Reinhard Winzen

The p38 MAP kinase pathway signals for cytokine- induced mRNA stabilization via MAP kinase- activated protein kinase 2 and an AU-rich region- targeted mechanism

Stabilization of mRNAs contributes to the strong and rapid induction of genes in the inflammatory response. The signaling mechanisms involved were investigated using a tetracycline‐controlled expression system to determine the half‐lives of interleukin (IL)‐6 and IL‐8 mRNAs. Transcript stability was low in untreated HeLa cells, but increased in cells expressing a constitutively active form of the MAP kinase kinase kinase MEKK1. Destabilization and signal‐induced stabilization was transferred to the stable β‐globin mRNA by a 161‐nucleotide fragment of IL‐8 mRNA which contains an AU‐rich region, as well as by defined AU‐rich elements (AREs) of the c‐fos and GM‐CSF mRNAs. Of the different MEKK1‐activated signaling pathways, no significant effects on mRNA degradation were observed for the SAPK/JNK, extracellular regulated kinase and NF‐κB pathways. Selective activation of the p38 MAP kinase (=SAPK2) pathway by MAP kinase kinase 6 induced mRNA stabilization. A dominant‐negative mutant of p38 MAP kinase interfered with MEKK1 and also IL‐1‐induced stabilization. Furthermore, an active form of the p38 MAP kinase‐activated protein kinase (MAPKAP K2 or MK2) induced mRNA stabilization, whereas a negative interfering MK2 mutant interfered with MAP kinase kinase 6‐induced stabilization. These findings indicate that the p38 MAP kinase pathway contributes to cytokine/stress‐induced gene expression by stabilizing mRNAs through an MK2‐dependent, ARE‐targeted mechanism.

Induction of Interleukin-8 Synthesis Integrates Effects on Transcription and mRNA Degradation from at Least Three Different Cytokine- or Stress-Activated Signal Transduction Pathways

ABSTRACT A hallmark of inflammation is the burst-like formation of certain proteins, initiated by cellular stress and proinflammatory cytokines like interleukin 1 (IL-1) and tumor necrosis factor, stimuli which simultaneously activate different mitogen-activated protein (MAP) kinases and NF-κB. Cooperation of these signaling pathways to induce formation of IL-8, a prototype chemokine which causes leukocyte migration and activation, was investigated by expressing active and inactive forms of protein kinases. Constitutively active MAP kinase kinase 7 (MKK7), an activator of the stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) pathway, induced IL-8 synthesis and transcription from a minimal IL-8 promoter. Furthermore, MKK7 synergized in both effects with NF-κB-inducing kinase (NIK). Activation of the IL-8 promoter by either of the kinases required functional NF-κB and AP-1 sites. While NIK and MKK7 did not affect degradation of IL-8 mRNA, an active form of MKK6, which selectively activates p38 MAP kinase, induced marked stabilization of the transcript and further increased IL-8 protein formation induced by NIK plus MKK7. Consistently, the MAP kinase kinase kinase MEKK1, which can activate NF-κB, SAPK/JNK, and p38 MAP kinases, most potently induced IL-8 formation. These results provide evidence that maximal IL-8 gene expression requires the coordinate action of at least three different signal transduction pathways which cooperate to induce mRNA synthesis and suppress mRNA degradation.

Distinct Domains of AU-Rich Elements Exert Different Functions in mRNA Destabilization and Stabilization by p38 Mitogen-Activated Protein Kinase or HuR

ABSTRACT AU-rich elements (AREs) control the expression of numerous genes by accelerating the decay of their mRNAs. Rapid decay and deadenylation of β-globin mRNA containing AU-rich 3′ untranslated regions of the chemoattractant cytokine interleukin-8 (IL-8) are strongly attenuated by activating the p38 mitogen-activated protein (MAP) kinase/MAP kinase-activated protein kinase 2 (MK2) pathway. Further evidence for a crucial role of the poly(A) tail is provided by the loss of destabilization and kinase-induced stabilization in ARE RNAs expressed as nonadenylated forms by introducing a histone stem-loop sequence. The minimal regulatory element in the IL-8 mRNA is located in a 60-nucleotide evolutionarily conserved sequence with a structurally and functionally bipartite character: a core domain with four AUUUA motifs and limited destabilizing function on its own and an auxiliary domain that markedly enhances destabilization exerted by the core domain and thus is essential for the rapid removal of RNA targets. A similar bipartite structure and function are observed for the granulocyte-macrophage colony-stimulating factor (GM-CSF) ARE. Stabilization in response to p38/MK2 activation is seen with the core domain alone and also after mutation of the AUUUA motifs in the complete IL-8 ARE. Stabilization by ARE binding protein HuR requires different sequence elements. Binding but no stabilization is observed with the IL-8 ARE. Responsiveness to HuR is gained by exchanging the auxiliary domain of the IL-8 ARE with that of GM-CSF or with a domain of the c-fos ARE, which results in even stronger responsiveness. These results show that distinct ARE domains differ in function with regard to destabilization, stabilization by p38/MK2 activation, and stabilization by HuR.

Functional Analysis of KSRP Interaction with the AU-Rich Element of Interleukin-8 and Identification of Inflammatory mRNA Targets†

ABSTRACT mRNA stability is a major determinant of inflammatory gene expression. Rapid degradation of interleukin-8 (IL-8) mRNA is imposed by a bipartite AU-rich element (ARE) in the 3′ untranslated region (R. Winzen et al., Mol. Cell. Biol. 24:4835-4847, 2004). Small interfering RNA-mediated knockdown of the ARE-binding protein KSRP resulted in stabilization of IL-8 mRNA or of a β-globin reporter mRNA containing the IL-8 ARE. Rapid deadenylation was impaired, indicating a crucial role for KSRP in this step of mRNA degradation. The two IL-8 ARE domains both contribute to interaction with KSRP, corresponding to the importance of both domains for rapid degradation. Exposure to the inflammatory cytokine IL-1 has been shown to stabilize IL-8 mRNA through p38 mitogen-activated protein (MAP) kinase and MK2. IL-1 treatment impaired the interaction of KSRP with the IL-8 ARE in a manner dependent on p38 MAP kinase but apparently independent of MK2. Instead, evidence that TTP, a target of MK2, can also destabilize the IL-8 ARE reporter mRNA is presented. In a comprehensive approach to identify mRNAs controlled by KSRP, two criteria were evaluated by microarray analysis of (i) association of mRNAs with KSRP in pulldown assays and (ii) increased amounts in KSRP knockdown cells. According to both criteria, a group of 100 mRNAs is controlled by KSRP, many of which are unstable and encode proteins involved in inflammation. These results indicate that KSRP functions as a limiting factor in inflammatory gene expression.

Stress-activated Protein Kinase/Jun N-terminal Kinase Is Required for Interleukin (IL)-1-induced IL-6 and IL-8 Gene Expression in the Human Epidermal Carcinoma Cell Line KB

The cytokine interleukin-1 (IL-1) is a major inflammatory hormone which activates a broad range of genes during inflammation. The signaling mechanisms triggered by IL-1 include activation of several distinct protein kinase systems. The stress-activated protein kinase (SAPK), also termed Jun N-terminal kinase (JNK), is activated particularly strongly by the cytokine. In an attempt to delineate its role in activation of gene expression by IL-1, we inhibited the IL-1-induced SAPK/JNK activity by stable overexpression of either a catalytically inactive mutant of SAPKβ (SAPKβ(K-R)) or antisense RNA to SAPKβ in human epidermal carcinoma cells. A detailed analysis of signal transduction in those cells showed that activation of neither NFκB nor p38 mitogen-activated protein kinase was affected, suggesting that we achieved specific blockade of the SAPK/JNK. In untransfected and vector-transfected KB cells, IL-1 induced a strong increase in expression of IL-6 and IL-8 mRNA, along with the synthesis of high amounts of the proteins. In two KB cell clones stably overexpressing the mutant SAPKβ(K-R), and three clones stably overexpressing antisense RNA to SAPKβ, expression of IL-6 and IL-8 in response to IL-1 was strongly reduced at both the mRNA and protein level. These data indicate that the SAPK/JNK pathway provides an indispensable signal for IL-1-induced expression of IL-6 and IL-8.

Interleukin-1 Activates Synthesis of Interleukin-6 by Interfering with a KH-type Splicing Regulatory Protein (KSRP)-dependent Translational Silencing Mechanism

Post-transcriptional mechanisms play an important role in the control of inflammatory gene expression. The heterogeneous nuclear ribonucleoprotein K homology (KH)-type splicing regulatory protein (KSRP) triggers rapid degradation of mRNAs for various cytokines, chemokines, and other inflammation-related proteins by interacting with AU-rich elements (AREs) in the 3′-untranslated mRNA regions. In addition to destabilizing mRNAs, AU-rich elements can restrict their translation. Evidence that KSRP also participates in translational silencing was obtained in a screen comparing the polysome profiles of cells with siRNA-mediated depletion of KSRP with that of control cells. Among the group of mRNAs showing increased polysome association upon KSRP depletion are those of interleukin (IL)-6 and IL-1α as well as other ARE-containing transcripts. Redistribution of IL-6 mRNA to polysomes was associated with increased IL-6 protein secretion by the KSRP-depleted cells. Silencing of IL-6 and IL-1α mRNAs depended on their 3′-untranslated regions. The sequence essential for translational control of IL-6 mRNA and its interaction with KSRP was located to an ARE. KSRP-dependent silencing was reversed by IL-1, a strong inducer of IL-6 mRNA and protein expression. The results identify KSRP as a protein involved in ARE-mediated translational silencing. They suggest that KSRP restricts inflammatory gene expression not only by enhancing degradation of mRNAs but also by inhibiting translation, both functions that are counteracted by the proinflammatory cytokine IL-1.

Inhibition of mRNA deadenylation and degradation by different types of cell stress

Abstract We have previously observed rapid and strong inhibition of mRNA deadenylation and degradation in response to UV-B light [Gowrishankar et al., Biol. Chem. 386 (2005), pp. 1287–1293]. Expression analysis using a microarray for inflammatory genes showed that UV-B light induces stabilization of all short-lived mRNAs assayed. Stabilization was observed in HeLa cells, as well as in the keratinocyte line HaCaT. It affected constitutively expressed mRNA species, as well as species induced by the inflammatory cytokine IL-1. Many of the latter encode proteins involved in inflammation, suggesting that stress-induced inhibition of mRNA deadenylation contributes to changes in inflammatory gene expression. Deadenylation and degradation of tet-off-expressed mRNAs were also inhibited upon exposure to H2O2. However, scavengers of reactive oxygen species did not interfere with UV-B-induced inhibition of degradation, arguing against the involvement of UV-induced H2O2 in these effects of UV-B light. Heat shock and hyperosmolarity also inhibited mRNA deadenylation and degradation, whereas γ-radiation did not. Thus, inhibition of mRNA deadenylation and degradation is a cellular response elicited by several but not all inducers of cell stress.

Inhibition of mRNA deadenylation and degradation by ultraviolet light.

Abstract Post-transcriptional mechanisms contribute to the changes in gene expression induced by cell stress. The effect of UV-B light on mRNA degradation in HeLa cells was investigated using a transcriptional chase system to determine the decay kinetics of tet-off vector-derived mRNAs containing or lacking a destabilizing AU-rich element. Degradation of both mRNAs was strongly inhibited in cells exposed to UV-B light. Removal of the poly(A)-tail, considered a crucial step in mRNA degradation, was strikingly impaired. UV light also inhibited deadenylation and degradation of endogenous mRNA of the chemoattractant cytokine interleukin (IL)-8. Both effects occurred rapidly and independently of newly induced genes. Importantly, stabilization of IL-8 mRNA was accompanied by a strong increase in the duration of IL-8 protein formation. Furthermore, general inhibition of protein synthesis, a hallmark of the response to cell stress, required far higher doses of UV-B than inhibition of mRNA deadenylation and degradation. The difference in sensitivity of cells to these effects of UV-B light establishes a dose range in which mRNA stabilization can lead to dramatically enhanced expression of proteins derived from normally unstable mRNAs, such as those of inflammatory cytokines, growth factors and proto-oncogenes, and thereby have a major impact on the response to UV light.

IL-1-induced Post-transcriptional Mechanisms Target Overlapping Translational Silencing and Destabilizing Elements in IκBζ mRNA

The inflammatory cytokine IL-1 induces profound changes in gene expression. This is contributed in part by activating translation of a distinct set of mRNAs, including IκBζ, as indicated by genome-wide analysis of changes in ribosomal occupancy in IL-1α-treated HeLa cells. Polysome profiling of IκBζ mRNA and reporter mRNAs carrying its 3′ UTR indicated poor translation in unstimulated cells. 3′ UTR-mediated translational silencing was confirmed by suppression of luciferase activity. Translational silencing was unaffected by replacing the poly(A) tail with a histone stem-loop, but lost under conditions of cap-independent internal initiation. IL-1 treatment of the cells caused profound shifts of endogenous and reporter mRNAs to polysome fractions and relieved suppression of luciferase activity. IL-1 also inhibited rapid mRNA degradation. Both translational activation and mRNA stabilization involved IRAK1 and -2 but occurred independently of the p38 MAPK pathway, which is known to target certain other post-transcriptional mechanisms. The translational silencing RNA element contains the destabilizing element but requires additional 5′ sequences and is impaired by mutations that leave destabilization unaffected. These differences in function are associated with differential changes in protein binding in vitro. Thus, rapid degradation occurs independently of the translational silencing effect. The results provide evidence for a novel mode of post-transcriptional control by IL-1, which impinges on the time course and pattern of IL-1-induced gene expression.

Interaction between the mRNA of the 55-kDa Tumor Necrosis Factor Receptor and Cellular Proteins POSSIBLE INVOLVEMENT IN POST-TRANSCRIPTIONAL REGULATION OF RECEPTOR EXPRESSION

Numerous effects of tumor necrosis factor are signaled by its 55-kDa receptors. Studying their expression we found that the level of receptor mRNA was decreased during the phorbol ester-induced differentiation of myelomonocytic cell lines. While only minor changes in transcription were noted, the half-life of receptor mRNA in the differentiated cells was markedly decreased, indicating the involvement of post-transcriptional regulation. In an electrophoretic mobility shift assay, formation of complexes between radiolabeled receptor mRNA and cellular proteins was observed. The decrease in receptor mRNA levels during phorbol ester-induced differentiation was paralleled by a change in the pattern of those complexes. Protein-RNA interaction was selective, as it was not competed by unrelated RNAs. Yet, certain mRNAs that contain AU-rich sequences, known to be involved in the control of their stability, did compete with the receptor mRNA, although the latter is devoid of such sequences. A region of 18 nucleotides within its coding region was found to contain an element essential for the formation of all complexes and sufficient for the formation of those with lower molecular mass. Adjacent bases were required in addition for the formation of the complexes with higher molecular mass. The results suggest that proteins interacting with this region of the 55-kDa tumor necrosis factor receptor mRNA contribute to the regulation of its expression.