RESUMO
The skeletal muscle is an endocrine organ that produces proteins and peptides, collectively termed as myokines. The temperature of skeletal muscles varies during exercise and/or with changes in ambient temperature. However, whether myokine secretion is regulated by heat stimulation is unclear. Thus, we aimed to explore the effects of environmental heat stimulation on myokine secretion. We initially investigated the secretome of C2C12 myotubes and identified several novel heat-responsive myokines. The concentration of C-C motif chemokine ligand 5 (CCL5) dramatically decreased by 0.3-fold in response to heat stress. After 3 h heat stimulation of C2C12 cells, the expression of heat shock protein 70 was induced, and the gene expression and secretion of CCL5 was significantly attenuated in C2C12 cells. We then examined the effects of acute heat stress on serum CCL5 levels in mice and Ccl5 gene expression in skeletal muscles. Mice were maintained at 23°C, exposed to 45°C for 30 min, and then returned to the 23°C chamber for recovery. The expression of Ccl5 in the skeletal muscle significantly decreased after 3 h of recovery. Serum CCL5 levels increased by approximately 1.9-fold after 30 min of heat exposure and then significantly decreased by approximately 0.7-fold after 23 h of recovery. This study suggests that heat stimulation decreases CCL5 secretion from the skeletal muscle in vitro and in vivo. Given its fundamental role in inflammation by recruiting several immune cells, CCL5 has a potential role in controlling inflammatory responses in the body after heat stimulation.
Assuntos
Fibras Musculares Esqueléticas , Músculo Esquelético , Animais , Camundongos , Ligantes , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Expressão Gênica , Quimiocinas/metabolismoRESUMO
Progranulin (PGRN) is a multifunctional growth factor expressed in central nervous system. Although PGRN expression is regulated by various stressors, its precise role(s) and regulatory mechanism(s) remain elusive. In this study, we used HT22 cells to investigate the physiological implications of oxidative stress-induced PGRN expression and the regulation of PGRN expression by oxidative stress. We observed that p38 MAP kinase was activated upon the addition of H2O2, and a selective p38 MAP kinase inhibitor attenuated PGRN induction by H2O2. To explore the physiological role(s) of the PGRN induction, we first confirmed H2O2-dependent responses of HT22 cells and found that the length and number of neurites were increased by H2O2. Pgrn knockdown experiments suggested that these changes were mediated by H2O2-induced PGRN expression, at least in part. Overall, the results suggested that an increase in oxidative stress in HT22 cells induced PGRN expression via p38 MAP kinase pathway, thereby controlling neurite outgrowth.
Assuntos
Peróxido de Hidrogênio , Progranulinas , Animais , Sistema de Sinalização das MAP Quinases , Crescimento NeuronalRESUMO
Evidence suggests that exercise can regulate skin functions such as promoting wound healing and inhibiting aging. Physical exercise modulates the secretion of proteins and peptides from skeletal muscles, called myokines, which play a role in transmitting exercise signals throughout the body. Therefore, exercise-regulated myokines may play a role in controlling skin functions; however, the precise mechanisms remain elusive. In this study, we focused on the recently identified CXC motif chemokine ligand 10 (CXCL10), an exercise-reduced myokine, and attempted to elucidate its role in regulating collagen synthesis in dermal fibroblasts. Mouse C2C12 myotubes were stimulated with or without electrical pulse stimulation (EPS) to induce contraction for 24 h, and conditioned medium was collected (EPS-CM or Ctrl-CM, respectively). The reduction in CXCL10 concentration by EPS was confirmed using ELISA. Next, mouse dermal fibroblasts were isolated from the dorsal skin of C57BL6/J mice (2 weeks old) and were stimulated with Ctrl-CM or EPS-CM for 24 h. EPS-CM treatment significantly increased collagen production compared to Ctrl-CM treatment. Even in the Ctrl-CM condition, the addition of an antagonist for CXCR3 (CXCL10 receptor) increased collagen production. In contrast, recombinant CXCL10 abolished EPS-CM-dependent collagen induction. Overall, this study raises the possibility that CXCL10 secretion from skeletal muscles may control collagen production in mouse dermal fibroblasts.
Assuntos
Quimiocina CXCL10/metabolismo , Colágeno/metabolismo , Fibroblastos/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Pele/metabolismo , Animais , Linhagem Celular , Estimulação Elétrica , Camundongos , Contração MuscularRESUMO
Skeletal muscles produce secretory factors termed as myokines, which alter physiological functions of target tissues. We recently identified C-X-C chemokine ligand 10 (CXCL10) as a novel myokine, which is downregulated in response to exercise. In the present study, we investigated whether the nutritional changes affect CXCL10 expression in mouse skeletal muscle. Expression of CXCL10 was evaluated in mice fed a normal diet or a high fat diet for 10 weeks. In animals fed on HFD, Cxcl10 expression was significantly induced in fast-twitched muscles, and was accompanied by increased blood glucose and free fatty acid levels. In vitro experiments using C2C12 myotubes suggested that the increased levels of glucose and palmitic acids directly enhanced CXCL10 expression. Interestingly, the effect of palmitic acids was attenuated by palmitoleic acids. Considering its potent angiostatic activity, induction of CXCL10 by nutritional changes may contribute to the impairment of microvascular networks in skeletal muscles.
Assuntos
Quimiocina CXCL10/metabolismo , Ácidos Graxos não Esterificados/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Músculo Esquelético/citologia , Animais , Relação Dose-Resposta a Droga , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismoRESUMO
Skeletal muscle is an endocrine organ that secretes several proteins, which are collectively termed myokines. Although many studies suggest that exercise regulates myokine secretion, the underlying mechanisms remain unclear and all the exercise-dependent myokines have not yet been identified. Therefore, in this study, we attempted to identify novel exercise-dependent myokines by using our recently developed in vitro contractile model. Differentiated C2C12 myotubes were cultured with or without electrical pulse stimulation (EPS) for 24â¯h to induce cell contraction, and the myokines secreted in conditioned medium were analyzed using a cytokine array. Although most myokine secretions were not affected by EPS, the secretion of Chemokine (C-C motif) ligand 5 (CCL5) (regulated on activation, normal T cell expressed and secreted (RANTES)) was significantly reduced by EPS. This was further confirmed by ELISA and quantitative PCR. Contraction-dependent calcium transients and activation of 5'-AMP activating protein kinase (AMPK) appears to be involved in this decrease, as the chelating Ca2+ by EGTA blocked contraction-dependent CCL5 reduction, whereas the pharmacological activation of AMPK significantly reduced it. However, Ccl5 gene expression was increased by AMPK activation, suggesting that AMPK-dependent CCL5 decrease occurred via post-transcriptional regulation. Finally, mouse experiments revealed that voluntary wheel-running exercise reduced serum CCL5 levels and Ccl5 gene expression in the fast-twitch muscles. Overall, our study provides the first evidence of an exercise-reducible myokine, CCL5, in the mouse skeletal muscle. Although further studies are required to understand the precise roles of the skeletal muscle cell contraction-induced decrease in CCL5, this decrease may explain some exercise-dependent physiological changes such as those in immune responses.
Assuntos
Quimiocina CCL5/fisiologia , Contração Muscular , Músculo Esquelético/fisiologia , Condicionamento Físico Animal , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Células Cultivadas , Quimiocina CCL5/genética , Citocinas/genética , Citocinas/metabolismo , Estimulação Elétrica , Expressão Gênica , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Accumulating evidence indicates that skeletal muscle secrets proteins referred to as myokines and that exercise contributes to their regulation. In this study, we propose that chemokine (C-X-C motif) ligand 10 (CXCL10) functions as a novel myokine. Initially, we stimulated differentiated C2C12 myotubes with or without electrical pulse stimulation (EPS) to identify novel myokines. Cytokine array analysis revealed that CXCL10 secretion was significantly reduced by EPS, which was further confirmed by enzyme-linked immunosorbent assay and quantitative polymerase chain reaction analysis. Treadmill experiments in mice identified significant reduction of Cxcl10 gene expression in the soleus muscle. Additionally, contraction-dependent p38 MAPK activation appeared to be involved in this reduction. Furthermore, C2C12 conditioned medium obtained after applying EPS could induce survival of MSS31, a vascular endothelial cell model, which was partially attenuated by the addition of recombinant CXCL10. Overall, our findings suggest CXCL10 as a novel exercise-reducible myokine, to control endothelial cell viability.
Assuntos
Quimiocina CXCL10/fisiologia , Teste de Esforço , Fibras Musculares Esqueléticas/fisiologia , Indutores da Angiogênese , Animais , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Sistema de Sinalização das MAP Quinases , Camundongos , Contração Muscular , Condicionamento Físico Animal , Reação em Cadeia da PolimeraseRESUMO
In skeletal muscle, sortilin plays a predominant role in the sorting of glucose transporter 4 (Glut4), thereby controlling glucose uptake. Moreover, our previous study suggested that the sortilin expression levels are also implicated in myogenesis. Despite the importance of sortilin in skeletal muscle, however, the regulation of sortilin expression has not been completely understood. In the present study, we analyzed if the sortilin expression is regulated by glucose in C2C12 myocytes and rat skeletal muscles in vivo. Sortilin protein expression was elevated upon C2C12 cell differentiation and was further enhanced in the presence of a high concentration of glucose. The gene expression and protein degradation of sortilin were not affected by glucose. On the other hand, rapamycin partially reduced sortilin induction by a high concentration of glucose, which suggested that sortilin translation could be regulated by glucose, at least in part. We also examined if the sortilin regulation by glucose was also observed in skeletal muscles that were obtained from fed or fasted rats. Sortilin expression in both gastrocnemius and extensor digitorum longus (EDL) muscle was significantly decreased by 17-18h of starvation. On the other hand, pathological levels of high blood glucose did not alter the sortilin expression in rat skeletal muscle. Overall, the present study suggests that sortilin protein levels are reduced under hypoglycemic conditions by post-transcriptional control in skeletal muscles.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Glicemia/análise , Diabetes Mellitus Experimental/metabolismo , Regulação para Baixo , Jejum/metabolismo , Músculo Esquelético/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/agonistas , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Diferenciação Celular , Linhagem Celular , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/patologia , Regulação para Baixo/efeitos dos fármacos , Privação de Alimentos , Glucose/metabolismo , Membro Posterior , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/metabolismo , Células Musculares/citologia , Células Musculares/efeitos dos fármacos , Células Musculares/metabolismo , Células Musculares/patologia , Músculo Esquelético/citologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/metabolismo , Ratos Wistar , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Extracellular glutamate concentration is a critical determinant of neuronal cell fate. We recently demonstrated that HT22 murine hippocampal cell viability was reduced by exposure to high concentrations of glutamate, whereas low concentrations promoted cell survival. Extracellular signal-regulated kinase (Erk)1/2 activation by glutamate is important for both glutamate-induced cell death and survival. In this study, we investigated the role of glutamate-induced or hydrogen peroxide (H2O2)-induced Erk1/2 activation in HT22 cell fate determination. Glutamate and H2O2 treatment similarly induced early (<1 h) Erk1/2 phosphorylation regardless of concentration. On the other hand, persistent Erk1/2 phosphorylation (16-24 h) was observed only in the presence of excess glutamate. Only the latter contributed to glutamate-induced cell death, which involved metabolic glutamate receptor 5. Our findings suggest that glutamate concentration modulates two distinct phases of Erk1/2 activation, which can explain the glutamate concentration-dependent determination of HT22 cell fate.
Assuntos
Morte Celular , Ácido Glutâmico/farmacologia , Hipocampo/metabolismo , Sistema de Sinalização das MAP Quinases , Animais , Linhagem Celular , Ativação Enzimática , Hipocampo/citologia , Camundongos , Fosforilação , Receptor de Glutamato Metabotrópico 5RESUMO
Muscle mass is determined between protein synthesis and protein degradation. Reduction of muscle mass leads to bedridden condition and attenuation of resistance to diseases. Moreover, bedridden condition leads to additional muscle loss due to disuse muscle atrophy. In our previous study (Sato et al. 2013), we showed that administered lysine (Lys), one of essential amino acid, suppressed protein degradation in skeletal muscle. In this study, we investigated that the mechanism of the suppressive effects of Lys on skeletal muscle proteolysis in C2C12 cell line. C2C12 myotubes were incubated in the serum-free medium containing 10 mM Lys or 20 mM Lys, and myofibrillar protein degradation was determined by the rates of 3-methylhistidine (MeHis) release from the cells. The mammalian target of rapamycin (mTOR) activity from the phosphorylation levels of p70-ribosormal protein S6 kinase 1 and eIF4E-binding protein 1 and the autophagic-lysosomal system activity from the ratio of LC3-II/I in C2C12 myotubes stimulated by 10 mM Lys for 0-3 h were measured. The rates of MeHis release were markedly reduced by addition of Lys. The autophagic-lysosomal system activity was inhibited upon 30 min of Lys supplementation. The activity of mTOR was significantly increased upon 30 min of Lys supplementation. The suppressive effect of Lys on the proteolysis by the autophagic-lysosomal system was maintained partially when mTOR activity was inhibited by 100 nM rapamycin, suggesting that some regulator other than mTOR signaling, for example, Akt, might also suppress the autophagic-lysosomal system. From these results, we suggested that Lys suppressed the activity of the autophagic-lysosomal system in part through activation of mTOR and reduced myofibrillar protein degradation in C2C12 myotubes.
Assuntos
Autofagia/efeitos dos fármacos , Lisina/farmacologia , Lisossomos/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteólise/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Fatores de Iniciação em Eucariotos , Regulação da Expressão Gênica/efeitos dos fármacos , Metilistidinas/farmacologia , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Fibras Musculares Esqueléticas/efeitos dos fármacos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miofibrilas/efeitos dos fármacos , Miofibrilas/metabolismo , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Fatores de Tempo , Proteínas com Motivo Tripartido , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Oxidative stress is recognized as one of the pathogenic mechanisms involved in neurodegenerative disease. However, recent evidence has suggested that regulation of cellular fate in response to oxidative stress appears to be dependent on the stress levels. In this study, using HT22 cells, we attempted to understand how an alteration in the oxidative stress levels would influence neuronal cell fate. HT22 cell viability was reduced with exposure to high levels of oxidative stress, whereas, low levels of oxidative stress promoted cell survival. Erk1/2 activation induced by a low level of oxidative stress played a role in this cell protective effect. Intriguingly, subtoxic level of H2O2 induced expression of a growth factor, progranulin (PGRN), and exogenous PGRN pretreatment attenuated HT22 cell death induced by high concentrations of H2O2 in Erk1/2-dependent manner. Together, our study indicates that two different cell protection mechanisms are activated by differing levels of oxidative stress in HT22 cells.
Assuntos
Hipocampo/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Doenças Neurodegenerativas/metabolismo , Estresse Oxidativo , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Granulinas , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Humanos , Peróxido de Hidrogênio/toxicidade , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/patologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , ProgranulinasRESUMO
Muscle contractile activity functions as a potent stimulus for acute interleukin (IL)-6 expression in working skeletal muscles. Recently, we established an "in vitro contraction model" using highly-developed contractile C2C12 myotubes by applying electric pulse stimulation (EPS). Herein, we characterize the effects of EPS-evoked contraction on IL-6 expression in contractile C2C12 myotubes. Both secretion and mRNA expression of IL-6 were significantly up-regulated by EPS in a frequency-dependent manner in contracting myotubes during a 24-h period, and the response was blunted by cyclosporine A, a calcineurin inhibitor. Longer time (~12h) was required for the induction of IL-6 after the initiation of EPS as compared to that of other contraction-inducible CXC chemokines such as CXCL1/KC, which were induced in less than 3 hours. Furthermore, these acute inducible CXC chemokines exhibited no autocrine effect on IL-6 expression. Importantly, contraction-dependent IL-6 up-regulation was markedly suppressed in the presence of high levels of glucose along with increased glycogen accumulations. Experimental manipulation of intracellular glycogen contents by modulating available glucose or pyruvate during a certain EPS period further established the suppressive effect of glycogen accumulations on contraction-induced IL-6 up-regulation, which appeared to be independent of calcineurin activity. We also document that EPS-evoked contractile activity improved insulin-responsiveness in terms of intracellular glycogen accumulations. Taken together, these data provide important insights into the regulation of IL-6 expression in response to contractile activity of muscle cells, which is difficult to examine using in vivo experimental techniques. Our present results thus expand the usefulness of our "in vitro contraction model".
Assuntos
Glicogênio/metabolismo , Resistência à Insulina , Interleucina-6/metabolismo , Contração Muscular , Fibras Musculares Esqueléticas/metabolismo , Regulação para Cima , Animais , Calcineurina/metabolismo , Inibidores de Calcineurina , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Quimiocinas CXC/antagonistas & inibidores , Quimiocinas CXC/metabolismo , Estimulação Elétrica , Glucose/metabolismo , Hiperglicemia/metabolismo , Hipoglicemiantes/metabolismo , Hipoglicemiantes/farmacologia , Imunossupressores/farmacologia , Insulina/metabolismo , Insulina/farmacologia , Interleucina-6/genética , Cinética , Camundongos , Contração Muscular/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/imunologia , RNA Mensageiro/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
The disturbance of intercellular communication is one of the hallmarks of aging. The goal of this study is to clarify the impact of chronological aging on extracellular vesicles (EVs), a key mode of communication in mammalian tissues. We focused on epidermal keratinocytes, the main cells of the outer protective layer of the skin which is strongly impaired in the skin of elderly. EVs were purified from conditioned medium of primary keratinocytes isolated from infant or aged adult skin. A significant increase of the relative number of EVs released from aged keratinocytes was observed whereas their size distribution was not modified. By small RNA sequencing, we described a specific microRNA (miRNA) signature of aged EVs with an increase abundance of miR-30a, a key regulator of barrier function in human epidermis. EVs from aged keratinocytes were found to be able to reduce the proliferation of young keratinocytes, to impact their organogenesis properties in a reconstructed epidermis model and to slow down the early steps of skin wound healing in mice, three features observed in aged epidermis. This work reveals that intercellular communication mediated by EVs is modulated during aging process in keratinocytes and might be involved in the functional defects observed in aged skin.
Assuntos
Vesículas Extracelulares , MicroRNAs , Idoso , Humanos , Animais , Camundongos , MicroRNAs/genética , Queratinócitos , Epiderme , Envelhecimento/genética , Mamíferos/genéticaRESUMO
Physical exercise triggers the release of several cytokines/chemokines from working skeletal muscles, but the underlying mechanism(s) by which skeletal muscles decipher and respond to highly complex contractile stimuli remains largely unknown. In an effort to investigate the regulatory mechanisms of the expressions of two contraction-inducible CXC chemokines, CXCL1/KC and CXCL5/LIX, in contracting skeletal muscle cells, we took advantage of our in vitro exercise model using highly developed contractile C(2)C(12) myotubes, which acquire properties similar to those of in vivo skeletal muscle via manipulation of Ca(2+) transients with electric pulse stimulation (EPS). Production of these CXC chemokines was immediately augmented by EPS-evoked contractile activity in a manner dependent on the activities of JNK and NF-kappaB, but not p38, ERK1/2, or calcineurin. Intriguingly, exposure of myotubes to cyclic mechanical stretch also induced expression of these CXC chemokines; however, a much longer period of stimulation (approximately 12 h) was required, despite rapid JNK phosphorylation. We also demonstrate herein that CXCL1/KC and CXCL5/LIX have the ability to raise intracellular Ca(2+) concentrations via CXCR2-mediated activation of pertussis toxin-sensitive Galpha(i) proteins in C(2)C(12) myoblasts, an action at least partially responsible for their migration and differentiation. Although we revealed a possible negative feedback regulation of their own production in response to the contractile activity in differentiated myotubes, exogenous administration of these CXC chemokines did not acutely influence either insulin-induced Akt phosphorylation or GLUT4 translocation in C(2)C(12) myotubes. Taken together, these data shed light on the fundamental characteristics of contraction-inducible CXC chemokine production and their potential roles in skeletal muscle cells.
Assuntos
Quimiocinas CXC/fisiologia , Contração Muscular/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Animais , Western Blotting , Cálcio/metabolismo , Linhagem Celular , Movimento Celular/fisiologia , Quimiocina CXCL1/genética , Quimiocina CXCL1/metabolismo , Quimiocina CXCL5/genética , Quimiocina CXCL5/metabolismo , Quimiocinas CXC/biossíntese , Estimulação Elétrica , Ensaio de Imunoadsorção Enzimática , Mecanorreceptores/fisiologia , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/fisiologia , NF-kappa B/metabolismo , Estimulação Física , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA/biossíntese , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Bioactivities of IGFs in various cells are often potentiated in the presence of other hormones. In previous studies we showed that pretreatment of rat FRTL-5 thyroid cells with TSH or other cAMP-generating agents markedly potentiated DNA synthesis induced by IGF-I. Under these conditions we found that phosphatidylinositol (PI) 3-kinase was activated in response to either cAMP or IGF stimulus, and both activation modes were indispensable for the potentiation of DNA synthesis. The present studies were undertaken to elucidate how cAMP and/or IGF-I stimulus regulated the G1 cyclin-cyclin dependent kinase (CDK)-inhibitor system, and to determine the roles of PI 3-kinase activation by cAMP or IGF-I stimulus in this system. We found that cAMP pretreatment enhanced IGF-I-dependent increases in cyclin D1, due to synergistic increases in mRNA and elevation of translation rates. Furthermore, cAMP pretreatment enhanced IGF-I-induced protein degradation of the CDK inhibitor, p27(Kip1). These changes well explained an increase in cyclin E, leading to marked activation of G1 CDKs, followed by retinoblastoma protein phosphorylation. Our results using a PI 3-kinase inhibitor showed that cAMP-dependent PI 3-kinase activation plays an important role in the increase in cyclin D1 translation. In contrast, IGF-I-dependent PI 3-kinase activation was required for the increase in cyclin D1 mRNA levels and degradation of p27(Kip1). Together, the present study elucidates the role of cAMP and IGF-I in differentially activating PI 3-kinase as a mediator of multiple molecular events. These events converge in the regulation of cyclin D1 and p27(Kip1), leading to cAMP-dependent potentiation of IGF-I-dependent CDK activation and DNA synthesis.
Assuntos
AMP Cíclico/farmacologia , Ciclina D1/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Northern Blotting , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Ciclina D1/genética , Quinases Ciclina-Dependentes/metabolismo , Replicação do DNA/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Immunoblotting , Imunoprecipitação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RatosRESUMO
Since cAMP blocks meiotic maturation of mammalian and amphibian oocytes in vitro and cyclic nucleotide phosphodiesterase 3A (PDE3A) is primarily responsible for oocyte cAMP hydrolysis, we generated PDE3A-deficient mice by homologous recombination. The Pde3a(-/-) females were viable and ovulated a normal number of oocytes but were completely infertile, because ovulated oocytes were arrested at the germinal vesicle stage and, therefore, could not be fertilized. Pde3a(-/-) oocytes lacked cAMP-specific PDE activity, contained increased cAMP levels, and failed to undergo spontaneous maturation in vitro (up to 48 hours). Meiotic maturation in Pde3a(-/-) oocytes was restored by inhibiting protein kinase A (PKA) with adenosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-cAMPS) or by injection of protein kinase inhibitor peptide (PKI) or mRNA coding for phosphatase CDC25, which confirms that increased cAMP-PKA signaling is responsible for the meiotic blockade. Pde3a(-/-) oocytes that underwent germinal vesicle breakdown showed activation of MPF and MAPK, completed the first meiotic division extruding a polar body, and became competent for fertilization by spermatozoa. We believe that these findings provide the first genetic evidence indicating that resumption of meiosis in vivo and in vitro requires PDE3A activity. Pde3a(-/-) mice represent an in vivo model where meiotic maturation and ovulation are dissociated, which underscores inhibition of oocyte maturation as a potential strategy for contraception.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Infertilidade Feminina , 3',5'-AMP Cíclico Fosfodiesterases/genética , Animais , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3 , Inibidores Enzimáticos/metabolismo , Feminino , Humanos , Infertilidade Feminina/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Fator Promotor de Maturação/metabolismo , Meiose/fisiologia , Mesotelina , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oócitos/citologia , Oócitos/fisiologia , Ovário/anatomia & histologia , Ovário/fisiologia , Ovulação/fisiologia , Sistemas do Segundo Mensageiro/fisiologiaRESUMO
Progranulin (PGRN) is a growth factor implicated in several neurodegenerative diseases, such as frontotemporal lobar degeneration. Despite its important role in the central nervous system (CNS), the mechanisms controlling PGRN expression in the CNS are largely unknown. Recent evidence, however, suggested that several stressors, such as hypoxia, acidosis, or oxidative stress, induce PGRN expression. The present study was mainly aimed at determining whether and, if so, how glucose deprivation affects PGRN expression in PC12 cells. Initially, it was found that glucose deprivation gradually induced PGRN gene expression in PC12 cells. To elucidate the underlying molecular mechanisms, several intracellular signalings that were modified in response to glucose deprivation were examined. Both adenosine monophosphate kinase (AMPK) activation and changes in osmotic pressure, which are modified by extracellular glucose concentration, had no effect on PGRN gene expression; on the other hand, p38 activation in response to glucose deprivation played an important role in inducing PGRN gene expression. It was also found that expression of sortilin, a PGRN receptor implicated in PGRN endocytosis, was dramatically reduced by glucose deprivation. In contrast to glucose-dependent regulation of PGRN gene expression, AMPK activation played a central role in reducing sortilin expression. Overall, the present study suggests that the PGRN-sortilin axis is modulated by glucose deprivation via two distinct mechanisms. As PGRN is neuroprotective, this system may represent a new neuroprotective mechanism activated by glucose deprivation in the CNS.
RESUMO
BACKGROUND: Oxidative stress is one of the mechanisms underlying pathogenesis in neurodegenerative diseases such as Alzheimer's disease. Generally, oxidative stress represents cell toxicity; however, we recently found that oxidative stress promotes the expression of growth factor progranulin (PGRN) in HT22 murine hippocampus cells, thereby protecting the HT22 cells. In this study, we attempted to clarify whether a similar system exists in the other neuronal cell model, rat pheochromocytoma (PC12) cells. RESULTS: After confirming that high concentrations of hydrogen peroxide (H2O2; 100-250 µM) initiate PC12 cell death, we analyzed growth factor expressional changes after H2O2 treatment. We found, intriguingly, that gene expression of brain-derived neurotrophic factor (BDNF), but not PGRN was significantly induced by H2O2. Although little expression of the high affinity BDNF receptor tropomyosin-related kinase TrkB was observed in PC12 cells, expression of low affinity neurotrophin receptor, p75NTR, was clearly observed. This BDNF signaling appeared to contribute to PC12 cell protection, since PC12 cell death was significantly attenuated by BDNF treatment. CONCLUSIONS: Based on our results, we conclude that the induction of BDNF by subtoxic levels of H2O2 and its signaling may have roles in PC12 cell protection.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/genética , Regulação da Expressão Gênica , Peróxido de Hidrogênio/farmacologia , Animais , Fator Neurotrófico Derivado do Encéfalo/agonistas , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Morte Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas do Tecido Nervoso , Estresse Oxidativo , Células PC12 , Progranulinas , Ratos , Receptor trkB/genética , Receptor trkB/metabolismo , Receptores de Fatores de Crescimento , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/metabolismo , Transdução de SinaisRESUMO
Nutrient availability is one of the most important signals regulating cellular fates including cell growth, differentiation, and death. Recent evidence suggests that the NAD(+)-dependent histone deacetylase sirtuin 1 (SIRT1) plays a prominent role in linking changes in nutritional availability with cellular fate regulation. SIRT1 expression is observed in neurons, yet the expressional and functional regulation of this protein is not fully understood. In the present study, we examined whether extracellular glucose concentration affects the expression and localization of SIRT1 in PC12 cells. Further, we examined levels of forkhead box O3a (FoxO3a), which is also controlled by changes in extracellular glucose concentration. We observed the total expression levels of SIRT1 and FoxO3a in PC12 cells were reduced when glucose availability increased via gene expressional control, at least in part. Nuclear localization of SIRT1 and FoxO3a was increased by glucose deprivation. Even though the changes in extracellular glucose concentration regulated SIRT1 and FoxO3a in a similar direction, the effects of nerve growth factor on these two proteins were completely different. Finally, we found the potent SIRT1 inhibitor enhanced glucose deprivation-induced cell death. Therefore, we propose that glucose deprivation-induced SIRT1 expression potentially plays a major role in protecting PC12 cells.
Assuntos
Glucose/deficiência , Neurônios/metabolismo , Sirtuína 1/metabolismo , Animais , Western Blotting , Imunofluorescência , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead , Células PC12 , Ratos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
We previously demonstrated that long-term pretreatment of rat FRTL-5 thyroid cells with TSH or cAMP-generating reagents potentiated IGF-I-dependent DNA synthesis. Under these conditions, cAMP treatment increased tyrosine phosphorylation of a 125-kDa protein (p125) and its association with a p85 regulatory subunit of phosphatidylinositol 3-kinase (p85 PI3K), which were suggested to mediate potentiation of DNA synthesis. This study was undertaken to identify p125 and to elucidate its roles in potentiation of DNA synthesis induced by IGF-I. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis revealed p125 to be a rat ortholog of human XB130, which we named PI3K-associated protein (PI3KAP). cAMP treatment elevated PI3KAP/XB130 mRNA and protein levels as well as tyrosine phosphorylation and interaction with p85 PI3K leading to increased PI3K activities associated with PI3KAP/XB130, supporting the role of PI3KAP/XB130 in DNA synthesis potentiation. Importantly, PI3KAP/XB130 knockdown attenuated cAMP-dependent potentiation of IGF-I-induced DNA synthesis. Furthermore, c-Src was associated with PI3KAP/XB130 and was activated in response to cAMP. Addition of Src family kinase inhibitors, PP1 or PP2, during cAMP treatment abolished tyrosine phosphorylation of PI3KAP/XB130 and its interaction with p85 PI3K. Finally, introduction of PI3KAP/XB130 into NIH3T3 fibroblasts lacking endogenous PI3KAP/XB130 enhanced IGF-I-induced DNA synthesis; however, a mutant Y72F incapable of binding to p85 PI3K did not show this response. Together, these data indicate that cAMP-dependent induction of PI3KAP/XB130, which is associated with PI3K, is required for enhancement of IGF mitogenic activities.
Assuntos
Proteínas de Transporte/metabolismo , AMP Cíclico/fisiologia , Fator de Crescimento Insulin-Like I/fisiologia , Mitógenos/fisiologia , Glândula Tireoide/citologia , Animais , Proteínas de Transporte/genética , Ciclo Celular , Proliferação de Células , Replicação do DNA , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Ratos , Ratos Wistar , Análise de Sequência de DNA , Tireotropina/fisiologia , Quinases da Família src/metabolismoRESUMO
Progranulin (PGRN) is a growth modulating factor released by a variety of cells. This molecule has gained the attention of the neuroscience community with recent discoveries of multifunctional roles of PGRN in normal brain and neurodegenerative disorders. We focus on novel roles of PGRN as a sex steroid-responsible gene in the developing and adult rodent brain. While the developing brain is feminine by default, hormone exposure, including androgen and estrogen, induces masculinization during the critical period. We have shown that PGRN is a sex steroid-responsible gene that may be involved in masculinization of the perinatal rat brain. We also found that in adult rats PGRN gene expression was up-regulated by estrogen in the hippocampus, suggesting that PGRN may mediate the mitogenic effects of estrogen in the active area of neurogenesis. Since it has been recently reported that mutations in PGRN gene are responsible for a type of frontotemporal lobar degeneration in humans, PGRN appears to be also involved in modulating neurodegeneration. Together, PGRN gene expression is induced by estrogen in both developing and adult brains, and it may play multifunctional roles in the organization of functional masculinization in the developing brain and the maintenance of adult brain function.