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1.
Acta Crystallogr F Struct Biol Commun ; 78(Pt 10): 348-353, 2022 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-36189718

RESUMO

The small molecule belumosudil was initially identified as a selective inhibitor of Rho-associated coiled-coil kinase 2 (ROCK2) and has recently been approved for the treatment of graft-versus-host disease. However, recent studies have shown that many of the phenotypes displayed upon treatment with belumosudil were due to CK2α inhibition. CK2α is in itself a very promising therapeutic target for a range of conditions and has recently been put forward as a potential treatment for COVID-19. Belumosudil presents a promising starting point for the development of future CK2α inhibitors as it provides a safe, potent and orally bioavailable scaffold. Therefore, several of the major hurdles in drug development have already been overcome. Here, the crystal structure of belumosudil bound to the ATP site of CK2α is presented. This crystal structure combined with modelling studies further elucidates how belumosudil could be developed into a selective and potent CK2α or ROCK2 inhibitor.


Assuntos
COVID-19 , Quinases Associadas a rho , Acetamidas , Trifosfato de Adenosina , Cristalografia por Raios X , Humanos , Quinases Associadas a rho/genética
2.
Int J Mol Sci ; 23(17)2022 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-36077069

RESUMO

Pancreatic cancer (PC) has a high mortality rate due to its poor prognosis and the possibility of surgical resection in patients with the disease. Importantly, adjuvant chemotherapy is necessary to improve PC prognosis. Chrysin, a natural product with anti-inflammatory, antioxidant, and anticancer properties, has been studied for several years. Our previous study demonstrated that chrysin induced G protein-coupled estrogen receptor (GPER) expression and regulated its activity in breast cancer. Herein, we investigated whether chrysin-induced GPER activation suppresses PC progression in MIA PaCa-2 cells and a xenograft model. To determine its mechanism of action, cytotoxicity and clonogenic assays, a FACS analysis, and Western blotting were performed. Furthermore, the delay in tumor growth was evaluated in the MIA PaCa-2-derived xenograft model. Tumor tissues were investigated by Western blotting, immunohistochemistry, and a proteomic analysis. Chrysin caused cell cycle arrest and significantly decreased cell viability. Following co-treatment with chrysin and 17ß-estradiol, the inhibitory effect of chrysin on cell proliferation was enhanced. In the xenograft model, chrysin and G1 (a GPER agonist) significantly delayed tumor growth and reduced both Ki-67 (a proliferation marker) and c-Myc expressions in tumor tissues. The proteomic analysis of tumor tissues identified that rho-associated coiled-coil containing protein kinase 1 (ROCK1), transgelin 2 (TAGLN2), and FCH and Mu domain containing endocytic adaptor 2 (FCHO2) levels were significantly reduced in chrysin-treated tumor tissues. High ROCK1, TAGLN2, and FCHO2 expressions were indicative of low overall PC survival as found using the Kaplan-Meier plotter. In conclusion, our results suggest that chrysin suppresses PC progression through the activation of GPER and reductions in ROCK1, TAGLN2, and FCHO2 expressions.


Assuntos
Neoplasias Pancreáticas , Receptores de Estrogênio , Linhagem Celular Tumoral , Proliferação de Células , Estrogênios/farmacologia , Flavonoides , Proteínas de Ligação ao GTP/metabolismo , Humanos , Neoplasias Pancreáticas/tratamento farmacológico , Proteômica , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Quinases Associadas a rho/metabolismo
3.
Int J Mol Sci ; 23(17)2022 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-36077249

RESUMO

Liver sinusoidal endothelial cells (LSECs) facilitate the efficient transport of macromolecules and solutes between the blood and hepatocytes. The efficiency of this transport is realized via transcellular nanopores, called fenestrations. The mean fenestration size is 140 ± 20 nm, with the range from 50 nm to 350 nm being mostly below the limits of diffraction of visible light. The cellular mechanisms controlling fenestrations are still poorly understood. In this study, we tested a hypothesis that both Rho kinase (ROCK) and myosin light chain (MLC) kinase (MLCK)-dependent phosphorylation of MLC regulates fenestrations. We verified the hypothesis using a combination of several molecular inhibitors and by applying two high-resolution microscopy modalities: structured illumination microscopy (SIM) and scanning electron microscopy (SEM). We demonstrated precise, dose-dependent, and reversible regulation of the mean fenestration diameter within a wide range from 120 nm to 220 nm and the fine-tuning of the porosity in a range from ~0% up to 12% using the ROCK pathway. Moreover, our findings indicate that MLCK is involved in the formation of new fenestrations-after inhibiting MLCK, closed fenestrations cannot be reopened with other agents. We, therefore, conclude that the Rho-ROCK pathway is responsible for the control of the fenestration diameter, while the inhibition of MLCK prevents the formation of new fenestrations.


Assuntos
Actinas , Cadeias Leves de Miosina , Actinas/metabolismo , Animais , Células Endoteliais/metabolismo , Hepatócitos/metabolismo , Fígado/metabolismo , Camundongos , Microscopia Eletrônica de Varredura , Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Fosforilação , Quinases Associadas a rho/metabolismo
4.
Eur Cytokine Netw ; 33(1): 13-24, 2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-36102857

RESUMO

Background: Asthma is an airway disease characterized by airflow limitation and various additional clinical manifestations. Repeated inflammatory stimulation of the airways leads to epithelial-mesenchymal transition (EMT) which aggravates subepithelial fibrosis during the process of airway remodelling and enhances resistance to corticosteroids and bronchodilators in refractory asthma. There is growing evidence that IL-27 modulates airway remodelling, however, the molecular mechanisms involving IL-27 and EMT are poorly understood. The objective of this study was to investigate the effects of IL-27 on ovalbumin (OVA)-challenged asthmatic mice in vivo and TGF-ß1-induced EMT in 16HBE cells in vitro. Methods: Airway inflammation, mucus secretion, and collagen deposition were analysed by conventional pathological techniques. The ratio of Th17 and Th9 cells in the spleen of mice was measured using flow cytometry, ELISA was performed for cytokine analysis to identify EMT-related molecules and signalling pathways, and other molecular and cellular techniques were used to explore the functional mechanism involving IL-27 and EMT. Results: Airway inflammation in asthmatic mice was significantly alleviated by IL-27, with downregulation of RhoA and ROCK, upregulation of E-cadherin, and a decrease of vimentin and α-SMA expression, compared to asthmatic mice. Moreover, the frequency of Th17 and Th9 cells in the spleen of asthmatic mice decreased following treatment with IL-27. In TGF-ß1-induced 16HBE cells, the addition of IL-27 was shown to inhibit EMT, based on the expression of E-cadherin, vimentin, and α-SMA. Conclusion: Intranasal administration of IL-27 attenuates airway inflammation and EMT in a murine model of allergic asthma possibly by downregulating the RhoA/ROCK signalling pathway.


Assuntos
Asma , Interleucina-27 , Remodelação das Vias Aéreas , Animais , Asma/tratamento farmacológico , Caderinas/metabolismo , Caderinas/farmacologia , Caderinas/uso terapêutico , Transição Epitelial-Mesenquimal/fisiologia , Inflamação/tratamento farmacológico , Camundongos , Fator de Crescimento Transformador beta1/metabolismo , Vimentina/farmacologia , Vimentina/uso terapêutico , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
5.
BMC Cancer ; 22(1): 1027, 2022 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-36175877

RESUMO

The protein Talin1 encoded by the TLN1 gene is a focal adhesion-related protein that binds to various cytoskeletal proteins and plays an important role in cell adhesion and movement. Recent studies have shown that it is overexpressed in prostate cancer, liver cancer, and oral squamous cell carcinoma, and is closely related to tumor progression and metastasis. This study integrated bioinformatics and functional analysis to reveal the prognosis and potential functions of TLN1 in AML. The results showed that the expression level of TLN1 was abnormally increased in AML and localized in the cell membrane and cytoplasm, and TLN1 is a significant prognostic indicator of overall survival (OS). Enrichment analysis of related genes showed that TLN1 is related to neutrophil mediated immunity, neutrophil activation and may regulate important signal pathways in hematological tumors including tyrosine kinase receptor, FLT3 and PIK3/AKT. The PPI network shows that TLN1 and MYH9 may be involved in the process of AML tumors together with PIP5K1C, ROCK1, S100A4, MY01A and WAC. Immune infiltration analysis explains that TLN1 is associated with multiple immune cells and may be an important immune marker in AML. Furthermore, molecular biology experiments confirmed that TLN1 is related to the proliferation, differentiation and cycle of AML cells. Silencing TLN1 can inhibit the proliferation of AML cells and promote differentiation through the Talin1/P-AKT/CREB signaling pathway.


Assuntos
Carcinoma de Células Escamosas , Leucemia Mieloide Aguda , Neoplasias Bucais , Proliferação de Células/genética , Proteínas do Citoesqueleto , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Masculino , Prognóstico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Proteína Tirosina Quinases , Talina/genética , Talina/metabolismo , Quinases Associadas a rho
6.
Clin Transl Med ; 12(10): e1036, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36178087

RESUMO

BACKGROUND: Emerging evidence provides mechanistic insights into the pathogenesis of pulmonary fibrosis (PF), and rare anti-PF therapeutic method has promising effect in its treatment. Rho-associated coiled-coil kinases (ROCK) inhibition significantly ameliorates bleomycin-induced PF and decreases macrophage infiltration, but the mechanism remains unclear. We established bleomycin and radiation-induced PF to identify the activity of WXWH0265, a newly designed unselective ROCK inhibitor in regulating macrophages. METHODS: Bleomycin-induced PF was induced by intratracheal instillation and radiation-induced PF was induced by bilateral thoracic irradiation. Histopathological techniques (haematoxylin and eosin, Masson's trichrome and immunohistochemistry) and hydroxyproline were used to evaluate PF severity. Western blot, quantitative real-time reverse transcription-polymerase chain reaction and flow cytometry were performed to explore the underlying mechanisms. Bone marrow-derived macrophages (BMDMs) were used to verify their therapeutic effect. Clodronate liposomes were applied to deplete macrophages and to identify the therapeutic effect of WXWH0265. RESULTS: Therapeutic administration of ROCK inhibitor ameliorates bleomycin-induced PF by inhibiting M2 macrophages polarisation. ROCK inhibitor showed no significant anti-fibrotic effect in macrophages-depleted mice. Treatment with WXWH0265 demonstrated superior protection effect in bleomycin-induced PF compared with positive drugs. In radiation-induced PF, ROCK inhibitor effectively ameliorated PF. Fibroblasts co-cultured with supernatant from various M2 macrophages phenotypes revealed that M2 macrophages stimulated by interleukin-4 promoted extracellular matrix production. Polarisation of M2 macrophages was inhibited by ROCK inhibitor treatment in vitro. The p-signal transducer and activator of transcription 3 (STAT3) in lung tissue and BMDMs was significantly decreased in PF in vivo and vitro after treated with ROCK inhibitors. CONCLUSION: Inhibiting ROCK could significantly attenuate bleomycin- and radiation-induced PF by regulating the macrophages polarisation via phosphorylation of STAT3. WXWH0265 is a kind of efficient unselective ROCK inhibitor in ameliorating PF. Furthermore, the results provide empirical evidence that ROCK inhibitor, WXWH0265 is a potential drug to prevent the development of PF.


Assuntos
Fibrose Pulmonar , Animais , Bleomicina/efeitos adversos , Ácido Clodrônico/efeitos adversos , Amarelo de Eosina-(YS)/efeitos adversos , Hidroxiprolina/efeitos adversos , Hidroxiprolina/metabolismo , Interleucina-4/efeitos adversos , Lipossomos/efeitos adversos , Macrófagos/metabolismo , Camundongos , Fosforilação , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/tratamento farmacológico , Fibrose Pulmonar/patologia , Fator de Transcrição STAT3/uso terapêutico , Quinases Associadas a rho/uso terapêutico
7.
Int J Mol Sci ; 23(18)2022 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-36142622

RESUMO

The purpose of this work was to demonstrate the use of the AQbD with the DOE approach to the methodical step-by-step development of a UHPLC method for the quantitative determination of the impurity profile of new CPL409116 substance (JAK/ROCK inhibitor) on the preclinical and clinical step of drug discovery studies. The critical method parameters (CMPs) have been tested extensively: the kind of stationary phase (8 different columns), pH of the aqueous mobile phase (2.6, 3.2, 4.0, 6.8), and start (20-25%) and stop (85-90%) percentage of organic mobile phase (ACN). The critical method attributes (CMAs) are the resolution between the peaks (≥2.0) and peak symmetry of analytes (≥0.8 and ≤1.8). In the screening step, the effects of different levels of CMPs on the CMAs were evaluated based on a full fractional design 22. The robustness tests were established from the knowledge space of the screening step and performed by application fractional factorial design 2(4-1). Method operable design region (MODR) was generated. The probability of meeting the specifications for the CMAs was calculated by Monte-Carlo simulations. In relation to literature such a complete AQbD approach including screening, optimization, and validation steps for the development of a new method for the quantitative determination of the full profile of nine impurities of an innovative pharmaceutical substance with the structure-based pre-development pointed out the novelty of our work. The final working conditions were as follows: column Zorbax Eclipse Plus C18, aqueous mobile phase 10 mM ± 1 mM aqueous solution of HCOOH, pH 2.6, 20% ± 1% of ACN at the start and 85% ± 1% of ACN at the end of the gradient, and column temperature 30 °C ± 2 °C. The method was validated in compliance with ICH guideline Q2(R1). The optimized method is specified, linear, precise, and robust. LOQ is on the reporting threshold level of 0.05% and LOD at 0.02% for all impurities.


Assuntos
Descoberta de Drogas , Quinases Associadas a rho , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida/métodos , Estranos , Nitrilas , Preparações Farmacêuticas , Reprodutibilidade dos Testes
8.
Atherosclerosis ; 358: 12-28, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36049290

RESUMO

Cardiovascular disease (CVD) continues to be the primary cause of global mortality. Vascular smooth muscle cells (VSMCs) are integral components of vascular structure and function, evident by their vital roles in modulating blood flow and pressure. Such roles exist due to the differentiated contractile phenotype of VSMCs. However, VSMCs may switch to a dedifferentiated, proliferative synthetic phenotype in a phenomenon known as phenotypic switching. This switch involves dramatic changes in VSMC migration, proliferation, gene expression programs, differentiation, cellular stiffness and extracellular matrix (ECM) deposition. In this review, we explore the role of the small GTPase Rho and its effector, Rho-associated kinase (ROCK), in phenotypic switching as well as apoptotic pathways in VSMCs. We critically dissect how RhoA promotes cell migration and proliferation as well as its role in modulating the expression of a battery of VSMC marker proteins. We also discuss how RhoA modulates apoptosis, induces dedifferentiation, increases vascular stiffness, or modifies ECM accumulation. These alterations in VSMC phenotypes contribute to multiple vascular dysfunctions, including hypertension and atherosclerosis. Understanding the molecular underpinnings and the signaling pathways involved in these altered phenotypes may provide novel avenues of drug design and other therapeutic interventions for the management of CVDs.


Assuntos
Músculo Liso Vascular , Quinases Associadas a rho , Proliferação de Células , Células Cultivadas , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fenótipo , Quinases Associadas a rho/metabolismo
9.
Life Sci Alliance ; 5(11)2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36096674

RESUMO

Cell-matrix adhesions are mainly provided by integrin-mediated focal adhesions (FAs). We previously found that Shp2 is essential for FA maturation by promoting ROCK2 activation at FAs. In this study, we further delineated the role of α-actinin-4 in the FA recruitment and activation of Shp2. We used the conditional immortalized mouse podocytes to examine the role of α-actinin-4 in the regulation of Shp2 and ROCK2 signaling. After the induction of podocyte differentiation, Shp2 and ROCK2 were strongly activated, concomitant with the formation of matured FAs, stress fibers, and interdigitating intracellular junctions in a ROCK-dependent manner. Gene knockout of α-actinin-4 abolished the Shp2 activation and subsequently reduced matured FAs in podocytes. We also demonstrated that gene knockout of ROCK2 impaired the generation of contractility and interdigitating intercellular junctions. Our results reveal the role of α-actinin-4 in the recruitment of Shp2 at FAs to potentiate ROCK2 activation for the maintenance of cellular contractility and cytoskeletal architecture in the cultured podocytes.


Assuntos
Adesões Focais , Podócitos , Actinina/genética , Actinina/metabolismo , Animais , Citoesqueleto/metabolismo , Adesões Focais/genética , Adesões Focais/metabolismo , Camundongos , Podócitos/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Transdução de Sinais/genética , Quinases Associadas a rho/genética
10.
J Cell Biol ; 221(10)2022 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-36102907

RESUMO

Reversible protein phosphorylation by kinases controls a plethora of processes essential for the proper development and homeostasis of multicellular organisms. One main obstacle in studying the role of a defined kinase-substrate interaction is that kinases form complex signaling networks and most often phosphorylate multiple substrates involved in various cellular processes. In recent years, several new approaches have been developed to control the activity of a given kinase. However, most of them fail to regulate a single protein target, likely hiding the effect of a unique kinase-substrate interaction by pleiotropic effects. To overcome this limitation, we have created protein binder-based engineered kinases that permit a direct, robust, and tissue-specific phosphorylation of fluorescent fusion proteins in vivo. We show the detailed characterization of two engineered kinases based on Rho-associated protein kinase (ROCK) and Src. Expression of synthetic kinases in the developing fly embryo resulted in phosphorylation of their respective GFP-fusion targets, providing for the first time a means to direct the phosphorylation to a chosen and tagged target in vivo. We presume that after careful optimization, the novel approach we describe here can be adapted to other kinases and targets in various eukaryotic genetic systems to regulate specific downstream effectors.


Assuntos
Proteínas , Quinases Associadas a rho , Quinases da Família src , Animais , Drosophila , Fosforilação , Engenharia de Proteínas , Proteínas/metabolismo , Transdução de Sinais , Especificidade por Substrato , Quinases Associadas a rho/metabolismo , Quinases da Família src/metabolismo
11.
ACS Appl Mater Interfaces ; 14(38): 42976-42987, 2022 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-36103264

RESUMO

Local stimuli differentiate monocytes into M2-like macrophages that mechanistically drive the pathologies in cancer and age-related macular degeneration (AMD). A photo-controlled nanodrug that halts macrophage polarization through Rho-associated kinase (ROCK) inhibition was developed. A small-molecule ROCK inhibitor, fasudil, was conjugated to a photo-responsive group and a short poly(ethylene glycol) (PEG) chain. This resulted in the novel amphiphilic prodrug, PEG-2-(4'-(di(prop-2-yn-1-yl)amino)-4-nitro-[1,1'-biphenyl]-yl)propan-1-ol (PANBP)-Fasudil, that spontaneously formed micelles. Ultraviolet (UV) irradiation of PEG-PANBP-Fasudil nanoparticles rapidly released fasudil. For visualization of linker degradation, a reporter nanoprobe was synthesized, in which 2-Me-4-OMe TokyoGreen (TG), a fluorophore that does not fluoresce in conjugation, was incorporated. Irradiation of nanoprobe-laden monocytes activated the reporter fluorophore. Cytokine stimulation differentiated monocytes into macrophages, while UV irradiation prevented polarization of PEG-PANBP-Fasudil nanoparticle-laden monocytes. Nanoarchitectonics-based design opens new possibilities for intracellular drug delivery and precise spatiotemporal immune cell modulation toward the development of new therapies.


Assuntos
Pró-Fármacos , Quinases Associadas a rho , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Citocinas/metabolismo , Liberação Controlada de Fármacos , Mercaptoetanol , Micelas , Polietilenoglicóis/metabolismo
12.
Int J Mol Sci ; 23(18)2022 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-36142844

RESUMO

We previously reported that lysophosphatidylinositol (LPI) functions as an endogenous agonist of GPR55, a novel cannabinoid receptor. However, the physiological roles of LPI-GPR55 have not yet been elucidated in detail. In the present study, we found that LPI induced morphological changes in GPR55-expressing HEK293 cells. LPI induced the cell rounding of GPR55-expressing HEK293 cells but not of empty-vector-transfected cells. LPI also induced the activation of small GTP-binding protein RhoA and increased stress fiber formation in GPR55-expressing HEK293 cells. The inhibition of RhoA and Rho kinase ROCK by the C3 exoenzyme and the ROCK inhibitor reduced LPI-induced cell rounding and stress fiber formation. These results clearly indicated that the LPI-induced morphological changes and the assembly of the cytoskeletons were mediated through the GPR55-RhoA-ROCK pathway.


Assuntos
Receptores Acoplados a Proteínas G , Quinases Associadas a rho , Células HEK293 , Humanos , Lisofosfolipídeos/metabolismo , Receptores de Canabinoides/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Fibras de Estresse/metabolismo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo
13.
Oxid Med Cell Longev ; 2022: 2198923, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36160709

RESUMO

Preeclampsia is regarded as an evolution-related disease that has only been observed in humans and our closest relatives, and the important factor contributing to its pathogenesis is endothelial dysregulation secondary to a stressed placenta. Hypoxia-inducible factor 1 subunit alpha (HIF1α), a highly conserved molecule in virtually all mammals, is regarded as a crucial regulator of the hypoxia adaptation and evolution. Persistent high expression of HIF1α in the placenta is one of the pathogenic mechanisms of preeclampsia. Therefore, human-specific molecules should link increased HIF1α to preeclampsia. We reported that urothelial cancer associated 1 (UCA1) is a potential mediator because it is a human-specific long noncoding RNA (lncRNA) that is upregulated in placental tissues and maternal serum from women with preeclampsia and is regulated by HIF1α. The cellular HIF1α-UCA1 pathway promoted the adaptation of trophoblasts to hypoxia by inducing vascular endothelial growth factor (VEGF) secretion and changes in the levels of key enzymes in glycolysis. On the other hand, circulating exosomal UCA1 secreted from stressed trophoblasts induced vascular endothelial dysfunction, especially excess ROS production, as measured by exosome extraction and a coculture system. At the molecular level, UCA1 physically bound to ubiquitin-specific peptidase 14 (USP14), which is a deubiquitinating enzyme, and UCA1 functioned as a scaffold to recruit USP14 to profilin 1 (PFN1), an actin-binding protein contributing to endothelial abnormalities and vascular diseases. This ternary complex inhibited the ubiquitination-dependent degradation of PFN1 and prolonged its half-life, further activating the RhoA/Rho-kinase (ROCK) pathway to induce ROS production in endothelial cells. Taken together, these observations suggest a role for the evolution-related UCA1 in the HIF1α-induced adaptive pathogenic mechanism of preeclampsia, promoting the survival of hypoxic trophoblasts and injuring maternal endothelial cells.


Assuntos
Pré-Eclâmpsia , RNA Longo não Codificante , Neoplasias da Bexiga Urinária , Enzimas Desubiquitinantes/metabolismo , Células Endoteliais/metabolismo , Feminino , Humanos , Hipóxia/metabolismo , Fator 1 Induzível por Hipóxia/metabolismo , Placenta/metabolismo , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/metabolismo , Gravidez , Profilinas/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Trofoblastos/metabolismo , Ubiquitina Tiolesterase , Proteases Específicas de Ubiquitina/metabolismo , Neoplasias da Bexiga Urinária/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP
14.
Arch Oral Biol ; 143: 105540, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36087522

RESUMO

OBJECTIVE: During enamel formation, transforming growth factor-beta (TGF-ß) isoforms exhibit different activities for gene expression, apoptosis, and endocytosis. This study aimed to investigate the differential response of TGF-ß isoforms to epithelial-mesenchymal transition (EMT) in enamel epithelial cells. DESIGN: Using a mouse enamel epithelial cell line (mHAT9d) cultured in the presence of each TGF-ß isoform, (1) the morphological changes in EMT were explored, (2) EMT-related genes were analyzed by next-generation sequencing (NGS), (3) TGF-ß pathway for EMT was identified by inhibition experiments, and (4) the expression of the TGF-ß receptor gene in response to the binding affinity of the TGF-ß isoform were analyzed. RESULTS: EMT was observed in mHAT9d cultured in the presence of TGF-ß1 and ß3 but not TGF-ß2. The expression of both epithelial and mesenchymal marker genes was observed in mHAT9d exhibiting EMT. NGS analysis suggested extracellular signal-regulated kinase (ERK) and Rho pathways as TGF-ß signaling pathways associated with EMT. However, EMT in mHAT9d cultured in the presence of TGF-ß1 or ß3 occurred even in presence of an ERK1/2 inhibitor and was suppressed by Rho-kinase inhibitor. The expression of co-receptors for TGF-ß signaling in mHAT9d cells reduced following stimulation with each TGF-ß isoform. In contrast, endoglin levels increased following TGF-ß1 or ß3 stimulation, but no change was noted in response to TGF-ß2. CONCLUSIONS: We propose that in TGF-ß-stimulated enamel epithelial cells, EMT mainly occurred via the Rho signaling pathway, and the differences in response across TGF-ß isoforms were due to their endoglin-mediated binding affinity for the TGF-ß receptor.


Assuntos
Transição Epitelial-Mesenquimal , Fator de Crescimento Transformador beta1 , Esmalte Dentário/metabolismo , Endoglina/metabolismo , Células Epiteliais/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Isoformas de Proteínas/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Fatores de Crescimento Transformadores/metabolismo , Quinases Associadas a rho/metabolismo
15.
Biomolecules ; 12(9)2022 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-36139129

RESUMO

The present study investigated whether Rho-associated protein kinase (RhoA/ROCK) signaling pathway inhibitor simvastatin inhibits matrix metalloproteinase 2 (MMP-2) activity in a rat ischemia-reperfusion injury (I/Ri) model by inhibiting the RhoA/ROCK pathway and reducing MMP-2 mRNA levels. Isolated rat hearts were subjected to aerobic perfusion or I/Ri control. The effect of simvastatin was assessed in hearts subjected to I/Ri. We determined cardiac mechanical function, the content of RhoA, phosphorylated myosin light chain subunit 1 (phospho-MYL9), troponin I, MMP-2, and MMP-2 mRNA in the heart homogenates, as well as MMP-2 activity in heart tissue. We showed that treatment with simvastatin caused improvement in the contractile function of the heart subjected to I/Ri which was accompanied by a decrease of MMP-2 activity in heart tissue along with inhibition of RhoA pathway, expressed in a reduction in both RhoA and its downstream product-phosphorylated myosin light chain (phospho-MYL9) in hearts treated with simvastatin. MMP-2 inactivation is not due to inhibition of MMP-2 m-RNA synthesis caused by inhibition of RhoA/ROCK pathway and is due, at least in part, to the direct drug action. The protective effect of simvastatin on systolic function in the acute ischemia-reperfusion model does not appear to be related to reduced MMP-2 activation, but other mechanisms related with the inhibition RhoA/ROCK pathway.


Assuntos
Metaloproteinase 2 da Matriz , Traumatismo por Reperfusão , Sinvastatina , Quinases Associadas a rho , Animais , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Cadeias Leves de Miosina/metabolismo , RNA Mensageiro , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/tratamento farmacológico , Sinvastatina/farmacologia , Troponina I/metabolismo , Proteínas rho de Ligação ao GTP , Quinases Associadas a rho/metabolismo
16.
Sheng Li Xue Bao ; 74(4): 555-562, 2022 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-35993207

RESUMO

This study aimed to investigate the effects of hypoxia on RhoA/Rho-kinase (ROCK) signaling pathway and autophagy in pulmonary artery smooth muscle cells (PASMCs), and to explore the underlying mechanism of Umbelliferone (Umb) in ameliorating chronic hypoxic pulmonary hypertension. PASMCs were cultured from Sprague-Dawley (SD) rats and randomly divided into control group, hypoxia group, hypoxia + Umb intervention group and normoxia + Umb intervention group. Alpha smooth muscle actin (α-SMA) and LC3 were assessed by immunofluorescence staining. Protein expression of RhoA, ROCK2, p-MYPT1, LC3-II, Beclin-1, p62, C-Caspase 3, Bax and Bcl-2 was analyzed by Western blotting. In in vivo study, SD rats were divided into control group, hypoxia group and hypoxia + Umb intervention group. Weight ratio of the right ventricle (RV)/left ventricle plus septum (LV+S) was detected, and pulmonary arterial morphological features were examined by HE staining. The results indicated that compared with the control group, the LC3-II/LC3-I ratio and expression of Beclin-1 were significantly increased, while p62 expression was significantly decreased, and the expressions of RhoA, ROCK2 and p-MYPT1 were significantly increased in PASMCs of hypoxia group (P < 0.05). The changes of LC3-II/LC3-I ratio, the expressions of Beclin-1, p62, RhoA, ROCK2 and p-MYPT1 in PASMCs were reversed by Umb treatment (P < 0.05). Consistently, the pulmonary arterial wall was thickened and the RV/(LV+S) ratio was increased in hypoxic rats, which were significantly improved by Umb treatment (P < 0.05). These results suggest that Umb can improve hypoxia-induced pulmonary hypertension by inhibiting the RhoA/ROCK signaling pathway and autophagy in PASMCs.


Assuntos
Hipertensão Pulmonar , Animais , Autofagia , Proteína Beclina-1/metabolismo , Proteína Beclina-1/farmacologia , Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/etiologia , Hipóxia/complicações , Miócitos de Músculo Liso/metabolismo , Artéria Pulmonar , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Umbeliferonas/metabolismo , Umbeliferonas/farmacologia , Quinases Associadas a rho/metabolismo , Quinases Associadas a rho/farmacologia
17.
Eur J Pharmacol ; 931: 175207, 2022 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-35987254

RESUMO

Current antipsychotics used to treat schizophrenia have associated problems, including serious side effects and treatment resistance. We recently identified a significant association of schizophrenia with exonic copy number variations in the Rho GTPase activating protein 10 (ARHGAP10) gene using genome-wide analysis. ARHGAP10 encodes a RhoGAP superfamily member that is involved in small GTPase signaling. In mice, Arhgap10 gene variations result in RhoA/Rho-kinase pathway activation. We evaluated the pharmacokinetics of fasudil and hydroxyfasudil using liquid chromatography-tandem mass spectrometry in mice. The antipsychotic effects of fasudil on hyperlocomotion, social interaction deficits, prepulse inhibition deficits, and novel object recognition deficits were also investigated in a MK-801-treated pharmacological mouse schizophrenia model. Fasudil and its major metabolite, hydroxyfasudil, were detected in the brain at concentrations above their respective Ki values for Rho-kinase after intraperitoneal injection of 10 mg kg-1 fasudil. Fasudil improved the hyperlocomotion, social interaction deficits, prepulse inhibition deficits, and novel object recognition deficits in MK-801-treated mice in a dose-dependent manner. Following oral administration of fasudil, brain hydroxyfasudil was detected at concentration above the Ki value for Rho-kinase whilst fasudil was undetectable. MK-801-induced hyperlocomotion was also improved by oral fasudil administration. These results suggest that fasudil has antipsychotic-like effects on the MK-801-treated pharmacological mouse schizophrenia model. There are two isoforms in Rho-kinase, and further investigation is needed to clarify the isoforms involved in the antipsychotic-like effects of fasudil in the MK-801-treated mouse schizophrenia model.


Assuntos
Antipsicóticos , Esquizofrenia , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/uso terapêutico , Animais , Antipsicóticos/farmacologia , Antipsicóticos/uso terapêutico , Variações do Número de Cópias de DNA , Modelos Animais de Doenças , Maleato de Dizocilpina/farmacologia , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Esquizofrenia/tratamento farmacológico , Quinases Associadas a rho
18.
Poult Sci ; 101(10): 102051, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35961254

RESUMO

Cryopreservation of rooster sperm leads to relatively low semen quality due to cytoskeletal damage during the freeze-thawing process. This study aimed to explore how the addition of RhoA recombinant protein affected the viability and subcellular structure of rooster sperm after freeze-thawing and elucidated the molecular mechanisms of sperm cryopreservation. Semen quality and acrosome integrity testing revealed that the addition of 0.5 µg/mL RhoA recombinant protein to the cryoprotectant fluid significantly increased sperm motility, survival rate, linearity, straight-line velocity, and acrosome integrity after freeze-thawing (P < 0.05). Ultrastructure analysis of cryopreserved sperm showed structural damage to the sperm plasma membrane, nuclear membrane, and tail. However, compared to the control, these structural changes were reduced upon the addition of RhoA recombinant protein to the cryoprotective fluid (P < 0.05). Western blotting revealed that the expression of Rho/RhoA-associated kinase and p-cofilin was increased, and cofilin expression was decreased after sperm cryopreservation with recombinant RhoA protein. Treatment with Y-27632, a ROCK antagonist, suppressed ROCK and p-cofilin expression and decreased semen quality, acrosome integrity, and ultrastructure integrity. In summary, we have demonstrated a cryoprotective effect in spermatozoa involving the Rho/ROCK pathway during freeze-thawing. Furthermore, the addition of 0.5 µg/mL RhoA recombinant protein to the cryoprotective fluid improved rooster semen quality and subcellular structural homeostasis after freeze-thawing via the Rho/ROCK pathway. This pathway may regulate the dynamic reorganization of the actin cytoskeleton by regulating the cofilin phosphorylation.


Assuntos
Crioprotetores , Preservação do Sêmen , Fatores de Despolimerização de Actina , Animais , Galinhas/fisiologia , Criopreservação/veterinária , Crioprotetores/farmacologia , Masculino , Proteínas Recombinantes , Sêmen , Análise do Sêmen/veterinária , Preservação do Sêmen/veterinária , Motilidade Espermática/fisiologia , Espermatozoides/fisiologia , Quinases Associadas a rho , Proteína rhoA de Ligação ao GTP
19.
Int J Biochem Cell Biol ; 151: 106281, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35995387

RESUMO

Excessive mitochondrial fission in podocytes serves as a central hub for the pathogenesis of diabetic nephropathy (DN), and the thromboxane/prostaglandin receptor (TP receptor) plays a potential role in DN. However, regulation of the TP receptor during mitochondrial dynamics disorder in podocytes remains unknown. Here, we firstly reported novel mechanistic details of TP receptor effects on mitochondrial dynamics in podocytes under diabetic conditions. Expression of the TP receptor was significantly upregulated in podocytes under diabetic conditions both in vivo and in vitro. S18886 attenuated podocyte mitochondrial fission, glomerular injury and renal dysfunction in diabetic mice. Furthermore, inhibition of the TP receptor by both genetic and pharmacological methods dramatically reduced mitochondrial fission and attenuated podocyte injury induced by high glucose through regulating dynamin-related protein 1 (Drp1) phosphorylation and its subsequent translocation to mitochondria. In contrast, TP receptor overexpression and application of TP receptor agonist U46619 in these podocytes showed the opposite effect on mitochondrial fission and podocyte injury. Furthermore, treatment with Y27632, an inhibitor of Rho-associated kinase1 (ROCK1), significantly blunted more fragmented mitochondria and reduced podocyte injuries in podocytes with TP receptor overexpression or after U46619 treatment. Finally, pharmacological inhibition of Drp1 alleviated excessive mitochondrial fragmentation and podocyte damage in TP receptor overexpressing podocytes. Our data suggests that increased expression of the TP receptor can occur in a human cultured podocyte cell line and in podocytes derived from streptozotocin (STZ)-induced diabetic mice, which contributes to mitochondrial excessive fission and podocyte injury via ROCK1-Drp1 signaling.


Assuntos
Diabetes Mellitus Experimental , Nefropatias Diabéticas , Doenças Mitocondriais , Podócitos , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/metabolismo , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/uso terapêutico , Animais , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/patologia , Dinaminas/metabolismo , Glucose/metabolismo , Glucose/farmacologia , Humanos , Camundongos , Doenças Mitocondriais/metabolismo , Dinâmica Mitocondrial , Prostaglandinas/metabolismo , Prostaglandinas/farmacologia , Prostaglandinas/uso terapêutico , Receptores de Prostaglandina/metabolismo , Receptores de Prostaglandina/uso terapêutico , Receptores de Tromboxanos/metabolismo , Receptores de Tromboxanos/uso terapêutico , Estreptozocina , Tromboxanos/metabolismo , Tromboxanos/farmacologia , Tromboxanos/uso terapêutico , Quinases Associadas a rho/metabolismo
20.
Curr Pharm Des ; 28(29): 2426-2435, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35909282

RESUMO

BACKGROUND: The clinical utility of Adriamycin (ADR) is limited due to its toxicity, particularly cardiotoxicity. Therefore, effective cardioprotective adjuvants to minimize ADR-induced acute cardiotoxicity are urgently needed. Our previous studies have demonstrated the protective roles of fasudil on tissue injury. Here, we further explore whether inhibition of Rho-kinase could alleviate the acute heart injury induced by ADR. METHODS: C57BL6 mice were randomly divided into the following four groups: ① ADR group; ② low-dose fasudil (ADR+L); ③ high-dose fasudil (ADR+H); and ④ control group (CON). Animals were injected i.p 20 mg/kg ADR once in group ①~③. Animals were injected i.p fasudil (2 or 10 mg/kg/day) daily for consecutive 6 days in groups ② and ③, respectively. Blood samples and heart tissues were collected for assays. H9C2 cells were treated with fasudil for 30 mins and then incubated with ADR for 24 hours. Cells were collected for immunohistochemistry and western blot study, respectively. RESULTS: In the mouse model, administration of fasudil significantly ameliorated ADR-induced cardiac damage, suppressed cell apoptosis and senescence, and ameliorated redox imbalance and DNA damage. In vitro, fasudil treatment ameliorated ADR-induced immunofluorescence reaction of 8-OHdG, decreased the expression of TUNEL cells and proteins of Bax, Caspase-3 and p53, and increased the expression of proteins of Bcl-2 and SIRT 1. CONCLUSION: Fasudil has a protective effect on ADR induced acute cardiotoxicity, which is partially attributed to its antioxidant, anti-senescence, and anti-apoptotic effects.


Assuntos
Traumatismos Cardíacos , Sirtuínas , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Animais , Antioxidantes/farmacologia , Apoptose , Cardiotoxicidade/tratamento farmacológico , Cardiotoxicidade/prevenção & controle , Caspase 3/metabolismo , Caspase 3/farmacologia , Senescência Celular , Doxorrubicina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sirtuínas/metabolismo , Sirtuínas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/farmacologia , Proteína X Associada a bcl-2/metabolismo , Proteína X Associada a bcl-2/farmacologia , Quinases Associadas a rho
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