FOXO signaling and skeletal muscle atrophy

FOXO 信号传导与骨骼肌萎缩

• 批准号: 8676431
• 负责人: Andrew Robert Judge
• 金额: $ 31.71万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8676431
• 关键词: Acetylation; Acetyltransferase; Acquired Immunodeficiency Syndrome; Address; Aging; Area; Atrophic; Bed rest; Binding; Boxing; CREB-binding protein; Cells; Chronic; Chronic Obstructive Airway Disease; DNA Binding; Data; Deacetylase; Deacetylation; Denervation; Diabetes Mellitus; Disease; Dominant-Negative Mutation; EP300 gene; Electrophoretic Mobility Shift Assay; Family; Gene Targeting; Genetic; Genetic Transcription; Goals; HDAC2 gene; HDAC4 gene; Health; Heart failure; IGF1 gene; Immobilization; In Vitro; Intervention; Lead; MLLT7 gene; Malignant Neoplasms; Measures; Mediating; Muscle; Muscle Fibers; Muscular Atrophy; Nuclear; Nutrient; Oligonucleotides; Pathway interactions; Phosphorylation; Physiological; Plasmids; Post-Translational Protein Processing; Protein Acetylation; Protein Family; Proteins; Regulation; Reporter; Research; Role; Sepsis; Signal Pathway; Signal Transduction; Signaling Molecule; Skeletal Muscle; Starvation; Stimulus; Testing; Transcriptional Activation; Transfection; Work; base; cell type; deprivation; human CREBBP protein; improved; in vivo; mRNA Expression; muscle form; mutant; p300/CBP-Associated Factor; programs; research study; response; skeletal muscle wasting; therapeutic target; tool; transcription factor; wasting

DESCRIPTION (provided by applicant): Skeletal muscle atrophy is a widespread physiological phenomenon and significant health problem associated with muscle disuse (immobilization, bedrest, denervation) and various diseases (cancer, sepsis, AIDS, diabetes, chronic heart failure, chronic obstructive pulmonary disease). However, our understanding of the signaling molecules that regulate muscle mass during an atrophy condition are ill defined. Therefore the long- range goal of our research program is to understand the regulation of signaling pathways that cause muscle atrophy during various conditions. Eventually improved understanding will lead to the identification of the most suitable targets for specific interventions. One family of proteins that is known to cause skeletal muscle atrophy is the FOXO family, and FOXO signaling is increased in a variety of atrophy conditions, including cast immobilization, sepsis, cancer and starvation. However, our understanding of the regulation of FOXO signaling in skeletal muscle is largely confined to the phosphorylation of FOXO factors via the IGF1/PI3K/Akt pathway. While this pathway is clearly important in the regulation of FOXO signaling, another mechanism of regulation, acetylation, appears to be equally important in regulating FOXO signaling, at least in cultured non-muscle cell. However, it is currently unknown whether acetylation of FOXO factors is a regulatory mechanism of FOXO signaling in skeletal muscle during any condition of muscle atrophy. The overall objective of the current proposal is to determine the role of acetyltransferase (HAT) proteins and deacetylase (HDAC) proteins, and acetylation and deacetylation of FOXO factors, in regulating FOXO signaling as it relates to skeletal muscle atrophy. To address this, in Aim 1 we will transfect C2C12 skeletal muscle cells with wild type (WT) or dominant negative (d.n.) HAT proteins and determine their regulation of endogenous FOXO signaling. We will subsequently select one HAT protein for further study in whole skeletal muscle during muscle disuse and cancer. In Aim 2 we will transfect C2C12 skeletal muscle cells with wild type (WT) or dominant negative (d.n.) HDAC proteins and determine their regulation of endogenous FOXO signaling. We will subsequently select one HDAC protein for further study in whole skeletal muscle during muscle disuse and cancer. In Aim 3 we will transfect C2C12 skeletal muscle cells with plasmids encoding FOXO acetylation mutants that mimic either the acetylated or deacetylated forms of FOXO and determine their regulation of FOXO signaling. In each of these Aims we will measure FOXO transcriptional activity, FOXO- DNA binding, FOXO cellular localization, post-translational modifications of FOXO factors, the transcription of a sub-set of known FOXO target genes, and skeletal muscle fiber cross sectional area. The findings from these experiments will lead to a greater understanding of the regulation of FOXO signaling as it relates to skeletal muscle atrophy.

描述(申请人提供)：骨骼肌萎缩是一种广泛的生理现象，与肌肉停用(固定、卧床、失神经)和各种疾病(癌症、败血症、艾滋病、糖尿病、慢性心力衰竭、慢性阻塞性肺疾病)有关。然而，我们对在萎缩条件下调节肌肉质量的信号分子的理解是模糊的。因此，我们研究计划的长期目标是了解在不同条件下导致肌肉萎缩的信号通路的调节。最终，增进了解将导致确定最适合具体干预措施的目标。已知的导致骨骼肌萎缩的一个蛋白质家族是FOXO家族，FOXO信号在各种萎缩情况下都会增加，包括石膏固定、败血症、癌症和饥饿。然而，我们对FOXO信号在骨骼肌中的调控在很大程度上仅限于通过IGF1/PI3K/Akt通路对FOXO因子的磷酸化。虽然这一途径显然在FOXO信号的调节中很重要，但另一种调节机制，乙酰化，似乎在调节FOXO信号方面也同样重要，至少在培养的非肌肉细胞中是这样。然而，目前尚不清楚FOXO因子的乙酰化是否是骨骼肌萎缩过程中FOXO信号的调节机制。本提案的总体目标是确定乙酰基转移酶(HAT)蛋白和脱乙酰基酶(HDAC)蛋白以及FOXO因子的乙酰化和去乙酰化在调节FOXO信号中的作用，因为FOXO信号与骨骼肌萎缩有关。为了解决这个问题，在目标1中，我们将野生型(WT)或显性阴性(D.N.)的C2C12骨骼肌细胞导入。HAT蛋白，并确定它们对内源性FOXO信号的调节。随后，我们将选择一种HAT蛋白，用于在肌肉废弃和癌症期间对整个骨骼肌进行进一步研究。在目标2中，我们将野生型(WT)或显性阴性(D.N.)的C2C12骨骼肌细胞导入。HDAC蛋白，并确定它们对内源性FOXO信号的调节。随后，我们将选择一种HDAC蛋白，用于在肌肉废弃和癌症期间对整个骨骼肌进行进一步研究。在目标3中，我们将用编码FOXO乙酰化突变体的质粒转染C2C12骨骼肌细胞，这些突变体模仿FOXO的乙酰化或去乙酰化形式，并确定它们对FOXO信号的调控。在每个目标中，我们将测量FOXO转录活性、FOXO-DNA结合、FOXO细胞定位、FOXO因子的翻译后修饰、已知FOXO靶基因的子集的转录以及骨骼肌纤维横截面积。这些实验的发现将使我们更好地理解FOXO信号的调节，因为它与骨骼肌萎缩有关。