Machado Juliano, Manfredi Leandro H, Silveira Wilian A, Gonçalves Dawit A P, Lustrino Danilo, Zanon Neusa M, Kettelhut Isis C, Navegantes Luiz C
Department of Physiology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP, Brazil.
Department of Physiology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP, Brazil; Department of Biochemistry/Immunology, Ribeirão Preto Medical School, University of São Paulo, Brazil.
Int J Biochem Cell Biol. 2016 Mar;72:40-50. doi: 10.1016/j.biocel.2015.12.011. Epub 2015 Dec 21.
Calcitonin gene-related peptide (CGRP) is a neuropeptide released by motor neuron in skeletal muscle and modulates the neuromuscular transmission by induction of synthesis and insertion of acetylcholine receptor on postsynaptic muscle membrane; however, its role in skeletal muscle protein metabolism remains unclear. We examined the in vitro and in vivo effects of CGRP on protein breakdown and signaling pathways in control skeletal muscles and muscles following denervation (DEN) in rats. In isolated muscles, CGRP (10(-10) to 10(-6)M) reduced basal and DEN-induced activation of overall proteolysis in a concentration-dependent manner. The in vitro anti-proteolytic effect of CGRP was completely abolished by CGRP8-37, a CGRP receptor antagonist. CGRP down-regulated the lysosomal proteolysis, the mRNA levels of LC3b, Gabarapl1 and cathepsin L and the protein content of LC3-II in control and denervated muscles. In parallel, CGRP elevated cAMP levels, stimulated PKA/CREB signaling and increased Foxo1 phosphorylation in both conditions. In denervated muscles and starved C2C12 cells, Rp-8-Br-cAMPs or PKI, two PKA inhibitors, completely abolished the inhibitory effect of CGRP on Foxo1, 3 and 4 and LC3 lipidation. A single injection of CGRP (100 μg kg(-1)) in denervated rats increased the phosphorylation levels of CREB and Akt, inhibited Foxo transcriptional activity, the LC3 lipidation as well as the mRNA levels of LC3b and cathepsin L, two bona fide targets of Foxo. This study shows for the first time that CGRP exerts a direct inhibitory action on autophagic-lysosomal proteolysis in control and denervated skeletal muscle by recruiting cAMP/PKA signaling, effects that are related to inhibition of Foxo activity and LC3 lipidation.
降钙素基因相关肽（CGRP）是一种由骨骼肌运动神经元释放的神经肽，可通过诱导突触后肌膜上乙酰胆碱受体的合成与插入来调节神经肌肉传递；然而，其在骨骼肌蛋白质代谢中的作用仍不清楚。我们研究了CGRP对大鼠对照骨骼肌和去神经支配（DEN）后肌肉中蛋白质分解及信号通路的体外和体内效应。在分离的肌肉中，CGRP（10⁻¹⁰至10⁻⁶M）以浓度依赖性方式降低基础及DEN诱导的整体蛋白水解激活。CGRP受体拮抗剂CGRP8 - 37完全消除了CGRP的体外抗蛋白水解作用。CGRP下调对照和去神经支配肌肉中的溶酶体蛋白水解、LC3b、Gabarapl1和组织蛋白酶L的mRNA水平以及LC3 - II的蛋白质含量。同时，在两种情况下CGRP均提高了cAMP水平，刺激了PKA/CREB信号传导并增加了Foxo1磷酸化。在去神经支配的肌肉和饥饿的C2C12细胞中，两种PKA抑制剂Rp - 8 - Br - cAMPs或PKI完全消除了CGRP对Foxo1、3和4以及LC3脂化的抑制作用。对去神经支配的大鼠单次注射CGRP（100μg kg⁻¹）可增加CREB和Akt的磷酸化水平，抑制Foxo转录活性、LC3脂化以及Foxo的两个真正靶标LC3b和组织蛋白酶L的mRNA水平。这项研究首次表明，CGRP通过募集cAMP/PKA信号传导对对照和去神经支配的骨骼肌中的自噬 - 溶酶体蛋白水解发挥直接抑制作用，这些作用与抑制Foxo活性和LC3脂化有关。
