Interleukin-6 Causes Dose dependent Increase In Proliferative Potential Of Satellite Cells In Vitro

Abstract

Recently, it has shown that Interleukin-6 (IL-6) knock-out mice undergo a blunted hypertrophic response compared with wild type mice following compensatory hypertrophy. Thus, IL-6 may be a key factor for an increase in proliferative potential of satellite cells. PURPOSE: To determine the effects and mechanisms of action of IL-6 on the proliferative potential of satellite cells. METHODS: Twenty male F344 rats (average 13 weeks) were used in this study. All of the major hind limb muscles of all rats were removed and trimmed of excess fat and connective tissue. Satellite cells were isolated according to previous methods (Machida et al., 2004). Isolated satellite cells were cultured on rat tail collagen coated plates in growth media. All cultures were maintained in 37°C humid air containing 5% CO2. Cultured cells were >90% desmin and MyoD positive cells using immunocytochemical analysis. Different concentrations of human recombinant IL-6 (0.01, 0.1, 1, 10, 100 ng/ml) were added to the culture medium. After 23 h, cultures were pulse-labeled with 10 μM BrdU in the medium for 1 h prior to fixation in methanol, and immunocytochemistry was used to estimate the number of BrdU positive cells. To investigate IL-6 signaling pathways related to increases in the proliferative potential of satellite cells, we used specific inhibitors to block JAK2 (AG490), STAT3 (STAT3 peptide), MEK (PD98059) and PI3K (LY294002). RESULTS: IL-6 at concentrations 0.01 to 1 ng/ml induced a dose-dependent increase cell proliferation. At 1 ng/ml IL-6, cell proliferation was 131% higher compared with control media (p < 0.05). In contrast, 10 ng/ml did not stimulate greater cell proliferation compared with control media. AG490, STAT3 peptide and LY294002 signaling inhibitors blocked cell proliferation at 6 h in response to stimulation with 1 ng/ml IL-6 (p < 0.05). AG490, STAT3 peptide, PD98059 and LY294002 inhibitors also blocked cell proliferation at 24 h in response to stimulation with 1 ng/ml IL-6 (p < 0.05). CONCLUSION: IL-6 can induce dose-dependent increases in proliferative potential of satellite cells through activation of the multiple signaling pathways. This work is supported by KAKENHI (21800054).