p38 MAPK regulates IL-1β induced IL-6 expression through mRNA stability in osteoblasts
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Data
2004-06-08
Autores
Patil, Chetan
Zhu, Xinsheng
Rossa Júnior, Carlos [UNESP]
Kim, Young Joon
Kirkwood, Keith L.
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Resumo
Osteoblast-derived IL-6 functions in coupled bone turnover by supporting osteoclastogenesis favoring bone resorption instead of bone deposition. Gene regulation of IL-6 is complex occurring both at transcription and post-transcription levels. The focus of this paper is at the level of mRNA stability, which is important in IL-6 gene regulation. Using the MC3T3-E1 as an osteoblastic model, IL-6 secretion was dose dependently decreased by SB203580, a p38 MAPK inhibitor. Steady state IL-6 mRNA was decreased with SB203580 (2 μM) ca. 85% when stimulated by IL-1β (1-5 ng/ ml). These effects require de novo protein synthesis as they were inhibited by cycloheximide. p38 MAPK had minor effects on proximal IL-6 promoter activity in reporter gene assays. A more significant effect on IL-6 mRNA stability was observed in the presence of SB203580. Western blot analysis confirmed that SB203580 inhibited p38 MAP kinase, in response to IL-1β in a dose dependent manner in MC3T3-E1 cells. Stably transfected MC3T3-E1 reporter cell lines (MC6) containing green fluorescent protein (GFP) with the 3′untranslated region of IL-6 were constructed. Results indicated that IL-1β, TNFα, LPS but not parathyroid hormone (PTH) could increase GFP expression of these reporter cell lines. Endogenous IL-6 and reporter gene eGFP-IL-6 3′UTR mRNA was regulated by p38 in MC6 cells. In addition, transient transfection of IL-6 3′UTR reporter cells with immediate upstream MAP kinase kinase-3 and -6 increased GFP expression compared to mock transfected controls. These results indicate that p38 MAPK regulates IL-1β-stimulated IL-6 at a post transcriptional mechanism and one of the primary targets of IL-6 gene regulation is the 3′UTR of IL-6.
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Palavras-chave
ARE, Gene expression, GFP, IL-1β, IL-6, MRNA Stability, Osteoblasts, p38 MAPK, 4 (4 fluorophenyl) 2 (4 methylsulfinylphenyl) 5 (4 pyridyl)imidazole, cycloheximide, green fluorescent protein, interleukin 1beta, interleukin 6, lipopolysaccharide, messenger RNA, mitogen activated protein kinase p38, synaptophysin, tumor necrosis factor alpha, animal cell, bone turnover, cell strain, cell strain MC 3T3 E1, cell strain MC6, controlled study, cytokine release, dose response, gene control, genetic analysis, genetic transfection, mouse, nonhuman, osteoblast, osteoclast, osteolysis, priority journal, protein expression, protein processing, protein synthesis, publication, regulatory mechanism, reporter gene, RNA stability, steady state, Western blotting, 3' Untranslated Regions, Animals, Base Sequence, Cell Line, Enzyme Inhibitors, Gene Expression Regulation, Genetic Vectors, Interleukin-1, Interleukin-6, Mice, Molecular Sequence Data, p38 Mitogen-Activated Protein Kinases, Promoter Regions (Genetics), Recombinant Proteins, RNA Stability, RNA, Messenger, Signal Transduction
Como citar
Immunological Investigations, v. 33, n. 2, p. 213-233, 2004.