G-CSF does not influence C2C12 myogenesis despite receptor expression in healthy and dystrophic skeletal muscle

Abstract

Granulocyte-colony stimulating factor (G-CSF) increases recovery of rodent skeletal muscles after injury, and increases muscle function in rodent models of neuromuscular disease. However, the mechanisms by which G-CSF mediates these effects are poorly understood. G-CSF acts by binding to the membrane spanning G-CSFR and activating multiple intracellular signaling pathways. Expression of the G-CSFR within the haematopoietic system is well known, but more recently it has been demonstrated to be expressed in other tissues. However, comprehensive characterization of G-CSFR expression in healthy and diseased skeletal muscle, imperative before implementing G-CSF as a therapeutic agent for skeletal muscle conditions, has been lacking. Here we show that the G-CSFR is expressed in proliferating C2C12 myoblasts, differentiated C2C12 myotubes, human primary skeletal muscle cell cultures and in mouse and human skeletal muscle. In mdx mice, a model of human Duchenne muscular dystrophy (DMD), G-CSF mRNA and protein was down-regulated in limb and diaphragm muscle, but circulating G-CSF ligand levels were elevated. G-CSFR mRNA in the muscles of mdx mice was up-regulated however steady-state levels of the protein were down-regulated. We show that G-CSF does not influence C2C12 myoblast proliferation, differentiation or phosphorylation of Akt, STAT3, and Erk1/2. Media change alone was sufficient to elicit increases in Akt, STAT3, and Erk1/2 phosphorylation in C2C12 muscle cells and suggest previous observations showing a G-CSF increase in phosphoprotein signaling be viewed with caution. These results suggest that the actions of G-CSF may require the interaction with other cytokines and growth factors in vivo, however these data provides preliminary evidence supporting the investigation of G-CSF for the management of muscular dystrophy.

Granulocyte Colony-Stimulating Factor Ameliorates Skeletal Muscle Dysfunction in Amyotrophic Lateral Sclerosis Mice and Improves Proliferation of SOD1-G93A Myoblasts in vitro

“…They also observed a stimulatory effect of G-CSF on the proliferation of healthy C2C12 in vitro and evidence was presented in support of G-CSF as a direct activator of various signaling pathways related to cell proliferation, including PI3K/Akt and MAPK signals. Wright et al [15], on the other hand, found that CSF3R is expressed not only in active myoblasts, but also in myotubes and in mature SKM tissues. They found no evidence that G-CSF would influence in vitro proliferation of healthy C2C12 myoblasts and, in fact, it did not activate the above-mentioned signaling pathways either.…”

“…CSF3R is expressed in regenerating myocytes [11] and enhances proliferation of satellite cells in vivo [11,47]. Administration of G-CSF facilitates the regeneration of cardiotoxin-injured SKM [48] and protects against muscle degeneration in MDX mice [16]. Some progress has also been made in order to investigate whether the effects of G-CSF on muscle cells can be observed in vitro where myoblasts are deprived of their natural niche.…”

“…Hara et al [11] presented data supporting the role of G-CSF as a direct activator of various signaling pathways leading to increased C2C12 proliferation. Wright et al [15], on the other hand, found no evidence that G-CSF would influence in vitro proliferation of healthy C2C12 myoblasts. Therefore, although the debate continues whether G-CSF can stimulate proliferation of wild-type myoblasts in vitro, it remains unstudied altogether whether G-CSF or its analogs can stimulate myoblasts whose proliferation capacity is diminished, such as those from SOD1-G93A mice.…”

“…G-CSF in the CNS increases neurogenesis and neuroplasticity as well as counteracts apoptosis [13]. Supporting its role in muscle biology in vivo, G-CSF has positive effects on muscle fiber regeneration after nerve lesion in Duchenne muscular dystrophy mice [16]. …”

“…However, recent evidence suggests that the effects of G-CSF may not be restricted to hematopoiesis, which may in the future expand the clinical application of its analogs. CSF3R is expressed in the central nervous system (CNS), including brain and spinal cord MNs, as well as in developing and regenerating SKM in vivo and in proliferating myoblasts and differentiated myotubes in vitro [11,12,13,14,15]. G-CSF in the CNS increases neurogenesis and neuroplasticity as well as counteracts apoptosis [13].…”