Skeletal muscle adapts to decreases in activity and load by undergoing atrophy. To identify candidate molecular mediators of muscle atrophy, we performed transcript profiling. Although many genes were up-regulated in a single rat model of atrophy, only a small subset was universal in all atrophy models. Two of these genes encode ubiquitin ligases: Muscle RING Finger 1 (MuRF1), and a gene we designate Muscle Atrophy F-box (MAFbx), the latter being a member of the SCF family of E3 ubiquitin ligases. Overexpression of MAFbx in myotubes produced atrophy, whereas mice deficient in either MAFbx or MuRF1 were found to be resistant to atrophy. These proteins are potential drug targets for the treatment of muscle atrophy.

Skeletal muscle atrophy occurs as a side effect of treatment with synthetic glucocorticoids such as dexamethasone (DEX) and is a hallmark of cachectic syndromes associated with increased cortisol levels. The E3 ubiquitin ligase MuRF1 (muscle RING finger protein 1) is transcriptionally upregulated by DEX treatment. Differentiated myotubes treated with DEX undergo depletion of myosin heavy chain protein (MYH), which physically associates with MuRF1. This loss of MYH can be blocked by inhibition of MuRF1 expression. When wild-type and MuRF1(-/-) mice are treated with DEX, the MuRF1(-/-) animals exhibit a relative sparing of MYH. In vitro, MuRF1 is shown to function as an E3 ubiquitin ligase for MYH. These data identify the mechanism by which MYH is depleted under atrophy conditions and demonstrate that inhibition of a single E3 ligase, MuRF1, is sufficient to maintain this important sarcomeric protein.

Loss of myofibrillar proteins is a hallmark of atrophying muscle. Expression of muscle RING-finger 1 (MuRF1), a ubiquitin ligase, is markedly induced during atrophy, and MuRF1 deletion attenuates muscle wasting. We generated mice expressing a Ring-deletion mutant MuRF1, which binds but cannot ubiquitylate substrates. Mass spectrometry of the bound proteins in denervated muscle identified many myofibrillar components. Upon denervation or fasting, atrophying muscles show a loss of myosin-binding protein C (MyBP-C) and myosin light chains 1 and 2 (MyLC1 and MyLC2) from the myofibril, before any measurable decrease in myosin heavy chain (MyHC). Their selective loss requires MuRF1. MyHC is protected from ubiquitylation in myofibrils by associated proteins, but eventually undergoes MuRF1-dependent degradation. In contrast, MuRF1 ubiquitylates MyBP-C, MyLC1, and MyLC2, even in myofibrils. Because these proteins stabilize the thick filament, their selective ubiquitylation may facilitate thick filament disassembly. However, the thin filament components decreased by a mechanism not requiring MuRF1.

Skeletal muscle size is regulated by anabolic (hypertrophic) and catabolic (atrophic) processes. We first characterized molecular markers of both hypertrophy and atrophy and identified a small subset of genes that are inversely regulated in these two settings (e.g. upregulated by an inducer of hypertrophy, insulin-like growth factor-1 (IGF-1), and down-regulated by a mediator of atrophy, dexamethasone). The genes identified as being inversely regulated by atrophy, as opposed to hypertrophy, include the E3 ubiquitin ligase MAFbx (also known as atrogin-1). We next sought to investigate the mechanism by which IGF-1 inversely regulates these markers, and found that the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/ mTOR) pathway, which we had previously characterized as being critical for hypertrophy, is also required to be active in order for IGF-1-mediated transcriptional changes to occur. We had recently demonstrated that the IGF1/PI3K/Akt pathway can block dexamethasoneinduced up-regulation of the atrophy-induced ubiquitin ligases MuRF1 and MAFbx by blocking nuclear translocation of a FOXO transcription factor. In the current study we demonstrate that an additional step of IGF1 transcriptional regulation occurs downstream of mTOR, which is independent of FOXO. Thus both the Akt/FOXO and the Akt/mTOR pathways are required for the transcriptional changes induced by IGF-1.Skeletal muscle mass and fiber size is regulated in response to changes in workload, activity, conditions such as AIDS, cancer, and aging, and by cachectic glucocorticoids such as dexamethasone (1-3). An increase in adult muscle mass and fiber size is called "hypertrophy" and is associated with increased protein synthesis (4). A decrease in mass, called "atrophy," is characterized by enhanced protein degradation (3,5,6).Hypertrophy in adult skeletal muscle is accompanied by the increased expression of insulin-like growth factor-1 (IGF-1) 1 (4, 7). When IGF-1 was overexpressed in the skeletal muscle of transgenic mice an increase in muscle size resulted (8, 9). Furthermore, addition of IGF-1 in vitro to differentiated muscle cells promotes myotube hypertrophy (10 -12), supporting the idea that IGF-1 is sufficient to induce hypertrophy. The binding of IGF-1 to its receptor triggers the activation of phosphatidylinositol 3-kinase (PI3K). PI3K phosphorylates the membrane phospholipid phosphatidylinositol 4,5-bisphosphate to produce phosphatidylinositol 3,4,5-trisphosphate (13, 14), creating a lipid binding site on the cell membrane for the serine/threonine kinase Akt (also called Akt1 and PKB, for protein kinase B) (15-17). The subsequent translocation of Akt to the membrane facilitates its phosphorylation and activation by the kinase PDK-1 (14, 18, 19). Cell growth and survival in a variety of tissues and cell types in response to IGF-1, insulin, and other growth factors is critically mediated by Akt (14, 19). Direct and indirect targets downstream of Akt include the mammalian target of rapamycin (mTOR), p70S6K, and PHAS-1...

Entry of quiescent cells into the cell cycle is driven by the cyclin D-dependent kinases Cdk4 and Cdk6. These kinases are negatively regulated by the INK4 cell cycle inhibitors. We report the generation of mice defective in P15 INK4b and P18 INK4c . Ablation of these genes, either alone or in combination, does not abrogate cell contact inhibition or senescence of mouse embryo ®broblasts in culture. However, loss of P15 INK4b , but not of P18 INK4c , confers proliferative advantage to these cells and makes them more sensitive to transformation by H-ras oncogenes. In vivo, ablation of P15 INK4b and P18 INK4c genes results in lymphoproliferative disorders and tumor formation. Mice lacking P18 INK4c have deregulated epithelial cell growth leading to the formation of cysts, mostly in the cortical region of the kidneys and the mammary epithelium. Loss of both P15 INK4b and P18 INK4c does not result in signi®cantly distinct phenotypic manifestations except for the appearance of cysts in additional tissues. These results indicate that P15 INK4b and P18 IKN4c are tumor suppressor proteins that act in different cellular lineages and/or pathways with limited compensatory roles.