Research ArticleLeukemia inhibitory factor-dependent increase in myoblast cell number is associated with phosphotidylinositol 3-kinase-mediated inhibition of apoptosis and not mitosis
Introduction
Leukemia inhibitory factor (LIF) is a pleiotropic cytokine belonging to the interleukin-6 (IL-6) family of cytokines that share similar activities and receptors [1]. LIF is expressed in multiple tissues and involved in many biological processes, but its increased expression in dystrophic and injured skeletal muscle [2], [3], [4] indicates an important role in skeletal muscle regeneration. LIF is actively involved in regeneration of skeletal muscle, with LIF knockout mice showing a decrease in the area occupied by regenerating myofibres after crush injury compared to wild-type, which is restored by administration of exogenous LIF [5]. Administration of LIF to the site of crush injury in wild-type mice increased the area occupied by regenerating fibres with an associated increase in average myofibre diameter [2], [5]. These original studies suggested that enhanced regeneration and increase in fibre size occurred, at least in part via stimulation of proliferation of the muscle forming myoblast cells, thus providing more cells to fuse to and increase the size of regenerating fibres.
Earliest descriptions of LIF as a possible mitogen for myoblasts suggested that LIF treatment increased the number of human and mouse derived primary myoblast cells present in culture in a dose-dependent manner after several days of culture, with the earliest increases noticeable after 6 days [6], [7]. It was also shown that short periods of exposure to LIF (4 h) produced similar increases in cell number after 10 days compared to periods of exposure for all 10 days [7]. LIF binds to a heterodimer of the LIF receptor (LIFR) and gp130 receptor subunits [8], which leads to activation of multiple signalling pathways within the target cell including signal transducer and activator of transcription-3 (STAT3), phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK) [9], [10], [11]. Although some of these signal transducers are shown to be activated shortly after LIF treatment (15–30 min), the increase in cell number is not observed until at least 48 h later [10]. This effect of LIF on cell number is consistently observed in myoblasts cultured under sub-optimal conditions where low serum concentrations are used (5% FBS) and frequently only after several days of culture [6], [7], [10], [12]. Mitogens generate rapid responses [13]. That this effect occurs under conditions which are not optimal for cell viability and after extended periods of culture suggests that LIF may be increasing cell number not by increasing replication but by promoting survival of myoblasts.
There is evidence to suggest that LIF promotes survival of myoblasts and other cell types [14], [15], [16]. It has been demonstrated that LIF treatment maintained viability of primary myoblasts over long periods of culture under sub-optimal growth conditions [14]. Similarly, LIF enhanced the survival of rhabdomyosarcoma cell lines treated with etoposide [15]. LIF treatment was also capable of inhibiting doxorubicin induced apoptosis of cardiac myocytes via activation of PI3K [16], a pathway shown to be involved in LIF-dependent increases in myoblast cell number [10]. Administration of LIF in conjunction with myoblast transfer therapy showed an increase in dystrophin expression of mdx mice [17], [18] and may be due to LIF promoting survival of the transferred myoblasts, which otherwise die within 24 h of injection in the host environment [19]. Therefore we hypothesise that LIF inhibits apoptosis of myoblasts and that this accounts for the enhanced viability and increased cell number previously observed.
Section snippets
Cell culture and reagents
C2C12 myoblast cells were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). Recombinant murine LIF was purchased from Millipore (Billerica, MA, USA). FGF-2 was obtained from Invitrogen (Carlsbad, California, USA). All reagents were obtained from Sigma-Aldrich (Castle Hill, NSW, Australia) unless otherwise stated.
MTT assay of cell number
An MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)-based assay was employed which is commonly used to measure cell viability or number. C2C12
LIF increased myoblast cell number over extended periods of culture
Using an MTT-based assay of cell number, we observed that when myoblasts were cultured under sub-optimal conditions (growth media containing 5% FBS) with concentrations of LIF ranging from 0.001 to 10 ng/mL, the number of cells present was increased after 5 days of culture, but not at earlier time points (Fig. 1). By day 5, concentrations as low as 0.1 ng/mL LIF significantly increased (P < 0.05) the number of cells present. 5 ng/mL LIF produced a maximum increase in cell number, which was
Discussion
These studies demonstrate the novel effect that LIF prevents apoptotic cell death of myoblasts, which results in increased cell number. Increases in myoblast cell number caused by LIF occurred after relatively long treatment periods, which suggested the possibility of an anti-apoptotic effect of LIF on myoblasts. LIF induced a rapid response that protected myoblasts from caspase-3 activation and DNA fragmentation. It is not surprising that LIF acts quickly to prevent these apoptotic changes as
Acknowledgments
The authors would like to acknowledge the support and funding of the Muscular Dystrophy Association of Australia (MDA).
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