p38 MAPK Regulates IL‐1β Induced IL‐6 Expression Through mRNA Stability in Osteoblasts

Abstract

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 (2μM) ca. 85% when stimulated by IL-1β (1 5ng/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 was 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.

IL-1β potently stabilizes IL-6 mRNA in human astrocytes

“…This mRNA stabilization translates into a very potent synergistic IL-6 protein expression. Based on pharmacological inhibitor data, the stabilization process seems to depend on PKC, but not on p38, contrary to the earlier described modes of IL-6 mRNA stabilization [12,[15][16][17]. Moreover, the prototypical mRNA-stabilizing protein HuR is not involved.…”

“…has been previously demonstrated to stabilize multiple mRNA species, such as COX-2 [42,43,[62][63][64], IL-8 [56] and NGF [65,66]. Importantly, it has already been reported that IL-1β stabilizes IL-6 mRNA in osteoblasts [16,35], fibroblasts [9], myofibroblasts [14], astrocytes [36] and fibroblast-like synoviocytes [12], although relatively little was known about the molecular mechanisms underlying this stabilization. Moreover, other studies found no stabilization of IL-6 mRNA after IL-1β induction, but instead found that TNF-α [11] or cAMP [67] were the stabilizing agents in this instance, indicating the cell type specificity of IL-1β-mediated mRNA stabilization.…”

“…Relatively little is known about the signaling pathways responsible for IL-6 mRNA stabilization. It has been found by multiple groups that the mitogen-activated protein kinase (MAPK) p38 stabilizes IL-6 mRNA [12,[15][16][17][18]. Another kinase often associated with mRNA stabilization is protein kinase C (PKC).…”

“…It has been shown that IL-1β stabilizes multiple mRNA species, including IL-6, in a variety of cell types [9,12,14,16,35,36]. A paradigm commonly used to study effects on mRNA stabilization is the blockade of new transcription by a transcriptional inhibitor, such as actinomycin D, followed by quantification of the mRNA levels at several time points after inhibition of transcription [37].…”

“…Importantly, both can be activated by IL-1β and have been convincingly implicated in COX-2 mRNA stabilization [21,31,[41][42][43][44][45][46]. Moreover, in multiple studies, IL-1β-induced mRNA stabilization of IL-6 was shown to be crucially dependent on p38 [12,16], whereas dependence on PKC has not yet been described. Therefore, we used pharmacological inhibitors of p38 and PKC to investigate if they are involved in the mRNA-stabilizing effect of IL-1β in our experimental system.…”