Attenuation of p38-Mediated miR-1/133 Expression Facilitates Myoblast Proliferation during the Early Stage of Muscle Regeneration

<div><p>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.</p> </div

miR-1/133 Expression and p38 Activity are Attenuated in Regenerating Muscle Tissues.

<p>(A) Hematoxylin and eosin staining of GAS muscle of mice at the indicated time points following freeze injury. (B) Real-time RT-PCR analysis of the time-course expression of miR-1 and miR-133 in GAS muscle of mice following freeze injury. (C and D) Western blot analysis of phospho-p38 (p-p38) in GAS muscle at the indicated time points following freeze injury. The densities of p-p38 bands were quantitated and normalized to total p38 (t-p38) or tubulin as indicated.</p

Sp1 and Cylin D1 expression are downregulated during Early Phase of Muscle Regeneration.

<p>(A and B) Real-time RT-PCR analysis of Sp1 and Cyclin D1 mRNA expression in C2C12 myoblasts treated with SB203580 or DMSO as a control for 24 hours (A) or 48 hours (B) as indicated. Error bars represent the SD of three independent experiments. (C) Western blot analysis of Sp1 and Cyclin D1 protein expression in C2C12 myoblasts treated with SB203580 or DMSO as a control for 24 hours or 48 hours as indicated. (D) Western blot analysis of Sp1, Cyclin D1 and p-p38 protein level in C2C12 myoblasts infected with Ad-MKK6E or Ad-GFP as a control for 24 hours or 48 hours as indicated. (E) Real-time RT-PCR analysis of Sp1 and Cyclin D1 mRNA expression in p38α^f/f myoblasts infected with Ad-Cre or Ad-GFP as a control as indicated. Error bars represent the SD of three independent experiments. (F–I) Real-time RT-PCR analysis of the expression of Sp1 and Cyclin D1 in GAS muscle of mice following freeze injury (F), in GAS muscle of mice following lidocaine injection (G), in Diaphragm muscle of mdx mice (H), in skeletal muscle of patients with BMD or DMD (I). SDs are shown as error bars (n≥3). (J–L) Western blot analysis of Sp1, Cyclin D1, and p-p38 in muscle regeneration mouse models, including freeze injury model (J), lidocaine injury model (K), and mdx mouse model (L). The relative intensity of each band was quantified with NIH image. Tubulin and t-p38 were used for normalization for Sp1/Cyclin D1, and p-p38 respectively. SDs are shown as error bars (n = 3). *p<0.05, **p<0.01.</p

miR-133 induces G1 phase arrest and suppresses myoblast proliferation by downregualtion of Cyclin D1.

<p>(A) Growth curves of C2C12 myoblasts were determined by MTT assay. Cells were transfected with miR-133 precursors or anti-miR-133 as indicated. Error bars represent the standard deviation of three independent experiments. (B and C) Proliferation of C2C12 myoblasts was evaluated by BrdU staining. Cells were transfected with miR-133 mimics. Representative images of cells were taken by fluorescence microscope (B). The percentage of BrdU positive cells was measured (C). Data shown are from a typical experiment performed in triplicate. (D) Cell cycle analysis of C2C12 myoblasts transfected with miR-133 precursors or anti-miR-133 as indicated. Data shown are from a typical experiment performed. (E) Western blot analysis of Cyclin D1 protein expression in C2C12 myoblasts transfected with miR-133 precursors or anti-miR-133 as indicated. (F) Identification of miR-133 regulatory element in the 3′UTR of mouse Sp1. (G) Western blot analysis of Sp1 protein level in HEK293T cells transfected with miR-133 precursors as indicated. (H) Evaluation of miR-133 effect on a reporter containing Sp1–3× MRE in C2C12 myoblasts transfected with miR-133 precursors or anti-miR-133 as indicated. Error bars represent the SD of three independent experiments. (I) Determination of miR-133 effect on a reporter containing Sp1–3′UTR in C2C12 myoblasts transfected with miR-133 precursors or miR-133 specific sponges as indicated. Error bars represent the SD of three independent experiments. (J) Western blot analysis of Sp1 protein expression in C2C12 myoblasts transfected with miR-133 precursors or anti-miR-133 as indicated. *p<0.05, **p<0.01.</p

miR-133 downregulates Cyclin D1 expression via direct targeting Sp1.

<p>(A) Determination of Sp1 effect on the promoter activity of Cyclin D1. C2C12 myoblasts were cotransfected with a reporter containing Cyclin D1 promoter, and Sp1 expression vector (MSCV-Sp1), or control vector (MSCV), or Sp1 specific siRNA (siSp1), or negative control siRNA (NC) as indicated. Error bars represent the SD of three independent experiments. (B and C) Real-time RT-PCR analysis of Sp1 and Cyclin D1 mRNA expression in C2C12 myoblasts transfected with MSCV-Sp1 or MSCV (B), or transfected with siSp1 or NC (C), as indicated. Error bars represent the SD of three independent experiments. (D and E) Western blot analysis of Sp1 and Cyclin D1 protein level in C2C12 myoblasts transfected with siSp1 or NC (D), or MSCV-Sp1 or MSCV.(E). (F and G) Growth curves of C2C12 myoblasts were determined by MTT assay. Cells were transfected with MSCV-Sp1 or MSCV (F), or transfected with siSp1 or NC (G). Error bars represent the SD of three independent experiments. (H) Cell cycle analysis of C2C12 myoblasts transfected with siSp1 or NC as indicated. Data shown are from a typical experiment performed. **p<0.01.</p

p38 Activity is Required for miR-1/133a Cluster Transcription.

<p>(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 p38α^f/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.</p

miR-1 induces G1 phase arrest and inhibits myoblast proliferation by direct targeting Cyclin D1.

<p>(A) Identification of miR-1 regulatory element in the 3′UTR of mouse Cyclin D1. (B) Evaluation of effect of miR-1 mimics and miR-133 mimics on reporters containing three different regions of Cyclin D1–3′UTR in C2C12 myoblasts transfected with miR-1 or miR-133 mimics as indicated. Error bars represent the SD of three independent experiments. (C) Western blot analysis of Cyclin D1 protein expression in C2C12 myoblasts transfected with miR-1 mimics or control oligos. (D) Cell cycle analysis of C2C12 myoblasts transfected with miR-1 mimics or control oligos. Data shown are from a typical experiment performed. (E) Growth curves of C2C12 myoblasts were determined by MTT assay. Cells were transfected with miR-1 mimics or control oligos. Error bars represent the SD of three independent experiments. (F and G) Proliferation of C2C12 myoblasts was evaluated by BrdU incorporation. Cells were transfected with miR-1 mimics. Representative images of cells were taken by fluorescence microscope (F). The percentage of BrdU positive cells was measured (G). Data shown are from a typical experiment performed in triplicate. **p<0.01.</p

Proproliferative FGF2 attenuates p38 activity and miR-1/133 expression in myoblasts.

<p>(A–D) Evaluation of FGF2 mRNA expression by real-time RT-PCR in regenerating muscle following freeze injury (A), or following lidocaine injection (B), or in the muscle of mdx mice (C) or patients with BMD or DMD (D). SDs are shown as error bars (n≥3). (E) Western blot analysis of p-p38, Sp1 and Cyclin D1 in C2C12 myoblasts treated with FGF2 or vehicle. (F) Real-time RT-PCR analysis of miR-1/133 expression in C2C12 myoblasts treated with FGF2 and FGF2 signaling inhibitor (PD173074), as indicated. Error bars represent the SD of three independent experiments. (G) Determination of the effect of FGF2 and PD173074 on enhancer activity of miR-1–2/miR-133a-1 or miR-1–1/miR-133a-2 cluster in C2C12 myoblasts, as indicated. Error bars represent the SD of three independent experiments. (H) Real-time RT-PCR analysis of Sp1 and Cyclin D1 mRNA expression in C2C12 myoblasts treated with FGF2 and PD173074 as indicated. Error bars represent the SD of three independent experiments. (I and J) Real-time RT-PCR analysis of miR-1/133 (I), Sp1 (J), and Cyclin D1 (J) expression in isolated GAS muscle treated with FGF2 and PD173074 as indicated. Error bars represent the SD of three independent experiments. (K) Schematic representation of a novel mechanism of FGF2 stimulated myoblast proliferation during muscle regeneration. FGF2-induced attenuation of p38-mediated miR-1/133 expression facilities myoblasts proliferation via derepressing miR-1/133 direct downstream targets Sp1/Cyclin D1. *p<0.05, **p<0.01.</p

Loss of p38α in adipose tissues causes minimal effects on BAT.

<p>(A) BT of Floxed and Fp38αKO mice maintained at RT (<i>n</i> = 6 per group). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (B and C) VO₂ (B) and VCO₂ (C) in Floxed and Fp38αKO mice maintained at RT (<i>n</i> = 4 per group). The values were normalized by LM. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (D) iBAT weight and relative iBAT weight to BW ratio (iBAT/BW) of Floxed (<i>n</i> = 10) and Fp38αKO (<i>n</i> = 8). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (E-G) Representative HE staining of iBAT (E), diameter and cross-sectional area of adipocytes in iBAT (F), and representative UCP-1 staining of iBAT (G) from Floxed and Fp38αKO mice maintained at RT. Bars: 100 μm. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (H) Representative EM images of iBAT from Floxed and Fp38αKO mice maintained at RT at low (top), medium (middle), and high (bottom) magnification, as indicated. (I) Relative mitDNA to nuDNA ratio in unilateral iBAT of Floxed (<i>n</i> = 5) and Fp38αKO (<i>n</i> = 9) mice maintained at RT. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (J) OCR of ADP, Oligomycin, FCCP, and Antimycin A/Rotenone-treated mitochondria derived from iBAT of Floxed and Fp38αKO mice exposed to cold for 2 d (<i>n</i> = 4 per group). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (K) Relative mRNA levels of UCP-1, PGC1α, DIO2, COX8B, and PRDM16 in iBAT from Floxed and Fp38αKO mice maintained at RT (<i>n</i> = 6 per group) or exposed to cold for 2 d (<i>n</i> = 8 per group). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (L and M) Representative western blots of UCP-1 in iBAT from Floxed and Fp38αKO mice maintained at RT (L) or exposed to cold for 2 d (M). (N) BT of Fp38αKO and Floxed mice exposed to cold for 4 h (<i>n</i> = 4 per group). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (O) Relative mRNA levels of UCP-1 and PGC1α in iBAT from Floxed (<i>n</i> = 7–8) and Fp38αKO (<i>n</i> = 8) mice exposed to cold for 4 h. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (P and Q) Representative western blots (P) and densitometry analysis (Q) of UCP-1 in iBAT from Floxed and Fp38αKO mice exposed to cold for 4 h. The densities of UCP-1 bands were quantitated and normalized to Hsp90 (<i>n</i> = 4 per group). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (R and S) Relative mRNA levels of PRDM16, DIO2, ELVOL3, COX8B, and CIDEA(R), ATGL, MGL, and HSL (S) in iBAT from Floxed (<i>n</i> = 7–8) and Fp38αKO (<i>n</i> = 6–8) mice exposed to cold for 4 h. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. Means ± SEM are shown. *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001. ADP, adenosine diphosphate; ATGL, adipose triglyceride lipase; BAT, brown adipose tissue; BT, body temperature; BW, body weight; CIDEA, cell death-inducing DNA fragmentation factor, alpha subunit-like effector A; COX8B, cytochrome c oxidase subunit 8B; DIO2, deiodinase 2; ELVOL3, elongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3, yeast)-like 3; EM, electron microscopy; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; HE staining, hematoxylin-eosin staining; HSL, hormone-sensitive lipase; iBAT, interscapular brown adipose tissue; IHC, immunohistochemistry; LM, lean mass; MGL, monoglyceride lipase; mitDNA, mitochondrial DNA; NS, not significant; nuDNA, nuclear DNA; OCR, oxygen consumption rate; PGC1α, peroxisome proliferative activated receptor gamma coactivator 1α; PRDM16, positive regulatory domain containing 16; RT, room temperature; UCP-1, uncoupling protein 1; VCO₂, carbon dioxide production; VO₂, oxygen consumption.</p

Adipocyte-specific deletion of p38α leads to a lean phenotype and increased glucose tolerance and insulin sensitivity.

<p>(A-C) Representative western blots of p38α and p-p38 in iBAT (A), iWAT (B), and eWAT (C) from Floxed and Fp38αKO mice as indicated. (D-F) Representative western blots of p38α in other tissues, including liver (D), skeletal muscle (E), and macrophages (F) from Floxed and Fp38αKO mice as indicated. (G) Cumulative gross energy intake and feces of Floxed and Fp38αKO mice for 24 h (<i>n</i> = 4 per group). Mice were maintained at RT. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (H) Growth curve of Floxed (<i>n</i> = 15) and Fp38αKO (<i>n</i> = 8–13) mice maintained at RT. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (I and J) BW, FM, and FM to BW ratio (FM/BW) of Floxed and Fp38αKO mice maintained at RT (<i>n</i> = 11 per group). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. (K and L) GTT (K, <i>n</i> = 8 per group) and ITT (L, <i>n</i> = 8 per group) in Floxed and Fp38αKO mice. AUCs were calculated. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004225#pbio.2004225.s010" target="_blank">S1 Data</a>. Means ± SEM are shown. *<i>p</i> < 0.05; **<i>p</i> < 0.01. AUC, area under curve; BW, body weight; eWAT, epididymal white adipose tissue; FM, fat mass; GTT, glucose tolerance test; iBAT, interscapular brown adipose tissue; ITT, insulin tolearance test; iWAT, inguinal white adipose tissue; NS, not significant; RT, room temperature.</p