RESUMEN

OBJECTIVE: To investigate the correlation between serum Rac1 enzyme (Rac1) level with asthma control, airway inflammatory response and lung function in asthmatic children. METHODS: A retrospective analysis was performed on 79 children with asthma who were diagnosed and treated in our hospital from June 2020 to January 2023. According to the severity of the disease, the children were divided into mild group (25 cases), moderate group (30 cases) and severe group (24 cases). 36 healthy children who underwent physical examination at the same period in our hospital were selected as the control group. The state of an illness, control level, serum mRNA Rac1, inflammatory factors, and lung function of the children in two groups were compared between the control group and the observation group. RESULTS: The Rac1 mRNA levels, forced vital capacity (FVC), forced expiratory volume in one second/FVC (FEV1/FVC), peak expiratory flow (PEF), and maximum mid-expiratory flow (MMEF) in the observation group were significantly lower than these in the control group (P < 0.05). The tumor necrosis factor-alpha (TNF-α), interleukin-5 (IL-5), IL-6, and IL-33 in the observation group were markedly higher than these in the control group (P < 0.05). As the state of an illness worsened, the Rac1 mRNA levels, FVC, FEV1/FVC, PEF, and MMEF gradually reduced (P < 0.05), while the levels of TNF-α, IL-5, IL-6, and IL-33 increased (P < 0.05). As the degree of disease control improved, the Rac1 mRNA levels, FVC, FEV1/FVC, PEF, and MMEF gradually elevated (P < 0.05), and the levels of TNF- α, IL-5, IL-6, and IL-33 showed the opposite trend (P < 0.05). Rac1 was negatively related to the levels of TNF-α, IL-5, IL-6 and IL-33 (P < 0.05), and positively to the levels of FVC, FEV1/FVC, PEF and MMEF (P < 0.001). Rac1 mRNA levels, FVC, FEV1/FVC, PEF and MMEF were protective factors, while TNF-α, IL-5, IL-6 and IL-33 were risk factors for the prognosis of children with asthma (P < 0.05). CONCLUSION: Children with asthma have obviously lower serum Rac1 mRNA levels, higher inflammatory factor levels and lower lung function. Serum Rac1 mRNA level may be associated with better asthma control, lower airway inflammatory response, better lung function and lower disease severity. It has important reference value for the evaluation of the state of an illness, efficacy and prognosis of children with bronchial asthma.

Asunto(s)

Asma , Proteína de Unión al GTP rac1 , Humanos , Asma/fisiopatología , Asma/genética , Asma/sangre , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rac1/genética , Femenino , Masculino , Niño , Estudios Retrospectivos , Capacidad Vital , Volumen Espiratorio Forzado , Pulmón/fisiopatología , Pruebas de Función Respiratoria , Factor de Necrosis Tumoral alfa/sangre , Estudios de Casos y Controles , Interleucina-33/sangre , Interleucina-33/genética , Preescolar , Interleucina-6/sangre , Adolescente , Índice de Severidad de la Enfermedad , Interleucina-5/sangre , ARN Mensajero/metabolismo

RESUMEN

BACKGROUND: Hepatocellular carcinoma (LIHC) has severe consequences due to late diagnosis and the lack of effective therapies. Currently, potential biomarkers for the diagnosis and prognosis of LIHC have not been systematically evaluated. Previous studies have reported that RAC1 is associated with the B cell receptor signaling pathway in various tumor microenvironments, but its relationship with LIHC remains unclear. We investigated the relationship between RAC1 and the prognosis and immune infiltration microenvironment of LIHC, exploring its potential as a prognostic biomarker for this type of cancer. METHODS: In this study, we analyzed data from The Cancer Genome Atlas (TCGA) using the Wilcoxon signed-rank test and logistic regression to assess the association between RAC1 expression and clinical characteristics in LIHC patients. Additionally, Kaplan-Meier and Cox regression methods were employed to confirm the impact of RAC1 expression levels on overall survival. Immunohistochemistry was used to validate RAC1 protein expression in LIHC. We constructed RAC1 knockdown LIHC cells and studied the effects of RAC1 protein on cell proliferation and migration at both cellular and animal levels. RESULTS: RAC1 expression levels were significantly elevated in LIHC tissues compared to normal tissues. High RAC1 expression was strongly associated with advanced pathological stages and was identified as an independent factor negatively affecting overall survival. At both cellular and animal levels, RAC1 knockdown significantly inhibited the proliferation and migration of LIHC cells. Furthermore, RAC1 expression was positively correlated with the infiltration of Th2 cells and macrophages in the tumor microenvironment, suggesting that RAC1 may contribute to the deterioration of the tumor immunosuppressive microenvironment and potentially lead to reduced patient survival. CONCLUSION: These findings indicate that RAC1 expression promotes LIHC proliferation and migration and influences the landscape of immune cell infiltration in the tumor microenvironment. Based on these results, RAC1 is proposed as a potential prognostic biomarker for LIHC, associated with both cancer progression and tumor immune cell infiltration.

Asunto(s)

Biomarcadores de Tumor , Carcinoma Hepatocelular , Neoplasias Hepáticas , Microambiente Tumoral , Proteína de Unión al GTP rac1 , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rac1/genética , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Humanos , Pronóstico , Masculino , Femenino , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Microambiente Tumoral/inmunología , Animales , Persona de Mediana Edad , Ratones , Proliferación Celular , Movimiento Celular , Línea Celular Tumoral , Linfocitos Infiltrantes de Tumor/inmunología , Ratones Desnudos

RESUMEN

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder distinguished by gradual depletion of motor neurons. RNA binding motif protein 5 (RBM5), an abundantly expressed RNA-binding protein, plays a critical role in the process of cellular death. However, little is known about the role of RBM5 in the pathogenesis of ALS. Here, we found that RBM5 was upregulated in ALS hSOD1G93A-NSC34 cell models and hSOD1G93A mice due to a reduction of miR-141-5p. The upregulation of RBM5 increased the apoptosis of motor neurons by inhibiting Rac1-mediated neuroprotection. In contrast, genetic knockdown of RBM5 rescued motor neurons from hSOD1G93A-induced degeneration by activating Rac1 signaling. The neuroprotective effect of RBM5-knockdown was significantly inhibited by the Rac1 inhibitor, NSC23766. These findings suggest that RBM5 could potentially serve as a therapeutic target in ALS by activating the Rac1 signalling.

Asunto(s)

Esclerosis Amiotrófica Lateral , Apoptosis , Neuronas Motoras , Proteínas Proto-Oncogénicas c-akt , Proteínas de Unión al ARN , Transducción de Señal , Proteína de Unión al GTP rac1 , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rac1/genética , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Apoptosis/fisiología , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Ratones , Humanos , Transducción de Señal/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratones Transgénicos , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa/genética , Masculino , Proteínas de Unión al ADN , Proteínas de Ciclo Celular , Proteínas Supresoras de Tumor

RESUMEN

Aberrant alternative splicing events play a critical role in cancer biology, contributing to tumor invasion, metastasis, epithelial-mesenchymal transition, and drug resistance. Recent studies have shown that alternative splicing is a key feature for transcriptomic variations in colorectal cancer, which ranks third among malignant tumors worldwide in both incidence and mortality. Long non-coding RNAs can modulate this process by acting as trans-regulatory agents, recruiting splicing factors, or driving them to specific targeted genes. LncH19 is a lncRNA dis-regulated in several tumor types and, in colorectal cancer, it plays a critical role in tumor onset, progression, and metastasis. In this paper, we found, that in colorectal cancer cells, the long non-coding RNA H19 can bind immature RNAs and splicing factors as hnRNPM and RBFOX2. Through bioinformatic analysis, we identified 57 transcripts associated with lncH19 and containing binding sites for both splicing factors, hnRNPM, and RBFOX2. Among these transcripts, we identified the mRNA of the GTPase-RAC1, whose alternatively spliced isoform, RAC1B, has been ascribed several roles in the malignant transformation. We confirmed, in vitro, the binding of the splicing factors to both the transcripts RAC1 and lncH19. Loss and gain of expression experiments in two colorectal cancer cell lines (SW620 and HCT116) demonstrated that lncH19 is required for RAC1B expression and, through RAC1B, it induces c-Myc and Cyclin-D increase. In vivo, investigation from biopsies of colorectal cancer patients showed higher levels of all the explored genes (lncH19, RAC1B, c-Myc and Cyclin-D) concerning the healthy counterpart, thus supporting our in vitro model. In addition, we identified a positive correlation between lncH19 and RAC1B in colorectal cancer patients. Finally, we demonstrated that lncH19, as a shuttle, drives the splicing factors RBFOX2 and hnRNPM to RAC1 allowing exon retention and RAC1B expression. The data shown in this paper represent the first evidence of a new mechanism of action by which lncH19 carries out its functions as an oncogene by prompting colorectal cancer through the modulation of alternative splicing.

Asunto(s)

Empalme Alternativo , Neoplasias Colorrectales , Regulación Neoplásica de la Expresión Génica , Factores de Empalme de ARN , ARN Largo no Codificante , Proteína de Unión al GTP rac1 , Humanos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/metabolismo , ARN Largo no Codificante/genética , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , Línea Celular Tumoral , Ribonucleoproteína Heterogénea-Nuclear Grupo M/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo M/genética , Proteínas Represoras/genética , Proteínas Represoras/metabolismo

RESUMEN

BACKGROUND: Hepatocellular carcinoma (HCC) is a malignant tumor with significant variability in prognosis among patients. Ras-related C3 botulinum toxin substrate 1 (RAC1) is a key focus in the area of cancer research. However, the molecular mechanisms of RAC1 in HCC remain incompletely elucidated. MATERIALS AND METHODS: In this study, bioinformatics analysis was used, and public databases were used to obtain information about HCC cases. The samples were categorized into two groups of high and low expression based on the expression level of RAC1 gene. The limma package was used to calculate the differentially expressed genes between the two groups, and univariate Cox regression analysis was used to screen the prognostic related factors. Consensus clustering analysis was performed using the ConsensusClusterPlus package to identify molecular subtypes of HCC patients. Immune cell infiltration and ESTIMATE scores were assessed using the single sample gene set enrichment analysis and ESTIMATE algorithms. The sensitivity of different isoforms to chemotherapeutic agents was predicted by the oncoPredict package. Finally, we also performed cell function experiments to validate the biological role of RAC1 in vitro. Initially, we classified patients into high and low expression groups based on RAC1 gene expression levels and identified 195 up-regulated genes and 107 down-regulated genes. Through univariate Cox regression analysis, we screened out 169 prognosis-related factors. Furthermore, HCC patients were categorized into two subtypes. Subsequently, Kaplan-Meier survival curves showed that there was a significant difference in prognosis between the two molecular subtypes. Further analysis indicated substantial differences in gene expression levels and TIDE scores between two molecular subtypes. Moreover, these two subtypes exhibited varying sensitivity to chemotherapy drugs, as evidenced by differences in IC50 values. In addition, we found that the silence of RAC1 could effectively inhibit the migration and invasion of HCC cells in vitro. CONCLUSION: This study sheds light on the molecular intricacies of RAC1 in HCC and identifies patient populations that may benefit from immunotherapeutic interventions, with potential implications for tailored treatment strategies.

Asunto(s)

Biomarcadores de Tumor , Carcinoma Hepatocelular , Biología Computacional , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas , Proteína de Unión al GTP rac1 , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/mortalidad , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/mortalidad , Pronóstico , Biología Computacional/métodos , Biomarcadores de Tumor/genética , Perfilación de la Expresión Génica , Línea Celular Tumoral , Estimación de Kaplan-Meier

RESUMEN

Hypoxia and low glucose abundance often occur simultaneously at sites of inflammation. In monocytes and macrophages, glucose-oxygen deprivation stimulates the assembly of the NLRP3 inflammasome to generate the proinflammatory cytokine IL-1ß. We found that concomitant glucose deprivation and hypoxia activated the NLRP3 inflammasome by constraining the function of HMG-CoA reductase (HMGCR), the rate-limiting enzyme of the mevalonate kinase pathway. HMGCR is involved in the synthesis of geranylgeranyl pyrophosphate (GGPP), which is required for the prenylation and lipid membrane integration of proteins. Under glucose-oxygen deprivation, GGPP synthesis was decreased, leading to reduced prenylation of the small GTPase Rac1, increased binding of nonprenylated Rac1 to the scaffolding protein IQGAP1, and enhanced activation of the NLRP3 inflammasome. In response to restricted oxygen and glucose supply, patient monocytes with a compromised mevalonate pathway due to mevalonate kinase deficiency or Muckle-Wells syndrome released more IL-1ß than did control monocytes. Thus, reduced GGPP synthesis due to inhibition of HMGCR under glucose-oxygen deprivation results in proinflammatory innate responses, which are normally kept in check by the prenylation of Rac1. We suggest that this mechanism is also active in inflammatory autoimmune conditions.

Asunto(s)

Glucosa , Hidroximetilglutaril-CoA Reductasas , Inflamasomas , Monocitos , Proteína con Dominio Pirina 3 de la Familia NLR , Proteína de Unión al GTP rac1 , Humanos , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rac1/genética , Monocitos/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Hidroximetilglutaril-CoA Reductasas/metabolismo , Hidroximetilglutaril-CoA Reductasas/genética , Inflamasomas/metabolismo , Glucosa/metabolismo , Fosfatos de Poliisoprenilo/metabolismo , Interleucina-1beta/metabolismo , Oxígeno/metabolismo , Prenilación de Proteína , Deficiencia de Mevalonato Quinasa/metabolismo , Deficiencia de Mevalonato Quinasa/genética , Ácido Mevalónico/metabolismo

RESUMEN

One of the most extensively studied members of the Ras superfamily of small GTPases, Rac1 is an intracellular signal transducer that remodels actin and phosphorylation signaling networks. Previous studies have shown that Rac1-mediated signaling is associated with hippocampal-dependent working memory and longer-term forms of learning and memory and that Rac1 can modulate forms of both pre- and postsynaptic plasticity. How these different cognitive functions and forms of plasticity mediated by Rac1 are linked, however, is unclear. Here, we show that spatial working memory in mice is selectively impaired following the expression of a genetically encoded Rac1 inhibitor at presynaptic terminals, while longer-term cognitive processes are affected by Rac1 inhibition at postsynaptic sites. To investigate the regulatory mechanisms of this presynaptic process, we leveraged new advances in mass spectrometry to identify the proteomic and post-translational landscape of presynaptic Rac1 signaling. We identified serine/threonine kinases and phosphorylated cytoskeletal signaling and synaptic vesicle proteins enriched with active Rac1. The phosphorylated sites in these proteins are at positions likely to have regulatory effects on synaptic vesicles. Consistent with this, we also report changes in the distribution and morphology of synaptic vesicles and in postsynaptic ultrastructure following presynaptic Rac1 inhibition. Overall, this study reveals a previously unrecognized presynaptic role of Rac1 signaling in cognitive processes and provides insights into its potential regulatory mechanisms.

Asunto(s)

Hipocampo , Memoria a Corto Plazo , Proteína de Unión al GTP rac1 , Animales , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rac1/genética , Hipocampo/metabolismo , Hipocampo/fisiología , Ratones , Memoria a Corto Plazo/fisiología , Terminales Presinápticos/metabolismo , Terminales Presinápticos/fisiología , Transducción de Señal , Masculino , Fosforilación , Neuropéptidos/metabolismo , Neuropéptidos/genética , Ratones Endogámicos C57BL , Plasticidad Neuronal/fisiología

RESUMEN

Coordinated activation and inhibition of F-actin supports the movements of morphogenesis. Understanding the proteins that regulate F-actin is important, since these proteins are mis-regulated in diseases like cancer. Our studies of C. elegans embryonic epidermal morphogenesis identified the GTPase CED-10/Rac1 as an essential activator of F-actin. However, we need to identify the GEF, or Guanine-nucleotide Exchange Factor, that activates CED-10/Rac1 during embryonic cell migrations. The two-component GEF, CED-5/CED-12, is known to activate CED-10/Rac1 to promote cell movements that result in the engulfment of dying cells during embryogenesis, and a later cell migration of the larval Distal Tip Cell. It is believed that CED-5/CED-12 powers cellular movements of corpse engulfment and DTC migration by promoting F-actin formation. Therefore, we tested if CED-5/CED-12 was involved in embryonic migrations, and got a contradictory result. CED-5/CED-12 definitely support embryonic migrations, since their loss led to embryos that died due to failed epidermal cell migrations. However, CED-5/CED-12 inhibited F-actin in the migrating epidermis, the opposite of what was expected for a CED-10 GEF. To address how CED-12/CED-5 could have two opposing effects on F-actin, during corpse engulfment and cell migration, we investigated if CED-12 harbors GAP (GTPase Activating Protein) functions. A candidate GAP region in CED-12 faces away from the CED-5 GEF catalytic region. Mutating a candidate catalytic Arginine in the CED-12 GAP region (R537A) altered the epidermal cell migration function, and not the corpse engulfment function. We interfered with GEF function by interfering with CED-5's ability to bind Rac1/CED-10. Mutating Serine-Arginine in CED-5/DOCK predicted to bind and stabilize Rac1 for catalysis, resulted in loss of both ventral enclosure and corpse engulfment. Genetic and expression studies strongly support that the GAP function likely acts on different GTPases. Thus, we propose CED-5/CED-12 support the cycling of multiple GTPases, by using distinct domains, to both promote and inhibit F-actin nucleation.

Asunto(s)

Actinas , Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Movimiento Celular , Animales , Actinas/metabolismo , Secuencias de Aminoácidos , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Epidermis/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Morfogénesis/genética , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rac1/genética

RESUMEN

Activation of the maternal immune system leads to a downstream cascade of proinflammatory events that culminate in the activation of spontaneous uterine contractions, which is associated with preterm birth. Ras-related C3 botulinum toxin substrate 1 (Rac1) is a crucial protein related to cell contraction and inflammation. The main purpose of this study was to explore the role and function of Rac1's regulation of inflammation through in- vivo and in-vitro experiments. Rac1 inhibitor was used in animal model of preterm birth and cells isolated from the uterine tissues of pregnant mice on gestational day 16 were transfected with adenovirus to knockdown or overexpress Rac1 and treated with the Calcium-calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93. The expression of Rac1, uterine contraction-associated proteins (CAPs) (COX-2 and Connexin43), and inflammatory cytokines, were assessed by Western blotting and RTPCR. LPS upregulated Rac1, COX-2 and Connexin43 expression in uterine smooth muscle cells (USMCs). The expression of inflammatory cytokines, COX-2, and Connexin43 was significantly decreased in shRac1-transfected cells compared with cells stimulated with LPS only. Rac1 overexpression led to an increase in the expression of inflammatory cytokines, COX-2, and Connexin43. Furthermore, after Rac1 overexpression, KN93 reduced the expression of uterine contraction-associated proteins and inflammatory cytokines. It is thought that the effect of Rac1 on inflammatory cytokine and contraction-associated protein expression in USMCs is mediated by CaMKII. Rac1 can modulate the expression of contraction-associated proteins and inflammatory cytokines through the CaMKII pathway. Rac1 could be an effective therapeutic target for improving the outcome of preterm birth.

Asunto(s)

Lipopolisacáridos , Miocitos del Músculo Liso , Neuropéptidos , Útero , Proteína de Unión al GTP rac1 , Animales , Femenino , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rac1/genética , Lipopolisacáridos/farmacología , Ratones , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Útero/metabolismo , Neuropéptidos/metabolismo , Neuropéptidos/genética , Embarazo , Inflamación/metabolismo , Contracción Uterina/efectos de los fármacos , Conexina 43/metabolismo , Conexina 43/genética

RESUMEN

Kinases are known to have kinase activity independent functions. To gain further insights into potential kinase-independent functions of SLK/STK2, we have developed a kinase-dead allele, SLKK63R using in vivo CRISPR/Cas technology. Our studies show that blastocysts homozygote for SLKK63R do not develop into viable mice. However, heterozygotes are viable and fertile with no overt phenotypes. Analyses of mouse embryonic fibroblasts show that expression of SLKK63R results in a 50% decrease in kinase activity in heterozygotes. In contrast to previous studies, our data show that SLK does not form homodimers and that the kinase defective allele does not act in a dominant negative fashion. Expression of SLKK63R leads to altered Rac1 and RhoA activity, increased stress fiber formation and delayed focal adhesion turnover. Our data support a previously observed role for SLK in cell migration and suggest that at least 50% kinase activity is sufficient for embryonic development.

Asunto(s)

Movimiento Celular , Fibroblastos , Proteínas Serina-Treonina Quinasas , Proteína de Unión al GTP rac1 , Animales , Femenino , Ratones , Movimiento Celular/genética , Sistemas CRISPR-Cas , Pérdida del Embrión/genética , Pérdida del Embrión/patología , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/citología , Fibroblastos/metabolismo , Fibroblastos/citología , Adhesiones Focales/metabolismo , Adhesiones Focales/genética , Neuropéptidos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rhoA/metabolismo , Proteína de Unión al GTP rhoA/genética

RESUMEN

Periventricular nodular heterotopia (PNH), the most common brain malformation diagnosed in adulthood, is characterized by the presence of neuronal nodules along the ventricular walls. PNH is mainly associated with mutations in the FLNA gene - encoding an actin-binding protein - and patients often develop epilepsy. However, the molecular mechanisms underlying the neuronal failure still remain elusive. It has been hypothesized that dysfunctional cortical circuitry, rather than ectopic neurons, may explain the clinical manifestations. To address this issue, we depleted FLNA from cortical pyramidal neurons of a conditional Flnaflox/flox mice by timed in utero electroporation of Cre recombinase. We found that FLNA regulates dendritogenesis and spinogenesis thus promoting an appropriate excitatory/inhibitory inputs balance. We demonstrated that FLNA modulates RAC1 and cofilin activity through its interaction with the Rho-GTPase Activating Protein 24 (ARHGAP24). Collectively, we disclose an uncharacterized role of FLNA and provide strong support for neural circuit dysfunction being a consequence of FLNA mutations.

Asunto(s)

Corteza Cerebral , Filaminas , Proteína de Unión al GTP rac1 , Animales , Ratones , Factores Despolimerizantes de la Actina/metabolismo , Corteza Cerebral/metabolismo , Filaminas/metabolismo , Filaminas/genética , Proteínas Activadoras de GTPasa/metabolismo , Proteínas Activadoras de GTPasa/genética , Ratones Transgénicos , Neurogénesis/fisiología , Neuronas/metabolismo , Neuropéptidos/metabolismo , Neuropéptidos/genética , Heterotopia Nodular Periventricular/genética , Heterotopia Nodular Periventricular/metabolismo , Heterotopia Nodular Periventricular/patología , Células Piramidales/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rac1/genética

RESUMEN

The small G-protein Ras-related C3 botulinum toxin substrate 1 (Rac1) promotes the formation of filamentous actin (F-actin). Actin is a major component of dendritic spines, and we previously found that alcohol alters actin composition and dendritic spine structure in the nucleus accumbens (NAc) and the dorsomedial striatum (DMS). To examine if Rac1 contributes to these alcohol-mediated adaptations, we measured the level of GTP-bound active Rac1 in the striatum of mice following 7 weeks of intermittent access to 20% alcohol. We found that chronic alcohol intake activates Rac1 in the DMS of male mice. In contrast, Rac1 is not activated by alcohol in the NAc and DLS of male mice or in the DMS of female mice. Similarly, closely related small G-proteins are not activated by alcohol in the DMS, and Rac1 activity is not increased in the DMS by moderate alcohol or natural reward. To determine the consequences of alcohol-dependent Rac1 activation in the DMS of male mice, we inhibited endogenous Rac1 by infecting the DMS of mice with an adeno-associated virus (AAV) expressing a dominant negative form of the small G-protein (Rac1-DN). We found that overexpression of AAV-Rac1-DN in the DMS inhibits alcohol-mediated Rac1 signaling and attenuates alcohol-mediated F-actin polymerization, which corresponded with a decrease in dendritic arborization and spine maturation. Finally, we provide evidence to suggest that Rac1 in the DMS plays a role in alcohol-associated goal-directed learning. Together, our data suggest that Rac1 in the DMS plays an important role in alcohol-dependent structural plasticity and aberrant learning.

Asunto(s)

Cuerpo Estriado , Ratones Endogámicos C57BL , Plasticidad Neuronal , Proteína de Unión al GTP rac1 , Animales , Masculino , Ratones , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rac1/genética , Plasticidad Neuronal/fisiología , Plasticidad Neuronal/efectos de los fármacos , Femenino , Cuerpo Estriado/metabolismo , Cuerpo Estriado/efectos de los fármacos , Etanol/farmacología , Aprendizaje/fisiología , Aprendizaje/efectos de los fármacos , Neuropéptidos/metabolismo , Neuropéptidos/genética , Espinas Dendríticas/metabolismo , Espinas Dendríticas/efectos de los fármacos

RESUMEN

Rabies virus (RABV) is highly lethal and triggers severe neurological symptoms. The neuropathogenic mechanism remains poorly understood. Ras-related C3 botulinum toxin substrate 1 (Rac1) is a Rho-GTPase that is involved in actin remodeling and has been reported to be closely associated with neuronal dysfunction. In this study, by means of a combination of pharmacological inhibitors, small interfering RNA, and specific dominant-negatives, we characterize the crucial roles of dynamic actin and the regulatory function of Rac1 in RABV infection, dominantly in the viral entry phase. The data show that the RABV phosphoprotein interacts with Rac1. RABV phosphoprotein suppress Rac1 activity and impedes downstream Pak1-Limk1-Cofilin1 signaling, leading to the disruption of F-actin-based structure formation. In early viral infection, the EGFR-Rac1-signaling pathway undergoes a biphasic change, which is first upregulated and subsequently downregulated, corresponding to the RABV entry-induced remodeling pattern of F-actin. Taken together, our findings demonstrate for the first time the role played by the Rac1 signaling pathway in RABV infection and may provide a clue for an explanation for the etiology of rabies neurological pathogenesis.IMPORTANCEThough neuronal dysfunction is predominant in fatal rabies, the detailed mechanism by which rabies virus (RABV) infection causes neurological symptoms remains in question. The actin cytoskeleton is involved in numerous viruses infection and plays a crucial role in maintaining neurological function. The cytoskeletal disruption is closely associated with abnormal nervous symptoms and induces neurogenic diseases. In this study, we show that RABV infection led to the rearrangement of the cytoskeleton as well as the biphasic kinetics of the Rac1 signal transduction. These results help elucidate the mechanism that causes the aberrant neuronal processes by RABV infection and may shed light on therapeutic development aimed at ameliorating neurological disorders.

Asunto(s)

Citoesqueleto de Actina , Actinas , Virus de la Rabia , Transducción de Señal , Proteína de Unión al GTP rac1 , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rac1/genética , Humanos , Citoesqueleto de Actina/metabolismo , Animales , Virus de la Rabia/fisiología , Actinas/metabolismo , Receptores ErbB/metabolismo , Receptores ErbB/genética , Quinasas p21 Activadas/metabolismo , Quinasas p21 Activadas/genética , Quinasas Lim/metabolismo , Quinasas Lim/genética , Internalización del Virus , Rabia/metabolismo , Rabia/virología , Línea Celular , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Factores Despolimerizantes de la Actina/metabolismo

RESUMEN

The activation of the angiopoietin (Angpt)-Tie system is linked to endothelial dysfunction during sepsis. Bacterial quorum-sensing molecules function as pathogen-associated molecular patterns. However, their impact on the endothelium and the Angpt-Tie system remains unclear. Therefore, this study investigated whether treatment with N-3-oxododecanoyl homoserine lactone (3OC12-HSL), a quorum-sensing molecule derived from Pseudomonas aeruginosa, impaired endothelial function in human umbilical vein endothelial cells. 3OC12-HSL treatment impaired tube formation even at sublethal concentrations, and immunocytochemistry analysis revealed that it seemed to reduce vascular endothelial-cadherin expression at the cell-cell interface. Upon assessing the mRNA expression patterns of genes associated with the Angpt-Tie axis, the expressions of Angpt2, Forkhead box protein O1, Tie1, and vascular endothelial growth factor 2 were found to be upregulated in the 3OC12-HSL-treated cells. Moreover, western blot analysis revealed that 3OC12-HSL treatment increased Angpt2 expression. A co-immunoprecipitation assay was conducted to assess the effect of 3OC12-HSL on the IQ motif containing GTPase activating protein 1 (IQGAP1) and Rac1 complex and the interaction between these proteins was consistently maintained regardless of 3OC12-HSL treatment. Next, recombinant human (rh)-Angpt1 was added to assess whether it modulated the effects of 3OC12-HSL treatment. rh-Angpt1 addition increased cellular viability, improved endothelial function, and reversed the overall patterns of mRNA and protein expression in endothelial cells treated with 3OC12-HSL. Additionally, it was related to the increased expression of phospho-Akt and the IQGAP1 and Rac1 complex. Collectively, our findings indicated that 3OC12-HSL from Pseudomonas aeruginosa can impair endothelial integrity via the activation of the Angpt-Tie axis, which appeared to be reversed by rh-Angpt1 treatment.

Asunto(s)

4-Butirolactona , Angiopoyetina 2 , Homoserina , Células Endoteliales de la Vena Umbilical Humana , Pseudomonas aeruginosa , Humanos , Pseudomonas aeruginosa/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Angiopoyetina 2/metabolismo , Angiopoyetina 2/genética , 4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacología , Homoserina/análogos & derivados , Homoserina/farmacología , Homoserina/metabolismo , Cadherinas/metabolismo , Cadherinas/genética , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rac1/genética , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O1/genética , Proteínas Activadoras de ras GTPasa/metabolismo , Proteínas Activadoras de ras GTPasa/genética , Transducción de Señal/efectos de los fármacos , Antígenos CD/metabolismo , Antígenos CD/genética , Angiopoyetina 1/metabolismo , Angiopoyetina 1/genética

RESUMEN

Endothelial cells (ECs) have essential roles in cardiac tissue repair after myocardial infarction (MI). To establish stage-specific and long-term effects of the ischemic injury on cardiac ECs, we analyzed their transcriptome at landmark time points after MI in mice. We found that early EC response at Day 2 post-MI centered on metabolic changes, acquisition of proinflammatory phenotypes, initiation of the S phase of cell cycle, and activation of stress-response pathways, followed by progression to mitosis (M/G2 phase) and acquisition of proangiogenic and mesenchymal properties during scar formation at Day 7. In contrast, genes involved in vascular physiology and maintenance of vascular tone were suppressed. Importantly, ECs did not return to pre-injury phenotypes after repair has been completed but maintained inflammatory, fibrotic and thrombotic characteristics and lost circadian rhythmicity. We discovered that the highest induced transcript is the mammalian-specific Sh2d5 gene that promoted migration and invasion of ECs through Rac1 GTPase. Our results revealed a synchronized, temporal activation of disease phenotypes, metabolic pathways, and proliferation in quiescent ECs after MI, indicating that precisely-timed interventions are necessary to optimize cardiac tissue repair and improve outcomes. Furthermore, long-term effects of acute ischemic injury on ECs may contribute to vascular dysfunction and development of heart failure.

Asunto(s)

Células Endoteliales , Perfilación de la Expresión Génica , Infarto del Miocardio , Animales , Infarto del Miocardio/genética , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Ratones , Células Endoteliales/metabolismo , Células Endoteliales/patología , Transcriptoma , Masculino , Ratones Endogámicos C57BL , Miocardio/metabolismo , Miocardio/patología , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rac1/genética , Modelos Animales de Enfermedad , Proliferación Celular , Movimiento Celular/genética

RESUMEN

Cell stiffness is regulated by dynamic interaction between ras-related C3 botulinum toxin substrate 1 (Rac1) and p21 protein-activated kinase 1 (PAK1) proteins, besides other biochemical and molecular regulators. In this study, we investigated how the Placental Growth Factor (PlGF) changes endometrial mechanics by modifying the actin cytoskeleton at the maternal interface. We explored the global effects of PlGF in endometrial stromal cells (EnSCs) using the concerted approach of proteomics, atomic force microscopy (AFM), and electrical impedance spectroscopy (EIS). Proteomic analysis shows PlGF upregulated RhoGTPases activating proteins and extracellular matrix organization-associated proteins in EnSCs. Rac1 and PAK1 transcript levels, activity, and actin polymerization were significantly increased with PlGF treatment. AFM further revealed an increase in cell stiffness with PlGF treatment. The additive effect of PlGF on actin polymerization was suppressed with siRNA-mediated inhibition of Rac1, PAK1, and WAVE2. Interestingly, the increase in cell stiffness by PlGF treatment was pharmacologically reversed with pravastatin, resulting in improved trophoblast cell invasion. Taken together, aberrant PlGF levels in the endometrium can contribute to an altered pre-pregnancy maternal microenvironment and offer a unifying explanation for the pathological changes observed in conditions such as pre-eclampsia (PE).

Asunto(s)

Endometrio , Factor de Crecimiento Placentario , Preeclampsia , Transducción de Señal , Proteína de Unión al GTP rac1 , Femenino , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rac1/genética , Humanos , Preeclampsia/metabolismo , Embarazo , Endometrio/metabolismo , Endometrio/patología , Factor de Crecimiento Placentario/metabolismo , Factor de Crecimiento Placentario/genética , Células del Estroma/metabolismo , Quinasas p21 Activadas/metabolismo , Quinasas p21 Activadas/genética , Microscopía de Fuerza Atómica

RESUMEN

The role of endothelial cells in promoting cancer cell extravasation to the brain during the interaction of cancer cells with the vasculature is not well characterised. We show that brain endothelial cells activate EGFR signalling in triple-negative breast cancer cells with propensity to metastasise to the brain. This activation is dependent on soluble factors secreted by brain endothelial cells, and occurs via the RAC1 GEF DOCK4, which is required for breast cancer cell extravasation to the brain in vivo. Knockdown of DOCK4 inhibits breast cancer cell entrance to the brain without affecting cancer cell survival or growth. Defective extravasation is associated with loss of elongated morphology preceding intercalation into brain endothelium. We also show that brain endothelial cells promote paracrine stimulation of mesenchymal-like morphology of breast cancer cells via DOCK4, DOCK9, RAC1 and CDC42. This stimulation is accompanied by EGFR activation necessary for brain metastatic breast cancer cell elongation which can be reversed by the EGFR inhibitor Afatinib. Our findings suggest that brain endothelial cells promote metastasis through activation of cell signalling that renders breast cancer cells competent for extravasation. This represents a paradigm of brain endothelial cells influencing the signalling and metastatic competency of breast cancer cells.

Asunto(s)

Neoplasias Encefálicas , Encéfalo , Células Endoteliales , Receptores ErbB , Transducción de Señal , Proteína de Unión al GTP rac1 , Receptores ErbB/metabolismo , Receptores ErbB/genética , Humanos , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rac1/genética , Femenino , Células Endoteliales/metabolismo , Células Endoteliales/patología , Línea Celular Tumoral , Animales , Encéfalo/metabolismo , Encéfalo/patología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/genética , Ratones , Proteínas Activadoras de GTPasa/metabolismo , Proteínas Activadoras de GTPasa/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/genética

RESUMEN

Disulfidptosis, a newly recognized cell death triggered by disulfide stress, has garnered attention for its potential role in osteoporosis (OP) pathogenesis. Although sulfide-related proteins are reported to regulate the balance of bone metabolism in OP, the precise involvement of disulfidptosis regulators remains elusive. Herein, leveraging the GSE56815 dataset, we conducted an analysis to delineate disulfidptosis-associated diagnostic clusters and immune landscapes in OP. Subsequently, vertebral bone tissues obtained from OP patients and controls were subjected to RNA sequencing (RNA-seq) for the validation of key disulfidptosis gene expression. Our analysis unveiled seven significant disulfidptosis regulators, including FLNA, ACTB, PRDX1, SLC7A11, NUBPL, OXSM, and RAC1, distinguishing OP samples from controls. Furthermore, employing a random forest model, we identified four diagnostic disulfidptosis regulators including FLNA, SLC7A11, NUBPL, and RAC1 potentially predictive of OP risk. A nomogram model integrating these four regulators was constructed and validated using the GSE35956 dataset, demonstrating promising utility in clinical decision-making, as affirmed by decision curve analysis. Subsequent consensus clustering analysis stratified OP samples into two different disulfidptosis subgroups (clusters A and B) using significant disulfidptosis regulators, with cluster B exhibiting higher disulfidptosis scores and implicating monocyte immunity, closely linked to osteoclastogenesis. Notably, RNA-seq analysis corroborated the expression patterns of two disulfidptosis modulators, PRDX1 and OXSM, consistent with bioinformatics predictions. Collectively, our study sheds light on disulfidptosis patterns, offering potential markers and immunotherapeutic avenues for future OP management.

Asunto(s)

Osteoporosis , Análisis de Secuencia de ARN , Proteína de Unión al GTP rac1 , Humanos , Osteoporosis/genética , Osteoporosis/inmunología , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo , Filaminas/genética , Femenino , Sistema de Transporte de Aminoácidos y+/genética , Sistema de Transporte de Aminoácidos y+/metabolismo , Nomogramas , Masculino , Peroxirredoxinas

RESUMEN

The spatial sorting of cells into appropriate tissue compartments is essential for embryogenesis and tissue development. Spatial cell sorting is controlled by the interplay between cell surface affinity and intracellular mechanical properties. However, intracellular signaling that can sufficiently sort cell populations remains unexplored. In this study, we engineered chimeric cadherins by replacing the cadherin intracellular domain with cytoskeletal regulators to test their ability to induce spatial cell sorting. Using a fibroblast-based reconstitution system, we observed that Rac1 and RhoA activity in the cadherin tail induced outward and inward sorting, respectively. In particular, RhoA activity embedded cells toward the inside of E-cadherin-expressing spheroids and tumor spheroids, leading to tissue invagination. Despite the simplicity of chimeric cadherin design, our results indicate that differences in cadherin intracellular activities can determine the direction of spatial cell sorting, even when cell surface affinity is not different, and provide new molecular tools to engineer tissue architectures.

Asunto(s)

Cadherinas , Proteína de Unión al GTP rac1 , Proteína de Unión al GTP rhoA , Cadherinas/metabolismo , Cadherinas/genética , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rac1/genética , Humanos , Proteína de Unión al GTP rhoA/metabolismo , Proteína de Unión al GTP rhoA/genética , Animales , Ratones , Fibroblastos/metabolismo , Fibroblastos/citología , Esferoides Celulares/metabolismo

RESUMEN

BACKGROUND: Recent studies have emphasized the critical role of Telocytes (TCs)-derived exosomes in organ tissue injury and repair. Our previous research showed a significant increase in ITGB1 within TCs. Pulmonary Arterial Hypertension (PAH) is marked by a loss of microvessel regeneration and progressive vascular remodeling. This study aims to investigate whether exosomes derived from ITGB1-modified TCs (ITGB1-Exo) could mitigate PAH. METHODS: We analyzed differentially expressed microRNAs (DEmiRs) in TCs using Affymetrix Genechip miRNA 4.0 arrays. Exosomes isolated from TC culture supernatants were verified through transmission electron microscopy and Nanoparticle Tracking Analysis. The impact of miR-429-3p-enriched exosomes (Exo-ITGB1) on hypoxia-induced pulmonary arterial smooth muscle cells (PASMCs) was evaluated using CCK-8, transwell assay, and inflammatory factor analysis. A four-week hypoxia-induced mouse model of PAH was constructed, and H&E staining, along with Immunofluorescence staining, were employed to assess PAH progression. RESULTS: Forty-five miRNAs exhibited significant differential expression in TCs following ITGB1 knockdown. Mus-miR-429-3p, significantly upregulated in ITGB1-overexpressing TCs and in ITGB1-modified TC-derived exosomes, was selected for further investigation. Exo-ITGB1 notably inhibited the migration, proliferation, and inflammation of PASMCs by targeting Rac1. Overexpressing Rac1 partly counteracted Exo-ITGB1's effects. In vivo administration of Exo-ITGB1 effectively reduced pulmonary vascular remodeling and inflammation. CONCLUSIONS: Our findings reveal that ITGB1-modified TC-derived exosomes exert anti-inflammatory effects and reverse vascular remodeling through the miR-429-3p/Rac1 axis. This provides potential therapeutic strategies for PAH treatment.

Asunto(s)

Exosomas , Integrina beta1 , MicroARNs , Telocitos , Proteína de Unión al GTP rac1 , MicroARNs/genética , MicroARNs/metabolismo , Animales , Exosomas/metabolismo , Exosomas/genética , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rac1/genética , Integrina beta1/metabolismo , Integrina beta1/genética , Ratones , Telocitos/metabolismo , Masculino , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Ratones Endogámicos C57BL , Hipertensión Arterial Pulmonar/metabolismo , Hipertensión Arterial Pulmonar/genética , Hipertensión Arterial Pulmonar/patología , Hipoxia/metabolismo , Hipoxia/genética , Hipoxia/complicaciones , Proliferación Celular/genética , Movimiento Celular/genética , Humanos , Remodelación Vascular/genética , Neuropéptidos