TNF induction of atrogin-1/MAFbx mRNA depends on Foxo4 expression but not AKT-Foxo1/3 signalling

Department of Physiology, University of Kentucky, Lexington, Kentucky 40536-0298, USA.
AJP Cell Physiology (Impact Factor: 3.78). 09/2008; 295(4):C986-93. DOI: 10.1152/ajpcell.00041.2008
Source: PubMed


Murine models of starvation-induced muscle atrophy demonstrate that reduced protein kinase B (AKT) function upregulates the atrophy-related gene atrogin-1/MAFbx (atrogin). The mechanism involves release of inhibition of Forkhead transcription factors, namely Foxo1 and Foxo3. Elevated atrogin mRNA also corresponds with elevated TNF in inflammatory catabolic states, including cancer and chronic heart failure. Exogenous tumor necrosis factor (TNF) increases atrogin mRNA in vivo and in vitro. We used TNF-treated C2C12 myotubes to test the hypothesis that AKT-Foxo1/3 signaling mediates TNF regulation of atrogin mRNA. Here we confirm that exposure to TNF increases atrogin mRNA (+125%). We also confirm that canonical AKT-mediated regulation of atrogin is active in C2C12 myotubes. Inhibition of phosphoinositol-3 kinase (PI3K)/AKT signaling with wortmannin reduces AKT phosphorylation (-87%) and increases atrogin mRNA (+340%). Activation with insulin-like growth factor (IGF) increases AKT phosphorylation (+126%) and reduces atrogin mRNA (-15%). Although AKT regulation is intact, our data suggest it does not mediate TNF effects on atrogin. TNF increases AKT phosphorylation (+50%) and stimulation of AKT with IGF does not prevent TNF induction of atrogin mRNA. Nor does TNF appear to signal through Foxo1/3 proteins. TNF has no effect on Foxo1/3 mRNA or Foxo1/3 nuclear localization. Instead, TNF increases nuclear Foxo4 protein (+55%). Small interfering RNA oligos targeted to two distinct regions of Foxo4 mRNA reduce the TNF-induced increase in atrogin mRNA (-34% and -32%). We conclude that TNF increases atrogin mRNA independent of AKT via Foxo4. These results suggest a mechanism by which inflammatory catabolic states may persist in the presence of adequate growth factors and nutrition.

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    • "c o m / l o c a t e / y b b r c and modulate inflammation and oxidative stress after downhill running. Recently, it is reported that these inflammatory mediators such as TNF-a and COX-2 up-regulate expression of catabolic condition inducer such as atrogin1/MAFbx and MuRF1 in skeletal muscle, and induce muscle protein degradation [13] [14] [15]. Hydrogen peroxide also induced expression of atrogin1/MAFbx and MuRF1 in skeletal muscle myotubes [16] [17]. "
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    Biochemical and Biophysical Research Communications 10/2013; 441(3). DOI:10.1016/j.bbrc.2013.10.119 · 2.30 Impact Factor
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    • "We cannot rule out the possibility that this response is secondary to other regulation events and thus not part of a primary response. It is still a matter of debate what the individual biological contributions of each FOXO gene are and under what circumstances they are activated, but murine in vitro and in vivo studies indicate that the FOXO genes should be considered negative regulators of muscle mass [20,35-37]. "
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    ABSTRACT: Limb immobilization causes a rapid loss of muscle mass and strength that requires appropriate rehabilitation to ensure restoration of normal function. Whereas the knowledge of muscle mass signaling with immobilization has increased in recent years, the molecular regulation in the rehabilitation of immobilization-induced muscle atrophy is only sparsely studied. To investigate the phosphorylation and expression of candidate key molecular muscle mass regulators after immobilization and subsequent rehabilitation we performed two separate studies. We immobilized the lower limb for 2 weeks followed by the in-house hospital standard physiotherapy rehabilitation (Study 1). Secondly, we conducted an intervention study using the same 2 weeks immobilization protocol during which protein/carbohydrate supplementation was given. This was followed by 6 weeks of rehabilitation in the form of resistance training and continued protein/carbohydrate supplementation (Study 2). We obtained muscle biopsies from the medial gastrocnemius prior to immobilization (PRE), post-immobilization (IMMO) and post-rehabilitation (REHAB) and measured protein expression and phosphorylation of Akt, mTOR, S6k, 4E-BP1, GSK3β, ubiquitin and MURF1 and mRNA expression of Atrogin-1, MURF1, FOXO1, 3 and 4 as well as appropriate housekeeping genes. In both studies, no changes in protein expression or phosphorylation for any measured protein were observed. In Study 1, FOXO3 and FOXO4 mRNA expression decreased after IMMO and REHAB compared to PRE, whereas other mRNAs remained unchanged. Interestingly, we found significant changes in expression of the putative housekeeping genes GAPDH, HADHA and S26 with immobilization in both studies. In neither study, the changes in muscle mass associated with immobilization and rehabilitation were accompanied by expected changes in expression of atrophy-related genes or phosphorylation along the Akt axis. Unexpectedly, we observed significant changes in several of the so-called housekeeping genes GAPDH, HADHA and S26 with immobilization in both studies, thereby questioning the usefulness of these genes for normalization of RNA data purposes in muscle immobilization studies.
    BMC Research Notes 03/2012; 5(1):166. DOI:10.1186/1756-0500-5-166
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    • "For example, multiple studies suggest that TNFα is an important regulator of protein balance in atrophying muscle (Frost et al, 2007; Li et al, 2003, 2005). Interestingly, in a recent study by Moylan et al (2008), treatment of cultured myotubes with TNF had no effect on FOXO1 or 3a expression and nuclear localization but instead upregulated the expression of FOXO4. In additional experiments in the same report, silencing of the FOXO4 gene reduced the TNF-induced expression of atrogin-1. "
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