ABSTRACT
p38 mitogen-activated protein kinase (MAPK) stabilises pro-inflammatory mediator mRNAs by inhibiting AU-rich element (ARE)-mediated decay. We show that in bone-marrow derived murine macrophages tristetraprolin (TTP) is necessary for the p38 MAPK-sensitive decay of several pro-inflammatory mRNAs, including cyclooxygenase-2 and the novel targets interleukin (IL)-6, and IL-1alpha. TTP(-/-) macrophages also strongly overexpress IL-10, an anti-inflammatory cytokine that constrains the production of the IL-6 despite its disregulation at the post-transcriptional level. TTP directly controls IL-10 mRNA stability, which is increased and insensitive to inhibition of p38 MAPK in TTP(-/-) macrophages. Furthermore, TTP enhances deadenylation of an IL-10 3'-untranslated region RNA in vitro.
Subject(s)
Inflammation Mediators/metabolism , Interleukin-10/genetics , Macrophages/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Tristetraprolin/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Base Sequence , In Vitro Techniques , Interleukin-10/antagonists & inhibitors , Interleukin-12 Subunit p40/biosynthesis , Interleukin-6/biosynthesis , MAP Kinase Signaling System , Mice , Mice, Inbred C57BL , Mice, Knockout , RNA Stability , Tristetraprolin/deficiency , Tristetraprolin/geneticsABSTRACT
Transcription factors belonging to the NF-kappaB family regulate inflammation by inducing pro-inflammatory molecules (e.g. interleukin (IL)-8) in response to cytokines (e.g. tumor necrosis factor (TNF) alpha, IL-1) or other stimuli. Several negative regulators of NF-kappaB, including the ubiquitin-editing enzyme A20, participate in the resolution of inflammatory responses. We report that Cezanne, a member of the A20 family of the deubiquitinating cysteine proteases, can be induced by TNFalpha in cultured cells. Silencing of endogenous Cezanne using small interfering RNA led to elevated NF-kappaB luciferase reporter gene activity and enhanced expression of IL-8 transcripts in TNFalpha-treated cells. Thus we conclude that endogenous Cezanne can attenuate NF-kappaB activation and the induction of pro-inflammatory transcripts in response to TNF receptor (TNFR) signaling. Overexpression studies revealed that Cezanne suppressed NF-kappaB nuclear translocation and transcriptional activity by targeting the TNFR signaling pathway at the level of the IkappaB kinase complex or upstream from it. These effects were not observed in a form of Cezanne that was mutated at the catalytic cysteine residue (Cys209), indicating that the deubiquitinating activity of Cezanne is essential for NF-kappaB regulation. Finally, we demonstrate that Cezanne can be recruited to activated TNFRs where it suppresses the build-up of polyubiquitinated RIP1 signal adapter proteins. Thus we conclude that Cezanne forms a novel negative feedback loop in pro-inflammatory signaling and that it suppresses NF-kappaB activation by targeting RIP1 signaling intermediaries for deubiquitination.
Subject(s)
Endopeptidases/metabolism , Gene Expression Regulation, Enzymologic , Inflammation , NF-kappa B/metabolism , Nuclear Pore Complex Proteins/metabolism , RNA-Binding Proteins/metabolism , Ubiquitin/metabolism , DNA-Binding Proteins , Endothelial Cells/cytology , Humans , Interleukin-8/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Lysine/chemistry , Models, Biological , Mutation , Nuclear Proteins/metabolism , Receptors, Tumor Necrosis Factor/metabolism , Signal Transduction , Tumor Necrosis Factor alpha-Induced Protein 3ABSTRACT
Heat shock protein (HSP) 27 has long been known to be a component of the p38 mitogen-activated protein kinase (MAPK) signaling pathway. p38 MAPK has important functions in the inflammatory response, but the role of HSP27 in inflammation has remained unknown. We have used small interfering RNAs to suppress HSP27 expression in HeLa cells and fibroblasts and found that it is required for pro-inflammatory cell signaling and the expression of pro-inflammatory genes. HSP27 is needed for the activation by interleukin (IL)-1 of TAK1 and downstream signaling by p38 MAPK, JNK, and their activators (MKK-3, -4, -6, -7) and IKKbeta. IL-1-induced ERK activation appears to be independent of HSP27. HSP27 is required for both IL-1 and TNF-induced signaling pathways for which the most upstream common signaling protein is TAK1. HSP27 is also required for IL-1-induced expression of the pro-inflammatory mediators, cyclooxygenase-2, IL-6, and IL-8. HSP27 functions to drive cyclooxygenase-2 and IL-6 expression by augmenting the activation of the kinase downstream of p38 MAPK, MK2, resulting in stabilization of cyclooxygenase-2 and IL-6 mRNAs. The mechanism may not occur in cells of myeloid lineage because HSP27 protein was undetectable in human monocytes and murine macrophages.
Subject(s)
Gene Expression Regulation , Heat-Shock Proteins/physiology , Inflammation/genetics , MAP Kinase Kinase Kinases/metabolism , Neoplasm Proteins/physiology , Signal Transduction , Cells, Cultured , Cyclooxygenase 2/genetics , Fibroblasts , HSP27 Heat-Shock Proteins , HeLa Cells , Humans , Interleukin-1/metabolism , Interleukin-6/genetics , Mitogen-Activated Protein Kinases/metabolism , Molecular Chaperones , RNA Stability , Tumor Necrosis Factor-alpha/metabolismABSTRACT
COX-2 (cyclo-oxygenase-2) mRNA is degraded rapidly in resting cells, but is stabilized by the mitogen-activated protein kinase p38 signalling pathway in response to pro-inflammatory stimuli. A conserved ARE (AU-rich element) of the COX-2 3' untranslated region, CR1 (conserved region 1), acts as a potent instability determinant, and mediates stabilization in response to p38 activation. A detailed structural and functional analysis of this element was performed in an attempt to identify RNA-binding proteins involved in the regulation of COX-2 mRNA stability. Destabilization of a beta-globin reporter mRNA was dependent upon two distinct AREs within CR1, each containing three copies of the sequence AUUUA. CR1 was shown to bind AUF-1 [ARE/poly(U)-binding/degradation factor-1] and/or AUF-2, HuR (Hu antigen R), TTP (tristetraprolin) and FBP1 (far-upstream-sequence-element-binding protein 1), yet these factors did not appear to account for the effects of CR1 upon mRNA stability. Mutant sequences were identified that were incapable of destabilizing a reporter mRNA, yet showed unimpaired binding of FBP1 and AUF-1 and/or -2. TTP was absent from the HeLa cell line used in this analysis. Finally, RNA interference experiments argued against a prominent role for HuR in the CR1-mediated regulation of mRNA stability. We conclude that at least one critical regulator of COX-2 mRNA stability is likely to remain unidentified at present.
Subject(s)
Antigens, Surface , Conserved Sequence/physiology , DNA-Binding Proteins/genetics , Heterogeneous-Nuclear Ribonucleoprotein D/genetics , Immediate-Early Proteins/genetics , Isoenzymes/genetics , Prostaglandin-Endoperoxide Synthases/genetics , RNA-Binding Proteins/genetics , Regulatory Sequences, Ribonucleic Acid/physiology , Repressor Proteins/genetics , 3' Untranslated Regions/genetics , 3' Untranslated Regions/metabolism , 3' Untranslated Regions/physiology , Base Composition , Base Sequence , Cell Line, Tumor , Conserved Sequence/genetics , Cyclooxygenase 2 , DNA Mutational Analysis , ELAV Proteins , ELAV-Like Protein 1 , Electrophoretic Mobility Shift Assay , HeLa Cells , Heterogeneous Nuclear Ribonucleoprotein D0 , Heterogeneous-Nuclear Ribonucleoprotein D/metabolism , Humans , Immediate-Early Proteins/metabolism , Isoenzymes/physiology , Membrane Proteins , Molecular Sequence Data , Prostaglandin-Endoperoxide Synthases/physiology , RNA Stability/genetics , RNA Stability/physiology , RNA, Neoplasm/genetics , RNA, Neoplasm/metabolism , RNA, Neoplasm/physiology , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/physiology , Regulatory Sequences, Ribonucleic Acid/genetics , Structure-Activity Relationship , TristetraprolinABSTRACT
The AU-rich element (ARE) is an important instability determinant for a large number of early-response-gene mRNAs. AREs also mediate the stabilization of certain pro-inflammatory mRNAs, such as tumour necrosis factor (TNF)-alpha and cyclo-oxygenase-2 (COX-2), in response to inflammatory stimuli. To understand how AREs control mRNA stability, it is necessary to identify trans-acting factors. We have purified a new ARE-binding protein and identified it as CArG box-binding factor-A (CBF-A). The amino acid sequence of CBF-A is highly similar to that of the ARE-binding protein AUF1. Recombinant CBF-A bound the COX-2 and TNF-alpha AREs, but not a non-specific control RNA. In contrast, in an electrophoretic-mobility-shift assay (EMSA) of crude RAW 264.7 macrophage-like cell extracts, an antiserum that recognizes both AUF1 and CBF-A failed to supershift complexes formed on the TNF-alpha ARE, but did supershift a complex specific for the COX-2 ARE. CBF-A exists as two isoforms, p37 and p42, that differ by a 47-amino-acid insertion close to the C-terminus. By expressing epitope-tagged isoforms of CBF-A it was shown that the p42 isoform binds the COX-2 ARE in EMSA of crude cell extracts. In a HeLa-cell tetracycline-regulated reporter system, overexpression of the p42 CBF-A isoform resulted in stabilization of a COX-2 ARE reporter mRNA. Epitope-tagged p42 CBF-A expressed in HeLa cells co-immunoprecipitated with endogenous COX-2 mRNA, but not glyceraldehyde-3-phosphate dehydrogenase mRNA, as shown by reverse-transcription PCR. The similarity between CBF-A and AUF1 suggests that CBF-A could be re-named AUF2.
