Year: 2015
Type: dissertation
Abstract: Satellite cells are muscle stem cells that reside in a niche between the basal lamina and \nsarcolemma of mature muscle fibers. Upon muscle injury, these cells are activated to \nmyoblasts that subsequently proliferate, migrate and differentiate into myotubes in order to \nfacilitate repair. Extracellular matrix (ECM) and growth factors are known to regulate certain \naspects of myogenesis however, a comprehensive study of the direct effects of niche ECM \nfactors on C2C12 myogenesis has not previously been conducted and forms the core of this \nstudy. \nWe first examined the role of hepatocyte growth factor (HGF) on C2C12 myogenesis. HGF \nis known to initiate activation of quiescent satellite cells in the niche and regulate various \naspects of differentiation. In this study, we determined that HGF has a dose-dependent dual \nrole in C2C12 myogenesis. HGF (2 ng/ml) significantly promoted cell division, but reduced \nmyogenic commitment and fusion. Conversely, 10 ng/ml HGF reduced proliferative \ncapability, but increased differentiation. This is potentially regulated by changes in c-Met \nexpression; analysis revealed significantly decreased c-Met expression in differentiating \ncells cultured with 2 ng/ml HGF, but increased expression in proliferating cells with 10 \nng/ml HGF. Furthermore, investigation into the mechanisms by which HGF affects \nmyogenesis, revealed that mitogen-activated protein kinase (MAPKs: ERK, JNK or p38K) \nand phosphatidylinositol-3-kinase (PI3K) inhibition abrogated the HGF-stimulated increase \nin cell number. Interestingly, PI3K and p38 kinase facilitated the negative effect of HGF on \nproliferation, while ERK inhibition abrogated the HGF-mediated decrease in differentiation. \nNext, we analyzed the effect of the satellite cell niche ECM on C2C12 myogenesis. Collagen \nIV and laminin, the major components of the basal lamina, bind to and interact with satellite \ncells via integrins and other cell surface proteins such as the tetraspanin, CD9. Matrigel \nsignificantly increased terminal fusion but had no effect on Pax7+ and MyoD+ cell numbers. \nCollagen IV, the second largest constituent of Matrigel, was observed to significantly \nincrease MyoD+ cell numbers and terminal fusion without effecting percentage Pax7+ cell \nnumbers. Furthermore collagen IV stimulated an increase in CD9 expression on \ndifferentiating cells, such that cells cultured on collagen IV required higher levels of \nneutralizing anti-CD9 monoclonal antibodies to reduce fusion. These results indicate, for the \n \nfirst time, that the interaction of collagen IV with CD9 is a critical mediator of skeletal \nmuscle fusion and that the observed pro-myogenic effect is accompanied, on a molecular \nlevel, by an increase in the number of committed MyoD positive cells. \nExtracellular matrix (ECM) and growth factors are known to have complex interactions that \nmay modulate their activity in vivo. Lastly, in an attempt to more closely mimic in vivo \nconditions, murine C2C12 myoblasts were cultured on collagen IV in HGF-supplemented \nmedia followed by assessment of differentiation and proliferation. Collagen IV was not able \nto negate the negative effect of HGF (2 ng/ml) on fusion but was able to restore normal \nMyHC expression. Due to a collagen IV stimulated increase in CD9 expression in \ndifferentiating myoblasts, cells cultured on collagen IV required higher levels of neutralizing \nanti-CD9 monoclonal antibodies to reduce fusion; an effect not observed when cells were \ndifferentiated in the presence of HGF alone. HGF (10 ng/ml) treated samples we unable to \ndemonstrate any fusion when CD9 was completely blocked suggesting that CD9 is a crucial \nco-factor in HGF (10 ng/ml)-induced fusion. These results show, for the first time, that \ncollagen IV is able to modulate certain aspects of the dual role of HGF on myogenesis. \nIn summary, we identified a novel dose-dependent dual role of HGF in myogenesis and \nuncovered that these effects are mediated by changes in c-Met expression and downstream \nMAPK and PI3K signalling. We showed, for the first time, that collagen IV is able to \npositively mediate C2C12 differentiation via a CD9-dependent pathway. Lastly, we revealed \nthat collagen IV is able to mediate the dose-dependent effects of HGF on C2C12 myogenesis.
Author: Nicholas Lee. Walker
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2550030770