Julie M. Tebo

Heterogeneity in control of mRNA stability by AU-rich elements.

AU-rich elements (AREs), located in the 3′-untranslated region of unstable cytokine and chemokine mRNAs, promote rapid decay of otherwise stable mRNAs and may mediate selective mRNA stabilization in response to stimulation with interleukin-1 (IL-1). AREs vary considerably, however, in both size and sequence context. To assess the heterogeneity involved in control of mRNA stability by ARE motifs, human mRNA sequences from IL-1α-stimulated HEK293 cells and T98G cells were screened for either instability or stability using both cDNA (950 ARE containing sequences) and Affymetrix oligonucleotide (U95Av2 GeneChip) array analysis. Although ARE-containing mRNAs exhibited a broad range of stability, IL-1α promoted stability in a subset of mRNAs that were unstable when transcriptionally induced by tumor necrosis factor α. Stabilization of granulocyte/macrophage-colony stimulating factor and IL-8 mRNAs by IL-1α was achieved only after 2 h of stimulation, required ongoing protein synthesis, and depended on the activation of p38 MAPK. In contrast, stabilization of Gro3 mRNA in response to IL-1α was achieved immediately and was insensitive to inhibitors of protein synthesis and p38 MAPK activation. In concert, these findings demonstrate that ARE sequences are functionally heterogeneous; only a subset of unstable mRNAs is sensitive to stabilization by IL-1α. Moreover, IL-1α promotes stabilization of unstable mRNAs through distinct mechanistic pathways that distinguish between specific mRNA sequences.

Regulation of chemokine expression by antiinflammatory cytokines.

The antiinflammatory cytokines IL-4 and IL-10 are well recognized as important negative regulators of proinflammatory gene expression in mononuclear phagocytes. The intracellular mechanisms that mediate these responses appear to be multifactorial. IL-4 is able to suppress transcriptional activation of IFNγ-and LPS-responsive genes; IL-4 activated STAT6 is required for the suppressive activity of IL-4. IL-4 and STAT6 appear to suppress trascription of select proiflammatory genes through the ability of STAT6 to sequenster coactivator molecules that may be required for the transcriptional action of STAT1 and NFκB. In contrast, IL-10 suppresses the expression of genes induced in lipopolysaccharide (LPS)-stimulated macrophages by modulating both the transcription and stability of specific mRNAs. AU-rich nucleotide sequence elements in the 3′ untranslated region IL-10-sensitive genesconfer sensitivity to IL-10-mediated destabilization. Thus mechanisms through which IL-10 and IL-4 acttodampen inflammatory responses are mechanistically distinct and involve diverse intracellular signaling pathways.

Regulation of macrophage gene expression by pro- and anti-inflammatory cytokines

The anti-inflammatory cytokines IL-4 and IL-10 are well recognized as important negative regulators of proinflammatory gene expression in mononuclear phagocytes. The intracellular mechanisms which mediate these responses appear to be multifactorial. IL-4 is able to markedly suppress transcriptional activation of IFNγ-responsive genes and the promoter sequences required for both IFNγ and IL-4 sensitivity are identical. IFNγ-activated STAT1 and IL-4-activated STAT6 can both form complexes on the same regulatory sequence element; while STAT1 functions to promote transcription, STAT6 is inactive. STAT6 is, however, required for the suppressive activity of IL-4. In this model, IL-4 appears to suppress IFNγ-inducible proinflammatory gene expression through the ability of STAT6 to compete with STAT1 for occupancy of promoter sites necessary for IFNγ-induced transcriptional initiation. In a second model, IL-10 suppresses the expression of genes induced in LPS-stimulated macrophages through a pathway involving destabilization of specific mRNAs. We have demonstrated that nucleotide sequences in the 3′-untranslated region of an IL-10-sensitive gene can both destabilize a stable reporter mRNA (CAT) and confer sensitivity to IL-10-mediated destabilization. Deletion and site-specific mutagenesis have mapped this to an AU-rich sequence motif similar to that found in many cytokine and growth factor mRNAs. IL-10 is able to modulate the activity of proteins capable of binding to this sequence and one or more of these may regulate the rate of mRNA degradation. Thus mechanisms through which IL-10 and IL-4 act to dampen inflammatory responses are mechanistically distinct and involve diverse intracellular signaling pathways.

Regulation of Chemokine mRNA Stability by Lipopolysaccharide and IL-10

IL-10 has been reported to inhibit the expression of LPS-induced proinflammatory cytokines and chemokines by altering the rate of specific mRNA decay although the molecular target(s) for its action remain unknown. In the present study, using primary peritoneal exudate macrophages and a cell culture model in which a tetracycline-responsive promoter controls transcription of CXC ligand 1 (KC) mRNA, we demonstrate that LPS promotes a time-dependent increase in KC mRNA stability. Although IL-10 had no direct effect on mRNA decay, this treatment antagonized the stabilizing action of LPS. The mechanisms involved were further explored using a cell-free mRNA degradation system. A 5′-capped, polyadenylated in vitro transcript derived from the 3′-untranslated region of KC mRNA exhibited time-dependent decay in the presence of protein extracts prepared from untreated RAW264.7 macrophages. Extracts prepared from LPS-treated RAW264.7 cells had reduced decay activity and this change was antagonized if the cells were costimulated with IL-10. A substrate in which the AU-rich element motifs were mutated exhibited minimal decay that did not vary using extracts prepared from cells treated with LPS or LPS and IL-10. A nonadenylated RNA substrate was also degraded and that activity was diminished by LPS. In concert, these findings demonstrate that KC mRNA stability is regulated by LPS-induced alterations in activities that govern both deadenylation and degradation of the mRNA body. The effects of IL-10 on KC mRNA stability reflect antagonism of the response to LPS.

IL-10 suppresses LPS-induced KC mRNA expression via a translation-dependent decrease in mRNA stability.

This study examines the mechanism of interleukin‐10 (IL‐10)‐mediated suppression of KC chemokine gene expression in mouse macrophages. Suppression of KC mRNA levels by IL‐10 occurred late in the time course of response to lipopolysaccharide (LPS). Equivalent IL‐10‐mediated suppression was observed when the agent was added 1 h before, simultaneous with, or 1 h after LPS. IL‐10 did not inhibit KC gene transcription but rather produced a decrease in the stability of KC mRNA. The suppressive action of IL‐10 was prevented in macrophages that were also treated with inhibitors of protein synthesis even when added 2 h after LPS and IL‐10. These results suggest that IL‐10 acts to destabilize LPS‐induced KC mRNA through a process that depends on coincident KC mRNA translation. J. Leukoc. Biol. 64: 33–39; 1998.

Chemokine and chemoattractant receptor expression : post-transcriptional regulation

The magnitude and character of the inflammatory process are determined in part via the trafficking of leukocytes into sites of injury and infection, and this process depends on proper control of the expression of genes encoding chemoattractant peptides and their receptors. Although these controls operate at multiple mechanistic levels, recent evidence indicates that post‐transcriptional events governing the half‐life of select mRNAs are important determinants. Adenine‐uridine rich elements (AREs) located within 3′ untranslated regions (UTRs) confer constitutive mRNA instability and in some cases, stabilization following stimulation by ligands of the Toll‐IL‐1 receptor (TIR) family. Although the importance of AREs in determining activity and mRNA half‐life is well‐recognized, the mechanistic scope and diversity remain poorly understood. Using the mouse KC or CXCL1 gene as a model, we have demonstrated that the abundance of mRNA and protein produced during an inflammatory response depends on multiple mechanistically distinct AREs present in the 3′ UTR of the mRNA. The mRNA encoding the receptor for N‐terminal formyl‐methionine‐containing peptides is also unstable and subject to stabilization in response to TIR ligands. These two models can, however, be readily distinguished from one another on the basis of specific stimulus sensitivity and the signaling pathways, through which such stimuli couple to the control of mRNA decay. These models demonstrate the substantial diversity operative in the post‐transcriptional regulation of inflammatory gene expression.

Oxidized LDL modulates activation of NFkappaB in mononuclear phagocytes by altering the degradation if IkappaBs.

Oxidized low density lipoprotein (oxLDL) is known to alter the expression of inflammatory gene products in mononuclear phagocytes. The mechanisms involved in this effect were studied by examining the activation of nuclear factor κB (NFκB), a transcription factor known to be important in controlling the expression of such genes. Pretreatment of peritoneal macrophages with oxLDL modulated the activation of NFκB in response to either lipopolysaccharide (LPS) or the combination of interferon‐γ (IFN‐γ) and interleukin‐2 (IL‐2). In macrophages pretreated with oxLDL the nuclear translocation of Rel family members (RelA and c‐Rel) is delayed (LPS) or markedly diminished (IFN‐γ/IL‐2) and results in delayed or reduced appearance of κB binding activity within the nucleus. These changes in NFκB activation result from alterations in the stimulus‐dependent degradation of IκBα and IκBβ. The effects of oxLDL on NFκB activation depend both on the degree of LDL oxidation (most potent with extensive oxidation) and on the time of exposure of the cells to the lipoprotein preparation (a minimal exposure of 6 h is required before inhibitory effects are observed). The modulation of IκB/NFκB function in cells exposed to oxLDL appears to be responsible for previously reported effects of oxLDL on chemoattractant cytokine gene expression where both inhibition and delay of such stimulus‐dependent events has been observed. J. Leukoc. Biol. 64: 667–674; 1998.

Toll IL-1 Receptors Differ in Their Ability to Promote the Stabilization of Adenosine and Uridine-Rich Elements Containing mRNA

Several ligands for Toll IL-1R (TIR) family are known to promote stabilization of a subset of short-lived mRNAs containing AU-rich elements (AREs) in their 3′ untranslated regions. It is now evident however, that members of the TIR family may use distinct intracellular signaling pathways to achieve a spectrum of biological end points. Using human embryonic kidney 293 cells transfected to express different TIRs we now report that signals initiated through IL-1R1 or TLR4 but not TLR3 can promote the stabilization of unstable chemokine mRNAs. Similar results were obtained when signaling from endogenous receptors was examined using a mouse endothelial cell line (H5V). The ability of TIR family members to stabilize ARE-containing mRNAs results from their differential use of signaling adaptors MyD88, MyD88 adaptor-like protein, Toll receptor IFN-inducing factor (Trif), and Trif-related adaptor molecule. Overexpression of MyD88 or MyD88 adaptor-like protein was able to promote enhanced stability of ARE-containing mRNA, whereas Trif and Trif-related adaptor molecule exhibited markedly reduced capacity. Hence the ability of TIRs to signal stabilization of mRNA appears to be linked to the MyD88-dependent signaling pathway.