Michael Novotny

IL-17 Enhances Chemokine Gene Expression through mRNA Stabilization

IL-17 plays an important role in host defense and autoimmunity via the induction of proinflammatory gene expression, particularly in combination with TNF-α. The molecular mechanisms by which IL-17 regulates such expression are not well understood. Using the mouse chemokine CXCL1 (KC) gene as a model, we have examined the effects of IL-17 alone or in combination with TNF-α on transcriptional and posttranscriptional events. Although treatment of mouse embryonic fibroblasts with IL-17 alone only modestly increased KC expression, the combination of IL-17 with TNF-α induced a synergistic response. IL-17 treatment exerted a strong posttranscriptional effect by extending the t1/2 of the highly unstable, TNF-α-induced KC mRNA. Using a tetracycline-regulated transgene in HeLa cells, we determined that IL-17 treatment alone promoted stabilization of KC mRNA in the absence of TNF-α. IL-17 treatment exerted little effect on KC transcription or NF-κB activation, suggesting that it primarily acts posttranscriptionally. We identified a number of other mRNAs whose t1/2 are prolonged in response to IL-17, suggesting that this is a common mechanism by which IL-17 promotes enhanced gene expression. Finally, activator of NF-κB1 protein (Act1), an adaptor protein recently implicated in IL-17 signaling, was necessary for IL-17-induced stabilization, and overexpression of Act1 resulted in stabilization of KC mRNA, indicating that events downstream of Act1 are sufficient to initiate this process. Thus, the synergy between TNF-α and IL-17 reflects their independent actions on KC gene expression; TNF-α serves as a stimulus to initiate transcription through activation of NF-κB, whereas IL-17 drives mRNA stabilization through an Act1-dependent pathway.

Treatment with IL-17 prolongs the half-life of chemokine CXCL1 mRNA via the adaptor TRAF5 and the splicing-regulatory factor SF2 (ASF)

Interleukin 17 (IL-17) promotes the expression of chemokines and cytokines via the induction of gene transcription and post-transcriptional stabilization of mRNA. We show here that IL-17 enhanced the stability of chemokine CXCL1 mRNA and other mRNAs through a pathway that involved the adaptor Act1, the adaptors TRAF2 or TRAF5 and the splicing factor SF2 (also known as alternative splicing factor (ASF)). TRAF2 and TRAF5 were necessary for IL-17 to signal the stabilization of CXCL1 mRNA. Furthermore, IL-17 promoted the formation of complexes of TRAF5-TRAF2, Act1 and SF2 (ASF). Overexpression of SF2 (ASF) shortened the half-life of CXCL1 mRNA, whereas depletion of SF2 (ASF) prolonged it. SF2 (ASF) bound chemokine mRNA in unstimulated cells, whereas the SF2 (ASF)-mRNA interaction was much lower after stimulation with IL-17. Our findings define an IL-17-induced signaling pathway that links to the stabilization of selected mRNA species through Act1, TRAF2-TRAF5 and the RNA-binding protein SF2 (ASF).

Tristetraprolin Regulates CXCL1 (KC) mRNA Stability

mRNAs encoding proinflammatory chemokines are regulated posttranscriptionally via adenine-uridine-rich sequences (AREs) located in the 3′ untranslated region of the message, which are recognized by sequence-specific RNA-binding proteins. One ARE binding protein, tristetraprolin (TTP), has been implicated in regulating the stability of several ARE-containing mRNAs, including those encoding TNF-α and GM-CSF. In the present report we examined the role of TTP in regulating the decay of the mouse chemokine KC (CXCL1) mRNA. Using tetR-regulated control of transcription in TTP-deficient HEK293 cells, KC mRNA half-life was markedly decreased in the presence of TTP. Deletion and site-specific mutagenesis were used to identify multiple AUUUA sequence determinants responsible for TTP sensitivity. Although a number of studies suggest that the destabilizing activity of TTP is subject to modulation in response to ligands of Toll/IL-1 family receptors, decay mediated by TTP in 293 cells was not sensitive to stimulation with IL-1α. Using primary macrophages from wild-type and TTP-deficient mice, KC mRNA instability was found to be highly dependent on TTP. Furthermore, LPS-mediated stabilization of KC mRNA is blocked by inhibition of the p38 MAPK in macrophages from wild-type but not TTP-deficient mice. These findings demonstrate that TTP is the predominant regulator of KC mRNA decay in mononuclear phagocytes acting via multiple 3′-untranslated region-localized AREs. Nevertheless, KC mRNA remains highly unstable in cells that do not express TTP, suggesting that additional determinants of instability and stimulus sensitivity may operate in cell populations where TTP is not expressed.

IL-17 Signaling for mRNA Stabilization Does Not Require TNF Receptor-Associated Factor 6

IL-17 alone is a relatively weak inducer of gene expression, but cooperates with other cytokines, including TNF-α, to generate a strong response in part via prolongation of mRNA t1/2. Because TNFR-associated factor 6 (TRAF6) has been reported to be essential for signaling by IL-17, we examined its involvement in IL-17-mediated mRNA stabilization. Although overexpression of TRAF6 in HeLa cells activates NF-κB, it does not stabilize transfected KC mRNA. Furthermore, a dominant-negative TRAF6 abrogates NF-κB activation, but does not block IL-17-induced chemokine mRNA stabilization. IL-17 can stabilize KC and MIP-2 mRNAs comparably in TNF-α-treated mouse embryo fibroblasts from TRAF6+/+ and TRAF6−/− mice. TRAF6 is known to couple upstream signals with activation of p38 MAPK and mitogen activated protein kinase activated protein kinase 2, both of which have been shown to be important for Toll/IL-1R-mediated mRNA stabilization in various cell types. Inhibition of p38 MAPK, however, does not block IL-17-induced KC mRNA stabilization, and IL-17 can stabilize KC mRNA equally in mouse embryo fibroblasts from both wild-type and mitogen activated protein kinase activated protein kinase 2/3 doubly-deficient mice. Finally, IL-17 can amplify the levels of multiple TNF-α-stimulated mRNAs in wild-type and TRAF6-deficient cells, but not in cells from Act1−/− mice. Collectively, these findings demonstrate the existence of a TRAF6/p38 MAPK-independent pathway that couples the IL-17R with enhanced mRNA stability. Because the most potent effects of IL-17 on gene expression are obtained in cooperation with other cytokines such as TNF-α, these findings suggest that this pathway is a major contributing mechanism for response to IL-17.

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.

Distinct Temporal Patterns of Macrophage-Inflammatory Protein-2 and KC Chemokine Gene Expression in Surgical Injury

In the present study the regulation of CXC chemokine expression was evaluated in full-thickness abdominal wounds in mice. During the first 24 h after injury, IL-1αβ, KC, macrophage-inflammatory protein (MIP)-2, and monocyte chemoattractant protein-1 were the predominant cytokines and chemokines produced; TNF-α was not detected. Chemokine mRNA expression and protein secretion occurred in two temporal stages. The first, which reached a maximum at 6 h, was associated with high levels of IL-1α and KC and low levels of MIP-2. This stage could be reproduced by intradermal injection of IL-1α or IL-1β and was partially blocked by injection of neutralizing Ab against IL-1α but not IL-1β. In animals depleted of circulating neutrophils, chemokine expression was reduced by nearly 70% during this stage. In the second stage, which peaked at 24 h after injury, modest but significant levels of IL-1β were detected in association with low levels of KC and high levels of MIP-2. This pattern of chemokine expression could not be mimicked by injection of IL-1α or IL-1β (even with prolonged exposure), although MIP-2 expression could be partially inhibited by intradermal injection of neutralizing Ab against IL-1β. Surprisingly, neutrophil depletion before injury resulted in sustained high levels of both KC and MIP-2 expression. These observations demonstrate that these two closely related chemokines are under distinct regulatory controls in vivo that are likely to reflect the temporally ordered participation of different cell types and/or extracellular stimuli and inhibitors.

IL-17 Regulates CXCL1 mRNA Stability via an AUUUA/Tristetraprolin-Independent Sequence

IL-17 contributes to inflammatory response in part by promoting enhanced expression of chemokines, such as CXCL1, by prolonging the t1/2 of this constitutively unstable mRNA. Although IL-17 is a weak stimulus for transcription of the CXCL1 gene, it strongly potentiates message accumulation via stabilization when the mRNA is transcribed in cells stimulated with TNF. In myeloid cells, LPS-induced CXCL1 mRNA stabilization is dependent on AUUUA-containing sequence motifs that are recognized by the RNA binding protein tristetraprolin (TTP). Using deletion and site-specific mutagenesis, we report that IL-17–mediated stabilization of CXCL1 mRNA in nonmyeloid cells depends on a sequence that does not contain the AUUUA motif. Furthermore, a specific two-nucleotide mutation within this region markedly abrogates sensitivity for IL-17–mediated stabilization. Consistent with this finding, the IL-17–sensitive sequence does not exhibit increased instability in the presence of TTP, and CXCL1 mRNA remains unstable and can be stabilized in response to treatment with IL-17 in embryo fibroblasts from mice in which the TTP gene has been deleted. Whereas the RNA binding protein KSRP has been shown to participate in regulating the instability of human CXCL8 mRNA, inhibitory RNA-based reduction in KSRP does not effect the instability mediated by the IL-17–sensitive sequence motif. These findings suggest that IL-17–mediated chemokine mRNA stabilization in nonmyeloid cells uses a mechanism that is distinct from that operating to control AU-rich mRNA stability in myeloid cells.

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.

Lipopolysaccharide Induces Formyl Peptide Receptor 1 Gene Expression in Macrophages and Neutrophils via Transcriptional and Posttranscriptional Mechanisms

Bacterial infection promotes the infiltration of inflammatory leukocytes mediated in part by receptors for formyl-methionine-terminated peptides. In this study, we show that LPS can markedly enhance the expression of the formyl peptide receptor gene (FPR1) in mouse macrophages and neutrophils by enhancing transcription and by stabilization of the mRNA. In untreated cells, FPR1 mRNA exhibits a half-life of ∼90 min and this is markedly increased (to >6 h) following stimulation with LPS. Although FPR1 mRNA levels remained elevated over baseline for >20 h after stimulation, the half-life of the message is prolonged only transiently. LPS-induced FPR1 mRNA expression is mediated in part by the intermediate production of secreted factors. First, the response to LPS is partially blocked by the translational inhibitor cycloheximide. Second, a heat-labile but polymyxin B-insensitive factor present in supernatants from LPS-treated cells stimulates enhanced expression of FPR1 mRNA and, like LPS, promotes stabilization of FPR1 mRNA. Furthermore, supernatants from LPS-treated wild-type macrophages can stimulate FPR1 mRNA expression in LPS-insensitive macrophages from TLR4-mutant mice. Elevated FPR1 mRNA expression is also induced in response to ligands for TLR2 and TLR3. TNF-α but not IL-1, IL-6, IFN-β, and IFN-γ can mimic the effects of LPS although other factors apparently also contribute. Collectively, these findings define a distinct molecular pattern of response to TLR stimulation in inflammatory phagocytes and demonstrate that regulation of FPR1 expression is achieved through both transcriptional and posttranscriptional mechanisms.

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.