Bart van der Burg

Distinct Domains of the RelA NF-κB Subunit Are Required for Negative Cross-talk and Direct Interaction with the Glucocorticoid Receptor

The RelA subunit of NF-κB and the glucocorticoid receptor mutually repress each others transcriptional activity, thus providing a mechanism for immunosuppression. Deletion analysis of the glucocorticoid receptor has shown that the DNA binding domain and the ligand binding domain are essential components for repression. Here, we show by deletions and point mutations that both the Rel homology domain and the transactivation domains of RelA are required for repression of the transcriptional activity of the glucocorticoid receptor in intact cells. However, only the Rel homology domain of RelA was found to associate with the glucocorticoid receptorin vitro. RelA mutants, not able to repress glucocorticoid receptor activity, but still able to dimerize, behaved as transdominant inhibitors of the repressive activity of wild type RelA. Furthermore, we show that the 13 S E1A protein is able to interfere with the transrepressive activity of RelA. We propose that negative cross-talk between the glucocorticoid receptor and RelA is due to direct interaction via the Rel homology domain of RelA and the DNA binding domain of the glucocorticoid receptor in combination with interference by the transactivation domains of RelA with the transcriptional activity of the glucocorticoid receptor.

Short-Chain Fatty Acids Stimulate Angiopoietin-Like 4 Synthesis in Human Colon Adenocarcinoma Cells by Activating Peroxisome Proliferator-Activated Receptor γ

ABSTRACT Angiopoietin-like protein 4 (ANGPTL4/FIAF) has been proposed as a circulating mediator between the gut microbiota and fat storage. Here, we show that transcription and secretion of ANGPTL4 in human T84 and HT29 colon adenocarcinoma cells is highly induced by physiological concentrations of short-chain fatty acids (SCFA). SCFA induce ANGPTL4 by activating the nuclear receptor peroxisome proliferator activated receptor γ (PPARγ), as demonstrated using PPARγ antagonist, PPARγ knockdown, and transactivation assays, which show activation of PPARγ but not PPARα and PPARδ by SCFA. At concentrations required for PPARγ activation and ANGPTL4 induction in colon adenocarcinoma cells, SCFA do not stimulate PPARγ in mouse 3T3-L1 and human SGBS adipocytes, suggesting that SCFA act as selective PPARγ modulators (SPPARM), which is supported by coactivator peptide recruitment assay and structural modeling. Consistent with the notion that fermentation leads to PPAR activation in vivo, feeding mice a diet rich in inulin induced PPAR target genes and pathways in the colon. We conclude that (i) SCFA potently stimulate ANGPTL4 synthesis in human colon adenocarcinoma cells and (ii) SCFA transactivate and bind to PPARγ. Our data point to activation of PPARs as a novel mechanism of gene regulation by SCFA in the colon, in addition to other mechanisms of action of SCFA.

Selection and Validation of Toxicogenomics Assays as Alternatives to Animal Tests

Toxicogenomics-based methods are providing promising perspectives in the reduction and replacement of toxicity testing of chemicals and pharmaceuticals using experimental animals. Transcriptomics using DNA arrays and related methods can be used to select signature profiles of genes that can predict the response of a cell or organism. These genes sets can also be used to identify the pathways involved in toxic effects of chemicals and, based on that knowledge, design relatively straightforward molecular screening tools measuring modulation of these key toxicity pathways. Many of these methods bear great promise but have not matured sufficiently to reach regulatory application and, therefore, their impact on replacement of experimental animals is still limited to scientific applications. To be able to profit to the full extent from the possibilities of these modern toxicogenomics and related molecular screening tools, essential hurdles towards regulatory acceptance need to be passed. One of the main obstacles towards regulatory acceptance is the need to validate these tests in a manner that is acceptable to regulators. Although the principles of validation are straightforward, in practice this aspect of test development is very often neglected and the efforts to reach a validated test grossly underestimated. In this chapter, the possibilities, requirements, and feasibility of validating toxicogenomics-based tests are discussed, to provide insight and guidance in this process.