Linda Vermeulen

Transcriptional activation of the NF-κB p65 subunit by mitogen- and stress-activated protein kinase-1 (MSK1)

Nuclear factor κB (NF‐κB) is one of the key regulators of transcription of a variety of genes involved in immune and inflammatory responses. NF‐κB activity has long been thought to be regulated mainly by IκB family members, which keep the transcription factor complex in an inactive form in the cytoplasm by masking the nuclear localization signal. Nowadays, the importance of additional mechanisms controlling the nuclear transcription potential of NF‐κB is generally accepted. We show that the mitogen‐activated protein kinase inhibitors SB203580 and PD98059 or U0126, as well as a potent mitogen‐ and stress‐ activated protein kinase‐1 (MSK1) inhibitor H89, counteract tumor necrosis factor (TNF)‐mediated stimulation of p65 transactivation capacity. Muta tional analysis of p65 revealed Ser276 as a target for phosphorylation and transactivation in response to TNF. Moreover, we identified MSK1 as a nuclear kinase for p65, since MSK1 associates with p65 in a stimulus‐dependent way and phosphorylates p65 at Ser276. This effect represents, together with phosphorylation of nucleosome components such as histone H3, an essential step leading to selective transcriptional activation of NF‐κB‐dependent gene expression.

Regulation of the transcriptional activity of the nuclear factor-κB p65 subunit

Abstract Nuclear factor-κB (NF-κB) is well known for its role in inflammation, immune response, control of cell division and apoptosis. The function of NF-κB is primarily regulated by IκB family members, which ensure cytoplasmic localisation of the transcription factor in the resting state. Upon stimulus-induced IκB degradation, the NF-κB complexes move to the nucleus and activate NF-κB-dependent transcription. Over the years, a second regulatory mechanism, independent of IκB, has become generally accepted. Changes in NF-κB transcriptional activity have been assigned to phosphorylation of the p65 subunit by a large variety of kinases in response to different stimuli. Here, we give an overview of the kinases and signalling pathways mediating this process and comment on the players involved in tumour necrosis factor-induced regulation of NF-κB transcriptional activity. Additionally, we describe how other posttranslational modifications, such as acetylation and methylation of transcription factors or of the chromatin environment, may also affect NF-κB transcriptional activity.

Crosstalk in Inflammation: The Interplay of Glucocorticoid Receptor-Based Mechanisms and Kinases and Phosphatases

Glucocorticoids (GCs) are steroidal ligands for the GC receptor (GR), which can function as a ligand-activated transcription factor. These steroidal ligands and derivatives thereof are the first line of treatment in a vast array of inflammatory diseases. However, due to the general surge of side effects associated with long-term use of GCs and the potential problem of GC resistance in some patients, the scientific world continues to search for a better understanding of the GC-mediated antiinflammatory mechanisms. The reversible phosphomodification of various mediators in the inflammatory process plays a key role in modulating and fine-tuning the sensitivity, longevity, and intensity of the inflammatory response. As such, the antiinflammatory GCs can modulate the activity and/or expression of various kinases and phosphatases, thus affecting the signaling efficacy toward the propagation of proinflammatory gene expression and proinflammatory gene mRNA stability. Conversely, phosphorylation of GR can affect GR ligand- and DNA-binding affinity, mobility, and cofactor recruitment, culminating in altered transactivation and transrepression capabilities of GR, and consequently leading to a modified antiinflammatory potential. Recently, new roles for kinases and phosphatases have been described in GR-based antiinflammatory mechanisms. Moreover, kinase inhibitors have become increasingly important as antiinflammatory tools, not only for research but also for therapeutic purposes. In light of these developments, we aim to illuminate the integrated interplay between GR signaling and its correlating kinases and phosphatases in the context of the clinically important combat of inflammation, giving attention to implications on GC-mediated side effects and therapy resistance.

A paradigm for gene regulation: inflammation, NF-kappaB and PPAR.

The onset of inflammatory gene expression is driven by the transcription factor NF-kappaB, whose transcriptional activity is regulated at multiple levels. First, NF-kappaB activity is regulated by cytoplasmic degradation of the IkappaB inhibitor and nuclear translocation. Second, the nuclear p65 transactivation potential can be further influenced by posttranslational modifications, such as phosphorylation and/or acetylation. The p65 phosphorylation is a process highly regulated by both cell- and stimulus-dependent activating kinases. Ser276 phosphorylation seems to be highly important considering its crucial role in the interaction with and the engagement of the cofactor CBP/p300. We have identified MSK1 as an acting kinase in the TNF-signalling pathway, where it is responsible for p65 phosphorylation at Ser276, as well as for H3 phosphorylation of Ser10 in IL-6 promoter-associated chromatin (Fig. 1) (Saccani et al., 2002; Vermeulen et al., 2002, 2003). To our knowledge, this was the first report that identifies one particular kinase involved in transcription factor phosphorylation and histone modification at the level of a single promoter in order to establish gene activation. The question of which element takes the initial step to recruit and to assemble the activated transcription complex still remains unanswered (Vanden Berghe et al., 2002). PPAR alpha negatively interferes with inflammatory gene expression by up-regulation of the cytoplasmic inhibitor molecule IkappaB alpha, thus establishing an autoregulatory loop (Fig. 1). This induction takes place in the absence of a PPRE, but requires the presence of NF-kappaB and Sp1 elements in the IkappaB alpha promoter sequence as well as DRIP250 cofactors. The detailed mechanism how PPAR can activate genes in a non-DNA-binding way needs further investigation; moreover, it is at present not clear whether this upregulation, unlike the inhibitory effect of glucocorticoids, is a cell type- or a PPAR-specific phenomenon.

The versatile role of MSKs in transcriptional regulation

Among the mitogen-activated protein kinase (MAPK) targets, MSKs (mitogen- and stress-activated protein kinases) comprise a particularly interesting protein family. Because MSKs can be activated by both extracellular-signal-regulated kinase and p38 MAPKs, they are activated by many physiological and pathological stimuli. About ten years after their original discovery, they have been recognized as versatile kinases regulating gene transcription at multiple levels. MSKs directly target transcription factors, such as cAMP-response-element-binding protein and nuclear factor-kappaB, thereby enhancing their transcriptional activity. They also induce histone phosphorylation, which is accompanied by chromatin relaxation and facilitated binding of additional regulatory proteins. Here, we review the current knowledge on MSK activation and its molecular targets, focusing on recent insights into the role of MSKs at multiple levels of transcriptional regulation.

Ser276 phosphorylation of NF-kB p65 by MSK1 controls SCF expression in inflammation.

Transcription of the mast cell growth factor SCF (stem cell factor) is upregulated in inflammatory conditions, and this is dependent upon NF-κB, as well as the MAP kinases p38 and ERK activation. We show here that the MAPK downstream nuclear kinase MSK1 induces NF-κB p65 Ser276 phosphorylation upon IL-1ß treatment, which was inhibited in cells transfected with a MSK1 kinase-dead (KD) mutant compared to the WT control. In addition, we show by ChIP experiments that MSK1 as well as MAPK inhibition abolishes binding of p65, of its coactivator CBP, and of MSK1 itself to the κB intronic enhancer site of the SCF gene. We show that interaction between NF-κB and CBP is prevented in cells transfected by a p65 S276C mutant. Finally, we demonstrate that both transfections of MSK1-KD and MSK1 siRNA - but not the WT MSK1 or control siRNA - downregulate the expression of SCF induced by IL-1ß. Our study provides therefore a direct link between MSK1-mediated phosphorylation of Ser276 p65 of NF-κB, allowing its binding to the SCF intronic enhancer, and pathophysiological SCF expression in inflammation.

Attenuation of Mitogen- and Stress-Activated Protein Kinase-1–Driven Nuclear Factor-κB Gene Expression by Soy Isoflavones Does Not Require Estrogenic Activity

We have analyzed in molecular detail how soy isoflavones (genistein, daidzein, and biochanin A) suppress nuclear factor-kappaB (NF-kappaB)-driven interleukin-6 (IL6) expression. In addition to its physiologic immune function as an acute stress cytokine, sustained elevated expression levels of IL6 promote chronic inflammatory disorders, aging frailty, and tumorigenesis. Our results in estrogen-unresponsive fibroblasts, mitogen- and stress-activated protein kinase (MSK) knockout cells, and estrogen receptor (ER)-deficient breast tumor cells show that phytoestrogenic isoflavones can selectively block nuclear NF-kappaB transactivation of specific target genes (in particular IL6), independently of their estrogenic activity. This occurs via attenuation of mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK) and ERK activity, which further down-regulates MSK-dependent NF-kappaB p65 and histone H3 phosphorylation. As constitutive NF-kappaB and MSK activity are hallmarks of aggressive metastatic ER-deficient breast cancer, the MSK signaling pathway may become an attractive target for chemotherapy.

Altered subcellular distribution of MSK1 induced by glucocorticoids contributes to NF‐κB inhibition

Glucocorticoids are widely used anti‐inflammatory and immunomodulatory agents, of which the action mechanism is mainly based on interference of hormone‐activated glucocorticoid receptor (GR) with the activity of transcription factors, such as nuclear factor‐κB (NF‐κB). In addition to the well described interaction‐based mutual repression mechanism between the GR and NF‐κB, additional mechanisms are at play, which help to explain the efficacy of glucocorticoid‐mediated gene repression. In this respect, we found that glucocorticoids counteract the recruitment of activated Mitogen‐ and Stress‐activated protein Kinase‐1 (MSK1) at inflammatory gene promoters resulting in the inhibition of NF‐κB p65 transactivation and of concurrent histone H3 phosphorylation. Additionally, we observed that activated GR can trigger redistribution of nuclear MSK1 to the cytoplasm through a CRM1‐dependent export mechanism, as a result of an interaction between liganded GR and activated MSK1. These findings unveil a novel aspect within the GR‐mediated NF‐κB‐targeting anti‐inflammatory mechanism.

Cooperation of NFκB and CREB to induce synergistic IL-6 expression in astrocytes

Astrocytes are critical players in the innate immune response of the central nervous system. Upon encountering proinflammatory stimuli, astrocytes produce a plethora of inflammatory mediators. Here, we have investigated how beta(2)-adrenergic receptor activation modulates proinflammatory gene expression in astrocytes. We have observed that treatment of human 1321N1 astrocytes with the beta-adrenergic agonist isoproterenol synergistically enhanced TNF-alpha-induced expression of the cytokine IL-6. The effect of isoproterenol was cAMP-dependent and mediated by the beta(2)-adrenergic subtype. Using pharmacological inhibitors and siRNA we showed that protein kinase A (PKA) is an indispensable mediator of the synergy. Simultaneous induction with isoproterenol and TNF-alpha was moreover associated with combined recruitment of CREB and p65 to the native IL-6 promoter. The role of CREB and NFkappaB in promoting the synergy was corroborated using IL-6 promoter point mutants, as well as via siRNA-mediated silencing of CREB and NFkappaB. Interestingly, whereas CREB and NFkappaB usually compete for the limiting cofactor CREB binding protein (CBP), we detected enhanced recruitment of CBP at the IL-6 promoter in our system. The transcriptional synergy seems to be a gene specific process, occurring at the IL-6 and COX-2 gene, but not at other typical NFkappaB-dependent genes such as IL-8, ICAM-1 or VCAM-1. As astrocytic IL-6 overexpression has been associated with neuroinflammatory and neurodegenerative processes, our findings might have important physiological consequences.

Medroxyprogesterone acetate downregulates cytokine gene expression in mouse fibroblast cells

Although medroxyprogesterone acetate (MPA) is used as an injectable contraceptive, in hormone replacement therapy (HRT) and in treatment of certain cancers, the steroid receptors and their target genes involved in the actions of MPA are not well understood. We show that MPA, like dexamethasone (dex), significantly represses tumour necrosis factor (TNF)-stimulated interleukin-6 (IL-6) protein production in mouse fibroblast (L929sA) cells. In addition, MPA repressed IL-6 and IL-8 promoter-reporter constructs at the transcriptional level, via interference with nuclear factor kappaB (NFkappaB) and activator protein-1 (AP-1). Furthermore, like dex, MPA does not affect NFkappaB DNA-binding activity. We also observed significant transactivation by MPA of a glucocorticoid response element (GRE)-driven promoter-reporter construct in both L929sA and COS-1 cells. The MPA-induced nuclear translocation of the glucocorticoid receptor (GR), as well as the antagonistic effects of RU486, strongly suggest that the actions of MPA in these cells are mediated at least in part via the GR.