action and may acidify the tumor intracellular compartment. In-vivo detection of acute tumor acidification could aid in cancer diagnosis and monitoring treatment response. Chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) has been used to measure tissue pH. Using a recently developed CEST-MRI method called amine/amide concentration independent detection (AACID), we have previously shown intracellular acidification caused by single dose of lonidamine. The current study aims to evaluate the intracellular acidification induced by a single dose of the clinically approved drug TPM. Brain tumors were induced in NU/NU mice by injecting 105 U87 human glioblastoma multiforme cells into the right frontal lobe. Using a 9.4T MRI scanner AACID measurements were acquired, before and after administration of TPM (dose: 120 mg/kg, intraperitoneal), 15 ± 2 days after tumor cell implantation. TPM administration induced acute intracellular acidification (average ± SD: baseline AACID = 1.14 ± 0.05; post AACID = 1.19 ± 0.05, paired ttest p = 0.02) in implanted brain tumors. In contrast, contralateral tissue showed no change in AACID value. These results suggest that topiramate can rapidly induce a tumor specific physiological change detectable by AACID CEST. This pH challenge paradigm could be exploited to aid in tumor detection and monitoring treatment response.
Assuntos
Neoplasias Encefálicas/patologia , Anidrases Carbônicas/metabolismo , Frutose/análogos & derivados , Glioblastoma/patologia , Espaço Intracelular/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Animais , Neoplasias Encefálicas/diagnóstico por imagem , Linhagem Celular Tumoral , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Frutose/farmacologia , Glioblastoma/diagnóstico por imagem , Humanos , Concentração de Íons de Hidrogênio , Espaço Intracelular/metabolismo , Imageamento por Ressonância Magnética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Topiramato , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Macropinocytosis is a normal cellular process by which cells internalize extracellular fluids and nutrients from their environment and is one strategy that Ras-transformed pancreatic cancer cells use to increase uptake of amino acids to meet the needs of rapid growth. Paradoxically, in non-Ras transformed medulloblastoma brain tumors, we have shown that expression and activation of the receptor tyrosine kinase TrkA overactivates macropinocytosis, resulting in the catastrophic disintegration of the cell membrane and in tumor cell death. The molecular basis of this uncontrolled form of macropinocytosis has not been previously understood. Here, we demonstrate that the overactivation of macropinocytosis is caused by the simultaneous activation of two TrkA-mediated pathways: (i) inhibition of RhoB via phosphorylation at Ser(185) by casein kinase 1, which relieves actin stress fibers, and (ii) FRS2-scaffolded Src and H-Ras activation of RhoA, which stimulate actin reorganization and the formation of lamellipodia. Since catastrophic macropinocytosis results in brain tumor cell death, improved understanding of the mechanisms involved will facilitate future efforts to reprogram tumors, even those resistant to apoptosis, to die.
Assuntos
Caseína Quinase I/metabolismo , Neoplasias Cerebelares/metabolismo , Meduloblastoma/metabolismo , Pinocitose , Receptor trkA/metabolismo , Proteína rhoB de Ligação ao GTP/metabolismo , Actinas/metabolismo , Morte Celular , Linhagem Celular Tumoral , Humanos , Fosforilação , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Serina/metabolismo , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Alzheimer's disease (AD) is characterized by the deposition of Beta-Amyloid (Aß) peptides in the brain. Aß peptides are generated by cleavage of the Amyloid Precursor Protein (APP) by the ß - and γ - secretase enzymes. Although this process is tightly linked to the internalization of cell surface APP, the compartments responsible are not well defined. We have found that APP can be rapidly internalized from the cell surface to lysosomes, bypassing early and late endosomes. Here we show by confocal microscopy and electron microscopy that this pathway is mediated by macropinocytosis. APP internalization is enhanced by antibody binding/crosslinking of APP suggesting that APP may function as a receptor. Furthermore, a dominant negative mutant of Arf6 blocks direct transport of APP to lysosomes, but does not affect classical endocytosis to endosomes. Arf6 expression increases through the hippocampus with the development of Alzheimer's disease, being expressed mostly in the CA1 and CA2 regions in normal individuals but spreading through the CA3 and CA4 regions in individuals with pathologically diagnosed AD. Disruption of lysosomal transport of APP reduces both Aß40 and Aß42 production by more than 30 %. Our findings suggest that the lysosome is an important site for Aß production and that altering APP trafficking represents a viable strategy to reduce Aß production.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Peptídeos beta-Amiloides/biossíntese , Lisossomos/metabolismo , Pinocitose , Fator 1 de Ribosilação do ADP/metabolismo , Fator 6 de Ribosilação do ADP , Actinas/metabolismo , Oxirredutases do Álcool/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Compartimento Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Sobrevivência Celular , Reagentes de Ligações Cruzadas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dextranos/metabolismo , Endossomos/metabolismo , Endossomos/ultraestrutura , Técnicas de Silenciamento de Genes , Hipocampo/metabolismo , Humanos , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/ultraestrutura , Camundongos , Proteínas Mutantes/metabolismo , Transporte Proteico , RNA Interferente Pequeno/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Increased lactate production through glycolysis in aerobic conditions is a hallmark of cancer. Some anticancer drugs have been designed to exploit elevated glycolysis in cancer cells. For example, lonidamine (LND) inhibits lactate transport, leading to intracellular acidification in cancer cells. Chemical exchange saturation transfer (CEST) is a novel MRI contrast mechanism that is dependent on intracellular pH. Amine and amide concentration-independent detection (AACID) and apparent amide proton transfer (APT*) represent two recently developed CEST contrast parameters that are sensitive to pH. The goal of this study was to compare the sensitivity of AACID and APT* for the detection of tumor-selective acidification after LND injection. Using a 9.4-T MRI scanner, CEST data were acquired in mice approximately 14 days after the implantation of 10(5) U87 human glioblastoma multiforme (GBM) cells in the brain, before and after the administration of LND (dose, 50 or 100 mg/kg). Significant dose-dependent LND-induced changes in the measured CEST parameters were detected in brain regions spatially correlated with implanted tumors. Importantly, no changes were observed in T1- and T2-weighted images acquired before and after LND treatment. The AACID and APT* contrast measured before and after LND injection exhibited similar pH sensitivity. Interestingly, LND-induced contrast maps showed increased heterogeneity compared with pre-injection CEST maps. These results demonstrate that CEST contrast changes after the administration of LND could help to localize brain cancer and monitor tumor response to chemotherapy within 1 h of treatment. The LND CEST experiment uses an anticancer drug to induce a metabolic change detectable by endogenous MRI contrast, and therefore represents a unique cancer detection paradigm which differs from other current molecular imaging techniques that require the injection of an imaging contrast agent or tracer.
Assuntos
Neoplasias Encefálicas/química , Neoplasias Encefálicas/diagnóstico , Indazóis/uso terapêutico , Imageamento por Ressonância Magnética/métodos , Imagem Molecular/métodos , Animais , Antineoplásicos/uso terapêutico , Meios de Contraste , Humanos , Concentração de Íons de Hidrogênio , Interpretação de Imagem Assistida por Computador/métodos , Camundongos , Camundongos Nus , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Células Tumorais CultivadasRESUMO
The neurotrophins are a family of closely related growth factors that regulate proliferation and differentiation in the developing and mature nervous systems. Neurotrophins stimulate a family of receptor tyrosine kinases (Trk receptors) and utilize an intracellular docking protein termed fibroblast growth factor (FGF) receptor substrate 2 (FRS2) as a major downstream adapter to activate Ras, phosphatidylinositide 3-kinase (PI3K), and mitogen-activated protein kinase (MAPK) signaling cascades. The goals of this study were twofold: first, to investigate the complexity of neurotrophin-induced FRS2 interactions in primary cortical neurons and to determine which pathway(s) are important in regulating neuronal growth and, second, to determine whether the related signaling adapter, FRS3, stimulates neuron growth comparable to FRS2. We find that neurotrophin treatment of primary cortical neurons stimulates the tyrosine phosphorylation of FRS2 and the subsequent recruitment of Shp2, Grb2, and Gab2. With FRS2 mutants deficient in Grb2 or Shp2 binding, we demonstrate that FRS2 binds Gab1 and Gab2 through Grb2, providing an alternative route to activate PI3 kinase and Shp2. Using recombinant adenoviruses expressing FRS2, we demonstrate that FRS2 overexpression promotes neurite outgrowth and branching in cortical neurons relative to controls. In contrast, overexpression of FRS3 does not stimulate neuronal growth. Moreover, we find that while loss of Shp2, but not Grb2, reduces brain-derived neurotrophic factor (BDNF)-induced MAPK activation, the loss of either pathway impairs neuronal growth. Collectively, these experiments demonstrate that FRS2 functions as an adapter of a multiprotein complex that is activated by the Trk receptors and that the activation of both Grb2- and Shp2-dependent pathways facilitates cortical neuronal growth.
Assuntos
Córtex Cerebral/metabolismo , Proteína Adaptadora GRB2/metabolismo , Proteínas de Membrana/metabolismo , Neurônios/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Animais , Células COS , Células Cultivadas , Córtex Cerebral/citologia , Chlorocebus aethiops , Proteína Adaptadora GRB2/genética , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana/genética , Camundongos , Neurônios/citologia , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 11/genéticaRESUMO
Tissue pH is an indicator of altered cellular metabolism in diseases including stroke and cancer. Ischemic tissue often becomes acidic due to increased anaerobic respiration leading to irreversible cellular damage. Chemical exchange saturation transfer (CEST) effects can be used to generate pH-weighted magnetic resonance imaging (MRI) contrast, which has been used to delineate the ischemic penumbra after ischemic stroke. In the current study, a novel MRI ratiometric technique is presented to measure absolute pH using the ratio of CEST-mediated contrast from amine and amide protons: amine/amide concentration-independent detection (AACID). Effects of CEST were observed at 2.75 parts per million (p.p.m.) for amine protons and at 3.50 p.p.m. for amide protons downfield (i.e., higher frequency) from bulk water. Using numerical simulations and in vitro MRI experiments, we showed that pH measured using AACID was independent of tissue relaxation time constants, macromolecular magnetization transfer effects, protein concentration, and temperature within the physiologic range. After in vivo pH calibration using phosphorus ((31)P) magnetic resonance spectroscopy ((31)P-MRS), local acidosis is detected in mouse brain after focal permanent middle cerebral artery occlusion. In summary, our results suggest that AACID represents a noninvasive method to directly measure the spatial distribution of absolute pH in vivo using CEST MRI.
Assuntos
Acidose Láctica , Amidas/análise , Aminas/análise , Isquemia Encefálica/metabolismo , Concentração de Íons de Hidrogênio , Imageamento por Ressonância Magnética/métodos , Acidose Láctica/diagnóstico , Acidose Láctica/metabolismo , Animais , Biomarcadores/análise , Calibragem , Simulação por Computador , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos QuímicosRESUMO
TrkA is a cell surface transmembrane receptor tyrosine kinase for nerve growth factor (NGF). TrkA has an NPXY motif and kinase regulatory loop similar to insulin receptor (INSR) suggesting that NGFâTrkA signaling might overlap with insulinâINSR signaling. During insulin or NGF stimulation TrkA, insulin receptor substrate-1 (IRS-1), INSR (and presumably other proteins) forms a complex in PC12 cells. In PC12 cells, tyrosine phosphorylation of INSR and IRS-1 is dependent upon the functional TrkA kinase domain. Moreover, expression of TrkA kinase-inactive mutant blocked the activation of Akt and Erk5 in response to insulin or NGF. Based on these data, we propose that TrkA participates in insulin signaling pathway in PC12 cells.
Assuntos
Insulina/metabolismo , Fator de Crescimento Neural/metabolismo , Receptor trkA/metabolismo , Transdução de Sinais , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Ativação Enzimática/efeitos dos fármacos , Glucose/metabolismo , Humanos , Insulina/farmacologia , Proteínas Substratos do Receptor de Insulina/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Dados de Sequência Molecular , Fator de Crescimento Neural/farmacologia , Células PC12 , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Receptor de Insulina/química , Receptor de Insulina/metabolismo , Receptor trkA/química , Transdução de Sinais/efeitos dos fármacosRESUMO
We previously demonstrated that the guanine nucleotide exchange factor, RasGrf1, binds nerve growth factor (NGF)-activated TrkA and facilitates neurotrophin-induced neurite outgrowth in PC12 cells. RasGrf1 can activate both Ras and Rac, via intrinsic Cdc25 and DH domains, respectively, suggesting that the activation of both could contribute to this process. Previous studies have assayed constitutive neurite outgrowth following RasGrf1 over-expression in PC12 cells, in either the absence or presence of ectopic H-Ras, and have suggested an essential role for either Ras or Rac depending on the presence of H-Ras over-expression. In contrast, in this study, we have addressed the mechanism of how RasGrf1 facilitates neurite outgrowth in response to the neurotrophins, NGF and BDNF. Using Ras/Rac activation assays and site-directed RasGrf1 mutants, we find that both Ras and Rac are essential to neurotrophin-induced neurite outgrowth. Moreover, we find that H-Ras over-expression rescues the loss of neurite outgrowth observed with a Rac minus mutant and that RasGrf1 differentially stimulates NGF-dependent activation of Rac or Ras, depending on cell type. Collectively, these studies clarify the mechanism of how RasGrf1 expression facilitates neurotrophin-induced neurite outgrowth. Moreover, they suggest that H-Ras over-expression should be used with caution to measure phenotypic responses.
Assuntos
Neuritos/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , ras-GRF1/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Células HEK293 , Humanos , Camundongos , Mutagênese Sítio-Dirigida , Mutação , Fator de Crescimento Neural/metabolismo , Neuritos/metabolismo , Células PC12 , Proteínas Proto-Oncogênicas p21(ras)/genética , Ratos , Receptor trkA/metabolismo , Receptor trkB/metabolismo , Regulação para Cima , ras-GRF1/genéticaRESUMO
Vesicular transport in neurons plays a vital role in neuronal function and survival. Nesca is a novel protein that we previously identified and herein describe its pattern of expression, subcellular localization and protein-protein interactions both in vitro and in vivo. Specifically, a large proportion of Nesca is in tight association with both actin and microtubule cytoskeletal proteins. Nesca binds to F-actin, microtubules, ßIII and acetylated α-tubulin, but not neurofilaments or the actin-binding protein drebrin, in in vitro-binding assays. Nesca co-immunoprecipitates with kinesin heavy chain (KIF5B) and kinesin light-chain motors as well as with the synaptic membrane precursor protein, syntaxin-1, and is a constituent of the post-synaptic density. Moreover, in vitro-binding assays indicate that Nesca directly binds KIF5B, kinesin light-chain and syntaxin-1. In contrast, Nesca does not co-immunoprecipitate with the kinesin motors KIF1B, KIF3A nor does it bind syntaxin-4 or the synaptosome-associated protein 25 kDa (SNAP-25) in vitro. Nesca expression in neurons is highly punctuate, co-stains with syntaxin-1, and is found in fractions containing markers of early endosomes and Golgi suggesting that it is involved in vesicular transport. Collectively, these data suggest that Nesca functions as an adapter involved in neuronal vesicular transport including vesicles containing soluble N-ethylmaleimide sensitive factor attachment protein receptors that are essential to exocytosis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cinesinas/metabolismo , Neurônios/metabolismo , Sintaxina 1/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Western Blotting , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Hipocampo/metabolismo , Humanos , Imuno-Histoquímica , Imunoprecipitação , Camundongos , Neurogênese/fisiologia , Densidade Pós-Sináptica/metabolismo , Transporte Proteico/fisiologia , Membranas Sinápticas/metabolismo , TransfecçãoRESUMO
BACKGROUND: FGF receptor substrates (FRS2 and FRS3) are key adaptor proteins that mediate FGF-FGFR signalling in benign as well as malignant tissue. Here we investigated FRS2 and FRS3 as a means of disrupting global FGF signalling in prostate cancer. METHODS: FRS2 and FRS3 manipulation was investigated in vitro using over-expression, knockdown and functional assays. FRS2 and FRS3 expression was profiled in cell lines and clinical tumors of different grades. RESULTS: In a panel of cell lines we observed ubiquitous FRS2 and FRS3 transcript and protein expression in both benign and malignant cells. We next tested functional redundancy of FRS2 and FRS3 in prostate cancer cells. In DU145 cells, specific FRS2 suppression inhibited FGF induced signalling. This effect was not apparent in cells stably over-expressing FRS3. Indeed FRS3 over-expression resulted in enhanced proliferation (p = 0.005) compared to control cells. Given this functional redundancy, we tested the therapeutic principle of dual targeting of FRS2 and FRS3 in prostate cancer. Co-suppression of FRS2 and FRS3 significantly inhibited ERK activation with a concomitant reduction in cell proliferation (p < 0.05), migration and invasion (p < 0.05). Synchronous knockdown of FRS2 and FRS3 with exposure to cytotoxic irradiation resulted in a significant reduction in prostate cancer cell survival compared to irradiation alone (p < 0.05). Importantly, this synergistic effect was not observed in benign cells. Finally, we investigated expression of FRS2 and FRS3 transcript in a cohort of micro-dissected tumors of different grades as well as by immunohistochemistry in clinical biopsies. Here, we did not observe any difference in expression between benign and malignant biopsies. CONCLUSIONS: These results suggest functional overlap of FRS2 and FRS3 in mediating mitogenic FGF signalling in the prostate. FRS2 and FRS3 are not over-expressed in tumours but targeted dual inhibition may selectively adversely affect malignant but not benign prostate cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias da Próstata/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Western Blotting , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células , Sobrevivência Celular/efeitos da radiação , Estudos de Coortes , Inativação Gênica , Humanos , Imuno-Histoquímica , Masculino , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , Neoplasias da Próstata/genéticaRESUMO
Paramagnetic chemical exchange saturation transfer (PARACEST) contrast agents are under development for biological target identification by magnetic resonance imaging. Image contrast associated with PARACEST agents can be generated by radiofrequency irradiation of the chemically shifted protons bound to a PARACEST contrast agent molecule or by direct irradiation of the on-resonance bulk water protons. The observed signal change in a magnetic resonance image after the administration of a PARACEST contrast agent is due to both altered relaxation time constants and the CEST effect. Despite high sensitivity in vitro, PARACEST agents have had limited success in vivo where sensitivity is reduced by the magnetization transfer effect from endogenous macromolecules. The purpose of this study was to demonstrate the in vivo detection of a PARACEST contrast agent using the on-resonance paramagnetic chemical exchange effect (OPARACHEE) in a mouse glioblastoma multiforme tumor model and to isolate the OPARACHEE effect from the changes in relaxation induced by the PARACEST agent. Three mice with tumors were imaged on a 9.4 T MRI scanner following tail vein injection of 150 µL 50 mM Tm(3+)-DOTAM-glycine-lysine. A fast low angle shot pulse sequence with a low power radiofrequency pulse train (WALTZ-16) as the preparation pulse was used to generate OPARACHEE contrast. To study the dynamics of agent uptake, reference images (without the preparation pulse) and OPARACHEE images were acquired continuously in an alternating fashion before, during and after agent injection. Signal intensity decreased by more than 10% in tumor in the control images after agent administration. Despite these changes, a clear OPARACHEE contrast of 1-5% was also observed in brain tumors after contrast agent injection and maintained in the hour following injection. This result is the first in vivo observation of OPARACHEE contrast in brain tumors with correction of T(1) and T(2) relaxation effects.
Assuntos
Neoplasias Encefálicas/diagnóstico , Meios de Contraste , Glioblastoma/diagnóstico , Imageamento por Ressonância Magnética , Animais , Meios de Contraste/análise , Meios de Contraste/farmacocinética , Modelos Animais de Doenças , Espectroscopia de Ressonância de Spin Eletrônica , Camundongos , Estrutura Molecular , Túlio/química , Túlio/farmacocinéticaRESUMO
Ectopic expression of the TrkA receptor tyrosine kinase in tumors of the nervous system can mediate nerve growth factor (NGF)-dependent cell death by apoptosis and /or autophagy. Herein, we demonstrate that TrkA can also induce cell death in medulloblastoma Daoy cells by a caspase-independent mechanism that involves the hyperstimulation of macropinocytosis. Specifically, NGF-stimulates the uptake of AlexaFluor546-dextran into lysosome-associated membrane protein-1 positive vacuoles which fuse with microtubule associated protein light chain 3 (LC3) positive autophagosomes, to form large intracellular vacuoles (> 1 mum), which then fuse with lysotracker positive lysosomes. While LC3 cleavage and the appearance of LC3 positive vacuoles suggest the induction of autophagy, siRNA reduced expression of four proteins essential to autophagy (beclin-1, Atg5, LC3 and Atg9) neither blocks NGF-induced vacuole formation nor cell death. TrkA activated cell death does not require p38, JNK or Erk1/2 kinases but does require activation of class III PI-3 kinase and is blocked by the casein kinase 1 (CK1) inhibitor, D4476. This inhibitor does not interfere with TrkA activation but does block NGF-dependent AlexaFluor546-dextran uptake and CK1 dependent phosphorylation of beta-catenin. Collectively, these data demonstrate that TrkA stimulates cell death by a novel mechanism involving CK1-dependent hyperstimulation of macropinocytosis.
Assuntos
Autofagia/efeitos dos fármacos , Fator de Crescimento Neural/farmacologia , Pinocitose/efeitos dos fármacos , Pinocitose/fisiologia , Receptor trkA/metabolismo , Análise de Variância , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 5 Relacionada à Autofagia , Proteína Beclina-1 , Linhagem Celular Tumoral , Citocromos c/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imunoprecipitação/métodos , Proteínas de Membrana Lisossomal/genética , Proteínas de Membrana Lisossomal/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/ultraestrutura , Meduloblastoma/patologia , Meduloblastoma/ultraestrutura , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia Eletrônica de Transmissão/métodos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção/métodosRESUMO
Fibroblast growth factor (FGF) mediated signaling is essential to many aspects of neural development. Activated FGF receptors signal primarily through the FGF receptor substrate (Frs) adapters, which include Frs2/Frs2alpha and Frs3/Frs2beta. While some studies suggest that Frs3 can compensate for the loss of Frs2 in transfected cells, the lack of an effective Frs3 specific antibody has prevented efforts to determine the role(s) of the endogenous protein. To this end, we have generated a Frs3 specific antibody and have characterized the pattern of Frs3 expression in the developing nervous system, its subcellular localization as well as its biochemical properties. We demonstrate that Frs3 is expressed at low levels in the ventricular zone of developing cortex, between E12 and E15, and it co-localizes with nestin and acetylated alpha-tubulin in radial processes in the ventricular/subventricular zones as well as with betaIII tubulin in differentiated cortical neurons. Subcellular fractionation studies demonstrate that endogenous Frs3 is both soluble and plasma membrane associated while Frs3 expressed in 293T cells associates exclusively with lipid rafts. Lastly, we demonstrate that neuronal Frs3 binds microtubules comparable to the microtubule-associated protein, MAP2, while Frs2 does not. Collectively, these data suggest that neuronal Frs3 functions as a novel microtubule binding protein and they provide the first biochemical evidence that neuronal Frs3 is functionally distinct from Frs2/Frs2alpha.