Doctoral Degrees (Physiological Sciences)
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Browsing Doctoral Degrees (Physiological Sciences) by browse.metadata.advisor "Myburgh, Kathryn"
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- ItemIL-6 and the skeletal muscle myoblast : a guiding hand in myoblast cell fate(Stellenbosch : Stellenbosch University, 2015-12) Steyn, Paul; Myburgh, Kathryn; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.ENGLISH ABSTRACT: Skeletal muscle myoblasts have the ability to proliferate rapidly and differentiate into myotubes capable of generating functional muscle fibres. Interleukin-6 (IL-6) is a cytokine that is prominent in the context of muscle exercise and injury and has been shown to be elevated to various concentrations. It has been postulated that IL-6 is responsible for an increase in myoblast proliferation by keeping cells in the synthesis phase of the cell cycle, but these studies only focussed on a narrow concentration range of IL-6. It is well known that IL-6 signals through the JAK/STAT/SOCS pathway but there are numerous players involved in this pathway and the exact mechanisms that govern myoblast fate have not been established. Thus the aim was to study myoblast fate at 2 physiologically relevant IL-6 concentrations with the intent of fully characterizing the JAK/STAT/SOCS pathway within the skeletal muscle niche. The main aim was to determine the effects of the 2 doses of IL-6 on cell cycle progression in myoblasts. In order to attain this aim a primary human myoblast culture needed to be established. Cell cycle analysis was performed via flow cytometry to assess the number of cells in specific cell cycle phases. The JAK/STAT/SOCS pathway, myogenic regulatory factors and the cytokine receptor were studied with the aid of western blotting and immunofluorescent staining. In order to assess mRNA expression of SOCS signalling molecules, MRFs, and the IL-6 receptor, qPCR was performed on myoblast cell lysates. It was found that IL-6 is capable of performing a dual role in increasing proliferation as well as differentiation and this dual role is dependent on the concentration of IL-6 present. High concentrations of IL-6 were capable of shifting a larger portion of cells to the pro-differentiation G0/G1 phase of the cell cycle whereas Low concentrations were able to facilitate cell cycle progression from the G0/G1 to the S-phase. The Low dose decreased the expression of the myogenic regulatory factors, MyoD and myogenin and increased PCNA expression. The High dose had an alternate effect by increasing MyoD and myogenin expression and decreasing PCNA expression. The two doses also signalled differently through the JAK/STAT/SOCS pathway. The Low dose was responsible for prolonged JAK1 activation and increased SOCS1 protein expression. The High dose had initial increases in JAK activation but prolonged JAK2 activation and elevated SOCS3 protein expression. Regulation of the IL-6 receptor was also altered as a result of IL-6 treatment, with the High dose decreasing receptor expression 24 hours after treatment and the Low dose increasing receptor expression at the same time point. From these data it can be concluded that IL-6 can guide myoblasts into a proliferative or differentiation path dependent on its concentration and achieves this through alternate paths through the JAK/STAT/SOCS pathway and receptor regulation.