RESUMO
Migration of endothelial and many other cells requires spatiotemporal regulation of protrusive and contractile cytoskeletal rearrangements that drive local cell shape changes. Unexpectedly, the small GTPase Rho, a crucial regulator of cell movement, has been reported to be active in both local cell protrusions and retractions, raising the question of how Rho activity can coordinate cell migration. Here, we show that Rho activity is absent in local protrusions and active during retractions. During retractions, Rho rapidly activated ezrin-radixin-moesin proteins (ERMs) to increase actin-membrane attachment, and, with a delay, nonmuscle myosin 2 (NM2). Rho activity was excitable, with NM2 acting as a slow negative feedback regulator. Strikingly, inhibition of SLK/LOK kinases, through which Rho activates ERMs, caused elongated cell morphologies, impaired Rho-induced cell contractions, and reverted Rho-induced blebbing. Together, our study demonstrates that Rho activity drives retractions by sequentially enhancing ERM-mediated actin-membrane attachment for force transmission and NM2-dependent contractility.
Assuntos
Actomiosina , Movimento Celular , Forma Celular , Proteínas do Citoesqueleto , Actomiosina/metabolismo , Humanos , Proteínas do Citoesqueleto/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas rho de Ligação ao GTP/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas de Membrana/metabolismo , Actinas/metabolismo , Animais , Miosina Tipo II/metabolismoRESUMO
Ovulation is critical for sexual reproduction and consists of the process of liberating fertilizable oocytes from their somatic follicle capsules, also known as follicle rupture. The mechanical force for oocyte expulsion is largely unknown in many species. Our previous work demonstrated that Drosophila ovulation, as in mammals, requires the proteolytic degradation of the posterior follicle wall and follicle rupture to release the mature oocyte from a layer of somatic follicle cells. Here, we identified actomyosin contraction in somatic follicle cells as the major mechanical force for follicle rupture. Filamentous actin (F-actin) and nonmuscle myosin II (NMII) are highly enriched in the cortex of follicle cells upon stimulation with octopamine (OA), a monoamine critical for Drosophila ovulation. Pharmacological disruption of F-actin polymerization prevented follicle rupture without interfering with the follicle wall breakdown. In addition, we demonstrated that OA induces Rho1 guanosine triphosphate (GTP)ase activation in the follicle cell cortex, which activates Ras homolog (Rho) kinase to promote actomyosin contraction and follicle rupture. All these results led us to conclude that OA signaling induces actomyosin cortex enrichment and contractility, which generates the mechanical force for follicle rupture during Drosophila ovulation. Due to the conserved nature of actomyosin contraction, this work could shed light on the mechanical force required for follicle rupture in other species including humans.
Assuntos
Actomiosina , Proteínas de Drosophila , Octopamina , Folículo Ovariano , Ovulação , Animais , Actomiosina/metabolismo , Ovulação/fisiologia , Folículo Ovariano/metabolismo , Folículo Ovariano/fisiologia , Feminino , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Octopamina/metabolismo , Actinas/metabolismo , Drosophila melanogaster/fisiologia , Miosina Tipo II/metabolismo , Epitélio/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Oócitos/metabolismo , Drosophila/fisiologiaRESUMO
Cardiac hypertrophy results from the heart reacting and adapting to various pathological stimuli and its persistent development is a major contributing factor to heart failure. However, the molecular mechanisms of cardiac hypertrophy remain unclear. Small GTPases in the Ras, Rho, Rab, Arf and Ran subfamilies exhibit GTPase activity and play crucial roles in regulating various cellular responses. Previous studies have shown that Ras, Rho and Rab are closely linked to cardiac hypertrophy and that their overexpression can induce cardiac hypertrophy. Here, we review the functions of small GTPases in cardiac hypertrophy and provide additional insights and references for the prevention and treatment of cardiac hypertrophy.
Assuntos
Cardiomegalia , Proteínas Monoméricas de Ligação ao GTP , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Humanos , Animais , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Blood platelets are produced by megakaryocytes (MKs), their parent cells, which are in the bone marrow. Once mature, MK pierces through the sinusoid vessel, and the initial protrusion further elongates as proplatelet or buds to release platelets. The mechanisms controlling the decision to initiate proplatelet and platelet formation are unknown. Here, we show that the mechanical properties of the microenvironment prevent proplatelet and platelet release in the marrow stroma while allowing this process in the bloodstream. Loss of marrow confinement following myelosuppression led to inappropriate proplatelet and platelet release into the extravascular space. We further used an inert viscoelastic hydrogel to evaluate the impact of compressive stress. Transcriptional analysis showed that culture in three-dimensional gel induced upregulation of genes related to the Rho-GTPase pathway. We found higher Rho-GTPase activation, myosin light chain phosphorylation and F-actin under mechanical constraints while proplatelet formation was inhibited. The use of latrunculin-A to decrease F-actin promoted microtubule-dependent budding and proplatelet extension inside the gel. Additionally, ex vivo exposure of intact bone marrow to latrunculin-A triggered proplatelet extensions in the interstitial space. In vivo, this confinement-mediated high intracellular tension is responsible for the formation of the peripheral zone, a unique actin-rich structure. Cytoskeleton reorganization induces the disappearance of the peripheral zone upon reaching a liquid milieu to facilitate proplatelet and platelet formation. Hence, our data provide insight into the mechanisms preventing ectopic platelet release in the marrow stroma. Identifying such pathways is especially important for understanding pathologies altering marrow mechanics such as chemotherapy or myelofibrosis.
Assuntos
Plaquetas , Megacariócitos , Plaquetas/metabolismo , Plaquetas/efeitos dos fármacos , Megacariócitos/metabolismo , Megacariócitos/efeitos dos fármacos , Megacariócitos/citologia , Animais , Camundongos , Actinas/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Cadeias Leves de Miosina/metabolismo , Camundongos Endogâmicos C57BL , Compostos Bicíclicos Heterocíclicos com Pontes , TiazolidinasRESUMO
OBJECTIVES: To investigate the effects and molecular mechanisms of inhibition of the Ras homolog gene (Rho)/Rho-associated coiled-coil forming protein kinase (ROCK) pathway on the proliferation and migration of airway smooth muscle cells involving myocardin (MYOCD). METHODS: Human airway smooth muscle cells were infected with the adenoviral vector Ad-ZsGreen-shRNA-hROCK1 in vitro. The cells were randomly divided into four groups: ROCK1 gene silencing control (shNC) group, shNC + arachidonic acid (AA, Rho/ROCK pathway activator) group, ROCK1 gene silencing (shROCK1) group, and shROCK1 + AA group (n=3 each). Quantitative real-time polymerase chain reaction and Western blot were used to detect the expression levels of ROCK1 and MYOCD mRNA and protein. ELISA was employed to measure the levels of globular actin and filamentous actin, while immunofluorescent staining and scratch assays were utilized to assess cell proliferation and migration. RESULTS: Compared to the shNC + AA group, the shROCK1 + AA group exhibited decreased levels of ROCK1 and MYOCD mRNA and protein expression, reduced expression levels of globular actin and filamentous actin, and diminished cell proliferation and migration capabilities (P<0.05). CONCLUSIONS: Inhibition of the Rho/ROCK pathway suppresses the proliferation and migration of airway smooth muscle cells, which may be associated with the downregulation of MYOCD.
Assuntos
Movimento Celular , Proliferação de Células , Miócitos de Músculo Liso , Transdução de Sinais , Transativadores , Quinases Associadas a rho , Quinases Associadas a rho/metabolismo , Quinases Associadas a rho/fisiologia , Quinases Associadas a rho/genética , Humanos , Miócitos de Músculo Liso/fisiologia , Miócitos de Músculo Liso/metabolismo , Células Cultivadas , Transativadores/genética , Transativadores/fisiologia , Transativadores/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Proteínas Nucleares/metabolismo , Proteínas rho de Ligação ao GTP/fisiologia , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Cerebral ischemia reperfusion injury is a severe neurological impairment that occurs after blood flow reconstruction in stroke, and microglia cell pyroptosis is one of its important mechanisms. Electroacupuncture has been shown to be effective in mitigating and alleviating cerebral ischemia reperfusion injury by inhibiting neuroinflammation, reducing cellular pyroptosis, and improving neurological function. In this experiment, we divided the rats into three groups, including the sham operation (Sham) group, the middle cerebral artery occlusion/reperfusion (MCAO/R) group, and the pre-electroacupuncture (EAC) group. Pre-electroacupuncture group was stimulated with electroacupuncture of a certain intensity on the Baihui (GV 20) and Dazhui (GV 14) of the rat once a day from the 7th day to the 1st day before the MCAO/R operation. The extent of cerebral infarction was detected by TTC staining. A modified Zea-Longa five-point scale scoring system was used to determine neurologic function in MCAO rats. The number of neurons and morphological changes were accessed by Nissl staining and HE staining. The cellular damage was detected by TUNEL staining. In addition, the expression levels of RhoA, pyrin, GSDMD, Caspase1, cleaved-Caspase1, Iba-1, CD206, and ROCK2 were examined by western blotting and immunofluorescence. The results found that pre-electroacupuncture significantly attenuated neurological impairment and cerebral infarction compared to the post-MCAO/R rats. In addition, pre-electroacupuncture therapy promoted polarization of microglia to the neuroprotective (M2) phenotype. In addition, pre-electroacupuncture inhibited microglia pyroptosis by inhibiting RhoA/pyrin/GSDMD signaling pathway, thereby reducing neuronal injury and increasing neuronal survival in the MCAO/R rats. Taken together, these results demonstrated that pre-acupuncture could attenuate cerebral ischemia-reperfusion injury by inhibiting microglial pyroptosis. Therefore, pre-electroacupuncture might be a potential preventive strategy for ischemic stroke patients.
Assuntos
Eletroacupuntura , Microglia , Ratos Sprague-Dawley , Traumatismo por Reperfusão , Transdução de Sinais , Animais , Eletroacupuntura/métodos , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/terapia , Traumatismo por Reperfusão/prevenção & controle , Microglia/metabolismo , Masculino , Transdução de Sinais/fisiologia , Ratos , Proteínas de Ligação a Fosfato/metabolismo , Infarto da Artéria Cerebral Média/terapia , Infarto da Artéria Cerebral Média/metabolismo , Piroptose/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Isquemia Encefálica/metabolismo , Isquemia Encefálica/terapia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Gasderminas , Proteínas rho de Ligação ao GTPRESUMO
BACKGROUND: Metastatic prostate cancer is a leading cause of cancer-related morbidity and mortality in men, yet the underlying molecular mechanisms are poorly understood. Plexins are transmembrane receptors for semaphorins with divergent roles in many forms of cancer. We recently found that a single clinically relevant specific amino acid change (Proline1597Leucine, (P1597L)), found in metastatic deposits of prostate cancer patients, converts PlexinB1 from a metastasis suppressor to a gene that drives prostate cancer metastasis in vivo. However, the mechanism by which PlexinB1(P1597L) promotes metastasis is not known. METHODS: Pull down assays using GST-RalGDS or -GSTRaf1-RBD were used to reveal the effect of mutant or wild-type PlexinB1 expression on Rap and Ras activity respectively. Protein-protein interactions were assessed in GST pulldown assays, Akt/ERK phosphorylation by immunoblotting and protein stability by treatment with cycloheximide. Rho/ROCK activity was monitored by measuring MLC2 phosphorylation and actin stress fiber formation. PlexinB1 function was measured using cell-collapse assays. RESULTS: We show here that the single clinically relevant P1597L amino acid change converts PlexinB1 from a repressor of Ras to a Ras activator. The PlexinB1(P1597L) mutation inhibits the RapGAP activity of PlexinB1, promoting a significant increase in Ras activity. The P1597L mutation also blocks PlexinB1-mediated reduction in Rho/ROCK activity, restraining the decrease in MLC2 phosphorylation and actin stress fiber formation induced by overexpression of wild-type PlexinB1. PlexinB1(P1597L) has little effect on the interaction of PlexinB1 with small GTPases or receptor tyrosine kinases and does not inhibit PlexinB1-stimulated Akt or ERK phosphorylation. These results indicate that the mutation affects Rho signalling via the Rap/Ras pathway. The PlexinB1(P1597L) mutation inhibits morphological cell collapse induced by wild-type PlexinB1 expression, suggesting that the mutation induces a loss of an inhibitory tumour suppressor function. CONCLUSION: These results suggest that the clinically relevant P1597L mutation in PlexinB1 may transform PlexinB1 from a suppressor to a driver of metastasis in mouse models of prostate cancer by reducing the RapGAP activity of PlexinB1, leading to Ras activation. These findings highlight the PlexinB1-Rap-Ras pathway for therapeutic intervention in prostate cancer.
Assuntos
Proteínas do Tecido Nervoso , Neoplasias da Próstata , Receptores de Superfície Celular , Humanos , Masculino , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo , Linhagem Celular Tumoral , Mutação , Proteínas ras/genética , Proteínas ras/metabolismo , Metástase Neoplásica , Animais , Fosforilação , Transdução de Sinais , Camundongos , Semaforinas/metabolismo , Semaforinas/genética , Proteínas rho de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/genéticaRESUMO
Rho/Rac of plant (ROP) GTPases are plant-specific proteins that function as molecular switches, activated by guanine nucleotide exchange factors (GEFs) and inactivated by GTPase-activating proteins (GAPs). The bryophyte Marchantia polymorpha contains single copies of ROP (MpROP), GEFs [ROPGEF and SPIKE (SPK)] and GAPs [ROPGAP and ROP ENHANCER (REN)]. MpROP regulates the development of various tissues and organs, such as rhizoids, gemmae and air chambers. The ROPGEF KARAPPO (MpKAR) is essential for gemma initiation, but the functions of other ROP regulatory factors are less understood. This study focused on two GAPs: MpROPGAP and MpREN. Mpren single mutants showed defects in thallus growth, rhizoid tip growth, gemma development, and air-chamber formation, whereas Mpropgap mutants showed no visible abnormalities. However, Mpropgap Mpren double mutants had more severe phenotypes than the Mpren single mutants, suggesting backup roles of MpROPGAP in processes involving MpREN. Overexpression of MpROPGAP and MpREN resulted in similar gametophyte defects, highlighting the importance of MpROP activation/inactivation cycling (or balancing). Thus, MpREN predominantly, and MpROPGAP as a backup, regulate gametophyte development, likely by controlling MpROP activation in M. polymorpha.
Assuntos
Marchantia , Proteínas de Plantas , Marchantia/genética , Marchantia/metabolismo , Marchantia/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Regulação da Expressão Gênica de Plantas , Mutação/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Ativadoras de GTPase/genética , Organogênese Vegetal/genética , Proteínas rho de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/genéticaRESUMO
Prenatal exposure to dibutyl phthalate (DBP) has been reported to cause erectile dysfunction (ED) in adult offspring rats. However, its underlying mechanisms are not fully understood. Previously, we found that DBP activates the RhoA/ROCK pathway in the male reproductive system. This study investigated how prenatal exposure to DBP activates the RhoA/ROCK signalling pathway, leading to ED in male rat offspring. Pregnant rats were stratified into DBP-exposed and NC groups, with the exposed group receiving 750 milligrams per kilogram per day (mg/kg/day) of DBP through gavage from days 14-18 of gestation. DBP exposure activated the RhoA/ROCK pathway in the penile corpus cavernosum (CC) of descendants, causing smooth muscle cell contraction, fibrosis, and apoptosis, all of which contribute to ED. In vitro experiments confirmed that DBP induces apoptosis and RhoA/ROCK pathway activation in CC smooth muscle cells. Treatment of DBP-exposed offspring with the ROCK inhibitor Y-27632 for 8 weeks significantly improved smooth muscle cell condition, erectile function, and reduced fibrosis. Thus, prenatal DBP exposure induces ED in offspring through RhoA/ROCK pathway activation, and the ROCK inhibitor Y-27632 shows potential as an effective treatment for DBP-induced ED.
Assuntos
Apoptose , Dibutilftalato , Disfunção Erétil , Efeitos Tardios da Exposição Pré-Natal , Ratos Sprague-Dawley , Transdução de Sinais , Quinases Associadas a rho , Animais , Dibutilftalato/toxicidade , Masculino , Quinases Associadas a rho/metabolismo , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Gravidez , Feminino , Transdução de Sinais/efeitos dos fármacos , Disfunção Erétil/induzido quimicamente , Disfunção Erétil/metabolismo , Ratos , Apoptose/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/metabolismo , Pênis/efeitos dos fármacos , Pênis/metabolismo , Fibrose , Piridinas/farmacologia , Piridinas/toxicidade , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Amidas , Proteínas rho de Ligação ao GTPRESUMO
BACKGROUND: Gastric cancer (GC) is a common cancer worldwide; however, its molecular and pathogenic mechanisms remain unclear. MicroRNAs (miRNAs), which target key genes in GC, are associated with tumor promotion or suppression. Therefore, identifying new miRNA mechanisms could improve the novel diagnostic and therapeutic strategies for patients with GC. METHODS: To explore the biological functions of miR-135b-5p in GC, bioinformatic analysis and in vitro functional assays, including colony formation, wound healing, Transwell, and EdU assays, were used to assess the proliferative, invasive, and migratory capacities of GC cells. Target genes were predicted using RNA-seq and online databases. Dual-luciferase reporter assay, fluorescence in situ hybridization and western blotting were used to confirm the regulatory relationship between miR-135b-5p and CLIP4. The role of CLIP4 in tumor progression was assessed using clinical samples and both in vitro and in vivo assays. The tumor-suppressive mechanism of CLIP4 in GC was elucidated using rescue assays. RESULTS: Our study identified that miR-135b-5p as one of the top three over-expressed miRNAs in GC tissues, with RT-qPCR confirming its upregulation. Functional analysis showed that upregulated miR-135b-5p promoted malignant phenotypes in GC cells. Mechanistic research indicated that miR-135b-5p acts as a cancer promoter by targeting CLIP4. Moreover, our study suggested that CLIP4 exerts its tumor-suppressive function by inhibiting the JAK2/STAT3 signaling pathway. CONCLUSION: This study reveals a novel mechanism by which miR-135b-5p exerts its tumor-promoting functions by targeting CLIP4. The tumor-suppressive function of CLIP4 by inactivating the JAK2/STAT3 pathway is also elucidated. Regulatory mechanism of CLIP4 by miR-135b-5p provides a promising novel therapeutic strategy for GC patients.
Assuntos
Regulação Neoplásica da Expressão Gênica , Janus Quinase 2 , MicroRNAs , Fator de Transcrição STAT3 , Transdução de Sinais , Neoplasias Gástricas , Animais , Humanos , Masculino , Camundongos , Carcinogênese/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Janus Quinase 2/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/metabolismo , MicroRNAs/genética , Proteínas rho de Ligação ao GTP , Fator de Transcrição STAT3/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Neoplasias Gástricas/metabolismoRESUMO
ARHGAP25, a crucial molecule in immunological processes, serves as a Rac-specific GTPase-activating protein. Its role in cell migration and phagocyte functions, affecting the outcome of complex immunological diseases such as rheumatoid arthritis, renders it a promising target for drug research. Despite its importance, our knowledge of its intracellular interactions is still limited. This study employed proteomic analysis of glutathione S-transferase (GST)-tag pulldowns and co-immunoprecipitation from neutrophilic granulocyte cell lysate, revealing 76 candidates for potential physical interactions that complement ARHGAP25's known profile. Notably, four small GTPases (RAC2, RHOG, ARF4, and RAB27A) exhibited high affinity for ARHGAP25. The ARHGAP25-RAC2 and ARHGAP25-RHOG interactions appeared to be affected by the activation state of the small GTPases, suggesting a GTP-GDP cycle-dependent interaction. In silico dimer prediction pinpointed ARHGAP25's GAP domain as a credible binding interface, suggesting its suitability for GTP hydrolysis. Additionally, a list of Fc receptor-related kinases, phosphatases, and three of the 14-3-3 members were identified as potential partners, with in silico predictions highlighting eight binding sites, presenting novel insight on a potential regulatory mechanism for ARHGAP25.
Assuntos
Proteínas Ativadoras de GTPase , Neutrófilos , Ligação Proteica , Humanos , Proteínas Ativadoras de GTPase/metabolismo , Neutrófilos/metabolismo , Proteômica/métodos , Proteínas 14-3-3/metabolismo , Proteína RAC2 de Ligação ao GTP , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Lung regeneration after fibrosis requires formation of functional new vasculature, which is essential for gas exchange and cellular cross-talk with other lung cells. It remains unknown how the lung vasculature can be regenerated without fibrosis. Here, we tested the role of N6-methyladenosine (m6A) modification of forkhead box protein O1 (Foxo1) mRNA in lung regeneration after pneumonectomy (PNX) in mice, a model for lung regrowth after surgical resection. Endothelial cell (EC)-specific knockout of methyltransferase-like 3 (Mettl3) and Foxo1 caused nonproductive intussusceptive angiogenesis (IA), which impaired regeneration and enhanced fibrosis. This nonproductive IA was characterized by enhanced endothelial proliferation and increased vascular splitting with increased numbers of pillar ECs. Endothelial-selective knockout of Mettl3 in mice stimulated nonproductive IA and up-regulation of profibrotic factors after PNX, promoting regeneration to fibrotic transition. EC-specific mutation of m6A modification sites in the Foxo1 gene in mice revealed that endothelial Mettl3 modified A504 and A2035 sites in the Foxo1 mRNA to maintain pro-regenerative endothelial glycolysis, ensuring productive IA and lung regeneration without fibrosis. Suppression of Mettl3-Foxo1 signaling stimulated a subset of hyperglycolytic and hyperproliferative 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (Pfkfb3)+, Ras homolog family member J (Rhoj)+, and platelet-derived growth factor subunit B (Pdgfb)+ ECs in both human and mouse lungs with fibrosis. Inhibiting this Pfkfb3+Rhoj+Pdgfb+ EC subset normalized IA, alleviated fibrosis, and restored regeneration in bleomycin (BLM)-injured mouse lungs. We found that m6A modification of Foxo1 in the mouse vasculature promoted lung regeneration over fibrosis after PNX and BLM injury.
Assuntos
Proteína Forkhead Box O1 , Pulmão , Metiltransferases , Regeneração , Animais , Humanos , Camundongos , Adenosina/análogos & derivados , Adenosina/metabolismo , Proliferação de Células , Células Endoteliais/metabolismo , Fibrose , Proteína Forkhead Box O1/metabolismo , Pulmão/irrigação sanguínea , Pulmão/metabolismo , Pulmão/patologia , Pulmão/fisiologia , Metiltransferases/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Fisiológica , Pneumonectomia , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
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
Rho GTPases are a family of highly conserved G proteins that regulate numerous cellular processes, including cytoskeleton organisation, migration, and proliferation. The 20 canonical Rho GTPases are regulated by â¼85 guanine nucleotide exchange factors (GEFs), with the largest family being the 71 Diffuse B-cell Lymphoma (Dbl) GEFs. Dbl GEFs promote GTPase activity through the highly conserved Dbl homology domain. The specificity of GEF activity, and consequently GTPase activity, lies in the regulation and structures of the GEFs themselves. Dbl GEFs contain various accessory domains that regulate GEF activity by controlling subcellular localisation, protein interactions, and often autoinhibition. This review focuses on the two phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3)-dependent Rac exchangers (P-Rex), particularly the structural basis of P-Rex1 autoinhibition and synergistic activation. First, we discuss structures that highlight the conservation of P-Rex catalytic and phosphoinositide binding activities. We then explore recent breakthroughs in uncovering the structural basis for P-Rex1 autoinhibition and detail the proposed minimal two-step model of how PI(3,4,5)P3 and Gßγ synergistically activate P-Rex1 at the membrane. Additionally, we discuss the further layers of P-Rex regulation provided by phosphorylation and P-Rex2-PTEN coinhibitory complex formation, although these mechanisms remain incompletely understood. Finally, we leverage the available data to infer how cancer-associated mutations in P-Rex2 destabilise autoinhibition and evade PTEN coinhibitory complex formation, leading to increased P-Rex2 GEF activity and driving cancer progression and metastasis.
Assuntos
Fatores de Troca do Nucleotídeo Guanina , Humanos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Animais , Ligação Proteica , Fosfatos de Fosfatidilinositol/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/química , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
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
Fatty liver, which is induced by abnormal lipid metabolism, is one of the most common causes of chronic liver disease globally and causes liver fibrosis. During this process, bone marrow-derived mesenchymal stromal cells (BMSCs) and hepatic stellate cells (HSCs) migrate toward the injured liver and participate in fibrogenesis by transdifferentiating into myofibroblasts. S100A8/A9 is a powerful inducer of cell migration and is involved in liver injury. But there are few reports about the effects of S100A8/A9 on BMSC/HSC migration. In the current study, we found that S100A8/A9 expression was increased during fatty liver injury/fibrogenesis. Moreover, S100A8/A9 expression had a positive correlation with fibrosis marker gene expressions in the injured liver. S100A8/A9 was mainly produced by neutrophils in the fibrotic liver. In vitro, neutrophil-secreted S100A8/A9 promoted BMSC/HSC migration via remodeling of microfilaments. Using specific siRNA and inhibitor, we proved that S100A8/A9-induced BMSC/HSC migration is dependent on TLR4/Rho GTPases signaling. Moreover, S100A8/A9 knock-down alleviated liver injury and fibrogenesis in vivo, while injection of S100A9 neutralizing antibody performed similar roles. We proved that S100A8/A9 was involved in liver injury and fibrogenesis via inducing BMSC/HSC migration. Our research reveals a new mechanism underlying BMSC/HSC migration in liver fibrosis and suggests S100A8/A9 as a potential therapeutic target of liver fibrosis. KEY MESSAGES: S100A8/A9 is secreted by neutrophils and increased in fatty liver injury. Neutrophil-secreted S100A8/A9 is a mediator of BMSC/HSC migration in vitro. S100A8/A9-induced BMSC/HSC migration is dependent on TLR4/Rho GTPases signaling. S100A8/A9 blockade alleviates liver injury and fibrogenesis in vivo.
Assuntos
Calgranulina A , Calgranulina B , Movimento Celular , Cirrose Hepática , Miofibroblastos , Neutrófilos , Receptor 4 Toll-Like , Animais , Masculino , Camundongos , Calgranulina A/metabolismo , Calgranulina A/genética , Calgranulina B/metabolismo , Calgranulina B/genética , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Células Estreladas do Fígado/metabolismo , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos C57BL , Miofibroblastos/metabolismo , Neutrófilos/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , HumanosRESUMO
Members of the Rho family of small monomeric GTPases regulate a plethora of critical cellular functions including gene expression, cell cycle progression, and the dynamic modeling of the actin cytoskeleton. Diversity among Rho family members is derived, in part, from variations in their subcellular distribution. Localization of newly synthesized (naïve) Rho proteins to target subcellular compartments is largely governed by lipid modifications, including posttranslational prenylation. Here, using well-established and widely available contemporary methodologies, detailed protocols by which to semiquantitatively evaluate the functional consequence of posttranslational prenylation in human trabecular meshwork cells are described. We propose the novel concept that posttranslational prenylation itself is a key regulator of mammalian Rho GTPase protein expression and turnover.
Assuntos
Malha Trabecular , Humanos , Malha Trabecular/metabolismo , Malha Trabecular/citologia , Células Cultivadas , Terpenos/metabolismo , Prenilação de Proteína , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/genética , Processamento de Proteína Pós-TraducionalRESUMO
Ras homolog gene family member C (RhoC) is a GTPase involved in cell migration, implicated in epithelial-mesenchymal transition and treatment resistance and metastasis of cancer. For example, RhoC has been shown to be involved in resistance to radiation in cervical carcinoma. Here, the effect of X-ray irradiation on RhoC expression in prostate cancer (PCa) xenografts was investigated in both xenografts in regression and relapse. Male BALB/cAnNRj-Foxn1nu/nu mice were inoculated with 4-6 million LNCaP-FGC cells and established xenografts were irradiated with X-rays (200 kV, 1 Gymin-1), 5, 10 or 15 Gy using a Gulmay Medical X-ray system. Expression of RhoC and Ki67, a known proliferation marker, was investigated in xenografts, given 15 Gy, 7 days (midst response as measured by size) or 3 weeks (relapse) post irradiation. Staining was quantified using the Halo software (v2.3.2089.34) with the Indica Labs - cytonuclear v1.6 algorithm. RhoC and Ki67 staining was divided into weak, medium, and strong staining and the percentage of cells stained, single and dual staining, was quantified. The HALO software was further used to classify the tissue in each section so that analysis of RhoC and Ki67 expression in cancer cells, stroma and necrotic areas could be done separately. The results showed that RhoC expression in cancer and stroma cells was significantly higher in relapsed xenografts than in those in regression. This was not seen for Ki67 staining, where the percentage of stained cells were the same in regressing and relapsing tumors. RhoC could be a useful biomarker to confirm relapse following external beam radiation therapy.
Assuntos
Antígeno Ki-67 , Camundongos Endogâmicos BALB C , Camundongos Nus , Recidiva Local de Neoplasia , Neoplasias da Próstata , Proteína de Ligação a GTP rhoC , Masculino , Animais , Neoplasias da Próstata/radioterapia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Neoplasias da Próstata/genética , Antígeno Ki-67/metabolismo , Antígeno Ki-67/genética , Humanos , Proteína de Ligação a GTP rhoC/metabolismo , Proteína de Ligação a GTP rhoC/genética , Camundongos , Recidiva Local de Neoplasia/metabolismo , Linhagem Celular Tumoral , Proteínas rho de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/genéticaRESUMO
The transitioning of neural stem cells (NSCs) between quiescent and proliferative states is fundamental for brain development and homeostasis. Defects in NSC reactivation are associated with neurodevelopmental disorders. Drosophila quiescent NSCs extend an actin-rich primary protrusion toward the neuropil. However, the function of the actin cytoskeleton during NSC reactivation is unknown. Here, we reveal the fine filamentous actin (F-actin) structures in the protrusions of quiescent NSCs by expansion and super-resolution microscopy. We show that F-actin polymerization promotes the nuclear translocation of myocardin-related transcription factor, a microcephaly-associated transcription factor, for NSC reactivation and brain development. F-actin polymerization is regulated by a signaling cascade composed of G protein-coupled receptor Smog, G protein αq subunit, Rho1 guanosine triphosphatase, and Diaphanous (Dia)/Formin during NSC reactivation. Further, astrocytes secrete a Smog ligand folded gastrulation to regulate Gαq-Rho1-Dia-mediated NSC reactivation. Together, we establish that the Smog-Gαq-Rho1 signaling axis derived from astrocytes, an NSC niche, regulates Dia-mediated F-actin dynamics in NSC reactivation.
Assuntos
Actinas , Astrócitos , Proteínas de Drosophila , Células-Tronco Neurais , Receptores Acoplados a Proteínas G , Transdução de Sinais , Animais , Actinas/metabolismo , Astrócitos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/citologia , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Citoesqueleto de Actina/metabolismo , Drosophila melanogaster/metabolismo , Proteínas rho de Ligação ao GTP/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.