RESUMEN
Interactions of developing T cells with Aire+ medullary thymic epithelial cells expressing high levels of MHCII molecules (mTEChi) are critical for the induction of central tolerance in the thymus. In turn, thymocytes regulate the cellularity of Aire+ mTEChi. However, it remains unknown whether thymocytes control the precursors of Aire+ mTEChi that are contained in mTEClo cells or other mTEClo subsets that have recently been delineated by single-cell transcriptomic analyses. Here, using three distinct transgenic mouse models, in which antigen presentation between mTECs and CD4+ thymocytes is perturbed, we show by high-throughput RNA-seq that self-reactive CD4+ thymocytes induce key transcriptional regulators in mTEClo and control the composition of mTEClo subsets, including Aire+ mTEChi precursors, post-Aire and tuft-like mTECs. Furthermore, these interactions upregulate the expression of tissue-restricted self-antigens, cytokines, chemokines, and adhesion molecules important for T-cell development. This gene activation program induced in mTEClo is combined with a global increase of the active H3K4me3 histone mark. Finally, we demonstrate that these self-reactive interactions between CD4+ thymocytes and mTECs critically prevent multiorgan autoimmunity. Our genome-wide study thus reveals that self-reactive CD4+ thymocytes control multiple unsuspected facets from immature stages of mTECs, which determines their heterogeneity.
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Autoantígenos/fisiología , Células Epiteliales/fisiología , Timocitos/fisiología , Timo , Animales , Linfocitos T CD4-Positivos , Proteínas de Unión al ADN , Epitelio/fisiología , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Histonas , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas del Tejido Nervioso , Transducción de SeñalRESUMEN
Pulmonary surfactant is a lipid-protein complex secreted by alveolar type II epithelial cells and is essential for the maintenance of the delicate structure of mammalian alveoli to promote efficient gas exchange across the air-liquid barrier. The Golgi apparatus plays an important role in pulmonary surfactant modification and secretory trafficking. However, the physiological function of the Golgi apparatus in the transport of pulmonary surfactants is unclear. In the present study, deletion of GM130, which encodes for a matrix protein of the cis-Golgi cisternae, was shown to induce the disruption of the Golgi structure leading to impaired secretion of lung surfactant proteins and lipids. Specifically, the results of in vitro and in vivo analysis indicated that the loss of GM130 resulted in trapping of Sftpa in the endoplasmic reticulum, Sftpb and Sftpc accumulation in the Golgi apparatus, and an increase in the compensatory secretion of Sftpd. Moreover, global and epithelial-specific GM130 knockout in mice resulted in an enlargement of alveolar airspace and an increase in alveolar epithelial autophagy; however, surfactant repletion partially rescued the enlarged airspace defects in GM130-deficient mice. Therefore, our results demonstrate that GM130 and the mammalian Golgi apparatus play a critical role in the control of surfactant protein secretion in pulmonary epithelial cells.
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Autoantígenos/fisiología , Aparato de Golgi/metabolismo , Proteínas de la Membrana/fisiología , Alveolos Pulmonares/metabolismo , Surfactantes Pulmonares/metabolismo , Animales , Autoantígenos/genética , Aparato de Golgi/patología , Aparato de Golgi/ultraestructura , Pulmón/patología , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones NoqueadosRESUMEN
The E2 component of the mitochondrial pyruvate dehydrogenase complex (PDC) is the key autoantigen in primary biliary cholangitis (PBC) and STAT3 is an inflammatory modulator that participates in the pathogenesis of many liver diseases. This study investigated whether PDC-E2 interacts with STAT3 in human cholangiocytes (NHC) and hepatocytes (Hep-G2) under cholestatic conditions induced by glyco-chenodeoxycholic acid (GCDC). GCDC induced PDC-E2 expression in the cytoplasmic and nuclear fraction of NHC, whereas in Hep-G2 cells PDC-E2 expression was induced only in the cytoplasmic fraction. GCDC-treatment stimulated phosphorylation of STAT3 in the cytoplasmic fraction of NHC. siRNA-mediated gene silencing of PDC-E2 reduced the expression of pY-STAT3 in NHC but not in HepG2 cells. Immunoprecipitation and a proximity ligation assay clearly demonstrated that GCDC enhanced pY-STAT3 binding to PDC-E2 in the nuclear and cytoplasmic fraction of NHC cells. Staining with Mitotracker revealed mitochondrial co-localization of PDC-E2/pS-STAT3 complexes in NHC and Hep-G2 cells. In cirrhotic PBC livers the higher expression of both PDC-E2 and pY-STAT3 was observed. The immunoblot analysis demonstrated the occurrence of double bands of PDC-E2 protein in control livers, which was associated with a lower expression of pY-STAT3. Our data indicate the interaction between PDC-E2 and phosphorylated STAT3 under cholestatic conditions, which may play a role in the development of PBC.
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Autoantígenos/metabolismo , Acetiltransferasa de Residuos Dihidrolipoil-Lisina/metabolismo , Proteínas Mitocondriales/metabolismo , Complejo Piruvato Deshidrogenasa/metabolismo , Factor de Transcripción STAT3/metabolismo , Autoantígenos/fisiología , Conductos Biliares/patología , Línea Celular , Acetiltransferasa de Residuos Dihidrolipoil-Lisina/fisiología , Células Epiteliales/metabolismo , Ácido Glicoquenodesoxicólico/farmacología , Células Hep G2 , Hepatocitos/metabolismo , Humanos , Immunoblotting/métodos , Inmunoprecipitación/métodos , Hígado/patología , Cirrosis Hepática Biliar/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/fisiología , Complejo Piruvato Deshidrogenasa/fisiología , Factor de Transcripción STAT3/fisiologíaRESUMEN
BACKGROUND: Cancerous inhibitor of protein phosphatase 2A (CIP2A) plays a critical role in the pathogenesis of various types of cancer. Here, we investigated whether manipulating CIP2A abundance could enhance the treatment effects of doxorubicin in MCF-7/ADR cells. METHODS: CIP2A silencing was achieved by specific siRNAs. Proliferation of breast cancer cell line MCF-7/ADR under effective doxorubicin concentrations after CIP2A silencing was examined by MTT assay. Wound healing assay was performed to quantify cell migration and caspase-3/-7 activities were measured for assessing the extent of apoptosis. RESULTS: First, our data confirmed that MCF-7/ADR cell proliferation was suppressed by doxorubicin in a dose-dependent manner. Additionally, knocking down of CIP2A could further decrease MCF-7 cell proliferation and migration, even in the presence of doxorubicin. Mechanistically, we have found that CIP2A silencing promoted cell apoptosis relative to doxorubicin alone or vehicle control groups. Lastly, phosphatase2A (PP2A) activity was potentiated and the autophagy markers, LC3B and Beclin1, were upregulated after knocking down CIP2A. CONCLUSION: Our findings support the potential benefits of using CIP2A inhibitor as a therapeutic agent to treat doxorubicin-resistant breast cancer.
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Antibióticos Antineoplásicos/farmacología , Autoantígenos/genética , Neoplasias de la Mama/tratamiento farmacológico , Doxorrubicina/farmacología , Resistencia a Antineoplásicos/genética , Silenciador del Gen , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Proteína Fosfatasa 2/metabolismo , Apoptosis , Autoantígenos/fisiología , Autofagia , Beclina-1/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Movimiento Celular , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/fisiología , Células MCF-7 , Proteínas de la Membrana/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , ARN Interferente Pequeño , Regulación hacia ArribaRESUMEN
Mechanistic target of rapamycin complex 1 (mTORC1) deficiency or chronic hyperactivation in pancreatic ß-cells leads to diabetes. mTORC1 complexes with La-related protein 1 (LARP1) to specifically regulate the expression of 5' terminal oligopyrimidine tract (5'TOP) mRNAs which encode proteins of the translation machinery and ribosome biogenesis. Here we show that LARP1 is the most expressed LARP in mouse islets and human ß-cells, being 2-4-fold more abundant than LARP1B, a member of the family that also interacts with mTORC1. Interestingly, ß-cells from diabetic patients have higher LARP1 and LARP1B expression. However, specific deletion of Larp1 gene in ß-cells (ß-Larp1KO mice) did not impair insulin secretion and glucose metabolism in male and female mice. High fat or high branched-chain amino acid (BCAA) diets did not disturb glucose homeostasis compared to control littermates up to 8 weeks; BCAA diet slightly impaired glucose tolerance in the ß-Larp1KO mice at 16 weeks. However, no differences in plasma insulin levels, non-fasting glycemia and ß-cell mass were observed in the ß-Larp1KO mice. In conclusion, LARP1 is the most abundant LARP in mouse islets and human ß-cells, and it is upregulated in diabetic subjects. However, genetically disruption of Larp1 gene did not impact glucose homeostasis in basal and diabetogenic conditions, suggesting no major role for LARP1 in ß-cells.
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Autoantígenos/fisiología , Células Secretoras de Insulina/fisiología , Proteínas de Unión al ARN/fisiología , Ribonucleoproteínas/fisiología , Animales , Autoantígenos/genética , Autoantígenos/metabolismo , Glucemia/metabolismo , Dieta Alta en Grasa , Femenino , Homeostasis , Humanos , Células Secretoras de Insulina/citología , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Ratones Noqueados , Unión Proteica , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Regulación hacia Arriba , Antígeno SS-BRESUMEN
The mammalian target of rapamycin (mTOR) is a critical regulator of cell growth, integrating multiple signalling cues and pathways. Key among the downstream activities of mTOR is the control of the protein synthesis machinery. This is achieved, in part, via the co-ordinated regulation of mRNAs that contain a terminal oligopyrimidine tract (TOP) at their 5'ends, although the mechanisms by which this occurs downstream of mTOR signalling are still unclear. We used RNA-binding protein (RBP) capture to identify changes in the protein-RNA interaction landscape following mTOR inhibition. Upon mTOR inhibition, the binding of LARP1 to a number of mRNAs, including TOP-containing mRNAs, increased. Importantly, non-TOP-containing mRNAs bound by LARP1 are in a translationally-repressed state, even under control conditions. The mRNA interactome of the LARP1-associated protein PABPC1 was found to have a high degree of overlap with that of LARP1 and our data show that PABPC1 is required for the association of LARP1 with its specific mRNA targets. Finally, we demonstrate that mRNAs, including those encoding proteins critical for cell growth and survival, are translationally repressed when bound by both LARP1 and PABPC1.
Asunto(s)
Autoantígenos/fisiología , Proteína I de Unión a Poli(A)/fisiología , Polirribosomas/metabolismo , Biosíntesis de Proteínas/fisiología , ARN Mensajero/metabolismo , Ribonucleoproteínas/fisiología , Serina-Treonina Quinasas TOR/fisiología , Regiones no Traducidas 5'/genética , Autoantígenos/genética , Regulación de la Expresión Génica , Genes Reporteros , Células HeLa , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Diana Mecanicista del Complejo 2 de la Rapamicina/antagonistas & inhibidores , Mutagénesis Sitio-Dirigida , Mutación Missense , Naftiridinas/farmacología , Mutación Puntual , Biosíntesis de Proteínas/genética , Interferencia de ARN , ARN Mensajero/genética , Proteínas de Unión al ARN/aislamiento & purificación , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleoproteínas/genética , Antígeno SS-BRESUMEN
Golgi matrix protein 130 (GM130), encoded by GOLGA2, is the classical marker of the Golgi apparatus. It plays important roles in various mitotic events, such as interacting with importin-alpha and liberating spindle assembly factor TPX2 to regulate mitotic spindle formation. A previous study showed that in vitro knockdown of GM130 could regulate the meiotic spindle pole assembly. In the current study, we found that knockout (KO) mice progressively died, had a small body size and were completely infertile. Furthermore, we constructed an oocyte-specific GM130 knockout mouse model (GM130-ooKO) driven by Gdf9-Cre. Through breeding assays, we found that the GM130-ooKO mice showed similar fecundity as control mice. During superovulation assays, the KO and GM130-ooKO mice had comparable numbers of ovulated eggs, oocyte maturation rates and normal polar bodies, similar to the control groups. Thus, this study indicated that deletion of GM130 might have a limited impact on the maturation and morphology of oocytes. This might due to more than one golgin sharing the same function, with others compensating for the loss of GM130.
Asunto(s)
Desarrollo Embrionario/fisiología , Meiosis/fisiología , Proteínas de la Membrana/deficiencia , Oocitos/citología , Oocitos/fisiología , Animales , Autoantígenos/genética , Autoantígenos/fisiología , Desarrollo Embrionario/genética , Femenino , Fertilidad/genética , Fertilidad/fisiología , Aparato de Golgi/fisiología , Trastornos del Crecimiento/genética , Trastornos del Crecimiento/fisiopatología , Infertilidad Femenina/genética , Infertilidad Femenina/fisiopatología , Masculino , Meiosis/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Ratones , Ratones Noqueados , Oogénesis/genética , Oogénesis/fisiología , Ovulación/genética , Ovulación/fisiología , EmbarazoRESUMEN
Pathological cardiac hypertrophy eventually leads to heart failure without adequate treatment. REGγ is emerging as 11S proteasome activator of 20S proteasome to promote the degradation of cellular proteins in a ubiquitin- and ATP-independent manner. Here, we found that REGγ was significantly upregulated in the transverse aortic constriction (TAC)-induced hypertrophic hearts and angiotensin II (Ang II)-treated cardiomyocytes. REGγ deficiency ameliorated pressure overload-induced cardiac hypertrophy were associated with inhibition of cardiac reactive oxygen species (ROS) accumulation and suppression of protein phosphatase 2A catalytic subunit α (PP2Acα) decay. Mechanistically, REGγ interacted with and targeted PP2Acα for degradation directly, thereby leading to increase of phosphorylation levels and nuclear export of Forkhead box protein O (FoxO) 3a and subsequent of SOD2 decline, ROS accumulation, and cardiac hypertrophy. Introducing exogenous PP2Acα or SOD2 to human cardiomyocytes significantly rescued the REGγ-mediated ROS accumulation of Ang II stimulation in vitro. Furthermore, treatment with superoxide dismutase mimetic, MnTBAP prevented cardiac ROS production and hypertrophy features that REGγ caused in vivo, thereby establishing a REGγ-PP2Acα-FoxO3a-SOD2 pathway in cardiac oxidative stress and hypertrophy, indicates modulating the REGγ-proteasome activity may be a potential therapeutic approach in cardiac hypertrophy-associated disorders.
Asunto(s)
Autoantígenos/fisiología , Cardiomegalia/metabolismo , Complejo de la Endopetidasa Proteasomal/fisiología , Especies Reactivas de Oxígeno/metabolismo , Animales , Animales Recién Nacidos , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos Cardíacos , Estrés Oxidativo , Cultivo Primario de Células , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Increasing evidence has revealed a close correlation between cancerous inhibitor of protein phosphatase 2A (CIP2A) and cancer progression. CIP2A has been shown to participate in diverse biological processes, such as development, tumorigenic transformation and chemoresistance. However, the functions of CIP2A in colorectal cancer (CRC) and its underlying mechanisms of action are not yet completely understood. The purpose of this study was to explore its clinical significance, function and relevant pathways in CRC. METHODS: Quantitative RT-PCR (qRT-PCR), immunohistochemistry (IHC), western blotting and enzyme-linked immunosorbent assay (ELISA) were used to identify the expression of CIP2A in CRC tissues, sera and CRC cell lines. The association between the expressions of CIP2A and patient survival was analyzed using the Kaplan-Meier curves. Additionally, the functional role of CIP2A in the cell lines was identified through small interfering RNA (siRNA)-mediated depletion of the protein followed by analyses of proliferation and xenograft growth in vivo using short hairpin (sh) RNAs. Effects of the C-myc inhibitor 10,058-F4 on the expressions of C-myc, and CIP2A in CRC cell lines and its potential mechanisms of action were investigated. Finally, the potential molecular pathways associated with CIP2A were screened using the phosphokinase array and identified through western blotting. RESULTS: CIP2A mRNA and protein levels were upregulated in CRC tissues compared to those of the corresponding normal tissues. It can be used as an independent prognostic indicator to determine overall survival (OS) and disease-free survival (DFS). Depletion of CIP2A substantially suppressed the growth of CRC cells and colony formation in vitro, and inhibited the growth of xenograft tumors in vivo. Additionally, the levels of CIP2A in the sera of patients with CRC were higher than those of the control subjects. Multivariate analyses revealed that the levels of CIP2A in the sera were not independent prognostic indicators in patients with CRC. Moreover, 10,058-F4 could effectively inhibit the growth of CRC cells in vitro, which could be correlated with an inhibition in the expressions of C-myc, CIP2A and its downstream regulatory anti-apoptotic proteins. Furthermore, the Human Phosphokinase Antibody Array was used to gain insights into the CIP2A-dependent intermediary signaling pathways. The results revealed that several signaling pathways were affected and the protein levels of p-p53 (S392), p-STAT5a (Y694), Cyclin D1, p-ERK1/2 and p-AKT (T308) had decreased in CIP2A-shRNA group based on the results of the western blot analysis. CONCLUSIONS: CIP2A could promote the development of CRC cells and predict poor prognosis in patients with CRC, suggesting that it may serve as a potential prognostic marker and therapeutic target against CRC. Video Abstract.
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Autoantígenos/fisiología , Neoplasias Colorrectales , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas de la Membrana/fisiología , Animales , Biomarcadores de Tumor/fisiología , Línea Celular Tumoral , Proliferación Celular , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/terapia , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones Endogámicos BALB C , Persona de Mediana Edad , Resultado del TratamientoRESUMEN
Angiotensin-[1-7] (Ang-[1-7]) antagonize the actions of the renin-angiotensin-system via the Mas receptor and thereby exert renoprotective effects. Murine recombinant angiotensin-converting enzyme (ACE)2 was reported to show renoprotective effects in an experimental Alport syndrome model; however, the protective effect of direct administration of Ang-[1-7] is unknown. Here, we used Col4a3-/- mice as a model of Alport syndrome, which were treated with saline or Ang- [1-7]; saline-treated wild-type mice were used as a control group. The mice were continuously infused with saline or Ang-[1-7] (25 µg/kg/h) using osmotic mini-pumps. Col4a3-/- mice showed increased α-smooth muscle actin (SMA), collagen, and fibronectin expression levels, which were attenuated by Ang-[1-7] treatment. Moreover, Ang-[1-7] alleviated activation of transforming growth factor-ß/Smad signaling, and attenuated the protein expression of ED-1 and heme oxygenase-1, indicating reduction of renal inflammation. Ang-[1-7] treatment further reduced the expression levels of inflammatory cytokines and adhesion molecules and attenuated apoptosis in human kidney cells. Finally, Ang-[1-7] downregulated TNF-α converting enzyme and upregulated ACE2 expression. Thus, treatment with Ang-[1-7] altered the ACE2-Ang-[1-7]-Mas receptor axis in the kidneys of Col4a3-/- mice to attenuate the nephropathy progression of Alport syndrome.
Asunto(s)
Lesión Renal Aguda/tratamiento farmacológico , Angiotensina I/farmacología , Antihipertensivos/farmacología , Autoantígenos/fisiología , Colágeno Tipo IV/fisiología , Modelos Animales de Enfermedad , Inflamación/prevención & control , Nefritis Hereditaria/complicaciones , Fragmentos de Péptidos/farmacología , Lesión Renal Aguda/etiología , Lesión Renal Aguda/patología , Animales , Apoptosis , Inflamación/etiología , Inflamación/patología , Masculino , Ratones , Ratones Noqueados , Nefritis Hereditaria/patologíaRESUMEN
La-related protein 6 (Larp6) is a conserved RNA-binding protein found across eukaryotes that has been suggested to regulate collagen biogenesis, muscle development, ciliogenesis, and various aspects of cell proliferation and migration. Zebrafish have two Larp6 family genes: larp6a and larp6b Viable and fertile single and double homozygous larp6a and larp6b zygotic mutants revealed no defects in muscle structure, and were indistinguishable from heterozygous or wild-type siblings. However, larp6a mutant females produced eggs with chorions that failed to elevate fully and were fragile. Eggs from larp6b single mutant females showed minor chorion defects, but chorions from eggs laid by larp6a;larp6b double mutant females were more defective than those from larp6a single mutants. Electron microscopy revealed defective chorionogenesis during oocyte development. Despite this, maternal zygotic single and double mutants were viable and fertile. Mass spectrometry analysis provided a description of chorion protein composition and revealed significant reductions in a subset of zona pellucida and lectin-type proteins between wild-type and mutant chorions that paralleled the severity of the phenotype. We conclude that Larp6 proteins are required for normal oocyte development, chorion formation and egg activation.
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Autoantígenos/genética , Autoantígenos/fisiología , Corion/fisiología , Oocitos/fisiología , Ribonucleoproteínas/genética , Ribonucleoproteínas/fisiología , Animales , Movimiento Celular , Proliferación Celular , Colágeno/fisiología , Proteínas del Huevo/fisiología , Femenino , Edición Génica , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Genoma , Genotipo , Heterocigoto , Homocigoto , Lectinas/fisiología , Masculino , Mutación , Oocitos/citología , Oogénesis/fisiología , Fenotipo , Pez Cebra , Zona Pelúcida/fisiología , Antígeno SS-BRESUMEN
Abdominal Aortic Aneurysm (AAA) is a severe cardiovascular disease, with high mortality rate after acute rupture of blood vessels. However, the underlying pathogenesis of different morbidity between men and women remains unclear. In the present study, we first selected four datasets including 68 AAA and 32 control samples from published data on GEO database, and analyzed them by data mining. The integrative analysis found a total of 368 differentially expressed genes in E2-related AAA. Next, regulatory mechanism networks among these target genes were predicted, and four genes were identified as key nodes in the network, which play a major role in the immune system. We focused on the role of monocytes/macrophages in the development of cardiovascular diseases to further explore the role of estrogen in the polarization of monocytes/macrophage, the mRNA level of the four genes was validated by RT-PCR in RAW264.7 cells treated with ß-estradiol (E2), diarylpropionitrile (DPN), 1,3,5-Tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT), fulvestrant or vehicle. The results showed that the mRNA level and protein level of TROVE2 was significantly increased in estrogen or estrogen receptor agonist-treated groups. Moreover, estrogen affected the transformation of macrophages to M2 phenotype by detecting M1- and M2-related indicator genes at the mRNA level. Flow cytometry demonstrated that the TROVE2 deficiency led to a notable decrease in the level of M2 phenotype marker protein CD206. In conclusion, our results suggest that E2 can promote the expression of TROVE2, which is closely related to the M2-phenotype transformation of macrophages.
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Aneurisma de la Aorta Abdominal/fisiopatología , Autoantígenos/fisiología , Estradiol/metabolismo , Activación de Macrófagos , ARN Citoplasmático Pequeño/fisiología , Ribonucleoproteínas/fisiología , Animales , Aneurisma de la Aorta Abdominal/metabolismo , Autoantígenos/metabolismo , Western Blotting , Estradiol/fisiología , Citometría de Flujo , Humanos , Ratones , Células RAW 264.7 , ARN Citoplasmático Pequeño/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Ribonucleoproteínas/metabolismo , TranscriptomaRESUMEN
AIMS: FL118, a novel camptothecin analogue, has been extensively studied for its superior antitumor potency. The aim of this research study is to explore its potential mechanism of action in anti- colorectal cancer (CRC). MAIN METHODS: The effect of FL118 on CRC cell proliferation was assessed using CCK-8 assay, while apoptosis was detected using Hoechst staining and Flow cytometry assays. The expression levels of CIP2A were analyzed using qRT-PCR. The expression of CIP2A, PP2A-C, Bax, cleaved caspase-3 and PARP were analyzed using western blotting analysis. The expressions of related proteins in CRC tissues were detected using immunohistochemical staining. TUNEL assay was used to detect apoptosis of tissue. Toxicity of FL118 in primary organs were examined using H&E staining. KEY FINDINGS: The results show that FL118 can inhibit the proliferation and clonogenic potential of CRC cells and increase the expression of pro-apoptosis proteins, Bax, cleaved caspase-3 and PARP. Microarray analyses found that FL118 treatment significantly decreases cancerous inhibition of protein phosphatase 2A (CIP2A). Further validation found that CIP2A is aberrantly upregulated in CRC tissues, and is positively correlated with the progression of CRC. In vitro findings confirm that FL118 mediates the downregulation of CIP2A, at both protein and mRNA levels. Co-treatment with Okadaic acid (OA) (a PP2A inhibitor) partially abolishes the anti-proliferative and pro-apoptotic effect of FL118. Consistently, in vivo experiment demonstrates that FL118 can effectively suppress tumorigenesis without any obvious toxic effects. SIGNIFICANCE: Collectively, these findings exhibit the anti-neoplastic effects of FL118 against CRC through the down regulation of CIP2A, which subsequently enhances the activity of PP2A.
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Autoantígenos/metabolismo , Benzodioxoles/farmacología , Indolizinas/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Proteína Fosfatasa 2/metabolismo , Animales , Apoptosis/efectos de los fármacos , Autoantígenos/fisiología , Benzodioxoles/metabolismo , Camptotecina/análogos & derivados , Camptotecina/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , China , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Indolizinas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/fisiología , Masculino , Proteínas de la Membrana/fisiología , Ratones , Ratones Endogámicos BALB C , Proteína Fosfatasa 2/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Long-palate, lung and nasal epithelium clone 1 (LPLUNC1) is a tumour suppressor gene in nasopharyngeal carcinoma (NPC), and low expression of LPLUNC1 is associated with poor prognosis. Our previous study showed that LPLUNC1 upregulates Prohibitin 1 (PHB1), a pleiotropic protein that functions as a tumour suppressor gene in various cancers. Low expression of PHB1 was also found to be associated with the poor prognosis of NPC patients. However, the mechanisms by which LPLUNC1 upregulates PHB1 and the potential role of PHB1 in NPC are unclear. Here, we found that LPLUNC1 stabilised PHB1 by inhibiting PHB1 ubiquitination, which is mediated by E3 ligase TRIM21. LPLUNC1 competitively impaired the binding of PHB1 to TRIM21 due to its stronger binding affinity to PHB1, suppressing the ubiquitination of PHB1. Therefore, our study indicates that PHB1 acted as a tumour suppressor gene by inhibiting NF-κB activity. Depletion of PHB1 significantly attenuated the anti-tumour effects of LPLUNC1 in NPC cells, and the inhibitory effect of LPLUNC1 on NF-κB activity was thus reversed. Together, our findings revealed a novel mechanism underlying the anticancer effect of LPLUNC1 and clarified that PHB1 may represent a novel, promising candidate tumour suppressor gene in NPC, with potential therapeutic target value.
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Autoantígenos/fisiología , Proteínas de Unión a Ácidos Grasos/fisiología , FN-kappa B/metabolismo , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas , Proteínas Represoras/metabolismo , Ribonucleoproteínas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor/fisiología , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/metabolismo , Carcinoma Nasofaríngeo/patología , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patología , Prohibitinas , Procesamiento Proteico-Postraduccional/genética , Estabilidad Proteica , Ribonucleoproteínas/genética , Transducción de Señal , Ubiquitinación/genéticaRESUMEN
The mechanism of self-recognition of the autoantigen TROVE2, a common biomarker in autoimmune diseases, has been studied with a quartz crystal microbalance with dissipation monitoring (QCM-D) and dual polarization interferometry (DPI). The complementarity and remarkable analytical features of both techniques has allowed new insights into the onset of systemic lupus erythematosus (SLE) to be achieved at the molecular level. The in vitro study for SLE patients and healthy subjects suggests that anti-TROVE2 autoantibodies may undergo an antibody bipolar bridging. An epitope-paratope-specific binding initially occurs to activate a hidden Fc receptor in the TROVE2 tertiary structure. This bipolar mechanism may contribute to the pathogenic accumulation of anti-TROVE2 autoantibody immune complex in autoimmune disease. Furthermore, the specific calcium-dependent protein-protein bridges point out at how the TRIM21/TROVE2 association might occur, suggesting that the TROVE2 protein could stimulate the intracellular immune signaling via the TRIM21 PRY-SPRY domain. These findings may help to better understand the origins of the specificity and affinity of TROVE2 interactions, which might play a key role in the SLE pathogenesis. This manuscript gives one of the first practical applications of two novel functions (-df/dD and Δh/molec) for the analysis of the data provided by QCM-D and DPI. In addition, it is the first time that QCM-D has been used for mapping hidden Fc receptors as well as linear epitopes in a protein tertiary structure. Graphical abstract á .
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Autoantígenos/fisiología , Interferometría/métodos , Lupus Eritematoso Sistémico/inmunología , Tecnicas de Microbalanza del Cristal de Cuarzo , ARN Citoplasmático Pequeño/fisiología , Ribonucleoproteínas/fisiología , Autoanticuerpos/inmunología , Autoantígenos/química , Autoantígenos/inmunología , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Conformación Proteica , ARN Citoplasmático Pequeño/química , ARN Citoplasmático Pequeño/inmunología , Ribonucleoproteínas/química , Ribonucleoproteínas/inmunologíaRESUMEN
Type XVII collagen (COL17) is a transmembranous protein that is mainly expressed in the epidermal basal keratinocytes. Epidermal-dermal attachment requires COL17 expression at the hemidesmosomes of the epidermal basement membrane zone because congenital COL17 deficiency leads to junctional epidermolysis bullosa and acquired autoimmunity to COL17 induces bullous pemphigoid. Recently, in addition to facilitating epidermal-dermal attachment, COL17 has been reported to serve as a niche for hair follicle stem cells, to regulate proliferation in the interfollicular epidermis and to be present along the non-hemidesmosomal plasma membrane of epidermal basal keratinocytes. This review focuses on the physiological properties of COL17 in the epidermis, its role in maintaining stem cells and its association with signalling pathways. We propose possible solutions to unanswered questions in this field.
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Autoantígenos/inmunología , Epidermis/patología , Epidermólisis Ampollosa de la Unión/genética , Hemidesmosomas/metabolismo , Queratinocitos/metabolismo , Colágenos no Fibrilares/fisiología , Penfigoide Ampolloso/inmunología , Uniones Adherentes/patología , Autoanticuerpos/inmunología , Autoantígenos/genética , Autoantígenos/fisiología , Línea Celular , Micropartículas Derivadas de Células/química , Epidermólisis Ampollosa de la Unión/patología , Proteínas de la Matriz Extracelular/fisiología , Predicción , Hemidesmosomas/ultraestructura , Humanos , Colágenos no Fibrilares/genética , Colágenos no Fibrilares/inmunología , Dominios Proteicos , Transducción de Señal , Nicho de Células Madre , Colágeno Tipo XVIIRESUMEN
When a cell migrates, the centrosome positions between the nucleus and the leading edge of migration via the microtubule system. The protein CrpF46 (centrosome-related protein F46) has a known role during mitosis and centrosome duplication. However, how CrpF46 efficiently regulates centrosome-related cell migration is unclear. Here, we report that knockdown of CrpF46 resulted in the disruption of microtubule arrangement, with impaired centrosomal reorientation, and slowed down cell migration. In cells that express low levels of CrpF46, stress fibers were weakened, which could be rescued by recovering Flag-CrpF46. We also found that CrpF46 interacted with non-muscle myosin high chain IIA (NMHC IIA) and that its three coiled-coil domains are pivotal for its binding to NMHC IIA. Additionally, analyses of phosphorylation of NMHC IIA and RLC (regulatory light chain) demonstrated that CrpF46 was associated with myosin IIA during filament formation. Indirect immunofluorescence images indicated that NM IIA filaments were inhibited when CrpF46 was under-expressed. Thus, CrpF46 regulates cell migration by centrosomal reorientation and altering the function of the actomyosin network by controlling specific phosphorylation of myosin.
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Actomiosina/metabolismo , Autoantígenos/fisiología , Movimiento Celular , Proteínas Motoras Moleculares/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Citoesqueleto de Actina/ultraestructura , Autoantígenos/genética , Autoantígenos/metabolismo , Línea Celular Tumoral , Polaridad Celular , Centrosoma , Células HeLa , Humanos , Microtúbulos/ultraestructuraRESUMEN
Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by the production of antibodies to components of the cell nucleus (antinuclear antibodies or ANAs) and the formation of immune complexes with nuclear antigens. These complexes can drive pathogenesis by depositing in the tissue to incite inflammation or induce cytokine production by cells of the innate immune system. While ANAs can bind to purified nuclear molecules, nuclear autoantigens in vivo most likely exist attached to other molecules or embedded in larger structures. Among these structures, microparticles (MPs) are membrane bound vesicles that are released from dead and dying cells by a blebbing process; MPs can also be released during activation of platelets. The presence of MPs in the blood or tissue culture media can be assayed by flow cytometry on the basis of light scattering as well as binding of marker antibodies to identify the cell of origin. As shown by biochemical analyses, MPs contain an ensemble of intracellular components including nuclear, cytoplasmic and membrane molecules. Because of the display of these molecules on the particle surface or in an otherwise accessible form, ANAs, including anti-DNA, can bind to particles. Levels of MPs are increased in the blood of patients with SLE, with flow cytometry demonstrating the presence of IgG-containing particles. In addition to forming immune complexes, MPs can directly stimulate immune responses. Together, these findings suggest an important role of particles in the pathogenesis of SLE and their utility as biomarkers.
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Autoantígenos/fisiología , Micropartículas Derivadas de Células/inmunología , Lupus Eritematoso Sistémico/inmunología , Apoptosis/inmunología , Biomarcadores/metabolismo , Micropartículas Derivadas de Células/química , Micropartículas Derivadas de Células/metabolismo , ADN/inmunología , Humanos , Inmunoensayo/métodosRESUMEN
Neuroblastoma is a neural crest-derived childhood tumor of the peripheral nervous system in which MycN amplification is a hallmark of poor prognosis. Here we show that MycN is expressed together with phosphorylation-stabilizing factor CIP2A in regions of the neural plate destined to form the CNS, but MycN is excluded from the neighboring neural crest stem cell domain. Interestingly, ectopic expression of MycN or CIP2A in the neural crest domain biases cells toward CNS-like neural stem cells that express Sox2. Consistent with this, some forms of neuroblastoma have been shown to share transcriptional resemblance with CNS neural stem cells. As high MycN/CIP2A levels correlate with poor prognosis, we posit that a MycN/CIP2A-mediated cell-fate bias may reflect a possible mechanism underlying early priming of some aggressive forms of neuroblastoma. In contrast to MycN, its paralogue cMyc is normally expressed in the neural crest stem cell domain and typically is associated with better overall survival in clinical neuroblastoma, perhaps reflecting a more "normal" neural crest-like state. These data suggest that priming for some forms of aggressive neuroblastoma may occur before neural crest emigration from the CNS and well before sympathoadrenal specification.
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Autoantígenos/fisiología , Proteínas de la Membrana/fisiología , Proteína Proto-Oncogénica N-Myc/fisiología , Cresta Neural/citología , Células-Madre Neurales/fisiología , Neuroblastoma/etiología , Autoantígenos/análisis , Movimiento Celular , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/análisis , Proteína Proto-Oncogénica N-Myc/análisis , Neuroblastoma/patología , Factores de Transcripción SOXB1/análisisRESUMEN
CIP2A is a recent identified oncogene that inhibits protein phosphatase 2A (PP2A) and stabilizes c-Myc in cancer cells. To investigate the potential oncogenic role and prognostic value of CIP2A, we comprehensively analyzed the CIP2A expression levels in pan-cancer and observed high expression level of CIP2A in majority cancer types, including hepatocellular carcinoma (HCC). Based on a validation cohort including 60 HCC and 20 non-tumorous tissue samples, we further confirmed the high mRNA and protein expression levels of CIP2A in HCC, and found high CIP2A mRNA expression level was associated with unfavorable overall and recurrence-free survival in patients with HCC. Mechanistic investigations revealed that inhibition of CIP2A significantly attenuated cellular proliferation in vitro and tumourigenicity in vivo. Bioinformatic analysis suggested that CIP2A might be involved in regulating cell cycle. Our experimental data further confirmed CIP2A knockdown induced cell cycle arrest at G1 phase. We found accumulated cellular senescence in HCC cells with CIP2A knockdown, companying expression changes of senescence associated proteins (p21, CDK2, CDK4, cyclin D1, MCM7 and FoxM1). Mechanistically, CIP2A knockdown repressed FoxM1 expression and induced FoxM1 dephosphorylation. Moreover, inhibition of PP2A by phosphatase inhibitor rescued the repression of FoxM1. Taken together, our results showed that CIP2A was highly expressed in HCC. Inhibition of CIP2A induced cell cycle arrest and promoted cellular senescence via repressing FoxM1 transcriptional activity, suggesting a potential anti-cancer target for patients with HCC.