Linlin Sun

Expression of CRM1 in human gliomas and its significance in p27 expression and clinical prognosis.

OBJECTIVEGliomas are the most common type of primary intracranial tumor. Although tumor grade predicts the clinical course of most patients, molecular characteristics of individual tumors have emerged as important prognostic factors for patients with gliomas. Reduced expression of p27 protein is known as an independent prognostic marker in a large variety of cancers and is associated with an unfavorable prognosis. It is believed that phosphorylation of p27 on Ser10 has been shown to be required for the binding of CRM1, a carrier protein for nuclear export. This study assessed whether CRM1, Ser10-phosphorylated p27, and p27 correlated with each other, with glioma pathological stage, and with patient outcome. METHODSImmunohistochemical and Western blot analysis were performed in 70 cases of human gliomas and normal brain tissues. Survival analyses were performed using the Kaplan-Meier method. RESULTSHigh CRM1 expression (80% of cancer cell nuclei stained) was observed in 70 specimens and was related to the grade of malignancy. A strong inverse correlation was evident between p27 levels and both Ser10-phosphorylated p27 (P < 0.001) and CRM1 level (P < 0.001). We also reviewed each grade of tumors separately and investigated whether CRM1 expression predicted patient survival within each subgroup. In brief, CRM1 overexpression was significantly associated with overall survival (P < 0.001). CONCLUSIONThe current results showed that CRM1 and p27 expression were associated with glioma grade and that high CRM1 protein expression might be related to poor outcome.

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.

Changes in mRNA for CAPON and Dexras1 in adult rat following sciatic nerve transection.

Peripheral nerve transection has been implicated to cause a production of neuronal nitric oxide synthase (nNOS), which may influence a range of post-axotomy processes necessary for neuronal survival and nerve regeneration. Carboxy-terminal post synaptic density protein/Drosophila disc large tumor suppressor/zonula occuldens-1 protein (PDZ) ligand of neuronal nitric oxide synthase (CAPON), as an adaptor, interacts with nNOS via the PDZ domain helping regulate nNOS activity at postsynaptic sites in neurons. And Dexras1, a small G protein mediating multiple signal transductions, has been reported to form a complex with CAPON and nNOS. A role for the physiologic linkage by CAPON of nNOS to Dexras1 has suggested that NO-mediated activation of Dexras1 is markedly enhanced by CAPON. We investigated the changes in mRNA for CAPON, Dexras1 and nNOS in the sciatic nerve, dorsal root ganglia and lumbar spinal cord of adult rat following sciatic axotomy by TaqMan quantitative real-time PCR and in situ hybridization combined with immunofluorescence. Signals of mRNA for CAPON and Dexras1 were initially expressed in these neural tissues mentioned, transiently increased at certain time periods after sciatic axotomy and finally recovered to the basal level. It was also found that nNOS mRNA underwent a similar change pattern during this process. These results suggest that CAPON as well as Dexras1 may be involved in the different pathological conditions including nerve regeneration, neuron loss or survival and even pain process, possibly via regulating the nNOS activity or through the downstream targets of Dexras1.

Expression of Pirh2, a p27Kip1 ubiquitin ligase, in hepatocellular carcinoma: correlation with p27Kip1 and cell proliferation ☆

p53-Induced ring-H2 protein (Pirh2), a recently identified ubiquitin-protein ligase, interacts with p27(Kip1) to promote ubiquitination of p27(Kip1) independently of p53. High Pirh2 and low p27(Kip1) immunoreactivity are associated with a poor prognosis in several cancers, including resistant phenotypes. In the present study, we investigated the role of Pirh2 and p27(Kip1) in human hepatocellular carcinoma (HCC) progression. Immunohistochemical analysis was performed on formalin-fixed paraffin sections of 87 specimens. Statistical analysis showed that expression of Pirh2 was negatively related to p27(Kip1) expression (r = 0.787; P < .05), and Pirh2 expression correlated significantly with histologic grade (P < .001), venous invasion (P = .004), tumor size (P = .024), and the presence of multiple tumor-bearing lymph nodes (P = .017), whereas p27(Kip1) expression correlated significantly with histologic grade (P < .001), venous invasion (P = .048), and cirrhosis (P = .028). By Kaplan-Meier analysis, the survival curves of low versus high expressers of Pirh2 and p27(Kip1) showed significant separation (P < .01). Molecular interaction could be demonstrated between Pirh2 and p27(Kip1) in three HCC cell lines. In vitro, following release of two HCC cell lines from serum starvation, the expression of Pirh2 was upregulated, whereas p27(Kip1) was downregulated. Our results suggest that Pirh2 mediates the degradation of p27(Kip1) and participates in cell proliferation in human HCC. These findings provide a rational framework for further development of Pirh2 inhibitors as a novel class of anti-tumor agents.

Src suppressed C kinase substrate regulates the lipopolysaccharide-induced TNF-α biosynthesis in rat astrocytes

The protein kinase C (PKC) is known to be a critical component in the signaling cascades that lead to astrocyte-activation. To further understand the mechanism of PKC signaling in astrocyte-activation, we investigated the effect of SSeCKS, a PKC substrate, on LPS-induced cytokine expression in astrocytes by RT-PCR and enzymelinked immunosorbent assay. Exposure of the cells to LPS induced rapid translocation of SSeCKS to the perinuclear sides, ERK activation and pronounced TNF-α production, which can be inhibited by the PKC inhibitor Gö6983. By using siRNA knockdown of SSeCKS expression, LPS-induced signaling events were partly inhibited, including ERK activation, inducible TNF-α biosynthesis and secretion. These results suggest that SSeCKS is involved in the LPS-induced TNF-α expression in astrocytes mediated by PKC.

Lipopolysaccharide‐evoked activation of p38 and JNK leads to an increase in ICAM‐1 expression in Schwann cells of sciatic nerves

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‐α and interleukins IL‐1β 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.

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.

Involvement of Src-suppressed C kinase substrate in experimental autoimmune encephalomyelitis: A link between release of astrocyte proinflammatory factor and oligodendrocyte apoptosis

Src‐suppressed C kinase substrate (SSeCKS) is involved in inflammation in the central nervous system (CNS), and plays a role in control of cell signaling and cytoskeletal arrangement. However, the expression and function of SSeCKS and its function in multiple sclerosis (MS) and its common animal model, experimental autoimmune encephalomyelitis (EAE) remained to be elucidated. In the present study, we first reported that SSeCKS was remarkably increased in astrocytes of EAE rats in vivo. TNF‐α and NO were significantly induced in astrocytes stimulated with LPS/IFN‐γ in vitro, which was blocked in astrocytes transfected with SSeCKS siRNA. These results indicated that SSeCKS played a role in the production of TNF‐α and NO in astrocytes with inflammatory stimulation. As excessive release of TNF‐α and NO were major mediators in autoimmune diseases and correlated with oligodendrocyte cell death, we further investigated whether SSeCKS participated in oligodendrocyte apoptosis. Conditioned media (CM) from astrocytes treated with LPS/IFN‐γ decreased oligodendrocyte cell viability, while siRNA targeted to SSeCKS in astrocytes inhibited oligodendrocyte cell death. The results from antibody neutralization and NO inhibition suggested that the oligodendrocyte apoptosis may be due to the production of astrocyte‐derived proinflammatory factors (TNF‐α and NO). These findings revealed that there was a pathogenic interaction between SSeCKS expression in astrocytes and oligodendrocyte apoptosis. Understanding the mechanism of SSeCKS in the pathogenesis of EAE may contribute to the development of new therapeutic strategies against EAE and MS.

Lipopolysaccharide induces expression of SSeCKS in rat lung microvascular endothelial cell

Src-suppressed C kinase substrate (SSeCKS) plays a role in membrane-cytoskeletal remodeling to regulate mitogenesis, cell differentiation, and motility. Previous study showed that lipopolysaccharide (LPS) induced a selective and strong expression of SSeCKS in the vascular endothelial cells of lung. Here we show that LPS stimulation elevated expression of SSeCKS mRNA and protein in Rat pulmonary microvascular endothelial cell (RPMVEC). LPS potentiated SSeCKS phosphorylation in a time- and dose-dependent manner, and partly induced translocation of SSeCKS from the cytosol to the membrane after LPS challenge. The PKC inhibitor, Calphostin C, significantly decreased LPS-induced phosphorylation of SSeCKS, inhibited SSeCKS translocation and actin cytoskeleton reorganization after LPS challenge, suggesting that PKC may play a role in LPS-induced SSeCKS translocation and actin rearrangement. We conclude that SSeCKS is located downstream of PKC and that SSeCKS and PKC are both necessary for LPS-induced stress fiber formation.

Expression of β-1,4-Galactosyltransferase-I in Rat during Inflammation

Abstractβ-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 (sLex) and sulfated sLex. 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.