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1.
Front Immunol ; 10: 1769, 2019.
Article in English | MEDLINE | ID: mdl-31447832

ABSTRACT

Glucocorticoids (GCs) act via the glucocorticoid receptor (NR3C1, GRα) to combat overshooting responses to infectious stimuli, including lipopolysaccharide (LPS). As such, GCs inhibit the activity of downstream effector cytokines, such as tumor necrosis factor (TNF). PPARα (NR1C1) is a nuclear receptor described to function on the crossroad between lipid metabolism and control of inflammation. In the current work, we have investigated the molecular mechanism by which GCs and PPARα agonists cooperate to jointly inhibit NF-κB-driven expression in A549 cells. We discovered a nuclear mechanism that predominantly targets Mitogen- and Stress-activated protein Kinase-1 activation upon co-triggering GRα and PPARα. In vitro GST-pull down data further support that the anti-inflammatory mechanism may additionally involve a non-competitive physical interaction between the p65 subunit of NF-κB, GRα, and PPARα. Finally, to study metabolic effector target cells common to both receptors, we overlaid the effect of GRα and PPARα crosstalk in mouse primary hepatocytes under LPS-induced inflammatory conditions on a genome-wide level. RNA-seq results revealed lipid metabolism genes that were upregulated and inflammatory genes that were additively downregulated. Validation at the cytokine protein level finally supported a consistent additive anti-inflammatory response in hepatocytes.


Subject(s)
Inflammation/immunology , PPAR alpha/immunology , Receptors, Glucocorticoid/immunology , A549 Cells , Animals , Dexamethasone/pharmacology , Glucocorticoids/pharmacology , Hepatocytes/drug effects , Hepatocytes/metabolism , Humans , Lipid Metabolism/genetics , Lipopolysaccharides , Male , Mice, Inbred C57BL , NF-kappa B/immunology , PPAR alpha/agonists
2.
Endocr Rev ; 39(5): 760-802, 2018 10 01.
Article in English | MEDLINE | ID: mdl-30020428

ABSTRACT

Peroxisome proliferator-activated receptor α (PPARα) is a nuclear receptor of clinical interest as a drug target in various metabolic disorders. PPARα also exhibits marked anti-inflammatory capacities. The first-generation PPARα agonists, the fibrates, have however been hampered by drug-drug interaction issues, statin drop-in, and ill-designed cardiovascular intervention trials. Notwithstanding, understanding the molecular mechanisms by which PPARα works will enable control of its activities as a drug target for metabolic diseases with an underlying inflammatory component. Given its role in reshaping the immune system, the full potential of this nuclear receptor subtype as a versatile drug target with high plasticity becomes increasingly clear, and a novel generation of agonists may pave the way for novel fields of applications.


Subject(s)
Inflammation/metabolism , Lipid Metabolism/physiology , PPAR alpha/metabolism , Animals , Humans , Liver/metabolism
3.
J Invest Dermatol ; 138(6): 1360-1370, 2018 06.
Article in English | MEDLINE | ID: mdl-29288652

ABSTRACT

Children with atopic dermatitis show an increased risk to develop asthma later in life, a phenomenon referred to as "atopic march," which emphasizes the need for secondary prevention therapies. This study aimed to investigate whether relief of skin inflammation by glucocorticoids and peroxisome proliferator-activated receptor agonists might influence the subsequent development of asthma in a murine model for the atopic march in which mice were repeatedly exposed to house dust mite via the skin, followed by exposure to house dust mite in lungs. To abrogate atopic dermatitis, mice received topical treatment with glucocorticoid receptor/peroxisome proliferator-activated receptor-γ agonists. Nuclear receptor ligand effects were assessed on primary keratinocytes and dendritic cells, as central players in skin inflammation. Prior house dust mite-induced skin inflammation aggravates allergic airway inflammation and induces a mixed T helper type 2/T helper type 17 response in the lungs. Cutaneous combined activation of glucocorticoid receptor/peroxisome proliferator-activated receptor-γ reduced skin inflammation to a higher extent compared to single activation. Additive anti-inflammatory effects were more prominent in dendritic cells, as compared to keratinocytes. Alleviation of allergic skin inflammation by activation of glucocorticoid receptor/peroxisome proliferator-activated receptor-γ appeared insufficient to avoid the allergic immune response in the lungs, but efficiently reduced asthma severity by counteracting the Th17 response. Glucocorticoid receptor/peroxisome proliferator-activated receptor-γ co-activation represents a potent remedy against allergic skin inflammation and worsening of atopic march.


Subject(s)
Asthma/prevention & control , Dermatitis, Atopic/drug therapy , Glucocorticoids/pharmacology , PPAR gamma/metabolism , Receptors, Glucocorticoid/metabolism , Administration, Cutaneous , Animals , Asthma/diagnosis , Asthma/immunology , Dendritic Cells , Dermatitis, Atopic/complications , Dermatitis, Atopic/immunology , Disease Models, Animal , Female , Glucocorticoids/therapeutic use , Humans , Keratinocytes , Lung/cytology , Lung/immunology , Mice , Mice, Inbred C57BL , PPAR gamma/agonists , Primary Cell Culture , Pyroglyphidae/immunology , Receptors, Glucocorticoid/agonists , Severity of Illness Index , Skin/cytology , Skin/drug effects , Skin/immunology , Th17 Cells/drug effects , Th17 Cells/immunology , Th2 Cells/drug effects , Th2 Cells/immunology
4.
Front Immunol ; 8: 1324, 2017.
Article in English | MEDLINE | ID: mdl-29163463

ABSTRACT

Synthetic glucocorticoids (GC) are the mainstay therapy for treatment of acute and chronic inflammatory disorders. Due to the high adverse effects associated with long-term use, GC pharmacology has focused since the nineties on more selective GC ligand-binding strategies, classified as selective glucocorticoid receptor (GR) agonists (SEGRAs) or selective glucocorticoid receptor modulators (SEGRMs). In the current study, GSK866 analogs with electrophilic covalent-binding warheads were developed with potential SEGRA properties to improve their clinical safety profile for long-lasting topical skin disease applications. Since the off-rate of a covalently binding drug is negligible compared to that of a non-covalent drug, its therapeutic effects can be prolonged and typically, smaller doses of the drug are necessary to reach the same level of therapeutic efficacy, thereby potentially reducing systemic side effects. Different analogs of SEGRA GSK866 coupled to cysteine reactive warheads were characterized for GR potency and selectivity in various biochemical and cellular assays. GR- and NFκB-dependent reporter gene studies show favorable anti-inflammatory properties with reduced GR transactivation of two non-steroidal GSK866 analogs UAMC-1217 and UAMC-1218, whereas UAMC-1158 and UAMC-1159 compounds failed to modulate cellular GR activity. These results were further supported by GR immuno-localization and S211 phospho-GR western analysis, illustrating significant GR phosphoactivation and nuclear translocation upon treatment of GSK866, UAMC-1217, or UAMC-1218, but not in case of UAMC-1158 or UAMC-1159. Furthermore, mass spectrometry analysis of tryptic peptides of recombinant GR ligand-binding domain (LBD) bound to UAMC-1217 or UAMC-1218 confirmed covalent cysteine-dependent GR binding. Finally, molecular dynamics simulations, as well as glucocorticoid receptor ligand-binding domain (GR-LBD) coregulator interaction profiling of the GR-LBD bound to GSK866 or its covalently binding analogs UAMC-1217 or UAMC-1218 revealed subtle conformational differences that might underlie their SEGRA properties. Altogether, GSK866 analogs UAMC-1217 and UAMC-1218 hold promise as a novel class of covalent-binding SEGRA ligands for the treatment of topical inflammatory skin disorders.

5.
Sci Rep ; 7(1): 8941, 2017 08 21.
Article in English | MEDLINE | ID: mdl-28827617

ABSTRACT

The transcriptional activity of the glucocorticoid receptor (GR) is co-determined by its ability to recruit a vast and varying number of cofactors. We here identify Striatin-3 (STRN3) as a novel interaction partner of GR that interferes with GR's ligand-dependent transactivation capacity. Remarkably, STRN3 selectively affects only GR-dependent transactivation and leaves GR-dependent transrepression mechanisms unhampered. We found that STRN3 down-regulates GR transactivation by an additional recruitment of the catalytic subunit of protein phosphatase 2A (PPP2CA) to GR. We hypothesize the existence of a functional trimeric complex in the nucleus, able to dephosphorylate GR at serine 211, a known marker for GR transactivation in a target gene-dependent manner. The presence of STRN3 appears an absolute prerequisite for PPP2CA to engage in a complex with GR. Herein, the C-terminal domain of GR is essential, reflecting ligand-dependency, yet other receptor parts are also needed to create additional contacts with STRN3.


Subject(s)
Autoantigens/metabolism , Calmodulin-Binding Proteins/metabolism , Down-Regulation , Protein Phosphatase 2/metabolism , Receptors, Glucocorticoid/chemistry , Receptors, Glucocorticoid/genetics , A549 Cells , Binding Sites , Cell Nucleus/metabolism , HEK293 Cells , HeLa Cells , Humans , Phosphorylation , Protein Interaction Maps , Protein Multimerization , Receptors, Glucocorticoid/metabolism , Transcriptional Activation
6.
J Nat Prod ; 80(5): 1505-1513, 2017 05 26.
Article in English | MEDLINE | ID: mdl-28489375

ABSTRACT

Species of the genus Laserpitium have been used traditionally to treat inflammation and infection. From the herb of Laserpitium zernyi, six new compounds were isolated and their structures elucidated (using IR, NMR, HRMS data) as derivatives of 8-daucene-2,4,10-triol (1, 2, and 4), 7-daucene-2,4,10-triol (3), a lapiferin derivative featuring a C-2 ester moiety (5), and a daucane featuring an exomethylene group at C-8 (6). Also isolated were the rare daucanes vaginatin (7) and laserpitin (8). In a search for selective glucocorticoid receptor (GR) modulators, the compounds were tested for their capacity to inhibit NF-κB and AP-1 pro-inflammatory factors and for a potential competitive effect on a dexamethasone (Dex)-induced GR-driven glucocorticoid response element (GRE) reporter gene. The new 2ß-angeloyloxy-10α-acetoxy-8-daucene-2,4,10-triol (2) significantly inhibited transactivation of both NF-κB and AP-1, while vaginatin (7) was the most active of the compounds tested in blocking AP-1. Both compounds competitively repressed Dex-induced GRE-driven promoter activities, indicative of a potential role for GR. In addition, a decreased potential to inhibit NF-κB was apparent in GR knockout A549 cells. In line with the transcriptional assays, compounds 2 and 7 also significantly lowered CCL-2 chemokine production, albeit to a lesser extent than Dex. The results suggest that daucanes may be interesting candidates in the search for compounds with GR-modulating activities.


Subject(s)
Anti-Inflammatory Agents/isolation & purification , Anti-Inflammatory Agents/pharmacology , Apiaceae/chemistry , Bridged Bicyclo Compounds/isolation & purification , Bridged Bicyclo Compounds/pharmacology , Dexamethasone/antagonists & inhibitors , Dexamethasone/chemistry , NF-kappa B/antagonists & inhibitors , Receptors, Glucocorticoid/antagonists & inhibitors , Receptors, Glucocorticoid/chemistry , Sesquiterpenes/isolation & purification , Sesquiterpenes/pharmacology , Anti-Inflammatory Agents/chemistry , Bridged Bicyclo Compounds/chemistry , Esters , Humans , Magnetic Resonance Spectroscopy , Molecular Structure , NF-kappa B/chemistry , Sesquiterpenes/chemistry , Transcription Factor AP-1 , Transcriptional Activation
7.
Nucleic Acids Res ; 44(22): 10539-10553, 2016 12 15.
Article in English | MEDLINE | ID: mdl-27576532

ABSTRACT

Adaptation to fasting involves both Glucocorticoid Receptor (GRα) and Peroxisome Proliferator-Activated Receptor α (PPARα) activation. Given both receptors can physically interact we investigated the possibility of a genome-wide cross-talk between activated GR and PPARα, using ChIP- and RNA-seq in primary hepatocytes. Our data reveal extensive chromatin co-localization of both factors with cooperative induction of genes controlling lipid/glucose metabolism. Key GR/PPAR co-controlled genes switched from transcriptional antagonism to cooperativity when moving from short to prolonged hepatocyte fasting, a phenomenon coinciding with gene promoter recruitment of phosphorylated AMP-activated protein kinase (AMPK) and blocked by its pharmacological inhibition. In vitro interaction studies support trimeric complex formation between GR, PPARα and phospho-AMPK. Long-term fasting in mice showed enhanced phosphorylation of liver AMPK and GRα Ser211. Phospho-AMPK chromatin recruitment at liver target genes, observed upon prolonged fasting in mice, is dampened by refeeding. Taken together, our results identify phospho-AMPK as a molecular switch able to cooperate with nuclear receptors at the chromatin level and reveal a novel adaptation mechanism to prolonged fasting.


Subject(s)
Adenylate Kinase/metabolism , Chromatin/metabolism , PPAR alpha/physiology , Receptors, Glucocorticoid/physiology , Animals , Base Sequence , Binding Sites , Cells, Cultured , Enhancer Elements, Genetic , Fasting , Hepatocytes/metabolism , Lipid Metabolism , Mice, Inbred C57BL , Mice, Knockout , Protein Transport , Sequence Analysis, DNA , Transcriptional Activation , Transcriptome
8.
Chem Commun (Camb) ; 51(48): 9868-71, 2015 Jun 18.
Article in English | MEDLINE | ID: mdl-25994553

ABSTRACT

A small library of 3-[(4-hydroxycarbamoylphenyl)aminomethyl]benzothiophenes was prepared and assessed as a novel class of HDAC6 inhibitors, leading to the identification of three representatives as potent and selective HDAC6 inhibitors. Further tests with regard to inflammatory responses indicated that HDAC6 inhibition can be uncoupled from transcriptional inhibition at the level of activated NF-κB, AP-1, and GR.


Subject(s)
Histone Deacetylase Inhibitors/chemical synthesis , Hydroxamic Acids/chemical synthesis , Thiophenes/chemical synthesis , Cell Line, Tumor , Histone Deacetylase 6 , Histone Deacetylase Inhibitors/chemistry , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylases/chemistry , Histone Deacetylases/metabolism , Humans , Hydroxamic Acids/chemistry , Models, Molecular , NF-kappa B/metabolism , Thiophenes/chemistry , Transcription Factor AP-1/metabolism , Tubulin/metabolism
9.
Molecules ; 20(1): 863-78, 2015 Jan 08.
Article in English | MEDLINE | ID: mdl-25580684

ABSTRACT

In this study a series of curcumin analogues were evaluated for their ability to inhibit the activation of NF-κΒ, a transcription factor at the crossroads of cancer-inflammation. Our novel curcumin analogue BAT3 was identified to be the most potent NF-κB inhibitor and EMSA assays clearly showed inhibition of NF-κB/DNA-binding in the presence of BAT3, in agreement with reporter gene results. Immunofluorescence experiments demonstrated that BAT3 did not seem to prevent nuclear p65 translocation, so our novel analogue may interfere with NF-κB/DNA-binding or transactivation, independently of IKK2 regulation and NF-κB-translocation. Gene expression studies on endogenous NF-κB target genes revealed that BAT3 significantly inhibited TNF-dependent transcription of IL6, MCP1 and A20 genes, whereas an NF-κB independent target gene heme oxygenase-1 remained unaffected. In conclusion, we demonstrate that BAT3 seems to inhibit different cancer-related inflammatory targets in the NF-κB signaling pathway through a different mechanism in comparison to similar analogues, previously reported.


Subject(s)
Curcumin/analogs & derivatives , Curcumin/pharmacology , Heterocyclic Compounds/pharmacology , NF-kappa B/metabolism , Signal Transduction/drug effects , Animals , Anti-Inflammatory Agents/pharmacology , Cell Line , Curcumin/chemistry , DNA/metabolism , Gene Expression/drug effects , Genes, Reporter , Humans , Inhibitory Concentration 50 , Mice , Protein Binding/drug effects , Protein Transport/drug effects , Structure-Activity Relationship , Transcription, Genetic/drug effects
10.
Cell Mol Life Sci ; 71(1): 143-63, 2014 Jan.
Article in English | MEDLINE | ID: mdl-23784308

ABSTRACT

Glucocorticoids (GCs) block inflammation via interference of the liganded glucocorticoid receptor (GR) with the activity of pro-inflammatory transcription factors NF-κB and AP-1, a mechanism known as transrepression. This mechanism is believed to involve the activity of GR monomers. Here, we explored how the GR monomer-favoring Compound A (CpdA) affects AP-1 activation and activity. Our results demonstrate that non-steroidal CpdA, unlike classic steroidal GCs, blocks NF-κB- but not AP-1-driven gene expression. CpdA rather sustains AP-1-driven gene expression, a result which could mechanistically be explained by the failure of CpdA to block upstream JNK kinase activation and concomitantly also phosphorylation of c-Jun. In concordance and in contrast to DEX, CpdA maintained the expression of the activated AP-1 target gene c-jun, as well as the production of the c-Jun protein. As for the underlying mechanism, GR is a necessary intermediate in the CpdA-mediated gene expression of AP-1-regulated genes, but seems to be superfluous to CpdA-mediated JNK phosphorylation prolongation. The latter phenomenon concurs with the inability of CpdA to stimulate DUSP1 gene expression. ChIP analysis demonstrates that DEX-activated GR, but not CpdA-activated GR, is recruited to AP-1-driven promoters. Furthermore, in mice we observed that CpdA instigates a strong enhancement of TNF-induced AP-1-driven gene expression. Finally, we demonstrate that this phenomenon coincides with an increased sensitivity towards TNF lethality, and implicate again a role for JNK2. In conclusion, our data support the hypothesis that a ligand-induced differential conformation of GR yields a different transcription factor cross-talk profile.


Subject(s)
NF-kappa B/metabolism , Receptors, Glucocorticoid/metabolism , Transcription Factor AP-1/metabolism , Animals , Cell Line, Tumor , Dexamethasone/pharmacology , Dual Specificity Phosphatase 1/genetics , Dual Specificity Phosphatase 1/metabolism , Gene Expression/drug effects , Humans , Interleukin-6/genetics , Interleukin-6/metabolism , JNK Mitogen-Activated Protein Kinases/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitogen-Activated Protein Kinase 9/deficiency , Mitogen-Activated Protein Kinase 9/genetics , Mitogen-Activated Protein Kinase 9/metabolism , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation/drug effects , Promoter Regions, Genetic , Proto-Oncogene Proteins c-jun/genetics , Proto-Oncogene Proteins c-jun/metabolism , RNA Interference , RNA, Small Interfering/metabolism , Receptors, Glucocorticoid/antagonists & inhibitors , Receptors, Glucocorticoid/genetics , Staurosporine/pharmacology , Transcription Factor AP-1/genetics , Transcriptional Activation/drug effects , Triazoles/pharmacology , Tumor Necrosis Factor-alpha/pharmacology
11.
PLoS One ; 8(7): e69115, 2013.
Article in English | MEDLINE | ID: mdl-23935933

ABSTRACT

Compound A possesses glucocorticoid receptor (GR)-dependent anti-inflammatory properties. Just like classical GR ligands, Compound A can repress NF-κB-mediated gene expression. However, the monomeric Compound A-activated GR is unable to trigger glucocorticoid response element-regulated gene expression. The heat shock response potently activates heat shock factor 1 (HSF1), upregulates Hsp70, a known GR chaperone, and also modulates various aspects of inflammation. We found that the selective GR modulator Compound A and heat shock trigger similar cellular effects in A549 lung epithelial cells. With regard to their anti-inflammatory mechanism, heat shock and Compound A are both able to reduce TNF-stimulated IκBα degradation and NF-κB p65 nuclear translocation. We established an interaction between Compound A-activated GR and Hsp70, but remarkably, although the presence of the Hsp70 chaperone as such appears pivotal for the Compound A-mediated inflammatory gene repression, subsequent novel Hsp70 protein synthesis is uncoupled from an observed CpdA-induced Hsp70 mRNA upregulation and hence obsolete in mediating CpdA's anti-inflammatory effect. The lack of a Compound A-induced increase in Hsp70 protein levels in A549 cells is not mediated by a rapid proteasomal degradation of Hsp70 or by a Compound A-induced general block on translation. Similar to heat shock, Compound A can upregulate transcription of Hsp70 genes in various cell lines and BALB/c mice. Interestingly, whereas Compound A-dependent Hsp70 promoter activation is GR-dependent but HSF1-independent, heat shock-induced Hsp70 expression alternatively occurs in a GR-independent and HSF1-dependent manner in A549 lung epithelial cells.


Subject(s)
Gene Expression Regulation , HSP70 Heat-Shock Proteins/genetics , Promoter Regions, Genetic , Receptors, Glucocorticoid/metabolism , Transcriptional Activation , Animals , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/metabolism , Anti-Inflammatory Agents/pharmacology , Cell Line , DNA-Binding Proteins/metabolism , Dose-Response Relationship, Drug , Female , Gene Expression Regulation/drug effects , HSP70 Heat-Shock Proteins/metabolism , HSP90 Heat-Shock Proteins/metabolism , Heat Shock Transcription Factors , Humans , Mice , Models, Biological , NF-kappa B/antagonists & inhibitors , NF-kappa B/metabolism , Protein Binding , Receptors, Glucocorticoid/agonists , Response Elements , Transcription Factors/metabolism
12.
Eur J Pharmacol ; 715(1-3): 1-9, 2013 Sep 05.
Article in English | MEDLINE | ID: mdl-23831393

ABSTRACT

The glucocorticoid receptor is a nuclear receptor, and can be activated by glucocorticoid ligands. Mitogen- and stress-activated protein kinase (MSK1), when activated by p38 and ERK mitogen-activated protein kinases (MAPKs), plays a major role in chromatin relaxation via phosphorylation of histone H3 S10. The glucocorticoid receptor can target MSK1 as part of its anti-inflammatory mechanism. Here, we studied the converse mechanism, i.e. the impact of MSK1 on glucocorticoid receptor-mediated transactivation. Upstream MSK1-activating kinases concentration-dependently enhanced glucocorticoid response element (GRE)-regulated promoter activity. Correspondingly, MSK1 inhibition, via H89, or combined p38 and ERK MAPK inhibition, via SB203580 and U0126, diminished maximally stimulated GRE-regulated promoter activity using high concentrations of glucocorticoids. Concomitantly, the combination of these agents does not seem to alter site-specific phosphorylations of murine glucocorticoid receptor S212 or S220. Paradoxically, we reveal that a sub-maximally activated GRE-mediated promoter activity, by using lower concentrations of glucocorticoids, is consistently enhanced by H89 or a combination of SB203580 and U0126, irrespective of the GRE promoter context. Furthermore, we show that the glucocorticoid-induced nucleocytoplasmic translocation of MSK1 occurs in a glucocorticoid concentration-dependent manner. The observed glucocorticoid concentration-dependent effect of MSK1 or MAPK inhibition on glucocorticoid receptor transactivation warrants further research into the applicability of combined glucocorticoid and kinase inhibitor strategies for anti-inflammatory purposes.


Subject(s)
Glucocorticoids/metabolism , Glucocorticoids/pharmacology , Response Elements/genetics , Ribosomal Protein S6 Kinases, 90-kDa/metabolism , Animals , Cell Line, Tumor , Cytoplasm/drug effects , Cytoplasm/metabolism , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Isoquinolines/pharmacology , MAP Kinase Kinase 6/genetics , MAP Kinase Kinase 6/metabolism , Mice , Protein Transport/drug effects , Receptors, Glucocorticoid/genetics , Receptors, Glucocorticoid/metabolism , Response Elements/drug effects , Ribosomal Protein S6 Kinases, 90-kDa/antagonists & inhibitors , Sulfonamides/pharmacology , Transcription, Genetic/drug effects , Transcriptional Activation/drug effects , Transfection , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/genetics , p38 Mitogen-Activated Protein Kinases/metabolism
13.
Cancer Lett ; 305(1): 21-31, 2011 Jun 01.
Article in English | MEDLINE | ID: mdl-21420233

ABSTRACT

Vimang is a standardized extract derived from Mango bark (Mangifera Indica L.), commonly used as anti-inflammatory phytomedicine, which has recently been used to complement cancer therapies in cancer patients. We have further investigated potential anti-tumour effects of glucosylxanthone mangiferin and indanone gallic acid, which are both present in Vimang extract. We observed significant anti-tumour effects of both Vimang constituents in the highly aggressive and metastatic breast cancer cell type MDA-MB231. At the molecular level, mangiferin and gallic acid both inhibit classical NFκB activation by IKKα/ß kinases, which results in impaired IκB degradation, NFκB translocation and NFκB/DNA binding. In contrast to the xanthone mangiferin, gallic acid further inhibits additional NFκB pathways involved in cancer cell survival and therapy resistance, such as MEK1, JNK1/2, MSK1, and p90RSK. This results in combinatorial inhibition of NFκB activity by gallic acid, which results in potent inhibition of NFκB target genes involved in inflammation, metastasis, anti-apoptosis and angiogenesis, such as IL-6, IL-8, COX2, CXCR4, XIAP, bcl2, VEGF. The cumulative NFκB inhibition by gallic acid, but not mangiferin, is also reflected at the level of cell survival, which reveals significant tumour cytotoxic effects in MDA-MB231 cells. Altogether, we identify gallic acid, besides mangiferin, as an essential anti-cancer component in Vimang extract, which demonstrates multifocal inhibition of NFκB activity in the cancer-inflammation network.


Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/metabolism , Gallic Acid/analogs & derivatives , Gallic Acid/pharmacology , Phytotherapy , Plant Extracts/pharmacology , Xanthones/pharmacology , Blotting, Western , Cell Line, Tumor , Cell Survival/drug effects , Electrophoretic Mobility Shift Assay , Female , Humans , Immunosuppressive Agents/pharmacology , Mangifera/chemistry , Plant Bark/chemistry , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects
14.
Mol Endocrinol ; 24(2): 310-22, 2010 Feb.
Article in English | MEDLINE | ID: mdl-19965930

ABSTRACT

Compound A (CpdA), a plant-derived phenyl aziridine precursor, was recently characterized as a fully dissociated nonsteroidal antiinflammatory agent, acting via activation of the glucocorticoid receptor, thereby down-modulating nuclear factor-kappaB-mediated transactivation, but not supporting glucocorticoid response element-driven gene expression. The present study demonstrates the effectiveness of CpdA in inhibiting the disease progress in experimental autoimmune encephalomyelitis (EAE), a well-characterized animal model of multiple sclerosis. CpdA treatment of mice, both early and at the peak of the disease, markedly suppressed the clinical symptoms of EAE induced by myelin oligodendrocyte glycoprotein peptide immunization. Attenuation of the clinical symptoms of EAE by CpdA was accompanied by reduced leukocyte infiltration in the spinal cord, reduced expression of inflammatory cytokines and chemokines, and reduced neuronal damage and demyelination. In vivo CpdA therapy suppressed the encephalogenicity of myelin oligodendrocyte glycoprotein peptide-specific T cells. Moreover, CpdA was able to inhibit TNF- and lipopolysaccharide-induced nuclear factor-kappaB activation in primary microglial cells in vitro, in a differential mechanistic manner as compared with dexamethasone. Finally, in EAE mice the therapeutic effect of CpdA, in contrast to that of dexamethasone, occurred in the absence of hyperinsulinemia and in the absence of a suppressive effect on the hypothalamic-pituitary-adrenal axis. Based on these results, we propose CpdA as a compound with promising antiinflammatory characteristics useful for therapeutic intervention in multiple sclerosis and other neuroinflammatory diseases.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Neuroprotective Agents/therapeutic use , Quaternary Ammonium Compounds/therapeutic use , Receptors, Glucocorticoid/agonists , Acetates , Animals , Anti-Inflammatory Agents, Non-Steroidal/administration & dosage , Anti-Inflammatory Agents, Non-Steroidal/adverse effects , Astrocytes/drug effects , Astrocytes/metabolism , Astrocytes/pathology , Cells, Cultured , Cytokines/genetics , Cytokines/metabolism , Demyelinating Diseases/pathology , Encephalomyelitis, Autoimmune, Experimental/chemically induced , Encephalomyelitis, Autoimmune, Experimental/pathology , Gene Expression Regulation/drug effects , Leukocytes/drug effects , Male , Mice , Mice, Inbred C57BL , Microglia/drug effects , Microglia/metabolism , Microglia/pathology , Multiple Sclerosis/drug therapy , NF-kappa B/metabolism , Neuroprotective Agents/administration & dosage , Neuroprotective Agents/adverse effects , Protein Transport/drug effects , Quaternary Ammonium Compounds/administration & dosage , Quaternary Ammonium Compounds/adverse effects , RNA, Messenger/metabolism , Spinal Cord/drug effects , Spinal Cord/pathology , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Time Factors , Tyramine/analogs & derivatives
15.
Arthritis Rheum ; 60(11): 3241-50, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19877072

ABSTRACT

OBJECTIVE: To investigate and compare the molecular mechanisms by which 2 glucocorticoid receptor (GR)-activating compounds, dexamethasone (DEX) and Compound A (CpdA), interfere with the NF-kappaB activation pathway in rheumatoid arthritis (RA) synovial cells. METHODS: Quantitative polymerase chain reaction was performed to detect the tumor necrosis factor alpha (TNFalpha)-induced cytokine gene expression of interleukin-1beta (IL-1beta) and to investigate the effects of DEX and CpdA in RA fibroblast-like synoviocytes (FLS) transfected with small interfering RNA (siRNA) against GR (siGR) compared with nontransfected cells. Immunofluorescence analysis was used to detect the subcellular distribution of NF-kappaB (p65) under the various treatment conditions, and active DNA-bound p65 was measured using a TransAM assay and by chromatin immunoprecipitation analysis of IL-1beta. Signaling pathways were studied via Western blotting of siGR-transfected cells, compared with nontransfected and nontargeting siRNA-transfected control cells, to detect the regulation of phospho-IKK, IkappaBalpha, phospho-p38, phospho-ERK, and phospho-JNK. RESULTS: Both DEX and CpdA efficiently inhibited IL-1beta gene expression in a GR-dependent manner. In addition, CpdA attenuated the TNFalpha-induced nuclear translocation and DNA binding of p65 in RA FLS, via the attenuation of IKK phosphorylation and subsequent IkappaBalpha degradation. CpdA also displayed profound effects on TNFalpha-induced MAPK activation. The effects of CpdA on TNFalpha-induced kinase activities occurred independently of the presence of GR. In sharp contrast, DEX did not affect TNFalpha-induced IKK phosphorylation, IkappaBalpha degradation, p65 nuclear translocation, or MAPK activation in RA FLS. CONCLUSION: DEX and CpdA display a dissimilar molecular mechanism of interaction with the NF-kappaB activation pathway ex vivo. A dual pathway, partially dependent and partially independent of GR (nongenomic), may explain the gene-inhibitory effects of CpdA in RA FLS.


Subject(s)
Arthritis, Rheumatoid/metabolism , Dexamethasone/pharmacology , Fibroblasts/metabolism , Glucocorticoids/pharmacology , NF-kappa B/antagonists & inhibitors , Receptors, Glucocorticoid/drug effects , Synovial Membrane/metabolism , Acetates , Arthritis, Rheumatoid/pathology , Cells, Cultured , Fibroblasts/drug effects , Fibroblasts/pathology , Humans , I-kappa B Kinase/metabolism , Interleukin-1beta/metabolism , Mitogen-Activated Protein Kinase Kinases/metabolism , NF-kappa B/metabolism , Quaternary Ammonium Compounds/pharmacology , RNA, Small Interfering/genetics , Receptors, Glucocorticoid/genetics , Receptors, Glucocorticoid/metabolism , Synovial Membrane/drug effects , Synovial Membrane/pathology , Tyramine/analogs & derivatives , eIF-2 Kinase/metabolism
17.
Proc Natl Acad Sci U S A ; 106(18): 7397-402, 2009 May 05.
Article in English | MEDLINE | ID: mdl-19376972

ABSTRACT

Glucocorticoid receptor alpha (GRalpha) and peroxisome proliferator-activated receptor alpha (PPARalpha) are transcription factors with clinically important immune-modulating properties. Either receptor can inhibit cytokine gene expression, mainly through interference with nuclear factor kappaB (NF-kappaB)-driven gene expression. The present work aimed to investigate a functional cross-talk between PPARalpha- and GRalpha-mediated signaling pathways. Simultaneous activation of PPARalpha and GRalpha dose-dependently enhances transrepression of NF-kappaB-driven gene expression and additively represses cytokine production. In sharp contrast and quite unexpectedly, PPARalpha agonists inhibit the expression of classical glucocorticoid response element (GRE)-driven genes in a PPARalpha-dependent manner, as demonstrated by experiments using PPARalpha wild-type and knockout mice. The underlying mechanism for this transcriptional antagonism relies on a PPARalpha-mediated interference with the recruitment of GRalpha, and concomitantly of RNA polymerase II, to GRE-driven gene promoters. Finally, the biological relevance of this phenomenon is underscored by the observation that treatment with the PPARalpha agonist fenofibrate prevents glucocorticoid-induced hyperinsulinemia of mice fed a high-fat diet. Taken together, PPARalpha negatively interferes with GRE-mediated GRalpha activity while potentiating its antiinflammatory effects, thus providing a rationale for combination therapy in chronic inflammatory disorders.


Subject(s)
Glucocorticoids/pharmacology , NF-kappa B/metabolism , PPAR alpha/metabolism , Receptors, Glucocorticoid/metabolism , Transcriptional Activation , Animals , Cell Line, Tumor , Dietary Fats/administration & dosage , Fenofibrate/pharmacology , Gene Expression/drug effects , Hepatocytes/drug effects , Hepatocytes/metabolism , Humans , Hyperinsulinism/chemically induced , Hyperinsulinism/prevention & control , Mice , Mice, Knockout , PPAR alpha/agonists , PPAR alpha/antagonists & inhibitors , PPAR alpha/genetics , Promoter Regions, Genetic , Receptors, Glucocorticoid/agonists
18.
Biochem Pharmacol ; 77(7): 1194-205, 2009 Apr 01.
Article in English | MEDLINE | ID: mdl-19150610

ABSTRACT

In the combat against inflammation, glucocorticoids (GCs) are a widespread therapeutic. These ligands of the glucocorticoid receptor (GR) inhibit the transactivation of various transcription factors, including nuclear factor-kappaB (NF-kappaB), and alter the composition of the pro-inflammatory enhanceosome, culminating in the repression of pro-inflammatory gene expression. However, pharmacological usage of GCs in long-term treatment is burdened with a detrimental side-effect profile. Recently, we discovered that GCs can lower NF-kappaB transactivation and pro-inflammatory gene expression by abolishing the recruitment of mitogen- and stress-activated protein kinase 1 (MSK1) (EC 2.7.11.1) to pro-inflammatory gene promoters and displacing a significant fraction of MSK1 to the cytoplasm. In our current investigation in L929sA fibroblasts, upon combining GCs and MSK1 inhibitors, we discovered a dose-dependent additive repression of pro-inflammatory gene expression, most likely due to diverse and multilayered repression mechanisms employed by GCs and MSK1 inhibitors. Therefore, the combined application of GCs and MSK1 inhibitors enabled a similar level of repression of pro-inflammatory gene expression, using actually a lower concentration of GCs and MSK1 inhibitors combined than would be necessary when using these inhibitors separately. Although H89 can inhibit both MSK1 and PKA, TNF does not activate PKA (EC 2.7.11.11) and as such PKA inhibition does not mediate H89-instigated repression of TNF-stimulated gene expression. Furthermore, the additional repressive effects of liganded GR and inhibition of MSK1, are not mediated via GR transactivation mechanisms. In conclusion, these results could entail a new therapeutic strategy using lower drug concentrations, potentially leading to a more beneficial side-effect profile.


Subject(s)
Glucocorticoids/pharmacology , Inflammation/drug therapy , Inflammation/enzymology , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 8/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Animals , Cells, Cultured , Drug Therapy, Combination , Glucocorticoids/therapeutic use , Isoquinolines/pharmacology , Isoquinolines/therapeutic use , Mice , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 8/metabolism , Protein Kinase Inhibitors/therapeutic use , Sulfonamides/pharmacology , Sulfonamides/therapeutic use
20.
EMBO J ; 27(12): 1682-93, 2008 Jun 18.
Article in English | MEDLINE | ID: mdl-18511904

ABSTRACT

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-kappaB (NF-kappaB). In addition to the well described interaction-based mutual repression mechanism between the GR and NF-kappaB, 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-kappaB 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-kappaB-targeting anti-inflammatory mechanism.


Subject(s)
Glucocorticoids/pharmacology , Ribosomal Protein S6 Kinases, 90-kDa/metabolism , Transcription Factor RelA/antagonists & inhibitors , Cell Line , Cytokines/genetics , Cytoplasm/enzymology , Enzyme Induction/drug effects , Gene Expression Regulation/drug effects , Histones/metabolism , Humans , Inflammation/genetics , Isoquinolines/pharmacology , Karyopherins/metabolism , Ligands , Phosphorylation/drug effects , Promoter Regions, Genetic/genetics , Protein Transport , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Glucocorticoid/metabolism , Serine/metabolism , Subcellular Fractions/enzymology , Sulfonamides/pharmacology , Tumor Necrosis Factor-alpha/pharmacology , Exportin 1 Protein
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