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1.
Proc Natl Acad Sci U S A ; 116(14): 6766-6774, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30877242

RESUMO

Focal adhesion kinase (FAK) is a key signaling molecule regulating cell adhesion, migration, and survival. FAK localizes into focal adhesion complexes formed at the cytoplasmic side of cell attachment to the ECM and is activated after force generation via actomyosin fibers attached to this complex. The mechanism of translating mechanical force into a biochemical signal is not understood, and it is not clear whether FAK is activated directly by force or downstream to the force signal. We use experimental and computational single-molecule force spectroscopy to probe the mechanical properties of FAK and examine whether force can trigger activation by inducing conformational changes in FAK. By comparison with an open and active mutant of FAK, we are able to assign mechanoactivation to an initial rupture event in the low-force range. This activation event occurs before FAK unfolding at forces within the native range in focal adhesions. We are also able to assign all subsequent peaks in the force landscape to partial unfolding of FAK modules. We show that binding of ATP stabilizes the kinase domain, thereby altering the unfolding hierarchy. Using all-atom molecular dynamics simulations, we identify intermediates along the unfolding pathway, which provide buffering to allow extension of FAK in focal adhesions without compromising functionality. Our findings strongly support that forces in focal adhesions applied to FAK via known interactions can induce conformational changes, which in turn, trigger focal adhesion signaling.


Assuntos
Trifosfato de Adenosina/química , Proteínas Aviárias/química , Proteína-Tirosina Quinases de Adesão Focal/química , Simulação de Dinâmica Molecular , Desdobramento de Proteína , Trifosfato de Adenosina/metabolismo , Animais , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Galinhas , Ativação Enzimática , Proteína-Tirosina Quinases de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Adesões Focais/enzimologia , Adesões Focais/genética , Mecanotransdução Celular/genética , Domínios Proteicos , Relação Estrutura-Atividade
2.
Mech Dev ; 156: 8-19, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30796970

RESUMO

Blood vessel maturation, which is characterized by the investment of vascular smooth muscle cells (vSMCs) around developing blood vessels, begins when vessels remodel into a hierarchy of proximal arteries and proximal veins that branch into smaller distal capillaries. The ultimate result of maturation is formation of the tunica media-the middlemost layer of a vessel that is composed of vSMCs and acts to control vessel integrity and vascular tone. Though many studies have implicated the role of various signaling molecules in regulating maturation, no studies have determined a role for hemodynamic force in the regulation of maturation in the mouse. In the current study, we provide evidence that a hemodynamic force-dependent mechanism occurs in the mouse because reduced blood flow mouse embryos exhibited a diminished or absent coverage of vSMCs around vessels, and in normal-flow embryos, extent of coverage correlated to the amount of blood flow that vessels were exposed to. We also determine that the cellular mechanism of force-induced maturation was not by promoting vSMC differentiation/proliferation, but instead involved the recruitment of vSMCs away from neighboring low-flow distal capillaries towards high-flow vessels. Finally, we hypothesize that hemodynamic force may regulate expression of specific signaling molecules to control vSMC recruitment to high-flow vessels, as reduction of flow results in the misexpression of Semaphorin 3A, 3F, 3G, and the Notch target gene Hey1, all of which are implicated in controlling vessel maturation. This study reveals another role for hemodynamic force in regulating blood vessel development of the mouse, and opens up a new model to begin elucidating mechanotransduction pathways regulating vascular maturation.


Assuntos
Vasos Sanguíneos/crescimento & desenvolvimento , Diferenciação Celular/genética , Desenvolvimento Embrionário/genética , Músculo Liso Vascular/crescimento & desenvolvimento , Animais , Artérias/crescimento & desenvolvimento , Artérias/metabolismo , Vasos Sanguíneos/metabolismo , Proliferação de Células/genética , Hemodinâmica , Mecanotransdução Celular/genética , Camundongos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo
3.
Proc Natl Acad Sci U S A ; 116(9): 3502-3507, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30755531

RESUMO

Breast cancer development is associated with increasing tissue stiffness over years. To more accurately mimic the onset of gradual matrix stiffening, which is not feasible with conventional static hydrogels, mammary epithelial cells (MECs) were cultured on methacrylated hyaluronic acid hydrogels whose stiffness can be dynamically modulated from "normal" (<150 Pascals) to "malignant" (>3,000 Pascals) via two-stage polymerization. MECs form and remain as spheroids, but begin to lose epithelial characteristics and gain mesenchymal morphology upon matrix stiffening. However, both the degree of matrix stiffening and culture time before stiffening play important roles in regulating this conversion as, in both cases, a subset of mammary spheroids remained insensitive to local matrix stiffness. This conversion depended neither on colony size nor cell density, and MECs did not exhibit "memory" of prior niche when serially cultured through cycles of compliant and stiff matrices. Instead, the transcription factor Twist1, transforming growth factor ß (TGFß), and YAP activation appeared to modulate stiffness-mediated signaling; when stiffness-mediated signals were blocked, collective MEC phenotypes were reduced in favor of single MECs migrating away from spheroids. These data indicate a more complex interplay of time-dependent stiffness signaling, spheroid structure, and soluble cues that regulates MEC plasticity than suggested by previous models.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias da Mama/genética , Mecanotransdução Celular/genética , Fosfoproteínas/genética , Fator de Crescimento Transformador beta/genética , Proteína 1 Relacionada a Twist/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/genética , Feminino , Humanos , Hidrogéis/química , Comunicação Parácrina/genética , Transdução de Sinais/genética , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia
4.
EMBO Rep ; 20(1)2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30538117

RESUMO

The mechanical properties of the tumor microenvironment are emerging as attractive targets for the development of therapies. Tamoxifen, an agonist of the G protein-coupled estrogen receptor (GPER), is widely used to treat estrogen-positive breast cancer. Here, we show that tamoxifen mechanically reprograms the tumor microenvironment through a newly identified GPER-mediated mechanism. Tamoxifen inhibits the myofibroblastic differentiation of pancreatic stellate cells (PSCs) in the tumor microenvironment of pancreatic cancer in an acto-myosin-dependent manner via RhoA-mediated contractility, YAP deactivation, and GPER signaling. This hampers the ability of PSCs to remodel the extracellular matrix and to promote cancer cell invasion. Tamoxifen also reduces the recruitment and polarization to the M2 phenotype of tumor-associated macrophages. Our results highlight GPER as a mechanical regulator of the tumor microenvironment that targets the three hallmarks of pancreatic cancer: desmoplasia, inflammation, and immune suppression. The well-established safety of tamoxifen in clinics may offer the possibility to redirect the singular focus of tamoxifen on the cancer cells to the greater tumor microenvironment and lead a new strategy of drug repurposing.


Assuntos
Neoplasias Pancreáticas/tratamento farmacológico , Células Estreladas do Pâncreas/efeitos dos fármacos , Receptores Estrogênicos/genética , Receptores Acoplados a Proteínas-G/genética , Tamoxifeno/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Polaridade Celular/efeitos dos fármacos , Humanos , Inflamação/tratamento farmacológico , Inflamação/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Mecanotransdução Celular/genética , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Células Estreladas do Pâncreas/patologia , Fosfoproteínas/genética , Microambiente Tumoral/efeitos dos fármacos
5.
Mem. Inst. Oswaldo Cruz ; 114: e180593, 2019. graf
Artigo em Inglês | LILACS | ID: biblio-1020079

RESUMO

BACKGROUND Cardiac physiology depends on coupling and electrical and mechanical coordination through the intercalated disc. Focal adhesions offer mechanical support and signal transduction events during heart contraction-relaxation processes. Talin links integrins to the actin cytoskeleton and serves as a scaffold for the recruitment of other proteins, such as paxillin in focal adhesion formation and regulation. Chagasic cardiomyopathy is caused by infection by Trypanosoma cruzi and is a debilitating condition comprising extensive fibrosis, inflammation, cardiac hypertrophy and electrical alterations that culminate in heart failure. OBJECTIVES Since mechanotransduction coordinates heart function, we evaluated the underlying mechanism implicated in the mechanical changes, focusing especially in mechanosensitive proteins and related signalling pathways during infection of cardiac cells by T. cruzi. METHODS We investigated the effect of T. cruzi infection on the expression and distribution of talin/paxillin and associated proteins in mouse cardiomyocytes in vitro by western blotting, immunofluorescence and quantitative real-time polymerase chain reaction (qRT-PCR). FINDINGS Talin and paxillin spatial distribution in T. cruzi-infected cardiomyocytes in vitro were altered associated with a downregulation of these proteins and mRNAs levels at 72 h post-infection (hpi). Additionally, we observed an increase in the activation of the focal adhesion kinase (FAK) concomitant with increase in β-1-integrin at 24 hpi. Finally, we detected a decrease in the activation of FAK at 72 hpi in T. cruzi-infected cultures. MAIN CONCLUSION The results suggest that these changes may contribute to the mechanotransduction disturbance evidenced in chagasic cardiomyopathy.


Assuntos
Animais , Camundongos , Trypanosoma cruzi/fisiologia , Cardiomiopatia Chagásica/metabolismo , Miócitos Cardíacos/parasitologia , Mecanotransdução Celular/genética , Western Blotting , Reação em Cadeia da Polimerase , Imunofluorescência , Paxilina/metabolismo
6.
Science ; 360(6388): 530-533, 2018 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-29724954

RESUMO

The somatosensory system relays many signals ranging from light touch to pain and itch. Touch is critical to spatial awareness and communication. However, in disease states, innocuous mechanical stimuli can provoke pathologic sensations such as mechanical itch (alloknesis). The molecular and cellular mechanisms that govern this conversion remain unknown. We found that in mice, alloknesis in aging and dry skin is associated with a loss of Merkel cells, the touch receptors in the skin. Targeted genetic deletion of Merkel cells and associated mechanosensitive Piezo2 channels in the skin was sufficient to produce alloknesis. Chemogenetic activation of Merkel cells protected against alloknesis in dry skin. This study reveals a previously unknown function of the cutaneous touch receptors and may provide insight into the development of alloknesis.


Assuntos
Canais Iônicos/fisiologia , Mecanotransdução Celular/fisiologia , Células de Merkel/fisiologia , Prurido/fisiopatologia , Pele/inervação , Tato , Animais , Deleção de Genes , Canais Iônicos/genética , Mecanotransdução Celular/genética , Células de Merkel/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Prurido/genética
7.
Biochem Biophys Res Commun ; 500(4): 937-943, 2018 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-29709477

RESUMO

Accumulating evidence has shown that matrix stiffening in cancer tissue by the deposition of extracellular matrix (ECM) is closely related with severe tumor progression. However, much less is known about the genes affected by matrix stiffness and its signaling for cancer progression. In the current research, we investigated the differential gene expression of a non-small lung adenocarcinoma cell line, H1299, cultured under the conditions of soft (∼0.5 kPa) and stiff (∼40 kPa) matrices, mimicking the mechanical environments of normal and cancerous tissues, respectively. For integrated transcriptome analysis, the genes identified by ECM stiffening were compared with 8248 genes retrieved from The Cancer Genome Atlas Lung Adenocarcinoma (TCGA). In stiff matrix, 29 genes were significantly upregulated, while 75 genes were downregulated. The screening of hazard ratios for these genes using the Kaplan-Meier Plotter identified 8 genes most closely associated with cancer progression under the condition of matrix stiffening. Among these genes, spindle pole body component 25 homolog (SPC25) was one of the most up-regulated genes in stiff matrix and tumor tissue. Knockdown of SPC25 in H1299 cells using shRNA significantly inhibited cell proliferation with downregulation of the expression of checkpoint protein, Cyclin B1, under the condition of stiff matrix whereas the proliferation rate in soft matrix was not affected by SPC25 silencing. Thus, our findings provide novel key molecules for studying the relationship of extracellular matrix stiffening and cancer progression.


Assuntos
Proliferação de Células/genética , Matriz Extracelular/química , Regulação Neoplásica da Expressão Gênica , Mecanotransdução Celular/genética , Proteínas Associadas aos Microtúbulos/genética , Mucosa Respiratória/metabolismo , Atlas como Assunto , Fenômenos Biomecânicos , Ciclo Celular/genética , Linhagem Celular Tumoral , Ciclina B1/genética , Ciclina B1/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Perfilação da Expressão Gênica , Células HEK293 , Dureza , Humanos , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/metabolismo , Anotação de Sequência Molecular , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Mucosa Respiratória/patologia , Transcriptoma
8.
PLoS One ; 13(1): e0188764, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29385174

RESUMO

Audible sound is a ubiquitous environmental factor in nature that transmits oscillatory compressional pressure through the substances. To investigate the property of the sound as a mechanical stimulus for cells, an experimental system was set up using 94.0 dB sound which transmits approximately 10 mPa pressure to the cultured cells. Based on research on mechanotransduction and ultrasound effects on cells, gene responses to the audible sound stimulation were analyzed by varying several sound parameters: frequency, wave form, composition, and exposure time. Real-time quantitative PCR analyses revealed a distinct suppressive effect for several mechanosensitive and ultrasound-sensitive genes that were triggered by sounds. The effect was clearly observed in a wave form- and pressure level-specific manner, rather than the frequency, and persisted for several hours. At least two mechanisms are likely to be involved in this sound response: transcriptional control and RNA degradation. ST2 stromal cells and C2C12 myoblasts exhibited a robust response, whereas NIH3T3 cells were partially and NB2a neuroblastoma cells were completely insensitive, suggesting a cell type-specific response to sound. These findings reveal a cell-level systematic response to audible sound and uncover novel relationships between life and sound.


Assuntos
Estimulação Acústica , Regulação da Expressão Gênica , Mecanotransdução Celular/genética , Som , Animais , Linhagem Celular , Camundongos , Reação em Cadeia da Polimerase em Tempo Real
9.
BMB Rep ; 51(3): 151-156, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29353600

RESUMO

The Hippo signaling pathway controls nuclear accumulation and stability of the transcriptional coregulator YAP and its paralog TAZ. The activity of Hippo-YAP signaling is influenced not only by biochemical signals, but also by cell shape and mechanical tension transmitted through cell-cell junctions and cell-matrix adhesions. Data accumulated thus far indicates that the actin cytoskeleton is a key mediator of the regulation of Hippo-YAP signaling by means of a variety of biochemical and mechanical cues. In this review, we have outlined the role of actin dynamics and actin-associated proteins in the regulation of Hippo-YAP signaling. In addition, we discuss actinmediated regulation of YAP/TAZ activity independent of the core Hippo kinases MST and LATS. Although our understanding of the link between Hippo-YAP signaling and the actin cytoskeleton is progressing rapidly, many open questions remain. [BMB Reports 2018; 51(3): 151-156].


Assuntos
Actinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Humanos , Mecanotransdução Celular/genética , Mecanotransdução Celular/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
10.
Proc Natl Acad Sci U S A ; 115(5): 992-997, 2018 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-29343642

RESUMO

While cell-based immunotherapy, especially chimeric antigen receptor (CAR)-expressing T cells, is becoming a paradigm-shifting therapeutic approach for cancer treatment, there is a lack of general methods to remotely and noninvasively regulate genetics in live mammalian cells and animals for cancer immunotherapy within confined local tissue space. To address this limitation, we have identified a mechanically sensitive Piezo1 ion channel (mechanosensor) that is activatable by ultrasound stimulation and integrated it with engineered genetic circuits (genetic transducer) in live HEK293T cells to convert the ultrasound-activated Piezo1 into transcriptional activities. We have further engineered the Jurkat T-cell line and primary T cells (peripheral blood mononuclear cells) to remotely sense the ultrasound wave and transduce it into transcriptional activation for the CAR expression to recognize and eradicate target tumor cells. This approach is modular and can be extended for remote-controlled activation of different cell types with high spatiotemporal precision for therapeutic applications.


Assuntos
Imunoterapia/métodos , Neoplasias/terapia , Animais , Fenômenos Biomecânicos , Sinalização do Cálcio , Genes Sintéticos , Engenharia Genética , Técnicas Genéticas , Células HEK293 , Humanos , Canais Iônicos/genética , Canais Iônicos/imunologia , Células Jurkat , Mecanotransdução Celular/genética , Mecanotransdução Celular/imunologia , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Neoplasias/genética , Neoplasias/imunologia , Biologia Sintética , Linfócitos T/imunologia , Ultrassom
11.
Cell Rep ; 21(10): 2714-2723, 2017 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-29212020

RESUMO

α-Actinins, a family of critical cytoskeletal actin-binding proteins that usually exist as anti-parallel dimers, play crucial roles in organizing the framework of the cytoskeleton through crosslinking the actin filaments, as well as in focal adhesion maturation. However, the molecular mechanisms underlying its functions are unclear. Here, by mechanical manipulation of single human α-actinin 1 using magnetic tweezers, we determined the mechanical stability and kinetics of the functional domains in α-actinin 1. Moreover, we identified the force-dependence of vinculin binding to α-actinin 1, with the demonstration that force is required to expose the high-affinity binding site for vinculin binding. Further, a role of the α-actinin 1 as molecular shock absorber for the cytoskeleton network is revealed. Our results provide a comprehensive analysis of the force-dependent stability and interactions of α-actinin 1, which sheds important light on the molecular mechanisms underlying its mechanotransmission and mechanosensing functions.


Assuntos
Actinina/metabolismo , Actinina/química , Citoesqueleto/metabolismo , Humanos , Cinética , Nanopartículas de Magnetita/química , Mecanotransdução Celular/genética , Mecanotransdução Celular/fisiologia , Vinculina/química
12.
Cell Rep ; 21(10): 2760-2771, 2017 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-29212024

RESUMO

Piezo2 is a mechanically activated ion channel required for touch discrimination, vibration detection, and proprioception. Here, we discovered that Piezo2 is extensively spliced, producing different Piezo2 isoforms with distinct properties. Sensory neurons from both mice and humans express a large repertoire of Piezo2 variants, whereas non-neuronal tissues express predominantly a single isoform. Notably, even within sensory ganglia, we demonstrate the splicing of Piezo2 to be cell type specific. Biophysical characterization revealed substantial differences in ion permeability, sensitivity to calcium modulation, and inactivation kinetics among Piezo2 splice variants. Together, our results describe, at the molecular level, a potential mechanism by which transduction is tuned, permitting the detection of a variety of mechanosensory stimuli.


Assuntos
Canais Iônicos/metabolismo , Mecanotransdução Celular/fisiologia , Processamento Alternativo/genética , Animais , Eletrofisiologia , Feminino , Células HEK293 , Humanos , Hibridização In Situ , Canais Iônicos/genética , Masculino , Mecanotransdução Celular/genética , Camundongos , Camundongos Endogâmicos C57BL , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Processamento de RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
13.
Nat Commun ; 8(1): 2234, 2017 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-29269857

RESUMO

Detection of water motion by the lateral line relies on mechanotransduction complexes at stereocilia tips. This sensory system is comprised of neuromasts, patches of hair cells with stereociliary bundles arranged with morphological mirror symmetry that are mechanically responsive to two opposing directions. Here, we find that transmembrane channel-like 2b (Tmc2b) is differentially required for mechanotransduction in the zebrafish lateral line. Despite similarities in neuromast hair cell morphology, three classes of these cells can be distinguished by their Tmc2b reliance. We map mechanosensitivity along the lateral line using imaging and electrophysiology to determine that a hair cell's Tmc2b dependence is governed by neuromast topological position and hair bundle orientation. Overall, water flow is detected by molecular machinery that can vary between hair cells of different neuromasts. Moreover, hair cells within the same neuromast can break morphologic symmetry of the sensory organ at the stereocilia tips.


Assuntos
Sistema da Linha Lateral/metabolismo , Mecanorreceptores/metabolismo , Mecanotransdução Celular/genética , Proteínas de Membrana/metabolismo , Movimento (Física) , Água , Proteínas de Peixe-Zebra/metabolismo , Animais , Sistema da Linha Lateral/fisiologia , Mecanorreceptores/fisiologia , Mecanotransdução Celular/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologia
14.
Int J Mol Sci ; 19(1)2017 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-29283422

RESUMO

Diabetes mellitus is associated with damage to tendons, which may result from cellular dysfunction in response to a hyperglycemic environment. Tenocytes express diminished levels of tendon-associated genes under hyperglycemic conditions. In contrast, mechanical stretch enhances tenogenic differentiation. However, whether hyperglycemia increases the non-tenogenic differentiation potential of tenocytes and whether this can be mitigated by mechanical stretch remains elusive. We explored the in vitro effects of high glucose and mechanical stretch on rat primary tenocytes. Specifically, non-tenogenic gene expression, adipogenic potential, cell migration rate, filamentous actin expression, and the activation of signaling pathways were analyzed in tenocytes treated with high glucose, followed by the presence or absence of mechanical stretch. We analyzed tenocyte phenotype in vivo by immunohistochemistry using an STZ (streptozotocin)-induced long-term diabetic mouse model. High glucose-treated tenocytes expressed higher levels of the adipogenic transcription factors PPARγ and C/EBPs. PPARγ was also highly expressed in diabetic tendons. In addition, increased adipogenic differentiation and decreased cell migration induced by high glucose implicated a fibroblast-to-adipocyte phenotypic change. By applying mechanical stretch to tenocytes in high-glucose conditions, adipogenic differentiation was repressed, while cell motility was enhanced, and fibroblastic morphology and gene expression profiles were strengthened. In part, these effects resulted from a stretch-induced activation of ERK (extracellular signal-regulated kinases) and a concomitant inactivation of Akt. Our results show that mechanical stretch alleviates the augmented adipogenic transdifferentiation potential of high glucose-treated tenocytes and helps maintain their fibroblastic characteristics. The alterations induced by high glucose highlight possible pathological mechanisms for diabetic tendinopathy. Furthermore, the beneficial effects of mechanical stretch on tenocytes suggest that an appropriate physical load possesses therapeutic potential for diabetic tendinopathy.


Assuntos
Adipócitos/efeitos dos fármacos , Diabetes Mellitus Experimental/terapia , Glucose/farmacologia , Mecanotransdução Celular/genética , Estresse Mecânico , Tenócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adipócitos/patologia , Adipogenia/efeitos dos fármacos , Adipogenia/genética , Animais , Fenômenos Biomecânicos , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Transdiferenciação Celular/efeitos dos fármacos , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Regulação da Expressão Gênica , Masculino , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , PPAR gama/genética , PPAR gama/metabolismo , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Estreptozocina , Tendões/efeitos dos fármacos , Tendões/metabolismo , Tendões/patologia , Tenócitos/metabolismo , Tenócitos/patologia
15.
Nat Commun ; 8(1): 2145, 2017 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-29247198

RESUMO

The shape of a cell within tissues can represent the history of chemical and physical signals that it encounters, but can information from cell shape regulate cellular phenotype independently? Using optimal control theory to constrain reaction-diffusion schemes that are dependent on different surface-to-volume relationships, we find that information from cell shape can be resolved from mechanical signals. We used microfabricated 3-D biomimetic chips to validate predictions that shape-sensing occurs in a tension-independent manner through integrin ß3 signaling pathway in human kidney podocytes and smooth muscle cells. Differential proteomics and functional ablation assays indicate that integrin ß3 is critical in transduction of shape signals through ezrin-radixin-moesin (ERM) family. We used experimentally determined diffusion coefficients and experimentally validated simulations to show that shape sensing is an emergent cellular property enabled by multiple molecular characteristics of integrin ß3. We conclude that 3-D cell shape information, transduced through tension-independent mechanisms, can regulate phenotype.


Assuntos
Forma Celular/fisiologia , Mecanotransdução Celular/fisiologia , Miócitos de Músculo Liso/fisiologia , Podócitos/fisiologia , Estresse Mecânico , Animais , Animais Recém-Nascidos , Células COS , Forma Celular/genética , Células Cultivadas , Cercopithecus aethiops , Proteínas do Citoesqueleto/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Humanos , Integrina beta3/genética , Integrina beta3/metabolismo , Mecanotransdução Celular/genética , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Podócitos/citologia , Podócitos/metabolismo , Proteômica/métodos , Ratos
16.
Stem Cell Res ; 25: 107-114, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29125993

RESUMO

Physical cues are major determinants of cellular phenotype and evoke physiological and pathological responses on cell structure and function. Cellular models aim to recapitulate basic functional features of their in vivo counterparts or tissues in order to be of use in in vitro disease modeling or drug screening and testing. Understanding how culture systems affect in vitro development of human pluripotent stem cell (hPSC)-derivatives allows optimization of cellular human models and gives insight in the processes involved in their structural organization and function. In this work, we show involvement of the mechanotransduction pathway RhoA/ROCK in the structural reorganization of hPSC-derived cardiomyocytes after adhesion plating. These structural changes have a major impact on the intracellular localization of SERCA2 pumps and concurrent improvement in calcium cycling. The process is triggered by cell interaction with the culture substrate, which mechanical cues drive sarcomeric alignment and SERCA2a spreading and relocalization from a perinuclear to a whole-cell distribution. This structural reorganization is mediated by the mechanical properties of the substrate, as shown by the process failure in hPSC-CMs cultured on soft 4kPa hydrogels as opposed to physiologically stiff 16kPa hydrogels and glass. Finally, pharmacological inhibition of Rho-associated protein kinase (ROCK) by different compounds identifies this specific signaling pathway as a major player in SERCA2 localization and the associated improvement in hPSC-CMs calcium handling ability in vitro.


Assuntos
Miócitos Cardíacos/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Humanos , Mecanotransdução Celular/genética , Mecanotransdução Celular/fisiologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética
17.
Biochem Biophys Res Commun ; 494(3-4): 663-667, 2017 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-29061307

RESUMO

Mechanical stimuli such as fluid shear and cyclic tension force induced extracellular adenosine triphosphate (ATP) release in osteoblasts. In particular, cyclic tension force-induced ATP enhances bone formation through P2X7 activation. Proline-rich tyrosine kinase 2 (PYK2) mediate osteoblasts differentiation is induced by mechanical stimuli. Furthermore, activation of PYK2 also was a response to integrin by mechanical stimuli. Extracellular matrix protein (ECMP)s, which are important factors for bone formation are expressed by osteoblasts. However, the effect of the interaction of 2'(3)-Ο-(4-Benzoylbenzoyl) adenosine-5'-triphosphate (BzATP), which is the agonist of the mechanosensitive receptor P2X7, with PYK2 on ECMP production is poorly understood. Thus, our purpose was to investigate the effects of PYK2 on BzATP-induced ECMP production in osteoblasts. BzATP increased phospho-PYK2 protein expression on days 3 and 7 of culture. Furthermore, the PYK2 inhibitor PF431394 inhibited the stimulatory effect of BzATP on the expression of type I collagen, bone sialoprotein and osteocalcin expression. PF431396 did not inhibit the stimulatory effect of BzATP on osteopontin (OPN) mRNA expression. These results suggest that mechanical stimuli activate P2X7 might induce ECMPs expression through PYK2 except in the case of OPN expression. Altogether, mechanical stimuli-induced ECMPs production might be implicated by extracellular ATP secretion or integrin via PYK2 activation.


Assuntos
Trifosfato de Adenosina/análogos & derivados , Proteínas da Matriz Extracelular/biossíntese , Matriz Extracelular/fisiologia , Quinase 2 de Adesão Focal/metabolismo , Mecanotransdução Celular/fisiologia , Osteoblastos/fisiologia , Piranos/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Células 3T3 BALB , Matriz Extracelular/efeitos dos fármacos , Mecanotransdução Celular/genética , Camundongos , Osteoblastos/efeitos dos fármacos , Piranos/agonistas
18.
Cancer Res ; 77(22): 6179-6189, 2017 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-28972074

RESUMO

In response to chemical stimuli from cancer cells, mesenchymal stem cells (MSC) can differentiate into cancer-associated fibroblasts (CAF) and promote tumor progression. How mechanical stimuli such as stiffness of the extracellular matrix (ECM) contribute to MSC phenotype in cancer remains poorly understood. Here, we show that ECM stiffness leads to mechano-signal transduction in MSC, which promotes mammary tumor growth in part through secretion of the signaling protein prosaposin. On a stiff matrix, MSC cultured with conditioned media from mammary cancer cells expressed increased levels of α-smooth muscle actin, a marker of CAF, compared with MSC cultured on a soft matrix. By contrast, MSC cultured on a stiff matrix secreted prosaposin that promoted proliferation and survival of mammary carcinoma cells but inhibited metastasis. Our findings suggest that in addition to chemical stimuli, increased stiffness of the ECM in the tumor microenvironment induces differentiation of MSC to CAF, triggering enhanced proliferation and survival of mammary cancer cells. Cancer Res; 77(22); 6179-89. ©2017 AACR.


Assuntos
Proliferação de Células , Neoplasias Mamárias Experimentais/metabolismo , Células-Tronco Mesenquimais/metabolismo , Saposinas/metabolismo , Animais , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Fibroblastos Associados a Câncer/efeitos dos fármacos , Fibroblastos Associados a Câncer/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular Tumoral , Meios de Cultivo Condicionados/farmacologia , Matriz Extracelular/metabolismo , Humanos , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Mecanotransdução Celular/efeitos dos fármacos , Mecanotransdução Celular/genética , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia de Fluorescência , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética
19.
Stem Cell Res ; 24: 69-76, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28843157

RESUMO

Epidermal growth factors (EGFs) e.g. EGF, heparin-binding EGF and transforming growth factor alpha and their receptors e.g. EGFR and ErbB2 control proinflammatory signaling and modulate proliferation in bone marrow stromal cells (BMSC). Interleukin-6 and interleukin-8 are EGF targets and participate in the inflammatory phase of bone regeneration via non-canonical wnt signaling. BMSC differentiation is also influenced by mechanical strain-related activation of ERK1/2 and AP-1, but the role of EGFR signaling in mechanotransduction is unclear. We investigated the effects of EGFR signaling in telomerase-immortalized BMSC, transfected with a luciferase reporter, comprising a mechanoresponsive AP1 element, using ligands, neutralizing antibodies and EGFR inhibitors on mechanotransduction and we found that EGF via EGFR increased the response to mechanical strain. Results were confirmed by qPCR analysis of mechanoresponsive genes. EGF-responsive interleukin-6 and interleukin-8 were synergistically enhanced by EGF stimulation and mechanical strain. We show here in immortalized and primary BMSC that EGFR signaling enhances mechanotransduction, indicating that the EGF system is a mechanosensitizer in BMSC. Alterations in mechanosensitivity and -adaptation are contributors to age-related diseases like osteoporosis and the identification of a suitable mechanosensitizer could be beneficial. The role of the synergism of these signaling cascades in physiology and disease remains to be unraveled.


Assuntos
Fator de Crescimento Epidérmico/metabolismo , Mecanotransdução Celular , Células-Tronco Mesenquimais/metabolismo , Idoso , Células Cultivadas , Feminino , Genes Reporter , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Ligantes , Masculino , Mecanotransdução Celular/efeitos dos fármacos , Mecanotransdução Celular/genética , Inibidores de Proteínas Quinases/farmacologia , Receptor ErbB-2/metabolismo , Transdução de Sinais , Estresse Mecânico , Telomerase/metabolismo , Fator de Transcrição AP-1/metabolismo
20.
J Neurogenet ; 31(3): 102-112, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28705044

RESUMO

Our ears are remarkable sensory organs, providing the important senses of balance and hearing. The complex structure of the inner ear, or 'labyrinth', along with the assorted neuroepithelia, have evolved to detect head movements and sounds with impressive sensitivity. The rub is that the inner ear is highly vulnerable to genetic lesions and environmental insults. According to National Institute of Health estimates, hearing loss is one of the most commonly inherited or acquired sensorineural diseases. To understand the causes of deafness and balance disorders, it is imperative to understand the underlying biology of the inner ear, especially the inner workings of the sensory receptors. These receptors, which are termed hair cells, are particularly susceptible to genetic mutations - more than two dozen genes are associated with defects in this cell type in humans. Over the past decade, a substantial amount of progress has been made in working out the molecular basis of hair-cell function using vertebrate animal models. Given the transparency of the inner ear and the genetic tools that are available, zebrafish have become an increasingly popular animal model for the study of deafness and vestibular dysfunction. Mutagenesis screens for larval defects in hearing and balance have been fruitful in finding key components, many of which have been implicated in human deafness. This review will focus on the genes that are required for hair-cell function in zebrafish, with a particular emphasis on mechanotransduction. In addition, the generation of new tools available for the characterization of zebrafish hair-cell mutants will be discussed.


Assuntos
Animais Geneticamente Modificados/genética , Células Ciliadas Auditivas/fisiologia , Mutação , Proteínas de Peixe-Zebra/genética , Animais , Genética , Mecanotransdução Celular/genética , Mecanotransdução Celular/fisiologia , Transporte Proteico/genética , Transmissão Sináptica/genética , Peixe-Zebra
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