Helmut Holtmann

Targeted Disruption of the Mouse Caspase 8 Gene Ablates Cell Death Induction by the TNF Receptors, Fas/Apo1, and DR3 and Is Lethal Prenatally

Homozygous targeted disruption of the mouse Caspase 8 (Casp8) gene was found to be lethal in utero. The Caspase 8 null embryos exhibited impaired heart muscle development and congested accumulation of erythrocytes. Recovery of hematopoietic colony-forming cells from the embryos was very low. In fibroblast strains derived from these embryos, the TNF receptors, Fas/Apo1, and DR3 were able to activate the Jun N-terminal kinase and to trigger IkappaB alpha phosphorylation and degradation. They failed, however, to induce cell death, while doing so effectively in wild-type fibroblasts. These findings indicate that Caspase 8 plays a necessary and nonredundant role in death induction by several receptors of the TNF/NGF family and serves a vital role in embryonal development.

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.

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.

The p38/MK2-driven exchange between tristetraprolin and HuR regulates AU-rich element-dependent translation.

TNF expression of macrophages is under stringent translational control that depends on the p38 MAPK/MK2 pathway and the AU–rich element (ARE) in the TNF mRNA. Here, we elucidate the molecular mechanism of phosphorylation-regulated translation of TNF. We demonstrate that translation of the TNF-precursor at the ER requires expression of the ARE–binding and -stabilizing factor human antigen R (HuR) together with either activity of the p38 MAPK/MK2 pathway or the absence of the ARE-binding and -destabilizing factor tristetraprolin (TTP). We show that phosphorylation of TTP by MK2 decreases its affinity to the ARE, inhibits its ability to replace HuR, and permits HuR-mediated initiation of translation of TNF mRNA. Since translation of TTPs own mRNA is also regulated by this mechanism, an intrinsic feedback control of the inflammatory response is ensured. The phosphorylation-regulated TTP/HuR exchange at target mRNAs provides a reversible switch between unstable/non-translatable and stable/efficiently translated mRNAs.

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.

c-Jun N-terminal kinase phosphorylates DCP1a to control formation of P bodies

JNK-mediated phosphorylation of the mRNA-decapping protein DCP1a disrupts P body structure, mRNA stability, and gene expression in response to stress and inflammatory stimuli.

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.