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Runx2, also known as Cbfa1, is a multifunctional transcription factor essential for osteoblast differentiation. It also plays major roles in chondrocyte maturation, mesenchymal stem cell differentiation, cleidocranial dysplasia, and the growth and metastasis of tumors. The present study was performed to investigate the functions of Runx2 in the differentiation and migration of Schwann cells and outgrowth of neurites after peripheral nervous system injury. In a model of sciatic nerve crush (SNC) injury, we found a gradual increase in the expression of Runx2, which reached a peak after 1 week. Immunofluorescence revealed increased expression of Runx2 in Schwann cells and axons after SNC injury. Runx2 and Oct-6 expression trends were consistent with each other in western blotting, and colocalization of Runx2 and Oct-6 was observed in immunofluorescence. In vitro, Runx2 promoted Schwann cell differentiation by activation of the Akt-GSK3β signaling pathway. In addition, Runx2 promoted the migration of Schwann cells and outgrowth of neurites. These findings suggest that Runx2 may be involved in neurite outgrowth and Schwann cell differentiation and migration after sciatic nerve injury.

Cell division cycle protein 37 (Cdc37), a molecular chaperone takes part in a series of cellular processes including cell signal transduction, cell cycle progression, cell proliferation, cell motility, oncogenesis and malignant progression. It can not only recruit immature protein kinases to HSP90 but also work alone. Cdc37 was reported to be associated with neurogenesis, neurite outgrowth, axon guidance and myelination. However, the roles of Cdc37 on Schwann cells (SC) after peripheral nerve injury (PNI) remain unknown. In this study, we found that the expression of Cdc37 increased and reached the peak at 1 week after sciatic nerve crush (SNC), which was consistent with that of proliferation cell nuclear antigen. Immunofluorescence verified that Cdc37 co-localized with SC in vivo and in vitro. Intriguingly, Cdc37 protein level was potentiated in the model of TNF-α-induced SC proliferation. Moreover, we found that Cdc37 silencing impaired proliferation of SC in vitro. Moreover, Cdc37 suppression attenuated kinase signaling pathways of Raf-ERK and PI3K/AKT which are crucial cell signaling for SC proliferation. Finally, we found that Cdc37 silencing inhibited SC migration in vitro. In conclusion, we demonstrated that the way Cdc37 contributed to SC proliferation is likely via activating kinase signaling pathways of Raf-ERK and PI3K/AKT, and CDC37 was also involved in SC migration after SNC.

Tripartite motif containing 32 (TRIM32), a member of the tripartite motif (TRIM) family, plays an indispensable role in myoblast proliferation. It also regulates neuron and skeletal muscle stem cell differentiation. Although it is of great importance, we know little about the roles of TRIM32 during peripheral nervous system injury. Here, we examined the dynamic changes of TRIM32 in acute sciatic nerve crush (SNC) model. After crush, TRIM32 rapidly increased and reached the climax at 1 week but then gradually declined to the normal level at 4 weeks post-injury. Meanwhile, we observed similar changes of Oct-6. What is more, we found co-localization of TRIM32 with S100 and Oct-6 in 1-week-injured tissues using double immunofluorescent staining. In further vitro experiments, enhancive expression of TRIM32 was detected during the process of cyclic adenosine monophosphate (cAMP)-induced Schwann cell differentiation and nerve growth factor (NGF)-induced PC12 cell neurite outgrowth. More interestingly, specific si-TRIM32-transfected RSC96 cells exhibited obvious reduction in the ability of migration. Taken together, we inferred that upregulated TRIM32 was not only involved in the differentiation and migration of Schwann cells but the neurite elongation after SNC.

Astrocyte-elevated gene-1 (AEG-1), also known as metadherin (MTDH) and lysine-rich CEACAM1 coisolated (LYRIC), has emerged as an important oncogene that regulates key cellular processes including apoptosis, migration, invasion, proliferation, and differentiation. It was reported that AEG-1 enhanced breast cancer cells migration in a NF-κB-dependent manner. Also, AEG-1 contributed cell proliferation through the PI3K-Akt/cyclin pathway. Besides, AEG-1 is implicated in diverse physiological and pathological diseases of the central nervous system (CNS), such as brain tumors, neuroblastomas, neurodegeneration, and neuronal development. However, the role of AEG-1 in the process of peripheral nervous regeneration after injury remains virtually unknown. In this study, we investigated the spatiotemporal expression of AEG-1 in a rat sciatic nerve crush model. At its peak expression, AEG-1 was expressed mainly in Schwann cells of the distal sciatic nerve segment from injury, but had few colocalizations in axons. Besides, the peak expression of AEG-1 was in parallel with proliferating cell nuclear antigen (PCNA). In vitro, we detected the increased expression of AEG-1 during the process of tumor necrosis factor α (TNF-α)-induced Schwann cell proliferation. Meanwhile, interference of AEG-1 inhibited both proliferation and migration of Schwann cells. In conclusion, we speculated that AEG-1 is involved in biochemical and physiological responses after sciatic nerve crush (SNC).

Dixdc1 (DIX domain containing-1), the mammalian homolog of Ccd1 (Coiled-coil-Dishevelled-Axin1), is a protein containing a coiled-coil domain and a Dishevelled-Axin (DIX) domain. As a novel component of the Wnt pathway, Dixdc1 has been reported to be able to promote neural progenitor proliferation and neuronal differentiation via Wnt/β-catenin signaling. But there still remains something unknown about Dixdc1 distribution and functions in the lesion and regeneration of the peripheral nervous system (PNS), so we tried to investigate dynamic changes of Dixdc1 expression in a rat sciatic nerve crush (SNC) model in this study. First of all, we detected SNC-induced increased levels of Dixdc1 in Schwann cells and interestingly identified parallel expression of PCNA (proliferation cell nuclear antigen) with Dixdc1. Besides, we observed up-regulated Dixdc1 during the process of TNF-α-induced Schwann cell proliferation. Also, we discovered that Dixdc1 could promote G1-S phase transition accompanied with the up-regulation of CyclinD1 and down-regulation of p21. More importantly, enhanced effects of Dixdc1 on cell proliferation were confirmed to be associated with PI3K activation. Not only blocking of the PI3K but Dixdc1 knockdown led to significantly decreased ability for proliferation, as well as down-regulation of CyclinD1 and up-regulation of p21. In summary, these data demonstrated that Dixdc1 might participate in Schwann cell proliferation by targeting CyclinD1 and p21 at least partially through the PI3K/AKT activation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Poly(C)-binding proteins (PCBPs), also known as RNA-binding proteins, interact in a sequence-specific fashion with single-stranded poly(C). It was reported that PCBP2 contributed to gastric cancer proliferation and survival through miR-34a, and knockdown of PCBP2 inhibited glioma proliferation through inhibition of cell cycle progression. In addition, PCBP2 might play a critical role in the regulation of cortical neurons apoptosis induced by hypoxia or ischemia. Because of the essential role of PCBP2 in nervous system and cell growth, we investigated the spatiotemporal expression of PCBP2 in a rat sciatic nerve crush (SNC) model. We detected the upregulated expression of PCBP2 in Schwann cell after SNC. Besides, the peak expression of PCBP2 was in parallel with proliferation cell nuclear antigen. In vitro, we observed increased expression of PCBP2 during the process of TNF-α-induced Schwann cell proliferation. Specially, PCBP2-specific siRNA-transfected Schwann cell showed significantly decreased ability for proliferation. Together, all these data indicated that the change of PCBP2 protein expression was associated with Schwann cell proliferation after the trauma of the peripheral nervous system.

Sam68 (Src-associated in mitosis of 68 kD), a KH domain RNA-binding protein, is not only important in signaling transduction cascades, but crucial in a variety of cellular processes. Sam68 is reported to be involved in the phospoinositide3-kinase (PI3K) and nuclear factor-kappa B (NF-κB) signaling pathways, and it is closely associated with cell proliferation, RNA metabolism, and tumor progression. However, we know little about the role of Sam68 during peripheral nervous system injury and regeneration. In this study, we investigated the expression of Sam68 and its biological significances in sciatic nerve crush. Interestingly, we found Sam68 had a co-localization with S100 (Schwann cell marker). Moreover, after crush, Sam68 had a spatiotemporal protein expression, which was in parallel with proliferation cell nuclear antigen (PCNA). In vitro, we also observed increased expression of Sam68 during the process of TNF-α-induced Schwann cell proliferation model. Besides, flow cytometry analyses, CCK-8, and EDU were all performed with the purpose of investigating the role of Sam68 in the regulation of Schwann cell proliferation. Even more importantly, we discovered that Sam68 could enhance the phosphorylation of Akt while LY294002 (a PI3K inhibitor) obviously reversed Sam68-induced cell proliferation. Finally, we detected the variance during regeneration progress through the rat walk footprint test. In summary, all these evidences demonstrated that Sam68 might participate in Schwann cell proliferation partially via PI3K/Akt pathway and also regulate regeneration after sciatic nerve crush., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Nur77, together with Nurr1 and NOR-1, constitutes the NR4A subgroup of orphan nuclear receptors and plays critical roles in cell proliferation, differentiation, migration, and apoptosis. Among them, Nur77 is universally well known to contribute to neurite outgrowth. However, information regarding its regulation and possible function in the peripheral nervous system is still limited. In this study, we performed a sciatic nerve injury model in adult rats and detected an increased expression of Nur77 in the sciatic nerve, which was similar to the expression of Oct-6. Immunofluorescence indicated that Nur77 was located in both axons and Schwann cells. In vitro, we observed enhanced expression of Nur77 during the process of both basic fibroblast growth factor (bFGF)-induced Schwann cells differentiation and nerve growth factor (NGF)-induced PC12 cell neurite outgrowth. In vitro and in vivo experiments indicated that inhibiting the function of Nur77 by specific short hairpin RNA could depress Schwann cells myelinization and axons regeneration. Collectively, all these results suggested that upregulation of Nur77 might be involved in Schwann cells differentiation and neurite elongation following sciatic nerve crush.

Nuclear factor (NF)45 (also known as interleukin enhancer-binding factor (ILF)2), is a transcription factor that interacts with NF90 to regulate gene expression. It has long been implicated in the regulation of cell proliferation. However, the role of NF45 in the process of peripheral nervous system regeneration after injury remains poorly understood. Herein, we investigated the spatiotemporal expression of NF45 in a rat sciatic nerve crush model. We detected the up-regulated expression of NF45 in Schwann cell after sciatic nerve crush. What's more, the expression of the cell proliferation marker proliferating cell nuclear antigen (PCNA) exhibited a similar tendency with that of NF45. In cell cultures, we observed increased expression of NF45 during the process of TNF-α-induced Schwann cell proliferation, whereas the protein level of p21 was down-regulated. Interference of NF45 led to enhanced expression of p21 and also impaired proliferation of Schwan cells. Taken together, our data implicated that NF45 was up-regulated in the sciatic nerve after crush, which was associated with proliferation of Schwann cell.

Ebp1, an ErbB3-binding protein, is the human homologue of the cell cycle-regulated mouse protein p38-2G4. Ebp1 was reported to inhibit the proliferation and induce the differentiation of human cancer cells. Its p48 isoform contributes to neuronal differentiation and growth factor specificity. However, the expression and role of Ebp1 in peripheral system lesions and repair are still unknown. Herein, we investigated the spatiotemporal pattern of Ebp1 expression following sciatic nerve crush. After crush, the level of Ebp1 protein was elevated gradually, peaked at day 5, and then declined to the normal at 4 weeks, which was similar to the expression of Oct-6. Furthermore, using double immunofluorescent staining, we found Ebp1 had a colocalization with S100 and Oct-6 in 5-day injured tissues. In vitro, we observed enhanced expression of Ebp1 during the process of cyclic adenosine monophosphate (cAMP)-induced Schwann cells differentiation. Interestingly, Ebp1-depleted SCs did not show significant morphologic change after the treatment of cAMP. Also, we observed a colocalization between Ebp1 and Cyclin D1 and that Ebp1-specific siRNA-transfected SCs had a decreased migration. Taken together, we speculated that Ebp1 was upregulated in the sciatic nerve after crush, which was involved in the differentiation and migration of Schwann cells.

Histone deacetylase 4 (HDAC4), a member of the class IIa HDACs subfamily, has emerged as a critical regulator of cell growth, differentiation, and migration in various cell types. It was reported that HDAC4 stimulated colon cell proliferation via repression of p21. Also, HDAC4 contributes to platelet-derived growth factor-BB-induced proliferation and migration of vascular smooth muscle cells. Furthermore, HDAC4 may play an important role in the regulation of neuronal differentiation and survival. However, the role of HDAC4 in the process of peripheral nervous system regeneration after injury remains virtually unknown. Herein, we investigated the spatiotemporal expression of HDAC4 in a rat sciatic nerve crush model. We found that sciatic nerve crush induced up-regulated expression of HDAC4 in Schwann cells. Moreover, the expression of the proliferation marker Ki-67 exhibited a similar tendency with that of HDAC4. In cell cultures, we observed increased expression of HDAC4 during the process of TNF-α-induced Schwann cell proliferation, whereas the protein level of p21 was down-regulated. Interference of HDAC4 led to enhanced expression of p21 and impaired proliferation of Schwan cells. Taken together, our findings implicated that HDAC4 was up-regulated in the sciatic nerve after crush, which was associated with proliferation of Schwann cells.

Ski-interacting protein (SKIP) is a highly conserved protein from yeast to Human. As an essential spliceosomal component and transcriptional co-regulator it plays an important role in preinitiation, splicing and polyadenylation. SKIP can also combine with Ski to overcome the G1 arrest and the growth-suppressive activities of pRb. Furthermore SKIP has the capacity to augment TGF-β dependent transcription. While the distribution and function of SKIP in peripheral nervous system lesion and regeneration remain unclear. Here, we investigated the spatiotemporal expression of SKIP in an acute sciatic nerve crush model in adult rats. Western Blot analysis revealed that SKIP was expressed in normal sciatic nerves. It gradually increased, reached a peak at 1 week after crush, and then returned to the normal level at 4 weeks. Besides, we observed that up-regulation of SKIP was approximately in parallel with Proliferating cell nuclear antigen (PCNA), and numerous Schwann cells (SCs) expressing SKIP were PCNA and Ki-67 positive. Collectively, we hypothesized peripheral nerve crush induced up-regulation of SKIP in the sciatic nerve, which was associated with SCs proliferation.

Bcl-2-associated athanogene-1 (BAG1), a co-chaperone for Hsp70/Hsc70, is a multifunctional protein, which has been shown to suppress apoptosis and enhance neuronal differentiation. However, the expression and roles of BAG1 in peripheral system lesions and repair are still unknown. In this study, we investigated the dynamic changes in BAG1 expression in an acute sciatic nerve crush model in adult rats. Western blot analysis revealed that BAG1 was expressed in normal sciatic nerves. BAG1 expression increased progressively after sciatic nerve crush, reached a peak 2 weeks post-injury, and then returned to the normal level 4 weeks post-injury. Spatially, we observed that BAG1 was mainly expressed in Schwann cells and that BAG1 expression increased in Schwann cells after injury. In vitro, we found that BAG1 expression increased during the cyclic adenosine monophosphate (cAMP)-induced Schwann cell differentiation process. BAG1-specific siRNA inhibited cAMP-induced Schwann cell differentiation. In conclusion, we speculated that BAG1 was upregulated in the sciatic nerve after crush, which was associated with Schwann cell differentiation.

Transcription Initiation Factor IIB (TFIIB), as a general transcription factor, plays an essential role in preinitiation complex assembly and transcription initiation by recruiting RNA polymerase II to the promoter. However, its distribution and function in peripheral system lesion and repair were still unknown. Here, we investigated the spatiotemporal expression of TFIIB in an acute sciatic nerve crush model in adult rats. Western blot analysis revealed that TFIIB was expressed in normal sciatic nerve. It gradually increased, reached a peak at the seventh day after crush, and then returned to the normal level at 4 weeks. We observed that TFIIB expressed mainly increased in Schwann cells and co-localized with Oct-6. In vitro, we induced Schwann cell differentiation with cyclic adenosine monophosphate (cAMP) and found that TFIIB expression was increased in the differentiated process. TFIIB-specific siRNA inhibited cAMP-induced Schwann cell morphological change and the expression of P0. Collectively, we hypothesized peripheral nerve crush-induced upregulation of TFIIB in the sciatic nerve was associated with Schwann cell differentiation.

Peroxisome proliferator-activated receptor-γ (PPAR-γ) has been found to play an essential role in cell proliferation, but whether it was involved in Schwann cells differentiation has never been studied. We have found in sciatic nerve injury that expression of PPAR-γ decreases mainly in Schwann cells, and it was also increased in differentiated Schwann cells. Further, activated PPAR-γ by the endogenous ligand 15 d-PGJ(2) increased expressions of PPAR-γ level and Schwann cell differentiation, and this effect may be protected by its antagonist GDW9662. These results indicate that PPAR-γ could promote Schwann cell differentiation, which plays an important role in peripheral nerve injury and regeneration.

Schwann cell precursors differentiating into a myelinating phenotype are critical for peripheral nerve development and regeneration. However, little is known about the underlying molecular mechanisms of Schwann cell differentiation. In the present study, we performed a cyclic adenosine monophosphate-induced Schwann cell differentiation model in vitro. Western blot analysis showed that p27(Kip1) expression was upregulated during the differentiation of Schwann cell, while the inhibition of p27(Kip1) expression by short hairpin RNA-mediated knockdown significantly abolished the expression of promyelinating markers and the alteration of cellular morphology. In addition, immunofluorescence revealed a decrease of p27(Kip1) nuclear staining and a concomitant increase of cytoplasmic staining in differentiated Schwann cells. In summary, our data indicated that p27(Kip1) was a positive regulator of Schwann cell differentiation in vitro.

Forkhead box-containing protein, class O 3 a (FOXO3a), an Akt downstream target, plays an important role in peripheral nervous system. FOXO3a shares the ability to be inhibited and translocated from the nucleus on phosphorylation by proteins such as Akt/PKB in the PI3K signaling pathway. To elucidate the expression and possible function of FOXO3a in lesion and repair, we performed an acute sciatic nerve crush model and studied differential expressions of FOXO3a. We observed that expressions of FOXO3a in Schwann cells (SCs) of the peripheral nervous system and cAMP-induced differentiation were dynamically regulated. Western blot analysis showed FOXO3a level significantly decreased post injury. Moreover, Immunofluorescence double labeling suggested the changes were striking especially in SCs. In vitro, Western blot analysis showed that the expression of FOXO3a was decreased in cAMP-induced differentiated primary SCs. The FOXO3a siRNA-transfected SCs treated by cAMP promote differentiation of SCs through the PI3K/Akt pathway. The results indicate that FOXO3a plays an important role during differentiation of SCs.

Tumor necrosis factor-alpha (TNF-alpha) derived from activated Schwann cells (SCs) plays a critical role as an inflammatory mediator in the peripheral nervous system disease. TNF-alpha could act as an autocrine mediator in SC activation. In this study, we found knockdown Src-suppressed protein kinase C substrate (SSeCKS) expression suppressed TNF-alpha production induced by TNF-alpha, overexpression of SSeCKS could promoted TNF-alpha autocrine in SCs. Such effects might be resulted in SSeCKS promoted p38 and JNK activation in SCs treated by TNF-alpha. Thus present data show that while SCs activation, SSeCKS may plays an important role in the release of inflammatory mediators.

Src-suppressed protein kinase C substrate (SSeCKS) plays an important role in the differentiation process. In regeneration of sciatic nerve injury, expression of SSeCKS decreases, mainly in Schwann cells. However, the function of SSeCKS in Schwann cells differentiation remains unclear. We observed that SSeCKS was decreased in differentiated Schwann cells. In long-term SSeCKS-reduced Schwann cells, cell morphology changed and myelin gene expression induced by cAMP was accelerated. Myelination was also enhanced in SSeCKS-suppressed Schwann cells co-culture with dorsal root ganglion (DRG). In addition, we found suppression of SSeCKS expression promoted Akt serine 473 phosphorylation in cAMP-treated Schwann cells. In summary, our data indicated that SSeCKS was a negative regulator of myelinating glia differentiation.