RESUMEN
Both chronic obstructive pulmonary disease (COPD) and asthma are severe respiratory diseases. Bitter receptor-mediated bronchodilation is a potential therapy for asthma, but the mechanism underlying the agonistic relaxation of airway smooth muscle (ASM) is not well defined. By exploring the ASM relaxation mechanism of bitter substances, we observed that pretreatment with the bitter substances nearly abolished the methacholine (MCh)-induced increase in the ASM cell (ASMC) calcium concentration, thereby suppressing the calcium-induced contraction release. The ASM relaxation was significantly inhibited by simultaneous deletion of three Gαt proteins, suggesting an interaction between Tas2R and AChR signaling cascades in the relaxation process. Biochemically, the Gαt released by Tas2R activation complexes with AChR and blocks the Gαq cycling of AChR signal transduction. More importantly, a bitter substance, kudinoside A, not only attenuates airway constriction but also significantly inhibits pulmonary inflammation and tissue remodeling in COPD rats, indicating its modulation of additional Gαq-associated pathological processes. Thus, our results suggest that Tas2R activation may be an ideal strategy for halting multiple pathological processes of COPD.
Asunto(s)
Asma , Músculo Liso , Enfermedad Pulmonar Obstructiva Crónica , Receptores Acoplados a Proteínas G , Activación Transcripcional , Animales , Asma/genética , Asma/metabolismo , Asma/fisiopatología , Broncodilatadores/farmacología , Calcio/metabolismo , Músculo Liso/efectos de los fármacos , Músculo Liso/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/genética , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Ratas , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transducción de SeñalRESUMEN
The thymus is the central immune organ, but it is known to progressively degenerate with age. As thymus degeneration is paralleled by the wasting of aging skeletal muscle, we speculated that the thymus may play a role in muscle wasting. Here, using thymectomized mice, we show that the thymus is necessary for skeletal muscle regeneration, a process tightly associated with muscle aging. Compared to control mice, the thymectomized mice displayed comparable growth of muscle mass, but decreased muscle regeneration in response to injury, as evidenced by small and sparse regenerative myofibers along with inhibited expression of regeneration-associated genes myh3, myod, and myogenin. Using paired box 7 (Pax7)-immunofluorescence staining and 5-Bromo-2'-deoxyuridine-incorporation assay, we determined that the decreased regeneration capacity was caused by a limited satellite cell pool. Interestingly, the conditioned culture medium of isolated thymocytes had a potent capacity to directly stimulate satellite cell expansion in vitro. These expanded cells were enriched in subpopulations of quiescent satellite cells (Pax7highMyoDlowEdUpos) and activated satellite cells (Pax7highMyoDhighEdUpos), which were efficiently incorporated into the regenerative myofibers. We thus propose that the thymus plays an essential role in muscle regeneration by directly promoting satellite cell expansion and may function profoundly in the muscle aging process.
Asunto(s)
Músculo Esquelético , Regeneración , Células Satélite del Músculo Esquelético , Timo , Animales , Diferenciación Celular , Proliferación Celular , Ratones , Desarrollo de Músculos/fisiología , Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/metabolismo , Proteína MioD/genética , Proteína MioD/metabolismo , Factor de Transcripción PAX7/genética , Factor de Transcripción PAX7/metabolismo , Regeneración/fisiología , Células Satélite del Músculo Esquelético/citología , Células Satélite del Músculo Esquelético/metabolismo , Timo/metabolismo , Cicatrización de HeridasRESUMEN
Genetic hearing loss is a common health problem with no effective therapy currently available. DFNA15, caused by mutations of the transcription factor POU4F3, is one of the most common forms of autosomal dominant non-syndromic deafness. In this study, we established a novel mouse model of the human DFNA15 deafness, with a Pou4f3 gene mutation (Pou4f3Δ) identical to that found in a familial case of DFNA15. The Pou4f3(Δ/+) mice suffered progressive deafness in a similar manner to the DFNA15 patients. Hair cells in the Pou4f3(Δ/+) cochlea displayed significant stereociliary and mitochondrial pathologies, with apparent loss of outer hair cells. Progression of hearing and outer hair cell loss of the Pou4f3(Δ/+) mice was significantly modified by other genetic and environmental factors. Using Pou4f3(-/+) heterozygous knockout mice, we also showed that DFNA15 is likely caused by haploinsufficiency of the Pou4f3 gene. Importantly, inhibition of retinoic acid signaling by the aldehyde dehydrogenase (Aldh) and retinoic acid receptor inhibitors promoted Pou4f3 expression in the cochlear tissue and suppressed the progression of hearing loss in the mutant mice. These data demonstrate Pou4f3 haploinsufficiency as the main underlying cause of human DFNA15 deafness and highlight the therapeutic potential of Aldh inhibitors for treatment of progressive hearing loss.
Asunto(s)
Aldehído Deshidrogenasa/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Células Ciliadas Auditivas/patología , Pérdida Auditiva/tratamiento farmacológico , Pérdida Auditiva/etiología , Proteínas de Homeodominio/genética , Factor de Transcripción Brn-3C/genética , Animales , Benzaldehídos/farmacología , Modelos Animales de Enfermedad , Haploinsuficiencia/genética , Pérdida Auditiva/genética , Pérdida Auditiva/patología , Proteínas de Homeodominio/metabolismo , Humanos , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Noqueados , Ruido/efectos adversos , Quinolinas/farmacología , Factor de Transcripción Brn-3C/metabolismo , Tretinoina/farmacología , para-Aminobenzoatos/farmacologíaRESUMEN
CD3+CD4-CD8- T cells (double-negative T cells; DNTs) have diverse functions in peripheral immune-related diseases by regulating immunological and inflammatory homeostasis. However, the functions of DNTs in the central nervous system remain unknown. Here, we found that the levels of DNTs were dramatically increased in both the brain and peripheral blood of stroke patients and in a mouse model in a time-dependent manner. The infiltrating DNTs enhanced cerebral immune and inflammatory responses and exacerbated ischemic brain injury by modulating the FasL/PTPN2/TNF-α signaling pathway. Blockade of this pathway limited DNT-mediated neuroinflammation and improved the outcomes of stroke. Our results identified a critical function of DNTs in the ischemic brain, suggesting that this unique population serves as an attractive target for the treatment of ischemic stroke.
Asunto(s)
Isquemia Encefálica/inmunología , Complejo CD3/inmunología , Accidente Cerebrovascular/inmunología , Subgrupos de Linfocitos T/inmunología , Anciano de 80 o más Años , Animales , Encéfalo/inmunología , Antígenos CD4/inmunología , Antígenos CD8/inmunología , Modelos Animales de Enfermedad , Femenino , Humanos , Inflamación/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Mutations in the myotubularin 1 (MTM1) gene can cause the fatal disease X-linked centronuclear myopathy (XLCNM), but the underlying mechanism is incompletely understood. In this report, using an Mtm1-/y disease model, we found that expression of the intragenic microRNA miR-199a-1 is up-regulated along with that of its host gene, dynamin 2 (Dnm2), in XLCNM skeletal muscle. To assess the role of miR-199a-1 in XLCNM, we crossed miR-199a-1-/- with Mtm1-/y mice and found that the resultant miR-199a-1-Mtm1 double-knockout mice display markers of improved health, as evidenced by lifespans prolonged by 30% and improved muscle strength and histology. Mechanistic analyses showed that miR-199a-1 directly targets nonmuscle myosin IIA (NM IIA) expression and, hence, inhibits muscle postnatal development as well as muscle maturation. Further analysis revealed that increased expression and phosphorylation of signal transducer and activator of transcription 3 (STAT3) up-regulates Dnm2/miR-199a-1 expression in XLCNM muscle. Our results suggest that miR-199a-1 has a critical role in XLCNM pathology and imply that this microRNA could be targeted in therapies to manage XLCNM.
Asunto(s)
Dinamina II/genética , MicroARNs/genética , Miopatías Estructurales Congénitas/genética , Animales , Sistemas CRISPR-Cas , Dinamina II/análisis , Femenino , Longevidad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/análisis , Fuerza Muscular , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Miopatías Estructurales Congénitas/patologíaRESUMEN
Metaflammation is a primary inflammatory complication of metabolic disorders characterized by altered production of many inflammatory cytokines, adipokines, and lipid mediators. Whereas multiple inflammation networks have been identified, the mechanisms by which metaflammation is initiated have long been controversial. As the mevalonate pathway (MVA) produces abundant bioactive isoprenoids and abnormal MVA has a phenotypic association with inflammation/immunity, we speculate that isoprenoids from the MVA may provide a causal link between metaflammation and metabolic disorders. Using a line with the MVA isoprenoid producer geranylgeranyl diphosphate synthase (GGPPS) deleted, we find that geranylgeranyl pyrophosphate (GGPP) depletion causes an apparent metaflammation as evidenced by abnormal accumulation of fatty acids, eicosanoid intermediates, and proinflammatory cytokines. We also find that GGPP prenylate cytochrome b5 reductase 3 (CYB5R3) and the prenylated CYB5R3 then translocate from the mitochondrial to the endoplasmic reticulum (ER) pool. As CYB5R3 is a critical NADH-dependent reductase necessary for eicosanoid metabolism in ER, we thus suggest that GGPP-mediated CYB5R3 prenylation is necessary for metabolism. In addition, we observe that pharmacological inhibition of the MVA pathway by simvastatin is sufficient to inhibit CYB5R3 translocation and induces smooth muscle death. Therefore, we conclude that the dysregulation of MVA intermediates is an essential mechanism for metaflammation initiation, in which the imbalanced production of eicosanoid intermediates in the ER serve as an important pathogenic factor. Moreover, the interplay of MVA and eicosanoid metabolism as we reported here illustrates a model for the coordinating regulation among metabolite pathways.
Asunto(s)
Citocromo-B(5) Reductasa/metabolismo , Eicosanoides/metabolismo , Retículo Endoplásmico/metabolismo , Mitocondrias/metabolismo , Fosfatos de Poliisoprenilo/metabolismo , Prenilación , Animales , Citocromo-B(5) Reductasa/genética , Eicosanoides/genética , Retículo Endoplásmico/genética , Ácido Mevalónico/metabolismo , Ratones , Ratones Noqueados , Mitocondrias/genética , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/genética , Simvastatina/farmacologíaRESUMEN
Neurite outgrowth requires coordinated cytoskeletal rearrangements in the growth cone and directional membrane delivery from the neuronal soma. As an essential Rho guanine nucleotide exchange factor (GEF), TRIO is necessary for cytoskeletal dynamics during neurite outgrowth, but its participation in the membrane delivery is unclear. Using co-localization studies, live-cell imaging, and fluorescence recovery after photobleaching analysis, along with neurite outgrowth assay and various biochemical approaches, we here report that in mouse cerebellar granule neurons, TRIO protein pools at the Golgi and regulates membrane trafficking by controlling the directional maintenance of both RAB8 (member RAS oncogene family 8)- and RAB10-positive membrane vesicles. We found that the spectrin repeats in Golgi-resident TRIO confer RAB8 and RAB10 activation by interacting with and activating the RAB GEF RABIN8. Constitutively active RAB8 or RAB10 could partially restore the neurite outgrowth of TRIO-deficient cerebellar granule neurons, suggesting that TRIO-regulated membrane trafficking has an important functional role in neurite outgrowth. Our results also suggest cross-talk between Rho GEF and Rab GEF in controlling both cytoskeletal dynamics and membrane trafficking during neuronal development. They further highlight how protein pools localized to specific organelles regulate crucial cellular activities and functions. In conclusion, our findings indicate that TRIO regulates membrane trafficking during neurite outgrowth in coordination with its GEF-dependent function in controlling cytoskeletal dynamics via Rho GTPases.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/metabolismo , Neuritas/metabolismo , Proyección Neuronal/fisiología , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Movimiento Celular , Cerebelo/metabolismo , Aparato de Golgi/enzimología , Aparato de Golgi/metabolismo , Conos de Crecimiento/metabolismo , Factores de Intercambio de Guanina Nucleótido/fisiología , Humanos , Proteínas de Transporte de Membrana/metabolismo , Ratones , Neuritas/fisiología , Neuronas/metabolismo , Fosfoproteínas/fisiología , Unión Proteica , Proteínas Serina-Treonina Quinasas/fisiología , Transporte de Proteínas , Transducción de Señal/fisiología , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rho/metabolismoRESUMEN
The Mediator complex functions as a control center, orchestrating diverse signaling, gene activities, and biological processes. However, how Mediator subunits determine distinct cell fates remains to be fully elucidated. Here, we show that Mediator MED23 controls the cell fate preference that directs differentiation into smooth muscle cells (SMCs) or adipocytes. Med23 deficiency facilitates SMC differentiation but represses adipocyte differentiation from the multipotent mesenchymal stem cells. Gene profiling revealed that the presence or absence of Med23 oppositely regulates two sets of genes: the RhoA/MAL targeted cytoskeleton/SMC genes and the Ras/ELK1 targeted growth/adipogenic genes. Mechanistically, MED23 favors ELK1-SRF binding to SMC gene promoters for repression, whereas the lack of MED23 favors MAL-SRF binding to SMC gene promoters for activation. Remarkably, the effect of MED23 on SMC differentiation can be recapitulated in zebrafish embryogenesis. Collectively, our data demonstrate the dual, opposing roles for MED23 in regulating the cytoskeleton/SMC and growth/adipogenic gene programs, suggesting its "Ying-Yang" function in directing adipogenesis versus SMC differentiation.
Asunto(s)
Adipocitos/citología , Diferenciación Celular , Complejo Mediador/metabolismo , Miocitos del Músculo Liso/citología , Animales , Células COS , Línea Celular , Chlorocebus aethiops , Citoesqueleto/genética , Citoesqueleto/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células HeLa , Humanos , Complejo Mediador/deficiencia , Complejo Mediador/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Pez Cebra/embriologíaRESUMEN
BACKGROUND: Allergic inflammation has long been implicated in asthmatic hyperresponsiveness of airway smooth muscle (ASM), but its underlying mechanism remains incompletely understood. Serving as G protein-coupled receptor agonists, several inflammatory mediators can induce membrane depolarization, contract ASM, and augment cholinergic contractile response. We hypothesized that the signal cascade integrating on membrane depolarization by the mediators might involve asthmatic hyperresponsiveness. OBJECTIVE: We sought to investigate the signaling transduction of inflammatory mediators in ASM contraction and assess its contribution in the genesis of hyperresponsiveness. METHODS: We assessed the capacity of inflammatory mediators to induce depolarization currents by electrophysiological analysis. We analyzed the phenotypes of transmembrane protein 16A (TMEM16A) knockout mice, applied pharmacological reagents, and measured the Ca2+ signal during ASM contraction. To study the role of the depolarization signaling in asthmatic hyperresponsiveness, we measured the synergistic contraction by methacholine and inflammatory mediators both ex vivo and in an ovalbumin-induced mouse model. RESULTS: Inflammatory mediators, such as 5-hydroxytryptamin, histamine, U46619, and leukotriene D4, are capable of inducing Ca2+-activated Cl- currents in ASM cells, and these currents are mediated by TMEM16A. A combination of multiple analysis revealed that a G protein-coupled receptor-TMEM16A-voltage-dependent Ca2+ channel signaling axis was required for ASM contraction induced by inflammatory mediators. Block of TMEM16A activity may significantly inhibit the synergistic contraction of acetylcholine and the mediators and hence reduces hypersensitivity. CONCLUSIONS: A G protein-coupled receptor-TMEM16A-voltage-dependent Ca2+ channel axis contributes to inflammatory mediator-induced ASM contraction and synergistically activated TMEM16A by allergic inflammatory mediators with cholinergic stimuli.
Asunto(s)
Anoctamina-1/metabolismo , Asma/metabolismo , Hiperreactividad Bronquial/metabolismo , Canales de Calcio/metabolismo , Contracción Muscular , Músculo Liso/fisiopatología , Transducción de Señal , Animales , Asma/fisiopatología , Biomarcadores/metabolismo , Hiperreactividad Bronquial/fisiopatología , Fenómenos Electrofisiológicos , Femenino , Cobayas , Masculino , Ratones , Ratones Noqueados , FenotipoRESUMEN
BACKGROUND & AIMS: The α subunit of the heterotrimeric G stimulatory protein (Gsa), encoded by the guanine nucleotide binding protein, α-stimulating gene (Gnas, in mice), is expressed ubiquitously and mediates receptor-stimulated production of cyclic adenosine monophosphate and activation of the protein kinase A signaling pathway. We investigated the roles of Gsa in vivo in smooth muscle cells of mice. METHODS: We performed studies of mice with Cre recombinase-mediated disruption of Gnas in smooth muscle cells (GsaSMKO and SM22-CreERT2, induced in adult mice by tamoxifen). Intestinal tissues were collected for histologic, biochemical, molecular, cell biology, and physiology analyses. Intestinal function was assessed in mice using the whole-gut transit time test. We compared gene expression patterns of intestinal smooth muscle from mice with vs without disruption of Gnas. Biopsy specimens from ileum of patients with chronic intestinal pseudo-obstruction and age-matched control biopsies were analyzed by immunohistochemistry. RESULTS: Disruption of Gnas in smooth muscle of mice reduced intestinal motility and led to death within 4 weeks. Tamoxifen-induced disruption of Gnas in adult mice impaired contraction of intestinal smooth muscle and peristalsis. More than 80% of these died within 3 months of tamoxifen exposure, with features of intestinal pseudo-obstruction characterized by chronic intestinal dilation and dysmotility. Gsa deficiency reduced intestinal levels of cyclic adenosine monophosphate and transcriptional activity of the cyclic adenosine monophosphate response element binding protein 1 (CREB1); this resulted in decreased expression of the forkhead box F1 gene (Foxf1) and protein, and contractile proteins, such as myosin heavy chain 11; actin, α2, smooth muscle, aorta; calponin 1; and myosin light chain kinase. We found decreased levels of Gsa, FOXF1, CREB1, and phosphorylated CREB1 proteins in intestinal muscle layers of patients with chronic intestinal pseudo-obstruction, compared with tissues from controls. CONCLUSIONS: Gsa is required for intestinal smooth muscle contraction in mice, and its levels are reduced in ileum biopsies of patients with chronic intestinal pseudo-obstruction. Mice with disruption of Gnas might be used to study human chronic intestinal pseudo-obstruction.
Asunto(s)
Cromograninas/genética , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Motilidad Gastrointestinal/genética , Seudoobstrucción Intestinal/metabolismo , Intestinos/fisiología , Contracción Muscular/genética , Músculo Liso/fisiología , Actinas/metabolismo , Adulto , Animales , Proteínas de Unión al Calcio/metabolismo , Cromograninas/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Femenino , Factores de Transcripción Forkhead/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Proteínas de Unión al GTP Heterotriméricas , Humanos , Íleon/metabolismo , Integrasas , Masculino , Ratones , Proteínas de Microfilamentos/metabolismo , Persona de Mediana Edad , Cadenas Pesadas de Miosina/metabolismo , Quinasa de Cadena Ligera de Miosina/metabolismo , CalponinasRESUMEN
Inheritance of the callipyge phenotype in sheep is an example of polar overdominance inheritance, an unusual mode of inheritance. To investigate the underlying molecular mechanism, we profiled the expression of the genes located in the Delta-like 1 homolog (Dlk1)-type III iodothyronine deiodinase (Dio3) imprinting region in mice. We found that the transcripts of the microRNA (miR) 379/miR-544 cluster were highly expressed in neonatal muscle and paralleled the expression of the Dlk1. We then determined the in vivo role of the miR-379/miR-544 cluster by establishing a mouse line in which the cluster was ablated. The maternal heterozygotes of young mutant mice displayed a hypertrophic tibialis anterior muscle, extensor digitorum longus muscle, gastrocnemius muscle, and gluteus maximus muscle and elevated expression of the DLK1 protein. Reduced expression of DLK1 was mediated by miR-329, a member of this cluster. Our results suggest that maternal expression of the imprinted miR-379/miR-544 cluster regulates paternal expression of the Dlk1 gene in mice. We therefore propose a miR-based molecular working model for polar overdominance inheritance.
Asunto(s)
Impresión Genómica , Péptidos y Proteínas de Señalización Intercelular/genética , MicroARNs/genética , Animales , Proteínas de Unión al Calcio , Femenino , Ratones , Familia de MultigenesRESUMEN
BACKGROUND & AIMS: Liver injury triggers a highly organized and ordered liver regeneration (LR) process. Once regeneration is complete, a stop signal ensures that the regenerated liver is an appropriate functional size. The inhibitors and stop signals that regulate LR are unknown, and only limited information is available about these mechanisms. METHODS: A 70% partial hepatectomy (PH) was performed in hepatocyte-specific PP2Acα-deleted (PP2Acα(-/-)) and control (PP2Acα(+/+)) mice. LR was estimated by liver weight, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and cell proliferation, and the related cellular signals were analyzed. RESULTS: We found that the catalytic subunit of PP2A was markedly upregulated during the late stage of LR. PP2Acα(-/-) mice showed prolonged LR termination, an increased liver size compared to the original mass and lower levels of serum ALT and AST compared with control mice. In these mice, cyclin D1 protein levels, but not mRNA levels, were increased. Mechanistically, AKT activated by the loss of PP2Acα inhibited glycogen synthase kinase 3ß (GSK3ß) activity, which led to the accumulation of cyclin D1 protein and accelerated hepatocyte proliferation at the termination stage. Treatment with the PI3K inhibitor wortmannin at the termination stage was sufficient to inhibit cyclin D1 accumulation and hepatocyte proliferation. CONCLUSIONS: PP2Acα plays an essential role in the proper termination of LR via the AKT/GSK3ß/Cyclin D1 pathway. Our findings enrich the understanding of the molecular mechanism that controls the termination of LR and provides a potential therapeutic target for treating liver injury.
Asunto(s)
Ciclina D1/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Hepatocitos/metabolismo , Regeneración Hepática/fisiología , Proteína Fosfatasa 2/metabolismo , Animales , Apoptosis/fisiología , Proliferación Celular/fisiología , Ratones , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/fisiologíaRESUMEN
KEY POINTS: Force production and maintenance in smooth muscle is largely controlled by myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. MYPT1 is the regulatory subunit of MLCP that biochemically inhibits MLCP activity via T694 or T852 phosphorylation in vitro. Here we separately investigated the contribution of these two phosphorylation sites in bladder smooth muscles by establishing two single point mutation mouse lines, T694A and T852A, and found that phosphorylation of MYPT1 T694, but not T852, mediates force maintenance via inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo. Our findings reveal the role of MYPT1 T694/T852 phosphorylation in vivo in regulation of smooth muscle contraction. ABSTRACT: Force production and maintenance in smooth muscle is largely controlled by different signalling modules that fine tune myosin regulatory light chain (RLC) phosphorylation, which relies on a balance between Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. To investigate the regulation of MLCP activity in vivo, we analysed the role of two phosphorylation sites on MYPT1 (regulatory subunit of MLCP) that biochemically inhibit MLCP activity in vitro. MYPT1 is constitutively phosphorylated at T694 by unidentified kinases in vivo, whereas the T852 site is phosphorylated by RhoA-associated protein kinase (ROCK). We established two mouse lines with alanine substitution of T694 or T852. Isolated bladder smooth muscle from T852A mice displayed no significant changes in RLC phosphorylation or force responses, but force was inhibited with a ROCK inhibitor. In contrast, smooth muscles containing the T694A mutation showed a significant reduction of force along with reduced RLC phosphorylation. The contractile responses of T694A mutant smooth muscle were also independent of ROCK activation. Thus, phosphorylation of MYPT1 T694, but not T852, is a primary mechanism contributing to inhibition of MLCP activity and enhancement of RLC phosphorylation in vivo. The constitutive phosphorylation of MYPT1 T694 may provide a mechanism for regulating force maintenance of smooth muscle.
Asunto(s)
Contracción Muscular , Músculo Liso/metabolismo , Quinasa de Cadena Ligera de Miosina/metabolismo , Vejiga Urinaria/metabolismo , Animales , Ratones , Ratones Endogámicos C57BL , Músculo Liso/fisiología , Quinasa de Cadena Ligera de Miosina/química , Quinasa de Cadena Ligera de Miosina/genética , Fosfatasa de Miosina de Cadena Ligera , Fosforilación , Mutación Puntual , Vejiga Urinaria/citología , Vejiga Urinaria/fisiologíaRESUMEN
Myosin light chain kinase (MLCK) has long been implicated in the myosin phosphorylation and force generation required for cell migration. Here, we surprisingly found that the deletion of MLCK resulted in fast cell migration, enhanced protrusion formation, and no alteration of myosin light chain phosphorylation. The mutant cells showed reduced membrane tether force and fewer membrane F-actin filaments. This phenotype was rescued by either kinase-dead MLCK or five-DFRXXL motif, a MLCK fragment with potent F-actin-binding activity. Pull-down and co-immunoprecipitation assays showed that the absence of MLCK led to attenuated formation of transmembrane complexes, including myosin II, integrins and fibronectin. We suggest that MLCK is not required for myosin phosphorylation in a migrating cell. A critical role of MLCK in cell migration involves regulating the cell membrane tension and protrusion necessary for migration, thereby stabilizing the membrane skeleton through F-actin-binding activity. This finding sheds light on a novel regulatory mechanism of protrusion during cell migration.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Membrana Celular/metabolismo , Yeyuno/metabolismo , Miocitos del Músculo Liso/metabolismo , Quinasa de Cadena Ligera de Miosina/metabolismo , Citoesqueleto de Actina/química , Actinas/química , Actinas/genética , Adenoviridae/genética , Secuencias de Aminoácidos , Animales , Membrana Celular/química , Movimiento Celular , Regulación de la Expresión Génica , Vectores Genéticos , Yeyuno/citología , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Miocitos del Músculo Liso/citología , Quinasa de Cadena Ligera de Miosina/química , Quinasa de Cadena Ligera de Miosina/genética , Fosforilación , Cultivo Primario de Células , Unión Proteica , Transducción de Señal , Tensión Superficial , TransfecciónRESUMEN
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.
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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
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.
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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
BACKGROUND & AIMS: The regulatory subunit of myosin light chain phosphatase, MYPT1, has been proposed to control smooth muscle contractility by regulating phosphorylation of the Ca(2+)-dependent myosin regulatory light chain. We generated mice with a smooth muscle-specific deletion of MYPT1 to investigate its physiologic role in intestinal smooth muscle contraction. METHODS: We used the Cre-loxP system to establish Mypt1-floxed mice, with the promoter region and exon 1 of Mypt1 flanked by 2 loxP sites. These mice were crossed with SMA-Cre transgenic mice to generate mice with smooth muscle-specific deletion of MYPT1 (Mypt1(SMKO) mice). The phenotype was assessed by histologic, biochemical, molecular, and physiologic analyses. RESULTS: Young adult Mypt1(SMKO) mice had normal intestinal motility in vivo, with no histologic abnormalities. On stimulation with KCl or acetylcholine, intestinal smooth muscles isolated from Mypt1(SMKO) mice produced robust and increased sustained force due to increased phosphorylation of the myosin regulatory light chain compared with muscle from control mice. Additional analyses of contractile properties showed reduced rates of force development and relaxation, and decreased shortening velocity, compared with muscle from control mice. Permeable smooth muscle fibers from Mypt1(SMKO) mice had increased sensitivity and contraction in response to Ca(2+). CONCLUSIONS: MYPT1 is not essential for smooth muscle function in mice but regulates the Ca(2+) sensitivity of force development and contributes to intestinal phasic contractile phenotype. Altered contractile responses in isolated tissues could be compensated by adaptive physiologic responses in vivo, where gut motility is affected by lower intensities of smooth muscle stimulation for myosin phosphorylation and force development.
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Señalización del Calcio/fisiología , Motilidad Gastrointestinal/fisiología , Intestinos/fisiología , Contracción Muscular/fisiología , Músculo Liso/fisiología , Quinasa de Cadena Ligera de Miosina/fisiología , Animales , Calcio/metabolismo , Señalización del Calcio/genética , Femenino , Motilidad Gastrointestinal/genética , Masculino , Ratones , Ratones Noqueados , Contracción Muscular/genética , Quinasa de Cadena Ligera de Miosina/deficiencia , Quinasa de Cadena Ligera de Miosina/genética , Fosfatasa de Miosina de Cadena LigeraRESUMEN
RATIONALE: Asthma is a chronic inflammatory disorder with a characteristic of airway hyperresponsiveness (AHR). Ca(2+)-activated Cl(-) [Cl((Ca))] channels are inferred to be involved in AHR, yet their molecular nature and the cell type they act within to mediate this response remain unknown. OBJECTIVES: Transmembrane protein 16A (TMEM16A) and TMEM16B are Cl((Ca)) channels, and activation of Cl((Ca)) channels in airway smooth muscle (ASM) contributes to agonist-induced airway contraction. We hypothesized that Tmem16a and/or Tmem16b encode Cl((Ca)) channels in ASM and mediate AHR. METHODS: We assessed the expression of the TMEM16 family, and the effects of niflumic acid and benzbromarone on AHR and airway contraction, in an ovalbumin-sensitized mouse model of chronic asthma. We also cloned TMEM16A from ASM and examined the Cl(-) currents it produced in HEK293 cells. We further studied the impacts of TMEM16A deletion on Ca(2+) agonist-induced cell shortening, and on Cl((Ca)) currents activated by Ca(2+) sparks (localized, short-lived Ca(2+) transients due to the opening of ryanodine receptors) in mouse ASM cells. MEASUREMENTS AND MAIN RESULTS: TMEM16A, but not TMEM16B, is expressed in ASM cells and its expression in these cells is up-regulated in ovalbumin-sensitized mice. Niflumic acid and benzbromarone prevent AHR and contraction evoked by methacholine in ovalbumin-sensitized mice. TMEM16A produces Cl((Ca)) currents with kinetics similar to native Cl((Ca)) currents. TMEM16A deletion renders Ca(2+) sparks unable to activate Cl((Ca)) currents, and weakens caffeine- and methacholine-induced cell shortening. CONCLUSIONS: Tmem16a encodes Cl((Ca)) channels in ASM and contributes to Ca(2+) agonist-induced contraction. In addition, up-regulation of TMEM16A and its augmented activation contribute to AHR in an ovalbumin-sensitized mouse model of chronic asthma. TMEM16A may represent a potential therapeutic target for asthma.
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Asma/metabolismo , Hiperreactividad Bronquial/metabolismo , Canales de Cloruro/metabolismo , Miocitos del Músculo Liso/metabolismo , Análisis de Varianza , Animales , Anoctamina-1 , Asma/genética , Asma/fisiopatología , Western Blotting/métodos , Hiperreactividad Bronquial/genética , Hiperreactividad Bronquial/fisiopatología , Canales de Cloruro/genética , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos C57BL , Técnicas de Placa-Clamp/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Regulación hacia Arriba/genéticaRESUMEN
BACKGROUND: The incidence of gallbladder diseases is as high as 20%, but whether gallbladder diseases contribute to hepatic disorders remains unknown. METHODS: Here, we established an animal model of gallbladder dysfunction and assessed the role of a diseased gallbladder in cholestasis-induced hepatic fibrosis (CIHF). RESULTS: Mice with smooth muscle-specific deletion of Mypt1, the gene encoding the main regulatory subunit of myosin light chain phosphatase (myosin phosphatase target subunit 1 [MYPT1]), had apparent dysfunction of gallbladder motility. This dysfunction was evidenced by abnormal contractile responses, namely, inhibited cholecystokinin 8-mediated contraction and nitric oxide-resistant relaxation. As a consequence, the gallbladder displayed impaired bile filling and biliary tract dilation comparable to the alterations in CIHF. Interestingly, the mutant animals also displayed CIHF features, including necrotic loci by the age of 1 month and subsequently exhibited progressive fibrosis and hyperplastic/dilated bile ducts. This pathological progression was similar to the phenotypes of the animal model with bile duct ligation and patients with CIHF. The characteristic biomarker of CIHF, serum alkaline phosphatase activity, was also elevated in the mice. Moreover, we observed that the myosin phosphatase target subunit 1 protein level was able to be regulated by several reagents, including lipopolysaccharide, exemplifying the risk factors for gallbladder dysfunction and hence CIHF. CONCLUSIONS: We propose that gallbladder dysfunction caused by myosin phosphatase target subunit 1 ablation is sufficient to induce CIHF in mice, resulting in impairment of the bile transport system.
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Colestasis , Modelos Animales de Enfermedad , Cirrosis Hepática , Fosfatasa de Miosina de Cadena Ligera , Animales , Fosfatasa de Miosina de Cadena Ligera/metabolismo , Fosfatasa de Miosina de Cadena Ligera/genética , Ratones , Cirrosis Hepática/fisiopatología , Cirrosis Hepática/genética , Colestasis/complicaciones , Enfermedades de la Vesícula Biliar/genética , Enfermedades de la Vesícula Biliar/fisiopatología , Enfermedades de la Vesícula Biliar/patología , Vesícula Biliar/patología , Vesícula Biliar/fisiopatología , Masculino , Ratones NoqueadosRESUMEN
Mutations in smooth muscle cell (SMC)-specific isoforms of α-actin and ß-myosin heavy chain, two major components of the SMC contractile unit, cause familial thoracic aortic aneurysms leading to acute aortic dissections (FTAAD). To investigate whether mutations in the kinase that controls SMC contractile function (myosin light chain kinase [MYLK]) cause FTAAD, we sequenced MYLK by using DNA from 193 affected probands from unrelated FTAAD families. One nonsense and four missense variants were identified in MYLK and were not present in matched controls. Two variants, p.R1480X (c.4438C>T) and p.S1759P (c.5275T>C), segregated with aortic dissections in two families with a maximum LOD score of 2.1, providing evidence of linkage of these rare variants to the disease (p = 0.0009). Both families demonstrated a similar phenotype characterized by presentation with an acute aortic dissection with little to no enlargement of the aorta. The p.R1480X mutation leads to a truncated protein lacking the kinase and calmodulin binding domains, and p.S1759P alters amino acids in the α-helix of the calmodulin binding sequence, which disrupts kinase binding to calmodulin and reduces kinase activity in vitro. Furthermore, mice with SMC-specific knockdown of Mylk demonstrate altered gene expression and pathology consistent with medial degeneration of the aorta. Thus, genetic and functional studies support the conclusion that heterozygous loss-of-function mutations in MYLK are associated with aortic dissections.