Mechanisms underlying the functional cooperation between PPARα and GRα to attenuate inflammatory responses

Glucocorticoids (GCs) act via the glucocorticoid receptor (NR3C1, GR alpha) 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). PPAR alpha (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 PPAR alpha agonists cooperate to jointly inhibit NF-kappa B-driven expression in A549 cells. We discovered a nuclear mechanism that predominantly targets Mitogen- and Stress-activated protein Kinase-1 activation upon co-triggering GR alpha and PPAR alpha. 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-kappa B, GR alpha, and PPAR alpha. Finally, to study metabolic effector target cells common to both receptors, we overlaid the effect of GR alpha and PPAR alpha 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

Unraveling the role of IL-17A in the intestinal immune response against the protozoan parasite Giardia muris by an RNA sequencing approach

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The increasing complexity of glucocorticoid receptor signaling and regulation

Glucocorticoids, although being one of the eldest drugs in the clinic and despite their widespread usage for the treatment of inflammatory and immune disorders and cancer, have not yet come of age when it comes to a full understanding of how they work. The majority of the biological actions of glucocorticoid hormones are explained by a wide diversity in the cellular action mechanism of the hormone-activated Glucocorticoid Receptor (GR). All molecular mechanisms described in the current overview are not only complex, exhibiting an astonishing degree of gene- and tissue-specificity, but on top of this they are also non-exclusive. This layering of mechanisms makes it extremely difficult for researchers to extract the crucial pieces of information that would assist in a rational design of drugs with an improved therapeutic profile, i.e. a satisfying and maintained therapeutic response in the absence of the many incapacitating glucocorticoid-associated side effects, such as diabetes, osteoporosis, muscle wasting, depression etc. In direct correlation with increased glucocorticoid usage as observed in the clinic, the impetus and desire to reveal all of these mechanisms -and most importantly, to try to integrate them in a sensible manner for the sake of finding better alternatives- has never been stronger

Coregulator profiling of the glucocorticoid receptor in lymphoid malignancies

Coregulators cooperate with nuclear receptors, such as the glucocorticoid receptor (GR), to enhance or repress transcription. These regulatory proteins are implicated in cancer, yet, their role in lymphoid malignancies, including multiple myeloma (MM) and acute lymphoblastic leukemia (ALL), is largely unknown. Here, we report the use and extension of the microarray assay for real-time nuclear receptor coregulator interactions (MARCoNI) technology to detect coregulator associations with endogenous GR in cell lysates. We use MARCoNI to determine the GR coregulator profile of glucocorticoid-sensitive (MM and ALL) and glucocorticoid-resistant (ALL) cells, and identify common and unique coregulators for different cell line comparisons. Overall, we identify SRC-1/2/3, PGC-1 alpha, RIP140 and DAX-1 as the strongest interacting coregulators of GR in MM and ALL cells and show that the interaction strength does not correlate with GR protein levels. Lastly, as a step towards patient samples, we determine the GR coregulator profile of peripheral blood mononuclear cells. We profile the interactions between GR and coregulators in MM and ALL cells and suggest to further explore the GR coregulator profile in hematological patient samples

Chromatin recruitment of activated AMPK drives fasting response genes co-controlled by GR and PPARα

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

Compound A influences gene regulation of the dexamethasone-activated glucocorticoid receptor by alternative cofactor recruitment

The glucocorticoid receptor (GR) is a transcription factor of which the underlying gene regulatory mechanisms are complex and incompletely understood. The non-steroidal anti-inflammatory Compound A (CpdA), a selective GR modulating compound in various cell models, has been shown to favour GR-mediated gene repression but not GR-mediated gene activation. Shifting balances towards only a particular subset of GR gene regulatory events may be of benefit in the treatment of inflammatory diseases. We present evidence to support that the combination of CpdA with Dexamethasone (DEX), a classic steroidal GR ligand, can shape GR function towards a unique gene regulatory profile in a cell type-dependent manner. The molecular basis hereof is a changed GR phosphorylation status concomitant with a change in the GR cofactor recruitment profile. We subsequently identified and confirmed the orphan nuclear receptor SHP as a coregulator that is specifically enriched at GR when CpdA and DEX are combined. Combining CpdA with DEX not only leads to stronger suppression of pro-inflammatory gene expression, but also enhanced anti-inflammatory GR target gene expression in epithelial cells, making ligand combination strategies in future a potentially attractive alternative manner of skewing and fine-tuning GR effects towards an improved therapeutic benefit

The autophagy receptor SQSTM1/p62 mediates anti-inflammatory actions of the selective NR3C1/glucocorticoid receptor modulator compound A (CpdA) in macrophages

Glucocorticoids are widely used to treat inflammatory disorders; however, prolonged use of glucocorticoids results in side effects including osteoporosis, diabetes and obesity. Compound A (CpdA), identified as a selective NR3C1/glucocorticoid receptor (nuclear receptor subfamily 3, group C, member 1) modulator, exhibits an inflammation-suppressive effect, largely in the absence of detrimental side effects. To understand the mechanistic differences between the classic glucocorticoid dexamethasone (DEX) and CpdA, we looked for proteins oppositely regulated in bone marrow-derived macrophages using an unbiased proteomics approach. We found that the autophagy receptor SQSTM1 but not NR3C1 mediates the anti-inflammatory action of CpdA. CpdA drives SQSTM1 upregulation by recruiting the NFE2L2 transcription factor to its promoter. In contrast, the classic NR3C1 ligand dexamethasone recruits NR3C1 to the Sqstm1 promoter and other NFE2L2-controlled gene promoters, resulting in gene downregulation. Both DEX and CpdA induce autophagy, with marked different autophagy characteristics and morphology. Suppression of LPS-induced Il6 and Ccl2 genes by CpdA in macrophages is hampered upon Sqstm1 silencing, confirming that SQSTM1 is essential for the anti-inflammatory capacity of CpdA, at least in this cell type. Together, these results demonstrate how off-target mechanisms of selective NR3C1 ligands may contribute to a more efficient anti-inflammatory therapy

Pharmacological levels of withaferin A (Withania somnifera) trigger clinically relevant anticancer effects specific to triple negative breast cancer cells

Withaferin A (WA) isolated from Withania somnifera (Ashwagandha) has recently become an attractive phytochemical under investigation in various preclinical studies for treatment of different cancer types. In the present study, a comparative pathway-based transcriptome analysis was applied in epithelial-like MCF-7 and triple negative mesenchymal MDA-MB-231 breast cancer cells exposed to different concentrations of WA which can be detected systemically in in vivo experiments. Whereas WA treatment demonstrated attenuation of multiple cancer hallmarks, the withanolide analogue Withanone (WN) did not exert any of the described effects at comparable concentrations. Pathway enrichment analysis revealed that WA targets specific cancer processes related to cell death, cell cycle and proliferation, which could be functionally validated by flow cytometry and real-time cell proliferation assays. WA also strongly decreased MDA-MB-231 invasion as determined by single-cell collagen invasion assay. This was further supported by decreased gene expression of extracellular matrix-degrading proteases (uPA, PLAT, ADAM8), cell adhesion molecules (integrins, laminins), pro-inflammatory mediators of the metastasis-promoting tumor microenvironment (TNFSF12, IL6, ANGPTL2, CSF1R) and concomitant increased expression of the validated breast cancer metastasis suppressor gene (BRMS1). In line with the transcriptional changes, nanomolar concentrations of WA significantly decreased protein levels and corresponding activity of uPA in MDA-MB-231 cell supernatant, further supporting its anti-metastatic properties. Finally, hierarchical clustering analysis of 84 chromatin writer-reader-eraser enzymes revealed that WA treatment of invasive mesenchymal MDA-MB-231 cells reprogrammed their transcription levels more similarly towards the pattern observed in non-invasive MCF-7 cells. In conclusion, taking into account that sub-cytotoxic concentrations of WA target multiple metastatic effectors in therapy-resistant triple negative breast cancer, WA-based therapeutic strategies targeting the uPA pathway hold promise for further (pre)clinical development to defeat aggressive metastatic breast cancer

The transcriptional co-regulation of hepatic lipid metabolism by GR and PPARα

The Glucocorticoid Receptor α (GRα) and Peroxisome Proliferator-Activated Receptor α (PPARα) are two Nuclear Receptors (NRs) with important roles in metabolism and inflammation control. The agonists of both GR and PPARα are also frequently used in the clinic for the treatment of inflammatory diseases and dyslipidemia, respectively. In contrast to fibrates, which activate PPARα and are relatively safe and well-tolerated drugs, the use of glucocorticoids (GCs) may lead to a multitude of side effects, including metabolic disturbances. The current work was initiated following a study, which demonstrated potential benefits of combining fibrates with GCs. More specifically, our group has shown that co-activation of PPARα and GR corrects GC-induced insulin resistance and glucose intolerance, which are among the typical side effects of GC-based therapies. Besides the potential utilization of these findings for the improvement of GC-based therapies, a better understanding of the cross talk between GR and PPARα is interesting from a basic science perspective as both factors participate in the metabolic adaptation to fasting. Thus, we embarked on a further characterization of the molecular mechanism behind the GR and PPARα cross talk in a genome wide manner, using primary hepatocyte cells as a model. We demonstrate that both factors share a significant fraction of their genomic binding sites and colocalize in the vicinity of lipid metabolic genes, which are cooperatively activated upon combined stimulation with GR- and PPARα-agonists. A subset of those genes switched from transcriptional antagonism to cooperativity when moving from short to prolonged hepatocyte fasting, a phenomenon found to be strongly dependent on the cellular nutritional status. We moreover found that this regulatory switch coincides with recruitment of phospho-AMPK at the proximal promoters of lipid metabolic genes. As such, active AMPK drives GR/PPARα-agonist-dependent cooperative transcription of genes responding to prolonged fasting. Finally, as the ability of exogenous GCs to induce accumulation of fatty acids in primary hepatocytes was completely abolished upon co-triggering PPARα, our results support that the observed cross-talk can also be recapitulated at the metabolite level