Effects of Interleukin-6, Leukemia Inhibitory Factor, and Ciliary Neurotrophic Factor on the Proliferation and Differentiation of Adult Human Myoblasts
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Our previous studies have demonstrated that ciliary neurotrophic factor, a member of the interleukin-6-type cytokine superfamily, could inhibit the differentiation of myoblasts into mature myotubes at a certain concentration. In this study, another two members, interleukin-6 and leukemia inhibitory factor, together with ciliary neurotrophic factor were tested their roles in the proliferation and differentiation of myoblasts derived from the adult human skeletal muscles, in order to confirm that these cytokines might be a new type of regulatory factors on the myoblasts. The results showed that the effects of interleukin-6, leukemia inhibitory factor, and ciliary neurotrophic factor on the proliferation and differentiation of adult human myoblasts were different. Leukemia inhibitory factor in the dose of 10 ng/ml could accelerate the cell proliferation. Leukemia inhibitory factor in the dose of 10 or 50 ng/ml and ciliary neurotrophic factor in the dose of 10 or 50 ng/ml could inhibit the myoblast differentiation. The inhibition mechanism might be that leukemia inhibitory factor and ciliary neurotrophic factor inhibited the expressions of transcription factor MyoD/myf5, which could regulate the myoblast differentiation. This study will provide the experimental and theoretic foundations for the basic and clinical researches about human myoblasts.
KeywordsMyoblasts Interleukin-6 Leukemia inhibitory factor Ciliary neurotrophic factor
The superfamily of interleukin-6-type cytokines regulates cell survival, proliferation, and differentiation, and plays an important role in anti-inflammation, hematopoietic, and neuronal differentiation. This subfamily includes ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), interleukin-6 (IL-6), interleukin-11 (IL-11), oncostatin M (OSM), cardiotrophin-1 (CT-1) and so on (Heinrich et al. 2003; Murakami et al. 2004). Among them, IL-6, LIF, and CNTF are extensively studied with their roles in the hematopoietic and neural development (Nakajima et al. 1996; Gregg and Weiss 2005; Emery et al. 2006). It has been shown that CNTF receptors are expressed in the skeletal muscle tissue (Sleeman et al. 2000) and LIF is a trophic factor for cadiocytes and cardiomyoblasts (Austin et al. 2000). For embryonic stem cells, LIF inhibits their differentiation and maintains them in the primary status (Davey and Zandstra 2006).
The myoblasts provide the sufficient source for muscle formation, which is potentially useful for treatment of the muscular atrophy and non-muscle diseases, such as neurodegenerative disorders (Gussoni et al. 1992; Mignon et al. 2005). Furthermore, these cells can be obtained from the patients per se, providing the advantage for clinical use. It had been reported that LIF promoted the proliferation of myoblasts (Spangenburg and Booth 2002) and blocked the myogenic differentiation of murine myoblasts (Jo et al. 2005). CNTF had been reported that it inhibited the human myoblasts to differentiate into mature myotubes (Chen et al. 2005). However, whether IL-6 and LIF also affect proliferation and differentiation of adult human myoblasts remains unclear. Since, IL-6 and LIF share many common functions with CNTF, it is thus possible that these two cytokines may also affect human myoblasts. In the present study, we have studied the effects of IL-6, LIF, and CNTF on the myoblasts derived from the adult human skeletal muscles and found that these cytokines could affect the proliferation and differentiation of myoblasts. The molecular mechanisms by which the three cytokines regulate the proliferation and differentiation of myoblasts were also discussed.
Source of Muscle
Mixed primary cultures were obtained from the temporal muscle of an adult human autopsy tissue suffered with cerebral tumor. The procedure of myoblast isolation and purification was performed as previously described (Rando and Blau 1994). Briefly, the muscle was placed in a few drops of cold D-Hank’s buffer to keep it moist, then was dissociated both enzymatically and mechanically by mincing the muscle into a coarse slurry with a razor blade in 2 ml of a solution of 2.4 U/ml dispase and 1% collagenase II, supplemented with CaCl2 to a final concentration of 2.5 mM. The slurry was then maintained at 37°C for 45 min with occasional mixing. To remove the enzymatic solution, the slurry was centrifuged and resuspended in a selective medium containing 80% Ham’s F-10 nutrient mixture, 20% fetal bovine serum (FBS), 2.5 ng/ml basic fibroblast growth factor (bFGF), penicillin G (200 U/ml), and streptomycin (200 μg/ml). Then, the suspension was plated on the 1% poly-l-lysin-coated dishes growing in a humidified incubator containing 5% CO2 at 37°C. This medium is selective for the growth of myoblasts while inhibiting fibroblast growth. After 2 h, supersuspension was collected and replaced into new lysine-coated dishes. Then supersuspension was removed into new dishes again after 24 h, and the procedure of the above continued for 3 days. After contiguous adhesion experiments, myoblasts would be highly purified, and the fibroblasts contaminated could shrink to the least extent.
After above primary purification cultivation, a selective medium containing growth medium (GM) for primary myoblasts was used. It consisted of 40% Ham’s F-10 nutrient mixture, 40% DMEM, and 20% FBS, supplemented with 2.5 ng/ml bFGF, penicillin G (200 U/ml), and streptomycin (200 μg/ml). This medium was selective for the growth of myoblasts while inhibiting fibroblast growth. In order to purify the primary cultured myoblasts further, myoblasts were serially diluted in GM and plated onto 96-well plates, and allowed to adhere overnight, after which the well containing the individual cell was marked. After 1-week proliferation, progenies derived from single one cell were passaged and seeded into one well of 24-well plate for 1 week, then of 6-well plate. After grown to subconfluence, these monoclonal cells were passaged and part of them were stained with anti-desmin antibody (see Immunocytochemistry assay). Only the desmin + clones were selected and seeded into the 25-cm2 tissue culture flasks in GM for further proliferation and passages.
IL-6/LIF/CNTF on the Proliferation and Differentiation of Myoblasts
Cloned myoblasts were seeded in the 21 wells of 24-well plates. The cell density was adjusted to 104/ml. For the proliferation experiment, GM was added into the wells as the control group. Cells in other groups were treated with different doses of IL-6, LIF, or CNTF, respectively. The concentration of cytokines was 0.1, 1, 10, and 50 ng/ml. Cell number in three wells were counted (n = 3) by trypan blue staining every 24 h for 7 days, and then cell proliferation curve was drafted. The different effects of IL-6, LIF, and CNTF on the day 5 in vitro (DIV) were analyzed and compared with each other. Cell cycle was analyzed by flow cytometry for further testing the cell proliferation. Briefly, cloned myoblasts were treated with cytokines, respectively for 48 h. Then they were trypsinized, spun, washed in cold phosphate-buffered salt (PBS) solution, and fixed in ethanol for 2 h. The cells were resuspended in 0.1% Triton X-100 in PBS with 0.2 mg/ml RNase A and 20 ng/ml propidium iodide solution. Cell percentage of G0/G1 phase, S phase, and G2-M phase among the whole cell cycle were recorded and analyzed. For differentiation experiment, GM was replaced with differentiation media (DM) containing DMEM with 2% horse serum, penicillin G (200 U/ml), and streptomycin (200 μg/ml). Different doses of IL-6, LIF, or CNTF were added into the culture media, respectively as above-mentioned. The extent of myoblast myogenic differentiation was measured by the number of multinucleated myotube forming cells containing more than three nuclei were regarded as multinucleated myotubes. The expressions of mature myogenic differentiation marker proteins, myogenin and myosin were examined by immunofluorescent staining, and the expression levels of myogenic regulatory factors, MyoD and myf5 were also tested by Western blot analysis.
Cells on coverslips were fixed by 4% paraformaldehyde, blocked with 10% normal goat serum and incubated with rabbit anti-desmin (1:500, Chemicon), mouse anti-myogenin (1:500, Santa Cruze), and mouse anti-myosin (1:500, Santa Cruze) primary antibodies and then with TRITC or FITC-conjugated secondary antibodies. Images were examined under confocal lazer scanning microscope (CLSM, Leica, Heideberg, Germany).
Western Blot Analysis
Cells were washed twice with 0.1 M PBS and lysed with 200 μl lysis buffer containing 0.01 M Tris–HCl, 0.15 M NaCl, 1 mM EDTA, 0.5% deoxycholic acid, 0.1% SDS, 1 mM Na3VO4, 1 mM PMSF, and 1% NP-40. Equal amounts of protein were loaded under reducing conditions onto a 10% SDS gel. After electrophoresis, the protein was blotted onto a polyvinylidene difluoride membrane. The membrane was blocked by skimmed milk and incubated with mouse anti-MyoD (1:1000, Santa Cruze) and rabbit anti-myf5 primary antibodies (1:1000, Santa Cruze). Signals were visualized by incubating with horse radish peroxidase-conjugated secondary antibody and enhanced chemiluminescence reagent (NENTM Life Science Products), then photographed using Kodak 1D image analysis software. Mouse anti-β-actin (1:1000, Santa Cruze) was used as inner parameter.
All data were expressed as the mean ± SEM. One-way analysis of variance (ANOVA SPSS software) was used to compare the differences among groups, followed by a Bonferroni (Dunn) comparison using least squares-adjusted means. Probability levels of <0.05 were considered statistically significant.
Isolation and Characterization of Myoblasts
IL-6/LIF/CNTF Promoted Proliferation of the Myoblasts
IL-6/LIF/CNTF on the Differentiation of Myoblasts
Activated muscle satellite cells were considered as myoblasts when cultured in vitro and these precursor cells are very useful for degenerative diseases. The chief problem during studies of adult human myoblasts is that how to obtain homogeneous cell population without fibroblasts mixture (Rando and Blau 1994; Yablonka-Reuveni et al. 1987; Fukada et al. 2004; Qu-Petersen et al. 2002). In the present study, the desmin + monoclone of individual myoblasts was used for experiments, and these cloned cells were homogeneous, proliferated for a large scale and for a long time with preserving their myogenic lineage-committed phenotypes. In order to confirm the function of IL-6, LIF, and CNTF on the proliferation and differentiation of myoblasts, different concentration of three factors were added into the culture media. The results showed that among these cytokines, certain concentration of LIF could promote the cell proliferation, and certain concentration of LIF and CNTF could inhibit the cell differentiation into matured myotubes by inhibiting the expression of myogenic regulatory factors.
Contrary with those derived from rodent muscle tissues, cell proliferation ability of adult human myoblasts in our study was lower, which indicated that different genus derived myoblasts might have different cell growth checkpoint mechanism. Many experiments have confirmed that LIF, as an activator of human telomerase reverse transcriptase gene (Ostenfeld et al. 2000; Caldwell 2001), played a broad role in promoting the proliferation of kinds of stem cells such as embryonic stem cells or neural stem cells (Metcalf 2003; Uchida et al. 2000). Our results suggested that addition of LIF into the cultivation system of adult human myoblasts; it could resolve the problem of long-term proliferation and passage of cells.
Activation and differentiation of myoblasts are regulated by basic helix-loop-helix (bHLH) transcription factors—MyoD, myf5, myogenin, and MRF4, which determine the myogenic destiny of myoblasts (Seale and Rudnicki 2000; Sabourin et al. 1999; Arnold and Winter 1998). In the present experiment, when culture media containing the high level (20%) serum converting into low level (2%), membranes of myoblasts could fuse into multi-nuclei myotubes with expressing myogenic differentiation specific proteins myosin and myogenin. When the differentiating myoblasts were treated with different doses of IL-6, LIF, or CNTF, certain concentration of LIF or CNTF could inhibit the cell fusion, resulting in the less formation of multi-nuclei myotubes apparently. At the same time, the expression of myosin and myogenin sharply decreased, accompanied with the down-regulation of MyoD and myf5 expression. IL-6 did not affect on the differentiation of human adult myoblasts. These results indicated that during the differentiation regulation of human adult myoblasts, LIF and CNTF played the similar negative roles, whereas, IL-6 could not inhibit the differentiation and maturation of myoblasts. There have been other reports confirming that IL-6 may promote the differentiation and maturation procedure of myoblasts (Okazaki et al. 1996).
Presently, it has been very clear that IL-6, LIF, or CNTF play their biologic functions through combining with corresponding receptor compounds. The structures of receptor compounds of these three factors are very distinct. IL-6 receptor compound is a dimer consisting of IL-6 receptor and gp130, LIF receptor compound contains LIF receptor and gp130, and CNTF one contains CNTF receptor, LIF receptor, and gp130 (Heinrich et al. 1998). From the compositions of these receptor compounds, it is found that LIF receptor exists in the compounds of CNTF receptor, as well as LIF one (März et al. 2002), whereas default in the IL-6 receptor compounds. This suggested that LIF receptor might offer the most critical contribution to the differentiation inhibition of human adult myoblasts (Spangenburg and Booth 2002).
After combination with receptor compounds on the cell membrane, LIF, or CNTF activated inner-cellular signal transduction pathway, resulting in transcription and expression of target genes (Kami and Senba 2002). Our result had showed that LIF and CNTF could inhibit the expressions of MRFs—MyoD and myf5, therefore inhibiting the differentiation of human adult myoblasts into mature myotubes. Either LIF or CNTF possessed the ability of differentiation limitation on myoblasts, however, it seemed that the mechanism was slightly different. LIF inhibit the expression of MyoD mainly, and CNTF affected the myf5 expression. The reasons interpreting this disparity need to be investigated further. In addition to above interpretations on the differentiation inhibition efficacy of cytokines, the promoted proliferation by them followed by increasing cell number could not be ignored.
In conclusion, present studies demonstrated that IL-6, LIF, and CNTF, members of IL-6-type cytokine superfamily, were important regulation factors on the proliferation and differentiation of myoblasts derived from adult human skeletal muscles. Three factors promoted the cell growth, and certain doses of LIF or CNTF inhibit the myogenic differentiation. The different signal transduction procedures of three factors should be the next study focus in order to explore the molecular mechanisms of regulating the proliferation and differentiation of myoblasts.
This work was supported by Grants from the Chinese National Basic Research 973 Program (2006CB500700), National Nature Science Foundation of China (30500255, 30470833), Key Grant of PLA (06G002) and Talent Training Plan of Beijing (20061D0501800255). We thank Dr. Yizheng Wang for critical reading of the manuscript.
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