RESUMO
Arrhythmogenic cardiomyopathy (ACM) is a cardiomyopathy that is predominantly inherited and characterized by cardiac arrhythmias and structural abnormalities. TMEM43 (transmembrane protein 43) is one of the well-known genetic culprits behind ACM. In this study, we successfully generated an induced pluripotent stem cell (iPSC) line, YCMi010-A, derived from a male patient diagnosed with ACM. Although these iPSCs harbored a heterozygous intronic splice variant, TMEM43 c.443-2A > G, they still displayed normal cellular morphology and were confirmed to express pluripotency markers. YCMi010-A iPSC line is a promising model for investigating the pathomechanisms associated with ACM and exploring potential therapeutic strategies.
Assuntos
Displasia Arritmogênica Ventricular Direita , Células-Tronco Pluripotentes Induzidas , Proteínas de Membrana , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Displasia Arritmogênica Ventricular Direita/genética , Displasia Arritmogênica Ventricular Direita/patologia , Displasia Arritmogênica Ventricular Direita/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Linhagem Celular , Adulto , Sítios de Splice de RNA/genética , Diferenciação CelularRESUMO
AIMS: Doxorubicin (DOX) is a widely used anthracycline anticancer agent; however, its irreversible effects on the heart can result in DOX-induced cardiotoxicity (DICT) after cancer treatment. Unfortunately, the pathophysiology of DICT has not yet been fully elucidated, and there are no effective strategies for its prevention or treatment. In this investigation, the novel role of transducin beta-like protein 1 (TBL1) in developing and regulating DICT was explored. METHODS AND RESULTS: We observed a reduction in TBL1 protein expression levels as well as cleavage events in the transplanted cardiac tissues of patients diagnosed with Dilated Cardiomyopathy and DICT. It was revealed that DOX selectively induces TBL1 cleavage at caspase-3 preferred sites-D125, D136, and D215. Interestingly, overexpression of the uncleaved TBL1 mutant (TBL1uclv) variant reduced apoptosis, effectively preventing DOX-induced cell death. We confirmed that cleaved TBL1 cannot form a complex with ß-catenin. As a result, Wnt reporter activity and Wnt target gene expression collectively indicate a decrease in Wnt/ß-catenin signalling, leading to DICT progression. Furthermore, the cleaved TBL1 triggered DOX-induced abnormal electrophysiological features and disrupted calcium homeostasis. However, these effects were improved in TBL1uclv-overexpressing human-induced pluripotent stem cell-derived cardiomyocytes. Finally, in a DICT mouse model, TBL1uclv overexpression inhibited the DICT-induced reduction of cardiac contractility and collagen accumulation, ultimately protecting cardiomyocytes from cell death. CONCLUSION: Our findings reveal that the inhibition of TBL1 cleavage not only mitigates apoptosis but also enhances cardiomyocyte function, even in the context of DOX administration. Consequently, this study's results suggest that inhibiting TBL1 cleavage may be a novel strategy to ameliorate DICT.
Assuntos
Apoptose , Cardiotoxicidade , Doxorrubicina , Miócitos Cardíacos , Via de Sinalização Wnt , beta Catenina , Doxorrubicina/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Miócitos Cardíacos/enzimologia , Via de Sinalização Wnt/efeitos dos fármacos , Humanos , Animais , Apoptose/efeitos dos fármacos , beta Catenina/metabolismo , beta Catenina/genética , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/patologia , Cardiomiopatia Dilatada/induzido quimicamente , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/enzimologia , Cardiomiopatia Dilatada/fisiopatologia , Masculino , Transducina/metabolismo , Transducina/genética , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/enzimologia , Células-Tronco Pluripotentes Induzidas/patologia , Feminino , Estudos de Casos e Controles , Antibióticos Antineoplásicos/farmacologia , Antibióticos Antineoplásicos/toxicidadeRESUMO
BACKGROUND: Doxorubicin (DOX) is an effective anticancer agent. However, the clinical outcomes of DOX-based therapies are severely hampered by their significant cardiotoxicity. PURPOSE: We investigated the beneficial effects of an ethanol extract of Cirsium setidens (CSE) on DOX-induced cardiomyotoxicity (DICT). METHODS: UPLC-TQ/MS analysis was used to identify CSE metabolite profiles. H9c2 rat cardiomyocytes and MDA-MB-231 human breast cancer cells were used to evaluate the effects of CSE on DICT-induced cell death. To elucidate the mechanism underlying it, AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor gamma co-activator l-alpha (PGC1-α), nuclear respiratory factor 1 (NRF1), NRF2, superoxide dismutase (SOD1), and SOD2 expression was detected using western blot analysis. The oxygen consumption rate (OCR), cellular ROS, and mitochondrial membrane potential were measured. Finally, we confirmed the cardioprotective effect of CSE against DICT in both C57BL/6 mice and human induced pluripotent stem cell-derived cardiomyocytes (hiPSCCMs) by observing various parameters, such as electrophysiological changes, cardiac fibrosis, and cardiac cell death. RESULTS: Chlorogenic acid and nicotiflorin were the major compounds in CSE. Our data demonstrated that CSE blocked DOX-induced cell death of H9c2 cells without hindrance of its apoptotic effects on MDA-MB-231 cells. DOX-induced defects of OCR and mitochondrial membrane potential were recovered in a CSE through upregulation of the AMPK-PGC1-α-NRF1 signaling pathway. CSE accelerated NRF1 translocation to the nucleus, increased SOD activity, and consequently blocked apoptosis in H9c2 cells. In mice treated with 400 mg/kg CSE for 4 weeks, electrocardiogram data, creatine kinase and lactate dehydrogenase levels in the serum, and cardiac fibrosis, were improved. Moreover, various electrophysiological features indicative of cardiac function were significantly enhanced following the CSE treatment of hiPSCCMs. CONCLUSION: Our findings demonstrate CSE that ameliorates DICT by protecting mitochondrial dysfunction via the AMP- PGC1α-NRF1 axis, underscoring the therapeutic potential of CSE and its underlying molecular pathways, setting the stage for future investigations into its clinical applications.
Assuntos
Proteínas Quinases Ativadas por AMP , Cardiotoxicidade , Cirsium , Doxorrubicina , Miócitos Cardíacos , Extratos Vegetais , Animais , Humanos , Masculino , Camundongos , Ratos , Proteínas Quinases Ativadas por AMP/metabolismo , Apoptose/efeitos dos fármacos , Cardiotoxicidade/tratamento farmacológico , Linhagem Celular Tumoral , Cirsium/química , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/efeitos dos fármacos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Extratos Vegetais/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismoRESUMO
Cardiac muscle troponin T protein binds to tropomyosin and regulates the calcium-dependent actin-myosin interaction on thin filaments in cardiomyocytes. Recent genetic studies have revealed that TNNT2 mutations are strongly linked to dilated cardiomyopathy (DCM). In this study, we generated YCMi007-A, a human induced pluripotent stem cell (hiPSC) line from a DCM patient with a p. Arg205Trp mutation in the TNNT2 gene. The YCMi007-A cells show high expression of pluripotent markers, normal karyotype, and differentiation into three germ layers. Thus, YCMi007-A-an established iPSC-could be useful for the investigation of DCM.
Assuntos
Cardiomiopatia Dilatada , Células-Tronco Pluripotentes Induzidas , Humanos , Cardiomiopatia Dilatada/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Troponina T/genética , Troponina T/metabolismo , Heterozigoto , MutaçãoRESUMO
Hypertrophic cardiomyopathy (HCM) is a common inherited cardiovascular disease and is characterized by hypertrophy of the left ventricle. We reprogrammed peripheral blood mononuclear cells (PBMCs) from a HCM patient into pluripotent stem cells (iPSC) (YCMi006-A) carrying a heterozygous c.1029C > G mutation in ACTA1. The YCMi006-A cells expressed high levels of pluripotent markers, had a normal 46XX karyotype and demonstrated the capacity to differentiate into derivatives of all three germ layers. This cell line can be a valuable tool for investigating the pathogenesis of HCM.
Assuntos
Cardiomiopatia Hipertrófica , Linhagem Celular , Células-Tronco Pluripotentes Induzidas , Cardiomiopatia Hipertrófica/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Leucócitos Mononucleares/metabolismo , Mutação/genéticaRESUMO
Dilated cardiomyopathy (DCM) is one of the leading causes of heart transplantation. The clinical feature of DCM is characterized by enlarged heart and impaired function of the left or both ventricles, while its etiology is varied. In this study, we generated YCMi005-A, a human-induced pluripotent stem cell (hiPSC) line from a patient with DCM carrying the missense mutation of p.Glu192Lys in the TPM1 genes. YCMi005-A, an established hiPSC, showed the normal karyotype (46, XX) and high expression of pluripotency markers. In addition, it was confirmed that YCMi005-A has the differentiation potential assessed by staining of three germ layer markers.
Assuntos
Cardiomiopatia Dilatada , Células-Tronco Pluripotentes Induzidas , Cardiomiopatia Dilatada/genética , Diferenciação Celular , Humanos , Mutação , Mutação de Sentido Incorreto , Tropomiosina/genéticaRESUMO
Dilated cardiomyopathy (DCM) is a heart muscle disease that causes heart failure and is the leading cause for heart transplantation. It is a heart muscle disease resulted from a variety of genetics, toxic, metabolic, and infectious causes. One of the most prevalent genetic causes of DCM is a protein-truncating variant in the Titin gene (TTNtv). We have generated a human-induced pluripotent stem cell (hiPSC) line from patients who underwent heart transplantation due to DCM carrying a TTNtv mutation (c.70051C > T, p.Arg23351Ter) at the age of 20. The generated hiPSCs showed normal karyotype (46, XY) and expression of pluripotency markers, and were differentiated towards cardiomyocytes successfully.
RESUMO
Cardiac laminopathy caused by mutations in the LMNA gene are common and highly penetrant with a poor prognosis. We have generated a novel human induced pluripotent stem cell(iPSC) lines YCMi003-A from a patient with dilated cardiomyopathy associated with genetic variant LMNA c.1090G > C; p.Asp364His. We reprogrammed patient-specific peripheral blood mononuclear cells using five episomal vectors Oct4, Sox2, Lin28, L-Myc, and Klf4. The reported iPSC line would be a useful model for in vitro modeling of cardiac laminopathy.
Assuntos
Cardiomiopatia Dilatada , Células-Tronco Pluripotentes Induzidas , Cardiomiopatia Dilatada/genética , Diferenciação Celular , Humanos , Fator 4 Semelhante a Kruppel , Lamina Tipo A/genética , Leucócitos Mononucleares , MutaçãoRESUMO
BACKGROUND: Driver genes of GBM may be crucial for the onset of isocitrate dehydrogenase (IDH)-wildtype (WT) glioblastoma (GBM). However, it is still unknown whether the genes are expressed in the identical cluster of cells. Here, we have examined the gene expression patterns of GBM tissues and patient-derived tumorspheres (TSs) and aimed to find a progression-related gene. METHODS: We retrospectively collected primary IDH-WT GBM tissue samples (n = 58) and tumor-free cortical tissue samples (control, n = 20). TSs are isolated from the IDH-WT GBM tissue with B27 neurobasal medium. Associations among the driver genes were explored in the bulk tissue, bulk cell, and a single cell RNAsequencing techniques (scRNAseq) considering the alteration status of TP53, PTEN, EGFR, and TERT promoter as well as MGMT promoter methylation. Transcriptomic perturbation by temozolomide (TMZ) was examined in the two TSs. RESULTS: We comprehensively compared the gene expression of the known driver genes as well as MGMT, PTPRZ1, or IDH1. Bulk RNAseq databases of the primary GBM tissue revealed a significant association between TERT and TP53 (p < 0.001, R = 0.28) and its association increased in the recurrent tumor (p < 0.001, R = 0.86). TSs reflected the tissue-level patterns of association between the two genes (p < 0.01, R = 0.59, n = 20). A scRNAseq data of a TS revealed the TERT and TP53 expressing cells are in a same single cell cluster. The driver-enriched cluster dominantly expressed the glioma-associated long noncoding RNAs. Most of the driver-associated genes were downregulated after TMZ except IGFBP5. CONCLUSIONS: GBM tissue level expression patterns of EGFR, TERT, PTEN, IDH1, PTPRZ1, and MGMT are observed in the GBM TSs. The driver gene-associated cluster of the GBM single cells were enriched with the glioma-associated long noncoding RNAs.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Neoplasias Encefálicas/genética , Glioblastoma/genética , Humanos , Isocitrato Desidrogenase/genética , Mutação/genética , Recidiva Local de Neoplasia , Prognóstico , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores , Estudos RetrospectivosRESUMO
An increasing number of studies indicate air pollutants infiltrate into the brain. We aimed to find the association of cumulative air pollution exposure in the main body of primary brain tumor: glioblastoma (GBM). In this double-cohort, retrospective analysis study with a protocol, we compared the health effect of air pollution on the GBM patients from the SEER (Surveillance, Epidemiology, and End Results Program) in 27 U.S. counties from 10 states and GBM patients of Severance cohort of Korea. From 2000 to 2015, 10621 GBM patients of the SEER were individually evaluated for the cumulative average exposure for each pollutant, and 9444 (88.9%) mortality events were reported. From 2011 to 2018, 398 GBM patients of the Severance with the same protocol showed 259 (65.1%) mortality events. The multi-pollutant models show that the association level of risk with CO is increased in the SEER (HR 1.252; 95% CI 1.141-1.373) with an increasing linear trend of relative death rate in the spline curve. The Severance GBM data showed such a statistically significant result of the health impact of CO on GBM patients. The overall survival gain of the less exposure group against CO was 2 and 3 months in the two cohorts. Perioperative exposure to CO may increase the risk of shorter survival of GBM patients of the SEER and the Severance cohort.
Assuntos
Poluentes Atmosféricos/efeitos adversos , Neoplasias Encefálicas/mortalidade , Monóxido de Carbono/efeitos adversos , Glioblastoma/mortalidade , Exposição por Inalação/efeitos adversos , Adulto , Idoso , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/cirurgia , Feminino , Glioblastoma/diagnóstico , Glioblastoma/cirurgia , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , República da Coreia/epidemiologia , Estudos Retrospectivos , Medição de Risco , Fatores de Risco , Programa de SEER , Fatores de Tempo , Estados Unidos/epidemiologiaRESUMO
The purpose of this study was to investigate the role of piceatannol (PT) in statin (rosuvastatin and simvastatin) resistance and tolerance and its association with PCSK9 expression via its p300 inhibitory (p300i) activity. An in vitro study was performed using HepG2 cells that were exposed to statins (rosuvastatin or simvastatin) with or without PT in delipidated serum (DLPS) medium. In the statin exposed conditions, PCSK9 expression was reduced following PT treatment when compared to HepG2 cells w/o PT treatment. Furthermore, no significant difference was observed in the expression of the transcription factors SREBP2 and HNF1α, which regulate PCSK9 expression. This resulted in low density lipoprotein receptor (LDLR) stabilization and reduced cellular cholesterol levels. This indicates that PT epigenetically controls statin-induced PCSK9 expression. Interestingly, PT attenuated p300 histone acetyltransferase (HAT) activity. Moreover, simulation of PT-p300 binding suggested that PT inhibits p300 as PT could be docked in the p300 HAT domain. Furthermore, inhibition of p300 HAT activity using C-646, a selective p300 inhibitor, or through an siRNA system effectively reduced PCSK9 induction upon statin exposure in HepG2 cells. The chromatin immunoprecipitation (ChIP) assays revealed that PT blocked the recruitment of p300 to the PCSK9 promoter region. In summary, PT attenuated statin-induced PCSK9 expression by inhibiting p300 HAT activity. Finally, co-administration of simvastatin and PT for 10 weeks further reduced plasma low-density lipoprotein-cholesterol (LDL-C) levels and stabilized the hepatic LDLR protein level compared with those resulting from single treatment of simvastatin in a high-fat diet-induced hypercholesterolemia mouse model. Our findings indicate that PT is a new nutraceutical candidate to reduce the statin resistance and tolerance that occurs in patients with hypercholesterolemia.
Assuntos
Hepatócitos/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Hipercolesterolemia/tratamento farmacológico , Pró-Proteína Convertase 9/metabolismo , Rosuvastatina Cálcica/farmacologia , Sinvastatina/farmacologia , Estilbenos/farmacologia , Fatores de Transcrição de p300-CBP/antagonistas & inibidores , Animais , LDL-Colesterol/sangue , Modelos Animais de Doenças , Regulação para Baixo , Resistência a Medicamentos , Células Hep G2 , Hepatócitos/enzimologia , Humanos , Hipercolesterolemia/sangue , Hipercolesterolemia/enzimologia , Hipercolesterolemia/genética , Masculino , Camundongos Endogâmicos C57BL , Pró-Proteína Convertase 9/genética , Estabilidade Proteica , Receptores de LDL/metabolismo , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
BACKGROUND: Gastric cancer is a considerable burden for worldwide patients. And diffuse gastric cancer is the most insidious subgroup with poor survival. The phenotypic characterization of the diffuse gastric cancer cell line can be useful for gastric cancer researchers. In this article, we aimed to characterize the diffuse gastric cancer cells with MRI and transcriptomic data. We hypothesized that gene expression pattern is associated with the phenotype of the cells and that the heterogeneous enhancement pattern and the high tumorigenicity of SNU484 can be modulated by the perturbation of the highly expressed gene. METHODS: We evaluated the 9.4 T magnetic resonance imaging and transcriptomic data of the orthotopic mice models from diffuse gastric cancer cells such as SNU484, Hs746T, SNU668, and KATO III. We included MKN74 as an intestinal cancer control cell. After comprehensive analysis integrating MRI and transcriptomic data, we selected CD34 and validated the effect by shRNA in the BALB/c nude mice models. RESULTS: SNU484, SNU668, Hs746T, and MKN74 formed orthotopic tumors by the 5 weeks after cell injection. The diffuse phenotype was found in the SNU484 and Hs746T. SNU484 was the only tumor showing the heterogeneous enhancement pattern on T2 images with a high level of CD34 expression. Knockdown of CD34 decreased the round-void shape in the H&E staining (P = 0.028), the heterogeneous T2 enhancement, and orthotopic tumorigenicity (100% vs 66.7%). The RNAseq showed that the suppressed CD34 is associated with the downregulated gene-sets of the extracellular matrix remodeling. CONCLUSION: Suppression of CD34 in the human-originated gastric cancer cell suggests that it is important for the round-void histologic shape, heterogeneous enhancement pattern on MRI, and the growth of gastric cancer cell line.
Assuntos
Antígenos CD34/genética , Perfilação da Expressão Gênica/métodos , Neoplasias Gástricas/diagnóstico por imagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Células Endoteliais da Veia Umbilical Humana , Humanos , Imageamento por Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Transplante de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Sequência de RNA , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologiaRESUMO
AIMS: Proprotein convertase subtilisin/kexin type-9 (PCSK9), a molecular determinant of low-density lipoprotein (LDL) receptor (LDLR) fate, has emerged as a promising therapeutic target for atherosclerotic cardiovascular diseases. However, the precise mechanism by which PCSK9 regulates the internalization and lysosomal degradation of LDLR is unknown. Recently, we identified adenylyl cyclase-associated protein 1 (CAP1) as a receptor for human resistin whose globular C-terminus is structurally similar to the C-terminal cysteine-rich domain (CRD) of PCSK9. Herein, we investigated the role of CAP1 in PCSK9-mediated lysosomal degradation of LDLR and plasma LDL cholesterol (LDL-C) levels. METHODS AND RESULTS: The direct binding between PCSK9 and CAP1 was confirmed by immunoprecipitation assay, far-western blot, biomolecular fluorescence complementation, and surface plasmon resonance assay. Fine mapping revealed that the CRD of PCSK9 binds with the Src homology 3 binding domain (SH3BD) of CAP1. Two loss-of-function polymorphisms found in human PCSK9 (S668R and G670E in CRD) were attributed to a defective interaction with CAP1. siRNA against CAP1 reduced the PCSK9-mediated degradation of LDLR in vitro. We generated CAP1 knock-out mice and found that the viable heterozygous CAP1 knock-out mice had higher protein levels of LDLR and lower LDL-C levels in the liver and plasma, respectively, than the control mice. Mechanistic analysis revealed that PCSK9-induced endocytosis and lysosomal degradation of LDLR were mediated by caveolin but not by clathrin, and they were dependent on binding between CAP1 and caveolin-1. CONCLUSION: We identified CAP1 as a new binding partner of PCSK9 and a key mediator of caveolae-dependent endocytosis and lysosomal degradation of LDLR.
Assuntos
Aterosclerose/genética , Proteínas de Transporte/genética , LDL-Colesterol/sangue , Mutação , Pró-Proteína Convertase 9/genética , Receptores de LDL/sangue , Animais , Aterosclerose/metabolismo , Proteínas de Transporte/metabolismo , DNA/genética , Análise Mutacional de DNA , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Knockout , Pró-Proteína Convertase 9/metabolismoRESUMO
The regulation of osteogenesis is important for bone formation and fracture healing. Despite advances in understanding the molecular mechanisms of osteogenesis, crucial modulators in this process are not well-characterized. Here we demonstrate that suppression of signal transducer and activator of transcription 5A (STAT5A) activates distal-less homeobox 5 (DLX5) in human bone marrow-derived stromal cells (hBMSCs) and enhances osteogenesis in vitro and in vivo. We show that STAT5A negatively regulates expression of Dlx5 in vitro and that STAT5A deletion results in increased trabecular and cortical bone mass and bone mineral density in mice. Additionally, STAT5A deletion prevents age-related bone loss. In a murine fracture model, STAT5A deletion was found to significantly enhance bone remodeling by stimulating the formation of a fracture callus. Our findings indicate that STAT5A inhibition enhances bone formation by promoting osteogenesis of BMSCs.
Assuntos
Fraturas Ósseas/genética , Proteínas de Homeodomínio/genética , Osteoblastos/metabolismo , Osteogênese/genética , Osteoporose/genética , Fator de Transcrição STAT5/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Animais , Densidade Óssea/genética , Diferenciação Celular , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Fêmur/lesões , Fêmur/metabolismo , Consolidação da Fratura/genética , Fraturas Ósseas/metabolismo , Fraturas Ósseas/patologia , Fraturas Ósseas/terapia , Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Sialoproteína de Ligação à Integrina/genética , Sialoproteína de Ligação à Integrina/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Knockout , Osteoblastos/citologia , Osteoporose/metabolismo , Osteoporose/patologia , Osteoporose/prevenção & controle , Cultura Primária de Células , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fator de Transcrição STAT5/antagonistas & inibidores , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/metabolismoRESUMO
Statins mediate the transactivation of PCSK9, which in turn limits their cholesterol-lowering effects via LDL receptor (LDLR) degradation. The objective of the present study was to investigate the mechanism of action by which Welsh onion (Allium fistulosum L. [family Amaryllidaceae]) extract (WOE) regulates LDLR and PCSK9. HepG2 cells were cultured under lipid depletion conditions using a medium supplemented with delipidated serum (DLPS). WOE (50, 100, 200, and 400 µg ml-1) significantly attenuated the DLPS-mediated increases in LDLR, PCSK9, and SREBP2 gene expression. While WOE treatment maintained the DLPS-mediated increases in LDLR protein expression, it dose-dependently and significantly attenuated the DLPS-mediated increases in the protein content of PCSK9. The suppression of PCSK9 was associated with the WOE-mediated reductions in SREBP2, but not HNF1α. WOE also dose-dependently reduced PCSK9 protein expression that was otherwise markedly induced by concomitant statin treatment. WOE-mediated PCSK9 inhibition contributed to LDLR lysosomal degradation suppression, and subsequent LDLR protein stabilization. HPLC analysis indicated that WOE contains kaempferol, quercetin, ferulic acid, and p-coumaric acid. Kaempferol and p-coumaric acid contributed to the maintenance of LDLR expression by inhibiting PCSK9 in lipid depleted HepG2 cells. Altogether, these findings suggest that WOE inhibits PCSK9 transcription and protein expression via the reduction of SREBP2, and decreased PCSK9 further contributes to LDLR degradation prevention and LDLR protein stabilization under conditions of lipoprotein deficiency. The PCSK9 inhibition-mediated mechanism of WOE was likely attributed to the action of kaempferol and p-coumaric acid present in WOE.
Assuntos
Metabolismo dos Lipídeos/efeitos dos fármacos , Cebolas/química , Extratos Vegetais/farmacologia , Pró-Proteína Convertase 9/genética , Receptores de LDL/genética , LDL-Colesterol/metabolismo , Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Humanos , Extratos Vegetais/química , Pró-Proteína Convertase 9/metabolismo , Receptores de LDL/metabolismo , Soro/química , Soro/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismoRESUMO
PURPOSE: Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the low density lipoprotein receptor (LDLR) and promotes degradation of the LDLR. Inhibition of PCSK9 either by reducing its expression or by blocking its activity results in the upregulation of the LDLR and subsequently lowers the plasma concentration of LDL-cholesterol. As a modality to inhibit PCSK9 action, we searched the chemical library for small molecules that block the binding of PCSK9 to the LDLR. MATERIALS AND METHODS: We selected 100 chemicals that bind to PCSK9 where the EGF-AB fragment of the LDLR binds via in silico screening of the ChemBridge chemical library, using the computational GOLD algorithm analysis. Effects of chemicals were evaluated using the PCSK9-LDLR binding assay, immunoblot analysis, and the LDL-cholesterol uptake assay in vitro, as well as the fast performance liquid chromatography assay for plasma lipoproteins in vivo. RESULTS: A set of chemicals were found that decreased the binding of PCSK9 to the EGF-AB fragment of the LDLR in a dose-dependent manner. They also increased the amount of the LDLR significantly and subsequently increased the uptake of fluorescence-labeled LDL in HepG2 cells. Additionally, one particular molecule lowered the plasma concentration of total cholesterol and LDL-cholesterol significantly in wild-type mice, while such an effect was not observed in Pcsk9 knockout mice. CONCLUSION: Our findings strongly suggest that in silico screening of small molecules that inhibit the protein-protein interaction between PCSK9 and the LDLR is a potential modality for developing hypercholesterolemia therapeutics.
Assuntos
Colesterol/sangue , Pró-Proteína Convertases/metabolismo , Receptores de LDL/metabolismo , Serina Endopeptidases/metabolismo , Bibliotecas de Moléculas Pequenas , Animais , LDL-Colesterol/sangue , Células Hep G2 , Humanos , Camundongos , Camundongos Knockout , Pró-Proteína Convertase 9RESUMO
The specific delivery of ribonucleic acid (RNA) interfering molecules to disease-related cells is still a critical blockade for in vivo systemic treatment. Here, this study suggests a robust delivery carrier for targeted delivery of RNA-interfering molecules using galactosylated magnetic nanovectors (gMNVs). gMNVs are an organic-inorganic polymeric nanomaterial composed of polycationics and magnetic nanocrystal for delivery of RNA-interfering molecules and tracking via magnetic resonance (MR) imaging. In particular, the surface of gMNVs was modified by galactosylgluconic groups for targeted delivering to asialoglycoprotein receptor (ASGPR) of hepatocytes. Moreover, the small interfering RNAs were used to regulate target proteins related with low-density lipoprotein level and in vivo MR imaging was conducted for tracking of nanovectors. The obtained results show that the prepared gMNVs demonstrate potential as a systemic theragnostic nanoplatform for RNA interference and MR imaging.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Galactose/química , Vetores Genéticos/genética , Metabolismo dos Lipídeos/genética , Imageamento por Ressonância Magnética/métodos , Nanopartículas de Magnetita/química , Interferência de RNA/efeitos dos fármacos , Animais , Receptor de Asialoglicoproteína/metabolismo , Vetores Genéticos/química , Vetores Genéticos/farmacologia , Vetores Genéticos/toxicidade , Células Hep G2 , Humanos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Cancer cells can express specific biomarkers, such as cell membrane proteins and signaling factors. Thus, finding biomarkers and delivering diagnostic agents are important in the diagnosis of cancer. In this study, we investigated a biomarker imaging agent for the diagnosis of hepatic cancers. The asialoglycoprotein receptor (ASGPr) was selected as a biomarker for hepatoma cells and the ASGPr-targetable imaging agent bearing a galactosyl group was prepared using manganese ferrite nanoparticles (MFNP) and galactosylgluconic acid. The utility of the ASGPr-targetable imaging agent, galactosylated MFNP (G-MFNP) was assessed by several methods in ASGPr-expressing HepG2 cells as target cells and ASGPr-deficient MCF7 cells. Physical and chemical properties of G-MFNP were examined using Fourier-transform infrared spectroscopy, dynamic light scattering, zeta potential analysis, and transmission electron microscopy. No significant cytotoxicity was observed in either cell line. Targeting ability was assessed using flow cytometry, magnetic resonance imaging, inductively coupled plasma atomic emission spectroscopy, absorbance analysis, dark-field microscopy, Prussian blue staining, and transmission electron microscopy. We demonstrated that G-MFNP target successfully and bind to ASGPr-expressing HepG2 cells specifically. We suggest that these results will be useful in strategies for cancer diagnoses based on magnetic resonance imaging.
Assuntos
Receptor de Asialoglicoproteína/metabolismo , Compostos Férricos/metabolismo , Galactose/metabolismo , Imageamento por Ressonância Magnética , Compostos de Manganês/metabolismo , Nanopartículas/química , Coloides/química , Fluorescência , Glicosilação , Células Hep G2 , Humanos , Células MCF-7 , Nanopartículas/ultraestrutura , Tamanho da Partícula , Espectrofotometria Atômica , Espectroscopia de Infravermelho com Transformada de Fourier , Eletricidade EstáticaRESUMO
PPARγ (peroxisome-proliferator-activated receptor γ) is a master transcription factor involved in adipogenesis through regulating adipocyte-specific gene expression. Recently, lipin1 was found to act as a key factor for adipocyte maturation and maintenance by modulating the C/EBPα (CCAAT/enhancer-binding protein α) and PPARγ network; however, the precise mechanism by which lipin1 affects the transcriptional activity of PPARγ is largely unknown. The results of the present study show that lipin1 activates PPARγ by releasing co-repressors, NCoR1 (nuclear receptor co-repressor 1) and SMRT (silencing mediator of retinoid and thyroid hormone receptor), from PPARγ in the absence of the ligand rosiglitazone. We also identified a novel lipin1 TAD (transcriptional activation domain), between residues 217 and 399, which is critical for the activation of PPARγ, but not PPARα. Furthermore, this TAD is unique to lipin1 since this region does not show any homology with the other lipin isoforms, lipin2 and lipin3. The activity of the lipin1 TAD is enhanced by p300 and SRC-1 (steroid receptor co-activator 1), but not by PCAF (p300/CBP-associated factor) and PGC-1α (PPAR co-activator 1α). The physical interaction between lipin1 and PPARγ occurs at the lipin1 C-terminal region from residues 825 to 926, and the VXXLL motif at residue 885 is critical for binding with and the activation of PPARγ. The action of lipin1 as a co-activator of PPARγ enhanced adipocyte differentiation; the TAD and VXXLL motif played critical roles, but the catalytic activity of lipin1 was not directly involved. Collectively, these data suggest that lipin1 functions as a key regulator of PPARγ activity through its ability to release co-repressors and recruit co-activators via a mechanism other than PPARα activation.
Assuntos
Proteínas Nucleares/fisiologia , PPAR gama/genética , Fosfatidato Fosfatase/fisiologia , Células 3T3-L1 , Adipócitos/citologia , Animais , Diferenciação Celular/efeitos dos fármacos , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , PPAR alfa/metabolismo , PPAR gama/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
OBJECTIVE: The clinical significance of the measurement of serum PCSK9 (proprotein subtilisin kexin type 9) is not well defined. This study investigated the association between serum PCSK9 levels and atherosclerosis assessed by carotid intima media thickness (IMT) in hypertensive patients. METHODS: A total of 126 hypertensive patients over the age of 45 were enrolled. The maximum carotid IMT (max-IMT) and the mean carotid IMT (mean-IMT) were measured at the time of enrollment. Clinical and laboratory parameters including serum PCSK9 were analyzed. RESULTS: Patients were divided into tertiles based on serum PCSK9 levels. After adjusting for age, sex, total cholesterol, HDL-cholesterol and triglyceride, max-IMT was significantly increased in the highest tertile of serum PCSK9 (0.969±0.033 vs 0.959±0.033 vs 1.077±0.033 mm, respectively; P=0.026). Mean-IMT showed a tendency to increase across the tertile groups (0.773±0.025 vs 0.790±0.026 vs 0.856±0.025 mm, respectively; P=0.059). Multivariate regression analysis revealed that serum PCSK9 was independently associated with carotid IMT (max-IMT: ß=0.212, P=0.016; mean-IMT: ß=0.184, P=0.04). CONCLUSION: The present study is the first to report the association between serum PCSK9 levels and carotid IMT in hypertensive patients. These results suggest that serum PCSK9 may have a certain role in early pathogenesis of atherosclerosis.