IL-6 and the skeletal muscle myoblast : a guiding hand in myoblast cell fate
| dc.contributor.advisor | Myburgh, Kathryn | en_ZA |
| dc.contributor.author | Steyn, Paul | en_ZA |
| dc.contributor.other | Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences. | en_ZA |
| dc.date.accessioned | 2015-12-14T07:41:52Z | |
| dc.date.available | 2016-03-02T03:00:04Z | |
| dc.date.issued | 2015-12 | |
| dc.description | Thesis (MSc)--Stellenbosch University, 2015. | en_ZA |
| dc.description.abstract | 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. | en_ZA |
| dc.description.abstract | AFRIKAANSE OPSOMMING: Skelet spier mioblaste het die vermoë om vinnig te vermenigvuldig en te onderskei tot miobuise wat funksionele spiervesels kan genereer. Interleukin-6 (IL-6) is 'n sitokiene wat prominent in die konteks van spier oefening en besering is en kan verhef word tot verskeie konsentrasies. Dit is aangevoer dat IL-6 verantwoordelik is vir 'n toename in mioblast verspreiding deur die behoud van die selle in die sintese fase van die selsiklus, maar hierdie studies het net gefokus op 'n noue verskeidenheid IL-6 konsentrasies. Dit is bekend dat IL-6 sein deur die JAK/STAT/SOCS pad, maar daar is talle spelers wat betrokke is in hierdie pad en die presiese meganismes wat mioblast lot beheer is nog nie vasgestel nie. Die doel was dus om mioblast lot bepaling te bestudeer teen twee IL-6 konsentrasies met die doel om die JAK/STAT/SOCS pad binne die skeletspier nis te karakteriseer. Die hoofdoel was om die gevolge van die twee dosisse van IL-6 op selsiklus progressie in mioblaste te bepaal. Om hierdie doel te bereik moes 'n primêre menslike mioblast kultuur gestig word. Selsiklus analise is uitgevoer via vloeisitometrie om die aantal selle in spesifieke sel siklus fases te evalueer. Die JAK/STAT/SOCS pad, miogeniese regulerende faktore en die sitokien reseptor was bestudeer met behulp van Western blotting en immunofluorescent kleuring. Ten einde om mRNA uitdrukking van SOCS sein molekules, MRFs, en die IL-6 reseptor te bepaal, is qPCR uitgevoer op mioblast sel lisate. Daar is gevind dat IL-6 in staat is om 'n dubbele rol te speel in die verhoging van mioblast proliferasie asook differensiasie en hierdie dubbele rol is afhanklik van die konsentrasie van IL-6 teenwoordig. Hoë konsentrasies van IL-6 was in staat om 'n groter gedeelte van die selle na die pro-differensiasie G0/G1 fase van die selsiklus te verskuif terwyl Lae konsentrasies in staat was om selsiklus progressie van die G0/G1 na die S-fase te fasiliteer. Die Lae dosis het die uitdrukking van die miogene regulerende faktore, MyoD en myogenin, verlaag asook om PCNA uitdrukking te verhoog. Die Hoë dosis het 'n alternatiewe effek gehad deur die verhoging van MyoD en myogenin uitdrukking en 'n daling in PCNA uitdrukking. Die twee dosisse het ook anders deur die JAK/ STAT/SOCS pad gesein. Die Lae dosis was verantwoordelik vir langdurige JAK1 aktivering sowel as toename in SOCS1 proteien vlakke. Die Hoë dosis het aanvanklike stygings in JAK1 aktivering gehad maar langdurige JAK2 aktivering, en SOCS3 proteien styging. Regulering van die IL-6 reseptor is ook verander as gevolg van IL-6 behandeling, met die Hoë dosis wat reseptor uitdrukking verminder het 24 uur na behandeling en die Lae dosis het reseptor uitdrukking op dieselfde tyd punt verhoog. Van hierdie data kan dit afgelei word dat IL-6 mioblaste kan lei tot 'n proliferatiewe of differensiasie pad afhanklik van sy konsentrasie en bereik dit deur alternatiewe paaie deur die JAK/STAT/SOCS weg en reseptor regulasie. | af_ZA |
| dc.description.version | Doctoral | en_ZA |
| dc.embargo.terms | 2016-03-01 | |
| dc.format.extent | 123 pages : illustrations (some color) | en_ZA |
| dc.identifier.uri | http://hdl.handle.net/10019.1/97709 | |
| dc.language.iso | en | en_ZA |
| dc.publisher | Stellenbosch : Stellenbosch University | en_ZA |
| dc.rights.holder | Stellenbosch University | en_ZA |
| dc.subject.lcsh | Myoblasts -- Growth | en_ZA |
| dc.subject.lcsh | Interleukin-6 -- Receptors | en_ZA |
| dc.subject.lcsh | Myoblasts -- Differentiation | en_ZA |
| dc.subject.lcsh | Cell cycle -- Regulation | en_ZA |
| dc.subject.lcsh | Myoblasts -- Physiological effect | en_ZA |
| dc.subject.name | UCTD | |
| dc.title | IL-6 and the skeletal muscle myoblast : a guiding hand in myoblast cell fate | en_ZA |
| dc.type | Thesis | en_ZA |