RESUMO
Targeting apoptosis in the ischemic penumbra is a rational therapeutic approach for restricting cerebral infarct volume after clinical stroke. The present work explored the capability of the obestatin peptide, as a novel approach to inhibit apoptotic signaling cascades on PC12 cells. According to the results, obestatin treatment significantly reduced nutrient deprivation-induced apoptotic cell death. The protective effects were related to the regulation of the anti-apoptotic protein, BCL-2, and the apoptotic protein caspase-3. This encompasses the control of apoptosis by the interplay between Akt, ERK1/2 and AMPK signaling pathways. The activation of Akt and AMPK was concomitant with the phosphorylation of their downstream targets, GSK3 and ACC, respectively. Besides, obestatin also causes FoxO1 nuclear export supporting the prevention of the apoptosome formation. The concurrent activation of Akt and AMPK by obestatin via the GPR39 receptor, supports a role for this system in the balance concerning the catabolic and the anabolic signaling to sustain cellular function and viability. Furthermore, these results provide both an insight into how the obestatin/GPR39 system regulates anti-apoptotic pathways, and a framework for ascertaining how this system can be optimally targeted in treatment of brain cell death after stroke.
Assuntos
Grelina , Acidente Vascular Cerebral , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Apoptose , Grelina/farmacologia , Quinase 3 da Glicogênio Sintase/metabolismo , Quinase 3 da Glicogênio Sintase/farmacologia , Sistema de Sinalização das MAP Quinases , Nutrientes , Células PC12 , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Although cell-based therapy is considered a promising method aiming at treating different muscular disorders, little clinical benefit has been reported. One of major hurdles limiting the efficiency of myoblast transfer therapy is the poor survival of the transplanted cells. Any intervention upon the donor cells focused on enhancing in vivo survival, proliferation, and expansion is essential to improve the effectiveness of such therapies in regenerative medicine. In the present work, we investigated the potential role of obestatin, an autocrine peptide factor regulating skeletal muscle growth and repair, to improve the outcome of myoblast-based therapy by xenotransplanting primary human myoblasts into immunodeficient mice. The data proved that short in vivo obestatin treatment of primary human myoblasts not only enhances the efficiency of engraftment, but also facilitates an even distribution of myoblasts in the host muscle. Moreover, this treatment leads to a hypertrophic response of the human-derived regenerating myofibers. Taken together, the activation of the obestatin/GPR39 pathway resulted in an overall improvement of the efficacy of cell engraftment within the host's skeletal muscle. These data suggest considerable potential for future therapeutic applications and highlight the importance of combinatorial therapies.
Assuntos
Grelina/metabolismo , Grelina/farmacologia , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Injeções Intramusculares , Camundongos , Camundongos SCID , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismoRESUMO
Obestatin/GPR39 signaling stimulates skeletal muscle repair by inducing the expansion of satellite stem cells as well as myofiber hypertrophy. Here, we describe that the obestatin/GPR39 system acts as autocrine/paracrine factor on human myogenesis. Obestatin regulated multiple steps of myogenesis: myoblast proliferation, cell cycle exit, differentiation and recruitment to fuse and form multinucleated hypertrophic myotubes. Obestatin-induced mitogenic action was mediated by ERK1/2 and JunD activity, being orchestrated by a G-dependent mechanism. At a later stage of myogenesis, scaffolding proteins ß-arrestin 1 and 2 were essential for the activation of cell cycle exit and differentiation through the transactivation of the epidermal growth factor receptor (EGFR). Upon obestatin stimulus, ß-arrestins are recruited to the membrane, where they functionally interact with GPR39 leading to Src activation and signalplex formation to EGFR transactivation by matrix metalloproteinases. This signalplex regulated the mitotic arrest by p21 and p57 expression and the mid- to late stages of differentiation through JNK/c-Jun, CAMKII, Akt and p38 pathways. This finding not only provides the first functional activity for ß-arrestins in myogenesis but also identify potential targets for therapeutic approaches by triggering specific signaling arms of the GPR39 signaling involved in myogenesis.
Assuntos
Arrestinas/fisiologia , Grelina/metabolismo , Desenvolvimento Muscular/genética , Receptores Acoplados a Proteínas G/metabolismo , Arrestinas/química , Arrestinas/genética , Arrestinas/metabolismo , Ciclo Celular , Diferenciação Celular , Proliferação de Células , Grelina/fisiologia , Humanos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/citologia , Fosforilação , Receptores Acoplados a Proteínas G/fisiologia , Transdução de Sinais , beta-Arrestina 1 , beta-ArrestinasRESUMO
The development of therapeutic strategies for skeletal muscle diseases, such as physical injuries and myopathies, depends on the knowledge of regulatory signals that control the myogenic process. The obestatin/GPR39 system operates as an autocrine signal in the regulation of skeletal myogenesis. Using a mouse model of skeletal muscle regeneration after injury and several cellular strategies, we explored the potential use of obestatin as a therapeutic agent for the treatment of trauma-induced muscle injuries. Our results evidenced that the overexpression of the preproghrelin, and thus obestatin, and GPR39 in skeletal muscle increased regeneration after muscle injury. More importantly, the intramuscular injection of obestatin significantly enhanced muscle regeneration by simulating satellite stem cell expansion as well as myofiber hypertrophy through a kinase hierarchy. Added to the myogenic action, the obestatin administration resulted in an increased expression of vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor 2 (VEGFR2) and the consequent microvascularization, with no effect on collagen deposition in skeletal muscle. Furthermore, the potential inhibition of myostatin during obestatin treatment might contribute to its myogenic action improving muscle growth and regeneration. Overall, our data demonstrate successful improvement of muscle regeneration, indicating obestatin is a potential therapeutic agent for skeletal muscle injury and would benefit other myopathies related to muscle regeneration.
Assuntos
Proliferação de Células/efeitos dos fármacos , Grelina/farmacologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Doenças Musculares/tratamento farmacológico , Regeneração/efeitos dos fármacos , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Injeções Intramusculares , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Camundongos , Desenvolvimento Muscular/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Células Satélites de Músculo Esquelético/efeitos dos fármacos , Células Satélites de Músculo Esquelético/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fatores de Crescimento do Endotélio Vascular/genética , Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The maintenance and repair of skeletal muscle are attributable to an elaborate interaction between extrinsic and intrinsic regulatory signals that regulate the myogenic process. In the present work, we showed that obestatin, a 23-amino acid peptide encoded by the ghrelin gene, and the GPR39 receptor are expressed in rat skeletal muscle and are up-regulated upon experimental injury. To define their roles in muscle regeneration, L6E9 cells were used to perform in vitro assays. For the in vivo assays, skeletal muscle tissue was obtained from male rats and maintained under continuous subcutaneous infusion of obestatin. In differentiating L6E9 cells, preproghrelin expression and correspondingly obestatin increased during myogenesis being sustained throughout terminal differentiation. Autocrine action was demonstrated by neutralization of the endogenous obestatin secreted by differentiating L6E9 cells using a specific anti-obestatin antibody. Knockdown experiments by preproghrelin siRNA confirmed the contribution of obestatin to the myogenic program. Furthermore, GPR39 siRNA reduced obestatin action and myogenic differentiation. Exogenous obestatin stimulation was also shown to regulate myoblast migration and proliferation. Furthermore, the addition of obestatin to the differentiation medium increased myogenic differentiation of L6E9 cells. The relevance of the actions of obestatin was confirmed in vivo by the up-regulation of Pax-7, MyoD, Myf5, Myf6, myogenin, and myosin heavy chain (MHC) in obestatin-infused rats when compared with saline-infused rats. These data elucidate a novel mechanism whereby the obestatin/GPR39 system is coordinately regulated as part of the myogenic program and operates as an autocrine signal regulating skeletal myogenesis.
Assuntos
Grelina/metabolismo , Músculo Esquelético/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Regulação para Cima , Animais , Comunicação Autócrina , Cardiotoxinas/toxicidade , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Expressão Gênica/efeitos dos fármacos , Grelina/genética , Grelina/farmacologia , Immunoblotting , Imuno-Histoquímica , Masculino , Músculo Esquelético/lesões , Músculo Esquelético/fisiopatologia , Doenças Musculares/induzido quimicamente , Doenças Musculares/patologia , Doenças Musculares/fisiopatologia , Proteína MyoD/genética , Proteína MyoD/metabolismo , Mioblastos Esqueléticos/citologia , Mioblastos Esqueléticos/efeitos dos fármacos , Mioblastos Esqueléticos/metabolismo , Miogenina/genética , Miogenina/metabolismo , Interferência de RNA , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/genética , Regeneração , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
The role of obestatin, a 23-amino-acid peptide encoded by the ghrelin gene, on the control of the metabolism of pre-adipocyte and adipocytes as well as on adipogenesis was determined. For in vitro assays, pre-adipocyte and adipocyte 3T3-L1 cells were used to assess the obestatin effect on cell metabolism and adipogenesis based on the regulation of the key enzymatic nodes, Akt and AMPK and their downstream targets. For in vivo assays, white adipose tissue (WAT) was obtained from male rats under continuous subcutaneous infusion of obestatin. Obestatin activated Akt and its downstream targets, GSK3α/ß, mTOR and S6K1, in 3T3-L1 adipocyte cells. Simultaneously, obestatin inactivated AMPK in this cell model. In keeping with this, ACC phosphorylation was also decreased. This fact was confirmed in vivo in white adipose tissue (omental, subcutaneous and gonadal) obtained from male rats under continuous sc infusion of obestatin (24 and 72 hrs). The relevance of obestatin as regulator of adipocyte metabolism was supported by AS160 phosphorylation, GLUT4 translocation and augment of glucose uptake in 3T3-L1 adipocyte cells. In contrast, obestatin failed to modify translocation of fatty acid transporters, FATP1, FATP4 and FAT/CD36, to plasma membrane. Obestatin treatment in combination with IBMX and DEX showed to regulate the expression of C/EBPα, C/EBPß, C/EBPδ and PPARγ promoting adipogenesis. Remarkable, preproghrelin expression, and thus obestatin expression, increased during adipogenesis being sustained throughout terminal differentiation. Neutralization of endogenous obestatin secreted by 3T3-L1 cells by anti-obestatin antibody decreased adipocyte differentiation. Furthermore, knockdown experiments by preproghrelin siRNA supported that obestatin contributes to adipogenesis. In summary, obestatin promotes adipogenesis in an autocrine/paracrine manner, being a regulator of adipocyte metabolism. These data point to a putative role in the pathogenesis of metabolic syndrome.
Assuntos
Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adipogenia/efeitos dos fármacos , Grelina/farmacologia , Células 3T3-L1 , Adenilato Quinase/metabolismo , Adipócitos/enzimologia , Tecido Adiposo Branco/efeitos dos fármacos , Tecido Adiposo Branco/enzimologia , Animais , Comunicação Autócrina/efeitos dos fármacos , Western Blotting , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Ativação Enzimática/efeitos dos fármacos , Proteínas de Transporte de Ácido Graxo/metabolismo , Grelina/metabolismo , Glucose/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Masculino , Camundongos , Comunicação Parácrina/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
Ghrelin synergizes with growth hormone-releasing hormone (GHRH) to potentiate growth hormone (GH) response through a mechanism not yet fully characterized. This study was conducted to analyze the role of GHRH as a potential ligand of the ghrelin receptor, GHS-R1a. The results show that hGHRH(1-29)NH(2) (GHRH) induces a dose-dependent calcium mobilization in HEK 293 cells stably transfected with GHS-R1a an effect not observed in wild-type HEK 293 cells. This calcium rise is also observed using the GHRH receptor agonists JI-34 and JI-36. Radioligand binding and cross-linking studies revealed that calcium response to GHRH is mediated by the ghrelin receptor GHS-R1a. GHRH activates the signaling route of inositol phosphate and potentiates the maximal response to ghrelin measured in inositol phosphate turnover. The presence of GHRH increases the binding capacity of (125)I-ghrelin in a dose dependent-fashion showing a positive binding cooperativity. In addition, confocal microscopy in CHO cells transfected with GHS-R1a tagged with enhanced green fluorescent protein shows that GHRH activates the GHS-R1a endocytosis. Furthermore, the selective GHRH-R antagonists, JV-1-42 and JMR-132, act also as antagonists of the ghrelin receptor GHS-R1a. Our findings suggest that GHRH interacts with ghrelin receptor GHS-R1a, and, in consequence, modifies the ghrelin-associated intracellular signaling pathway. This interaction may represent a form of regulation, which could play a putative role in the physiology of GH regulation and appetite control.
Assuntos
Hormônio Liberador de Hormônio do Crescimento/fisiologia , Receptores de Grelina/agonistas , Transdução de Sinais , Sinalização do Cálcio , Linhagem Celular , Endocitose , Hormônio do Crescimento , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Humanos , Fosfatos de Inositol/metabolismo , Ligação Proteica , Receptores de Grelina/genética , Receptores de Grelina/metabolismo , TransfecçãoRESUMO
BACKGROUND: A therapeutic approach for the treatment of glucocorticoid-induced skeletal muscle atrophy should be based on the knowledge of the molecular mechanisms determining the unbalance between anabolic and catabolic processes and how to re-establish this balance. Here, we investigated whether the obestatin/GPR39 system, an autocrine signalling system acting on myogenesis and with anabolic effects on the skeletal muscle, could protect against chronic glucocorticoid-induced muscle atrophy. METHODS: In this study, we used an in vivo model of muscle atrophy induced by the synthetic glucocorticoid dexamethasone to examine the liaison molecules that define the interaction between the glucocorticoid receptor and the obestatin/GPR39 systems. The findings were extended to in vitro effects on human atrophy using human KM155C25 myotubes. RESULTS: KLF15 and FoxO transcription factors were identified as direct targets of obestatin signalling in the control of proteostasis in skeletal muscle. The KLF15-triggered gene expression program, including atrogenes and FoxOs, was regulated via KLF15 ubiquitination by the E3 ubiquitin ligase NEDD4. Additionally, a specific pattern of FoxO post-translational modification, including FoxO4 phosphorylation by Akt pathway, was critical in the regulation of the ubiquitin-proteasome system. The functional cooperativity between Akt and NEDD4 in the regulation of FoxO and KLF15 provides integrated cues to counteract muscle proteostasis and re-establish protein synthesis. CONCLUSIONS: The effective control of FoxO activity in response to glucocorticoid is critical to counteract muscle-related pathologies. These results highlight the potential of the obestatin/GPR39 system to fine-tune the effects of glucocorticoids on skeletal muscle wasting.
Assuntos
Transdução de Sinais , Grelina , Glucocorticoides , Humanos , Fatores de Transcrição Kruppel-Like , Músculo Esquelético , Atrofia Muscular/induzido quimicamente , Atrofia Muscular/tratamento farmacológico , Ubiquitina-Proteína Ligases Nedd4 , Receptores Acoplados a Proteínas G/genéticaRESUMO
Obestatin was identified as a gut peptide encoded by the ghrelin gene that interacts with the G protein-coupled receptor, GPR39. In this work, a sequential analysis of its transmembrane signalling pathway has been undertaken to characterize the intracellular mechanisms responsible for Akt activation. The results show that Akt activation requires the phosphorylation of T308 in the A-loop by the phosphoinositide-dependent kinase 1 (PDK1) and S473 within the HM by the mammalian target of rapamycin (mTOR) kinase complex 2 (mTORC2: Rictor, mLST8, mSin1, mTOR kinase) with participation neither of G(i)(/o)-protein nor Gbetagamma dimers. Obestatin induces the association of GPR39/beta-arrestin 1/Src signalling complex resulting in the transactivation of the epidermal growth factor receptor (EGFR) and downstream Akt signalling. Upon administration of obestatin, phosphorylation of mTOR (S2448) and p70S6K1 (T389) rise with a time course that parallels that of Akt activation. Based on the experimental data obtained, a signalling pathway involving a beta-arrestin 1 scaffolding complex and EGFR to activate Akt signalling is proposed.
Assuntos
Arrestinas/metabolismo , Receptores ErbB/genética , Hormônios Peptídicos/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias Gástricas/tratamento farmacológico , Arrestinas/antagonistas & inibidores , Arrestinas/genética , Receptores ErbB/metabolismo , Grelina , Humanos , Immunoblotting , Imunoprecipitação , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , RNA Interferente Pequeno/farmacologia , Transdução de Sinais , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Ativação Transcricional , Células Tumorais Cultivadas , beta-Arrestina 1 , beta-ArrestinasRESUMO
Obestatin, the ghrelin-associated peptide, showed to activate MAPK signaling with no effect on Akt nor cell proliferating activity in rat tumor somatotroph cells (growth cells, GC). A sequential analysis of the obestatin transmembrane signaling pathway indicated a route involving the consecutive activation of G(i), PI3k, novel PKCepsilon, and Src for ERK1/2 activation. Furthermore, obestatin treatment triggers growth hormone (GH) release in the first 30min, being more acute at 15min. At 1h, obestatin treated cells showed the same levels in GH secretion than controls. Added to this functionality, obestatin was secreted by GC cells. Based on the capacity to stimulate GH release from somatotroph cells, obestatin may act directly in the pituitary through an autocrine/paracrine mechanism.
Assuntos
Grelina/farmacologia , Hormônio do Crescimento/metabolismo , Somatotrofos/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Ativação Enzimática , Camundongos , Proteína Quinase 3 Ativada por Mitógeno/biossíntese , Fosfatidilinositol 3-Quinases/biossíntese , Proteína Quinase C-épsilon/biossíntese , Ratos , Somatotrofos/enzimologia , Somatotrofos/metabolismo , Quinases da Família src/biossínteseRESUMO
BACKGROUND: This study was performed to test the therapeutic potential of obestatin, an autocrine anabolic factor regulating skeletal muscle repair, to ameliorate the Duchenne muscular dystrophy (DMD) phenotype. METHODS AND RESULTS: Using a multidisciplinary approach, we characterized the ageing-related preproghrelin/GPR39 expression patterns in tibialis anterior (TA) muscles of 4-, 8-, and 18-week-old mdx mice (n = 3/group) and established the effects of obestatin administration at this level in 8-week-old mdx mice (n = 5/group). The findings were extended to in vitro effects on human immortalized DMD myotubes. An analysis of TAs revealed an age-related loss of preproghrelin expression, as precursor of obestatin, in mdx mice. Administration of obestatin resulted in a significant increase in tetanic specific force (33.0% ± 1.5%, P < 0.05), compared with control mdx mice. Obestatin-treated TAs were characterized by reduction of fibres with centrally located nuclei (10.0% ± 1.2%, P < 0.05) together with an increase in the number of type I fibres (25.2% ± 1.7%, P < 0.05) associated to histone deacetylases/myocyte enhancer factor-2 and peroxisome proliferator-activated receptor-gamma coactivator 1α axis, and down-regulation of ubiquitin E3-ligases by inactivation of FoxO1/4, indexes of muscle atrophy. Obestatin reduced the level of contractile damage and tissue fibrosis. These observations correlated with decline in serum creatine kinase (58.8 ± 15.2, P < 0.05). Obestatin led to stabilization of the sarcolemma by up-regulation of utrophin, α-syntrophin, ß-dystroglycan, and α7ß1-integrin proteins. These pathways were also operative in human DMD myotubes. CONCLUSIONS: These results highlight the potential of obestatin as a peptide therapeutic for preserving muscle integrity in DMD, thus allowing a better efficiency of gene or cell therapy in a combined therapeutic approach.
Assuntos
Grelina/uso terapêutico , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/fisiopatologia , Fenótipo , Animais , Biomarcadores , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Endogâmicos mdx , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Força Muscular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Distrofia Muscular de Duchenne/diagnóstico , Oxirredução/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Proteólise , Ratos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Sarcolema/efeitos dos fármacos , Sarcolema/metabolismoRESUMO
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
RESUMO
Ghrelin, the endogenous ligand for the growth hormone secretagogue receptor type 1a (GHS-R1a), is a 28 amino acid residue with a post-translational octanoyl modification on Ser3. Despite the biomedical interest in this hormone, the fine details of its regulation and the mechanisms controlling its secretion are largely unknown. The present study analyzes the molecular steps involved in the full lysophosphatidic acid (LPA) receptor-mediated activation of the mitogenic extracellular signal-regulated kinase (ERK) pathway and its consequent role as an inhibitor of ghrelin secretion in the gastric adenocarcinoma cell line AGS. ERK1/2 phosphorylation mediated by LPA proceeds via activation of the type 2 LPA receptor, activation of the nonreceptor tyrosine kinase c-Src, and subsequent transactivation of the epidermal growth factor receptor. Furthermore, LPA-induced ERK activation was found to be independent of matrix metalloproteinases; thus, c-Src acted as the scaffold-transactivating epidermal growth factor receptor. Finally, a correlation was observed between the mitogenic effects of LPA and ghrelin secretion in the human gastric adenocarcinoma cell line AGS. These data suggest a possible physiological role of LPA in ghrelin secretion. The relationship found between LPA and ghrelin secretion might explain the low circulating levels of ghrelin observed in obese patients, as a bona fide reflex of the energetic stores.
Assuntos
Adenocarcinoma/metabolismo , Grelina/metabolismo , Lisofosfolipídeos/farmacologia , Sistema de Sinalização das MAP Quinases , Neoplasias Gástricas/metabolismo , Adenocarcinoma/enzimologia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Grelina/genética , Humanos , Modelos Biológicos , RNA Mensageiro/metabolismo , Neoplasias Gástricas/enzimologiaRESUMO
BACKGROUND: Many pathological states characterized by muscle atrophy are associated with an increase in circulating glucocorticoids and poor patient prognosis, making it an important target for treatment. The development of treatments for glucocorticoid-induced and wasting disorder-related skeletal muscle atrophy should be designed based on how the particular transcriptional program is orchestrated and how the balance of muscle protein synthesis and degradation is deregulated. Here, we investigated whether the obestatin/GPR39 system, an autocrine/paracrine signaling system acting on myogenesis and with anabolic effects on the skeletal muscle, could protect against glucocorticoid-induced muscle cell atrophy. METHODS: In the present study, we have utilized mouse C2C12 myotube cultures to examine whether the obestatin/GPR39 signaling pathways can affect the atrophy induced by the synthetic glucocorticoid dexamethasone. We have extended these findings to in vitro effects on human atrophy using human KM155C25 myotubes. RESULTS: The activation of the obestatin/GPR39 system protects from glucocorticoid-induced atrophy by regulation of Akt, PKD/PKCµ, CAMKII and AMPK signaling and its downstream targets in the control of protein synthesis, ubiquitin-proteasome system and autophagy-lysosome system in mouse cells. We compared mouse and human myotube cells in their response to glucocorticoid and identified differences in both the triggering of the atrophic program and the response to obestatin stimulation. Notably, we demonstrate that specific patterns of post-translational modifications of FoxO4 and FoxO1 play a key role in directing FoxO activity in response to obestatin in human myotubes. CONCLUSIONS: Our findings emphasize the function of the obestatin/GPR39 system in coordinating a variety of pathways involved in the regulation of protein degradation during catabolic conditions.
Assuntos
Autofagia/efeitos dos fármacos , Grelina/farmacologia , Glucocorticoides/farmacologia , Lisossomos/metabolismo , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Animais , Linhagem Celular , Humanos , Camundongos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Obestatin/GPR39 signaling stimulates skeletal muscle growth and repair by inducing both G-protein-dependent and -independent mechanisms linking the activated GPR39 receptor with distinct sets of accessory and effector proteins. In this work, we describe a new level of activity where obestatin signaling plays a role in the formation, contractile properties and metabolic profile of skeletal muscle through determination of oxidative fiber type. Our data indicate that obestatin regulates Mef2 activity and PGC-1α expression. Both mechanisms result in a shift in muscle metabolism and function. The increase in Mef2 and PGC-1α signaling activates oxidative capacity, whereas Akt/mTOR signaling positively regulates myofiber growth. Taken together, these data indicate that the obestatin signaling acts on muscle fiber-type program in skeletal muscle.
Assuntos
Grelina/farmacologia , Desenvolvimento Muscular/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Animais , Linhagem Celular , Fatores de Transcrição MEF2/metabolismo , Masculino , Camundongos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismoRESUMO
Ghrelin regulates GH secretion and energy homeostasis through the GH secretagogue receptor type-1a (GHS-R1a). This G-protein coupled receptor shows the peculiarity to transduce information provided not just by ghrelin as well as by adenosine through a supposed binding site different from the characterized ghrelin-binding pocket. Indeed, adenosine triggers intracellular calcium rise through a distinct signaling pathway to the one described for ghrelin, although it fails to stimulate GH secretion. Despite multiple active conformations of GHS-R1a, suggested as an explanation for a ligand-dependent activation of the downstream signaling, the concept of adenosine as agonist for GHS-R1a has been re-evaluated. The results revealed that calcium rise of both ghrelin and adenosine appears to be mediated by receptors that did not show the same sensitivity to protein kinase C (PKC) activity in GHS-R1a-transfected HEK 293 cells (HEK-GHS-R1a cells). The binding analyses showed the same number of adenosine-binding sites in both HEK 293 (B(max) = 2.01 +/- 0.15 fmol/cell) and HEK-GHS-R1a cells (B(max) = 1.90 +/- 0.11 fmol/cell). This binding was unaltered by different GHS-R1a antagonists. Western blot analysis showed a similar endogenous expression of endogenous adenosine receptor type-2b and -3 in both cell lines. The K(d) values for adenosine were 1.78 microM in HEK 293 cells and 6.30 microM in HEK-GHS-R1a cells, pointing to a modification of agonist affinity induced by overexpression of the GHS-R1a. Additionally, adenosine failed to induce the GHS-R1a endocytosis, although it attenuates the ghrelin-induced GHS-R1a endocytosis. In conclusion, adenosine is not an agonist of the GHS-R1a and its action is mediated by the endogenous adenosine receptor type-2b and -3, which is able to partially use the intracellular signaling machinery of HEK-GHS-R1a cells.
Assuntos
Adenosina/metabolismo , Rim/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Arvicolinae , Western Blotting/métodos , Células CHO , Cálcio/metabolismo , Linhagem Celular , Cricetinae , Humanos , Rim/embriologia , Microscopia Confocal , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ensaio Radioligante , Receptor A2B de Adenosina/genética , Receptor A3 de Adenosina/genética , Receptores Acoplados a Proteínas G/genética , Receptores de Grelina , Transfecção/métodosRESUMO
The growth hormone secretagogue receptor, GHSR1a, mediates the biological activities of ghrelin, which includes the secretion of growth hormone, as well as the stimulation of appetite, food intake and maintenance of energy homeostasis. Mapping phosphorylation sites on GHSR1a and knowledge of how these sites control specific functional consequences unlocks new strategies for the development of therapeutic agents targeting individual functions. Herein, we have identified the phosphorylation of different sets of sites within GHSR1a which engender distinct functionality of ß-arrestins. More specifically, the Ser(362), Ser(363) and Thr(366) residues at the carboxyl-terminal tail were primarily responsible for ß-arrestin 1 and 2 binding, internalization and ß-arrestin-mediated proliferation and adipogenesis. The Thr(350) and Ser(349) are not necessary for ß-arrestin recruitment, but are involved in the stabilization of the GHSR1a-ß-arrestin complex in a manner that determines the ultimate cellular consequences of ß-arrestin signaling. We further demonstrated that the mitogenic and adipogenic effect of ghrelin were mainly dependent on the ß-arrestin bound to the phosphorylated GHSR1a. In contrast, the ghrelin function on GH secretion was entirely mediated by G protein signaling. Our data is consistent with the hypothesis that the phosphorylation pattern on the C terminus of GHSR1a determines the signaling and physiological output.
Assuntos
Complexos Multiproteicos/metabolismo , Receptores de Grelina/metabolismo , Transdução de Sinais/fisiologia , beta-Arrestinas/metabolismo , Células HEK293 , Humanos , Complexos Multiproteicos/genética , Fosforilação/fisiologia , Domínios Proteicos , Receptores de Grelina/genética , beta-Arrestinas/genéticaRESUMO
Obestatin, a 23-amino acid peptide encoded by the ghrelin gene, and the GPR39 receptor were reported to be involved in the control of mitogenesis of gastric cancer cell lines; however, the relationship between the obestatin/GPR39 system and gastric cancer progression remains unknown. In the present study, we determined the expression levels of the obestatin/GPR39 system in human gastric adenocarcinomas and explored their potential functional roles. Twenty-eight patients with gastric adenocarcinomas were retrospectively studied, and clinical data were obtained. The role of obestatin/GPR39 in gastric cancer progression was studied in vitro using the human gastric adenocarcinoma AGS cell line. Obestatin exogenous administration in these GPR39-bearing cells deregulated the expression of several hallmarks of the epithelial-mesenchymal transition (EMT) and angiogenesis. Moreover, obestatin signaling promoted phenotypic changes via GPR39, increasingly impacting on the cell morphology, proliferation, migration and invasion of these cells. In healthy human stomachs, obestatin expression was observed in the neuroendocrine cells and GPR39 expression was localized mainly in the chief cells of the oxyntic glands. In human gastric adenocarcinomas, no obestatin expression was found; however, an aberrant pattern of GPR39 expression was discovered, correlating to the dedifferentiation of the tumor. Altogether, our data strongly suggest the involvement of the obestatin/GPR39 system in the pathogenesis and/or clinical outcome of human gastric adenocarcinomas and highlight the potential usefulness of GPR39 as a prognostic marker in gastric cancer.
Assuntos
Adenocarcinoma/genética , Receptores Acoplados a Proteínas G/metabolismo , Neoplasias Gástricas/genética , Proliferação de Células , Feminino , Humanos , Masculino , Transdução de SinaisRESUMO
Human retinal pigmented epithelial cell (hRPE) proliferation plays a significant role in various proliferative diseases associated to the retina that leads to loss of vision, such as proliferative vitreoretinopathy. In the current study, the role of the bovine vitreous lipid factor (bVLF) in hRPE cell proliferation has been investigated. bVLF is a bioactive lipid isolated from the bovine vitreous body with strong Ca(2+)-mobilizing activity in fibroblast. In the first approach, the effects of bVLF on Ca(2+)-mobilizing activity were investigated in hRPE. The results showed that bVLF induced, in a dose-dependent manner, a Ca(2+) mobilization from PA-sensitive intracellular stores [non-Ins(1,4,5)P(3)-sensitive stores], in which extracellular Ca(2+) participated. The increase in intracellular Ca(2+) was associated with a dose-dependent inhibiting effect on cell proliferation. At a dose of 10 microg/mL, bVLF caused a 26% or a 44% inhibition in hRPE cell proliferation during the 3- or the 6-day culture periods, respectively. These effects appear to be specific in hRPE cells, since EFGR-T17 fibroblast cells treated with equivalent amounts of bVLF did not show any inhibiting effects. This inhibitory action was not associated to apoptotic/necrotic processes. Furthermore, bVLF inhibited EGF-, bFGF-, IGF-I-, PDGF-, HGF- and VEGF-induced proliferation of the hRPE cells. Moreover, this inhibitory response was also observed in FBS-induced hRPE cell proliferation. bVLF, at a concentration of 10 microg/mL, induced 16% inhibition of proliferation during a culture period of 3 days. This inhibitory action was greater during the 6-day culture period, exceeding 40%. With regard to this action, the results showed that bVLF has a potent inhibitory effect on ERK1/2 activation, and plays a key role in the control of hRPE cell proliferation. These observations contribute to the knowledge of inhibitory factors responsible for keeping antiproliferative environment that preserve the RPE-associated activities in normal states. It advances the interesting possibility that this factor or a factor with characteristics common to bVLF might be involved in the pathogenesis of abnormal proliferative eye processes.
Assuntos
Células Epiteliais/citologia , Fosfolipídeos/fisiologia , Epitélio Pigmentado Ocular/citologia , Retina/metabolismo , Animais , Apoptose , Western Blotting , Cálcio/metabolismo , Bovinos , Proliferação de Células , Separação Celular , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática , Citometria de Fluxo , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Fosfatos de Inositol/metabolismo , Metabolismo dos Lipídeos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Necrose , Fosfatos/metabolismo , Fosfolipídeos/metabolismo , Transdução de Sinais , Fatores de Tempo , Fosfolipases Tipo C/metabolismoRESUMO
2-aminoethoxydiphenyl borate (2-APB) has been widely used as a blocker of the IP3 receptor and TRP channels, including store-operated calcium channels. We now show in monolayers of normal rat kidney cells (NRK/49F) that 2-APB completely and reversibly blocks gap junctional intercellular communication at concentrations similar to that required for inhibition of PGF2alpha-induced increases in intracellular calcium. Gap junctional conductances between NRK cells were estimated with single-electrode patch-clamp measurements and were fully blocked by 2-APB (50 microM), when applied extracellularly but not via the patch pipette. Half maximal inhibition (IC50) of electrical coupling in NRK cells was achieved at 5.7 microM. Similar results were obtained for human embryonic kidney epithelial cells (HEK293/tsA201) with an IC50 of 10.3 microM. Using 2-APB as an electrical uncoupler of monolayer cells, we could thus measure inward rectifier potassium, L-type calcium, and calcium-dependent chloride membrane currents in confluent NRK monolayers, with properties similar to those in dissociated NRK cells in the absence of 2-APB. The electrical uncoupling action described here is a new 2-APB property that promises to provide a powerful pharmacological tool to study single-cell properties in cultured confluent monolayers and intact tissues by electrical and chemical uncoupling of the cells without the need of prior dissociation.