Akirin2 regulates proliferation and differentiation of porcine skeletal muscle satellite cells via ERK1/2 and NFATc1 signaling pathways

Akirin2, a novel nuclear factor, plays an important role in myogenesis. To investigate the role of Akirin2 in proliferation and differentiation of porcine skeletal muscle satellite cells, Akirin2 overexpression and Akirin2 silence technologies were employed. Our results showed that overexpression of Akirin2 markedly enhanced the proliferation and differentiation of porcine skeletal muscle satellite cells, whereas silencing of Akirin2 got the opposite results. Furthermore, our results showed that Akirin2 affected proliferation and differentiation of porcine skeletal muscle satellite cells through extracellular-signal regulated kinase-1/2 (ERK1/2) and NFATc1 signaling pathways. These results indicate that Akirin2 can effectively promote skeletal muscle satellite cells proliferation and differentiation, acting through ERK1/2- and NFATc1-dependent mechanisms.

Scientific RepoRts | 7:45156 | DOI: 10.1038/srep45156 Akirin2 promotes proliferation of porcine skeletal muscle satellite cells. Porcine skeletal muscle satellite cells were subjected to cell proliferation analysis after transfection of pcDNA3.1(+ )-pAkirin2 plasmid or Akirin2 siRNA for 24 h. The results showed that overexpression of Akirin2 could promote the mRNA (Fig. 3A) and protein expressions of Akirin2 (Fig. 3B) and the cell proliferation ( Fig. 4A and B), whereas silencing of Akirin2 inhibited the expression of Akirin2 ( Fig. 3C and D) and the cell proliferation ( Fig. 4C and D). Taken together, these findings show that Akirin2 functions in promoting the proliferation of porcine skeletal muscle satellite cells.
Akirin2 promotes differentiation of porcine skeletal muscle satellite cells. To investigate the role of Akirin2 in differentiation of porcine skeletal muscle satellite cells, we performed overexpression or silencing of Akirin2 in differentiating cells and measured the protein level of myogenic marker MHC. As shown in Fig. 5, overexpression of Akirin2 significantly increased, whereas silencing of Akirin2 significantly decreased, the protein level of MHC, suggesting that Akirin2 functions in promoting the differentiation of porcine skeletal muscle satellite cells.

Involvement of ERK1/2 signaling pathway in Akirin2-induced proliferation and differentiation of porcine skeletal muscle satellite cells.
To investigate whether Akirin2 affects the ERK1/2 signaling pathway, we probed for phospho-ERK1/2 levels in lysates from Akirin2-overexpressed porcine skeletal muscle satellite cells. The results indicated that overexpression of Akirin2 activated ERK1/2 in proliferating cells (Fig. 6). RNA was extracted from the proliferating porcine skeletal muscle satellite cells on the days 1, 2, 3, and 4. Akirin2 mRNA expression was analyzed by real-time quantitative PCR. The amount of Akirin2 mRNA was normalized to the amount of GAPDH mRNA. Data were presented as means ± SE (n = 3). *P < 0.05, **P < 0.01 and ***P < 0.001 as compared with the control group (1 d). RNA was extracted from the differentiating porcine skeletal muscle satellite cells on the days 2, 4, and 6. Akirin2 (A) and myogenin (B) mRNA expression was analyzed by real-time quantitative PCR. The amount of Akirin2 and myogenin mRNA was normalized to the amount of GAPDH mRNA. Data were presented as means ± SE (n = 3). ***P < 0.001 as compared with the control group (2 d). A similar result was also observed in differentiating cells (data not shown). To determine whether Akirin2 affects proliferation and differentiation of porcine skeletal muscle satellite cells through the ERK1/2 signaling pathway, porcine skeletal muscle satellite cells were treated with specific ERK inhibitor PD98059 and Akirin2 overexpression. The results indicated that inhibition of ERK1/2 signaling pathway significantly eliminated the proliferation (Figs 4A,B and 7) and differentiation (Figs 5A and 8) promotion by Akirin2 overexpression.
Involvement of NFATc1 signaling pathway in Akirin2-induced proliferation and differentiation of porcine skeletal muscle satellite cells. To investigate whether Akirin2 affects the NFATc1 signaling pathway, we probed for NFATc1 levels in lysates from Akirin2-overexpressed porcine skeletal muscle satellite cells. The results showed that overexpression of Akirin2 increased the protein expression of NFATc1 in proliferating cells (Fig. 9). A similar result was also observed in differentiating cells (data not shown). To verify whether NFATc1 signaling pathway is involved in Akirin2-induced proliferation and differentiation promotion of porcine Interaction effects between ERK1/2 and NFATc1 signaling pathways in porcine skeletal muscle satellite cells. Porcine skeletal muscle satellite cells were treated with inhibitors PD98059 or CsA for 1 h before transfection with pcDNA3.1(+ )-Akirin2 plasmid for 24 h in proliferation medium and for 72 h in differentiation medium, respectively. The results of Western blot indicated that PD98059 and CsA inhibited NFATc1 and phosphorylation of ERK1/2, respectively, in both proliferation and differentiation medium (Fig. 12).

Discussion
The process of myogenesis is controlled by several myogenic regulatory factors (MyoG, MyoD, Myf5, Myf6, and so on) which further regulate the expression of many muscle specific genes 21 . This process is also guided by various environmental cues 3,4 . There is some evidence that many peptidic factors are able to regulate skeletal (A,C) Cell proliferation was evaluated by EdU proliferation assay. The percentage of EdU-positive cells was quantified. Results were presented as mean ± SE (n = 6). (B,D) Western blot analysis of extracts from porcine skeletal muscle satellite cells by using anti-phospho-Histone H3 antibody. Results were presented as means ± SE (n = 3). *P < 0.05 and ***P < 0.001 as compared with the control group. muscle cells proliferation or differentiation through distinct signaling pathways 9,22 . Akirin2 has been identified as a potential regulator of myogenesis 15 . Here, we addressed the questions whether Akirin2 might participate in regulation of skeletal muscle satellite cells proliferation and differentiation. The results of our work demonstrated that Akirin2 promotes proliferation and differentiation of porcine skeletal muscle satellite cells.
Phospho-Histone H3 protein was used as a mitotic cell cycle biomarker for cell proliferation 23 . MHC is the late differentiation markers of myoblasts 24,25 . To study the effects of Akirin2 on the proliferation and differentiation of porcine skeletal muscle satellite cells, we used two approaches: over-expression and RNA interference with Akirin2. Our results showed that overexpression of Akirin2 significantly increased the abundance of phospho-Histone-H3, a S/G2 and G2/M phase marker protein. Furthermore, overexpression of Akirin2 significantly increased the protein expression levels of MHC. Moreover, silencing of Akirin2 decreased the proliferation and differentiation of porcine skeletal muscle satellite cells. Our in vitro study revealed that Akirin2 plays an important role in proliferation and differentiation of porcine skeletal muscle satellite cells. However, Sun et al. reported that Akirin2 could promote the proliferation but not the differentiation of duck myoblasts 26 . Those findings suggest that the function of Akirin2 in skeletal myogenesis exists the cells or species difference.
There is a lot of evidence to indicate that ERK1/2 pathway is involved in regulating the proliferation of muscle cells [27][28][29] . To gain insight into the mechanisms by which Akirin2 stimulates the proliferation and differentiation of porcine skeletal muscle satellite cells, we evaluated the signaling events. We found that Akirin2 increased the phosphorylation level of ERK1/2 in proliferating porcine skeletal muscle satellite cells. To test the functional role of ERK1/2 activation induced by Akirin2 in porcine skeletal muscle satellite cells proliferation, we next explored the effects of ERK1/2 inhibitor (PD98059) on Akirin2-induced proliferation promotion of porcine skeletal muscle satellite cells. Inhibition of the ERK1/2 pathway by PD98059 decreased Akirin2-induced proliferation promotion of porcine skeletal muscle satellite cells. These results suggest that the ERK1/2 pathway mediates the stimulatory effects of Akirin2 on the proliferation of porcine skeletal muscle satellite cells.
We have also demonstrated that Akirin2 promotes the myogenic differentiation of porcine skeletal muscle satellite cells. The stimulatory effect was characterized by increasing protein expression level of MHC, a myogenic differentiation-related protein. Previous studies suggested that the effect of MAPK on muscle cells differentiation is controversial. Some studies suggest that activation of the ERK pathway prevents skeletal muscle differentiation [29][30][31] , and other studies believe that it functions at two stages of skeletal muscle differentiation 10,32 . However, recent data indicate that ERK1/2 may positively regulate myogenic differentiation 6,28,33 . In this study, we showed that Akirin2 promotes differentiation of porcine skeletal muscle satellite cells through ERK1/2 signaling pathway.
Calcineurin (CaN) has been reported to be a possible candidate in the signaling of skeletal muscle cellular growth, and plays an important role in regulating cell proliferation 34 and differentiation [35][36][37] . CaN has also been reported to affect the muscle regeneration by association with NFATc1, a downstream target of CaN signaling 37 . As Akirin2 affects both the proliferation and differentiation of porcine skeletal muscle satellite cells, we hypothesized that Akirin2 affects the proliferation and differentiation of porcine skeletal muscle satellite cells via the NFATc1 signaling pathway. The immunosuppressive drug CsA is a well-known inhibitor of CaN, and thus inhibits NFAT activity by blocking its dephosphorylation 38 . CsA has also been reported to inhibit myoblast differentiation 35,36 . Here we demonstrated that CsA inhibited proliferation and differentiation of porcine skeletal muscle satellite cells, and Akirin2 promoted the NFATc1 protein expression in porcine skeletal muscle satellite cells during both proliferation and differentiation stages.
In the present study, we demonstrated that Akirin2 activated both ERK1/2 and NFATc1 signaling pathway in porcine skeletal muscle satellite cells. So we speculated that ERK1/2 and NFATc1 signaling pathways might crosstalk with each other. This speculation was partly supported by the data of the present study. Further study is warranted to verify the speculation more thoroughly.
In summary, the present study demonstrated that Akirin2 plays an important role in proliferation and differentiation of porcine skeletal muscle satellite cells, and further revealed that Akirin2 promotes proliferation and differentiation of porcine skeletal muscle satellite cells through ERK1/2 and NFATc1 signaling pathways. However, it is necessary to further investigation of the role of Akirin2 in skeletal muscle development by in vivo study.

Materials and Methods
Ethics statement. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of Sichuan Agricultural University. All experimental protocols were approved by the Animal Care Advisory Committee of Sichuan Agricultural University.  Isolation of porcine skeletal muscle satellite cells. Porcine skeletal muscle satellite cells were isolated from 3-day-old male Duroc × Yorkshire × Landrace (DLY) pigs as described previously 39 with some modifications. Briefly, skeletal muscles were digested with 0.2% collagenase type II (Sigma) and then filtered successively through 200-mesh and 400-mesh cell sieves. The collected cells were purified by differential adhesion method. The resulting mononuclear cells were cultured in DMEM/F12 (Invitrogen) supplemented with 20% FBS, 100 U/mL penicillin and 100 μ g/μ L streptomycin at 37 °C in a humidified 5% CO 2 atmosphere. The cells were identified by immunofluorescence with anti-Pax7 antibody (PAX7, DSHB, USA) (data not shown).

Reagents. The calcineurin inhibitor cyclosporin
Cell culture. For stimulation experiments, cells were treated with recombinant plasmid pcDNA3.1(+ )-pAkirin2, and PD98059 (ERK1/2 inhibitor) or cyclosporin (CsA, CaN inhibitor) were added 1 h before the treatment. Myogenic differentiation was induced by changing the medium to DMEM/F12 supplemented with 2% horse serum and penicillin/streptomycin. The siRNA and plasmid transfection. A pair of 21-nucleotide siRNA sequences targeting Akirin2 was designed and synthesized by GenePharm (Shanghai, China). The sense strand of the Akirin2 siRNA was 5′ -GCUGUACUUCUGAUGCACATT-3′ , and the antisense strand was 5′ -UGUGCAUCAGAAGUACAGCTT-3′ .   The sense strand of the negative control siRNA was 5′ -UUCUCCGAACGUGUCACGUTT-3′ , and the antisense strand was 5′ -ACGUGACACGUUCGGAGAATT-3′ . The siRNA was dissolved in DEPC-treated water, and the final concentration was 50 nM. The pcDNA3.1(+ )-pAkirin2 plasmid was constructed by our lab 20 . The lipofectamine 2000 (Invitrogen, California, USA) was used to transfect the porcine skeletal muscle satellite cells according to the manufacturer's instruction. Cell proliferation analysis. To investigate cell proliferation, markers of two phases of the cell cycle were analyzed by using EdU (5-ethynyl-2′ -deoxyuridine) for synthesis phase and phospho-histone H3 for G2-M phase. EdU proliferation assay was performed as described by Chen et al. 40    Western blotting. Protein was extracted from porcine skeletal muscle satellite cells using RIPA cell lysis buffer (Pierce, Rockford, IL, USA) supplemented with protease inhibitor cocktail (Sigma). Protein concentrations were assessed by BCA protein assay kit (Pierce). Equal amounts of protein were loaded onto 10% sodium dodecyl sulfate-polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was blocked in 3% non-fat milk in TBS-0.1% Tween-20 for 1 h and incubated overnight with primary antibody at 4 °C, followed by horseradish peroxidase-linked secondary antibodies (Santa Cruz Biotechnology) for 1 h at 37 °C. The bound antibodies were visualized with a Clarity TM Western ECL Substrate (Bio-Rad, Hercules, CA, USA) using a ChemiDoc XRS Imager System (Bio-Rad). Housekeeping protein GAPDH was used as a control for equal protein loading. The density of the protein bands was determined using Gel-Pro Analyzer 4.2 software (Media Cybernetics, Rockville, MD, USA).
Statistical analysis. All data, expressed as mean ± SE, were subjected to one-way ANOVA analysis or Tukey test using SPSS 11.0 software and P < 0.05 was considered significant.