RESUMO
Nanorobots are safe and exhibit powerful functionalities, including delivery, therapy, and diagnosis. Therefore, they are in high demand for the development of new cancer therapies. Although many studies have contributed to the progressive development of the nanorobot system for anticancer drug delivery, these systems still face some critical limitations, such as potentially toxic materials in the nanorobots, unreasonable sizes for passive targeting, and the lack of several essential functions of the nanorobot for anticancer drug delivery including sensing, active targeting, controlling drug release, and sufficient drug loading capacity. Here, we developed a multifunctional nanorobot system capable of precise magnetic control, sufficient drug loading for chemotherapy, light-triggered controlled drug release, light absorption for photothermal therapy, enhanced magnetic resonance imaging, and tumor sensing. The developed nanorobot system exhibits an in vitro synergetic antitumor effect of photothermal therapy and chemotherapy and outstanding tumor-targeting efficiency in both in vitro and in vivo environments. The results of this study encourage further explorations of an efficient active drug delivery system for cancer treatment and the development of nanorobot systems for other biomedical applications.
Assuntos
Sistemas de Liberação de Medicamentos , Hipertermia Induzida , Nanoestruturas , Neoplasias/terapia , Fototerapia , Robótica , Linhagem Celular Tumoral , Humanos , Neoplasias/metabolismo , Neoplasias/patologiaRESUMO
RATIONALE: Histone deacetylases (HDACs) are closely involved in cardiac reprogramming. Although the functional roles of class I and class IIa HDACs are well established, the significance of interclass crosstalk in the development of cardiac hypertrophy remains unclear. OBJECTIVE: Recently, we suggested that casein kinase 2α1-dependent phosphorylation of HDAC2 leads to enzymatic activation, which in turn induces cardiac hypertrophy. Here we report an alternative post-translational activation mechanism of HDAC2 that involves acetylation of HDAC2 mediated by p300/CBP-associated factor/HDAC5. METHODS AND RESULTS: Hdac2 was acetylated in response to hypertrophic stresses in both cardiomyocytes and a mouse model. Acetylation was reduced by a histone acetyltransferase inhibitor but was increased by a nonspecific HDAC inhibitor. The enzymatic activity of Hdac2 was positively correlated with its acetylation status. p300/CBP-associated factor bound to Hdac2 and induced acetylation. The HDAC2 K75 residue was responsible for hypertrophic stress-induced acetylation. The acetylation-resistant Hdac2 K75R showed a significant decrease in phosphorylation on S394, which led to the loss of intrinsic activity. Hdac5, one of class IIa HDACs, directly deacetylated Hdac2. Acetylation of Hdac2 was increased in Hdac5-null mice. When an acetylation-mimicking mutant of Hdac2 was infected into cardiomyocytes, the antihypertrophic effect of either nuclear tethering of Hdac5 with leptomycin B or Hdac5 overexpression was reduced. CONCLUSIONS: Taken together, our results suggest a novel mechanism by which the balance of HDAC2 acetylation is regulated by p300/CBP-associated factor and HDAC5 in the development of cardiac hypertrophy.
Assuntos
Cardiomegalia/metabolismo , Histona Desacetilases/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação , Animais , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Camundongos , Mutação , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/metabolismo , Fosforilação , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição de p300-CBP/genéticaRESUMO
RATIONALE: Small heterodimer partner (SHP; NR0B2) is an atypical orphan nuclear receptor that lacks a conventional DNA-binding domain. Through interactions with other transcription factors, SHP regulates diverse biological events, including glucose metabolism in liver. However, the role of SHP in adult heart diseases has not yet been demonstrated. OBJECTIVE: We aimed to investigate the role of SHP in adult heart in association with cardiac hypertrophy. METHODS AND RESULTS: The roles of SHP in cardiac hypertrophy were tested in primary cultured cardiomyocytes and in animal models. SHP-null mice showed a hypertrophic phenotype. Hypertrophic stresses repressed the expression of SHP, whereas forced expression of SHP blocked the development of hypertrophy in cardiomyocytes. SHP reduced the protein amount of Gata6 and, by direct physical interaction with Gata6, interfered with the binding of Gata6 to GATA-binding elements in the promoter regions of natriuretic peptide precursor type A. Metformin, an antidiabetic agent, induced SHP and suppressed cardiac hypertrophy. The metformin-induced antihypertrophic effect was attenuated either by SHP small interfering RNA in cardiomyocytes or in SHP-null mice. CONCLUSIONS: These results establish SHP as a novel antihypertrophic regulator that acts by interfering with GATA6 signaling. SHP may participate in the metformin-induced antihypertrophic response.
Assuntos
Cardiomegalia/prevenção & controle , Fator de Transcrição GATA6/metabolismo , Miócitos Cardíacos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais , Animais , Fator Natriurético Atrial/genética , Fator Natriurético Atrial/metabolismo , Sítios de Ligação , Cardiomegalia/genética , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Modelos Animais de Doenças , Fator de Transcrição GATA6/genética , Regulação da Expressão Gênica , Genótipo , Células HEK293 , Humanos , Masculino , Metformina/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Fenótipo , Regiões Promotoras Genéticas , Interferência de RNA , Ratos , Ratos Sprague-Dawley , Receptores Citoplasmáticos e Nucleares/deficiência , Receptores Citoplasmáticos e Nucleares/genética , Transdução de Sinais/efeitos dos fármacos , TransfecçãoRESUMO
The coupling of autophagy and endoplasmic reticulum (ER) stress has been implicated in a variety of biological processes; however, little is known regarding the involvement of the autophagy/ER stress pathway in early embryogenesis or the underlying mechanism(s). Here, we showed that the developmental competence of in vitro-produced (IVP) bovine embryos was highly dependent on the autophagy/ER stress balance. Although relative abundances of autophagy-associated gene transcripts, including LC3, Atg5, and Atg7 transcripts, were high in oocytes and throughout the early stages of preattachment development, extensive autophagosome formation was only detected in fertilized embryos. Using an inducer and inhibitor of autophagy, we showed that transient elevation of autophagic activity during early preattachment development greatly increased the blastocyst development rate, trophectoderm cell numbers, and blastomere survival; these same parameters were reduced by both inhibition and prolonged induction of autophagy. Interestingly, the induction of autophagy reduced ER stress and associated damage, while the developmental defects in autophagy-inhibited embryos were significantly alleviated by ER stress inhibitor treatment, indicating that autophagy is a negative regulator of ER stress in early embryos. Collectively, these results suggest that early embryogenesis of IVP bovine embryos depends on an appropriate balance between autophagy and ER stress. These findings may increase our understanding of important early developmental events by providing compelling evidence concerning the tight association between autophagy and ER stress, and may contribute to the development of strategies for the production of IVP bovine blastocysts with high developmental competence.
Assuntos
Autofagia/fisiologia , Desenvolvimento Embrionário/fisiologia , Estresse do Retículo Endoplasmático/fisiologia , Animais , Autofagia/genética , Blastômeros/citologia , Blastômeros/metabolismo , Bovinos , Contagem de Células , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Desenvolvimento Embrionário/genética , Feminino , Fertilização in vitro , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Associadas aos Microtúbulos/genética , Modelos Biológicos , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Trofoblastos/citologia , Trofoblastos/metabolismo , Enzimas Ativadoras de Ubiquitina/genéticaRESUMO
Exosomes are nanosized extracellular vesicles secreted by various cell types, including those of the immune system, such as natural killer (NK) cells. They play a role in intercellular communication by transporting signal molecules between the cells. Recent studies have reported that NK cell-derived exosomes (NK-exo) contain cytotoxic proteins-induced cell death. However, the characteristics and potential functions of NK-exo, especially for the liver cancer are poorly understood. In this study, we investigated the anti-tumor effects of NK-exo in the primary liver cancer, hepatocellular carcinoma (HCC), using the orthotopic and subcutaneous tumor model. We found that NK-exo expressed both typical exosomal markers (e.g. CD63, CD81, and Alix) and cytotoxic proteins (e.g. perforin, granzyme B, FasL, and TRAIL). NK-exo were selectively taken up by HCC cells (e.g. Hep3B, HepG2, and Huh 7). Interestingly, Hep3B cells induced the highest cytotoxicity compared with HepG2 and Huh7 cells, and substantially enhanced the apoptosis by NK-exo. Furthermore, we demonstrated that NK-exo inhibited the phosphorylation of serine/threonine protein kinases (e.g. AKT and ERK1/2), and enhanced the activation of specific apoptosis markers (e.g. caspase-3, -7, -8, -9, and PARP) in Hep3B cells. NK-exo also exhibit the active targeting ability and potent therapeutic effects in both orthotopic and subcutaneous HCC mouse models. Overall, these results suggest that NK-exo indicate strong anti-tumor effects in HCC, which are mediated by novel regulatory mechanisms involved in serine/threonine kinase pathway-associated cell proliferation and caspase activation pathway-associated apoptosis.
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Antineoplásicos , Carcinoma Hepatocelular , Exossomos , Neoplasias Hepáticas , Animais , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Exossomos/metabolismo , Humanos , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Neoplasias Hepáticas/metabolismo , Camundongos , Modelos Animais , Serina/metabolismoRESUMO
Macrophages (MΦs) have the capability to sense chemotactic cues and to home tumors, therefore presenting a great approach to engineer these cells to deliver therapeutic agents to treat diseases. However, current cell-based drug delivery systems usually use commercial cell lines that may elicit an immune response when injected into a host animal. Furthermore, premature off-target drug release also remains an enormous challenge. Here, we isolated and differentiated MΦs from the spleens of BALB/c mice and developed dual-targeting MΦ-based microrobots, regulated by chemotaxis and an external magnetic field, and had a precise spatiotemporal controlled drug release at the tumor sites in response to the NIR laser irradiation. These microrobots were prepared by coloading citric acid (CA)-coated superparamagnetic nanoparticles (MNPs) and doxorubicin (DOX)-containing thermosensitive nanoliposomes (TSLPs) into the MΦs. CA-MNPs promoted a magnetic targeting function to the microrobots and also permitted photothermal heating in response to the NIR irradiation, triggering drug release from TSLPs. In vitro experiments showed that the microrobots effectively infiltrated tumors in 3D breast cancer tumor spheroids, particularly in the presence of the magnetic field, and effectively induced tumor cell death, further enhanced by the NIR laser irradiation. In vivo experiments confirmed that the application of the magnetic field and NIR laser could markedly inhibit the growth of tumors with a subtherapeutic dose of DOX and a single injection of the microrobots. In summary, the study proposes a strategy for the effective anticancer treatment using the developed microrobots.
Assuntos
Doxorrubicina , Nanopartículas , Animais , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Macrófagos , Camundongos , Camundongos Endogâmicos BALB C , FototerapiaRESUMO
The demethylation of histone lysine residues, one of the most important modifications in transcriptional regulation, is associated with various physiological states. KDM2B is a demethylase of histones H3K4, H3K36, and H3K79 and is associated with the repression of transcription. Here, we present a novel mechanism by which KDM2B demethylates serum response factor (SRF) K165 to negatively regulate muscle differentiation, which is counteracted by the histone methyltransferase SET7. We show that KDM2B inhibited skeletal muscle differentiation by inhibiting the transcription of SRF-dependent genes. Both KDM2B and SET7 regulated the balance of SRF K165 methylation. SRF K165 methylation was required for the transcriptional activation of SRF and for the promoter occupancy of SRF-dependent genes. SET7 inhibitors blocked muscle cell differentiation. Taken together, these data indicate that SRF is a nonhistone target of KDM2B and that the methylation balance of SRF as maintained by KDM2B and SET7 plays an important role in muscle cell differentiation.
Assuntos
Diferenciação Celular , Proteínas F-Box/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Músculo Esquelético/metabolismo , Fator de Resposta Sérica/metabolismo , Sítios de Ligação , Biomarcadores , Diferenciação Celular/genética , Linhagem Celular , Células Cultivadas , Proteínas F-Box/genética , Regulação da Expressão Gênica , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Metilação , Modelos Biológicos , Músculo Esquelético/citologia , Mioblastos Esqueléticos/citologia , Mioblastos Esqueléticos/metabolismo , Ligação Proteica , Elementos de Resposta , Transcrição GênicaRESUMO
We described a magnetic chitosan microscaffold tailored for applications requiring high biocompatibility, biodegradability, and monitoring by real-time imaging. Such magnetic microscaffolds exhibit adjustable pores and sizes depending on the target application and provide various functions such as magnetic actuation and enhanced cell adhesion using biomaterial-based magnetic particles. Subsequently, we fabricated the magnetic chitosan microscaffolds with optimized shape and pore properties to specific target diseases. As a versatile tool, the capability of the developed microscaffold was demonstrated through in vitro laboratory tasks and in vivo therapeutic applications for liver cancer therapy and knee cartilage regeneration. We anticipate that the optimal design and fabrication of the presented microscaffold will advance the technology of biopolymer-based microscaffolds and micro/nanorobots.
Assuntos
Materiais Biocompatíveis , Quitosana , CartilagemRESUMO
Although great efforts have been undertaken to develop a nanoparticle-based drug delivery system (DDS) for the treatment of solid tumors, the therapeutic outcomes are still limited. Immune cells, which possess an intrinsic ability to phagocytose nanoparticles and are recruited by tumors, can be exploited to deliver nanotherapeutics deep inside the tumors. Photothermal therapy using near-infrared light is a promising noninvasive approach for solid tumor ablation, especially when combined with chemotherapy. In this study, we design and evaluate a macrophage-based, multiple nanotherapeutics DDS, involving the phagocytosis by macrophages of both small-sized gold nanorods and anticancer drug-containing nanoliposomes. The aim is to treat solid tumors, utilizing the tumor-infiltrating properties of macrophages with synergistic photothermal-chemotherapy. Using a 3D cancer spheroid as an in vitro solid tumor model, we show that tumor penetration and coverage of the nanoparticles are both markedly enhanced when the macrophages are used. In addition, in vivo experiments involving both local and systemic administrations in breast tumor-bearing mice demonstrate that the proposed DDS can effectively target and kill the tumors, especially when the synergistic therapy is used. Consequently, this immune cell-based theranostic strategy may represent a potentially important advancement in the treatment of solid tumors.
Assuntos
Antineoplásicos/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Doxorrubicina/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Macrófagos/imunologia , Fotoquimioterapia , Animais , Antineoplásicos/química , Neoplasias da Mama/imunologia , Linhagem Celular Tumoral , Doxorrubicina/química , Sistemas de Liberação de Medicamentos/instrumentação , Feminino , Ouro/administração & dosagem , Ouro/química , Humanos , Raios Infravermelhos , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/química , Fagocitose , Fármacos Fotossensibilizantes/administração & dosagem , Fármacos Fotossensibilizantes/químicaRESUMO
An effective nanoparticle-based drug delivery platform holds great promise for non-invasive cancer therapy. This study explores the breast tumor regression in vivo by synergistic photothermal-chemotherapy based on liposomal nanocomplex (folic acid-gold nanorods-anticancer drug-liposome). The proposed liposomal nanocomplex can enhance the tumor targeting by functionalizing folic acid (FA) molecules on the surface of liposome that encapsulates both gold nanorods (AuNRs) and the doxorubicin (DOX) to combine the photothermal therapy and the chemotherapy, respectively. Herein, 7-nm gold nanorods were fabricated and co-encapsulated with DOX into nanoliposomes functionalized with FA, with an average diameter of 154 nm, for active targeting to the cancer cells. The experimental results showed that the FA targeting liposomes had better cellular uptake than the non-targeting liposomes (AuNRs-DOX-LPs). Especially, upon 5 min exposure to near infrared (NIR) irradiation (808 nm) triggered DOX release could be achieved to 46.38% in 60 min at pH 5.5. In addition, in vitro cell proliferation assays indicated that, with synergistic photothermal-chemotherapy, the targeting liposomes could significantly enhance the toxicity towards the cancer cells with the IC50 value of 1.90 ± 0.12 µg mL-1. Furthermore, in vivo experiments on the breast tumor-bearing mice showed that the targeting liposomes could effectively inhibit the growth of the tumors using the combined strategy.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Receptores de Folato com Âncoras de GPI/metabolismo , Ácido Fólico/metabolismo , Terapia de Alvo Molecular/métodos , Neoplasias/terapia , Animais , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Terapia Combinada/métodos , Doxorrubicina/química , Doxorrubicina/metabolismo , Composição de Medicamentos/métodos , Liberação Controlada de Fármacos , Feminino , Ácido Fólico/química , Ouro/química , Concentração de Íons de Hidrogênio , Raios Infravermelhos , Injeções Subcutâneas , Lipossomos/administração & dosagem , Lipossomos/química , Terapia com Luz de Baixa Intensidade/métodos , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/patologia , Camundongos , Nanotubos/química , Ligação ProteicaRESUMO
After the publication of this article, the authors noticed an error in one of the grant numbers (2015R1A2A1A05001708) in the Acknowledgements section.
RESUMO
AIMS: Previously, we reported that phosphorylation of histone deacetylase 2 (HDAC2) and the resulting activation causes cardiac hypertrophy. Through further study of the specific binding partners of phosphorylated HDAC2 and their mechanism of regulation, we can better understand how cardiac hypertrophy develops. Thus, in the present study, we aimed to elucidate the function of one such binding partner, heat shock protein 70 (HSP70). METHODS AND RESULTS: Primary cultures of rat neonatal ventricular cardiomyocytes and H9c2 cardiomyoblasts were used for in vitro cellular experiments. HSP70 knockout (KO) mice and transgenic (Tg) mice that overexpress HSP70 in the heart were used for in vivo analysis. Peptide-precipitation and immunoprecipitation assay revealed that HSP70 preferentially binds to phosphorylated HDAC2 S394. Forced expression of HSP70 increased phosphorylation of HDAC2 S394 and its activation, but not that of S422/424, whereas knocking down of HSP70 reduced it. However, HSP70 failed to phosphorylate HDAC2 in the cell-free condition. Phosphorylation of HDAC2 S394 by casein kinase 2α1 enhanced the binding of HSP70 to HDAC2, whereas dephosphorylation induced by the catalytic subunit of protein phosphatase 2A (PP2CA) had the opposite effect. HSP70 prevented HDAC2 dephosphorylation by reducing the binding of HDAC2 to PP2CA. HSP70 KO mouse hearts failed to phosphorylate S394 HDAC2 in response to isoproterenol infusion, whereas Tg overexpression of HSP70 increased the phosphorylation and activation of HDAC2. 2-Phenylethynesulfonamide (PES), an HSP70 inhibitor, attenuated cardiac hypertrophy induced either by phenylephrine in neonatal ventricular cardiomyocytes or by aortic banding in mice. PES reduced HDAC2 S394 phosphorylation and its activation by interfering with the binding of HSP70 to HDAC2. CONCLUSION: These results demonstrate that HSP70 specifically binds to S394-phosphorylated HDAC2 and maintains its phosphorylation status, which results in HDAC2 activation and the development of cardiac hypertrophy. Inhibition of HSP70 has possible application as a therapeutic.
Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Histona Desacetilase 2/metabolismo , Hipertrofia Ventricular Esquerda/enzimologia , Miócitos Cardíacos/enzimologia , Função Ventricular Esquerda , Remodelação Ventricular , Animais , Sítios de Ligação , Linhagem Celular , Modelos Animais de Doenças , Ativação Enzimática , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Proteínas de Choque Térmico HSP70/deficiência , Proteínas de Choque Térmico HSP70/genética , Hipertrofia Ventricular Esquerda/genética , Hipertrofia Ventricular Esquerda/fisiopatologia , Hipertrofia Ventricular Esquerda/prevenção & controle , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Fosforilação , Ligação Proteica , Proteína Fosfatase 2/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Sulfonamidas/farmacologia , Função Ventricular Esquerda/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacosRESUMO
Cardiac hypertrophy occurs in response to increased hemodynamic demand and can progress to heart failure. Identifying the key regulators of this process is clinically important. Though it is thought that the phosphorylation of histone deacetylase (HDAC) 2 plays a crucial role in the development of pathological cardiac hypertrophy, the detailed mechanism by which this occurs remains unclear. Here, we performed immunoprecipitation and peptide pull-down assays to characterize the functional complex of HDAC2. Protein phosphatase (PP) 2 A was confirmed as a binding partner of HDAC2. PPP2CA, the catalytic subunit of PP2A, bound to HDAC2 and prevented its phosphorylation. Transient overexpression of PPP2CA specifically regulated both the phosphorylation of HDAC2 S394 and hypertrophy-associated HDAC2 activation. HDAC2 S394 phosphorylation was increased in a dose-dependent manner by PP2A inhibitors. Hypertrophic stresses, such as phenylephrine in vitro or pressure overload in vivo, caused PPP2CA to dissociate from HDAC2. Forced expression of PPP2CA negatively regulated the hypertrophic response, but PP2A inhibitors provoked hypertrophy. Adenoviral delivery of a phosphomimic HDAC2 mutant, adenovirus HDAC2 S394E, successfully blocked the anti-hypertrophic effect of adenovirus-PPP2CA, implicating HDAC2 S394 phosphorylation as a critical event for the anti-hypertrophic response. PPP2CA transgenic mice were protected against isoproterenol-induced cardiac hypertrophy and subsequent cardiac fibrosis, whereas simultaneous expression of HDAC2 S394E in the heart did induce hypertrophy. Taken together, our results suggest that PP2A is a critical regulator of HDAC2 activity and pathological cardiac hypertrophy and is a promising target for future therapeutic interventions.
Assuntos
Cardiomegalia/metabolismo , Histona Desacetilase 2/metabolismo , Miócitos Cardíacos/metabolismo , Proteína Fosfatase 2/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Histona Desacetilase 2/genética , Camundongos , Fosforilação , Proteína Fosfatase 2/antagonistas & inibidores , Ratos , Ratos Sprague-DawleyRESUMO
The BMB Reports would like to correct in the ACKNOWLEDGEMENTS of BMB Rep. 45(12), 713-718 titled "Ganglioside GM1 influences the proliferation rate of mouse induced pluripotent stem cells".
RESUMO
Vascular calcification (VC) is often associated with cardiovascular and metabolic diseases. However, the molecular mechanisms linking VC to these diseases have yet to be elucidated. Here we report that MDM2-induced ubiquitination of histone deacetylase 1 (HDAC1) mediates VC. Loss of HDAC1 activity via either chemical inhibitor or genetic ablation enhances VC. HDAC1 protein, but not mRNA, is reduced in cell and animal calcification models and in human calcified coronary artery. Under calcification-inducing conditions, proteasomal degradation of HDAC1 precedes VC and it is mediated by MDM2 E3 ubiquitin ligase that initiates HDAC1 K74 ubiquitination. Overexpression of MDM2 enhances VC, whereas loss of MDM2 blunts it. Decoy peptide spanning HDAC1 K74 and RG 7112, an MDM2 inhibitor, prevent VC in vivo and in vitro. These results uncover a previously unappreciated ubiquitination pathway and suggest MDM2-mediated HDAC1 ubiquitination as a new therapeutic target in VC.
Assuntos
Histona Desacetilase 1/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Calcificação Vascular/metabolismo , Animais , Cálcio , Regulação da Expressão Gênica , Histona Desacetilase 1/genética , Humanos , Masculino , Camundongos , Músculo Liso Vascular/citologia , Proteínas Proto-Oncogênicas c-mdm2/genética , Ratos , UbiquitinaçãoRESUMO
Skeletal muscle atrophy results from the net loss of muscular proteins and organelles and is caused by pathologic conditions such as nerve injury, immobilization, cancer, and other metabolic diseases. Recently, ubiquitination-mediated degradation of skeletal-muscle-specific transcription factors was shown to be involved in muscle atrophy, although the mechanisms have yet to be defined. Here we report that ret finger protein (RFP), also known as TRIM27, works as an E3 ligase in Pax7-induced degradation of MyoD. Muscle injury induced by sciatic nerve transection up-regulated RFP and RFP physically interacted with both Pax7 and MyoD. RFP and Pax7 synergistically reduced the protein amounts of MyoD but not the mRNA. RFP-induced reduction of MyoD protein was blocked by proteasome inhibitors. The Pax7-induced reduction MyoD was attenuated by RFP siRNA and by MG132, a proteasome inhibitor. RFPΔR, an RFP construct that lacks the RING domain, failed to reduce MyoD amounts. RFP ubiquitinated MyoD, but RFPΔR failed to do so. Forced expression of RFP, but not RFPΔR, enhanced Pax7-induced ubiquitination of MyoD, whereas RFP siRNA blocked the ubiquitination. Sciatic nerve injury-induced muscle atrophy as well the reduction in MyoD was attenuated in RFP knockout mice. Taken together, our results show that RFP works as a novel E3 ligase in the Pax7-mediated degradation of MyoD in response to skeletal muscle atrophy.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Atrofia Muscular/patologia , Proteína MyoD/metabolismo , Proteínas Nucleares/metabolismo , Fator de Transcrição PAX7/metabolismo , Animais , Linhagem Celular , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Células HEK293 , Humanos , Leupeptinas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/patologia , Músculo Esquelético/fisiologia , Atrofia Muscular/metabolismo , Proteína MyoD/química , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Fator de Transcrição PAX7/química , Inibidores de Proteases/farmacologia , Ligação Proteica , Proteólise/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Regeneração , Ubiquitina-Proteína Ligases , Ubiquitinação/efeitos dos fármacosRESUMO
Gangliosides are ubiquitous components of the membranes of mammalian cells that are thought to play important roles in various cell functions such as cell-cell interaction, cell adhesion, cell differentiation, growth control, and signaling. However, the role that gangliosides play in the immune rejection response after xenotransplantation is not yet clearly understood. In this study, the regulatory effects of human leukocytes on ganglioside expression in primary cultured micro-pig aortic endothelial cells (PAECs) were investigated. To determine the impact of human leukocytes on the expression of gangliosides in PAECs, we performed high-performance thin layer chromatography (HPTLC) in PAECs incubated with FBS, FBS containing human leukocytes, human serum containing human leukocytes, and FBS containing TNF-α. Both HPTLC and immunohistochemistry analyses revealed that PAECs incubated with FBS predominantly express the gangliosides GM3, GM1, and GD3. However, the expression of GM1 significantly decreased in PAECs incubated for 5 h with TNF-α (10 ng/mL), 10% human serum containing human leukocytes, and 10% FBS containing human leukocytes. Taken together, these results suggest that human leukocytes induced changes in the expression profile of ganglioside GM1 similar to those seen upon treatment of PAECs with TNF-α. This finding may be relevant for designing future therapeutic strategies intended to prolong xenograft survival.
RESUMO
Gangliosides play important roles in the control of several biological processes, including proliferation and transmembrane signaling. In this study, we demonstrate the effect of ganglioside GM1 on the proliferation of mouse induced pluripotent stem cells (miPSCs). The proliferation rate of miPSCs was lower than in mouse embryonic stem cells (mESCs). Fluorescence activated cell sorting analysis showed that the percentage of cells in the G2/M phase in miPSCs was lower than that in mESCs. GM1 was expressed in mESCs, but not miPSCs. To confirm the role of GM1 in miPSC proliferation, miPSCs were treated with GM1. GM1-treated miPSCs exhibited increased cell proliferation and a larger number of cells in the G2/M phase. Furthermore, phosphorylation of mitogen-activated protein kinases was increased in GM1- treated miPSCs.