RESUMO
Satellite cells (SCs) are adult muscle stem cells that are mobilized when muscle homeostasis is perturbed. Here we show that RhoA in SCs is indispensable to have correct muscle regeneration and hypertrophy. In particular, the absence of RhoA in SCs prevents a correct SC fusion both to other RhoA-deleted SCs (regeneration context) and to growing control myofibers (hypertrophy context). We demonstrated that RhoA is dispensable for SCs proliferation and differentiation; however, RhoA-deleted SCs have an inefficient movement even if their cytoskeleton assembly is not altered. Proliferative myoblast and differentiated myotubes without RhoA display a decreased expression of Chordin, suggesting a crosstalk between these genes for myoblast fusion regulation. These findings demonstrate the importance of RhoA in SC fusion regulation and its requirement to achieve an efficient skeletal muscle homeostasis restoration.
Assuntos
Fusão Celular , Fibras Musculares Esqueléticas , Células Satélites de Músculo Esquelético , Proteína rhoA de Ligação ao GTP , Humanos , Comunicação Celular , Hipertrofia/metabolismo , Células Satélites de Músculo Esquelético/fisiologia , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/fisiologiaRESUMO
Epithelia must eliminate apoptotic cells to preserve tissue barriers and prevent inflammation.1 Several different mechanisms exist for apoptotic clearance, including efferocytosis2,3 and apical extrusion.4,5 We found that extrusion was the first-line response to apoptosis in cultured monolayers and in zebrafish epidermis. During extrusion, the apoptotic cell elicited active lamellipodial protrusions and assembly of a contractile extrusion ring in its neighbors. Depleting E-cadherin compromised both the contractile ring and extrusion, implying that a cadherin-dependent pathway allows apoptotic cells to engage their neighbors for extrusion. We identify RhoA as the cadherin-dependent signal in the neighbor cells and show that it is activated in response to contractile tension from the apoptotic cell. This mechanical stimulus is conveyed by a myosin-VI-dependent mechanotransduction pathway that is necessary both for extrusion and to preserve the epithelial barrier when apoptosis was stimulated. Earlier studies suggested that release of sphingosine-1-phosphate (S1P) from apoptotic cells might define where RhoA was activated. However, we found that, although S1P is necessary for extrusion, its contribution does not require a localized source of S1P in the epithelium. We therefore propose a unified view of how RhoA is stimulated to engage neighbor cells for apoptotic extrusion. Here, tension-sensitive mechanotransduction is the proximate mechanism that activates RhoA specifically in the immediate neighbors of apoptotic cells, but this also must be primed by S1P in the tissue environment. Together, these elements provide a coincidence detection system that confers robustness on the extrusion response.
Assuntos
Apoptose , Células Epiteliais/citologia , Mecanotransdução Celular , Peixe-Zebra , Proteína rhoA de Ligação ao GTP/fisiologia , Animais , Caderinas/genética , Lisofosfolipídeos , Esfingosina/análogos & derivadosRESUMO
BACKGROUND: Emerging evidence has noted the important participation of microRNAs (miRNAs) in several human diseases including cancer. This research was launched to probe the function of miR-381 in bladder cancer (BCa) progression. METHODS: Twenty-eight patients with primary BCa were included in this study. Cancer tissues and the adjacent normal tissues were obtained. Aberrantly expressed miRNAs in BCa tissues were analyzed using miRNA microarrays. miR-381 expression in the bladder and paired tumor tissues, and in BCa and normal cell lines was determined. The target relationship between miR-381 and BMI1 was predicted online and validated through a luciferase assay. Gain-of-functions of miR-381 and BMI1 were performed to identify their functions on BCa cell behaviors as well as tumor growth in vivo. The involvement of the Rho/ROCK signaling was identified. RESULTS: miR-381 was poor regulated in BCa tissues and cells (all p < 0.05). A higher miR-381 level indicated a better prognosis of patients with BCa. Artificial up-regulation of miR-381 inhibited proliferation, invasion, migration, resistance to apoptosis, and tumor formation ability of BCa T24 and RT4 cells (all p < 0.05). miR-381 was found to directly bind to BMI1 and was negatively correlated with BMI1 expression. Overexpression of BMI1 partially blocked the tumor suppressing roles of miR-381 in cell malignancy and tumor growth (all p < 0.05). In addition, miR-381 led to decreased RhoA phosphorylation and ROCK2 activation, which were also reversed by BMI1 (all p < 0.05). Artificial inhibition of the Rho/ROCK signaling blocked the functions of BMI1 in cell growth and metastasis (all p < 0.05). CONCLUSION: The study evidenced that miR-381 may act as a beneficiary biomarker in BCa patients. Up-regulation of miR-381 suppresses BCa development both in vivo and in vitro through BMI1 down-regulation and the Rho/ROCK inactivation.
Assuntos
MicroRNAs/fisiologia , Complexo Repressor Polycomb 1/fisiologia , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Quinases Associadas a rho/fisiologia , Proteína rhoA de Ligação ao GTP/fisiologia , Proliferação de Células , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Transdução de SinaisRESUMO
Parkinson's disease (PD) is a complex and widespread neurodegenerative disease characterized by depletion of midbrain dopaminergic (DA) neurons. Key issues are the development of therapies that can stop or reverse the disease progression, identification of dependable biomarkers, and better understanding of the pathophysiological mechanisms of PD. RhoA-ROCK signals appear to have an important role in PD symptoms, making it a possible approach for PD treatment strategies. Activation of RhoA-ROCK (Rho-associated coiled-coil containing protein kinase) appears to stimulate various PD risk factors including aggregation of alpha-synuclein (αSyn), dysregulation of autophagy, and activation of apoptosis. This manuscript reviews current updates about the biology and function of the RhoA-ROCK pathway and discusses the possible role of this signaling pathway in causing the pathogenesis of PD. We conclude that inhibition of the RhoA-ROCK signaling pathway may have high translational potential and could be a promising therapeutic target in PD.
Assuntos
Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/etiologia , Transdução de Sinais , Quinases Associadas a rho/fisiologia , Proteína rhoA de Ligação ao GTP/fisiologia , Animais , Axônios/metabolismo , Humanos , Microglia/metabolismo , Transdução de Sinais/efeitos dos fármacos , alfa-Sinucleína/metabolismo , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/química , Proteína rhoA de Ligação ao GTP/agonistas , Proteína rhoA de Ligação ao GTP/antagonistas & inibidoresRESUMO
Embryo polarization is critical for mouse development; however, neither the regulatory clock nor the molecular trigger that it activates is known. Here, we show that the embryo polarization clock reflects the onset of zygotic genome activation, and we identify three factors required to trigger polarization. Advancing the timing of transcription factor AP-2 gamma (Tfap2c) and TEA domain transcription factor 4 (Tead4) expression in the presence of activated Ras homolog family member A (RhoA) induces precocious polarization as well as subsequent cell fate specification and morphogenesis. Tfap2c and Tead4 induce expression of actin regulators that control the recruitment of apical proteins on the membrane, whereas RhoA regulates their lateral mobility, allowing the emergence of the apical domain. Thus, Tfap2c, Tead4, and RhoA are regulators for the onset of polarization and cell fate segregation in the mouse.
Assuntos
Relógios Biológicos/fisiologia , Blastocisto/fisiologia , Polaridade Celular/fisiologia , Proteínas de Ligação a DNA/fisiologia , Proteínas Musculares/fisiologia , Fator de Transcrição AP-2/fisiologia , Fatores de Transcrição/fisiologia , Proteína rhoA de Ligação ao GTP/fisiologia , Actinas/metabolismo , Animais , Relógios Biológicos/genética , Blastocisto/citologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Polaridade Celular/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/genética , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Feminino , Técnicas de Silenciamento de Genes , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Musculares/genética , Interferência de RNA , Fatores de Transcrição de Domínio TEA , Fator de Transcrição AP-2/genética , Fatores de Transcrição/genética , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
BACKGROUND: Diabetic nephropathy (dNP), now the leading cause of ESKD, lacks efficient therapies. Coagulation protease-dependent signaling modulates dNP, in part via the G protein-coupled, protease-activated receptors (PARs). Specifically, the cytoprotective protease-activated protein C (aPC) protects from dNP, but the mechanisms are not clear. METHODS: A combination of in vitro approaches and mouse models evaluated the role of aPC-integrin interaction and related signaling in dNP. RESULTS: The zymogen protein C and aPC bind to podocyte integrin-ß3, a subunit of integrin-αvß3. Deficiency of this integrin impairs thrombin-mediated generation of aPC on podocytes. The interaction of aPC with integrin-αvß3 induces transient binding of integrin-ß3 with G α13 and controls PAR-dependent RhoA signaling in podocytes. Binding of aPC to integrin-ß3via its RGD sequence is required for the temporal restriction of RhoA signaling in podocytes. In podocytes lacking integrin-ß3, aPC induces sustained RhoA activation, mimicking the effect of thrombin. In vivo, overexpression of wild-type aPC suppresses pathologic renal RhoA activation and protects against dNP. Disrupting the aPC-integrin-ß3 interaction by specifically deleting podocyte integrin-ß3 or by abolishing aPC's integrin-binding RGD sequence enhances RhoA signaling in mice with high aPC levels and abolishes aPC's nephroprotective effect. Pharmacologic inhibition of PAR1, the pivotal thrombin receptor, restricts RhoA activation and nephroprotects RGE-aPChigh and wild-type mice.Conclusions aPC-integrin-αvß3 acts as a rheostat, controlling PAR1-dependent RhoA activation in podocytes in diabetic nephropathy. These results identify integrin-αvß3 as an essential coreceptor for aPC that is required for nephroprotective aPC-PAR signaling in dNP.
Assuntos
Nefropatias Diabéticas/prevenção & controle , Integrina beta3/fisiologia , Podócitos/fisiologia , Proteína C/fisiologia , Proteína rhoA de Ligação ao GTP/fisiologia , Animais , Citoproteção , Receptor de Proteína C Endotelial/fisiologia , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/fisiologia , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Receptor PAR-1/fisiologiaRESUMO
Endometriosis is a benign gynaecological disease appearing with pelvic pain, rising dysmenorrhoea and infertility seriously impacting on 10% of reproductive-age females. This research attempts to demonstrate the function and molecular mechanism of RhoA/ROCK pathway on epithelial-mesenchymal transition (EMT) and proliferation in endometriosis. The expression of Rho family was abnormally changed in endometriotic lesions; in particular, RhoA and ROCK1/2 were significantly elevated. Overexpression of RhoA in human eutopic endometrial epithelial cells (eutopic EECs) enhanced the cell mobility, epithelial-mesenchymal transition (EMT) and proliferation, and RhoA knockdown exhibited the opposite function. Oestrogen up-regulated the RhoA activity and expression of RhoA and ROCK1/2. RhoA overexpression reinforced the effect of oestrogen on promoting EMT and proliferation, and RhoA knockdown impaired the effect of oestrogen. oestrogen receptor α (ERα) was involved with the regulation of oestrogen on EMT and proliferation and up-regulated RhoA activity and expression of RhoA and ROCK1/2. The function of ERα was modulated by the change in RhoA expression. Furthermore, phosphorylated ERK that was enhanced by oestrogen and ERα promoted the protein expression of RhoA/ROCK pathway. Endometriosis mouse model revealed that oestrogen enhanced the size and weight of endometriotic lesions. The expression of RhoA and phosphorylated ERK in mouse endometriotic lesions was significantly elevated by oestrogen. We conclude that abnormal activated RhoA/ROCK pathway in endometriosis is responsible for the function of oestrogen/ERα/ERK signalling, which promoted EMT and proliferation and resulted in the development of endometriosis.
Assuntos
Endometriose/patologia , Endométrio/patologia , Transição Epitelial-Mesenquimal/fisiologia , Estrogênios/fisiologia , Transdução de Sinais/fisiologia , Quinases Associadas a rho/fisiologia , Proteína rhoA de Ligação ao GTP/fisiologia , Adulto , Animais , Células Cultivadas , Modelos Animais de Doenças , Endometriose/cirurgia , Endométrio/efeitos dos fármacos , Endométrio/transplante , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Estradiol/farmacologia , Receptor alfa de Estrogênio/efeitos dos fármacos , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/fisiologia , Feminino , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Cistos Ovarianos/etiologia , Cistos Ovarianos/cirurgia , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinases Associadas a rho/biossíntese , Quinases Associadas a rho/genética , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/biossíntese , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Inhalation of crystalline silica (SiO2) is a risk factor of systemic autoimmune diseases such as systemic sclerosis (SSc) and fibrotic pulmonary disorders such as silicosis. A defect of apoptotic cell clearance (i.e., efferocytosis, a key process in the resolution of inflammation) is reported in macrophages from patients with fibrotic or autoimmune diseases. However, the precise links between SiO2 exposure and efferocytosis impairment remain to be determined. Answering to this question may help to better link innate immunity and fibrosis. In this study, we first aim to determine whether SiO2 might alter efferocytosis capacities of human and mouse macrophages. We secondly explore possible mechanisms explaining efferocytosis impairment, with a specific focus on macrophage polarization and on the RhoA/ROCK pathway, a key regulator of cytoskeleton remodeling and phagocytosis. Human monocyte-derived macrophages (MDM) and C57BL/6J mice exposed to SiO2 and to CFSE-positive apoptotic Jurkat cells were analyzed by flow cytometry to determine their efferocytosis index (EI). The effects of ROCK inhibitors (Y27632 and Fasudil) on EI of SiO2-exposed MDM and MDM from SSc patients were evaluated in vitro. Our results demonstrated that SiO2 significantly decreased EI of human MDM in vitro and mouse alveolar macrophages in vivo. In human MDM, this SiO2-associated impairment of efferocytosis, required the expression of the membrane receptor SR-B1 and was associated with a decreased expression of M2 polarization markers (CD206, CD204, and CD163). F-actin staining, RhoA activation and impairment of efferocytosis, all induced by SiO2, were reversed by ROCK inhibitors. Moreover, the EI of MDM from SSc patients was similar to the EI of in vitro- SiO2-exposed MDM and Y27632 significantly increased SSc MDM efferocytosis capacities, suggesting a likewise activation of the RhoA/ROCK pathway in SSc. Altogether, our results demonstrate that SiO2 exposure may contribute to the impairment of efferocytosis capacities of mouse and human macrophages but also of MDM in SiO2-associated autoimmune diseases and fibrotic disorders such as SSc; in this context, the silica/RhoA/ROCK pathway may constitute a relevant therapeutic target.
Assuntos
Apoptose/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Escleroderma Sistêmico/induzido quimicamente , Dióxido de Silício/toxicidade , Animais , Feminino , Humanos , Células Jurkat , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Quinases Associadas a rho/fisiologia , Proteína rhoA de Ligação ao GTP/fisiologiaRESUMO
Sickle cell disease (SCD) is associated with overactive bladder (OAB). Detrusor overactivity, a component of OAB, is present in an SCD mouse, but the molecular mechanisms for this condition are not well-defined. We hypothesize that nitric oxide (NO)/ ras homolog gene family (Rho) A/Rho-associated kinase (ROCK) dysregulation is a mechanism for detrusor overactivity and that NO-releasing nanoparticles (NO-nps), a novel NO delivery system, may serve to treat this condition. Male adult SCD transgenic, combined endothelial NO synthases (eNOSs) and neuronal NOS (nNOS) gene-deficient (dNOS-/-), and wild-type (WT) mice were used. Empty nanoparticle or NO-np was injected into the bladder, followed by cystometric studies. The expression levels of phosphorylated eNOS (Ser-1177), protein kinase B (Akt) (Ser-473), nNOS (Ser-1412), and myosin phosphatase target subunit 1 (MYPT1) (Thr-696) were assessed in the bladder. SCD and dNOS-/- mice had a greater (P < 0.05) number of voiding and nonvoiding contractions compared with WT mice, and they were normalized by NO-np treatment. eNOS (Ser-1177) and AKT (Ser-473) phosphorylation were decreased (P < 0.05) in the bladder of SCD compared with WT mice and reversed by NO-np. Phosphorylated MYPT1, a marker of the RhoA/ROCK pathway, was increased (P < 0.05) in the bladder of SCD mice compared with WT and reversed by NO-np. nNOS phosphorylation on positive (Ser-1412) regulatory site was decreased (P < 0.05) in the bladder of SCD mice compared with WT and was not affected by NO-np. NO-nps did not affect any of the measured parameters in WT mice. In conclusion, dysregulation of NO and RhoA/ROCK pathways is associated with detrusor overactivity in SCD mice; NO-np reverses these molecular derangements in the bladder and decreases detrusor overactivity. SIGNIFICANCE STATEMENT: Voiding abnormalities commonly affect patients with sickle cell disease (SCD) but are problematic to treat. Clarification of the science for this condition in an animal model of SCD may lead to improved interventions for it. Our findings suggest that novel topical delivery of a vasorelaxant agent nitric oxide into the bladder of these mice corrects overactive bladder by improving deranged bladder physiology regulatory signaling.
Assuntos
Nanopartículas/uso terapêutico , Óxido Nítrico/fisiologia , Bexiga Urinária Hiperativa/tratamento farmacológico , Quinases Associadas a rho/fisiologia , Anemia Falciforme/complicações , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Óxido Nítrico Sintase/fisiologia , Fosforilação , Transdução de Sinais/fisiologia , Proteína rhoA de Ligação ao GTP/fisiologiaRESUMO
Activity-dependent remodeling of excitatory connections underpins memory formation in the brain. Serotonin receptors are known to contribute to such remodeling, yet the underlying molecular machinery remains poorly understood. Here, we employ high-resolution time-lapse FRET imaging in neuroblastoma cells and neuronal dendrites to establish that activation of serotonin receptor 5-HT4 (5-HT4R) rapidly triggers spatially-restricted RhoA activity and G13-mediated phosphorylation of cofilin, thus locally boosting the filamentous actin fraction. In neuroblastoma cells, this leads to cell rounding and neurite retraction. In hippocampal neurons in situ, 5-HT4R-mediated RhoA activation triggers maturation of dendritic spines. This is paralleled by RhoA-dependent, transient alterations in cell excitability, as reflected by increased spontaneous synaptic activity, apparent shunting of evoked synaptic responses, and enhanced long-term potentiation of excitatory transmission. The 5-HT4R/G13/RhoA signaling thus emerges as a previously unrecognized molecular pathway underpinning use-dependent functional remodeling of excitatory synaptic connections.
Assuntos
Actinas/metabolismo , Espinhas Dendríticas/fisiologia , Receptores 5-HT4 de Serotonina/fisiologia , Sinapses/fisiologia , Proteína rhoA de Ligação ao GTP/fisiologia , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/fisiologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Feminino , Potenciação de Longa Duração , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores 5-HT4 de Serotonina/genética , Transdução de Sinais/genética , Transmissão Sináptica/fisiologiaRESUMO
Collective cell migration plays crucial roles in tissue remodeling, wound healing, and cancer cell invasion. However, its underlying mechanism remains unknown. Previously, we showed that the RhoA-targeting guanine nucleotide exchange factor Solo (ARHGEF40) is required for tensile force-induced RhoA activation and proper organization of keratin-8/keratin-18 (K8/K18) networks. Here, we demonstrate that Solo knockdown significantly increases the rate at which Madin-Darby canine kidney cells collectively migrate on collagen gels. However, it has no apparent effect on the migratory speed of solitary cultured cells. Therefore, Solo decelerates collective cell migration. Moreover, Solo localized to the anteroposterior regions of cell-cell contact sites in collectively migrating cells and was required for the local accumulation of K8/K18 filaments in the forward areas of the cells. Partial Rho-associated protein kinase (ROCK) inhibition or K18 or plakoglobin knockdown also increased collective cell migration velocity. These results suggest that Solo acts as a brake for collective cell migration by generating pullback force at cell-cell contact sites via the RhoA-ROCK pathway. It may also promote the formation of desmosomal cell-cell junctions related to K8/K18 filaments and plakoglobin.
Assuntos
Movimento Celular/fisiologia , Transdução de Sinais/fisiologia , Proteínas rho de Ligação ao GTP/fisiologia , Quinases Associadas a rho/fisiologia , Amidas/farmacologia , Animais , Polaridade Celular , Colágeno , Citoesqueleto/fisiologia , Desmossomos/fisiologia , Cães , Géis , Técnicas de Silenciamento de Genes , Queratina-18/antagonistas & inibidores , Queratina-18/genética , Queratina-18/fisiologia , Queratina-8/antagonistas & inibidores , Queratina-8/genética , Queratina-8/fisiologia , Células Madin Darby de Rim Canino , Piridinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Estresse Mecânico , Imagem com Lapso de Tempo , gama Catenina/antagonistas & inibidores , gama Catenina/genética , gama Catenina/fisiologia , Proteínas rac1 de Ligação ao GTP/fisiologia , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/fisiologiaRESUMO
The specification of an axon and its subsequent outgrowth are key steps during neuronal polarization, a prerequisite to wire the brain. The Rho-guanosine triphosphatase (GTPase) RhoA is believed to be a central player in these processes. However, its physiological role has remained undefined. Here, genetic loss- and gain-of-function experiments combined with time-lapse microscopy, cell culture, and in vivo analysis show that RhoA is not involved in axon specification but confines the initiation of neuronal polarization and axon outgrowth during development. Biochemical analysis and super-resolution microscopy together with molecular and pharmacological manipulations reveal that RhoA restrains axon growth by activating myosin-II-mediated actin arc formation in the growth cone to prevent microtubules from protruding toward the leading edge. Through this mechanism, RhoA regulates the duration of axon growth and pause phases, thus controlling the tightly timed extension of developing axons. Thereby, this work unravels physiologically relevant players coordinating actin-microtubule interactions during axon growth.
Assuntos
Axônios/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Axônios/fisiologia , Encéfalo/embriologia , Encéfalo/metabolismo , Polaridade Celular/fisiologia , Feminino , Mutação com Ganho de Função/genética , Cones de Crescimento/metabolismo , Mutação com Perda de Função/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microtúbulos/metabolismo , Miosina Tipo II/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismo , Proteína rhoA de Ligação ao GTP/fisiologiaRESUMO
SCOPE: Podocytes are a component of glomerular filtration barrier with interdigitating foot processes. The podocyte function depends on the dynamics of actin cytoskeletal and focal adhesion crucial for foot process structure. This study investigates the renoprotective effects of eucalyptol on the F-actin cytoskeleton formation and focal adhesion assembly in glucose-loaded podocytes and diabetic kidneys. METHODS AND RESULTS: Eucalyptol at 1-20 µm reverses the reduction of cellular level of F-actin, ezrin, cortactin, and Arp2/3 in 33 mm glucose-loaded mouse podocytes, and oral administration of 10 mg kg-1 eucalyptol elevates tissue levels of actin cytoskeletal proteins reduced in db/db mouse kidneys. Eucalyptol inhibits podocyte morphological changes, showing F-actin cytoskeleton formation in cortical regions and agminated F-actin along the cell periphery. Eucalyptol induces focal adhesion proteins of paxillin, vinculin, talin1, FAK, and Src in glucose-exposed podocytes and diabetic kidneys. Additionally, GTP-binding Rac1, Cdc42, Rho A, and ROCK are upregulated in glucose-stimulated podocytes and diabetic kidneys, which is attenuated by supplying eucalyptol. Rho A gene depletion partially diminishes GSK3ß induction of podocytes by glucose. CONCLUSION: Eucalyptol ameliorates F-actin cytoskeleton formation and focal adhesion assembly through blockade of the Rho signaling pathway, entailing partial involvement of GSK3ß, which may inhibit barrier dysfunction of podocytes and resultant proteinuria.
Assuntos
Citoesqueleto de Actina/efeitos dos fármacos , Nefropatias Diabéticas/tratamento farmacológico , Eucaliptol/farmacologia , Adesões Focais/efeitos dos fármacos , Glucose/toxicidade , Podócitos/efeitos dos fármacos , Citoesqueleto de Actina/fisiologia , Animais , Células Cultivadas , Membrana Basal Glomerular/fisiologia , Glicogênio Sintase Quinase 3 beta/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína rhoA de Ligação ao GTP/fisiologiaRESUMO
Cells respond and adapt to their physical environments and to the mechanical forces that they experience. The translation of physical forces into biochemical signalling pathways is known as mechanotransduction. In this review, we focus on two aspects of mechanotransduction. First, we consider how forces exerted on cell adhesion molecules at the cell surface regulate the RhoA signalling pathway by controlling the activities of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). In the second part of the review, we discuss how the nucleus contributes to mechanotransduction as a physical structure connected to the cytoskeleton. We focus on recent studies that have either severed the connections between the nucleus and the cytoskeleton, or that have entirely removed the nucleus from cells. These actions reduce the levels of active RhoA, thereby altering the mechanical properties of cells and decreasing their ability to generate tension and respond to external mechanical forces. This article is part of a discussion meeting issue 'Forces in cancer: interdisciplinary approaches in tumour mechanobiology'.
Assuntos
Membrana Celular/fisiologia , Núcleo Celular/fisiologia , Mecanotransdução Celular/fisiologia , Transdução de Sinais/fisiologia , Proteína rhoA de Ligação ao GTP/fisiologia , HumanosRESUMO
Cell motility and migration play critical roles in various physiological processes and disease states. Here, we show that the BBBsome, a macromolecule composed of eight Bardet-Biedl syndrome (BBS) proteins including BBS1, is a critical determinant of cell migration and wound healing. Fibroblast cells derived from mice or humans harboring a homozygous missense mutation (BBS1M390R/M390R) that disrupt the BBSome exhibit defects in migration and wound healing. Furthermore, we demonstrate that BBS1M390R/M390R mice have significantly delayed wound closure. In line with this, we provide data suggesting that BBS1M390R/M390R fibroblasts have impaired platelet-derived growth factor-AA (PDGF) receptor-α signaling, a key regulator of directional cell migration acting as a chemoattractant during postnatal migration responses such as wound healing. In addition, we show that BBS1M390R/M390R fibroblasts have upregulated RhoA expression and activity. The relevance of RhoA upregulation is demonstrated by the ability of RhoA-kinase inhibitor Y27632 to partially rescue the migration defect of BBS1M390R/M390R fibroblasts cells. We also show that accumulation of RhoA protein in BBS1M390R/M390R fibroblasts cells is associated with reduction and inactivation of the ubiquitin ligase Cullin-3. Consistent with this, Cullin-3 inhibition with MLN4924 is sufficient to reduce migration of normal fibroblasts. These data implicate the BBSome in cell motility and tissue repair through a mechanism that involves PDGF receptor signaling and Cullin-3-mediated control of RhoA.
Assuntos
Síndrome de Bardet-Biedl , Movimento Celular/fisiologia , Proteínas Culina/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Transdução de Sinais/fisiologia , Proteína rhoA de Ligação ao GTP/fisiologia , Animais , Síndrome de Bardet-Biedl/genética , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Proteínas Culina/antagonistas & inibidores , Ciclopentanos/farmacologia , Inibidores Enzimáticos/farmacologia , Feminino , Técnicas de Introdução de Genes/métodos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pirimidinas/farmacologia , Proteína rhoA de Ligação ao GTP/antagonistas & inibidoresRESUMO
Muscle differentiation is controlled by adhesion and growth factor-dependent signalling through common effectors that regulate muscle-specific transcriptional programs. Here we report that mDiaphanous1, an effector of adhesion-dependent RhoA-signalling, negatively regulates myogenesis at the level of Myogenin expression. In myotubes, over-expression of mDia1ΔN3, a RhoA-independent mutant, suppresses Myogenin promoter activity and expression. We investigated mDia1-interacting proteins that may counteract mDia1 to permit Myogenin expression and timely differentiation. Using yeast two-hybrid and mass-spectrometric analysis, we report that mDia1 has a stage-specific interactome, including Prohibitin2, MyoD, Akt2, and ß-Catenin, along with a number of proteosomal and mitochondrial components. Of these interacting partners, Prohibitin2 colocalises with mDia1 in cytoplasmic punctae in myotubes. We mapped the interacting domains of mDia1 and Phb2, and used interacting (mDia1ΔN3/Phb2 FL or mDia1ΔN3/Phb2-Carboxy) and non-interacting pairs (mDia1H + P/Phb2 FL or mDia1ΔN3/Phb2-Amino) to dissect the functional consequences of this partnership on Myogenin promoter activity. Co-expression of full-length as well as mDia1-interacting domains of Prohibitin2 reverse the anti-myogenic effects of mDia1ΔN3, while non-interacting regions do not. Our results suggest that Prohibitin2 sequesters mDia1, dampens its anti-myogenic activity and fine-tunes RhoA-mDia1 signalling to promote differentiation. Overall, we report that mDia1 is multi-functional signalling effector whose anti-myogenic activity is modulated by a differentiation-dependent interactome. The data have been deposited to the ProteomeXchange with identifier PXD012257.
Assuntos
Forminas/fisiologia , Regiões Promotoras Genéticas , Proteínas Repressoras/fisiologia , Proteína rhoA de Ligação ao GTP/fisiologia , Animais , Diferenciação Celular , Citoplasma/metabolismo , Células HEK293 , Humanos , Camundongos , Desenvolvimento Muscular , Fibras Musculares Esqueléticas/metabolismo , Mutação , Proteína MyoD/metabolismo , Miogenina/metabolismo , Proibitinas , Ligação Proteica , Domínios Proteicos , Transdução de Sinais , Técnicas do Sistema de Duplo-HíbridoRESUMO
Glioblastomas strongly invade the brain by infiltrating into the white matter along myelinated nerve fiber tracts even though the myelin protein Nogo-A prevents cell migration by activating inhibitory RhoA signaling. The mechanisms behind this long-known phenomenon remained elusive so far, precluding a targeted therapeutic intervention. This study demonstrates that the prevalent activation of AKT in gliomas increases the ER protein-folding capacity and enables tumor cells to utilize a side effect of RhoA activation: the perturbation of the IRE1α-mediated decay of SPARC mRNA. Once translation is initiated, glioblastoma cells rapidly secrete SPARC to block Nogo-A from inhibiting migration via RhoA. By advanced ultramicroscopy for studying single-cell invasion in whole, undissected mouse brains, we show that gliomas require SPARC for invading into white matter structures. SPARC depletion reduces tumor dissemination that significantly prolongs survival and improves response to cytostatic therapy. Our finding of a novel RhoA-IRE1 axis provides a druggable target for interfering with SPARC production and underscores its therapeutic value.
Assuntos
Neoplasias Encefálicas/genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Proteínas de Neoplasias/fisiologia , Proteínas Nogo/biossíntese , Osteonectina/biossíntese , Biossíntese de Proteínas , Substância Branca/patologia , Proteína rhoA de Ligação ao GTP/fisiologia , Animais , Ligação Competitiva , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Invasividade Neoplásica , Proteínas Nogo/genética , Osteonectina/genética , Domínios Proteicos , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Receptores de Esfingosina-1-Fosfato/fisiologia , Células Tumorais Cultivadas , Substância Branca/metabolismoRESUMO
BACKGROUND: Nucleus accumbens dopamine 1 receptor medium spiny neurons (D1-MSNs) play a critical role in the development of depression-like behavior in mice. Social defeat stress causes dendritic morphological changes on this MSN subtype through expression and activation of early growth response 3 (EGR3) and the Rho guanosine triphosphatase RhoA. However, it is unknown how RhoA inhibition affects electrophysiological properties underlying stress-induced susceptibility. METHODS: A novel RhoA-specific inhibitor, Rhosin, was used to inhibit RhoA activity following chronic social defeat stress. Whole-cell electrophysiological recordings of D1-MSNs were performed to assess synaptic and intrinsic consequences of Rhosin treatment on stressed mice. Additionally, recorded cells were filled and analyzed for their morphological properties. RESULTS: We found that RhoA inhibition prevents both D1-MSN hyperexcitability and reduced excitatory input to D1-MSNs caused by social defeat stress. Nucleus accumbens-specific RhoA inhibition is capable of blocking susceptibility caused by D1-MSN EGR3 expression. Lastly, we found that Rhosin enhances spine density, which correlates with D1-MSN excitability, without affecting overall dendritic branching. CONCLUSIONS: These findings demonstrate that pharmacological inhibition of RhoA during stress drives an enhancement of total spine number in a subset of nucleus accumbens neurons that prevents stress-related electrophysiological deficits and promotes stress resiliency.
Assuntos
Plasticidade Neuronal , Neurônios/fisiologia , Núcleo Accumbens/fisiologia , Compostos Orgânicos/administração & dosagem , Receptores de Dopamina D1/fisiologia , Resiliência Psicológica , Estresse Psicológico/fisiopatologia , Proteína rhoA de Ligação ao GTP/fisiologia , Animais , Proteína 3 de Resposta de Crescimento Precoce/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Núcleo Accumbens/citologia , Núcleo Accumbens/efeitos dos fármacos , Resiliência Psicológica/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/antagonistas & inibidoresRESUMO
Prior to the cytokinesis, the cell-matrix interactions should be disrupted, and the mitotic cells round up. Prerequisite of mitosis, the centrosomes duplicate, spindle fibers are generated and move away from each other to opposite sides of the cells marking the cell poles. Later, an invagination in the plasma membrane is formed a few minutes after anaphase. This furrow ingression is driven by a contractile actomyosin ring, whose assembly is regulated by RhoA GTPase. At the completion of cytokinesis, the two daughter cells are still connected by a thin intercellular bridge, which is subjected to abscission, as the terminal step of cytokinesis. Here, it is overviewed, how syndecan-4, a transmembrane, heparan sulfate proteoglycan, can contribute to these processes in a phosphorylation-dependent manner.
Assuntos
Divisão Celular , Proteoglicanas de Heparan Sulfato/fisiologia , Actinas/química , Animais , Citocinese , Humanos , Mitose , Sindecana-4/fisiologia , Proteína rhoA de Ligação ao GTP/fisiologiaRESUMO
The prevalence of high blood pressure (also known as hypertension) has steadily increased over the last few decades. Known as a silent killer, hypertension increases the risk for cardiovascular disease and can lead to stroke, heart attack, kidney failure and associated sequela. While numerous hypertensive therapies are currently available, it is estimated that only half of medicated patients exhibit blood pressure control. This signifies the need for a better understanding of the underlying cause of disease and for more effective therapies. While blood pressure homeostasis is very complex and involves the integrated control of multiple body systems, smooth muscle contractility and arterial resistance are important contributors. Strong evidence from pre-clinical animal models and genome-wide association studies indicate that smooth muscle contraction and BP homeostasis are governed by the small GTPase RhoA and its downstream target, Rho kinase. In this review, we summarize the signaling pathways and regulators that impart tight spatial-temporal control of RhoA activity in smooth muscle cells and discuss current therapeutic strategies to target these RhoA pathway components. We also discuss known allelic variations in the RhoA pathway and consider how these polymorphisms may affect genetic risk for hypertension and its clinical manifestations.