Cell Proliferation, Differentiation, Apoptosis Under A Serial Stretches And The Underlying Mechanisms

Mechanical force is essential for orthodontics, in which the adjustment of the soft tissue, especially the muscle is the key to the treatment. During the treatment, Mechanical forces induce changes in the structure, composition, and function of tissues such as bone, vessel and muscle. Molecularly, the mechanical force signal is transduced into the cell, resulting in the cell proliferation, differentiation and apoptosis, which is vital for the adjustment.Recently, responses to stretch have been intensely studied in load-sensitive cells, which include fibroblasts, osteoblasts, endothelial cells, smooth muscle cells and skeletal muscle cells. Apoptosis has been found to be induced in myocytes by inappropriate mechanical stimuli under pathological conditions or during clinical treatments. Although, apoptosis is intensively studied in muscle tissue and muscle cells, most of those studies just focus on muscle cell apoptosis in atrophy. The mechanisms responsible for muscle cell apoptosis under stretches are rarely studied.ROS is abundantly induced in muscle cells by stretch which is one of the key factors responsible for apoptosis and related muscle damages. Up to now, how ROS regulate JNK and NFkappaB discriminately and coordinately is poorly understood. Since there should be a coordinated regulation of these two pathways, it is of great value to elucidate how ROS regulate the crosstalk between NFkappaB and JNK pathway under the mechanical forces.[Aims] The present study explores the effects of different mechanical forces on cellfunction, and the dynamic changes of ROS under different mechanical forces and how they are involved in determination of myoblast fate and integration of both NFkappaB and JNK signal pathway.[Methods and results](1) Correlation between cyclic stretch magnitudes and cell fate.To study the detailed effects of the cyclic stretches of a serial of magnitudes on cell growth and differentiation, we first evaluated cell viability upon serial cyclic stretches (0%, 5%, 10%, 15%, 20% /10 cycles / min). When the extension magnitude is about 5%, cell growth was facilitated. In contrast, myogenic medium induced cell differentiation under this condition was inhibited as seen by myogenin expression. With the extension magnitudes exceeding 15% 10cycles/min, cell viability decreased. Since both proliferation and cell death affected the MTT results, we then tested whether apoptosis was involved in the process. To this end, DNA fragmentation analysis was applied. Consistent with the decreased cell viability, we observed obvious DNA fragmentation under cyclic stretches of over 10% extension, suggesting apoptosis contributed at least partially to the observed cell viability. In addition, we analyzed the cleavage of PARP, another marker of apoptosis, which has been shown in muscle cell apoptosis. Consistently, abundant PARP cleavage was observed when the cyclic stretches were over 15%.(2) Relationship among ROS production, NFkappaB activity and JNK1 activation.Considering the paradoxical roles of JNK and NFkappaB in ROS induced apoptosis, we examined ROS generation, NFkappaB activity and phosphorylated JNK1 levels under serial stretches. With the stretch magnitudes increased, consistent with the increased apoptosis, JNK1 were gradually activated, with no obvious changes of JNK1 expression. In contrast, NFkappaB activity increased with the extension rate increasing within the magnitudes no more than 10%, and it began to decline when the extension rate is over 15%. To confirm the relationship between ROS and JNK activation, we pre-treated cells with ROS scavenger NAC at serial concentrations 4 hrs before cells undergoing a 24 hrs of 20%/10cycles/min stretch. ROS production was significantly inhibited by NAC treatment, with the ROS level decreased to nearly the baseline when the NAC concentration was 100μM. 10μM NAC treatment began to inhibit the JNK activation, and with the NAC concentration increased, JNK activation decreased gradually. In contrast, 10μM NAC treatment increased the activity of NFkappaB. With the NAC concentration higher, NFkappaB activity began to decline from the climax, and became lower than the baseline when NAC concentration was 1000μM. All of these suggested that JNK activation occurred only when ROS production accumulated sufficiently while NFkappaB was activated at a much lower threshold of ROS abundance.(3) Low magnitude of cyclic stretch increased the expression of miR-146a in differentiating C2C12 cellsFrom the above data, we deduce that low magnitude of cyclic stretch may affect cell differentiation. To this end, we examined cyclic stretch effects on differentiation. Remarkably, cyclic stretch reduced the expression of myogenin, an indicator of differentiation. Then, we examined the expression level of miR-146a, which is a known target of NFkappaB, by miRNA qRT-PCR. Strikingly, compared with the control group, miR-146a in cyclic stretch treated group was nearly 4 folds higher. miR-146a inhibited Numb expression through its 3'UTR. To elucidate the potential role of miR-146a in muscle differentiation and proliferation, putative targets of this miRNA were predicted by the online tool Targetscan 4.2. There are nearly 200 potential targets of miR-146a, among which Numb has been reported to be involved in myogenesis. To confirm the regulation of Numb by miR-146a, we then analyzed the expression of Numb in differentiating C2C12 cells under cyclic stretch. Consistent with the increase of miR-146a, we found a decline of Numb protein level after cyclic stretch. Furthermore, transfection of siRNA-like miR-146a also repressed the expression of Numb in C2C12 cells.(4) Under high mechanical forces, JNK1 activation contributed to the NFkappaB inhibition upon excessive ROS generation.Usually, ROS are the inducer of NFkappaB activation. Strikingly, in contrast to the continual increase of activated JNK1, here we did not observe a steady increase of NFkappaB activity with ROS gradual accumulation. NFkappaB activity began to decline from the climax when the magnitudes of stretch exceeding 15%. The reverse relationship between NFkappaB and JNK at intensive stretches prompted us to assume that JNK activation might contribute to the decline of NFkappaB activity. To this end, we pretreated cells with a specific JNK chemical inhibitor or specific RNAi 24 hrs before cells undergoing a 24hrs of 20% cyclic stretch. Using the RNAi technique, we efficiently reduced the expression of JNK1 to about 30%. Compared with the control, inhibition of JNK signal increased the NFkappaB activity, and consistently cell viability increased. To confirm whether inhibition of NFkappaB activity was critical for the cell death induced by JNK activation, we further blocked the activation of NFkappaB by its inhibitor aspirin in combination with JNK inhibition. 5mM aspirin nearly blocked the activation of NFkappaB as indicated by the luciferase assay. Upon 20% stretch, compared with the group treated with JNK inhibitor only, combination of JNK inhibitor and NFkappaB inhibitor decreased the cell viability significantly, suggesting that NFkappaB inhibition played an important role in JNK activation induced cell death.(5) Under high mechanical forces, JNK1 activation blocked the nuclear translocation of NFkappaB and the transcription of Bcl2.Since nuclear translocation and DNA binding ability are the two main factors affecting NFkappaB transcriptional activity, we firstly analyzed the nuclear levels of p65 under different conditions. Nuclear p65 was rarely detectable upon 20% extension stretch. In contrast, blockade of JNK activity by RNAi or chemical inhibitor rescued the nuclear localized p65. Furthermore, we observed the expression of Bcl2, which is one of the most important anti-apoptotic target genes of NFkappaB. Upon 20% extension stretch, consistent with the increase of nuclear NFkappaB by JNK blockade, Bcl2 expression was upregulated. Furthermore, we could see that the chemical inhibitor of JNK functioned stronger than JNK1 siRNA, which might be explained by the fact that other JNK isoforms were inhibited simultaneously by the chemical inhibitor or alternatively the knockdown efficiency in our study was compromised. Using the cyclic stretch model, we observed that ROS generation accumulated progressively and cell death occurred under excessive stretches. NFkappaB transcriptional activity increased when there was minor to moderate ROS production, and began to decline when ROS generation continued. In contrast, obvious JNK activation just occurred when ROS generation was abundant enough under severe stretches. Through blocking JNK activation by a specific chemical inhibitor or JNK1 specific RNAi, we concluded that JNK activation was responsible for the inhibition of NFkappaB activity, which subsequently functioned importantly in apoptosis.【Conclusion】In this study, we serially studied the effects of cyclic stretch on cell proliferation, differentiation and apoptosis. We found that under low magnitude mechanical forces, cell growth was facilitated while cell differentiation was inhibited. In this process, NFkappaB activation and miR146a expression functions importantly. Under high magnitude mechanical forces, increased ROS induced by mechanical forces activates JNK1, which in turn repress NFkappaB and its target Bcl2, resulting in cell apoptosis. Our observation here may facilitate our manipulation of mechanical forces in Sports Medicine and Ortho Medicine.