Extracellular matrix factors influence myoblast activation, differentiation and fusion.
Date
2015
Authors
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Abstract
Satellite cells are muscle stem cells that reside in a niche between the basal lamina and
sarcolemma of mature muscle fibers. Upon muscle injury, these cells are activated to
myoblasts that subsequently proliferate, migrate and differentiate into myotubes in order to
facilitate repair. Extracellular matrix (ECM) and growth factors are known to regulate certain
aspects of myogenesis however, a comprehensive study of the direct effects of niche ECM
factors on C2C12 myogenesis has not previously been conducted and forms the core of this
study.
We first examined the role of hepatocyte growth factor (HGF) on C2C12 myogenesis. HGF
is known to initiate activation of quiescent satellite cells in the niche and regulate various
aspects of differentiation. In this study, we determined that HGF has a dose-dependent dual
role in C2C12 myogenesis. HGF (2 ng/ml) significantly promoted cell division, but reduced
myogenic commitment and fusion. Conversely, 10 ng/ml HGF reduced proliferative
capability, but increased differentiation. This is potentially regulated by changes in c-Met
expression; analysis revealed significantly decreased c-Met expression in differentiating
cells cultured with 2 ng/ml HGF, but increased expression in proliferating cells with 10
ng/ml HGF. Furthermore, investigation into the mechanisms by which HGF affects
myogenesis, revealed that mitogen-activated protein kinase (MAPKs: ERK, JNK or p38K)
and phosphatidylinositol-3-kinase (PI3K) inhibition abrogated the HGF-stimulated increase
in cell number. Interestingly, PI3K and p38 kinase facilitated the negative effect of HGF on
proliferation, while ERK inhibition abrogated the HGF-mediated decrease in differentiation.
Next, we analyzed the effect of the satellite cell niche ECM on C2C12 myogenesis. Collagen
IV and laminin, the major components of the basal lamina, bind to and interact with satellite
cells via integrins and other cell surface proteins such as the tetraspanin, CD9. Matrigel
significantly increased terminal fusion but had no effect on Pax7+ and MyoD+ cell numbers.
Collagen IV, the second largest constituent of Matrigel, was observed to significantly
increase MyoD+ cell numbers and terminal fusion without effecting percentage Pax7+ cell
numbers. Furthermore collagen IV stimulated an increase in CD9 expression on
differentiating cells, such that cells cultured on collagen IV required higher levels of
neutralizing anti-CD9 monoclonal antibodies to reduce fusion. These results indicate, for the
first time, that the interaction of collagen IV with CD9 is a critical mediator of skeletal
muscle fusion and that the observed pro-myogenic effect is accompanied, on a molecular
level, by an increase in the number of committed MyoD positive cells.
Extracellular matrix (ECM) and growth factors are known to have complex interactions that
may modulate their activity in vivo. Lastly, in an attempt to more closely mimic in vivo
conditions, murine C2C12 myoblasts were cultured on collagen IV in HGF-supplemented
media followed by assessment of differentiation and proliferation. Collagen IV was not able
to negate the negative effect of HGF (2 ng/ml) on fusion but was able to restore normal
MyHC expression. Due to a collagen IV stimulated increase in CD9 expression in
differentiating myoblasts, cells cultured on collagen IV required higher levels of neutralizing
anti-CD9 monoclonal antibodies to reduce fusion; an effect not observed when cells were
differentiated in the presence of HGF alone. HGF (10 ng/ml) treated samples we unable to
demonstrate any fusion when CD9 was completely blocked suggesting that CD9 is a crucial
co-factor in HGF (10 ng/ml)-induced fusion. These results show, for the first time, that
collagen IV is able to modulate certain aspects of the dual role of HGF on myogenesis.
In summary, we identified a novel dose-dependent dual role of HGF in myogenesis and
uncovered that these effects are mediated by changes in c-Met expression and downstream
MAPK and PI3K signalling. We showed, for the first time, that collagen IV is able to
positively mediate C2C12 differentiation via a CD9-dependent pathway. Lastly, we revealed
that collagen IV is able to mediate the dose-dependent effects of HGF on C2C12 myogenesis.
Description
Doctor of Philosophy in Biochemistry.
Keywords
Extracellular matrix., Myoblast transfer therapy., Myoblasts., Satellite cells., Theses -- Biochemistry., Muscle cells.