RESUMEN
The Ras homology (Rho) family of GTPases serves various functions, including promotion of cell migration, adhesion, and transcription, through activation of effector molecule targets. One such pair of effectors, the Rho-associated coiled-coil kinases (ROCK1 and ROCK2), induce reorganization of actin cytoskeleton and focal adhesion through substrate phosphorylation. Studies on ROCK knockout mice have confirmed that ROCK proteins are essential for embryonic development, but their physiological functions in adult mice remain unknown. In this study, we aimed to examine the roles of ROCK1 and ROCK2 proteins in normal adult mice. Tamoxifen (TAM)-inducible ROCK1 and ROCK2 single and double knockout mice (ROCK1flox/flox and/or ROCK2flox/flox;Ubc-CreERT2) were generated and administered a 5-day course of TAM. No deaths occurred in either of the single knockout strains, whereas all of the ROCK1/ROCK2 double conditional knockout mice (DcKO) had died by Day 11 following the TAM course. DcKO mice exhibited increased lung tissue vascular permeability, thickening of alveolar walls, and a decrease in percutaneous oxygen saturation compared with noninducible ROCK1/ROCK2 double-floxed control mice. On Day 3 post-TAM, there was a decrease in phalloidin staining in the lungs in DcKO mice. On Day 5 post-TAM, immunohistochemical analysis also revealed reduced staining for vascular endothelial (VE)-cadherin, ß-catenin, and p120-catenin at cell-cell contact sites in vascular endothelial cells in DcKO mice. Additionally, VE-cadherin/ß-catenin complexes were decreased in DcKO mice, indicating that ROCK proteins play a crucial role in maintaining lung function by regulating cell-cell adhesion.
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Células Endoteliales , Ratones Noqueados , Quinasas Asociadas a rho , Animales , Quinasas Asociadas a rho/metabolismo , Quinasas Asociadas a rho/genética , Ratones , Células Endoteliales/metabolismo , Uniones Intercelulares/metabolismo , Pulmón/metabolismo , Pulmón/patología , Cadherinas/metabolismo , Cadherinas/genética , beta Catenina/metabolismo , beta Catenina/genética , Masculino , Antígenos CDRESUMEN
Prostaglandin E2 (PGE2) is a key player in a plethora of physiological and pathological events. Nevertheless, little is known about the dynamics of PGE2 secretion from a single cell and its effect on the neighboring cells. Here, by observing confluent Madin-Darby canine kidney (MDCK) epithelial cells expressing fluorescent biosensors, we demonstrate that calcium transients in a single cell cause PGE2-mediated radial spread of PKA activation (RSPA) in neighboring cells. By in vivo imaging, RSPA was also observed in the basal layer of the mouse epidermis. Experiments with an optogenetic tool revealed a switch-like PGE2 discharge in response to the increasing cytoplasmic Ca2+ concentrations. The cell density of MDCK cells correlated with the frequencies of calcium transients and the following RSPA. The extracellular signal-regulated kinase (ERK) activation also enhanced the frequency of RSPA in MDCK and in vivo. Thus, the PGE2 discharge is regulated temporally by calcium transients and ERK activity.
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Calcio , Quinasas MAP Reguladas por Señal Extracelular , Ratones , Animales , Perros , Dinoprostona , Riñón , FosforilaciónRESUMEN
Allergic contact dermatitis (ACD) is a type IV hypersensitivity mainly mediated by Th1/Th17 immune response. Topical corticosteroid is currently the first-line treatment for allergic contact dermatitis (ACD) and systemic administration of immunosuppressive drugs are used in patients with severe disseminated cases. However, increased risk of adverse effects has limited their use. Thus, the development of a novel immunosuppressant for ACD with low toxicity is a challenging issue. In this study, we began our study by using a murine contact hypersensitivity (CHS) model of ACD to examine the immunosuppressive effects of DYRK1B inhibition. We found that mice treated with a selective DYRK1B inhibitor show reduced ear inflammation. In addition, a significant reduction of Th1 and Th17 cells in the regional lymph node upon DYRK1B inhibition was observed by FACS analysis. Studies in vitro further revealed that DYRK1B inhibitor does not only suppressed Th1 and Th17 differentiation, but also promotes regulatory T cells (Treg) differentiation. Mechanistically, FOXO1 signaling was enhanced due to the suppression of FOXO1Ser329 phosphorylation in the presence of DYRK1B inhibitor. Therefore, these findings suggest that DYRK1B regulates CD4 T cell differentiation through FOXO1 phosphorylation and DYRK1B inhibitor has a potential as a novel agent for treatment of ACD.
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Dermatitis Alérgica por Contacto , Células Th17 , Animales , Ratones , Células Th17/patología , Inflamación , Linfocitos T CD4-Positivos/patología , Inmunosupresores/uso terapéutico , InmunidadRESUMEN
Active inflammation generally promotes immune activation. However, in the tumor microenvironment (TME), active inflammation occurs in parallel with immunosuppression, and both contribute to tumor growth. Why inflammation does not lead to immune activation in TME remains unclear. In this study, using the immune checkpoint inhibitor-insensitive mouse cancer model and single-cell RNA sequencing, we show that PGE2-EP2/EP4 signaling simultaneously promotes active inflammation by inducing expression of the NF-κB genes in myeloid cells and elicits immunosuppression by driving the mregDC (mature DC enriched in immunoregulatory molecules)-Treg (regulatory T cell) axis for Treg recruitment and activation in the tumor. Importantly, the EP2/EP4 expression level is strongly correlated with the gene signatures of both active inflammation and the mregDC-Treg axis and has significant prognosis value in various human cancers. Thus, PGE2-EP2/EP4 signaling functions as the key regulatory node linking active inflammation and immunosuppression in TME, which can be targeted by EP2 and EP4 antagonists for cancer therapeutics.
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Dinoprostona , Subtipo EP4 de Receptores de Prostaglandina E , Animales , Dinoprostona/metabolismo , Terapia de Inmunosupresión , Inflamación , Ratones , Subtipo EP2 de Receptores de Prostaglandina E/genética , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Linfocitos T Reguladores/metabolismo , Microambiente TumoralRESUMEN
Keratinocyte differentiation is an intricate process that is regulated by multiple mediators. Using cultured human keratinocytes, we found that lysophosphatidic acid (LPA) induced the differentiation of a previously unsuspected keratinocyte subpopulation expressing the extracellular matrix protein, thrombospondin-1 (THBS1). This action of LPA was mediated by the RHO/ROCK-SRF signaling downstream of LPA1 and LPA5 receptors and required ERK activity. Suppression of THBS1 in vitro suggested a migratory role of THBS1+ keratinocytes. Moreover, we analyzed publicly deposited single-cell RNA sequencing dataset and identified Thbs1-expressing keratinocytes in the mouse wound skin. Immunohistochemistry analysis revealed that Thbs1+ keratinocytes were apparently differentiated from basal keratinocytes upon wounding, subsequently polarized and migrated suprabasally toward the wound front, and eventually underwent terminal differentiation in the neo-epidermis. Importantly, inhibition of Erk activity suppressed Thbs1+ keratinocyte differentiation in wound healing. Based on these findings, we suggest that THBS1+ keratinocyte is a migratory keratinocyte subpopulation that facilitates epidermal wound healing.
RESUMEN
Cancer and chronic infections often increase levels of the bioactive lipid, lysophosphatidic acid (LPA), that we have demonstrated acts as an inhibitory ligand upon binding LPAR5 on CD8 T cells, suppressing cytotoxic activity and tumor control. This study, using human and mouse primary T lymphocytes, reveals how LPA disrupts antigen-specific CD8 T cell:target cell immune synapse (IS) formation and T cell function via competing for cytoskeletal regulation. Specifically, we find upon antigen-specific T cell:target cell formation, IP3R1 localizes to the IS by a process dependent on mDia1 and actin and microtubule polymerization. LPA not only inhibited IP3R1 from reaching the IS but also altered T cell receptor (TCR)induced localization of RhoA and mDia1 impairing F-actin accumulation and altering the tubulin code. Consequently, LPA impeded calcium store release and IS-directed cytokine secretion. Thus, targeting LPA signaling in chronic inflammatory conditions may rescue T cell function and promote antiviral and antitumor immunity.
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Linfocitos T CD8-positivos , Sinapsis Inmunológicas , Infecciones , Lisofosfolípidos , Neoplasias , Animales , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/inmunología , Humanos , Sinapsis Inmunológicas/efectos de los fármacos , Sinapsis Inmunológicas/inmunología , Infecciones/inmunología , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Lisofosfolípidos/metabolismo , Lisofosfolípidos/farmacología , Ratones , Neoplasias/inmunología , Receptores del Ácido Lisofosfatídico/metabolismoRESUMEN
Here, we describe a protocol for single-cell isolation from the primary culture of normal human epidermal keratinocytes derived from neonatal foreskin. The cell culture conditions have been optimized for inducing expression of keratinocyte differentiation markers. Cells are cultured in the absence or presence of a bioactive lipid lysophosphatidic acid (LPA). Single cells are isolated by Fluidigm C1 system. This is followed by cDNA library preparation using Takara SMART-Seq v4 Ultra and Illumina Nextera XT kit for RNA sequencing. For complete details on the use and execution of this protocol, please refer to Siriwach et al. (2022).1.
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Técnicas de Cultivo de Célula , Queratinocitos , Recién Nacido , Humanos , Biblioteca de Genes , Análisis de Secuencia de ARN/métodos , Separación CelularRESUMEN
Importance: Moyamoya disease (MMD), a progressive vasculopathy leading to narrowing and ultimate occlusion of the intracranial internal carotid arteries, is a cause of childhood stroke. The cause of MMD is poorly understood, but genetic factors play a role. Several familial forms of MMD have been identified, but the cause of most cases remains elusive, especially among non-East Asian individuals. Objective: To assess whether ultrarare de novo and rare, damaging transmitted variants with large effect sizes are associated with MMD risk. Design, Setting, and Participants: A genetic association study was conducted using whole-exome sequencing case-parent MMD trios in a small discovery cohort collected over 3.5 years (2016-2019); data were analyzed in 2020. Medical records from US hospitals spanning a range of 1 month to 1.5 years were reviewed for phenotyping. Exomes from a larger validation cohort were analyzed to identify additional rare, large-effect variants in the top candidate gene. Participants included patients with MMD and, when available, their parents. All participants who met criteria and were presented with the option to join the study agreed to do so; none were excluded. Twenty-four probands (22 trios and 2 singletons) composed the discovery cohort, and 84 probands (29 trios and 55 singletons) composed the validation cohort. Main Outcomes and Measures: Gene variants were identified and filtered using stringent criteria. Enrichment and case-control tests assessed gene-level variant burden. In silico modeling estimated the probability of variant association with protein structure. Integrative genomics assessed expression patterns of MMD risk genes derived from single-cell RNA sequencing data of human and mouse brain tissue. Results: Of the 24 patients in the discovery cohort, 14 (58.3%) were men and 18 (75.0%) were of European ancestry. Three of 24 discovery cohort probands contained 2 do novo (1-tailed Poisson P = 1.1 × 10-6) and 1 rare, transmitted damaging variant (12.5% of cases) in DIAPH1 (mammalian diaphanous-1), a key regulator of actin remodeling in vascular cells and platelets. Four additional ultrarare damaging heterozygous DIAPH1 variants (3 unphased) were identified in 3 other patients in an 84-proband validation cohort (73.8% female, 77.4% European). All 6 patients were non-East Asian. Compound heterozygous variants were identified in ena/vasodilator-stimulated phosphoproteinlike protein EVL, a mammalian diaphanous-1 interactor that regulates actin polymerization. DIAPH1 and EVL mutant probands had severe, bilateral MMD associated with transfusion-dependent thrombocytopenia. DIAPH1 and other MMD risk genes are enriched in mural cells of midgestational human brain. The DIAPH1 coexpression network converges in vascular cell actin cytoskeleton regulatory pathways. Conclusions and Relevance: These findings provide the largest collection to date of non-East Asian individuals with sporadic MMD harboring pathogenic variants in the same gene. The results suggest that DIAPH1 is a novel MMD risk gene and impaired vascular cell actin remodeling in MMD pathogenesis, with diagnostic and therapeutic ramifications.
Asunto(s)
Forminas/genética , Enfermedad de Moyamoya/genética , Adulto , Edad de Inicio , Moléculas de Adhesión Celular/genética , Niño , Preescolar , Estudios de Cohortes , Simulación por Computador , Exoma/genética , Femenino , Variación Genética , Humanos , Lactante , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Enfermedad de Moyamoya/diagnóstico por imagen , Fenotipo , Análisis de Secuencia de ARN , Población Blanca , Secuenciación del ExomaRESUMEN
In this issue of Structure, Nojima et al. (2021) report the structure of the PGE2-EP4-Gs complex by cryo-electron microscopy. This work shows unique modes of ligand binding, transduction mechanism, and G protein coupling of EP4, and serves as a starting point for development of more selective drugs.
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Aspirina , AMP Cíclico , Microscopía por Crioelectrón , Proteínas de Unión al GTP , Subtipo EP4 de Receptores de Prostaglandina E/metabolismoRESUMEN
AIMS: Cardiac hypertrophy is a compensatory response to pressure overload, leading to heart failure. Recent studies have demonstrated that Rho is immediately activated in left ventricles after pressure overload and that Rho signalling plays crucial regulatory roles in actin cytoskeleton rearrangement during cardiac hypertrophic responses. However, the mechanisms by which Rho and its downstream proteins control actin dynamics during hypertrophic responses remain not fully understood. In this study, we identified the pivotal roles of mammalian homologue of Drosophila diaphanous (mDia) 1, a Rho-effector molecule, in pressure overload-induced ventricular hypertrophy. METHODS AND RESULTS: Male wild-type (WT) and mDia1-knockout (mDia1KO) mice (10-12 weeks old) were subjected to a transverse aortic constriction (TAC) or sham operation. The heart weight/tibia length ratio, cardiomyocyte cross-sectional area, left ventricular wall thickness, and expression of hypertrophy-specific genes were significantly decreased in mDia1KO mice 3 weeks after TAC, and the mortality rate was higher at 12 weeks. Echocardiography indicated that mDia1 deletion increased the severity of heart failure 8 weeks after TAC. Importantly, we could not observe apparent defects in cardiac hypertrophic responses in mDia3-knockout mice. Microarray analysis revealed that mDia1 was involved in the induction of hypertrophy-related genes, including immediate early genes, in pressure overloaded hearts. Loss of mDia1 attenuated activation of the mechanotransduction pathway in TAC-operated mice hearts. We also found that mDia1 was involved in stretch-induced activation of the mechanotransduction pathway and gene expression of c-fos in neonatal rat ventricular cardiomyocytes (NRVMs). mDia1 regulated the filamentous/globular (F/G)-actin ratio in response to pressure overload in mice. Additionally, increases in nuclear myocardin-related transcription factors and serum response factor were perturbed in response to pressure overload in mDia1KO mice and to mechanical stretch in mDia1 depleted NRVMs. CONCLUSION: mDia1, through actin dynamics, is involved in compensatory cardiac hypertrophy in response to pressure overload.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Forminas/metabolismo , Insuficiencia Cardíaca/metabolismo , Hipertrofia Ventricular Izquierda/metabolismo , Miocitos Cardíacos/metabolismo , Disfunción Ventricular Izquierda/metabolismo , Función Ventricular Izquierda , Remodelación Ventricular , Citoesqueleto de Actina/ultraestructura , Anciano , Anciano de 80 o más Años , Animales , Aorta/fisiopatología , Aorta/cirugía , Presión Arterial , Células Cultivadas , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Forminas/genética , Regulación de la Expresión Génica , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/fisiopatología , Humanos , Hipertrofia Ventricular Izquierda/genética , Hipertrofia Ventricular Izquierda/fisiopatología , Hipertrofia Ventricular Izquierda/prevención & control , Ligadura , Masculino , Mecanotransducción Celular , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Miocitos Cardíacos/ultraestructura , Ratas Sprague-Dawley , Disfunción Ventricular Izquierda/genética , Disfunción Ventricular Izquierda/fisiopatologíaRESUMEN
The discovery of >60 monogenic causes of nephrotic syndrome (NS) has revealed a central role for the actin regulators RhoA/Rac1/Cdc42 and their effectors, including the formin INF2. By whole-exome sequencing (WES), we here discovered bi-allelic variants in the formin DAAM2 in four unrelated families with steroid-resistant NS. We show that DAAM2 localizes to the cytoplasm in podocytes and in kidney sections. Further, the variants impair DAAM2-dependent actin remodeling processes: wild-type DAAM2 cDNA, but not cDNA representing missense variants found in individuals with NS, rescued reduced podocyte migration rate (PMR) and restored reduced filopodia formation in shRNA-induced DAAM2-knockdown podocytes. Filopodia restoration was also induced by the formin-activating molecule IMM-01. DAAM2 also co-localizes and co-immunoprecipitates with INF2, which is intriguing since variants in both formins cause NS. Using in vitro bulk and TIRF microscopy assays, we find that DAAM2 variants alter actin assembly activities of the formin. In a Xenopus daam2-CRISPR knockout model, we demonstrate actin dysregulation in vivo and glomerular maldevelopment that is rescued by WT-DAAM2 mRNA. We conclude that DAAM2 variants are a likely cause of monogenic human SRNS due to actin dysregulation in podocytes. Further, we provide evidence that DAAM2-associated SRNS may be amenable to treatment using actin regulating compounds.
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Actinas/metabolismo , Variación Genética , Proteínas de Microfilamentos/genética , Síndrome Nefrótico/genética , Proteínas de Unión al GTP rho/genética , Alelos , Animales , Animales Modificados Genéticamente , Movimiento Celular/genética , Citoplasma/metabolismo , Forminas/metabolismo , Humanos , Riñón/metabolismo , Glomérulos Renales/metabolismo , Mutación Missense , Podocitos/metabolismo , Seudópodos/metabolismo , ARN Interferente Pequeño/metabolismo , Secuenciación del Exoma , XenopusRESUMEN
BACKGROUND: IL-23 is the key cytokine for generation of pathogenic IL-17-producing helper T (TH17) cells, which contribute critically to autoimmune diseases. However, how IL-23 generates pathogenic TH17 cells remains to be elucidated. OBJECTIVES: We sought to examine the involvement, molecular mechanisms, and clinical implications of prostaglandin (PG) E2-EP2/EP4 signaling in induction of IL-23-driven pathogenic TH17 cells. METHODS: The role of PGE2 in induction of pathogenic TH17 cells was investigated in mouse TH17 cells in culture in vitro and in an IL-23-induced psoriasis mouse model in vivo. Clinical relevance of the findings in mice was examined by using gene expression profiling of IL-23 and PGE2-EP2/EP4 signaling in psoriatic skin from patients. RESULTS: IL-23 induces Ptgs2, encoding COX2 in TH17 cells, and produces PGE2, which acts back on the PGE receptors EP2 and EP4 in these cells and enhances IL-23-induced expression of an IL-23 receptor subunit gene, Il23r, by activating signal transducer and activator of transcription (STAT) 3, cAMP-responsive element binding protein 1, and nuclear factor κ light chain enhancer of activated B cells (NF-κB) through cyclic AMP-protein kinase A signaling. This PGE2 signaling also induces expression of various inflammation-related genes, which possibly function in TH17 cell-mediated pathology. Combined deletion of EP2 and EP4 selectively in T cells suppressed accumulation of IL-17A+ and IL-17A+IFN-γ+ pathogenic Th17 cells and abolished skin inflammation in an IL-23-induced psoriasis mouse model. Analysis of human psoriatic skin biopsy specimens shows positive correlation between PGE2 signaling and the IL-23/TH17 pathway. CONCLUSIONS: T cell-intrinsic EP2/EP4 signaling is critical in IL-23-driven generation of pathogenic TH17 cells and consequent pathogenesis in the skin.
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Inflamación/inmunología , Psoriasis/inmunología , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Células Th17/inmunología , Animales , Células Cultivadas , AMP Cíclico/metabolismo , Dinoprostona/metabolismo , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Humanos , Imiquimod , Interleucina-23/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Subtipo EP2 de Receptores de Prostaglandina E/genética , Subtipo EP4 de Receptores de Prostaglandina E/genética , Transducción de SeñalRESUMEN
The skin barrier protects the body from water loss, allergens, and pathogens. Profilaggrin is produced by differentiated keratinocytes and is processed into filaggrin monomers. These monomers cross-link keratin filaments and are also decomposed to natural moisturizing factors in the stratum corneum for skin hydration and barrier function. Deficits in FLG expression impair skin barrier function and underlie skin diseases such as dry skin and atopic dermatitis. However, intrinsic factors that regulate FLG expression and their mechanisms of action remain unknown. Here, we show that lysophosphatidic acid induces FLG expression in human keratinocytes via the LPAR1 and LPAR5 receptors and the downstream RHO-ROCK-SRF pathway. Comprehensive gene profiling analysis further showed that lysophosphatidic acid not only induces FLG expression but also facilitates keratinocyte differentiation. Moreover, lysophosphatidic acid treatment significantly up-regulated FLG production in a three-dimensional culture model of human skin and promoted barrier function in mouse skin in vivo. Thus, our work shows a previously unsuspected role for lysophosphatidic acid and its downstream signaling in the maintenance of skin homeostasis, which may serve as a novel therapeutic target for skin barrier dysfunction.
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Proteínas de Filamentos Intermediarios/metabolismo , Queratinocitos/citología , Lisofosfolípidos/farmacología , Receptores del Ácido Lisofosfatídico/genética , Animales , Diferenciación Celular/genética , Células Cultivadas , Proteínas Filagrina , Regulación de la Expresión Génica , Homeostasis/genética , Humanos , Queratinocitos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores del Ácido Lisofosfatídico/metabolismo , Absorción Cutánea/genética , Fenómenos Fisiológicos de la Piel/efectos de los fármacos , Fenómenos Fisiológicos de la Piel/genética , Regulación hacia ArribaRESUMEN
Formin is one of the two major classes of actin binding proteins (ABPs) with nucleation and polymerization activity. However, despite advances in our understanding of its biochemical activity, whether and how formins generate specific architecture of the actin cytoskeleton and function in a physiological context in vivo remain largely obscure. It is also unknown how actin filaments generated by formins interact with other ABPs in the cell. Here, we combine genetic manipulation of formins mammalian diaphanous homolog1 (mDia1) and 3 (mDia3) with superresolution microscopy and single-molecule imaging, and show that the formins mDia1 and mDia3 are dominantly expressed in Sertoli cells of mouse seminiferous tubule and together generate a highly dynamic cortical filamentous actin (F-actin) meshwork that is continuous with the contractile actomyosin bundles. Loss of mDia1/3 impaired these F-actin architectures, induced ectopic noncontractile espin1-containing F-actin bundles, and disrupted Sertoli cell-germ cell interaction, resulting in impaired spermatogenesis. These results together demonstrate the previously unsuspected mDia-dependent regulatory mechanism of cortical F-actin that is indispensable for mammalian sperm development and male fertility.
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Actinas/metabolismo , Proteínas Portadoras/metabolismo , Fertilidad , Células de Sertoli/metabolismo , Espermatogénesis , Actomiosina/metabolismo , Uniones Adherentes/metabolismo , Animales , Adhesión Celular , Células Cultivadas , Forminas , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Polimerizacion , Túbulos Seminíferos/metabolismo , Espermatozoides/citología , Espermatozoides/metabolismoRESUMEN
One of the main research areas in biology from the mid-1980s through the 1990s was the elucidation of signaling pathways governing cell responses. These studies brought, among other molecules, the small GTPase Rho to the epicenter. Rho signaling research has since expanded to all areas of biology and medicine. Here, we describe how Rho emerged as a key molecule governing cell morphogenesis and movement, how it was linked to actin reorganization, and how the study of Rho signaling has expanded from cultured cells to whole biological systems. We then give an overview of the current research status of Rho signaling in development, brain, cardiovascular system, immunity and cancer, and discuss the future directions of Rho signaling research, with emphasis on one Rho effector, ROCK*. *The Rho GTPase family. Rho family GTPases have now expanded to contain 20 members. Amino acid sequences of 20 Rho GTPases found in human were aligned and the phylogenetic tree was generated by ClustalW2 software (EMBL-EBI) based on NJ algorithm. The subfamilies of the Rho GTPases are highlighted by the circle and labeled on the right side. Rho cited in this review refers to the original members of Rho subfamily, RhoA, RhoB and RhoC, that are C3 substrates, and, unless specified, not to other members of the Rho subfamily such as Rac, Cdc42, and Rnd.
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Encéfalo/inmunología , Sistema Cardiovascular/inmunología , Proteínas de Neoplasias/inmunología , Neoplasias/inmunología , Transducción de Señal/inmunología , Proteínas de Unión al GTP rho/inmunología , Quinasas Asociadas a rho/inmunología , Animales , Encéfalo/patología , Sistema Cardiovascular/patología , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/historia , Neoplasias/genética , Neoplasias/patología , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/historia , Quinasas Asociadas a rho/genética , Quinasas Asociadas a rho/historiaRESUMEN
DIAPH1 encodes human DIA1, a formin protein that elongates unbranched actin. The c.3634+1G>T DIAPH1 mutation causes autosomal dominant nonsyndromic sensorineural hearing loss, DFNA1, characterized by progressive deafness starting in childhood. The mutation occurs near the C-terminus of the diaphanous autoregulatory domain (DAD) of DIA1, which interacts with its N-terminal diaphanous inhibitory domain (DID), and may engender constitutive activation of DIA1. However, the underlying pathogenesis that causes DFNA1 is unclear. We describe a novel patient-derived DIAPH1 mutation (c.3610C>T) in two unrelated families, which results in early termination prior to a basic amino acid motif (RRKR1204-1207) at the DAD C-terminus. The mutant DIA1(R1204X) disrupted the autoinhibitory DID-DAD interaction and was constitutively active. This unscheduled activity caused increased rates of directional actin polymerization movement and induced formation of elongated microvilli. Mice expressing FLAG-tagged DIA1(R1204X) experienced progressive deafness and hair cell loss at the basal turn and had various morphological abnormalities in stereocilia (short, fused, elongated, sparse). Thus, the basic region of the DAD mediates DIA1 autoinhibition; disruption of the DID-DAD interaction and consequent activation of DIA1(R1204X) causes DFNA1.
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Proteínas Adaptadoras Transductoras de Señales/genética , Regulación de la Expresión Génica , Pérdida Auditiva Sensorineural/genética , Animales , Niño , Modelos Animales de Enfermedad , Femenino , Forminas , Pérdida Auditiva Sensorineural/patología , Humanos , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Análisis de Secuencia de ADNRESUMEN
The lipid mediator sphingosine 1-phosphate (S1P) regulates a wide range of cellular activities, including vascular maturation, angiogenesis, and immune-cell trafficking. Among the five known receptors for S1P (S1PR1-S1PR5), S1PR1 is a critical regulator of lymphocyte trafficking: its signaling is required for lymphocyte egress from lymphoid organs, while its down-modulation by agonist-induced internalization is a prerequisite for lymphocyte entry into lymphoid organs from the bloodstream. Despite the importance of S1PR1 down-regulation in determining lymphocyte behavior, the molecular mechanism of its internalization in lymphocytes has not been defined. Here we show that agonist-induced S1PR1 internalization in T cells occurs via clathrin-mediated endocytosis and is regulated by moesin, an ezrin-radixin-moesin (ERM) family member. In S1P-stimulated T cells, S1PR1 relocalized within clathrin-coated vesicles (CCVs) and early endosomes, and S1PR1 internalization was blocked when clathrin was pharmacologically inhibited. Stimulating moesin-deficient T cells with S1P failed to induce S1PR1 internalization and CCV formation. Furthermore, treating moesin-deficient mice with FTY720, an S1P receptor agonist known to internalize S1PR1, caused delayed lymphopenia, and lymphocytes isolated from FTY720-treated moesin-deficient mice still responded to S1P ex vivo in chemotaxis assays. These results reveal a novel role for moesin in regulating clathrin-dependent S1PR1 internalization through CCV formation.
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Linfocitos T CD4-Positivos/metabolismo , Vesículas Cubiertas por Clatrina/metabolismo , Endocitosis/genética , Proteínas de Microfilamentos/fisiología , Receptores de Lisoesfingolípidos/metabolismo , Animales , Células Cultivadas , Quimiotaxis de Leucocito/genética , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Esfingosina-1-FosfatoRESUMEN
Rho GTPase is a master regulator controlling cytoskeleton in multiple contexts such as cell migration, adhesion and cytokinesis. Of several Rho GTPases in mammals, the best characterized is the Rho subfamily including ubiquitously expressed RhoA and its homologs RhoB and RhoC. Upon binding GTP, Rho exerts its functions through downstream Rho effectors, such as ROCK, mDia, Citron, PKN, Rhophilin and Rhotekin. Until recently, our knowledge about functions of Rho and Rho effectors came mostly from in vitro studies utilizing cultured cells, and their physiological roles in vivo were largely unknown. However, gene-targeting studies of Rho and its effectors have now unraveled their tissue- and cell-specific roles and provide deeper insight into the physiological function of Rho signaling in vivo. In this article, we briefly describe previous studies of the function of Rho and its effectors in vitro and then review and discuss recent studies on knockout mice of Rho and its effectors.
Asunto(s)
Proteínas de Unión al GTP rho/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Proteínas Portadoras/metabolismo , Forminas , Proteínas de Unión al GTP , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Ratones Noqueados , Proteína Quinasa C/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Unión al GTP rho/genética , Quinasas Asociadas a rho/metabolismoRESUMEN
mDia is an actin nucleator and polymerization factor regulated by the small GTPase Rho and consists of three isoforms. Here, we found that mice lacking mDia1 and mDia3, two isoforms expressed in the brain, in combination (mDia-DKO mice) show impaired left-right limb coordination during locomotion and aberrant midline crossing of axons of corticospinal neurons and spinal cord interneurons. Given that mice lacking Ephrin-B3-EphA4 signaling show a similar impairment in locomotion, we examined whether mDia is involved in Ephrin-B3-EphA4 signaling for axon repulsion. In primary cultured neurons, mDia deficiency impairs growth cone collapse and axon retraction induced by chemo-repellants including EphA ligands. In mDia-DKO mice, the Ephrin-B3-expressing midline structure in the spinal cord is disrupted, and axons aberrantly cross the spinal cord midline preferentially through the region devoid of Ephrin-B3. Therefore, mDia plays multiple roles in the proper formation of the neural network in vivo.
Asunto(s)
Axones/fisiología , Proteínas Portadoras/fisiología , Efrina-B3/metabolismo , Médula Espinal/fisiología , Animales , Proteínas Portadoras/genética , Células Cultivadas , Miembro Anterior/fisiología , Forminas , Marcha/fisiología , Miembro Posterior/fisiología , Interneuronas/fisiología , Locomoción/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/fisiología , Receptor EphA4/metabolismo , Transducción de Señal/fisiología , Médula Espinal/citologíaRESUMEN
This article reports a case of macrocephalic sperm head syndrome, which is defined as the presence of a very high percentage of spermatozoa with enlarged heads and multiple flagellae, together with detailed morphological analysis. After a couple presented with infertility, sperm analysis showed severe teratozoospermia and almost all of the spermatozoa had macrocephaly with multiple tails. The morphological analysis revealed that most of the sperm heads contained several nuclei and had a similar number of tails as that of nuclei. However, detailed analysis revealed that there were a very few spermatozoa with an almost normal morphology. After genetic counselling, intracytoplasmic sperm injection was performed using a few spermatozoa that had an almost normal morphology, resulting in pregnancy and successful delivery. Even in macrocephalic sperm head syndrome, which may be caused by meiotic division failure, pregnancy is possible if some spermatozoa with almost normal morphology can be utilized, although there may be genetic risks.