Ruolo della ciclina D3 nel controllo della funzionalità delle cellule staminali del muscolo scheletrico in vivo
Author(s)
Ferretti, Roberta
Date Issued
May 31, 2013
Type
Doctoral Thesis
Abstract
The irreversible exit from the cell cycle is a necessary requisite for the differentiation of muscle
cell precursors. Cell cycle progression and exit from the cell cycle are positively controlled by
cell cycle activators, such as the cyclins and cyclin dependent kinases (CDK), and negatively
controlled by cell cycle inhibitors, such as the CDK inhibitor p21 and the retinoblastoma (Rb)
gene product. In this context, cyclin D3, whose expression is induced by the master myogenic
regulatory factor MyoD during the transition from the proliferation to the differentiation stage,
represents an exception. Moreover, cyclin D3 is the only cyclin that accumulates in post-mitotic
myocytes by forming kinase-inactive complexes with CDK4 and p21 and mediating their
interaction with hypophosphorylated pRb.
Cyclin D3 protein is expressed at high levels in mouse skeletal muscle in vivo during the first 14
days after birth, but its expression declines to a low level in adult muscle. This expression pattern
is similar to that of different proliferation and differentiation regulatory genes expressed in the
activated satellite cells, which are involved in the post-natal muscle growth. Upon injury, cyclin
D3 expression is induced in adult muscle, together with that of activated satellite cell markers,
suggesting a physiological role for cyclin D3 in regenerative myogenesis.
To elucidate the role of cyclin D3 in adult myogenesis, we used a cyclin D3 knock-out mouse
model. We found that adult cyclin D3 knockout mice are phenotipically carachterized by
reduced body size and, in particular, by a reduced muscle mass.
Immunohistochemical analysis performed on cyclin D3-/- adult skeletal muscle shows a reduced
number of myofibers and a reduced myofiber size as compared to wild-type mice, togheter with a
reduced number of myofiber-associated quiescent satellite cells.
Moreover, cyclin D3 deficiency affects proliferation of myogenic precursor cells. In fact, cyclin
D3-/- primary myoblasts in culture show an evident proliferative defect and are characterized by a
significant decrease of the S-phase cell proportion in concomitance with a significant increase of
the G1 and G2/M-phase cell population compared with control wild-type myoblasts. However,
cyclin D3-/- myoblasts, although characterized by an evident proliferative deficit, are capable of
myogenic differentiation.
The proliferative deficit of the activated myogenic precursors is observed also by analyzing
satellite cells associated with single muscle fibers ex-vivo. After 72h in culture, the clusters
formed by activated satellite cells resident on myofibers derived from cyclin D3-/- mice display a
reduced number of cells, and contain an increased percentage of cells expressing the
differentiation marker myogenin, and a decreased percentage of cells expressing Pax7 (cells
destined to self-renewal), compared with wild-type controls.
Similar results are obtained from in vivo experiments performed by inducing muscle damage in
WT and cyclin D3-/- mice. Indeed, the regenerative process of the cyclin D3-/- muscle is
characterized by reduced proliferation of the activated satellite cells, precocious differentiation,
and decreased propensity to self-renew. At the end of regeneration, the size of regenerated fiber is
smaller in cyclin D3-null muscle than in wild type, and the number of Pax7+ cells repopulating
the satellite cell niche was reduced.
The results shown in this thesis provide the first evidence that cyclin D3 plays a unique
functional role, which is not vicariated by the others D type cyclins, in regulating the correct
balance between proliferation, differentiation and selfrenewal of skeletal muscle precursor cell
Additional information
Dottorato di ricerca in Genetica e biologia cellulare
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