RESUMO
Accumulating evidence supports the role of the DNA damage response (DDR) in the negative regulation of tumorigenesis. Here, we found that DDR signaling poises a series of epigenetic events, resulting in activation of pro-tumorigenic genes but can go as far as reactivation of the pluripotency gene OCT4. Loss of DNA methylation appears to be a key initiating event in DDR-dependent OCT4 locus reactivation although full reactivation required the presence of a driving oncogene, such as Myc and macroH2A downregulation. Using genetic-lineage-tracing experiments and an in situ labeling approach, we show that DDR-induced epigenetic reactivation of OCT4 regulates the resistance to chemotherapy and contributes to tumor relapse both in mouse and primary human cancers. In turn, deletion of OCT4 reverses chemoresistance and delays the relapse. Here, we uncovered an unexpected tumor-promoting role of DDR in cancer cell reprogramming, providing novel therapeutic entry points for cancer intervention strategies.
Assuntos
Carcinogênese/genética , Metilação de DNA/genética , Neoplasias/genética , Fator 3 de Transcrição de Octâmero/genética , Animais , Reprogramação Celular/genética , Dano ao DNA/genética , Epigênese Genética/genética , Regulação Neoplásica da Expressão Gênica , Histonas/genética , Humanos , Camundongos , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-myc/genética , Recidiva , Transdução de Sinais/genéticaRESUMO
Collapsing focal segmental glomerulosclerosis (FSGS), also known as collapsing glomerulopathy (CG), is the most aggressive variant of FSGS and is characterized by a rapid progression to kidney failure. Understanding CG pathogenesis represents a key step for the development of targeted therapies. Previous work implicated the telomerase protein component TERT in CG pathogenesis, as transgenic TERT expression in adult mice resulted in a CG resembling that seen in human primary CG and HIV-associated nephropathy (HIVAN). Here, we used the telomerase-induced mouse model of CG (i-TERTci mice) to identify mechanisms to inhibit CG pathogenesis. Inactivation of WIP1 phosphatase, a p53 target acting in a negative feedback loop, blocked disease initiation in i-TERTci mice. Repression of disease initiation upon WIP1 deficiency was associated with senescence enhancement and required transforming growth factor-ß functions. The efficacy of a pharmacologic treatment to reduce disease severity in both i-TERTci mice and in a mouse model of HIVAN (Tg26 mice) was then assessed. Pharmacologic inhibition of WIP1 enzymatic activity in either the telomerase mice with CG or in the Tg26 mice promoted partial remission of proteinuria and ameliorated kidney histopathologic features. Histological as well as high-throughput sequencing methods further showed that selective inhibition of WIP1 does not promote kidney fibrosis or inflammation. Thus, our findings suggest that targeting WIP1 may be an effective therapeutic strategy for patients with CG.
Assuntos
Nefropatia Associada a AIDS , Glomerulosclerose Segmentar e Focal , Insuficiência Renal , Telomerase , Adulto , Humanos , Camundongos , Animais , Glomerulosclerose Segmentar e Focal/patologia , Telomerase/uso terapêutico , Nefropatia Associada a AIDS/patologia , Proteinúria , Insuficiência Renal/complicações , Modelos Animais de DoençasRESUMO
BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most fatal cancers. Due to limited strategies for effective treatments, patients with advanced HCC have a very poor prognosis. This study aims to identify new insights in HCC to develop novel strategies for HCC management. METHODS: The role of WIP1 (wild type p53 induced protein phosphatase1) in HCC was analyzed in HCC cells, xenograft model, DEN (Diethylnitrosamine) induced mice liver cancer model with WIP1 knockout mice, and TCGA database. DNA damage was evaluated by Gene Set Enrichment Analysis, western blotting, comet assay, and Immunofluorescence. RESULTS: High expression of WIP1 is associated with the poor prognosis of patients with HCC. Genetically and chemically suppression of WIP1 drastically reduced HCC cell proliferation. Besides, WIP1 knockout retarded DEN induced mice hepato-carcinogenesis. Mechanically, WIP1 inhibition induced DNA damage by increasing H2AX phosphorylation (γH2AX). Therefore, suppression of WIP1 and PARP induced synthetic lethality in HCC in vitro and in vivo by augmenting DNA damage. CONCLUSION: WIP1 plays an oncogenic effect in HCC development, and targeting WIP1-dependent DNA damage repair alone or in combination with PARP inhibition might be a reasonable strategy for HCC management. Video abstract.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Camundongos , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases , Proteína Fosfatase 2C/genética , Proteína Fosfatase 2C/metabolismo , Mutações Sintéticas LetaisRESUMO
The tumor suppressor Smad4, a key mediator of the TGF-ß/BMP pathways, is essential for development and tissue homeostasis. Phosphorylation of Smad4 in its linker region catalyzed by the mitogen-activated protein kinase (MAPK) plays a pivotal role in regulating its transcriptional activity and stability. In contrast, roles of Smad4 dephosphorylation as a control mechanism of TGF-ß/BMP signaling and the phosphatases responsible for its dephosphorylation remain so far elusive. Here, we identify Wip1 as a Smad4 phosphatase. Wip1 selectively binds and dephosphorylates Smad4 at Thr277, a key MAPK phosphorylation site, thereby regulating its nuclear accumulation and half-life. In Xenopus embryos, Wip1 limits mesoderm formation and favors neural induction by inhibiting TGF-ß/BMP signals. Wip1 restrains TGF-ß-induced growth arrest, migration, and invasion in human cells and enhances the tumorigenicity of cancer cells by repressing the antimitogenic activity of Smad4. We propose that Wip1-dependent dephosphorylation of Smad4 is critical for the regulation of TGF-ß signaling.
Assuntos
Proteína Fosfatase 2C/metabolismo , Transdução de Sinais , Proteína Smad4/metabolismo , Fator de Crescimento Transformador beta , Proteínas de Xenopus/metabolismo , Animais , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Proteína Fosfatase 2C/genética , Proteína Smad4/genética , Fator de Crescimento Transformador beta/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis/metabolismoRESUMO
Cancer stem cells (CSCs) are involved in the initiation and progression of human malignancies by enabling cancer tissue self-renewal capacity and constituting the therapy-resistant population of tumor cells. However, despite the exhausting characterization of CSC genetics, epigenetics, and kinase signaling, eradication of CSCs remains an unattainable goal in most human malignancies. While phosphatases contribute equally with kinases to cellular phosphoregulation, our understanding of phosphatases in CSCs lags severely behind our knowledge about other CSC signaling mechanisms. Many cancer-relevant phosphatases have recently become druggable, indicating that further understanding of the CSC phosphatases might provide novel therapeutic opportunities. This review summarizes the current knowledge about fundamental, but yet poorly understood involvement of phosphatases in the regulation of major CSC signaling pathways. We also review the functional roles of phosphatases in CSC self-renewal, cancer progression, and therapy resistance; focusing particularly on hematological cancers and glioblastoma. We further discuss the small molecule targeting of CSC phosphatases and their therapeutic potential in cancer combination therapies.
Assuntos
Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Transdução de Sinais , Autorrenovação Celular/genética , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/genética , Epigênese Genética/genética , Humanos , Modelos Biológicos , Neoplasias/genética , Neoplasias/terapia , Células-Tronco Neoplásicas/enzimologia , Monoéster Fosfórico Hidrolases/classificaçãoRESUMO
BACKGROUND: Ovarian clear cell carcinoma (OCCC) is one of the most lethal types of ovarian cancer. Early-stage OCCC can be cured by surgery; however, advanced-stage disease shows poor prognosis due to chemoresistance unlike the more common high-grade serous carcinoma. METHODS: We explored the differential roles of the Wip1-p38-p53 DNA damage response pathway in respective early- or advanced-stage OCCC by immunohistochemistry of Wip1, phospho-p38, p53, and phospho-p53 from consecutive 143 patients. RESULTS: High Wip1 expression correlated with positive p53 (p=0.011), which in turn correlated with low nuclear phospho-p38 expression (p=0.0094). In the early stages, positive p53 showed trends toward worse overall survival (OS) (p=0.062), whereas in the advanced stages, high Wip1 correlated with worse OS (p=0.0012). The univariate and multivariate analyses of prognostic factors indicated that high Wip1 was significant and independent for worse OS (p=0.011) in the advanced stages, but not in the early stages. Additionally, high Wip1 showed trends toward shorter treatment-free interval (TFI) in the advanced stages, but not in the early stages (p=0.083 vs. 0.93). Furthermore, high Wip1 was significantly associated with positive p53 only in the patients with shorter TFI (<6 months), but not in those with longer TFI (≥6 months) (p=0.036 vs. 0.34). CONCLUSIONS: Wip1 appears to play a crucial role for the prognosis of OCCC through chemoresistance specifically in the advanced stages, implicating that Wip1 possibly serves as a reasonable therapeutic target for improving chemoresistance and poor prognosis of advanced-stage OCCC.
Assuntos
Carcinoma , Proteína Fosfatase 2C/genética , Proteína Supressora de Tumor p53 , Dano ao DNA , Humanos , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Proteína Fosfatase 2C/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
There has been a consensus that actin plays an important role in scission of the clathrin-coated pits (CCPs) together with large GTPases of the dynamin family in metazoan cells. However, the recruitment, regulation and functional interdependence of actin and dynamin during this process remain inadequately understood. Here, based on small-scale screening and in vivo live-imaging techniques, we identified a novel set of molecules underlying CCP scission in the multicellular organism Caenorhabditis elegans We found that loss of Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP-1) impaired CCP scission in a manner that is independent of the C. elegans homolog of WASP/N-WASP (WSP-1) and is mediated by direct binding to G-actin. Moreover, the cortactin-binding domain of WIP-1 serves as the binding interface for DBN-1 (also known in other organisms as Abp1), another actin-binding protein. We demonstrate that the interaction between DBN-1 and F-actin is essential for Dynamin-1 (DYN-1) recruitment at endocytic sites. In addition, the recycling regulator RME-1, a homolog of mammalian Eps15 homology (EH) domain-containing proteins, is increasingly recruited at the arrested endocytic intermediates induced by F-actin loss or DYN-1 inactivation, which further stabilizes the tubular endocytic intermediates. Our study provides new insights into the molecular network underlying F-actin participation in the scission of CCPs.This article has an associated First Person interview with the first author of the paper.
Assuntos
Actinas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas do Citoesqueleto/metabolismo , Dinamina I/metabolismo , Vesículas Transportadoras/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Intestinos/efeitos dos fármacos , Intestinos/patologiaRESUMO
Downregulation of the p53 tumor suppressor in cancers is frequently accompanied by the upregulation of Wip1 (a phosphatase) and Mdm2 (an E3 ubiquitin ligase). Mdm2 binds and ubiquitinates p53, promoting its degradation by the proteasome. As the p53/Mdm2 interaction is alleviated by the phosphorylation of the serine-15 (S15) residue of p53, Wip1, which can directly dephosphorylate phospho-S15, facilitates the Mdm2-mediated degradation of p53. Here, we found that p21WAF1/CIP1, previously shown to bind p53 and Mdm2, reduces the cellular levels of p53 protein by decreasing its stability. This is accompanied by a decrease in p53-S15 phosphorylation levels. In agreement, p21 promotes the p53/Wip1 interaction. Additionally, p21 interacts with Wip1, forming a trimeric complex of p53, p21, and Wip1. Studies using a p21 deletion mutant that cannot bind p53 revealed that the p53/p21 complex is more efficient than p53 alone in facilitating the binding of p53 to Wip1 and Mdm2. These findings indicate that p21 is a novel negative regulator of p53 stability and therefore, may be used as a target to restore p53 activity by preventing the action of Wip1 and Mdm2 on p53.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Neoplasias/metabolismo , Proteína Fosfatase 2C/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Humanos , Neoplasias/patologia , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Proteólise , Transdução de SinaisRESUMO
RESEARCH QUESTION: What role does wild-type p53-induced phosphatase 1 (WIP1) play in the regulation of primordial follicle development? DESIGN: WIP1 expression was detected in the ovaries of mice of different ages by western blotting and immunohistochemical staining. Three-day-old neonatal mouse ovaries were cultured in vitro with or without the WIP1 inhibitor GSK2830371 (10 µM) for 4 days. Ovarian morphology, follicle growth and follicle classification were analysed and the PI3K-AKT-mTOR signal pathway and the WIP1-p53-related mitochondrial apoptosis pathway evaluated. RESULTS: WIP1 expression was downregulated with age. Primordial follicles were significantly decreased in the GSK2830371-treated group, without a significant increase in growing follicles. The ratio of growing follicles to primordial follicles was not significantly different between the control and GSK2830371 groups, and no significant variation was observed in the PI3K-AKT-mTOR signal pathway. The inhibition of WIP1 phosphatase accelerated primordial follicle atresia by activating the p53-BAX-caspase-3 pathway. CONCLUSIONS: These findings reveal that WIP1 participates in regulating primordial follicle development and that inhibiting WIP1 phosphatase leads to massive primordial follicle loss via interaction with the p53-BAX-caspase-3 pathway. This might also provide valuable information for understanding decreased ovarian reserve during ovarian ageing.
Assuntos
Aminopiridinas/farmacologia , Dipeptídeos/farmacologia , Folículo Ovariano/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Contagem de Células , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Folículo Ovariano/citologia , Folículo Ovariano/fisiologia , Ovário/citologia , Ovário/efeitos dos fármacos , Gravidez , Proteína Fosfatase 2C/antagonistas & inibidoresRESUMO
WT P53-Induced Phosphatase 1 (WIP1) is a member of the magnesium-dependent serine/threonine protein phosphatase (PPM) family and is induced by P53 in response to DNA damage. In several human cancers, the WIP1 protein is overexpressed, which is generally associated with a worse prognosis. Although WIP1 is an attractive therapeutic target, no potent, selective, and bioactive small-molecule modulator with favorable pharmacokinetics has been reported. Phosphatase enzymes are among the most challenging targets for small molecules because of the difficulty of achieving both modulator selectivity and bioavailability. Another major obstacle has been the availability of robust and physiologically relevant phosphatase assays that are suitable for high-throughput screening. Here, we describe orthogonal biochemical WIP1 activity assays that utilize phosphopeptides from native WIP1 substrates. We optimized an MS assay to quantify the enzymatically dephosphorylated peptide reaction product in a 384-well format. Additionally, a red-shifted fluorescence assay was optimized in a 1,536-well format to enable real-time WIP1 activity measurements through the detection of the orthogonal reaction product, Pi We validated these two optimized assays by quantitative high-throughput screening against the National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection and used secondary assays to confirm and evaluate inhibitors identified in the primary screen. Five inhibitors were further tested with an orthogonal WIP1 activity assay and surface plasmon resonance binding studies. Our results validate the application of miniaturized physiologically relevant and orthogonal WIP1 activity assays to discover small-molecule modulators from high-throughput screens.
Assuntos
Ativadores de Enzimas/química , Fosfopeptídeos/química , Proteína Fosfatase 2C/química , Bibliotecas de Moléculas Pequenas/química , Ativadores de Enzimas/isolamento & purificação , Ativadores de Enzimas/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , Proteína Fosfatase 2C/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/isolamento & purificação , Bibliotecas de Moléculas Pequenas/farmacologia , Especificidade por Substrato , Proteína Supressora de Tumor p53/químicaRESUMO
Accumulation of DNA damage has been detected in the spinal cord of patients as well as in the G93A mouse model of amyotrophic lateral sclerosis (ALS). Wild-type p53-induced phosphatase 1 (Wip1) is a p53-inducible serine/threonine phosphatase that terminates DNA-damage responses via dephosphorylation of DNA-damage response proteins, namely ataxia-telangiectasia mutated (ATM) kinase, checkpoint kinase 2, and p53, thus enhancing cell proliferation. However, the role of Wip1, DNA-damage responses, and their interaction in ALS development remains to be elucidated. Here, we showed that Wip1 expression levels were substantially decreased in ALS motor neurons compared with wild-type controls both in vivo and in vitro. The DNA-damage response was activated in superoxide dismutase 1 (SOD1) G93A-transfected cells. However, increased expression of Wip1 improved cell viability and inhibited the DNA-damage response in mutated SOD1G93A cells. Further studies demonstrated that decreased Wip1 expression reduced cell viability and further activated the DNA-damage response in chronic H2O2-treated NSC34 cells. In contrast, Wip1 promoted cell survival and suppressed DNA damage-induced apoptosis during persistent DNA damage conditions. Over-expression of Wip1 in the central nervous system (CNS) can delay the onset of disease symptoms, extended the survival, decreased MN loss improved motor function and inhibit the DNA-damage response in SOD1 G93A mice. Furthermore, homeodomain-interacting protein kinase 2 (HIPK2) promoted the degradation of Wip1 via the ubiquitin-proteasome system during chronic stress. These findings indicate that persistent accumulation of DNA damage and subsequent chronic activation of the downstream DNA damage-response ATM and p53 pro-apoptotic signaling pathways may trigger neuronal dysfunction and neuronal death in ALS. Wip1 may play a protective role by targeting the DNA-damage response in ALS motor neurons. Importantly, these findings provide a novel direction for therapeutic options for patients with ALS.
Assuntos
Esclerose Lateral Amiotrófica/patologia , Dano ao DNA/fisiologia , Neurônios Motores/metabolismo , Proteína Fosfatase 2C/metabolismo , Transdução de Sinais/fisiologia , Esclerose Lateral Amiotrófica/metabolismo , Animais , Apoptose/fisiologia , Regulação para Baixo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios Motores/patologiaRESUMO
Spatial learning and memory are typically assessed to evaluate hippocampus-dependent cognitive and memory functions in vivo. Protein phosphorylation and dephosphorylation by kinases and phosphatases play critical roles in spatial learning and memory. Here we report that the Wip1 phosphatase is essential for spatial learning, with knockout mice lacking Wip1 phosphatase exhibiting dysfunctional spatial cognition. Aberrant phosphorylation of the Wip1 substrates p38, ATM, and p53 were observed in the hippocampi of Wip1-/- mice, but only p38 inhibition reversed impairments in long-term potentiation in Wip1-knockout mice. p38 inhibition consistently ameliorated the spatial learning dysfunction caused by Wip1 deficiency. Our results demonstrate that deletion of Wip1 phosphatase impairs hippocampus-dependent spatial learning and memory, with aberrant downstream p38 phosphorylation involved in this process and providing a potential therapeutic target.
Assuntos
Memória , Proteína Fosfatase 2C/fisiologia , Aprendizagem Espacial , Animais , Hipocampo/enzimologia , Hipocampo/fisiologia , Potenciação de Longa Duração , Masculino , Camundongos Knockout , Teste do Labirinto Aquático de Morris , Fosforilação , Proteína Fosfatase 2C/genética , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Osteosarcoma (OS) is a primary malignant bone tumor with high morbidity. Developing new therapeutic approaches with neoadjuvant is of great interest in OS treatment. Reportedly, ataxia telangiectasia mutated (ATM)/ataxia telangiectasia and radiation resistance gene 3 related (ATR)-p53 signaling is considered as a critical DNA damage signaling pathway sensitizing cancer cells to chemotherapies; while wild-type p53-induced phosphatase 1 (WIP1), an oncogene overexpressed in diverse cancers, has been regarded as a critical inhibitor in the ATM/ATR-p53 DNA damage signaling pathway. Herein, the expression of WIP1 in OS tissues and cell lines was examined; to investigate the mechanism of WIP1 abnormal upregulation, online tools were used to predict the upstream regulatory microRNAs (miRNAs) targeting WIP1. Among the candidate miRNAs, the expression and detailed function of miR-590 were validated. Through binding to the 3'-untranslated region of WIP1, miR-590 inhibited WIP1 expression and, therefore, enhanced the effect of Dox on OS cell proliferation and apoptosis through downstream ATM-p53 signaling. Moreover, RELA could bind to the promoter region of miR-590 to inhibit its expression, thereby affecting downstream WIP1 and ATM-p53 signaling. The expression of p65 was upregulated in OS tissues, indicating that the effect of p65 inhibition on cell viability, apoptosis, and related mechanisms could be partially restored by miR-590 inhibition. Taken together, these results showed that p65-mediated miR-590/WIP1/ATM-p53 modulation might be a novel target to enhance the cellular effect of Dox on OS cell lines.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Neoplasias Ósseas/metabolismo , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , MicroRNAs/antagonistas & inibidores , Osteossarcoma/metabolismo , Proteína Fosfatase 2C/metabolismo , Fator de Transcrição RelA/metabolismo , Regiões 3' não Traduzidas , Antibióticos Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Doxorrubicina/uso terapêutico , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Osteossarcoma/tratamento farmacológico , Osteossarcoma/patologia , Regiões Promotoras Genéticas , Proteína Fosfatase 2C/genética , RNA Mensageiro/genética , Fator de Transcrição RelA/genética , Transfecção , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima/genéticaRESUMO
BACKGROUND/AIMS: Excessive apoptosis of trophoblasts, induced by sustained hypoxia, leads to abnormal placentation and is strongly linked to pregnancy complications such as preeclampsia (PE). Wild-type p53-induced phosphatase (Wip1) positively regulates cellular survival in tumor cells through the p38 and p53 pathways, but its expression pattern and effects in trophoblasts have yet to be reported. This study clarified the effect of Wip1 on the regulatory mechanism of p53-dependent apoptosis in trophoblasts, and thus increases understanding of the etiology of PE. METHODS: In normal and PE placentas, Wip1 mRNA and protein levels were determined by RT-qPCR and Western blotting respectively, while localization of Wip1 in placental tissues and in HTR8/SVneo cells was determined by immunohistochemistry and immunofluorescence. Two in vitro trophoblastic PE models were established by subjecting HTR8/SVneo cells to either hypoxia intervention in incubator (HII) or simulated ischemic buffer (SIB). Wip1 was suppressed in the aforementioned PE models by specific inhibitor or shRNA, and apoptosis was then assessed by flow cytometry, while further validation was done by measurement of cleaved-caspase 9 expression by Western blotting. The p38 inhibitor SB202190, Mdm2 inhibitor NVP-CGM097, and proteasome inhibitor MG-132 were administered in PE models, either in combination or alone, to determine the regulatory order of the component signal molecules of the feedback loop. The impact of Wip1 on p53-Mdm2 interaction was examined by coimmunoprecipitation. Lastly, the upregulation of the p38-Wip1 loop was confirmed in human placentas from pregnancies complicated by PE, using Western blotting. RESULTS: Wip1 expression was significantly elevated in human PE placentas and in vitro trophoblastic PE models; this is opposite to the pattern observed in tumor cells. Inhibition of Wip1 rescued hypoxia-induced p38 activation, cleavage of caspase 9 and apoptosis but significantly compromised p53-Mdm2 binding, while p-p53Ser15 was increased. Inhibition of Mdm2 degradation resulted in p53 destabilization and p38-Wip1 loop down-regulation, while degradation of the p53-Mdm2 complex resulted in p53 accumulation and p38-Wip1 loop hyperactivation. However, the p53-Mdm2 interaction was found to be more important in the regulation of the p38-Wip1 loop than Mdm2 stability. CONCLUSION: Trophoblastic p53 homeostasis is maintained by the p38-Wip1 feedback regulatory loop in response to hypoxic stress, which is dysregulated in the placentas of pregnancies complicated by PE, and thereby leads to excessive apoptosis.
Assuntos
Pré-Eclâmpsia/metabolismo , Proteínas da Gravidez/metabolismo , Proteína Fosfatase 2C/metabolismo , Proteostase , Transdução de Sinais , Trofoblastos/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Adulto , Apoptose , Linhagem Celular , Feminino , Humanos , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/patologia , Gravidez , Proteínas da Gravidez/genética , Proteína Fosfatase 2C/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Trofoblastos/patologia , Proteína Supressora de Tumor p53/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
BACKGROUND: Allergic asthma is one of the most common diseases worldwide, resulting in a burden of diseases. No available therapeutic regimens can cure asthma thus far. OBJECTIVE: We sought to identify new molecular targets for TH9 cell-mediated allergic airway inflammation. METHODS: Wild-type p53-induced phosphatase 1 (Wip1) gene knockout mice, Wip1 inhibitor-treated mice, and ovalbumin-induced allergic airway inflammation mouse models were used to characterize the roles of Wip1 in allergic airway inflammation. The induction of TH cell subsets in vitro, real-time PCR, immunoblots, luciferase assays, and chromatin immunoprecipitation assays were used to determine the regulatory pathways of Wip1 in TH9 differentiation. RESULTS: Here we demonstrate that Wip1-deficient mice are less prone to allergic airway inflammation, as indicated by the decreased pathologic alterations in lungs. Short-term treatment with a Wip1-specific inhibitor significantly ameliorates allergic inflammation progression. Intriguingly, Wip1 selectively impaired TH9 but not TH1, TH2, and TH17 cell differentiation. Biochemical assays show that Wip1 deficiency increases c-Jun/c-Fos activity in a c-Jun N-terminal kinase-dependent manner and that c-Jun/c-Fos directly binds to Il9 promoter and inhibits Il9 transcription. CONCLUSION: Wip1 controls TH9 cell development through regulating c-Jun/c-Fos activity on the Il9 promoter and is important for the pathogenesis of allergic airway inflammation. These findings shed light on the previously unrecognized roles of Wip1 in TH9 cell differentiation. The inhibitory effects of a Wip1 inhibitor on the pathogenesis of allergic airway inflammation can have important implications for clinical application of Wip1 inhibitors in allergy therapies.
Assuntos
Asma/imunologia , Proteína Fosfatase 2C/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Interleucina-9/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
WIP1, as a critical phosphatase, plays many important roles in various physiological and pathological processes through dephosphorylating different substrate proteins. However, the functions of WIP1 in adipogenesis and fat accumulation are not clear. Here, we report that WIP1-deficient mice show impaired body weight growth, dramatically decreased fat mass, and significantly reduced triglyceride and leptin levels in circulation. This dysregulation of adipose development caused by the deletion of WIP1 occurs as early as adipogenesis. In contrast, lentivirus-mediated WIP1 phosphatase overexpression significantly increases the adipogenesis of pre-adipocytes via an enzymatic activity-dependent mechanism. PPARγ is a master gene of adipogenesis, and the phosphorylation of PPARγ at serine 112 strongly inhibits adipogenesis; however, very little is known about the negative regulation of this phosphorylation. Here, we show that WIP1 phosphatase plays a pro-adipogenic role by interacting directly with PPARγ and dephosphorylating p-PPARγ S112 in vitro and in vivo.
Assuntos
Adipogenia , PPAR gama/metabolismo , Fosfosserina/metabolismo , Proteína Fosfatase 2C/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Adiposidade , Animais , Peso Corporal , Linhagem Celular , Tamanho Celular , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Leptina/sangue , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Ligação Proteica , Proteína Fosfatase 2C/deficiência , Triglicerídeos/sangueRESUMO
Cell cycle checkpoints activated by DNA double-strand breaks (DSBs) are essential for the maintenance of the genomic integrity of proliferating cells. Following DNA damage, cells must detect the break and either transiently block cell cycle progression, to allow time for repair, or exit the cell cycle. Reversal of a DNA-damage-induced checkpoint not only requires the repair of these lesions, but a cell must also prevent permanent exit from the cell cycle and actively terminate checkpoint signalling to allow cell cycle progression to resume. It is becoming increasingly clear that despite the shared mechanisms of DNA damage detection throughout the cell cycle, the checkpoint and its reversal are precisely tuned to each cell cycle phase. Furthermore, recent findings challenge the dogmatic view that complete repair is a precondition for cell cycle resumption. In this Commentary, we highlight cell-cycle-dependent differences in checkpoint signalling and recovery after a DNA DSB, and summarise the molecular mechanisms that underlie the reversal of DNA damage checkpoints, before discussing when and how cell fate decisions after a DSB are made.
Assuntos
Pontos de Checagem do Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Processamento de Proteína Pós-Traducional/genética , Proteínas de Ciclo Celular/genética , Divisão Celular , DNA/genética , Humanos , Transdução de Sinais/genéticaRESUMO
Mice null for wild-type p53-induced phosphatase 1 (WIP1) display defects in testis development and spermatogenesis, resulting in reduced fertility. However, the molecular mechanism underlying these abnormalities in the testis remains uncharacterized. We report that the phosphatase activity of WIP1 increases Wnt activity through Nemo-like kinase (NLK). WIP1 directly interacted with NLK, which is highly homologous to p38 MAPK, a WIP1 substrate, and dephosphorylated its activation site. The WIP1-mediated inhibition of NLK activity markedly decreased the phosphorylation of lymphoid enhancer-binding factor 1 (LEF1), enhancing its interaction with ß-catenin. Additionally, WIP1 depletion impaired germ cell development, as evidenced by the expression of Oct4 and the germ cell-specific markers Ddx4, Nanos3 and Dnd1 during the development of germ cells from Oct4-GFP transgenic (OG2) mouse embryonic stem cells (mESCs). The expression of WIP1, whose level was significantly lower after the differentiation of germ cells from mESCs, occurred in parallel with the expression of germ cell development markers and SRY-box 17 (Sox17), a downstream target of Wnt. These results indicate that WIP1 is essential for germ cell development, which is known to require Wnt activity.
Assuntos
Células Germinativas/citologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Proteína Fosfatase 2C/metabolismo , Proteínas Wnt/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/metabolismo , Células HEK293 , Humanos , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/metabolismo , Fosforilação , Proteína Fosfatase 2C/genética , Proteínas Serina-Treonina Quinases , Testículo/citologia , Testículo/metabolismo , beta Catenina/metabolismoRESUMO
OBJECTIVE: The aims of the present study were to detect the interaction of Wip1 and NF-κB P65 in retina of an LPS-induced astrocytes activation model. METHODS: The interaction between Wip1 and nuclear factor kappa B (NF-κB) P65 was observed in lipopolysaccharide (LPS)-stimulated primary rat astrocytes derived from retina. The expressions of Wip1 and NF-κB P65 were evaluated using Western blot and RT-PCR. Small interfering RNA (siRNA) against Wip1 was transfected into astrocytes to clarify the phosphorylation status and nuclear translocation of NF-κB P65 and expressions of these proinflammatory factors. Meanwhile, expression of Wip1 was assessed following treatment with NF-κB inhibitor. RESULTS: Wip1 and phospho-NF-κB P65 (p-P65) expressions were significantly increased and colocalization in astrocytes after LPS treatment. The expression of p-P65 was augmented by transfected with Wip1 siRNA followed by LPS. Furthermore, pre-treatment with Wip1 siRNA further enhanced LPS-induced NF-κB P65 translocation into the nuclei and proinflammatory cytokine release. Finally, inhibition of NF-κB decreases Wip1 expression and transcription in primary astrocytes. CONCLUSION: These data provide a mechanism for the role for a negative feedback loop of Wip1 and NF-κB in LPS-induced astrocytic activation.
Assuntos
Astrócitos/metabolismo , Fator de Transcrição RelA/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Células Cultivadas , Citocinas/genética , Lipopolissacarídeos/farmacologia , Proteína Fosfatase 2C , RatosRESUMO
Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), is a chronically intestinal autoimmune disease, the pathological mechanisms of which are not very clear. Wild type p-53 induced phosphatase 1 (Wip1), a serine/threonine protein phosphatase, has been reported to negatively regulate the inflammation in sepsis. However, the role of Wip1 in IBD is not very clear. Therefore, colonic tissues and peripheral blood from patients with IBD and healthy controls were collected to analyzed mRNA and protein expression of Wip1 using the method of qPCR and immunohistochemistry. Immune cells of neutrophils, CD4+ T cells, CD8+ T cells, B cells, monocytes and intestinal epithelial cells were isolated to analyze Wip1 expression by means of qPCR. Expression of Wip 1 was analyzed after the cells were stimulated by various of cytokines. DSS-colitis model was induced on the wild type (WT) and Wip 1 knock out (Wip1-/-) mice, and cytokines expression were analyzed in intestinal lamina propria from WT and Wip1-/- mice. Neutrophil specific markers were examined in intestinal lamina propria from WT and Wip1-/- mice. Moreover, neutrophils were isolated from bone marrow of WT and Wip1-/- mice to examine neutrophil migration with or without inhibitors of signaling pathway in vitro. The expression of Wip 1 mRNA and protein were found to be significantly decreased in patients with active IBD compared with healthy controls. And Wip 1 was mainly expressed on neutrophils from peripheral blood and colonic tissues. Moreover, expression of Wip1 was significantly decreased on neutrophils after the stimulation of TNF-α and IFN-γ. Wip1-/- mice were more susceptible to DSS induced colitis compared to WT mice, and expressed more pro-inflammatory cytokines (e.g. TNF-α, IFN-γ, IL-17A, etc). Moreover, expression of neutrophil specific markers (e.g. Ly6G, CD11b, elastase, etc) was also increased in Wip1-/- mice. The migration of neutrophils from the bone marrow of Wip1 mice was marked increased, which was mediated by MAPK-P38 signaling pathway. Wip1 plays an importantly protective role in the pathogenesis of IBD. Therefore, Wip1 may be used a new targets in the treatment for IBD in the future.