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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 2, 2004 as doi:10.1096/fj.03-1039fje. |
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* Skeletal Muscle Research Laboratory, School of Medical Sciences, Royal Melbourne Institute of Technology, Bundoora; and
Centre for Physical Activity and Nutrition, School of Exercise and Nutrition Sciences, Deakin University, Burwood, Australia
1Correspondence: Skeletal Muscle Research Laboratory, School of Medical Sciences, RMIT University, P.O. Box 71, Bundoora 3083, Victoria, Australia. E-mail: mark.febbraio{at}rmit.edu.au
SPECIFIC AIMS
We recently proposed a model hypothesizing that muscle contraction could activate interleukin-6 (IL-6) transcription through activation of nuclear factor of activated T cells (NFAT) and that low glycogen would activate p38 mitogen-activated protein kinase (MAPK), leading to a potentiation of IL-6 gene transcription in skeletal muscle (M. A. Febbraio and Pedersen, FASEB J. 16, 1335–1347, 2002). In the present study we tested the hypothesis that the transcription of IL-6 during concentric exercise was activated by NFAT, but enhanced with low glycogen due to increased nuclear localization and phosphorylation of p38 MAPK. To do this, we isolated nuclear fractions of muscle samples collected from human subjects before and after exercise and probed them for total protein or phosphorylated proteins using Western blot analyses. We incubated L6 myotubes in ionomycin (a compound known to induce IL-6 mRNA) with or without the pyridinylimidazole p38 MAPK inhibitor SB203580 to determine whether phosphorylation of p38 MAPK at the nucleus may result in regulation of IL-6 gene transcription.
PRINCIPAL FINDINGS
1. Low muscle glycogen results in phosphorylation of nuclear p38 MAPK
We were able to decrease intramuscular glycogen content from 375 ± 35 (CON) to 163 ± 27 (LCHO) mmol glucosyl u·kg-1 dry mass prior to exercise by having subjects perform glycogen-depleting exercise, then consume either a high-carbohydrate or high-fat diet for 24 h. We show that low intramuscular glycogen per se does not translocate p38 MAPK to the nucleus, but rather phosphorylates p38 MAPK that resides in the nucleus. Although speculative, we suggest that kinases upstream of p38 MAPK, such as MKK3 and MKK6, may have a functional binding domain that inhibits phosphorylation when bound to glycogen.
2. Muscle contraction increases phosphorylation of c-jun amino-terminal kinase (JNK) at the nucleus but does not appear to increase the nuclear abundance of NFAT or nuclear factor kappa ß (NF-
B)
Nuclear JNK 1/2 phosphorylation was increased by contraction in both CON and LG, a result consistent with previous experiments that have measured JNK activity in crude muscle homogenates from humans subjected to 60 min of cycling exercise. However, we did not detect any effect of either lowered glycogen or muscle contraction on the nuclear abundance of NFAT or NF-B. These results would argue against a role for these nuclear transcription factors being upstream activators for IL-6 gene transcription during concentric muscle contractile activity because of the observation that IL-6 mRNA increased with exercise, and this increase was potentiated by low intramuscular glycogen.
3. The increase in the phosphorylation of nuclear p38 MAPK is an upstream regulator of IL-6 mRNA
The most important finding of this study was that the increase in phosphorylation of p38 MAPK in the nucleus due to low glycogen was associated with the magnitude of the increase in IL-6 mRNA (Fig. 1
). This relationship suggests that phosphorylation of p38 MAPK at the nucleus may result in regulation of corepressors or coactivators binding to the 5' flanking region of the IL-6 gene. To determine whether this relationship was causal or a random association, it was necessary to perform tissue culture experiments. We hypothesized that ionomycin (a calcium ionophore) would phosphorylate p38 MAPK in L6 myotubes. We found that treatment of our cells with ionomycin had no effect on total nuclear p38 MAPK, but that it phosphorylated nuclear p38 MAPK. Moreover, this resulted in a marked increase in IL-6 mRNA. Inhibition of p38 MAPK in the nucleus using SB203580 not only reduced the phosphorylation of nuclear p38 MAPK, but completely ablated the increase in IL-6 mRNA (Fig. 2
). To our knowledge, no studies have examined the transcription factors that may bind to the IL-6 promoter region in muscle cells to activate the gene, but based on our results, we suggest that candidate transcription factors may be mediated by p38 MAPK. It is important to note that the ß-isoform of p38 MAPK contains an LPS kinase domain; therefore, even though our antibody detected both 
- and ß-isoforms, it is more likely that ß-isoform of p38 MAPK would be the isoform that may lead to the activation of IL-6.
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CONCLUSIONS
We have shown that dietary manipulation that resulted in low intramuscular glycogen was associated with the phosphorylation of the nuclear p38 MAPK. Phosphorylation of p38 MAPK in the nuclear fraction prior to exercise was related to the exercise-induced fold increase in IL-6 mRNA. This evidence, along with our observation that nuclear p38 MAPK phosphorylation alters IL-6 mRNA levels in L6 myotubes, provides good evidence that one role of phosphorylation of p38 MAPK in the nucleus is to participate in the regulation of corepressors or coactivators binding to the promoter region of the IL-6 gene in skeletal muscle cells.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1039fje;
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