In Ae Seo

Interleukin‐6 is required for the early induction of glial fibrillary acidic protein in Schwann cells during Wallerian degeneration

Signal transducer and activator of transcription 3 (STAT3) regulates gene transcription in response to cytokines and growth factors. In the central nervous system, STAT3 plays a role in neuroprotection and reactive gliosis after lesions. During peripheral nerve regeneration, a nerve injury‐induced up‐regulation of cytokines and growth factors accompanies STAT3 activation in sensory neurons and Schwann cells (SCs) even though its molecular details and functions are unknown. We then analyzed the ligands and functions of STAT3 activation in RT4 schwannoma cells and adult SCs in vitro and in vivo. We have identified that interleukin‐6 (IL‐6), but not ciliary neurotrophic factor, leukemia inhibitory factor, or ligands for receptor tyrosine kinases, activates STAT3 in SCs. The IL‐6/STAT3 signaling in primary SCs and RT4 cells induced the gene expression of glial fibrillary acidic protein (GFAP), which is known to be required for the proper regeneration of the injured nerves. Finally, the GFAP induction in the sciatic nerves after injury was significantly delayed in IL‐6‐deficient mice. These findings indicate that IL‐6 plays an important role in STAT3‐dependent GFAP induction in SCs during peripheral nerve regeneration.

Nidogen is a prosurvival and promigratory factor for adult Schwann cells

Schwann cells provide a favorable microenvironment for successful regeneration of the injured peripheral nerve. Even though the roles of extracellular matrix proteins in the Schwann cell physiology have long been studied, the precise function of nidogen, a ubiquitous component of the basal lamina, in Schwann cells is unknown. In this study, we show that the protein and mRNA messages for nidogens are up‐regulated in the sciatic nerve after sciatic nerve transection. We demonstrate that recombinant nidogen‐1 increased the process formation of Schwann cells cultured from adult rat sciatic nerves and that nidogen‐1 prevented Schwann cells from serum‐deprivation‐induced death. In addition, nidogen‐1 promoted spontaneous migration of Schwann cells in two‐independent migration assays. The Schwann cell responses to the recombinant nidogen‐1 were specific because the nidogen‐binding ectodomain of tumor endothelial marker 7 inhibited the nidogen responses without affecting Schwann cell response to laminin. Finally, we found that β1 subunit‐containing integrins play a key role in the nidogen‐induced process formation, survival, and migration of Schwann cells. Altogether, these results indicate that nidogen has a prosurvival and promigratory activity on Schwann cells in the peripheral nerve.

Netrin induces down-regulation of its receptor, Deleted in Colorectal Cancer, through the ubiquitin–proteasome pathway in the embryonic cortical neuron

The proper regulation of temporal and spatial expression of the axon guidance cues and their receptors is critical for the normal wiring of nervous system during development. Netrins, a family of secreted guidance cues, are involved in the midline crossing of spinal commissural axons and in the guidance of cortical efferents. Axons normally lose the responsiveness to their attractants when they arrive at their targets, where the attractant is produced. However the molecular mechanism is still unknown. We investigated the molecular mechanism of down‐regulation of netrin‐1 signaling in the embryonic cortical neurons. Netrin‐1 induced the ubiquitination and proteolytic cleavage of Deleted in Colorectal Cancer (DCC), a transmembrane receptor for netrin, in dissociated cortical neurons. A dramatic decrease of DCC level particularly on the cell surface was also observed after netrin‐1 stimulation. Specific ubiquitin–proteasome inhibitors prevented the netrin‐induced DCC cleavage and decrease of cell surface DCC. We suggest that the ligand‐mediated down‐regulation of DCC might participate in the loss of netrin‐responsiveness in the developing nervous system.

A novel mechanism of methylglyoxal cytotoxicity in neuroglial cells.

Methylglyoxal (MGO) is an endogenous dicarbonyl compound that is highly produced in hyperglycemic conditions. It forms advanced glycation endproducts that are believed to contribute, as etiological factors, to the pathophysiology of diabetic complications. In addition, MGO suppresses cell viability through the induction of apoptosis in vitro. In this study, we have, for the first time, demonstrated the effect of MGO on the gp130 cytokine‐induced signal transducer and activator of transcription 3 (STAT3) responses in RT4 schwannoma, PC12 pheochromocytoma and U87MG glioma cells. At dose that very mildly affects cell viability, MGO rapidly induces endocytotic degradation of gp130, which involves the di‐leucine internalization motif in the cytoplasmic domain of gp130, without affecting other growth factor receptors. Concomitant inhibition of basal and interleukin‐6‐induced STAT3 activation was observed following pre‐treatment with MGO. The inhibitory effect of MGO on the gp130/STAT3 signaling was prevented by the pre‐treatment with an advanced glycation endproduct scavenger aminoguanidine. Finally, these deleterious effects of MGO on STAT3 signaling led to down‐regulation of a STAT3 target gene, Bcl‐2, and sensitized cellular toxicity induced by H2O2 and etoposide. Our data indicate that MGO affects cell viability via desensitization of gp130/STAT3 signaling, which is the key signaling pathway for cell survival, and thereby promotes cytotoxicity.

Identification of the basement membrane protein nidogen as a candidate ligand for tumor endothelial marker 7 in vitro and in vivo

Tumor endothelial marker 7 (TEM7) is a novel transmembrane protein that is highly expressed in the tumor endothelium. However, the ligands and functions of TEM7 are unknown at present. Using a recombinant ectodomain of TEM7 as a probe, we could identify the saturable interaction between nidogen and TEM7 in vitro. A cell overlay binding assay showed that recombinant nidogen interact with full length TEM7 on cell surface. Finally, nidogen/TEM7 interaction enhanced cell spreading in TEM7 transfected 293T cells. This novel nidogen/TEM7 interaction may provide an important molecular tool with potential therapeutic applications in the anti‐angiogenic therapies.

Cell type-specific Stat3 activation by gp130-related cytokines in the peripheral nerves

Nerve injury-induced activation of signal transducer and activator of transcription 3 (STAT3) in sensory neurons and Schwann cells has been implicated in peripheral nerve regeneration. In this study, we investigated the role of gp130-related cytokines including interleukin-6 (IL-6), ciliary neurotrophic factor (CNTF), and leukemia inhibitory factor (LIF) in STAT3 activation in dorsal root ganglion neurons, Schwann cells, and endoneurial fibroblasts. We found that IL-6, but not CNTF or LIF, activated STAT3 in Schwann cells. However, CNTF and LIF, but not IL-6, activated STAT3 in dorsal root ganglion neurons. Furthermore, LIF was the primary activator of STAT3 in endoneurial fibroblasts. These findings indicate that gp130 cytokines may have cell type-specific roles in peripheral nerve regeneration.

Capsaicin inhibits the IL-6/STAT3 pathway by depleting intracellular gp130 pools through endoplasmic reticulum stress.

Capsaicin has been shown to have anti-carcinogenic effects on various tumor cells through multiple mechanisms. It was recently reported that capsaicin inhibited interleukin-6 (IL-6)-induced activation of signal transducer and activator of transcription 3 (STAT3), an anti-apoptotic transcription factor. Here we demonstrate that capsaicin induced downregulation of the IL-6 receptor gp130 within 2h in glial tumors. The downregulation of gp130 was not caused by enhanced degradation of gp130 or by inhibition of mRNA transcription. The downregulation was attributed to translation inhibition of gp130, which was associated with activation of endoplasmic reticulum (ER) stress. The depletion of the intracellular pool of gp130 by capsaicin and an ER stress inducer led to an immediate loss of the IL-6 response due to the short half-life of membrane localized gp130. These results suggest a novel mechanism for the anti-tumor effect of capsaicin.

Janus Kinase 2 Inhibitor AG490 Inhibits the STAT3 Signaling Pathway by Suppressing Protein Translation of gp130

The binding of interleukin-6 (IL-6) cytokine family ligands to the gp130 receptor complex activates the Janus kinase (JAK)/ signal transducer and activator of transcription 3 (STAT3) signal transduction pathway, where STAT3 plays an important role in cell survival and tumorigenesis. Constitutive activation of STAT3 has been frequently observed in many cancer tissues, and thus, blocking of the gp130 signaling pathway, at the JAK level, might be a useful therapeutic approach for the suppression of STAT3 activity, as anticancer therapy. AG490 is a tyrphostin tyrosine kinase inhibitor that has been extensively used for inhibiting JAK2 in vitro and in vivo. In this study, we demonstrate a novel mechanism associated with AG490 that inhibits the JAK/STAT3 pathway. AG490 induced downregulation of gp130, a common receptor for the IL-6 cytokine family compounds, but not JAK2 or STAT3, within three hours of exposure. The downregulation of gp130 was not caused by enhanced degradation of gp130 or by inhibition of mRNA transcription. It most likely occurred by translation inhibition of gp130 in association with phosphorylation of the eukaryotic initiation factor-2alpha . The inhibition of protein synthesis of gp130 by AG490 led to immediate loss of mature gp130 in cell membranes, due to its short half-life, thereby resulting in reduction in the STAT3 response to IL-6. Taken together, these results suggest that AG490 blocks the STAT3 activation pathway via a novel pathway.

Down-regulation of UNC5 homologue expression after the spinal cord injury in the adult rat

UNC5A-D are transmembrane receptors for netrin that plays an important role in the development of the spinal cord. However, their function in the adult nervous system remains to be established. In the present study, the mRNA expression of UNC5 family was investigated in the adult rat spinal cord following a dorsal hemisection injury. Reverse transcription-polymerase chain reaction (RT-PCR) revealed that mRNA expression of rat UNC5 family was down-regulated in the spinal cord within 3 days after the dorsal hemisection. Unc5b-d mRNA expressions returned to near normal level after a week, whereas the down-regulation of Unc5a mRNA expression was maintained until 1 month after the injury. In situ hybridization and immunofluorescent showed that both neurons and non-neuronal cells express Unc5a and b mRNAs in the adult spinal cord, and the significant long lasting down-regulation of Unc5a mRNA expression was also noticed following the spinal cord injury. This result suggests netrin/UNC5 involvement in the injury response of the adult spinal cord of the rats.

Nidogen plays a role in the regenerative axon growth of adult sensory neurons through Schwann cells.

We previously reported that nidogen is an extracellular matrix protein regulating Schwann cell proliferation and migration. Since Schwann cells play a critical role in peripheral nerve regeneration, nidogen may play a role in it via regulation of Schwann cells. Here, we demonstrate direct evidence that nidogen induces elongation of regenerative axon growth of adult sensory neurons, and that the effect is Schwann cell dependent. Continuous infusion of recombinant ectodomain of tumor endothelial marker 7, which specifically blocks nidogen function in Schwann cells, suppressed regenerative neurite growth in a sciatic nerve axotomy model. Taken together, it is likely that nidogen is required for proper regeneration of peripheral nerves after injury.