Zhengming Zhou

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.

FBP1 and p27kip1 Expression After Sciatic Nerve Injury: Implications for Schwann Cells Proliferation and Differentiation

Far Upstream Element (FUSE) Binding Protein 1 (FBP1), first identified as a single‐stranded DNA (ssDNA) binding protein that binds to the FUSE, could modulate c‐myc mRNA levels and also has been shown to regulate tumor cell proliferation and replication of virus. Typically, FBP1 could active the translation of p27kip1 (p27) and participate in tumor growth. However, the expression and roles of FBP1 in peripheral system lesions and repair are still unknown. In our study, we found that FBP1 protein levels was relatively higher in the normal sciatic nerves, significantly decreased and reached a minimal level at Day 3, and then returned to the normal level at 4 weeks. Spatially, we observed that FBP1 had a major colocation in Schwann cells and FBP1 was connected with Ki‐67 and Oct‐6. In vitro, we detected the decreased level of FBP1 and p27 in the TNF‐α‐induced Schwann cells proliferation model, while increased expression in cAMP‐induced Schwann cells differentiation system. Specially, FBP1‐specific siRNA‐transfected SCs did not show fine and longer morphological change after cAMP treatment and had a decreased motility compared with normal. At 3 days after cAMP treatment and SC/neuron co‐cultures, p27 was transported to cytoplasm to form CDK4/6‐p27 to participate in SCs differentiation. In conclusion, we speculated that FBP1 and p27 were involved in SCs proliferation and the following differentiation in the sciatic nerve after crush by transporting p27 from nucleus to cytoplasm. J. Cell. Biochem. 115: 130–140, 2014.

Dynamic Changes of Jab1 and p27kip1 Expression in Injured Rat Sciatic Nerve

Jun activation domain-binding protein (Jab1) is a multifunctional protein that participates in affecting signaling pathway, controlling cell proliferation and apoptosis, and regulating genomic instability and DNA repair, and acts as a key subunit of COP9 signalosome. p27kip1, a member of the Cip/Kip family of cyclin-dependent kinase inhibitors, was shown to inhibit the enzymatic activity of cyclin–CDK complexes, resulting in cell-cycle arrest at G1. Recent studies have shown that Jab1 directly binds to p27kip1 and induces nuclear export and subsequent degradation in a variety of human cancers, while the association and function of Jab1 and p27kip1 in nervous system lesion and regeneration remain unclear. Here, we performed a sciatic nerve injury model in adult rats and studied the dynamic changes of Jab1 and p27kip1 expression by Western blot. Sciatic nerve crush (SNC) resulted in a significant upregulation of Jab1 and a downregulation of p27kip1. Besides, we observed that Jab1 was expressed widely in Schwann cells (SCs) and had few co-localization in axons by double immunofluorescence staining. In addition, the peak expression of Jab1 was parallel with proliferating cell nuclear antigen (PCNA), and numerous SCs expressing Jab1 were PCNA-positive. Results obtained by co-immunoprecipitation and double labeling further showed their interaction in the sciatic nerve. Thus, these results suggested that Jab1 and p27kip1 may be involved in the pathophysiology of sciatic nerve after SNC.

Temporal-Spatial Expressions of Spy1 in Rat Sciatic Nerve After Crush

As a novel cell cycle protein, Spy1 enhances cell proliferation, promotes the G1/S transition as well as inhibits apoptosis in response to UV irradiation. Spy1 levels are tightly regulated during mammary development, and overexpression of Spy1 accelerates tumorigenesis in vivo. But little is known about the role of Spy1 in the pathological process of damage and regeneration of the peripheral nervous system. Here we established a rat sciatic nerve crush (SNC) model to examine the spatiotemporal expression of Spy1. Spy1 expression was elevated gradually after sciatic nerve crush and peaked at day 3. The alteration was due to the increased expression of Spy1 in axons and Schwann cells after SNC. Spy1 expression correlated closely with Schwann cells proliferation in sciatic nerve post injury. Furthermore, Spy1 largely localized in axons in the crushed segment, but rarely co-localized with GAP43. These findings suggested that Spy1 participated in the pathological process response to sciatic nerve injury and may be associated with Schwann cells proliferation and axons regeneration.

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.

β-1,4-Galactosyltransferase I Promotes Tumor Necrosis Factor-α Autocrine via the Activation of MAP Kinase Signal Pathways in Schwann Cells

Recent studies have demonstrated that aberrant galactosylation is associated with some inflammation diseases. β-1,4-Galactosyltransferase-I (β-1,4-GalT I), which transferred galactose to the terminal N-acetylglucosamine of N- and O-linked glycans in a β-1,4-linkage, was considered to be the major galactosyltransferase among the seven members of the subfamily responsible for β4 galactosylation. To elucidate the expression and possible function of β-1,4-GalT I in the peripheral nervous system (PNS) inflammatory diseases, we performed a tumor necrosis factor-alpha (TNF-α) autocrine inflammatory model in Schwann cells (SCs). In this study, we found that silencing of β-1,4-GalT I suppressed TNF-α autocrine, while overexpression of β-1,4-GalT I promoted TNF-α autocrine in TNF-α-treated SCs. Meanwhile, anti-TNFR1 antibody suppressed the expression of β-1,4-GalT I, and TNF-α autocrine. β-1,4-GalT I conferred its effect by promoting ERK, JNK, and P38 MAP kinase signal pathways activation in TNF-α-induced SCs. Thus, the present data shows that during SCs activation, β-1,4-GalT I may play an important role in the release of inflammatory mediators.

Expressions of Forkhead Class Box O 3a on Crushed Rat Sciatic Nerves and Differentiated Primary Schwann cells

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.

SSeCKS Promotes Tumor Necrosis Factor-α Autocrine via Activating p38 and JNK Pathways in Schwann Cells

Tumor necrosis factor-alpha (TNF-α) derived from activated Schwann cells (SCs) plays a critical role as an inflammatory mediator in the peripheral nervous system disease. TNF-α 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-α production induced by TNF-α, overexpression of SSeCKS could promoted TNF-α autocrine in SCs. Such effects might be resulted in SSeCKS promoted p38 and JNK activation in SCs treated by TNF-α. Thus present data show that while SCs activation, SSeCKS may plays an important role in the release of inflammatory mediators.