ARTICLE |
Correspondence to: Katsuya Kami, Dept. of Health Science, Osaka Univ. of Health and Sport Sciences, Noda 1558-1, Kumatori-cho, Sennan-gun, Osaka 590-0496, Japan. E-mail: kami@ouhs.ac.jp
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Summary |
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The biological actions of interleukin-6 (IL-6), leukemia inhibitory factor (LIF), and ciliary neurotrophic factor (CNTF) are mediated via respective functional receptor complexes consisting of a common signal-transducing component, gp130, and other specific receptor components, IL-6 receptor (IL-6R), LIF receptor ß (LIFR), and CNTF receptor
(CNTFR). IL-6, LIF, and CNTF are implicated in skeletal muscle regeneration. However, the cell populations that express these receptor components in regenerating muscles are unknown. Using in situ hybridization histochemistry, we examined spatiotemporal expression patterns of gp130, IL-6R, LIFR, and CNTFR mRNAs in regenerating muscles after muscle contusion. At the early stages of regeneration (from 3 hr to Day 2 post contusion), significant signals for gp130 and LIFR mRNAs were detected in myonuclei and/or nuclei of muscle precursor cells (mpcs) and in mononuclear cells located in extracellular spaces between myofibers after muscle contusion, but IL-6R mRNA was expressed only in mononuclear cells. At Day 7 post contusion, signals for gp130, LIFR, and IL-6R mRNAs were not detected in newly formed myotubes, whereas the CNTFR mRNA level was upregulated in myotubes. These findings suggest that the upregulation of receptor subunits in distinct cell populations plays an important role in the effective regeneration of both myofibers and motor neurons. (J Histochem Cytochem 48:12031213, 2000)
Key Words:
leukemia inhibitory factor, receptor ß, ciliary neurotrophic factor, receptor , interleukin-6 receptor
, gp130, muscle regeneration
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Introduction |
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SKELETAL MUSCLES are able to regenerate after injury. At the early stage of muscle regeneration, the injured myofibers degenerate and cell debris is removed by phagocytosis. In parallel with this degeneration and removal of necrotic tissues, quiescent muscle precursor cells (mpcs), also called satellite cells, are activated, proliferate, and fuse with other mpcs to form new multinucleated myotubes, and eventually replace the disrupted myofibers. This process is regulated in part by growth factors and cytokines, which are locally produced and released from injured myofibers and inflammatory mononuclear cells at the site of muscle injury (
A line of evidence suggests that interleukin-6 (IL-6), leukemia inhibitory factor (LIF), and ciliary neurotrophic factor (CNTF), which are members of the IL-6 family of cytokines consisting of IL-6, LIF, CNTF, interleukin-11 (IL-11), oncostatin M (OM), and cardiotrophin-1 (CT-1), are implicated in the degenerative and regenerative reactions of myofibers. Proteolysis of skeletal muscle proteins can be initiated by IL-6 in vitro and in vivo (
The biological actions of IL-6, LIF, and CNTF are mediated via their respective functional receptor complexes ( (IL-6R), and gp130. LIF receptor ß (LIFR) is a transmembrane signaling subunit, and LIF signaling is mediated by the heterodimerization of LIFR and gp130. The complex of gp130 and LIFR is also utilized by other members of the IL-6 family of cytokines, such as CNTF, OM, and CT-1. The action of CNTF is mediated via a tripartite complex consisting of a ligand-binding component, CNTF receptor
(CNTFR), gp130, and LIFR. IL-6R and CNTFR also exist in soluble forms (sIL-6R and sCNTFR). IL-6 bound to sIL-6R elicits an IL-6-specific response in cells that express gp130 on their surfaces, and the complex with CNTF and sCNTFR acts on the cells that express gp130 and LIFR. There are no data showing IL-6R expression in adult skeletal muscles. On the other hand, expression of gp130, LIFR, and CNTFR is detected in intact muscles and this receptor expression is increased in denervated muscles (
In the present study, to determine target cells for LIF, IL-6, and CNTF and to further clarify the roles of these cytokines in muscle regeneration, we examined the spatiotemporal expression patterns of gp130, IL-6R, LIFR, and CNTFR mRNAs in regenerating muscles using in situ hybridization (ISH) histochemistry. Muscle regeneration is induced by muscle contusion, a model of muscle crush injury that has been used to induce degenerative and regenerative reactions in both myofibers and intramuscular nerves (
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Materials and Methods |
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Muscle Contusion and Tissue Preparation
Adult (200250 g bw) Wistar rats were housed in individual cages and fed commercial pellets and water ad libitum. Muscle contusion was achieved without skin incision as described previously (
Probes
A plasmid vector containing 3.0 kbp at the XhoI site of pSP72 (provided by Dr. Kishimoto, Osaka University) was digested with Pvu II and was self-ligated. The resultant plasmid containing 500 bp of the 3' end of cytoplasmic domain was linearized at a SacI or XhoI site for the antisense and sense gp130 probes, respectively. A plasmid vector containing ~2.5 kbp at the EcoRI site of PUC18 (provided by Dr. Kishomoto, Osaka University) was digested with Hind III. The resultant cDNA fragment of 600 bp was inserted into pBS and linearized at the XhoI or XbaI site for the antisense and sense probes for IL-6 mRNA, respectively. Characterization of the LIFR probe has been described in a previous report (
Three oligonucleotide probes for CNTFR were complementary to nucleotides 129164, 470500, and 11141500 of the published rat CNTFR sequence (
In Situ Hybridization
Frozen cross-sections of gastrocnemius muscle 12 µm thick were thawed on 3-amino propylethoxysilane-coated slides, then hybridized with radiolabeled probes, as follows. Sections were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 15 min, then rinsed in 2 x SSC (0.3 M NaCl in 30 mM Na citrate). The sections were incubated with 1 µg/ml proteinase K (Sigma Chemical; St Louis, MO) at 37C for 5 min. After two rinses in 2 x SSC, the sections were dehydrated in 70, 80, 90, 95, and 100% ethanol, then air-dried. The sections were then incubated with hybridization buffer [50% formamide, 4 x SSC, 0.12 M phosphate buffer, pH 7.4, 1 x Denhardt's solution, 0.2% SDS, 0.25 mg/ml tRNA, 10% dextran sulfate, 100 mM dithiothreitol (DTT)] containing the radiolabeled antisense oligonucleotide probes at 37C for 16 hr to detect the signals of the myogenin and CNTFR mRNAs. After hybridization, the sections were washed with three changes of 1 x SSC at 55C for 20 min each. Other series of sections were incubated with hybridization buffer containing the radiolabeled antisense or sense cRNA probes at 52C for 16 hr to detect the gp130, IL-6R, and LIFR mRNA signals. After hybridization, the sections were washed at 65C for 30 min with 50% formamide in 2 x SSC and 10 mM DTT. After incubation with RNase buffer (0.4 M NaCl, 10 mM Tris-HCl, pH 8.0, 50 mM EDTA-2Na) at 37C for 10 min, the sections were digested at 37C for 30 min with 25 µg/ml ribonuclease A (Sigma Chemical) in RNase buffer, then washed in RNase buffer for 10 min, 2 x SSC for 15 min, and 0.1 x SSC for 15 min at 37C, followed by dehydration in 80, 90, 95, and 100% ethanol, and then air-dried. To visualize the signals for the specific mRNAs, the sections were dipped in Ilford K15 autoradiography emulsion diluted 2:3 with distilled water at 45C. After exposure at 4C for 4 weeks, they were developed in Kodak D19 for 4 min at 20C, then fixed in 24% sodium thiosulfate solution for 5 min. Lastly, the sections were stained with hematoxylin and eosin.
Semiquantitative analysis was performed to make a general survey of the expression of gp130, IL-6R, LIFR, and CNTFR mRNAs in various regions of rat gastrocnemius muscle after muscle contusion. The relative signal level in each hybridized section was inspected and graded visually. Upregulated hybridization signal was designated "+" and background hybridization signal "-".
Specificity of Hybridization Signals
To ascertain the specificity of the observed hybridization signals for gp130, IL-6R, and LIFR mRNAs, adjacent serial sections were hybridized with antisense or sense probes for each mRNA. Sense probes did not display specific hybridization signals in cells in regenerating muscles, whereas antisense probes detected abundant hybridization signals for gp130, IL-6R, and LIFR mRNAs (data not shown). In addition to the specificities of nucleotide sequences designed as probes for CNTFR and myogenin mRNAs, we observed hybridization signals as follows. The CNTFR oligonucleotide probe revealed significantly increased signals in myonuclei of denervated myofibers and axotomized spinal motorneurons. These cells were previously shown to upregulate CNTFR mRNA in the same situation (
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Results |
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Discrimination of Cell Types Expressing Receptor mRNAs
Specific hybridization signals for gp130, LIFR, IL-6R, and CNTFR mRNAs were detected in nuclei of several cell populations in regenerating muscles. We defined cell types showing significant hybridization signals for these receptor mRNAs as follows. Myonuclei were easily distinguished when they were located in the center of the cytoplasm of myofibers at 3 hr post contusion. It was hard to distinguish myonuclei when they were located at the edge of myofibers. We observed the relationship between myofibers and nuclei at higher magnification. Nuclei located within myofibers were defined as myonuclei/nuclei of mpcs. Nuclei located in extracellular spaces between myofibers were defined as mononuclear cells. Newly formed myotubes were confirmed as small myofibers with centrally located myonuclei and signals for myogenin mRNA.
Effect of Muscle Contusion on gp130 Expression
Signals for gp130 mRNA were at background level in intact myofibers, but intense signals were detected in the endothelium-like cells of blood vessels (Fig 1A1C). At 3 hr after muscle contusion, gp130 mRNA expression was upregulated in injured muscle tissues (Fig 1D). gp130 mRNA was expressed in mononuclear cells located in extracellular spaces between myofibers (Fig 1E). Signals for gp130 mRNA were also detected in a myonucleus located in the center of the cytoplasm of a myofiber (Fig 1F) and in myonuclei/nuclei of mpcs at 3 hr postcontusion (Fig 1G). On Day 1 postcontusion, myonuclei/nuclei of mpcs expressing gp130 mRNA were still detected (Fig 2A2C), and then obvious signals for gp130 mRNA were observed in many mononuclear cells on Day 2 postcontusion (Fig 2D and Fig 2E). Low levels of mononuclear cells expressing gp130 mRNA were observed in extracellular spaces between myotubes until Day 7 post contusion, although gp130 mRNA expression in myonuclei/nuclei of mpcs decreased after Day 2 post contusion (data not shown).
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Myogenin, a member of the MyoD family, is first upregulated in myonuclei/nuclei of mpcs at 6 hr postcontusion (
Effect of muscle contusion on LIFR expression
Signals for LIFR mRNA were at background level in intact myofibers (Fig 3A) but, as in the case of gp130, intense signals were detected in the endothelium-like cells of blood vessels (Fig 3B). At 3 hr after muscle contusion, upregulation of the LIFR mRNA level was observed in injured muscle tissues (Fig 3C). Signals for LIFR mRNA were detected in myonuclei/nuclei of mpcs (Fig 3D). At Day 1 post contusion, several mononuclear cells located in extracellular spaces between myofibers expressed LIFR mRNA (Fig 3F). LIFR mRNA expression in myonuclei/nuclei of mpcs decreased after Day 2 post contusion, but a few mononuclear cells expressing LIFR mRNA were observed at low levels until Day 7 post contusion (data not shown).
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To determine whether expression of LIFR mRNA can be detected in newly formed myotubes, adjacent serial sections were hybridized with probes for LIFR and myogenin mRNAs (Fig 3G3J). At Day 7 postcontusion, some myotubes expressed myogenin mRNA (Fig 3J), but LIFR mRNA expression in these myotubes was not detected (Fig 3H).
Effect of Muscle Contusion on IL-6R Expression
Signals for IL-6R mRNA were at background level in intact myofibers (data not shown). Upregulation of IL-6R mRNA was first observed at low levels in injured muscle tissues at 3 hr post contusion (data not shown), and then signals for IL-6R mRNA increased at Day 1 postcontusion (Fig 3A and Fig 3B). Obvious myogenin mRNA expression has been observed in myonuclei/nuclei of mpcs at Day 1 post-contusion (
To determine whether IL-6R mRNA was expressed in myonuclei/nuclei of mpcs after contusion, adjacent serial sections were hybridized with probes for IL-6R and myogenin mRNAs (Fig 4A4D). On Day 1 post contusion, some myonuclei/nuclei of mpcs expressed myogenin mRNA (Fig 4C and Fig 4D). In contrast to myogenin expression, signals for IL-6R mRNA were not detected in myonuclei/nuclei of mpcs (Fig 4A and Fig 4B), but significant signals for IL-6R mRNA were detected in mononuclear cells located in extracellular spaces between myofibers (Fig 4B). IL-6R mRNA expression throughout the regeneration process was detected only in mononuclear cells and was never observed in myonuclei/nuclei of mpcs. The level of IL-6R mRNA peaked at Days 1 and 2 post contusion, and only a small number of IL-6R mRNA-positive mononuclear cells were observed at low levels until Day 7 post contusion (data not shown).
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To confirm whether expression of IL-6R mRNA was present in newly formed myotubes, adjacent serial sections were hybridized with probes for IL-6R and myogenin mRNAs (Fig 4E4H). Myogenin mRNA expression was observed in some newly formed myotubes (Fig 4H). However, signals for IL-6R mRNA were not detected in these myotubes (Fig 4F).
Effect of Muscle Contusion on CNTFR Expression
Signals for CNTFR mRNA were at background level in intact myofibers (data not shown). To determine whether CNTFR mRNA was expressed in myonuclei/nuclei of mpcs after contusion, adjacent serial sections were hybridized with probes for CNTFR and myogenin mRNAs (Fig 5A5D). At Day 1 postcontusion, some myonuclei/nuclei of mpcs expressed myogenin mRNA (Fig 5C and Fig 5D), but signals for CNTFR mRNA were not detected in myonuclei/nuclei of mpcs in injured myofibers that expressed myogenin mRNA (Fig 5A and Fig 5B). Furthermore, CNTFR mRNA expression was not observed in mononuclear cells throughout the regeneration process (data not shown).
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To determine whether expression of CNTFR mRNA was detected in newly formed myotubes, adjacent serial sections were hybridized with probes for CNTFR and myogenin mRNAs (Fig 5E5H). At Day 7 post contusion, signals for myogenin mRNA were observed in some newly formed myotubes (Fig 5H), and these myotubes also expressed CNTFR mRNA at significant levels (Fig 5F).
The cell populations that expressed gp130, LIFR, IL-6R, and CNTFR mRNAs in intact and injured muscles after muscle contusion are summarized in Table 1.
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Discussion |
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Constitutive expression of gp130 and LIFR mRNAs in adult intact skeletal muscles has been detected by Northern blotting (
At 3 hr post contusion, gp130 and LIFR expression began to be upregulated in myonuclei/nuclei of mpcs and increased levels were maintained until Day 2 postcontusion. However, both mRNAs were apparently downregulated in the newly formed myotubes. There are many data showing that LIF is upregulated in regenerating myofibers and that this contributes to the proliferation of myoblasts in vitro (
Several observations suggest the participation of IL-6 in myofiber degeneration (
Mononuclear cells located in extracellular spaces between myofibers express gp130, LIFR, and IL-6R mRNAs. In this study, we did not determine the cell types of these mononuclear cells. However, macrophages, fibroblasts, neutrophils, and mpcs, all of which have been extensively observed in injured muscles, may be candidates for these mononuclear cells (
CNTFR expression was not detected at significant levels in myonuclei/nuclei of mpcs in intact and injured myofibers nor in mononuclear cells throughout the process of muscle regeneration. However, the level of CNTFR mRNA was significantly upregulated in some of the myotubes, whereas both gp130 and LIFR, the other components of functional CNTF receptors, diminished in myotubes. These findings suggest that myotubes are not targets for CNTF action. The effect of CNTF on myotube differentiation (
In conclusion, this study has demonstrated that expression of gp130, IL-6R, LIFR, and CNTFR mRNAs in regenerating muscles is induced in distinct cell populations, such as myonuclei/nuclei of mpcs, mononuclear cells, and myotubes. These receptor subunits probably mediate actions of LIF, IL-6, and CNTF to facilitate the effective degenerative and regenerative reactions of myofibers and reinnervation on myotubes in regenerating muscles.
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Acknowledgments |
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cDNAs for gp130 and IL-6R were generous gifts from Dr T. Kishimoto (Osaka University). Rat LIFR cDNA was a generous gift from Dr M. Minami and Dr M. Sato (Kyoto University).
Received for publication October 30, 1999; accepted April 5, 2000.
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