RESUMEN
Nickel pollution is a recognized factor contributing to lung cancer. Understanding the molecular mechanisms of its carcinogenic effects is crucial for lung cancer prevention and treatment. Our previous research identified the downregulation of a long noncoding RNA, maternally expressed gene 3 (MEG3), as a key factor in transforming human bronchial epithelial cells (HBECs) into malignant cells following nickel exposure. In our study, we found that deletion of MEG3 also reduced the expression of RhoGDIß. Notably, artificially increasing RhoGDIß levels counteracted the malignant transformation caused by MEG3 deletion in HBECs. This indicates that the reduction in RhoGDIß contributes to the transformation of HBECs due to MEG3 deletion. Further exploration revealed that MEG3 downregulation led to enhanced c-Jun activity, which in turn promoted miR-200c transcription. High levels of miR-200c subsequently increased the translation of AUF1 protein, stabilizing SOX2 messenger RNA (mRNA). This stabilization affected the regulation of miR-137, SP-1 protein translation, and the suppression of RhoGDIß mRNA transcription and protein expression, leading to cell transformation. Our study underscores the co-regulation of RhoGDIß expression by long noncoding RNA MEG3, multiple microRNAs (miR-200c and miR-137), and RNA-regulated transcription factors (c-Jun, SOX2, and SP1). This intricate network of molecular events sheds light on the nature of lung tumorigenesis. These novel findings pave the way for developing targeted strategies for the prevention and treatment of human lung cancer based on the MEG3/RhoGDIß pathway.
Asunto(s)
Neoplasias Pulmonares , MicroARNs , ARN Largo no Codificante , Humanos , Proliferación Celular/genética , Transformación Celular Neoplásica/genética , Regulación hacia Abajo , Células Epiteliales/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , MicroARNs/genética , MicroARNs/metabolismo , Níquel , Inhibidor beta de Disociación del Nucleótido Guanina rho/antagonistas & inhibidores , Inhibidor beta de Disociación del Nucleótido Guanina rho/genética , Inhibidor beta de Disociación del Nucleótido Guanina rho/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , ARN Mensajero , Factores de Transcripción SOXB1/genética , Ribonucleoproteína Nuclear Heterogénea D0/genética , Ribonucleoproteína Nuclear Heterogénea D0/metabolismoRESUMEN
Small G proteins (e.g., Rac1) play critical regulatory roles in islet ß-cell function in health (physiological insulin secretion) and in metabolic stress (cell dysfunction and demise). Multiple regulatory factors for these G proteins, such as GDP dissociation inhibitors (GDIs), have been implicated in the functional regulation of these G proteins. The current set of investigations is aimed at understanding impact of chronic hyperglycemic stress on the expression and subcellular distribution of three known isoforms of RhoGDIs (RhoGDIα, RhoGDIß, and RhoGDIγ) in insulin-secreting ß-cells. The data accrued in these studies revealed that the expression of RhoGDIß, but not RhoGDIα or RhoGDIγ, is increased in INS-1 832/13 cells, rat islets, and human islets. Hyperglycemic stress also promoted the cleavage of RhoGDIß, leading to its translocation to the nuclear compartment. We also report that RhoGDIα, but not RhoGDIγ, is associated with the nuclear compartment. However, unlike RhoGDIß, hyperglycemic conditions exerted no effects on RhoGDIα's association with nuclear fraction. Based on these observations, and our earlier findings of the translocation of Rac1 to the nuclear compartment under the duress of metabolic stress, we conclude that the RhoGDIß-Rac1 signaling module promotes signals from the cytosolic to the nucleus, culminating in accelerated ß-cell dysfunction under metabolic stress.
Asunto(s)
Células Secretoras de Insulina , Inhibidor beta de Disociación del Nucleótido Guanina rho , Animales , Humanos , Ratas , Proteínas de Unión al GTP/metabolismo , Células Secretoras de Insulina/metabolismo , Inhibidor alfa de Disociación del Nucleótido Guanina rho/metabolismo , Inhibidor beta de Disociación del Nucleótido Guanina rho/metabolismo , Inhibidor gamma de Disociación del Nucleótido Guanina rho/metabolismoRESUMEN
Leukemia is a type of disease in which hematopoietic stem cells proliferate clonally at the genetic level. We discovered previously by high-resolution mass spectrometry that diallyl disulfide (DADS), which is one of the effective ingredients of garlic, reduces the performance of RhoGDI2 from APL HL-60 cells. Although RhoGDI2 is oversubscribed in several cancer categories, the effect of RhoGDI2 in HL-60 cells has remained unexplained. We aimed to investigate the influence of RhoGDI2 on DADS-induced differentiation of HL-60 cells to elucidate the association among the effect of inhibition or over-expression of RhoGDI2 with HL-60 cell polarization, migration and invasion, which is important for establishing a novel generation of inducers to elicit leukemia cell polarization. Co-transfection with RhoGDI2-targeted miRNAs apparently decreases the malignant biological behavior of cells and upregulates cytopenias in DADS-treated HL-60 cell lines, which increases CD11b and decreases CD33 and mRNA levels of Rac1, PAK1 and LIMK1. Meanwhile, we generated HL-60 cell lines with high-expressing RhoGDI2. The proliferation, migration and invasion capacity of such cells were significantly increased by the treated with DADS, while the reduction capacity of the cells was decreased. There was a reduction in CD11b and an increase in CD33 production, as well as an increase in the mRNA levels of Rac1, PAK1 and LIMK1. It also confirmed that inhibition of RhoGDI2 attenuates the EMT cascade via the Rac1/Pak1/LIMK1 pathway, thereby inhibiting the malignant biological behavior of HL-60 cells. Thus, we considered that inhibition of RhoGDI2 expression might be a new therapeutic direction for the treatment of human promyelocytic leukemia. The anti-cancer property of DADS against HL-60 leukemia cells might be regulated by RhoGDI2 through the Rac1-Pak1-LIMK1 pathway, which provides new evidence for DADS as a clinical anti-cancer medicine.
Asunto(s)
Leucemia , Inhibidor beta de Disociación del Nucleótido Guanina rho , Humanos , Compuestos Alílicos/farmacología , Diferenciación Celular/efectos de los fármacos , Disulfuros/farmacología , Células HL-60/efectos de los fármacos , Células HL-60/metabolismo , Leucemia/metabolismo , Leucemia/patología , Quinasas Lim/genética , Quinasas Lim/metabolismo , Quinasas p21 Activadas/metabolismo , Quinasas p21 Activadas/farmacología , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rac1/farmacología , Inhibidor beta de Disociación del Nucleótido Guanina rho/efectos de los fármacos , Inhibidor beta de Disociación del Nucleótido Guanina rho/metabolismo , ARN Mensajero , Invasividad Neoplásica/genética , Invasividad Neoplásica/patologíaRESUMEN
RhoGDI2 is a guanine nucleotide dissociation inhibitor (GDI) specific for the Rho family of small GTPases. It is highly expressed in hematopoietic cells but is also present in a large array of other cell types. RhoGDI2 has been implicated in multiple human cancers and immunity regulation, where it can display a dual role. Despite its involvement in various biological processes, we still do not have a clear understanding of its mechanistic functions. This review sheds a light on the dual opposite role of RhoGDI2 in cancer, highlights its underappreciated role in immunity and proposes ways to explain its intricate regulatory functions.
Asunto(s)
Inmunidad , Neoplasias , Inhibidor beta de Disociación del Nucleótido Guanina rho , Humanos , Neoplasias/metabolismo , Inhibidor beta de Disociación del Nucleótido Guanina rho/metabolismoRESUMEN
BACKGROUND: Faecal biomarkers have emerged as important tools in managing of inflammatory bowel disease [IBD], which includes Crohn's disease [CD] and ulcerative colitis [UC]. AIM: To identify new biomarkers of gut inflammation in the stools of IBD patients using a proteomic approach. METHODS: Proteomic analysis of stools was performed in patients with both active CD and CD in remission and in controls by 2-DIGE and MALDI-TOF/TOF MS. An ELISA was used to confirm results in a second cohort of IBD patients and controls. RESULTS: 2-DIGE analysis detected 70 spots in the stools of patients with active CD or patients in remission CD and in controls. MALDI-TOF/TOF MS analysis identified 21 proteins with Chymotrypsin C, Gelsolin and Rho GDP-dissociation inhibitor 2 [RhoGDI2] best correlating with the levels of intestinal inflammation. Results were confirmed in a second cohort of IBD patients and controls [57 CD, 60 UC, 31 controls]. The identified faecal markers significantly correlated with the severity of intestinal inflammation in IBD patients [SES-CD in CD, Mayo endoscopic subscore in UC] [CD; Chymotrypsin-C: r = 0.64, p < 0.001; Gelsolin: r = 0.82, p < 0.001; RhoGDI2: r = 0.64, p < 0.001; UC; Chymotrypsin-C: r = 0.76, p < 0.001; Gelsolin: r = 0.75, p < 0.001; RhoGDI2: r = 0.63, p < 0.001]. Moreover, ROC analysis showed that Gelsolin [p < 0.0002] and RhoGDI2 [p < 0.0001] in CD, and RhoGDI2 [p = 0.0004] in UC, have higher sensitivity and specificity than faecal calprotectin in discriminating between patients and controls. CONCLUSIONS: We show for the first time that 2-DIGE is a reliable method to detect proteins in human stools. Three novel faecal biomarkers of gut inflammation have been identified that display good specificity and sensitivity for identifying IBD and significantly correlate with IBD severity.
Asunto(s)
Colitis Ulcerosa , Enfermedad de Crohn , Enfermedades Inflamatorias del Intestino , Humanos , Quimotripsina/metabolismo , Gelsolina/metabolismo , Proteómica , Inhibidor beta de Disociación del Nucleótido Guanina rho/metabolismo , Mucosa Intestinal/metabolismo , Enfermedades Inflamatorias del Intestino/complicaciones , Enfermedades Inflamatorias del Intestino/diagnóstico , Enfermedades Inflamatorias del Intestino/metabolismo , Colitis Ulcerosa/diagnóstico , Colitis Ulcerosa/metabolismo , Enfermedad de Crohn/diagnóstico , Enfermedad de Crohn/metabolismo , Biomarcadores/análisis , Inflamación/metabolismo , Complejo de Antígeno L1 de Leucocito/análisis , Heces/química , Índice de Severidad de la EnfermedadRESUMEN
BACKGROUND: Rho GDP dissociation inhibitor 2 (RhoGDI2) has been shown to contribute to the aggressive phenotypes of human cancers, such as tumor metastasis and chemoresistance. OBJECTIVE: This study aimed to assess the effects of RhoGDI2 on tumor progression and chemoresistance in pancreatic cancer cells. METHODS: The expression of RhoGDI2 in pancreatic cancer cells was detected by Western blot analysis. Gain-of-function and loss-of-function approaches were done to examine the malignant phenotypes of the RhoGDI2-expressing or RhoGDI2-depleting cells. The correlation between RhoGDI2 and Snail was also analyzed. RESULTS: Differential expression of RhoGDI2 protein in pancreatic cancer cell lines was identified. Gain-of-function and loss-of-function experiments showed that RhoGDI2 induced the malignant phenotypes of pancreatic cancer cells, including proliferation, migration, invasion, and gemcitabine (GEM) chemoresistance. The upregulation of RhoGDI2 stimulated the expression of Snail, resulting in the altered expression of epithelial marker E-cadherin and mesenchymal marker Vimentin, which were characteristics of the tumorigenic activity of epithelial-mesenchymal transition. The expression of RhoGDI2 and Snail was upregulated in clinical tumor samples, and higher expression of RhoGDI2 or Snail was significantly associated with poor patient survival in pancreatic ductal adenocarcinoma (PDAC). CONCLUSION: The findings indicated that RhoGDI2 promoted GEM resistance and tumor progression in pancreatic cancer and that RhoGDI2 might be a potential therapeutic target in patients with PDAC.
Asunto(s)
Neoplasias Pancreáticas , Inhibidor beta de Disociación del Nucleótido Guanina rho , Transición Epitelial-Mesenquimal/genética , Humanos , Neoplasias Pancreáticas/metabolismo , Fenotipo , Inhibidor beta de Disociación del Nucleótido Guanina rho/genética , Inhibidor beta de Disociación del Nucleótido Guanina rho/metabolismo , Neoplasias PancreáticasRESUMEN
The fusion gene of ABL1 is closely related to tumor proliferation, invasion, and migration. It has been reported recently that ABL1 itself is required for T-cell acute lymphoblastic leukemia (T-ALL) cell migration induced by CXCL12. Further experiments revealed that ABL1 inhibitor Nilotinib inhibited leukemia cell migration induced by CXCL12, indicating the possible application of Nilotinib in T-ALL leukemia treatment. However, the interacting proteins of ABL1 and the specific mechanisms of their involvement in this process need further investigation. In the present study, ABL1 interacting proteins were characterized and their roles in the process of leukemia cell migration induced by CXCL12 were investigated. Co-immunoprecipitation in combination with mass spectrometry analysis identified 333 proteins that interact with ABL1, including Cofilin1. Gene ontology analysis revealed that many of them were enriched in the intracellular organelle or cytoplasm, including nucleic acid binding components, transfectors, or co-transfectors. Kyoto Encyclopedia of Genes and Genomes analysis showed that the top three enriched pathways were translation, glycan biosynthesis, and metabolism, together with human diseases. ABL1 and Cofilin1 were in the same complex. Cofilin1 binds the SH3 domain of ABL1 directly; however, ABL1 is not required for the phosphorylation of Cofilin1. Molecular docking analysis shows that ABL1 interacts with Cofilin1 mainly through hydrogen bonds and ionic interaction between amino acid residues. The mobility of leukemic cells was significantly decreased by Cofilin1 siRNA. These results demonstrate that Cofilin1 is a novel ABL1 binding partner. Furthermore, Cofilin1 participates in the migration of leukemia cells induced by CXCL12. These data indicate that ABL1 and Cofilin1 are possible targets for T-ALL treatment.
Asunto(s)
Movimiento Celular/inmunología , Cofilina 1/inmunología , Cofilina 1/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteínas Proto-Oncogénicas c-abl/inmunología , Proteínas Proto-Oncogénicas c-abl/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Quimiocina CXCL12/farmacología , Cofilina 1/genética , Biología Computacional , Citoesqueleto/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos DBA , Simulación del Acoplamiento Molecular , Dominios Proteicos , Proteínas Proto-Oncogénicas c-abl/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-abl/genética , Pirimidinas/farmacología , Linfocitos T/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Inhibidor beta de Disociación del Nucleótido Guanina rho/metabolismoRESUMEN
BACKGROUND: KDM6A, a histone demethylase, is frequently mutated in bladder cancer (BCa). However, the role and detailed molecular mechanism of KDM6A involved in bladder cancer progression remains unknown. METHODS: Tissue specimens were used to determine the expression levels and prognostic values of KDM6A and ARHGDIB. The MTT, colony formation, wound healing and Transwell migration and invasion assays were employed to detect the BCa cell proliferation, migration and invasion, respectively. Chemotaxis of macrophages was used to evaluate the ability of KDM6A to recruit macrophages. A subcutaneous tumour model and tail vein tumour injection in nude mice were used to assess the role of KDM6A in vivo. RNA sequencing, qPCR, Western blot, ChIP and phalloidin staining assay were performed to investigate the molecular functions of KDM6A. Dual-luciferase reporter assay was used to determine the effects of KDM6A and FOXA1 on the promoters of the ARHGDIB and KDM6A. RESULTS: We showed that the KDM6A inhibited the motility and invasiveness of the BCa cells. Mechanistically, KDM6A promotes the transcription of ARHGDIB by demethylating histone H3 lysine di/trimethylation (H3K27me2/3) and consequently leads to inhibition of Rac1. EZH2, which catalyses the methylation of H3K27, functions to silence ARHGDIB expression, and an EZH2 inhibitor can neutralize the metastatic effect caused by KDM6A deficiency. Furthermore, we demonstrated that FOXA1 directly binds to the KDM6A promoter and thus transactivates KDM6A, leading to diminished metastatic potential. CONCLUSION: Our findings establish the critical role of the FOXA1-KDM6A-ARHGDIB axis in restraining the malignancy of BCa and identify KDM6A and EZH2 as potential therapeutic targets in the management of BCa.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/fisiología , Histona Demetilasas/metabolismo , Neoplasias de la Vejiga Urinaria/patología , Proteína de Unión al GTP rac1/biosíntesis , Inhibidor beta de Disociación del Nucleótido Guanina rho/metabolismo , Animales , Movimiento Celular/fisiología , Xenoinjertos , Humanos , Ratones , Ratones Desnudos , Invasividad Neoplásica/patologíaRESUMEN
There are three RhoGDIs in mammalian cells, which were initially defined as negative regulators of Rho family small GTPases. However, it is now accepted that RhoGDIs not only maintain small GTPases in their inactive GDP-bound form but also act as chaperones for small GTPases, targeting them to specific intracellular membranes and protecting them from degradation. Studies to date with RhoGDIs have usually focused on the interactions between the "typical" or "classical" small GTPases, such as the Rho, Rac, and Cdc42 subfamily members, and either the widely expressed RhoGDI-1 or the hematopoietic-specific RhoGDI-2. Less is known about the third member of the family, RhoGDI-3 and its interacting partners. RhoGDI-3 has a unique N-terminal extension and is found to localize in both the cytoplasm and the Golgi. RhoGDI-3 has been shown to target RhoB and RhoG to endomembranes. In order to facilitate a more thorough understanding of RhoGDI function, we undertook a systematic study to determine all possible Rho family small GTPases that interact with the RhoGDIs. RhoGDI-1 and RhoGDI-2 were found to have relatively restricted activity, mainly binding members of the Rho and Rac subfamilies. RhoGDI-3 displayed wider specificity, interacting with the members of Rho, Rac, and Cdc42 subfamilies but also forming complexes with "atypical" small Rho GTPases such as Wrch2/RhoV, Rnd2, Miro2, and RhoH. Levels of RhoA, RhoB, RhoC, Rac1, RhoH, and Wrch2/RhoV bound to GTP were found to decrease following coexpression with RhoGDI-3, confirming its role as a negative regulator of these small Rho GTPases.
Asunto(s)
Inhibidor alfa de Disociación del Nucleótido Guanina rho/metabolismo , Inhibidor beta de Disociación del Nucleótido Guanina rho/metabolismo , Inhibidor gamma de Disociación del Nucleótido Guanina rho/metabolismo , Secuencia de Aminoácidos , Membrana Celular/metabolismo , Proteínas de Unión al GTP/metabolismo , Inhibidores de Disociación de Guanina Nucleótido/química , Células HEK293 , Humanos , Proteínas de Unión al GTP Monoméricas/metabolismo , Unión Proteica , Proteínas de Unión al GTP rho/química , Inhibidor alfa de Disociación del Nucleótido Guanina rho/fisiología , Inhibidor beta de Disociación del Nucleótido Guanina rho/fisiología , Inhibidor gamma de Disociación del Nucleótido Guanina rho/fisiología , Inhibidores de la Disociación del Nucleótido Guanina rho-Específico/metabolismo , Inhibidores de la Disociación del Nucleótido Guanina rho-Específico/fisiologíaRESUMEN
BACKGROUND: ARHGDIB, a Rho GDP dissociation inhibitor protein, has been reported playing critical roles in regulation of multiple biological responses. However, whether ARHGDIB serves as a valuable biomarker in cancer is little known so far, especially in breast cancer. OBJECTIVE: In this study, we aimed to investigate the importance of ARHGDIB in breast cancer, including but not limited to biomarker-like role, as well as potential mechanisms. METHODS: Total 100 breast cancer samples and 100 benign breast disease samples were enrolled and underwent detailed pathological assessment and IHC analysis. Human breast cancer cell lines and epithelial cell line were subjected to siRNA-mediated knock-down, RT-qPCR, western blot, MTT staining, cell cycle assay, transwell analysis respectively. RESULTS: We observed the expression of ARHGDIB is significantly higher in human breast cancer tissues compared with the benign tissues. ARHGDIB expression was positively correlated with tumor size, lymph node metastasis and TNM stage in breast cancer patients. Moreover, ARHGDIB depletion decreased proliferation, migration and invasion of breast cancer cells. Furthermore, we found ARHGDIB mediated epithelial-mesenchymal transition, and MMP2 is the key downstream effector of ARHGDIB. CONCLUSIONS: Hence, our results suggested the significance and predictive role of ARHGDIB in breast cancer. High expression of ARHGDIB indicated the poor prognosis for breast cancer patients.
Asunto(s)
Neoplasias de la Mama/metabolismo , Inhibidor beta de Disociación del Nucleótido Guanina rho/metabolismo , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Femenino , Humanos , Inmunohistoquímica , Metástasis Linfática , Persona de Mediana Edad , Invasividad Neoplásica , Estadificación de Neoplasias , Pronóstico , Transfección , Inhibidor beta de Disociación del Nucleótido Guanina rho/biosíntesis , Inhibidor beta de Disociación del Nucleótido Guanina rho/genéticaRESUMEN
BACKGROUND: The impact of donor-specific anti-HLA antibodies (DSA) on antibody-mediated rejection (AMR) and kidney allograft failure is well established. However, the relevance of non-HLA antibodies remains unclear. METHODS: We investigated 13 pretransplant non-HLA antibodies and their association with histology of AMR (AMRh) and kidney allograft failure. We included single kidney recipients (n = 203) with AMRh, according to the Banff 2017 classification and matched AMRh-free controls (n = 219). Non-HLA antibodies were assessed using multiplex Luminex assay. RESULTS: Of the selected non-HLA antibodies (against agrin, adipocyte plasma membrane-associated protein, Rho GDP-dissociation inhibitor 2 [ARHGDIB], Rho guanine nucleotide exchange factor 6, angiotensin-II type 1 receptor, endothelin type A receptor, lamin B1, BPI fold-containing family B member 1, peroxisomal trans-2-enoyl-coenzyme A reductase, phospholipase A2 receptor, protein kinase C zeta type, tubulin beta-4B class IVb, vimentin), only antibodies against ARHGDIB (adjusted median fluorescence intensity [aMFI] ≥ 1000), a minor histocompatibility antigen, associated with graft failure, in univariate and multivariate models (hazard ratio = 2.7; 95% confidence interval [CI],1.3-5.4; P = 0.007). There was a 19.5-fold (95% CI, 6.0-63.9; P < 0.0001) increased risk of graft failure in patients positive for both DSA and anti-ARHGDIB antibodies (aMFI ≥ 1000) versus patients negative for both DSA and anti-ARHGDIB antibodies, compared with a 4.4-fold (95% CI, 2.4-8.2; P < 0.0001) increased risk in patients with only DSA, and a 4.1-fold (95% CI, 1.4-11.7; P = 0.009) increased risk in patients with only anti-ARHGDIB antibodies above 2000 aMFI. AMRh associated with increased intrarenal expression of the ARHGDIB gene. In the absence of AMRh and DSA, anti-ARHGDIB antibodies were not clearly associated with graft failure. CONCLUSIONS: The presence of pretransplant anti-ARHGDIB antibodies has an additive effect in patients with DSA on the risk of graft failure via AMRh. Other investigated non-HLA antibodies, including antibodies against angiotensin-II type 1 receptor, did not contribute to risk stratification and could not explain the histology of AMR in the absence of DSA.
Asunto(s)
Autoanticuerpos/sangre , Rechazo de Injerto/epidemiología , Fallo Renal Crónico/cirugía , Trasplante de Riñón/efectos adversos , Inhibidor beta de Disociación del Nucleótido Guanina rho/metabolismo , Adulto , Anciano , Aloinjertos/inmunología , Aloinjertos/patología , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Autoantígenos/metabolismo , Biopsia/estadística & datos numéricos , Estudios de Casos y Controles , Femenino , Estudios de Seguimiento , Perfilación de la Expresión Génica/estadística & datos numéricos , Rechazo de Injerto/diagnóstico , Rechazo de Injerto/inmunología , Rechazo de Injerto/patología , Supervivencia de Injerto/inmunología , Prueba de Histocompatibilidad/métodos , Prueba de Histocompatibilidad/estadística & datos numéricos , Humanos , Riñón/inmunología , Riñón/patología , Fallo Renal Crónico/sangre , Fallo Renal Crónico/inmunología , Masculino , Persona de Mediana Edad , Periodo Preoperatorio , Estudios Prospectivos , Medición de Riesgo/métodos , Medición de Riesgo/estadística & datos numéricos , Trasplante Homólogo/efectos adversos , Inhibidor beta de Disociación del Nucleótido Guanina rho/inmunologíaRESUMEN
BACKGROUND AND AIMS: The physiological role of Rho-specific guanine nucleotide dissociation inhibitor (RhoGDI) in vascular remodeling remains unknown. We investigated the function of RhoGDI in angiotensin II (Ang II)-induced vascular remodeling in cultured human aortic vascular smooth muscle cells (HA-VSMCs) and in an Ang II-infusion vascular remodeling mouse model. METHODS: For in vitro assays of HA-VSMCs, proliferation was assessed by BrdU and EdU assays and immunofluorescence analysis of ki-67 expression. RhoGDI1 and RhoGDI2 function and expression were assessed by RNAi, Western blotting and real-time RT-PCR. RhoGDI ubiquitination and SUMOylation levels were evaluated by co-immunoprecipitation and Western blotting. The functions of proteosomal-mediated degradation, ubiquitination, SUMOylation and Ang II receptors were assessed using specific inhibitors. To evaluate the in vivo effects of Ang II and RhoGDI, H & E staining, Masson's trichrome staining, and immunostaining were employed. RESULTS: Ang II treatment of HA-VSMCs for 6 or 48â¯h promoted RhoGDI1 and RhoGDI2 protein degradation and reduced cell proliferation, which was reversed by proteosome inhibition. In contrast, treatment with Ang II for 12 or 24â¯h induced dose-dependent cell proliferation without affecting RhoGDI expression. RNA interference of either RhoGDI1 or RhoGDI2 blocked proliferation induced by 12 or 24â¯h treatment of Ang II. Moreover, Ang II-dependent degradation at 6 and 48â¯h correlated with RhoGDI ubiquitination and inversely correlated with RhoGDI SUMOylation and cell proliferation. Treatment with specific inhibitors suggests that ubiquitin and SUMO competitively bind to RhoGDI1 and RhoGDI2 to reciprocally regulate RhoGDI stability and HA-VSMC proliferation. Furthermore, inhibition of the Ang II receptor 1 (AT1 receptor), but not the Ang II receptor 2, blocked Ang II-dependent RhoGDI stabilization and proliferation at 12 and 24â¯h. In mice, Ang II infusion increased the intima-media thickness, collagen and myofiber production and VSMC proliferation, and these effects were shown to be dependent on RhoGDI1, RhoGDI2 and AT1 receptor. Ang II infusion exerted no significant effect on RhoGDI1 and RhoGDI2 protein levels, which were decreased after AT1 receptor inhibition. CONCLUSIONS: Together, the results of this study reveal a novel mechanism by which Ang II regulates RhoGDI stability by SUMOylation and ubiquitination via AT1 receptor activation and thus affects VSMC proliferation and vascular remodeling.
Asunto(s)
Angiotensina II/farmacología , Proliferación Celular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Receptor de Angiotensina Tipo 1/agonistas , Ubiquitinación , Remodelación Vascular/efectos de los fármacos , Inhibidor alfa de Disociación del Nucleótido Guanina rho/metabolismo , Inhibidor beta de Disociación del Nucleótido Guanina rho/metabolismo , Animales , Células Cultivadas , Humanos , Masculino , Ratones Endogámicos C57BL , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Neointima , Complejo de la Endopetidasa Proteasomal/metabolismo , Estabilidad Proteica , Proteolisis , Receptor de Angiotensina Tipo 1/metabolismo , Transducción de Señal , Sumoilación , Inhibidor alfa de Disociación del Nucleótido Guanina rho/genética , Inhibidor beta de Disociación del Nucleótido Guanina rho/genéticaRESUMEN
Cytoskeletal reorganization driven by Rho GTPases plays a crucial role in the migration of T cells, which are key regulators of immunity. The molecular mechanisms that control actin cytoskeleton remodeling during T cell movement have only partially been clarified as the function of many modulators has not been evaluated in these cells. Here, we report a new function of RhoGDI2 by showing that this protein positively regulates Rho GTPase activation during T cell adhesion and migration. RhoGDI2 knockdown significantly reduced T cell adhesion and migration. Furthermore, RhoGDI2 knockdown decreased the activation of Rac1 and Cdc42, 2 members of Rho GTPases, and the remodeling of the actin cytoskeleton. Upon P-selectin glycoprotein ligand-1 engagement, RhoGDI2 was phosphorylated at Y24 and Y153 by kinases related to ß2 integrin outside-in signaling, Src, c-Abl, and Syk, resulting in the accumulation of RhoGDI2 at the cell membrane. Subsequent phosphorylation of S31 induced the opening of RhoGDI2 and the release of Rho GTPases, whereas phosphorylation of Y153 might promote the activation of Rho GTPases by recruiting Vav1. Moreover, the disruption of lipid rafts with methyl-ß-cyclodextrin blocked the interaction between integrins and RhoGDI2, reducing the level of phosphorylated RhoGDI2 and the activation of downstream Rho GTPases. Based on these observations, RhoGDI2 is a target of intergrin outside-in signaling that activates Rho GTPases during T cell adhesion and migration, and RhoGDI2-mediated signal transduction is based on the lipid rafts integrity.
Asunto(s)
Antígenos CD18/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Transducción de Señal , Linfocitos T/inmunología , Linfocitos T/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Inhibidor beta de Disociación del Nucleótido Guanina rho/metabolismo , Adhesión Celular/genética , Adhesión Celular/inmunología , Quimiotaxis de Leucocito/genética , Quimiotaxis de Leucocito/inmunología , Humanos , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Glicoproteínas de Membrana/metabolismo , Microdominios de Membrana/metabolismo , Fosforilación , Inhibidor beta de Disociación del Nucleótido Guanina rho/deficienciaRESUMEN
Although overexpression of the non-canonical NFκB subunit p52 has been observed in several tumors, the function and mechanism of p52 in bladder cancer (BC) are less well understood. Here, we aimed at understanding the role and mechanism underlying p52 regulation of BC invasion. Human p52 was stably knockdown with shRNA targeting p52 in two bladder cancer cell lines (T24 and UMUC3). Two constitutively expressing constructs, p52 and p100, were stably transfected in to T24 or UMUC3, respectively. The stable transfectants were used to determine function and mechanisms responsible for p52 regulation of BC invasion. We demonstrate that p52 mediates human BC invasion. Knockdown of p52 impaired bladder cancer invasion by reduction of rhogdiß mRNA stability and expression. Positively regulation of rhogdiß mRNA stability was mediated by p52 promoting AUF1 protein degradation, consequently resulting in reduction of AUF1 binding to rhogdiß mRNA. Further studies indicated that AUF1 protein degradation was mediated by upregulating USP8 transcription, which was modulated by its negative regulatory transcription factor Sp1. Moreover, we found that p52 upregulated miR-145, which directly bound to the 3'-UTR of sp1 mRNA, leading to downregulation of Sp1 protein translation. Our results reveal a comprehensive pathway that p52 acts as a positive regulator of BC invasion by initiating a novel miR-145/Sp1/USP8/AUF1/RhoGDIß axis. These findings provide insight into the understanding of p52 in the pathology of human BC invasion and progression, which may be useful information in the development of preventive and therapeutic approaches for using p52 as a potential target.
Asunto(s)
Endopeptidasas/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo D/metabolismo , MicroARNs/metabolismo , Subunidad p52 de NF-kappa B/metabolismo , Estabilidad del ARN , Factor de Transcripción Sp1/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Neoplasias de la Vejiga Urinaria/patología , Inhibidor beta de Disociación del Nucleótido Guanina rho/metabolismo , Endopeptidasas/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Regulación Neoplásica de la Expresión Génica , Ribonucleoproteína Nuclear Heterogénea D0 , Ribonucleoproteína Heterogénea-Nuclear Grupo D/genética , Humanos , MicroARNs/genética , Subunidad p52 de NF-kappa B/genética , Biosíntesis de Proteínas , Proteolisis , Factor de Transcripción Sp1/genética , Células Tumorales Cultivadas , Ubiquitina Tiolesterasa/genética , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/metabolismo , Inhibidor beta de Disociación del Nucleótido Guanina rho/química , Inhibidor beta de Disociación del Nucleótido Guanina rho/genéticaRESUMEN
OBJECTIVE: This study aimed to investigate the effect of adenosine (Ado) on the growth of ovarian cancer and to explore the related mechanisms. METHODS: The effect of Ado on the proliferation of A2780 human ovarian cancer cells was examined according to the MTT method. Moreover, the nude mouse model of subcutaneous A2780 xenograft was constructed, and then, Ado and cisplatin were administered intraperitoneally to investigate the effect of Ado on tumor growth in vivo. Immunohistochemistry (IHC) was carried out to study the effect of Ado on the expression of Rho-specific guanine nucleotide dissociation inhibitor 2 (RhoGDI2) in the subcutaneous xenografts. Afterwards, the commercially constructed RhoGDI2 siRNA plasmid was transfected into A2780 cells, and tube formation assay was conducted to determine the effect of down-regulating RhoGDI2 expression on the regulation of angiogenesis in ovarian cancer by Ado. Besides, Western blotting was performed to detect the effect of RhoGDI2 down-regulation on the regulation of matrix metalloproteinase 2 (MMP-2), MMP-9, vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF-ß), tumor necrosis factor (TNF-α), and platelet endothelial cell adhesion molecule-1 (PECAM-1 or CD31) expression in ovarian cancer cells by Ado. RESULTS: The relative viability of cells subsequent to Ado treatment proved to be both concentration- and time dependent. IHC results showed that Ado evidently enhanced the RhoGDI2 protein expression. In addition, interference with RhoGDI2 outstandingly attenuated the ability of Ado to suppress tumor cell invasion and induce angiogenesis in vitro. Furthermore, molecular mechanism studies indicated that Ado remarkably inhibited the expression of MMP-2, MMP-9, VEGF, TGF-ß, TNF-α, and CD31, while interference with RhoGDI2 restored the expression of the above-mentioned angiogenic factors. CONCLUSION: Ado inhibits the growth of A2780 human ovarian cancer cells through inhibiting tumor cell invasion and angiogenesis in a RhoGDI2-dependent manner.
Asunto(s)
Adenosina/farmacología , Antineoplásicos/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Inhibidor beta de Disociación del Nucleótido Guanina rho/biosíntesis , Adenosina/química , Animales , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Neoplasias Ováricas/metabolismo , Inhibidor beta de Disociación del Nucleótido Guanina rho/genética , Inhibidor beta de Disociación del Nucleótido Guanina rho/metabolismoRESUMEN
Interferon gamma (IFN-γ) is a dimeric soluble cytokine and the only type II interferon. Accumulated evidence suggests that IFN-γ inhibits tumor progression. This study investigated the effects of IFN-γ on the proliferation and migration of pancreatic cancer (PC) cells and the underlying mechanism. IFN-γ treatment decreased the expression and secretion of CXCL8 in BxPC-3 PC cells, suppressed the proliferation and migration of these cells, and enhanced their apoptosis, as determined by increased levels of cleaved Caspase-8 and Bax together with reduced expression of Bcl-2. These effects were abolished by overexpression of CXCL8. Moreover, IFN-γ treatment downregulated RhoGDI2 expression. Depletion of RhoGDI2 and Rac1 by using small interfering RNAs and inhibition of NF-κB by BMS-345541 (an IκB kinase [IKK] inhibitor) suppressed expression of CXCL8. Our results indicate that IFN-γ inhibits the proliferation and migration of PC cells by suppressing CXCL8 expression via a RhoGDI2/Rac1/NF-κB signaling pathway.
Asunto(s)
Antineoplásicos/farmacología , Movimiento Celular/efectos de los fármacos , Interferón gamma/farmacología , Interleucina-8/metabolismo , FN-kappa B/antagonistas & inhibidores , Neoplasias Pancreáticas/tratamiento farmacológico , Proteína de Unión al GTP rac1/metabolismo , Inhibidor beta de Disociación del Nucleótido Guanina rho/biosíntesis , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , FN-kappa B/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Transducción de Señal/efectos de los fármacos , Proteína de Unión al GTP rac1/deficiencia , Inhibidor beta de Disociación del Nucleótido Guanina rho/metabolismoRESUMEN
Our recent studies demonstrate that X-linked inhibitor of apoptosis protein (XIAP) is essential for regulating colorectal cancer invasion. Here, we discovered that RhoGDIß was a key XIAP downstream effector mediating bladder cancer (BC) invasion in vitro and in vivo. We found that both XIAP and RhoGDIß expressions were consistently elevated in BCs of N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-treated mice in comparison to bladder tissues from vehicle-treated mice and human BCs in comparison to the paired adjacent normal bladder tissues. Knockdown of XIAP attenuated RhoGDIß expression and reduced cancer cell invasion, whereas RhoGDIß expression was attenuated in BBN-treated urothelium of RING-deletion knockin mice. Mechanistically, XIAP stabilized RhoGDIß mRNA by its positively regulating nucleolin mRNA stability via Erks-dependent manner. Moreover, ectopic expression of GFP-RhoGDIß in T24T(shXIAP) cells restored its lung metastasis in nude mice. Our results demonstrate that XIAP-regulated Erks/nucleolin/RhoGDIß axis promoted BC invasion and lung metastasis.
Asunto(s)
Proteínas Inhibidoras de la Apoptosis/biosíntesis , Neoplasias Pulmonares/secundario , Fosfoproteínas/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Neoplasias de la Vejiga Urinaria/patología , Proteína Inhibidora de la Apoptosis Ligada a X/biosíntesis , Inhibidor beta de Disociación del Nucleótido Guanina rho/genética , Animales , Línea Celular Tumoral , Femenino , Células HCT116 , Humanos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Invasividad Neoplásica , ARN Mensajero/genética , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Inhibidor beta de Disociación del Nucleótido Guanina rho/metabolismo , NucleolinaRESUMEN
Our most recent studies demonstrate that RhoGDIß is able to promote human bladder cancer (BC) invasion and metastasis in an X-link inhibitor of apoptosis protein-dependent fashion accompanied by increased levels of matrix metalloproteinase (MMP)-2 protein expression. We also found that RhoGDIß and MMP-2 protein expressions are consistently upregulated in both invasive BC tissues and cell lines. In the present study, we show that knockdown of RhoGDIß inhibited MMP-2 protein expression accompanied by a reduction of invasion in human BC cells, whereas ectopic expression of RhoGDIß upregulated MMP-2 protein expression and promoted invasion as well. The mechanistic studies indicated that MMP-2 was upregulated by RhoGDIß at the transcriptional level by increased specific binding of the transcription factor Sp1 to the mmp-2 promoter region. Further investigation revealed that RhoGDIß overexpression led to downregulation of miR-200c, whereas miR-200c was able directly to target 3'-UTR of jnk2mRNA and attenuated JNK2 protein translation, which resulted in attenuation of Sp1mRNA and protein expression in turn, inhibiting Sp1-dependent mmp-2 transcription. Collectively, our studies demonstrate that RhoGDIß overexpression inhibits miR-200c abundance, which consequently results in increases of JNK2 protein translation, Sp1 expression, mmp-2 transcription, and BC invasion. These findings, together with our previous results showing X-link inhibitor of apoptosis protein mediating mRNA stabilization of both RhoGDIß and mmp-2, reveal the nature of the MMP-2 regulatory network, which leads to MMP-2 overexpression and BC invasion.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Metaloproteinasa 2 de la Matriz/genética , MicroARNs/genética , Proteína Quinasa 9 Activada por Mitógenos/genética , Invasividad Neoplásica/genética , Factor de Transcripción Sp1/genética , Neoplasias de la Vejiga Urinaria/genética , Inhibidor beta de Disociación del Nucleótido Guanina rho/genética , Línea Celular Tumoral , Regulación hacia Abajo , Técnicas de Silenciamiento del Gen , Humanos , Metaloproteinasa 2 de la Matriz/metabolismo , MicroARNs/metabolismo , Proteína Quinasa 9 Activada por Mitógenos/metabolismo , Invasividad Neoplásica/patología , Biosíntesis de Proteínas , Factor de Transcripción Sp1/metabolismo , Regulación hacia Arriba , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/patología , Inhibidor beta de Disociación del Nucleótido Guanina rho/metabolismoRESUMEN
BACKGROUND: The importance of the mTOR complex 2 (mTORC2) signaling complex in tumor progression is becoming increasingly recognized. HER2-amplified breast cancers use Rictor/mTORC2 signaling to drive tumor formation, tumor cell survival and resistance to human epidermal growth factor receptor 2 (HER2)-targeted therapy. Cell motility, a key step in the metastatic process, can be activated by mTORC2 in luminal and triple negative breast cancer cell lines, but its role in promoting metastases from HER2-amplified breast cancers is not yet clear. METHODS: Because Rictor is an obligate cofactor of mTORC2, we genetically engineered Rictor ablation or overexpression in mouse and human HER2-amplified breast cancer models for modulation of mTORC2 activity. Signaling through mTORC2-dependent pathways was also manipulated using pharmacological inhibitors of mTOR, Akt, and Rac. Signaling was assessed by western analysis and biochemical pull-down assays specific for Rac-GTP and for active Rac guanine nucleotide exchange factors (GEFs). Metastases were assessed from spontaneous tumors and from intravenously delivered tumor cells. Motility and invasion of cells was assessed using Matrigel-coated transwell assays. RESULTS: We found that Rictor ablation potently impaired, while Rictor overexpression increased, metastasis in spontaneous and intravenously seeded models of HER2-overexpressing breast cancers. Additionally, migration and invasion of HER2-amplified human breast cancer cells was diminished in the absence of Rictor, or upon pharmacological mTOR kinase inhibition. Active Rac1 was required for Rictor-dependent invasion and motility, which rescued invasion/motility in Rictor depleted cells. Rictor/mTORC2-dependent dampening of the endogenous Rac1 inhibitor RhoGDI2, a factor that correlated directly with increased overall survival in HER2-amplified breast cancer patients, promoted Rac1 activity and tumor cell invasion/migration. The mTORC2 substrate Akt did not affect RhoGDI2 dampening, but partially increased Rac1 activity through the Rac-GEF Tiam1, thus partially rescuing cell invasion/motility. The mTORC2 effector protein kinase C (PKC)α did rescue Rictor-mediated RhoGDI2 downregulation, partially rescuing Rac-guanosine triphosphate (GTP) and migration/motility. CONCLUSION: These findings suggest that mTORC2 uses two coordinated pathways to activate cell invasion/motility, both of which converge on Rac1. Akt signaling activates Rac1 through the Rac-GEF Tiam1, while PKC signaling dampens expression of the endogenous Rac1 inhibitor, RhoGDI2.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Transducción de Señal , Proteína de Unión al GTP rac1/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/mortalidad , Línea Celular Tumoral , Movimiento Celular/genética , Modelos Animales de Enfermedad , Femenino , Amplificación de Genes , Xenoinjertos , Humanos , Ratones , Ratones Transgénicos , Metástasis de la Neoplasia , Estadificación de Neoplasias , Pronóstico , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína Asociada al mTOR Insensible a la Rapamicina/genética , Proteína Asociada al mTOR Insensible a la Rapamicina/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Inhibidor beta de Disociación del Nucleótido Guanina rho/genética , Inhibidor beta de Disociación del Nucleótido Guanina rho/metabolismoRESUMEN
Rho GDP-dissociation inhibitor ß (RhoGDIß), a regulator of the Rho family of proteins, is expressed abundantly in the hematopoietic cell lineage. During apoptosis of hematopoietic cells, RhoGDIß is cleaved by caspase3 at Asp19 and this cleaved form (Δ19RhoGDIß) has been implicated in the apoptotic pathway. To clarify the role of RhoGDIß in hematopoietic cells, the present study performed immunoblotting and immunofluorescence staining to examine the expression of RhoGDIß and ∆19RhoGDIß during phorbol 12myristate 13acetate (PMA)stimulated differentiation of human THP1 monocytic cells to macrophages. During differentiation of the THP1 cells to macrophages, the expression of RhoGDIß remained stable; however, the expression of Δ19RhoGDIß increased, particularly in wellspreading, nonapoptotic cells, which differentiated into macrophages. These results suggested that Δ19RhoGDIß has an apoptosisindependent role in the PMAinduced differentiation of THP1 cells to macrophages.