Wim Vanden Berghe

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.

The nuclear factor-kappa B engages CBP/p300 and histone acetyltransferase activity for transcriptional activation of the interleukin-6 gene promoter.

Expression of the pleiotropic cytokine interleukin (IL)-6 can be stimulated by the proinflammatory cytokine tumor necrosis factor (TNF) and the microbial alkaloid staurosporine (STS). In this report, the transcriptional mechanisms were thoroughly investigated. Whereas transcription factors binding to the activator protein-1-, cAMP-responsive element-, and CAAT enhancer-binding protein-responsive sequences are necessary for gene activation by STS, nuclear factor (NF)-κB alone is responsible and sufficient for inducibility by TNF, which reveals distinct signaling pathways for both compounds. At the cofactor level, cAMP-responsive element-binding protein-binding protein (CBP) or p300 potentiate basal and induced IL-6 promoter activation via multiple protein-protein interactions with all transcription factors bound to the promoter DNA. However, the strongest promoter activation relies on the p65 NF-κB subunit, which specifically engages CBP/p300 for maximal transcriptional stimulation by its histone acetyltransferase activity. Moreover, treatment of chromatin-integrated promoter constructions with the histone deacetylase inhibitor trichostatin A exclusively potentiates TNF-dependent (i.e. NF-κB-mediated) gene activation, while basal or STS-stimulated IL-6 promoter activity remains completely unchanged. Similar observations were recorded with other natural NF-κB-driven promoters, namely IL-8 and endothelial leukocyte adhesion molecule (ELAM). We conclude that, within an “enhanceosome-like” structure, NF-κB is the central mediator of TNF-induced IL-6 gene expression, involving CBP/p300 and requiring histone acetyltransferase activity.

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.

Mechanisms of anti-inflammatory action and of immunosuppression by glucocorticoids: negative interference of activated glucocorticoid receptor with transcription factors

Glucocorticoids are the most widely used anti-inflammatory and immunomodulatory agents, whose mechanism of action is based mainly on interference with the activity of transcription factors, such as nuclear factor kappaB (NF-kappaB) and activator protein-1 (AP-1). The precise molecular mechanisms of gene repression by glucocorticoids are a controversial matter, due to the existence of many conflicting hypotheses. We discuss the three main paradigms reported in the literature, namely the inhibitor kappaB-alpha (IkappaB-alpha) upregulatory model, the protein-protein interaction model and the competition model.

Withaferin a strongly elicits IkappaB kinase beta hyperphosphorylation concomitant with potent inhibition of its kinase activity

The transcription factor NFκB plays a critical role in normal and pathophysiological immune responses. Therefore, NFκB and the signaling pathways that regulate its activation have become a major focus of drug development programs. Withania somnifera (WS) is a medicinal plant that is widely used in Palestine for the treatment of various inflammatory disorders. In this study we show that the leave extract of WS, as well as its major constituent withaferin A (WA), potently inhibits NFκB activation by preventing the tumor necrosis factor-induced activation of IκB kinase β via a thioalkylation-sensitive redox mechanism, whereas other WS-derived steroidal lactones, such as withanolide A and 12-deoxywithastramonolide, are far less effective. To our knowledge, this is the first communication of IκB kinase β inhibition by a plant-derived inhibitor, coinciding with MEK1/ERK-dependent Ser-181 hyperphosphorylation. This prevents IκB phosphorylation and degradation, which subsequently blocks NFκB translocation, NFκB/DNA binding, and gene transcription. Taken together, our results indicate that pure WA or WA-enriched WS extracts can be considered as a novel class of NFκB inhibitors, which hold promise as novel anti-inflammatory agents for treatment of various inflammatory disorders and/or cancer.

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.

How glucocorticoid receptors modulate the activity of other transcription factors: A scope beyond tethering

The activity of the glucocorticoid receptor (GR), a nuclear receptor transcription factor belonging to subclass 3C of the steroid/thyroid hormone receptor superfamily, is typically triggered by glucocorticoid hormones. Apart from driving gene transcription via binding onto glucocorticoid response elements in regulatory regions of particular target genes, GR can also inhibit gene expression via transrepression, a mechanism largely based on protein:protein interactions. Hereby GR can influence the activity of other transcription factors, without contacting DNA itself. GR is known to inhibit the activity of a growing list of immune-regulating transcription factors. Hence, GCs still rule the clinic for treatments of inflammatory disorders, notwithstanding concomitant deleterious side effects. Although patience is a virtue when it comes to deciphering the many mechanisms GR uses to influence various signaling pathways, the current review is testimony of the fact that groundbreaking mechanistic work has been accumulating over the past years and steadily continues to grow.

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.

Epigenetic impact of dietary polyphenols in cancer chemoprevention: lifelong remodeling of our epigenomes

Cancer, as one of the non-communicable diseases, remains one of the leading causes of death around the world. Recently, epigenetic changes in DNA methylation patterns at CpG sites (epimutations) or deregulated chromatin states of tumor promoting genes and noncoding RNAs emerged as major governing factors in tumor progression and cancer drug sensitivity. Furthermore, various environmental factors such as nutrition, behavior, stress, and toxins remodel our epigenomes lifelong in a beneficial or detrimental way. Since epigenetic marks (epimutations) are reversible in contrast to genetic defects, chemopreventive nutritional polyphenols (soy, genistein, resveratrol, catechin, curcumin) are currently evaluated for their ability to reverse adverse epigenetic marks in cancer (stem) cells to attenuate tumorigenesis-progression, prevent metastasis or sensitize for drug sensitivity. Although polyphenols in fruit and vegetables may help to reduce the risk of cancer, few protective effects have been firmly established, presumably because of inappropriate timing or dosing of diet exposure or due to confounding factors such as smoking and alcohol. In this review will discuss the possible epigenetic contributions of dietary polyphenols in cancer chemoprevention.

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.