RESUMEN

Arpin was discovered as an inhibitor of the Arp2/3 complex localized at the lamellipodial tip of fibroblasts, where it regulated migration steering. Recently, we showed that arpin stabilizes the epithelial barrier in an Arp2/3-dependent manner. However, the expression and functions of arpin in endothelial cells (EC) have not yet been described. Arpin mRNA and protein are expressed in EC and downregulated by pro-inflammatory cytokines. Arpin depletion in Human Umbilical Vein Endothelial Cells causes the formation of actomyosin stress fibers leading to increased permeability in an Arp2/3-independent manner. Instead, inhibitors of ROCK1 and ZIPK, kinases involved in the generation of stress fibers, normalize the loss-of-arpin effects on actin filaments and permeability. Arpin-deficient mice are viable but show a characteristic vascular phenotype in the lung including edema, microhemorrhage, and vascular congestion, increased F-actin levels, and vascular permeability. Our data show that, apart from being an Arp2/3 inhibitor, arpin is also a regulator of actomyosin contractility and endothelial barrier integrity.

Asunto(s)

Actomiosina , Permeabilidad Capilar , Células Endoteliales de la Vena Umbilical Humana , Animales , Humanos , Actomiosina/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Ratones , Serpinas/metabolismo , Serpinas/genética , Ratones Noqueados , Quinasas Asociadas a rho/metabolismo , Quinasas Asociadas a rho/genética , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/genética , Fibras de Estrés/metabolismo , Células Endoteliales/metabolismo , Proteínas Portadoras

RESUMEN

Cellular Communication Network Factor 2, CCN2, is a profibrotic cytokine implicated in physiological and pathological processes in mammals. The expression of CCN2 is markedly increased in dystrophic muscles. Interestingly, diminishing CCN2 genetically or inhibiting its function improves the phenotypes of chronic muscular fibrosis in rodent models. Elucidating the cell-specific mechanisms behind the induction of CCN2 is a fundamental step in understanding its relevance in muscular dystrophies. Here, we show that the small lipids LPA and 2S-OMPT induce CCN2 expression in fibro/adipogenic progenitors (FAPs) through the activation of the LPA1 receptor and, to a lower extent, by also the LPA6 receptor. These cells show a stronger induction than myoblasts or myotubes. We show that the LPA/LPARs axis requires ROCK kinase activity and organized actin cytoskeleton upstream of YAP/TAZ signaling effectors to upregulate CCN2 levels, suggesting that mechanical signals are part of the mechanism behind this process. In conclusion, we explored the role of the LPA/LPAR axis on CCN2 expression, showing a strong cytoskeletal-dependent response in muscular FAPs.

Asunto(s)

Adipogénesis , Factor de Crecimiento del Tejido Conjuntivo , Lisofosfolípidos , Animales , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/genética , Ratones , Lisofosfolípidos/metabolismo , Comunicación Celular , Transducción de Señal , Receptores del Ácido Lisofosfatídico/metabolismo , Receptores del Ácido Lisofosfatídico/genética , Células Madre/metabolismo , Células Madre/citología , Regulación de la Expresión Génica , Quinasas Asociadas a rho/metabolismo , Quinasas Asociadas a rho/genética , Diferenciación Celular , Músculo Esquelético/metabolismo , Músculo Esquelético/citología , Humanos , Citoesqueleto de Actina/metabolismo

RESUMEN

The small GTPase RhoA and the downstream Rho kinase (ROCK) regulate several cell functions and pathological processes in the vascular system that contribute to the age-dependent risk of cardiovascular disease, including endothelial dysfunction, excessive permeability, inflammation, impaired angiogenesis, abnormal vasoconstriction, decreased nitric oxide production and apoptosis. Frailty is a loss of physiological reserve and adaptive capacity with advanced age and is accompanied by a pro-inflammatory and pro-oxidative state that promotes vascular dysfunction and thrombosis. This review summarises the role of the RhoA/Rho kinase signalling pathway in endothelial dysfunction, the acquisition of the pro-thrombotic state and vascular ageing. We also discuss the possible role of RhoA/Rho kinase signalling as a promising therapeutic target for the prevention and treatment of age-related cardiovascular disease.

Asunto(s)

Enfermedades Cardiovasculares , Trombosis , Enfermedades Vasculares , Humanos , Quinasas Asociadas a rho/genética , Células Endoteliales

RESUMEN

Resistance to radio and chemotherapy in Glioblastoma (GBM) is correlated with its malignancy, invasiveness, and aggressiveness. The Rho GTPase pathway plays important roles in these processes, but its involvement in the GBM response to genotoxic treatments remains unsolved. Inhibition of this signaling pathway has emerged as a promising approach for the treatment of CNS injuries and diseases, proving to be a strong candidate for therapeutic approaches. To this end, Rho-associated kinases (ROCK), classic downstream effectors of small Rho GTPases, were targeted for pharmacological inhibition using Y-27632 in GBM cells, expressing the wild-type or mutated p53 gene, and exposed to genotoxic stress by gamma ionizing radiation (IR) or cisplatin (PT). The use of the ROCK inhibitor (ROCKi) had opposite effects in these cells: in cells expressing wild-type p53, ROCKi reduced survival and DNA repair capacity (reduction of γH2AX foci and accumulation of strand breaks) after stress promoted by IR or PT; in cells expressing the mutant p53 protein, both treatments promoted longer survival and more efficient DNA repair, responses further enhanced by ROCKi. The target DNA repair mechanisms of ROCK inhibition were, respectively, an attenuation of NHEJ and NER pathways in wild-type p53 cells, and a stimulation of HR and NER pathways in mutant p53 cells. These effects were accompanied by the formation of reactive oxygen species (ROS) induced by genotoxic stress only in mutant p53 cells but potentiated by ROCKi and reversed by p53 knockdown. N-acetyl-L-cysteine (NAC) treatment or Rac1 knockdown completely eliminated ROCKi's p53-dependent actions, since ROCK inhibition specifically elevated Rac-GTP levels only in mutant p53 cells. Combining IR or PT and ROCKi treatments broadens our understanding of the sensitivity and resistance of, respectively, GBM expressing wild-type or mutant p53 to genotoxic agents. Our proposal may be a determining factor in improving the efficiency and assertiveness of CNS antitumor therapies based on ROCK inhibitors. SIGNIFICANCE: The use of ROCK inhibitors in association with radio or chemotherapy modulates GBM resistance and sensitivity depending on the p53 activity, suggesting the potential value of this protein as therapeutic target for tumor pre-sensitization strategies.

Asunto(s)

Glioblastoma , Humanos , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Quinasas Asociadas a rho/genética , Quinasas Asociadas a rho/metabolismo , Daño del ADN , Línea Celular Tumoral

RESUMEN

OBJECTIVE: Acute appendicitis represents one of the most common causes of acute intra-abdominal emergencies worldwide. In this case-control study, we aimed to investigate associations of Rho-kinase gene expression and polymorphisms with acute appendicitis in a Turkish population. We also aimed to study the effects of gender on these parameters. METHODS: A total of 93 unrelated patients with acute appendicitis and 93 healthy controls in the Department of Emergency Medicine, Erciyes University, between June 2019 and June 2021 were included in this study. Genomic DNA was isolated from peripheral leukocytes, and the LightCycler 480 II real-time polymerase chain reaction was utilized to detect Rho-kinase1 gene rs35996865 and Rho-kinase2 gene rs2230774 (Thr431Asn) polymorphisms. Quantitative real-time polymerase chain reaction was applied to determine Rho-kinase1 and Rho-kinase2 gene expressions. RESULTS: There was a marked increase in Rho-kinase1, but not in Rho-kinase2, mRNA expression, and this increase was evident only in male patients (p=0.0008). No significant differences were found in allele and genotype frequencies for Rho-kinase1 gene rs35996865 and Rho-kinase2 gene rs2230774 polymorphisms between the patients with acute appendicitis and the control group. CONCLUSIONS: Our data imply that Rho-kinase1 (rs35996865) and Rho-kinase2 (rs2230774) gene variants are not risk factors for the development of acute appendicitis in the Turkish population. However, increased mRNA expression of the Rho-kinase1 gene in males indicated that Rho-kinase1 is involved in the pathogenesis of acute appendicitis in a gender-specific way.

Asunto(s)

Apendicitis , Quinasas Asociadas a rho , Adulto , Humanos , Masculino , Quinasas Asociadas a rho/genética , Apendicitis/genética , Estudios de Casos y Controles , Predisposición Genética a la Enfermedad , Enfermedad Aguda , Expresión Génica , ARN Mensajero

RESUMEN

OBJECTIVE: Sepsis is a complex and serious medical condition resulting from the activation of an innate host response to infections. The etiology of sepsis is complex and can be influenced by genetic susceptibility. The purpose of the present study was to investigate a possible association of Rho-kinase 1 (ROCK1) gene polymorphism with sepsis in a Turkish population. METHODS: The study group consisted of 100 unrelated patients with sepsis and 100 healthy controls. Genomic DNA was isolated from peripheral leukocytes from EDTA-containing blood using the QIAamp DNA Blood Mini Kit. ROCK1 gene rs35996865 and rs112130712 (Lys1054Arg) polymorphisms were analyzed in genomic DNA using the LightCycler 480 II real-time polymerase chain reaction. RESULTS: There were no significant differences in allele and genotype frequencies for ROCK1 gene rs35996865 polymorphism between the patients with sepsis and control group (p>0.05). Additionally, no association was detected between the rs35996865 polymorphism and mortality in the patient group. No polymorphism was detected with ROCK1 gene rs112130712 (Lys1054Arg) in our study groups. CONCLUSIONS: Our data showed that there is no marked association between the rs35996865 polymorphism and sepsis. Therefore, these results suggest that ROCK1 gene rs35996865 polymorphism is not risk factor for the development of sepsis in the Turkish population.

Asunto(s)

Sepsis , Quinasas Asociadas a rho , Estudios de Casos y Controles , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Polimorfismo de Nucleótido Simple , Sepsis/enzimología , Sepsis/genética , Quinasas Asociadas a rho/genética

RESUMEN

Colorectal cancer (CRC) has a high incidence and mortality worldwide. Microsatellite instability (MSI) is crucial in CRC, with distinct molecular and clinicopathological features in patients. Nowadays, it is a predictive marker for immunotherapy. We proposed to evaluate the 5-year outcome of MSI status in 1002 Brazilian CRC, and associate it with genetic ancestry, molecular and clinicopathological features. MSI evaluation was performed using molecular markers. MSI+ tumors were analyzed for alterations in 23 MSI-targeted genes. Genetic ancestry was evaluated using an Ancestry-Informative markers panel. MSI status was analyzed in relation to CRC specific survival and other clinical and genetic variables. MSI+ status was observed in 10.5% of cases. MSI+ status was significantly associated with the anatomic site right colon, mucinous histological type, clinical stage II, histological grade III/undifferentiated, no recurrence of disease, and live cases without cancer. No association of MSI status with genetic ancestry components was observed. MSI-targeted genes analyses showed the most frequently altered genes: ATM, EGFR, MRE11, ROCK1, and TGFBRII. There was a statistically significant difference in cancer-specific survival between cases according to MSI status. This study constitutes the most comprehensive analyses of the MSI impact on the Brazilian CRC. MSI+ frequency in Brazilian CRC agreed with the literature and was associated with several clinicopathological features related with less aggressive tumors, independently of their genetic ancestry.

Asunto(s)

Neoplasias Colorrectales , Inestabilidad de Microsatélites , Brasil , Neoplasias Colorrectales/patología , Humanos , Repeticiones de Microsatélite , Pronóstico , Quinasas Asociadas a rho/genética , Quinasas Asociadas a rho/uso terapéutico

RESUMEN

PURPOSE: Breast cancer (BC) is one of the most common malignant tumors for women. The role and potential mechanisms of long non-coding RNA plasmacytoma variant translocation 1 (lncRNA PVT1) were explored in BC cell migration and invasion. METHODS: PVT1, miR-148a-3p and Rho­associated, coiled­coil containing protein kinase 1 (ROCK1) mRNA expressions were detected using real-time fluorescent quantitative polymerase chain reaction (qRT-PCR). The ROCK1 protein expression was detected by Western blotting. The relationship of PVT1, miR-148a-3p and ROCK1 was analyzed by Dual Luciferase activity, RNA immunoprecipitation (RIP) and Spearman correlation analysis. Cell invasion and migration were detected by Transwell assay. RESULTS: Upregulation of PVT1 and ROCK1, and downregulation of miR-148a-3p were observed in BC tissues and cell lines. According to the analysis of Dual Luciferase activity, RIP and Spearman correlation analysis, miR-148a-3p directly binds to PVT1, and ROCK1 is a target of miR-148a-3p. In addition, PVT1 regulated the cells migration and invasion by regulating miR-148a-3p and ROCK1 expression. CONCLUSION: These data demonstrated that PVT1 was upregulated and facilitated to the cell migration and invasion of BC by the regulation of miR-148a-3p and ROCK1, indicating that PVT1 may be a potential biomarker of BC diagnosis and treatment.

Asunto(s)

Neoplasias de la Mama , MicroARNs , ARN Largo no Codificante/genética , Neoplasias de la Mama/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Luciferasas/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , ARN Largo no Codificante/metabolismo , Quinasas Asociadas a rho/genética , Quinasas Asociadas a rho/metabolismo

RESUMEN

BACKGROUND: This study evaluated the effect of treatment with TKI-258 on apoptosis, involving Rho GTPases and their effectors in SCC-4 cells of oral squamous cell carcinoma. METHODS: Markers of cell death and apoptosis were analyzed in control and TKI-258-treated SCC-4 cells by flow cytometry. The involvement of Rho GTPases and effectors in the induction of apoptosis by TKI-258 was evaluated by quantification of cleaved PARP. Also, gene expression analysis of those proteins was performed. RESULTS: The treatment with TKI-258 led to a significant increase in cell death (7-AAD) and apoptosis (annexin V and cleaved PARP). When Rho GTPases were stimulated with LPA and inhibited with toxin A Clostridium difficile, the percentage of apoptotic cells increased and decreased, respectively. A similar effect was found when the treatment was with TKI-258 combined with LPA and toxin A. Treatment with TKI-258 significantly increased RhoA gene expression, while RhoB, RhoC, Rac1, and Cdc42 decreased significantly. ROCKs inhibitors (Y-27632 and HA-1077) reduced apoptosis compared with control. TKI-258 combined with Y-27632 or HA-1077 led to an increase in apoptosis compared with inhibitors only. Treatment with TKI-258 led to an increase in ROCK1 and ROCK2 gene expression, and a decrease in PAK1 and PAK2 gene expression. CONCLUSIONS: TKI-258 stimulates apoptosis in SCC-4 cells of oral squamous cell carcinoma. Possibly, RhoA GTPase and their effectors ROCKs participate in the signaling pathway inhibited by TKI-258. CLINICAL RELEVANCE: Therapies with multi-target inhibitors, such as TKI-258, may be promising alternatives for the clinical treatment of oral squamous cell carcinoma.

Asunto(s)

Carcinoma de Células Escamosas , Neoplasias de Cabeza y Cuello , Neoplasias de la Boca , Apoptosis , Bencimidazoles , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/genética , Línea Celular , Humanos , Neoplasias de la Boca/tratamiento farmacológico , Neoplasias de la Boca/genética , Quinolonas , Transducción de Señal , Carcinoma de Células Escamosas de Cabeza y Cuello , Quinasas Asociadas a rho/genética , Proteína de Unión al GTP rhoA/genética

RESUMEN

INTRODUCTION AND OBJECTIVES: Hepatocellular carcinoma (HCC) is the second most lethal cancer around the world, with poor survival rate and high metastasis rate in patients. Long noncoding RNAs (lncRNAs) have been reported to modulate the initiation and development of liver cancer. We aimed to investigate the role of lncRNA MAGI2-AS3 in HCC and underlying mechanisms. MATERIALS AND METHODS: The expression levels of MAGI2-AS3 in plasma of HCC patients and the control participants were measured by qPCR. Hep3B and MHCC97-H cells were transfected with MAGI2-AS3 and ROCK2 expression vectors. Cell migration and invasion of HCC cells transfected with the vectors were investigated by transwell assay. In addition, flow cytometry and western blot were performed for apoptosis detection. RESULTS: We found that MAGI2-AS3 was downregulated in plasma of early stage HCC patients compared to healthy controls. After surgical resection, the expression levels of MAGI2-AS3 were increased compared to pretreatment levels on the day of discharge. During the follow-up, MAGI2-AS3 was downregulated in patients developed distant recurrence, but not in other patients compared to the levels measured on the day of discharge. In HCC cells, overexpression of MAGI2-AS3 mediated the downregulation of ROCK2. Cell invasion and migration assay showed that overexpression of MAGI2-AS3 mediated the decreased cell invasion and migration rate, while ROCK2 played an opposite role and attenuated the effects of overexpression of MAGI2-AS3. CONCLUSION: Our study indicated that MAGI2-AS3 was downregulated in the distant recurrence of HCC after surgical resection and affected the invasion and migration of HCC cells via ROCK2.

Asunto(s)

Carcinoma Hepatocelular/cirugía , Movimiento Celular , Hepatectomía/efectos adversos , Neoplasias Hepáticas/cirugía , ARN Largo no Codificante/metabolismo , Quinasas Asociadas a rho/metabolismo , Adulto , Anciano , Carcinoma Hepatocelular/enzimología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/secundario , Estudios de Casos y Controles , Línea Celular Tumoral , Regulación hacia Abajo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , ARN Largo no Codificante/genética , Transducción de Señal , Resultado del Tratamiento , Quinasas Asociadas a rho/genética

RESUMEN

Opa-interacting protein 5 antisense transcript 1 (OIP5-AS1) is one kind of cytoplasmic long non-coding RNA (lncRNA), which has been demonstrated to play a critical function in multiple cancers. However, the detailed mechanism of OIP5-AS1 in the regulation of cervical cancer progression is still obscure. Here, we demonstrated that lncRNA OIP5-AS1 was upregulated in cervical cancer and was correlated with poor prognosis by bioinformatics studies. OIP5-AS1 depletion inhibited cell proliferation and promoted cell apoptosis in cervical cancer cells. Furthermore, we clarified that ROCK1 was the downstream effector of OIP5-AS1 and OIP5-AS1 acted as a molecular sponge of miR-143-3p. Finally, we verified that OIP5-AS1 exerted its function in the regulation of cervical cancer progression via interacting with miR-143-3p to regulate ROCK1 expression. Our study revealed novel mechanisms about how lncRNA OIP5-AS1 executed its function in cervical cancer and thus provided potential therapeutic targets for the disease.

Asunto(s)

Apoptosis/fisiología , Proliferación Celular/fisiología , MicroARNs/metabolismo , ARN Largo no Codificante/metabolismo , Neoplasias del Cuello Uterino/patología , Quinasas Asociadas a rho/metabolismo , Apoptosis/genética , Western Blotting , Línea Celular Tumoral , Proliferación Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/genética , ARN Largo no Codificante/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba , Neoplasias del Cuello Uterino/metabolismo , Quinasas Asociadas a rho/genética

RESUMEN

Opa-interacting protein 5 antisense transcript 1 (OIP5-AS1) is one kind of cytoplasmic long non-coding RNA (lncRNA), which has been demonstrated to play a critical function in multiple cancers. However, the detailed mechanism of OIP5-AS1 in the regulation of cervical cancer progression is still obscure. Here, we demonstrated that lncRNA OIP5-AS1 was upregulated in cervical cancer and was correlated with poor prognosis by bioinformatics studies. OIP5-AS1 depletion inhibited cell proliferation and promoted cell apoptosis in cervical cancer cells. Furthermore, we clarified that ROCK1 was the downstream effector of OIP5-AS1 and OIP5-AS1 acted as a molecular sponge of miR-143-3p. Finally, we verified that OIP5-AS1 exerted its function in the regulation of cervical cancer progression via interacting with miR-143-3p to regulate ROCK1 expression. Our study revealed novel mechanisms about how lncRNA OIP5-AS1 executed its function in cervical cancer and thus provided potential therapeutic targets for the disease.

Asunto(s)

Humanos , Femenino , Neoplasias del Cuello Uterino/patología , Apoptosis/fisiología , MicroARNs/metabolismo , Proliferación Celular/fisiología , Quinasas Asociadas a rho/metabolismo , ARN Largo no Codificante/metabolismo , Regulación Neoplásica de la Expresión Génica , Regulación hacia Arriba , Neoplasias del Cuello Uterino/metabolismo , Western Blotting , Apoptosis/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , MicroARNs/genética , Línea Celular Tumoral , Proliferación Celular/genética , Quinasas Asociadas a rho/genética , ARN Largo no Codificante/genética

RESUMEN

Over the last decade, the rho-associated kinases and several metastasis-associated microRNAs have emerged as important contributors of tumor invasion. However, despite prominence, our understanding of their involvement in the metastatic potential of Ewing Sarcoma (EWS) is incomplete. The expression profiles of ROCK1 or ROCK2 and miR-124-3p, miR-138-5p, miR-139-5p, miR-335-5p and miR-584-5p (all of which were previously predicted or validated to regulate these kinases) were evaluated through qRT-PCR and associated with clinical parameters. In vitro assays to evaluate colony formation and invasion/migration capacieties were performed on SK-ES-1 cells transfected with pre-miR mimics. ROCK1 expression was significantly reduced in EWS tissues, though there was no association with pathological parameters. miR-124-3p, miR-139-5p and miR-335-3p were also found significantly downregulated and positively correlated with ROCK1. Stratification indicated an association between lower levels of miR-139-5p and miR-584-5p with disease progression (p < 0.05), while reduced expression of the former and miR-124-3p were associated with reduced survival. In vitro miR-139-5p overexpression yielded inconsistent results: while mir-139-5p restoration significantly reduced invasion, the clonogenic capacity of cells was increased. Our study demonstrated that down-regulation of miR-124-3p, miR-139-5p and miR-584-5p are associated with disease progression in EWS and may serve as a risk assessment biomarkers though, as seen for mir-139-5p, their specific role remain to be elucidated for considering tailoring treatment options.

Asunto(s)

Neoplasias Óseas/patología , MicroARNs/genética , Sarcoma de Ewing/patología , Quinasas Asociadas a rho/genética , Adolescente , Adulto , Neoplasias Óseas/genética , Línea Celular Tumoral , Movimiento Celular/genética , Niño , Preescolar , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Invasividad Neoplásica/genética , Sarcoma de Ewing/genética , Adulto Joven

RESUMEN

PTEN-induced kinase 1 (PINK1) mutations can cause early-onset Parkinson's disease and patients are likely to develop cognitive decline, depression, and dementia. Several neurophysiological studies have demonstrated PINK1 deficiency impairs striatal and hippocampal presynaptic plasticity. Dendritic spine postsynaptic abnormalities are common in neurological diseases; however, whether PINK1 silencing modifies dendritic spine dynamics of hippocampal neurons is unclear. To address this question, confocal images of mouse cultured hippocampal neurons transfected with plasmids to silence PINK1 were analyzed. These studies revealed that PINK1 silencing increased density of thin spines and reduced head size of stubby spines. Immunoblotting analysis uncovered that PINK1 silencing decreased expression of postsynaptic density proteins (PSD95 and Shank) and glutamate receptors (NR2B and mGluR5). We also found PINK1 silencing regulated dendritic spine morphology by actin regulatory proteins (RhoGAP29 and ROCK2) and regulated neuronal survival by decreased Akt activation. These results suggest PINK1 may regulate postsynaptic plasticity in hippocampal neurons generating presymptomatic alterations in dendritic spines that eventually could lead to the neurodegeneration and cognitive decline often seen in Parkinson's disease.

Asunto(s)

Espinas Dendríticas/metabolismo , Plasticidad Neuronal , Proteínas Quinasas/metabolismo , Animales , Células Cultivadas , Espinas Dendríticas/ultraestructura , Homólogo 4 de la Proteína Discs Large/metabolismo , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Hipocampo/citología , Hipocampo/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/metabolismo , Proteínas Quinasas/genética , Receptores de Glutamato/genética , Receptores de Glutamato/metabolismo , Quinasas Asociadas a rho/genética , Quinasas Asociadas a rho/metabolismo

RESUMEN

Inhibition of histone deacetylase 6 (HDAC6) was shown to support axon growth on the nonpermissive substrates myelin-associated glycoprotein (MAG) and chondroitin sulfate proteoglycans (CSPGs). Though HDAC6 deacetylates α-tubulin, we find that another HDAC6 substrate contributes to this axon growth failure. HDAC6 is known to impact transport of mitochondria, and we show that mitochondria accumulate in distal axons after HDAC6 inhibition. Miro and Milton proteins link mitochondria to motor proteins for axon transport. Exposing neurons to MAG and CSPGs decreases acetylation of Miro1 on Lysine 105 (K105) and decreases axonal mitochondrial transport. HDAC6 inhibition increases acetylated Miro1 in axons, and acetyl-mimetic Miro1 K105Q prevents CSPG-dependent decreases in mitochondrial transport and axon growth. MAG- and CSPG-dependent deacetylation of Miro1 requires RhoA/ROCK activation and downstream intracellular Ca2+ increase, and Miro1 K105Q prevents the decrease in axonal mitochondria seen with activated RhoA and elevated Ca2+ These data point to HDAC6-dependent deacetylation of Miro1 as a mediator of axon growth inhibition through decreased mitochondrial transport.

Asunto(s)

Histona Desacetilasa 6/genética , Mitocondrias/metabolismo , Neuronas/metabolismo , Proteínas de Unión al GTP rho/genética , Quinasas Asociadas a rho/genética , Acetilación/efectos de los fármacos , Animales , Transporte Axonal/efectos de los fármacos , Transporte Axonal/genética , Calcio/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/farmacología , Femenino , Ganglios Espinales/citología , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Regulación de la Expresión Génica , Histona Desacetilasa 6/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Glicoproteína Asociada a Mielina/farmacología , Neuronas/citología , Neuronas/efectos de los fármacos , Cultivo Primario de Células , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Proteínas de Unión al GTP rho/metabolismo , Quinasas Asociadas a rho/metabolismo

RESUMEN

Cytoskeletal organization, actin-myosin contractility and the cell membrane together regulate cell morphology in response to the cell environment, wherein the extracellular matrix (ECM) is an indispensable component. Plasticity in cell shape enables cells to adapt their migration mode to their surroundings. GH3 endocrine cells respond to different ECM proteins, acquiring different morphologies: a rounded on collagen I-III (C I-III) and an elongated on collagen IV (C IV). However, the identities of the molecules that participate in these responses remain unknown. Considering that actin-myosin contractility is crucial to maintaining cell shape, we analyzed the participation of MLCK and ROCK in the acquisition of cell shape, the generation of cellular tension and the cell motility mode. We found that a rounded shape with high cortical tension depends on MLCK and ROCK, whereas in cells with an elongated shape, MLCK is the primary protein responsible for cell spreading. Further, in cells with a slow and directionally persistent motility, MLCK predominates, while rapid and erratic movement is ROCK-dependent. This behavior also correlates with GTPase activation. Cells on C I-III exhibited higher Rho-GTPase activity than cells on C IV and vice versa with Rac-GTPase activity, showing a plastic response of GH3 cells to their environment, leading to the generation of different cytoskeleton and membrane organizations and resulting in two movement strategies, rounded and fibroblastoid-like.

Asunto(s)

Adhesión Celular/genética , Movimiento Celular/genética , Contracción Muscular/genética , Péptidos/genética , Quinasas Asociadas a rho/genética , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Actinas/genética , Actinas/metabolismo , Animales , Membrana Celular/genética , Membrana Celular/metabolismo , Forma de la Célula/genética , Matriz Extracelular/genética , Contracción Muscular/fisiología , Péptidos/metabolismo , Fosforilación , Ratas , Transducción de Señal/genética , Quinasas Asociadas a rho/biosíntesis

RESUMEN

BACKGROUND: ROCK-1 expression is associated with the malignant character of tumors, while inhibiting this molecule results in a significant suppression of tumor metastasis. Likewise, transforming growth factor beta (TGF-ß) is associated with this malignancy by having the ability to induce epithelial-mesenchymal transition (EMT). Metformin, a drug used in the treatment of diabetes, has previously been shown to inhibit EMT in breast cancer cells. OBJECTIVE: The aim of this study is to evaluate the TGF-ß1 action model for induction of EMT and the action of metformin and ROCK-1 inhibitor (Y27632) in EMT process in breast cancer cell lines. METHOD: MCF-7 and MDA-MB-231 cell lines were treated with metformin and Y27632, after induction of EMT by TGF-ß1, to examine the effects on cell migration as well as the protein expression of the ROCK-1 markers, vimentin, E-cadherin, CD44 and CD24 by immunocitochemistry. RESULTS: There was a lower protein expression of ROCK-1, vimentin, CD44 and CD24 in both cell lines after treatment with metformin and Y27632. In MDA-MB-231 cells, E-cadherin expression was increased in all treatment groups. Treatment of MDA-MB-231 cell line with metformin and Y27632 significantly reduced the invasion of these cells. CONCLUSION: This study confirms the benefits of metformin and Y27632 as potential therapeutic agents in mammary tumors, by blocking EMT process and metastatic potential.

Asunto(s)

Amidas/farmacología , Antineoplásicos/farmacología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Metformina/farmacología , Piridinas/farmacología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Células MCF-7 , Estructura Molecular , Relación Estructura-Actividad , Células Tumorales Cultivadas , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/genética , Quinasas Asociadas a rho/metabolismo

RESUMEN

The assembly of the musculoskeletal system in Drosophila relies on the integration of chemical and mechanical signaling between the developing muscles with ectodermal cells specialized as "tendon cells." Mechanical tension generated at the junction of flight muscles and tendon cells of the notum epithelium is required for muscle morphogenesis, and is balanced by the epithelium in order to not deform. We report that Drosophila Rho kinase (DRok) is necessary in tendon cells to assemble stable myotendinous junctions (MTJ), which are required for muscle morphogenesis and survival. In addition, DRok is required in tendon cells to maintain epithelial shape and cell orientation in the notum, independently of chascon (chas). Loss of DRok function in tendon cells results in mis-orientation of tendon cell extensions and abnormal accumulation of Thrombospondin and ßPS-integrin, which may cause abnormal myotendinous junction formation and muscle morphogenesis. This role does not depend exclusively on nonmuscular Myosin-II activation (Myo-II), indicating that other DRok targets are key in this process. We propose that DRok function in tendon cells is key to promote the establishment of MTJ attachment and to balance mechanical tension generated at the MTJ by muscle compaction.

Asunto(s)

Proteínas de Drosophila/genética , Drosophila/genética , Morfogénesis , Músculo Esquelético/metabolismo , Tendones/metabolismo , Quinasas Asociadas a rho/genética , Animales , Drosophila/crecimiento & desarrollo , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Cadenas beta de Integrinas/genética , Cadenas beta de Integrinas/metabolismo , Músculo Esquelético/crecimiento & desarrollo , Tendones/crecimiento & desarrollo , Tórax/crecimiento & desarrollo , Trombospondinas/genética , Trombospondinas/metabolismo , Quinasas Asociadas a rho/metabolismo

RESUMEN

Cynopoecilines comprise a diversified clade of small killifishes occurring in the Atlantic Forest, one of the most endangered biodiversity hotspots in the world. They are found in temporary pools of savannah-like and dense forest habitats, and most of them are highly threatened with extinction if not already extinct. The greatest gap in our knowledge of cynopoecilines stems from the absence of an integrative approach incorporating molecular phylogenetic data of species still found in their habitats with phylogenetic data taken from the rare and possibly extinct species without accessible molecular information. An integrative analysis combining 115 morphological characters with a multigene dataset of 2,108 bp comprising three nuclear loci (GLYT1, ENC1, Rho), provided a robust phylogeny of cynopoeciline killifishes, which was herein used to attain an accurate phylogenetic placement of nearly extinct species. The analysis indicates that the most recent common ancestor of the Cynopoecilini lived in open vegetation habitats of the Atlantic Forest of eastern Brazil and was a miniature species, reaching between 25 and 28 mm of standard length. The rare cases of cynopoecilines specialized in inhabiting pools within dense forests are interpreted as derived from four independent evolutionary events. Shifts in habitat usage and biogeographic patterns are tentatively associated to Cenozoic paleogeographic events, but the evolutionary history of cynopoecilines may be partially lost by a combination of poor past sampling and recent habitat decline. A sharp evolutionary shift directed to increased body size in a clade encompassing the genera Campellolebias and Cynopoecilus may be related to a parallel acquisition of an internally-fertilizing reproductive strategy, unique among aplocheiloid killifishes. This study reinforces the importance of adding morphological information to molecular databases as a tool to understand the biological complexity of organisms under intense pressure from loss of habitat.

Asunto(s)

ADN Mitocondrial/genética , Proteínas de Peces/genética , Peces Killi/genética , Filogenia , Reproducción/genética , Animales , Biodiversidad , Evolución Biológica , Tamaño Corporal , Brasil , Conservación de los Recursos Naturales , Ecosistema , Bosques , Expresión Génica , Proteínas de Transporte de Glicina en la Membrana Plasmática/genética , Peces Killi/anatomía & histología , Peces Killi/clasificación , Proteínas de Microfilamentos/genética , Proteínas Nucleares/genética , Estaciones del Año , Quinasas Asociadas a rho/genética

RESUMEN

The GTPases belonging to the Rho family control the actin cytoskeleton rearrangements needed for particle internalization during phagocytosis. ROCK and mDia1 are downstream effectors of RhoA, a GTPase involved in that process. Coxiella burnetii, the etiologic agent of Q fever, is internalized by the host´s cells in an actin-dependent manner. Nevertheless, the molecular mechanism involved in this process has been poorly characterized. This work analyzes the role of different GTPases of the Rho family and some downstream effectors in the internalization of C. burnetii by phagocytic and non-phagocytic cells. The internalization of C. burnetii into HeLa and RAW cells was significantly inhibited when the cells were treated with Clostridium difficile Toxin B which irreversibly inactivates members of the Rho family. In addition, the internalization was reduced in HeLa cells that overexpressed the dominant negative mutants of RhoA, Rac1 or Cdc42 or that were knocked down for the Rho GTPases. The pharmacological inhibition or the knocking down of ROCK diminished bacterium internalization. Moreover, C. burnetii was less efficiently internalized in HeLa cells overexpressing mDia1-N1, a dominant negative mutant of mDia1, while the overexpression of the constitutively active mutant mDia1-ΔN3 increased bacteria uptake. Interestingly, when HeLa and RAW cells were infected, RhoA, Rac1 and mDia1 were recruited to membrane cell fractions. Our results suggest that the GTPases of the Rho family play an important role in C. burnetii phagocytosis in both HeLa and RAW cells. Additionally, we present evidence that ROCK and mDia1, which are downstream effectors of RhoA, are involved in that process.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Bacterianas/metabolismo , Coxiella burnetii/metabolismo , Fagocitosis , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas Bacterianas/genética , Línea Celular , Células HeLa , Humanos , Ratones , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo , Quinasas Asociadas a rho/genética , Proteína de Unión al GTP rhoA/genética