RESUMO
BACKGROUND: Amyotrophic lateral sclerosis (ALS) is characterized by progressive motor neuron (MN) degeneration, leading to neuromuscular junction (NMJ) dismantling and severe muscle atrophy. The nuclear receptor interaction protein (NRIP) functions as a multifunctional protein. It directly interacts with calmodulin or α-actinin 2, serving as a calcium sensor for muscle contraction and maintaining sarcomere integrity. Additionally, NRIP binds with the acetylcholine receptor (AChR) for NMJ stabilization. Loss of NRIP in muscles results in progressive motor neuron degeneration with abnormal NMJ architecture, resembling ALS phenotypes. Therefore, we hypothesize that NRIP could be a therapeutic factor for ALS. METHODS: We used SOD1 G93A mice, expressing human SOD1 with the ALS-linked G93A mutation, as an ALS model. An adeno-associated virus vector encoding the human NRIP gene (AAV-NRIP) was generated and injected into the muscles of SOD1 G93A mice at 60 days of age, before disease onset. Pathological and behavioral changes were measured to evaluate the therapeutic effects of AAV-NRIP on the disease progression of SOD1 G93A mice. RESULTS: SOD1 G93A mice exhibited lower NRIP expression than wild-type mice in both the spinal cord and skeletal muscle tissues. Forced NRIP expression through AAV-NRIP intramuscular injection was observed in skeletal muscles and retrogradely transduced into the spinal cord. AAV-NRIP gene therapy enhanced movement distance and rearing frequencies in SOD1 G93A mice. Moreover, AAV-NRIP increased myofiber size and slow myosin expression, ameliorated NMJ degeneration and axon terminal denervation at NMJ, and increased the number of α-motor neurons (α-MNs) and compound muscle action potential (CMAP) in SOD1 G93A mice. CONCLUSIONS: AAV-NRIP gene therapy ameliorates muscle atrophy, motor neuron degeneration, and axon terminal denervation at NMJ, leading to increased NMJ transmission and improved motor functions in SOD1 G93A mice. Collectively, AAV-NRIP could be a potential therapeutic drug for ALS.
Assuntos
Esclerose Lateral Amiotrófica , Dependovirus , Modelos Animais de Doenças , Terapia Genética , Camundongos Transgênicos , Neurônios Motores , Atrofia Muscular , Animais , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/terapia , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Terapia Genética/métodos , Atrofia Muscular/genética , Atrofia Muscular/terapia , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Dependovirus/genética , Camundongos , Humanos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Junção Neuromuscular/metabolismo , Junção Neuromuscular/patologia , Vetores Genéticos/administração & dosagem , Degeneração Neural/genética , Degeneração Neural/terapia , Masculino , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismoRESUMO
Pigment epithelium-derived factor (PEDF) is widely recognized as a neuroprotective factor expressed in the retina and has shown therapeutic potential in several retinal diseases. Our study aimed to identify the neuroprotective fragment in PEDF and investigate its protective activity in retinas under ischemia-reperfusion (IR) condition. We synthesized a series of shorter synthetic peptides, 6-mer (Ser93-Gln98) and its d-form variant (6 dS) derived from the 44-mer (Val78-Thr121; a PEDF neurotrophic fragment), to determine their cytoprotective activity in IR injury, which was induced in rat retinas by injection of saline into the anterior chamber to increase the intraocular pressure (IOP) followed by reperfusion. We found the cytoprotective effect of 6-mer on glutamate-treated Neuro-2a cells and tert-butyl hydroperoxide (tBHP)-treated 661W cells were 2.6-fold and 1.5-fold higher than the 44-mer, respectively. The cytoprotective effect was blocked by a chemical inhibitor atglistatin and blocking antibody targeting PEDF receptor (PEDF-R). IR induced several impairments in retina, including cell apoptosis, activation of microglia/macroglia, degeneration of retinal capillaries, reduction in electroretinography (ERG) amplitudes, and retinal atrophy. Such IR injuries were ameliorated by treatment with 6-mer and 6 dS eye drops. Also, the neuroprotective activity of 6-mer and 6 dS in ischemic retinas were dramatically reversed by atglistatin preconditioning. Taken together, our data demonstrate smallest neuroprotective fragment of PEDF has potential to treat retinal degeneration-related diseases.
Assuntos
Proteínas do Olho , Fatores de Crescimento Neural , Traumatismo por Reperfusão , Retina , Retinite , Serpinas , Animais , Ratos , Coelhos , Fatores de Crescimento Neural/administração & dosagem , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/metabolismo , Proteínas do Olho/administração & dosagem , Proteínas do Olho/química , Proteínas do Olho/metabolismo , Serpinas/administração & dosagem , Serpinas/química , Serpinas/metabolismo , Retina/metabolismo , Retina/patologia , Traumatismo por Reperfusão/metabolismo , Citoproteção , Apoptose , Neurônios/metabolismo , Retinite/tratamento farmacológico , Retinite/metabolismo , Administração Tópica , Peptídeos/administração & dosagem , Peptídeos/metabolismoRESUMO
PURPOSE: The purpose of this study was to extensively evaluate the efficacy of integrin αvß3 antagonists for the treatment of experimental dry eye (EDE). METHODS: Vitronectin, an αvß3 ligand, was used to induce tumor necrosis factor-α gene expression in human THP-1 macrophages. To induce EDE, C57BL/6 mice were housed in a low-humidity controlled environment chamber and injected subcutaneously with scopolamine for 7 days. Subsequently, αvß3 antagonists, including RGDfD, c(RGDfD), c(RGDiD), c(RGDfK), ATN-161, SB273005, and cilengitide, were administered topically to EDE animals under controlled environment chamber conditions. Corneal epithelial damage in EDE was assessed by fluorescein staining. The density of conjunctival goblet cells and secretion of tears was measured by period acid-Schiff staining and phenol red-impregnated cotton threads, respectively. Inflammation markers, including tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, IL-17A, and metalloproteinase (MMP)-9, in the pooled cornea and conjunctiva tissues were examined by real-time polymerase chain reaction. RESULTS: The inhibitory effects of αvß3 antagonists on the vitronectin-induced tumor necrosis factor-α gene expression and integrin-mediated inflammatory signaling were validated in THP-1 macrophages. αvß3 antagonists ameliorated the impairment of the corneal epithelial barrier with varying therapeutic efficacies, compared with vehicle-treated mice. c(RGDfD) and c(RGDiD) significantly protected against goblet cell loss, tear reduction, and proinflammatory gene expression in EDE. CONCLUSIONS: Topical applications of αvß3 antagonists yield therapeutic benefits in EDE by promoting corneal epithelial defect healing and reducing inflammation. Antagonistic targeting αvß3 may be a novel promising strategy to treat patients with dry eye disease.
Assuntos
Síndromes do Olho Seco , Integrina alfaVbeta3 , Humanos , Animais , Camundongos , Integrina alfaVbeta3/metabolismo , Integrina alfaVbeta3/uso terapêutico , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Vitronectina/metabolismo , Vitronectina/farmacologia , Vitronectina/uso terapêutico , Camundongos Endogâmicos C57BL , Síndromes do Olho Seco/metabolismo , Lágrimas/metabolismo , Túnica Conjuntiva/patologia , Córnea/patologia , Inflamação/metabolismo , Modelos Animais de DoençasRESUMO
Purpose: To evaluate the efficacy of a pigment epithelium-derived factor (PEDF)-derived short peptide 29-mer, on the treatment and prevention of experimental dry eye (EDE). Methods: C57BL/6 mice were housed in a low humidity controlled environment chamber for 14 days to induce EDE. The 29-mer was administered topically to their eyes, for treatment or dosing, from the point of housing in the controlled environment chamber. The efficacy of the 29-mer on EDE was evaluated in terms of corneal epithelial integrity, tear secretion, and the density of conjunctival goblet cells. PEDF and inflammatory factors, including tumor necrosis factor-α, IL-1ß, IL-6, monocyte chemotactic protein (MCP)-1, matrix metalloproteinase-9, and macrophage infiltration, were examined by real-time polymerase chain reaction, Western blotting, and immunostaining. The involvement of the PEDF receptor/PNPLA2 on the 29-mer effects was evaluated by a specific inhibitor, atglistatin. Rabbit corneal epithelial cells were exposed to hyperosmotic medium to induce inflammatory responses. Results: The levels of PEDF protein increased in the corneal epithelium of EDE, compared with the nonstressed mice. The 29-mer showed a therapeutic effect on EDE and prevented the development of EDE, accompanied by amelioration of the inflammatory factors. The 29-mer effects of inflammatory relief were dramatically reversed by atglistatin. The 29-mer also suppressed the expression of matrix metalloproteinase-9 and proinflammatory cytokines in rabbit corneal epithelial cells induced by hyperosmolarity. Conclusions: Through this animal study, we provide a proof of concept of the anti-inflammatory domain of PEDF having potential to treat dry eye disease. Translational Relevance: This study shows the 29-mer has novel potential as an ophthalmic drop treatment for dry eye disease.
Assuntos
Síndromes do Olho Seco , Metaloproteinase 9 da Matriz , Animais , Anti-Inflamatórios/uso terapêutico , Citocinas/metabolismo , Citocinas/uso terapêutico , Modelos Animais de Doenças , Síndromes do Olho Seco/tratamento farmacológico , Síndromes do Olho Seco/metabolismo , Síndromes do Olho Seco/patologia , Proteínas do Olho , Inflamação/tratamento farmacológico , Interleucina-6/uso terapêutico , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quimioatraentes de Monócitos/uso terapêutico , Fatores de Crescimento Neural , Compostos de Fenilureia , Coelhos , Serpinas , Fator de Necrose Tumoral alfa/uso terapêuticoRESUMO
BACKGROUND: Breast carcinoma-amplified sequence 2 (BCAS2) regulates ß-catenin gene splicing. The conditional knockout of BCAS2 expression in the forebrain (BCAS2 cKO) of mice confers impaired learning and memory along with decreased ß-catenin expression. Because ß-catenin reportedly regulates adult neurogenesis, we wondered whether BCAS2 could regulate adult neurogenesis via ß-catenin. METHODS: BCAS2-regulating neurogenesis was investigated by characterizing BCAS2 cKO mice. Also, lentivirus-shBCAS2 was intracranially injected into the hippocampus of wild-type mice to knock down BCAS2 expression. We evaluated the rescue effects of BCAS2 cKO by intracranial injection of adeno-associated virus encoding BCAS2 (AAV-DJ8-BCAS2) and AAV-ß-catenin gene therapy. RESULTS: To show that BCAS2-regulating adult neurogenesis via ß-catenin, first, BCAS2 cKO mice showed low SRY-box 2-positive (Sox2+) neural stem cell proliferation and doublecortin-positive (DCX+) immature neurons. Second, stereotaxic intracranial injection of lentivirus-shBCAS2 knocked down BCAS2 in the hippocampus of wild-type mice, and we confirmed the BCAS2 regulation of adult neurogenesis via ß-catenin. Third, AAV-DJ8-BCAS2 gene therapy in BCAS2 cKO mice reversed the low proliferation of Sox2+ neural stem cells and the decreased number of DCX+ immature neurons with increased ß-catenin expression. Moreover, AAV-ß-catenin gene therapy restored neuron stem cell proliferation and immature neuron differentiation, which further supports BCAS2-regulating adult neurogenesis via ß-catenin. In addition, cells targeted by AAV-DJ8 injection into the hippocampus included Sox2 and DCX immature neurons, interneurons, and astrocytes. BCAS2 may regulate adult neurogenesis by targeting Sox2+ and DCX+ immature neurons for autocrine effects and interneurons or astrocytes for paracrine effects. CONCLUSIONS: BCAS2 can regulate adult neurogenesis in mice via ß-catenin.
Assuntos
Células-Tronco Neurais , beta Catenina , Animais , Hipocampo , Camundongos , Proteínas de Neoplasias/metabolismo , Células-Tronco Neurais/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
BACKGROUND: Pigment epithelial-derived factor (PEDF), a 50 kDa secreted glycoprotein, exhibits distinct effects on a range of cell types. PEDF has been shown to inhibit vascular endothelial growth factor (VEGF)-mediated angiogenesis and widely accepted as a promising agent for treatment eye diseases related to neovascularization. A pool of short peptide fragments derived from PEDF reportedly manifests angioinhibitory activity. This study aims to determine the minimal PEDF fragment which can exert the anti-VEGF effect. METHODS: A series of shorter synthetic peptides, derived from the 34-mer (PEDF amino acid positions Asp44-Asn77), were synthesized. An MTT assay was used to evaluate the ability of the 34-mer-derived peptides to inhibit VEGF-induced proliferation of multiple myeloma RPMI8226 cells. Cell apoptosis was monitored by annexin V-FITC staining. Western blot analysis was used to detect phosphorylated kinases, including c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), and the expression of apoptosis-associated proteins, including p53, bax and caspase-3. VEGF-mediated angiogenesis of human umbilical vein endothelial cells (HUVECs), rat aortic ring and mouse cornea were used to detect the angioinhibitory activity of the PEDF-derived peptides. RESULTS: The MTT assay showed that the anti-VEGF effect of a 7-mer (Asp64-Ser70) was 1.5-fold greater than the 34-mer. In addition, massive apoptosis (37%) was induced by 7-mer treatment. The 7-mer induced JNK phosphorylation in RPMI8226 cells. Cell apoptosis and apoptosis-associated proteins induced by the 7-mer were blocked by pharmacological inhibition of JNK, but not p38 MAPK. Moreover, the 7-mer prevented VEGF-mediated angiogenesis of endothelial cells (ECs), including tube formation, aortic EC spreading and corneal neovascularization in mice. CONCLUSIONS: This is the first study to show that the PEDF 7-mer peptide manifests anti-VEGF activity, further establishing its potential as an anti-angiogenic agent.
Assuntos
Inibidores da Angiogênese/farmacologia , Proteínas do Olho/farmacologia , Fatores de Crescimento Neural/farmacologia , Peptídeos/farmacologia , Serpinas/farmacologia , Animais , Linhagem Celular Tumoral , Células Endoteliais/metabolismo , Proteínas do Olho/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Fatores de Crescimento Neural/metabolismo , Ratos , Serpinas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Cell signaling mediated by the αv integrin plays a pivotal role in macrophage activation in various inflammatory processes, but its involvement in the pathogenesis of dry eye disease (DED) remains unclear. In a murine model of DED, we found increased αv integrin expression in ocular surface macrophages. The αv integrins inhibitor c(RGDfK) ameliorated the corneal damage caused by DED, suggesting a pathogenic role for αv integrin. Because tear hyperosmolarity induces ocular inflammation in DED, a hyperosmolar culture of murine bone marrow-derived macrophages (BMDMs) is used to reproduce inflammation in vitro. However, the expression of proinflammatory cytokine mRNA was minimal, even though αv integrin was induced. In searching for components that are involved in αv integrin-mediated inflammation but that are missing from the culture model, we showed that the levels of vitronectin (VTN), a binding ligand of αv integrins, were increased in the tear fluid and conjunctival stroma of DED animals. The addition of VTN prominently enhanced hyperosmolarity-induced inflammation in BMDMs. Mechanistically, we showed that VTN/αv integrins mediated NF-κB activation to induce inflammatory gene expression in the BMDMs. Our findings indicate that interaction the of VTN with αv integrins is a crucial step in the inflammatory process in DED and suggests a novel therapeutic target.
Assuntos
Síndromes do Olho Seco/metabolismo , Inflamação/metabolismo , Integrina alfaV/metabolismo , Macrófagos/metabolismo , Vitronectina/metabolismo , Animais , Linhagem Celular , Citocinas/metabolismo , Olho/metabolismo , Feminino , Regulação da Expressão Gênica/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/fisiologia , Células THP-1 , Lágrimas/metabolismoRESUMO
BACKGROUND: Nuclear receptor interaction protein (NRIP) co-localizes with acetylcholine receptor (AChR) at the neuromuscular junction (NMJ), and NRIP deficiency causes aberrant NMJ architecture. However, the normal physiological and pathophysiological roles of NRIP in NMJ are still unclear. METHODS: We investigated the co-localization and interaction of NRIP with AChR-associated proteins using immunofluorescence and immunoprecipitation assay, respectively. The binding affinity of AChR-associated proteins was analysed in muscle-restricted NRIP knockout mice and NRIP knockout muscle cells (C2C12). We further collected the sera from 43 patients with myasthenia gravis (MG), an NMJ disorder. The existence and features of anti-NRIP autoantibody in sera were studied using Western blot and epitope mapping. RESULTS: NRIP co-localized with AChR, rapsyn and α-actinin 2 (ACTN2) in gastrocnemius muscles of mice; and α-bungarotoxin (BTX) pull-down assay revealed NRIP with rapsyn and ACTN2 in complexes from muscle tissues and cells. NRIP directly binds with α subunit of AChR (AChRα) in vitro and in vivo to affect the binding affinity of AChR with rapsyn and rapsyn with ACTN2. In 43 patients with MG (age, 58.4 ± 14.5 years; female, 55.8%), we detected six of them (14.0%) having anti-NRIP autoantibody. The presence of anti-NRIP autoantibody correlated with a more severe type of MG when AChR autoantibody existed (P = 0.011). The higher the titre of anti-NRIP autoantibody, the more severe MG severity (P = 0.032). The main immunogenic region is likely on the IQ motif of NRIP. We also showed the IgG subclass of anti-NRIP autoantibody mainly to be IgG1. CONCLUSIONS: NRIP is a novel AChRα binding protein and involves structural NMJ formation, which acts as a scaffold to stabilize AChR-rapsyn-ACTN2 complexes. Anti-NRIP autoantibody is a novel autoantibody in MG and plays a detrimental role in MG with the coexistence of anti-AChR autoantibody.
Assuntos
Acetilcolina , Miastenia Gravis , Animais , Feminino , Humanos , Camundongos , Músculo Esquelético , Junção Neuromuscular , Receptores ColinérgicosRESUMO
BACKGROUND: Breast cancer amplified sequence 2 (BCAS2) plays crucial roles in pre-mRNA splicing and androgen receptor transcription. Previous studies suggested that BCAS2 is involved in double-strand breaks (DSB); therefore, we aimed to characterise its mechanism and role in prostate cancer (PCa). METHODS: Western blotting and immunofluorescence microscopy were used to assay the roles of BCAS2 in the DSBs of PCa cells and apoptosis in Drosophila, respectively. The effect of BCAS2 dosage on non-homologous end joining (NHEJ) and homologous recombination (HR) were assayed by precise end-joining assay and flow cytometry, respectively. Glutathione-S-transferase pulldown and co-immunoprecipitation assays were used to determine whether and how BCAS2 interacts with NBS1. The expression of BCAS2 and other proteins in human PCa was determined by immunohistochemistry. RESULTS: BCAS2 helped repair radiation-induced DSBs efficiently in both human PCa cells and Drosophila. BCAS2 enhanced both NHEJ and HR, possibly by interacting with NBS1, which involved the BCAS2 N-terminus as well as both the NBS1 N- and C-termini. The overexpression of BCAS2 was significantly associated with higher Gleason and pathology grades and shorter survival in patients with PCa. CONCLUSION: BCAS2 promotes two DSB repair pathways by interacting with NBS1, and it may affect PCa progression.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/fisiologia , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias da Próstata/metabolismo , Animais , Apoptose/genética , DNA/efeitos da radiação , Enzimas Reparadoras do DNA/metabolismo , Drosophila/genética , Humanos , Masculino , Gradação de Tumores , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologiaRESUMO
BACKGROUND: Cardiomyopathy is a common and lethal complication in patients with limb-girdle muscular dystrophy (LGMD), one of the most prevalent forms of muscular dystrophy. The pathogenesis underlying LGMD-related cardiomyopathy remains unclear. NRIP (gene name DCAF6), a Ca2+-dependent calmodulin binding protein, was reduced in dystrophic muscles from LGMD patients. Mice lacking NRIP exhibit a myopathic phenotype resembling that in LGMD patients, making NRIP deficiency a potential culprit leading to cardiomyopathy. This study aimed to determine if NRIP deficiency leads to cardiomyopathy and to explore the underlying molecular mechanisms. METHODS AND RESULTS: NRIP expression was reduced in both human and mouse failing hearts. Muscle-specific NRIP knockout (MCK-Cre::Dcaf6flox/flox) mouse heart and isolated cardiomyocytes exhibited markedly reduced contractility. Transmission electron microscopy revealed abnormal sarcomere structures and mitochondrial morphology in MCK-Cre::Dcaf6flox/flox hearts. Protein co-immunoprecipitation and confocal imaging revealed that NRIP interacts with α-actinin 2 (ACTN2) at the Z-disc. We found that NRIP facilitated ACTN2-mediated F-actin bundling, and that NRIP deficiency resulted in reduced binding between Z-disc proteins ACTN2 and Cap-Z. In addition, NRIP-deficiency led to increased mitochondrial ROS and impaired mitochondrial respiration/ATP production owing to elevated cellular NADH/NAD+ ratios. Treatment with mitochondria-directed antioxidant mitoTEMPO or NAD+ precursor nicotinic acid restored mitochondrial function and cardiac contractility in MCK-Cre::Dcaf6flox/flox mice. CONCLUSIONS: NRIP is essential to maintain sarcomere structure and mitochondrial/contractile function in cardiomyocytes. Our results revealed a novel role for NRIP deficiency in the pathogenesis of LGMD and heart failure. Targeting NRIP, therefore, could be a powerful new approach to treat myocardial dysfunction in LGMD and heart failure patients.
Assuntos
Cardiomiopatias/metabolismo , Mitocôndrias Cardíacas/metabolismo , Proteína 1 de Interação com Receptor Nuclear/metabolismo , Sarcômeros/metabolismo , Actinina/metabolismo , Actinas/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Cardiomiopatias/fisiopatologia , Respiração Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Insuficiência Cardíaca/genética , Homeostase/efeitos dos fármacos , Humanos , Masculino , Camundongos , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/ultraestrutura , Modelos Biológicos , Contração Miocárdica/efeitos dos fármacos , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , NAD/metabolismo , Niacina/farmacologia , Proteína 1 de Interação com Receptor Nuclear/química , Fenótipo , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Espécies Reativas de Oxigênio/metabolismo , Sarcômeros/efeitos dos fármacos , Sarcômeros/ultraestruturaRESUMO
BACKGROUND: Tendon stem/progenitor cells (TSPC) exhibit a low proliferative response to heal tendon injury, leading to limited regeneration outcomes. Exogenous growth factors that activate TSPC proliferation have emerged as a promising approach for treatment. Here, we evaluated the pigment epithelial-derived factor (PEDF)-derived short peptide (PSP; 29-mer) for treating acute tendon injury and to determine the timing and anatomical features of CD146- and necleostemin-positive TSPC in the tendon healing process. METHODS: Tendon cells were isolated from rabbit Achilles tendons, stimulated by the 29-mer and analyzed for colony-forming capacity. The expression of the TSPC markers CD146, Oct4, and nestin, induced by the 29-mer, was examined by immunostaining and western blotting. Tendo-Achilles injury was induced in rats by full-thickness insertion of an 18-G needle and immediately treated topically with an alginate gel, loaded with 29-mer. The distribution of TSPC in the injured tendon and their proliferation were monitored using immunohistochemistry with antibodies to CD146 and nucleostemin and by BrdU labeling. RESULTS: TSPC markers were enriched among the primary tendon cells when stimulated by the 29-mer. The 29-mer also induced the clonogenicity of CD146+ TSPC, implying TSPC stemness was retained during TSPC expansion in culture. Correspondingly, the expanded TSPC differentiated readily into tenocyte-like cells after removal of the 29-mer from culture. 29-mer/alginate gel treatment caused extensive expansion of CD146+ TSPC in their niche on postoperative day 2, followed by infiltration of CD146+/BrdU- TSPC into the injured tendon on day 7. The nucleostemin+ TSPC were located predominantly in the healing region of the injured tendon in the later phase (day 7) and exhibited proliferative capacity. By 3 weeks, 29-mer-treated tendons showed more organized collagen fiber regeneration and higher tensile strength than control tendons. In culture, the mitogenic effect of the 29-mer was found to be mediated by the phosphorylation of ERK2 and STAT3 in nucleostemin+ TSPC. CONCLUSIONS: The anatomical analysis of TSPC populations in the wound healing process supports the hypothesis that substantial expansion of resident TSPC by exogenous growth factor is beneficial for tendon healing. The study suggests that synthetic 29-mer peptide may be an innovative therapy for acute tendon rupture.
Assuntos
Tendão do Calcâneo/fisiopatologia , Proteínas do Olho/metabolismo , Fatores de Crescimento Neural/metabolismo , Peptídeos/metabolismo , Regeneração/genética , Serpinas/metabolismo , Células-Tronco/metabolismo , Traumatismos dos Tendões/terapia , Animais , Humanos , Coelhos , Ratos , Células-Tronco/citologiaRESUMO
BACKGROUND: Nuclear receptor interaction protein (NRIP) is a calcium/calmodulin (CaM) binding protein. Nuclear receptor interaction protein interacts with CaM to activate calcineurin and CaMKII signalling. The conventional NRIP knockout mice (global knockout) showed muscular abnormality with reduction of muscle oxidative functions and motor function defects. METHODS: To investigate the role of NRIP on neuromuscular system, we generated muscle-restricted NRIP knockout mice [conditional knockout (cKO)]. The muscle functions (including oxidative muscle markers and muscle strength) and lumbar motor neuron functions [motor neuron number, axon denervation, neuromuscular junction (NMJ)] were tested. The laser-captured microdissection at NMJ of skeletal muscles and adenovirus gene therapy for rescued effects were performed. RESULTS: The cKO mice showed muscular abnormality with reduction of muscle oxidative functions and impaired motor performances as global knockout mice. To our surprise, cKO mice also displayed motor neuron degeneration with abnormal architecture of NMJ. Specifically, the cKO mice revealed reduced motor neuron number with small neuronal size in lumbar spinal cord as well as denervating change, small motor endplates, and decreased myonuclei number at NMJ in skeletal muscles. To explore the mechanisms, we screened various muscle-derived factors and found that myogenin is a potential candidate that myogenin expression was lower in skeletal muscles of cKO mice than wild-type mice. Because NRIP and myogenin were colocalized around acetylcholine receptors at NMJ, we extracted RNA from synaptic and extrasynaptic regions of muscles using laser capture microdissection and showed that myogenin expression was especially lower at synaptic region in cKO than wild-type mice. Notably, overexpression of myogenin using intramuscular adenovirus encoding myogenin treatment rescued abnormal NMJ architecture and preserved motor neuron death in cKO mice. CONCLUSIONS: In summary, we demonstrated that deprivation of NRIP decreases myogenin expression at NMJ, possibly leading to abnormal NMJ formation, denervation of acetylcholine receptor, and subsequent loss of spinal motor neuron. Overexpression of myogenin in cKO mice can partially rescue abnormal NMJ architecture and motor neuron death. Therefore, muscular NRIP is a novel trophic factor supporting spinal motor neuron via stabilization of NMJ by myogenin expression.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Neurônios Motores/metabolismo , Miogenina/genética , Junção Neuromuscular/metabolismo , Proteínas Nucleares/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Biomarcadores , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , Distrofias Musculares/patologia , Miogenina/metabolismo , Degeneração Neural , Proteínas Nucleares/metabolismo , Fenótipo , Transdução GenéticaRESUMO
Hepatic stem/progenitor cells (HPC) have been considered as a potential cell source of an alternative to liver transplantation. Production of large numbers of autologous HPC from small pieces of live tissue is crucial for the application of HPC-based liver therapy. In this study, we demonstrated that a synthetic 44-amino acid peptide (44-mer) derived from pigment epithelium-derived factor (PEDF) can facilitate the production of a large number of actively dividing HPC from normal adult rat livers in a 35-day culture period. The phenotypic properties of HPC were characterized by morphological observation, colony formation and high expression of classical HPC markers including epithelial cell adhesion molecule (EpCAM), leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) and tumor-associated calcium signal transducer (TROP2). The 44-mer stimulated HPC proliferation in vitro and in mouse livers injured by a single intraperitoneal injection of carbon tetrachloride. In addition, the 44-mer induced the phosphorylation of ERK1/2 and STAT3 in HPC. Blocking the activity of ERK or STAT3 with pharmacological inhibitors attenuated the effects of the 44-mer on the induction of HPC proliferation. The long-term expanded HPC still possessed a bipotent ability to differentiate towards bile duct cells and mature hepatocytes. These results imply that the PEDF peptide may be a simple and effective agent to improve HPC-based liver therapy.
RESUMO
Both nuclear receptor interaction protein (NRIP) and DNA damage binding protein 2 (DDB2) belong to the Cullin 4 (CUL4)-DDB1 binding protein family and are androgen receptor (AR)-interacting proteins. Here, we investigated the expression patterns of the NRIP, DDB2 and AR proteins in human prostate cancer tissues and found that the expression levels of NRIP and AR were higher, but the DDB2 level was lower, in prostate cancer tissues than in non-neoplastic controls, suggesting NRIP as a candidate tumor promoter and DDB2 as a tumor suppressor in prostate cancer. Furthermore, both NRIP and DDB2 shared the same AR binding domain; they were competitors for the AR, but not for DDB1 binding, in the AR-DDB2-DDB1-CUL4A complex. Conclusively, NRIP stabilizes the AR protein by displacing DDB2 from the AR-DDB2 complex. Consistent with our hypothesis, a specific expression pattern with high levels of NRIP and AR, together with a low level of DDB2, was found more frequently in the human prostate cancer tissues with a cribriform pattern than in non-cribriform tumors, suggesting that disruption of the balance between NRIP and DDB2 may change AR protein homeostasis and contribute to pathogenesis in certain aggressive types of prostate cancer.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Receptores Androgênicos/metabolismo , Western Blotting , Proteínas Culina/metabolismo , Humanos , Imuno-Histoquímica , Imunoprecipitação , Masculino , Estabilidade Proteica , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Breast carcinoma amplified sequence 2 (BCAS2) is a core component of the hPrP19 complex that controls RNA splicing. Here, we performed an exon array assay and showed that ß-catenin is a target of BCAS2 splicing regulation. The regulation of dendrite growth and morphology by ß-catenin is well documented. Therefore, we generated conditional knockout (cKO) mice to eliminate the BCAS2 expression in the forebrain to investigate the role of BCAS2 in dendrite growth. BCAS2 cKO mice showed a microcephaly-like phenotype with a reduced volume in the dentate gyrus (DG) and low levels of learning and memory, as evaluated using Morris water maze analysis and passive avoidance, respectively. Golgi staining revealed shorter dendrites, less dendritic complexity and decreased spine density in the DG of BCAS2 cKO mice. Moreover, the cKO mice displayed a short dendrite length in newborn neurons labeled by DCX, a marker of immature neurons, and BrdU incorporation. To further examine the mechanism underlying BCAS2-mediated dendritic malformation, we overexpressed ß-catenin in BCAS2-depleted primary neurons and found that the dendritic growth was restored. In summary, BCAS2 is an upstream regulator of ß-catenin gene expression and plays a role in dendrite growth at least partly through ß-catenin.
Assuntos
Dendritos/metabolismo , Dendritos/patologia , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Prosencéfalo/anormalidades , Prosencéfalo/metabolismo , beta Catenina/metabolismo , Animais , Comportamento Animal/fisiologia , Disfunção Cognitiva/genética , Disfunção Cognitiva/metabolismo , Disfunção Cognitiva/patologia , Proteína Duplacortina , Éxons , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Células MCF-7 , Camundongos , Camundongos Knockout , Microcefalia/genética , Microcefalia/metabolismo , Microcefalia/patologia , Proteínas de Neoplasias/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Splicing de RNARESUMO
Fibrogenesis is induced by repeated injury to the liver and reactive regeneration and leads eventually to liver cirrhosis. Pigment epithelium derived factor (PEDF) has been shown to prevent liver fibrosis induced by carbon tetrachloride (CCl4). A 44 amino acid domain of PEDF (44-mer) was found to have a protective effect against various insults to several cell types. In this study, we investigated the capability of synthetic 44-mer to protect against liver injury in mice and in primary cultured hepatocytes. Acute liver injury, induced by CCl4, was evident from histological changes, such as cell necrosis, inflammation and apoptosis, and a concomitant reduction of glutathione (GSH) and GSH redox enzyme activities in the liver. Intraperitoneal injection of the 44-mer into CCl4-treated mice abolished the induction of AST and ALT and markedly reduced histological signs of liver injury. The 44-mer treatment can reduce hepatic oxidative stress as evident from lower levels of lipid hydroperoxide, and higher levels of GSH. CCl4 caused a reduction of Bcl-xL, PEDF and PPARγ, which was markedly restored by the 44-mer treatment. Consequently, the 44-mer suppressed liver fibrosis induced by repeated CCl4 injury. Furthermore, our observations in primary culture of rat hepatocytes showed that PEDF and the 44-mer protected primary rat hepatocytes against apoptosis induced by serum deprivation and TGF-ß1. PEDF/44-mer induced cell protective STAT3 phosphorylation. Pharmacological STAT3 inhibition prevented the antiapoptotic action of PEDF/44-mer. Among several PEDF receptor candidates that may be responsible for hepatocyte protection, we demonstrated that PNPLA2 was essential for PEDF/44-mer-mediated STAT3 phosphorylation and antiapoptotic activity by using siRNA to selectively knockdown PNPLA2. In conclusion, the PEDF 44-mer protects hepatocytes from single and repeated CCl4 injury. This protective effect may stem from strengthening the counter oxidative stress capacity and induction of hepatoprotective factors.
Assuntos
Proteínas do Olho/química , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Fatores de Crescimento Neural/química , Serpinas/química , Animais , Apoptose , Tetracloreto de Carbono , Linhagem Celular , Cricetinae , Proteínas do Olho/metabolismo , Feminino , Glutationa/metabolismo , Células Hep G2 , Humanos , Injeções Intraperitoneais , Peróxidos Lipídicos/química , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Crescimento Neural/metabolismo , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Ratos , Proteínas Recombinantes/química , Serpinas/metabolismoRESUMO
Previously, we showed that BCAS2 is essential for Drosophila viability and functions in pre-mRNA splicing. In this study, we provide strong evidence that BCAS2 regulates the activity of Delta-Notch signaling via Delta pre-mRNA splicing. Depletion of dBCAS2 reduces Delta mRNA expression and leads to accumulation of Delta pre-mRNA, resulting in diminished transcriptions of Delta-Notch signaling target genes, such as cut and E(spl)m8. Furthermore, ectopic expression of human BCAS2 (hBCAS2) and Drosophila BCAS2 (dBCAS2) in a dBCAS2-deprived fly can rescue dBCAS2 depletion-induced wing damage to the normal phenotypes. These rescued phenotypes are correlated with the restoration of Delta pre-mRNA splicing, which affects Delta-Notch signaling activity. Additionally, overexpression of Delta can rescue the wing deformation by deprivation of dBCAS2; and the depletion of dBCAS2 can restore the aberrant eye associated with Delta-overexpressing retinas; providing supporting evidence for the regulation of Delta-Notch signaling by dBCAS2. Taken together, dBCAS2 participates in Delta pre-mRNA splicing that affects the regulation of Delta-Notch signaling in Drosophila wing development.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Proteínas de Neoplasias/metabolismo , Precursores de RNA/metabolismo , Receptores Notch/metabolismo , Animais , Drosophila/crescimento & desenvolvimento , Proteínas de Drosophila/genética , Olho/metabolismo , Humanos , Proteínas de Neoplasias/genética , Fenótipo , Plasmídeos/genética , Plasmídeos/metabolismo , Precursores de RNA/genética , Splicing de RNA , Receptores Notch/genética , Transdução de Sinais , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/metabolismoRESUMO
The papillomavirus E2 protein is involved in the maintenance of persistent infection and known to bind either to cellular factors or directly to mitotic chromosomes in order to partition the viral genome into the daughter cells. However, how the HPV-16 E2 protein acts to facilitate partitioning of the viral genome remains unclear. In this study, we found that serine 243 of HPV-16 E2, located in the hinge region, is crucial for chromosome binding during mitosis. Bromodomain protein 4 (Brd4) has been identified as a cellular binding target through which the E2 protein of bovine papillomavirus type 1 (BPV-1) tethers the viral genome to mitotic chromosomes. Mutation analysis showed that, when the residue serine 243 was substituted by glutamic acid or aspartic acid, whose negative charges mimic the effect of constitutive phosphorylation, the protein still can interact with Brd4 and colocalize with Brd4 in condensed metaphase and anaphase chromosomes. However, substitution by the polar uncharged residues asparagine or glutamine abrogated Brd4 and mitotic chromosome binding. Moreover, following treatment with the inhibitor JQ1 to release Brd4 from the chromosomes, Brd4 and E2 formed punctate foci separate from the chromosomes, further supporting the hypothesis that the association of the HPV-16 E2 protein with the chromosomes is Brd4-dependent. In addition, the S243A E2 protein has a shorter half-life than the wild type, indicating that phosphorylation of the HPV-16 E2 protein at serine 243 also increases its half-life. Thus, phosphorylation of serine 243 in the hinge region of HPV-16 E2 is essential for interaction with Brd4 and required for host chromosome binding.
Assuntos
Proteínas de Ligação a DNA/química , Papillomavirus Humano 16/genética , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas Virais/química , Serina/química , Fatores de Transcrição/metabolismo , Anáfase , Animais , Ácido Aspártico/química , Células COS , Proteínas de Ciclo Celular , Chlorocebus aethiops , Genoma Viral , Ácido Glutâmico/química , Glutamina/química , Células HEK293 , Humanos , Metáfase , Mutação , Fenótipo , Fosforilação , Ligação ProteicaRESUMO
Limbal epithelial stem cell (LSC) transplantation is a prevalent therapeutic method for patients with LSC deficiency. The maintenance of stem cell characteristics in the process of culture expansion is critical for the success of ocular surface reconstruction. Pigment epithelial-derived factor (PEDF) increased the numbers of holoclone in LSC monolayer culture and preserved the stemness of LSC in suspension culture by evidence of ΔNp63α, Bmi-1, and ABCG2 expression. BrdU pulse-labeling assay also demonstrated that PEDF stimulated LSCs proliferation. In air-lift culture of limbal equivalent, PEDF was capable of increasing the numbers of ΔNp63α-positive cells. The mitogenic effect of PEDF was found to be mediated by the phosphorylations of p38 MAPK and STAT3 in LSCs. Synthetic 44-mer PEDF (residues 78-121) was as effective as the full length PEDF in LSC expansion in suspension culture and limbal equivalent formation, as well as the activation of p38 MAPK and STAT3. In mice subjecting to mechanical removal of cornea epithelium, 44-mer PEDF facilitated corneal wound healing. Microscopically, 44-mer PEDF advanced the early proliferative response in limbus, increased the proliferation of ΔNp63α-positive cells both in limbus and in epithelial healing front, and assisted the repopulation of limbus in the late phase of wound healing. In conclusion, the capability of expanding LSC in cell culture and in animal indicates the potential of PEDF and its fragment (e.g., 44-mer PEDF) in ameliorating limbal stem cell deficiency; and their uses as therapeutics for treating corneal wound.
Assuntos
Epitélio Corneano/patologia , Proteínas do Olho/farmacologia , Limbo da Córnea/citologia , Fatores de Crescimento Neural/farmacologia , Serpinas/farmacologia , Células-Tronco/citologia , Cicatrização/efeitos dos fármacos , Animais , Bromodesoxiuridina/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Epitélio Corneano/efeitos dos fármacos , Imunofluorescência , Humanos , Camundongos , Mitógenos/farmacologia , Células NIH 3T3 , Peptídeos/farmacologia , Coelhos , Fator de Transcrição STAT3/metabolismo , Células-Tronco/efeitos dos fármacos , Células-Tronco/enzimologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Here, we show that dBCAS2 (CG4980, human Breast Carcinoma Amplified Sequence 2 ortholog) is essential for the viability of Drosophila melanogaster. We find that ubiquitous or tissue-specific depletion of dBCAS2 leads to larval lethality, wing deformities, impaired splicing, and apoptosis. More importantly, overexpression of hBCAS2 rescues these defects. Furthermore, the C-terminal coiled-coil domain of hBCAS2 binds directly to CDC5L and recruits hPrp19/PLRG1 to form a core complex for splicing in mammalian cells and can partially restore wing damage induced by knocking down dBCAS2 in flies. In summary, Drosophila and human BCAS2 share a similar function in RNA splicing, which affects cell viability.