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GRN mutations cause frontotemporal dementia (GRN-FTD) due to deficiency in progranulin (PGRN), a lysosomal and secreted protein with unclear function. Here, we found that Grn-/- mice exhibit a global deficiency in bis(monoacylglycero)phosphate (BMP), an endolysosomal phospholipid we identified as a pH-dependent PGRN interactor as well as a redox-sensitive enhancer of lysosomal proteolysis and lipolysis. Grn-/- brains also showed an age-dependent, secondary storage of glucocerebrosidase substrate glucosylsphingosine. We investigated a protein replacement strategy by engineering protein transport vehicle (PTV):PGRN-a recombinant protein linking PGRN to a modified Fc domain that binds human transferrin receptor for enhanced CNS biodistribution. PTV:PGRN rescued various Grn-/- phenotypes in primary murine macrophages and human iPSC-derived microglia, including oxidative stress, lysosomal dysfunction, and endomembrane damage. Peripherally delivered PTV:PGRN corrected levels of BMP, glucosylsphingosine, and disease pathology in Grn-/- CNS, including microgliosis, lipofuscinosis, and neuronal damage. PTV:PGRN thus represents a potential biotherapeutic for GRN-FTD.
Assuntos
Produtos Biológicos/uso terapêutico , Encéfalo/metabolismo , Doenças por Armazenamento dos Lisossomos/terapia , Progranulinas/uso terapêutico , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Endossomos/metabolismo , Feminino , Demência Frontotemporal/sangue , Demência Frontotemporal/líquido cefalorraquidiano , Gliose/complicações , Gliose/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Inflamação/patologia , Metabolismo dos Lipídeos , Lipofuscina/metabolismo , Lisossomos/metabolismo , Macrófagos/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/metabolismo , Degeneração Neural/patologia , Fenótipo , Progranulinas/deficiência , Progranulinas/metabolismo , Receptores Imunológicos/metabolismo , Receptores da Transferrina/metabolismo , Distribuição TecidualRESUMO
Mechanical force modulates development, influences tissue homeostasis, and contributes to disease. Forces sculpt tissue-level behaviors and direct cell fate by engaging mechanoreceptors and by altering organization of the cytoskeleton and actomyosin contractility to stimulate mechanotransduction mechanisms that alter transcription. Nevertheless, how force specifically leverages mechanotransduction pathways to control transcriptional regulation of cell fate remains unclear. Here we review recent findings specifically in the context of epithelial-to-mesenchymal transitions that have revealed conserved mechanisms whereby extracellular force, mediated through cell-extracellular matrix and cell-cell junctional complexes, induces transcriptional reprogramming to alter cell and tissue fate. We focus on the interplay between tissue mechanics and the epithelial-to-mesenchymal transitions that occur during embryonic development and cancer malignancy. We describe the adhesion-linked cellular machinery that mediates mechano-transduction and elaborate on how these force-linked networks stimulate key transcriptional programs that induce an epithelial-to-mesenchymal phenotypic transition, thereby providing an overview of how mechanical signals can be translated into a change in cell fate.
Assuntos
Desenvolvimento Embrionário , Transição Epitelial-Mesenquimal , Mecanotransdução Celular , Neoplasias/patologia , Animais , Retroalimentação Fisiológica , Humanos , Transdução de SinaisRESUMO
The past 25 years of genomics research first revealed which genes are encoded by the human genome and then a detailed catalogue of human genome variation associated with many diseases. Despite this, the function of many genes and gene regulatory elements remains poorly characterized, which limits our ability to apply these insights to human disease. The advent of new CRISPR functional genomics tools allows for scalable and multiplexable characterization of genes and gene regulatory elements encoded by the human genome. These approaches promise to reveal mechanisms of gene function and regulation, and to enable exploration of how genes work together to modulate complex phenotypes.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Genoma Humano/genética , Estudo de Associação Genômica Ampla/métodos , Genômica/métodos , Polimorfismo de Nucleotídeo Único , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes/genética , Estudos de Associação Genética/métodos , Genômica/tendências , HumanosRESUMO
All cells sense and integrate mechanical and biochemical cues from their environment to orchestrate organismal development and maintain tissue homeostasis. Mechanotransduction is the evolutionarily conserved process whereby mechanical force is translated into biochemical signals that can influence cell differentiation, survival, proliferation and migration to change tissue behavior. Not surprisingly, disease develops if these mechanical cues are abnormal or are misinterpreted by the cells - for example, when interstitial pressure or compression force aberrantly increases, or the extracellular matrix (ECM) abnormally stiffens. Disease might also develop if the ability of cells to regulate their contractility becomes corrupted. Consistently, disease states, such as cardiovascular disease, fibrosis and cancer, are characterized by dramatic changes in cell and tissue mechanics, and dysregulation of forces at the cell and tissue level can activate mechanosignaling to compromise tissue integrity and function, and promote disease progression. In this Commentary, we discuss the impact of cell and tissue mechanics on tissue homeostasis and disease, focusing on their role in brain development, homeostasis and neural degeneration, as well as in brain cancer.
Assuntos
Encéfalo/crescimento & desenvolvimento , Encéfalo/patologia , Matriz Extracelular/metabolismo , Homeostase , Animais , Fenômenos Biomecânicos , Humanos , Mecanotransdução Celular , Microambiente TumoralRESUMO
The role of extrinsic factors in maintaining self-renewal of embryonic stem cells (ESCs) has been extensively studied since the cells' isolation, but the necessity for cell-secreted factors in self-renewal has remained undefined to date. Although it is generally accepted that addition of leukemia inhibitory factor (LIF) together with either serum or bone morphogenetic protein 4 (BMP4) is sufficient to maintain mouse ESCs (mESCs) in a self-renewing state, this does not preclude the possibility that autocrine factors are also required. Here we make use of a microfluidic perfusion device that is able to globally diminish diffusible autocrine signaling by applying continuous media flow to deplete cell-secreted factors. We demonstrate mESC culture for several days under continuous microfluidic perfusion and show that cell-secreted factors are removed and can be recovered downstream. We find that perturbing cell-secreted signaling causes mESCs to exit their stable self-renewing state in defined conditions that normally support self-renewal and to exhibit properties characteristic of epiblast cells. This state change is not due to the presence of the known autocrine differentiation inducer fibroblast growth factor 4, but, remarkably, it can be prevented by global remodeling of the extracellular matrix (ECM). We also find that cell-secreted matrix remodeling proteins are removed under perfusion and that inhibition of extracellular matrix remodeling causes mESCs to differentiate. Taken together, our data indicate that LIF and BMP4 are not sufficient to maintain self-renewal and that cell-secreted factors are necessary to continuously remodel the ECM and thereby prevent differentiation, revealing a previously undescribed level of mESC regulation through the use of microfluidic perfusion technology.
Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Matriz Extracelular/metabolismo , Transdução de Sinais , Animais , Proliferação de Células , Células Cultivadas , Regulação para Baixo , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Camundongos , Modelos Biológicos , Perfusão , SolubilidadeRESUMO
While a variety of natural and synthetic matrices have been used to influence embryonic stem cell (ESC) self-renewal or differentiation, and ESCs also deposit a rich matrix of their own, the mechanisms behind how extracellular matrix affects cell fate are largely unexplored. The ESC matrix is continuously remodeled by matrix metalloproteinases (MMPs), a process that we find is enhanced by the presence of mouse embryonic fibroblast feeders in a paracrine manner. Matrix remodeling by MMPs aids in the self-renewal of ESCs, as inhibition of MMPs inhibits the ability of ESCs to self-renew. We also find that addition of the interstitial collagenase MMP1 is sufficient to maintain long-term leukemia inhibitory factor (LIF)-independent mouse ESC (mESC) self-renewal in a dose-dependent manner. This remarkable ability is due to the presence of endogenously produced self-renewal-inducing signals, including the LIF-family ligand ciliary neurotrophic factor, that are normally trapped within the ECM and become exposed upon MMP-induced matrix remodeling to signal through JAK and Stat3. These results uncover a new role for feeder cells in maintaining self-renewal and show that mESCs normally produce sufficient levels of autocrine-acting pro-self-renewal ligands.
Assuntos
Células-Tronco Embrionárias/citologia , Matriz Extracelular/fisiologia , Fator de Transcrição STAT3/metabolismo , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Matriz Extracelular/metabolismo , Janus Quinases/metabolismo , Fator Inibidor de Leucemia/metabolismo , Metaloproteinase 13 da Matriz/metabolismo , Camundongos , Comunicação ParácrinaRESUMO
CRISPR genome editing approaches theoretically enable researchers to define the function of each human gene in specific cell types, but challenges remain to efficiently perform genetic perturbations in relevant models. In this work, we develop a library cloning protocol that increases sgRNA uniformity and greatly reduces bias in existing genome-wide libraries. We demonstrate that our libraries can achieve equivalent or better statistical power compared to previously reported screens using an order of magnitude fewer cells. This improved cloning protocol enables genome-scale CRISPR screens in technically challenging cell models and screen formats.
Assuntos
Sistemas CRISPR-Cas , RNA Guia de Sistemas CRISPR-Cas , Humanos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Biblioteca Gênica , Edição de Genes , Clonagem MolecularRESUMO
Kidney disease is highly heritable; however, the causal genetic variants, the cell types in which these variants function, and the molecular mechanisms underlying kidney disease remain largely unknown. To identify genetic loci affecting kidney function, we performed a GWAS using multiple kidney function biomarkers and identified 462 loci. To begin to investigate how these loci affect kidney function, we generated single-cell chromatin accessibility (scATAC-seq) maps of the human kidney and identified candidate cis-regulatory elements (cCREs) for kidney podocytes, tubule epithelial cells, and kidney endothelial, stromal, and immune cells. Kidney tubule epithelial cCREs explained 58% of kidney function SNP-heritability and kidney podocyte cCREs explained an additional 6.5% of SNP-heritability. In contrast, little kidney function heritability was explained by kidney endothelial, stromal, or immune cell-specific cCREs. Through functionally informed fine-mapping, we identified putative causal kidney function variants and their corresponding cCREs. Using kidney scATAC-seq data, we created a deep learning model (which we named ChromKid) to predict kidney cell type-specific chromatin accessibility from sequence. ChromKid and allele specific kidney scATAC-seq revealed that many fine-mapped kidney function variants locally change chromatin accessibility in tubule epithelial cells. Enhancer assays confirmed that fine-mapped kidney function variants alter tubule epithelial regulatory element function. To map the genes which these regulatory elements control, we used CRISPR interference (CRISPRi) to target these regulatory elements in tubule epithelial cells and assessed changes in gene expression. CRISPRi of enhancers harboring kidney function variants regulated NDRG1 and RBPMS expression. Thus, inherited differences in tubule epithelial NDRG1 and RBPMS expression may predispose to kidney disease in humans. We conclude that genetic variants affecting tubule epithelial regulatory element function account for most SNP-heritability of human kidney function. This work provides an experimental approach to identify the variants, regulatory elements, and genes involved in polygenic disease.
RESUMO
Alphavirus budding from the plasma membrane occurs through the specific interaction of the nucleocapsid core with the cytoplasmic domain of the E2 glycoprotein (cdE2). Structural studies of the Sindbis virus capsid protein (CP) have suggested that these critical interactions are mediated by the binding of cdE2 into a hydrophobic pocket in the CP. Several molecular genetic studies have implicated amino acids Y400 and L402 in cdE2 as important for the budding of alphaviruses. In this study, we characterized the role of cdE2 residues in structural polyprotein processing, glycoprotein transport, and capsid interactions. Along with hydrophobic residues, charged residues in the N terminus of cdE2 were critical for the effective interaction of cores with cdE2, a process required for virus budding. Mutations in the C-terminal signal sequence region of cdE2 affected E2 protein transport to the plasma membrane, while nonbudding mutants that were defective in cdE2-CP interaction accumulated E2 on the plasma membrane. The interaction of cdE2 with cytoplasmic cores purified from infected cells and in vitro-assembled core-like particles suggests that cdE2 interacts with assembled cores to mediate budding. We hypothesize that these cdE2 interactions induce a change in the organization of the nucleocapsid core upon binding leading to particle budding and priming of the nucleocapsid cores for disassembly that is required for virus infection.
Assuntos
Infecções por Alphavirus/virologia , Proteínas do Capsídeo/metabolismo , Sindbis virus/fisiologia , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Liberação de Vírus , Proteínas do Capsídeo/genética , Linhagem Celular , Humanos , Ligação Proteica , Estrutura Terciária de Proteína , Sindbis virus/química , Sindbis virus/genética , Proteínas do Envelope Viral/genéticaRESUMO
Protocols to differentiate human pluripotent stem cells have advanced in terms of cell type specificity and tissue-level complexity over the past 2 decades, which has facilitated human disease modeling in the most relevant cell types. The ability to generate induced PSCs (iPSCs) from patients further enables the study of disease mutations in an appropriate cellular context to reveal the mechanisms that underlie disease etiology and progression. As iPSC-derived disease models have improved in robustness and scale, they have also been adopted more widely for use in drug screens to discover new therapies and therapeutic targets. Advancement in genome editing technologies, in particular the discovery of CRISPR-Cas9, has further allowed for rapid development of iPSCs containing disease-causing mutations. CRISPR-Cas9 technologies have now evolved beyond creating single gene edits, aided by the fusion of inhibitory (CRISPRi) or activation (CRISPRa) domains to a catalytically dead Cas9 protein, enabling inhibition or activation of endogenous gene loci. These tools have been used in CRISPR knockout, CRISPRi, or CRISPRa screens to identify genetic modifiers that synergize or antagonize with disease mutations in a systematic and unbiased manner, resulting in identification of disease mechanisms and discovery of new therapeutic targets to accelerate drug discovery research. However, many technical challenges remain when applying large-scale functional genomics approaches to differentiated PSC populations. Here we review current technologies in the field of iPSC disease modeling and CRISPR-based functional genomics screens and practical considerations for implementation across a range of modalities, applications, and disease areas, as well as explore CRISPR screens that have been performed in iPSC models to-date and the insights and therapies these screens have produced.
RESUMO
Variants in the leucine-rich repeat kinase 2 (LRRK2) gene are associated with increased risk for familial and sporadic Parkinson's disease (PD). Pathogenic variants in LRRK2, including the common variant G2019S, result in increased LRRK2 kinase activity, supporting the therapeutic potential of LRRK2 kinase inhibitors for PD. To better understand the role of LRRK2 in disease and to support the clinical development of LRRK2 inhibitors, quantitative and high-throughput assays to measure LRRK2 levels and activity are needed. We developed and applied such assays to measure the levels of LRRK2 as well as the phosphorylation of LRRK2 itself or one of its substrates, Rab10 (pT73 Rab10). We observed increased LRRK2 activity in various cellular models of disease, including iPSC-derived microglia, as well as in human subjects carrying the disease-linked variant LRRK2 G2019S. Capitalizing on the high-throughput and sensitive nature of these assays, we detected a significant reduction in LRRK2 activity in subjects carrying missense variants in LRRK2 associated with reduced disease risk. Finally, we optimized these assays to enable analysis of LRRK2 activity following inhibition in human peripheral blood mononuclear cells (PBMCs) and whole blood, demonstrating their potential utility as biomarkers to assess changes in LRRK2 expression and activity in the clinic.
Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Biomarcadores , Ativação Enzimática , Ensaios Enzimáticos/métodos , Ensaios Enzimáticos/normas , Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Leucócitos Mononucleares/metabolismo , Camundongos , Neuroglia/metabolismo , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Human genetic data indicate that microglial dysfunction contributes to the pathology of Alzheimer's disease (AD), exemplified by the identification of coding variants in triggering receptor expressed on myeloid cells 2 (TREM2) and, more recently, in PLCG2, a phospholipase-encoding gene expressed in microglia. Although studies in mouse models have implicated specific Trem2-dependent microglial functions in AD, the underlying molecular mechanisms and translatability to human disease remain poorly defined. In this study, we used genetically engineered human induced pluripotent stem cell-derived microglia-like cells to show that TREM2 signals through PLCγ2 to mediate cell survival, phagocytosis, processing of neuronal debris, and lipid metabolism. Loss of TREM2 or PLCγ2 signaling leads to a shared signature of transcriptional dysregulation that underlies these phenotypes. Independent of TREM2, PLCγ2 also signals downstream of Toll-like receptors to mediate inflammatory responses. Therefore, PLCγ2 activity regulates divergent microglial functions via distinct TREM2-dependent and -independent signaling and might be involved in the transition to a microglial state associated with neurodegenerative disease.
Assuntos
Inflamação/metabolismo , Glicoproteínas de Membrana/metabolismo , Microglia/metabolismo , Fosfolipase C gama/metabolismo , Receptores Imunológicos/metabolismo , Transdução de Sinais/fisiologia , Animais , Sobrevivência Celular/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Fagocitose/fisiologia , Fosfolipase C gama/genética , Receptores Imunológicos/genéticaRESUMO
Loss-of-function (LOF) variants of TREM2, an immune receptor expressed in microglia, increase Alzheimer's disease risk. TREM2 senses lipids and mediates myelin phagocytosis, but its role in microglial lipid metabolism is unknown. Combining chronic demyelination paradigms and cell sorting with RNA sequencing and lipidomics, we find that wild-type microglia acquire a disease-associated transcriptional state, while TREM2-deficient microglia remain largely homeostatic, leading to neuronal damage. TREM2-deficient microglia phagocytose myelin debris but fail to clear myelin cholesterol, resulting in cholesteryl ester (CE) accumulation. CE increase is also observed in APOE-deficient glial cells, reflecting impaired brain cholesterol transport. This finding replicates in myelin-treated TREM2-deficient murine macrophages and human iPSC-derived microglia, where it is rescued by an ACAT1 inhibitor and LXR agonist. Our studies identify TREM2 as a key transcriptional regulator of cholesterol transport and metabolism under conditions of chronic myelin phagocytic activity, as TREM2 LOF causes pathogenic lipid accumulation in microglia.
Assuntos
Encéfalo/metabolismo , Colesterol/metabolismo , Macrófagos/metabolismo , Glicoproteínas de Membrana/genética , Microglia/metabolismo , Bainha de Mielina/metabolismo , Fagocitose/genética , Receptores Imunológicos/genética , Acetil-CoA C-Acetiltransferase/antagonistas & inibidores , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Animais , Ésteres do Colesterol/metabolismo , Modelos Animais de Doenças , Citometria de Fluxo , Humanos , Células-Tronco Pluripotentes Induzidas , Metabolismo dos Lipídeos/genética , Lipidômica , Receptores X do Fígado/agonistas , Camundongos , Camundongos Knockout , Camundongos Knockout para ApoE , RNA-SeqRESUMO
Glioblastoma multiforme (GBMs) are recurrent lethal brain tumours. Recurrent GBMs often exhibit mesenchymal, stem-like phenotypes that could explain their resistance to therapy. Analyses revealed that recurrent GBMs have increased tension and express high levels of glycoproteins that increase the bulkiness of the glycocalyx. Studies showed that a bulky glycocalyx potentiates integrin mechanosignalling and tissue tension and promotes a mesenchymal, stem-like phenotype in GBMs. Gain- and loss-of-function studies implicated integrin mechanosignalling as an inducer of GBM growth, survival, invasion and treatment resistance, and a mesenchymal, stem-like phenotype. Mesenchymal-like GBMs were highly contractile and expressed elevated levels of glycoproteins that expanded their glycocalyx, and they were surrounded by a stiff extracellular matrix that potentiated integrin mechanosignalling. Our findings suggest that there is a dynamic and reciprocal link between integrin mechanosignalling and a bulky glycocalyx, implying a causal link towards a mesenchymal, stem-like phenotype in GBMs. Strategies to ameliorate GBM tissue tension offer a therapeutic approach to reduce mortality due to GBM.
Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Glicocálix/metabolismo , Integrinas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Neoplásicas/metabolismo , Animais , Antineoplásicos Alquilantes/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Sobrevivência Celular/efeitos dos fármacos , Retroalimentação Fisiológica/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos Nus , Células-Tronco Neoplásicas/efeitos dos fármacos , Tensão Superficial , Temozolomida/uso terapêutico , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Biomechanical and biochemical cues within a tissue collaborate across length scales to direct cell fate during development and are critical for the maintenance of tissue homeostasis. Loss of tensional homeostasis in a tissue not only accompanies malignancy but may also contribute to oncogenic transformation. High mechanical stress in solid tumors can impede drug delivery and may additionally drive tumor progression and promote metastasis. Mechanistically, biomechanical forces can drive tumor aggression by inducing a mesenchymal-like switch in transformed cells so that they attain tumor-initiating or stem-like cell properties. Given that cancer stem cells have been linked to metastasis and treatment resistance, this raises the intriguing possibility that the elevated tissue mechanics in tumors could promote their aggression by programming their phenotype toward that exhibited by a stem-like cell.Significance: Recent findings argue that mechanical stress and elevated mechanosignaling foster malignant transformation and metastasis. Prolonged corruption of tissue tension may drive tumor aggression by altering cell fate specification. Thus, strategies that could reduce tumor mechanics might comprise effective approaches to prevent the emergence of treatment-resilient metastatic cancers. Cancer Discov; 7(11); 1224-37. ©2017 AACR.
Assuntos
Fenômenos Biomecânicos , Carcinogênese/genética , Neoplasias/genética , Estresse Mecânico , Matriz Extracelular/genética , Matriz Extracelular/patologia , Humanos , Metástase Neoplásica , Neoplasias/patologiaRESUMO
Metastasis requires tumor cells to navigate through a stiff stroma and squeeze through confined microenvironments. Whether tumors exploit unique biophysical properties to metastasize remains unclear. Data show that invading mammary tumor cells, when cultured in a stiffened three-dimensional extracellular matrix that recapitulates the primary tumor stroma, adopt a basal-like phenotype. Metastatic tumor cells and basal-like tumor cells exert higher integrin-mediated traction forces at the bulk and molecular levels, consistent with a motor-clutch model in which motors and clutches are both increased. Basal-like nonmalignant mammary epithelial cells also display an altered integrin adhesion molecular organization at the nanoscale and recruit a suite of paxillin-associated proteins implicated in invasion and metastasis. Phosphorylation of paxillin by Src family kinases, which regulates adhesion turnover, is similarly enhanced in the metastatic and basal-like tumor cells, fostered by a stiff matrix, and critical for tumor cell invasion in our assays. Bioinformatics reveals an unappreciated relationship between Src kinases, paxillin, and survival of breast cancer patients. Thus adoption of the basal-like adhesion phenotype may favor the recruitment of molecules that facilitate tumor metastasis to integrin-based adhesions. Analysis of the physical properties of tumor cells and integrin adhesion composition in biopsies may be predictive of patient outcome.
Assuntos
Adesão Celular/fisiologia , Integrinas/metabolismo , Paxilina/metabolismo , Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Matriz Extracelular/metabolismo , Feminino , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Metástase Neoplásica/fisiopatologia , Fosforilação , Transdução de SinaisRESUMO
Regenerative medicine is predicated on understanding the mechanisms regulating development and applying these conditions to direct stem cell fate. Embryogenesis is guided by cell-cell and cell-matrix interactions, but it is unclear how these physical cues influence stem cells in culture. We used human embryonic stem cells (hESCs) to examine whether mechanical features of the extracellular microenvironment could differentially modulate mesoderm specification. We found that, on a hydrogel-based compliant matrix, hESCs accumulate ß-catenin at cell-cell adhesions and show enhanced Wnt-dependent mesoderm differentiation. Mechanistically, Src-driven ubiquitination of E-cadherin by Cbl-like ubiquitin ligase releases P120-catenin to facilitate transcriptional activity of ß-catenin, which initiates and reinforces mesoderm differentiation. By contrast, on a stiff hydrogel matrix, hESCs show elevated integrin-dependent GSK3 and Src activity that promotes ß-catenin degradation and inhibits differentiation. Thus, we found that mechanical features of the microenvironmental matrix influence tissue-specific differentiation of hESCs by altering the cellular response to morphogens.