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1.
Mol Biol Rep ; 51(1): 837, 2024 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-39042337

RESUMEN

BACKGROUND: MiR-21-5p is a highly expressed microRNA that plays an important role in various cancer-promoting processes, including anchorage-independent growth, invasion, migration metastasis, and drug resistance in lung cancer. Studies indicate that miR-21-5p may contribute to these processes by promoting epithelial-mesenchymal transition (EMT). Ras homolog gene family member B (RhoB), a gene downregulated by miR-21-5p, has also been linked to EMT in lung cancer. However, the role of the miR-21-5p/RhoB axis in EMT regulation in lung adenocarcinoma remains unclear. In this study, we aimed to investigate the regulatory role of the miR-21-5p/RhoB axis in EMT and related in vitro functional characteristics such as migration, invasion, cisplatin resistance, and the formation of tumor spheroids. METHODS AND RESULTS: A549 cells were transfected with the miR-21-5p inhibitor, RhoB siRNA, and their corresponding negative controls. Wound healing, transwell invasion, Methyl thiazole tetrazolium (MTT), and sphere formation assays were also performed to evaluate the migration, invasion, cisplatin resistance, and anchorage-independent growth of A549 cells. RT-qPCR was used to determine the mRNA expression levels of EMT markers. MiR-21-5p knockdown inhibited migration, invasion, cisplatin resistance, and sphere formation while upregulating E-cadherin and downregulating Slug. Furthermore, RhoB silencing restored EMT and related in vitro functional characteristics in A549 cells. CONCLUSIONS: Knockdown of miR-21-5p inhibits EMT and related in vitro functional characteristics by upregulating RhoB, suggesting that miR-21-5p may promote EMT through downregulation of RhoB.


Asunto(s)
Adenocarcinoma del Pulmón , Movimiento Celular , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares , MicroARNs , Proteína de Unión al GTP rhoB , Humanos , Transición Epitelial-Mesenquimal/genética , MicroARNs/genética , MicroARNs/metabolismo , Proteína de Unión al GTP rhoB/genética , Proteína de Unión al GTP rhoB/metabolismo , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/patología , Adenocarcinoma del Pulmón/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Movimiento Celular/genética , Células A549 , Resistencia a Antineoplásicos/genética , Cisplatino/farmacología , Regulación hacia Arriba/genética , Proliferación Celular/genética , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Invasividad Neoplásica/genética , Cadherinas/genética , Cadherinas/metabolismo
2.
J Dev Orig Health Dis ; 14(5): 670-677, 2023 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-38073570

RESUMEN

Increasing evidence shows that maternal hyperglycemia inhibits cardiomyocyte (CM) proliferation and promotes cell apoptosis during fetal heart development, which leads to cardiac dysplasia. Accumulating evidence suggests that the overexpression of miR-21 in CMs has a protective role in cardiac function. Therefore, we investigated whether miR-21 can rescue CM injury caused by high glucose. First, we performed biological function analysis of miR-21-5p overexpression in H9c2 cells treated with high glucose. We found that the proliferation of H9c2 cells treated with high glucose decreased significantly and was rescued after overexpression of miR-21-5p. CCK-8 and EdU incorporation assays were performed to assess cell proliferation. The cell proliferation of the miR-21-5p mimic transfection group was improved compared with that of the NC mimic group (*p < 0.05, miR-21-5p mimics vs. NC mimics) when the proliferation of H9c2 cells was reduced by high glucose (****p < 0.0001, high glucose (HG) vs. normal glucose (NG)). Then, we verified the targeted and negative regulation of miR-21-5p on Rhob using a dual-luciferase activity assay and RT-qPCR, respectively. We further demonstrated that miR-21-5p regulates Rhob to rescue the inhibition of CM proliferation induced by high glucose. The CCK-8 results showed that the cell proliferation of the siRNA-Rhob group was higher than that of the NC mimic group (***p < 0.001) and that of the cotransfection group with Up-Rhob plasmids and miR-21-5p mimics was lower than that of the miR-21-5p mimic group (*p < 0.05). Conclusion: Overexpression of miR-21-5p rescues the inhibition of high glucose-induced CM proliferation through regulation of Rhob.


Asunto(s)
Glucosa , MicroARNs , Miocitos Cardíacos , Apoptosis/genética , Proliferación Celular , Glucosa/toxicidad , Glucosa/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Miocitos Cardíacos/metabolismo , Sincalida/metabolismo , Regulación hacia Arriba , Proteína de Unión al GTP rhoB/metabolismo , Animales , Ratas
3.
Eur J Cell Biol ; 102(4): 151355, 2023 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-37639782

RESUMEN

Small GTPases are highly regulated proteins that control essential signaling pathways through the activity of their effector proteins. Among the RHOA subfamily, RHOB regulates peculiar functions that could be associated with the control of the endocytic trafficking of signaling proteins. Here, we used an optimized assay based on tripartite split-GFP complementation to localize GTPase-effector complexes with high-resolution. The detection of RHOB interaction with the Rhotekin Rho binding domain (RBD) that specifically recognizes the active GTP-bound GTPase, is performed in vitro by the concomitant addition of recombinant GFP1-9 and a GFP nanobody. Analysis of RHOB-RBD complexes localization profiles combined with immunostaining and live cell imaging indicated a serum-dependent reorganization of the endosomal and membrane pool of active RHOB. We further applied this technology to the detection of RHO-effector complexes that highlighted their subcellular localization with high resolution among the different cellular compartments.


Asunto(s)
Transducción de Señal , Proteína de Unión al GTP rhoB , Proteína de Unión al GTP rhoB/genética , Proteína de Unión al GTP rhoB/química , Proteína de Unión al GTP rhoB/metabolismo , GTP Fosfohidrolasas/metabolismo , Membrana Celular/metabolismo , Guanosina Trifosfato/metabolismo , Proteína de Unión al GTP rhoA/metabolismo
4.
Eur J Cell Biol ; 102(2): 151313, 2023 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-36996579

RESUMEN

The small GTPase RhoB is distinguished from other Rho proteins by its unique subcellular localization in endosomes, multivesicular bodies, and nucleus. Despite high sequence homology with RhoA and RhoC, RhoB is mainly associated with tumor suppressive function, while RhoA and RhoC support oncogenic transformation in most malignancies. RhoB regulates the endocytic trafficking of signaling molecules and cytoskeleton remodeling, thereby controlling growth, apoptosis, stress response, immune function, and cell motility in various contexts. Some of these functions may be ascribed to RhoB's unique subcellular localization to endocytic compartments. Here we describe the pleiotropic roles of RhoB in cancer suppression in the context of its subcellular localization, and we discuss possible therapeutic avenues to pursue and highlight priorities for future research.


Asunto(s)
Neoplasias , Proteína de Unión al GTP rhoB , Humanos , Proteína de Unión al GTP rhoB/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Transducción de Señal , Movimiento Celular
5.
Am J Pathol ; 193(5): 579-590, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-36740183

RESUMEN

RhoB protein belongs to the Rho GTPase family, which plays an important role in governing cell signaling and tissue morphology. Its expression is known to have implications in pathologic processes of diseases. In particular, the role of RhoB in rectal cancer is not well understood. Investigation in the regulation and communication of this protein, detected by immunohistochemical staining on the microscope, can help gain insightful information leading to optimal disease treatment options. Herein, deep learning-based image analysis and the decomposition of multiway arrays were used to study the predictive factor of RhoB in two cohorts of patients with rectal cancer having survival rates of <5 and >5 years. The results show distinctions between the tensor decomposition factors of the two cohorts.


Asunto(s)
Neoplasias del Recto , Proteína de Unión al GTP rhoB , Humanos , Proteína de Unión al GTP rhoB/química , Proteína de Unión al GTP rhoB/metabolismo , Transducción de Señal , Biopsia
6.
Traffic ; 24(4): 162-176, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-36562184

RESUMEN

The control of intracellular membrane trafficking by Rho GTPases is central to cellular homeostasis. How specific guanine nucleotide exchange factors and GTPase-activating proteins locally balance GTPase activation in this process is nevertheless largely unclear. By performing a microscopy-based RNAi screen, we here identify the RhoGEF protein Solo as a functional counterplayer of DLC3, a RhoGAP protein with established roles in membrane trafficking. Biochemical, imaging and optogenetics assays further uncover Solo as a novel regulator of endosomal RhoB. Remarkably, we find that Solo and DLC3 control not only the activity, but also total protein levels of RhoB in an antagonistic manner. Together, the results of our study uncover the first functionally connected RhoGAP-RhoGEF pair at endomembranes, placing Solo and DLC3 at the core of endocytic trafficking.


Asunto(s)
Proteínas de Unión al GTP rho , Proteína de Unión al GTP rhoB , Factores de Intercambio de Guanina Nucleótido Rho/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Proteína de Unión al GTP rhoB/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Aparato de Golgi/metabolismo , Endosomas/metabolismo
7.
Biochem Pharmacol ; 206: 115321, 2022 12.
Artículo en Inglés | MEDLINE | ID: mdl-36306821

RESUMEN

The Rho subfamily members of Rho GTPases, RhoA, RhoB, and RhoC, are key regulators of signal transduction in a variety of cellular processes, including regulation of actomyosin and microtubule dynamics, cell shape, cell adhesion, cell division, cell migration, vesicle/membrane trafficking, and cell proliferation. Traditionally, the focus of research on RhoA/B/C has been on tumor biology, as dysregulation of expression or function of these proteins plays an important role in the pathogenesis of various cancer entities. However, RhoA, RhoB, and RhoC are also important in the context of vascular biology and pathology because they influence endothelial barrier function, vascular smooth muscle contractility and proliferation, vascular function and remodelling as well as angiogenesis. In this context, RhoA/B/C exploit numerous effector molecules to transmit their signals, and their activity is regulated by a variety of guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs) that enable precise spatiotemporal activation often in concert with other Rho GTPases. Although their protein structure is very similar, different mechanisms of regulation of gene expression, different localization, and to some extent different interaction with RhoGAPs and RhoGEFs have been observed for RhoA/B/C. In this review, we aim to provide a current overview of the Rho subfamily as regulators of vascular biology and pathology, analyzing database information and existing literature on expression, protein structure, and interaction with effectors and regulatory proteins. In this setting, we will also discuss recent findings on Rho effectors, RhoGEFs, RhoGAPs, as well as guanine nucleotide dissociation inhibitors (RhoGDIs).


Asunto(s)
Proteína de Unión al GTP rhoA , Proteína de Unión al GTP rhoB , Proteína rhoC de Unión a GTP/metabolismo , Proteína de Unión al GTP rhoB/genética , Proteína de Unión al GTP rhoB/metabolismo , Proteína de Unión al GTP rhoA/genética , Proteínas de Unión al GTP rho/genética , Movimiento Celular , Biología
8.
Cells ; 11(19)2022 10 06.
Artículo en Inglés | MEDLINE | ID: mdl-36231106

RESUMEN

Bovine mammary epithelial cells (bMECs) are part of the first line of defense against pathogens. In recent studies, bta-miR-223 has been reported to activate congenital and innate immunity against inflammatory damage during the pathogenesis of mastitis in dairy cows. The purpose of this study was to identify the regulatory mechanism of bta-miR-223 and its downstream target genes in inflammatory bMECs. A double luciferase reporter gene assay demonstrated that ras homolog family member B (RHOB) was the target gene of bta-miR-223. To further elucidate the role of bta-miR-223 in congenital immune responses, bta-miR-223 mimics (mimic/inhibitor) were transfected into bMECs stimulated with lipopolysaccharide (LPS), which activates the Toll-like receptor 4/nuclear factor-κB (TLR4/NF-κB) signaling pathway. Real-time quantitative PCR (qPCR) and Western blot were used to detect the expression of related genes and proteins, and enzyme-linked immunosorbent assay (ELISA) was used to detect secreted inflammatory factors. Results showed that bta-miR-223 expression during inflammation in bMECs reduced the secretion of inflammatory factors by targeting RHOB and deactivation of NF-κB gene activity. Silencing RHOB inhibited LPS-induced inflammatory response in bMECs. Overall, bta-miR-223 attenuated LPS-induced inflammatory response, and acted as a negative feedback regulator via targeting RHOB, providing a novel avenue for mastitis treatment.


Asunto(s)
Mastitis , MicroARNs , Proteína de Unión al GTP rhoB/genética , Animales , Bovinos , Células Epiteliales/metabolismo , Femenino , Humanos , Lipopolisacáridos/metabolismo , Lipopolisacáridos/farmacología , Mastitis/metabolismo , MicroARNs/metabolismo , FN-kappa B/metabolismo , Receptor Toll-Like 4/metabolismo
9.
Microbiome ; 10(1): 149, 2022 09 16.
Artículo en Inglés | MEDLINE | ID: mdl-36114582

RESUMEN

BACKGROUND: The pathogenesis of inflammatory bowel diseases (IBD) is multifactorial, and diagnostic and treatment strategies for IBD remain to be developed. RhoB regulates multiple cell functions; however, its role in colitis is unexplored. RESULTS: Here, we found RhoB was dramatically increased in colon tissues of ulcerative colitis (UC) patients and mice with DSS-induced colitis. Compared with wild type mice, RhoB+/- and RhoB-/- mice developed milder DSS-induced colitis and increased goblet cell numbers and IEC proliferation. Decreased RhoB promoted goblet cell differentiation and epithelial regeneration through inhibiting Wnt signaling pathway and activating p38 MAPK signaling pathway. Moreover, increased SCFA-producing bacteria and SCFA concentrations were detected in intestinal microbiome of both RhoB+/- and RhoB-/- mice and upregulated SCFA receptor expression was also observed. CONCLUSIONS: Taken together, a higher level of RhoB is associated with UC, which also contributes to UC development through modulating cell signaling and altering intestinal bacterial composition and metabolites. These observations suggest that RhoB has potential as a biomarker and a treatment target for UC. Video Abstract.


Asunto(s)
Colitis Ulcerosa , Colitis , Microbioma Gastrointestinal , Proteína de Unión al GTP rhoB/metabolismo , Animales , Biomarcadores , Colitis/inducido químicamente , Colitis/patología , Colitis Ulcerosa/metabolismo , Colitis Ulcerosa/microbiología , Colitis Ulcerosa/patología , Sulfato de Dextran , Humanos , Ratones , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
10.
Endocrinology ; 163(11)2022 10 11.
Artículo en Inglés | MEDLINE | ID: mdl-36047434

RESUMEN

Endometrial decidualization refers to a series of morphological changes and functional remodeling of the uterine endometrium to accept the embryo under the effect of estrogen and progesterone secreted by ovaries after ovulation. During decidualization, endometrial stromal cells (ESCs) proliferate and differentiate into decidual stromal cells, undergoing cytoskeletal rearrangement-mediated morphological changes and expressing decidualization markers, such as insulin-like growth factor-binding protein-1 and prolactin. Ras homology (Rho) proteins, a family of small G proteins, are well known as regulators of cellular morphology and involved in multiple other cellular processes. In this study, we found ras homolog family member B (RHOB) was the most significantly upregulated gene in the Rho protein family after the in vitro decidualization of human primary ESCs. RhoB expression was induced mainly by 3',5'-cyclic adenosine 5'-monophosphate (cAMP) / protein kinase A (PKA) / cyclic adenosine monophosphate-response element binding protein signaling and partly by progesterone signaling. Knockdown of RhoB in ESCs greatly inhibited actin cytoskeletal rearrangement, cell morphological transformation, and upregulation of insulin-like growth factor-binding protein-1, suggesting an indispensable role of RhoB in decidualization. Mechanistically, the downstream target of RhoB was semaphorin3A (Sema3A), which mediated its signaling via interacting with the receptor, plexinA4. More importantly, decreased expression of RhoB, Sema3A, and plexinA4 were detected in deciduas from patients with unexplained spontaneous miscarriage. Collectively, our results indicate that RhoB/Sema3A/plexinA4 signaling plays a positive role in endometrial decidualization and relates to unexplained spontaneous miscarriage, which is worthy of further exploration so as to provide new insights into therapeutic strategies for pregnancy diseases associated with poor decidualization.


Asunto(s)
Proteínas de Unión al GTP Monoméricas , Receptores de Superficie Celular , Semaforina-3A , Células del Estroma , Proteína de Unión al GTP rhoB , Aborto Espontáneo/metabolismo , Actinas/metabolismo , Adenosina Monofosfato/metabolismo , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Decidua/metabolismo , Endometrio/metabolismo , Estrógenos/farmacología , Femenino , Humanos , Proteínas de Unión al GTP Monoméricas/metabolismo , Embarazo , Progesterona/metabolismo , Prolactina/metabolismo , Receptores de Superficie Celular/metabolismo , Semaforina-3A/metabolismo , Células del Estroma/metabolismo , Proteína de Unión al GTP rhoB/metabolismo
11.
Blood Adv ; 6(17): 5184-5197, 2022 09 13.
Artículo en Inglés | MEDLINE | ID: mdl-35819450

RESUMEN

Megakaryocytes are large cells in the bone marrow that give rise to blood platelets. Platelet biogenesis involves megakaryocyte maturation, the localization of the mature cells in close proximity to bone marrow sinusoids, and the formation of protrusions, which are elongated and shed within the circulation. Rho GTPases play important roles in platelet biogenesis and function. RhoA-deficient mice display macrothrombocytopenia and a striking mislocalization of megakaryocytes into bone marrow sinusoids and a specific defect in G-protein signaling in platelets. However, the role of the closely related protein RhoB in megakaryocytes or platelets remains unknown. In this study, we show that, in contrast to RhoA deficiency, genetic ablation of RhoB in mice results in microthrombocytopenia (decreased platelet count and size). RhoB-deficient platelets displayed mild functional defects predominantly upon induction of the collagen/glycoprotein VI pathway. Megakaryocyte maturation and localization within the bone marrow, as well as actin dynamics, were not affected in the absence of RhoB. However, in vitro-generated proplatelets revealed pronouncedly impaired microtubule organization. Furthermore, RhoB-deficient platelets and megakaryocytes displayed selective defects in microtubule dynamics/stability, correlating with reduced levels of acetylated α-tubulin. Our findings imply that the reduction of this tubulin posttranslational modification results in impaired microtubule dynamics, which might contribute to microthrombocytopenia in RhoB-deficient mice. Importantly, we demonstrate that RhoA and RhoB are localized differently and have selective, nonredundant functions in the megakaryocyte lineage.


Asunto(s)
Megacariocitos , Trombocitopenia , Proteína de Unión al GTP rhoB/metabolismo , Animales , Plaquetas/metabolismo , Megacariocitos/metabolismo , Ratones , Microtúbulos/metabolismo , Trombocitopenia/genética , Tubulina (Proteína)/metabolismo
12.
Mol Cancer ; 21(1): 112, 2022 05 10.
Artículo en Inglés | MEDLINE | ID: mdl-35538494

RESUMEN

BACKGROUND: Although gemcitabine has been considered as the first-line drug for advanced pancreatic cancer (PC), development of resistance to gemcitabine severely limits the effectiveness of this chemotherapy, and the underlying mechanism of gemcitabine resistance remains unclear. Various factors, such as ATP binding cassette (ABC) transporters, microRNAs and their downstream signaling pathways are included in chemoresistance to gemcitabine. This study investigated the potential mechanisms of microRNAs and ABC transporters related signaling pathways for PC resistance to gemcitabine both in vivo and in vitro. METHODS: Immunohistochemistry and Western blotting were applied to detect the expression of ABC transporters. Molecular docking analysis was performed to explore whether gemcitabine interacted with ABC transporters. Gain-of-function and loss-of-function analyses were performed to investigate the functions of hsa-miR-3178 in vitro and in vivo. Bioinformatics analysis, Western blotting and dual-luciferase reporter assay were used to confirm the downstream regulatory mechanisms of hsa-miR-3178. RESULTS: We found that P-gp, BCRP and MRP1 were highly expressed in gemcitabine-resistant PC tissues and cells. Molecular docking analysis revealed that gemcitabine can bind to the ABC transporters. Hsa-miR-3178 was upregulated in gemcitabine resistance PANC-1 cells as compared to its parental PANC-1 cells. Moreover, we found that hsa-miR-3178 promoted gemcitabine resistance in PC cells. These results were also verified by animal experiments. RhoB was down-regulated in gemcitabine-resistant PC cells and it was a downstream target of hsa-miR-3178. Kaplan-Meier survival curve showed that lower RhoB expression was significantly associated with poor overall survival in PC patients. Rescue assays demonstrated that RhoB could reverse hsa-miR-3178-mediated gemcitabine resistance. Interestingly, hsa-miR-3178 promoted gemcitabine resistance in PC by activating the PI3K/Akt pathway-mediated upregulation of ABC transporters. CONCLUSIONS: Our results indicate that hsa-miR-3178 promotes gemcitabine resistance via RhoB/PI3K/Akt signaling pathway-mediated upregulation of ABC transporters. These findings suggest that hsa-miR-3178 could be a novel therapeutic target for overcoming gemcitabine resistance in PC.


Asunto(s)
Transportadoras de Casetes de Unión a ATP , Desoxicitidina , MicroARNs , Neoplasias Pancreáticas , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Proteína de Unión al GTP rhoB , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Línea Celular Tumoral , Desoxicitidina/análogos & derivados , Resistencia a Antineoplásicos , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Simulación del Acoplamiento Molecular , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Proteína de Unión al GTP rhoB/metabolismo , Gemcitabina , Neoplasias Pancreáticas
13.
Cell Biol Int ; 46(7): 1074-1088, 2022 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-35347804

RESUMEN

TOX high mobility group box family member 3 (TOX3) can function as tumor suppressor or oncogene in different tumors, while ras homolog family member B (RhoB) is a well-known tumor suppressor. The expression and role of TOX3 in colorectal cancer (CRC) are unknown. This study aimed to investigate the expression of TOX3 in CRC and the role of TOX3/mitogen-activated protein kinase (MAPK)/RhoB signaling in the proliferation and apoptosis of CRC cells. We showed that TOX3 messenger RNA (mRNA) and protein expression levels were significantly upregulated in CRC tissues and cell lines. High TOX3 expression was associated with high T stage, nodal invasion, and advanced tumor stage. Disease-free survival (DFS) was shortened for CRC patients with high expression of TOX3, while overall survival showed no significant difference. TOX3 promoted proliferation, inhibited apoptosis, and decreased the sensitivity to oxaliplatin of CRC cells. In addition, the inhibition of TOX3 led to the upregulation of RhoB, and RhoB overexpression suppressed the proliferation and promoted apoptosis of CRC cells. Moreover, TOX3 overexpression upregulated MAPK signaling, while MAPK signaling inhibitor U0126 induced CRC cell proliferation arrest or apoptosis, and attenuated the inhibition of RhoB in TOX3 overexpression cells. In addition, the overexpression of TOX3 increased tumor volume in nude mice. In conclusion, TOX3 may be an oncogene in CRC and can predict DFS in CRC patients. TOX3/MAPK/RhoB signaling plays an important role in the modulation of proliferation and apoptosis of CRC cells.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Neoplasias Colorrectales , Proteínas Quinasas Activadas por Mitógenos , Transactivadores/metabolismo , Animales , Apoptosis/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Neoplasias Colorrectales/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Ratones Desnudos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteína de Unión al GTP rhoB/genética , Proteína de Unión al GTP rhoB/metabolismo
14.
FASEB J ; 36(4): e22254, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35294066

RESUMEN

Overwhelming inflammation in the setting of acute critical illness induces capillary leak resulting in hypovolemia, edema, tissue dysoxia, organ failure and even death. The tight junction (TJ)-dependent capillary barrier is regulated by small GTPases, but the specific regulatory molecules most active in this vascular segment under such circumstances are not well described. We set out to identify GTPase regulatory molecules specific to endothelial cells (EC) that form TJs. Transcriptional profiling of confluent monolayers of TJ-forming human dermal microvascular ECs (HDMECs) and adherens junction only forming-human umbilical vein EC (HUVECs) demonstrate ARHGEF12 is basally expressed at higher levels and is only downregulated in HDMECs by junction-disrupting tumor necrosis factor (TNF). HDMECs depleted of ArhGEF12 by siRNA demonstrate a significantly exacerbated TNF-induced decrease in trans-endothelial electrical resistance and disruption of TJ continuous staining. ArhGEF12 is established as a RhoA-GEF in HUVECs and its knock down would be expected to reduce RhoA activity and barrier disruption. Pulldown of active GEFs from HDMECs depleted of ArhGEF12 and treated with TNF show decreased GTP-bound Rap1A after four hours but increased GTP-bound RhoA after 12 h. In cell-free assays, ArhGEF12 immunoprecipitated from HDMECs is able to activate both Rap1A and RhoA, but not act on Rap2A-C, RhoB-C, or even Rap1B which shares 95% sequence identity with Rap1A. We conclude that in TJ-forming HDMECs, ArhGEF12 selectively activates Rap1A to limit capillary barrier disruption in a mechanism independent of cAMP-mediated Epac1 activation.


Asunto(s)
Factores de Intercambio de Guanina Nucleótido , Proteína de Unión al GTP rhoA , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Guanosina Trifosfato , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Factor de Necrosis Tumoral alfa/farmacología , Proteínas de Unión al GTP rap1/genética , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismo , Proteína de Unión al GTP rhoB/metabolismo
15.
Small GTPases ; 13(1): 196-204, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-34304710

RESUMEN

The Ras homologous (Rho) protein family of GTPases (RhoA, RhoB and RhoC) are the members of the Ras superfamily and regulate cellular processes such as cell migration, proliferation, polarization, adhesion, gene transcription and cytoskeletal structure. Rho GTPases function as molecular switches that cycle between GTP-bound (active state) and GDP-bound (inactive state) forms. Leukaemia-associated RhoGEF (LARG) is a guanine nucleotide exchange factor (GEF) that activates RhoA subfamily GTPases by promoting the exchange of GDP for GTP. LARG is selective for RhoA subfamily GTPases and is an essential regulator of cell migration and invasion. Here, we describe the mechanisms by which LARG is regulated to facilitate the understanding of how LARG mediates functions like cell motility and to provide insight for better therapeutic targeting of these functions.


Asunto(s)
Leucemia , Proteína de Unión al GTP rhoA , Humanos , Factores de Intercambio de Guanina Nucleótido Rho/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Factores de Intercambio de Guanina Nucleótido ras/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Proteína de Unión al GTP rhoB/metabolismo , Proteínas ras/metabolismo , Guanosina Trifosfato , Proteínas de Unión al GTP rho/metabolismo
16.
Viruses ; 13(11)2021 10 20.
Artículo en Inglés | MEDLINE | ID: mdl-34834920

RESUMEN

Zika virus (ZIKV) is a re-emerging flavivirus that has caused large-scale epidemics. Infection during pregnancy can lead to neurologic developmental abnormalities in children. There is no approved vaccine or therapy for ZIKV. To uncover cellular pathways required for ZIKV that can be therapeutically targeted, we transcriptionally upregulated all known human coding genes with an engineered CRISPR-Cas9 activation complex in human fibroblasts deficient in interferon (IFN) signaling. We identified Ras homolog family member V (RhoV) and WW domain-containing transcription regulator 1 (WWTR1) as proviral factors, and found them to play important roles during early ZIKV infection in A549 cells. We then focused on RhoV, a Rho GTPase with atypical terminal sequences and membrane association, and validated its proviral effects on ZIKV infection and virion production in SNB-19 cells. We found that RhoV promotes infection of some flaviviruses and acts at the step of viral entry. Furthermore, RhoV proviral effects depend on the complete GTPase cycle. By depleting Rho GTPases and related proteins, we identified RhoB and Pak1 as additional proviral factors. Taken together, these results highlight the positive role of RhoV in ZIKV infection and confirm CRISPR activation as a relevant method to identify novel host-pathogen interactions.


Asunto(s)
Proteínas de Unión al GTP/metabolismo , Proteínas de Neoplasias/metabolismo , Infección por el Virus Zika/enzimología , Virus Zika/fisiología , Proteína de Unión al GTP rhoB/metabolismo , Células A549 , Sistemas CRISPR-Cas , Proteínas de Unión al GTP/genética , Humanos , Proteínas de Neoplasias/genética , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ/genética , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ/metabolismo , Internalización del Virus , Replicación Viral , Virus Zika/genética , Infección por el Virus Zika/genética , Infección por el Virus Zika/virología , Quinasas p21 Activadas/genética , Quinasas p21 Activadas/metabolismo , Proteína de Unión al GTP rhoB/genética
17.
Front Immunol ; 12: 747020, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34557203

RESUMEN

Background: Ischemia-reperfusion injury (IRI) remains an inevitable and major challenge in renal transplantation. The current study aims to obtain deep insights into underlying mechanisms and seek prognostic genes as potential therapeutic targets for renal IRI (RIRI). Methods: After systematically screening the Gene Expression Omnibus (GEO) database, we collected gene expression profiles of over 1,000 specimens from 11 independent cohorts. Differentially expressed genes (DEGs) were identified by comparing allograft kidney biopsies taken before and after reperfusion in the discovery cohort and further validated in another two independent transplant cohorts. Then, graft survival analysis and immune cell analysis of DEGs were performed in another independent renal transplant cohort with long-term follow-ups to further screen out prognostic genes. Cell type and time course analyses were performed for investigating the expression pattern of prognostic genes in more dimensions utilizing a mouse RIRI model. Finally, two novel genes firstly identified in RIRI were verified in the mouse model and comprehensively analyzed to investigate potential mechanisms. Results: Twenty DEGs upregulated in the process of RIRI throughout different donor types (living donors, cardiac and brain death donors) were successfully identified and validated. Among them, upregulation of 10 genes was associated with poor long-term allograft outcomes and exhibited strong correlations with prognostic immune cells, like macrophages. Furthermore, certain genes were found to be only differentially expressed in specific cell types and remained with high expression levels even months after RIRI in the mouse model, which processed the potential to serve as therapeutic targets. Importantly, two newly identified genes in RIRI, Btg2 and Rhob, were successfully confirmed in the mouse model and found to have strong connections with NF-κB signaling. Conclusions: We successfully identified and validated 10 IRI-associated prognostic genes in renal transplantation across different donor types, and two novel genes with crucial roles in RIRI were recognized for the first time. Our findings offered promising potential therapeutic targets for RIRI in renal transplantation.


Asunto(s)
Proteínas Inmediatas-Precoces/genética , Trasplante de Riñón/efectos adversos , Daño por Reperfusión/genética , Transcriptoma , Proteínas Supresoras de Tumor/genética , Proteína de Unión al GTP rhoB/genética , Animales , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Pronóstico
18.
Life Sci Alliance ; 4(9)2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34187934

RESUMEN

Epidermal growth factor receptor (EGFR) and human EGFR 2 (HER2) phosphorylation drives HER2-positive breast cancer cell proliferation. Enforced activation of phosphatases for those receptors could be a therapeutic option for HER2-positive breast cancers. Here, we report that degradation of an endosomal small GTPase, RhoB, by the ubiquitin ligase complex cullin-3 (CUL3)/KCTD10 is essential for both EGFR and HER2 phosphorylation in HER2-positive breast cancer cells. Using human protein arrays produced in a wheat cell-free protein synthesis system, RhoB-GTP, and protein tyrosine phosphatase receptor type H (PTPRH) were identified as interacting proteins of connector enhancer of kinase suppressor of Ras1 (CNKSR1). Mechanistically, constitutive degradation of RhoB, which is mediated by the CUL3/KCTD10 E3 complex, enabled CNKSR1 to interact with PTPRH at the plasma membrane resulting in inactivation of EGFR phosphatase activity. Depletion of CUL3 or KCTD10 led to the accumulation of RhoB-GTP at the plasma membrane followed by its interaction with CNKSR1, which released activated PTPRH from CNKSR1. This study suggests a mechanism of PTPRH activation through the exclusive binding of RhoB-GTP to CNKSR1.


Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína de Unión al GTP rhoB/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/etiología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas Portadoras , Línea Celular Tumoral , Proteínas Cullin/metabolismo , Receptores ErbB/agonistas , Receptores ErbB/genética , Receptores ErbB/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Persona de Mediana Edad , Modelos Biológicos , Fosforilación , Canales de Potasio con Entrada de Voltaje/metabolismo , Pronóstico , Análisis por Matrices de Proteínas , Unión Proteica , Proteolisis , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/metabolismo
19.
FASEB J ; 35(6): e21627, 2021 06.
Artículo en Inglés | MEDLINE | ID: mdl-33948992

RESUMEN

Capillary endothelial cells (ECs) maintain a semi-permeable barrier between the blood and tissue by forming inter-EC tight junctions (TJs), regulating selective transport of fluid and solutes. Overwhelming inflammation, as occurs in sepsis, disrupts these TJs, leading to leakage of fluid, proteins, and small molecules into the tissues. Mechanistically, disruption of capillary barrier function is mediated by small Rho-GTPases, such as RhoA, -B, and -C, which are activated by guanine nucleotide exchange factors (GEFs) and disrupted by GTPase-activating factors (GAPs). We previously reported that a mutation in a specific RhoB GAP (p190BRhoGAP) underlays a hereditary capillary leak syndrome. Tumor necrosis factor (TNF) treatment disrupts TJs in cultured human microvascular ECs, a model of capillary leak. This response requires new gene transcription and involves increased RhoB activation. However, the specific GEF that activates RhoB in capillary ECs remains unknown. Transcriptional profiling of cultured tight junction-forming human dermal microvascular endothelial cells (HDMECs) revealed that 17 GEFs were significantly induced by TNF. The function of each candidate GEF was assessed by short interfering RNA depletion and trans-endothelial electrical resistance screening. Knockown of ArhGEF10 reduced the TNF-induced loss of barrier which was phenocopied by RhoB or dual ArhGEF10/RhoB knockdown. ArhGEF10 knockdown also reduced the extent of TNF-induced RhoB activation and disruption at tight junctions. In a cell-free assay, immunoisolated ArhGEF10 selectively catalyzed nucleotide exchange to activate RhoB, but not RhoA or RhoC. We conclude ArhGEF10 is a TNF-induced RhoB-selective GEF that mediates TJ disruption and barrier loss in human capillary endothelial cells.


Asunto(s)
Dermis/metabolismo , Endotelio Vascular/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Uniones Estrechas/fisiología , Proteína de Unión al GTP rhoB/metabolismo , Permeabilidad Capilar , Dermis/citología , Dermis/efectos de los fármacos , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Factores de Intercambio de Guanina Nucleótido Rho/genética , Transducción de Señal , Factor de Necrosis Tumoral alfa/farmacología , Proteína de Unión al GTP rhoB/genética
20.
Nat Commun ; 12(1): 2587, 2021 05 10.
Artículo en Inglés | MEDLINE | ID: mdl-33972537

RESUMEN

Host cells use several anti-bacterial pathways to defend against pathogens. Here, using a uropathogenic Escherichia coli (UPEC) infection model, we demonstrate that bacterial infection upregulates RhoB, which subsequently promotes intracellular bacteria clearance by inducing LC3 lipidation and autophagosome formation. RhoB binds with Beclin 1 through its residues at 118 to 140 and the Beclin 1 CCD domain, with RhoB Arg133 being the key binding residue. Binding of RhoB to Beclin 1 enhances the Hsp90-Beclin 1 interaction, preventing Beclin 1 degradation. RhoB also directly interacts with Hsp90, maintaining RhoB levels. UPEC infections increase RhoB, Beclin 1 and LC3 levels in bladder epithelium in vivo, whereas Beclin 1 and LC3 levels as well as UPEC clearance are substantially reduced in RhoB+/- and RhoB-/- mice upon infection. We conclude that when stimulated by UPEC infections, host cells promote UPEC clearance through the RhoB-Beclin 1-HSP90 complex, indicating RhoB may be a useful target when developing UPEC treatment strategies.


Asunto(s)
Autofagosomas/metabolismo , Beclina-1/metabolismo , Infecciones por Escherichia coli/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Infecciones Urinarias/metabolismo , Escherichia coli Uropatógena/crecimiento & desarrollo , Proteína de Unión al GTP rhoB/metabolismo , Animales , Autofagosomas/genética , Autofagosomas/ultraestructura , Beclina-1/genética , Línea Celular , Epitelio/metabolismo , Epitelio/microbiología , Infecciones por Escherichia coli/genética , Infecciones por Escherichia coli/microbiología , Femenino , Técnicas de Silenciamiento del Gen , Proteínas HSP90 de Choque Térmico/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica de Transmisión , Proteínas Asociadas a Microtúbulos/metabolismo , Unión Proteica , Estabilidad Proteica , ARN Interferente Pequeño , Proteínas Recombinantes , Vejiga Urinaria/metabolismo , Vejiga Urinaria/microbiología , Infecciones Urinarias/genética , Infecciones Urinarias/microbiología , Escherichia coli Uropatógena/patogenicidad , Proteína de Unión al GTP rhoB/genética
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