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TNF- regulates early differentiation of C2C12 myoblasts in an autocrine fashion¹

Department of Medicine and
^* Department of Molecular and Cell Biology, Baylor College of Medicine, Houston, Texas 77030, USA

²Correspondence: Pulmonary Medicine, Suite 520B, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. E-mail: yiping{at}bcm.tmc.edu

SPECIFIC AIMS

We hypothesize that skeletal muscle-synthesized tumor necrosis factor (TNF-) has a physiological role in muscle adaptation. The present study was designed to determine whether TNF- expression in myocytes is regulated by myogenic stimuli and whether myocyte-expressed TNF- promotes myogenic differentiation.

PRINCIPAL FINDINGS

1. TNF- expression in C2C12 myoblasts is up-regulated by serum restriction
We analyzed the expression of TNF- in C2C12 myoblasts with the RNA protection assay (RPA). Using a multiple cytokine probe set, we detected TNF- mRNA in undifferentiated C2C12 myoblasts, a cell line originated from mouse skeletal muscle (Fig. 1A ). To evaluate whether TNF- expression is regulated by myogenic stimuli, we induced C2C12 myoblast differentiation by serum restriction and observed a marked increase in TNF- mRNA level. TNF- mRNA level peaked at around 10 h after serum restriction (273% of the basal level), then gradually returned to predifferentiation level between 24 and 48 h (Fig. 1B ). Western blot analysis of cell lysates of C2C12 myoblasts confirmed an increase in the TNF- protein level at 10 h postserum restriction (Fig. 1C ).

View larger version (21K): [in this window] [in a new window]   Figure 1. TNF- expression in C2C12 myoblasts and its up-regulation by serum restriction. A) TNF- mRNA was detected in undifferentiated C2C12 myoblasts by RPA using a multiple cytokine probe set, including probes for housekeeping genes GAPDH and L32. The identities of RNase-protected bands on the autoradiograph were determined using the labeled probe set (left lane). B) TNF- expression in C2C12 myoblasts is up-regulated by serum restriction. Confluent C2C12 myoblasts were induced to differentiate by serum restriction. Total RNA was isolated at the times indicated and mRNA was analyzed by RPA. The RNase-protected bands were quantified by densitometry and data were normalized against the housekeeping gene GAPDH. Means ± SE are shown and analyzed by ANOVA (indicated by the P value). Asterisks indicate a difference from control (0 h) determined by Dunn’s multiple comparison test (P<0.05). C) TNF- peptide level is up-regulated by serum restriction. TNF- peptide present in C2C12 myoblast lysates was analyzed by Western blot using a TNF- antibody after serum restriction. Lane 1 is recombinant mouse TNF- used as a reference.

2. NF-B activity undergoes a TNF--dependent increase after serum restriction
TNF- is a potent activator of NF-B in myoblasts. To verify whether the increased TNF- expression is associated with an increase of TNF- activity, we evaluated NF-B activity in C2C12 myoblast after serum restriction by EMSA. We observed an increase of NF-B binding activity in nuclear extracts that was similar to the magnitude and time course of the increase in TNF- expression induced by serum restriction. The components of the NF-B–DNA complexes were identified by supershift assay to be a p65/p50 heterodimer and a p50/p50 homodimer. To determine whether increased endogenous TNF- is responsible for the activation of NF-B by serum restriction, we added a TNF--neutralizing antibody to the low serum differentiation medium and observed a blockade of NF-B activation. These data indicate that TNF- is responsible for the activation of NF-B induced by serum restriction.

3. TNF- stimulates MHCf expression
We evaluated the effect of endogenous and exogenous TNF- on the expression of MHCf (a differentiation marker) during the 24 h after serum restriction by using Western blot analysis. We observed an induction of MHCf expression by serum restriction. A TNF--neutralizing antibody added to the differentiation medium inhibited MHCf expression (Fig. 2 ). Conversely, recombinant mouse TNF- added to the differentiation medium stimulated the expression of MHCf in a dose-dependent fashion whereas total protein content remained unchanged. These results indicate that endogenous TNF- is required for the normal differentiation of myoblasts.

View larger version (17K): [in this window] [in a new window]   Figure 2. Endogenous TNF- is required for normal expression of MHCf. A TNF--neutralizing antibody (5 µg/ml) or the vehicle was incubated with confluent C2C12 myoblasts in low serum differentiation medium for 24 h. MHCf content in the total cell lysates was determined by Western blot analysis using an anti-MHCf monoclonal antibody and quantified by densitometry. A representative blot is shown in the inset. Data from three separate experiments were analyzed by a paired t test.

4. TNF- effect on MHCf content depends on the differentiation stage
We further monitored TNF-’s effect on MHCf content over a 72 h period after serum restriction. We found that TNF- reduced MHCf content at 72 h in a dose-dependent fashion after an early increase in MHCf content at 24 h. TNF--induced loss of MHCf content at the late stage of differentiation, when myotubes have formed, is similar to its effect on differentiated myotubes reported previously. We demonstrated in an earlier study that accelerated protein degradation, not slower synthesis, is responsible for the loss of MHCf.

5. NF-B mediates TNF- stimulation of MHCf expression
We evaluated NF-B involvement in the regulation of MHCf expression by selectively inhibiting NF-B with a genetic approach. We had earlier created a stable C2C12 cell line that overexpresses a dominant negative mutant of NF-B inhibitor protein I-B, I-BN, which lacks the amino-terminal 36 amino acid residues containing serine-32 and serine-36 required for its phosphorylation, degradation, and subsequent release of NF-B. Using this cell line, we observed that TNF- was unable to stimulate MHCf expression due to the blockade of NF-B activation. In control C2C12 myoblasts transfected with the empty vector pCMV4, TNF- stimulated MHCf expression as it did in the parent C2C12 cells. These results demonstrate that NF-B mediates TNF- stimulation of MHCf expression.

6. TNF- stimulates SRF binding and expression of the skeletal muscle -actin gene
Using EMSA, we evaluated the effect of TNF- on the binding of SRF to SRE1 of the skeletal muscle -actin gene promoter. We observed a rapid increase of SRF binding activity in nuclear extracts prepared from C2C12 myoblasts treated with recombinant mouse TNF-. The binding of SRF increased 142% in 5 min of TNF- treatment and gradually returned to the control level at 60 min. The specificity of SRF binding to SRE1 was confirmed by a successful competition with unlabeled probe in 100-fold excess and a supershift assay using an anti-SRF antibody.

To investigate whether TNF- stimulation of SRF binding to the skeletal muscle -actin gene promoter is accompanied by an up-regulation of the gene expression, we examined skeletal muscle -actin gene expression in rat skeletal myoblasts after serum restriction with or without the presence of recombinant mouse TNF-. We used rat skeletal myoblasts instead of C2C12 in this experiment because C2C12 poorly expresses the -actin gene in comparison to primary myoblasts. Northern blot analysis revealed that TNF- stimulates expression of the skeletal muscle -actin mRNA at 1.5 and 3 h after serum restriction. Such a rapid increase in skeletal muscle -actin mRNA expression by TNF- is consistent with the rapid increase of SRF binding activity induced by TNF-.

CONCLUSIONS

Production of TNF- is tightly regulated at the transcription level in a tissue-specific manner. Saghizadeh and colleagues detected TNF- expression in human and rat skeletal muscle using RT-PCR whereas a previous attempt using Northern blot failed to do so. In the present study, we show that C2C12 myoblasts express TNF- mRNA at a level detectable by RPA, a method that is more suitable for quantitative analysis than RT-PCR. Using RPA, we show for the first time that in addition to a basal level presence, TNF- expression is up-regulated markedly by a differentiation stimulus, serum restriction. These data suggest that increased expression of TNF- in myocytes is an early event during myogenesis.

Our data further demonstrate that endogenous TNF- stimulates specific muscle gene expression and is required for the normal differentiation of myoblasts. In addition, we show that TNF- promotes myoblast differentiation by activating NF-B and SRF. These data establish a physiological role for TNF- in myogenesis as summarized in Fig. 3 .

View larger version (4K): [in this window] [in a new window]   Figure 3. Schematic diagram of the hypothesized physiological role of muscle-synthesized TNF- in myogenesis. Myogenic stimuli (such as stimuli for differentiation) up-regulate TNF- expression. TNF- stimulates NF-B and SRF activity, which in turn up-regulate expression of muscle genes.

We observed a differentiation stage dependency or time dependency of TNF- effect. TNF- increases MHCf content only during a window of time—the first 24 h after serum restriction—that overlaps with the increase of TNF- expression and NF-B activation. TNF- reduces MHCf content after 48 h of differentiation when myoblasts have fused to form myotubes. The seemingly opposing effects of TNF- on MHCf content at early and late stages of differentiation indicate that TNF- effect on myocytes has two components: simulating muscle gene expression during the early stage of differentiation and stimulating muscle protein degradation during the late stage of differentiation. TNF- stimulates the ubiquitin-proteasome system, which is responsible the degradation of muscle proteins, including MHC. Our data reiterate the concept that chronic elevation of TNF- is harmful to myocytes as we demonstrated previously. It appears that TNF- expression is tightly regulated so that it rises transiently during the initial hours of differentiation to stimulate muscle gene expression, then returns to the basal level to avoid the catabolic effect during later stages of differentiation

There have been opposing reports on whether NF-B activity increases or decreases during myoblast differentiation. The increase in NF-B activity during differentiation observed in the present study agrees with two earlier reports. Our data further reveal a detailed time course and the mechanism of the increase in NF-B activity, and thus provide greater evidence to support the concept that NF-B activity increases transiently during the early stage of myoblast differentiation.

We demonstrate here that NF-B mediates TNF- stimulation of MHCf expression. We previously showed that exposure of the C2C12 cell line overexpressing I-BN to 6 ng/ml of TNF- for up to 72 h does not cause apoptosis. Therefore, the lack of response of this cell line to TNF- cannot be attributed to apoptosis induced by NF-B blockade. Two previous studies demonstrated a dependency of the expression of MHC on NF-B by using an inhibitor of NF-B, PTDC, which is an antioxidant and has other pharmacological effects. By using a selective dominant negative inhibitor, we demonstrate more convincingly the importance of NF-B in regulating MHC expression.

We further discovered in the present study that TNF- rapidly enhances SRF binding to SRE and that the increase of SRF binding is accompanied by a rapid increase of SRF-controlled -actin gene expression. These results demonstrate that TNF- stimulates muscle gene expression during differentiation through multiple factors. The ability of TNF- to stimulate SRF binding appears to be a unique property of TNF- in comparison to growth factors. In most cell types, the DNA binding activity of SRF does not change with growth factor treatment. Thus, SRF binding was thought to be constitutive. It is generally believed that regulation of SRF activity by growth factors is achieved by influencing the activities of SRF ternary complex factors.

The catabolic effect and other cytotoxicities of TNF- have long masked the proposed physiological role of TNF- in myogenesis. When TNF-’s effect on MHC protein level is examined only at late stages of differentiation, it may give a misleading impression that TNF- inhibits MHC expression. TNF-’s effect is dose dependent: it induces apoptosis or necrosis at high doses, causes inflammatory or cachectic responses at medium doses, and has a tissue-remodeling effect at low doses. We had found that prolonged incubation with repeated doses of TNF- at 10 ng/ml or higher kills cultured myocytes. An early study reported a negative effect of TNF- on muscle gene expression when treating myoblasts with repeated doses of 25 ng/ml of TNF- that is several times higher than the serum levels found in humans with catabolic diseases. Such results may be due to the cytotoxicity of the extremely high level of TNF-.

The new findings presented in the current report may have several implications in the physiology and pathology of skeletal muscle. As a physiological factor synthesized in myocytes, TNF- may be important in muscle adaptation in response to exercise or muscle repair in injury. On the other hand, chronically elevated serum TNF- due to inflammatory diseases may constantly stimulate skeletal muscle satellite cell differentiation and deplete satellite cells. This scenario may contribute to the chronic atrophic effect of TNF- in addition to its known catabolic effect in differentiated myocytes.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0632fje ; to cite this article, use FASEB J. (April 6, 2001) 10.1096/fj.00-0632fje

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This Article Full Text (PDF) All Versions of this Article: 15/8/1413 00-0632fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by LI, Y.-P. Articles by SCHWARTZ, R. J. Search for Related Content PubMed PubMed Citation Articles by LI, Y.-P. Articles by SCHWARTZ, R. J.