RESUMEN
The dramatic reorganization of chromatin during mitosis is perhaps one of the most fundamental of all cell processes. It remains unclear how epigenetic histone modifications, despite their crucial roles in regulating chromatin architectures, are dynamically coordinated with chromatin reorganization in controlling this process. We have developed and characterized biosensors with high sensitivity and specificity based on fluorescence resonance energy transfer (FRET). These biosensors were incorporated into nucleosomes to visualize histone H3 Lys-9 trimethylation (H3K9me3) and histone H3 Ser-10 phosphorylation (H3S10p) simultaneously in the same live cell. We observed an anticorrelated coupling in time between H3K9me3 and H3S10p in a single live cell during mitosis. A transient increase of H3S10p during mitosis is accompanied by a decrease of H3K9me3 that recovers before the restoration of H3S10p upon mitotic exit. We further showed that H3S10p is causatively critical for the decrease of H3K9me3 and the consequent reduction of heterochromatin structure, leading to the subsequent global chromatin reorganization and nuclear envelope dissolution as a cell enters mitosis. These results suggest a tight coupling of H3S10p and H3K9me3 dynamics in the regulation of heterochromatin dissolution before a global chromatin reorganization during mitosis.
Asunto(s)
Técnicas Biosensibles/métodos , Ensamble y Desensamble de Cromatina , Código de Histonas , Proteínas Bacterianas , Ensamble y Desensamble de Cromatina/genética , Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas Fluorescentes Verdes , Células HEK293 , Heterocromatina/genética , Heterocromatina/metabolismo , Código de Histonas/genética , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Proteínas Luminiscentes , Mitosis , Modelos Biológicos , Análisis de la Célula Individual/métodosRESUMEN
The focal nature of atherosclerotic lesions suggests an important role of local hemodynamic environment. Recent studies have demonstrated significant roles of Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in mediating mechanotransduction and vascular homeostasis. The objective of this study is to investigate the functional role of YAP/TAZ in the flow regulation of atheroprone endothelial phenotypes and the consequential development of atherosclerotic lesions. We found that exposure of cultured endothelial cells (ECs) to the atheroprone disturbed flow resulted in YAP/TAZ activation and translocation into EC nucleus to up-regulate the target genes, including cysteine-rich angiogenic inducer 61 (CYR61), connective tissue growth factor (CTGF), and ankyrin repeat domain 1 (ANKRD1). In contrast, the athero-protective laminar flow suppressed YAP/TAZ activities. En face analysis of mouse arteries demonstrated an increased nuclear localization of YAP/TAZ and elevated levels of the target genes in the endothelium in atheroprone areas compared with athero-protective areas. YAP/TAZ knockdown significantly attenuated the disturbed flow induction of EC proliferative and proinflammatory phenotypes, whereas overexpression of constitutively active YAP was sufficient to promote EC proliferation and inflammation. In addition, treatment with statin, an antiatherosclerotic drug, inhibited YAP/TAZ activities to diminish the disturbed flow-induced proliferation and inflammation. In vivo blockade of YAP/TAZ translation by morpholino oligos significantly reduced endothelial inflammation and the size of atherosclerotic lesions. Our results demonstrate a critical role of the activation of YAP/TAZ by disturbed flow in promoting atheroprone phenotypes and atherosclerotic lesion development. Therefore, inhibition of YAP/TAZ activation is a promising athero-protective therapeutic strategy.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Aterosclerosis/metabolismo , Aterosclerosis/patología , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fosfoproteínas/metabolismo , Reología , Animales , Apolipoproteínas E/deficiencia , Aterosclerosis/tratamiento farmacológico , Arteria Carótida Común/patología , Ciclo Celular , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Proteína 61 Rica en Cisteína/metabolismo , Técnicas de Silenciamiento del Gen , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Inflamación/patología , Ratones , Proteínas Musculares/metabolismo , Proteínas Nucleares/metabolismo , Fenotipo , Proteínas Represoras/metabolismo , Transactivadores , Factores de Transcripción , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Proteínas Señalizadoras YAPRESUMEN
Mechanosensation is perhaps the last sensory modality not understood at the molecular level. Ion channels that sense mechanical force are postulated to play critical roles in a variety of biological processes including sensing touch/pain (somatosensation), sound (hearing), and shear stress (cardiovascular physiology); however, the identity of these ion channels has remained elusive. We previously identified Piezo1 and Piezo2 as mechanically activated cation channels that are expressed in many mechanosensitive cell types. Here, we show that Piezo1 is expressed in endothelial cells of developing blood vessels in mice. Piezo1-deficient embryos die at midgestation with defects in vascular remodeling, a process critically influenced by blood flow. We demonstrate that Piezo1 is activated by shear stress, the major type of mechanical force experienced by endothelial cells in response to blood flow. Furthermore, loss of Piezo1 in endothelial cells leads to deficits in stress fiber and cellular orientation in response to shear stress, linking Piezo1 mechanotransduction to regulation of cell morphology. These findings highlight an essential role of mammalian Piezo1 in vascular development during embryonic development.
Asunto(s)
Sistema Cardiovascular/embriología , Desarrollo Embrionario/fisiología , Células Endoteliales/metabolismo , Canales Iónicos/metabolismo , Mecanotransducción Celular/fisiología , Animales , Sistema Cardiovascular/citología , Células Endoteliales/citología , Canales Iónicos/genética , Ratones , Ratones TransgénicosRESUMEN
Endothelial cells (ECs) are constantly exposed to xenobiotics and endobiotics or their metabolites, which perturb EC function, as well as to shear stress, which plays a crucial role in vascular homeostasis. Pregnane X receptor (PXR) is a nuclear receptor and a key regulator of the detoxification of xeno- and endobiotics. Here we show that laminar shear stress (LSS), the atheroprotective flow, activates PXR in ECs, whereas oscillatory shear stress, the atheroprone flow, suppresses PXR. LSS activation of PXR in cultured ECs led to the increased expression of a PXR target gene, multidrug resistance 1 (MDR1). An in vivo study using rats showed that the expression of MDR1 was significantly higher in the endothelium from the descending thoracic aorta, where flow is mostly laminar, than from the inner curvature of aortic arch, where flow is disturbed. Functionally, LSS-activated PXR protects ECs from apoptosis triggered by doxorubicin via the induction of MDR1 and other detoxification genes. PXR also suppressed the expression of proinflammatory adhesion molecules and monocyte adhesion in response to TNF-α and lipopolysaccharide. Overexpression of a constitutively active PXR in rat carotid arteries potently attenuated proinflammatory responses. In addition, cDNA microarray revealed a large number of the PXR-activated endothelial genes whose products are responsible for major steps of detoxification, including phase I and II metabolizing enzymes and transporters. These detoxification genes in ECs are induced by LSS in ECs in a PXR-dependent manner. In conclusion, our results indicate that PXR represents a flow-activated detoxification system to protect ECs against damage by xeno- and endobiotics.
Asunto(s)
Perfilación de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Inactivación Metabólica/genética , Receptores de Esteroides/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Apoptosis/efectos de los fármacos , Hidrocarburo de Aril Hidroxilasas/genética , Hidrocarburo de Aril Hidroxilasas/metabolismo , Western Blotting , Arterias Carótidas/metabolismo , Línea Celular Tumoral , Células Cultivadas , Citocromo P-450 CYP1B1 , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptor X de Pregnano , Interferencia de ARN , Ratas , Ratas Sprague-Dawley , Receptores de Esteroides/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estrés Mecánico , Xenobióticos/metabolismo , Xenobióticos/farmacologíaRESUMEN
Endothelial cells (ECs) respond to changes in mechanical forces, leading to the modulation of signaling networks and cell function; an example is the inhibition of EC proliferation by steady laminar flow. MicroRNAs (miRs) are short noncoding 20-22 nucleotide RNAs that negatively regulate the expression of target genes at the posttranscriptional level. This study demonstrates that miRs are involved in the flow regulation of gene expression in ECs. With the use of microRNA chip array, we found that laminar shear stress (12 dyn/cm(2), 12 h) regulated the EC expression of many miRs, including miR-19a. We further showed that stable transfection of miR-19a significantly decreased the expression of a reporter gene controlled by a conserved 3'-untranslated region of the cyclinD1 gene and also the protein level of cyclin D1, leading to an arrest of cell cycle at G1/S transition. Laminar flow suppressed cyclin D1 protein level, and this suppressive effect was diminished when the endogenous miR-19a was inhibited. In conclusion, we demonstrated that miR-19a plays an important role in the flow regulation of cyclin D1 expression. These results revealed a mechanism by which mechanical forces modulate endothelial gene expression.
Asunto(s)
Ciclina D1/metabolismo , Células Endoteliales/metabolismo , Regulación de la Expresión Génica/fisiología , MicroARNs/metabolismo , Flujo Sanguíneo Regional/fisiología , Venas Umbilicales/citología , Emparejamiento Base , Secuencia de Bases , Fenómenos Biomecánicos , Western Blotting , Cartilla de ADN/genética , Citometría de Flujo , Humanos , MicroARNs/genética , Análisis por Micromatrices , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Venas Umbilicales/fisiologíaRESUMEN
Interstitial flow in and around bone tissue is oscillatory in nature and affects the mechanical microenvironment for bone cell growth and formation. We investigated the role of oscillatory shear stress (OSS) in modulating the proliferation of human osteoblast-like MG63 cells and its underlying mechanisms. Application of OSS (0.5 +/- 4 dynes/cm(2)) to MG63 cells induced sustained activation of phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR/p70S6K (p70S6 kinase) signaling cascades and hence cell proliferation, which was accompanied by increased expression of cyclins A and D1, cyclin-dependent protein kinases-2, -4, and -6, and bone formation-related genes (c-fos, Egr-1, and Cox-2) and decreased expression of p21(CIP1) and p27(KIP1). OSS-induced activation of PI3K/Akt/mTOR/p70S6K and cell proliferation were inhibited by specific antibodies or small interference RNAs of alpha(v)beta(3) and beta(1) integrins and by dominant-negative mutants of Shc (Shc-SH2) and focal adhesion kinase (FAK) (FAK(F397Y)). Co-immunoprecipitation assay showed that OSS induces sustained increases in association of Shc and FAK with alpha(v)beta(3) and beta(1) integrins and PI3K subunit p85, which were abolished by transfecting the cells with FAK(F397Y) or Shc-SH2. OSS also induced sustained activation of ERK, which was inhibited by the specific PI3K inhibitor LY294002 and was required for OSS-induced activation of mTOR/p70S6K and proliferation in MG63 cells. Our findings provide insights into the mechanisms by which OSS induces osteoblast-like cell proliferation through activation of alpha(v)beta(3) and beta(1) integrins and synergistic interactions of FAK and Shc with PI3K, leading to the modulation of downstream ERK and Akt/mTOR/p70S6K pathways.
Asunto(s)
Integrina alfaVbeta3/metabolismo , Integrina beta1/metabolismo , Osteoblastos/citología , Osteoblastos/enzimología , Reología , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Transducción de Señal , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Proliferación Celular , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Regulación de la Expresión Génica , Humanos , Complejos Multiproteicos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Estrés Mecánico , Serina-Treonina Quinasas TORRESUMEN
During endoderm formation, cell identity and tissue morphogenesis are tightly controlled by cell-intrinsic and cell-extrinsic factors such as biochemical and physical inputs. While the effects of biochemical factors are well studied, the physical cues that regulate cell division and differentiation are poorly understood. RNA sequencing analysis demonstrated increases of endoderm-specific gene expression in hPSCs cultured on soft substrate (Young's modulus, 3 ± 0.45 kPa) in comparison with hard substrate (Young's modulus, 165 ± 6.39 kPa). Further analyses revealed that multiple long noncoding RNAs (lncRNAs) were up-regulated on soft substrate; among them, LINC00458 was identified as a stiffness-dependent lncRNA specifically required for hPSC differentiation toward an early endodermal lineage. Gain- and loss-of-function experiments confirmed that LINC00458 is functionally required for hPSC endodermal lineage specification induced by soft substrates. Our study provides evidence that mechanical cues regulate the expression of LINC00458 and induce differentiation of hPSC into hepatic lineage progenitors.
Asunto(s)
Endodermo/citología , Endodermo/metabolismo , Regulación del Desarrollo de la Expresión Génica , ARN Largo no Codificante/genética , Proteína Smad2/genética , Proteína smad3/genética , Animales , Diferenciación Celular/genética , Línea Celular , Linaje de la Célula/genética , Células Cultivadas , Matriz Extracelular , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Modelos Biológicos , Especificidad de Órganos/genética , Interferencia de ARN , TranscriptomaRESUMEN
Bacterial adherence to epithelial cells is a key virulence trait of pathogenic bacteria. The type 1 fimbriae and the P-fimbriae of uropathogenic Escherichia coli (UPEC) have both been described to be important for the establishment of urinary tract infections (UTI). To explore the interactions between the host and bacterium responsible for the different environments of UPEC invasion, we examined the effect of pH and osmolarity on UPEC strain J96 fimbrial expression, and subsequent J96-induced interleukin-8 (IL-8) expression in different uroepithelial cells. The J96 strain grown in high pH with low osmolarity condition was favorable for the expression of type 1 fimbriae; whereas J96 grown in low pH with high osmolarity condition was beneficial for P fimbriae expression. Type 1 fimbriated J96 specifically invaded bladder 5637 epithelial cells and induced IL-8 expression. On the contrary, P fimbriated J96 invaded renal 786-O epithelial cells and induced IL-8 expression effectively. Type 1 fimbriated J96-induced IL-8 induction involved the p38, as well as ERK, JNK pathways, which leads to AP-1-mediated gene expression. P fimbriated J96-induced augmentation of IL-8 expression mainly involved p38-mediated AP-1 and NF-kappaB transcriptional activation. These results indicate that different expression of fimbriae in J96 trigger differential IL-8 gene regulation pathways in different uroepithelial cells.
Asunto(s)
Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Regulación Bacteriana de la Expresión Génica , Interleucina-8/biosíntesis , Sistema Urinario/metabolismo , Sistema Urinario/microbiología , Escherichia coli Uropatógena/metabolismo , Adhesinas Bacterianas/química , Línea Celular , Línea Celular Tumoral , Humanos , Concentración de Iones de Hidrógeno , FN-kappa B/metabolismo , Invasividad Neoplásica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción AP-1/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The pulsatile nature of blood pressure and flow creates hemodynamic stimuli in the forms of cyclic stretch and shear stress, which exert continuous influences on the constituents of the blood vessel wall. Vascular smooth muscle cells (VSMCs) use multiple sensing mechanisms to detect the mechanical stimulus resulting from pulsatile stretch and transduce it into intracellular signals that lead to modulations of gene expression and cellular functions, e.g., proliferation, apoptosis, migration, and remodeling. The cytoskeleton provides a structural framework for the VSMC to transmit mechanical forces between its luminal, abluminal, and junctional surfaces, as well as its interior, including the focal adhesion sites, the cytoplasm, and the nucleus. VSMCs also respond differently to the surrounding structural environment, e.g., two-dimensional versus three-dimensional matrix. In vitro studies have been conducted on cultured VSMCs on deformable substrates to elucidate the molecular mechanisms by which the cells convert mechanical inputs into biochemical events, eventually leading to functional responses. The knowledge gained from research on mechanotransduction in vitro, in conjunction with verifications under in vivo conditions, will advance our understanding of the physiological and pathological processes involved in vascular remodeling and adaptation in health and disease.
Asunto(s)
Mecanotransducción Celular/fisiología , Músculo Liso Vascular/citología , Músculo Liso Vascular/fisiología , Miocitos del Músculo Liso/fisiología , Animales , Humanos , Estrés MecánicoRESUMEN
Blood vessels are constantly exposed to hemodynamic forces in the form of cyclic stretch and shear stress due to the pulsatile nature of blood pressure and flow. Endothelial cells (ECs) are subjected to the shear stress resulting from blood flow and are able to convert mechanical stimuli into intracellular signals that affect cellular functions, e.g., proliferation, apoptosis, migration, permeability, and remodeling, as well as gene expression. The ECs use multiple sensing mechanisms to detect changes in mechanical forces, leading to the activation of signaling networks. The cytoskeleton provides a structural framework for the EC to transmit mechanical forces between its luminal, abluminal and junctional surfaces and its interior, including the cytoplasm, the nucleus, and focal adhesion sites. Endothelial cells also respond differently to different modes of shear forces, e.g., laminar, disturbed, or oscillatory flows. In vitro studies on cultured ECs in flow channels have been conducted to investigate the molecular mechanisms by which cells convert the mechanical input into biochemical events, which eventually lead to functional responses. The knowledge gained on mechano-transduction, with verifications under in vivo conditions, will advance our understanding of the physiological and pathological processes in vascular remodeling and adaptation in health and disease.
Asunto(s)
Células Endoteliales/fisiología , Resistencia al Corte , Estrés Mecánico , Proteínas de Unión al GTP/sangre , Proteínas de Unión al GTP/metabolismo , Expresión Génica , Humanos , Integrinas/sangre , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/sangre , Canales de Potasio Calcio-Activados/sangre , Canales de Potasio Calcio-Activados/metabolismo , Proteínas Tirosina Quinasas Receptoras/sangre , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Acoplados a Proteínas G/sangre , Estados UnidosRESUMEN
The phenotype of smooth muscle cells (SMCs) plays an important role in vascular function in health and disease. We investigated the mechanism of modulation of SMC phenotype (from contractile to synthetic) induced by the synergistic action of a growth factor (platelet-derived growth factor, PDGF-BB) and a cytokine (interleukin, IL-1beta). Human aortic SMCs grown on polymerized collagen showed high expression levels of contractile markers (smooth muscle alpha-actin, myosin heavy chain, and calponin). These levels were not significantly affected by PDGF-BB and IL-1beta individually, but decreased markedly after the combined usage of PDGF-BB and IL-1beta. PDGF/IL-1beta costimulation also induced a sustained phosphorylation of Akt and p70 ribosomal S6 kinase (p70S6K). The effects of PDGF/IL-1beta costimulation on contractile marker expression and Akt and p70S6K phosphorylation were blocked by the phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 and by adenovirus expressing a dominant-negative Akt, and they were mimicked by constitutively active Akt. PDGF-BB/IL-1beta induced a sustained phosphorylation of PDGF receptor (PDGFR)-beta and its association with IL-1 receptor (IL-1R1). Such activation and association of receptors were blocked by a PDGFR-beta neutralizing antibody (AF385), an IL-1R1 antagonist (IL-1ra), as well as a specific inhibitor of PDGFR-beta phosphorylation (AG1295); these agents also eliminated the PDGF-BB/IL-1beta-induced signaling and phenotypic modulation. PDGF-BB/IL-1beta inhibited the polymerized collagen-induced serum response factor DNA binding activity in the nucleus, and this effect was mediated by the PDGFR-beta/IL-1R1 association and phosphatidylinositol 3-kinase/Akt/p70S6K pathway. Our findings provide insights into the mechanism of SMC phenotypic modulation from contractile to synthetic, e.g., in atherosclerosis.