Genetic analysis of p38 MAP kinases in myogenesis: fundamental role of p38alpha in abrogating myoblast proliferation

Abstract

The p38 mitogen-activated protein kinase (MAPK) pathway plays a critical role in skeletal muscle differentiation. However, the relative contribution of the four p38 MAPKs (p38alpha, p38beta, p38gamma and p38delta) to this process is unknown. Here we show that myoblasts lacking p38alpha, but not those lacking p38beta or p38delta, are unable to differentiate and form multinucleated myotubes, whereas p38gamma-deficient myoblasts exhibit an attenuated fusion capacity. The defective myogenesis in the absence of p38alpha is caused by delayed cell-cycle exit and continuous proliferation in differentiation-promoting conditions. Indeed, activation of JNK/cJun was enhanced in p38alpha-deficient myoblasts leading to increased cyclin D1 transcription, whereas inhibition of JNK activity rescued the proliferation phenotype. Thus, p38alpha controls myogenesis by antagonizing the activation of the JNK proliferation-promoting pathway, before its direct effect on muscle differentiation-specific gene transcription. More importantly, in agreement with the defective myogenesis of cultured p38alpha(Delta/Delta) myoblasts, neonatal muscle deficient in p38alpha shows cellular hyperproliferation and delayed maturation. This study provides novel evidence of a fundamental role of p38alpha in muscle formation in vitro and in vivo.

Figure 1

Expression and activation pattern of p38 MAPKs in primary myoblasts. (A) Myoblasts were cultured in GM until subconfluence, and then shifted to DM for the indicated times (hours). Expression of p38α, β, γ and δ mRNA was analyzed by RT–PCR. 18S expression was used as control. (B) Analysis of p38 MAPKs protein expression in primary myoblasts by Western blotting with p38 isoform-specific antibodies (see Supplementary Figure 1). (C) p38 phosphorylation in primary myoblasts (top) and C2C12 cells (bottom) was analyzed by Western blotting using a specific anti-phospho-p38 antibody. (D) p38α and p38γ kinase assays in primary myoblasts. (E) Comparative analysis of muscle differentiation-specific gene markers in primary myoblasts and C2C12 cells by RT–PCR: myogenin (early marker); MCK and MRF4 (late markers).

Figure 2

p38α deficiency reduces the expression of early and late muscle differentiation-specific gene products in differentiating primary myoblasts. WT (p38α^Δ/+) and p38α-deficient (p38α^Δ/Δ) primary myoblasts were cultured as in Figure 1. (A) Comparative qRT–PCR mRNA analysis of myogenin (left), MCK (center) and MHC-2X (right). (B) ChIP analysis was performed using MyoD, RNA pol II and MEF2 antibodies, and subjected to PCR with primers corresponding to the myogenin and MCK promoter regions. (C) Myoblasts were transfected with p-Myogenin-Luc, pMCK-Luc, p4RE-tk-Luc vectors or an empty vector (control), in the absence or presence of a p38α expression plasmid, and incubated in DM for 36 h. Luciferase activities are expressed relative to the activity found for WT myoblasts.

Figure 3

p38α deficiency abrogates multinucleated myotube formation. WT and p38α^Δ/Δ (A) and p38γ^Δ/Δ (B) myoblasts were switched to DM to induce myoblast fusion. Cells were immunostained for eMHC to define nuclei inside myotubes. Several parameters were analyzed: the percentage of nuclei within eMHC-positive cells (% fusion); the number of uninucleated cells; the number of myotubes. (C) p38 phosphorylation is reduced in p38α^Δ/Δ myoblasts. Western blotting analysis of phospho-p38 levels in p38-deficient and WT myoblasts in DM (hours). (Top) WT versus p38α^Δ/Δ, (bottom) WT versus p38β^Δ/Δ, p38γ^Δ/Δ and p38δ^Δ/Δ.

Figure 4

Delayed cell-cycle exit of p38α^Δ/Δ primary myoblasts. Myoblasts were cultured in GM and then shifted to DM for the indicated times (hours), and incubated for 1 h with BrdU. (A) WT, p38α^Δ/Δ and p38α^Δ/Δ myoblasts infected with a p38α-expressing retrovirus. (B) WT and p38β^Δ/Δ, p38γ^Δ/Δ and p38δ^Δ/Δ. Cells were fixed and immunostained against BrdU, and positive cells were quantified.

Figure 5

Altered expression of cell-cycle regulators in p38α^Δ/Δ myoblasts. WT and p38α^Δ/Δ myoblasts were cultured as in Figure 4. Analysis of cyclin D1, cyclin E, cyclin B1 and p21 expression by Western blotting (A) and qRT–PCR (B). Quantification of the immunoblots by scanning densitometry (corrected by tubulin expression) is shown. (C) Analysis of phosphorylated and total Rb. (D) Overexpression of MKK6E in C2C12 cells regulates cyclin D1 and myogenin mRNA expression and pRb phosphorylation in GM. Confirmation of p38 phosphorylation by MKK6E is shown.

Figure 6

Increased JNK activity contributes to the enhanced proliferation of p38α-deficient myoblasts. WT and p38α^Δ/Δ myoblasts were cultured as in Figure 5. (A) Phosphorylated JNK and Ser63-phosphorylated cJun were analyzed by Western blotting using anti-phospho-specific antibodies. Numbers show quantification of phospho-proteins normalized to total JNK and cJun levels. (B) qPCR analysis of the relative cJun mRNA levels (top). ChIP analysis was performed using an anti-cJun antibody, and PCR was performed with primers corresponding to the cyclin D1 promoter region (bottom). (C) p38α^Δ/Δ primary myoblasts were cultured in DM for the indicated hours, in the absence or presence of D-JNKI1 and SP600125 (JNK inhibitors), and incubated for 1 h with BrdU. BrdU-positive cells were quantified as in Figure 4. The effect of D-JNKI1 and SP600125 on JNK activation is shown in Supplementary Figure 6B. (D) Cells were cultured as in (C), and mRNA for the indicated myogenic markers was analyzed by qPCR. (E) p38α activity is necessary for myogenic differentiation, independent of cell-cycle withdrawal. Myoblasts cultured in GM were shifted to DM for 24 h and then cultured for an additional 24 h period in DM alone or DM supplemented with SB203580. mRNA for the indicated myogenic markers was analyzed by qPCR. (F) Reduced MKP-1 levels in p38α-deficient myoblasts. Myoblast extracts used in (A) were analyzed by Western blotting using anti-MKP-1 and anti-tubulin antibodies.

Figure 7

Reduced fiber size and increased cell proliferation in p38α^Δ/Δ neonatal muscles. (A) p38α expression in neonatal muscles analyzed by RT–PCR (left) and Western blotting using an anti-p38α-specific antibody (right). (B, C) The number of PCNA- and Pax7-positive cells (arrowhead) was quantified on p38α^Δ/+ and p38α^Δ/Δ neonatal muscle sections. Scale bar, 20 μm. (D) Top: HE staining showing reduced myofiber size in p38α^Δ/Δ muscle; bottom: presence of small immature myofibers stained strongly with an anti-embryonic MHC antibody. Scale bar, 20 μm. (E) Frequency histograms of myofiber size in p38α^Δ/+ and p38α^Δ/Δ neonatal muscle. A Mann–Whitney non-parametric test was used for comparisons between groups. Data are means±s.e.m. P<0.001.

Figure 8

Muscle structure is not altered in p38α^Δ/Δ neonatal muscles. Representative transmission electron micrographs of longitudinal and transverse p38α^Δ/+ and p38α^Δ/Δ sections showed no morphological abnormalities in myofibrils and muscle sarcomeres. (A, B) Longitudinal sections. Scale bar, 5 μM. (C, D) Higher magnification of longitudinal sections. Scale bar, 500 nM. (E, F) Transversal sections. Scale bar, 500 nM.