RESUMO
The co-chaperone p50/Cdc37 is an important partner for Hsp90, assisting in molecular chaperone activities, particularly with regard to the regulation of protein kinases. Analysis of the structure of Hsp90-Cdc37-kinase complexes demonstrates the way in which Cdc37 interacts with and controls the folding of a large proportion of intracellular protein kinases. This co-chaperone thus stands at the hub of a multitude of intracellular signaling networks. Indeed, the influence of Cdc37 reaches beyond the housekeeping pathways of protein folding into the regulation of a wide range of cellular processes. This co-chaperone has attracted attention as a potential intermediate in carcinogenesis. Cdc37 is an attractive potential target in cancer due to (1) high expression in a number of tumor types and (2) control of multiple signaling pathways. These properties indicate (3) a potential for selectivity due to its elevated expression in malignant cells and (4) robustness, as the co-chaperone may control multiple growth signaling pathways and thus be less prone to evolution of resistance than less versatile oncoproteins. Cdc37 may also be involved in other aspects of pathophysiology and has been shown to be secreted in exosomes. Protein aggregation disorders have been linked to age-related declines in molecular chaperones and co-chaperones. Cdc37 also appears to be a potential agent in longevity due to its links to protein folding and autophagy, and it will be informative to study the role of Cdc37 maintenance/decline in aging organisms.
Assuntos
Proteínas de Ciclo Celular , Chaperoninas , Chaperoninas/genética , Chaperoninas/química , Chaperoninas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas Quinases/metabolismo , Ligação ProteicaRESUMO
Accumulating evidence suggests that Rab GTPases representing the largest branch of Ras superfamily have recently emerged as the core factors for the regulation of osteoclastogenesis through modulating vesicular transport amongst specific subcellular compartments. Among these, Rab34 GTPase has been identified to be important for the post-Golgi secretory pathway and for phagocytosis; nevertheless, its specific role in osteoclastogenesis has been completely obscure. Here, upon the in vitro model of osteoclast formation derived from murine macrophages like RAW-D cells or bone marrow-derived macrophages, we reveal that Rab34 regulates osteoclastogenesis bidirectionally. More specifically, Rab34 serves as a negative regulator of osteoclast differentiation by promoting the lysosome-induced proteolysis of two osteoclastogenic surface receptors, c-fms and RANK, via the axis of early endosomes-late endosomes-lysosomes, leading to alleviate the transcriptional activity of two of the master regulator of osteoclast differentiation, c-fos and NFATc-1, eventually attenuating osteoclast differentiation and bone resorption. Besides, Rab34 plays a crucial role in modulating the secretory network of lysosome-related proteases including matrix metalloprotease 9 and Cathepsin K across the ruffled borders of osteoclasts, contributing to the regulation of bone resorption.
Assuntos
Reabsorção Óssea , Osteogênese , Animais , Reabsorção Óssea/metabolismo , Diferenciação Celular , Camundongos , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/metabolismo , Ligante RANK/metabolismo , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Rab11a, which ubiquitously localizes to early and recycling endosomes, is required for regulating the vesicular transport of cellular cargos. Interestingly, our previous study revealed that Rab11a served as a negative regulator of osteoclastogenesis by facilitating the lysosomal proteolysis of (1) colony-stimulating factor-1 (c-fms) receptor and (2) receptor activator of nuclear factor-κB (RANK) receptor, thereby resulting in inhibition of osteoclast (OC) differentiation, maturation, and bone-resorbing activity. However, the molecular mechanisms of how Rab11a negatively affected osteoclastogenesis were largely unknown. Heat shock protein (HSP90), including two isoforms HSP90α and HSP90ß, necessitates the stability, maturation, and activity of a broad range of its clients, and is essentially required for a vast array of signal transduction pathways in nonstressful conditions. Furthermore, cumulative evidence suggests that HSP90 is a vital element of the vesicular transport network. Indeed, our recent study revealed that HSP90, a novel effector protein of Rab11b, modulated Rab11b-mediated osteoclastogenesis. In this study, we also found that Rab11a interacted with both HSP90α and HSP90ß in OCs. Upon blockade of HSP90 ATPase activity by a specific inhibitor(17-allylamino-demethoxygeldanamycin), we showed that (1) the ATPase domain of HSP90 was a prerequisite for the interaction between HSP90 and Rab11a, and (2) the interaction of HSP90 to Rab11a sufficiently maintained the inhibitory effects of Rab11a on osteoclastogenesis. Altogether, our findings undoubtedly indicate a novel role of HSP90 in regulating Rab11a-mediated osteoclastogenesis.
Assuntos
Proteínas de Choque Térmico HSP90 , Osteoclastos , Proteínas rab de Ligação ao GTP , Humanos , Adenosina Trifosfatases/metabolismo , Diferenciação Celular , Endossomos , Proteínas de Choque Térmico HSP90/metabolismo , Osteoclastos/metabolismo , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Osteogênese , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Heat shock protein 90 (Hsp90), although one of the most essential intracellular chaperones, can also play key roles in the extracellular milieu. Here, we review the properties of extracellular Hsp90 in cellular homeostasis in the heat shock response (HSR), focusing on cells of the central nervous system. Hsp90 can be secreted by microglia as well as other cell types by non-canonical pathways of secretion. The chaperone may then influence the behavior of distant cells and can for instance protect neuronal cells from the oxidative burst accompanying phagocytosis by microglia of beta-amyloid fibrils. A mechanism involving activation of the transcription factor Nrf2, and induction of the antioxidant response is reported. We review the potential role of extracellular Hsp90, Nrf2 and transcellular chaperone signaling in the non-cell-intrinsic HSR.
Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Neurônios/metabolismo , Antioxidantes/metabolismo , Humanos , Microglia/metabolismo , Chaperonas Moleculares/metabolismo , Estresse Oxidativo , Fagocitose , Transdução de SinaisRESUMO
Cells respond to protein-damaging (proteotoxic) stress by activation of the Heat Shock Response (HSR). The HSR provides cells with an enhanced ability to endure proteotoxic insults and plays a crucial role in determining subsequent cell death or survival. The HSR is, therefore, a critical factor that influences the toxicity of protein stress. While named for its vital role in the cellular response to heat stress, various components of the HSR system and the molecular chaperone network execute essential physiological functions as well as responses to other diverse toxic insults. The effector molecules of the HSR, the Heat Shock Factors (HSFs) and Heat Shock Proteins (HSPs), are also important regulatory targets in the progression of neurodegenerative diseases and cancers. Modulation of the HSR and/or its extended network have, therefore, become attractive treatment strategies for these diseases. Development of effective therapies will, however, require a detailed understanding of the HSR, important features of which continue to be uncovered and are yet to be completely understood. We review recently described and hallmark mechanistic principles of the HSR, the regulation and functions of HSPs, and contexts in which the HSR is activated and influences cell fate in response to various toxic conditions.
Assuntos
Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico/fisiologia , Proteostase/fisiologia , Animais , Sobrevivência Celular/fisiologia , Humanos , Chaperonas Moleculares/metabolismo , Neoplasias/patologia , Neoplasias/terapia , Doenças Neurodegenerativas/fisiopatologia , Doenças Neurodegenerativas/terapiaRESUMO
Rab11b, abundantly enriched in endocytic recycling compartments, is required for the establishment of the machinery of vesicle trafficking. Yet, no report has so far characterized the biological function of Rab11b in osteoclastogenesis. Using in vitro model of osteoclasts differentiated from murine macrophages like RAW-D cells or bone marrow-derived macrophages, we elucidated that Rab11b served as an inhibitory regulator of osteoclast differentiation sequentially via (i) abolishing surface abundance of RANK and c-Fms receptors; and (ii) attenuating nuclear factor of activated T-cells c1 (NFATc-1) upstream signaling cascades, following RANKL stimulation. Rab11b was localized in early and late endosomes, Golgi complex, and endoplasmic reticulum; moreover, its overexpression enlarged early and late endosomes. Upon inhibition of lysosomal function by a specific blocker, chloroquine (CLQ), we comprehensively clarified a novel function of lysosomes on mediating proteolytic degradation of c-Fms and RANK surface receptors, drastically ameliorated by Rab11b overexpression in RAW-D cell-derived osteoclasts. These findings highlight the key role of Rab11b as an inhibitor of osteoclastogenesis by directing the transport of c-Fms and RANK surface receptors to lysosomes for degradation via the axis of early endosomes-late endosomes-lysosomes, thereby contributing towards the systemic equilibrium of the bone resorption phase.
Assuntos
Osteoclastos/metabolismo , Osteogênese , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/citologia , Proteólise , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Members of matrix metalloproteinase (MMP) family promote cancer cell migration, invasion, and metastasis through alteration of the tumor milieu, intracellular signaling pathways, and transcription. We examined gene expression signatures of colon adenocarcinoma cell lines with different metastatic potentials and found that rapidly metastatic cells powerfully expressed genes encoding MMP3 and MMP9. The non-proteolytic PEX isoform and proteolytic isoforms of MMPs were significantly expressed in the metastatic cells in vitro. Knockdown of MMP3 attenuated cancer cell migration and invasion in vitro and lung metastasis in vivo. Profound nuclear localization of MMP3/PEX was found in tumor-stroma marginal area. In contrast, MMP9 was localized in central area of subcutaneous tumors. Overexpression of the PEX isoform of MMP3 promoted proliferation and migration of the rapidly metastatic cells in vitro. Taken together, the non-proteolytic PEX isoform of MMPs locating in cell nuclei involves proliferation, migration, and subsequent metastasis of aggressive adenocarcinoma cells.
Assuntos
Adenocarcinoma/patologia , Neoplasias do Colo/patologia , Hemopexina/metabolismo , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 3 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Aloenxertos/metabolismo , Análise de Variância , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Expressão Gênica , Técnicas de Silenciamento de Genes , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Invasividade Neoplásica , Metástase NeoplásicaRESUMO
Head and neck squamous cell carcinoma (HNSCC) poses a significant challenge clinically, as it can invade facial bones and cause bone pain that is undertreated and poorly understood. Here we studied HNSCC bone pain (HNSCC-BP) in an intratibial mouse xenograft model that uses a human HNSCC cell line (SAS cells). These mice develop HNSCC-BP associated with an upregulation of phosphorylated ERK1/2 (pERK1/2), which is a molecular indicator of neuron excitation in the dorsal root ganglia (DRGs) of sensory nerve cell bodies. Our experiments demonstrated that the inhibition of monocarboxylate transporter 4 (MCT4) by short hairpin (shRNA) transduction suppressed the HNSCC-BP, the lactate level in bone marrow, and the pERK1/2 expression in DRG. The sensory nerves also expressed increased levels of the acid-sensing receptor TRPV1. DRG neurons co-cultured with SAS cells showed increased neurite outgrowth, and were inhibited by MCT4 silencing with shRNA. Collectively, our results show that HNSCC induced an acidic bone microenvironment that evokes HNSCC-BP via MCT4 expression.
Assuntos
Doenças Ósseas/metabolismo , Doenças Ósseas/patologia , Transportadores de Ácidos Monocarboxílicos/metabolismo , Dor/metabolismo , Dor/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Animais , Linhagem Celular Tumoral , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Transportadores de Ácidos Monocarboxílicos/genética , Canais de Cátion TRPV/metabolismoRESUMO
This study identified tumorigenic processes most dependent on murine heat shock protein 72 (HSP72) in the mouse mammary tumor virus-PyMT mammary tumor model, which give rise to spontaneous mammary tumors that exhibit HSP72-dependent metastasis to the lung. RNA-seq expression profiling of Hspa1a/Hspa1b (Hsp72) WT and Hsp72-/- primary mammary tumors discovered significantly lower expression of genes encoding components of the extracellular matrix (ECM) in Hsp72 knockout mammary tumors compared to WT controls. In vitro studies found that genetic or chemical inhibition of HSP72 activity in cultured collagen-expressing human or murine cells also reduces mRNA and protein levels of COL1A1 and several other ECM-encoding genes. In search of a possible mechanistic basis for this relationship, we found HSP72 to support the activation of the tumor growth factor-ß-suppressor of mothers against decapentaplegic-3 signaling pathway and evidence of suppressor of mothers against decapentaplegic-3 and HSP72 coprecipitation, suggesting potential complex formation. Human COL1A1 mRNA expression was found to have prognostic value for HER2+ breast tumors over other breast cancer subtypes, suggesting a possible human disease context where targeting HSP72 may have a therapeutic rationale. Analysis of human HER2+ breast tumor gene expression data using a gene set comprising ECM-related gene and protein folding-related gene as an input to the statistical learning algorithm, Galgo, found a subset of these genes that can collectively stratify patients by relapse-free survival, further suggesting a potential interplay between the ECM and protein-folding genes may contribute to tumor progression.
Assuntos
Matriz Extracelular , Proteínas de Choque Térmico HSP72 , Animais , Humanos , Matriz Extracelular/metabolismo , Feminino , Camundongos , Proteínas de Choque Térmico HSP72/metabolismo , Proteínas de Choque Térmico HSP72/genética , Linhagem Celular Tumoral , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , Regulação Neoplásica da Expressão Gênica , Camundongos Knockout , Cadeia alfa 1 do Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Transdução de Sinais , Metástase NeoplásicaRESUMO
Preserving and regulating cellular homeostasis in the light of changing environmental conditions or developmental processes is of pivotal importance for single cellular and multicellular organisms alike. To counteract an imbalance in cellular homeostasis transcriptional programs evolved, called the heat shock response, unfolded protein response, and integrated stress response, that act cell-autonomously in most cells but in multicellular organisms are subjected to cell-nonautonomous regulation. These transcriptional programs downregulate the expression of most genes but increase the expression of heat shock genes, including genes encoding molecular chaperones and proteases, proteins involved in the repair of stress-induced damage to macromolecules and cellular structures. Sixty-one years after the discovery of the heat shock response by Ferruccio Ritossa, many aspects of stress biology are still enigmatic. Recent progress in the understanding of stress responses and molecular chaperones was reported at the 12th International Symposium on Heat Shock Proteins in Biology, Medicine and the Environment in the Old Town Alexandria, VA, USA from 28th to 31st of October 2023.
Assuntos
Proteínas de Choque Térmico , Medicina , Biologia , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico/genética , Chaperonas Moleculares/metabolismoRESUMO
The Cellular Communication Network Factor (CCN) family is composed of six members: CCN1/CYR61, CCN2/CTGF, CCN3/NOV, CCN4/WISP1, CCN5/WISP2, and CCN6/WISP3. The second member, CCN2/CTGF is a matricellular protein that promotes extracellular matrix (ECM) synthesis and controls angiogenesis. On the other hand, moonlighting/matrix metalloproteinase 3 (MMP3) is an ECM-degrading enzyme that also functions as an intracellular transcription factor. Importantly, extracellular MMP3 is uptaken into cells, translocating into nuclei, and transcriptionally activating CCN2/CTGF gene in cancer and chondrocytes. Thus, the MMP3-CTGF axis balances the matrix metabolism and turnover in the tissue and tumor microenvironments. We established an MMP3 knockout cell line using the CRISPR/Cas9 system, demonstrating the sequential regulatory events of the MMP3-CCN2 axis in the microenvironment. Notably, our protocol is useful for generation of CCN knockout cells as well. Here we serve a protocol of the CRISPR/Cas9-based gene targeting in cultured cells for investigating cellular communication network.
Assuntos
Edição de Genes , Metaloproteinase 3 da Matriz , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 3 da Matriz/metabolismo , Sistemas CRISPR-Cas/genética , Regulação da Expressão Gênica , Comunicação Celular/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismoRESUMO
Heat shock proteins (HSPs) are key stress proteins induced in cells exposed to proteotoxic insult and are critical for thermotolerance. The dynamic network of chaperone interactions, known as the chaperome, contributes significantly to the proteotoxic cell response and the malignant phenotype in cancer. We identified a potent microRNA, miR-570 that could bind the 3'untranslated regions of multiple HSP mRNAs and inhibit HSP synthesis. Here, we will introduce the transfection and thermotolerance methods for analysis of miR-570 targeting the HSP chaperone network.
Assuntos
MicroRNAs , Termotolerância , Termotolerância/genética , Temperatura Alta , Chaperonas Moleculares/genética , Proteínas de Choque Térmico/metabolismo , MicroRNAs/genética , TransfecçãoRESUMO
Protein homeostasis involves a number of overlapping mechanisms, including the autophagy program, that can lead to the resolution of protein damage. We aimed in this study to examine mechanisms of autophagy in the proteotoxic stress response. We found that such stress results in a rapid elevation in the rate of autophagy in mammalian cells. Induction of this process occurred coincidentally with the increased release of extracellular vesicles (EVs) into the extracellular microenvironment. We next found that purified EVs that had been released from stressed cells were capable of directly increasing autophagic flux in recipient cells. The EVs contained a range of cargo proteins, including HSP70, BAG3, and activated transcription factor phospho-NRF2 (pNRF2). NRF2 regulates the activation of both the oxidative stress response and autophagy genes. Both heat shock and exposure of cells to proteotoxic stress-induced EVs increased the intracellular levels of pNRF2 in cells. Heat shock-induced proteotoxicity also led to increases in the levels of proteins in the oxidative stress response, including HO-1 and NQO1, as well as the key autophagy proteins LC3, ATG5, and ATG7, known to be regulated by NRF2. Increases in these autophagy proteins were dependent on the expression of NRF2 and were ablated by NRF2 knockdown.
Assuntos
Vesículas Extracelulares , Fator 2 Relacionado a NF-E2 , Autofagia/genética , Vesículas Extracelulares/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/fisiologia , Estresse Proteotóxico , HumanosRESUMO
Cell communication network factor 2 (CCN2), also known as connective tissue growth factor (CTGF), is protein inducible in response to TGFß/Smad signal or the transcriptional activity of matrix metalloproteinase 3 (MMP3). We discovered that MMP3 in exosomes is transferable to recipient cells and then translocates into cell nuclei to transactivate the CCN2/CTGF gene. Exosomes and liposomes enable molecular transfection to recipient cells in vitro and in vivo. These small vesicles are surrounded by lipid membranes and carry proteins, RNA, DNA, and small chemicals. Here we define the exosome-based transfection as "exofection." In addition, spinfection increases the efficiencies of transfection, exofection, and viral infection, thus being compatible with various molecular transfer protocols. Here, we provide protocols, tips, and practical examples of transfection, spinfection, exofection, fluorescence microscopy, and luciferase assays to analyze the CCNs gene expression mechanisms.
Assuntos
Metaloproteinase 3 da Matriz , Transdução de Sinais , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 3 da Matriz/metabolismo , Transdução de Sinais/fisiologia , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Transfecção , Luciferases/genética , Luciferases/metabolismo , Fator de Crescimento Transformador beta/genéticaRESUMO
Cellular Communication Network (CCN) proteins are growth factors that play key roles in many pathophysiological events, including bone formation, wound healing, and cancer. CCN factors and fragments generated by metalloproteinases-dependent cleavage are often associated with extracellular matrix (ECM) or small extracellular vesicles (sEVs) such as exosomes or matrix-coated vesicles. We provide reliable methods and protocols for Western blotting to analyze CCN factors and fragments in cells, sEVs, and vesicle-free fractions.
Assuntos
Exossomos , Vesículas Extracelulares , Exossomos/metabolismo , Proteínas de Sinalização Intercelular CCN/metabolismo , Comunicação Celular , Matriz Extracelular/metabolismo , Vesículas Extracelulares/metabolismo , Western BlottingRESUMO
Cellular Communication Network (CCN) proteins are secretory growth factors often associated with extracellular matrix (ECM) and extracellular vesicles (EVs) such as exosomes or matrix-coated vesicles. CCN factors and fragments loaded on/in EVs may play key roles in cell communication networks in cancer biology, bone and cartilage metabolism, wound healing, and tissue regeneration. CCN proteins and EVs/exosomes are found in body fluids, such as blood, urine, milk, and supernatants of the two-dimensionally (2D) cultured cells and three-dimensionally (3D) cultured tissues, such as spheroids or organoids. More than ten methods to isolate exosomes or EVs have been developed with different properties. Here, we introduce comprehensive protocols for polymer-based precipitation, affinity purification, ultracentrifugation methods combined with the ultrafiltration method for isolating CCN-loaded exosomes/EVs from 2D and 3D cultured tissues, and proteome analysis using mass spectrometry for comprehensive analysis of CCN proteins.
Assuntos
Exossomos , Vesículas Extracelulares , Exossomos/metabolismo , Proteoma/metabolismo , Ultracentrifugação/métodos , Vesículas Extracelulares/metabolismo , Comunicação CelularRESUMO
Heat Shock Proteins (HSPs) and their co-chaperones have well-established roles in regulating proteostasis within the cell, the nature of which continues to emerge with further study. To date, HSPs have been shown to be integral to protein folding and re-folding, protein transport, avoidance of protein aggregation, and modulation of protein degradation. Many cell signaling events are mediated by the chemical modification of proteins post-translationally that can alter protein conformation and activity, although it is not yet known whether the changes in protein conformation induced by post-translational modifications (PTMs) are also dependent upon HSPs and their co-chaperones for subsequent protein re-folding. We discuss what is known regarding roles for HSPs and other molecular chaperones in cell signaling events with a focus on oncogenic signaling. We also propose a hypothesis by which Hsp70 and Hsp90 may co-operate to facilitate cell signaling events that may link PTMs with the cellular protein folding machinery.
Assuntos
Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Neoplasias/patologia , Proteostase , Transdução de Sinais , Animais , Humanos , Neoplasias/metabolismoRESUMO
Delivery of exogenous heat shock protein 90α (Hsp90α) and/or its induced expression in neural tissues has been suggested as a potential strategy to combat neurodegenerative disease. However, within a neurodegenerative context, a pro-inflammatory response to extracellular Hsp90α (eHsp90α) could undermine strategies to use it for therapeutic intervention. The aim of this study was to investigate the biological effects of eHsp90α on microglial cells, the primary mediators of inflammatory responses in the brain. Transcriptomic profiling by RNA-seq of primary microglia and the cultured EOC2 microglial cell line treated with eHsp90α showed the chaperone to stimulate activation of innate immune responses in microglia that were characterized by an increase in NF-kB-regulated genes. Further characterization showed this response to be substantially lower in amplitude than the effects of other inflammatory stimuli such as fibrillar amyloid-ß (fAß) or lipopolysaccharide (LPS). Additionally, the toxicity of conditioned media obtained from microglia treated with fAß was attenuated by addition of eHsp90α. Using a co-culture system of microglia and hippocampal neuronal cell line HT22 cells separated by a chamber insert, the neurotoxicity of medium conditioned by microglia treated with fAß was reduced when eHsp90α was also added. Mechanistically, eHsp90α was shown to activate Nrf2, a response which attenuated fAß-induced nitric oxide production. The data thus suggested that eHsp90α protects against fAß-induced oxidative stress. We also report eHsp90α to induce expression of macrophage receptor with collagenous structure (Marco), which would permit receptor-mediated endocytosis of fAß.
Assuntos
Microglia , Doenças Neurodegenerativas , Peptídeos beta-Amiloides/toxicidade , Meios de Cultivo Condicionados/farmacologia , Proteínas de Choque Térmico HSP90 , Proteínas de Choque Térmico/metabolismo , Humanos , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/toxicidade , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Doenças Neurodegenerativas/metabolismo , Óxido Nítrico/metabolismo , Estresse OxidativoRESUMO
The dynamic network of chaperone interactions known as the chaperome contributes significantly to the proteotoxic cell response and the malignant phenotype. To bypass the inherent redundancy in the network, we have used a microRNA (mir) approach to target multiple members of the chaperome simultaneously. We identified a potent microRNA, miR-570 that could bind the 3'untranslated regions of multiple HSP mRNAs and inhibit HSP synthesis. Transfection of cells with this miR species reduced expression of multiple HSPs, inhibited the heat shock response and reduced tumor cell growth while acted additively in combination with cytotoxic drugs. As overexpression of miR-570 elicited tumor suppressive effects, we inferred that this miR could play a potential role in inhibiting tumorigenesis and cancer cell growth. In accordance with this hypothesis, we determined a significant role for miR-570 in regulating markers of mammary tumor progression, including cell motility and invasion. Our data provide a proof of the principle that the tumor chaperome can be targeted by microRNAs suggesting a potential therapeutic avenue towards cancer therapy.
Assuntos
Regulação Neoplásica da Expressão Gênica , MicroRNAs , Linhagem Celular Tumoral , Movimento Celular , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Invasividade Neoplásica/genética , Regiões não TraduzidasRESUMO
Researchers have developed several three-dimensional (3D) culture systems, including spheroids, organoids, and tumoroids with increased properties of cancer stem cells (CSCs), also called cancer-initiating cells (CICs). Drug resistance is a crucial issue involving recurrence in cancer patients. Many studies on anti-cancer drugs have been reported using 2D culture systems, whereas 3D cultured tumoroids have many advantages for assessing drug sensitivity and resistance. Here, we aimed to investigate whether Cisplatin (a DNA crosslinker), Imatinib (a multiple tyrosine kinase inhibitor), and 5-Fluorouracil (5-FU: an antimetabolite) alter the tumoroid growth of metastatic colorectal cancer (mCRC). Gene expression signatures of highly metastatic aggregative CRC (LuM1 cells) vs. low-metastatic, non-aggregative CRC (Colon26 and NM11 cells) were analyzed using microarray. To establish a 3D culture-based multiplexing reporter assay system, LuM1 was stably transfected with the Mmp9 promoter-driven ZsGreen fluorescence reporter gene, which was designated as LuM1/m9 cells and cultured in NanoCulture Plate®, a gel-free 3D culture device. LuM1 cells highly expressed mRNA encoding ABCG2 (a drug resistance pump, i.e., CSC/CIC marker), other CSC/CIC markers (DLL1, EpCAM, podoplanin, STAT3/5), pluripotent stem cell markers (Sox4/7, N-myc, GATA3, Nanog), and metastatic markers (MMPs, Integrins, EGFR), compared to the other two cell types. Hoechst efflux stem cell-like side population was increased in LuM1 (7.8%) compared with Colon26 (2.9%), both of which were markedly reduced by verapamil treatment, an ABCG2 inhibitor. Smaller cell aggregates of LuM1 were more sensitive to Cisplatin (at 10 µM), whereas larger tumoroids with increased ABCG2 expression were insensitive. Notably, Cisplatin (2 µM) and Imatinib (10 µM) at low concentrations significantly promoted tumoroid formation (cell aggregation) and increased Mmp9 promoter activity in mCRC LuM1/m9, while not cytotoxic to them. On the other hand, 5-FU significantly inhibited tumoroid growth, although not completely. Thus, drug resistance in cancer with increased stem cell properties was modeled using the gel-free 3D cultured tumoroid system. The tumoroid culture is useful and easily accessible for the assessment of drug sensitivity and resistance.