A Preliminary Study On The Role Of SSeCKS In Astrocyte Activation

Background and ObjectiveA dysregulated inflammatory response in the central nervous system (CNS), characterized by activation of glia cells and infiltration of peripheral inflammatory cells, lies in the central of many neuropathological conditions. As the most abundant cell type in the CNS, astrocytes maintain the homeostatic environment of the CNS and play an important role in regulation of immune responses by functioning as a source of proinflammatory cytokines. These cytokines act on the CNS to induce expression of multiple genes such as chemokines, other cytokines, adhesion molecules, that drive and regulate inflammatory and bactericidal effector pathways, however, overexpression of these bioactive molecules results in a shape change (stellation) resembling a reactive gliosis and the formation of the inhibiting environment which blocks axonal outgrowth. Thus modulation of the inflammatory response is equally important in order to ensure preservation of immune homeostasis.A set of intracellular signaling cascades are required to these activation process, recent studies confirmed the critical role of protein kinase C (PKC) in regulating astrocytes activation, including cell differentiation, proliferation, migration, especially biosynthesis. These functions were achieved by locally anchoring the kinases to specific organelles or subcellular structures.It is thought that the proteins such as protein kinase A anchoring proteins (AKAPs) and several classes of protein kinase C targeting proteins serve as linker proteins to position certain kinases to these immobile structures. PKC-targeting proteins such as substrates interacting with C kinase and receptors of activated C kinases similarly localize PKC. Example is rodent SSeCKS, which was originally identified as a negative mitotic regulator, is a major in vivo substrate of PKC. As a member of the AKAP family, the ability of its regulatory subunit to bind to PKC, protein kinase A, calmodulin, andβ2-adrenergic receptors, suggests its function to assemble a multiprotein signaling complex at the sites of its localization. Evidence exists that the phosphorylation by PKCαaffects the intracellular distribution of SSeCKS. Recently, SSeCKS has been shown to be a major lipopolysaccharide (LPS) response protein which markedly upregulated in several organs, including lung, heart, kidney, brain et al, indicating a possible role of SSeCKS in inflammatory process. More recently, SSeCKS is shown to be expressed in astrocytes and provide a stabilizing signal for BBB integrity under physiological conditions. Such observations have led to the proposition that SSeCKS might be a regulator in PKC-mediated astrocytes activation.In the current study, we examined SSeCKS expression in various regions of adult rat brain after intraperitoneal (IP) administration of the endotoxin, and the possible role of the molecule in activation of astrocytes.Methods 1. The model was preparated in rat by LPS injection intraperitoneally.2. The brain tissue content of SSeCKS mRNA and protein were assayed by Real-time PCR and western blot, and the location of SSeCKS protein in the brain tissues were detected by double immumofluorescence.3. Cultured astrocytes were stimulated by LPS and IL-1β. The expression of SSeCKS was detected by Real-time PCR and western blot analysis. Immunofluorescent staining with confocal laser-scanning fluorescence microscope was used to observe the effects of LPS, as well as PKC activity, on the distribution of SSeCKS.4. SSeCKS-siRNA and nonspecific-siRNA plasmids were transfected into astrocytes. Methods of RT-PCR, Western blot, ELISA, FACS (fluorescence-activated cell sorting), BrdU staining and immunoprecipitation were used to investigate the role of SSeCKS in biosynthesis and proliferation of astrocytes.Results1. 1) Administration of LPS resulted in remarkable upregulation of SSeCKS, as well as its phosphorylation in rat brain tissues.2) In the control group, SSeCKS staining dispersed in the brain tissue, and was intensive in neuronal cell bodies. LPS injection resulted in obvious increase of SSeCKS-immunoreactivity as compared to the control, especially in astrocytes.3) In vitro, stimulation such as LPS, IL-1βand TNF-αlead to the alteration of both SSeCKS expression and phosphorylation. Besides, SSeCKS translocated rapidly to the perinucleus and the foot processes of the membrane, these effects could be blocked by pretreatment with PKC inhibitors.2. 1) PKC-ERK1/2 signal transduction pathway is involved in LPS-induced TNF-αproduction in astrocytes.2) Transient transfection of SSeCKS-siRNA suppressed LPS-stimulated TNF-αexpression and secretion.3) SSeCKS-siRNA treatment resulted in significant decrease in LPS-induced ERK phosphorylation.3. 1) TNF-αevoked rapid proliferation of astrocytes. This effect was substantially inhibited by pretreatment with classic PKC inhibitor G?6976 and completely abolished by broad-spectrum PKC inhibitor G?6983 or PI3K inhibitor LY294002. The novel PKCδinhibitor rotterin had no significant effect on TNF-αinduced astrocyte-proliferation.2) There is a high degree of correlation (r = 0.905, P = 0.02) between astrocyte-proliferation and relative SSeCKS phosphorylation (pSSeCKS value).3) Inhibition of PI3K activity had no effect on PKC-mediated SSeCKS activation, and pretreatment of astrocytes with PKC inhibitor didn't affect TNF-αinduced Akt phosphorylation.Conclusions1. SSeCKS dispersely distributes in rat brain tissues. intraperitoneal administration of LPS alters SSeCKS expression and its phosphorylation, indicating that SSeCKS is an inflammatory response molecule participating in the inflammatory process of CNS.2. Exposure of astrocyte cultures to inflammatory factors results in obviously changes of SSeCKS expression and translocation. These effects are regulated by PKC, suggesting that SSeCKS is a down stream regulator in PKC mediated signal transduction.3. PKC-ERK1/2 signalling pathway was involved in LPS-induced TNF-αproduction in astrocytes.4. Down regulation of SSeCKS expression decreases LPS-induced ERK activation and TNF-αproduction in astrocytes, indicating that SSeCKS may serves as an intermediary mediator in regulating LPS-induced PKC-ERK1/2 signalling in astrocytes.5. SSeCKS participates in TNF-α-induced astrocyte-proliferation. The rapid phosphorylation of SSeCKS after TNF-αstimulation may contribute to this process.6. Activation of PKC-SSeCKS and PI3K-Akt contribute to TNF-α-induced astrocyte-proliferation. Although PKC and PI3K are relatively independent, crossing linking might exist between these two signal transduction pathways.