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Attenuation of p38-mediated miR-1/133 expression facilitates myoblast proliferation during the early stage of muscle regeneration.

Zhang D, Li X, Chen C, Li Y, Zhao L, Jing Y, Liu W, Wang X, Zhang Y, Xia H, Chang Y, Gao X, Yan J, Ying H - PLoS ONE (2012)

Bottom Line: Additionally, we show that both miR-1 and miR-133 reduce Cyclin D1 expression and repress myoblast proliferation by inducing G1 phase arrest.Finally, we reveal that proproliferative FGF2, which is elevated during muscle regeneration, attenuates p38 signaling and miR-1/133 expression.Taken together, our results suggest that downregulation of p38-mediated miR-1/133 expression by FGF2 and subsequent upregulation of Sp1/Cyclin D1 contribute to the increased myoblast proliferation during the early stage of muscle regeneration.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, China.

ABSTRACT
Myoblast proliferation following myotrauma is regulated by multiple factors including growth factors, signal pathways, transcription factors, and miRNAs. However, the molecular mechanisms underlying the orchestration of these regulatory factors remain unclear. Here we show that p38 signaling is required for miR-1/133a clusters transcription and both p38 activity and miR-1/133 expression are attenuated during the early stage of muscle regeneration in various animal models. Additionally, we show that both miR-1 and miR-133 reduce Cyclin D1 expression and repress myoblast proliferation by inducing G1 phase arrest. Furthermore, we demonstrate that miR-133 inhibits mitotic progression by targeting Sp1, which mediates Cyclin D1 transcription, while miR-1 suppresses G1/S phase transition by targeting Cyclin D1. Finally, we reveal that proproliferative FGF2, which is elevated during muscle regeneration, attenuates p38 signaling and miR-1/133 expression. Taken together, our results suggest that downregulation of p38-mediated miR-1/133 expression by FGF2 and subsequent upregulation of Sp1/Cyclin D1 contribute to the increased myoblast proliferation during the early stage of muscle regeneration.

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p38 Activity is Required for miR-1/133a Cluster Transcription.(A and B) Real-time RT-PCR analysis of miR-1/133 expression in C2C12 myoblasts treated with SB203580 for 24 hours (A) or 48 hours (B) as indicated. (C and D) Enhancer activity of miR-1–2/miR-133a-1 or miR-1–1/miR-133a-2 cluster in C2C12 myoblasts treated with SB203580 for 24 hours (C) or infected with MKK6E (D). (E and F) Real-time RT-PCR analysis of miR-1/133 expression in p38αf/f myoblasts infected with Ad-Cre or Ad-GFP as a control (E) or in C2C12 myoblasts infected with adenoviral MKK6E (Ad-MKK6E) or Ad-GFP (F). Error bars represent the SD of three independent experiments. *p<0.05, **p<0.01.
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pone-0041478-g002: p38 Activity is Required for miR-1/133a Cluster Transcription.(A and B) Real-time RT-PCR analysis of miR-1/133 expression in C2C12 myoblasts treated with SB203580 for 24 hours (A) or 48 hours (B) as indicated. (C and D) Enhancer activity of miR-1–2/miR-133a-1 or miR-1–1/miR-133a-2 cluster in C2C12 myoblasts treated with SB203580 for 24 hours (C) or infected with MKK6E (D). (E and F) Real-time RT-PCR analysis of miR-1/133 expression in p38αf/f myoblasts infected with Ad-Cre or Ad-GFP as a control (E) or in C2C12 myoblasts infected with adenoviral MKK6E (Ad-MKK6E) or Ad-GFP (F). Error bars represent the SD of three independent experiments. *p<0.05, **p<0.01.

Mentions: Although miR-1 and miR-133 have been studied during muscle regeneration, the upstream signalings and detailed mechanisms remain to be further clarified. As previously reported, p38 activity is required for modulating cell cycle exit and myogenesis [22], [28]. Based on our finding that both the expression of miR-1/133 and the activity of p38 were attenuated at the early stages of muscle regeneration, we speculated that p38 signaling might be one of the upstream signalings regulating miR-1/133 expression. To test our hypothesis, we investigated the relationship between p38 activity and the transcription of miR-1/133 in vitro. Interestingly, we found the expression of miR-1/133 was gradually reduced in C2C12 myoblasts treated with SB203580, an inhibitor of p38α and p38β (Figure 2A, B), indicating that the activity of p38 is required for the transcription of miR-1/133. Since miR-1 and miR-133 have several family members, which are located on different chromosomes as clusters with each other or with miR-206, to identify which family members are regulated by p38 activity, we determined the SB203580 effect on the transcription of miR-1–2/miR-133a-1, miR-1–1/miR-133a-2, and miR-133b in C2C12 myoblasts. As shown in Figure 2C, SB203580 only suppressed the activity of the enhancers of miR-1–2/miR-133a-1 and miR-1–1/miR-133a-2, but not that of miR-133b (data not shown), suggesting that p38 signaling selectively regulates the transcription of miR-1/miR-133a clusters. In agreement of these results, we found MKK6E, a constitutively active activator of p38, was capable of increasing the miR-1/miR-133a enhancer activity (Figure 2D). In addition, we also measured the expression level of miR-1/miR-133 in p38αf/f myoblasts infected with adenoviral Cre (Ad-Cre). As expected, we observed lower expression of miR-1/133 in myoblasts lacking p38α than that in control cells infected with adenoviral GFP (Ad-GFP) (Figure 2E). Moreover, we observed an increase in the expression level of miR-1/133 by adenoviral MKK6E infection, which provided further support for our hypothesis (Figure 2F). Taken together, these results indicate that p38 signaling is able to control the transcription of miR-1/miR-133a clusters.


Attenuation of p38-mediated miR-1/133 expression facilitates myoblast proliferation during the early stage of muscle regeneration.

Zhang D, Li X, Chen C, Li Y, Zhao L, Jing Y, Liu W, Wang X, Zhang Y, Xia H, Chang Y, Gao X, Yan J, Ying H - PLoS ONE (2012)

p38 Activity is Required for miR-1/133a Cluster Transcription.(A and B) Real-time RT-PCR analysis of miR-1/133 expression in C2C12 myoblasts treated with SB203580 for 24 hours (A) or 48 hours (B) as indicated. (C and D) Enhancer activity of miR-1–2/miR-133a-1 or miR-1–1/miR-133a-2 cluster in C2C12 myoblasts treated with SB203580 for 24 hours (C) or infected with MKK6E (D). (E and F) Real-time RT-PCR analysis of miR-1/133 expression in p38αf/f myoblasts infected with Ad-Cre or Ad-GFP as a control (E) or in C2C12 myoblasts infected with adenoviral MKK6E (Ad-MKK6E) or Ad-GFP (F). Error bars represent the SD of three independent experiments. *p<0.05, **p<0.01.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3404058&req=5

pone-0041478-g002: p38 Activity is Required for miR-1/133a Cluster Transcription.(A and B) Real-time RT-PCR analysis of miR-1/133 expression in C2C12 myoblasts treated with SB203580 for 24 hours (A) or 48 hours (B) as indicated. (C and D) Enhancer activity of miR-1–2/miR-133a-1 or miR-1–1/miR-133a-2 cluster in C2C12 myoblasts treated with SB203580 for 24 hours (C) or infected with MKK6E (D). (E and F) Real-time RT-PCR analysis of miR-1/133 expression in p38αf/f myoblasts infected with Ad-Cre or Ad-GFP as a control (E) or in C2C12 myoblasts infected with adenoviral MKK6E (Ad-MKK6E) or Ad-GFP (F). Error bars represent the SD of three independent experiments. *p<0.05, **p<0.01.
Mentions: Although miR-1 and miR-133 have been studied during muscle regeneration, the upstream signalings and detailed mechanisms remain to be further clarified. As previously reported, p38 activity is required for modulating cell cycle exit and myogenesis [22], [28]. Based on our finding that both the expression of miR-1/133 and the activity of p38 were attenuated at the early stages of muscle regeneration, we speculated that p38 signaling might be one of the upstream signalings regulating miR-1/133 expression. To test our hypothesis, we investigated the relationship between p38 activity and the transcription of miR-1/133 in vitro. Interestingly, we found the expression of miR-1/133 was gradually reduced in C2C12 myoblasts treated with SB203580, an inhibitor of p38α and p38β (Figure 2A, B), indicating that the activity of p38 is required for the transcription of miR-1/133. Since miR-1 and miR-133 have several family members, which are located on different chromosomes as clusters with each other or with miR-206, to identify which family members are regulated by p38 activity, we determined the SB203580 effect on the transcription of miR-1–2/miR-133a-1, miR-1–1/miR-133a-2, and miR-133b in C2C12 myoblasts. As shown in Figure 2C, SB203580 only suppressed the activity of the enhancers of miR-1–2/miR-133a-1 and miR-1–1/miR-133a-2, but not that of miR-133b (data not shown), suggesting that p38 signaling selectively regulates the transcription of miR-1/miR-133a clusters. In agreement of these results, we found MKK6E, a constitutively active activator of p38, was capable of increasing the miR-1/miR-133a enhancer activity (Figure 2D). In addition, we also measured the expression level of miR-1/miR-133 in p38αf/f myoblasts infected with adenoviral Cre (Ad-Cre). As expected, we observed lower expression of miR-1/133 in myoblasts lacking p38α than that in control cells infected with adenoviral GFP (Ad-GFP) (Figure 2E). Moreover, we observed an increase in the expression level of miR-1/133 by adenoviral MKK6E infection, which provided further support for our hypothesis (Figure 2F). Taken together, these results indicate that p38 signaling is able to control the transcription of miR-1/miR-133a clusters.

Bottom Line: Additionally, we show that both miR-1 and miR-133 reduce Cyclin D1 expression and repress myoblast proliferation by inducing G1 phase arrest.Finally, we reveal that proproliferative FGF2, which is elevated during muscle regeneration, attenuates p38 signaling and miR-1/133 expression.Taken together, our results suggest that downregulation of p38-mediated miR-1/133 expression by FGF2 and subsequent upregulation of Sp1/Cyclin D1 contribute to the increased myoblast proliferation during the early stage of muscle regeneration.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, China.

ABSTRACT
Myoblast proliferation following myotrauma is regulated by multiple factors including growth factors, signal pathways, transcription factors, and miRNAs. However, the molecular mechanisms underlying the orchestration of these regulatory factors remain unclear. Here we show that p38 signaling is required for miR-1/133a clusters transcription and both p38 activity and miR-1/133 expression are attenuated during the early stage of muscle regeneration in various animal models. Additionally, we show that both miR-1 and miR-133 reduce Cyclin D1 expression and repress myoblast proliferation by inducing G1 phase arrest. Furthermore, we demonstrate that miR-133 inhibits mitotic progression by targeting Sp1, which mediates Cyclin D1 transcription, while miR-1 suppresses G1/S phase transition by targeting Cyclin D1. Finally, we reveal that proproliferative FGF2, which is elevated during muscle regeneration, attenuates p38 signaling and miR-1/133 expression. Taken together, our results suggest that downregulation of p38-mediated miR-1/133 expression by FGF2 and subsequent upregulation of Sp1/Cyclin D1 contribute to the increased myoblast proliferation during the early stage of muscle regeneration.

Show MeSH
Related in: MedlinePlus