Michael Kracht

Multiple control of interleukin-8 gene expression

Interleukin (IL)‐8, a prototypic human chemokine, was detected more than a decade ago as the founding member of the chemokine superfamily. One of the most remarkable properties of IL‐8 is the variation of its expression levels. In healthy tissues, IL‐8 is barely detectable, but it is rapidly induced by ten‐ to 100‐fold in response to proinflammatory cytokines such as tumor necrosis factor or IL‐1, bacterial or viral products, and cellular stress. Recently, significant advances in the understanding of signaling pathways, which coordinately regulate IL‐8 transcription as well as mRNA stabilization in response to external stimuli, have been made. Maximal IL‐8 amounts are generated by a combination of three different mechanisms: first, derepression of the gene promoter; second, transcriptional activation of the gene by nuclear factor‐κB and JUN‐N‐terminal protein kinase pathways; and third, stabilization of the mRNA by the p38 mitogen‐activated protein kinase pathway. In that way, cells are able to rapidly increase and at the same time, to fine‐tune the amount of IL‐8 secreted and thereby control the extent of leukocytes attracted to sites of tissue injury.

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.

Interleukin-1 (IL-1) Pathway

IL-1α and IL-1β orchestrate the proinflammatory response by multiple tightly controlled mechanisms. The interleukin-1 (IL-1) family of cytokines comprises 11 proteins (IL-1F1 to IL-1F11) encoded by 11 distinct genes in humans and mice. IL-1–type cytokines are major mediators of innate immune reactions, and blockade of the founding members IL-1α or IL-1β by the interleukin-1 receptor antagonist (IL-1RA) has demonstrated a central role of IL-1 in a number of human autoinflammatory diseases. IL-1α or IL-1β rapidly increase messenger RNA expression of hundreds of genes in multiple different cell types. The potent proinflammatory activities of IL-1α and IL-1β are restricted at three major levels: (i) synthesis and release, (ii) membrane receptors, and (iii) intracellular signal transduction. This pathway summarizes extracellular and intracellular signaling of IL-1α or IL-1β, including positive- and negative-feedback mechanisms that amplify or terminate the IL-1 response. In response to ligand binding of the receptor, a complex sequence of combinatorial phosphorylation and ubiquitination events results in activation of nuclear factor κB signaling and the JNK and p38 mitogen-activated protein kinase pathways, which, cooperatively, induce the expression of canonical IL-1 target genes (such as IL-6, IL-8, MCP-1, COX-2, IκBα, IL-1α, IL-1β, MKP-1) by transcriptional and posttranscriptional mechanisms. Of note, most intracellular components that participate in the cellular response to IL-1 also mediate responses to other cytokines (IL-18 and IL-33), Toll-like-receptors (TLRs), and many forms of cytotoxic stresses.

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.

The Interleukin-1 Receptor Accessory Protein (IL-1RAcP) Is Essential for IL-1-induced Activation of Interleukin-1 Receptor-associated Kinase (IRAK) and Stress-activated Protein Kinases (SAP Kinases)

Interleukin-1 (IL-1) is a central mediator of the immune system involved in acute and chronic inflammatory responses. Although the sequences of two types of IL-1 receptors are known, the exact molecular events resulting in signal transduction and coupling to downstream signaling elements remain unclear. The recently cloned IL-1 receptor accessory protein (IL-1RAcP) has been suggested as a co-receptor molecule for IL-1RI, supported by the observation that its expression correlates to IL-1 responsiveness. We transfected the EL-4 subline D6/76 with IL-1RAcP cDNA. This cell line is an IL-1 non-responder expressing IL-1RI but lacking constitutive IL-1RAcP expression. The expression of IL-1RAcP in EL-4 D6/76 was sufficient to restore IL-1-induced activation of interleukin-1 receptor-associated kinase and of stress-activated protein kinases, translocation of the transcription factors NFκB and IL-1 NF to the nucleus, and induction of IL-2 mRNA synthesis. These results proved that IL-1RAcP is an indispensible molecule in the IL-1 receptor signal transduction complex, necessary to link events on the plasma membrane level to downstream signaling pathways, allowing IL-1-dependent activation of transcription factors and gene expression.

IκB-independent control of NF-κB activity by modulatory phosphorylations

Abstract Activation of the transcription factor nuclear factor κB (NF-κB) requires its release from inhibitor of NF-κB (IκB) proteins in the cytoplasm. Much work has focussed on the identification of pathways regulating this cytosolic rate-limiting step of NF-κB activation. However, there is increasing evidence for another complex level of NF-κB activation, which involves modulatory phosphorylations of the DNA-binding subunits. These phosphorylations can control several functions of NF-κB, including DNA binding and transactivation properties, as well as interactions between the transcription factor and regulatory proteins. Although their overall impact on NF-κB function has yet to be determined, modifications of this factor will very probably provide a mechanism to fine tune NF-κB function.

Protein synthesis inhibitors reveal differential regulation of mitogen-activated protein kinase and stress-activated protein kinase pathways that converge on Elk-1.

Inhibitors of protein synthesis, such as anisomycin and cycloheximide, lead to superinduction of immediate-early genes. We demonstrate that these two drugs activate intracellular signaling pathways involving both the mitogen-activated protein kinase (MAPK) and stress-activated protein kinase (SAPK) cascades. The activation of either pathway correlates with phosphorylation of the c-fos regulatory transcription factor Elk-1. In HeLa cells, anisomycin stabilizes c-fos mRNA when protein synthesis is inhibited to only 50%. Under these conditions, anisomycin, in contrast to cycloheximide, rapidly induces kinase activation and efficient Elk-1 phosphorylation. However, full inhibition of translation by either drug leads to prolonged activation of SAPK activity, while MAPK induction is transient. This correlates with prolonged Elk-1 phosphorylation and c-fos transcription. Elk-1 induction and c-fos activation are also observed in KB cells, in which anisomycin strongly induces SAPKs but not MAPKs. Purified p54 SAPK alpha efficiently phosphorylates the Elk-1 C-terminal domain in vitro and comigrates with anisomycin-activated kinases in in-gel kinase assays. Thus, Elk-1 provides a potential convergence point for the MAPK and SAPK signaling pathways. The activation of signal cascades and control of transcription factor function therefore represent prominent processes in immediate-early gene superinduction.

Targeting innate immunity protein kinase signalling in inflammation

Inflammation is an evolutionarily conserved host reaction that is initiated in response to trauma, tissue damage and infection. It leads to changes in tissue homeostasis and blood flow, immune-cell activation and migration, and secretion of cytokines and mediators in a spatio-temporally coordinated manner. Progress in understanding of the mechanisms of the inflammatory response has identified various protein kinases that act as essential signalling components and therefore represent potential therapeutic targets. This article summarizes advances in the identification and validation of such targets, and discusses key issues for the development of small-molecule kinase inhibitors as a new generation of oral anti-inflammatory drugs, including feedback loops, inhibitor specificity and combination therapy.

NF-κB: A multifaceted transcription factor regulated at several levels

NF‐κB is a generic name for an evolutionarily conserved transcription‐factor system that contributes to the mounting of an effective immune response but is also involved in the regulation of cell proliferation, development, and apoptosis. The implication of NF‐κB in central biological processes and its extraordinary connectivity to other signaling pathways raise a need for highly controlled regulation of NF‐κB activity at several levels. While all NF‐κB activation pathways share a central and critical proteasome‐mediated step that leads to the degradation of inhibitory proteins and the release of DNA‐binding subunits, there is evidence for a downstream level of NF‐κB regulation that employs several mechanisms. These include promoter‐specific exchange of dimers and modification of the transactivating p65 subunit by phosphorylation, acetylation, ubiquitination, or prolyl isomerization. The signaling pathways and enzymes controlling this second level of regulation and their potential use as therapeutic targets for the treatment of NF‐κB‐associated pathologies are discussed here.

NFκB activation by Fas is mediated through FADD, caspase-8, and RIP and is inhibited by FLIP

Fas (APO-1/CD95) is the prototypic death receptor, and the molecular mechanisms of Fas-induced apoptosis are comparably well understood. Here, we show that Fas activates NFκB via a pathway involving RIP, FADD, and caspase-8. Remarkably, the enzymatic activity of the latter was dispensable for Fas-induced NFκB signaling pointing to a scaffolding-related function of caspase-8 in nonapoptotic Fas signaling. NFκB was activated by overexpressed FLIPL and FLIPS in a cell type–specific manner. However, in the context of Fas signaling both isoforms blocked FasL-induced NFκB activation. Moreover, down-regulation of both endogenous FLIP isoforms or of endogenous FLIPL alone was sufficient to enhance FasL-induced expression of the NFκB target gene IL8. As NFκB signaling is inhibited during apoptosis, FasL-induced NFκB activation was most prominent in cells that were protected by Bcl2 expression or caspase inhibitors and expressed no or minute amounts of FLIP. Thus, protection against Fas-induced apoptosis in a FLIP-independent manner converted a proapoptotic Fas signal into an inflammatory NFκB-related response.