The role of Homer1b/c in neuronal apoptosis following LPS-induced neuroinflammation.

Homer, also designated Vesl, is one member of the newly found postsynaptic density scaffold proteins, playing a vital role in maintaining synaptic integrity, regulating intracellular calcium mobilization, and being critical for the regulation of cellular apoptosis. However, its function in the inflamed central nervous system (CNS) is not fully elucidated. Here, we investigated the role of Homer1b/c, a long form of Homer1, in lipopolysaccharide (LPS) induced neuroinflammation in CNS. Western blot analysis indicated that LPS administration significantly increased the expression of Homer1b/c in rat brain. Moreover, double immunofluorescent staining suggested Homer1b/c was mainly distributed in the cytoplasm of neurons and had a close association with cleaved caspase-3 level in neurons in rat brain after LPS injection. In vitro studies indicated that up-regulation of Homer1b/c might be related to the subsequent apoptosis in neurons treated by conditioned media (CM), collected from LPS-stimulated mixed glial cultures (MGC). We also found down-regulation of Homer1b/c partly blocked the increase of cleaved caspase-3 and the proportion of Bax/Bcl-2 in neurons induced by MGC-CM. Taken together, these findings suggested that Homer1b/c might promote neuronal apoptosis via the Bax/Bcl-2 dependent pathway during neuroinflammation in CNS, and inhibiting Homer1b/c expression might provide a novel neuroprotective strategy against the inflammation-related neuronal apoptosis.

Asparagine-linked glycosylation of bone morphogenetic protein-2 is required for secretion and osteoblast differentiation.

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.

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.

Matrix metalloproteinase-1 (MMP-1) expression in rat spinal cord injury model.

Matrix metalloproteinase-1 (MMP-1), a member of the matrix metalloproteinases family, plays an integral role in extracellular matrix degradation and has been reportedly involved in the regulation of the brain or spinal cord traumatic neurovascular remodeling. Although the critical involvement of MMP-1 in the metastasis of tumors has been extensively documented, the role of MMP-1 in the pathology of neurological diseases remains largely elusive. In the present study, we established an adult rat spinal cord injury (SCI) model and investigated a potential role of MMP-1 in the pathological process of SCI. Using Western blot analysis, we identified notable expression change of MMP-1 after SCI. Immunohistochemistry showed that MMP-1 was distributed widely in rat spinal cord. Double immunofluorescence staining revealed that MMP-1 immunoreactivity was predominantly increased in neurons and astrocytes following SCI. Moreover, after injury, colocalization of MMP-1/active caspase-3 in neurons (NeuN-positive), and colocalization of MMP-1/PCNA in astrocytes (GFAP-positive) were clearly observed. We also examined the protein expression of PCNA, active caspase-3, Bcl-2, and Bax and found that the expression of the proteins was closely correlated with that of MMP-1. Taken together, our findings indicate that MMP-1 might play an important role in the regulation of neuronal apoptosis and astrocyte proliferation after SCI.

ß-1,4-Galactosyltransferase-involved in lipopolysaccharide-induced adhesion of Schwann cells.

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 ß-1,4-galactosyltransferase-I (ß-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 ß-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 ß-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 ß-1,4-GalT-I, the adhesion of Schwann cells on laminin was increased as well. CONCLUSIONS: These results suggested that ß-1,4-GalT-I may play an important role in adhesion of Schwann cells during inflammation.

Lipopolysaccharide-induced upregulation of tumor necrosis factor-alpha (TNF-alpha) in rat spinal cord.

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 sites 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 cord of controls and rats under systemic challenge with LPS. The Enzyme-linked immunosorbent (ELISA) assay showed that the protein level of TNF-alpha reached peak at 6 h. Double immunofluorescence revealed that LPS-induced expression of TNF-alpha exclusively located in a subpopulation of neurons, microglia and macrophages. These observations have demonstrated the production of this proinflammatory cytokine by spinal neurons, 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.

High expression of VRK1 is related to poor prognosis in glioma.

Vaccinia-related kinase 1 (VRK1) is a member of the vaccinia-related kinase (VRK) family of serine/threonine protein kinases, which phosphorylates several transcription factors and has been postulated to be involved in regulation of cell proliferation. However, it remains unclear whether aberrant expression of VRK1 is related to the development of glioma. In this study, we aimed to investigate the clinical significance of VRK1 expression in human glioma and its biological function in glioma cells. Western blot and immunohistochemical analysis revealed that VRK1 was highly expressed in glioma tissues and cell lines. In addition, the expression level of VRK1 was positively correlated with glioma pathological grade, as well as Ki-67 expression. Kaplan-Meier analysis revealed that patients with high VRK1 expression was associated with a poorer prognosis. To determine whether VRK1 could regulate the proliferation of glioma cells, we transfected glioma cells with interfering RNA target VRK1, then investigated cell proliferation with cell counting kit (CCK) -8, flow cytometry assays and colony formation analyses. Our results indicated that knockdown of VRK1 would inhibit the proliferation of glioma cells. Besides, reduced expression of VRK1 could induce the apoptosis of glioma cells. On the basis of these findings, we suggested that VRK1 might be a promising prognostic biomarker of glioma.

[The role of PKC to TNF-alpha induced beta-1, 4-galactosyltransferase-I expression in endothelial cells].

AIM: To investigate the role of protein kinase C (PKC) to regulate the expression of beta-1, 4-galactosyltransferase-I(beta-1, 4-GalT-I)and the influence to cytoskeleton and adherence ability of human umbilical vein endothelial cells(HUVECs) when stimulated by tumor necrosis factor (TNF-alpha). METHODS: Cultured HUVECs were pretreated by various PKC inhibitors or PMA, an excitomotor of PKC respectively for 30 minutes, then stimulated by TNF-alpha for 4 hours, beta-1, 4-GalT-I expression were detected by RT-PCR and Western blot, expression of beta-1, 4-galactosylated carbohydrate chains and cytoskeleton were assayed by immunofluorescence, adherence ability of HUVECs was observed by endothelial-monocyte cell adherence test. RESULTS: Up-regulated expression of beta-1, 4-GalT-I and beta-1, 4-galactosylated carbohydrate chains in HUVECs stimulated by TNF-alpha were suppressed by PKC inhibitors and increased by PMA. F-actin and beta-1, 4-GalT-I were partly co-localized in HUVECs, PKC inhibitor inhibited the effect of TNF-alpha on the distribution of f-actin and beta-1, 4-GalT-I. Adherence ability of HUVECs enhanced by LPS was significantly suppressed by PKC inhibitor. CONCLUSION: PKC signal transduction pathway may participate in regulating beta-1, 4-GalT-I expression in endothelial cells (EC) stimulated by TNF-alpha, furthermore, polytypes of PKC may participate in this regulating process; PKC might regulate cytoskeleton reorganization and adherence ability of EC through beta-1, 4-GalT-I during inflammation.

Identification and potential role of PSD-95 in Schwann cells.

Postsynaptic density-95 (PSD-95) is one of neuronal nitric oxide synthase (nNOS)-anchoring proteins and plays an important role in specifying the sites of reaction of nitric oxide (NO) in the nervous system. The present study aims to investigate the presence of PSD-95 in rat Schwann cells (SCs) and the association of PSD-95 and nNOS with serum-induced SCs proliferation. The expression of both molecules downregulated significantly after 48 h of serum deprivation, and increased gradually to the peak at 12 h, ultimately returned to the control level at 48 h after serum stimulation. The association of PSD-95 with nNOS was observed in Ki67 and BrdU-positive SCs. The selective nNOS inhibitor arrested the cell cycle progress and decreased the proliferating cell nuclear antigen (PCNA) levels. These findings suggested that PSD-95 and nNOS may collectively participate in the proliferation of SCs, providing further evidence for the role of NO during peripheral nerve regeneration.

Evaled expression of ICAM-1 and its ligands in the rat spinal cord following lipopolysaccharide intraspinal injection.

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.