RESUMO
BACKGROUND: Studies have recently shown that RHOA mutations play a crucial role in angioimmunoblastic T-cell lymphoma (AITL) pathogenesis. We aimed to pool data from these studies to provide a comparison of clinicopathological features between the RHOA mutant and RHOA wild-type groups in the AITL population. METHODS: We searched PubMed and Web of Science for the keywords "RHOA AND lymphoma" and selected only studies reporting the clinical significance of RHOA mutations in AITL. We calculated the odds ratios (OR) or the mean difference with 95% CI using a random effect model. RESULTS: Our pooled results showed a significant association between RHOA mutations and a T-follicular helper cell (TFH) phenotype, especially CD10 (OR, 5.16; 95% CI, 2.32-11.46), IDH2 mutations (OR, 10.70; 95% CI, 4.22-27.15), and TET2 mutations (OR, 7.03; 95% CI, 2.14-23.12). Although DNMT3A together with TET2 and IDH2 mutations are epigenetic gene alterations, we found an insignificant association between RHOA and DNMT3A mutations (OR, 1.72; 95% CI, 0.73-4.05). No significant associations of RHOA mutations with other clinicopathological features and overall survival were found. CONCLUSIONS: RHOA mutations are strongly correlated with a T-follicular helper cell phenotype and epigenetic mutations such as TET2 and IDH2. Further studies with large AITL samples should be conducted to validate the relationship of TET2, DNMT3A, and RHOA co-mutations.
Assuntos
Biomarcadores Tumorais/genética , Linfadenopatia Imunoblástica/diagnóstico , Linfoma de Células T/diagnóstico , Proteína rhoA de Ligação ao GTP/genética , Biomarcadores Tumorais/análise , DNA Metiltransferase 3A/genética , Proteínas de Ligação a DNA/genética , Dioxigenases/genética , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Humanos , Linfadenopatia Imunoblástica/genética , Linfadenopatia Imunoblástica/patologia , Isocitrato Desidrogenase/genética , Linfoma de Células T/genética , Linfoma de Células T/patologia , Mutação , Células T Auxiliares Foliculares/patologia , Proteína rhoA de Ligação ao GTP/análiseRESUMO
Rho GTPases are well known for regulating cell morphology and intracellular interactions. They can either be oncogenic or tumor suppressors. However, these proteins are associated with the acquirement of malignant features by cancer cells. It has been reported that the overexpression of protein markers of Rho family members such as RhoA and Rac1 is linked with carcinogenesis and the progression of a variety of human tumors. In this paper, the expression of RhoA and Rac1 activity in various types of breast cancers cell lines is evaluated. These cells are preconditioned by mechanically stretching them to simulate the extracellular physical forces placed upon on cancer cells. It is observed that stretching the cancer cells induces significantly higher expression of RhoA and Rac1 markers when compared to non-stretched cells and stretched control cells in vitro. This stretching strategy helps to detect and quantify the signal when it is too weak to be detected. Furthermore, stretching enhances the assay by leading to overexpression of markers and makes the assay more sensitive. It is hypothesized that this inexpensive and relatively sensitive assay can potentially aid in the development of a diagnostic tool for cancer screening.
Assuntos
Neoplasias da Mama , Fenômenos Fisiológicos Celulares/fisiologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/fisiopatologia , Contagem de Células , Linhagem Celular Tumoral , Feminino , Humanos , Estresse Mecânico , Proteínas rac1 de Ligação ao GTP/análise , Proteína rhoA de Ligação ao GTP/análiseRESUMO
Extracellular vesicles, such as microvesicles (MVs), were identified as important players in tumor progression and acquisition of an aggressive phenotype. Tissue factor (TF) is a transmembrane protein that initiates the blood coagulation cascade. In tumor cells, TF has been associated with aggressiveness and cancer progression. Previous studies demonstrate that TF is incorporated into MVs secreted by tumor cells; however, it is unknown whether TF is actively involved in the release of MVs. Here, we investigated the influence of TF expression on the release of MVs. TF silencing was achieved through CRISPR/Cas9 approaches in the human breast cancer cell line, MDA-MB-231. TF knockout in MDA-MB-231â¯cells efficiently reduced TF-dependent signaling and procoagulant activity. Remarkably, silencing of TF caused a significant decrease in the number of MVs released by MDA-MB-231â¯cells. We also observed an increase in actin-positive membrane projections in TF knockout cells and a reduction in RhoA expression when compared to TF-expressing cells. Treatment of MDA-MB-231â¯cells with the RhoA-ROCK signaling pathway inhibitor, fasudil, significantly reduced the release of MVs. Taken together, our results suggest a novel and relevant role for TF in tumor biology by playing an active role in the MVs secretion.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/patologia , Tromboplastina/metabolismo , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Vesículas Extracelulares/genética , Fator VIIa/análise , Fator VIIa/metabolismo , Feminino , Inativação Gênica , Humanos , Transdução de Sinais , Tromboplastina/genética , Quinases Associadas a rho/análise , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/análise , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Clostridium difficile infection (CDI) is the leading cause of gastroenteritis-associated death in the United States. The major virulent factors of C. difficile are toxin A (TcdA) and toxin B (TcdB). Toxicity is mediated by the glucosyltransferase domains on TcdA and TcdB wherein a glucose is transferred from UDP-glucose to Ras homolog family member A (RhoA) receptor. This modification results in disruption of critical cell signaling events. Vaccination against these toxins is considered the best way to combat the CDI. In order to produce non-toxic TcdA and TcdB antigens, their glucosyltransferase domains were genetically mutated to inactivate the toxin activity. We have developed a reverse phase ultra performance liquid chromatographic (RP-UPLC) method to measure this glucosyltransferase activity by separating RhoA and glucosylated RhoA. Glucosylated RhoA and RhoA have a retention time (RT) of 31.25 and 31.95min. We determine for the first time the glucosyltransferase kinetics (Km and kcat) of both full length TcdA and TcdB to RhoA and demonstrate that the genetically mutated TcdA and TcdB show no glucosyltransferase activity. Furthermore, two-dimensional electron microscopy (2D EM) data demonstrates that the overall global structures of mutated toxins do not change compared to native toxins.
Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Cromatografia Líquida de Alta Pressão , Clostridioides difficile/metabolismo , Enterotoxinas/metabolismo , Glucosiltransferases/metabolismo , Proteína rhoA de Ligação ao GTP/análise , Glicosilação , Humanos , Cinética , Espectrometria de Massas , Microscopia Eletrônica , Uridina Difosfato Glucose/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
PURPOSE: The role of exercise to prevent or reverse aging-induced cognitive decline has been widely reported. This neuroprotection is associated with changes in the synaptic structure plasticity. However, the mechanisms of exercise-induced synaptic plasticity in the aging brain are still unclear. Thus, the aim of the present study is to investigate the aging-related alterations of Rho-GTPase and the modulatory influences of exercise training. METHODS: Young and old rats were used in this study. Old rats were subjected to different schedules of aerobic exercise (12 m/min, 60 min/d, 3d/w or 5d/w) or kept sedentary for 12 w. After 12 w of aerobic exercise, the synapse density in the cortex and hippocampus was detected with immunofluorescent staining using synaptophysin as a marker. The total protein levels of RhoA, Rac1, Cdc42 and cofilin in the cortex and hippocampus were detected with Western Blot. The activities of RhoA, Rac1 and Cdc42 were determined using a pull down assay. RESULTS: We found that synapse loss occurred in aging rats. However, the change of expression and activity of RhoA, Rac1 and Cdc42 was different in the cortex and hippocampus. In the cortex, the expression and activity of Rac1 and Cdc42 was greatly increased with aging, whereas there were no changes in the expression and activity of RhoA. In the hippocampus, the expression and activity of Rac1 and Cdc42 was greatly decreased and there were no changes in the expression and activity of RhoA. As a major downstream substrate of the Rho GTPase family, the increased expression of cofilin was only observed in the cortex. High frequency exercise ameliorated all aging-related changes in the cortex and hippocampus. CONCLUSIONS: These data suggest that aerobic exercise reverses synapse loss in the cortex and hippocampus in aging rats, which might be related to the regulation of Rho GTPases.
Assuntos
Envelhecimento/fisiologia , Condicionamento Físico Animal/fisiologia , Transdução de Sinais/fisiologia , Sinapses/fisiologia , Proteínas rac1 de Ligação ao GTP/fisiologia , Proteína rhoA de Ligação ao GTP/fisiologia , Fatores de Despolimerização de Actina/análise , Fatores de Despolimerização de Actina/fisiologia , Animais , Western Blotting , Córtex Cerebral/química , Córtex Cerebral/fisiologia , Imunofluorescência , Hipocampo/química , Hipocampo/fisiologia , Masculino , Ratos , Ratos Wistar , Proteína cdc42 de Ligação ao GTP/análise , Proteína cdc42 de Ligação ao GTP/fisiologia , Proteínas rac1 de Ligação ao GTP/análise , Proteína rhoA de Ligação ao GTP/análiseRESUMO
Aberrant activation of Wnt and base excision repair (BER) signaling pathways are implicated in tumor progression and chemotherapy resistance in gastric adenocarcinoma. This study was conducted to clarify the role of E2F6 and RhoA, components of the Wnt signaling pathway, and SMUG1, a component of the BER pathway in gastric adenocarcinoma. Expression levels and clinicopathological significance of three biomarkers, namely E2F6, RhoA, and SMUG1, as potential signaling molecules involved in tumorigenesis and aggressive behavior, were examined using tissue microarray. Our analysis showed a relative increase in the expression of E2F6 in gastric adenocarcinoma with no lymph node metastasis (χ 2, P = 0.04 and OR, P = 0.08), while overexpression of RhoA and SMUG1 was found more often in the diffuse subtype of gastric adenocarcinoma as compared to the intestinal subtype (χ 2, P = 0.05, OR, P = 0.08 and χ 2, P = 0.001, OR, P = 0.009, respectively). Higher expression of RhoA was frequently seen in tumors with vascular invasion (χ 2, P = 0.01 and OR, P = 0.01). In addition, increased expression of SMUG1 was found more often in poorly differentiated tumors (χ 2, P = 0.01 and OR, P = 0.01). The distinct phenotype of E2F6Low/SMUG1High was more common in poorly differentiated tumors (P = 0.04) and with omental involvement (P = 0.01). The RhoAHigh/SMUG1High expression pattern was significantly more often found in diffuse subtype compared to the intestinal subtype (P = 0.001) as well as in poorly differentiated tumors (P = 0.004). The E2F6Low/SMUG1High and RhoAHigh/SMUG1High phenotypes can be considered as aggressive phenotypes of gastric adenocarcinoma. Our findings also demonstrated the synergistic effect of RhoA and SMUG1 in conferring tumor aggressiveness in diffuse subtype of gastric adenocarcinoma.
Assuntos
Adenocarcinoma/patologia , Reparo do DNA , Fator de Transcrição E2F6/análise , Neoplasias Gástricas/patologia , Uracila-DNA Glicosidase/análise , Via de Sinalização Wnt , Proteína rhoA de Ligação ao GTP/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise Serial de TecidosRESUMO
OBJECTIVE: To study the possible effect of antepartum taurine supplementation in regulating the activity of Rho family factors and promoting the proliferation of neural stem cells in neonatal rats with fetal growth restriction (FGR), and to provide a basis for antepartum taurine supplementation to promote brain development in children with FGR. METHODS: A total of 24 pregnant Sprague-Dawley rats were randomly divided into three groups: control, FGR, and taurine (n=8 each ). A rat model of FGR was established by food restriction throughout pregnancy. RT-PCR, immunohistochemistry, and Western blot were used to measure the expression of the specific intracellular markers for neural stem cells fatty acid binding protein 7 (FABP7), Rho-associated coiled-coil containing protein kinase 2 (ROCK2), ras homolog gene family, member A (RhoA), and Ras-related C3 botulinum toxin substrate (Rac). RESULTS: The FGR group had significantly lower OD value of FABP7-positive cells and mRNA and protein expression of FABP7 than the control group, and the taurine group had significantly higher OD value of FABP7-positive cells and mRNA and protein expression of FABP7 than the FGR group (P<0.05). The FGR group had significantly higher mRNA expression of RhoA and ROCK2 than the control group. The taurine group had significantly higher mRNA expression of RhoA and ROCK2 than the control group and significantly lower expression than the FGR group (P<0.05). The FGR group had significantly lower mRNA expression of Rac than the control group. The taurine group had significantly higher mRNA expression of Rac than the FGR and control groups (P<0.05). The FGR group had significantly higher protein expression of RhoA and ROCK2 than the control group. The taurine group had significantly lower protein expression of RhoA and ROCK2 than the FGR group (P<0.05). CONCLUSIONS: Antepartum taurine supplementation can promote the proliferation of neural stem cells in rats with FGR, and its mechanism may be related to the regulation of the activity of Rho family factors.
Assuntos
Proliferação de Células/efeitos dos fármacos , Retardo do Crescimento Fetal/tratamento farmacológico , Células-Tronco Neurais/efeitos dos fármacos , Taurina/farmacologia , Quinases Associadas a rho/genética , Proteína rhoA de Ligação ao GTP/genética , Animais , Animais Recém-Nascidos , Peso Corporal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Proteína 7 de Ligação a Ácidos Graxos/análise , Feminino , Masculino , Células-Tronco Neurais/fisiologia , Ratos , Ratos Sprague-Dawley , Quinases Associadas a rho/análise , Proteína rhoA de Ligação ao GTP/análiseRESUMO
OBJECTIVE: To explore the apoptosis of HepG2 cells infected by Listeria monocytogenes EGD strain (Lm-EGD) as well as Rho family small GTPases RhoA expression. METHODS: HepG2 cells were infected with Lm-EGD (MOI=10 and MOI=100) and collected 1 hour and 20 hours after infection. After harvesting, the apoptosis of HepG2 cells was determined by flow cytometry combined with annexin V-FITC/PI assay. RhoA and caspase 3 mRNAs were analyzed by reverse-transcription PCR. The caspase 3 activity was detected by colorimetric assay. And Western blotting was used to detect RhoA expression in HepG2 cells. RESULTS: Lm invasion promoted HepG2 cell apoptosis and down-regulated RhoA mRNA and protein expression. Additionally, caspase 3 expression was up-regulated following Lm infection. CONCLUSION: Lm infection could promote host cell apoptosis and down-regulate RhoA expression.
Assuntos
Apoptose , Listeria monocytogenes/patogenicidade , Proteína rhoA de Ligação ao GTP/fisiologia , Caspase 3/metabolismo , Regulação para Baixo , Células Hep G2 , Humanos , RNA Mensageiro/análise , Proteína rhoA de Ligação ao GTP/análise , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Surface nanofeatures and bioactive ion chemical modification are centrally important in current titanium (Ti) oral implants for enhancing osseointegration. However, it is unclear whether the addition of bioactive ions definitively enhances the osteogenic capacity of a nanostructured Ti implant. We systematically investigated the osteogenesis process of human multipotent adipose stem cells triggered by bioactive ions in the nanostructured Ti implant surface. Here, we report that bioactive ion surface modification (calcium [Ca] or strontium [Sr]) and resultant ion release significantly increase osteogenic activity of the nanofeatured Ti surface. We for the first time demonstrate that ion modification actively induces focal adhesion development and expression of critical adhesionrelated genes (vinculin, talin, and RHOA) of human multipotent adipose stem cells, resulting in enhanced osteogenic differentiation on the nanofeatured Ti surface. It is also suggested that fibronectin adsorption may have only a weak effect on early cellular events of mesenchymal stem cells (MSCs) at least in the case of the nanostructured Ti implant surface incorporating Sr. Moreover, results indicate that Sr overrides the effect of Ca and other important surface factors (i.e., surface area and wettability) in the osteogenesis function of various MSCs (derived from human adipose, bone marrow, and murine bone marrow). In addition, surface engineering of nanostructured Ti implants using Sr ions is expected to exert additional beneficial effects on implant bone healing through the proper balancing of the allocation of MSCs between adipogenesis and osteogenesis. This work provides insight into the future surface design of Ti dental implants using surface bioactive ion chemistry and nanotopography.
Assuntos
Cálcio/química , Implantes Dentários , Materiais Dentários/química , Células-Tronco Mesenquimais/fisiologia , Nanoestruturas/química , Estrôncio/química , Titânio/química , Adipogenia/fisiologia , Tecido Adiposo/citologia , Adsorção , Fosfatase Alcalina/análise , Animais , Bioengenharia , Adesão Celular/fisiologia , Diferenciação Celular/fisiologia , Proliferação de Células , Células Cultivadas , Fibronectinas/química , Humanos , Teste de Materiais , Camundongos , Células-Tronco Multipotentes/fisiologia , Osteogênese/fisiologia , Propriedades de Superfície , Talina/análise , Vinculina/análise , Molhabilidade , Proteína rhoA de Ligação ao GTP/análiseRESUMO
BACKGROUND: The 5-year survival rate of patients with hepatocellular cancer (HCC) was very low because of invasion and metastasis in the early stage. Biomarkers might help predict early occurrence of invasion and metastasis. Accumulating evidence has shown that deleted in liver cancer-1 (DLC1) may be considered as a metastasis suppressor gene in numerous solid and hematological cancers. However, its prognostic role and mechanisms that regulate and coordinate these activities remain poorly understood. METHODS: With the method of immunohistochemistry, the expression of DLC-1 as well as Rho A, ROCK2, moesin had been characterized in 80 HCC tissues and adjacent noncancerous tissues. The correlation between their expression and their relationships with clinicopathological characteristics of HCC were also investigated. In addition, the prognostic value of DLC1 expression within the tumor tissues was assessed by Cox regression and Kaplan-Meier analysis. RESULTS: DLC1 expression was significantly lower in HCC tissues than in adjacent noncancerous tissues, and DLC-1 expression was found to be negatively correlated with tumor differentiation, TNM stage and lymph node metastasis. Furthermore, DLC-1 expression was found to inversely correlate with Rho A, ROCK2 and moesin which were all highly expressed in HCC tissues. Kaplan-Meier analysis showed that significantly longer 5-year survival rate was seen in HCC patients with higher DLC1 expression, compared to those with lower expression of DLC1. Multivariate Cox proportional hazard analyses revealed that DLC1 was an independent factor affecting the overall survival probability. CONCLUSION: DLC1 could be served as a tumor suppressor gene in the progression especially in the invasion and metastasis of HCC. DLC1 perhaps played its role by regulating the expression of Rho A, ROCK2 and moesin. Evaluation of the expression of DLC-1 might be a good prognostic marker for patients with HCC.
Assuntos
Biomarcadores Tumorais/análise , Carcinoma Hepatocelular/química , Proteínas Ativadoras de GTPase/análise , Neoplasias Hepáticas/química , Proteínas Supressoras de Tumor/análise , Adulto , Idoso , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/secundário , Carcinoma Hepatocelular/terapia , Diferenciação Celular , Progressão da Doença , Regulação para Baixo , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/terapia , Metástase Linfática , Masculino , Proteínas dos Microfilamentos/análise , Pessoa de Meia-Idade , Análise Multivariada , Estadiamento de Neoplasias , Valor Preditivo dos Testes , Modelos de Riscos Proporcionais , Fatores de Tempo , Quinases Associadas a rho/análise , Proteína rhoA de Ligação ao GTP/análiseRESUMO
Ras homologue gene family member A (RhoA) is involved in tumor mobility, invasion, and metastasis. We detected RhoA expression in vulvar squamous cell carcinoma (VSCC) tissue, measured RhoA expression in the VSCC cell phenotype, and measured the expression of the relevant molecules after RhoA small interfering RNA (siRNA) transfection in SW962 cells. RhoA has a higher expression level in VSCC than normal vulva skin tissue and was positively associated with the International Federation of Gynecology and Obstetrics (FIGO) stage and differentiation; besides, VSCC patients with lymph node metastasis had higher positive RhoA expression. RhoA messenger RNA and protein expression was significantly reduced in the RhoA siRNA transfectants as compared with the negative control (NC) and mock-transfected cells (p < 0.05). The RhoA siRNA transfectants lead to low growth, G1 arrest, high apoptosis, low migration and invasion (p < 0.05), and suppressed lamellipodia formation as compared to NC and mock-transfected cells. Besides, matrix metalloproteinase-2 (MMP2), MMP9, and cyclinA1 protein expression was downregulated, while that of Bax was upregulated in the RhoA siRNA transfectants (p < 0.05). SW962 cell proliferation rates were significantly lovastatin dose-dependent. Lovastatin caused G1 arrest, high apoptosis, low migration and invasion (p < 0.05), and suppression of lamellipodia formation. Similar to the RhoA siRNA transfectants, lovastatin treatment downregulated RhoA, MMP2, MMP9, and cyclinA1 protein expression, while upregulating that of Bax as compared to that of the NC (p < 0.05). Abnormal RhoA expression in vulvar carcinoma is involved in tumor proliferation and invasion and may be a treatment target. The RhoA inhibitor lovastatin alters VSCC cell migration and proliferation and may be effective for treating VSCC.
Assuntos
Biomarcadores Tumorais/fisiologia , Carcinoma de Células Escamosas/patologia , Neoplasias Vulvares/patologia , Proteína rhoA de Ligação ao GTP/fisiologia , Adulto , Idoso , Biomarcadores Tumorais/análise , Carcinoma de Células Escamosas/etiologia , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Humanos , Imuno-Histoquímica , Lovastatina/farmacologia , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Neoplasias Vulvares/etiologia , Proteína rhoA de Ligação ao GTP/análise , Proteína rhoA de Ligação ao GTP/antagonistas & inibidoresRESUMO
Vascular disorders, including hypertension, atherosclerosis and restenosis, arise from dysregulation of vascular smooth muscle cell (VSMC) differentiation, which can be controlled by regulatory factors. The present study investigated the regulatory mechanism of the phenotypic transformation of human VSMCs by NELIN in order to evaluate its potential as a preventive and therapeutic of vascular disorders. An in vitro model of NELINoverexpressing VSMCs was prepared by transfection with a lentiviral (LV) vector (NELINVSMCs) and NELIN was slienced using an a lentiviral vector with small interfering (si)RNA in another group (LVNELINsiRNAVSMCs). The effects of NELIN overexpression or knockdown on the phenotypic transformation of human VSMCs were observed, and its regulatory mechanism was studied. Compared with the control group, cells in the NELINVSMCs group presented a contractile phenotype with a significant increase of NELIN mRNA, NELIN protein, smooth muscle (SM)αactin and total Ras homolog gene family member A (RhoA) protein expression. The intranuclear translocation of SMαactinserum response factor (SMαactinSRF) occurred in these cells simultaneously. Following exposure to Rho kinsase inhibitor Y27632, SRF and SMαactin expression decreased. However, cells in the LVNELINsiRNAVSMCs group presented a synthetic phenotype, and the expression of NELIN mRNA, NELIN protein, SMαactin protein and total RhoA protein was decreased. The occurrence of SRF extranuclear translocation was observed. In conclusion, the present study suggested that NELIN was able to activate regulatory factors of SMαactin, RhoA and SRF successively in human VSMCs cultured in vitro. Furthermore, NELINinduced phenotypic transformation of human VSMCs was regulated via the RhoA/SRF signaling pathway. The results of the present study provide a foundation for the use of NELIN in preventive and therapeutic treatment of vascular remodeling diseases, including varicosity and atherosclerosis.
Assuntos
Proteínas dos Microfilamentos/metabolismo , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Actinas/análise , Actinas/metabolismo , Amidas/farmacologia , Diferenciação Celular , Células Cultivadas , Técnicas de Silenciamento de Genes , Humanos , Masculino , Proteínas dos Microfilamentos/análise , Proteínas dos Microfilamentos/genética , Pessoa de Meia-Idade , Relaxantes Musculares Centrais/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Fenótipo , Transporte Proteico/efeitos dos fármacos , Piridinas/farmacologia , Fator de Resposta Sérica/análise , Fator de Resposta Sérica/metabolismo , Regulação para Cima , Proteína rhoA de Ligação ao GTP/análise , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
OBJECTIVE: To observe the protective effect of salidroside against brain injury in rats exposed to hypobaric hypoxia, and investigate the molecular mechanism of salidroside in the prevention of hypobaric hypoxia-induced brain injury. METHODS: Rats were placed in experiment module simulating 6000 m altitude to establish acute hypobaric hypoxia-induced brain injury models. Their respiratory frequency was observed and recorded. Cell apoptosis in the hippocampal dentate gyrus (DG) was detected by TUNEL assay; the expressions of Ras homolog family member A (RhoA), phosphorylated extracellular signal-regulated kinase (p-ERK) and phosphorylated c-Jun N-terminal kinase (p-JNK) were detected by Western blotting. RESULTS: After acute exposure to 6000 m altitude, the respiratory frequency of the rats increased remarkably. The simulation of hypobaric hypoxia induced cell apoptosis in hippocampal DG region, and salidroside intervention inhibited the process of cell apoptosis. The expressions of RhoA, p-ERK, p-JNK decreased after hypobaric hypoxia exposure. Salidroside intervention reversed RhoA expression and raised the levels of p-ERK and p-JNK. CONCLUSION: Acute exposure to hypobaric hypoxia can induce cell apoptosis in rat hippocampal DG, and salidroside can protect the cells from the exposure-induced apoptosis.
Assuntos
Doença da Altitude/tratamento farmacológico , Glucosídeos/uso terapêutico , Hipóxia Encefálica/tratamento farmacológico , Fenóis/uso terapêutico , Doença da Altitude/patologia , Animais , Apoptose , Citoproteção , Giro Denteado/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Hipóxia Encefálica/patologia , Masculino , Ratos , Ratos Sprague-Dawley , Respiração , Proteína rhoA de Ligação ao GTP/análiseRESUMO
Adhesion of the junctional epithelium (JE) to the tooth surface is crucial for maintaining periodontal health. Although odontogenic ameloblast-associated protein (ODAM) is expressed in the JE, its molecular functions remain unknown. We investigated ODAM function during JE development and regeneration and its functional significance in the initiation and progression of periodontitis and peri-implantitis. ODAM was expressed in the normal JE of healthy teeth but absent in the pathologic pocket epithelium of diseased periodontium. In periodontitis and peri-implantitis, ODAM was extruded from the JE following onset with JE attachment loss and detected in gingival crevicular fluid. ODAM induced RhoA activity and the expression of downstream factors, including ROCK (Rho-associated kinase), by interacting with Rho guanine nucleotide exchange factor 5 (ARHGEF5). ODAM-mediated RhoA signaling resulted in actin filament rearrangement. Reduced ODAM and RhoA expression in integrin ß3- and ß6-knockout mice revealed that cytoskeleton reorganization in the JE occurred via integrin-ODAM-ARHGEF5-RhoA signaling. Fibronectin and laminin activated RhoA signaling via the integrin-ODAM pathway. Finally, ODAM was re-expressed with RhoA in regenerating JE after gingivectomy in vivo. These results suggest that ODAM expression in the JE reflects a healthy periodontium and that JE adhesion to the tooth surface is regulated via fibronectin/laminin-integrin-ODAM-ARHGEF5-RhoA signaling. We also propose that ODAM could be used as a biomarker of periodontitis and peri-implantitis.
Assuntos
Proteínas de Transporte/metabolismo , Inserção Epitelial/metabolismo , Periodontite/metabolismo , Proteínas/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Dente/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Amiloide , Animais , Proteínas de Transporte/análise , Linhagem Celular , Inserção Epitelial/patologia , Fibronectinas/análise , Fibronectinas/metabolismo , Humanos , Integrinas/análise , Integrinas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Laminina/análise , Laminina/metabolismo , Camundongos , Proteínas de Neoplasias , Periodontite/patologia , Proteínas/análise , Fatores de Troca de Nucleotídeo Guanina Rho/análise , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/análiseRESUMO
BACKGROUND & AIMS: Patients with cirrhosis display hypocontractility of splanchnic vessels because of dysregulation of vasoactive proteins, such as decreased effect of RhoA/ROCK and increased activity of ß-Arrestin-2 and eNOS. However, it is unknown whether the dysregulation of vasoactive proteins is displayed in other vessels. We investigated whether expression of vasoactive proteins can be evaluated in gastric mucosa vessels. METHODS: Biopsies from the gastric mucosa of 111 patients with cirrhosis were collected at three different centres and from 13 controls. Forty-nine patients had received TIPS. Portal pressure gradient was measured in 49 patients with TIPS and in 16 patients without TIPS. Biopsies from the antrum were conserved in formaldehyde for immunohistochemistry or shock-frozen for PCR and Western blot. RESULTS: The mucosal transcription of vascular markers (αSMA, CD31) was higher in cirrhotic patients than controls, which was confirmed by immunohistochemistry. On average, relative mucosal levels of RhoA and ROCK were lower, while ß-Arrestin-2 levels were higher in cirrhotic patients compared to controls. Transcriptional levels of eNOS increased with presence of ascites and grade of oesophageal varices. Patients with TIPS showed less pronounced markers of vascular dysfunction in gastric mucosa. CONCLUSION: This is the first evidence that the expression of vasoactive proteins in mucosa from the gastric antrum of patients with cirrhosis reflects their vascular dysfunction and possibly changes after therapeutic interventions.
Assuntos
Arrestinas/análise , Mucosa Gástrica/química , Hipertensão Portal/etiologia , Cirrose Hepática/complicações , Óxido Nítrico Sintase Tipo III/análise , Antro Pilórico/química , Quinases Associadas a rho/análise , Proteína rhoA de Ligação ao GTP/análise , Adulto , Idoso , Arrestinas/genética , Biópsia , Western Blotting , Estudos de Casos e Controles , Dinamarca , Feminino , Humanos , Hipertensão Portal/diagnóstico , Hipertensão Portal/genética , Hipertensão Portal/metabolismo , Hipertensão Portal/fisiopatologia , Hipertensão Portal/cirurgia , Imuno-Histoquímica , Cirrose Hepática/diagnóstico , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Cirrose Hepática/fisiopatologia , Masculino , Pessoa de Meia-Idade , Óxido Nítrico Sintase Tipo III/genética , Reação em Cadeia da Polimerase , Pressão na Veia Porta , Derivação Portossistêmica Transjugular Intra-Hepática , RNA Mensageiro/análise , Circulação Esplâncnica , Adulto Jovem , beta-Arrestina 2 , beta-Arrestinas , Quinases Associadas a rho/genética , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
O mecanismo pelo qual uma célula responde a algum dano no seu material genético é extremamente importante. Isto ocorre pela rápida ativação da maquinaria de reparo de danos no DNA, a qual é composta por uma rede intrincada de sinalização proteica, culminando no reparo do DNA; porém se o dano for irreparável ocorre ativação de mecanismos de morte celular. RhoA,e Rac1 pertencem a família das pequenas proteínas sinalizadoras Rho GTPases, as quais atuam como interruptores moleculares ciclando entre estado ativo (ligada a GTP) e inativo (ligada a GDP). Os componentes desta família estão relacionados ao controle dos mais diversos processos celulares como, por exemplo, remodelamento do citoesqueleto, migração, adesão, endocitose, progressão do ciclo celular e oncogênese. No entanto, apesar das proteínas Rho GTPases estarem envolvidas em um amplo espectro de atividades biológicas, há poucas informações sobre seu papel na manutenção da integridade genômica quando células são submetidas a algum agente genotóxico. Para investigar o envolvimento das GTPases RhoA e Rac1 nas respostas de células submetidas a radiação gama, foram gerados, a partir de células de carcinoma de cervix humano - HeLa, sublinhagens clonais mutantes de RhoA e Rac1 expressando exogenamente RhoA constitutivamente ativa (HeLa-RhoA V14), RhoA dominante negativa (HeLa-RhoA N19), Rac1 constitutivamente ativa (HeLa-Rac1 V12) e Rac1 dominante negativa (HeLa-Rac N17). Após estas linhagens celulares serem expostas a diferentes doses de radiação gama, observamos que ambas GTPases, RhoA e Rac1, são ativadas em resposta aos efeitos da radiação. Além disso, a modulação da atividade destas enzimas, através das mutações, levou a uma alteração das respostas celulares frente aos danos no DNA, como uma redução da capacidade de reparar quebras simples e duplas nas fitas do DNA. Por outro lado, a deficiência de RhoA ou Rac1 GTPase levou a uma redução da ativação de Chk1 e Chk2 ou da fosforilação da histona H2AX, respectivamente, prejudicando os mecanismos de detecção de danos no DNA e levando as células a permanecerem mais tempo nos pontos de checagem G1/S e/ou G2/M do ciclo celular. Esses fatores contribuíram de modo expressivo para a redução da proliferação e sobrevivência celular levando as células à morte. Por fim, ensaios celulares de reparo de danos de um DNA exógeno através de mecanismos de Recombinação Homóloga (HR) e Recombinação Não-Homóloga de extremidades (NHEJ), demonstraram que a inibição da atividade de RhoA reduz significativamente a eficiência de ambas vias de reparo. Desta maneira, este trabalho demonstra e reforça a existência de mais um viés de atuação das pequenas GTPases RhoA e Rac1, agora em células HeLa, nas respostas celulares aos danos induzidos por exposição a radiação gama, modulando a sobrevivência, proliferação e indiretamente modulando resposta ao reparo do DNA através da via de Recombinação Homóloga e Não-Homóloga
The mechanism by which a cell responds to DNA damage is extremely important. This occurs by a quick activation of the DNA damage repair machinery, which consists of an intricate protein signaling network culminating in DNA repair. But if the damages are irreparable occurs there is activation of cell death mechanisms. RhoA and Rac1 belong to family of small Rho GTPases, signaling proteins that act as molecular switches cycling between the active state (GTP-bound) and inactive state (GDP-bound). Members of this family are implicated in the control of diverse cellular process such as cytoskeletal remodeling, migration, adhesion, endocytosis, cell cycle progression, and oncogenesis. However, despite Rho proteins are involved in a broad spectrum of biological activities, there is just a few information about their roles in the maintenance of genomic integrity, that is, when the cells are subjected to some kinf of genotoxic agent. To investigate the involvement of the GTPases RhoA and Rac1 in cellular responses to gamma radiation, we generated from human cervix carcinoma cells - HeLa, clonal sublines of RhoA and Rac1 mutants, exogenous and stably expressing the constitutively active RhoA (HeLa-RhoA V14), the dominant negative RhoA (HeLa-RhoA N19), the constitutively active Rac1 (HeLa-Rac1 V12) and the dominant negative Rac1 (HeLa-Rac1 N17). After all these cell lines have been exposed to different doses of gamma radiation, we found that both GTPases, RhoA and Rac1, are activated in response to the radiation effects. Furthermore, the modulation of two enzymes activity, by using the mutant clones, led to a change in cellular responses to the DNA damage, as the reduction in the capacity of repairing DNA single and double strand breaksr. On the other hand, the deficiency of RhoA or Rac1 GTPase led to a reduction of Chk1 and Chk2 activation, or on the phosphorylation of histone H2AX, respectively, hindering the mechanisms of DNA damage detection and arresting cells in the G1/S and/or G2/M checkpoints of cell cycle. These factors significantly contributed to the reduction of cell proliferation and survival, leading cells to death. Finally, cellular assays of DNA damage repair of exogenous DNA by Homologous Recombination (HR) and Non-Homologous End Joining (NHEJ), demonstrated that RhoA inhibition significantly reduced the repair efficiency of both pathways. Thus, this work demonstrates and reinforces the existence of other biological functions of small GTPases RhoA and Rac1 in HeLa cells, by regulating cellular responses to DNA damage induced by exposure to gamma radiation, modulating the survival, proliferation and indirectly modulating the response to DNA damage repair pathway through the Homologous Recombination and Non-Homologous Recombination
Assuntos
GTP Fosfo-Hidrolases/análise , Proteínas rac1 de Ligação ao GTP/análise , Proteína rhoA de Ligação ao GTP/análise , Reparo do DNA por Junção de Extremidades/genética , Células HeLa , Recombinação Homóloga/genética , RadiaçãoRESUMO
PURPOSE OF INVESTIGATION: To evaluate the distribution of GTPases RhoA, RhoB, and Cdc42 in cervical intraepithelial neoplasias (CIN) and invasive neoplasias of the uterine cervix. MATERIALS AND METHODS: samples of neoplastic lesions of the uterine cervix of 44 patients were classified in: CIN I (n = 10), CIN II (n = 10), CIN III (n = 09), and invasive carcinoma (n = 15). Antibodies anti-RhoA, anti-RhoB, and anti-Cdc42 were used and staining was classified as: negative, mild, moderate, and intense positive. RESULTS: When compared with dysplastic cells, superficial cells showed: higher expression of RhoB in CIN I (p = 0.0018), and lower expression of Cdc42 in CIN I (p = 0.0225). The authors observed higher expression of RhoA (p = 0.0002) and RhoB (p = 0.0046) in CIN dysplastic cells when compared with invasive carcinoma cells. CONCLUSIONS: GTPases Rho may be involved with the regulation of biological processes, important to the progression of cervical neoplasias. Probably, RhoA is important for maintenance of cell differentiation and RhoB protects cells from malignant cervical neoplasia.
Assuntos
Displasia do Colo do Útero/patologia , Neoplasias do Colo do Útero/patologia , Proteína rhoA de Ligação ao GTP/fisiologia , Proteína rhoB de Ligação ao GTP/fisiologia , Adulto , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Invasividade Neoplásica , Estadiamento de Neoplasias , Neoplasias do Colo do Útero/enzimologia , Proteína cdc42 de Ligação ao GTP/análise , Proteína rhoA de Ligação ao GTP/análise , Proteína rhoB de Ligação ao GTP/análise , Displasia do Colo do Útero/enzimologiaRESUMO
Previously, we identified a microRNA (miRNA) signature for endothelial cells (ECs) subjected to unidirectional shear stress (USS). MiR-155, a multifunctional miRNA that has been implicated in atherosclerosis, was among the shear stress-responsive miRNAs. Here, we examined the role of miR-155 in modulating EC phenotype and function. In vitro, increased miR-155 levels in human ECs induced changes in morphology and filamentous (F)-actin organization. In addition, ECs transfected with miR-155 mimic were less migratory and less proliferative and had less apoptosis compared with control ECs. In mouse aorta, miR-155 expression was increased in the intima of thoracic aorta, where blood flow produces steady and unidirectional shear stress, compared with the intima of the lower curvature of the aortic arch, which is associated with oscillatory and low shear stress. These differences in miR-155 expression were associated with distinct changes in EC morphology and F-actin. The effects of miR-155 in vitro were mediated through suppression of two key regulators of the EC cytoskeleton organization: RhoA and myosin light chain kinase (MYLK). A novel direct interaction between miR-155 and the MYLK 3'UTR was verified by luciferase-MYLK 3'UTR reporter assays. Furthermore, the intensity of immunofluorescence staining for RhoA and MYLK in mouse aorta correlated inversely with miR-155 expression. In conclusion, a prominent effect of the multifunctional miR-155 in ECs is modulation of phenotype through alterations in RhoA, MYLK expression, and actin cytoskeleton organization.
Assuntos
Citoesqueleto de Actina/ultraestrutura , Células Endoteliais/enzimologia , Células Endoteliais/ultraestrutura , MicroRNAs/fisiologia , Quinase de Cadeia Leve de Miosina/metabolismo , Animais , Aorta/química , Aorta/metabolismo , Aterosclerose , Imunofluorescência , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Quinase de Cadeia Leve de Miosina/análise , Fenótipo , Transfecção , Proteína rhoA de Ligação ao GTP/análiseRESUMO
Previously, we reported that repeated oral treatment with etoposide (ETP) causes attenuation of oral morphine analgesia through upregulation of ileal P-glycoprotein (P-gp) mediated by Ras homolog gene family, member A (RhoA) activation. However, the detailed mechanism of the increase in ileal P-gp via RhoA activation remains unknown. Recently, it has been reported that ezrin-radixin-moesin (ERM) proteins, linking several plasma-membrane proteins to the actin cytoskeleton, are involved in the membrane localization and functional activity of P-gp. Moreover, the cross-linking activities of ERM are known to be regulated by RhoA and Rho-associated coiled-coil containing kinase (ROCK). Here, we examined the involvement of ERM in the changes in expression of P-gp via RhoA and ROCK in ileal membrane induced by ETP. Repeated oral treatment with ETP significantly increased the ileal membrane localization of ERM and phosphorylated ERM (p-ERM) in association with upregulation of P-gp and activation of RhoA and ROCK. Interestingly, coadministration of rosuvastatin (inhibitor of RhoA activation) and fasudil (ROCK inhibitor) prevented increments in the activation and phosphorylation of ERM, respectively. In conclusion, upregulation of ileal membrane localization of ERM and p-ERM via activation of RhoA/ROCK induced by ETP treatment may be involved in the regulation of ileal membrane localization of P-gp.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Analgésicos Opioides/uso terapêutico , Antineoplásicos Fitogênicos/uso terapêutico , Etoposídeo/uso terapêutico , Morfina/uso terapêutico , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/análise , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Analgesia , Analgésicos Opioides/farmacologia , Animais , Antineoplásicos Fitogênicos/farmacologia , Proteínas do Citoesqueleto/análise , Proteínas do Citoesqueleto/metabolismo , Interações Medicamentosas , Etoposídeo/farmacologia , Fluorbenzenos/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Íleo/efeitos dos fármacos , Íleo/metabolismo , Proteínas de Membrana/análise , Proteínas de Membrana/metabolismo , Camundongos , Proteínas dos Microfilamentos/análise , Proteínas dos Microfilamentos/metabolismo , Morfina/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Rosuvastatina Cálcica , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Regulação para Cima/efeitos dos fármacos , Quinases Associadas a rho/análise , Proteína rhoA de Ligação ao GTP/análiseRESUMO
BACKGROUND: We have recently reported that RhoA may regulate the invasion and metastasis of breast cancer cells as an upstream signal of ezrin in vitro. In this study, we examined the relationship of RhoA signaling activity with ezrin expression in breast cancer and its prognostic significance in patients with breast cancer. METHODS: Paraffin tumor sections of breast cancer were collected retrospectively from 487 patients diagnosed between 2001 and 2004. Immunohistochemical methods were used to detect the expression of RhoA, phosphorylated (activated) RhoA, and ezrin. RESULTS: Ezrin overexpression was detectable in 15.2% of 487 invasive breast cancers. The majority (85.1%) of ezrin-overexpressing tumors coexpressed phosphorylated RhoA; 78.8% of tumors with phosphorylated RhoA cooverexpressed ezrin. Patients whose cancers showed overexpression of ezrin or expression of phosphorylated RhoA had shorter survival rates. CONCLUSIONS: RhoA activation is important in human breast cancer due to its upregulation of ezrin; thus, agents that target phosphorylated RhoA may be useful in the treatment of tumors with ezrin overexpression.