An investigation into the P13-K/AKT signalling pathway in TNF-a-induced muscle proeolysis in L6 myotubes

[摘要] Introduction: Skeletal muscle atrophy is a mitigating complication that is characterized bya reduction in muscle fibre cross-sectional area as well as protein content, reduced force,elevated fatigability and insulin resistance. It seems to be a highly ordered and regulatedprocess and signs of this condition are often seen in inflammatory conditions such as cancer,AIDS, diabetes and chronic heart failure (CHF). It has long been understood that animbalance between protein degradation (increase) and protein synthesis (decrease) bothcontribute to the overall loss of muscle protein. Although the triggers that cause atrophy aredifferent, the loss of muscle mass in each case involves a common phenomenon that inducesmuscle proteolysis. It is becoming evident that interactions among known proteolytic systems(ubiquitin-proteosome) are actively involved in muscle proteolysis during atrophy. Factorssuch as TNF-α and ROS are elevated in a wide variety of chronic inflammatory diseases inwhich skeletal muscle proteolysis presents a lethal threat. There is an increasing body ofevidence that implies TNF-α may play a critical role in skeletal muscle atrophy in a number ofclinical settings but the mechanisms mediating its effects are not completely understood. It isalso now apparent that the transcription factor NF-κB is a key intracellular signal transducerin muscle catabolic conditions. This study investigated the various proposed signallingpathways that are modulated by increasing levels of TNF-α in a skeletal muscle cell line, inorder to synthesize our current understanding of the molecular regulation of muscle atrophy.Materials and Methods: L6 (rat skeletal muscle) cells were cultured under standardconditions where after reaching ± 60-65% confluency levels, differentiation was induced for amaximum of 8 days. During the last 2 days, myotubes were incubated with increasingconcentrations of recombinant TNF-α (1, 3, 6 and 10 ng/ml) for a period of 40 minutes, 24and 48 hours. The effects of TNF-α on proliferation and cell viability were measured by MTTassay and Trypan Blue exclusion technique. Morphological assessment of cell death wasconducted using the Hoechst 33342 and Propidium Iodide staining method. Detection ofapoptosis was assessed by DNA isolation and fragmentation assay. The HE stain was used forthe measurement of cell size. In order to determine the source and amount of ROS production,MitoTracker Red CM-H2 X ROS was utilised. Ubiquitin expression was assessed byimmunohistochemistry. PI3-K activity was calculated by using an ELISA assay and theexpression of signalling proteins was analysed by Western Blotting using phospho-specific and total antibodies. Additionally, the antioxidant Oxiprovin was used to investigate thequantity of ROS production in TNF-α-induced muscle atrophy.Results and Discussion: Incubation of L6 myotubes with increasing concentrations ofrecombinant TNF-α revealed that the lower concentrations of TNF-α used were not toxic tothe cells but data analysis of cell death showed that 10 ng/ml TNF-α induced apoptosis andnecrosis. Long-term treatment with TNF-α resulted in an increase in the upregulation of TNF-α receptors, specifically TNF-R1. The transcription factors NF-κB and FKHR were rapidlyactivated thus resulting in the induction of the ubiquitin-proteosome pathway. Activation ofthis pathway produced significant increases in the expression of E3 ubiquitin ligases MuRF-1and MAFbx. Muscle fibre diameter appeared to have decreased with increasing TNF-αconcentrations in part due to the suppressed activity of the PI3-K/Akt pathway as well assignificant reductions in differentiation markers. Western blot analysis also showed thatcertain MAPKs are activated in response to TNF-α. No profound changes were observed withROS production. Finally, the use Oxiprovin significantly lowered cell viability and ROSproduction. These findings suggest that TNF-α may elicit strong catabolic effects on L6myotubes in a dose and time dependent manner.Conclusion: These observations suggest that TNF-α might have beneficial effects inskeletal muscle in certain circumstances. This beneficial effect however is limited by severalaspects which include the concentration of TNF-α, cell type, time of exposure, cultureconditions, state of the cell (disturbed or normal) and the cells stage of differentiation. Theeffect of TNF-α can be positive or negative depending on the concentration and time pointsanalysed. This action is mediated by various signal transduction pathways that are thought tocooperate with each other. More understanding of these pathways as well as their subsequentupstream and downstream constituents is obligatory to clarify the central mechanism/s thatcontrol physiological and pathophysiological effects of TNF-α in skeletal muscle.

[发布机构] Stellenbosch University