title: The role of TNF-alpha and its receptors in the production of Src-suppressed C kinase substrate by rat primary type-2 astrocytes.
abstract: Src-suppressed C kinase substrate (SSeCKS), an in vivo and in vitro protein kinase C substrate, is a major lipopolysaccharide (LPS) response protein which markedly upregulated in several organs, including brain, lung, heart, kidney, etc., indicating a possible role of SSeCKS in inflammatory process. In the central nervous system (CNS), astrocytes play a pivotal role in immunity as immunocompetent cells by secreting cytokines and inflammatory mediators, there are two types of astrocytes. Type-1 astrocytes can secrete TNF-alpha when stimulated with lipopolysaccharide (LPS), while the responses of type-2 astrocytes during inflammation are unknown. So we examined the expression change of SSeCKS mRNA in type-2 astrocytes after exposure to TNF-alpha and LPS. Real-time PCR showed that TNF-alpha or LPS affected SSeCKS mRNA expression in a time- and dose-dependent manner. Now that LPS induces SSeCKS expression in type-2 astrocytes and type-1 astrocytes are well known to play a pivotal role in immunity, we compared SSeCKS mRNA expression in type-1 astrocytes with type-2 astrocytes after LPS stimulation. Real-time PCR showed that SSeCKS mRNA level was higher in normal untreated type-2 astrocytes than that in normal untreated type-1 astrocytes, increased significantly after 0.1-100 ng/ml LPS stimulation in type-2 astrocytes, but increased weakly after 10-100 ng/ml LPS stimulation in type-1 astrocytes. By using siRNA knockdown of SSeCKS expression, LPS-induced TNF-alpha synthesis and secretion in type-2 astrocytes were partly inhibited, which indicated that SSeCKS played a role in the TNF-alpha biosynthesis in type-2 astrocytes during the stimulation with LPS. RT-PCR analysis revealed that TNFR1 and TNFR2 were present in normal untreated type-2 astrocytes and that TNF-alpha, TNFR1 and TNFR2 increased in type-2 astrocytes after exposure to TNF-alpha or LPS. Immunocytochemistry showed that TNFR1 was expressed in the cytoplasm, nucleus and processes of normal untreated type-2 astrocytes and distributed mainly in the cytoplasm and processes after exposure to LPS. TNFR2 was mainly expressed in the nucleus of normal untreated type-2 astrocytes and distributed mainly in the processes of type-2 astrocytes after exposure to LPS. Both anti-TNFR1 and anti-TNFR2 antibodies suppressed SSeCKS mRNA expression induced by TNF-alpha or LPS. From these results, we conclude that TNF-alpha signaling via both TNFR1 and TNFR2 translocated from nucleus to cytoplasm or processes is sufficient to induce SSeCKS mRNA. In addition, we observed that not only exogenous TNF-alpha but also TNF-alpha produced by type-2 astrocytes affected SSeCKS mRNA production in type-2 astrocytes. These results suggest that an autocrine loop involving TNF-alpha contributes to the production of SSeCKS mRNA in response to inflammation. In addition, SSeCKS production was also drastically suppressed by U0126 (ERK inhibitor), SB203580 (p38 inhibitor), or SP600125 (SAPK/JNK inhibitor), which indicated that type-2 astrocytes which regulated SSeCKS expression after LPS stimulation were via ERK, SAPK/JNK, and P38MAP kinase signal pathway.
authors.affiliation: Jiangsu Key Laboratory of Neuroregeneration, Nantong University, Nantong, Jiangsu Province, PR China.
authors.forename: Meijuan
authors.initials: M
authors.lastname: Yan
authors.forename: Chunlin
authors.initials: C
authors.lastname: Xia
authors.forename: Chun
authors.initials: C
authors.lastname: Cheng
authors.forename: Xiaoyi
authors.initials: X
authors.lastname: Shao
authors.forename: Shuqiong
authors.initials: S
authors.lastname: Niu
authors.forename: Haiou
authors.initials: H
authors.lastname: Liu
authors.forename: Aiguo
authors.initials: A
authors.lastname: Shen
mesh_terms: D054758:A Kinase Anchor Proteins / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000145:classification / Q000187:drug effects / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000378:metabolism; D005732:Gangliosides / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D018124:Receptors, Tumor Necrosis Factor / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*; D001613:beta 2-Microglobulin / Q000235:genetics / Q000378:metabolism
chemical_list: D054758:A Kinase Anchor Proteins; C102361:Akap12 protein, rat; D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors; D005732:Gangliosides; D005904:Glial Fibrillary Acidic Protein; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha; D001613:beta 2-Microglobulin; C062875:ganglioside A2B5