RESUMO
White adipose tissue (WAT) controls energy storage, expenditure, and endocrine function. Rho-kinase (ROCK) is related to impaired thermogenesis, downregulation of preadipocyte differentiation, and adipokine production. Furthermore, WAT ROCK responds to metabolic stress from high-fat diets or diabetes. However, ROCK distribution in adipose depots and its response to aging and sex remain unclear. Thus, we aim to investigate ROCK function in adipose tissue of rodent and human in response to aging and sex. We observed specific differences in the ROCK1/2 distribution in inguinal WAT (ingWAT), perigonadal WAT (pgWAT), and brown adipose tissue of male and female rodents. However, ROCK2 expression was lower in female ingWAT compared with males, a fact that was not observed in the other depots. In the pgWAT and ingWAT of male and female rodents, ROCK activity increased during development. Moreover, middle-aged female rodents and humans showed downregulation in ROCK activity after acute physical exercise. Interestingly, ROCK levels were associated with several inflammatory markers both in rats and humans WAT (Nfkb1, Tnf, Il1b, Il6, and Mcp1). Induction of cell senescence by etoposide elevates ROCK activity in human preadipocytes; however, silencing ROCK1/2 demonstrates improvement in the inflammatory and cell senescence state. Using public databases, several pathways were strongly associated with ROCK modulation in WAT. In summary, WAT ROCK increases with development in association with inflammatory markers. Further, ROCK activity was attenuated by acute physical exercise, implicating it as a possible therapeutic target for metabolism improvement mediated by adipose tissue inflammatory state changes.
Assuntos
Roedores , Quinases Associadas a rho , Humanos , Ratos , Masculino , Feminino , Animais , Pessoa de Meia-Idade , Quinases Associadas a rho/fisiologia , Obesidade/metabolismo , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Envelhecimento , Tecido AdiposoRESUMO
Rho kinase (ROCK) is implicated in the development of pulmonary arterial hypertension (PAH) in which abnormal pulmonary vascular smooth muscle (VSM) contractility and remodeling lead to right heart failure. Pharmacologic ROCK inhibitors block experimental pulmonary hypertension (PH) development in rodents but can have off-target effects and do not distinguish between the two ROCK forms, ROCK1 and ROCK2, encoded by separate genes. An earlier study using gene knock out (KO) in mice indicated that VSM ROCK2 is required for experimental PH development, but the role of ROCK1 is not well understood. Here we investigated the in vivo role of ROCK1 in PH development by generating a VSM-targeted homozygous ROCK1 gene KO mouse strain. Adult control mice exposed to Sugen5416 (Su)/hypoxia treatment to induce PH had significantly increased right ventricular systolic pressures (RVSP) and RV hypertrophy versus normoxic controls. In contrast, Su/hypoxia-exposed VSM ROCK1 KO mice did not exhibit significant RVSP elevation, and RV hypertrophy was blunted. Su/hypoxia-induced pulmonary small vessel muscularization was similarly elevated in both control and VSM ROCK1 KO animals. siRNA-mediated ROCK1 knock-down (KD) in human PAH pulmonary arterial SM cells (PASMC) did not affect cell growth. However, ROCK1 KD led to reduced AKT and MYPT1 signaling in serotonin-treated PAH PASMC. The findings suggest that like VSM ROCK2, VSM ROCK1 actively contributes to PH development, but in distinction acts via nonproliferative pathways to promote hypoxemia, and thus may be a distinct therapeutic target in PH.
Assuntos
Hipertensão Arterial Pulmonar , Quinases Associadas a rho , Animais , Hipertrofia Ventricular Direita/genética , Hipóxia/complicações , Camundongos , Camundongos Knockout , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/metabolismo , Hipertensão Arterial Pulmonar/genética , Artéria Pulmonar/metabolismo , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismo , Quinases Associadas a rho/fisiologiaRESUMO
Stress granules (SGs) contain mRNAs and proteins stalled in translation during stress; these are increasingly being implicated in diseases, including neurological disorders and cancer. The dysregulated assembly, persistence, disassembly and clearance of SGs contribute to the process of senescence. Senescence has long been a mysterious player in cellular physiology and associated diseases. The systemic process of aging has been pivotal in the development of various neurological disorders like age-related neuropathy, Alzheimer's disease and Parkinson's disease. Glioma is a cancer of neurological origin with a very poor prognosis and high rate of recurrence, SGs have only recently been implicated in its pathogenesis. Senescence has long been established to play an antitumorigenic role, however, relatively less studied is its protumorigenic importance. Here, we have evaluated the existing literature to assess the crosstalk of the two biological phenomena of senescence and SG formation in the context of tumorigenesis. In this review, we have attempted to analyze the contribution of senescence in regulating diverse cellular processes, like, senescence associated secretory phenotype (SASP), microtubular reorganization, telomeric alteration, autophagic clearance and how intricately these phenomena are tied with the formation of SGs. Finally, we propose that interplay between senescence, its contributing factors and the genesis of SGs can drive tumorigenicity of gliomas, which can potentially be utilized for therapeutic intervention.
Assuntos
Neoplasias Encefálicas/etiologia , Senescência Celular/fisiologia , Glioma/etiologia , Grânulos de Estresse/fisiologia , Autofagia , Neoplasias Encefálicas/patologia , DNA Helicases/fisiologia , Progressão da Doença , Glioma/patologia , Humanos , Microtúbulos/química , Proteínas de Ligação a Poli-ADP-Ribose/fisiologia , RNA Helicases/fisiologia , Proteínas com Motivo de Reconhecimento de RNA/fisiologia , Telômero , Quinases Associadas a rho/fisiologiaRESUMO
Background: High mobility group box 1 (HMGB1) causes microvascular endothelial cell barrier dysfunction during acute lung injury (ALI) in sepsis, but the mechanisms have not been well understood. We studied the roles of RAGE and Rho kinase 1 (ROCK1) in HMGB1-induced human pulmonary endothelial barrier disruption. Methods: In the present study, the recombinant human high mobility group box 1 (rhHMGB1) was used to stimulate human pulmonary microvascular endothelial cells (HPMECs). The endothelial cell (EC) barrier permeability was examined by detecting FITC-dextran flux. CCK-8 assay was used to detect cell viability under rhHMGB1 treatments. The expression of related molecules involved in RhoA/ROCK1 pathway, phosphorylation of myosin light chain (MLC), F-actin, VE-cadherin and ZO-1 of different treated groups were measured by pull-down assay, western blot and immunofluorescence. Furthermore, we studied the effects of Rho kinase inhibitor (Y-27632), ROCK1/2 siRNA, RAGE-specific blocker (FPS-ZM1) and RAGE siRNA on endothelial barrier properties to elucidate the related mechanisms. Results: In the present study, we demonstrated that rhHMGB1 induced EC barrier hyperpermeability in a dose-dependent and time-dependent manner by measuring FITC-dextran flux, a reflection of the loss of EC barrier integrity. Moreover, rhHMGB1 induced a dose-dependent and time-dependent increases in paracellular gap formation accompanied by the development of stress fiber rearrangement and disruption of VE-cadherin and ZO-1, a phenotypic change related to increased endothelial contractility and endothelial barrier permeability. Using inhibitors and siRNAs directed against RAGE and ROCK1/2, we systematically determined that RAGE mediated the rhHMGB1-induced stress fiber reorganization via RhoA/ROCK1 signaling activation and the subsequent MLC phosphorylation in ECs. Conclusion: HMGB1 is capable of disrupting the endothelial barrier integrity. This study demonstrates that HMGB1 activates RhoA/ROCK1 pathway via RAGE, which phosphorylates MLC inducing stress fiber formation at short time, and HMGB1/RAGE reduces AJ/TJ expression at long term independently of RhoA/ROCK1 signaling pathway.
Assuntos
Permeabilidade Capilar/fisiologia , Células Endoteliais/metabolismo , Proteína HMGB1/fisiologia , Receptor para Produtos Finais de Glicação Avançada/fisiologia , Quinases Associadas a rho/fisiologia , Células Cultivadas , Humanos , Cadeias Leves de Miosina/fisiologia , Transdução de Sinais/fisiologiaRESUMO
PI3K signalling is required for activation, differentiation, and trafficking of T cells. PI3Kδ, the dominant PI3K isoform in T cells, has been extensively characterised using PI3Kδ mutant mouse models and PI3K inhibitors. Furthermore, characterisation of patients with Activated PI3K Delta Syndrome (APDS) and mouse models with hyperactive PI3Kδ have shed light on how increased PI3Kδ activity affects T cell functions. An important function of PI3Kδ is that it acts downstream of TCR stimulation to activate the major T cell integrin, LFA-1, which controls transendothelial migration of T cells as well as their interaction with antigen-presenting cells. PI3Kδ also suppresses the cell surface expression of CD62L and CCR7 which controls the migration of T cells across high endothelial venules in the lymph nodes and S1PR1 which controls lymph node egress. Therefore, PI3Kδ can control both entry and exit of T cells from lymph nodes as well as the recruitment to and retention of T cells within inflamed tissues. This review will focus on the regulation of adhesion receptors by PI3Kδ and how this contributes to T cell trafficking and localisation. These findings are relevant for our understanding of how PI3Kδ inhibitors may affect T cell redistribution and function.
Assuntos
Classe I de Fosfatidilinositol 3-Quinases/fisiologia , Linfócitos T/fisiologia , Animais , Adesão Celular , Movimento Celular , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Humanos , Sinapses Imunológicas/fisiologia , Integrinas/fisiologia , Antígeno-1 Associado à Função Linfocitária/fisiologia , Camundongos , Doenças da Imunodeficiência Primária/etiologia , Transdução de Sinais/fisiologia , Quinases Associadas a rho/fisiologiaAssuntos
Angiotensina II/fisiologia , Hipóxia/fisiopatologia , Artéria Cerebral Média/fisiologia , Vasoconstrição/fisiologia , Animais , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Feminino , Canais de Potássio/fisiologia , Gravidez , Proteína Quinase C/fisiologia , Ratos Sprague-Dawley , Transdução de Sinais , Quinases Associadas a rho/fisiologiaRESUMO
ABSTRACT: Vascular hypo-reactivity plays a critical role inducing organ injury during hemorrhagic shock. 17ß-estradiol (E2) can induce vasodilation to increase blood flow in various vascular beds. This study observed whether E2 can restore vascular hypo-reactivity induced by hemorrhagic shock, and whether E2 effects are associated with RhoA-Rho kinase (ROCK)-myosin light chain kinase phosphatase (MLCP) pathway. The hemorrhagic shock model (40â±â2âmm Hg for 1âh, resuscitation for 4âh) was established in ovary intact sham operation (OVI), ovariectomized (OVX), and OVX plus E2 supplement female mice. Intestinal microvascular loop was used to assess blood flow in vivo, mRNA expression and vascular reactivity in vitro. Hemorrhagic shock significantly reduced norepinephrine microvascular reactivity. Decreased microvascular reactivity was exacerbated by OVX and reversed by E2 supplement. U-46619 (RhoA agonist) increased microvascular reactivity, and C3 transferase (an ADP ribosyl transferase that selectively induces RhoA ribosylation) or Y-27632 (ROCK inhibitor) inhibited sham mice microvascular reactivity. Similarly, U-46619 increased microvascular reactivity in OVI and OVX mice following hemorrhagic shock, which was abolished by Y-27632 or concomitant incubation of okadaic acid (OA) (MLCP inhibitor) and Y-27632. In OVX plus E2 supplement mice with hemorrhagic shock, Y-27632 inhibited microvascular reactivity, which was abolished by concomitant U-46619 application. Lastly, hemorrhagic shock remarkably decreased intestinal loop blood flow, RhoA and ROCK mRNA expressions in vascular tissues in OVX females, but not in OVI females, which were reversed by E2 supplement. These results indicate that estrogen improves microvascular reactivity during hemorrhagic shock, and RhoA-ROCK signaling pathway may mediate E2 effects.
Assuntos
Estradiol/uso terapêutico , Estrogênios/uso terapêutico , Choque Hemorrágico/tratamento farmacológico , Transdução de Sinais/fisiologia , Vasoconstrição/fisiologia , Quinases Associadas a rho/fisiologia , Animais , Feminino , Camundongos , Choque Hemorrágico/fisiopatologiaRESUMO
Systemic lupus erythematosus is a chronic inflammatory disease, in which treatment is still limited due to suboptimal efficacy and toxicities associated with the available therapies. JAK kinases are well known to play an important role in systemic lupus erythematous. There is growing evidence that ROCK kinases are also important in disease development. In this paper, we present the results of the development of CPL409116, a dual JAK and ROCK inhibitor. The studies we performed demonstrate that this molecule is an effective JAK and ROCK inhibitor which efficiently blocks disease progression in NZBWF1/J mouse models of systemic lupus erythematous.
Assuntos
Inibidores de Janus Quinases/uso terapêutico , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Lúpus Eritematoso Sistêmico/enzimologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Progressão da Doença , Feminino , Inibidores de Janus Quinases/farmacologia , Janus Quinases/fisiologia , Camundongos Transgênicos , Piperidinas/farmacologia , Piperidinas/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Resultado do Tratamento , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/fisiologiaRESUMO
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
BACKGROUND: Bronchopulmonary dysplasia remains one of the most common complications of prematurity, despite significant improvements in perinatal care. Functional modeling of human lung development and disease, like BPD, is limited by our ability to access the lung and to maintain relevant progenitor cell populations in culture. METHODS: We supplemented Rho/SMAD signaling inhibition with mTOR inhibition to generate epithelial basal cell-like cell lines from tracheal aspirates of neonates. RESULTS: Single-cell RNA-sequencing confirmed the presence of epithelial cells in tracheal aspirates obtained from intubated neonates. Using Rho/SMAD/mTOR triple signaling inhibition, neonatal tracheal aspirate-derived (nTAD) basal cell-like cells can be expanded long term and retain the ability to differentiate into pseudostratified airway epithelium. CONCLUSIONS: Our data demonstrate that neonatal tracheal aspirate-derived epithelial cells can provide a novel ex vivo human cellular model to study neonatal lung development and disease. IMPACT: Airway epithelial basal cell-like cell lines were derived from human neonatal tracheal aspirates. mTOR inhibition significantly extends in vitro proliferation of neonatal tracheal aspirate-derived basal cell-like cells (nTAD BCCs). nTAD BCCs can be differentiated into functional airway epithelium. nTAD BCCs provide a novel model to investigate perinatal lung development and diseases.
Assuntos
Células Epiteliais/efeitos dos fármacos , Proteínas Smad/antagonistas & inibidores , Serina-Treonina Quinases TOR/antagonistas & inibidores , Traqueia/citologia , Quinases Associadas a rho/antagonistas & inibidores , Sequência de Bases , Líquidos Corporais/citologia , Displasia Broncopulmonar , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Células Epiteliais/química , Células Epiteliais/citologia , Humanos , Recém-Nascido , Cultura Primária de Células , Análise de Célula Única , Sirolimo/farmacologia , Proteínas Smad/fisiologia , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Sucção , Serina-Treonina Quinases TOR/fisiologia , Quinases Associadas a rho/fisiologiaRESUMO
Although the actomyosin cytoskeleton has been implicated in clathrin-mediated endocytosis, a clear requirement for actomyosin in clathrin-independent endocytosis (CIE) has not been demonstrated. We discovered that the Rho-associated kinase ROCK2 is required for CIE of MHCI and CD59 through promotion of myosin II activity. Myosin IIA promoted internalization of MHCI and myosin IIB drove CD59 uptake in both HeLa and polarized Caco2 intestinal epithelial cells. In Caco2 cells, myosin IIA localized to the basal cortex and apical brush border and mediated MHCI internalization from the basolateral domain, while myosin IIB localized at the basal cortex and apical cell-cell junctions and promoted CD59 uptake from the apical membrane. Atomic force microscopy demonstrated that myosin IIB mediated apical epithelial tension in Caco2 cells. Thus, specific cargoes are internalized by ROCK2-mediated activation of myosin II isoforms to mediate spatial regulation of CIE, possibly by modulation of local cortical tension.
Assuntos
Endocitose/fisiologia , Miosina Tipo II/metabolismo , Quinases Associadas a rho/metabolismo , Citoesqueleto de Actina/metabolismo , Actomiosina/metabolismo , Junções Aderentes/fisiologia , Antígenos CD59/metabolismo , Células CACO-2 , Caderinas/metabolismo , Clatrina/metabolismo , Proteínas do Citoesqueleto/fisiologia , Citoesqueleto/metabolismo , Células Epiteliais/citologia , Células HeLa , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Miosina Tipo II/fisiologia , Miosina não Muscular Tipo IIA/metabolismo , Miosina não Muscular Tipo IIB/metabolismo , Isoformas de Proteínas/metabolismo , Quinases Associadas a rho/fisiologiaRESUMO
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
The proliferation and differentiation of cultured epithelial cells may be modified by Rho-associated kinase (ROCK) inhibition and extracellular Ca2+ concentration. However, it was not known whether a combination would influence the behavior of cultured epithelial cells through changes in the phosphorylation of non-muscle myosin light chain II (MLC). Here we show that the combination of ROCK inhibition with Ca2+ elevation regulated the phosphorylation of MLC and improved both cell expansion and cell-cell adhesion during the culture of human nasal mucosal epithelial cell sheets. During explant culture, Ca2+ enhanced the adhesion of nasal mucosal tissue, while ROCK inhibition downregulated MLC phosphorylation and promoted cell proliferation. During cell sheet culture, an elevation of extracellular Ca2+ promoted MLC phosphorylation and formation of cell-cell junctions, allowing the harvesting of cell sheets without collapse. Moreover, an in vitro grafting assay revealed that ROCK inhibition increased the expansion of cell sheets three-fold (an effect maintained when Ca2+ was also elevated), implying better wound healing potential. We suggest that combining ROCK inhibition with elevation of Ca2+ could facilitate the fabrication of many types of cell graft.
Assuntos
Amidas/farmacologia , Cálcio/farmacologia , Adesão Celular/efeitos dos fármacos , Técnicas de Cultura de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Miosina Tipo II/metabolismo , Mucosa Nasal/citologia , Piridinas/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Cálcio/metabolismo , Cálcio/fisiologia , Transplante de Células/métodos , Células Cultivadas , Sinergismo Farmacológico , Humanos , Fosforilação/efeitos dos fármacos , Estimulação Química , Transplantes , Cicatrização , Quinases Associadas a rho/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
OBJECTIVE: Many studies have already suggested the role of long non-coding RNAs (lncRNAs) in Alzheimer's disease (AD), but the functions of lncRNA Taurine Upregulated Gene 1 (TUG1) in AD have been scarcely discussed. This study aims to verify how TUG1 affects hippocampal neurons in AD through modulation of microRNA-15a (miR-15a)/Rho-associated protein kinase 1 (ROCK1). METHOD: AD mice was modeled through injection of ß-amyloid 25-35 (Aß25-35) into the lateral ventricle. After modeling, the mice were injected with altered TUG1 and/or miR-15a agomir lentiviruses. The spatial learning ability and memory ability of mice were detected through Morris water maze test. Hippocampal neuronal apoptosis and oxidative stress indicators in AD mice were then detected. The hippocampal neuron AD model was induced by Aß25-35. Next, the neurons were, respectively, transfected with altered TUG1 vector and/or miR-15a mimics to determine the proliferation inhibition and apoptosis of hippocampal neurons. The interactions between TUG1 and miR-15a, and between miR-15a and ROCK1 were assessed using bioinformatic prediction, dual luciferase reporter gene assay and RNA-pull-down assay. RESULTS: In the animal models, Aß25-35-induced mice exhibited decreased spatial learning and memory ability, obvious pathological injury, promoted hippocampal neuronal apoptosis and decreased antioxidant ability. TUG1 silencing and miR-15a elevation improved spatial learning ability and memory ability, ameliorated pathological injury, depressed neuronal apoptosis, and strengthened antioxidant ability of hippocampal neurons in AD mice. In cellular models, Aß25-35-treated hippocampal neurons presented inhibited neuronal viability and promoted neuronal apoptosis. TUG1 silencing and miR-15a elevation increased viability and limited apoptosis of Aß25-35-treated hippocampal neurons. TUG1 specifically bound to miR-15a, and miR-15a targeted ROCK1. CONCLUSION: Collectively, this study reveals that TUG1 knockdown restricts apoptosis of hippocampal neurons in AD by elevating miR-15a and suppressing ROCK1 expression, and provides a new therapeutic target for AD treatment.
Assuntos
Doença de Alzheimer/terapia , Apoptose , Hipocampo/patologia , MicroRNAs/fisiologia , RNA Longo não Codificante/fisiologia , Quinases Associadas a rho/fisiologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/farmacologia , Animais , Células Cultivadas , Feminino , Hipocampo/metabolismo , Aprendizagem , Masculino , Memória , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The intestinal epithelial apical junctional complex, which includes tight and adherens junctions, contributes to the intestinal barrier function via their role in regulating paracellular permeability. Myosin light chain II (MLC-2), has been shown to be a critical regulatory protein in altering paracellular permeability during gastrointestinal disorders. Previous studies have demonstrated that phosphorylation of MLC-2 is a biochemical marker for perijunctional actomyosin ring contraction, which increases paracellular permeability by regulating the apical junctional complex. The phosphorylation of MLC-2 is dominantly regulated by myosin light chain kinase- (MLCK-) and Rho-associated coiled-coil containing protein kinase- (ROCK-) mediated pathways. In this review, we aim to summarize the current state of knowledge regarding the role of MLCK- and ROCK-mediated pathways in the regulation of the intestinal barrier during normal homeostasis and digestive diseases. Additionally, we will also suggest potential therapeutic targeting of MLCK- and ROCK-associated pathways in gastrointestinal disorders that compromise the intestinal barrier.
Assuntos
Mucosa Intestinal/fisiologia , Quinase de Cadeia Leve de Miosina/metabolismo , Quinases Associadas a rho/metabolismo , Animais , Humanos , Mucosa Intestinal/metabolismo , Quinase de Cadeia Leve de Miosina/fisiologia , Permeabilidade , Junções Íntimas/metabolismo , Quinases Associadas a rho/fisiologiaRESUMO
Leptomeningeal anastomoses (LMAs) are pial collaterals that perfuse the penumbra and important for stroke outcome. We previously showed LMAs from SHRs (spontaneously hypertensive rats) were vasoconstricted compared with normotensive Wistar rats. Here, we investigated mechanisms by which hypertension causes LMA vasoconstriction. SHRs were treated with the ACE (angiotensin-converting enzyme) inhibitor captopril, an Ang II (angiotensin II)-independent antihypertensive agent hydralazine, or vehicle for 5 weeks in drinking water (n=8/group). A group of Wistar rats (n=8) had regular drinking water served as controls. Blood pressure was measured twice weekly by tail-cuff. LMAs were isolated and studied under pressurized conditions. Vasoreactivity of LMAs, including myogenic responses, reactivity to Rho-kinase inhibitor Y-27632, and nitric oxide were measured. Both captopril and hydralazine lowered blood pressure in SHRs similar to Wistar. However, only captopril normalized LMA increased tone compared with untreated SHRs (15±2% versus 50±3%; P<0.01) that was similar to Wistar (16±2%) but not hydralazine (38±6%; P>0.05). Vasodilatory response of LMAs to Y-27632 was impaired in SHRs compared with Wistar (28±3% versus 81±4%; P<0.01) that was restored by captopril (84±5%; P<0.01) and partially hydralazine (59±4%). LMAs from all groups constricted similarly to NOS (NO synthase) inhibition; however, the vasodilatory response of LMAs to the nitric oxide donor sodium nitroprusside was impaired in SHRs compared with Wistar rats (29±4% versus 80±2%; P<0.01) that was restored by captopril (84±4%; P<0.01), not hydralazine (38±8%; P>0.05). These results suggest that ACE inhibition during chronic hypertension reversed vascular dysfunction and hyperconstriction of LMAs that could improve stroke outcome by increasing collateral perfusion.
Assuntos
Angiotensina II/fisiologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Captopril/farmacologia , Circulação Colateral/efeitos dos fármacos , Hidralazina/farmacologia , Hipertensão/tratamento farmacológico , Pia-Máter/irrigação sanguínea , Vasoconstrição/efeitos dos fármacos , Vasodilatadores/farmacologia , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Animais , Benzimidazóis/farmacologia , Compostos de Bifenilo/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Captopril/uso terapêutico , Doença Crônica , Hidralazina/uso terapêutico , Hipertensão/genética , Hipertensão/fisiopatologia , Masculino , Distribuição Aleatória , Ratos , Ratos Endogâmicos SHR , Ratos Wistar , Receptor Tipo 1 de Angiotensina/biossíntese , Receptor Tipo 1 de Angiotensina/genética , Tetrazóis/farmacologia , Sistema Vasomotor/efeitos dos fármacos , Quinases Associadas a rho/fisiologiaRESUMO
This study sought to derive an enhanced understanding of the complex intracellular interactions that drive bone loss in postmenopausal osteoporosis. We applied an in-vitro multicellular niche to recapitulate cell-cell signalling between osteocytes, osteoblasts and osteoclasts to investigate (1) how estrogen-deficient and mechanically loaded osteocytes regulate osteoclastogenesis and (2) whether ROCK-II inhibition affects these mechanobiological responses. We report that mechanically stimulated and estrogen-deficient osteocytes upregulated RANKL/OPG and M-CSF gene expression, when compared to those treated with 10 nM estradiol. Osteoclast precursors (RAW 264.7) cultured within this niche underwent significant reduction in osteoclastogenic gene expression (CTSK), and there was an increasing trend in the area covered by TRAP+ osteoclasts (24% vs. 19.4%, p = 0.06). Most interestingly, upon treatment with the ROCK-II inhibitor, RANKL/OPG and M-CSF gene expression by estrogen-deficient osteocytes were downregulated. Yet, this inhibition of the pro-osteoclastogenic factors by osteocytes did not ultimately reduce the differentiation of osteoclast precursors. Indeed, TRAP and CTSK gene expressions in osteoclast precursors were upregulated, and there was an increased trend for osteoclast area (30.4% vs. 24%, p = 0.07), which may have been influenced by static osteoblasts (MC3T3-E1) that were included in the niche. We conclude that ROCK-II inhibition can attenuate bone loss driven by osteocytes during estrogen deficiency.
Assuntos
Amidas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Estradiol/deficiência , Osteoclastos/efeitos dos fármacos , Osteócitos/efeitos dos fármacos , Piridinas/farmacologia , Animais , Células Cultivadas , Técnicas de Cocultura , Estradiol/farmacologia , Camundongos , Modelos Biológicos , Osteoclastos/fisiologia , Osteócitos/fisiologia , Osteogênese/efeitos dos fármacos , Pós-Menopausa/efeitos dos fármacos , Pós-Menopausa/fisiologia , Inibidores de Proteínas Quinases/farmacologia , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Nicho de Células-Tronco/efeitos dos fármacos , Nicho de Células-Tronco/fisiologia , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/fisiologiaRESUMO
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.