C-Jun N-terminal Kinase Signaling Pathway Is Involved In Myostatin-regulated Proliferation And Differentiation Inhibition Of Myoblast

Myostatin, a member of the transforming growth factorβ(TGF-β) superfamily, is a negative regulator of skeletal muscle growth. The aim of this study was to investigate the involvement of c-Jun N-terminal kinase (JNK) signaling pathway in myostatin's function as a negative regulator of muscle growth using mouse myoblast C2C12 cells as a model. This study consists of five parts of experiments as follows:Exp. 1 Effect of myostatin on JNK signaling pathwayTo investigate whether JNK signaling pathway is activated in response to myostatin treatment and to determine the optimal concentration of myostatin, C2C12 cells were stimulated with myostatin at different concentrations for 1 h. Cell lysates were immunoblotted with anti-phospho-JNK antibody. Western blot analysis showed that the maximal activation of JNK was present after 50 ng/mL of myostatin treatment. To further investigate whether myostatin could activate JNK signaling pathway in both proliferating and differentiating C2C12 cells and to know whether the activation of JNK by myostatin has the time effect, the cells grown either in proliferation medium or in differentiation medium were stimulated with 50 ng/mL myostatin for different time intervals (0 min, 20 min, 40 min, 1 h, 2 h, and 4 h). Cell lysates collected at different time intervals were immunoblotted with multiple antibodies. Western blot analysis showed that the maximal activation of JNK was present after 1 h of myostatin treatment in proliferating cells. In differentiating cells, there was an initial activation of JNK within 20 min of myostatin stimulation. This level of JNK activation then declined, but was observed to be activated subsequently at 1 h of myostain treatment and persisted for up to 4 h. The JNK activation by myostatin was further supported by the increased phosphorylation level of c-Jun, a downstream molecule in this pathway, in a manner similar to JNK. Total levels of JNK proteins remained unchanged in these experiments. This is the first evidence to reveal the activation of JNK signaling pathway by myostatin in both proliferating and differentiating C2C12 cells.Exp. 2 Role of ActRIIB in myostatin-induced JNK activationTo obtain the small interference RNA (siRNA) which can effectively down-regulate ActRIIB gene expression, three siRNAs specific to ActRIIB (Ambion siRNA ID 59935, ID 60120, or ID 162114) were transfected into C2C12 cells, respectively, under the optimal Lipofectamine transfection conditions determined by using positive control GAPDH siRNA. After a transfection of 48 h, the ActRIIB mRNA abundance was determined using real-time quantitative PCR. The expression levels of ActRIIB mRNA in the cells transfected with Ambion siRNA ID 59935, ID 60120, and ID 162114 were decreased to 46.68%, 27.46%, and 60.92%, respectively, compared to the negative control siRNA. To examine whether ActRIIB is involved in myostatin-induced JNK activation, C2C12 cells were transfected for 48 h with Ambion siRNA ID 162114 or Negative Control siRNA under the optimal Lipofectamine transfection conditions, followed by myostatin (50 ng/mL) stimulation for 1 h. Cell lysates were used to probe for phospho-JNK levels. Western blot analysis showed that knock down of ActRIIB significantly reduced the myostatin-induced JNK activation. The present work demonstrates ActRIIB is involved in the activation of JNK by myostatin.Exp. 3 Role of TAK1-MKK4 cascade in myostatin-induced JNK activationTo obtain the highly effective TAK1 siRNA and MKK4 siRNA, three siRNAs specific to TAK1 (Ambion siRNA ID 94455, ID 94549, or ID 189006) and three siRNAs targeted against MKK4 (Ambion siRNA ID 64447, ID 64539, or ID 186583) were transfected into C2C12 cells, respectively, under the optimal Lipofectamine transfection conditions determined in Exp. 2. Forty-eight hours after the transfection, the mRNA levels of TAK1 and MKK4 were determined using real-time quantitative PCR. The expression levels of TAK1 mRNA in the cells transfected with Ambion siRNA ID 94455, ID 94549, and ID 189006 were decreased 33.34%, 46.73%, and 79.97%, respectively, compared to the negative control siRNA. The MKK4 mRNA levels in the cells transfected with Ambion siRNA ID 64447, ID 64539, and ID 186583 were specifically knocked down by 85.19%, 79.18%, and 69.59%, respectively, compared with cells transfected with the negative control siRNA. To determine whether myostatin-induced activation of JNK is achieved through TAK1 and MKK4, C2C12 cells were transfected for 48 h with Ambion siRNA ID 189006, Ambion siRNA ID 64447, or Negative Control siRNA, followed by myostatin (50 ng/mL) stimulation for 1 h. Cell lysates were immunoblotted with multiple antibodies. Western blot analysis showed that knockdown expression of TAK1 by siRNA targeted at TAK1 reduced the myostatin-induced JNK activation, which indicates that TAK1 is involved in myostatin-induced JNK activation. Western blot also showed that JNK could not be activated by myostatin when the MKK4 expression was blocked, suggesting that myostatin-induced JNK activation was probably achieved only through MKK4. Taken together, these results indicate that the TAK1-MKK4 cascade is involved in myostatin-induced activation of JNK. Exp. 4 Role of JNK signaling pathway in myostatin-regulated proliferation and differentiation inhibition of myoblastTo determine whether the JNK signaling pathway is involved in myostatin-induced proliferation inhibition, C2C12 cells, pretreated with 10μmol/L SP600125 for 1 h or left untreated, were evaluated its viabilities by MTS assay after 24 h of myostatin (50 ng/mL) treatment. Cell proliferation assay showed that an approximately 20% reduction of cell viability was observed in myostatin-treated C2C12 cells compared to the untreated control, although the difference was not statistically significant (P = 0.28). Cell proliferation assay also showed that SP600125, a specific inhibitor of JNK, almost eliminated the growth inhibitory role of myostatin in C2C12 cells, suggesting that JNK plays a role in myostatin-mediated proliferation inhibition. It has been reported that myostatin-induced myoblast inhibition is associated with upregulating p21 gene expression. To evaluate the effect of myostatin on p21 mRNA abundance, cells grown to approximately 80% confluence were treated with recombinant mouse myostatin (50 ng/mL) for 2 h, 4 h, and 6 h. Real-time quantitative PCR showed that the maximal upregulation of p21 was detected at 4 h of myostatin treatment. To evaluate the effect of JNK on myostatin-induced upregulation of p21, approximately 80% confluent C2C12 cells were treated for 4 h without or with SP600125 and/or myostatin. Real-time quantitative PCR showed that the p21 mRNA levels in myostatin treatment increased up to approximately 3.5 times as compared to the untreated control. This increase in p21 mRNA levels was partially inhibited in cells pretreated with the JNK inhibitor SP600125. A similar result was also observed in p21 protein levels. Myostatin was reported to inhibit myoblast differentiation by downregulating expression of differentiation-related genes. To investigate whether JNK signaling pathway contributes to myostatin-induced downregulation of differentiation-related gene expression, approximately 80% confluent C2C12 cells were cultured in differentiation medium for 24 h and then treated with or without myostatin (50 ng/mL) for another 24 h in the presence or absence of 10μmol/L SP600125. The mRNA and protein levels of myogenin in myostatin stimulation reduced 40% and 80%, respectively, compared to the untreated control. There were a 25% (P=0.13) reduction of MyoD mRNA level and a 63% reduction of MyoD protein level in myostatin-treated C2C12 cells compared to the untreated control. The reductions of myogenin and MyoD in both transcriptional and protein levels were partially rescued in cells pretreated with the JNK inhibitor SP600125. These results indicate that JNK signaling pathway plays an important role in myostatin-mediated proliferation and differentiation inhibition of myoblast.Exp. 5 Effect of siRNA targeted against MKK4 on myostatin-induced downregulation of differentiation marker gene expressionBased on the good silencing efficiency of Ambion siRAN ID 64447 in differentiation condition, the MKK4-specific siRNA duplexes was used to continue further study. To evaluate the effect of MKK4 siRNA on myostatin-induced downregulation of differentiation marker gene expression, mouse C2C12 cells were transfected with siRNA duplexes targeted against MKK4 (Ambion siRNA ID 64447) or negative control siRNA. Cells were cultivated for another 48 h in DMEM/10%FBS medium, followed by myostatin (50 ng/mL) stimulation for another 48 h in DMEM/2% horse serum medium. Cell lysates were immunoblotted with multiple antibodies. Western blot analysis showed that the protein levels of myogenin and MyoD in 48 h of myostatin stimulation reduced approximately 82.54% and 90.02%, respectively, compared to the untreated control. The reductions of myogenin and MyoD in protein levels were rescued when the MKK4 expression was significantly knocked down by MKK4 siRNA, with a 40.42% reduction of myogenin protein level and a 24.38% reduction of MyoD protein level. On the one hand, this work further demonstrate that JNK signaling pathway plays an important role in myostatin-regulated differentiation inhibition of myoblast, and on the other hand this work may lead to new strategies for blocking myostatin activity.In summary, this study provided the first evidence for the involvement of JNK pathway in myostatin signaling. Firstly, this study demonstrated that myostatin could activate JNK signaling pathway in both proliferating and differentiating C2C12 cells. Secondly, this study demonstrated that ActRIIB and TAK1-MKK4 cascade were involved in myostatin-induced JNK activation. Finally, the activation of JNK signaling pathway was demonstrated to participate in myostatin's function as a negative regulator of muscle growth. Furthermore, the present study provided evidence that siRNA targeted against MKK4 attenuates myostatin-induced downregulation of differentiation marker gene expression, which may lead to new strategies for blocking myostatin activity.