RESUMEN
Force generation by the molecular motor myosin II (MII) at the actin cortex is a universal feature of animal cells. Despite its central role in driving cell shape changes, the mechanisms underlying MII regulation at the actin cortex remain incompletely understood. Here we show that myosin light chain kinase (MLCK) promotes MII turnover at the mitotic cortex. Inhibition of MLCK resulted in an alteration of the relative levels of phosphorylated regulatory light chain (RLC), with MLCK preferentially creating a short-lived pRLC species and Rho-associated kinase (ROCK) preferentially creating a stable ppRLC species during metaphase. Slower turnover of MII and altered RLC homeostasis on MLCK inhibition correlated with increased cortex tension, driving increased membrane bleb initiation and growth, but reduced bleb retraction during mitosis. Taken together, we show that ROCK and MLCK play distinct roles at the actin cortex during mitosis; ROCK activity is required for recruitment of MII to the cortex, while MLCK activity promotes MII turnover. Our findings support the growing evidence that MII turnover is an essential dynamic process influencing the mechanical output of the actin cortex.
Asunto(s)
Actinas , Proteínas de Unión al Calcio , Miosina Tipo II , Quinasa de Cadena Ligera de Miosina , Humanos , Actinas/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/fisiología , División del Núcleo Celular , Proteínas del Citoesqueleto/metabolismo , Células HeLa , Mitosis/fisiología , Cadenas Ligeras de Miosina/metabolismo , Miosina Tipo II/metabolismo , Miosina Tipo II/fisiología , Quinasa de Cadena Ligera de Miosina/metabolismo , Quinasa de Cadena Ligera de Miosina/fisiología , Fosforilación , Quinasas Asociadas a rho/metabolismoRESUMEN
Striated preferentially expressed gene (SPEG), a member of the myosin light chain kinase family, is localized at the level of triad surrounding myofibrils in skeletal muscles. In humans, SPEG mutations are associated with centronuclear myopathy and cardiomyopathy. Using a striated muscle-specific Speg-knockout (KO) mouse model, we have previously shown that SPEG is critical for triad maintenance and calcium handling. Here, we further examined the molecular function of SPEG and characterized the effects of SPEG deficiency on triad and focal adhesion proteins. We used yeast two-hybrid assay, and identified desmin, an intermediate filament protein, to interact with SPEG and confirmed this interaction by co-immunoprecipitation. Using domain-mapping assay, we defined that Ig-like and fibronectin III domains of SPEG interact with rod domain of desmin. In skeletal muscles, SPEG depletion leads to desmin aggregates in vivo and a shift in desmin equilibrium from soluble to insoluble fraction. We also profiled the expression and localization of triadic proteins in Speg-KO mice using western blot and immunofluorescence. The amount of RyR1 and triadin were markedly reduced, whereas DHPRα1, SERCA1 and triadin were abnormally accumulated in discrete areas of Speg-KO myofibers. In addition, Speg-KO muscles exhibited internalized vinculin and ß1 integrin, both of which are critical components of the focal adhesion complex. Further, ß1 integrin was abnormally accumulated in early endosomes of Speg-KO myofibers. These results demonstrate that SPEG-deficient skeletal muscles exhibit several pathological features similar to those seen in MTM1 deficiency. Defects of shared cellular pathways may underlie these structural and functional abnormalities in both types of diseases.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Desmina/metabolismo , Adhesiones Focales/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Musculares/metabolismo , Proteínas Musculares/fisiología , Músculo Esquelético/patología , Miopatías Estructurales Congénitas/patología , Quinasa de Cadena Ligera de Miosina/fisiología , Animales , Calcio/metabolismo , Moléculas de Adhesión Celular/genética , Desmina/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Mutación , Miopatías Estructurales Congénitas/etiología , Miopatías Estructurales Congénitas/metabolismoRESUMEN
The intestinal epithelial apical junctional complex, which includes tight and adherens junctions, contributes to the intestinal barrier function via their role in regulating paracellular permeability. Myosin light chain II (MLC-2), has been shown to be a critical regulatory protein in altering paracellular permeability during gastrointestinal disorders. Previous studies have demonstrated that phosphorylation of MLC-2 is a biochemical marker for perijunctional actomyosin ring contraction, which increases paracellular permeability by regulating the apical junctional complex. The phosphorylation of MLC-2 is dominantly regulated by myosin light chain kinase- (MLCK-) and Rho-associated coiled-coil containing protein kinase- (ROCK-) mediated pathways. In this review, we aim to summarize the current state of knowledge regarding the role of MLCK- and ROCK-mediated pathways in the regulation of the intestinal barrier during normal homeostasis and digestive diseases. Additionally, we will also suggest potential therapeutic targeting of MLCK- and ROCK-associated pathways in gastrointestinal disorders that compromise the intestinal barrier.
Asunto(s)
Mucosa Intestinal/fisiología , Quinasa de Cadena Ligera de Miosina/metabolismo , Quinasas Asociadas a rho/metabolismo , Animales , Humanos , Mucosa Intestinal/metabolismo , Quinasa de Cadena Ligera de Miosina/fisiología , Permeabilidad , Uniones Estrechas/metabolismo , Quinasas Asociadas a rho/fisiologíaRESUMEN
In adult Hydra, epitheliomuscle cells form the monolayered ecto- and endodermal epithelia. Their basal myonemes function as a longitudinal and circular muscle, respectively. Based on the observation that a Rho/Rock pathway, controlling the cell shape changes during detachment of Hydra buds, is not involved in body movement, at least two actomyosin compartments must exist in these cells: a basal one for body movement and a cortical one for cell shape changes. We therefore analyzed the regional and subcellular localization of the Ser19-phosphorylated myosin regulatory light chain (pMLC20). Along the body column, pMLC20 was detected strongly in the basal myonemes and weakly in the apical cell compartments of ectodermal epitheliomuscle cells. In cells of the bud base undergoing morphogenesis, pMLC20 was localized to intracellular stress fibers as well as to the apical and additionally to the lateral cortical compartment. Pharmacological inhibition revealed that pMLC20 is induced in these compartments by at least two independent pathways. In myonemes, MLC is phosphorylated mainly by myosin light chain kinase (MLCK). In contrast, the cortical apical and lateral MLC phosphorylation in constricting ectodermal cells of the bud base is stimulated via the Rho/ROCK pathway.
Asunto(s)
Actomiosina/metabolismo , Contracción Muscular/fisiología , Cadenas Ligeras de Miosina/metabolismo , Citoesqueleto de Actina/metabolismo , Actomiosina/fisiología , Animales , Forma de la Célula , Células Epiteliales/metabolismo , Hydra/metabolismo , Quinasa de Cadena Ligera de Miosina/metabolismo , Quinasa de Cadena Ligera de Miosina/fisiología , Fosforilación , Transducción de Señal , Fibras de Estrés/metabolismo , Quinasas Asociadas a rho/metabolismoRESUMEN
Myosin activation contributes to the contractile forces that induce disturbances in the vascular endothelial integrity and promote protein-rich edema of the underlying tissues. Myosin light chain kinase (MLCK) and Rho-associated protein kinase (ROCK) have been reported to phosphorylate myosin regulatory light chains (RLC) for myosin activation. However, the relative contribution and roles of these kinases are debatable and not understood in very detail. In this study, using a combinational inhibitory analysis of MLCK, ROCK, and their antagonist, myosin light chain phosphatase (MLCP), we show that the MLCK-dependent RLC mono-(Ser19)phosphorylation (P-RLC) is sufficient to induce the FITC-dextran hyperpermeability in EA.hy926 endothelial cells (EC) in response to thrombin. However, MLCK relies on the ROCK assistance that attenuates MLCP activity. On the other hand, MLCK supplies P-RLC myosin as an intermediate substrate to ROCK thus adding to a faster accumulation of di-(Thr18/Ser19)phosphorylated RLC (PP-RLC) by the latter kinase. ROCK also produces P-RLC but is solely responsible for the thrombin-induced PP-RLC generation in EA.hy926 EC and other cell types. Still, as a direct myosin activator, ROCK contributes less to endothelial hyperpermeability than MLCK. Our findings are consistent with a concerted complementary mutual interplay between ROCK and MLCK to activate endothelial myosin and elicit the thrombin-mediated EC barrier dysfunction.
Asunto(s)
Células Endoteliales/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Quinasa de Cadena Ligera de Miosina/fisiología , Quinasas Asociadas a rho/fisiología , Células Cultivadas , Humanos , Contracción Muscular/fisiología , Cadenas Ligeras de Miosina/metabolismo , Fosfatasa de Miosina de Cadena Ligera/metabolismo , Transducción de SeñalRESUMEN
Sympathetic tone may influence force potentiation, that is, the stimulation-induced increase in skeletal muscle mechanical function associated with myosin phosphorylation, although the mechanism for this effect remains unknown. The purpose of this study was to examine the influence of epinephrine on concentric twitch force potentiation of wild-type and skeletal myosin light-chain kinase devoid mouse muscle (skMLCK-/- ). To this end, concentric twitch force was assessed before and after a potentiating stimulus (PS) to determine the peak and the duration of potentiation in the absence (-EPI) and presence (+EPI) of 1 µmol/L epinephrine in both genotypes. Twitch force of wild-type and skMLCK-/- muscles was increased by up to 31 and 35% and 18 and 23% in the -EPI and EPI conditions, respectively (all data n = 8, P < 0.05). In wild-type muscles, the PS increased RLC phosphorylation from 0.14 ± 0.05 (rest) to 0.66 ± 0.08 mol phos mol RLC; by 480 sec RLC phosphorylation had returned to baseline (all data n = 4 each time point, P < 0.05). Neither resting nor peak levels of RLC phosphorylation were altered by +EPI, although the duration of RLC phosphorylation was prolonged. In skMLCK-/- muscles, RLC phosphorylation was not elevated above constituent levels by stimulation in either the -EPI or +EPI condition. Thus, given the similarity in potentiation responses between genotypes our data suggest that the influence of epinephrine on potentiation was independent of skMLCK catalyzed phosphorylation of the RLC, although the clinical significance of this pathway for skeletal muscle function remains to be identified.
Asunto(s)
Epinefrina/fisiología , Contracción Muscular , Músculo Esquelético/fisiología , Quinasa de Cadena Ligera de Miosina/fisiología , Miosinas/metabolismo , Animales , Epinefrina/administración & dosificación , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Esquelético/efectos de los fármacos , Cadenas Ligeras de Miosina/metabolismo , Quinasa de Cadena Ligera de Miosina/genética , FosforilaciónRESUMEN
Mucosal surfaces are lined by epithelial cells. In the intestine, the epithelium establishes a selectively permeable barrier that supports nutrient absorption and waste secretion while preventing intrusion by luminal materials. Intestinal epithelia therefore play a central role in regulating interactions between the mucosal immune system and luminal contents, which include dietary antigens, a diverse intestinal microbiome, and pathogens. The paracellular space is sealed by the tight junction, which is maintained by a complex network of protein interactions. Tight junction dysfunction has been linked to a variety of local and systemic diseases. Two molecularly and biophysically distinct pathways across the intestinal tight junction are selectively and differentially regulated by inflammatory stimuli. This review discusses the mechanisms underlying these events, their impact on disease, and the potential of using these as paradigms for development of tight junction-targeted therapeutic interventions.
Asunto(s)
Uniones Estrechas/fisiología , Animales , Humanos , Inmunidad Mucosa , Interleucina-13/fisiología , Mucosa Intestinal/anatomía & histología , Membrana Mucosa/anatomía & histología , Quinasa de Cadena Ligera de Miosina/fisiología , Permeabilidad , Uniones Estrechas/química , Proteína de la Zonula Occludens-1/fisiologíaRESUMEN
Tumor necrosis factor (TNF)-α, a key mediator of intestinal inflammation, causes an increase in intestinal epithelial tight junction (TJ) permeability by activating myosin light chain kinase (MLCK; official name MYLK3) gene. However, the precise signaling cascades that mediate the TNF-α-induced activation of MLCK gene and increase in TJ permeability remain unclear. Our aims were to delineate the upstream signaling mechanisms that regulate the TNF-α modulation of intestinal TJ barrier function with the use of in vitro and in vivo intestinal epithelial model systems. TNF-α caused a rapid activation of both canonical and noncanonical NF-κB pathway. NF-κB-inducing kinase (NIK) and mitogen-activated protein kinase kinase-1 (MEKK-1) were activated in response to TNF-α. NIK mediated the TNF-α activation of inhibitory κB kinase (IKK)-α, and MEKK1 mediated the activation of IKK complex, including IKK-ß. NIK/IKK-α axis regulated the activation of both NF-κB p50/p65 and RelB/p52 pathways. Surprisingly, the siRNA induced knockdown of NIK, but not MEKK-1, prevented the TNF-α activation of both NF-κB p50/p65 and RelB/p52 and the increase in intestinal TJ permeability. Moreover, NIK/IKK-α/NF-κB p50/p65 axis mediated the TNF-α-induced MLCK gene activation and the subsequent MLCK increase in intestinal TJ permeability. In conclusion, our data show that NIK/IKK-α/regulates the activation of NF-κB p50/p65 and plays an integral role in the TNF-α-induced activation of MLCK gene and increase in intestinal TJ permeability.
Asunto(s)
Quinasa I-kappa B/metabolismo , Quinasa 1 de Quinasa de Quinasa MAP/metabolismo , FN-kappa B/metabolismo , Uniones Estrechas/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Animales , Células CACO-2 , Células Cultivadas , Humanos , Intestino Delgado/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Quinasa de Cadena Ligera de Miosina/genética , Quinasa de Cadena Ligera de Miosina/fisiología , FN-kappa B/antagonistas & inhibidores , Subunidad p50 de NF-kappa B/metabolismo , Subunidad p52 de NF-kappa B/metabolismo , Permeabilidad , Regiones Promotoras Genéticas/fisiología , ARN Interferente Pequeño/metabolismo , Factor de Transcripción ReIA/metabolismo , Transfección , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Myosin light chain kinase (MLCK) expression is downregulated in breast cancer, including invasive ductal carcinoma compared with ductal breast carcinoma in situ and metastatic breast tumors. However, little is known about how loss of MLCK expression contributes to tumor progression. MLCK is a component of the actin cytoskeleton and its known role is the phosphorylation of the regulatory light chain of myosin II. To gain insights into the role of MLCK in breast cancer, we perturbed its function using small interfering RNA (siRNA) or pharmacological inhibition in untransformed breast epithelial cells (MCF10A). Loss of MLCK by siRNAs led to increased cell migration and invasion, disruption of cell-cell adhesions and enhanced formation of focal adhesions at the leading edge of migratory cells. In addition, downregulation of MLCK cooperated with HER2 in MCF10A cells to promote cell migration and invasion and low levels of MLCK is associated with a poor prognosis in HER2-positive breast cancer patients. Associated with these altered migratory behaviors were increased expression of epidermal growth factor receptor and activation of extracellular signal-regulated kinase and c-Jun N-terminal kinase signaling pathways in MLCK downregulated MCF10A cells. By contrast, inhibition of the kinase function of MLCK using pharmacological agents inhibited cell migration and invasion, and did not affect cellular adhesions. Our results show that loss of MLCK contributes to the migratory properties of epithelial cells resulting from changes in cell-cell and cell-matrix adhesions, and increased epidermal growth factor receptor signaling. These findings suggest that decreased expression of MLCK may have a critical role during tumor progression by facilitating the metastatic potential of tumor cells.
Asunto(s)
Neoplasias de la Mama/patología , Mama/patología , Receptores ErbB/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Quinasa de Cadena Ligera de Miosina/fisiología , Citoesqueleto de Actina/metabolismo , Neoplasias de la Mama/enzimología , Adhesión Celular , Agregación Celular , Línea Celular Tumoral , Movimiento Celular , Células Cultivadas , Células Epiteliales/patología , Femenino , Humanos , Invasividad Neoplásica , ARN Interferente Pequeño/genéticaRESUMEN
This review discusses and summarizes the results of molecular and cellular investigations of myosin light chain kinase (MLCK, MYLK1), the key regulator of cell motility. The structure and regulation of a complex mylk1 gene and the domain organization of its products is presented. The interactions of the mylk1 gene protein products with other proteins and posttranslational modifications of the mylk1 gene protein products are reviewed, which altogether might determine the role and place of MLCK in physiological and pathological reactions of cells and entire organisms. Translational potential of MLCK as a drug target is evaluated.
Asunto(s)
Proteínas de Unión al Calcio/fisiología , Quinasa de Cadena Ligera de Miosina/fisiología , Animales , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Regulación de la Expresión Génica , Humanos , Quinasa de Cadena Ligera de Miosina/química , Quinasa de Cadena Ligera de Miosina/genética , Dominios Proteicos , Procesamiento Proteico-PostraduccionalRESUMEN
While is widely acknowledged that nonmuscle myosin II (NMMII) enables stress fibers (SFs) to generate traction forces against the extracellular matrix, little is known about how specific NMMII isoforms and functional domains contribute to SF mechanics. Here we combine biophotonic and genetic approaches to address these open questions. First, we suppress the NMMII isoforms MIIA and MIIB and apply femtosecond laser nanosurgery to ablate and investigate the viscoelastic retraction of individual SFs. SF retraction dynamics associated with MIIA and MIIB suppression qualitatively phenocopy our earlier measurements in the setting of Rho kinase (ROCK) and myosin light chain kinase (MLCK) inhibition, respectively. Furthermore, fluorescence imaging and photobleaching recovery reveal that MIIA and MIIB are enriched in and more stably localize to ROCK- and MLCK-controlled central and peripheral SFs, respectively. Additional domain-mapping studies surprisingly reveal that deletion of the head domain speeds SF retraction, which we ascribe to reduced drag from actomyosin crosslinking and frictional losses. We propose a model in which ROCK/MIIA and MLCK/MIIB functionally regulate common pools of SFs, with MIIA crosslinking and motor functions jointly contributing to SF retraction dynamics and cellular traction forces.
Asunto(s)
Miosina Tipo II/química , Miosina Tipo II/fisiología , Quinasa de Cadena Ligera de Miosina/fisiología , Fibras de Estrés/química , Fibras de Estrés/fisiología , Quinasas Asociadas a rho/fisiología , Secuencia de Aminoácidos , Módulo de Elasticidad/fisiología , Humanos , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/fisiología , Datos de Secuencia Molecular , Músculo Esquelético/química , Músculo Esquelético/fisiología , Quinasa de Cadena Ligera de Miosina/química , Isoformas de Proteínas/química , Isoformas de Proteínas/fisiología , Estructura Terciaria de Proteína , Estrés Mecánico , Relación Estructura-Actividad , Viscosidad , Quinasas Asociadas a rho/químicaRESUMEN
The effect of phosphorylation on the conformation of the regulatory light chain (cRLC) region of myosin in ventricular trabeculae from rat heart was determined by polarized fluorescence from thiophosphorylated cRLCs labelled with bifunctional sulforhodamine (BSR). Less than 5% of cRLCs were endogenously phosphorylated in this preparation, and similarly low values of basal cRLC phosphorylation were measured in fresh intact ventricle from both rat and mouse hearts. BSR-labelled cRLCs were thiophosphorylated by a recombinant fragment of human cardiac myosin light chain kinase, which was shown to phosphorylate cRLCs specifically at serine 15 in a calcium- and calmodulin-dependent manner, both in vitro and in situ. The BSR-cRLCs were exchanged into demembranated trabeculae, and polarized fluorescence intensities measured for each BSR-cRLC in relaxation, active isometric contraction and rigor were combined with RLC crystal structures to calculate the orientation distribution of the C-lobe of the cRLC in each state. Only two of the four C-lobe orientation populations seen during relaxation and active isometric contraction in the unphosphorylated state were present after cRLC phosphorylation. Thus cRLC phosphorylation alters the equilibrium between defined conformations of the cRLC regions of the myosin heads, rather than simply disordering the heads as assumed previously. cRLC phosphorylation also changes the orientation of the cRLC C-lobe in rigor conditions, showing that the orientation of this part of the myosin head is determined by its interaction with the thick filament even when the head is strongly bound to actin. These results suggest that cRLC phosphorylation controls the contractility of the heart by modulating the interaction of the cRLC region of the myosin heads with the thick filament backbone.
Asunto(s)
Cadenas Ligeras de Miosina/química , Procesamiento Proteico-Postraduccional , Animales , Humanos , Miocardio/química , Miocardio/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Quinasa de Cadena Ligera de Miosina/química , Quinasa de Cadena Ligera de Miosina/fisiología , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosforilación , Conformación Proteica , Ratas WistarRESUMEN
The role of myosin light chain kinase (MLCK) in inducing podosomes was examined by confocal and electron microscopy. Removal of myosin from the actin core of podosomes using blebbistatin, a myosin inhibitor, resulted in the formation of smaller podosomes. Downregulation of MLCK by the transfection of MLCK small interfering RNA (siRNA) led to the failure of podosome formation. However, ML-7, an inhibitor of the kinase activity of MLCK, failed to inhibit podosome formation. Based on our previous report (Thatcher et al. J.Pharm.Sci. 116 116-127, 2011), we outlined the important role of the actin-binding activity of MLCK in producing smaller podosomes.
Asunto(s)
Quinasa de Cadena Ligera de Miosina/fisiología , Forbol 12,13-Dibutirato/farmacología , Podosomas/efectos de los fármacos , Podosomas/ultraestructura , Actinas/metabolismo , Animales , Azepinas/farmacología , Células Cultivadas , Regulación hacia Abajo , Microscopía Inmunoelectrónica , Quinasa de Cadena Ligera de Miosina/antagonistas & inhibidores , Quinasa de Cadena Ligera de Miosina/genética , Quinasa de Cadena Ligera de Miosina/metabolismo , Naftalenos/farmacología , Podosomas/genética , Unión Proteica , ARN Interferente Pequeño , RatasRESUMEN
The mammalian oocyte undergoes two rounds of asymmetric cell divisions during meiotic maturation and fertilization. Acentric spindle positioning and cortical polarity are two major factors involved in asymmetric cell division, both of which are thought to depend on the dynamic interaction between myosin II and actin filaments. Myosin light chain kinase (MLCK), encoded by the Mylk1 gene, could directly phosphorylate and activate myosin II. To determine whether MLCK was required for oocyte asymmetric division, we specifically disrupted the Mylk1 gene in oocytes by Cre-loxP conditional knockout system. We found that Mylk1 mutant female mice showed severe subfertility. Unexpectedly, contrary to previously reported in vitro findings, our data showed that oocyte meiotic maturation including spindle organization, polarity establishment, homologous chromosomes separation, and polar body extrusion were not affected in Mylk1(fl/fl);GCre(+) females. Follicular development, ovulation, and early embryonic development up to compact morula occurred normally in Mylk1(fl/fl);GCre(+) females, but deletion of MLCK caused delayed morula-to-blastocyst transition. More than a third of embryos were at morula stage at 3.5 Days Postcoitum in vivo. The delayed embryos could develop further to early blastocyst stage in vitro on Day 4 when most control embryos reached expanded blastocysts. Our findings provide evidence that MLCK is linked to timely blastocyst formation, though it is dispensable for oocyte meiotic maturation.
Asunto(s)
Blastocisto/fisiología , Fertilidad/genética , Mórula/fisiología , Quinasa de Cadena Ligera de Miosina/genética , Quinasa de Cadena Ligera de Miosina/fisiología , Oocitos/fisiología , Folículo Ovárico/fisiología , Animales , Cromosomas de los Mamíferos/genética , Femenino , Fertilidad/fisiología , Fertilización/genética , Eliminación de Gen , Infertilidad/genética , Infertilidad/fisiopatología , Meiosis/genética , Ratones , Ratones Endogámicos C57BL , Cuerpos Polares/fisiología , Embarazo , Huso Acromático/genética , Huso Acromático/fisiologíaRESUMEN
UNLABELLED: Intestinal barrier dysfunction is an important contributor to alcoholic liver disease (ALD). Translocated microbial products trigger an inflammatory response in the liver and contribute to steatohepatitis. Our aim was to investigate mechanisms of barrier disruption after chronic alcohol feeding. A Lieber-DeCarli model was used to induce intestinal dysbiosis, increased intestinal permeability, and liver disease in mice. Alcohol feeding for 8 weeks induced intestinal inflammation in the jejunum, which is characterized by an increased number of tumor necrosis factor alpha (TNF-α)-producing monocytes and macrophages. These findings were confirmed in duodenal biopsies from patients with chronic alcohol abuse. Intestinal decontamination with nonabsorbable antibiotics restored eubiosis, decreased intestinal inflammation and permeability, and reduced ALD in mice. TNF-receptor I (TNFRI) mutant mice were protected from intestinal barrier dysfunction and ALD. To investigate whether TNFRI on intestinal epithelial cells mediates intestinal barrier dysfunction and ALD, we used TNFRI mutant mice carrying a conditional gain-of-function allele for this receptor. Reactivation of TNFRI on intestinal epithelial cells resulted in increased intestinal permeability and liver disease that is similar to wild-type mice after alcohol feeding, suggesting that enteric TNFRI promotes intestinal barrier dysfunction. Myosin light-chain kinase (MLCK) is a downstream target of TNF-α and was phosphorylated in intestinal epithelial cells after alcohol administration. Using MLCK-deficient mice, we further demonstrate a partial contribution of MLCK to intestinal barrier dysfunction and liver disease after chronic alcohol feeding. CONCLUSION: Dysbiosis-induced intestinal inflammation and TNFRI signaling in intestinal epithelial cells mediate a disruption of the intestinal barrier. Therefore, intestinal TNFRI is a crucial mediator of ALD.
Asunto(s)
Disbiosis/complicaciones , Hepatopatías Alcohólicas/etiología , Receptores Tipo I de Factores de Necrosis Tumoral/fisiología , Animales , Enteritis/complicaciones , Mucosa Intestinal/fisiología , Enfermedades del Yeyuno/complicaciones , Ratones , Ratones Endogámicos C57BL , Quinasa de Cadena Ligera de Miosina/fisiología , Permeabilidad , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Myosin light chain phosphatase with its regulatory subunit, myosin phosphatase target subunit 1 (MYPT1) modulates Ca(2+)-dependent phosphorylation of myosin light chain by myosin light chain kinase, which is essential for smooth muscle contraction. The role of MYPT1 in vascular smooth muscle was investigated in adult MYPT1 smooth muscle specific knock-out mice. MYPT1 deletion enhanced phosphorylation of myosin regulatory light chain and contractile force in isolated mesenteric arteries treated with KCl and various vascular agonists. The contractile responses of arteries from knock-out mice to norepinephrine were inhibited by Rho-associated kinase (ROCK) and protein kinase C inhibitors and were associated with inhibition of phosphorylation of the myosin light chain phosphatase inhibitor CPI-17. Additionally, stimulation of the NO/cGMP/protein kinase G (PKG) signaling pathway still resulted in relaxation of MYPT1-deficient mesenteric arteries, indicating phosphorylation of MYPT1 by PKG is not a major contributor to the relaxation response. Thus, MYPT1 enhances myosin light chain phosphatase activity sufficient for blood pressure maintenance. Rho-associated kinase phosphorylation of CPI-17 plays a significant role in enhancing vascular contractile responses, whereas phosphorylation of MYPT1 in the NO/cGMP/PKG signaling module is not necessary for relaxation.
Asunto(s)
Músculo Liso Vascular/fisiología , Quinasa de Cadena Ligera de Miosina/fisiología , Animales , Presión Sanguínea/fisiología , Femenino , Hipertensión/etiología , Hipertensión/fisiopatología , Péptidos y Proteínas de Señalización Intracelular , Masculino , Arterias Mesentéricas/fisiología , Ratones , Ratones Noqueados , Proteínas Musculares/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Quinasa de Cadena Ligera de Miosina/deficiencia , Quinasa de Cadena Ligera de Miosina/genética , Fosfatasa de Miosina de Cadena Ligera , Óxido Nítrico/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Transducción de Señal , Vasoconstricción/fisiología , Vasodilatación/fisiologíaRESUMEN
Smooth muscle contraction initiated by myosin regulatory light chain (RLC) phosphorylation is dependent on the relative activities of Ca(2+)-calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). We have investigated the physiological role of the MLCP regulatory subunit MYPT1 in bladder smooth muscle containing a smooth muscle-specific deletion of MYPT1 in adult mice. Deep-sequencing analyses of mRNA and immunoblotting revealed that MYPT1 depletion reduced the amount of PP1cδ with no compensatory changes in expression of other MYPT1 family members. Phosphatase activity towards phosphorylated smooth muscle heavy meromyosin was proportional to the amount of PP1cδ in total homogenates from wild-type or MYPT1-deficient tissues. Isolated MYPT1-deficient tissues from MYPT1(SM-/-) mice contracted with moderate differences in response to KCl and carbachol treatments, and relaxed rapidly with comparable rates after carbachol removal and only 1.5-fold slower after KCl removal. Measurements of phosphorylated proteins in the RLC signalling and actin polymerization modules during contractions revealed moderate changes. Using a novel procedure to quantify total phosphorylation of MYPT1 at Thr696 and Thr853, we found substantial phosphorylation in wild-type tissues under resting conditions, predicting attenuation of MLCP activity. Reduced PP1cδ activity in MYPT1-deficient tissues may be similar to the attenuated MLCP activity in wild-type tissues resulting from constitutively phosphorylated MYPT1. Constitutive phosphorylation of MYPT1 Thr696 and Thr853 may thus represent a physiological mechanism acting in concert with agonist-induced MYPT1 phosphorylation to inhibit MLCP activity. In summary, MYPT1 deficiency may not cause significant derangement of smooth muscle contractility because the effective MLCP activity is not changed.
Asunto(s)
Músculo Liso/fisiología , Quinasa de Cadena Ligera de Miosina/fisiología , Fosfatasa de Miosina de Cadena Ligera/fisiología , Vejiga Urinaria/fisiología , Animales , Secuenciación de Nucleótidos de Alto Rendimiento , Masculino , Ratones Transgénicos , Contracción Muscular , Fosforilación , ARN Mensajero/genéticaRESUMEN
Smooth muscle contraction is activated primarily by phosphorylation at Ser19 of the regulatory light chain subunits (LC20) of myosin II, catalysed by Ca(2+)/calmodulin-dependent myosin light chain kinase. Ca(2+)-independent contraction can be induced by inhibition of myosin light chain phosphatase, which correlates with diphosphorylation of LC20 at Ser19 and Thr18, catalysed by integrin-linked kinase (ILK) and zipper-interacting protein kinase (ZIPK). LC20 diphosphorylation at Ser19 and Thr18 has been detected in mammalian vascular smooth muscle tissues in response to specific contractile stimuli (e.g. endothelin-1 stimulation of rat renal afferent arterioles) and in pathophysiological situations associated with hypercontractility (e.g. cerebral vasospasm following subarachnoid hemorrhage). Comparison of the effects of LC 20 monophosphorylation at Ser19 and diphosphorylation at Ser19 and Thr18 on contraction and relaxation of Triton-skinned rat caudal arterial smooth muscle revealed that phosphorylation at Thr18 has no effect on steady-state force induced by Ser19 phosphorylation. On the other hand, the rates of dephosphorylation and relaxation are significantly slower following diphosphorylation at Thr18 and Ser19 compared to monophosphorylation at Ser19. We propose that this diphosphorylation mechanism underlies the prolonged contractile response of particular vascular smooth muscle tissues to specific stimuli, e.g. endothelin-1 stimulation of renal afferent arterioles, and the vasospastic behavior observed in pathological conditions such as cerebral vasospasm following subarachnoid hemorrhage and coronary arterial vasospasm. ILK and ZIPK may, therefore, be useful therapeutic targets for the treatment of such conditions.
Asunto(s)
Músculo Liso Vascular/fisiología , Miosina Tipo II/química , Miosina Tipo II/fisiología , Vasoconstricción/genética , Lesión Renal Aguda/tratamiento farmacológico , Animales , Catálisis , Vasoespasmo Coronario/tratamiento farmacológico , Proteínas Quinasas Asociadas a Muerte Celular/fisiología , Proteínas Quinasas Asociadas a Muerte Celular/uso terapéutico , Endotelina-1/farmacología , Humanos , Hipertensión/tratamiento farmacológico , Microcirculación/efectos de los fármacos , Microcirculación/genética , Terapia Molecular Dirigida , Quinasa de Cadena Ligera de Miosina/fisiología , Fosfatasa de Miosina de Cadena Ligera/fisiología , Fosforilación , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Serina-Treonina Quinasas/uso terapéutico , Ratas , Circulación Renal/efectos de los fármacos , Circulación Renal/genética , Vasoespasmo Intracraneal/tratamiento farmacológicoRESUMEN
Aberrant elevation in the levels of the pro-inflammatory cytokine interleukin-1ß (IL-1ß) contributes to neuroinflammatory diseases. Blood-brain barrier (BBB) dysfunction is a hallmark phenotype of neuroinflammation. It is known that IL-1ß directly induces BBB hyperpermeability but the mechanisms remain unclear. Claudin-5 (Cldn5) is a tight junction protein found at endothelial cell-cell contacts that are crucial for maintaining brain microvascular endothelial cell (BMVEC) integrity. Transcriptional regulation of Cldn5 has been attributed to the transcription factors ß-catenin and forkhead box protein O1 (FoxO1), and the signaling molecules regulating their nuclear translocation. Non-muscle myosin light chain kinase (nmMlck, encoded by the Mylk gene) is a key regulator involved in endothelial hyperpermeability, and IL-1ß has been shown to mediate nmMlck-dependent barrier dysfunction in epithelia. Considering these factors, we tested the hypothesis that nmMlck modulates IL-1ß-mediated downregulation of Cldn5 in BMVECs in a manner that depends on transcriptional repression mediated by ß-catenin and FoxO1. We found that treating BMVECs with IL-1ß induced barrier dysfunction concomitantly with the nuclear translocation of ß-catenin and FoxO1 and the repression of Cldn5. Most importantly, using primary BMVECs isolated from mice null for nmMlck, we identified that Cldn5 repression caused by ß-catenin and FoxO1 in IL-1ß-mediated barrier dysfunction was dependent on nmMlck.
Asunto(s)
Barrera Hematoencefálica/fisiopatología , Claudina-5/genética , Células Endoteliales/fisiología , Factores de Transcripción Forkhead/fisiología , Interleucina-1beta/fisiología , Quinasa de Cadena Ligera de Miosina/fisiología , beta Catenina/fisiología , Animales , Antígenos CD/metabolismo , Encéfalo/irrigación sanguínea , Cadherinas/metabolismo , Células Cultivadas , Claudina-5/metabolismo , Regulación hacia Abajo , Endotelio Vascular/fisiopatología , Proteína Forkhead Box O1 , Ratones , Microvasos/patología , Secuencias Reguladoras de Ácidos Nucleicos , Transducción de Señal , Activación TranscripcionalRESUMEN
BACKGROUND: Vascular hyporeactivity plays an important role in the pathogenesis of severe shock. Previous studies have shown that postshock mesenteric lymph (PSML) blockage ameliorates the vascular reactivity and calcium sensitivity, and RhoA is involved in the regulation of vascular reactivity after hemorrhagic shock. Therefore, the present study tested whether small GTPase RhoA mediates the improvement of the vascular reactivity and calcium sensitivity in superior mesenteric artery (SMA) of rats with PSML drainage. MATERIALS AND METHODS: The hemorrhagic shock model (blood pressure to 40 ± 2 mm Hg) was established, and PSML was drained from immediate hypotension for 3 h, after which SMA was isolated, and the vascular reactivity and calcium sensitivity were tested in the presence of RhoA agonist (U-46619) or inhibitor (C3 transferase). The protein expressions of small GTPase RhoA and phospho-RhoA were also examined in SMA. RESULTS: The hemorrhagic shock resulted in a significant decrease in the SMA reactivity and calcium sensitivity, which was enhanced by the application of U-46619 to the SMA. In contrast, the PSML drainage ameliorated the deleterious effect of the hemorrhagic shock on the SMA. This beneficial effect of the PSML drainage was abolished by C3 transferase. Western blotting revealed that the expressions of the RhoA and phospho-RhoA in SMA tissue obtained from the shock group were significantly decreased, and the PSML drainage markedly enhanced these protein expressions. CONCLUSIONS: RhoA is an important contributor to the PSML drainage-induced amelioration of the vascular reactivity and calcium sensitivity in rats with hemorrhagic shock.