RESUMO
The effects of repetitive magnetic stimulation (rMS) have predominantly been studied in excitable cells, with limited research in non-excitable cells. This study aimed to investigate the impact of rMS on macrophages, which are crucial cells in the innate immune defense. THP-1-derived macrophages subjected to a 5 min session of 10 Hz rMS exhibited increased Nrf2 activation and decreased Keap1 expression. We found that activation of the Nrf2 signaling pathway relied on rMS-induced phosphorylation of p62. Notably, rMS reduced the intracellular survival of Staphylococcus aureus in macrophages. Silencing Nrf2 using siRNA in THP-1-derived macrophages or utilizing Nrf2 knockout in alveolar macrophages abolished this effect. Additionally, rMS attenuated the expression of IL-1ß and TNF-α inflammatory genes by S. aureus and inhibited p38 MAPK activation. These findings highlight the capacity of rMS to activate the non-canonical Nrf2 pathway, modulate macrophage function, and enhance the host's defense against bacterial infection.
RESUMO
High spinal cord injuries (SCI) induce the deafferentation of phrenic motoneurons, leading to permanent diaphragm paralysis. This involves secondary injury associated with pathologic and inflammatory processes at the site of injury, and at the level of phrenic motoneurons. In the present study, we evaluated the antioxidant response in phrenic motoneurons involving the AMPK-Nrf2 signaling pathway following C2 spinal cord lateral hemi-section in rats. We showed that there is an abrupt reduction in the expression of phosphorylated AMPK and Nrf2 at one hour post-injury in phrenic motoneurons. A rebound is then observed at one day post-injury, reflecting a return to homeostasis condition. In the total spinal cord around phrenic motoneurons, the increase in phosphorylated AMPK and Nrf2 occurred at three days post-injury, showing the differential antioxidant response between phrenic motoneurons and other cell types. Taken together, our results display the implication of the AMPK-Nrf2 signaling pathway in phrenic motoneurons' response to oxidative stress following high SCI. Harnessing this AMPK-Nrf2 signaling pathway could improve the antioxidant response and help in spinal rewiring to these deafferented phrenic motoneurons to improve diaphragm activity in patients suffering high SCI.
RESUMO
Repetitive transcranial magnetic stimulation (rTMS) is a promising, innovative, and non-invasive therapy used clinically. Efficacy of rTMS has been demonstrated to ameliorate psychiatric disorders and neuropathic pain through neuromodulation of affected neural circuits. However, little is known about the mechanisms and the specific neural circuits via which rTMS facilitates these functional effects. The aim of this study was to begin revealing the mechanisms by which rTMS may tap into existing neural circuits, by using a well characterized spinal motor circuit - the phrenic circuit. Here we hypothesized that rTMS can be used to enhance phrenic motoneuron excitability in anesthetized Sprague Dawley rats. Multiple acute rTMS protocols were used revealing 10 Hz rTMS protocol induced a robust, long-lasting increase in phrenic motoneuron excitability, functionally evaluated by diaphragm motor evoked potentials (59.1 ± 21.1 % of increase compared to baseline 60 min after 10 Hz protocol against 6.0 ± 5.8 % (p = 0.007) for Time Control, -5.8 ± 7.4 % (p < 0.001) for 3 Hz, and 5.2 ± 12.5 % (p = 0.008) for 30 Hz protocols). A deeper analyze allowed to discriminate "responder" and "non-responder" subgroups among 10 Hz rTMS treated animals. Intravenous injections of GABAA and GABAB receptor agonists prior to 10 Hz rTMS treatment, abolished the enhanced phrenic motoneuron excitability, suggesting GABAergic input plays a mechanistic role in rTMS-induced phrenic excitability. These data demonstrate that a single high frequency rTMS protocol at 10 Hz increases phrenic motoneuron excitability, mediated by a local GABAergic "disinhibition". By understanding how rTMS can be used to affect neural circuits non-invasively we can begin to harness the therapeutic potential of this neuromodulatory strategy to promote recovery after disease or injury to the central nervous system.
Assuntos
Potencial Evocado Motor/fisiologia , Agonistas de Receptores de GABA-A/farmacologia , Agonistas dos Receptores de GABA-B/farmacologia , Neurônios Motores/fisiologia , Rede Nervosa/fisiologia , Nervo Frênico/fisiologia , Estimulação Magnética Transcraniana , Animais , Diafragma/efeitos dos fármacos , Diafragma/fisiologia , Potencial Evocado Motor/efeitos dos fármacos , Feminino , Neurônios Motores/efeitos dos fármacos , Rede Nervosa/efeitos dos fármacos , Rede Nervosa/metabolismo , Nervo Frênico/efeitos dos fármacos , Nervo Frênico/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Cervical spinal cord injury (SCI) results in permanent life-altering motor and respiratory deficits. Other than mechanical ventilation for respiratory insufficiency secondary to cervical SCI, effective treatments are lacking and the development of animal models to explore new therapeutic strategies are needed. The aim of this work was to demonstrate the feasibility of using a mouse model of partial cervical spinal hemisection at the second cervical metameric segment (C2) to investigate the impact of 6 weeks training on forced exercise wheel system on locomotor/respiratory plasticity muscles. To measure run capacity locomotor and respiratory functions, incremental exercise tests and diaphragmatic electromyography were done. In addition, muscle fiber type composition and capillary distribution were assessed at 51 days following chronic C2 injury in diaphragm, extensor digitorum communis (EDC), tibialis anterior (TA) and soleus (SOL) muscles. Six-week exercise training increased the running capacity of trained SCI mice. Fiber type composition in EDC, TA and SOL muscles was not modified by our protocol of exercise. The vascularization was increased in all muscle limbs in SCI trained group. No increase in diaphragmatic electromyography amplitude of the diaphragm muscle on the side of SCI was observed, while the contraction duration was significantly decreased in sedentary group compared to trained group. Cross-sectional area of type IIa myofiber in the contralateral diaphragm side of SCI was smaller in trained group. Fiber type distribution between contralateral and ipsilateral diaphragm in SCI sedentary group was affected, while no difference was observed in trained group. In addition, the vascularization of the diaphragm side contralateral to SCI was increased in trained group. All these results suggest an increase in fatigue resistance and a contribution to the running capacity in SCI trained group. Our exercise protocol could be a promising non-invasive strategy to sustain locomotor and respiratory muscle plasticity following SCI.
Assuntos
Medula Cervical/lesões , Exercício Físico , Músculos/fisiopatologia , Traumatismos da Medula Espinal/terapia , Animais , Medula Cervical/fisiopatologia , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Recuperação de Função Fisiológica , Traumatismos da Medula Espinal/fisiopatologiaAssuntos
COVID-19 , Doenças Cardiovasculares/terapia , Sistema Cardiovascular/fisiopatologia , Diabetes Mellitus Tipo 2/terapia , Metabolismo Energético , Exercício Físico , Estilo de Vida Saudável , Sistema Imunitário/imunologia , Prognóstico , Comportamento de Redução do Risco , Animais , Doenças Cardiovasculares/imunologia , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/fisiopatologia , Diabetes Mellitus Tipo 2/imunologia , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Terapia por Exercício , Fatores de Risco de Doenças Cardíacas , Humanos , Medição de RiscoRESUMO
High spinal cord injuries (SCI) lead to permanent respiratory insufficiency, and the search for new therapeutics to restore this function is essential. To date, the most documented preclinical model for high SCI is the rat cervical C2 hemisection. However, molecular studies with this SCI model are limited due to the poor availability of genetically modified specimens. The aim of this work was to evaluate the pathophysiology of respiratory activity following a cervical C2 injury at different times post-injury in a C57BL/6 mouse model. No significant spontaneous recovery of diaphragmatic activity was observed up to 30 days post-injury in eupneic condition. However, during a respiratory challenge, i.e. mild asphyxia, a partial restoration of the injured diaphragm was observed at 7 days post-injury, corresponding to the crossed phrenic phenomenon. Interestingly, the diaphragmatic recording between 2 respiratory bursts on the injured side showed an amplitude increase between 1-7 days post-injury, reflecting a change in phrenic motoneuronal excitability. This increase in inter-burst excitability returned to pre-injured values when the crossed phrenic phenomenon started to be effective at 7 days post-injury. Taken together, these results demonstrate the ability of the mouse respiratory system to express long-lasting plasticity following a C2 cervical hemisection and genetically modified animals can be used to study the pathophysiological effects on these plasticity phenomena.
Assuntos
Medula Cervical/lesões , Diafragma/fisiopatologia , Neurônios Motores/fisiologia , Nervo Frênico/fisiopatologia , Traumatismos da Medula Espinal/fisiopatologia , Animais , Modelos Animais de Doenças , Eletromiografia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Inflammation plays a crucial role in the defense response of the innate immune system against pathogen infection. In this study, we selected 4 compounds for their potential or proven anti-inflammatory and/or anti-microbial properties to test on our in vitro model of bacteria-infected THP-1-derived macrophages. We first compared the capacity of sulforaphane (SFN), wogonin (WG), oltipraz (OTZ), and dimethyl fumarate (DMF) to induce the nuclear factor erythroid 2-related factor 2 (Nrf2), a key regulator of the antioxidant, anti-inflammatory response pathways. Next, we performed a comparative evaluation of the antioxidant and anti-inflammatory efficacies of the 4 selected compounds. THP-1-derived macrophages and LPS-stimulated macrophages were treated with each compound and expression levels of genes coding for inflammatory cytokines IL-1ß, IL-6, and TNF-α were quantified by RT-qPCR. Moreover, expression levels of genes coding for M1 (IL-23, CCR7, IL-1ß, IL-6, and TNF-α) and M2 (PPARγ, MRC1, CCL22, and IL-10) markers were determined in classically-activated M1 macrophages treated with each compound. Finally, the effects of each compound on the intracellular bacterial survival of gram-negative E. coli and gram-positive S. aureus in THP-1-derived macrophages and PBMC-derived macrophages were examined. Our data confirmed the anti-inflammatory and antioxidant effects of SFN, WG, and DMF on LPS-stimulated THP-1-derived macrophages. In addition, SFN or WG treatment of classically-activated THP-1-derived macrophages reduced expression levels of M1 marker genes, while SFN or DMF treatment upregulated the M2 marker gene MRC1. This decrease in expression of M1 marker genes may be correlated with the decrease in intracellular S. aureus load in SFN- or DMF-treated macrophages. Interestingly, an increase in intracellular survival of E. coli in SFN-treated THP-1-derived macrophages that was not observed in PBMC-derived macrophages. Conversely, OTZ exhibited pro-oxidant and proinflammatory properties, and affected intracellular survival of E. coli in THP-1-derived macrophages. Altogether, we provide new potential therapeutic alternatives in treating inflammation and bacterial infection.
Assuntos
Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Infecções por Escherichia coli/tratamento farmacológico , Inflamação/tratamento farmacológico , Ativação de Macrófagos/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/imunologia , Estresse Oxidativo/efeitos dos fármacos , Infecções Estafilocócicas/tratamento farmacológico , Fumarato de Dimetilo/farmacologia , Escherichia coli/efeitos dos fármacos , Infecções por Escherichia coli/imunologia , Flavanonas/farmacologia , Humanos , Inflamação/imunologia , Isotiocianatos/farmacologia , Leucócitos Mononucleares , Ativação de Macrófagos/imunologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Estresse Oxidativo/imunologia , Pirazinas/farmacologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/efeitos dos fármacos , Sulfóxidos , Células THP-1 , Tionas , TiofenosRESUMO
Macrophages are active contributors to the innate immune defense system. As macrophage activation is clearly affected by the surrounding microenvironment, the present study investigated the effect of sulforaphane (SFN) on the bactericidal activity of macrophages and the underlying molecular mechanisms involved in this process. Human THP1derived macrophages, primary human peripheral blood mononuclear cellderived macrophages, and primary mouse bone marrow derivedmacrophages (BMDMs) pretreated with SFN or DMSO were utilized in a model of Staphylococcus aureus infection. The results suggested that SFN pretreatment of macrophages effectively repressed the intracellular survival of S. aureus through modulation of p38/JNK signaling and decreased S. aureusinduced caspases3/7dependent cell apoptosis, potentially through downregulation of microRNA (miR)1425p and miR146a5p. As SFN is a wellknown activator of nuclear factor erythroid 2related factor 2 (Nrf2), Nrf2/ BMDMs were used to demonstrate that the SFNmediated inhibitory effect was independent of Nrf2. Nevertheless, an increase in intracellular bacterial survival in Nrf2deficient macrophages was observed. In addition, SFN pretreatment suppressed S. aureusinduced transcriptional expression of genes coding for the proinflammatory cytokines interleukin (IL)1ß, IL6, and tumor necrosis factorα (TNFα), as well as for the M1 markers CC motif chemokine receptor 7, IL23 and inducible nitric oxide synthase (iNOS). Western blot analysis indicated that S. aureus challenge activated p38 mitogenactivated protein kinase (MAPK) (p38) and cJun Nterminal kinase (JNK) MAPK signaling pathways, while SFN pretreatment prevented p38 and JNK phosphorylation. Pretreatment with 2 specific inhibitors of p38 and JNK, SB203580 and SP600125, respectively, resulted in a decrease in S. aureusinduced proinflammatory gene expression levels compared with those observed in the SFNpretreated macrophages. Furthermore, THP1derived macrophages pretreated with SB203580 or SP600125 prior to bacterial infection exhibited a significant inhibition in intracellular S. aureus survival. In conclusion, we hypothesize that concomitant targeting of the p38/JNKinflammatory response and the S. aureusinduced apoptosis with SFN may be a promising therapeutic approach in S. aureus infection.
Assuntos
Inflamação/tratamento farmacológico , Isotiocianatos/farmacologia , MAP Quinase Quinase 4/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Animais , Antibacterianos/farmacologia , Apoptose/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/genética , Inflamação/metabolismo , MAP Quinase Quinase 4/metabolismo , Macrófagos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/metabolismo , Sulfóxidos , Células THP-1RESUMO
High cervical spinal cord injuries lead to permanent respiratory deficits. One preclinical model of respiratory insufficiency in adult rats is the C2 partial injury which causes unilateral diaphragm paralysis. This model allows the investigation of a particular population of respiratory bulbospinal axons which cross the midline at C3-C6 spinal segment, namely the crossed phrenic pathway. Transcranial magnetic stimulation (TMS) is a non-invasive technique that can be used to study supraspinal descending respiratory pathways in the rat. Interestingly, a lateral C2 injury does not affect the amplitude and latency of the largest motor-evoked potential recorded from the diaphragm (MEPdia) ipsilateral to the injury in response to a single TMS pulse, compared to a sham animal. Although the rhythmic respiratory activity on the contralateral diaphragm is preserved at 7 days post-injury, no diaphragm activity can be recorded on the injured side. However, a profound reorganization of the MEPdia evoked by TMS can be observed. The MEPdia is reduced on the non-injured rather than the injured side. This suggests an increase in ipsilateral phrenic motoneurons excitability. Moreover, correlations between MEPdia amplitude and spontaneous contralateral diaphragmatic activity were observed. The larger diaphragm activity correlated with a larger MEPdia on the injured side, and a smaller MEPdia on the non-injured side. This suggests, for the first time, the occurrence of a functional neuroplasticity process involving changes in motoneuron excitability balance between the injured and non-injured sides at a short post-lesional delay.
Assuntos
Vértebras Cervicais/fisiopatologia , Respiração , Traumatismos da Medula Espinal/fisiopatologia , Estimulação Magnética Transcraniana , Animais , Asfixia/complicações , Asfixia/fisiopatologia , Diafragma/fisiopatologia , Potencial Evocado Motor , Masculino , Modelos Biológicos , Ratos Sprague-Dawley , Traumatismos da Medula Espinal/complicaçõesRESUMO
Respiratory related diseases associated with the neuronal control of breathing represent life-threatening issues and to date, no effective therapeutics are available to enhance the impaired function. The aim of this study was to determine whether a preclinical respiratory model could be used for further studies to develop a non-invasive therapeutic tool applied to rat diaphragmatic neuronal circuitry. Transcranial magnetic stimulation (TMS) was performed on adult male Sprague-Dawley rats using a human figure-of-eight coil. The largest diaphragmatic motor evoked potentials (MEPdia) were recorded when the center of the coil was positioned 6 mm caudal from Bregma, involving a stimulation of respiratory supraspinal pathways. Magnetic shielding of the coil with mu metal reduced magnetic field intensities and improved focality with increased motor threshold and lower amplitude recruitment curve. Moreover, transynaptic neuroanatomical tracing with pseudorabies virus (applied to the diaphragm) suggest that connections exist between the motor cortex, the periaqueductal grey cell regions, several brainstem neurons and spinal phrenic motoneurons (distributed in the C3-4 spinal cord). These results reveal the anatomical substrate through which supraspinal stimulation can convey descending action potential volleys to the spinal motoneurons (directly or indirectly). We conclude that MEPdia following a single pulse of TMS can be successfully recorded in the rat and may be used in the assessment of respiratory supraspinal plasticity. Supraspinal non-invasive stimulations aimed to neuromodulate respiratory circuitry will enable new avenues of research into neuroplasticity and the development of therapies for respiratory dysfunction associated with neural injury and disease (e.g. spinal cord injury, amyotrophic lateral sclerosis).
Assuntos
Diafragma/inervação , Modelos Animais de Doenças , Transtornos Respiratórios/terapia , Mecânica Respiratória/fisiologia , Estimulação Magnética Transcraniana/métodos , Análise de Variância , Animais , Diafragma/fisiologia , Potencial Evocado Motor/fisiologia , Comunicação Interdisciplinar , Masculino , Técnicas de Rastreamento Neuroanatômico , Ratos , Ratos Sprague-Dawley , Transtornos Respiratórios/fisiopatologia , Estatísticas não Paramétricas , Estimulação Magnética Transcraniana/instrumentaçãoRESUMO
A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed to improve/ameliorate the respiratory failure following high cervical spinal cord injury (SCI). Here we propose a murine pre-clinical model of high SCI at the cervical 2 (C2) metameric level to study diverse post-lesional respiratory neuroplasticity. The technique consists of a surgical partial injury at the C2 level, which will induce a hemiparalysis of the diaphragm due to a deafferentation of the phrenic motoneurons from the respiratory centers located in the brainstem. The contralateral side of the injury remains intact and allows the animal recovery. Unlike other SCIs which affect the locomotor function (at the thoracic and lumbar level), the respiratory function does not require animal motivation and the quantification of the deficit/recovery can be easily performed (diaphragm and phrenic nerve recordings, whole body ventilation). This pre-clinical C2 SCI model is a powerful, useful, and reliable pre-clinical model to study various respiratory and non-respiratory neuroplasticity events at different levels (molecular to physiology) and to test diverse putative therapeutic strategies which might improve the respiration in SCI patients.
Assuntos
Modelos Animais de Doenças , Plasticidade Neuronal/fisiologia , Sistema Respiratório/fisiopatologia , Traumatismos da Medula Espinal/fisiopatologia , Animais , Masculino , Camundongos , Ratos , Ratos Sprague-DawleyRESUMO
Focal adhesion kinase (FAK) plays an important role in signal transduction pathways initiated at sites of integrin-mediated cell adhesion to the extracellular matrix. Thus, FAK is involved in many aspects of the metastatic process including adhesion, migration and invasion. Recently, several small molecule inhibitors which target FAK catalytic activity have been developed by pharmaceutical companies. The current study was aimed at addressing whether inhibiting FAK targeting to focal adhesions (FA) represents an efficient alternative strategy to inhibit FAK downstream pathways. Using a mutagenesis approach to alter the targeting domain of FAK, we constructed a FAK mutant that fails to bind paxillin. Inhibiting FAK-paxillin interactions led to a complete loss of FAK localization at FAs together with reduced phosphorylation of FAK and FAK targets such as paxillin and p130Cas. This in turn resulted in altered FA dynamics and inhibition of cell adhesion, migration and invasion. Moreover, the migration properties of cells expressing the FAK mutant were reduced as compared to FAK-/- cells. This was correlated with a decrease in both phospho-Src and phospho-p130Cas levels at FAs. We conclude that targeting FAK-paxillin interactions is an efficient strategy to reduce FAK signalling and thus may represent a target for the development of new FAK inhibitors.
Assuntos
Proteína Substrato Associada a Crk/genética , Fibroblastos/efeitos dos fármacos , Quinase 1 de Adesão Focal/genética , Regulação da Expressão Gênica , Paxilina/genética , Quinases da Família src/genética , Animais , Sítios de Ligação , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proteína Substrato Associada a Crk/metabolismo , Embrião de Mamíferos , Fibroblastos/citologia , Fibroblastos/metabolismo , Quinase 1 de Adesão Focal/antagonistas & inibidores , Quinase 1 de Adesão Focal/metabolismo , Adesões Focais/efeitos dos fármacos , Adesões Focais/metabolismo , Humanos , Camundongos , Mutagênese Sítio-Dirigida , Paxilina/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Recombinantes de Fusão , Transdução de Sinais , Quinases da Família src/metabolismoRESUMO
Cell migration is a highly complex process that requires the coordinated formation of membrane protrusion and focal adhesions (FAs). Focal adhesion kinase (FAK), a major signaling component of FAs, is involved in the disassembly process of FAs through phosphorylation and dephosphorylation of its tyrosine residues, but the role of such phosphorylations in nascent FA formation and turnover near the cell front and in cell protrusion is less well understood. In the present study, we demonstrate that, depending on the phosphorylation status of Tyr-925 residue, FAK modulates cell migration via two specific mechanisms. FAKâ»/â» mouse embryonic fibroblasts (MEFs) expressing nonphosphorylatable Y925F-FAK show increased interactions between FAK and unphosphorylated paxillin, which lead to FA stabilization and thus decreased FA turnover and reduced cell migration. Conversely, MEFs expressing phosphomimetic Y925E-FAK display unchanged FA disassembly rates, show increase in phosphorylated paxillin in FAs, and exhibit increased formation of nascent FAs at the cell leading edges. Moreover, Y925E-FAK cells present enhanced cell protrusion together with activation of the p130(CAS)/Dock180/Rac1 signaling pathway. Together, our results demonstrate that phosphorylation of FAK at Tyr-925 is required for FAK-mediated cell migration and cell protrusion.
Assuntos
Extensões da Superfície Celular/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Adesões Focais/metabolismo , Transdução de Sinais/fisiologia , Tirosina/metabolismo , Animais , Células Cultivadas , Proteína Substrato Associada a Crk/metabolismo , Fibroblastos/citologia , Fibroblastos/fisiologia , Proteína-Tirosina Quinases de Adesão Focal/genética , Humanos , Camundongos , Camundongos Knockout , Paxilina/metabolismo , Fosforilação , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Cell adhesion and migration are key determinants in tumor metastasis. Adherence of tumor cell to the extracellular matrix is mediated via integrin containing focal adhesions (FAs). Binding of integrins to ECM triggers phosphorylation of two major components of FAs, focal adhesion kinase (FAK) and Src, activating downstream signaling pathway which leads to FA disassembly and cell migration. In this paper, we analyze how phosphorylation of FAK regulates its trafficking at FAs in living human astrocytoma cells. Upon pervanadate-induced FAK phosphorylation, phosphorylated FAK appeared highly expressed at newly formed membrane ruffles. This effect was abolished in presence of the specific Src inhibitor PP2. Our findings demonstrate that upon phosphorylation, FAK delocalizes from FAs to membrane ruffles.
RESUMO
Keratin 19 is a member of the cytokeratin family that is critical for maintenance of cellular architecture and organization, especially of epithelia. The pancreas has three distinct cell types, ductal, acinar, and islet, each with different functions. Embryologically, the pancreatic and duodenal homeobox 1 (PDX1) homeodomain protein is critical for the initiation of all pancreatic lineages; however, the later differentiation of the endocrine pancreas is uniquely dependent upon high PDX1 expression, whereas PDX1 is down-regulated in the ductal and acinar cell lineages. We find that this down-regulation may be required for normal ductal expression of cytokeratin K19. The K19 promoter-reporter gene assay demonstrates that ectopic PDX1 inhibits K19 reporter gene activity in primary pancreatic ductal cells. This is reinforced by our findings that retrovirally mediated stable transduction of PDX1 in primary pancreatic ductal cells suppresses K19 expression, and short interfering RNA to PDX1 in Min6 insulinoma cells results in the induction of normally undetectable K19. Complementary functional and biochemical approaches led to the unexpected finding that a multimeric complex of PDX1 and two members of the TALE homeodomain factor family, MEIS1a and PBX1b, regulates K19 gene transcription through a specific cis-regulatory element (-341 to -325) upstream of the K19 transcription start site. These data suggest a unifying mechanism whereby PDX1, myeloid ecotropic viral insertion site (MEIS), and pre-B-cell leukemia transcription factor 1 (PBX) may regulate ductal and acinar lineage specification during pancreatic development. Specifically, concomitant PDX1 suppression and MEIS isoform expression result in proper ductal and acinar lineage specification. Furthermore, PDX1 may inhibit the ductal differentiation program in the pancreatic endocrine compartment, particularly beta cells.
Assuntos
Proteínas de Homeodomínio/fisiologia , Queratina-19/genética , Pâncreas/metabolismo , Regiões Promotoras Genéticas , Transativadores/fisiologia , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA , Genes Reporter , Insulinoma/genética , Insulinoma/patologia , Camundongos , Proteína Meis1 , Proteínas de Neoplasias/fisiologia , Pâncreas/citologiaRESUMO
The genetic basis of pancreatic ductal adenocarcinoma, which constitutes the most common type of pancreatic malignancy, involves the sequential activation of oncogenes and inactivation of tumor suppressor genes. Among the pivotal genetic alterations are Ki-RAS oncogene activation and p53 tumor suppressor gene inactivation. We explain that the combination of these genetic events facilitates pancreatic carcinogenesis as revealed in novel three-dimensional cell (spheroid cyst) culture and in vivo subcutaneous and orthotopic xenotransplantation models. N-cadherin, a member of the classic cadherins important in the regulation of cell-cell adhesion, is induced in the presence of Ki-RAS mutation but subsequently downregulated with the acquisition of p53 mutation as revealed by gene microarrays and corroborated by reverse transcription-PCR and Western blotting. N-cadherin modulates the capacity of pancreatic ductal cells to migrate and invade, in part via complex formation with keratinocyte growth factor receptor and neural cell adhesion molecule and in part via interaction with p120-catenin. However, modulation of these complexes by Ki-RAS and p53 leads to enhanced cell migration and invasion. This preferentially induces the downstream effector AKT over mitogen-activated protein kinase to execute changes in cellular behavior. Thus, we are able to define molecules that in part are directly affected by Ki-RAS and p53 during pancreatic ductal carcinogenesis, and this provides a platform for potential new molecularly based therapeutic interventions.
Assuntos
Caderinas/metabolismo , Movimento Celular , Proteína Oncogênica p21(ras)/metabolismo , Ductos Pancreáticos/citologia , Ductos Pancreáticos/patologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Junções Aderentes/patologia , Animais , Carcinoma Ductal Pancreático/patologia , Técnicas de Cultura de Células , Transformação Celular Neoplásica , Cistos/patologia , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica , Camundongos , Mutação/genética , Invasividade Neoplásica/patologia , Moléculas de Adesão de Célula Nervosa/metabolismo , Proteína Oncogênica p21(ras)/genética , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Supressora de Tumor p53/genéticaRESUMO
To define the genetic requirements for pancreatic ductal adenocarcinoma (PDA), we have targeted concomitant endogenous expression of Trp53(R172H) and Kras(G12D) to the mouse pancreas, revealing the cooperative development of invasive and widely metastatic carcinoma that recapitulates the human disease. The primary carcinomas and metastases demonstrate a high degree of genomic instability manifested by nonreciprocal translocations without obvious telomere erosion-hallmarks of human carcinomas not typically observed in mice. No mutations were discovered in other cardinal tumor suppressor gene pathways, which, together with previous results, suggests that there are distinct genetic pathways to PDA with different biological behaviors. These findings have clear implications for understanding mechanisms of disease pathogenesis, and for the development of detection and targeted treatment strategies.
Assuntos
Carcinoma Ductal Pancreático/patologia , Instabilidade Cromossômica/genética , Mutação de Sentido Incorreto , Proteína Supressora de Tumor p53/genética , Proteínas ras/genética , Animais , Caderinas/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Centrossomo/patologia , Aberrações Cromossômicas , Análise Citogenética , Progressão da Doença , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Rearranjo Gênico/genética , Genes Supressores de Tumor , Proteínas de Homeodomínio/genética , Integrases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Mutantes , Camundongos Transgênicos , Metástase Neoplásica , Proteínas Oncogênicas v-erbB/metabolismo , Proteínas Proto-Oncogênicas p21(ras) , Análise de Sobrevida , Telômero/genética , Transativadores/genética , Translocação GenéticaRESUMO
BACKGROUND & AIMS: The Ki-RAS oncogene is altered in pancreatic ductal neoplasms. Pancreatic ductal cells (PDCs) were purified from cytokeratin 19 (K19)-Ki-RAS(G12V) transgenic mice and control littermates to identify properties of Ki-Ras activation in a cell-type-specific context. Because Ki-RAS mutation has prognostic significance in patients treated with radiation, we studied the influence of Ki-RAS status on radiation survival. METHODS: Pancreatic ductal fragments from mice with Ki-RAS(G12V) mutation or wild-type (WT)-Ki-RAS were cultured. Growth curves, electron microscopy, flow cytometry, and analysis of signaling and cell-cycle proteins were established. Farnesyltransferase inhibitor (FTI) treatment with R115777 before and after irradiation was used to determine the effect of Ki-Ras farnesylation on cell survival. RESULTS: PDCs from WT and K19-Ki-RAS(G12V) mice had features of ductal cells with formation of 3-dimensional structures on collagen without differences in morphology, growth, and cell-cycle distribution. This may result from up-regulation of p16INK4 and p27(Kip1) and lack of hyperstimulation of the mitogen-activated protein kinase pathway in Ki-RAS(G12V) PDCs. No differences in radiation survival between Ki-RAS(G12V) PDCs and WT PDCs were observed. However, Ki-RAS(G12V) PDCs expressing mutant p53(V143A) had enhanced survival compared with WT PDCs transduced with p53(V143A). R115777 treatment sensitized Ki-RAS(G12V) PDCs and Ki-RAS(G12V)/p53(V143A) PDCs, but not WT PDCs. CONCLUSIONS: Novel characterization of murine WT PDCs and Ki-RAS(G12V) PDCs is described. Induction of cell-cycle regulators and lack of mitogen-activated protein kinase hyperstimulation likely are responsible for constraining activated Ki-RAS(G12V)-mediated proliferation. Because its activation was required for sensitization by an FTI, R115777 may be useful against pancreatic tumors expressing oncogenic Ki-Ras.
Assuntos
Divisão Celular/efeitos da radiação , Genes ras/efeitos da radiação , Ductos Pancreáticos/efeitos da radiação , Prenilação de Proteína/fisiologia , Radioterapia/efeitos adversos , Animais , Proteínas de Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/efeitos da radiação , Divisão Celular/genética , Células Cultivadas , Células Epiteliais/efeitos da radiação , Genes ras/genética , Camundongos , Camundongos Transgênicos , Modelos Animais , MutaçãoRESUMO
Oncogenic Ras induces premature senescence in primary cells. Such an oncogene-induced senescence involves activation of tumor suppressor genes that provide a checkpoint mechanism against malignant transformation. In mouse, the ARF-p53 pathway mediates Ha-Ras(G12V)-induced senescence, and p19(ARF-/-) and p53(-/-) cells undergo transformation upon Ras activation. In addition, mouse cells, unlike human cells, express constitutively active telomerase and have long telomeres. However, it is unclear how Ras activation affects human cells of epithelial origin with p53 mutation and/or telomerase activation. In order to address this question, Ha-Ras(G12V) was expressed ectopically in primary as well as hTERT-immortalized human esophageal keratinocytes stably expressing dominant-negative p53 mutants. In human esophageal keratinocytes, we found that Ha-Ras(G12V) induced senescence regardless of p53 status and telomerase activation. Ras activation resulted in changes of cellular morphology, activation of senescence-associated beta-galactosidase, and suppression of cell proliferation, all coupled with reduction in the hyperphosphorylated form of the retinoblastoma protein (pRb). Furthermore, Ha-Ras(G12V) upregulated p16(INK4a) and downregulated cyclin-dependent kinase Cdk4 in human esophageal keratinocytes. Thus, Ras-mediated senescence may involve distinct mechanisms between human and mouse cells. Inactivation of the pRb pathway may be necessary for Ras to overcome senescence and transform human esophageal epithelial cells.