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1.
Med Sci Monit ; 25: 7605-7616, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31599230

RESUMO

BACKGROUND The aim of this study was to explore the impact of Ras homolog C/Rho-associated coiled-protein kinase (Rho/ROCK) signaling pathways intervention on biological characteristics of the human multiple myeloma cell lines RPMI-8226 and U266 cells, and to investigate the expression of RhoC, ROCK1, and ROCK2 in RPMI-8226 and U266 cells. MATERIAL AND METHODS RPMI8226 and U266 cell lines were treated by 5-aza-2-deoxycytidine (5-Aza-Dc), trichostatin A (TSA), RhoA inhibitor CCG-1423, Rac1 inhibitor NSC23766, and ROCK inhibitor fasudil. Cell proliferation was examined by Cell Counting Kit-8 (CCK-8) assay and clone formation. Cell apoptosis was examined by flow cytometry and TUNEL assay. The mRNA and protein expressions of RhoC, ROCK1, and ROCK2 were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blot, respectively. RESULTS CCG-1423, NSC23766, and fasudil could significantly inhibit the proliferation of RPMI8226 and U266 cells. The inhibitory effect was dose- and time-dependent within a certain concentration range (P<0.05). After treatment with CCG-1423, NSC23766, and fasudil for 24 hours, the apoptosis rates of RPMI8226 and U266 cells were significantly higher than those of the control group, which were dose-dependent (P<0.05). Compared with the control group, the mRNA and protein expressions of RhoC, ROCK1, and ROCK2 in RPMI8226 and U266 cells were significantly decreased with single 5-Aza-Dc or TSA treatment. However, the effects were obviously stronger after combined treatment of 5-Aza-CdR and TSA (P<0.05). CONCLUSIONS We found that 5-Aza-Dc and TSA can effectively decrease the mRNA and protein expressions of RhoC, ROCK1, and ROCK2. Furthermore, Rho and ROCK inhibitors significantly inhibit cell growth and induce cell apoptosis in the human multiple myeloma cell lines RPMI-8226 and U266.


Assuntos
Apoptose , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Apoptose/efeitos dos fármacos , Azacitidina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Ácidos Hidroxâmicos/farmacologia , Mieloma Múltiplo/genética , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/genética
2.
Invest Ophthalmol Vis Sci ; 60(10): 3659-3668, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31469406

RESUMO

Purpose: The purpose of this study was to characterize the ability of applied electrical fields (EFs) to direct retinal ganglion cell (RGC) axon growth as well as to assess whether Rho GTPases play a role in translating electrical cues to directional cues. Methods: Full-thickness, early postnatal mouse retina was cultured in electrotaxis chambers and exposed to EFs of varying strengths (50-200 mV/mm). The direction of RGC axon growth was quantified from time-lapsed videos. The rate of axon growth and responsiveness to changes in EF polarity were also assessed. The effect of toxin B, a broad-spectrum inhibitor of Rho GTPase signaling, and Z62954982, a selective inhibitor of Rac1, on EF-directed growth was determined. Results: In the absence of an EF, RGC axons demonstrated indiscriminate directional growth from the explant edge. Retinal cultures exposed to an EF of 100 and 200 mV/mm showed markedly asymmetric growth, with 74.2% and 81.2% of axons oriented toward the cathode, respectively (P < 0.001). RGC axons responded to acute changes in EF polarity by redirecting their growth toward the "new" cathode. This galvanotropic effect was partially neutralized by toxin B and Rac1 inhibitor Z62954982. Conclusions: RGC axons exhibit cathode-directed growth in the presence of an EF. This effect is mediated in part by the Rho GTPase signaling cascade.


Assuntos
Axônios/fisiologia , Terapia por Estimulação Elétrica , Campos Eletromagnéticos , Células Ganglionares da Retina/fisiologia , Animais , Polaridade Celular/fisiologia , Inibidores Enzimáticos/farmacologia , Camundongos , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/metabolismo
3.
Inflammopharmacology ; 27(6): 1309-1318, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31168686

RESUMO

SB-706375 is a selective receptor antagonist of human urotensin-II (hU-II), which can block the aorta contraction induced by hU-II in rats. The effect of SB-706375 on myocardial ischaemia-reperfusion (I/R) injury is unclear. The major objective of this study was to investigate whether SB-706375 has a protective effect on myocardial I/R injury in rats and explore its possible mechanisms. Isolated hearts of Adult Sprague-Dawley were perfused in a Langendorff apparatus, and haemodynamic parameters, lactate dehydrogenase (LDH), creatine phosphokinase-MB (CK-MB), cardiac troponin I (cTnI), RhoA, and the protein expressions of U-II receptor (UTR), receptor-interacting protein 3 (RIP3), Rho-associated coiled-coil-containing protein kinase 1 (ROCK1) and Rho-associated coiled-coil-containing protein kinase 2 (ROCK2) were assessed. We found that SB-706375 (1 × 10-6 and 1 × 10-5 mol/L) significantly inhibited the changes of haemodynamic parameters and reduced LDH and CK-MB activities and also cTnI level in the coronary effluents in the heart subjected to myocardial I/R injury. Further experiments studies showed that SB-706375 obviously prevented myocardial I/R increased RhoA activity and UTR, RIP3, ROCK1, and ROCK2 protein expressions. ROCK inhibition abolished the improving effect of SB-706375 on myocardial I/R-induced haemodynamic change in the isolated perfused rat heart. These findings suggested that SB-706375 provides cardio-protection against I/R injury in isolated rats by blocking UTR-RhoA/ROCK-RIP3 pathway.


Assuntos
Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/patologia , Pirrolidinas/farmacologia , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Sulfonamidas/farmacologia , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Quinases Associadas a rho/antagonistas & inibidores , Animais , Feminino , Masculino , Necrose , Ratos , Ratos Sprague-Dawley , Proteína Serina-Treonina Quinases de Interação com Receptores/fisiologia , Receptores Acoplados a Proteínas-G/análise , Transdução de Sinais/fisiologia , Proteínas rho de Ligação ao GTP/fisiologia , Quinases Associadas a rho/fisiologia
4.
Cells ; 8(6)2019 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-31185627

RESUMO

Neuronal migration is essential for the orchestration of brain development and involves several contiguous steps: interkinetic nuclear movement (INM), multipolar-bipolar transition, locomotion, and translocation. Growing evidence suggests that Rho GTPases, including RhoA, Rac, Cdc42, and the atypical Rnd members, play critical roles in neuronal migration by regulating both actin and microtubule cytoskeletal components. This review focuses on the spatiotemporal-specific regulation of Rho GTPases as well as their regulators and effectors in distinct steps during the neuronal migration process. Their roles in bridging extracellular signals and cytoskeletal dynamics to provide optimal structural support to the migrating neurons will also be discussed.


Assuntos
Neurônios/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Junções Aderentes/metabolismo , Animais , Movimento Celular , Células Ependimogliais/química , Células Ependimogliais/citologia , Células Ependimogliais/metabolismo , Humanos , Neurogênese , Neurônios/citologia , Proteína cdc42 de Ligação ao GTP/antagonistas & inibidores , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/antagonistas & inibidores
5.
J Vasc Surg ; 69(5): 1581-1589.e1, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31010523

RESUMO

OBJECTIVE: Current drug-eluting stent (DES) treatment is promising, but it still has the drawback of in-stent restenosis, which remains a clinically relevant problem. Efforts should be made to discover new signaling molecules and novel potential targets for the prevention of arterial restenosis. In this study, we fabricated a novel DES targeting the RhoA pathway and further examined this promising strategy in vitro and in a rabbit carotid model. METHODS: Active RhoA expression is correlated with the synthetic smooth muscle phenotype, and the RhoA inhibitor rhosin suppresses this phenotypic modulation at both transcriptional and translational levels. We further demonstrated that the RhoA inhibitor rhosin might act through the YAP pathway in smooth muscle cell phenotype modulation by a gain-of-function assay. Moreover, we fabricated a RhoA inhibitor-eluting stent and tested it in a rabbit carotid model. RESULTS: Compared with a bare-metal stent, the RhoA inhibitor-eluting stent significantly attenuated neointimal formation at 6 months. However, overexpression of YAP by lentivirus blocked the antirestenosis effect of the RhoA inhibitor-eluting stent and repressed smooth muscle-specific genes. CONCLUSIONS: RhoA inhibitor-eluting stents attenuate neointimal formation through inhibition of the YAP signaling pathway. This novel DES may represent a potential strategy for the treatment of in-stent restenosis.


Assuntos
Angioplastia com Balão/instrumentação , Proliferação de Células/efeitos dos fármacos , Stents Farmacológicos , Inibidores Enzimáticos/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Compostos Orgânicos/farmacologia , Proteínas Proto-Oncogênicas c-yes/metabolismo , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Angioplastia com Balão/efeitos adversos , Animais , Apoptose/efeitos dos fármacos , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/enzimologia , Artérias Carótidas/patologia , Células Cultivadas , Constrição Patológica , Masculino , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/enzimologia , Miócitos de Músculo Liso/patologia , Neointima , Proteínas Proto-Oncogênicas c-yes/genética , Coelhos , Ratos , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Proteínas rho de Ligação ao GTP/metabolismo
6.
Biomed Pharmacother ; 109: 1610-1619, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30551415

RESUMO

RhoA/Rho-associated coiled-coil forming protein serine/threonine kinase (ROCK) has appeared as a potential therapeutic target in numerous diseases, because of its preventing action on various enzymes providing antioxidant and cytoprotective action. Progression and pathophysiology of diabetic nephropathy have also shown potential involvement of oxidative stress and inflammatory pathways. In the present study, we investigated the effect of kaempferol on hyperglycemia-induced activation of RhoA kinase and associated inflammatory signaling cascade. Currently there is only small literature available on the mechanism of anti-diabetic and nephroprotective action of this compound, which creates a void. Therefore, we focused here on the investigation of molecular mechanisms for kaempferol by means of in vitro testing, using rat (NRK-52E) and human renal tubular epithelial cells (RPTEC). Our findings suggest that kaempferol inhibits hyperglycemia-induced activation of RhoA and decreased oxidative stress, pro-inflammatory cytokines (TNF-α and IL-1ß) and fibrosis (TGF-ß1 expression, extracellular matrix protein expression) in NRK-52E and RPTEC cells. Therefore, kaempferol can be used as a potential therapeutic for the treatment of diabetic nephropathy.


Assuntos
Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/metabolismo , Mediadores da Inflamação/metabolismo , Quempferóis/uso terapêutico , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Nefropatias Diabéticas/patologia , Relação Dose-Resposta a Droga , Humanos , Mediadores da Inflamação/antagonistas & inibidores , Quempferóis/farmacologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Quinases Associadas a rho/antagonistas & inibidores
7.
J Invest Dermatol ; 139(6): 1227-1236, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30579854

RESUMO

Desmoplakin (DP) is an obligate component of desmosomal cell-cell junctions that links the adhesion plaque to the cytoskeletal intermediate filament network. While a central role for DP in maintaining the structure and stability of the desmosome is well established, recent work has indicated that DP's functions may extend beyond cell-cell adhesion. In our study, we show that loss of DP results in a significant increase in cellular migration, as measured by scratch wound assays, Transwell migration assays, and invasion assays. Loss of DP causes dramatic changes in actin cytoskeleton morphology, including enhanced protrusiveness, and an increase in filopodia length and number. Interestingly, these changes are also observed in single cells, indicating that control of actin morphology is a cell-cell adhesion-independent function of DP. An investigation of cellular signaling pathways uncovered aberrant Rac and p38 mitogen-activated protein kinase (MAPK) activity in DP knockdown cells, restoration of which is sufficient to rescue DP-dependent changes in both cell migration and actin cytoskeleton morphology. Taken together, these data highlight a previously uncharacterized role for the desmosomal cytolinker DP in coordinating cellular migration via p38 MAPK and Rac signaling.


Assuntos
Movimento Celular/fisiologia , Desmoplaquinas/metabolismo , Transdução de Sinais/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/metabolismo , Amidas/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Desmoplaquinas/genética , Técnicas de Silenciamento de Genes , Humanos , Filamentos Intermediários/metabolismo , Piridinas/farmacologia , Pironas/farmacologia , Quinolinas/farmacologia , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Zearalenona/análogos & derivados , Zearalenona/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas rho de Ligação ao GTP/antagonistas & inibidores
8.
Exp Cell Res ; 371(2): 426-434, 2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30201453

RESUMO

Although parallel alignment of fibroblasts to the tension lines of scar has been evidenced in vivo, how scar contracture generates directional contraction remains largely unclear due to the lack of effective in vitro model. Fibroblast populated collagen lattice (FPCL), a widely used in vitro model, fails to mimic scar contracture since it produces concentric contraction with the random orientation of fibroblast. We hypothesized that a novel FPCL model with fibroblast alignment might produce directional contraction and then simulate scar contracture better. Here, we showed that although direct current electric fields (DCEFs) enabled fibroblasts aligned perpendicularly to the field vector, it also promoted electrotactic migration of fibroblast in FPCL. By contrast, biphasic pulse direct current electric fields (BPDCEFs), featured by reversal of the EF direction periodically, abolished the electrotactic migration, but induced fibroblast alignment in a pulse frequency dependent manner. Specifically, BPDCEF at a pulse frequency of 0.0002 Hz induced fibroblast alignment comparable to that induced by DCEF under the same field strength (300 mV/mm), leading to an enhanced contraction of FPCL along the direction of cell alignment. FPCL pretreated by BPDCEF showed an elliptical contraction whereas it was concentric in control FPCL. Further study revealed that F-actin redistributions acted as a key mechanism for the induction of fibroblasts alignment by BPDCEF. Cytochalasin D, an inhibitor of actin dynamics, abolished F-actins redistribution, and significantly suppressed the fibroblasts alignment and the directional contraction of FPCL. Importantly, BPDCEF significantly increased RhoA activity in fibroblasts, while this response was attenuated by C3 transferase pre-treatment, a potent inhibitor of RhoA, caused F-actin depolymerization and actin filament bundle randomly distributed. Taken together, our study suggests a crucial role for fibroblast orientation in scar contracture, and provides a novel FPCL model that may be feasible and effective for investigating scar contracture in vitro.


Assuntos
Eletricidade , Fibroblastos/citologia , Modelos Biológicos , Tecidos Suporte , ADP Ribose Transferases/farmacologia , Actinas/antagonistas & inibidores , Actinas/genética , Actinas/metabolismo , Animais , Animais Recém-Nascidos , Fenômenos Biomecânicos , Toxinas Botulínicas/farmacologia , Movimento Celular , Cicatriz/genética , Cicatriz/metabolismo , Cicatriz/patologia , Colágeno/química , Citocalasina D/farmacologia , Feminino , Fibroblastos/metabolismo , Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Cultura Primária de Células , Ratos , Pele/citologia , Pele/metabolismo , Tensão Superficial , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo
9.
Naunyn Schmiedebergs Arch Pharmacol ; 391(11): 1275-1283, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30073384

RESUMO

Rho/Rho-kinase (ROCK) signaling contributes to neuroinflammation, epileptogenesis, and seizures in convulsive-type epilepsies. However, this pathway has not been investigated in absence epilepsy. We investigated RhoA activity in genetic absence epilepsy rats from Strasburg (GAERS) and the effects of ROCK inhibitors Y-27632 and fasudil on spike-and-wave discharges (SWDs) of GAERS. ROCK level and activity were measured by Western blot analysis in the brain areas involved in absence seizures (i.e., cortex and thalamus) and hippocampus. Male GAERS were stereotaxically implanted with bilateral cortical electrodes for electroencephalogram (EEG) recordings and/or guide cannula into the right ventricle. ROCK inhibitors were administered by intraperitoneal injection (1-10 mg/kg for Y-27632 or fasudil) or intracerebroventricular injection (7-20 nmol/5 µl for Y-27632 or 10-100 nmol/5 µl for fasudil). EEG was recorded under freely moving conditions. Compared with Wistar rats, GAERS exhibited increased RhoA activity in the somatosensory cortex but not in the thalamus or hippocampus. The single systemic administration of Y-27632 and fasudil partially suppressed the duration and frequency of absence seizure, respectively. However, local brain administration caused a widespread suppressive effect on the total seizure duration, number of seizures, and the average individual seizure length. In summary, Rho/ROCK signaling may be involved in the pathophysiology of absence epilepsy. Furthermore, ROCK inhibitors can control the expression of absence seizure in GAERS, thus indicating that Y-27632 and fasudil have the potential to be used as novel anti-absence drugs.


Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Amidas/farmacologia , Encéfalo/efeitos dos fármacos , Epilepsia Tipo Ausência/fisiopatologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Encéfalo/fisiologia , Eletroencefalografia/efeitos dos fármacos , Masculino , Ratos Wistar , Proteínas rho de Ligação ao GTP/fisiologia
10.
Methods Mol Biol ; 1821: 283-295, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30062420

RESUMO

Human pluripotent stem cells indefinitely proliferate and survive in culture while retaining genomic integrity, providing a unique opportunity to study human molecular biology. Here, we introduced an RNA interference-based protocol of inducible gene silencing in human embryonic stem cells, which has several advantages in handling simplicity/convenience, cost/time performance, and applicability. Using this method, we had succeeded to elucidate the isoform-unique roles of Rho-family small GTPases in human embryonic stem cells.


Assuntos
Inativação Gênica , Células-Tronco Embrionárias Humanas/enzimologia , Lentivirus , Interferência de RNA , Transdução Genética/métodos , Proteínas rho de Ligação ao GTP , Células HEK293 , Células-Tronco Embrionárias Humanas/citologia , Humanos , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo
11.
Blood ; 132(6): 565-576, 2018 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-29891535

RESUMO

Aging-associated remodeling of the immune system impairs its functional integrity and contributes to increased morbidity and mortality in the elderly. Aging of hematopoietic stem cells (HSCs), from which all cells of the adaptive immune system ultimately originate, might play a crucial role in the remodeling of the aged immune system. We recently reported that aging of HSCs is, in part, driven by elevated activity of the small RhoGTPase Cdc42 and that aged HSCs can be rejuvenated in vitro by inhibition of the elevated Cdc42 activity in aged HSCs with the pharmacological compound CASIN. To study the quality of immune systems stemming selectively from young or aged HSCs, we established a HSC transplantation model in T- and B-cell-deficient young RAG1-/- hosts. We report that both phenotypic and functional changes in the immune system on aging are primarily a consequence of changes in the function of HSCs on aging and, to a large extent, independent of the thymus, as young and aged HSCs reconstituted distinct T- and B-cell subsets in RAG1-/- hosts that mirrored young and aged immune systems. Importantly, aged HSCs treated with CASIN reestablished an immune system similar to that of young animals, and thus capable of mounting a strong immune response to vaccination. Our studies further imply that epigenetic signatures already imprinted in aged HSCs determine the transcriptional profile and function of HSC-derived T and B cells.


Assuntos
Envelhecimento/imunologia , Senescência Celular/imunologia , Células-Tronco Hematopoéticas/imunologia , Subpopulações de Linfócitos/imunologia , Animais , Feminino , Perfilação da Expressão Gênica , Genes RAG-1 , Sobrevivência de Enxerto , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Subpopulações de Linfócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doadores de Tecidos , Vacinação , Vacinas de DNA/imunologia , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/fisiologia
12.
J R Soc Interface ; 15(143)2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29899159

RESUMO

Cell migration is an essential part of many (patho)physiological processes, including keratinocyte re-epithelialization of healing wounds. Physical forces and mechanical cues from the wound bed (in addition to biochemical signals) may also play an important role in the healing process. Previously, we explored this possibility and found that polyacrylamide (PA) gel stiffness affected human keratinocyte behaviour and that mechanical deformations in soft (approx. 1.2 kPa) PA gels produced by neighbouring cells appeared to influence the process of de novo epithelial sheet formation. To clearly demonstrate that keratinocytes do respond to such deformations, we conducted a series of experiments where we observed the response of single keratinocytes to a prescribed local substrate deformation that mimicked a neighbouring cell or evolving multicellular aggregate via a servo-controlled microneedle. We also examined the effect of adding either Y27632 or blebbistatin on cell response. Our results indicate that keratinocytes do sense and respond to mechanical signals comparable to those that originate from substrate deformations imposed by neighbouring cells, a finding that could have important implications for the process of keratinocyte re-epithelialization that takes place during wound healing. Furthermore, the Rho/ROCK pathway and the engagement of NM II are both essential to substrate deformation-directed keratinocyte migration.


Assuntos
Movimento Celular , Queratinócitos/metabolismo , Transdução de Sinais , Cicatrização , Amidas/farmacologia , Linhagem Celular , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Queratinócitos/patologia , Piridinas/farmacologia , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismo
13.
Pulm Pharmacol Ther ; 50: 111-122, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29673911

RESUMO

BACKGROUND: Hypoxic pulmonary arterial hypertension (PAH) is a crippling disease with limited therapeutic methods. The imbalance of T helper 17 cell (Th17)/regulatory T cell (Treg) plays an important role in the development of Hypoxic PAH. However, whether targeting the ras homolog family member A-Rho kinase (RhoA-ROCK) pathway (activation and inhibition) by lysophosphatidic acid (LPA) and fasudil (FSD) regulate T-cell homeostasis in Hypoxic PAH remain unknown. OBJECTIVE: To examine the effects of LPA and FSD on hypoxic pulmonary vascular remodeling and homeostasis of Th17/Treg cells in Hypoxic PAH. METHODS: Rats were exposed to hypoxia (10 ±â€¯0.5% O2) to induce Hypoxic PAH. The experiments consists of two parts. Forty rats were randomly divided into four groups (n = 10): normoxia group, normoxia + LPA group, hypoxia group and hypoxia + LPA group. Thirty rats were randomly divided into another three groups (n = 10): normoxia group, hypoxia group, and hypoxia + FSD group. Rats in normoxia + LPA group and hypoxia + LPA group were intraperitoneally injected 40 µg/kg LPA daily. Rats in hypoxia + FSD group were intraperitoneally injected 30 mg/kg fasudil daily. The effects of LPA and FSD on the development of hypoxic PAH and right ventricle (RV) hypertrophy, on pulmonary vascular remodeling, and on changes of Th17/Treg cells and levels of interleukin-17 (IL-17) and IL-10 were examined. RESULTS: PAH and RV hypertrophy occurred in rats exposed to hypoxia. LPA exacerbated hypoxic pulmonary vascular remodeling and FSD inhibited it. LPA increased Th17/Treg imbalance in peripheral blood and spleen. However, after treatment with FSD, hypoxic PAH rats showed an obvious reduction of Th17 cells as well as an increase of Treg cells. LPA increased the expression of phosphorylated-signal transducer and activator of transcription 3 (p-STAT3) and reduced the p-STAT5 in peripheral blood and spleen in hypoxic PAH rats. The expression of p-STAT3 and p-STAT5 in hypoxic PAH rats treated with FSD showed opposite changes. LPA increased the expression of IL-17 and reduced the IL-10 in small intrapulmonary arteries and serum in hypoxic PAH. However, the expression of IL-17 and IL-10 in hypoxic PAH rats treated with FSD showed opposite changes. CONCLUSIONS: Activation and inhibition of RhoA-ROCK pathway by LPA and FSD modulated the homeostasis of Th17/Treg cells via regulating STAT3/STAT5 phosphorylation in hypoxic PAH. Thus, Apart from influence of pulmonary vascular remodeling, regulation of Th17/Treg homeostasis by RhoA-ROCK pathway play a key role in hypoxic PAH.


Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/patologia , Hipóxia/patologia , Lisofosfolipídeos/farmacologia , Linfócitos T Reguladores/patologia , Células Th17/patologia , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Quinases Associadas a rho/antagonistas & inibidores , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Hipertensão Pulmonar/metabolismo , Hipóxia/metabolismo , Interleucina-10/metabolismo , Interleucina-17/metabolismo , Masculino , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/patologia , Distribuição Aleatória , Ratos , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo , Células Th17/efeitos dos fármacos , Células Th17/metabolismo , Remodelação Vascular/efeitos dos fármacos , Proteínas rho de Ligação ao GTP/metabolismo
14.
J Neurosci ; 38(10): 2589-2604, 2018 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-29440387

RESUMO

Nogo receptor type 1 (NgR1) is known to inhibit neuronal regeneration in the CNS. Previously, we have shown that lateral olfactory tract usher substance (LOTUS) interacts with NgR1 and inhibits its function by blocking its ligand binding. Therefore, LOTUS is expected to have therapeutic potential for the promotion of neuronal regeneration. However, it remains unknown whether the soluble form of LOTUS (s-LOTUS) also has an inhibitory action on NgR1 function as a candidate for therapeutic agents. Here, we show that s-LOTUS inhibits NgR1-mediated signaling by inhibiting the molecular interaction between NgR1 and its coreceptor, p75 neurotrophin receptor (p75NTR). In contrast to the membrane-bound form of LOTUS, s-LOTUS did not block ligand binding to NgR1. However, we identified p75NTR as a novel LOTUS binding partner and found that s-LOTUS suppressed the interaction between p75NTR and NgR1. s-LOTUS inhibited myelin-associated inhibitor (MAI)-induced RhoA activation in murine cortical neurons. Functional analyses revealed that s-LOTUS inhibited MAI-induced growth cone collapse and neurite outgrowth inhibition in chick DRG neurons. In addition, whereas olfactory bulb neurons of lotus-KO mice are sensitive to MAI due to a lack of LOTUS expression, treatment with s-LOTUS inhibited MAI-induced growth cone collapse in these neurons. Finally, we observed that s-LOTUS promoted axonal regeneration in optic nerve crush injury of mice (either sex). These findings suggest that s-LOTUS inhibits NgR1-mediated signaling, possibly by interfering with the interaction between NgR1 and p75NTR Therefore, s-LOTUS may have potential as a therapeutic agent for neuronal regeneration in the damaged CNS.SIGNIFICANCE STATEMENT Nogo receptor type 1 (NgR1) is a receptor well known to inhibit neuronal regeneration in the CNS. Because the membrane-bound form of lateral olfactory tract usher substance (LOTUS) antagonizes NgR1 through a cis-type molecular interaction between LOTUS and NgR1, the soluble form of LOTUS (s-LOTUS) is expected to be a therapeutic agent for neuronal regeneration. In our present study, we show that s-LOTUS inhibits the interaction between NgR1 and p75NTR, NgR1 ligand-induced RhoA activation, growth cone collapse, and neurite outgrowth inhibition and promotes axonal regeneration. Our results indicate that s-LOTUS inhibits NgR1-mediated signaling through a trans-type molecular interaction between LOTUS and NgR1 and, therefore, s-LOTUS may have therapeutic potential for neuronal regeneration.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Receptor Nogo 1/efeitos dos fármacos , Receptores de Fator de Crescimento Neural/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Embrião de Galinha , Feminino , Cones de Crescimento/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Glicoproteína Associada a Mielina/antagonistas & inibidores , Compressão Nervosa , Regeneração Nervosa/efeitos dos fármacos , Neuritos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Receptor Nogo 1/metabolismo , Bulbo Olfatório/citologia , Bulbo Olfatório/efeitos dos fármacos , Proteínas rho de Ligação ao GTP/antagonistas & inibidores
15.
Am J Physiol Renal Physiol ; 315(3): F445-F453, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29465305

RESUMO

It is well known that proteinuria following urinary tract obstruction is mainly of a tubular nature. However, it is unknown whether there are also changes in glomerular permeability. In this study, we compared glomerular sieving coefficients (θ) of polydisperse fluorescein isothiocyanate (FITC)-Ficoll 70/400 following a 120- or 180-min unilateral ureteral obstruction (UUO) in anesthetized Sprague-Dawley rats. Samples were collected from the obstructed kidney at 5, 15, and 30 min postrelease and analyzed by means of high-pressure size-exclusion chromatography. After 120-min UUO, mean θ for Ficoll70Å was increased ( P < 0.01) from 2.2 ± 0.5 × 10-5 (baseline) to 10.6 ± 10 × 10-5 15 min postrelease (highest value). After 180-min UUO, mean θ for Ficoll70Å was further increased ( P < 0.001) from 1.4 ± 0.5 × 10-5 (baseline) to 40 ± 10 × 10-5 at 5 min postrelease (highest value). Administration of a reactive oxygen species (ROS) scavenger (Tempol; 1 mg·kg-1·min-1) partly abrogated the permeability effects following 120-min UUO but not after 180 min. Moreover, administration of the RhoA kinase inhibitor Y-27632, the nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester, or Rac-1 inhibition did not ameliorate glomerular hyperpermeability following 180-min UUO. We show, for the first time, that acute UUO results in marked elevations in glomerular permeability. In addition, our data suggest a time-dependent pathophysiology of UUO-induced hyperpermeability, where reactive oxygen species generation may play an important role in the early stages.


Assuntos
Antioxidantes/farmacologia , Óxidos N-Cíclicos/farmacologia , Inibidores Enzimáticos/farmacologia , Taxa de Filtração Glomerular/efeitos dos fármacos , Glomérulos Renais/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Estresse Oxidativo/efeitos dos fármacos , Obstrução Ureteral/tratamento farmacológico , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Amidas/farmacologia , Aminoquinolinas/farmacologia , Animais , Pressão Arterial/efeitos dos fármacos , Modelos Animais de Doenças , Frequência Cardíaca/efeitos dos fármacos , Glomérulos Renais/enzimologia , Glomérulos Renais/fisiopatologia , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase Tipo III/metabolismo , Proteinúria/tratamento farmacológico , Proteinúria/enzimologia , Proteinúria/fisiopatologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Marcadores de Spin , Fatores de Tempo , Obstrução Ureteral/enzimologia , Obstrução Ureteral/fisiopatologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismo
16.
Small GTPases ; 9(3): 203-215, 2018 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-27548350

RESUMO

The 20 members of the Rho GTPase family are key regulators of a wide-variety of biological activities. In response to activation, they signal via downstream effector proteins to induce dynamic alterations in the organization of the actomyosin cytoskeleton. In this review, post-translational modifications, mechanisms of dysregulation identified in human pathological conditions, and the ways that Rho GTPases might be targeted for chemotherapy will be discussed.


Assuntos
Doença/genética , Inibidores Enzimáticos/farmacologia , Mutação , Processamento de Proteína Pós-Traducional , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Humanos , Ubiquitinação , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/química
17.
Arch Toxicol ; 92(1): 323-336, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28924833

RESUMO

Bacterial protein toxins became valuable molecular tools for the targeted modulation of cell functions in experimental pharmacology and attractive therapeutics because of their potent and specific mode of action in human cells. C2IN-C3lim, a recombinant fusion toxin (~50 kDa) of the Rho-inhibiting C3lim from Clostridium (C.) limosum and a non-toxic portion of the C. botulinum C2 toxin (C2IN), is selectively internalized into the cytosol of monocytic cells where C3lim specifically ADP-ribosylates Rho A and -B, thereby inhibiting Rho-mediated signaling. Thus, we hypothesized that these unique features make C2IN-C3lim an attractive molecule for the targeted pharmacological down-regulation of Rho-mediated functions in monocytes. The analysis of the actin structure and the Rho ADP-ribosylation status implied that C2IN-C3lim entered the cytosol of primary human monocytes from healthy donors ex vivo within 1 h. Moreover, it inhibited the fMLP-induced chemotaxis of human monocytes in a Boyden chamber model ex vivo. Similarly, in a 3-dimensional ex vivo model of extravasation, single cell analysis revealed that C2IN-C3lim-treated cells were not able to move. In a clinically relevant mouse model of blunt chest trauma, the local application of C2IN-C3lim into the lungs after thorax trauma prevented the trauma-induced recruitment of monocytes into the lungs in vivo. Thus, C2IN-C3lim might be an attractive lead compound for novel pharmacological strategies to avoid the cellular damage response caused by monocytes in damaged tissue after trauma and during systemic inflammation. The results suggest that the pathophysiological role of clostridial C3 toxins might be a down-modulation of the innate immune system.


Assuntos
ADP Ribose Transferases/genética , Toxinas Botulínicas/genética , Quimiotaxia/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Proteínas Recombinantes de Fusão/farmacologia , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Animais , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Humanos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Macrófagos/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Monócitos/citologia , Proteínas Recombinantes de Fusão/genética , Traumatismos Torácicos/tratamento farmacológico , Ferimentos não Penetrantes/tratamento farmacológico , Proteínas rho de Ligação ao GTP/metabolismo
18.
Biomed Res Int ; 2017: 2320519, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29201898

RESUMO

Platelets (PLTs) are produced by megakaryocytes (MKs) that completed differentiation and endomitosis. Endomitosis is an important process in which the cell replicates its DNA without cytokinesis and develops highly polyploid MK. In this study, to gain a better PLTs production, four small molecules (Rho-Rock inhibitor (RRI), nicotinamide (NIC), Src inhibitor (SI), and Aurora B inhibitor (ABI)) and their combinations were surveyed as MK culture supplements for promoting polyploidization. Three leukemia cell lines as well as primary mononuclear cells were chosen in the function and mechanism studies of the small molecules. In an optimal culture method, cells were treated with different small molecules and their combinations. The impact of the small molecules on megakaryocytic surface marker expression, polyploidy, proliferation, and apoptosis was examined for the best MK polyploidization supplement. The elaborate analysis confirmed that the combination of SI and RRI together with our MK induction system might result in efficient ploidy promotion. Our experiments demonstrated that, besides direct downregulation on the expression of cytoskeleton protein actin, SI and RRI could significantly enhance the level of cyclins through the suppression of p53 and p21. The verified small molecule combination might be further used in the in vitro PLT manufacture and clinical applications.


Assuntos
Aurora Quinase B/genética , Plaquetas/metabolismo , Megacariócitos/metabolismo , Bibliotecas de Moléculas Pequenas/administração & dosagem , Actinas/genética , Aurora Quinase B/antagonistas & inibidores , Plaquetas/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Megacariócitos/efeitos dos fármacos , Niacinamida/administração & dosagem , Poliploidia , Inibidores de Proteínas Quinases/administração & dosagem , Proteína Supressora de Tumor p53/genética , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/genética , Quinases da Família src/antagonistas & inibidores
19.
Medicina (B Aires) ; 77(6): 497-504, 2017.
Artigo em Espanhol | MEDLINE | ID: mdl-29223942

RESUMO

Rho GTPases are molecular switches that control the different cellular processes. Deregulation of these proteins is associated to transformation and malignant progression in several cancer types. Given the evidence available of the role of Rho GTPases in cancer it is suggested that these proteins can serve as potential therapeutic targets. This review focuses on the strategies used to develop Rho GTPases modulators and their potential use in therapeutic settings.


Assuntos
Antineoplásicos/uso terapêutico , Terapia de Alvo Molecular/métodos , Neoplasias/tratamento farmacológico , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Humanos , Neoplasias/enzimologia , Proteínas rho de Ligação ao GTP/fisiologia
20.
Nature ; 551(7679): 227-231, 2017 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-29088697

RESUMO

Copy-number variants of chromosome 16 region 16p11.2 are linked to neuropsychiatric disorders and are among the most prevalent in autism spectrum disorders. Of many 16p11.2 genes, Kctd13 has been implicated as a major driver of neurodevelopmental phenotypes. The function of KCTD13 in the mammalian brain, however, remains unknown. Here we delete the Kctd13 gene in mice and demonstrate reduced synaptic transmission. Reduced synaptic transmission correlates with increased levels of Ras homolog gene family, member A (RhoA), a KCTD13/CUL3 ubiquitin ligase substrate, and is reversed by RhoA inhibition, suggesting increased RhoA as an important mechanism. In contrast to a previous knockdown study, deletion of Kctd13 or kctd13 does not increase brain size or neurogenesis in mice or zebrafish, respectively. These findings implicate Kctd13 in the regulation of neuronal function relevant to neuropsychiatric disorders and clarify the role of Kctd13 in neurogenesis and brain size. Our data also reveal a potential role for RhoA as a therapeutic target in disorders associated with KCTD13 deletion.


Assuntos
Encéfalo/metabolismo , Proteínas de Transporte/metabolismo , Deleção de Genes , Transmissão Sináptica/genética , Proteínas de Peixe-Zebra/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/psicologia , Transtorno Autístico/genética , Transtorno Autístico/psicologia , Encéfalo/anatomia & histologia , Encéfalo/citologia , Encéfalo/patologia , Região CA1 Hipocampal/metabolismo , Região CA1 Hipocampal/patologia , Proteínas de Transporte/genética , Deleção Cromossômica , Transtornos Cromossômicos/genética , Transtornos Cromossômicos/psicologia , Cromossomos Humanos Par 16/genética , Proteínas Culina/metabolismo , Feminino , Deficiência Intelectual/genética , Deficiência Intelectual/psicologia , Masculino , Camundongos , Herança Multifatorial/genética , Neurogênese/genética , Tamanho do Órgão/genética , Reprodutibilidade dos Testes , Transmissão Sináptica/efeitos dos fármacos , Complexos Ubiquitina-Proteína Ligase , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas rho de Ligação ao GTP/antagonistas & inibidores
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