p38 MAPK regulates IL-1β induced IL-6 expression through mRNA stability in osteoblasts
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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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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
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Immunological Investigations, v. 33, n. 2, p. 213-233, 2004.
