Junling Yang

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.

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.

Elevated β1,4-galactosyltransferase-I induced by the intraspinal injection of lipopolysaccharide

Abstractβ1,4-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.

SSeCKS is a Suppressor in Schwann Cell Differentiation and Myelination

Src-suppressed protein kinase C substrate (SSeCKS) plays an important role in the differentiation process. In regeneration of sciatic nerve injury, expression of SSeCKS decreases, mainly in Schwann cells. However, the function of SSeCKS in Schwann cells differentiation remains unclear. We observed that SSeCKS was decreased in differentiated Schwann cells. In long-term SSeCKS-reduced Schwann cells, cell morphology changed and myelin gene expression induced by cAMP was accelerated. Myelination was also enhanced in SSeCKS-suppressed Schwann cells co-culture with dorsal root ganglion (DRG). In addition, we found suppression of SSeCKS expression promoted Akt serine 473 phosphorylation in cAMP-treated Schwann cells. In summary, our data indicated that SSeCKS was a negative regulator of myelinating glia differentiation.

Peripheral Nerve Lesion Induces an Up-regulation of Spy1 in Rat Spinal Cord

Spy1, as a member of the Speedy/RINGO family and a novel activator of cyclin-dependent kinases, was shown to promote cell cycle progression and cell survival in response to DNA damage. While its expression and roles in nervous system lesion and repair were still unknown. Here, we performed an acute sciatic nerve injury model in adult rats and studied the dynamic changes of Spy1 expression in lumbar spinal cord. Temporally, Spy1 expression was increased shortly after sciatic nerve crush and peaked at day 2. Spatially, Spy1 was widely expressed in the lumbar spinal cord including neurons and glial cells. While after injury, Spy1 expression was increased predominantly in astrocytes and microglia, which were largely proliferated. Moreover, there was a concomitant up-regulation of CDK2 activity and down-regulation of p27. Collectively, we hypothesized peripheral nerve injury induced an up-regulation of Spy1 in lumbar spinal cord, which was associated with glial proliferation.

Dynamic changes of ICAM-1 expression in peripheral nervous system following sciatic nerve injury

Abstract The injuries of the peripheral nerve are the results of trauma, disease, and surgical procedures that require section of peripheral nerves to gain access to the surgical site. Unlike elements of the central nervous system, peripheral nerves can regenerate when damaged. Recent studies provide evidence that intercellular adhesion molecule-1 (ICAM-1) is involved not only in inflammation, but also in cell recruitment during Wallerian degeneration after peripheral nerve injury. In our studies, two different injury models (crush and transect) for peripheral nerve regeneration were used. We investigated the dynamic changes of ICAM-1 expression in peripheral nervous system, including sciatic nerves and the dorsal root ganglia (DRG) following sciatic nerve injury. Double labeling demonstrated the presence of ICAM-1 within both Schwann cells and nerve blood vein endothelia cells in sciatic nerve after injury. In ipsilateral DRG, a few ICAM-1 signals were found in satellite cells, and ICAM-1 staining was mainly distributed in neuron. The results implied that ICAM-1 might be correlated with the function of Schwann cells about myelinogenesis and 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.

Involvement of Src-Suppressed C Kinase Substrate in Neuronal Death Caused by the Lipopolysaccharide-Induced Reactive Astrogliosis

Src-suppressed C kinase substrate (SSeCKS), a protein kinase C substrate, is a major lipopolysaccharide (LPS) response protein, regulating the inflammatory process. In the process of spinal inflammatory diseases by LPS intraspinal injection, expression of SSeCKS in the spinal cord was increased, mainly in active astrocytes and neurons. Induced SSeCKS was colabeled with terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick-end labeling (an apoptosis maker) in the late inflammation processes. These results indicated that SSeCKS might correlate with the inflammatory reaction and late neurodegeneration after LPS injection. A cell type-specific action for SSeCKS was further studied within C6 cells and PC12 cells. Knockdown of SSeCKS by small-interfering RNAs (siRNAs) blocked the LPS-induced inducible nitric oxide synthase (iNOS) expression in C6 cells, while overexpression SSeCKS enhanced iNOS expression. SSeCKS is also participated in regulation of PC12 cell viability. Loss of SSeCKS rescued PC12 cell viability, and excessive SSeCKS exacerbated the cell death upon conditioned medium and tumor necrosis factor-alpha exposure. This study delineates that SSeCKS may be important for host defenses in spinal inflammation and suggests a valuable molecular mechanism by which astrocytes modify neuronal viability during pathological states.