The present study demonstrates for the first time that TNF-α signaling is required for p38 activation and p38-dependent signaling events during muscle regeneration, and it provides morphological and functional evidence that TNF-α signaling is required for normal muscle regeneration, thus suggesting a critical role for TNF-α in regulating muscle regeneration.
Given the negative image of TNF-α in skeletal muscle metabolism because of its involvement in muscle protein breakdown, inflammatory myopathies, and insulin resistance (29
), it appears counterintuitive that expression of TNF-α and its receptors by myofibers would increase dramatically during injury-induced regeneration (7
), which, along with TNF-α released by infiltrating inflammatory cells, brings about an unusually high level of TNF-α receptor-mediated signaling. Several of the TNF-α-activated signaling molecules are involved in the regulation of myogenesis, including members of the MAPK family and NF-κB. Members of the MAPK family have different roles in myogenesis. p38MAPK is recognized as a necessary and sufficient “switch” that turns on the differentiation program (2
), while ERK1/2 stimulates the proliferation of myocytes (8
). On the other hand, the role of JNK in myogenesis is not well defined. Both inhibitory and stimulatory effects on myogenesis have been reported for JNK (20
). NF-κB also influences myogenic differentiation (19
). The dependence of p38 activation on TNF-α is remarkable, considering that the activation of ERK1/2, JNK, and NF-κB was not TNF-α dependent during muscle regeneration. These factors can be activated by multiple cytokines and growth factors in the absence of TNF-α signaling. Although p38 activation has been established as a key event in myogenic differentiation, the upstream signal for p38 activation during muscle regeneration was not identified. We have demonstrated herein for the first time that TNF-α receptors are required for p38 activation during muscle regeneration. Our observations suggest that the purpose of the surge of TNF-α synthesis in injured muscle fibers is to activate p38 and thus to turn on myogenic differentiation. Considering the importance of p38 activation in myogenic differentiation, the role of TNF-α in muscle regeneration appears to be more significant than previously thought.
The myogenic program is a highly complex signaling network, and p38 is involved in several key steps of the program. We show in the present study that multiple p38-mediated steps of the myogenic program are TNF-α signaling dependent, including MEF-2 phosphorylation, induction of myogenin and p21 expression, and suppression of cyclin D1 expression. MEF-2 phosphorylation is required for the expression of the majority of muscle-specific genes that are synergistically activated by MEF-2 and MyoD (31
). Myogenin plays a key role in executing the myogenic differentiation program (33
). On the other hand, p21 and cyclin D1 are cell cycle regulators for cell cycle exit that are critical so that differentiation can take place (41
). Although we measured these signaling events in intact muscle, they actually take place in activated satellite cells, not in myofibers and infiltrating inflammatory cells, which are terminally differentiated cells. These data consistently demonstrate an impairment of myogenic differentiation in the absence of TNF-α signaling during muscle regeneration.
The morphological abnormalities observed in regenerating p55−/−p75−/− soleus muscle, including more severe and persistent inflammation, dystrophic calcification, and the formation of smaller myotubes, confirm that muscle regeneration is impaired in the absence of TNF-α signaling. The morphological abnormalities can result from impairment of various aspects of muscle regeneration, including impairment of myogenesis due to the lack of p38 activation.
The results of force-frequency studies provide functional evidence that supports the histological data. In , the force-frequency curve for undamaged soleus (day 0) was shifted slightly to the right in p55−/−p75−/− relative to WT. This response suggests that constitutive TNF-α signaling influences the contractile properties of soleus muscle. Most likely, TNF modulates the expression of one or more regulatory proteins that affect the force-frequency characteristic. A partial listing of candidate proteins includes the voltage-sensitive dihydropyridine receptor, ryanodine-sensitive sarcoplasmic reticulum (SR) Ca2+ release channel, SR Ca2+-ATPase, myosin heavy chains, myosin light chains, troponin C, and tropomyosin. This issue, while intriguing, was not the focus of our present study. No attempt has been made to identify the proteins responsible for differences between groups at day 0.
Inflammation is a key response to muscle injury (44
) by virtue of its role in phagocytosis and satellite cell proliferation and differentiation (3
). Because an important function of TNF-α signaling is to amplify inflammatory response, the presence of more severe inflammation in injured p55−/−
soleus appears counterintuitive. Yet, despite the presence of more severe inflammation, regeneration in p55−/−
soleus was still impaired. These observations suggest that there is a compensatory increase of inflammation to promote regeneration as a result of the deficiency in muscle regeneration in p55−/−
muscle; nevertheless, with the lack of TNF-α signaling, increased inflammation is still ineffective in promoting regeneration. Thus TNF-α signaling appears to be a key component of inflammation that promotes muscle regeneration. TNF-α is known to stimulate phagocytosis (30
) and a chemotactic response (45
), which facilitate muscle regeneration. The present study shows that in addition to the previously known effects, TNF-α signaling is required for p38-mediated myogenesis.
That skeletal muscle develops in TNF-α receptor-knockout mice almost normally, although with different mechanical properties (7
), does not automatically preclude TNF-α from being a physiological regulator of myogenesis, because a compensatory mechanism may replace the role played by TNF-α. Previous studies showed that in TNF-α-null mice, any role played by TNF-α could be performed effectively by the upregulation of other cytokines. In the absence of TNF-α, a number of cytokines, including IL-12, INF-γ, and IL-1, are upregulated (12
). The networking of these cytokines is capable of activating macrophages and modulating myoblast proliferation and fusion (12
). An analogy can be found in MyoD-knockout mice. MyoD-knockout mice still develop muscle because of a redundancy in the role of Myf-5 (40
). A possible explanation for the difference observed between newborn animals and adult animals that undergo muscle regeneration is that in newborn animals, the compensatory mechanism plays out over time, whereas injury-induced regeneration is an acute response, especially with regard to regeneration induced by snake venom, in which degeneration and inflammation develop more quickly than other types of injury (25
), so that a role of TNF-α signaling can more readily be appreciated.
In summary, the present study provides new evidence that TNF-α has an important physiological role in regulating skeletal muscle regeneration. These data are helpful in sorting out the details of the mechanism that initiates muscle regeneration, particularly myogenesis, in response to injury. The results described herein also have clinical implications. TNF-α has long been considered a therapeutic target for inflammatory diseases. Anti-TNF-α strategies have gained popularity clinically in treating a variety of inflammatory conditions. Our data suggest that long-term use of anti-TNF-α reagents may impair skeletal muscle adaptation. Thus caution should be exercised in using anti-TNF-α strategies.