Myocyte-derived Tnfsf14 is a survival factor necessary for myoblast differentiation and skeletal muscle regeneration

Adult skeletal muscle tissue has a uniquely robust capacity for regeneration, which gradually declines with aging or is compromised in muscle diseases. The cellular mechanisms regulating adult myogenesis remain incompletely understood. Here we identify the cytokine tumor necrosis factor superfamily member 14 (Tnfsf14) as a positive regulator of myoblast differentiation in culture and muscle regeneration in vivo. We find that Tnfsf14, as well as its cognate receptors herpes virus entry mediator (HVEM) and lymphotoxin β receptor (LTβR), are expressed in both differentiating myocytes and regenerating myofibers. Depletion of Tnfsf14 or either receptor inhibits myoblast differentiation and promotes apoptosis. Our results also suggest that Tnfsf14 regulates myogenesis by supporting cell survival and maintaining a sufficient pool of cells for fusion. In addition, we show that Akt mediates the survival and myogenic function of Tnfsf14. Importantly, local knockdown of Tnfsf14 is found to impair injury-induced muscle regeneration in a mouse model, affirming an important physiological role for Tnfsf14 in myogenesis in vivo. Furthermore, we demonstrate that localized overexpression of Tnfsf14 potently enhances muscle regeneration, and that this regenerative capacity of Tnfsf14 is dependent on Akt signaling. Taken together, our findings reveal a novel regulator of skeletal myogenesis and implicate Tnfsf14 in future therapeutic development.Mature skeletal muscle tissue contains a resident population of stem cells that imparts a great capacity for regeneration. Upon injury, these quiescent satellite cells are reactivated and begin to proliferate.^{1, 2} Effective myogenesis depends on the daughter myoblasts successfully differentiating and fusing with each other to regenerate the characteristic multinucleated skeletal myofibers. This involves a number of highly regulated steps, including activation of myogenic genes, migration, cell–cell adhesion and alignment, and finally membrane fusion.^{3, 4, 5} The fundamental principles underlying each step are well-conserved across species.⁶ Pathologies may result from dysregulation of these processes, including the suite of muscular dystrophies, cachexia and sarcopenia. However, the complex signaling mechanisms underlying skeletal myogenesis are still not fully understood.It has long been accepted that the secreted factors influencing muscle cell regeneration in vivo are largely of immune cell origin; indeed, immune cells have been reported to reach concentrations over 100 000 cells/mm³ in regenerating muscle tissue.⁷ Recently, however, muscle cells are being revealed as prolific secretors of a wide variety of cytokines and growth factors,^{8, 9, 10, 11} including several that attract immune cells to regenerating muscle.⁷ Secretome studies show that myoblasts secrete different factors during proliferation than during differentiation, and even at different time points throughout differentiation.^{8, 10, 11} Another study identified numerous chemokine mRNAs expressed by differentiating myoblasts, which may be involved in regulating cell migration during myogenesis.⁹ However, functions of the newly identified muscle-secreted cytokines are mostly unexplored. Using RNAi, we conducted the first functional screen of cytokines for their impact on myogenic differentiation in C2C12 myoblasts, which allowed us to identify potential regulators of myogenesis in distinct functional groups.¹² These results suggest the intriguing possibility that muscle cell-secreted proteins have a previously under-appreciated role in modulating muscle development and regeneration.The function of cytokines in myogenesis is relevant to our understanding of not only basic muscle physiology, but also the diseases that negatively affect the health of muscle tissue, such as cachexia. Cachexia is characterized by extreme wasting of lean body mass and often occurs with an underlying chronic illness, such as cancer or congestive cardiac failure.¹³ Muscle atrophy during cachexic states ultimately stems from ubiquitin-mediated breakdown of myofibrils.¹⁴ Significantly, a well-documented association exists between cachexia and the dysregulation of cytokines, most notably the pro-inflammatory cytokines tumor necrosis factor alpha (TNFα), interleukin-1 (IL-1) and interleukin-6 (IL-6).^{14, 15}Tumor necrosis factor superfamily member 14 (Tnfsf14), also known as LIGHT (homologous to lymphotoxins, shows inducible expression, and competes with herpes simplex virus glycoprotein D for herpes virus entry mediator (HVEM), a receptor expressed by T lymphocytes), exists in two main forms: a type II transmembrane glycoprotein that projects extracellularly, and a soluble cytokine formed by cleavage of the extracellular portion of the protein off of the cell membrane.¹⁶ Through its receptors in the TNF receptor (TNFR) superfamily, HVEM (TNFRSF14) and lymphotoxin β receptor (LTβR), Tnfsf14 signaling is involved in lymphoid organ development and organization, as well as innate and adaptive immune responses.^{17, 18, 19} In recent years, Tnfsf14 has also emerged as a promising candidate for cancer immunotherapy.²⁰Tnfsf14 regulates cell survival and apoptosis in lymphocytes and tumor cells, and the cellular context determines whether Tnfsf14 is pro-survival or pro-apoptosis.^{20, 21, 22} Neither the expression nor the function of Tnfsf14 or its receptors has been reported in skeletal muscles. Our current study uncovers Tnfsf14 as a critical regulator of myoblast differentiation and muscle regeneration by governing myoblast survival, and implicates Tnfsf14 in potential therapeutic development for maintenance of muscle health.     

Hepatocyte growth factor is a novel stimulator of glucose uptake and metabolism in skeletal muscle cells

Skeletal muscle plays a major role in glucose and lipid metabolism. Active hepatocyte growth factor (HGF) is present in the extracellular matrix in skeletal muscle. However, the effects of HGF on glucose and lipid metabolism in skeletal muscle are completely unknown. We therefore examined the effects of HGF on deoxyglucose uptake (DOGU), glucose utilization, and fatty acid oxidation (FAO) in skeletal muscle cells. HGF significantly enhanced DOGU in mouse soleus muscles in vitro. Furthermore, HGF significantly increased: (i) DOGU in a time- and dose-dependent manner; (ii) glucose utilization; and (iii) plasma membrane expression of Glut-1 and Glut-4 in the rat skeletal muscle model of L6 myotubes. HGF-mediated effect on DOGU was dependent on the activation of phosphatidylinositol 3-kinase signaling pathway. On the other hand, HGF markedly and significantly decreased FAO in L6 myotubes without affecting the activities of carnitine palmitoyltransferase I and II. Collectively, these results indicate that HGF is a potent activator of glucose transport and metabolism and also a strong inhibitor of FAO in rodent myotubes. HGF, through its ability to stimulate glucose transport and metabolism and to impair FAO, may participate in the regulation of glucose disposal in skeletal muscle.     

Myodulin is a novel potential angiogenic factor in skeletal muscle

We examined the expression and function of a gene we previously cloned from its downregulation in a muscle atrophy model. The encoded protein was named myodulin because of sequence homologies with the cartilage-specific chondromodulin-I (ChM-I) protein, its restricted expression in skeletal muscle tissue, and its modulating properties on vascular endothelial cells described here. We investigated the expression of myodulin in muscle fibers and cultured muscle cells. Myodulin RNA messengers were found in muscle fibers and their tendon extensions. Overexpression of myodulin fused to a FLAG peptide showed evidence of a muscle cell surface protein. Myodulin functions were assessed from similarities with chondromodulin-I. Coculture experiments using C(2)C(12) mouse myoblasts or myotubes, which stably overexpress myodulin, with H5V mouse cardiac vascular endothelial cells revealed that myodulin had a very active role in the invasive action of endothelial cells, without any evidence of extracellular myodulin secretion. Our results suggest that myodulin may be a muscle angiogenic factor operating through direct cell-to-cell interactions. This role is consistent with the correlation between modulations in myodulin expression and modifications in muscle microvascularization associated with activity-dependent muscle mass variations.     

Nerve growth factor in skeletal tissues of the embryonic chick

Summary This study demonstrates, via immunohistochemistry and bioassay, the presence of NGF in embryonic bone and cartilage of the chick. Embryos were killed on days 6–9 of incubation at 12 h intervals, and on days 10–18 at 24 h intervals. Paraffin-embedded sections of hind limbs or buds were immunostained with a polyclonal antibody against NGF and the biotin-avidin-horseradish peroxidase technique. Immunostaining was positive in both bone and cartilage, with cartilage staining more intensely. For bioassay, bones from the hind limbs of 9- and 12-day embryos were fast-frozen, lyophilized, and homogenized with Medium 199 (M199). Dorsal root ganglia from 8-day embryos were cultured for 24–36 h with rooster plasma, M199, and varying concentrations of bone homogenate. Significant neurite outgrowth was seen, with the greatest response elicited by 12-day bone homogenate. Addition of anti-NGF to the cultures abolished neurite outgrowth. The results indicate that NGF is present in cartilage and bone of the chick embryo; it may determine the density of sympathetic innervation to the developing skeletal tissues.     

There is selective accumulation of a growth factor in chicken skeletal muscle. II. Transferrin accumulation in dystrophic fast muscle

Transferrin or a transferrin-like protein, with ability to stimulate myogenesis and terminal differentiation in vitro, is found in fast chicken muscle during embryonic development. After hatching, however, transferrin is no longer accumulated or is only weakly accumulated by fast muscles like the pectoralis major and the posterior latissimus dorsi but continues to be accumulated by slow muscles like the anterior latissimus dorsi. In congenic lines of chickens bearing the gene for muscular dystrophy, however, adult fast muscles do not lose the ability to accumulate transferrin. While transferrin is found selectively in adult normal and dystrophic muscle it does not appear to be synthesized by muscle cells. Immunocytochemical localization shows that transferrin is accumulated not so much by muscle fibers as it is by single cells in the muscle interstitial space. The relationship between transferrin presence and growth patterns in adult skeletal muscle is not currently understood but evidence suggests that transferrin stimulation of myogenesis observed in vitro may be mediated in vivo by non-muscle cells dwelling within the muscle interstitial space. These cells may act as transferrin-uptake sources for subsequent satellite cell stimulation.     

Fibroblast growth factor-16 is a growth factor for embryonic brown adipocytes

In rat embryos, fibroblast growth factor (FGF)-16 is predominantly expressed in brown adipose tissue. To elucidate the role of FGF-16, we examined the expression of FGF-16 mRNA in rat embryonic brown adipose tissue at different developmental stages by Northern blotting analysis and in situ hybridization. FGF-16 mRNA was expressed abundantly in brown adipose tissue during embryonic day 17. 5, embryonic days 17.5-19.5, and thereafter at lower levels into the neonatal period. The expression profile of FGF-16 mRNA well corresponds to the proliferative profile of embryonic brown adipose tissue reported. We also examined the mitogenic activity of recombinant rat FGF-16 for primary brown adipocytes prepared from rat embryonic brown adipose tissue. FGF-16 showed significant mitogenic activity for primary brown adipocytes. The mitogenic activity was found to be exerted by binding and activating FGF receptor-4 in the brown adipose tissue. As a great induction of proliferation of rat brown adipose tissue during cold acclimation was reported, we also examined the expression of FGF-16 mRNA in the brown adipose tissue during cold acclimation by Northern blotting analysis. The expression of FGF-16 mRNA was not increased, but rather decreased. The expression profile of FGF-16 mRNA and the mitogenic activity of FGF-16 reported here indicate that FGF-16 is a unique growth factor involved in proliferation of embryonic brown adipose tissue.     

Synaptic contacts between embryonic Xenopus neurons and myotubes formed from a rat skeletal muscle cell line

Acetylcholine receptors (AChRs) accumulate at the junctional region during early development. In an attempt to characterize this process of AChR accumulation, we combined embryonic Xenopus neurons with myotubes formed from a rat skeletal muscle cell line. Xenopus neurons in culture are known to induce AChR accumulation in Xenopus muscles [Anderson, M. J., Cohen, M. W., and Zorychta, E. (1977). J. (London), 268, 731–756]. Rat myotubes, however, do not exhibit AChR accumulation in culture even when they are functionally innervated by the fetal rat spinal cord explant [Kidokoro, Y. (1980) Develop. Biol., 78, 231–241]. Establishment of synaptic transmission was examined electrophysiologically by recording synaptic potentials, while the distribution of AChR clusters was visualized using fluorescent α-bungarotoxin. Our results indicate that embryonic Xenopus neurons formed functional synaptic contacts but did not cause AChR accumulation in L6-myotubes. It seems that the ability of a nerve to cause AChR accumulation is separate from that to form the functional synapse. We also found that the mean amplitude of synaptic potentials in L6-myotubes interacted with Xenopus neurons was about half of that in L6-myotubes innervated by fetal rat spinal cord explants. Possible explanations for this finding are discussed.     

Insulin-like growth factor 1 is required for survival of transit-amplifying neuroblasts and differentiation of otic neurons

Neurons that connect mechanosensory hair cell receptors to the central nervous system derive from the otic vesicle from where otic neuroblasts delaminate and form the cochleovestibular ganglion (CVG). Local signals interact to promote this process, which is autonomous and intrinsic to the otic vesicle. We have studied the expression and activity of insulin-like growth factor-1 (IGF-1) during the formation of the chick CVG, focusing attention on its role in neurogenesis. IGF-1 and its receptor (IGFR) were detected at the mRNA and protein levels in the otic epithelium and the CVG. The function of IGF-1 was explored in explants of otic vesicle by assessing the formation of the CVG in the presence of anti-IGF-1 antibodies or the receptor competitive antagonist JB1. Interference with IGF-1 activity inhibited CVG formation in growth factor-free media, revealing that endogenous IGF-1 activity is essential for ganglion generation. Analysis of cell proliferation cell death, and expression of the early neuronal antigens Tuj-1, Islet-1/2, and G4 indicated that IGF-1 was required for survival, proliferation, and differentiation of an actively expanding population of otic neuroblasts. IGF-1 blockade, however, did not affect NeuroD within the otic epithelium. Experiments carried out on isolated CVG showed that exogenous IGF-1 induced cell proliferation, neurite outgrowth, and G4 expression. These effects of IGF-1 were blocked by JB1. These findings suggest that IGF-1 is essential for neurogenesis by allowing the expansion of a transit-amplifying neuroblast population and its differentiation into postmitotic neurons. IGF-1 is one of the signals underlying autonomous development of the otic vesicle.     

Upregulation of endogenous heparin-binding EGF-like growth factor and its role as a survival factor in skeletal myotubes.

To investigate the role of heparin-binding EGF-like growth factor (HB-EGF) in skeletal muscle, we studied its function in skeletal myotubes in vitro using mouse C2C12 cells. Expression levels of membrane-anchored HB-EGF (proHB-EGF) protein were increased specifically during their differentiation among epidermal growth factor receptor (EGFR) ligands. Production levels of EGFR on the cell surface were constant. Tyrosine phosphorylation of EGFR, however, was constitutively increased during differentiation. Quenching of endogenous HB-EGF significantly rendered myotubes sensitive to apoptotic cell death induced by hypoxic stress, suggesting that proHB-EGF in the skeletal muscle is specifically upregulated to function as a survival factor.     

Nerve growth factor and fibroblast growth factor influence post-fusion expression of Na-channels in cultured rat skeletal muscle

We have examined effects of nerve growth factor (NGF) and fibroblast growth factor (FGF) on the density of tetrodotoxin (TTX)-sensitive Na-channels in cultured rat skeletal muscle. Measurements were made of specific binding of [3H]saxitoxin (STX) and the frequency and rate of rise of spontaneously occurring action potentials, the physiological expression of Na-channel density. Cells were transferred to various growth conditions at 6 days in vitro, and measurements were made beginning 24 hr later. Both growth factors (GF) caused dose-related increases in Na-channels compared with myotubes maintained in normal, serum-supplemented growth medium. Maximum effects occurred with a concentration of NGF of 50 ng/ml and FGF of 15 ng/ml. Scatchard analysis of specific STX binding showed an increase in Bmax with no significant change in Kd. Similar increases occurred on rate of rise and frequency spontaneous action potential. Treatment of cultures with cycloheximide or actinomycin D, inhibitors of protein and RNA synthesis, completely prevented the increase in STX-binding induced by GF treatment. The results indicate that NGF and FGF have important effects on regulation of excitable cell gene products after differentiation.     

The induction of skeletal muscle hypertrophy by a ski transgene is promoter-dependent

The chicken c-ski gene expresses at least three alternatively spliced messages. Transgenic mice expressing proteins from cDNA corresponding to two of these messages (FB27 and FB29) under the control of a murine sarcoma virus (MSV) long terminal repeat (LTR) express the transgene in skeletal muscle and develop a muscular phenotype. Both a biologically active form of c-ski and the MSV LTR are required for the development of the muscular phenotype. The normal c-ski gene linked to two other tissue-specific promoters failed to induce muscle growth in transgenic mice, as did an inactive mutant of c-ski expressed under the control of the MSV LTR.     

Insulin-like growth factor signaling regulates zebrafish embryonic growth and development by promoting cell survival and cell cycle progression

Although much is known about the global effects of insulin-like growth factor 1 receptor (IGF1R)-mediated signaling on fetal growth and the clinical manifestations resulting from IGF/IGF1R deficiencies, we have an incomplete understanding of the cellular actions of this essential pathway during vertebrate embryogenesis. In this study, we inhibited IGF1R signaling during zebrafish embryogenesis using antisense morpholino oligonucleotides or a dominant-negative IGF1R fusion protein. IGF1R inhibition resulted in reduced embryonic growth, arrested development and increased lethality. IGF1R-deficient embryos had significant defects in the retina, inner ear, motoneurons and heart. No patterning abnormalities, however, were found in the brain or other embryonic tissues. At the cellular level, IGF1R inhibition increased caspase 3 activity and induced neuronal apoptosis. Coinjection of antiapoptotic bcl2-like mRNA attenuated the elevated apoptosis and rescued the retinal and motoneuron defects, but not the developmental arrest. Subsequent cell cycle analysis indicated an increased percentage of cells in G1 and a decreased percentage in S phase in IGF1R-deficient embryos independent of apoptosis. These results provide novel insight into the cellular basis of IGF1R function and show that IGF1R signaling does not function as an anteriorizing signal but regulates embryonic growth and development by promoting cell survival and cell cycle progression.     

Elongation factor 2 in the liver and skeletal muscle of mice is decreased by starvation

We examined whether starvation affected the amount of EF-2 protein as well as the level of its mRNA in the liver and skeletal muscle of mice, to understand the molecular mechanism for nutritional adaptation of protein-turnover. Although the amount of EF-2 was diminished by starvation in each of the tissues examined, the amount of EF-2 mRNA did not decrease in parallel with the protein.     

Growth factor control of skeletal muscle differentiation: commitment to terminal differentiation occurs in G1 phase and is repressed by fibroblast growth factor

Analysis of MM14 mouse myoblasts demonstrates that terminal differentiation is repressed by pure preparations of both acidic and basic fibroblast growth factor (FGF). Basic FGF is approximately 30-fold more potent than acidic FGF and it exhibits half maximal activity in clonal assays at 0.03 ng/ml (2 pM). FGF repression occurs only during the G1 phase of the cell cycle by a mechanism that appears to be independent of ongoing cell proliferation. When exponentially growing myoblasts are deprived of FGF, cells become postmitotic within 2-3 h, express muscle-specific proteins within 6-7 h, and commence fusion within 12-14 h. Although expression of these three terminal differentiation phenotypes occurs at different times, all are initiated by a single regulatory "commitment" event in G1. The entire population commits to terminal differentiation within 12.5 h of FGF removal as all cells complete the cell cycle and move into G1. Differentiation does not require a new round of DNA synthesis. Comparison of MM14 behavior with other myoblast types suggests a general model for skeletal muscle development in which specific growth factors serve the dual role of stimulating myoblast proliferation and directly repressing terminal differentiation.     

Regulation of skeletal muscle satellite cell proliferation by bovine pituitary fibroblast growth factor

Satellite cells in skeletal muscle have been implicated in muscle growth processes and regeneration. However, very little is known about the regulation of their proliferation and differentiation. The effect of fibroblast growth factor (FGF) on the proliferation of myogenic cells from adult rat skeletal muscle, presumably satellite cells, has been examined, and FGF has been found to be a potent mitogen for these cells. The mitogenic properties of serum were also documented and studied in conjunction with FGF. Even under conditions of maximal stimulation by serum, the addition of FGF caused a substantial increase in proliferation of satellite cells. The additive nature of the FGF and serum-stimulatory activity suggests that FGF-like molecules are not the active agents in serum and that more than one pathway may be involved in stimulating satellite cell proliferation.     

Distinct neurotrophic factors from skeletal muscle and the central nervous system interact synergistically to support the survival of cultured embryonic spinal motor neurons

Motor neurons isolated from 6-day-old embryonic chick spinal cords require muscle extract for survival in culture; however, it was found, that some motor neurons, identified by retrograde labeling with rhodamine, will survive in mixed spinal cell cultures in the absence of the extract. The motor neuron survival-promoting activity produced by spinal cells is soluble and differs from the factor present in muscle extract, the two activities acting in a synergistic manner: the spinal cell activity potentiated that of muscle to decrease its ED50 by an order of magnitude, the motor neuronal survival (30%) seen in the presence of both factors being more than the sum of their individual activities. This synergism was shown to be restricted to the action of the spinal cell factor on motor neurons, no effect of the factor being noted with sympathetic neurons. As a series of defined growth and survival factors present in the central nervous system (nerve growth factor, brain-derived neurotrophic factor, acidic and basic fibroblast growth factors) had no effect on motor neuron survival, we conclude that the molecule responsible for the motor neuron survival-promoting activity of the spinal cells is a previously undefined factor.     

Acidic and basic fibroblast growth factor mRNAs are expressed by skeletal muscle satellite cells

We postulated that Fibroblast Growth Factor (FGF) involved in fetal or regenerative morphogenesis of skeletal muscle originated from this tissue. Using a bovine retina cDNA probe encoding acidic FGF, we showed that growing muscles from bovine fetuses express this mRNA, but that this expression is reduced in neonate muscles. Cultures of proliferating satellite cells isolated from adult rat muscles expressed aFGF mRNA strongly but bFGF mRNA weakly; these mRNAs disappeared in cells differentiated into myotubes. 10(-7)M 12-O-tetradecanoyl phorbol -13-acetate (TPA) increased aFGF mRNA expression in both proliferating and differentiated satellite cells. Contrastingly, proliferating L6 myogenic cells only expressed aFGF mRNA significantly under TPA treatment. Therefore, the satellite cells did seem to be a possible source for FGF, especially aFGF, which might regulate the myogenic process.