RESUMO
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
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
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
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
Diets rich in monounsaturated cis-FFA (cis FFA) are associated with a significant reduction of cardiovascular risk. Although several different mechanisms have been proposed to explain this protective effect, the biochemical processes involved have not been fully elucidated. It has been shown that upon their incorporation into the plasma membrane, cis FFA induce a marked perturbation of the lipid domains, altering membrane fluidity as well as lipid-lipid and lipid-protein interactions in the bilayer plane. During the last few years, several lines of evidence have shown that these perturbations disrupt the activity of several membrane proteins and enzymatic systems. As a result, several critical transmembrane signaling systems, including the Ins(1,4,5)P(3)/DAG/[Ca(2+)](i), the cAMP/PKA, and the voltage-operated Ca(2+) influx are strongly inhibited by cis FFA in different experimental models. Furthermore, this inhibition is associated with alterations in the timing of the cell cycle as well as in the final steps of the secretory pathway. We propose that this complex set of biological actions exerted by cis FFA at the plasma membrane may contribute to explain the protective roles that these molecules appear to exert on the vascular wall.
Assuntos
Membrana Celular/química , Membrana Celular/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Ácidos Graxos Insaturados/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/efeitos dos fármacos , Ácido Oleico/farmacologiaRESUMO
ß-Arrestins were identified as scaffold-proteins that have the capacity to desensitize G protein-coupled receptors. However, it has been found that ß-arrestins activate signaling pathways independent of G protein activation. The diversity of these signaling pathways has also been recognized for receptor tyrosine kinase. The aim of the present study was to validate the ß-arrestin-dependent signaling mechanism(s) responsible for regulation of adipogenesis. Two signal models were selected, ghrelin and insulin, based on its ß-arrestin-associated Akt activity. Herein, we found that ß-arrestin 1 and 2 were essential molecules for adipocyte differentiation. More specifically, the role of these scaffolding proteins was demonstrated by depletion of ß-arrestin 1 and 2 during ghrelin-induced adipogenesis in 3T3-L1 cells, which decreased the adipocyte differentiation and the expression levels of master regulators of early, the CCAAT/enhancer-binding protein ß (C/EBPß) and the CCAAT/enhancer-binding protein δ (C/EBPδ), and terminal, the peroxisome proliferator-activated receptor (PPARγ) and the CCAAT/enhancer-binding protein α (C/EBPα), adipogenesis. Accordingly ghrelin-induced Akt activity and its downstream targets, the mammalian target of rapamycin complex 1 (mTORC1) and the ribosomal protein S6 kinase beta-1 (S6K1), were inhibited by ß-arrestin 1 and 2 siRNAs. By contrast, assays performed during insulin-activated adipogenesis showed an intensifying effect on the adipocyte differentiation as well as on the expression of C/EBPß, C/EBPδ, PPARγ and C/EBPα. The increase in insulin-induced adipogenesis by ß-arrestin knock-down was concomitant to a decrease in the insulin receptor susbtrate-1 (IRS-1) serine phosphorylation, proving the loss of the negative feedback loop on IRS-1/phosphoinositide 3-kinase (PI3K)/Akt. Therefore, ß-arrestins control the extent and intensity of the lipogenic and adipogenic factors associated to Akt signaling, although the mechanistic and functional principles that underlie the connection between signaling and ß-arrestins are specifically associated to each receptor type.