RESUMO
Rho-associated coiled-coil kinase (ROCK) is involved in multiple cellular activities regulating the actin cytoskeleton, such as cell morphology, adhesion, and migration. The inhibition of ROCK is a feasible strategy to suppress breast cancer metastasis. Herein, based on Belumosudil, a series of pyrazolo[1,5-a]pyrimidine derivatives as selective ROCK2 inhibitors were designed and synthesized. Through systematic investigation of SARs, the piperazine analog 7u was identified with optimum ROCK2 inhibitory activity (IC50 = 36.8 nM) and excellent selectivity over the isoform protein ROCK1 (>250-fold). Intriguingly, upon treatment with 7u, the arrangement of the MDA-MB-231 cytoskeleton was affected accompanied by the alteration of morphology. Furthermore, cell scratch and transwell assays indicated that 7u inhibited MDA-MB-231 cell migration and invasion in a dose-dependent manner. Ultimately, the binding model of 7u with ROCK2 well accounted for the superior activities of 7u as a promising ROCK2 inhibitor with the potential application in breast cancer metastasis treatment.
Assuntos
Antineoplásicos , Neoplasias da Mama , Movimento Celular , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores de Proteínas Quinases , Pirazóis , Pirimidinas , Quinases Associadas a rho , Humanos , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismo , Movimento Celular/efeitos dos fármacos , Pirimidinas/farmacologia , Pirimidinas/química , Pirimidinas/síntese química , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/síntese química , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Relação Estrutura-Atividade , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Feminino , Pirazóis/farmacologia , Pirazóis/química , Pirazóis/síntese química , Estrutura Molecular , Proliferação de Células/efeitos dos fármacos , Linhagem Celular Tumoral , Descoberta de Drogas , Simulação de Acoplamento MolecularRESUMO
Human T-lymphotropic virus type 1 (HTLV-1) is a RNA virus belonging to Retroviridae family and is associated with the development of various diseases, including adult T-cell leukemia/lymphoma (ATLL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Aside from HAM/TSP, HTLV-1 has been implicated in the development of several disorders that mimic auto-inflammation. T-cell migration is important topic in the context of HTLV-1 associated diseases progression. The primary objective of this case-control study was to assess the relationship between increased mRNA expression in virus migration following HTLV-1 infection. PBMCs from 20 asymptomatic patients and 20 healthy subjects were analyzed using real-time PCR to measure mRNA expression of LFA1, MLCK, RAC1, RAPL, ROCK1, VAV1 and CXCR4. Also, mRNA expression of Tax and HBZ were evaluated. Mean expression of Tax and HBZ in ACs (asymptomatic carriers) was 0.7218 and 0.6517 respectively. The results revealed a noteworthy upregulation of these genes involved in T-cell migration among ACs patients in comparison to healthy individuals. Considering the pivotal role of gene expression alterations associated with the progression into two major diseases (ATLL or HAM/TSP), analyzing the expression of these genes in the ACs group can offer probable potential diagnostic markers and aid in monitoring the condition of ACs.
Assuntos
Movimento Celular , Infecções por HTLV-I , Vírus Linfotrópico T Tipo 1 Humano , Humanos , Vírus Linfotrópico T Tipo 1 Humano/genética , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Masculino , Feminino , Adulto , Estudos de Casos e Controles , Pessoa de Meia-Idade , Infecções por HTLV-I/imunologia , Infecções por HTLV-I/virologia , Infecções por HTLV-I/genética , Produtos do Gene tax/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismo , Leucócitos/metabolismo , Leucócitos/imunologia , Proteínas Proto-Oncogênicas c-vav/genética , Proteínas Proto-Oncogênicas c-vav/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Antígeno-1 Associado à Função Linfocitária/genética , Proteínas dos Retroviridae , Fatores de Transcrição de Zíper de Leucina BásicaRESUMO
BACKGROUND: Helicobacter pylori infection is one of the most common chronic bacterial infections, especially in developing countries. MicroRNA-148a is involved in the regulation of various genes, including Rock1, which is altered in gastric cancer. Decreased expression of mir-148a leads to tumor metastasis and increased Rock1 gene expression in gastric cancer. This study aimed to investigate the expression of these genes in biopsies collected from patients with H. pylori induced gastritis. METHODS: Informed consent forms were gotten from the studied patients with gastritis who needed endoscopy. Gastric biopsies were taken by a gastroenterologist from patients with inflammation. Rapid urease test, stool antigen detection, and histopathological staining were used to determine the H. pylori infected patients. Real time PCR was used to evaluate the miRNA and Rock1 expression levels. RESULTS: The Rock1 expression level in biopsies that were positive for H. pylori was significantly increased compared to our control gastritis group that were H. pylori-negative, but the results were not statistically significant. Moreover, the mir-148a expression level in H. pylori-positive patients with gastritis was increased compared to our control group. However, the results were not statistically significant. We did not find a significant relation between the expression levels of Rock1 and mir-148a in samples with gastritis infected or uninfected by H. pylori. This result may be due to the small sample size. CONCLUSION: We suggest that this test should be carried out with more samples, and the comparison should be done between biopsies with inflammation and no inflammation in a patient.
Assuntos
Gastrite , Infecções por Helicobacter , Helicobacter pylori , MicroRNAs , Quinases Associadas a rho , Humanos , Gastrite/microbiologia , Gastrite/patologia , Gastrite/metabolismo , MicroRNAs/metabolismo , MicroRNAs/genética , Infecções por Helicobacter/patologia , Quinases Associadas a rho/metabolismo , Quinases Associadas a rho/genética , Helicobacter pylori/isolamento & purificação , Biópsia , Masculino , Feminino , Pessoa de Meia-Idade , Adulto , Mucosa Gástrica/patologia , Mucosa Gástrica/microbiologia , IdosoRESUMO
Purpose: The Rho-associated protein kinase and myosin light chain kinase (ROCK/MYLK) pathway undeniably plays a pivotal role in the pathophysiology of primary open-angle glaucoma (POAG). In our study, we utilized both ocular hypertension (OHT) rabbit models and clinical investigations to gain invaluable insights that propel the development of novel treatments targeting proteins and genes associated with the trabecular meshwork (TM), thereby offering promising avenues for the management of POAG. Methods: Following microbead injections into the anterior chamber of the ocular cavity of rabbits, we observed elevated histiocyte numbers and immune scores for MYLK-4/ pMLC-2, alongside a reduction in the void space within the TM. Notably, treatment was performed with 0.1% ITRI-E-(S)-4046, a compound with dual kinase inhibitor (highly specific inhibitor of ROCK1/2 and MYLK4), significantly reduced intraocular pressure (IOP; P < 0.05) and expanded the void space within the TM (P < 0.0001) compared with OHT rabbits. In clinical investigations, we utilized whole transcriptome sequencing to analyze gene expression specifically related to the TM, obtained from patients (5 early-onset and 5 late-onset) undergoing trabeculectomy. Results: Our findings revealed 103 differential expression genes (DEGs) out of 265 molecules associated with the Rho family GTPase pathway, exhibiting a P value of 1.25E-10 and a z-score of -2.524. These results underscore significant differences between the early-onset and late-onset POAG and highlight the involvement of the ROCK/MYLK pathway. Conclusions: These findings underscore the critical involvement of the ROCK/MYLK pathway in both OHT-related and different onsets of POAG, providing valuable insights into the TM-related molecular mechanisms underlying the disease.
Assuntos
Modelos Animais de Doenças , Glaucoma de Ângulo Aberto , Pressão Intraocular , Quinase de Cadeia Leve de Miosina , Hipertensão Ocular , Malha Trabecular , Quinases Associadas a rho , Animais , Malha Trabecular/metabolismo , Malha Trabecular/patologia , Quinases Associadas a rho/genética , Coelhos , Hipertensão Ocular/genética , Hipertensão Ocular/fisiopatologia , Hipertensão Ocular/metabolismo , Pressão Intraocular/fisiologia , Humanos , Glaucoma de Ângulo Aberto/genética , Glaucoma de Ângulo Aberto/metabolismo , Glaucoma de Ângulo Aberto/fisiopatologia , Quinase de Cadeia Leve de Miosina/genética , Quinase de Cadeia Leve de Miosina/metabolismo , Masculino , Feminino , Transdução de Sinais , Idoso , Pessoa de Meia-IdadeAssuntos
Valva Aórtica , Calcinose , Transdução de Sinais , Quinases Associadas a rho , Humanos , Quinases Associadas a rho/metabolismo , Calcinose/etiologia , Calcinose/metabolismo , Valva Aórtica/patologia , Valva Aórtica/metabolismo , Valvopatia Aórtica/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Estenose da Valva Aórtica/etiologia , Estenose da Valva Aórtica/metabolismoRESUMO
Acute lung injury (ALI) is a significant health condition with notable rates of morbidity and mortality globally. Long non-coding ribose nucleic acids (lncRNAs) play vital roles in mitigating various inflammation-related diseases, including ALI. The study aimed to investigate the functional role and molecular mechanisms of lncRNA SNHG1 on ALI in lipopolysaccharide (LPS)-treated A549 cells and in LPS-induced ALI mice. The expression of SNHG1 was initially examined in LPS-treated A549 cells. We further demonstrated the critical function of SNHG1 through various cellular assessments following SNHG1 knockdown, including cell counting kit (CCK)-8 assay, flow cytometry analysis, as well as enzyme-linked immunosorbent assay (ELISA). Reducing SNHG1 levels hindered the negative effects of LPS on cell viability, apoptosis, and inflammation. Moreover, SNHG1 acted as a negative regulator for miR-199a-3p, which targeted downstream ROCK2. Depletion of miR-199a-3p or enhanced expression of ROCK2 abolished the protective effects of SNHG1 knockdown on LPS-induced apoptosis and inflammation. Consistently, silencing SNHG1 alleviated LPS-induced lung injury in mice, demonstrating its potential therapeutic benefits in managing ALI. Overall, this study sheds light on the role of SNHG1 in modulating inflammation and apoptosis in ALI through the miR-199a-3p/ROCK2 pathway, offering new insights for the treatment of this condition.
Assuntos
Lesão Pulmonar Aguda , Técnicas de Silenciamento de Genes , Lipopolissacarídeos , MicroRNAs , RNA Longo não Codificante , Quinases Associadas a rho , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/genética , MicroRNAs/metabolismo , MicroRNAs/genética , Animais , Camundongos , Humanos , Quinases Associadas a rho/metabolismo , Quinases Associadas a rho/genética , Masculino , Células A549 , Transdução de Sinais , Camundongos Endogâmicos C57BL , Apoptose/genéticaRESUMO
RhoA (ras homolog family member A) is a small G-protein that transduces intracellular signaling to regulate a broad range of cellular functions such as cell growth, proliferation, migration, and survival. RhoA serves as a proximal downstream effector of numerous G protein-coupled receptors (GPCRs) and is also responsive to various stresses in the heart. Upon its activation, RhoA engages multiple downstream signaling pathways. Rho-associated coiled-coil-containing protein kinase (ROCK) is the first discovered and best characterized effector or RhoA, playing a major role in cytoskeletal arrangement. Many other RhoA effectors have been identified, including myocardin-related transcription factor A (MRTF-A), Yes-associated Protein (YAP) and phospholipase Cε (PLCε) to regulate transcriptional and post-transcriptional processes. The role of RhoA signaling in the heart has been increasingly studied in last decades. It was initially suggested that RhoA signaling pathway is maladaptive in the heart, but more recent studies using cardiac-specific expression or deletion of RhoA have revealed that RhoA activation provides cardioprotection against stress through various mechanisms including the novel role of RhoA in mitochondrial quality control. This review summarizes recent advances in understanding the role of RhoA in the heart and its signaling pathways to prevent progression of heart disease.
Assuntos
Transdução de Sinais , Proteína rhoA de Ligação ao GTP , Humanos , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Miocárdio/metabolismo , Quinases Associadas a rho/metabolismo , Quinases Associadas a rho/genéticaRESUMO
Hippocampal area CA2 has garnered attention in recent times owing to its significant involvement in social memory and distinctive plasticity characteristics. Research has revealed that the CA2 region demonstrates a remarkable resistance to plasticity, particularly in the Schaffer Collateral (SC)-CA2 pathway. In this study we investigated the role of Nogo-A, a well-known axon growth inhibitor and more recently discovered plasticity regulator, in modulating plasticity within the CA2 region. The findings demonstrate that blocking Nogo-A in male rat hippocampal slices facilitates the establishment of both short-term and long-term plasticity in the SC-CA2 pathway, while having no impact on the Entorhinal Cortical (EC)-CA2 pathway. Additionally, the study reveals that inhibiting Nogo-A enables association between the SC and EC pathways. Mechanistically, we confirm that Nogo-A operates through its well-known co-receptor, p75 neurotrophin receptor (p75NTR), and its downstream signaling factor such as Rho-associated protein kinase (ROCK), as their inhibition also allows plasticity induction in the SC-CA2 pathway. Additionally, the induction of long-term depression (LTD) in both the EC and SC-CA2 pathways led to persistent LTD, which was not affected by Nogo-A inhibition. Our study demonstrates the involvement of Nogo-A mediated signaling mechanisms in limiting synaptic plasticity within the CA2 region.
Assuntos
Região CA2 Hipocampal , Plasticidade Neuronal , Proteínas Nogo , Sinapses , Animais , Proteínas Nogo/metabolismo , Masculino , Plasticidade Neuronal/fisiologia , Sinapses/fisiologia , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Região CA2 Hipocampal/fisiologia , Região CA2 Hipocampal/metabolismo , Região CA2 Hipocampal/efeitos dos fármacos , Ratos Sprague-Dawley , Ratos , Quinases Associadas a rho/metabolismo , Quinases Associadas a rho/antagonistas & inibidores , Córtex Entorrinal/fisiologia , Córtex Entorrinal/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Vias Neurais/fisiologia , Proteínas da Mielina/metabolismo , Proteínas da Mielina/genética , Proteínas do Tecido Nervoso , Receptores de Fatores de CrescimentoRESUMO
INTRODUCTION: To explore the mechanisms of labor by investigating the autophagy of placental and fetal membranes tissue in normal pregnant women. METHODS: Placenta and fetal membranes were collected from women with singleton pregnancies without any medical complications and from women who delivered vaginally (labor-initiated group; L group) or by caesarean section (labor-noninitiated group; NL group). Autophagosomes were observed by transmission electron microscopy (TEM). Immunofluorescence and western blotting (WB) were used to detect protein levels of the autophagy markers LC3A and LC3B. TEM, immunohistochemistry (IHC), and WB were used to compare autophagy in different parts of the placenta and fetal membranes in the L and NL groups. The expression of LC3B/LC3A, ROCK1, and ROCK2 in the placenta of nonpregnant and pregnant rats was detected by WB and IHC. RESULTS: TEM and IHC results showed an increase in the number of autophagosomes and autophagic lysosomes in the L group, and WB results indicated an increase in the LC3B/A ratio between the placenta and fetal membranes in the L group. Autophagy was significantly increased on the maternal side of the placenta in the L group, and the level of autophagy became higher near rupture in the fetal membranes and near the point where the umbilical cord joins the placenta in the L group. The LC3B/A ratio increased and ROCK1 and ROCK2 levels decreased in postnatal rats. DISCUSSION: Autophagy can occur in the placenta and fetal membranes and its activity is higher at the onset of labor, suggesting a role in labor.
Assuntos
Autofagia , Proteínas Associadas aos Microtúbulos , Placenta , Quinases Associadas a rho , Feminino , Gravidez , Humanos , Autofagia/fisiologia , Placenta/metabolismo , Placenta/ultraestrutura , Quinases Associadas a rho/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Animais , Ratos , Adulto , Início do Trabalho de Parto , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Membranas Extraembrionárias/metabolismo , Trabalho de Parto/metabolismo , Ratos Sprague-DawleyRESUMO
Integrin activation resulting in enhanced adhesion to the extracellular matrix plays a key role in fundamental cellular processes. Although integrin activation has been extensively studied in circulating cells such as leukocytes and platelets, much less is known about the regulation and functional impact of integrin activation in adherent cells such as smooth muscle. Here, we show that two different asthmagenic cytokines, IL-13 and IL-17A, activate type I and IL-17 cytokine receptor families, respectively, to enhance adhesion of airway smooth muscle. These cytokines also induce activation of ß1 integrins detected by the conformation-specific antibody HUTS-4. Moreover, HUTS-4 binding is increased in the smooth muscle of patients with asthma compared to nonsmokers without lung disease, suggesting a disease-relevant role for integrin activation in smooth muscle. Indeed, integrin activation induced by the ß1-activating antibody TS2/16, the divalent cation manganese, or the synthetic peptide ß1-CHAMP that forces an extended-open integrin conformation dramatically enhances force transmission in smooth muscle cells and airway rings even in the absence of cytokines. We demonstrate that cytokine-induced activation of ß1 integrins is regulated by a common pathway of NF-κB-mediated induction of RhoA and its effector Rho kinase, which in turn stimulates PIP5K1γ-mediated synthesis of PIP2 at focal adhesions, resulting in ß1 integrin activation. Taken together, these data identify a pathway by which type I and IL-17 cytokine receptor family stimulation induces functionally relevant ß1 integrin activation in adherent smooth muscle and help to explain the exaggerated force transmission that characterizes chronic airway diseases such as asthma.
Assuntos
Asma , Integrina beta1 , Interleucina-13 , Interleucina-17 , Músculo Liso , NF-kappa B , Quinases Associadas a rho , Humanos , Integrina beta1/metabolismo , Interleucina-17/metabolismo , Músculo Liso/metabolismo , NF-kappa B/metabolismo , Quinases Associadas a rho/metabolismo , Interleucina-13/metabolismo , Asma/metabolismo , Transdução de Sinais , Adesão Celular , Miócitos de Músculo Liso/metabolismo , AnimaisRESUMO
Long-term synaptic plasticity is critical for adaptive function of the brain, but presynaptic mechanisms of functional plasticity remain poorly understood. Here, we show that changes in synaptic efficacy induced by activation of the cannabinoid type-1 receptor (CB1R), one of the most widespread G-protein coupled receptors in the brain, requires contractility of the neuronal actomyosin cytoskeleton. Specifically, using a synaptophysin-pHluorin probe (sypH2), we show that inhibitors of non-muscle myosin II (NMII) ATPase as well as one of its upstream effectors Rho-associated kinase (ROCK) prevent the reduction of synaptic vesicle release induced by CB1R activation. Using 3D STORM super-resolution microscopy, we find that activation of CB1R induces a redistribution of synaptic vesicles within presynaptic boutons in an actomyosin dependent manner, leading to vesicle clustering within the bouton and depletion of synaptic vesicles from the active zone. We further show, using sypH2, that inhibitors of NMII and ROCK specifically restore the release of the readily releasable pool of synaptic vesicles from the inhibition induced by CB1R activation. Finally, using slice electrophysiology, we find that activation of both NMII and ROCK is necessary for the long-term, but not the short-term, form of CB1R induced synaptic plasticity at excitatory cortico-striatal synapses. We thus propose a novel mechanism underlying CB1R-induced plasticity, whereby CB1R activation leads to a contraction of the actomyosin cytoskeleton inducing a reorganization of the functional presynaptic vesicle pool, preventing vesicle release and inducing long-term depression.
Assuntos
Actomiosina , Plasticidade Neuronal , Terminações Pré-Sinápticas , Receptor CB1 de Canabinoide , Vesículas Sinápticas , Quinases Associadas a rho , Animais , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/efeitos dos fármacos , Receptor CB1 de Canabinoide/metabolismo , Receptor CB1 de Canabinoide/antagonistas & inibidores , Actomiosina/metabolismo , Quinases Associadas a rho/metabolismo , Plasticidade Neuronal/fisiologia , Plasticidade Neuronal/efeitos dos fármacos , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/efeitos dos fármacos , Camundongos , Ratos , Masculino , Miosina Tipo II/metabolismoRESUMO
Rho-associated protein kinase-2 (ROCK2) is a critical player in many cellular processes and was incriminated in cardiovascular and neurological disorders. Recent evidence has shown that non-selective pharmacological blockage of ROCKs ameliorates behavioral alterations in a mouse model of 16p11.2 haploinsufficiency. We had revealed that 16p11.2-deficient mice also display cerebrovascular abnormalities, including endothelial dysfunction. To investigate whether genetic blockage of ROCK2 also exerts beneficial effects on cognition and angiogenesis, we generated mice with both 16p11.2 and Rock2 haploinsufficiency (16p11.2df/+;Rock2+/-). We find that Rock2 heterozygosity on a 16p11.2df/+ background significantly improved recognition memory. Furthermore, brain endothelial cells from 16p11.2df/+;Rock2+/- mice display improved angiogenic capacity compared to cells from 16p11.2df/+ littermates. Overall, this study implicates Rock2 gene as a modulator of 16p11.2-associated alterations, highlighting its potential as a target for treatment of autism spectrum disorders.
Assuntos
Transtorno Autístico , Deleção Cromossômica , Transtornos Cromossômicos , Cromossomos Humanos Par 16 , Modelos Animais de Doenças , Quinases Associadas a rho , Animais , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismo , Cromossomos Humanos Par 16/genética , Camundongos , Transtorno Autístico/genética , Transtornos Cromossômicos/genética , Heterozigoto , Reconhecimento Psicológico/fisiologia , Células Endoteliais/metabolismo , Haploinsuficiência , Masculino , Camundongos Endogâmicos C57BL , Anormalidades Craniofaciais/genética , Deficiência Intelectual/genéticaRESUMO
Structural neuroplasticity such as neurite extension and dendritic spine dynamics is enhanced by brain-derived neurotrophic factor (BDNF) and impaired by types of inhibitory molecules that induce growth cone collapse and actin depolymerization, for example, myelin-associated inhibitors, chondroitin sulfate proteoglycans, and negative guidance molecules. These inhibitory molecules can activate RhoA/rho-associated coiled-coil containing protein kinase (ROCK) signaling (known to restrict structural plasticity). Intermittent hypoxia (IH) and high-intensity interval training (HIIT) are known to upregulate BDNF that is associated with improvements in learning and memory and greater functional recovery following neural insults. We investigated whether the RhoA/ROCK signaling pathway is also modulated by IH and HIIT in the hippocampus, cortex, and lumbar spinal cord of male Wistar rats. The gene expression of 25 RhoA/ROCK signaling pathway components was determined following IH, HIIT, or IH combined with HIIT (30 min/day, 5 days/wk, 6 wk). IH included 10 3-min bouts that alternated between hypoxia (15% O2) and normoxia. HIIT included 10 3-min bouts alternating between treadmill speeds of 50 cm·s-1 and 15 cm·s-1. In the hippocampus, IH and HIIT significantly downregulated Acan and NgR2 mRNA that are involved in the inhibition of neuroplasticity. However, IH and IH + HIIT significantly upregulated Lingo-1 and NgR3 in the cortex. This is the first time IH and HIIT have been linked to the modulation of plasticity-inhibiting pathways. These results provide a fundamental step toward elucidating the interplay between the neurotrophic and inhibitory mechanisms involved in experience-driven neural plasticity that will aid in optimizing physiological interventions for the treatment of cognitive decline or neurorehabilitation.NEW & NOTEWORTHY Intermittent hypoxia (IH) and high-intensity interval training (HIIT) enhance neuroplasticity and upregulate neurotrophic factors in the central nervous system (CNS). We provide evidence that IH and IH + HIIT also have the capacity to regulate genes involved in the RhoA/ROCK signaling pathway that is known to restrict structural plasticity in the CNS. This provides a new mechanistic insight into how these interventions may enhance hippocampal-related plasticity and facilitate learning, memory, and neuroregeneration.
Assuntos
Treinamento Intervalado de Alta Intensidade , Hipocampo , Ratos Wistar , Transdução de Sinais , Quinases Associadas a rho , Animais , Masculino , Quinases Associadas a rho/metabolismo , Quinases Associadas a rho/genética , Hipocampo/metabolismo , Transdução de Sinais/fisiologia , Ratos , Hipóxia/metabolismo , Hipóxia/fisiopatologia , Córtex Cerebral/metabolismo , Córtex Cerebral/fisiologia , Plasticidade Neuronal/fisiologia , Proteína rhoA de Ligação ao GTP/metabolismo , Medula Espinal/metabolismo , Medula Espinal/fisiologia , Proteínas rho de Ligação ao GTPRESUMO
We previously reported that the sustained component of contraction induced by depolarizing stimulation by high K+ concentration in rat caudal arterial smooth muscle involves a Ca2+-induced Ca2+ sensitization mechanism whereby Ca2+ entry through voltage-gated Ca2+ channels activates proline-rich tyrosine kinase 2 (Pyk2), leading to activation of RhoA/Rho-associated kinase (ROCK). In the present study, we investigated a potential role for Pyk2-mediated RhoA/ROCK activation in contraction mediated by elevation of cytosolic free Ca2+ concentration ([Ca2+]i) induced by a Ca2+ ionophore, ionomycin, rather than by depolarizing stimulation. Ionomycin (60 µM) induced slow and sustained contraction of rat caudal arterial smooth muscle due to influx of Ca2+. Pre-treatment with a myosin light chain kinase (MLCK) inhibitor, ML-9 (30 µM), inhibited both the early phase (4 min) and the sustained phase (30 min) of ionomycin-induced contraction. On the other hand, a ROCK inhibitor, HA-1077 (3 µM), and Pyk2 inhibitors, sodium salicylate (10 mM) and PF-431396 (3 µM), suppressed only the sustained phase of ionomycin-induced contraction. A calmodulin (CaM) inhibitor, W-7 (150 µM), but not W-5 (150 µM), suppressed the early phase of contraction. Early or sustained increase of ionomycin-induced 20 kDa light chain of myosin (LC20) phosphorylation was inhibited by each inhibitor in a manner similar to the attenuation of contraction. These results indicate that the early phase of ionomycin-induced contraction is mediated by MLCK activation by [Ca2+]i elevation, whereas the sustained phase of ionomycin-induced contraction involves RhoA/ROCK activation and inhibition of myosin light chain phosphatase (MLCP) through CaM-independent Pyk2 activation by [Ca2+]i elevation.
Assuntos
Cálcio , Ionomicina , Contração Muscular , Quinases Associadas a rho , Animais , Ionomicina/farmacologia , Masculino , Contração Muscular/efeitos dos fármacos , Cálcio/metabolismo , Quinases Associadas a rho/metabolismo , Quinases Associadas a rho/antagonistas & inibidores , Quinase de Cadeia Leve de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , Ratos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Músculo Liso Vascular/metabolismo , Quinase 2 de Adesão Focal/metabolismo , Ionóforos de Cálcio/farmacologia , Proteína rhoA de Ligação ao GTP/metabolismo , Ratos Sprague-Dawley , Ratos Wistar , Calmodulina/metabolismoRESUMO
GNAO1 encodes G protein subunit alpha O1 (Gαo). Pathogenic variations in GNAO1 cause developmental delay, intractable seizures, and progressive involuntary movements from early infancy. Because the functional role of GNAO1 in the developing brain remains unclear, therapeutic strategies are still unestablished for patients presenting with GNAO1-associated encephalopathy. We herein report that siRNA-mediated depletion of Gnao1 perturbs the expression of transcripts associated with Rho GTPase signaling in Neuro2a cells. Consistently, siRNA treatment hampered neurite outgrowth and extension. Growth cone formation was markedly disrupted in monolayer neurons differentiated from iPSCs from a patient with a pathogenic variant of Gαo (p.G203R). This variant disabled neuro-spherical assembly, acquisition of the organized structure, and polarized signals of phospho-MLC2 in cortical organoids from the patient's iPSCs. We confirmed that the Rho kinase inhibitor Y27632 restored these morphological phenotypes. Thus, Gαo determines the self-organizing process of the developing brain by regulating the Rho-associated pathway. These data suggest that Rho GTPase pathway might be an alternative target of therapy for patients with GNAO1-associated encephalopathy.
Assuntos
Diferenciação Celular , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP , Células-Tronco Pluripotentes Induzidas , Neurônios , Transdução de Sinais , Proteínas rho de Ligação ao GTP , Humanos , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Neurônios/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Proteínas rho de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/genética , Camundongos , Animais , Quinases Associadas a rho/metabolismo , Organoides/metabolismo , Amidas/farmacologia , PiridinasRESUMO
Fluoride exposure has been implicated as a potential risk factor for hypertension, but the underlying mechanisms remain unclear. This study investigated the role of the RhoA/ROCK signaling pathway in fluoride-induced hypertension. Male Wistar rats were divided into different groups and exposed to varying concentrations of sodium fluoride (NaF) or sodium chloride (NaCl) via drinking water. The rats' blood pressure was measured, and their aortic tissue was utilized for high-throughput sequencing analysis. Additionally, rat and A7r5 cell models were established using NaF and/or Fasudil. The study evaluated the effects of fluoride exposure on blood pressure, pathological changes in the aorta, as well as the protein/mRNA expression levels of phenotypic transformation indicators (a-SMA, calp, OPN) in vascular smooth muscle cells (VSMCs), along with the RhoA/ROCK signaling pathway (RhoA, ROCK1, ROCK2, MLC/p-MLC). The results demonstrated that fluoride exposure in rats led to increased blood pressure. High-throughput sequencing analysis revealed differential gene expression associated with vascular smooth muscle contraction, with the RhoA/ROCK signaling pathway emerging as a key regulator. Pathological changes in the rat aorta, such as elastic membrane rupture and collagen fiber deposition, were observed following NaF exposure. However, fasudil, a ROCK inhibitor, mitigated these pathological changes. Both in vitro and in vivo models confirmed the activation of the RhoA/ROCK signaling pathway and the phenotypic transformation of VSMCs from a contractile to a synthetic state upon fluoride exposure. Fasudil effectively inhibited the activities of ROCK1 and ROCK2 and attenuated the phenotypic transformation of VSMCs. In conclusion, fluoride has the potential to induce hypertension through the activation of the RhoA/ROCK signaling pathway and phenotypic changes in vascular smooth muscle cells. These results provide new insights into the mechanism of fluoride-induced hypertension.
Assuntos
Hipertensão , Músculo Liso Vascular , Ratos Wistar , Transdução de Sinais , Quinases Associadas a rho , Animais , Quinases Associadas a rho/metabolismo , Masculino , Hipertensão/induzido quimicamente , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologia , Ratos , Transdução de Sinais/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/metabolismo , Fluoreto de Sódio/toxicidade , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Fenótipo , Pressão Sanguínea/efeitos dos fármacos , Fluoretos/toxicidade , Proteínas rho de Ligação ao GTPRESUMO
Glaucoma affects approximately 80 million individuals worldwide, a condition for which current treatment options are inadequate. The primary risk factor for glaucoma is elevated intraocular pressure. Intraocular pressure is determined by the balance between the secretion and outflow of aqueous humor. Here we show that using the RNA interference tool CasRx based on shH10 adenovirus-associated virus can reduce the expression of the aqueous humor circulation related genes Rock1 and Rock2, as well as aquaporin 1 and ß2 adrenergic receptor in female mice. This significantly reduced intraocular pressure in female mice and provided protection to the retina ganglion cells, ultimately delaying disease progression. In addition, we elucidated the mechanisms by which the knockdown of Rock1 and Rock2, or aquaporin 1 and ß2 adrenergic receptor in female mice, reduces the intraocular pressure and secures the retina ganglion cells by single-cell sequencing.
Assuntos
Aquaporina 1 , Sistemas CRISPR-Cas , Glaucoma , Pressão Intraocular , Células Ganglionares da Retina , Quinases Associadas a rho , Animais , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/patologia , Quinases Associadas a rho/metabolismo , Quinases Associadas a rho/genética , Feminino , Aquaporina 1/metabolismo , Aquaporina 1/genética , Camundongos , Glaucoma/genética , Glaucoma/metabolismo , Humor Aquoso/metabolismo , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Interferência de RNA , HumanosRESUMO
Descemet's Stripping Only (DSO) is a surgical technique that utilizes the peripheral corneal endothelial cell (CEnC) migration for wound closure. Ripasudil, a Rho-associated protein kinase (ROCK) inhibitor, has shown potential in DSO treatment; however, its mechanism in promoting CEnC migration remains unclear. We observed that ripasudil-treated immortalized normal and Fuchs endothelial corneal dystrophy (FECD) cells exhibited significantly enhanced migration and wound healing, particularly effective in FECD cells. Ripasudil upregulated mRNA expression of Snail Family Transcriptional Repressor (SNAI1/2) and Vimentin (VIM) while decreasing Cadherin (CDH1), indicating endothelial-to-mesenchymal transition (EMT) activation. Ripasudil activated Rac1, driving the actin-related protein complex (ARPC2) to the leading edge, facilitating enhanced migration. Ex vivo studies on cadaveric and FECD Descemet's membrane (DM) showed increased migration and proliferation of CEnCs after ripasudil treatment. An ex vivo DSO model demonstrated enhanced migration from the DM to the stroma with ripasudil. Coating small incision lenticule extraction (SMILE) tissues with an FNC coating mix and treating the cells in conjunction with ripasudil further improved migration and resulted in a monolayer formation, as detected by the ZO-1 junctional marker, thereby leading to the reduction in EMT. In conclusion, ripasudil effectively enhanced cellular migration, particularly in a novel ex vivo DSO model, when the stromal microenvironment was modulated. This suggests ripasudil as a promising adjuvant for DSO treatment, highlighting its potential clinical significance.
Assuntos
Movimento Celular , Distrofia Endotelial de Fuchs , Quinases Associadas a rho , Humanos , Quinases Associadas a rho/metabolismo , Quinases Associadas a rho/antagonistas & inibidores , Movimento Celular/efeitos dos fármacos , Distrofia Endotelial de Fuchs/patologia , Distrofia Endotelial de Fuchs/tratamento farmacológico , Isoquinolinas/farmacologia , Sulfonamidas/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio Corneano/efeitos dos fármacos , Endotélio Corneano/metabolismo , Endotélio Corneano/patologia , Lâmina Limitante Posterior/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Ceratoplastia Endotelial com Remoção da Lâmina Limitante Posterior/métodos , Proliferação de Células/efeitos dos fármacos , Modelos Biológicos , Cicatrização/efeitos dos fármacosAssuntos
Antibacterianos , Cimentos Ósseos , Pé Diabético , Cicatrização , Quinases Associadas a rho , Pé Diabético/tratamento farmacológico , Cicatrização/efeitos dos fármacos , Humanos , Antibacterianos/uso terapêutico , Cimentos Ósseos/uso terapêutico , Cimentos Ósseos/farmacologia , Quinases Associadas a rho/metabolismo , Masculino , FemininoRESUMO
BACKGROUND: Dysregulation of Rho-associated coiled coil-containing protein kinases (ROCKs) is involved in the metastasis and progression of various malignant tumors. However, how one of the isomers, ROCK1, regulates glycolysis in tumor cells is incompletely understood. Here, we attempted to elucidate how ROCK1 influences pancreatic cancer (PC) progression by regulating glycolytic activity. METHODS: The biological function of ROCK1 was analyzed in vitro by establishing a silenced cell model. Coimmunoprecipitation confirmed the direct binding between ROCK1 and c-MYC, and a luciferase reporter assay revealed the binding of c-MYC to the promoter of the PFKFB3 gene. These results were verified in animal experiments. RESULTS: ROCK1 was highly expressed in PC tissues and enriched in the cytoplasm, and its high expression was associated with a poor prognosis. Silencing ROCK1 inhibited the proliferation and migration of PC cells and promoted their apoptosis. Mechanistically, ROCK1 directly interacted with c-MYC, promoted its phosphorylation (Ser 62) and suppressed its degradation, thereby increasing the transcription of the key glycolysis regulatory factor PFKFB3, enhancing glycolytic activity and promoting PC growth. Silencing ROCK1 increased gemcitabine (GEM) sensitivity in vivo and in vitro. CONCLUSIONS: ROCK1 promotes glycolytic activity in PC cells and promotes PC tumor growth through the c-MYC/PFKFB3 signaling pathway. ROCK1 knockdown can inhibit PC tumor growth in vivo and increase the GEM sensitivity of PC tumors, providing a crucial clinical therapeutic strategy for PC.