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1.
FEBS J ; 2020 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-32563195

RESUMO

Renal fibrosis is a common pathological feature of progressive chronic kidney disease (CKD). It is indicated that transforming growth factor-ß1 (TGF-ß1) plays as a central mediator in renal fibrosis. The present study aimed to investigate the role of δ-opioid receptor (DOR) on renal fibrosis of the rat renal proximal tubular epithelial cell line (NRK-52E) induced by TGF-ß1 and to elucidate its underlying mechanism, as well as its involvement in signaling pathways. Cells were treated with TGF-ß1 (10 ng·mL-1 ), along with a specific DOR agonist (UFP-512) or naltrindole (a DOR antagonist). Cell viability and morphology, as well as cell migration, were measured after drug administration. Western blotting was employed to examine the extracellular matrix (ECM) protein Fibronectin, and the tubular epithelial-mesenchymal transition (EMT) markers (E-cadherin and α-smooth muscle actin (α-SMA)), signal transducer (p-Smad3), and EMT-regulatory gene (Snail). The expression level of phosphorylated Akt and p38 was also examined. Our results showed that TGF-ß1 induced fibroblastic appearance and increased the expression of Fibronectin, α-SMA, P-Smad3, and Snail, while it decreased the expression of E-cadherin in NRK-52E cells. Moreover, TGF-ß1 induced the activation of Akt and p38 MAPK signaling pathways. DOR activation was found to efficiently block morphological changes and cell migration, as long as the expression changes of Fibronectin, E-cadherin, α-SMA, P-Smad3, Snail, P-Akt, and P-p38 were induced by TGF-ß1. These findings suggest that DOR may serve as an antifibrotic factor for renal proximal tubule cells by inhibiting the fibrosis process via TGF-ß/Smad, Akt, and p38 MAPK signaling pathways.

2.
Cancer Manag Res ; 12: 375-383, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32021450

RESUMO

Background: Nuclear receptor suppressor of variegation, enhancer of zeste, and trithorax (SET) domain-containing 2 (NSD2), is a well-known histone lysine methyltransferase (HMTase). The aim of this study was to investigate the biological role of NSD2 in clear cell renal cell carcinoma (ccRCC). Methods: GEO and OncoLnc databases were used to identify NSD2 expression and estimate its clinical value in ccRCC. Immunohistochemistry (IHC) was applied to further evaluate NSD2 protein level in ccRCC tissues. The expression of NSD2 in different cell lines and the transfection efficiency were determined by quantitative real-time PCR and Western blot analysis. The effect of NSD2 and the underlying mechanism in ccRCC progression were investigated via MTT, flow cytometry, Western blotting and xenograft tumor assays. Results: NSD2 was over-expressed in both ccRCC tissues and cell lines. NSD2 expression could discriminate ccRCC samples from normal samples, and moreover, high NSD2 expression was characterized with a short overall survival (OS) time. Additionally, knockdown of NSD2 suppressed proliferation and induced apoptosis of cancer cells by inhibiting Akt/Erk signaling and regulating Bcl-2 and Bax expression. Meanwhile, up-regulation of NSD2 contributed to the opposite effects. Silencing of NSD2 reduced xenograft tumor growth in vivo. Conclusion: NSD2 serves as an oncogenic factor in the progression of ccRCC via activation of Akt/Erk signaling.

3.
Front Physiol ; 10: 1572, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-32038276

RESUMO

Hypoxic injury is one of the most important factors in progressive kidney disorders. Since we have found that δ-opioid receptor (DOR) is neuroprotective against hypoxic stress through a differential regulation of mitogen-activated protein kinases (MAPKs) and anti-inflammatory cytokines, we asked if DOR that is highly expressed in the kidney can modulate renal MAPKs and anti-inflammatory cytokines under hypoxia. We exposed cultured rat kidney epithelial cells (NRK-52E) to prolonged hypoxia (1% O2) with applications of specific DOR agonist or/and antagonist to examine if DOR affects hypoxia-induced changes in MAPKs and anti-inflammatory cytokines. The results showed that endogenous DOR expression remained unchanged under hypoxia, while DOR activation with UFP-512 (a specific DOR agonist) reversed the hypoxia-induced up-regulation of ERK1/2 and p38 phosphorylation. DOR inhibition with naltrindole had no appreciable effect on the hypoxia-induced changes in ERK1/2 phosphorylation, but increased p38 phosphorylation. DOR inhibition with naltrindole attenuated the effects of DOR activation on the changes in ERK1/2 and p38 phosphorylation in hypoxia. Moreover, DOR activation/inhibition differentially affected the expression of transcriptional repressor B-cell lymphoma 6 (Bcl-6), anti-inflammatory cytokines tristetraprolin (TTP), and interleukin-10 (IL-10). Taken together, our novel data suggest that DOR activation differentially regulates ERK1/2, p38, Bcl-6, TTP, and IL-10 in the renal cells under hypoxia.

4.
Cell Physiol Biochem ; 46(4): 1483-1492, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29689570

RESUMO

BACKGROUND/AIMS: Mitogen-activated protein kinases (MAPKs) are involved in the cellular response to hypoxia and their dysregulation may contribute to the progression and pathology of diverse human renal diseases. Recent studies suggest that the regulation of MAPK responses to hypoxic stress may be different in different cells, even within the same organ. However, it is unclear if MAPKs are differentially regulated in different renal cells in hypoxia. This work was carried out to clarify this fundamental issue. METHODS: We cultured normal rat kidney epithelial (NRK-52E) cells, human kidney epithelial (HK-2) cells and human renal cell adenocarcinoma (769-P) cells simultaneously under normoxia and hypoxia (1% O2) for 24-72 hours. The protein levels of P-ERK1/2, ERK1/2, P-p38, p38 and eEF2K were detected by western blotting. The morphology of all cells was examined using light microscopy. RESULTS: Under the same hypoxic condition, P-ERK1/2 was up-regulated in all renal cells. Meanwhile,P-p38 in NRK-52E cells was markedly increased after hypoxia for 24-72 hours, while it appeared to show no appreciable change in HK-2 and 769-P cells exposed to hypoxia for 24-48 hours and significantly decreased in these cells after 72 hours hypoxia. On the other hand, hypoxia markedly down-regulated the expression of eukaryotic elongation factor-2 kinase (eEF2K) in all three cells. Under microscopy, NRK-52E cells had no visible injury after 72 hours hypoxia, while HK-2 and 769-P cells were mostly damaged under the same condition. CONCLUSIONS: Our data suggest that in response to prolonged hypoxic stress, ERK1/2 and p38 are differentially regulated in three renal cells, while eEF2K is largely down-regulated in all of these cells.


Assuntos
Hipóxia Celular , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Linhagem Celular , Regulação para Baixo , Quinase do Fator 2 de Elongação/genética , Quinase do Fator 2 de Elongação/metabolismo , Humanos , Rim/citologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Ratos , Transdução de Sinais
5.
Cell Transplant ; 26(8): 1441-1451, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28901193

RESUMO

Hypoxia is a common cause of kidney injury and a major issue in kidney transplantation. Mitogen-activated protein kinases (MAPKs) are involved in the cellular response to hypoxia, but the precise roles of MAPKs in renal cell reactions to hypoxic stress are not well known yet. This work was conducted to investigate the regulation of extracellular signal-regulated kinase-1 and -2 (ERK1/2) and p38 and their signaling-relevant molecules in kidney epithelial cells exposed to prolonged hypoxia. Rat kidney epithelial cells Normal Rat Kidney (NRK)-52E were exposed to hypoxic conditions (1% O2) for 24 to 72 h. Cell morphology was examined by light microscopy, and cell viability was checked by 3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxypheny]-2-[4-sulfophenyl]-2H-tetrazolium (MTS). The expression of ERK1/2 and p38 MAPK, as well as their signaling-related molecules, was measured by Western blot and real-time polymerase chain (RT-PCR) reaction. At the 1% oxygen level, cell morphology had no appreciable changes compared to the control up to 72 h of exposure under light microscopy, whereas the results of MTS showed a slight but significant reduction in cell viability after 72 h of hypoxia. On the other hand, ERK1/2 and p38 phosphorylation remarkably increased in these cells after 24 to 72 h of hypoxia. In sharp contrast, the expression of transcription factor B-cell lymphoma 6 (Bcl-6) was significantly downregulated in response to hypoxic stress. Other intracellular molecules relevant to the ERK1/2 and p38 signaling pathway, such as protein kinase A, protein kinase C, Bcl-2, nuclear factor erythroid 2-related factor 2, tristetraprolin, and interleukin-10(IL-10), had no significant alterations after 24 to 72 h of hypoxic exposure. We conclude that hypoxic stress increases the phosphorylation of both ERK1/2 and p38 but decreases the level of Bcl-6 in rat kidney epithelial cells.


Assuntos
Hipóxia Celular , Células Epiteliais/metabolismo , Rim/metabolismo , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Apoptose , Proliferação de Células , Regulação para Baixo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Ratos , Transdução de Sinais , Regulação para Cima
6.
Cell Transplant ; 26(1): 95-102, 2017 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-27524795

RESUMO

The aim of this study was to investigate follicular helper T (Tfh) cell response and its difference between renal graft and spleen in a rat renal transplantation model undergoing chronic allograft nephropathy (CAN). Orthotopical kidney transplantations were performed on Fischer (F344) rats and transplanted to Lewis rats, using syngeneic Lewis-Lewis grafts as controls. Tissue samples were collected at 8 weeks posttransplantation. The status of Tfh cell response was assessed by measuring the levels of transcription factor B-cell lymphoma 6 (Bcl-6), interleukin-21 (IL-21), chemokine receptor type 5 (CXCR5), and B-cell-activating factor belonging to the TNF family (BAFF). Tfh cell response was upregulated in both renal graft and spleen of the CAN group compared to the control group. However, Tfh cell response of the spleen was weaker than that of the graft, which was possibly related to the upregulation of splenic Treg activation. Also, the difference between two tissues was partially associated with the different expressions of tristetraprolin (TTP)/IL-10. Our data help improve our understanding of the role of Tfh cell response in the body with CAN and may provide a valuable clue for better treatment of CAN.


Assuntos
Aloenxertos/patologia , Nefropatias/metabolismo , Nefropatias/patologia , Baço/patologia , Aloenxertos/metabolismo , Animais , Western Blotting , Interleucina-10/metabolismo , Interleucinas/metabolismo , Rim/metabolismo , Rim/patologia , Rim/cirurgia , Transplante de Rim , Masculino , Ratos , Ratos Endogâmicos F344 , Ratos Endogâmicos Lew , Receptores CXCR5 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/metabolismo , Tristetraprolina/metabolismo
7.
Cell Physiol Biochem ; 39(3): 1051-67, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27544204

RESUMO

Tissue hypoxia/ischemia is a pathological feature of many human disorders including stroke, myocardial infarction, hypoxic/ischemic nephropathy, as well as cancer. In the kidney, the combination of limited oxygen supply to the tissues and high oxygen demand is considered the main reason for the susceptibility of the kidney to hypoxic/ischemic injury. In recent years, increasing evidence has indicated that a reduction in renal oxygen tension/blood supply plays an important role in acute kidney injury, chronic kidney disease, and renal tumorigenesis. However, the underlying signaling mechanisms, whereby hypoxia alters cellular behaviors, remain poorly understood. Mitogen-activated protein kinases (MAPKs) are key signal-transducing enzymes activated by a wide range of extracellular stimuli, including hypoxia/ischemia. There are four major family members of MAPKs: the extracellular signal-regulated kinases-1 and -2 (ERK1/2), the c-Jun N-terminal kinases (JNK), p38 MAPKs, and extracellular signal-regulated kinase-5 (ERK5/BMK1). Recent studies, including ours, suggest that these MAPKs are differentially involved in renal responses to hypoxic/ischemic stress. This review will discuss their changes in hypoxic/ischemic pathophysiology with acute kidney injury, chronic kidney diseases and renal carcinoma.


Assuntos
Carcinogênese/genética , Carcinoma de Células Renais/genética , Regulação da Expressão Gênica , Hipóxia/genética , Neoplasias Renais/genética , Traumatismo por Reperfusão/genética , Animais , Carcinogênese/metabolismo , Carcinogênese/patologia , Carcinoma de Células Renais/enzimologia , Carcinoma de Células Renais/patologia , Humanos , Hipóxia/enzimologia , Hipóxia/patologia , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Rim/enzimologia , Rim/patologia , Neoplasias Renais/enzimologia , Neoplasias Renais/patologia , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/genética , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
8.
Oncol Lett ; 11(2): 1474-1480, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26893763

RESUMO

MicroRNAs (miRNAs) are involved in tumorigenesis. However, little is known about their role in renal cell carcinoma (RCC). In the present study, the function of the miRNA miR-877 in RCC was investigated, and its expression levels in blood and paired RCC tissues were measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Bioinformatics analysis predicted eukaryotic elongation factor-2 kinase (eEF2K) to be the potential mRNA target of miR-877, which was verified by luciferase assay. The expression levels of eEF2K in RCC tissues were evaluated by western blot analysis and qPCR. The proliferation and migration abilities of RCC cells were measured by MTT and in vitro wound healing assays, respectively. The present results indicated that the expression levels of miR-877 were downregulated in blood and paired RCC tissues, whereas the expression levels of eEF2K were upregulated in RCC tissues. In addition, overexpression of miR-877 and knockdown of eEF2K significantly reduced the proliferation and migration abilities of RCC cells in vitro. Furthermore, miR-877 affected the eEF2K/eEF2 signaling pathway in these cells. In conclusion, the present study has demonstrated that miR-877 suppresses the proliferation and migration abilities of RCC cells by modulating the eEF2K/eEF2 signaling cascade. Therefore, miR-877 may be considered a potential biomarker for the diagnosis of RCC.

9.
BMC Nephrol ; 16: 182, 2015 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-26525294

RESUMO

BACKGROUND: Chronic antibody-mediated rejection is a major issue that affects long-term renal allograft survival. Since follicular helper T (Tfh) cells promote the development of antigen-specific B cells in alloimmune responses, we investigated the potential roles of Tfh cells, B cells and their alloimmune-regulating molecules in the pathogenesis of chronic renal allograft rejection in this study. METHODS: The frequency of Tfh, B cells and the levels of their alloimmune-regulating molecules including chemokine receptor type 5 (CXCR5), inducible T cell co-stimulator (ICOS), programmed death-1 (PD-1), ICOSL, PDL-1 and interleukin-21 (IL-21), of peripheral blood were comparatively measured in 42 primary renal allograft recipients within 1-3 years after transplantation. Among them, 24 patients had definite chronic rejection, while other 18 patients had normal renal function. RESULTS: Tfh-cell ratio was significantly increased with PD-1 down-regulation in the patients with chronic renal allograft rejection, while B cells and the alloimmune-regulating molecules studied did not show any appreciable change in parallel. CONCLUSIONS: The patients with chronic renal allograft rejection have a characteristic increase in circulating Tfh cells with a decrease in PD-1 expression. These pathological changes may be a therapeutic target for the treatment of chronic renal allograft rejection and can be useful as a clinical index for monitoring conditions of renal transplant.


Assuntos
Linfócitos B/imunologia , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Transplante de Rim/efeitos adversos , Receptor de Morte Celular Programada 1/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Adulto , Aloenxertos/imunologia , Aloenxertos/transplante , Doença Crônica , Feminino , Rejeição de Enxerto/etiologia , Humanos , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento
10.
Science ; 340(6130): 359-63, 2013 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-23470732

RESUMO

Cell-cell fusion is critical for the conception, development, and physiology of multicellular organisms. Although cellular fusogenic proteins and the actin cytoskeleton are implicated in cell-cell fusion, it remains unclear whether and how they coordinate to promote plasma membrane fusion. We reconstituted a high-efficiency, inducible cell fusion culture system in the normally nonfusing Drosophila S2R+ cells. Both fusogenic proteins and actin cytoskeletal rearrangements were necessary for cell fusion, and in combination they were sufficient to impart fusion competence. Localized actin polymerization triggered by specific cell-cell or cell-matrix adhesion molecules propelled invasive cell membrane protrusions, which in turn promoted fusogenic protein engagement and plasma membrane fusion. This de novo cell fusion culture system reveals a general role for actin-propelled invasive membrane protrusions in driving fusogenic protein engagement during cell-cell fusion.


Assuntos
Actinas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Moléculas de Adesão Celular/metabolismo , Comunicação Celular , Fusão Celular , Glicoproteínas de Membrana/metabolismo , Animais , Proteínas de Caenorhabditis elegans/genética , Moléculas de Adesão Celular/genética , Técnicas de Cultura de Células , Linhagem Celular , Extensões da Superfície Celular/metabolismo , Extensões da Superfície Celular/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Imunoglobulinas/genética , Imunoglobulinas/metabolismo , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo
11.
J Cell Biol ; 199(1): 169-85, 2012 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-23007650

RESUMO

The p21-activated kinases (PAKs) play essential roles in diverse cellular processes and are required for cell proliferation, apoptosis, polarity establishment, migration, and cell shape changes. Here, we have identified a novel function for the group I PAKs in cell-cell fusion. We show that the two Drosophila group I PAKs, DPak3 and DPak1, have partially redundant functions in myoblast fusion in vivo, with DPak3 playing a major role. DPak3 is enriched at the site of fusion colocalizing with the F-actin focus within a podosome-like structure (PLS), and promotes actin filament assembly during PLS invasion. Although the small GTPase Rac is involved in DPak3 activation and recruitment to the PLS, the kinase activity of DPak3 is required for effective PLS invasion. We propose a model whereby group I PAKs act downstream of Rac to organize the actin filaments within the PLS into a dense focus, which in turn promotes PLS invasion and fusion pore initiation during myoblast fusion.


Assuntos
Extensões da Superfície Celular/metabolismo , Drosophila/citologia , Drosophila/enzimologia , Mioblastos/citologia , Mioblastos/metabolismo , Quinases Ativadas por p21/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Animais , Fusão Celular , Drosophila/metabolismo
12.
Dev Cell ; 20(5): 623-38, 2011 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-21571220

RESUMO

Dynamic rearrangements of the actin cytoskeleton play a key role in numerous cellular processes. In Drosophila, fusion between a muscle founder cell and a fusion competent myoblast (FCM) is mediated by an invasive, F-actin-enriched podosome-like structure (PLS). Here, we show that the dynamics of the PLS is controlled by Blown fuse (Blow), a cytoplasmic protein required for myoblast fusion but whose molecular function has been elusive. We demonstrate that Blow is an FCM-specific protein that colocalizes with WASP, WIP/Solitary, and the actin focus within the PLS. Biochemically, Blow modulates the stability of the WASP-WIP complex by competing with WASP for WIP binding, leading to a rapid exchange of WASP, WIP and G-actin within the PLS, which, in turn, actively invades the adjacent founder cell to promote fusion pore formation. These studies identify a regulatory protein that modulates the actin cytoskeletal dynamics by controlling the stability of the WASP-WIP complex.


Assuntos
Actinas/metabolismo , Ligação Competitiva , Proteínas de Drosophila/metabolismo , Proteínas Musculares/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Actinas/química , Animais , Células Cultivadas , Drosophila , Feminino , Masculino
13.
J Cell Biol ; 191(5): 1013-27, 2010 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-21098115

RESUMO

Recent studies in Drosophila have implicated actin cytoskeletal remodeling in myoblast fusion, but the cellular mechanisms underlying this process remain poorly understood. Here we show that actin polymerization occurs in an asymmetric and cell type-specific manner between a muscle founder cell and a fusion-competent myoblast (FCM). In the FCM, a dense F-actin-enriched focus forms at the site of fusion, whereas a thin sheath of F-actin is induced along the apposing founder cell membrane. The FCM-specific actin focus invades the apposing founder cell with multiple finger-like protrusions, leading to the formation of a single-channel macro fusion pore between the two muscle cells. Two actin nucleation-promoting factors of the Arp2/3 complex, WASP and Scar, are required for the formation of the F-actin foci, whereas WASP but not Scar promotes efficient foci invasion. Our studies uncover a novel invasive podosome-like structure (PLS) in a developing tissue and reveal a previously unrecognized function of PLSs in facilitating cell membrane juxtaposition and fusion.


Assuntos
Extensões da Superfície Celular/ultraestrutura , Drosophila/ultraestrutura , Fusão de Membrana/fisiologia , Mioblastos/ultraestrutura , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Animais , Membrana Celular/metabolismo , Extensões da Superfície Celular/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas dos Microfilamentos/metabolismo , Mioblastos/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/metabolismo
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