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.
Highlights
Skeletal muscle atrophy is a devastating condition that is characteristic of neuromuscular diseases such as Duchenne muscular dystrophy (DMD) and many chronic diseases including cancer, heart disease, chronic obstructive pulmonary disease (COPD), sepsis and AIDS (Jagoe and Goldberg, 2001)
Similar forms were observed in BAF/3 cells stably expressing the G-CSFR protein used as a positive control (Figures 1B,C), but not in HEK293T cells used as a negative control since they are known not to express G-CSFR (Debruin et al, 2010) (Figure 1C)
G-CSFR expression has been observed on various cell types outside of the haematopoietic system leading to speculation that G-CSFR may be expressed in skeletal muscle
Summary
Skeletal muscle atrophy is a devastating condition that is characteristic of neuromuscular diseases such as Duchenne muscular dystrophy (DMD) and many chronic diseases including cancer, heart disease, chronic obstructive pulmonary disease (COPD), sepsis and AIDS (Jagoe and Goldberg, 2001). G-CSFR is highly expressed on haematopoietic stem cells and various myeloid cells (Nagata and Fukunaga, 1991), and on other cell types including neural tissue (Yata et al, 2007; Pitzer et al, 2008, 2010), liver stem cells (Piscaglia et al, 2007) and cardiac muscle (Li et al, 2006, 2007; Takano et al, 2006; Ueda et al, 2006; Shimoji et al, 2010). These observations suggest that G-CSF may regulate cells outside of the haematopoietic system
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