RESUMO
Prominin-1 (Prom1) is a major cell surface marker of cancer stem cells, but its physiological functions in the liver have not been elucidated. We analyzed the levels of mRNA transcripts in serum-starved primary WT (Prom1+/+ ) and KO (Prom1-/- ) mouse hepatocytes using RNA sequencing (RNA-seq) data, and found that CREB target genes were downregulated. This initial observation led us to determine that Prom1 deficiency inhibited cAMP response element-binding protein (CREB) activation and gluconeogenesis, but not cyclic AMP (cAMP) accumulation, in glucagon-, epinephrine-, or forskolin-treated liver tissues and primary hepatocytes, and mitigated glucagon-induced hyperglycemia. Because Prom1 interacted with radixin, Prom1 deficiency prevented radixin from localizing to the plasma membrane. Moreover, systemic adenoviral knockdown of radixin inhibited CREB activation and gluconeogenesis in glucagon-treated liver tissues and primary hepatocytes, and mitigated glucagon-elicited hyperglycemia. Based on these results, we conclude that Prom1 regulates hepatic PKA signaling via radixin functioning as an A kinase-anchored protein (AKAP).
Assuntos
Gluconeogênese , Glucose , Antígeno AC133/genética , Antígeno AC133/metabolismo , Animais , Proteínas do Citoesqueleto , Gluconeogênese/genética , Glucose/metabolismo , Hepatócitos , Fígado/metabolismo , Proteínas de Membrana , CamundongosRESUMO
Mitsugumin 53 (MG53) is an E3 ligase that interacts with and ubiquitinates insulin receptor substrate-1 (IRS-1) in skeletal muscle; thus, an MG53-IRS-1 interaction disruptor (MID), which potentially sensitizes insulin signaling with an elevated level of IRS-1 in skeletal muscle, is an excellent candidate for treating insulin resistance. To screen for an MID, we developed a bimolecular luminescence complementation system using an N-terminal luciferase fragment fused with IRS-1 and a C-terminal luciferase fragment fused with an MG53 C14A mutant that binds to IRS-1 but does not have E3 ligase activity. An MID, which was discovered using the bimolecular luminescence complementation system, disrupted the molecular association of MG53 with IRS-1, thus abolishing MG53-mediated IRS-1 ubiquitination and degradation. Thus, the MID sensitized insulin signaling and increased insulin-elicited glucose uptake with an elevated level of IRS-1 in C2C12 myotubes. These data indicate that this MID holds promise as a drug candidate for treating insulin resistance.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas Substratos do Receptor de Insulina/metabolismo , Insulina/metabolismo , Proteínas dos Microtúbulos/metabolismo , Músculo Esquelético/metabolismo , Proteínas Nucleares/metabolismo , Mapas de Interação de Proteínas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Transcrição/metabolismo , Células Cultivadas , Humanos , Resistência à Insulina , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Proteólise , Transdução de Sinais/efeitos dos fármacos , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
The aim of the present study was to examine the inhibitory roles and mechanisms of hirsutenone (HTN) in the regulation of osteoclastogenesis. Gene levels were compared to assure the effects of HTN on osteoclastogenesis in mouse splenocytes/CD4+ T cells, mouse macrophage-like cell line RAW264.7 (preosteoclast), MG63 (osteoblast), and RPMI1788 (B cell) cells. The mechanism by which HTN regulates the degradation of tumor necrosis factor receptor-associated factor 6 (TRAF6) and inhibits inhibitor of kappaB (IκB) and nuclear factor-kappaB (NF-κB) signaling was examined by Western blotting and luciferase reporter assays. Our results demonstrated that HTN effectively downregulated the expression of interferon γ (IFNγ), interleukin-22 (IL-22), IL-1ß, and tartrate-resistant acid phosphatase (TRAP) in splenocyte-/CD4+-RAW264.7 co-culture system. Moreover, receptor activator of nuclear factor-κB ligand (RANKL) and CD25 expression were also significantly inhibited in MG63 and CD4+ single culture system, suggesting an additional independent effect of HTN on osteoclastogenesis. Notably, TRAF6 was markedly degraded along with a decrease in nuclear factor of activated T-cells (NFATc) and NF-κB activities in RAW264.7 cells. Finally, we concluded that HTN directly or indirectly inhibits osteoclastogenesis via the inhibition of NF-κB signaling by promoting TRAF6 degradation, and plays a crucial role in suppressing the expression of RANKL and cytokines expressed in IFNγ-producing T-helper 1 (Th1) cells. These findings suggest that HTN may be a promising therapeutic candidate for diseases resulting from bone loss.
Assuntos
Catecóis/farmacologia , Diarileptanoides/farmacologia , Interferon gama/antagonistas & inibidores , NF-kappa B/antagonistas & inibidores , Osteoclastos/efeitos dos fármacos , Células Th1/efeitos dos fármacos , Alnus/química , Animais , Linfócitos T CD4-Positivos/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Citocinas/antagonistas & inibidores , Citocinas/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Osteogênese/efeitos dos fármacos , Casca de Planta/química , Ligante RANK/genética , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos , Baço/química , Baço/citologia , Células-Tronco/efeitos dos fármacos , Fosfatase Ácida Resistente a Tartarato/biossíntese , Fosfatase Ácida Resistente a Tartarato/genéticaRESUMO
Since detergent-resistant lipid rafts play important roles in multidrug resistance (MDR), their comprehensive proteomics could provide new insights to understand the underlying molecular mechanism of MDR in cancer cells. In the present work, lipid rafts were isolated from MCF-7 and adriamycin-resistant MCF-7/ADR cells and their proteomes were analyzed by label-free quantitative proteomics. Polymerase I and transcript release factor (PTRF)/cavin-1 was measured to be upregulated along with multidrug-resistant P-glycoprotein, caveolin-1, and serum deprivation protein response/cavin-2 in the lipid rafts of MCF-7/ADR cells. PTRF knockdown led to reduction in the amount of lipid rafts on the surface of MCF7/ADR cells as determined by cellular staining with lipid raft-specific dyes such as S-laurdan2 and FITC-conjugated cholera toxin B. PTRF knockdown also reduced MDR in MCF-7/ADR cells. These data indicate that PTRF is necessary for MDR in cancer cells via the fortification of lipid rafts.
Assuntos
Neoplasias da Mama/genética , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Ligação a RNA/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antibióticos Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cavéolas/efeitos dos fármacos , Cavéolas/metabolismo , Caveolina 1/genética , Caveolina 1/metabolismo , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Corantes Fluorescentes , Técnicas de Silenciamento de Genes , Humanos , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/genética , Microdomínios da Membrana/metabolismo , Proteínas de Ligação a Fosfato , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/metabolismoRESUMO
PURPOSE: To compare the fracture load and failure mode of the monolithic lithium disilicate crown (e.max group) and 2 types of veneered zirconia crowns, hand layer (ZV group) and heat pressed (ZP group), as a posterior implant-supported restoration. METHODS: A total of 24 all-ceramic crowns for molar tooth were fabricated using the computer-aided design/computer-assisted manufacture (CAD/CAM) system. The e.max group crowns and zirconia copings for ZV and ZP groups were fabricated using a Cerec milling unit. The ZV group was fabricated using a hand-layer veneering method, and the ZP group using a heat-pressing method. All crowns were luted to the abutments, which were connected to implant fixtures, using resin cement. Fracture load was measured using the universal testing machine, and the fracture surface was evaluated by scanning electron microscopy. RESULTS: The ZP group showed significantly higher fracture load (5229.3 N) compared with the e.max group (3852.1 N) and ZV group (3100.3 N). All fractures in the ZV group occurred in the veneered layer. CONCLUSION: Monolithic CAD/CAM lithium disilicate crowns are applicable to posterior implant-supported restorations because the fracture load was higher than the average occlusal force.
Assuntos
Desenho Assistido por Computador , Coroas , Porcelana Dentária/química , Prótese Dentária Fixada por Implante , Falha de Restauração Dentária , Facetas Dentárias , Zircônio/química , Cimentação/métodos , Colagem Dentária , Implantes Dentários , Planejamento de Prótese Dentária , Análise do Estresse Dentário/instrumentação , Humanos , Teste de Materiais , Microscopia Eletrônica de Varredura , Dente Molar , Cimentos de Resina/química , Estresse Mecânico , Propriedades de SuperfícieRESUMO
PURPOSE: The purpose of this study was to evaluate the success rate of implants and vertical bone gain of edentulous posterior maxilla using ultrasonic piezoelectric vibration and hydraulic pressure, namely the hydrodynamic piezoelectric internal sinus elevation (HPISE) technique through a crestal approach. MATERIALS AND METHODS: A total of 250 maxillary sinuses were augmented using HPISE and 353 implants (averaging 11.8 mm in length and 4.5 mm in diameter), with 12 different systems, were placed simultaneously with or without additional bone grafting. Plain radiograms and cone beam computed tomograms were taken in all patients to evaluate sinus augmentation. RESULTS: Membrane perforation was recorded at 10 of the 353 implant sites. The perforation rate was 2.83%. The total success rate of implantation was 97.2% after an average of 69.3 weeks of loading. CONCLUSION: The crestally approached sinus augmentation using ultrasonic piezoelectric vibration and hydraulic pressure is an additional method of maxillary sinus augmentation.
Assuntos
Piezocirurgia/métodos , Levantamento do Assoalho do Seio Maxilar/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Substitutos Ósseos/uso terapêutico , Transplante Ósseo/métodos , Tomografia Computadorizada de Feixe Cônico/métodos , Implantação Dentária Endóssea/métodos , Implantes Dentários , Planejamento de Prótese Dentária , Feminino , Seguimentos , Humanos , Complicações Intraoperatórias , Arcada Edêntula/patologia , Arcada Edêntula/cirurgia , Arcada Parcialmente Edêntula/patologia , Arcada Parcialmente Edêntula/cirurgia , Masculino , Maxila/patologia , Maxila/cirurgia , Pessoa de Meia-Idade , Procedimentos Cirúrgicos Minimamente Invasivos , Mucosa Nasal/lesões , Osseointegração/fisiologia , Osteogênese/fisiologia , Pressão , Estudos Retrospectivos , Resultado do Tratamento , Vibração , Adulto JovemRESUMO
PURPOSE: The aim of this retrospective analysis was to report on the clinical outcome of immediate provisionalization using one-piece narrow-diameter implants. MATERIALS AND METHODS: The dental records of patients who received narrow implants were reviewed. Narrow-diameter (3.0-mm) one-piece implants were used to support restorations of missing maxillary lateral incisors and mandibular incisors. All implants were placed in a one-stage procedure according to the protocol recommended by the manufacturer, with immediate placement of provisional restorations. Following an average healing period of 3 months in the mandible and 5 months in the maxilla, the definitive prostheses were fabricated. The survival rate of the implants was analyzed, and radiographic evaluation was performed. RESULTS. Thirty-six patients (20 men and 16 women), aged from 42 to 72 years (average age of 53 years), were treated with 62 one-piece narrow implants. A success rate of 100% was observed over a period up to 33 months (mean, 23 ± 4.3 months). Among these, 8 implants were placed in maxillary lateral incisor positions and 54 implants were placed in mandibular incisor areas. Forty-four implants supported fixed partial prostheses, and 18 implants supported single crowns. The majority of the implants were 15 mm in length. Mean marginal bone loss at the 12-month follow-up visit was 0.53 ± 0.37 mm (range, 0 to 1.4 mm). CONCLUSIONS: The results obtained in this retrospective analysis suggest that the one-piece narrow-diameter implant can predictably restore missing maxillary lateral incisors and mandibular incisor with narrow interdental spaces and labiolingual widths.
Assuntos
Implantes Dentários para Um Único Dente , Prótese Dentária Fixada por Implante , Carga Imediata em Implante Dentário/métodos , Adulto , Idoso , Perda do Osso Alveolar/etiologia , Coroas , Planejamento de Prótese Dentária , Restauração Dentária Temporária , Prótese Parcial Fixa , Prótese Parcial Temporária , Feminino , Seguimentos , Humanos , Incisivo , Masculino , Mandíbula/cirurgia , Maxila/cirurgia , Pessoa de Meia-Idade , Estudos Retrospectivos , Análise de Sobrevida , Resultado do Tratamento , Cicatrização/fisiologiaRESUMO
Mitsugumin 53 (MG53) is an E3 ligase that induces insulin receptor substrate-1 (IRS-1) ubiquitination and degradation in skeletal muscle. We previously demonstrated that the pharmaceutical disruption of the MG53-IRS-1 interaction improves insulin sensitivity by abrogating IRS-1 ubiquitination and increasing IRS-1 levels in C2C12 myotubes. Here, we developed a novel MG53-IRS-1 interaction disruptor (MID-00935) that ameliorates insulin resistance in diet-induced obese (DIO) mice. MID-00935 disrupted the molecular interaction of MG53 and IRS-1, abrogated MG53-induced IRS-1 ubiquitination and degradation and improved insulin signaling in C2C12 myotubes. Oral administration of MID-00935 increased insulin-induced IRS-1, Akt, and Erk phosphorylation via increasing IRS-1 levels in the skeletal muscle of DIO mice. In DIO mice, MID-00935 treatment lowered fasting blood glucose levels and improved glucose disposal in glucose and insulin tolerance tests. These results suggest that MID-00935 may be a potential muscle-targeting drug candidate for treating insulin resistance.
Assuntos
Proteínas de Transporte/antagonistas & inibidores , Proteínas Substratos do Receptor de Insulina/antagonistas & inibidores , Resistência à Insulina , Insulina/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Obesidade/metabolismo , Animais , Proteínas de Transporte/metabolismo , Células HEK293 , Humanos , Proteínas Substratos do Receptor de Insulina/metabolismo , Proteínas de Membrana , Fibras Musculares Esqueléticas/patologia , Obesidade/induzido quimicamente , Obesidade/tratamento farmacológico , Obesidade/patologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Paradoxical observations have been made regarding the role of caveolin-1 (Cav-1) during cellular senescence. For example, caveolin-1 deficiency prevents reactive oxygen species-induced cellular senescence despite mitochondrial dysfunction, which leads to senescence. To resolve this paradox, we re-addressed the role of caveolin-1 in cellular senescence in human diploid fibroblasts, A549, HCT116, and Cav-1-/- mouse embryonic fibroblasts. Cav-1 deficiency (knockout or knockdown) induced cellular senescence via a p53-p21-dependent pathway, downregulating the expression level of the cardiolipin biosynthesis enzymes and then reducing the content of cardiolipin, a critical lipid for mitochondrial respiration. Our results showed that Cav-1 deficiency decreased mitochondrial respiration, reduced the activity of oxidative phosphorylation complex I (CI), inactivated SIRT1, and decreased the NAD+ /NADH ratio. From these results, we concluded that Cav-1 deficiency induces premature senescence via mitochondrial dysfunction and silent information regulator 2 homologue 1 (SIRT1) inactivation.
Assuntos
Caveolina 1/genética , Senescência Celular/genética , Fibroblastos/metabolismo , Mitocôndrias/metabolismo , Sirtuína 1/genética , Células A549 , Animais , Cardiolipinas/biossíntese , Caveolina 1/deficiência , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Embrião de Mamíferos , Fibroblastos/patologia , Regulação da Expressão Gênica , Células HCT116 , Humanos , Camundongos , Mitocôndrias/patologia , NAD/metabolismo , Fosforilação Oxidativa , Cultura Primária de Células , Transdução de Sinais , Sirtuína 1/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
BCL-2 interacting cell death suppressor (BIS), which is ubiquitously expressed, has important roles in various cellular processes, such as apoptosis, the cellular stress response, migration and invasion and protein quality control. In particular, BIS is highly expressed in skeletal and cardiac muscles, and BIS gene mutations result in human myopathy. In this study, we show that mRNA and protein levels of BIS were markedly increased during skeletal myogenesis in C2C12 cells and mouse satellite cells. BIS knockdown did not prevent the early stage of skeletal myogenesis, but did induce muscle atrophy and a decrease in the diameter of myotubes. BIS knockdown significantly suppressed the expression level of myosin heavy chain (MyHC) without changing the expression levels of myogenic marker proteins, such as Mgn, Cav-3 and MG53. In addition, BIS endogenously interacted with MyHC, and BIS knockdown induced MyHC ubiquitination and degradation. From these data, we conclude that molecular association of MyHC and BIS is necessary for MyHC stabilization in skeletal muscle.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Músculo Esquelético/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas Reguladoras de Apoptose/genética , Linhagem Celular , Células Cultivadas , Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Desenvolvimento Muscular/genética , Ligação Proteica , Estabilidade Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Satélites de Músculo Esquelético/metabolismoRESUMO
It is well recognized that regulating the hair follicle cycle in association with Wnt signaling is one of the most interesting targets for promoting hair regrowth. In this study, we examined whether selected herbal medicines processed by decoction and fermentation promote hair growth by upregulating the number and size of hair follicles and Wnt signaling, including activation of ß-catenin and Akt in telogen-synchronized C57BL/6N mice. The results revealed that the fermented extract after decoction (FDE) more effectively promoted hair growth than that of a nonfermented extract (DE). Notably, FDE effectively enhanced formation of hair follicles with clearer differentiation between the inner and outer root sheath, which is observed during the anagen phase. Mechanistic evidence was found for increased ß-catenin and Akt phosphorylation levels in dorsal skin tissue along with elevated expression of hair regrowth-related genes, such as Wnt3/10a/10b, Lef1, and fibroblast growth factor 7. In conclusion, our findings suggest that FDE plays an important role in regulating the hair cycle by increasing expression of hair regrowth-related genes and activating downstream Wnt signaling targets.
RESUMO
We previously demonstrated that cell-surface gC1qR is a key regulator of lamellipodia formation and cancer metastasis. Here, we screened a monoclonal mouse antibody against gC1qR to prevent cell migration by neutralizing cell-surface gC1qR. The anti-gC1qR antibody prevented growth factor-stimulated lamellipodia formation, cell migration and focal adhesion kinase activation by inactivating receptor tyrosine kinases (RTKs) in various cancer cells such as A549, MDA-MB-231, MCF7 and HeLa cells. The antibody neutralization of cell-surface gC1qR also inhibited angiogenesis because the anti-gC1qR antibody prevented growth factor-stimulated RTK activation, lamellipodia formation, cell migration and tube formation in HUVEC. In addition, we found that A549 tumorigenesis was reduced in a xenograft mouse model by following the administration of the anti-gC1qR antibody. With these data, we can conclude that the antibody neutralization of cell-surface gC1qR could be a good therapeutic strategy for cancer treatment.
Assuntos
Anticorpos/farmacologia , Proteínas de Transporte/antagonistas & inibidores , Membrana Celular/metabolismo , Transformação Celular Neoplásica/metabolismo , Proteínas Mitocondriais/antagonistas & inibidores , Pseudópodes/efeitos dos fármacos , Animais , Carcinogênese , Proteínas de Transporte/imunologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Mitocondriais/imunologia , Proteínas Mitocondriais/metabolismo , Fosforilação , Transdução de SinaisRESUMO
Mitsugumin 53 (MG53) negatively regulates skeletal myogenesis by targeting insulin receptor substrate 1 (IRS-1). Here, we show that MG53 is an ubiquitin E3 ligase that induces IRS-1 ubiquitination with the help of an E2-conjugating enzyme, UBE2H. Molecular manipulations that disrupt the E3-ligase function of MG53 abolish IRS-1 ubiquitination and enhance skeletal myogenesis. Skeletal muscles derived from the MG53-/- mice show an elevated IRS-1 level with enhanced insulin signalling, which protects the MG53-/- mice from developing insulin resistance when challenged with a high-fat/high-sucrose diet. Muscle samples derived from human diabetic patients and mice with insulin resistance show normal expression of MG53, indicating that altered MG53 expression does not serve as a causative factor for the development of metabolic disorders. Thus, therapeutic interventions that target the interaction between MG53 and IRS-1 may be a novel approach for the treatment of metabolic diseases that are associated with insulin resistance.