Lihua Kang

Involvement of CLEC16A in activation of astrocytes after LPS treated.

CLEC16A, C-type lectin domain family 16, member A was recently found to be associated with inflation process in the autoimmune diseases. In this study, we elucidated the dynamic expression changes and localization of CLEC16A in lipopolysaccharide (LPS)-induced neuroinflammatory processes in adult rats. CLEC16A expression was strongly induced in active astrocytes in inflamed cerebral cortex. In vitro studies indicated that the up-regulation of CLEC16A may be involved in the subsequent astrocyte activation following LPS challenge. And Knock-down of CLEC16A in cultured primary astrocytes by siRNA showed that CLEC16A was required for the activation of astrocytes induced by LPS. Collectively, these results suggested CLEC16A may be important in host defense in astrocyte-mediated immune response. Understanding the cell signal pathway may provide a novel strategy against inflammatory and immune reaction in neuroinflammtion in CNS.

Lipopolysaccharide-evoked HSPA12B expression by activation of MAPK cascade in microglial cells of the spinal cord

The 70-kDa family of heat shock proteins (HSP70), in particular, plays a vital role in cellular protection and has been detected in various tissues subject to stress. HSPA12B is the newest member of the HSP70 family but is distinct from the HSP70 family. In this study, we elucidated the dynamic expression changes and localization of HSPA12B in lipopolysaccharide (LPS)-induced neuroinflammatory processes in adult rats. HSPA12B expression was strongly induced in active microglial cells in inflamed spinal cord. In vitro studies indicated that the up-regulation of HSPA12B may be involved in the subsequent microglia activation following LPS challenge. The elevated HSPA12B expression was regulated by activation of MAPK-p38 and ERK1/2 pathways, less contribution of the SAPK/JNK pathway in microglial cells. Collectively, these results suggested HSPA12B may be important for host defense in microglia-mediated immune response. Understanding the cell signal pathway may provide a novel strategy against inflammatory and immune reaction in neuroinflammtion in CNS.

The Role of HSPA12B in Regulating Neuronal Apoptosis

Heat shock protein A12B (HSPA12B) is the newest member of a recently defined subfamily of proteins distantly related to the 70-kDa family of heat shock proteins (HSP70) family. HSP70s play a crucial role in protecting cells, tissues, organs and animals from various noxious conditions. Here we studied the dynamic expression changes and localization of HSPA12B after middle cerebral artery occlusion (MCAO) with reperfusion induced ischemic insult processes in adult rats. Apoptosis, as indicated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, was also increased in the peri-ischemic cortex compared to non-ischemic hemisphere. The expression of HSPA12B was strongly induced in the ischemic hemisphere of MCAO reperfusion rats in vivo. In vitro studies indicated that the up-regulation of HSPA12B may be involved in oxygen-glucose deprivation-induced PC12 cell death. And knockdown of HSPA12B in cultured differentiated PC12 cells by siRNA showed that HSPA12B inhibited the expression of active caspase-3. Collectively, these results suggested that HSPA12B may be required for protecting neurons from ischemic insults.

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.

Involvement of CtBP2 in LPS-induced microglial activation

CtBP2 (C-terminal binding protein 2), which is widely expressed during developmental processes and differentiation, acts as a transcriptional repressor following recruitment to target promoters through repressors or other co-repressor proteins. In this study, we elucidated the dynamic expression changes and localization of CtBP2 in lipopolysaccharide (LPS)-induced neuroinflammatory processes in adult rats. CtBP2 expression was strongly induced in active glia cells (microglia and astrocytes) in inflamed spinal cord. In vitro studies indicated that the up-regulation of CtBP2 may be involved in the subsequent microglia activation following LPS exposure. And the knock-down of CtBP2 in microglia cell line HAPI by siRNA showed that CtBP2 increased the activation of microglia induced by LPS. Collectively, these results suggested CtBP2 may be important in host defense in microglia-mediated immune response. Understanding the cell signal pathway may provide a novel strategy against inflammatory and immune reaction in neuroinflammation in central nervous system.

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.

Müller Glia Cells Activation in Rat Retina After Optic Nerve Injury: Spatiotemporal Correlation with Transcription Initiation Factor IIB

Transcription initiation factor IIB (TFIIB) is an ideal factor to localize core promoters and plays a central role in the assembly of the pre-initiation complex. Previous studies showed that the assembly of TFIIB played an important role in rat ischemic brain injury. To elucidate the expression and possible functions of TFIIB in retina lesion and repair, we performed an optic nerve crush (ONC) model in adult rats. Western blot analysis and immunohistochemistry showed a significant upregulation of TFIIB in retina after ONC. Immunofluorescent labeling indicated that TFIIB was localized mainly in the Müller glia cells (MGCs); colocalization of TFIIB and proliferating cell nuclear antigen (PCNA) in the injured retina suggested that TFIIB might participate in MGCs proliferation. In addition, we also examined the expression of the retinal progenitor markers (Nestin and Pax6) whose changes were correlated with the expression of TFIIB. In vitro, we induced MGCs differentiation with brain nerve growth factor (BNGF) and found that TFIIB expression was increased in the differentiated process, which was collected with the expression of PCNA, Nestin, and Pax6. Additionally, knocking TFIIB down with siRNA inhibited the expression of PCNA, Nestin, and Pax6. Collectively, we hypothesized ONC-induced upregulation of TFIIB in the retina was associated with MGCs activation and differentiation.

Epigenetic Regulation of Werner Syndrome Gene in Age-Related Cataract

Purpose. To examine the promoter methylation and histone modification of WRN (Werner syndrome gene), a DNA repair gene, and their relationship with the gene expression in age-related cataract (ARC) lens. Methods. We collected the lenses after cataract surgery from 117ARC patients and 39 age-matched non-ARC. WRN expression, DNA methylation and histone modification around the CpG island were assessed. The methylation status of Human-lens-epithelium cell (HLEB-3) was chemically altered to observe the relationship between methylation and expression of WRN. Results. The WRN expression was significantly decreased in the ARC anterior lens capsules comparing with the control. The CpG island of WRN promoter in the ARC anterior lens capsules displayed hypermethylation comparing with the controls. The WRN promoter was almost fully methylated in the cortex of ARC and control lens. Acetylated H3 was lower while methylated H3-K9 was higher in ARC anterior lens capsules than that of the controls. The expression of WRN in HLEB-3 increased after demethylation of the cells. Conclusions. A hypermethylation in WRN promoter and altered histone modification in anterior lens capsules might contribute to the ARC mechanism. The data suggest an association of altered DNA repair capability in lens with ARC pathogenesis.

Expression Profiling of DNA Methylation and Transcriptional Repression Associated Genes in Lens Epithelium Cells of Age-Related Cataract

In our previous research, the formation and development of age-related cataract (ARC) is associated with DNA hypermethylation of some genes in lens epithelial cells (LECs). This study aimed to investigate the expression profile of DNA methylation- and transcriptional repression-associated genes in LECs of ARC. The expression levels of the genes were first evaluated by microarray analysis. The results were further confirmed by Quantitative Real-Time PCR (qRT-PCR) and Western blot assay. The mRNA and protein levels of 5 genes increased in LECs of ARCs compared with the controls. These data provided a global perspective on expression of DNA methylation- and transcriptional repression-associated genes. The study supports the notion that the epigenetic modification of macromolecules in LECs might contribute to ARC pathogenesis.

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.