TNFR2 knockdown triggers apoptosis-induced proliferation in primarily cultured Schwann cells
Introduction
Schwann cells, which form the myelin sheath of axons, play a key role in the conduction of action potentials and the maintenance of axonal transportation (Tian et al., 2016; Wang et al., 2012). After sciatic nerve injury, Schwann cells undergo significant proliferation in the distal segments to form bands of Bungner, which promote axon regeneration (Liang et al., 2012; Zhou et al., 2014). However, previous studies demonstrated that Schwann cells in the distal segments also occurred massive apoptosis (Boyle et al., 2005; Frost et al., 2016). It is not clear whether the proliferation of Schwann cells in the distal segments is related to the cell apoptosis.
Following massive stress-induced apoptosis, surviving cells undergo compensatory proliferation to maintain tissue homeostasis, which is termed apoptosis-induced proliferation (AiP) (Fogarty et al., 2016). This phenomenon was firstly described in Drosophila imaginal disc cells, which could undergo compensatory proliferation and thereby repair tissue damage even after a 60% loss of its cells by X-ray radiation (Haynie and Bryant, 1977). Such compensatory proliferation was also been demonstrated in other regenerative models, including hydra, planarians, zebrafish, and xenopus (Ryoo and Bergmann, 2012). In mammals, apoptotic myoblasts of mice trigger AiP to promote the fusion of normal myoblasts, which plays a key role in muscle development and repair (Hochreiter-Hufford et al., 2013). AiP can also promote proliferation of tumor cells and contribute to human tumor repopulation after cancer radiotherapy (Huang et al., 2011). Thus, we wonder whether AiP is responsible for the proliferation of Schwann cells in the distal segments from the sciatic nerve injury site.
Tumor necrosis factor-α (TNF-α), which binds to either TNF receptor 1 (TNFR1) or receptor 2 (TNFR2), is a pro-inflammatory cytokine involved in multiple biological responses including inflammatory reaction, cell death and proliferation. TNFR1 is expressed in most cells, while the expression of TNFR2 is limited to few cell types, including Schwann cells (Montgomery and Bowers, 2012). In this study, we tried to verify the hypothesis that apoptotic Schwann cells could promote the cell proliferation and figure out the role of TNF-α receptors in this phenomenon. Considering the co-expression of TNFR1/2 and self-secretion of TNF-α in Schwann cells (Yuan et al., 2012), we utilized small interfering RNA (siRNA) to transfect the primary cultured Schwann cells, and knock down the expression of TNFR1 and TNFR2, respectively. Then we evaluated the apoptosis rate and proliferation rate of Schwann cells. In addition, we established the sciatic nerve injury model on wild-type and TNF-α knock-out mice, and tried to confirm the role of TNF-α and TNFR in Schwann cell apoptosis and proliferation in vivo. We expect the finding of AiP in Schwann cells may be beneficial to develop more approaches to promote axon regeneration and improve the functional recovery after peripheral nerve injury.
Section snippets
Primary culture of Schwann cells and siRNA transfection
Sciatic nerves of newborn Sprague-Dawley rats were harvested to isolate Schwann cells, which were further purified with anti-Thy1.1 (Sigma, St Louis, MO) and rabbit complement (Invitrogen, Carlsbad, CA) to remove the fibroblasts. As described in previous study (Zhou et al., 2014), almost all the cells in the final preparation were Schwann cells, which was confirmed by anti-S100b immunostaining. Dulbecco’s modified Eagle’s medium (DMEM) mixed with 10% fetal bovine serum (Invitrogen) at 37 °C was
Knockdown of TNFR1 and TNFR2, respectively
To study the possible mechanisms of AiP in the Schwann cells, we first prepared primary Schwann cell cultures with an extremely high purity (about 98%), which was revealed by S100 labeling (Fig. 1A). To determine the possible roles of TNFR1 and TNFR2 in AiP, we designed three siRNAs specific for TNFR1 or TNFR2, respectively. qRT-PCR results showed all three siRNAs remarkably reduced the mRNA expression of either TNFR1 or TNFR2, when compared with control groups (Fig. 1B, 1C). The differences of
Discussion
Recent studies have identified AiP in various models, including hydra, planarians, zebrafish, xenopus, mouse and humans (Ryoo and Bergmann, 2012). AiP can not only promote tissue regeneration but also contribute to tumor repopulation. After sciatic nerve injury, Schwann cells in the distal segments can both undergo apoptosis and proliferation, the correlation between the two processes hasn’t been determined. The present study verified the Schwann cell apoptosis could induce the cell
Conclusions
Our findings suggest that Schwann cells can undergo compensatory proliferation after massive cell apoptosis, which is achieved by TNFR2 knockdown. We also found that the Schwann cells of TNF-α knock-out mice occurred much less apoptosis and proliferation than that of wild-type mice in the distal segments of injury site, which indicating that TNF-α and TNFR were essential in the massive apoptosis and the apoptosis-induced proliferation of Schwann cells after sciatic nerve injury. The phenomenon
Author contributions
Z.G. and C.M., designed and performed most of the experiments, analyzed all data, prepared figures and wrote the manuscript; J.Q. and H.X. performed part of the experiments; X.H. and S.Z. contributed to experimental design, data interpretation, and writing the paper. All authors read and approved the final manuscript.
Fundings
This study was supported by the National Key Basic Research Program of China, No. 2017YFA0104700; the National Natural Science Foundation of China, No. 31730031, 81571198, 81671230, 81571209 and 81870975; the Science and Technology Planning Project of Nantong (MS12017023-9).
Conflict of interest
The authors declare no competing interests.
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These authors contributed equally.