RESUMEN

Disuse induces adaptations in skeletal muscle, which lead to muscle deterioration. Hindlimb-unloading (HU) is a well-established model to investigate cellular mechanisms responsible for disuse-induced skeletal muscle dysfunction. In myosin heavy chain (MHC) type IIB fibers HU induces a reduction in contraction speed (Vo) and a reduction in the relative myosin light chain 3f (MLC3f) protein content compared with myosin light chain 1f (MLC1f) protein. This study tested the hypothesis that increasing the relative MLC3f protein content via rAd-MLC3f vector delivery would attenuate the HU-induced decline in Vo in single MHC type IIB fibers. Fischer-344 rats were randomly assigned to one of three groups: control, HU for 7 days, and HU for 7 days plus rAd-MLC3f. The semimembranosus muscles were injected with rAd-MLC3f (3.75 x 1011-5 x 1011 ifu/ml) at four days after the initiation of HU. In single MHC type IIB fibers the relative MLC3f content decreased by 25% (12.00±0.60% to 9.06±0.66%) and Vo was reduced by 29% (3.22±0.14fl/s vs. 2.27±0.08fl/s) with HU compared to the control group. The rAd-MLC3f injection resulted in an increase in the relative MLC3f content (12.26±1.19%) and a concomitant increase in Vo (2.90±0.15fl/s) of MHC type IIB fibers. A positive relationship was observed between the percent of MLC3f content and Vo. Maximal isometric force and specific tension were reduced with HU by 49% (741.45±44.24µN to 379.09±23.77µN) and 33% (97.58±4.25kN/m2 to 65.05±2.71kN/m2), respectively compared to the control group. The rAd-MLC3f injection did not change the HU-induced decline in force or specific tension. Collectively, these results indicate that rAd-MLC3f injection rescues hindlimb unloading-induced decline in Vo in MHC type IIB single muscle fibers.

Asunto(s)

Adaptación Fisiológica , Contracción Muscular , Fibras Musculares Esqueléticas/metabolismo , Trastornos Musculares Atróficos/prevención & control , Cadenas Ligeras de Miosina/biosíntesis , Adenoviridae , Animales , Vectores Genéticos , Suspensión Trasera , Masculino , Fibras Musculares Esqueléticas/patología , Trastornos Musculares Atróficos/genética , Trastornos Musculares Atróficos/metabolismo , Cadenas Ligeras de Miosina/genética , Ratas , Ratas Endogámicas F344 , Transducción Genética

RESUMEN

Chronic increased workload of the human heart causes ventricular hypertrophy, re-expression of the atrial essential myosin light chain (hALC-1), and improved contractile function. Although hALC-1 is an important positive inotropic regulator of the human heart, little is known about its regulation. Therefore, we investigated the role of the sex hormone 17ß-estradiol (E2) on hALC-1 gene expression, the underlying molecular mechanisms, and the impact of this regulatory process on cardiac contractile function. We showed that E2 attenuated hALC-1 expression in human atrial tissues of both sexes and in human ventricular AC16 cells. E2 induced the nuclear translocation of estrogen receptor alpha (ERα) and hALC-1 in AC16 cells, where they cooperatively regulate the transcriptional activity of hALC-1 gene promoter. E2-activated ERα required the estrogen response element (ERE) motif within the hALC-1 gene promoter to reduce its transcriptional activity (vehicle: 15.55 ± 4.80 vs. E2: 6.51 ± 3.69; ~2 fold). This inhibitory effect was potentiated in the presence of hALC-1 (vehicle: 11.13 ± 3.66 vs. E2: 2.18 ± 1.10; ~5 fold), and thus, hALC-1 acts as a co-repressor of ERα-mediated transcription. Yeast two-hybrid screening of a human heart cDNA library revealed that ERα interacts physically with hALC-1 in the presence of E2. This interaction was confirmed by Co-Immunoprecipitation and immunofluorescence in human atrium. As a further novel effect, we showed that chronic E2-treatment of adult mouse cardiomyocytes overexpressing hALC-1 resulted in reduced cell-shortening amplitude and twitching kinetics of these cells independent of Ca2+ activation levels. Together, our data showed that the expression of hALC-1 gene is, at least partly, regulated by E2/ERα, while hALC-1 acts as a co-repressor. The inotropic effect of hALC-1 overexpression in cardiomyocytes can be significantly repressed by E2.

Asunto(s)

Estradiol/metabolismo , Receptor alfa de Estrógeno/metabolismo , Regulación de la Expresión Génica/genética , Contracción Miocárdica/fisiología , Cadenas Ligeras de Miosina/biosíntesis , Animales , Western Blotting , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Inmunoprecipitación , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Miocitos Cardíacos/metabolismo , Cadenas Ligeras de Miosina/genética , Reacción en Cadena de la Polimerasa , Técnicas del Sistema de Dos Híbridos

RESUMEN

The role of myosin light chain II (MLC­II) in cellular differentiation of rat mandibular condylar chondrocytes (MCCs) induced by cyclical uniaxial compressive stress (CUCS) remains unclear. In the current study, a four­point bending system was used to apply CUCS to primary cultured MCCs from rats. It was identified that CUCS stimulated features of cellular differentiation including morphological alterations, cytoskeleton rearrangement and overproduction of proteoglycans. Furthermore, CUCS promoted runt­related transcription factor­2 (RUNX2) expression at mRNA (P<0.01) and protein levels (P<0.05) and elevated alkaline phosphatase (ALP) activity (P<0.01), which are both markers of osteogenic differentiation. Under conditions of stress, western blotting indicated that the ratio of phosphorylated MLC­II to total MLC­II was increased significantly (P<0.05). Silencing MLC­II by RNA interference reduced ALP activity (P<0.01), and eliminated RUNX2 mRNA expression (P<0.01). Addition of the MLC kinase inhibitor, ML­7, reduced the CUCS­associated upregulation of RUNX2 expression (P<0.01) and ALP activity (P<0.01). The data indicated that CUCS promoted cellular differentiation of rat primary MCCs, and this was suggested to be via the phosphorylation of MLC­II.

Asunto(s)

Miosinas Cardíacas/genética , Condrocitos/citología , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Cóndilo Mandibular/crecimiento & desarrollo , Cadenas Ligeras de Miosina/genética , Osteogénesis/genética , Fosfatasa Alcalina/antagonistas & inhibidores , Fosfatasa Alcalina/genética , Animales , Azepinas/administración & dosificación , Miosinas Cardíacas/antagonistas & inhibidores , Miosinas Cardíacas/biosíntesis , Diferenciación Celular/genética , Condrocitos/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Citoesqueleto/genética , Citoesqueleto/metabolismo , Regulación del Desarrollo de la Expresión Génica , Cóndilo Mandibular/citología , Cóndilo Mandibular/metabolismo , Cadenas Ligeras de Miosina/antagonistas & inhibidores , Cadenas Ligeras de Miosina/biosíntesis , Naftalenos/administración & dosificación , Fosforilación , Presión , Interferencia de ARN , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas

RESUMEN

Selenium (Se), a nutritionally essential trace element, is associated with health and disease. Selenoprotein T (SelT) was identified as a redoxin protein with a selenocystein, localizing in the endoplasmic reticulum. The myosin light chain kinase (MLCK) and myosin light chain (MLC) play key roles in the contraction process of smooth muscle. The present study was to detect the effect and mechanism of SelT on the contraction process of gastric smooth muscle. The WT rats were fed with different Se concentration diets, and Se and Ca(2+) concentrations were detected in the gastric smooth muscle. Western blot and qPCR were performed to determine SelT, CaM, MLCK, and MLC expressions. MLCK activity was measured by identifying the rates of [γ-32P]ATP incorporated into the MLC. The results showed Se and Ca(2+) concentrations were enhanced with Se intake in gastric smooth muscle tissues. With increasing Se, SelT, CaM, MLCK and MLC expressions increased, and MLCK and MLC activation improved in gastric smooth muscle tissue. The SelT RNA interference experiments showed that Ca(2+) release, MLCK activation, and MLC phosphorylation were regulated by SelT. Se affected the gastric smooth muscle constriction by regulating Ca(2+) release, MLCK activation, and MLC phosphorylation through SelT. Se plays a major role in regulating the contraction processes of gastric smooth muscle with the SelT.

Asunto(s)

Señalización del Calcio/efectos de los fármacos , Mucosa Gástrica/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Contracción Muscular/efectos de los fármacos , Músculo Liso/metabolismo , Quinasa de Cadena Ligera de Miosina/metabolismo , Selenio/farmacología , Selenoproteínas/biosíntesis , Animales , Activación Enzimática/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Cadenas Ligeras de Miosina/biosíntesis , Ratas , Ratas Wistar

RESUMEN

With the advent of high-throughput DNA sequencing, the number of identified cardiomyopathy-causing mutations has increased tremendously. As the majority of these mutations affect myofilament proteins, there is a need to understand their functional consequence on contraction. Permeabilized myofilament preparations coupled with protein exchange protocols are a common method for examining into contractile mechanics. However, producing large quantities of myofilament proteins can be time consuming and requires different approaches for each protein of interest. In the present study, we describe a unified automated method to produce troponin C, troponin T, and troponin I as well as myosin light chain 2 fused to a His6-tag followed by a tobacco etch virus (TEV) protease site. TEV protease has the advantage of a relaxed P1' cleavage site specificity, allowing for no residues left after proteolysis and preservation of the native sequence of the protein of interest. After expression in Esherichia coli, cells were lysed by sonication in imidazole-containing buffer. The His6-tagged protein was then purified using a HisTrap nickel metal affinity column, and the His6-tag was removed by His6-TEV protease digestion for 4 h at 30°C. The protease was then removed using a HisTrap column, and complex assembly was performed via column-assisted sequential desalting. This mostly automated method allows for the purification of protein in 1 day and can be adapted to most soluble proteins. It has the advantage of greatly increasing yield while reducing the time and cost of purification. Therefore, production and purification of mutant proteins can be accelerated and functional data collected in a faster, less expensive manner.

Asunto(s)

Miosinas Cardíacas/biosíntesis , Endopeptidasas , Histidina , Miofibrillas , Cadenas Ligeras de Miosina/biosíntesis , Oligopéptidos , Proteínas Recombinantes/biosíntesis , Troponina C/biosíntesis , Troponina I/biosíntesis , Troponina T/biosíntesis , Automatización , Miosinas Cardíacas/genética , Cromatografía de Afinidad , Dextranos , Escherichia coli/genética , Humanos , Cadenas Ligeras de Miosina/genética , Níquel , Proteínas Recombinantes/genética , Sefarosa , Troponina C/genética , Troponina I/genética , Troponina T/genética

RESUMEN

Cav1.3 L-type Ca(2+) channel is known to be highly expressed in neurons and neuroendocrine cells. However, we have previously demonstrated that the Cav1.3 channel is also expressed in atria and pacemaking cells in the heart. The significance of the tissue-specific expression of the channel is underpinned by our previous demonstration of atrial fibrillation in a Cav1.3 null mutant mouse model. Indeed, a recent study has confirmed the critical roles of Cav1.3 in the human heart (Baig, S. M., Koschak, A., Lieb, A., Gebhart, M., Dafinger, C., Nürnberg, G., Ali, A., Ahmad, I., Sinnegger-Brauns, M. J., Brandt, N., Engel, J., Mangoni, M. E., Farooq, M., Khan, H. U., Nürnberg, P., Striessnig, J., and Bolz, H. J. (2011) Nat. Neurosci. 14, 77-84). These studies suggest that detailed knowledge of Cav1.3 may have broad therapeutic ramifications in the treatment of cardiac arrhythmias. Here, we tested the hypothesis that there is a functional cross-talk between the Cav1.3 channel and a small conductance Ca(2+)-activated K(+) channel (SK2), which we have documented to be highly expressed in human and mouse atrial myocytes. Specifically, we tested the hypothesis that the C terminus of Cav1.3 may translocate to the nucleus where it functions as a transcriptional factor. Here, we reported for the first time that the C terminus of Cav1.3 translocates to the nucleus where it functions as a transcriptional regulator to modulate the function of Ca(2+)-activated K(+) channels in atrial myocytes. Nuclear translocation of the C-terminal domain of Cav1.3 is directly regulated by intracellular Ca(2+). Utilizing a Cav1.3 null mutant mouse model, we demonstrate that ablation of Cav1.3 results in a decrease in the protein expression of myosin light chain 2, which interacts and increases the membrane localization of SK2 channels.

Asunto(s)

Canales de Calcio Tipo L/metabolismo , Núcleo Celular/metabolismo , Regulación de la Expresión Génica/fisiología , Miocitos Cardíacos/metabolismo , Transcripción Genética/fisiología , Transporte Activo de Núcleo Celular/fisiología , Animales , Canales de Calcio Tipo L/genética , Miosinas Cardíacas/biosíntesis , Miosinas Cardíacas/genética , Núcleo Celular/genética , Atrios Cardíacos/citología , Atrios Cardíacos/metabolismo , Humanos , Ratones , Ratones Noqueados , Miocitos Cardíacos/citología , Cadenas Ligeras de Miosina/biosíntesis , Cadenas Ligeras de Miosina/genética , Estructura Terciaria de Proteína

RESUMEN

BACKGROUND: Nuclear reprogramming inculcates pluripotent capacity by which de novo tissue differentiation is enabled. Yet, introduction of ectopic reprogramming factors may desynchronize natural developmental schedules. This study aims to evaluate the effect of imposed transgene load on the cardiogenic competency of induced pluripotent stem (iPS) cells. METHODS AND RESULTS: Targeted inclusion and exclusion of reprogramming transgenes (c-MYC, KLF4, OCT4, and SOX2) was achieved using a drug-inducible and removable cassette according to the piggyBac transposon/transposase system. Pulsed transgene overexpression, before iPS cell differentiation, hindered cardiogenic outcomes. Delayed in counterparts with maintained integrated transgenes, transgene removal enabled proficient differentiation of iPS cells into functional cardiac tissue. Transgene-free iPS cells generated reproducible beating activity with robust expression of cardiac α-actinin, connexin 43, myosin light chain 2a, α/ß-myosin heavy chain, and troponin I. Although operational excitation-contraction coupling was demonstrable in the presence or absence of transgenes, factor-free derivatives exhibited an expedited maturing phenotype with canonical responsiveness to adrenergic stimulation. CONCLUSIONS: A disproportionate stemness load, caused by integrated transgenes, affects the cardiogenic competency of iPS cells. Offload of transgenes in engineered iPS cells ensures integrity of cardiac developmental programs, underscoring the value of nonintegrative nuclear reprogramming for derivation of competent cardiogenic regenerative biologics.

Asunto(s)

Células Madre Pluripotentes Inducidas/citología , Transgenes , Actinina/biosíntesis , Animales , Miosinas Cardíacas/biosíntesis , Diferenciación Celular , Separación Celular , Reprogramación Celular , Conexina 43/biosíntesis , Electrofisiología , Fibroblastos/metabolismo , Citometría de Flujo , Técnicas Genéticas , Factor 4 Similar a Kruppel , Ratones , Microscopía Electrónica , Cadenas Pesadas de Miosina/biosíntesis , Cadenas Ligeras de Miosina/biosíntesis , Troponina I/biosíntesis

RESUMEN

Breast cancer is the leading cause of cancer death in women worldwide which is closely related to metastasis. But the exact molecular mechanism on metastasis is still not fully understood; we now report that both MRTF-A and STAT3 play important role in breast cancer migration of MDA-MB-231 cells. Moreover, MRTF-A and STAT3 synergistically increased MDA-MB-231 cell migration by promoting the expression of migration markers Myl-9 and Cyr-61. Importantly, we identified a detailed molecular mechanism of MDA-MB-231 cell migration controlled via physical interaction between MRTF-A and STAT3, which synergistically promote the transactivity of the migration marker Myl-9 and Cyr-61 by CArG box binding. Interestingly, the two signaling pathways RhoA-MRTF-A and JAK-STAT3 across talk to regulate MDA-MB-231 cell migration. Our data thus provide important and novel insights into the roles of MRTF-A and STAT3 in regulating MDA-MB-231 cell migration.

Asunto(s)

Biomarcadores de Tumor/biosíntesis , Neoplasias de la Mama/metabolismo , Movimiento Celular , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/biosíntesis , Factor de Transcripción STAT3/biosíntesis , Transactivadores/biosíntesis , Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proteína 61 Rica en Cisteína/biosíntesis , Proteína 61 Rica en Cisteína/genética , Femenino , Humanos , Cadenas Ligeras de Miosina/biosíntesis , Cadenas Ligeras de Miosina/genética , Proteínas de Neoplasias/genética , Elementos de Respuesta , Factor de Transcripción STAT3/genética , Transactivadores/genética , Activación Transcripcional/genética

RESUMEN

Rho-associated coil kinase (ROCK) inhibitors reportedly prevent neurodegeneration, and abnormal ROCK activation in the central nervous system induces neurite collapse and retraction. However, it is unclear whether the ROCK inhibitor Y-27632 directly protects hippocampal neurons from excitotoxicity. Here, we determined the effects of Y-27632 on neuroprotection following kainic acid (KA)-induced seizures in mice and during glutamate-induced excitotoxicity in HT22 cells. One day after Y-27632 injection, mice were treated with KA and killed 1-2 days later. Fluoro-Jade B and rapid Golgi staining showed that Y-27632 protected against KA-induced neurodegeneration and neurite dystrophy. Y-27632 inhibited increases in hippocampal RhoA and ROCK2 in KA-treated mice as determined by western blot analysis. Immunohistochemical analysis revealed ROCK2-positive neurons and astrocytes in the KA-treated hippocampus. In HT22 cells, Y-27632 also protected neurons and neurite formation during glutamate-induced excitotoxicity in vitro. These results indicate that ROCK inhibition modulates neurite growth and protects neurons from excitotoxicity-induced cell death.

Asunto(s)

Amidas/uso terapéutico , Anticonvulsivantes/uso terapéutico , Hipocampo/efectos de los fármacos , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Neuronas/efectos de los fármacos , Neurotoxinas/toxicidad , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridinas/uso terapéutico , Convulsiones/prevención & control , Quinasas Asociadas a rho/antagonistas & inhibidores , Amidas/farmacología , Animales , Anticonvulsivantes/farmacología , Astrocitos/efectos de los fármacos , Astrocitos/enzimología , Muerte Celular , Células Cultivadas/efectos de los fármacos , Convulsivantes/antagonistas & inhibidores , Convulsivantes/toxicidad , Proteína Ácida Fibrilar de la Glía , Ácido Glutámico/toxicidad , Hipocampo/citología , Hipocampo/enzimología , Hipocampo/patología , Ácido Kaínico/toxicidad , Masculino , Ratones , Ratones Endogámicos ICR , Cadenas Ligeras de Miosina/biosíntesis , Cadenas Ligeras de Miosina/genética , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Neuritas/efectos de los fármacos , Neuronas/enzimología , Neuronas/ultraestructura , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Convulsiones/inducido químicamente , Método Simple Ciego , Proteínas de Unión al GTP rho/biosíntesis , Proteínas de Unión al GTP rho/genética , Proteína de Unión al GTP rhoA

RESUMEN

MicroRNA-200c (miR-200c) has been shown to suppress epithelial-mesenchymal transition (EMT), which is attributed mainly to targeting of ZEB1/ZEB2, repressors of the cell-cell contact protein E-cadherin. Here we demonstrated that modulation of miR-200c in breast cancer cells regulates cell migration, cell elongation, and transforming growth factor ß (TGF-ß)-induced stress fiber formation by impacting the reorganization of cytoskeleton that is independent of the ZEB/E-cadherin axis. We identified FHOD1 and PPM1F, direct regulators of the actin cytoskeleton, as novel targets of miR-200c. Remarkably, expression levels of FHOD1 and PPM1F were inversely correlated with the level of miR-200c in breast cancer cell lines, breast cancer patient samples, and 58 cancer cell lines of various origins. Furthermore, individual knockdown/overexpression of these target genes phenocopied the effects of miR-200c overexpression/inhibition on cell elongation, stress fiber formation, migration, and invasion. Mechanistically, targeting of FHOD1 by miR-200c resulted in decreased expression and transcriptional activity of serum response factor (SRF), mediated by interference with the translocation of the SRF coactivator mycocardin-related transcription factor A (MRTF-A). This finally led to downregulation of the expression and phosphorylation of the SRF target myosin light chain 2 (MLC2) gene, required for stress fiber formation and contractility. Thus, miR-200c impacts on metastasis by regulating several EMT-related processes, including a novel mechanism involving the direct targeting of actin-regulatory proteins.

Asunto(s)

Neoplasias de la Mama/patología , Proteínas Fetales/metabolismo , MicroARNs/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Neoplasias de la Mama/metabolismo , Miosinas Cardíacas/biosíntesis , Línea Celular Tumoral , Movimiento Celular , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Femenino , Forminas , Regulación Neoplásica de la Expresión Génica , Humanos , Cadenas Ligeras de Miosina/biosíntesis , Invasividad Neoplásica , Proteínas de Fusión Oncogénica/metabolismo , Factor de Respuesta Sérica/biosíntesis , Fibras de Estrés/metabolismo , Transactivadores , Factor de Crecimiento Transformador beta/metabolismo

RESUMEN

CD3 is a complex of polypeptides which form part of the T cell receptor. Normal human peripheral pan T cells, express not only CD3, but the mRNA for myosin regulatory light chains MYL9, MYL12A, and MYL12B are also significantly expressed. In the Jurkat wild strain, an acute T cell leukemia cell line, CD3 on the surface and MYL9 mRNA are not expressed, while both MYL12A mRNA and MYL12B mRNA are expressed. Jurkat-I, a new clone was established by the transfection of the MYL9 gene into the Jurkat wild strain. As a result, the level of CD3 expressed on the surface of Jurkat-I cells was significantly higher than those in the Jurkat wild strain. Phorbol 12-myristate 13-acetate increased the surface CD3 levels in Jurkat wild strain cells without resulting in MYL9 gene expression, indicating that protein kinase C is partially involved in the expression of CD3 on the surface. These results suggest that surface CD3 expression proceeds through both MYL9-dependent and MYL9-independent pathways (i.e. the protein kinase C- dependent pathway) in Jurkat cells.

Asunto(s)

Complejo CD3/biosíntesis , Regulación de la Expresión Génica/fisiología , Cadenas Ligeras de Miosina/biosíntesis , Proteína Quinasa C/metabolismo , Transducción de Señal/fisiología , Complejo CD3/genética , Carcinógenos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Células Jurkat , Cadenas Ligeras de Miosina/genética , Proteína Quinasa C/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Transducción de Señal/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología

RESUMEN

We show constitutive activation of Rho kinase (ROCK) in cells bearing oncogenic forms of KIT, FLT3, and BCR-ABL, which is dependent on PI3K and Rho GTPase. Genetic or pharmacologic inhibition of ROCK in oncogene-bearing cells impaired their growth as well as the growth of acute myeloid leukemia patient-derived blasts and prolonged the life span of mice bearing myeloproliferative disease. Downstream from ROCK, rapid dephosphorylation or loss of expression of myosin light chain resulted in enhanced apoptosis, reduced growth, and loss of actin polymerization in oncogene-bearing cells leading to significantly prolonged life span of leukemic mice. In summary we describe a pathway involving PI3K/Rho/ROCK/MLC that may contribute to myeloproliferative disease and/or acute myeloid leukemia in humans.

Asunto(s)

Transformación Celular Neoplásica , Proteínas de Fusión bcr-abl/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia/metabolismo , Trastornos Mieloproliferativos/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Factor de Células Madre/metabolismo , Tirosina Quinasa 3 Similar a fms/metabolismo , Quinasas Asociadas a rho/metabolismo , Actinas/metabolismo , Animales , Apoptosis/genética , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Proteínas de Fusión bcr-abl/biosíntesis , Proteínas de Fusión bcr-abl/genética , Humanos , Leucemia/mortalidad , Leucemia/patología , Leucemia Mieloide Aguda/mortalidad , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Trastornos Mieloproliferativos/mortalidad , Trastornos Mieloproliferativos/patología , Cadenas Ligeras de Miosina/biosíntesis , Cadenas Ligeras de Miosina/genética , Cadenas Ligeras de Miosina/metabolismo , Fosfatidilinositol 3-Quinasas/biosíntesis , Fosforilación , Proteínas Tirosina Quinasas/biosíntesis , Proteínas Tirosina Quinasas/genética , Interferencia de ARN , ARN Interferente Pequeño , Transducción de Señal/genética , Factor de Células Madre/biosíntesis , Factor de Células Madre/genética , Tirosina Quinasa 3 Similar a fms/biosíntesis , Tirosina Quinasa 3 Similar a fms/genética , Proteínas de Unión al GTP rho/biosíntesis , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/genética

RESUMEN

Skeletal muscle atrophy commonly occurs in acute and chronic disease. The expression of the muscle-specific E3 ligases atrogin-1 (MAFbx) and muscle RING finger 1 (MuRF1) is induced by atrophy stimuli such as glucocorticoids or absence of IGF-I/insulin and subsequent Akt signaling. We investigated whether glycogen synthase kinase-3ß (GSK-3ß), a downstream molecule in IGF-I/Akt signaling, is required for basal and atrophy stimulus-induced expression of atrogin-1 and MuRF1, and myofibrillar protein loss in C(2)C(12) skeletal myotubes. Abrogation of basal IGF-I signaling, using LY294002, resulted in a prominent induction of atrogin-1 and MuRF1 mRNA and was accompanied by a loss of myosin heavy chain fast (MyHC-f) and myosin light chains 1 (MyLC-1) and -3 (MyLC-3). The synthetic glucocorticoid dexamethasone (Dex) also induced the expression of both atrogenes and likewise resulted in the loss of myosin protein abundance. Genetic ablation of GSK-3ß using small interfering RNA resulted in specific sparing of MyHC-f, MyLC-1, and MyLC-3 protein levels after Dex treatment or impaired IGF-I/Akt signaling. Interestingly, loss of endogenous GSK-3ß suppressed both basal and atrophy stimulus-induced atrogin-1 and MuRF1 expression, whereas pharmacological GSK-3ß inhibition, using CHIR99021 or LiCl, only reduced atrogin-1 mRNA levels in response to LY294002 or Dex. In conclusion, our data reveal that myotube atrophy and myofibrillar protein loss are GSK-3ß dependent, and demonstrate for the first time that basal and atrophy stimulus-induced atrogin-1 mRNA expression requires GSK-3ß enzymatic activity, whereas MuRF1 expression depends solely on the physical presence of GSK-3ß.

Asunto(s)

Glucógeno Sintasa Quinasa 3/metabolismo , Músculo Esquelético/enzimología , Atrofia Muscular/enzimología , Mioblastos/enzimología , Animales , Línea Celular , Cromonas/farmacología , Dexametasona/farmacología , Inhibidores Enzimáticos/farmacología , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3 beta , Factor I del Crecimiento Similar a la Insulina/antagonistas & inhibidores , Cloruro de Litio/farmacología , Ratones , Morfolinas/farmacología , Proteínas Musculares/biosíntesis , Músculo Esquelético/efectos de los fármacos , Atrofia Muscular/tratamiento farmacológico , Mioblastos/efectos de los fármacos , Mioblastos/fisiología , Cadenas Pesadas de Miosina/biosíntesis , Cadenas Ligeras de Miosina/biosíntesis , Piridinas/farmacología , Pirimidinas/farmacología , ARN Interferente Pequeño/metabolismo , Proteínas Ligasas SKP Cullina F-box/biosíntesis , Transducción de Señal/efectos de los fármacos , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas/biosíntesis

RESUMEN

Although myocyte cell transplantation studies have suggested a promising therapeutic potential for myocardial infarction, a major obstacle to the development of clinical therapies for myocardial repair is the difficulties associated with obtaining relatively homogeneous ventricular myocytes for transplantation. Human embryonic stem cells (hESCs) are a promising source of cardiomyocytes. Here we report that retinoid signaling regulates the fate specification of atrial versus ventricular myocytes during cardiac differentiation of hESCs. We found that both Noggin and the pan-retinoic acid receptor antagonist BMS-189453 (RAi) significantly increased the cardiac differentiation efficiency of hESCs. To investigate retinoid functions, we compared Noggin+RAi-treated cultures with Noggin+RA-treated cultures. Our results showed that the expression levels of the ventricular-specific gene IRX-4 were radically elevated in Noggin+RAi-treated cultures. MLC-2V, another ventricular-specific marker, was expressed in the majority of the cardiomyocytes in Noggin+RAi-treated cultures, but not in the cardiomyocytes of Noggin+RA-treated cultures. Flow cytometry analysis and electrophysiological studies indicated that with 64.7 ± 0.88% (mean ±s.e.m) cardiac differentiation efficiency, 83% of the cardiomyocytes in Noggin+RAi-treated cultures had embryonic ventricular-like action potentials (APs). With 50.7 ± 1.76% cardiac differentiation efficiency, 94% of the cardiomyocytes in Noggin+RA-treated cultures had embryonic atrial-like APs. These results were further confirmed by imaging studies that assessed the patterns and properties of the Ca(2+) sparks of the cardiomyocytes from the two cultures. These findings demonstrate that retinoid signaling specifies the atrial versus ventricular differentiation of hESCs. This study also shows that relatively homogeneous embryonic atrial- and ventricular-like myocyte populations can be efficiently derived from hESCs by specifically regulating Noggin and retinoid signals.

Asunto(s)

Diferenciación Celular/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Atrios Cardíacos/metabolismo , Ventrículos Cardíacos/metabolismo , Miocitos Cardíacos/metabolismo , Retinoides/metabolismo , Transducción de Señal , Potenciales de Acción/efectos de los fármacos , Secuencia de Bases , Western Blotting , Miosinas Cardíacas/biosíntesis , Miosinas Cardíacas/genética , Proteínas Portadoras/farmacología , Línea Celular , Células Madre Embrionarias/citología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Atrios Cardíacos/embriología , Ventrículos Cardíacos/embriología , Proteínas de Homeodominio/biosíntesis , Proteínas de Homeodominio/genética , Humanos , Miocitos Cardíacos/efectos de los fármacos , Cadenas Ligeras de Miosina/biosíntesis , Cadenas Ligeras de Miosina/genética , Reacción en Cadena de la Polimerasa , Retinoides/farmacología

RESUMEN

OBJECTIVES: To investigate the effects of estrogen, raloxifene, and levormeloxifene on the expression of Rho-kinase signaling molecules in urethral smooth muscle cells (USMCs). METHODS: USMCs were isolated from female rats. Expression of calponin and estrogen receptors α (ERα) was detected by immunofluorescence staining. Cells were treated with estrogen, raloxifene, or levormeloxifene at 0, 1, 10, and 100 nmol/L for 48 h and then processed for Western blotting with antibodies against RhoA, Rho kinase I and II (Rock-I and Rock-II), myosin light chain (MLC), phosphorylated MLC, and ß-actin. Protein expression was quantitated by densitometry, followed by statistical analysis with ß-actin as control. RESULTS: USMCs expressed calponin and ERα. Treatment of USMCs with estrogen, raloxifene or levormeloxifene resulted in decreased expression of RhoA, Rock-I, Rock-II, and p-MLC in a dosage-dependent manner. CONCLUSIONS: Estrogen, raloxifene, and levormeloxifene may affect urinary continence by inhibiting the expression of Rho-kinase signaling molecules.

Asunto(s)

Estrógenos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Pirrolidinas/farmacología , Clorhidrato de Raloxifeno/farmacología , Uretra/efectos de los fármacos , Quinasas Asociadas a rho/biosíntesis , Animales , Proteínas de Unión al Calcio/biosíntesis , Proteínas de Unión al Calcio/genética , Células Cultivadas/efectos de los fármacos , Células Cultivadas/enzimología , Células Cultivadas/metabolismo , Inducción Enzimática/efectos de los fármacos , Receptor alfa de Estrógeno/biosíntesis , Receptor alfa de Estrógeno/genética , Femenino , Proteínas de Microfilamentos/biosíntesis , Proteínas de Microfilamentos/genética , Miocitos del Músculo Liso/enzimología , Miocitos del Músculo Liso/metabolismo , Cadenas Ligeras de Miosina/biosíntesis , Cadenas Ligeras de Miosina/genética , Pirrolidinas/toxicidad , Clorhidrato de Raloxifeno/toxicidad , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Uretra/citología , Incontinencia Urinaria/fisiopatología , Quinasas Asociadas a rho/genética , Proteína de Unión al GTP rhoA/biosíntesis , Proteína de Unión al GTP rhoA/genética , Calponinas

RESUMEN

BACKGROUND: The smooth muscle actin binding proteins Caldesmon and Tropomyosin (Tm) promote thin filament assembly by stabilizing actin polymerization, however, whether filament assembly affects either the stability or activation of these and other smooth muscle regulatory proteins is not known. METHODS: Measurement of smooth muscle regulatory protein levels in wild type zebrafish larvae following antisense knockdown of smooth muscle actin (Acta2) and myosin heavy chain (Myh11) proteins, and in colourless mutants that lack enteric nerves. Comparison of intestinal peristalsis in wild type and colourless larvae. KEY RESULTS: Knockdown of Acta2 led to reduced levels of phospho-Caldesmon and Tm. Total Caldesmon and phospho-myosin light chain (p-Mlc) levels were unaffected. Knockdown of Myh11 had no effect on the levels of either of these proteins. Phospho-Caldesmon and p-Mlc levels were markedly reduced in colourless mutants that have intestinal motility comparable with wild type larvae. CONCLUSIONS & INFERENCES: These in vivo findings provide new information regarding the activation and stability of smooth muscle regulatory proteins in zebrafish larvae and their role in intestinal peristalsis in this model organism.

Asunto(s)

Citoesqueleto de Actina/fisiología , Intestinos/citología , Intestinos/fisiología , Proteínas Musculares/metabolismo , Músculo Liso/citología , Músculo Liso/fisiología , Proteínas de Pez Cebra/metabolismo , Pez Cebra/fisiología , Animales , Proteínas de Unión a Calmodulina/biosíntesis , Proteínas de Unión a Calmodulina/genética , Sistema Nervioso Entérico/fisiología , Inmunohistoquímica , Mucosa Intestinal/metabolismo , Larva , Proteínas de Microfilamentos/metabolismo , Microscopía Confocal , Morfolinas/farmacología , Músculo Liso/metabolismo , Mutación/fisiología , Cadenas Pesadas de Miosina/biosíntesis , Cadenas Pesadas de Miosina/genética , Cadenas Ligeras de Miosina/biosíntesis , Cadenas Ligeras de Miosina/genética , Peristaltismo/fisiología , ARN sin Sentido , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Tropomiosina/biosíntesis , Tropomiosina/genética

RESUMEN

BACKGROUND AND OBJECTIVE: The junctional epithelium attaches to the tooth enamel at the dentogingival junction. The attachment mechanisms of the junctional epithelium have been studied histologically, but the molecular functions of the junctional epithelium have not been elucidated. The aim of this study was to perform a comprehensive analysis of gene expression in the junctional epithelium and to search for specific genetic markers of the junctional epithelium. MATERIAL AND METHODS: A comprehensive analysis of genes expressed in the mouse junctional epithelium and oral gingival epithelium was performed using laser microdissection and microarray analysis. To extract high-quality RNA from these tissues, we made frozen sections using a modified film method. Confirmation of the differential expression of selected genes was performed by quantitative real-time PCR and immunohistochemistry. RESULTS: The modified method produced RNA of sufficient quality for microarray analysis. The result of microarray analysis showed that 841 genes were up-regulated in the junctional epithelium compared with the oral gingival epithelium, and five were increased more than 50-fold in the junctional epithelium. These five genes were secretory leukocyte protease inhibitor (Slpi), keratin 17 (Krt17), annexin A1 (Anxa1), myosin light peptide 6 (Myl6) and endoplasmic reticulum protein 29 (Erp29). In particular, Slpi expression in the junctional epithelium was approximately 100-fold higher than in the oral gingival epithelium by real-time PCR. Additionally, immunohistochemistry indicated that the Slpi protein is highly expressed in the junctional epithelium. CONCLUSION: We developed a method for generating fresh-frozen tissue sections suitable for extraction of good-quality RNA. We determined that Slpi is characteristically expressed in the junctional epithelium. Our results provide a substantial advance in the analysis of gene expression in the junctional epithelium.

Asunto(s)

Inserción Epitelial/metabolismo , Perfilación de la Expresión Génica/métodos , Inhibidor Secretorio de Peptidasas Leucocitarias/biosíntesis , Animales , Anexina A1/biosíntesis , Anexina A1/genética , Retículo Endoplásmico , Inserción Epitelial/enzimología , Secciones por Congelación , Encía/metabolismo , Proteínas de Choque Térmico/biosíntesis , Proteínas de Choque Térmico/genética , Queratina-17/biosíntesis , Queratina-17/genética , Láseres de Gas , Ratones , Microdisección/métodos , Cadenas Ligeras de Miosina/biosíntesis , Cadenas Ligeras de Miosina/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Inhibidor Secretorio de Peptidasas Leucocitarias/genética

RESUMEN

The participation of nonmuscle myosin in force maintenance is controversial. Furthermore, its regulation is difficult to examine in a cellular context, as the light chains of smooth muscle and nonmuscle myosin comigrate under native and denaturing electrophoresis techniques. Therefore, the regulatory light chains of smooth muscle myosin (SM-RLC) and nonmuscle myosin (NM-RLC) were purified, and these proteins were resolved by isoelectric focusing. Using this method, intact mouse aortic smooth muscle homogenates demonstrated four distinct RLC isoelectric variants. These spots were identified as phosphorylated NM-RLC (most acidic), nonphosphorylated NM-RLC, phosphorylated SM-RLC, and nonphosphorylated SM-RLC (most basic). During smooth muscle activation, NM-RLC phosphorylation increased. During depolarization, the increase in NM-RLC phosphorylation was unaffected by inhibition of either Rho kinase or PKC. However, inhibition of Rho kinase blocked the angiotensin II-induced increase in NM-RLC phosphorylation. Additionally, force for angiotensin II stimulation of aortic smooth muscle from heterozygous nonmuscle myosin IIB knockout mice was significantly less than that of wild-type littermates, suggesting that, in smooth muscle, activation of nonmuscle myosin is important for force maintenance. The data also demonstrate that, in smooth muscle, the activation of nonmuscle myosin is regulated by Ca(2+)-calmodulin-activated myosin light chain kinase during depolarization and a Rho kinase-dependent pathway during agonist stimulation.

Asunto(s)

Músculo Liso/fisiología , Miosinas/metabolismo , Miosina Tipo IIB no Muscular/metabolismo , Secuencia de Aminoácidos , Angiotensina II/farmacología , Animales , Western Blotting , Pollos , Electroforesis en Gel Bidimensional , Regulación de la Expresión Génica/fisiología , Espectrometría de Masas , Ratones , Ratones Transgénicos , Contracción Muscular/fisiología , Músculo Liso/efectos de los fármacos , Cadenas Ligeras de Miosina/biosíntesis , Cadenas Ligeras de Miosina/química , Cadenas Ligeras de Miosina/metabolismo , Miosinas/química , Miosinas/genética , Miosina Tipo IIB no Muscular/química , Miosina Tipo IIB no Muscular/genética , Fosforilación/efectos de los fármacos , Proteínas Recombinantes , Quinasas Asociadas a rho/antagonistas & inhibidores

RESUMEN

Previous studies have suggested that mesenchymal stem cells (MSCs) can differentiate into smooth muscle-like cells. However their functionalities remain questionable. The aim of this study was to investigate the functionality of MSCs differentiated into smooth muscle (SM) in vitro by SM-inducing medium. MSCs have been isolated from rat bone marrow and cultured in SM-inducing medium. After 21 days in culture, messenger RNA and specific SM proteins such as myosin heavy chain and myosin light chain 2 were expressed in the in vitro differentiated MSCs to a similar level of that in freshly isolated SM cells (SMCs). At the electrophysiological level, MSCs presented an outward K+ current with an IK(DR) component and IK(Ca) component. In vitro differentiation induced an enhancement of the IK(Ca) current to a level similar to that observed in aortic SMCs. Calcium homeostasis measurements revealed that both differentiated and undifferentiated MSCs responded to extracellular adenosine triphosphate (ATP) in a similar fashion to SMCs. However MSCs failed to contract in response to ATP. This data shows that despite specific SM protein expression and modification of electrophysiological properties similar to that of aortic SMCs, MSCs cultured in differentiation medium failed to display contractile properties. These results underline the necessity to find the ideal cultured conditions to induce complete SMC function.

Asunto(s)

Diferenciación Celular/fisiología , Potenciales de la Membrana/fisiología , Células Madre Mesenquimatosas/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Adenosina Trifosfato/farmacología , Animales , Calcio/metabolismo , Miosinas Cardíacas/biosíntesis , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Homeostasis/fisiología , Potenciales de la Membrana/efectos de los fármacos , Células Madre Mesenquimatosas/citología , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Cadenas Pesadas de Miosina/biosíntesis , Cadenas Ligeras de Miosina/biosíntesis , Ratas , Factores de Tiempo

RESUMEN

BACKGROUND: Dilated cardiomyopathy is a naturally occurring disease in humans and dogs. Human studies have shown increased levels of myosin heavy chain (MHC)-beta in failing ventricles and the left atria (LA) and of ventricular light chain (VLC)-2 in the right atria in dilated cardiomyopathy. METHODS AND RESULTS: This study evaluates the levels of MHC-beta in all heart chambers in prolonged canine right ventricular pacing. In addition, we determined whether levels of VLC2 were altered in these hearts. Failing hearts demonstrated significantly increased levels of MHC-beta in the right atria, right atrial appendage, LA, left atrial appendage (LAA), and right ventricle compared with controls. Significant levels of VLC2 were detected in the right atria of paced hearts. Differences in MHC-beta expression were observed between the LA and the LAA of paced and control dogs. MHC-beta expression was significantly greater in the LA of paced and control dogs compared with their respective LAA. CONCLUSIONS: The cardiac myosin isoform shifts in this study were similar to those observed in end-stage human heart failure and more severe than those reported in less prolonged pacing models, supporting the use of this model for further study of end-stage human heart failure. The observation of consistent differences between sampling sites, especially LA versus LAA, indicates the need for rigorous sampling consistency in future studies.

Asunto(s)

Función del Atrio Derecho/fisiología , Cardiomiopatías/metabolismo , Cadenas Pesadas de Miosina/biosíntesis , Cadenas Ligeras de Miosina/biosíntesis , Miosina Tipo IIB no Muscular/biosíntesis , Animales , Cardiomiopatías/genética , Perros , Regulación de la Expresión Génica/fisiología , Cadenas Pesadas de Miosina/genética , Cadenas Ligeras de Miosina/genética , Miosina Tipo IIB no Muscular/genética