p38 MAPK-induced nuclear factor-kappaB activity is required for skeletal muscle differentiation: role of interleukin-6

Abstract

p38 MAPK and nuclear factor-kappaB (NF-kappaB) signaling pathways have been implicated in the control of skeletal myogenesis. However, although p38 is recognized as a potent activator of myoblast differentiation, the role of NF-kappaB remains controversial. Here, we show that p38 is activated only in differentiating myocytes, whereas NF-kappaB activity is present both in proliferation and differentiation stages. NF-kappaB activation was found to be dependent on p38 activity during differentiation, being NF-kappaB an effector of p38, thus providing a novel mechanism for the promyogenic effect of p38. Activation of p38 in C2C12 cells induced the activity of NF-kappaB, in a dual way: first, by reducing IkappaBalpha levels and inducing NF-kappaB-DNA binding activity and, second, by potentiating the transactivating activity of p65-NF-kappaB. Finally, we show that interleukin (IL)-6 expression is induced in C2C12 differentiating myoblasts, in a p38- and NF-kappaB-dependent manner. Interference of IL-6 mRNA reduced, whereas its overexpression increased, the extent of myogenic differentiation; moreover, addition of IL-6 was able to rescue significantly the negative effect of NF-kappaB inhibition on this process. This study provides the first evidence of a crosstalk between p38 MAPK and NF-kappaB signaling pathways during myogenesis, with IL-6 being one of the effectors of this promyogenic mechanism.

Figure 1.

NF-κB is activated in a dual phase during myogenesis, whereas p38 activity is only detected during differentiation. C2C12 myoblasts were cultured in GM until 80% confluence and then switched to lowserum DM. (A) Myogenic differentiation is induced in cells cultured in DM but not in GM. Protein lysates were obtained from C2C12 cells grown in GM and DM for the indicated time points (h) and analyzed by Western blotting with antibodies against MHC and α-tubulin (as loading control), respectively. (B) p38 MAPK phosphorylation is induced during C2C12 myoblast differentiation. Cell lysates were obtained from C2C12 myoblasts in GM or DM (as in A) and activation of p38 was determined by immunoblotting, by using a specific anti-phospho-p38 antibody (P-p38). An anti-p38 antibody was used to confirm that the amount of p38 protein was unchanged in all lanes. (C) NF-κB-DNA binding activity shows a biphasic profile during C2C12 myogenesis. NEs were obtained from cells cultured in GM or DM and incubated with a radiolabeled double-stranded oligonucleotide containing the NF-κB binding site, and DNA-protein binding activity was analyzed by EMSA. (D) NEs from C2C12 cells in GM or DM (24 h) were incubated in the absence or presence of specific antibodies for p65 and p50, respectively; an unspecific antibody was used as control. The specific NF-κB-DNA complex induced in DM is indicated by an arrow.

Figure 2.

p38 MAPK and NF-κBare both activated during myogenic differentiation. C2C12 myoblasts were cultured in GM until 80% confluence and then switched to lowserum DM or to serum-free DMEM, used as control for nondifferentiation conditions. (A) Myogenic differentiation is induced in cells cultured in DM but not in serum-free DMEM. Total RNA obtained from C2C12 cells grown in GM, DM, or DMEM for 24 h was analyzed by Northern blotting by using p21, myogenin, α-actin, and GAPDH cDNA probes, respectively. (B) p38 MAPK phosphorylation is induced during C2C12 myoblast differentiation. C2C12 myoblasts proliferating in GM were shifted to DM or DMEM for 24 h, and protein lysates obtained. Activation of p38 was determined by immunoblotting, by using an anti-P-p38 antibody (P-p38). An anti-p38 antibody was used to confirm that the amount of p38 protein was unchanged in all lanes. (C) NF-κB-DNA binding activity is induced during C2C12 myoblast differentiation. Left, NE were obtained from cells cultured in DM or DMEM as in Figure 1, A and B, and incubated with a radiolabeled double-stranded oligonucleotide containing the NF-κB binding site, and DNA-protein binding activity was analyzed by EMSA. Middle, EMSA of cells cultured in DM containing or not the NF-κB inhibitor PDTC. Right, NE from C2C12 cells in DM were incubated with the labeled NF-κB oligonucleotide in the absence or presence of a 150-fold molar excess of unlabeled competitors: NF-κB (specific competitor) and p53 (unspecific competitor) and analyzed by EMSA. The specific NF-κB-DNA complex induced in DM is indicated by an arrow. (D) IκBα protein level is reduced during C2C12 differentiation. Total cellular extracts were obtained from C2C12 cells cultured in DM or DMEM as described above, and IκBα protein levels were analyzed by immunoblotting by using an anti-IκBα antibody. Bottom, immunoblotting with an anti-α-tubulin antibody to confirm equal loading in all lanes. (E) NF-κB transcriptional activity is induced during C2C12 myoblast differentiation. C2C12 cells were transiently transfected with a luciferase reporter gene construct controlled by olimerized κB-binding sites, p6×NF-κB-Luc, with or without an expression plasmid for the superepressor IκBα(S32,36A). Cells were maintained in DMEM or DM for 36 h after transfection, in the absence or presence of PDTC or BAY11-7085. Luciferase activities are expressed relative to the activity of the reporter construct in cells cultured in DMEM, which was given a value of 1. Error bars represent the SE of the mean value.

Figure 3.

p38 and NF-κB activities are required for C2C12 myoblast differentiation and myotube formation. (A) Inhibition of p38 or NF-κB activity abrogates myoblast differentiation. C2C12 cells were cultured in DM for 24 h, in the absence or presence of the p38 inhibitor SB203580 or the NF-κB-inhibitor PDTC. Myogenin and α-actin expression was analyzed by Northern blotting by using the corresponding cDNA probes. Also, GAPDH mRNA expression was analyzed as internal loading control. (B) Muscle-specific promoter activity is abrogated by inhibition of p38 or NF-κB activity. C2C12 myoblasts were transiently transfected with the muscle-specific promoter-reporter plasmid MCK-Luc, together with an expression plasmid for the superepressor IκBα or with an empty vector and cultured in DM for 36, in the presence or absence of SB203580 or PDTC, respectively. Luciferase activities are expressed relative to the activity of MCK-Luc in DM, which was given a value of 100. Error bars represent the SE of the mean value. (C) Myoblast fusion is inhibited by p38 or NF-κB inhibitors. C2C12 cells were cultured for 3 d in the absence or presence of SB203580 or PDTC, and myoblast fusion was analyzed by examining the presence of plurinucleated myotubes.

Figure 4.

NF-κB activation in differentiating C2C12 cells is dependent on p38 MAPK activity. (A) p38 activation precedes activation of NF-κB during myoblast differentiation. C2C12 myoblasts were cultured in GM and then shifted to DM or DMEM for the indicated periods of time (12-48 h). Total cellular extracts or NEs were prepared, as detailed in MATERIALS AND METHODS, and p38 or NF-κB activity analyzed by Western blotting (top) or EMSA (bottom), respectively, as in Figure 1. (B) Induction of NF-κB-DNA binding activity in C2C12 differentiating cells requires functional p38 MAPK. NEs were prepared from C2C12 cells cultured in DM (or DMEM) for 24 h, in the absence or presence of 10 μM SB203580, or PDTC (used as control), and binding of nuclear proteins to a consensus labeled NF-κB oligonucleotide analyzed by EMSA. (C) Induction of NF-κB-dependent transcription during C2C12 myoblast differentiation requires functional p38 MAPK. C2C12 cells were transiently transfected with the p6×NF-κB-Luc reporter plasmid, with or without coexpression of IκBα(S32,36A), used as control for NF-κB activity. After transfection, cells were maintained in DM (or DMEM) for 36 h in the absence or presence of SB203580 or PDTC. Luciferase activities are expressed relative to the activity of MCK-Luc in DMEM, which was given a value of 1. Error bars represent the SE of the mean value. (D) Inhibition of p38 activity increases IκBα protein levels in C2C12 differentiating cells. Whole cell extracts were obtained from C2C12 cells cultured in DMEM, DM, or DM containing SB203580, and IκBα levels analyzed by immunoblotting by using an anti-IκBα antibody. Bottom, immunoblotting with an anti-α-tubulin antibody to confirm equal loading.

Figure 5.

Deliberate activation of p38 induces NF-κB-dependent transcription in C2C12 myoblasts cultured in serum-free medium. The reporter construct p6×NF-κB-Luc was transiently transfected into C2C12 cells, in the presence or absence of expression plasmids for MKK6(E), MKK6(A), IκBα(S32,36A) or empty vector, respectively, and luciferase activities were analyzed after 36 h in DMEM. When indicated, SB203580 was added to DMEM. pRSV-lacZ plasmid was used as a transfection control, and the corresponding β-galactosidase activities were used to normalize the luciferase values. Luciferase activities are expressed relative to the activity of the reporter plasmid (cotransfected with empty vector) in DMEM, which was given a value of 1. Error bars represent the SE of the mean value.

Figure 6.

NF-κB-DNA binding activity is induced by ectopic activation of p38 MAPK in C2C12 cells. (A) MKK6(E)-expressing C2C12 cells show constitutive p38 phosphorylation. C2C12 cells were transfected with either empty vector or an expression plasmid for MKK6(E) and cultured for 24 h in DMEM. p38 activation was analyzed by immunoblotting by using an anti-P-p38 antibody. An anti-p38 antibody was used to ensure equal p38 levels. (B) Constitutive p38 activation reduces IκBα protein levels. C2C12 cells were transfected with either empty vector or an expression plasmid for MKK6(E) and cultured for 24 h in DMEM, in the absence or presence of SB203580. IκBα protein levels were analyzed by immunoblotting by using an anti-IκBα antibody. An anti-α-tubulin antibody was used as loading control. (C) Deliberate activation of p38 MAPK induces NF-κB (p65/p50) binding to DNA. C2C12 cells were transfected with MKK6(E) or with an empty vector, in the absence or presence of SB203580 (top left), or plasmids encoding constitutively active forms of MEKK1 or AKT (bottom/left panel). NE were prepared after 24 h in DMEM, in the absence or presence of SB203580, and analyzed by EMSA. The specificity of NF-κBDNA binding activity was analyzed by incubating NE from MKK6(E)-transfected C2C12 cells with the labeled NF-κB double-stranded oligonucleotide in the absence or presence of a 150-fold molar excess of unlabeled competitors: NF-κB (specific competitor) and p53 (unspecific competitor) (top right). The composition of the NF-κB heterodimer was analyzed by EMSA, after incubating NE from MKK6-transfected C2C12 cells in the absence or presence of different antibodies directed against the indicated members of the Rel/NF-κB family, respectively (bottom right).

Figure 7.

p65-NF-κB transactivating activity is induced by ectopic activation of p38 MAPK: role of CBP. (A) C2C12 cells were transiently cotransfected with a GAL4 responsive-luciferase reporter (p5×GAL4-Luc) and GAL4-p65 or GAL-DBD, together with expression plasmids for MKK6(E) or MAKT (as positive control for GAL4-p65 activity). (B) p5×GAL4-Luc was cotransfected with a GAL4-MEF2C expression plasmid (as positive control for p38 activity), with or without MKK6(E). (C) p5×GAL4-Luc was cotransfected with GAL-DBD or GAL4-p65, and with MKK6(E) and/or CBP. Luciferase activities were analyzed after 36 h in DMEM. pRSV-lacZ plasmid was used as a transfection control, and the corresponding β-galactosidase activities were used to normalize the luciferase values. Luciferase activities are expressed relative to the activity of the GAL4-p65 or pGAL4-MEF2C plasmids, respectively, which were given a value of 1. Error bars represent the SE of the mean value.

Figure 8.

Constitutive activation of p38 accelerates myogenic differentiation in DMEM in a NF-κB-dependent manner. (A) p38-induced muscle-specific gene expression is reduced by inhibition of NF-κB activity. C2C12 cells were transfected with an empty vector or with the MKK6(E) plasmid, with or without IκBα(S32/36A) coexpression, and maintained in DMEM for 24 h, in the absence or presence of SB203580. Myogenin mRNA expression was analyzed by RT-PCR as an index of myogenic differentiation. GAPDH and luciferase expression were analyzed as controls for RT-PCR and transfection efficiency, respectively. (B) p38-induced muscle-specific promoter activity is reduced by inhibition of NF-κB activity. C2C12 myoblast were transiently transfected with MCK-Luc, together with expression plasmids for MKK6(E), IκBα(S32/36A) or an empty vector, and cultured in DMEM, in the presence or absence of SB203580. Luciferase activities are expressed relative to the activity of MCK-Luc, which was given a value of 1. pRSV-lacZ plasmid was used as a transfection control, and the corresponding β-galactosidase activities were used to normalize the luciferase values. Error bars represent the SE of the mean value.

Figure 9.

IL-6 expression is induced during myoblast differentiation in a p38- and NF-κB-dependent manner. (A) Analysis of IL-6 expression in myoblast differentiation. C2C12 cells were cultured in DMEM or DM for 24 h, in the absence or presence of SB203580, PDTC, or BAY11-7085. IL-6 mRNA was analyzed by RT-PCR using the corresponding cDNA probe. Also, GAPDH mRNA expression was analyzed as internal control. (B) IL-6 promoter activity is induced by p38 and NF-κB. C2C12 myoblasts were transiently transfected with an IL-6 promoter-reporter plasmid (see MATERIALS AND METHODS), together with expression plasmids for MKK6(E), IκBα(S32/36A), or p65 (or with an empty vector, as control), and cultured in DMEM for 36, in the presence or absence of SB203580, respectively. Luciferase activities are expressed relative to the activity of pIL-6-Luc transfected with an empty vector, which was given a value of 1. Error bars represent the SE of the mean value.

Figure 10.

Ablation of IL-6 results in down-regulation of muscle differentiation-specific genes. C2C12 cells were transfected with control firefly luciferase or with IL-6 siRNAs, respectively. After transfection, cells were cultured for 24 h in DM and the expression of IL-6, myogenin, α-actin, and GAPDH (used as control) mRNA was analyzed by RT-PCR. As shown in the figure, IL-6 mRNA levels were strongly decreased when cells were transfected with IL-6 siRNAs. Myogenin and α-actin expression levels decreased significantly, whereas no change in GAPDH expression was observed in cells transfected with IL-6 siRNAs.

Figure 11.

Exogenous IL-6 promotes myoblast differentiation. (A) Addition of IL-6 increases the expression of muscle differentiation-specific genes. C2C12 cell were cultured in DMEM for 24 h in the absence or presence of recombinant murine IL-6. Myogenin, skeletal α-actin and GAPDH mRNA expression was analyzed by RT-PCR. (B) Ectopic overexpression of IL-6 increases MCK-dependent transcription. C2C12 myoblasts were transiently transfected with an MCK promoter-reporter plasmid, together with an expression plasmid for murine IL-6 (1:1 and 1:2 ratio MCK-Luc/IL-6), and cultured in DMEM for 36 h. Luciferase activities are expressed relative to the activity of pMCK-Luc transfected with an empty vector, which was given a value of 1. Error bars represent the SE of the mean value. (C) Effect of exogenous IL-6 on the negative action of p38 and NF-κB inhibitors on myogenin expression. C2C12 cells were cultured in DMEM or DM for 24 h, containing or not the p38 inhibitor SB203580 or the NF-κB-inhibitor BAY11-7085, and in the absence or presence of recombinant IL-6. Myogenin and GAPDH expression was analyzed by RT-PCR.