Induce Schwann Cell Proliferation Research Articles

Melatonin Induced Schwann Cell Proliferation and Dedifferentiation Through NF-ĸB, FAK-Dependent but Src-Independent Pathways.

Peripheral nerve injury (PNI) is a common condition that compromises motor and sensory functions. Peripheral nerves are known to have regenerative capability and the pineal hormone, melatonin, is known to aid nerve regeneration. However, the role of Schwann cells and the pathways involved remain unclear. Thus, the aim of this study is to identify the effects of melatonin on Schwann cell proliferation, dedifferentiation, and the involvement of nuclear factor kappa light chain enhancer of activated B cells (NF-ĸB), focal adhesion kinase (FAK) and proto-oncogene tyrosine-protein kinase, Src pathways in this process. Schwann cells was treated with melatonin and its proliferation and dedifferentiation were identified using MTT assay and immunofluorescence staining for SRY (sex determining region Y)-box 2 (SOX2). Next, the protein expressions of NF-ĸB, FAK and Src pathways were identified by Western blot. MTT results confirmed increased proliferation of Schwann cells with melatonin treatment, and it was highest at 10 μM melatonin. Immunofluorescent staining revealed an increase in the green fluorescence staining for SOX2 in melatonin-treated cells, showing enhanced dedifferentiation. Western blot assay revealed melatonin increased phospho-NF-ĸB (PNF-ĸB), IKK-α, FAK (D2R2E), phospho-FAK (Tyr 576/577 and Tyr 397) protein expressions as compared with control. However, Src (32G6), Lyn (C13F9), Fyn, Csk (C74C1) protein expressions were not increased as compared with control. Melatonin promotes Schwann cell proliferation and dedifferentiation via NF-ĸB, FAK-dependent but Src-independent pathways.

Evaluation of Epigallocatechin-3-gallate Modified Collagen Membrane and Concerns on Schwann Cells.

Collagen is an essential component of the extracellular matrix (ECM) and is a suitable material for nerve repair during tissue remodeling for fracture repair. Epigallocatechin-3-gallate (EGCG), an extract of green tea, shows various biological activities that are beneficial to nerve repair. Here, we developed modified collagen containing different concentrations of EGCG (0.0064%, 0.064%, and 0.64%, resp.) to induce Schwann cell proliferation and differentiation. Cell Counting Kit-8 test, live/dead assay, and SEM showed that collagen cross-linked by EGCG induced Schwann cell proliferation. Real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blotting revealed that EGCG-modified collagen induced Schwann cell differentiation and downregulated reactive oxygen species (ROS) levels by downregulating the MAPK P38 signaling pathway. Our results indicate that collagen cross-linked with an appropriate concentration of EGCG induces the proliferation and differentiation of Schwann cells. The EGCG-modified collagen membrane may be applicable for nerve repair and guided tissue regeneration applications.

Upregulation of AEG-1 Involves in Schwann Cell Proliferation and Migration After Sciatic Nerve Crush.

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).

Neuroblastoma in dialog with its stroma: NTRK1 is a regulator of cellular cross-talk with Schwann cells.

In neuroblastoma, the most common solid tumor of childhood, excellent prognosis is associated with extensive Schwann cell (SC) content and high-level expression of the neurotrophin receptor, NTRK1/TrkA, which is known to mediate neuroblastoma cell differentiation. We hypothesized that both stromal composition and neuroblastic differentiation are based on bidirectional neuroblastoma-SC interaction. Reanalysis of microarray data from human SY5Y neuroblastoma cells stably transfected with either NTRK1 or NTRK2 revealed upregulation of the mRNA for the SC growth factor, NRG1, in NTRK1-positive cells. Media conditioned by NTRK1-expressing neuroblastoma cells induced SC proliferation and migration, while antibody-based NRG1 neutralization significantly decreased these effects. Vice versa, NRG1-stimulated SC secreted the NTRK1-specific ligand, NGF. SC-conditioned medium activated the NTRK1 receptor in a neuroblastoma cell culture model conditionally expressing NTRK1 and induced differentiation markers in NTRK1-expressing cells. NTRK1 induction in neuroblastoma xenografts mixed with primary SC also significantly reduced tumor growth in vivo. We propose a model for NTRK1-mediated and NRG1-dependent attraction of adjacent SC, which in turn induce neuroblastic differentiation by secretion of the NTRK1-specific ligand, NGF. These findings have implications for understanding the mature and less malignant neuroblastoma phenotype associated with NTRK1 expression, and could assist the development of new therapeutic strategies for neuroblastoma differentiation.

Effect of carboxymethylated chitosan on the biosynthesis of NGF and activation of the Wnt/β-catenin signaling pathway in the proliferation of Schwann cells

The proliferation of Schwann cells around injured peripheral nerves supports the process of Wallerian degeneration and is critical for axonal regeneration. In this publication, carboxymethylated chitosan (CMCS) was studied to determine its capacity (i) to induce proliferation and secretion of nerve growth factor (NGF) and (ii) to activate Wingless-type(Wnt) protein/β-catenin signaling pathways in rat Schwann cells. CMCS was found to induce Schwann cell proliferation and NGF synthesis in Schwann cell in a dose and time dependent manner. CMCS was shown to activate factors in the Wnt/β-catenin signaling pathway, including Dvl-1, β-catenin, Tcf4, Lef1, C-myc, and Cyclin D1 which are active in the proliferation of Schwann cells and biosynthesis of NGF of Schwann cell. Overall, this study suggests that CMCS can promote the proliferation of cultured Schwann cells and synthesis of NGF by activating the Wnt/β-catenin signaling pathway.

Neuron Regeneration and Proliferation Effects of Danshen and Tanshinone IIA

This study evaluates the proliferative effects of danshen and its monomer extract, tanshinone IIA, on Schwann cell proliferation. A piece of silicone rubber was guided across a 15-mm gap in the sciatic nerve of a rat. This nerve gap was then filled with different concentrations of danshen (0–100 mg/mL). The results showed that danshen increased the expressions of uPA, cyclin D1, E and ERK, JNK, and P38 MAP kinases via the FGF-2 signaling pathway in a dose-dependent manner. RSC96, Schwann cells were also administered with danshen (0, 20, 40, 60, 80, and 100 μg/mL) and tanshinone IIA (0, 2, 4, 6, 8, and 10 μg/mL). In lower concentrations, danshen and tanshinone IIA exhibited an apparent effect on Schwann cells. Similar effects were also demonstrated in the FGF-2-uPA regulating cascade and cell cycle proliferative protein results. Schwann cell migration was elevated as well. We used MAPK-signaling chemical inhibitors and identified the proliferative effects of danshen and tanshinone IIA as MAPK-signaling dependent. The results from the in vitro systems indicate that danshen and tanshinone IIA can be used to induce Schwann cell proliferation, and in vivo results potentially suggest that danshen and tanshinone IIA might enhance neuron regeneration.

NF1 loss disrupts Schwann cell–axonal interactions: a novel role for semaphorin 4F

Neurofibromatosis type 1 (NF1) patients develop neurofibromas, tumors of Schwann cell origin, as a result of loss of the Ras-GAP neurofibromin. In normal nerves, Schwann cells are found tightly associated with axons, while loss of axonal contact is a frequent and important early event in neurofibroma development. However, the molecular basis of this physical interaction or how it is disrupted in cancer remains unclear. Here we show that loss of neurofibromin in Schwann cells is sufficient to disrupt Schwann cell/axonal interactions via up-regulation of the Ras/Raf/ERK signaling pathway. Importantly, we identify down-regulation of semaphorin 4F (Sema4F) as the molecular mechanism responsible for the Ras-mediated loss of interactions. In heterotypic cocultures, Sema4F knockdown induced Schwann cell proliferation by relieving axonal contact-inhibitory signals, providing a mechanism through which loss of axonal contact contributes to tumorigenesis. Importantly, Sema4F levels were strongly reduced in a panel of human neurofibromas, confirming the relevance of these findings to the human disease. This work identifies a novel role for the guidance-molecules semaphorins in the mediation of Schwann cell/axonal interactions, and provides a molecular mechanism by which heterotypic cell-cell contacts control cell proliferation and suppress tumorigenesis. Finally, it provides a new approach for the development of therapies for NF1.

Secretory products of central nervous system glial cells induce Schwann cell proliferation and protect from cytokine‐mediated death

There continues to be interest in Schwann cells (SC) as a possible source of myelinating cells for transplantation into the central nervous system (CNS) of patients with multiple sclerosis (MS) and spinal cord injury. It has been suggested that CNS glial cells interfere with SC migration, survival, maturation, and clinically significant remyelination in the CNS. To investigate the effects of CNS glial cells on SC, we examined the effects of serum-free supernatants obtained from rat mixed CNS glial cultures on rat neonatal SC cultures. Supernatants from 1-, 3-, and 5-day CNS glial cultures induced proliferation of SC assayed at 5 days in vitro but did not induce SC differentiation as measured by induction of surface expression of galactolipids (GalL). High concentrations of cAMP simulate many of the effects of axolemma on SC; CNS glial cell supernatants did not inhibit cAMP induction of SC differentiation. CNS glial cell supernatants had no apparent effect on SC viability at 48 hr as measured by trypan blue exclusion. We have previously demonstrated that incubation of SC with transforming growth factor-beta1 (TGF-beta1) + tumor necrosis factor-alpha (TNF-alpha) induces SC death via apoptosis. We now show that CNS glial supernatants inhibits TGF-beta1/TNF-alpha-induced SC death. Our data show that soluble products of CNS glial cells do not induce or inhibit SC differentiation or increase cell death but have the potential to increase proliferation of SC and their resistance to cytokine-mediated death, and thus may affect the outcome of SC transplantation into the CNS.

Chronic nerve compression induces concurrent apoptosis and proliferation of Schwann cells.

Chronic nerve compression (CNC), as in carpal tunnel syndrome, is a common cause of peripheral nerve dysfunction in humans. Previous studies using animal models have demonstrated progressive demyelination and a slowing of nerve conduction velocity. To characterize the Schwann cell response to CNC, we evaluated total Schwann cell number, apoptosis, and proliferation in an animal model of CNC. Design-based stereologic techniques revealed a striking transient increase in Schwann cell number following CNC. Schwann cell number increased sixfold relative to the normal nerve at the site of compression at 1 month and then slowly declined toward control levels. Nevertheless, assays of apoptosis (TUNEL and an antipoly-ADP-ribose polymerase labeling assays) revealed extensive Schwann cell apoptosis at 2 weeks postcompression, which is during the time when Schwann cell number was increasing. Electron microscopic analysis confirmed that these dramatic changes in Schwann cells occurred in the absence of axon degeneration and axonal swelling and before there were any detectable alterations in nerve conduction velocity. Counts of bromodeoxyuridine-labeled Schwann cells revealed that proliferation occurred concurrently with ongoing apoptosis. To define further the possible mitogenic properties of mechanical stimuli on Schwann cells, we used an in-vitro model to deliver shear stress in the form of laminar fluid flow to pure populations of Schwann cells and confirmed that mechanical stimuli induce Schwann cell proliferation. Our findings indicate that chronic nerve compression induces Schwann cell turnover with minimal axonal injury and support the idea that mechanical stimuli have a direct mitogenic effect on Schwann cells.

IL‐1β and Interferon‐γ Induce Schwann Cell Proliferation And Death

Schwann cell apoptosis (programmed cell death) has been recently observed during the development and in rare pathological conditions of the peripheral nervous system (PNS) characterized by demyelination‐remyelination. However, the relevance of the phenomenon, and the characterization of the involved molecules are still controversial. This study has been designed to detect in Schwann cell tissue culture whether the administration of different doses of the two pro‐inflammatory cytokines IL‐1β and IFN‐γ, singly or in combination, can induce Schwann cell proliferation and/or death via apoptosis. Our results indicate that the administration of IL‐1β and IFN‐γ induced cell proliferation and concomitant apoptosis in Schwann cells that is numerically related to the different type and dosage of the two pro‐inflammatory cytokines. Moreover, Schwann cell apoptosis was maximal within 24 hours following the administration of 50 U/mL of IFN‐γ, and we did not observe any synergistic action of the two pro‐inflammatory molecules. These findings provide further “in vitro” evidence that cytokines can interact with Schwann cells through a mechanism that is probably mediated by the modulation in time and concentrations of these different pro‐inflammatory molecules.

Stimulation des Wachstums Schwannscher Zellen und der axonalen Regeneration peripherer Nerven durch das Immunsuppressivum FK 506

Nerve allografts are highly antigenic and, thus, require the continuous use of immunosuppressive drugs. The immunosuppressant Cyclosporine A was used in most studies. More recently, the immunosuppressive effect of FK 506 on peripheral nerve allografts and xenografts has been studied, and the drug has been found to prevent rejection successfully. However, neurotoxic complications have been noted in the central and peripheral nervous system although an increased rate of axonal regeneration has also been shown. Schwann cells were cultured from the sciatic nerve of the rat. The effect of 100 microM FK 506 administered daily on these cultures was assessed over a period of seven days and compared to an untreated control group of cultures. To visualize a direct effect of FK 506 on Schwann cells, the changes in intracellular calcium were recorded using fluorescence imaging of primary rat Schwann cell cultures loaded with Fluo-3. Regeneration of autologous nerve grafts was assessed in a rat model clinically and morphometrically after daily administration of 0.6 mg FK 506/kg body weight. FK 506 increases the number of Schwann cells in culture significantly compared to the control group, while the fibrocyte population is decreased. FK 506 caused a transient increase of intracellular calcium levels of cultured cells. A significantly higher axon count was observed in the FK 506-treated grafts after two weeks of regeneration compared with controls. Additionally, less myelin debris was evident in the FK 506 treated group after two weeks compared with the control group. Good regeneration was noted in all grafts after six weeks of regeneration. The increased axon counts and decreased myelin debris in the FK 506 grafts after two weeks indicate an accelerated Wallerian degeneration and increased axon sprouting into the graft initially. FK 506 promotes axonal regeneration through binding to FKBP-12, thus activating GAP-43 (growth associated protein) and the TGF beta 1-pathway (transforming growth factor). In addition, the increase of the intracellular calcium may induce Schwann cell proliferation via Calmodulin which in turn promotes axonal regeneration.

Two mitogenic regions of myelin basic protein interact with different receptors to induce Schwann cell proliferation in a cAMP dependent process.

Previous studies have shown that myelin basic protein (MBP) is mitogenic for Schwann cells (SCs) in the presence of elevated intracellular cAMP. Two mitogenic regions of MBP have been identified: one mitogenic region within the first 44 residues of the aminoterminus (1-44) and the other mitogenic region within the terminal 15 residues of the carboxyl end of the molecule (152-167). Unlike the mitogenic effect of a myelin enriched fraction (MEF), the mitogenic effect of MBP was not reduced by the addition of the lysosomal inhibitor, ammonium chloride. These data indicate that MBP causes SC proliferation by direct interaction of MBP with a surface receptor. Using Scatchard analysis of the binding of MBP to SCs, we report that treatment with forskolin does not cause the upregulation of receptors for MBP. Moreover, MBP blocks the cross-linking of 125I-bFGF with two fibroblast growth factor (FGF) receptors having apparent molecular weights of 140 kDa and 120 kDa, respectively. Since neither TGF-beta nor PDGF-BB displaced cell surface bound 125I-MBP, we conclude that MBP binds to the FGF receptor rather than other growth factor receptors. Furthermore, only MBP interacted with ganglioside GM1, whereas MBP did not interact with this ganglioside. These results are consistent with the view that ganglioside GM1 mediates the mitogenic effects of MBP, while the FGF receptor mediates the mitogenic effect of MBP. Intracellular cAMP of SCs was transiently increased after the addition of macrophage conditioned medium, suggesting that macrophages may produce factors in vivo which can transiently elevate intracellular cAMP levels, allowing a wave of SC proliferation in response to MBP-related mitogens.

Antibodies to interleukin-6 inhibit Schwann cell proliferation induced by unfractionated cytokines.

We have previously demonstrated that unfractionated cytokines induce proliferation of neonatal rat Schwann cells in vitro. Incubation of Schwann cells with natural or recombinant human interleukin-1 (IL-1) does not induce Schwann cell proliferation, but antibodies to IL-1 inhibit Schwann cell proliferation induced by unfractionated cytokines of human origin. In this study we investigated the effect of interleukin-6 (IL-6), another monokine, on proliferation of rat Schwann cells. Incubation of Schwann cells with recombinant (r) mouse IL-6 and two types of human rIL-6 did not induce proliferation of Schwann cells or of endoneurial fibroblasts. However, antibodies to rhIL-6 inhibited Schwann cell proliferation induced by unfractionated cytokines but had no effect on fibroblast proliferation. Addition of excess rhIL-6 to a mixture of unfractionated cytokines plus anti-rhIL-6 abrogated the inhibitory effect of the anti-rhIL-6. Therefore, IL-6 is a co-mitogen for neonatal Schwann cells in vitro.

Antibodies to interleukin-1 inhibit cytokine-induced proliferation of neonatal rat Schwann cells in vitro

Unfractionated cytokines have been shown to induce in vitro proliferation of neonatal rat Schwann cells but the nature of the mitogen(s) is not known. A mixture of rabbit antibodies specific for recombinant interleukin-1α (IL-1α) and interleukin-1β (IL-1β) inhibited Schwann cell proliferation induced by unfractionated human cytokines whereas antibodies to interleukin-2 (IL-2) and control IgG did not. However, purified human IL-1 and recombinant human IL-1α or β did not induce Schwann cell proliferation on their own.

Schwann cell responses to cyclic AMP: Proliferation, change in shape, and appearance of surface galactocerebroside

Surface galactocerebro side (galC) was induced on cultured Schwann cells by two analogues of cyclic adenosine 3′,5′-monophosphate (cAMP), dibutyryl cAMP and 8-bromo cAMP (as previously reported by Sobue and Pleasure 31) and also by forskolin, a potent adenylate cyclase activator. These reagents also induced a morphological transition of many of the Schwann cells, from an elongated spindle shape of flattened cells extending fenestrated cytoplasmic sheets. Surface galC and these changes in Schwann cell shape were not elicited by rising the extracellular cAMP concentration, nor by many compounds known to promote the differentiation of other cell types, suggesting that intracellular cAMP is the unique signal for their induction. The cAMP analogues also induced Schwann cell proliferation (as previously reported by Raff et al. 26), as did forskolin. The concentrations of cAMP analogues and forskolin eliciting largest increases in numbers of Schwann cells in the cultures were 10-fold lower than the concentrations required for optimal induction of Schwann cell surface galC.