Direct effects of Tumor Necrosis Factor Alpha(TNF-α) on L6 Myotubes

Tumor necrosis factor (TNF)-α is a pleiotropic cytokine responsible for a diverse range of signaling events within cells. We studied direct effects of TNF-α on skeletal muscle protein content. Incubation of the L6 myotubes with 1 to 10 U/ml of TNF-α resulted in a significant increase of total and myofibrillar protein contents as compared with the control. However, incubation with lower (0.001-0.1 U/ ml) or higher (100-300 U/ml) doses of TNF-α resulted in a decrease of protein content. These findings suggest that TNF-α may elicit both anabolic and catabolic effects on L6 myotubes in a dose dependent manner. The anabolic effect of TNF-α was mediated at least in part by mitogen activated protein kinase (MAPK), especially by an extracellular-regulated kinases (ERK). This divergent effect of TNF-α may be crucial to elucidate the complexity of TNF-α action on the skeletal muscle. The proinflammatory cytokine tumor necrosis factor (TNF) -α is a pleiotropic cytokine with a broad range of diverse actions that include proliferative effects as cell growth and differentiation [16], inflammatory effects, mediation of immune responses [23], and destructive cellular outcomes such as apoptotic and necrotic cell death mechanisms [3]. The expression level of TNF-α elevated in conditions associated with muscle pathology such as inflammatory myopathy [20], Duchenne muscular dystrophy [21], and cancer cachexia [24]. Actions of TNF-α are mediated by its binding to the specific receptors, TNF-α receptors 1 and 2 (TNFR-1 and TNFR-2), expressed on the surface of most cell types [4]. TNF-α stimulates a complex array of post receptor signaling events, mostly through three major pathways. One pathway stimulates apoptosis via interaction with the TNF-α receptor complex and the Fas-associated protein with death domain. The second pathway activates the Jun-N-terminal kinase, a mitogen activated protein kinase (MAPK) pathway, and the third pathway activates the nuclear factor κ B (NF-κB), a primary mediator of transcriptional control and a major candidate for catabolic signaling [6]. In addition, activation of phosphatidylinositol 3 (PI3)-kinase-Akt/PKB pathway via TNFR-2 was recently reported in fibroblasts [15], cardiac [13], and retinal cells [10]. A divergent effect of TNF-α on skeletal muscle protein has been reported, mostly using the C2C12 mouse skeletal muscle cell line. In this study, we examined the direct effects of different TNF-α concentrations on total muscle protein content as well as myofibrillar protein content of the L6 rat skeletal muscle cells. We also studied the effects of various kinase inhibitors to clarify the intracellular signaling pathways of TNF-α stimulation.