RESUMO
There are still gaps in our understanding of the complex processes by which p53 suppresses tumorigenesis. Here we describe a novel role for p53 in suppressing the mevalonate pathway, which is responsible for biosynthesis of cholesterol and nonsterol isoprenoids. p53 blocks activation of SREBP-2, the master transcriptional regulator of this pathway, by transcriptionally inducing the ABCA1 cholesterol transporter gene. A mouse model of liver cancer reveals that downregulation of mevalonate pathway gene expression by p53 occurs in premalignant hepatocytes, when p53 is needed to actively suppress tumorigenesis. Furthermore, pharmacological or RNAi inhibition of the mevalonate pathway restricts the development of murine hepatocellular carcinomas driven by p53 loss. Like p53 loss, ablation of ABCA1 promotes murine liver tumorigenesis and is associated with increased SREBP-2 maturation. Our findings demonstrate that repression of the mevalonate pathway is a crucial component of p53-mediated liver tumor suppression and outline the mechanism by which this occurs.
Assuntos
Ácido Mevalônico/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Animais , Linhagem Celular , Colesterol/metabolismo , Feminino , Genes Supressores de Tumor , Células HCT116 , Hepatócitos/metabolismo , Humanos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/genética , Regiões Promotoras Genéticas , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Terpenos/metabolismoRESUMO
Missense mutations in the p53 tumor suppressor inactivate its antiproliferative properties but can also promote metastasis through a gain-of-function activity. We show that sustained expression of mutant p53 is required to maintain the prometastatic phenotype of a murine model of pancreatic cancer, a highly metastatic disease that frequently displays p53 mutations. Transcriptional profiling and functional screening identified the platelet-derived growth factor receptor b (PDGFRb) as both necessary and sufficient to mediate these effects. Mutant p53 induced PDGFRb through a cell-autonomous mechanism involving inhibition of a p73/NF-Y complex that represses PDGFRb expression in p53-deficient, noninvasive cells. Blocking PDGFRb signaling by RNA interference or by small molecule inhibitors prevented pancreatic cancer cell invasion in vitro and metastasis formation in vivo. Finally, high PDGFRb expression correlates with poor disease-free survival in pancreatic, colon, and ovarian cancer patients, implicating PDGFRb as a prognostic marker and possible target for attenuating metastasis in p53 mutant tumors.
Assuntos
Carcinoma Ductal Pancreático/metabolismo , Metástase Neoplásica , Neoplasias Pancreáticas/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Carcinoma Ductal Pancreático/patologia , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Humanos , Camundongos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteína Supressora de Tumor p53/genéticaRESUMO
MDM2 and MDMX, negative regulators of the tumor suppressor p53, can work separately and as a heteromeric complex to restrain p53's functions. MDM2 also has pro-oncogenic roles in cells, tissues, and animals that are independent of p53. There is less information available about p53-independent roles of MDMX or the MDM2-MDMX complex. We found that MDM2 and MDMX facilitate ferroptosis in cells with or without p53. Using small molecules, RNA interference reagents, and mutant forms of MDMX, we found that MDM2 and MDMX, likely working in part as a complex, normally facilitate ferroptotic death. We observed that MDM2 and MDMX alter the lipid profile of cells to favor ferroptosis. Inhibition of MDM2 or MDMX leads to increased levels of FSP1 protein and a consequent increase in the levels of coenzyme Q10, an endogenous lipophilic antioxidant. This suggests that MDM2 and MDMX normally prevent cells from mounting an adequate defense against lipid peroxidation and thereby promote ferroptosis. Moreover, we found that PPARα activity is essential for MDM2 and MDMX to promote ferroptosis, suggesting that the MDM2-MDMX complex regulates lipids through altering PPARα activity. These findings reveal the complexity of cellular responses to MDM2 and MDMX and suggest that MDM2-MDMX inhibition might be useful for preventing degenerative diseases involving ferroptosis. Furthermore, they suggest that MDM2/MDMX amplification may predict sensitivity of some cancers to ferroptosis inducers.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ferroptose/genética , Metabolismo dos Lipídeos/genética , PPAR alfa/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Proteínas de Ciclo Celular/genética , Glioblastoma/fisiopatologia , Células HCT116 , Humanos , Mutação , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/genética , Interferência de RNA , Ratos , Proteína Supressora de Tumor p53/metabolismo , Ubiquinona/análogos & derivados , Ubiquinona/metabolismoRESUMO
p53 is a frequent target for mutation in human tumors, and mutant p53 proteins can actively contribute to tumorigenesis. We employed a three-dimensional culture model in which nonmalignant breast epithelial cells form spheroids reminiscent of acinar structures found in vivo, whereas breast cancer cells display highly disorganized morphology. We found that mutant p53 depletion is sufficient to phenotypically revert breast cancer cells to a more acinar-like morphology. Genome-wide expression analysis identified the mevalonate pathway as significantly upregulated by mutant p53. Statins and sterol biosynthesis intermediates reveal that this pathway is both necessary and sufficient for the phenotypic effects of mutant p53 on breast tissue architecture. Mutant p53 associates with sterol gene promoters at least partly via SREBP transcription factors. Finally, p53 mutation correlates with highly expressed sterol biosynthesis genes in human breast tumors. These findings implicate the mevalonate pathway as a therapeutic target for tumors bearing mutations in p53.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Ácido Mevalônico/metabolismo , Proteína Supressora de Tumor p53/genética , Linhagem Celular Tumoral , Feminino , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Redes e Vias Metabólicas/efeitos dos fármacos , Mutação , Prenilação , Regiões Promotoras Genéticas , Sinvastatina/farmacologia , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismoRESUMO
The c.453delC (p.Thr152Profs*14) frameshift mutation in KCNH2 is associated with an elevated risk of Long QT syndrome (LQTS) and fatal arrhythmia. Nevertheless, the loss-of-function mechanism underlying this mutation remains unexplored and necessitates an understanding of electrophysiology. To gain insight into the mechanism of the LQT phenotype, we conducted whole-cell patch-clamp and immunoblot assays, utilizing both a heterologous expression system and patient-derived induced pluripotent stem cell-cardiomyocytes (iPSC-CMs) with 453delC-KCNH2. We also explored the site of translational reinitiation by employing LC/MS mass spectrometry. Contrary to the previous assumption of early termination of translation, the findings of this study indicate that the 453delC-KCNH2 leads to an N-terminally truncated hERG channel, a potential from a non-canonical start codon, with diminished expression and reduced current (IhERG). The co-expression with wildtype KCNH2 produced heteromeric hERG channel with mild dominant-negative effect. Additionally, the heterozygote patient-derived iPSC-CMs exhibited prolonged action potential duration and reduced IhERG, which was ameliorated with the use of a hERG activator, PD-118057. The results of our study offer novel insights into the mechanisms involved in congenital LQTS associated with the 453delC mutation of KCNH2. The mutant results in the formation of less functional N-terminal-truncated channels with reduced amount of membrane expression. A hERG activator is capable of correcting abnormalities in both the heterologous expression system and patient-derived iPSC-CMs.
Assuntos
Células-Tronco Pluripotentes Induzidas , Síndrome do QT Longo , Humanos , Miócitos Cardíacos/metabolismo , Mutação da Fase de Leitura , Células-Tronco Pluripotentes Induzidas/metabolismo , Canais de Potássio Éter-A-Go-Go/genética , Canal de Potássio ERG1/genética , Canal de Potássio ERG1/metabolismo , Heterozigoto , Mutação , Síndrome do QT Longo/genética , Síndrome do QT Longo/metabolismoRESUMO
Benzyl butyl phthalate (BBP) is a widely used plasticizer that poses various potential health hazards. Although BBP has been extensively studied, the direct mechanism underlying its toxicity in male germ cells remains unclear. Therefore, we investigated BBP-mediated male germ cell toxicity in GC-1 spermatogonia (spg), a differentiated mouse male germ cell line. This study investigated the impact of BBP on reactive oxygen species (ROS) generation, apoptosis, and autophagy regulation, as well as potential protective measures against BBP-induced toxicity. A marked dose-dependent decrease in GC-1 spg cell proliferation was observed following treatment with BBP at 12.5⯵M. Exposure to 50⯵M BBP, approximating the IC50 of 53.9⯵M, markedly increased cellular ROS generation and instigated apoptosis, as evidenced by augmented protein levels of both intrinsic and extrinsic apoptosis-related markers. An amount of 50⯵M BBP induced marked upregulation of autophagy regulator proteins, p38 MAPK, and extracellular signal-regulated kinase and substantially downregulated the phosphorylation of key kinases involved in regulating cell proliferation, including phosphoinositide 3-kinase, protein kinase B, mammalian target of rapamycin (mTOR), c-Jun N-terminal kinase. The triple combination of N-acetylcysteine, parthenolide, and 3-methyladenine markedly restored cell proliferation, decreased BBP-induced apoptosis and autophagy, and restored mTOR phosphorylation. This study provides new insights into BBP-induced male germ cell toxicity and highlights the therapeutic potential of the triple inhibitors in mitigating BBP toxicity.
Assuntos
Acetilcisteína , Adenina , Apoptose , Autofagia , Proliferação de Células , Ácidos Ftálicos , Espécies Reativas de Oxigênio , Sesquiterpenos , Masculino , Animais , Camundongos , Ácidos Ftálicos/toxicidade , Autofagia/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Sesquiterpenos/farmacologia , Acetilcisteína/farmacologia , Adenina/análogos & derivados , Adenina/farmacologia , Adenina/toxicidade , Proliferação de Células/efeitos dos fármacos , Linhagem Celular , Plastificantes/toxicidade , Espermatogônias/efeitos dos fármacosRESUMO
Mutations within the SCN5A gene, which encodes the α-subunit 5 (NaV1.5) of the voltage-gated Na+ channel, have been linked to three distinct cardiac arrhythmia disorders: long QT syndrome type 3, Brugada syndrome (BrS), and cardiac conduction disorder. In this study, we have identified novel missense mutations (p.A385T/R504T) within SCN5A in a patient exhibiting overlap arrhythmia phenotypes. This study aims to elucidate the functional consequences of SCN5A mutants (p.A385T/R504T) to understand the clinical phenotypes. Whole-cell patch-clamp technique was used to analyze the NaV1.5 current (INa) in HEK293 cells transfected with the wild-type and mutant SCN5A with or without SCN1B co-expression. The amplitude of INa was not altered in mutant SCN5A (p.A385T/R504T) alone. Furthermore, a rightward shift of the voltage-dependent inactivation and faster recovery from inactivation was observed, suggesting a gain-of-function state. Intriguingly, the coexpression of SCN1B with p.A385T/R504T revealed significant reduction of INa and slower recovery from inactivation, consistent with the loss-of-function in Na+ channels. The SCN1B dependent reduction of INa was also observed in a single mutation p.R504T, but p.A385T co-expressed with SCN1B showed no reduction. In contrast, the slower recovery from inactivation with SCN1B was observed in A385T while not in R504T. The expression of SCN1B is indispensable for the electrophysiological phenotype of BrS with the novel double mutations; p.A385T and p.R504T contributed to the slower recovery from inactivation and reduced current density of NaV1.5, respectively.
RESUMO
The protein product of the CDKN1A gene, p21, has been extensively characterized as a negative regulator of the cell cycle. Nevertheless, it is clear that p21 has manifold complex and context-dependent roles that can be either tumor suppressive or oncogenic. Most well studied as a transcriptional target of the p53 tumor suppressor protein, there are other means by which p21 levels can be regulated. In this study, we show that pharmacological inhibition or siRNA-mediated reduction of O-GlcNAc transferase (OGT), the enzyme responsible for glycosylation of intracellular proteins, increases expression of p21 in both p53-dependent and p53-independent manners in nontransformed and cancer cells. In cells harboring WT p53, we demonstrate that inhibition of OGT leads to p53-mediated transactivation of CDKN1A, while in cells that do not express p53, inhibiting OGT leads to increased p21 protein stabilization. p21 is normally degraded by the ubiquitin-proteasome system following ubiquitination by, among others, the E3 ligase Skp-Cullin-F-box complex; however, in this case, we show that blocking OGT causes impairment of the Skp-Cullin-F-box ubiquitin complex as a result of disruption of the FoxM1 transcription factor-mediated induction of Skp2 expression. In either setting, we conclude that p21 levels induced by OGT inhibition correlate with cell cycle arrest and decreased cancer cell proliferation.
Assuntos
Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21 , Proteína Forkhead Box M1 , N-Acetilglucosaminiltransferases , Proteínas Quinases Associadas a Fase S , Proteína Supressora de Tumor p53 , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Proteínas Culina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Proteína Forkhead Box M1/metabolismo , Humanos , N-Acetilglucosaminiltransferases/antagonistas & inibidores , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , RNA Interferente Pequeno , Proteínas Quinases Associadas a Fase S/metabolismo , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina/metabolismoRESUMO
Parkinson's disease (PD) is a progressive neurodegenerative motor disorder without an available therapeutic to halt the formation of Lewy bodies for preventing dopaminergic neuronal loss in the nigrostriatal pathway. Since oxidative-stress-mediated damage has been commonly reported as one of the main pathological mechanisms in PD, we assessed the efficacy of a novel NOX inhibitor from AptaBio Therapeutics (C-6) in dopaminergic cells and PD mouse models. The compound reduced the cytotoxicity and enhanced the cell viability at various concentrations against MPP+ and α-synuclein preformed fibrils (PFFs). Further, the levels of ROS and protein aggregation were significantly reduced at the optimal concentration (1 µM). Using two different mouse models, we gavaged C-6 at two different doses to the PD sign-displaying transgenic mice for 2 weeks and stereotaxically PFF-injected mice for 5 weeks. Our results demonstrated that both C-6-treated mouse models showed alleviated motor deficits in pole test, hindlimb clasping, crossbeam, rotarod, grooming, and nesting analyses. We also confirmed that the compound treatment reduced the levels of protein aggregation, along with phosphorylated-α-synuclein, in the striatum and ventral midbrain and further dopaminergic neuronal loss. Taken together, our results strongly suggest that NOX inhibition can be a potential therapeutic target for PD.
Assuntos
Doença de Parkinson , alfa-Sinucleína , Animais , Modelos Animais de Doenças , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Camundongos , Camundongos Transgênicos , Degeneração Neural/patologia , Doença de Parkinson/metabolismo , Agregados Proteicos , alfa-Sinucleína/metabolismoRESUMO
Three-dimensional (3D) printing is perceived as an innovative tool for change in tissue engineering and regenerative medicine based on research outcomes on the development of artificial organs and tissues. With advances in such technology, research is underway into 3D-printed artificial scaffolds for tissue recovery and regeneration. In this study, we fabricated artificial scaffolds by coating bone demineralized and decellularized extracellular matrix (bdECM) onto existing 3D-printed polycaprolactone/tricalcium phosphate (PCL/TCP) to enhance osteoconductivity and osteoinductivity. After injecting adipose-derived stem cells (ADSCs) in an aggregate form found to be effective in previous studies, we examined the effects of the scaffold on ossification during mandibular reconstruction in beagle dogs. Ten beagles were divided into two groups: group A (PCL/TCP/bdECM + ADSC injection; n = 5) and group B (PCL/TCP/bdECM; n = 5). The results were analyzed four and eight weeks after intervention. Computed tomography (CT) findings showed that group A had more diffuse osteoblast tissue than group B. Evidence of infection or immune rejection was not detected following histological examination. Goldner trichrome (G/T) staining revealed rich ossification in scaffold pores. ColI, Osteocalcin, and Runx2 gene expressions were determined using real-time polymerase chain reaction. Group A showed greater expression of these genes. Through Western blotting, group A showed a greater expression of genes that encode ColI, Osteocalcin, and Runx2 proteins. In conclusion, intervention group A, in which the beagles received the additional ADSC injection together with the 3D-printed PCL/TCP coated with bdECM, showed improved mandibular ossification in and around the pores of the scaffold.
Assuntos
Tecido Adiposo/citologia , Fosfatos de Cálcio/química , Matriz Extracelular/fisiologia , Mandíbula/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Poliésteres/química , Células-Tronco/citologia , Alicerces Teciduais/química , Adipócitos/citologia , Animais , Regeneração Óssea/efeitos dos fármacos , Cães , Osteoblastos/efeitos dos fármacos , Impressão Tridimensional , Engenharia Tecidual/métodosRESUMO
The derivation of human embryonic stem cells (hESCs) by somatic cell nuclear transfer (SCNT) has prompted a re-emerging interest in using such cells for therapeutic cloning. Despite recent advancements in derivation protocols, the functional potential of CHA-NT4 derived cells is yet to be elucidated. For this reason, this study sought to differentiate CHA-NT4 cells toward an endothelial lineage in order to evaluate in vitro and in vivo functionality. To initial differentiation, embryoid body formation of CHA-NT4 was mediated by concave microwell system which was optimized for hESC-endothelial cell (EC) differentiation. The isolated CD31+ cells exhibited hallmark endothelial characteristics in terms of morphology, tubule formation, and ac-LDL uptake. Furthermore, CHA-NT4-derived EC (human nuclear transfer [hNT]-ESC-EC) transplantation in hind limb ischemic mice rescued the hind limb and restored blood perfusion. These findings suggest that hNT-ESC-EC are functionally equivalent to hESC-ECs, warranting further study of CHA-NT4 derivatives in comparison to other well established pluripotent stem cell lines. This revival of human SCNT-ESC research may lead to interesting insights into cellular behavior in relation to donor profile, mitochondrial DNA, and oocyte quality. Stem Cells 2019;37:623-630.
Assuntos
Diferenciação Celular/genética , Células Endoteliais/transplante , Células-Tronco Embrionárias Humanas/transplante , Células-Tronco Pluripotentes Induzidas/transplante , Animais , Membro Posterior/patologia , Membro Posterior/transplante , Humanos , Isquemia/terapia , Camundongos , Técnicas de Transferência NuclearRESUMO
Critical limb ischemia is one of the most common types of peripheral arterial disease. Preclinical development of ischemia therapeutics relies on the availability of a relevant and reproducible in vivo disease model. Thus, establishing appropriate animal disease models is essential for the development of new therapeutic strategies. Currently, the most commonly employed model of hindlimb ischemia is the surgical induction method with ligation of the femoral artery and its branches after skin incision. However, the efficiency of the method is highly variable depending on the availability of skilled technicians. In addition, after surgical procedures, animals can quickly and spontaneously recover from damage, limiting observations of the therapeutic effect of potential agents. The aim of this study was to develop a hindlimb ischemia mouse model with similarities to human ischemic disease. To that end, a photochemical reaction was used to induce thrombosis in the hindlimb. After the photochemical reaction was induced by light irradiation, thrombotic plugs and adjacent red blood cell stasis were observed in hindlimb vessels in the light-irradiated zone. Additionally, the photochemically induced thrombosis maintained the ischemic condition and did not cause notable side effects in mice.
Assuntos
Eritrosina , Isquemia/fisiopatologia , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica , Trombose/fisiopatologia , Animais , Velocidade do Fluxo Sanguíneo , Modelos Animais de Doenças , Membro Posterior , Isquemia/induzido quimicamente , Luz , Masculino , Camundongos Endogâmicos ICR , Processos Fotoquímicos , Fluxo Sanguíneo Regional , Trombose/induzido quimicamente , Fatores de TempoRESUMO
Proliferative and migratory abilities of fibroblasts are essential for wound healing at the skin surface. Cytoplasmic linker-associated protein-2 (CLASP2) was originally found to interact with cytoplasmic linker protein (CLIP)-170. CLASP2 plays an important role in microtubule stabilization and the microtubule-stabilizing activity of CLASP2 depends on its interactions with end binding (EB)-1 and CLIP-170. Although the microtubule-stabilizing role of CLASP2 is well established, the effects of CLASP2 on the migration and proliferation of fibroblasts remain unclear in the context of wound healing. Therefore, we tested the utilization of CLASP2 as a directly applied protein drug to improve wound healing by promoting the migration of effector cells, including skin fibroblasts, to the site of repair or injury using an in vivo excisional wound mouse model and in vitro Hs27 skin fibroblast model. Epidermal growth factor, which is a recognized contributor to cell proliferation and migration, was used as positive control. In vitro and in vivo, CLASP2 treatment significantly enhanced cell migration and accelerated wound closure. Furthermore, in vivo, the CLASP2-treated animal group displayed enhanced epidermal repair and collagen deposition. Next, we studied the mechanism of CLASP2 for wound healing. Increasing the abundance of intracellular free CLASP2 in skin fibroblasts by supplying exogenous CLASP2 seemed to stabilize microtubules through an interaction between CLASP2 and CLIP-170, as well as EB1. Exogenous CLASP2 also showed direct binding with IQGAP1, increasing both cyclic adenosine monophosphate activity and phosphorylation of glycogen synthase kinase 3ß, which in turn reinstated the binding between free CLASP2 and IQGAP1. In summary, exogenous CLASP2 increased Hs27 skin fibroblast migration by interacting with IQGAP1 and other cytoskeletal linker proteins, such as CLIP-170 and EB1. Our results strongly suggest that CLASP2 can be developed in wound healing drugs for skin repair and/or regenerating cosmetic products.
Assuntos
Fibroblastos/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Ferimentos e Lesões/patologia , Animais , Movimento Celular , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Células Cultivadas , Modelos Animais de Doenças , Fibroblastos/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Cicatrização/fisiologiaRESUMO
Basic fibroblast growth factor (bFGF) supplementation is critical to maintain the pluripotency of human pluripotent stem cells (hPSCs) through activation of PI3K/AKT, rather than MEK/ERK pathway. Thus, elaborate molecular mechanisms that preserve PI3K/AKT signaling upon bFGF stimulation may exist in hPSCs. Protein arginine methyltransferase 8 (PRMT8) was expressed and then its level gradually decreased during spontaneous differentiation of human embryonic stem cells (hESCs). PRMT8 loss- or gain-of-function studies demonstrated that PRMT8 contributed to longer maintenance of hESC pluripotency, even under bFGF-deprived conditions. Direct interaction of membrane-localized PRMT8 with p85, a regulatory subunit of PI3K, was associated with accumulation of phosphoinositol 3-phosphate and consequently high AKT activity. Furthermore, the SOX2 induction, which was controlled by the PRMT8/PI3K/AKT axis, was linked to mesodermal lineage differentiation. Thus, we propose that PRMT8 in hESCs plays an important role not only in maintaining pluripotency but also in controlling mesodermal differentiation through bFGF signaling toward the PI3K/AKT/SOX2 axis. Stem Cells 2017;35:2037-2049.
Assuntos
Linhagem da Célula , Células-Tronco Embrionárias Humanas/metabolismo , Proteínas de Membrana/metabolismo , Mesoderma/citologia , Fosfatidilinositol 3-Quinases/metabolismo , Células-Tronco Pluripotentes/citologia , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Biológicos , Fenótipo , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: NADPH oxidases (Nox) is a major enzyme system contributing to oxidative stress, which plays an important role in the pathogenesis of diabetic kidney disease (DKD). We have shown an elevation of renal Nox1, Nox2, and Nox4 in diabetic mice. APX-115, a pan-Nox inhibitor, attenuated the progression of DKD in mice. As the standard diabetic mice cannot fully mimic human DKD, the present study was aimed to show the dose-dependent effect and to provide a confirmatory evidence of APX-115 in attenuating DKD in diabetic rats. METHOD: Type 1 diabetes was induced by a single 60 mg/kg intraperitoneal injection of streptozotocin in Sprague-Dawley rats. 0.5, 5, or 30 mg APX-115/kg/day or losartan 1 mg/kg/day were administered orally to diabetic rats for 8 weeks. RESULTS: APX-115 treatment showed an improvement in kidney function and tubular and podocyte -injury, as well as attenuation of inflammation, fibrosis, and oxidative stress as much as losartan, a comparative drug and mainstay treatment in DKD. Therapeutic effect of APX-115 was exhibited in a dose-dependent manner; a dose of 30 mg/kg displayed a superior efficacy. CONCLUSION: This finding verified the pre-clinical data of APX-115 in protecting against DKD, which is important to bring APX-115 toward the next stage of drug development.
Assuntos
Diabetes Mellitus Tipo 1/tratamento farmacológico , Nefropatias Diabéticas/tratamento farmacológico , NADPH Oxidases/antagonistas & inibidores , Pirazóis/farmacologia , Piridinas/farmacologia , Animais , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/enzimologia , Diabetes Mellitus Tipo 1/patologia , Nefropatias Diabéticas/enzimologia , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/prevenção & controle , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Rim/efeitos dos fármacos , Rim/enzimologia , Rim/patologia , Losartan/farmacologia , Masculino , NADPH Oxidases/metabolismo , Podócitos/efeitos dos fármacos , Podócitos/patologia , Substâncias Protetoras/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Recent studies have suggested that renal Nox is important in the progression of diabetic nephropathy. Therefore, we investigated the effect of a novel pan-NOX-inhibitor, APX-115, on diabetic nephropathy in type 2 diabetic mice. Eight- week-old db/m and db/db mice were treated with APX-115 for 12 weeks. APX-115 was administered by oral gavage at a dose of 60 mg/kg per day. To compare the effects of APX-115 with a dual Nox1/Nox4 inhibitor, db/db mice were treated with GKT137831 according to the same protocol. APX-115 significantly improved insulin resistance in diabetic mice, similar to GKT137831. Oxidative stress as measured by plasma 8-isoprostane level was decreased in the APX-115 group compared with diabetic controls. All lipid profiles, both in plasma and tissues improved with Nox inhibition. APX-115 treatment decreased Nox1, Nox2, and Nox4 protein expression in the kidney. APX-115 decreased urinary albumin excretion and preserved creatinine level. In diabetic kidneys, APX-115 significantly improved mesangial expansion, but GKT137831 did not. In addition, F4/80 infiltration in the adipose tissue and kidney decreased with APX-115 treatment. We also found that TGF-ß stimulated ROS generation in primary mouse mesangial cells (pMMCs) from wild-type, Nox1 KO, and Duox1 KO mice, but did not induce Nox activity in pMMCs from Nox2 knockout (KO), Nox4 KO, or Duox2 KO mice. These results indicate that activating Nox2, Nox4, or Duox2 in pMMCs is essential for TGF-ß-mediated ROS generation. Our findings suggest that APX-115 may be as effective or may provide better protection than the dual Nox1/Nox4 inhibitor, and pan-Nox inhibition with APX-115 might be a promising therapy for diabetic nephropathy.
Assuntos
Diabetes Mellitus Tipo 2/complicações , Nefropatias Diabéticas/prevenção & controle , Inibidores Enzimáticos/farmacologia , NADPH Oxidases/antagonistas & inibidores , Pirazóis/farmacologia , Piridinas/farmacologia , Animais , Western Blotting , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/genética , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Peroxidação de Lipídeos/efeitos dos fármacos , Lipídeos/sangue , Masculino , Células Mesangiais/efeitos dos fármacos , Células Mesangiais/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Tamanho do Órgão/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Pirazolonas , Piridonas , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Na(+)/Ca(2+) exchanger current (INCX) triggered by spontaneous Ca(2+) release from sarcoplasmic reticulum (SR) has been suggested as one of the cardiac pacemaker mechanisms ("Ca(2+) clock model"). In human embryonic stem cell-derived cardiomyocytes (hESC-CMs) showing spontaneous action potentials (APs), we found that substantial population (35 %) showed regular oscillation of inward currents (SICs) in nystatin-perforated voltage clamp between -40 and 40 mV (-80 ± 10.6 pA, at -20 mV). SICs were similarly observed between nodal, atrial, and ventricular hESC-CMs. Oscillations of [Ca(2+)]i synchronized with SICs were observed under voltage clamp. SICs were eliminated by lowering [Ca(2+)]e, L-type Ca(2+) channel (VOCCL) blocker (nifedipine, 10 µM), ryanodine receptor (RyR) agonist (caffeine, 10 mM), or NCX inhibitor (1 µM SN-6 and 10 µM KB-R7943). Plasma membrane expression of NCX1 was confirmed using immunofluorescence confocal microcopy. Both caffeine and SN-6 slowed the pacemaker potential but did not abolish the AP generation. The inhibitors of funny current (3 µM ivabradine) or voltage-gated K(+) channel currents (1 µM E4031 and 10 µM chromanol-293B) also did not abolish but slowed the pacemaker potential. In a computational model of cardiac pacemaker by Maltsev and Lakatta (2009), after modifying the spatial distribution of RyR, VOCCL, and NCX by using our multiparameter adjust algorithm, we could successfully reproduce spontaneous SR Ca(2+) release and SICs under voltage clamp. It was proposed that, under the membrane depolarization activating VOCCL, oscillatory Ca(2+) releases via RyR induce sharp increases in subsarcolemmal [Ca(2+)]i and inward INCX (SICs). Since the hESC-CMs without SICs still showed spontaneous APs, the putative "Ca(2+) clock" would provide a redundant pacemaker or augmenting mechanism in hESC-CMs.
Assuntos
Potenciais de Ação , Sinalização do Cálcio , Células-Tronco Embrionárias/citologia , Miócitos Cardíacos/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Diferenciação Celular , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Humanos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismoRESUMO
Labeling of stem cells aims to distinguish transplanted cells from host cells, understand in vivo fate of transplanted cells, particularly important in stem cell therapy. Adipose-derived mesenchymal stem cells (ASCs) are considered as an emerging therapeutic option for tissue regeneration, but much remains to be understood regarding the in vivo evidence. In this study, a simple and efficient cell labeling method for labeling and tracking of stem cells was developed based on bio-orthogonal copper-free click chemistry, and it was applied in a mouse hindlimb ischemia model. The human ASCs were treated with tetra-acetylated N-azidoacetyl-d-mannosamine (Ac4ManNAz) to generate glycoprotein with unnatural azide groups on the cell surface, and the generated azide groups were fluorescently labeled by specific binding of dibenzylcyclooctyne-conjugated Cy5 (DBCO-Cy5). The safe and long-term labeling of the hASCs by this method was first investigated in vitro. Then the DBCO-Cy5-hASCs were transplanted into the hindlimb ischemia mice model, and we could monitor and track in vivo fate of the cells using optical imaging system. We could clearly observe the migration potent of the hASCs toward the ischemic lesion. This approach to design and tailor new method for labeling of stem cells may be useful to provide better understanding on the therapeutic effects of transplanted stem cells into the target diseases.
Assuntos
Rastreamento de Células/métodos , Isquemia/terapia , Células-Tronco Mesenquimais/citologia , Tecido Adiposo/citologia , Animais , Azidas/química , Química Click/métodos , Modelos Animais de Doenças , Corantes Fluorescentes/química , Membro Posterior , Humanos , Imageamento Tridimensional , Isquemia/patologia , Transplante de Células-Tronco Mesenquimais , CamundongosRESUMO
As wound contraction in the cutaneous layer occurs rapidly in mice, mechanical means are typically used to deliberately expose the wound to properly investigate healing by secondary intention. Previously, silicon rings and splinting models were attempted to analyze histological recovery but prevention of surrounding epidermal cell migration and subsequent closure was minimal. Here, we developed an ideal chimney wound model to evaluate epidermal regeneration in murine under hESC-EC transplantation through histological analysis encompassing the three phases of regeneration: migration, proliferation, and remodeling. Human embryonic stem cell derived endothelial cells (hESC-EC) were transplanted due to possessing a well-known therapeutic effect in angiogenesis which also enhances epidermal repair to depict the process of regeneration. Following a standard 1 mm biopsy punch, a chimney manufactured by modifying a 1.7 mL microtube was simply inserted into the excisional wound to complete the modeling process. Under this model, the excisional wound remained fully exposed for 14 days and even after 4 weeks, only a thin transparent layer of epidermal tissue covered the wound site. This approach is able to more accurately depict epidermal repair in relation to histology while also being a user-friendly and cost-effective way to mimic human recovery in rodents and evaluate epithelial repair induced by a form of therapy.
Assuntos
Células Endoteliais/metabolismo , Células-Tronco Embrionárias Humanas/transplante , Regeneração/fisiologia , Transplante de Células-Tronco/métodos , Cicatrização/fisiologia , Ferimentos Penetrantes/fisiopatologia , Animais , Colágeno Tipo VIII/metabolismo , Análise Custo-Benefício , Modelos Animais de Doenças , Células Endoteliais/citologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Ferimentos Penetrantes/terapiaRESUMO
The future of safe cell-based therapy rests on overcoming teratoma/tumor formation, in particular when using human pluripotent stem cells (hPSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Because the presence of a few remaining undifferentiated hPSCs can cause undesirable teratomas after transplantation, complete removal of these cells with no/minimal damage to differentiated cells is a prerequisite for clinical application of hPSC-based therapy. Having identified a unique hESC signature of pro- and antiapoptotic gene expression profile, we hypothesized that targeting hPSC-specific antiapoptotic factor(s) (i.e., survivin or Bcl10) represents an efficient strategy to selectively eliminate pluripotent cells with teratoma potential. Here we report the successful identification of small molecules that can effectively inhibit these antiapoptotic factors, leading to selective and efficient removal of pluripotent stem cells through apoptotic cell death. In particular, a single treatment of hESC-derived mixed population with chemical inhibitors of survivin (e.g., quercetin or YM155) induced selective and complete cell death of undifferentiated hPSCs. In contrast, differentiated cell types (e.g., dopamine neurons and smooth-muscle cells) derived from hPSCs survived well and maintained their functionality. We found that quercetin-induced selective cell death is caused by mitochondrial accumulation of p53 and is sufficient to prevent teratoma formation after transplantation of hESC- or hiPSC-derived cells. Taken together, these results provide the "proof of concept" that small-molecule targeting of hPSC-specific antiapoptotic pathway(s) is a viable strategy to prevent tumor formation by selectively eliminating remaining undifferentiated pluripotent cells for safe hPSC-based therapy.