Huiguang Yang

Tumor necrosis factor-alpha inhibits Schwann cell proliferation by up-regulating Src-suppressed protein kinase C substrate expression.

Src‐suppressed protein kinase C substrate (SSeCKS) is a protein kinase C substrate protein, which plays an important role in mitogenic regulatory activity. In the early stage of nerve injury, expression of SSeCKS in the PNS increases, mainly in Schwann cells (SCs). However, the exact function of SSeCKS in the regulation of SC proliferation remains unclear. In this study, we found that tumor necrosis factor‐alpha (TNF‐α) induced both SSeCKS α isoform expression and SC growth arrest in a dose‐dependent manner. By knocking down SSeCKS α isoform expression, TNF‐α‐induced growth arrest in SCs was partially rescued. Concurrently, the expression of cyclin D1 was reduced and the activity of extracellular signal‐regulated kinase 1/2 was decreased. A luciferase activity assay showed that cyclin D1 expression was regulated by SSeCKS at the transcription level. In addition, the cell fragments assay and immunofluorescence revealed that TNF‐α prevented the translocation of cyclin D1 into the nucleus, while knocking down SSeCKS α isoform expression prompted cyclin D1 redistribution to the nucleus. In summary, our data indicate that SSeCKS may play a critical role in TNF‐α‐induced SC growth arrest through inhibition of cyclin D1 expression thus preventing its nuclear translocation.

The member of high temperature requirement family HtrA2 participates in neuronal apoptosis after intracerebral hemorrhage in adult rats

The members of high-temperature requirement (HtrA) family are evolutionarily conserved serine proteases that combine a trypsin-like protease domain with at least one PDZ interaction domain. HtrA2, a special one, is mainly located in mitochondria and required for maintaining homeostasis. Once released into cytoplasm, HtrA2 contributes to apoptosis via caspase-dependent and -independent pathways. Accumulating evidence has showed its pro-apoptotic effect in cancers and central nervous system (CNS) diseases. However, the distribution and function of HtrA2 in CNS diseases remains to be further explored. To investigate HtrA2’s roles in the pathophysiology of intracerebral hemorrhage (ICH), an ICH rat model was established and assessed by behavioral tests. Western blot and immunohistochemistry revealed a remarkable up-regulation of HtrA2 surrounding the hematoma after ICH; and immunofluorescence showed HtrA2 was strikingly increased in neurons, but not in astrocytes and oligodendrocytes. Terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick-end labeling staining suggested the involvement of HtrA2 in neuronal apoptosis after ICH. Additionally, HtrA2 co-localized with active-caspase-3 around the hematoma and the expression of active-caspase-3 was parallel with that of HtrA2 in a time-dependent manner. Furthermore, hemin was used to stimulus a neuronal cell line PC12 to mimic ICH model in vitro. We analyzed the relationship of HtrA2 with X-linked inhibitor of apoptosis protein (XIAP) in PC12 cells by Western blot, immunofluorescence and co-immunoprecipitation. The connection of HtrA2 with XIAP was strengthened in apoptotic cells after hemin treatment. Thus, we speculated that HtrA2 might exert an important function in regulating caspase-dependent neuronal apoptosis through interacting with XIAP following ICH.

β-1,4-Galactosyltransferase I involved in Schwann cells proliferation and apoptosis induced by tumor necrosis factor-alpha via the activation of MAP kinases signal pathways

Abstractβ-1,4-galactosyltransferase-I (β-1,4-GalT-I) plays a critical role in the initiation and maintenance of peripheral nervous system inflammatory reaction. However, the exact function of β-1,4-GalT-I in the regulation of SCs proliferation and apoptosis remains unclear. In this study, we found that low concentration of tumor necrosis factor-alpha (TNF-α) induced SCs proliferation, while high concentration of TNF-α induced SCs apoptosis. Meanwhile, the expressions of β-1,4-GalT-I, TNFR1, and TNFR2 were changed following. When β-1,4-GalT I overexpression, low concentration of TNF-α-induced SCs proliferation was partially repressed. Concurrently, the activity of ERK1/2 was decreased. While knocking down β-1,4-GalT I expression, high concentration of TNF-α-induced SCs apoptosis was partially rescued. Consistent with this, the activity of P38 and JNK were decreased. We also found anti-TNFR2 antibody suppressed low concentration of TNF-α-induced SCs proliferation, while anti-TNFR1 antibody inhibited high concentration of TNF-α-induced SCs apoptosis. Thus, present data show that β-1,4-GalT I may play an important role in SCs proliferation and apoptosis induced by TNF-α via different signal pathways and TNFR.

SSeCKS is a Suppressor in Schwann Cell Differentiation and Myelination

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.

The Cyclin-Dependent Kinase Inhibitor p27 Kip1 is a Positive Regulator of Schwann Cell Differentiation In Vitro

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 p27Kip1 expression was upregulated during the differentiation of Schwann cell, while the inhibition of p27Kip1 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 p27Kip1 nuclear staining and a concomitant increase of cytoplasmic staining in differentiated Schwann cells. In summary, our data indicated that p27Kip1 was a positive regulator of Schwann cell differentiation in vitro.

Cyclin D3/CDK11p58 Complex Involved in Schwann Cells Proliferation Repression Caused by Lipopolysaccharide

Schwann cells proliferation is the main characterize of kinds PNS inflammation diseases. It has been well documented that cyclin D3 /CDK11p58 complex inhibits cell function through multiple mechanisms, but the mechanism of cyclin D3/CDK11p58 complex exerts its repressive role in the Schwann cells proliferation remains to be identified. In the present investigation, we demonstrated that the expression of CDK11p58 were upregulated in the inflammation caused by LPS, a main part of bactria. Cyclin D3 and the 58-kDa isoform of cyclin-dependent kinase 11 (CDK11p58) interacted with each other mainly in nuclear region, repressed Schwann cells proliferation and induced cell apoptosis. Overexpression of CDK11p58 expression might enhance this process, while silence of cyclin D3 reverting it. This work demonstrates for the first time the role of cyclin D3/CDK11p58 complex in repressing the Schwann cells proliferation and inducing its apoptosis.

Dynamic change of Numbl expression after sciatic nerve crush and its role in Schwann cell differentiation.

Numbl, as a conserved homolog of Drosophila Numb, has been implicated in early development of the nervous system, but its expression and roles in nervous system lesion and repair remained unknown. Here, we performed an acute sciatic nerve injury model in adult rats and studied the dynamic changes of Numbl expression in the sciatic nerve. Temporally, Numbl expression was sharply decreased after sciatic nerve crush and reached a valley at day 7. Spatially, Numbl was widely expressed in the normal sciatic nerve, including axons and Schwann cells, whereas, after injury, Numbl expression was decreased predominantly in Schwann cells. In vitro, we induced Schwann cell differentiation with cAMP and found that Numbl expression was decreased in the differentiated process. Depletion of Numbl could promote Schwann cell differentiation. In addition, we demonstrated that in vitro myelination was suppressed by overexpression of Numbl in Schwann cells. Collectively, we hypothesized peripheral nerve injury induced a downregulation of Numbl in the sciatic nerve, which was associated with Schwann cell differentiation.

The Relationship Between Src-Suppressed C Kinase Substrate and β-1,4 Galactosyltransferase-I in the Process of Lipopolysaccharide-Induced TNF-α Secretion in Rat Primary Astrocytes

Src-suppressed C kinase substrate (SSeCKS), a protein kinase C substrate, is a major lipopolysaccharide (LPS) response protein. In addition, β-1,4 Galactosyltransferase-I (β-1,4-GalT-I) also plays an important role in the inflammation reactions of nervous system. It was reported that both SSeCKS and β-1,4-GalT-I were involved in the LPS-induced tumor necrosis factor-alpha (TNF-α) expression in rat primary astrocytes. However, the functional interaction between SSeCKS and β-1,4-GalT-I in the LPS-induced TNF-α secretion remains unclear. Therefore, in this study, using the inflammation model of astrocytes treated by LPS in vitro, we found that the changed expressions of SSeCKS and β-1,4-GalT-I participated in LPS-induced TNF-α secretion through p38, JNK, and ERK signal transduction pathways in rat primary astrocytes. Knockdown by small-interfering RNAs (siRNAs) or overexpression of SSeCKS and β-1,4-GalT-I could influence Mitogen-activated protein kinases (MAPKs) signaling pathways activation and TNF-α secretion. Besides, we confirmed that knockdown of SSeCKS could prevent the induction of β-1,4-GalT-I in this process. Inversely, β-1,4-GalT-I had no significant effect on SSeCKS expression in the same way. In summary, our data indicated that SSeCKS could regulate LPS-induced TNF-α secretion through β-1,4-GalT-I in rat primary astrocytes.

Expression of β-1,4-Galactosyltransferase I in Rat Schwann Cells

Glycosylation is one of the most important post‐translational modifications. It is clear that the single step of β‐1,4‐galactosylation is performed by a family of β‐1,4‐galactosyltransferases (β‐1,4‐GalTs), and that each member of this family may play a distinct role in different tissues and cells. In the present study, real‐time PCR revealed that the β‐1,4‐GalT I mRNA reached peaks at 2 weeks after sciatic nerve crush and 3 days after sciatic nerve transection. Combined in situ hybridization for β‐1,4‐GalT I mRNA and immunohistochemistry for S100 showed that β‐1,4‐GalT I mRNAs were mainly located in Schwann cells after sciatic nerve injury. In conclusion, β‐1,4‐GalT I might play important roles in Schwann cells during the regeneration and degeneration of the injured sciatic nerve. In other pathology, such as inflammation, we found that LPS administration affected β‐1,4‐GalT I mRNA expression in sciatic nerve in a time‐ and dose‐dependent manner, and β‐1,4‐GalT I mRNA is expressed mainly in Schwann cells. These results indicated that β‐1,4‐GalT I plays an important role in the inflammation reaction induced by intraperitoneal injection of LPS. Similarly, we found that β‐1,4‐GalT I in Schwann cells in vitro was affected in a time‐ and concentration‐dependent manner in response to LPS stimulation. All these results suggest that β‐1,4‐GalT I play an important role in Schwann cells in vivo and vitro during pathology. In addition, β‐1,4‐GalT I production was drastically suppressed by U0126 (ERK inhibitor), SB203580 (p38 inhibitor), or SP600125 (SAPK/JNK inhibitor), which indicated that Schwann cells which regulated β‐1,4‐GalT I expression after LPS stimulation were via ERK, SAPK/JNK, and P38 MAP kinase signal pathways. J. Cell. Biochem. 108: 75–86, 2009.

SCY1-Like 1-Binding Protein 1 (SCYL1BP1) Suppressed Sciatic Nerve Regeneration by Enhancing the RhoA Pathway

SCY1-like 1-binding protein 1 (SCYL1BP1) is first identified as an interacting protein with SCYL1. Since SCYL1BP1 is a soluble protein with coiled-coil domains known to be relevant with transcriptional regulation, it has been found to activate the transcription of murine double minute 2 (MDM2) and participate in neurite outgrowth and regeneration. However, the role and mechanism of SCYL1BP1 in peripheral nerve system lesion and repair are still unknown. Here in vitro, our work demonstrated that SCYL1BP1 inhibited cAMP-induced primary Schwann cell differentiation and suppressed nerve growth factor-mediated neurite outgrowth in PC12 cells by enhancing the RhoA pathway. Furthermore, we found that pretreatment with a Rho kinase inhibitor Y-27632 resulted in partial rescue of Schwann cell differentiation and neurite outgrowth. In vivo experiments showed that SCYL1BP1 could also suppress nerve fiber regeneration. In conclusion, we speculated that SCYL1BP1 participated in Schwann cell (SC) differentiation and neurite outgrowth in the sciatic nerve after crush by regulating the RhoA pathway.