Karyopherin α2 (KPNA2) plays a central role in nucleocytoplasmic transport. It is involved in controlling the flow of genetic information and the modulation of diverse cellular activities. Here we explored the KPNA2′s roles during the pathophysiological processes of intracerebral hemorrhage (ICH). An ICH rat model was built and evaluated according to behavioral testing. Using Western blot, immunohistochemistry, and immunofluorescence, significant upregulation of KPNA2 was found in neurons in brain areas surrounding the hematoma following ICH. Increasing KPNA2 level was found to be accompanied by the upregulation of active caspase-3, Bax, and decreased expression of Bcl-2. Besides, KPNA2 co-localized well with active caspase-3 in neurons, indicating its potential role in neuronal apoptosis. What’s more, knocking down KPNA2 by RNA-interference in PC12 cells reduced active caspase-3 expression. Thus, KPNA2 may play a role in promoting the brain secondary damage following ICH.

Protein ubiquitination is a dynamic two-way process that can be reversed or regulated by deubiquitinating enzymes (DUB). USP11, located on the X chromosome, 6 is a member of USP subclass of the DUB family. Here, we demonstrate that USP11 may be involved in neuronal apoptosis in the processes of intracerebral hemorrhage (ICH). From the results of Western blot, immunohistochemistry, and immunofluorescence, we obtained a significant up-regulation of USP11 in neurons adjacent to the hematoma following ICH. Increasing USP11 level was found to be accompanied by the up-regulation of active caspase-3, Fas receptor (Fas), Fas ligand (FasL), and active caspase-8. Besides, USP11 co-localized well with active caspase-3 in neurons, indicating its potential role in neuronal apoptosis. What is more, knocking down USP11 by RNA-interference in PC12 cells reduced active caspase-3 expression. Thus, USP11 may play a role in promoting the brain secondary damage following ICH.

Protein tyrosine phosphatase 1B (PTP1B), a member of the protein tyrosine phosphatase family, attaches to the endoplasmic reticulum (ER) via its C-terminal tail. Previous studies have reported that PTP1B participates in various signal transduction pathways in many human diseases, including diabetes, cancers, osteoporosis, and obesity. It also plays an important role in the ER stress. ER stress induced by spinal cord injury (SCI) was reported to result in cell apoptosis. Till now, the role of PTP1B in the injury of the central nervous system remains unknown. In the present study, we built an adult rat SCI model to investigate the potential role of PTP1B in SCI. Western blot analysis detected a notable alteration of PTP1B expression after SCI. Immunohistochemistry indicated that PTP1B expressed at a low level in the normal spinal cord and greatly increased after SCI. Double immunofluorescence staining revealed that PTP1B immunoreactivity was predominantly increased in neurons following SCI. In addition, SCI resulted in a significant alteration in the level of active caspase-3, caspase-12, and 153/C/EBP homologous transcription factor protein, which were correlated with the upregulation of PTP1B. Co-localization of PTP1B/active caspase-3 was also detected in neurons. Taken together, our findings elucidated the PTP1B expression in the SCI for the first time. These results suggested that PTP1B might be deeply involved in the injury response and probably played an important role in the neuro-pathological process of SCI.

Bone morphogenetic protein-2 (BMP-2), a glycosylated protein, has been demonstrated to play a key role in osteoblast differentiation. However, the function of its glycosylation is incompletely understood. In this study, we examined the role that N-linked glycans (NLG) play in the secretion of BMP-2. Blocking the addition of NLGs or inhibiting initial glycan processing prevented the secretion of BMP-2. To identify the specific glycosylation sites, we abolished potential sites of N-linked glycosylation (Asn-Xaa-Ser/Thr) in BMP-2 by mutating the Asn residues to Gln individually or in combination, expressed the BMP-2 mutants in Chinese hamster ovary (CHO) and human embryonic kidney 293T (HEK293T) cells and determined their glycosylation state by using peptide:N-glycosidase F and endoglycosidase H digestion. We found that human BMP-2 contains three NLG on N135, N200 and N338. Elimination of N-glycosylation by mutation of N135 (N135Q) abolished the BMP-2 secretion from CHO cells. Overexpression of the BMP-2 mutant N135Q elicited endoplasmic reticulum (ER) stress and retention within the ER in CHO cells, indicating that N-glycosylation is required for folding of human BMP-2. Furthermore, we demonstrated that glycosylation at N135 was necessary for BMP-2-induced osteoblast differentiation in MC3T3-E1 cells. Taken together, these data provide further evidence of the critical role that individual NLG may play an important role in determining BMP-2 folding, secretion and function.

After traumatic injury, peripheral nerves can spontaneously regenerate through highly sophisticated and dynamic processes that are regulated by multiple cellular elements and molecular factors. Despite evidence of morphological changes and of expression changes of a few regulatory genes, global knowledge of gene expression changes and related biological processes during peripheral nerve injury and regeneration is still lacking. Here we aimed to profile global mRNA expression changes in proximal nerve segments of adult rats after sciatic nerve transection. According to DNA microarray analysis, the huge number of genes was differentially expressed at different time points (0.5 h-14 d) post nerve transection, exhibiting multiple distinct temporal expression patterns. The expression changes of several genes were further validated by quantitative real-time RT-PCR analysis. The gene ontology enrichment analysis was performed to decipher the biological processes involving the differentially expressed genes. Collectively, our results highlighted the dynamic change of the important biological processes and the time-dependent expression of key regulatory genes after peripheral nerve injury. Interestingly, we, for the first time, reported the presence of olfactory receptors in sciatic nerves. Hopefully, this study may provide a useful platform for deeply studying peripheral nerve injury and regeneration from a molecular-level perspective.

Objective and design The carbohydrate moieties of glycoprotein are associated with some inflammatory diseases by affecting a wide range of biological functions of cells. This study aimed to investigate the role of β-1,4-galactosyltransferase-I (β-1,4-GalT-I) in adhesion of Schwann cells during inflammation. Subjects A rat Schwann cell line, RSC 96 was used. Methods We used western blotting to detect the expression of β-1,4-GalT-I. Flow cytomety was used to measure the galactosylation of glycoproteins on cell surfaces. Immunofluorescent staining was used to examine the expression of α6 integrin, focal adhesion kinase (FAK) and F-actin. Tyrosine phosphorylation of FAK was detected by immunoprecipitation. An adhesion assay was performed to investigate the adhesion of Schwann cells. One-way ANOVA was used to compare differences between the operated and the control group. Results Schwann cell adhesion was induced by LPS stimulation and was accompanied by upregulation of β-1,4-GalT-I expression and galactosylation of glycoproteins. There was a change of localization of FAK and cytoskeleton organization in LPS treated cells compared with control cells. The pretreated cells enhanced tyrosine phosphorylation of FAK compared with control cells in the adhesion process. With the increased cell surface expression of α6 integrin and β-1,4-GalT-I, the adhesion of Schwann cells on laminin was increased as well. Conclusions These results suggested that β-1,4-GalT-I may play an important role in adhesion of Schwann cells during inflammation.

The proinflammatory and lipopolysaccharide (LPS)-inducible cytokine tumor necrosis factor alpha (TNF-alpha) has been shown to enhance primary sensory nociceptive signaling. However, the precise cellular site of TNF-alpha synthesis is still a matter of controversy. Therefore, we focused our study on TNF-alpha protein synthesis and expression patterns in spinal dorsal horn of naives and rats under intrathecal challenge with LPS. The enzyme-linked immunosorbent (ELISA) assay showed that the protein level of TNF-alpha reached peak at 8 h. Double immunofluorescence revealed that LPS-induced expression of TNF-alpha exclusively located in a subpopulation of microglia, which increased at 8 h in the rat spinal dorsal horn (the injected side). Positive staining of TNF receptor 1 (TNFR1) were also found in microglia. These observations have demonstrated the production of this proinflammatory cytokine by central nerve glia especially microglia. Synthesized TNF-alpha might directly act on microglia via TNFR1, but the inherent mechanisms remain unknown. Further studies are needed to confirm the pathogenic role of tumor necrosis factor in the early stage of inflammation.

β1,4-Galactosyltransferase-I (β1,4-GalT-I) is one of the best studied glycosyltransferases. Previous studies demonstrated that β1,4-GalT-I was a major galactosyltransferase responsible for selectin-ligand biosynthesis and that inflammatory responses of β1,4-GalT-I deficient mice were impaired. In this study, we investigate the expression of β1,4-GalT-I in lipopolysaccharide (LPS)-induced neuroinflammatory processes. The results of this study demonstrated that β1,4-GalT-I was strongly induced by intraspinal administration of LPS. More than 90% galactose-containing glycans and β1,4-GalT-I were expressed in immune cells. The ELISA assay shows focal injection LPS also induces TNF-α alteration. Double staining indicated β1,4-GalT-I overlapped with TNF-α. Moreover, RT-PCR for β1,4-GalT-I mRNA showed that β1,4-GalT-I mRNA in microglia in vitro was affected in a dose- and time dependent manner in response to LPS or TNF-α stimulation. All these results indicated that the increase of β1,4-GalT-I might attribute to the effect of TNF-α excreting during inflammation. E-selectin, which ligand was modified by β1,4-GalT-I, was correlated with galactose-containing glycans following injecting LPS into spinal cord. We therefore suggest that β1,4-GalT-I may play an important role in regulating immune cell migration into the inflammatory site.

β4 Galactosylation of glycoproteins is one of the most important post-translational modifications. Recent studies have demonstrated that aberrant galactosylation associates with some inflammation diseases. β-1,4-galactosyltransferase-I (β-1,4-GalT-I), which transfers galactose to the terminal N-acetylglucosamine of N- and O-linked glycans in a β-1,4- linkage, considered to be the major galactosyltransferse among the seven members of the subfamily responsible for β4 galactosylation. In the present study, we investigated the expression of β-1,4-GalT-I in Schwann cells under Lipopolysaccharide (LPS) treatment. RT-PCR revealed that the β-1,4-GalT-I mRNA was significant increased as early as 2 h after LPS stimulation. Immunofluorescence showed that β-1,4-GalT-I was located in Golgi apparatus and membrane of Schwann cells. With the 1 μg/ml LPS treatment, expression levels of β-1,4-GalT-I was much higher compared with control group. In addition, lectin blot indicated that the β4 galactosylation of glycoproteins such as integrin α5 was enhanced, which may due to the induced β-1,4-GalT-I expression. These results suggested that β-1,4-GalT-I may play an important role in adhesion and migration of Schwann cells during inflammation.

β-1,4-Galactosyltransferase-I (β-1,4-GalT-I) is one of the best studied glycosyltransferases. Previous studies demonstrated that β-1,4-GalT-I was a major galactosyltransferase responsible for selectin-ligand biosynthesis and that inflammatory responses of β-1,4-GalT-I deficient mice were impaired. Our previous study suggest that β-1,4-GalT-I may play an important role in regulating immune cell migration into the inflammatory site. In this study, we investigate β-1,4-GalT-I may play an important role in mediating microgliosis. The results of this study demonstrated that β-1,4-GalT-I was strongly induced in the ventral midbrain by intranigral injection of LPS. Most galactose-containing glycans and β-1,4-GalT-I were expressed in microglia. Moreover, an Ab against β-1,4-GalT-I attenuated both LPS-induced microglial activation and phagocytosis. We therefore suggest that β-1,4-GalT-I may play an important role in regulating immune cell migration into the inflammatory site and mediating microgliosis.

Lipopolysaccharide is a major constituent of the outer membrane of Gram-negative bacteria. It activates monocytes and macrophages to produce cytokines such as tumor necrosis factor-alpha and interleukins IL-1beta and IL-6. These cytokines appear to be responsible for the neurotoxicity observed in peripheral nervous system inflammatory disease. It has been reported that, in the central nervous system, the expression level of intercellular adhesion molecule-1 (ICAM-1) was dramatically upregulated in response to LPS, as well as many inflammatory cytokines. ICAM-1 contributes to multiple processes seen in central nervous system inflammatory disease, for example migration of leukocytes to inflammatory sites, and adhesion of polymorphonuclear cells and monocytes to central nervous system cells. In the present study, we found that lipopolysacharide evoked ICAM-1 mRNA and protein expression early at 1 h post-injection, and the most significant increase was seen at 4 h. Immunofluorescence double-labeling suggested that most of the ICAM-1-positive staining was located in Schwann cells. Using Schwann cell cultures, we demonstrated that ICAM-1 expression in Schwann cells is regulated by mitogen-activated protein kinases, especially the p38 and stress-activated protein kinase/c-Jun N-terminal kinase pathways. Thus, it is thought that upregulation of ICAM-1 expression in Schwann cells may be important for host defenses after peripheral nervous system injury, and reducing the biosynthesis of ICAM-1 and other cytokines by blocking the cell signal pathway might provide a new strategy against inflammatory and immune reaction after peripheral nerve injury.

The early stage of inflammation involves the adhesion and transmigration of leukocytes across the blood–brain barrier (BBB) to the normally sequestered central nervous system (CNS). This process is regulated by the expression of a series of adhesion molecules. One of the most well-known components is intercellular adhesion molecule-1 (ICAM-1). It was described as a ligand of the membrane-bound integrin receptors lymphocyte function-associated antigen-1 (LFA-1) and monocyte adhesion molecules-1 (Mac-1) on leukocytes, and was involved in the adhesion and transmigration of leukocytes. Studies have demonstrated the upregulation of ICAM-1 in many tissues after lipopolysaccharide (LPS) stimulation, for example. In the CNS, recent studies just focus on the relatively acute effects in brain tissues, but neglected the possibly existed differences between the brain and the spinal cord following traumatic lesions. Our data demonstrated the upregulation of ICAM-1, LFA-1, and Mac-1 in the spinal cords of LPS intraspinal injected rats, and the location of ICAM-1 in microglia cells. These results suggested a possible role of this molecule in microglia-mediated immune response and antigen presenting in CNS immune diseases.

Cell-surface carbohydrate chains are known to contribute to cell migration, interaction, and proliferation. β-1,4-galactosyltransferase-I (β-1,4-GalT-I), which is one of the best-studied glycosyltransferases, plays a key role in the synthesis of type 2 chains in N-glycans and the core 2 branch in O-glycans. Recently, it has been reported that skin wound healing is significantly delayed in β-1,4-GalT-I−/− mice. However, the expression of β-1,4-GalT-I and its biological function in the skin wound-healing process remain to be elucidated. We used real-time polymerase chain reaction to demonstrate that the expression of β-1,4-GalT-I mRNA reached plateau values at 12 hours after skin was injured and remained elevated until 11 days after the injury. Furthermore, lectin blotting showed that β-1,4-galactosylated carbohydrate chains were also increased after skin injury. A double-staining method combining lectin-fluorescent staining with RCA-I and immunofluorescence was first used to determine the cellular localization of β-1,4-galactosylated carbohydrate chains. Morphological analysis showed that the chains were primarily expressed in neutrophils and partially expressed in macrophages, endothelial cells, and collagen. Our results suggest that β-1,4-GalT-I and β-1,4-galactosylated carbohydrate chains participate in leukocyte recruitment, angiogenesis, and collagen deposition in the skin wound-healing process.

β-1,4-Galactosyltransferase-I (β-1,4-GalT-I) which is one of the best-studied glycosyltransferases, plays a key role in the synthesis of selectin ligands such as sialy Lewis (sLe x ) and sulfated sLe x . Previous studies showed that inflammatory responses of β-1,4-GalT-I-deficient mice were impaired because of the defect in selectin-ligand biosynthesis. However, the expression of β-1,4-GalT-I during inflammation and its biological function remains to be elucidated. Real-time PCR showed that intraperitoneal administration of LPS strongly induced β-1,4-GalT-I mRNA expression in the lung, heart, liver, spleen, kidney, lymph node, hippocampus, and testis, as well as in the cerebral cortex. In the rat lung, liver and testis, LPS stimulation of β-1,4-GalT-I mRNA expression is time-dependent and biphasic. Lectin-fluorescent staining with RCA-I showed that LPS induced expression of galactose-containing glycans in rat lung and liver to the higher lever. Morphology analysis observed that galactose-containing glycans and β-1,4-GalT-I mRNA was mostly expressed in neutrophils, macrophages and endothelial cells. These findings indicated that β-1,4-GalT-I may play an important role in the inflammation reaction.