Elsevier

Experimental Cell Research

Volume 316, Issue 6, 1 April 2010, Pages 1002-1009
Experimental Cell Research

Research Article
Leukemia inhibitory factor-dependent increase in myoblast cell number is associated with phosphotidylinositol 3-kinase-mediated inhibition of apoptosis and not mitosis

https://doi.org/10.1016/j.yexcr.2009.11.022Get rights and content

Abstract

Leukemia inhibitory factor (LIF) is an important regulator of skeletal muscle regeneration and has been suggested to be mitogenic for myogenic cells because it has been shown to increase the quantity of myoblast cells grown in culture over extended periods of time. Using the established C2C12 murine myoblast cell line, we observed that LIF treatment did not significantly increase the rate at which myoblasts synthesise DNA under conditions which increased cell quantity by 73% above control, whilst the known mitogen fibroblast growth factor-2 significantly increased DNA synthesis under these conditions. Consequently, we examined the capacity of LIF to prevent apoptotic cell death. LIF treatment significantly reduced staurosporine-induced apoptotic DNA fragmentation by 37% compared to control and also reduced the proteolytic activation of caspase-3 by 40% compared to control. This effect of LIF was completely abolished by addition of the phosphatidylinositol 3-kinase inhibitor wortmannin, indicating that the phosphatidylinositol 3-kinase signalling pathway, previously shown to be linked to LIF-dependent increases in cell number, is necessary in mediating the anti-apoptotic effects of LIF. LIF treatment was also associated with increased levels of Bcl-xL and XIAP transcripts compared to control. Therefore, we suggest that the role of LIF in skeletal muscle regeneration and myogenesis is that of a survival factor rather than a mitogen.

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).

References (43)

  • M. Sandri et al.

    Apoptosis of skeletal muscles during development and disease

    Int. J. Biochem. Cell Biol.

    (1999)
  • D.P. Gearing et al.

    Molecular cloning and expression of cDNA encoding a murine myeloid leukaemia inhibitory factor (LIF)

    EMBO J.

    (1987)
  • J.B. Kurek et al.

    Leukemia inhibitory factor and interleukin-6 are produced by diseased and regenerating skeletal muscle

    Muscle Nerve

    (1996)
  • K.A. Reardon et al.

    Increased levels of leukemia inhibitory factor mRNA in muscular dystrophy and human muscle trauma

    Muscle Nerve

    (2000)
  • J.B. Kurek et al.

    The role of leukemia inhibitory factor in skeletal muscle regeneration

    Muscle Nerve

    (1997)
  • D.P. Gearing et al.

    The IL-6 signal transducer, gp130: an oncostatin M receptor and affinity converter for the LIF receptor

    Science

    (1992)
  • L.A. Megeney et al.

    bFGF and LIF signaling activates STAT3 in proliferating myoblasts

    Developmental Genetics

    (1996)
  • E.E. Spangenburg et al.

    Multiple signaling pathways mediate LIF-induced skeletal muscle satellite cell proliferation

    Am. J. Physiol. Cell. Physiol.

    (2002)
  • X. Wang et al.

    Effects of interleukin-6, leukemia inhibitory factor, and ciliary neurotrophic factor on the proliferation and differentiation of adult human myoblasts

    Cell. Mol. Neurobiol.

    (2008)
  • D.J. Milasincic et al.

    Stimulation of C2C12 myoblast growth by basic fibroblast growth factor and insulin-like growth factor 1 can occur via mitogen-activated protein kinase-dependent and -independent pathways

    Mol. Cell. Biol.

    (1996)
  • J.D. White et al.

    Leukaemia inhibitory factor increases myoblast replication and survival and affects extracellular matrix production: combined in vivo and in vitro studies in post-natal skeletal muscle

    Cell. Tissue Res.

    (2001)
  • Cited by (24)

    • The cytokine interleukin-11 crucially links bone formation, remodeling and resorption

      2021, Cytokine and Growth Factor Reviews
      Citation Excerpt :

      LIF stimulates bone resorption by a mechanism involving prostaglandin [84] but also inhibitory actions of LIF on basal resorption rates have been identified [85]. Although skeletal muscle cells secrete several members of the IL-6-type cytokines including IL-6, LIF and OSM, the most abundant myokine is CNTF, which suppresses osteoblast differentiation and bone formation on the periosteum [86–89]. In addition to this CNTF is secreted by osteoblasts, osteocytes and osteoclasts.

    • Interval exercise training increases LIF expression and prevents myocardial infarction-induced skeletal muscle atrophy in rats

      2018, Life Sciences
      Citation Excerpt :

      Indeed, exogenous LIF intervention could induce human myoblast proliferation, an effect that likely occurs via induction of cell proliferation-associated factors c-Myc and JunB. In addition, LIF treatment could also inhibit staurosporine-induced myoblast cellapoptosis and increase the transcriptional levels of Bcl-xL and XIAP [12]. A study showed that the circulatory level of LIF is decreased quickly in mouse after myocardial infarction (MI) [13].

    • Cell-specific paracrine actions of IL-6 family cytokines from bone, marrow and muscle that control bone formation and resorption

      2016, International Journal of Biochemistry and Cell Biology
      Citation Excerpt :

      In contrast the periosteal surface exists in apposition to the muscle, which has recently come to prominence as a possible source of paracrine factors that influence bone structure, particularly cortical bone mass (DiGirolamo et al., 2013). Skeletal muscle secretes several members of the IL-6 cytokine family including leukemia inhibitory factor (LIF) (Hunt et al., 2010; Hunt et al., 2013), IL-6 (Hiscock et al., 2004), and OSM (Hojman et al., 2011). These factors are now recognised as myokines (muscle-derived cytokines) that influence local muscle cell homeostasis (Hunt and White, 2016) and enter the circulation as endocrine factors with effects on liver, adipose tissue, the immune system, cancer growth and pancreas function (Pedersen and Febbraio, 2012).

    • Myomir dysregulation and reactive oxygen species in aged human satellite cells

      2016, Biochemical and Biophysical Research Communications
      Citation Excerpt :

      It is worth mentioning, we previously showed apoptotic commitment in elderly myoblasts [17]. The down-regulation of PI3K and LIF along with the up-regulation of IL-18 in elderly myoblasts and it is involved in apoptosis and tissue destruction [38,39]. This situation seems further exacerbated by ubiquitin proteasome system pathway.

    View all citing articles on Scopus
    View full text