Chemsex and its impact on gay and bisexual men who have sex with men: Findings from an online survey in Belgium.

OBJECTIVES: The study aimed to estimate the prevalence of sexualized drug use, or chemsex, in a wide group of gay, bisexual and other men who have sex with men (gbMSM) in Belgium. It examined which drugs gbMSM used before and during sex, the frequency with which they used it, whether they experienced non-consensual sex, and evaluated the impact of chemsex on the health and professional and social lives of respondents. METHODS: We conducted an online survey among Belgian gbMSM. Five specific questions on chemsex were included in the survey. These questions assessed whether gbMSM engaged in chemsex. If they did, they were asked to provide information on which drugs they had used and how often. We asked whether their and their partners' boundaries had been respected and whether and how engaging in chemsex had affected a range of life domains. RESULTS: Of 836 respondents, 258 (30.9%) self-reported having had sex under the influence of substances ("chemsex") in the previous 6 months. Of these, 227 (88%) were considered chemsex users according to the definition used for the analysis. Poppers (73%), gamma-hydroxybutyrate/gamma-butyrolactone (GHB/GBL; 69%) and cathinones (68%) were the most commonly reported substances. Almost half of respondents (45%) engaging in chemsex were high on drugs during at least half of their sexual encounters. Nearly 1 in 10 (9%) reported that boundaries had not been respected, suggesting non-consensual sexual activities. The 82 respondents experiencing negative impacts from their chemsex use reported that mental health (65%), physical health (40%) and sexual health (38%) were most impacted. DISCUSSION: Our findings provide a snapshot of the current chemsex situation in Belgium. The frequency of chemsex is concerning because several health, professional and social aspects are impacted. Almost 1 in 10 respondents using chemsex reported that chemsex impacted consent around sex, requiring additional efforts to eradicate these non-consensual sexual experiences. Therefore, larger scale research focusing on respected boundaries and consent during chemsex and the frequency of drug use for sex seems required. Additionally, sensitisation, as well as care and support programmes are critical.

Preconception nutraceutical food supplementation can prevent oxidative and epigenetic DNA alterations induced by ovarian stimulation for IVF and increases pregnancy rates.

BACKGROUND: It is hypothesized that oxidative and epigenetic alterations to DNA induced by ovarian stimulation for in vitro fertilization (IVF) may be associated with an increased risk of diseases and cancer in the offspring and could possibly be attenuated by preconception food supplementation. METHODS: In a prospective randomised open-label trial, 62 patients were randomly assigned to either 30 days of preconception treatment with the nutraceutical Fertility woman ® duo (Nutriphyt, Beernem, Belgium) (group 1), this nutraceutical complemented with selenomethionine (group 2), or folic acid only (group 3). Biochemical and epigenetic effects and pregnancy rates were assessed. RESULTS: In all 3 groups the level of DNA oxidative damage, estimated by the concentration of 8-hydroxy- 2-deoxyguanosine over creatinine in early morning urine, and the concentration of homocysteine in the blood decreased after treatment. In group 2, the degree of methylation of the cancer-associated CpG2 dinucleotide of the human Telomerase Reverse Transcriptase (hTERT) promoter region, assessed by pyrosequence in follicular cells obtained at oocyte pick-up, was 18% lower than that of group 3. The pregnancy rate, including the transfer of fresh and frozen embryos, was significantly higher in group 2 (50%) than in group 3 (6%) with the result in group 1 being intermediate (30%). CONCLUSION: The results suggest that preconception food supplementation using a specific nutraceutical significantly reduces oxidative and epigenetic DNA changes to follicular cells of women treated by IVF, and may optimize gene expression in the oocytes, thus increasing the pregnancy rate per cycle of ovarian stimulation.

Current levels of gonorrhoea screening in MSM in Belgium may have little effect on prevalence: a modelling study.

There is considerable uncertainty as to the effectiveness of Neisseria gonorrhoeae (NG) screening in men who have sex with men. It is important to ensure that screening has benefits that outweigh the risks of increased antibiotics resistance. We develop a mathematical model to estimate the effectiveness of screening on prevalence. Separable Temporal Exponential family Random Graph Models are used to model the sexual relationships network, both with main and casual partners. Next, the transmission of Gonorrhoea is simulated on this network. The models are implemented using the R package 'statnet', which we adapted among other things to incorporate infection status at the pharynx, urethra and rectum separately and to distinguish between anal sex, oral sex and rimming. The different screening programmes compared are no screening, 3.5% of the population screened, 32% screened and 50% screened. The model simulates day-by-day evolution for 10 years of a population of 10 000. If half of the population would be screened, the prevalence in the pharynx decreases from 11.9% to 10.2%. We conclude that the limited impact of screening on NG prevalence may not outweigh the increased risk of antibiotic resistance.

The risk for behavioural deficits is determined by the maternal immune response to prenatal immune challenge in a neurodevelopmental model.

BACKGROUND: Schizophrenia is a highly disabling psychiatric disorder with a proposed neurodevelopmental basis. One mechanism through which genetic and environmental risk factors might act is by triggering persistent brain inflammation, as evidenced by long-lasting neuro-immunological disturbances in patients. Our goal was to investigate whether microglia activation is a neurobiological correlate to the altered behaviour in the maternal immune activation (MIA) model, a well-validated animal model with relevance to schizophrenia. A recent observation in the MIA model is the differential maternal body weight response to the immune stimulus, correlated with a different behavioural outcome in the offspring. Although it is generally assumed that the differences in maternal weight response reflect differences in cytokine response, this has not been investigated so far. Our aim was to investigate whether (i) the maternal weight response to MIA reflects differences in the maternal cytokine response, (ii) the differential behavioural phenotype of the offspring extends to depressive symptoms such as anhedonia and (iii) there are changes in chronic microglia activation dependent on the behavioural phenotype. METHODS: Based on a dose-response study, MIA was induced in pregnant rats by injecting 4mg/kg Poly I:C at gestational day 15. Serum samples were collected to assess the amount of TNF-α in the maternal blood following MIA. MIA offspring were divided into weight loss (WL; n=14) and weight gain (WG; n=10) groups, depending on the maternal body weight response to Poly I:C. Adult offspring were behaviourally phenotyped for prepulse inhibition, locomotor activity with and without amphetamine and MK-801 challenge, and sucrose preference. Finally, microglia activation was scored on CD11b- and Iba1-immunohistochemically stained sections. RESULTS: Pregnant dams that lost weight following MIA showed increased levels of TNF-α compared to controls, unlike dams that gained weight following MIA. Poly I:C WL offspring showed the most severe behavioural outcome. Poly I:C WG offspring, on the other hand, did not show clear behavioural deficits. Most interestingly a reduced sucrose preference indicative of anhedonia was found in Poly I:C WL but not Poly I:C WG offspring compared to controls. Finally, there were no significant differences in microglia activation scores between any of the investigated groups. CONCLUSIONS: The individual maternal immune response to MIA is an important determinant of the behavioural outcome in offspring, including negative symptoms such as anhedonia. We failed to find any significant difference in the level of microglia activation between Poly I:C WL, Poly I:C WG and control offspring.

A venue-based HIV prevalence and behavioural study among men who have sex with men in Antwerp and Ghent, Flanders, Belgium, October 2009 to March 2010.

This venue-based, cross-sectional study reports on human immunodeficiency virus (HIV) prevalence and behaviour of 649 men who have sex with men (MSM) in Antwerp and Ghent, Flanders, Belgium, from October 2009 to March 2010. Using time-location sampling, we found that HIV prevalence in MSM who attended different types of venue ranged from a high of 14.5% (95% CI: 8.9­20.1; n=22 in cruising venues to 4.9% (95% CI: 1.9­7.9; n=10) in more general gay venues to 1.4% (95% CI: 0.0­3.6; n=3) at younger MSM venues. Of those who tested HIV positive (n=35, five were unaware of their HIV status or self-reported as being HIV negative. One in five respondents were of non-Belgian nationality. The results showed relatively high rates of testing for HIV (52.2%; 95 % CI: 47.8­56.2; n=288) and other sexually transmitted infections (STIs) (57.4%; 95% CI: 52.6­62.0; n=248) in the last 12 months. A majority of the men (n=233) used condoms consistently during their last anal sexual contact with a casual partner; however, HIV-positive men who were aware of their serostatus (n=30) reported less condom use with casual partners. This is the first such study in Belgium and the results constitute the evidence base for local, targeted interventions. Furthermore, our findings underscore the need for European cross-border cooperation to prevent HIV infection and other STIs among MSM.

Deguelin inhibits expression of IkappaBalpha protein and induces apoptosis of B-CLL cells in vitro.

We investigated if deguelin, a naturally occurring rotenoid, was able to inhibit nuclear factor kappa B (NF-kappaB)-binding protein (IkappaBalpha) expression and to induce apoptosis in B-cell chronic lymphocytic leukemia (B-CLL) cells in vitro. Deguelin-induced cell death in the majority of B-CLL cells and was found to be more toxic toward B-CLL cells than to the normal mononuclear or B-cells, suggesting selectivity towards the malignant cells. Deguelin was found to reduce IkappaBalpha protein expression, and thus interacts with the NFkappaB pathway. The induced apoptosis was characterized by processing of caspase-9 and -3 and poly-(ADP)-ribose-polymerase cleavage. Exposure of B-CLL cells to deguelin resulted in Bcl2-associated protein (Bax) conformational changes and downregulation of the key survival protein myeloid cell leukemia sequence 1 (Mcl-1), which is associated with response to treatment in B-CLL patients. Deguelin retained its ability to induce apoptosis in B-CLL cells in the presence of interleukin-4, a pro-survival cytokine in B-CLL, and when cultured with 50% human serum. These data indicate that deguelin is able to induce apoptosis in B-CLL cells in the presence of pro-survival signals and thus merits further investigation for clinical application either as a single agent or in combination with other anticancer agents.

Cross-talk between nuclear receptors and nuclear factor kappaB.

A variety of studies have shown that some activated nuclear receptors (NRs), especially the glucorticoid receptor, the estrogen receptor and peroxisome proliferator-activated receptor, can inhibit the activity of the transcription factor nuclear factor kappaB (NF-kappaB), which plays a key role in the control of genes involved in inflammation, cell proliferation and apoptosis. This review describes the molecular mechanisms of cross-talk between NRs and NF-kappaB and the biological relevance of this cross-talk. The importance and mechanistic aspects of selective NR modulation are discussed. Also included are future research prospects, which will lead to a new era in the field of NR research with the aim of specifically inhibiting NF-kappaB-driven gene expression for anti-inflammatory, anti-tumor and immune-modulatory purposes.

Signal transduction by tumor necrosis factor and gene regulation of the inflammatory cytokine interleukin-6.

Interleukin (IL)-6 is a multifunctional cytokine that can be induced by a plethora of chemical or physiological compounds, including the inflammatory cytokines tumor necrosis factor (TNF) and IL-1. The molecule TNF has a trimeric configuration and thus binds to membrane-bound, cellular receptors to initiate cell death mechanisms and signaling pathways leading to gene induction. Previously, we showed that induced clustering of the intracellular domains of the p55 TNF receptor, or of their respective 'death domains' only, is sufficient to activate the nuclear factor kappa B (NF-kappa B) and several mitogen-activated protein kinase (MAPK) pathways. NF-kappa B is the exclusive transcription factor for induction of the IL-6 gene in response to TNF and functions as the final trigger to activate a multiprotein complex, a so-called 'enhanceosome', at the level of the IL-6 promoter. Furthermore, the enhanceosome displays histone acetylation activity, which turned out to be essential for IL-6 gene activation via NF-kappa B. However, activation of NF-kappa B alone is not sufficient for IL-6 gene induction in response to TNF, as inhibition of the coactivated extracellular signal-regulated kinase and p38 MAPK pathways blocks TNF-mediated gene expression. Nevertheless, the transactivating NF-kappa B subunit p65 is not a direct target of MAPK phosphorylation. Thus, we postulated that other components of the enhanceosome complex are sensitive to MAPK cascades and found that MAPK activity is unequivocally linked to the histone acetylation capacity of the enhanceosome to stimulate gene expression in response to TNF. In contrast, glucocorticoid repression of TNF-driven IL-6 gene expression does not depend on abrogation of histone acetyltransferase activity, but originates from interference of the liganded glucocorticoid receptor with the contacts between NF-kappa B p65 and the promoter configuration around the TATA box.

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.

Glucocorticoids repress NF-kappaB-driven genes by disturbing the interaction of p65 with the basal transcription machinery, irrespective of coactivator levels in the cell.

Glucocorticoids (GCs) are used to combat inflammatory diseases. Their beneficial effect relies mainly on the inhibition of NF-kappaB- and/or AP-1-driven proinflammatory gene expression. Previously, we have shown that GCs repress tumor necrosis factor-induced IL-6 gene expression by an NF-kappaB-dependent nuclear mechanism without changing the DNA-binding capacity of NF-kappaB or the expression levels of the cytoplasmic inhibitor of NF-kappaB (IkappaB-alpha). In the present work, we investigate the effect of GC repression on different natural and/or recombinant NF-kappaB-driven reporter gene constructs in the presence of increasing amounts of various coactivator molecules, such as CREB-binding protein (CBP), p300, and SRC-1. We found that GCs maintain their repressive capacities, irrespective of the amount of cofactor present in the cell. Similar results were obtained for the reciprocal transrepression of a GC receptor (GR) element-driven reporter gene by p65. We demonstrate that neither the expression levels of p65 and CBP nor their physical association are affected by activated GR. Using Gal4 chimeras, we show that repression by GCs is specific for p65-mediated transactivation, ruling out competition for limiting nuclear factors as the major underlying mechanism of gene repression. In addition, the transactivation potential of a point-mutated Gal4-p65 variant with a decreased CBP interaction capability is still repressed by GR. Finally, we present evidence that the specificity of GC repression on p65-driven gene expression is codetermined by the TATA box context.

Peroxisome proliferator-activated receptor alpha negatively regulates the vascular inflammatory gene response by negative cross-talk with transcription factors NF-kappaB and AP-1.

Interleukin-6 (IL-6) is a pleiotropic cytokine, whose plasma levels are elevated in inflammatory diseases such as atherosclerosis. We have previously reported that peroxisome proliferator-activated receptor alpha (PPARalpha) ligands (fibrates) lower elevated plasma concentrations of IL-6 in patients with atherosclerosis and inhibit IL-1-stimulated IL-6 secretion by human aortic smooth muscle cells (SMC). Here, we show that aortic explants isolated from PPARalpha-null mice display an exacerbated response to inflammatory stimuli, such as lipopolysaccharide (LPS), as demonstrated by increased IL-6 secretion. Furthermore, fibrate treatment represses IL-6 mRNA levels in LPS-stimulated aortas of PPARalpha wild-type, but not of PPARalpha-null mice, demonstrating a role for PPARalpha in this fibrate action. In human aortic SMC, fibrates inhibit IL-1-induced IL-6 gene expression. Furthermore, activation of PPARalpha represses both c-Jun- and p65-induced transcription of the human IL-6 promoter. Transcriptional interference between PPARalpha and both c-Jun and p65 occurs reciprocally, since c-Jun and p65 also inhibit PPARalpha-mediated activation of a PPAR response element-driven promoter. This transcriptional interference occurs independent of the promoter context as demonstrated by cotransfection experiments using PPARalpha, p65, and c-Jun Gal4 chimeras. Overexpression of the transcriptional coactivator cAMP-responsive element-binding protein-binding protein (CBP) does not relieve PPARalpha-mediated transcriptional repression of p65 and c-Jun. Finally, glutathione S-transferase pull-down experiments demonstrate that PPARalpha physically interacts with c-Jun, p65, and CBP. Altogether these data indicate that fibrates inhibit the vascular inflammatory response via PPARalpha by interfering with the NF-kappaB and AP-1 transactivation capacity involving direct protein-protein interaction with p65 and c-Jun.

The nuclear factor-kappaB 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)-kappaB 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-kappaB 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-kappaB-mediated) gene activation, while basal or STS-stimulated IL-6 promoter activity remains completely unchanged. Similar observations were recorded with other natural NF-kappaB-driven promoters, namely IL-8 and endothelial leukocyte adhesion molecule (ELAM). We conclude that, within an "enhanceosome-like" structure, NF-kappaB is the central mediator of TNF-induced IL-6 gene expression, involving CBP/p300 and requiring histone acetyltransferase activity.

Dissociated glucocorticoids with anti-inflammatory potential repress interleukin-6 gene expression by a nuclear factor-kappaB-dependent mechanism.

Synthetic glucocorticoids (GCs) remain among the most effective agents for the management of chronic inflammatory diseases. However, major side effects severely limit their therapeutic use. Physiologic and therapeutic activities of GCs are mediated by a nuclear receptor belonging to a superfamily of ligand-inducible transcription factors that, in addition to directly regulating their cognate gene programs, can also mutually interfere with other signaling pathways. We recently identified selective ligands of the glucocorticoid receptor that dissociate transactivation from activator protein 1 transrepression, and most importantly retain in vivo anti-inflammatory activity. To further document the mechanisms of action sustaining the observed in vivo activity, we report here on the interference of dissociated GCs with nuclear factor kappaB (NF-kappaB)-driven gene activation. We show that dissociated GCs repress tumor necrosis factor-induced interleukin-6 gene expression by an NF-kappaB-dependent mechanism, without changing the expression level of inhibitor kappaB. The DNA-binding activity of induced NF-kappaB also remained unchanged after stimulation of cells with the various compounds. Evidence for a direct nuclear mechanism of action was obtained by analysis of cell lines constitutively expressing a fusion protein between the DNA-binding domain of the yeast Gal4 protein and the transactivating p65 subunit of NF-kappaB, which was able to efficiently repress a Gal4-dependent luciferase reporter gene upon addition of the dissociated compounds. We therefore conclude that, in addition to dissociating transactivation from activator protein 1 transrepression, dissociated GCs mediate inhibition of NF-kappaB signaling by a mechanism that is independent of inhibitor kappaB induction.

The zinc finger protein A20 inhibits TNF-induced NF-kappaB-dependent gene expression by interfering with an RIP- or TRAF2-mediated transactivation signal and directly binds to a novel NF-kappaB-inhibiting protein ABIN.

The zinc finger protein A20 is a tumor necrosis factor (TNF)- and interleukin 1 (IL-1)-inducible protein that negatively regulates nuclear factor-kappa B (NF-kappaB)-dependent gene expression. However, the molecular mechanism by which A20 exerts this effect is still unclear. We show that A20 does not inhibit TNF- induced nuclear translocation and DNA binding of NF-kappaB, although it completely prevents the TNF- induced activation of an NF-kappaB-dependent reporter gene, as well as TNF-induced IL-6 and granulocyte macrophage-colony stimulating factor gene expression. Moreover, NF-kappaB activation induced by overexpression of the TNF receptor-associated proteins TNF receptor-associated death domain protein (TRADD), receptor interacting protein (RIP), and TNF recep- tor-associated factor 2 (TRAF2) was also inhibited by expression of A20, whereas NF-kappaB activation induced by overexpression of NF-kappaB-inducing kinase (NIK) or the human T cell leukemia virus type 1 (HTLV-1) Tax was unaffected. These results demonstrate that A20 inhibits NF-kappaB-dependent gene expression by interfering with a novel TNF-induced and RIP- or TRAF2-mediated pathway that is different from the NIK-IkappaB kinase pathway and that is specifically involved in the transactivation of NF-kappaB. Via yeast two-hybrid screening, we found that A20 binds to a novel protein, ABIN, which mimics the NF-kappaB inhibiting effects of A20 upon overexpression, suggesting that the effect of A20 is mediated by its interaction with this NF-kappaB inhibiting protein, ABIN.

N-acetyl-L-cysteine inhibits primary human T cell responses at the dendritic cell level: association with NF-kappaB inhibition.

N-acetyl-L-cysteine (NAC) is an antioxidant molecule endowed with immunomodulatory properties. To investigate the effect of NAC on the induction phase of T cell responses, we analyzed its action on human dendritic cells (DC) derived from adherent PBMC cultured with IL-4 and granulocyte-macrophage CSF. We first found that NAC inhibited the constitutive as well as the LPS-induced activity of the transcription factor NF-kappaB. In parallel, NAC was shown to down-regulate the production of cytokines by DC as well as their surface expression of HLA-DR, CD86 (B7-2), and CD40 molecules both at the basal state and upon LPS activation. NAC also inhibited DC responses induced by CD40 engagement. The inhibitory effects of NAC were not due to nonspecific toxicity as neither the viability of DC nor their mannose receptor-mediated endocytosis were modified by NAC. Finally, we found that the addition of NAC to MLR between naive T cells and allogeneic DC resulted in a profound inhibition of alloreactive responses, which could be attributed to a defect of DC as APC-independent T cell responses were not inhibited by NAC. Altogether, our results suggest that NAC might impair the generation of primary immune responses in humans through its inhibitory action on DC.

p38 and extracellular signal-regulated kinase mitogen-activated protein kinase pathways are required for nuclear factor-kappaB p65 transactivation mediated by tumor necrosis factor.

Interleukin-6 (IL-6) is a pleiotropic cytokine, which is involved in inflammatory and immune responses, acute phase reactions, and hematopoiesis. In the mouse fibrosarcoma cell line L929, the nuclear factor (NF)-kappaB plays a crucial role in IL-6 gene expression mediated by tumor necrosis factor (TNF). The levels of the activated factor do not, however, correlate with the variations of IL-6 gene transcription; therefore, other factors and/or regulatory mechanisms presumably modulate the levels of IL-6 mRNA production. Upon analysis of various deletion and point-mutated variants of the human IL-6 gene promoter coupled to a reporter gene, we screened for possible cooperating transcription factors. Even the smallest deletion variant, containing almost exclusively a NF-kappaB-responsive sequence preceding the IL-6 minimal promoter, as well as a recombinant construction containing multiple kappaB-motifs, could still be stimulated with TNF. We observed that the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 was able to repress TNF-stimulated expression of the IL-6 gene, as well as of a kappaB-dependent reporter gene construct, without affecting the levels of NF-kappaB binding to DNA. Furthermore, we clearly show that, using a nuclear Gal4 "one-hybrid" system, the MAPK inhibitors SB203580 and PD0980589 have a direct repressive effect on the transactivation potential of the p65 kappaB subunit. Therefore, we conclude that, in addition to cytoplasmic activation and DNA binding of NF-kappaB, the p38 and extracellular signal-regulated kinase MAPK pathways act as necessary cooperative mechanisms to regulate TNF-induced IL-6 gene expression by modulating the transactivation machinery.

Recombination signal sequence binding protein Jkappa is constitutively bound to the NF-kappaB site of the interleukin-6 promoter and acts as a negative regulatory factor.

Analysis by electrophoretic mobility shift assays (EMSA) of the different proteins associated with the kappaB sequence of the interleukin-6 (IL-6) promoter (IL6-kappaB) allowed us to detect a specific complex formed with the recombination signal sequence binding protein Jkappa (RBP-Jkappa). Single-base exchanges within the oligonucleotide sequence defined the critical base pairs involved in the interaction between RBP-Jkappa and the IL6-kappaB motif. Binding analysis suggests that the amount of RBP-Jkappa protein present in the nucleus is severalfold higher than the total amount of inducible NF-kappaB complexes but that the latter bind DNA with a 10-fold-higher affinity. A reporter gene study was performed to determine the functional implication of this binding; we found that the constitutive occupancy of the IL6-kappaB site by the RBP-Jkappa protein was responsible for the low basal levels of IL-6 promoter activity in L929sA fibrosarcoma cells and that RBP-Jkappa partially blocked access of NF-kappaB complexes to the IL-6 promoter. We propose that such a mechanism could be involved in the constitutive repression of the IL-6 gene under normal physiological conditions.

Glucocorticoid-mediated repression of nuclear factor-kappaB-dependent transcription involves direct interference with transactivation.

Glucocorticoids exert multiple anti-inflammatory activities, one of which is the inhibition of transcription dependent on the nuclear factor (NF)-kappaB. It has been suggested that the effect of dexamethasone (DEX), a glucocorticoid analog, is attributed to an increased production of the inhibitory IkappaB molecule, which in turn would bind and remove activated, DNA-bound NF-kappaB complexes in the cell nucleus. Upon investigating DEX-mediated repression of interleukin-6 expression induced by tumor necrosis factor, DEX treatment was found to act directly on NF-kappaB-dependent transcription, without changing the expression level of IkappaB. Neither the mRNA of IkappaB nor the protein was significantly elevated by a combined treatment with tumor necrosis factor and DEX of murine endothelial or fibroblast cells. The DNA-binding activity of induced NF-kappaB also remained unchanged after stimulation of cells with DEX. Evidence for a direct nuclear mechanism of action was obtained by analysis of cell lines stably expressing a fusion protein between the DNA-binding domain of the yeast Gal4 protein and the transactivating p65 subunit of NF-kappaB. Expression of a Gal4-dependent luciferase reporter gene activated by this nuclear fusion protein was also strongly repressed after addition of DEX. Because the DNA-binding activity of the Gal4 fusion protein was not affected by DEX, it can be concluded that the reduction of gene activation was caused by interference of the activated glucocorticoid receptor with the transactivation potential of the NF-kappaB p65 subunit.

The p38/RK mitogen-activated protein kinase pathway regulates interleukin-6 synthesis response to tumor necrosis factor.

Tumour necrosis factor (TNF) is a pleiotropic cytokine, the activities of which include effects on gene expression, cell growth and cell death. The biological signalling mechanisms which are responsible for these TNF effects remain largely unknown. Here we demonstrate that the stress-responsive p38 mitogen-activated protein (MAP) kinase is involved in TNF-induced cytokine expression. TNF Treatment of cell activated the p38 MAP kinase pathway, as revealed by increased phosphorylation of p38 MAP kinase itself, activation of the substrate protein MAPKAP kinase-2, and culminating in the phosphorylation of the heat shock protein 27 (hsp27). Pretreatment of cells with the highly specific p38 MAP kinase inhibitor SB203580 completely blocked this TNF-induced activation of MAPKAP kinase-2 and hsp27 phosphorylation. Under the same conditions, SB203580 also completely inhibited TNF-induced synthesis of interleukin (IL)-6 and expression of a reporter gene that was driven by a minimal promoter containing two NF-Kappa B elements. However, neither TNF-induced DNA binding of TNF-Kappa B nor TNF-induced phosphorylation of its subunits was modulated by SB203580, suggesting that NF-Kappa B is not a direct target for the p38 MAP kinase pathway. Interestingly, TNF-induced cytotoxicity was not affected by SB203580, indicating that p38 MAP kinase might be an interesting target to interfere selectively with TNF-induced gene activation.