The role of MMP-14 and MMP-2 in mediating myoblast fusion.
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Date
2016
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Abstract
Satellite cells are muscle precursor cells that have the ability to self-renew, proliferate
and differentiate into myoblasts that eventually elongate and fuse to form myotubes
which are vital for regeneration and repair of muscle. Satellite cells reside in a niche,
between the sarcolemma of the muscle fiber and the basal lamina, which consists of
mostly collagen IV, proteoglycans and laminin. Matrigel is a gelatinous protein mixture
that consists primarily of collagen IV and laminin and therefore resembles the basal
lamina. Matrix Metalloproteinases (MMPs) are zinc endopeptidases, proteolytic
peptidases which break peptide bonds within their substrates. MMP-14 (membrane
bound) also known as membrane-type 1 matrix metalloproteinase (MT1-MMP) is one of
the major matrix metalloproteinases (MMPs) involved in muscle repair and
regeneration, together with MMP-2 (secreted). MMP-2 is a secreted gelatinase A, which
is activated by MMP-14. MMP-2 is also known to be activated by nitric oxide (NO),
therefore allowing active MMP-2 to release growth factors such as Hepatocyte Growth
Factor (HGF) from the extracellular matrix (ECM). There are two forms of MMP-2,
intracellular MMP-2 and extracellular (secreted) MMP-2. Secreted MMP-2 contains a
peptide signal that helps direct it outside the cell, while intracellular MMP-2 lacks this
feature and is therefore retained within the cell. Intracellular MMP-2 activity is known
to be a major cause of muscular atrophy. Secreted MMP-2 is known to degrade ECM
components, facilitating satellite cell mobility and release of growth factors such as HGF,
aiding in muscle regeneration. MMP-2 can cleave collagen IV due to the presence of a
fibronectin-like domain within its catalytic domain; this is not the case with MMP-14.
MMP-14 and MMP-2 together degrade collagens, fibronectin, laminin-2/4 and other
adhesion molecules. This clears the path for the myoblast to align and fuse to form
myotubes which then finally align to form mature muscle fibers. The levels of MMP-14
and MMP-2 must be regulated; low levels can cause muscular dystrophy. The current
study analysed expression levels, activity and role of MMP-14 and MMP-2 in C2C12
myoblast differentiation. C2C12 myoblasts first proliferated (Day 0), then aligned and
elongated (Days 1-2) and then finally fused into myotubes (Days 3-5) during
differentiation. MMP-14 and MMP-2 protein levels were high during the elongation
period and also during fusion of C2C12 myoblasts. MMP-14 was localised at the focal
adhesions, where actin filaments terminate during myoblast proliferation and fusion. Inhibition of MMPs using BB94 (10 μM) was observed to significantly reduce C2C12
myoblasts fusion. Secreted MMP-2 seems to play a vital role in the C2C12 differentiation,
as activity was seen during myogenesis; when neutralised with an antibody, an 18%
decrease in fusion was observed. Matrigel promoted an increase of MMP-2 expression
within the cell during fusion (day 5 of differentiation), while no intracellular MMP-2
protein was observed at day 2 of differentiation. Levels of secreted MMP-2 increased
significantly from day 2 to day 5 of differentiation; however, the presence of Matrigel
significantly reduced levels of secreted MMP-2 detected in conditioned media at day 5
compared to uncoated conditions. The decrease is, in part, due to the fact that MMP-2
was found to bind to Matrigel. In conclusion, MMP-14 and MMP-2 play an important
role in C2C12 myoblast elongation and fusion. This study provides further insight into
the role of MMPs in myogenesis and lays the foundation for future work.
Description
Masters Degree. University of KwaZulu-Natal, Pietermaritzburg.