Dariusz Ratman

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.

Selective modulation of the glucocorticoid receptor can distinguish between transrepression of NF-κB and AP-1

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.

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.

Activation of the Glucocorticoid Receptor in Acute Inflammation: the SEDIGRAM Concept

Since the 1950s, glucocorticoids (GCs) have been a mainstay therapy for acute and chronic inflammatory disorders, although adverse effects limit their chronic use. Following the notion that the anti-inflammatory therapeutic and metabolic endocrine adverse effects of GCs may be based on different glucocorticoid receptor (GR)-dependent mechanisms, subsequent attempts to separate these mechanisms by trying to develop selective GR agonists and modulators (SEGRAMs) with an improved therapeutic benefit have yielded only a few molecules effective in clinical use. Recent new insights into the pro- and anti-inflammatory activities of GR support a more sophisticated drug discovery model. Here, we suggest that the way forward may include a need to redefine the pharmacological SEGRAM concept into selective monomerizing GR agonists and modulators (SEMOGRAMs) and selective dimerizing GR agonists or modulators (SEDIGRAMs) for selective therapeutic applications against chronic or acute inflammatory disorders, respectively.

Hypoxia-inducible Lipid Droplet-associated (HILPDA) Is a Novel Peroxisome Proliferator-activated Receptor (PPAR) Target Involved in Hepatic Triglyceride Secretion

Background: PPARα is an important regulator of hepatic lipid metabolism via target gene regulation. Results: HILPDA is regulated by PPARα via an upstream PPRE. Targeted overexpression of HILPDA increases hepatic triglyceride storage via reduction of TG secretion. Conclusion: HILPDA is a novel PPARα target involved in hepatic triglyceride secretion. Significance: HILPDA might be a potential target in the treatment of non-alcoholic fatty liver disease. Peroxisome proliferator-activated receptors (PPARs) play major roles in the regulation of hepatic lipid metabolism through the control of numerous genes involved in processes such as lipid uptake and fatty acid oxidation. Here we identify hypoxia-inducible lipid droplet-associated (Hilpda/Hig2) as a novel PPAR target gene and demonstrate its involvement in hepatic lipid metabolism. Microarray analysis revealed that Hilpda is one of the most highly induced genes by the PPARα agonist Wy14643 in mouse precision cut liver slices. Induction of Hilpda mRNA by Wy14643 was confirmed in mouse and human hepatocytes. Oral dosing with Wy14643 similarly induced Hilpda mRNA levels in livers of wild-type mice but not Ppara−/− mice. Transactivation studies and chromatin immunoprecipitation showed that Hilpda is a direct PPARα target gene via a conserved PPAR response element located 1200 base pairs upstream of the transcription start site. Hepatic overexpression of HILPDA in mice via adeno-associated virus led to a 4-fold increase in liver triglyceride storage, without any changes in key genes involved in de novo lipogenesis, β-oxidation, or lipolysis. Moreover, intracellular lipase activity was not affected by HILPDA overexpression. Strikingly, HILPDA overexpression significantly impaired hepatic triglyceride secretion. Taken together, our data uncover HILPDA as a novel PPAR target that raises hepatic triglyceride storage via regulation of triglyceride secretion.

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.

Analysis of the protective immune response following intramuscular vaccination of calves against the intestinal parasite Cooperia oncophora.

Recently we reported the successful vaccination of calves against Cooperia oncophora with a double domain activation-associated secreted protein, purified from the excretory-secretory material of adult stage parasites. In an attempt to elucidate the immune mechanisms involved in protection, the humoral and cell-mediated immune responses following vaccination and infection were compared with non-vaccinated control animals. Antigen-specific IgG1, IgG2 and IgA levels were significantly increased in sera of vaccinated animals post vaccination, whereas no effect was observed for IgM. Antigen-specific intestinal IgG1 levels were significantly increased in the vaccinated animals, whereas no differences were observed for antigen-specific IgA, IgM and IgG2 levels. Upon re-stimulation in vitro with the vaccine antigen, a significant proliferation of both αβ- and γδ-T cells, and B cells, collected from mesenteric lymph nodes, was only observed in vaccinated animals. RNA-seq analysis of intestinal tissue yielded a list of 67 genes that were differentially expressed in vaccinated animals following challenge infection, amongst which were several cell adhesion molecules, lectins and glycosyl transferases. A correlation analysis between all immunological and parasitological parameters indicated that intestinal anti-double domain activation-associated secreted protein IgG1 levels correlated negatively with cumulative faecal egg counts and positively with the proportion of L4s and L5s. The proportion of immature stages was also positively correlated with the proliferation of αβ T cells. Worm length was negatively correlated with the transcript levels of several lectins and cell adhesion molecules. Overall, the results indicate that intramuscular administration of the vaccine resulted in an immune memory response particularly characterised by increased antigen-specific IgG1 levels in the intestinal mucosa.

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-1α, 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.

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

ABSTRACT 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.