Signaling via the bHLH-PAS proteins AhR and HIF

This thesis concerns some mechanistic properties of the basic helix-loop-helix/PAS (bHLH-PAS) factors aryl hydrocarbon receptor (AhR) and hypoxia-inducible factor 1 alpha (HIF-1α). The bHLH-PAS family of proteins is a family of factors that controls a variety of developmental and physiological events. A common feature for this family of proteins is that they act mainly as intracellular transcription factors. They bind to DNA as a heterodimeric complex, usually together with a bHLH-PAS protein belonging to the aryl hydrocarbon receptor nuclear translocator (ARNT) subfamily. The AhR bind ligands that are environmental pollutants, as well as possibly physiological compounds occurring in the diet. Known functions of the ligand-activated AhR include activation of genes involved in xenobiotic metabolism and an ubiquitin ligase activity targeting nuclear receptors (such as the estrogen receptor) and beta-catenin. HIF-1α mediates signal transduction and gene regulation in cells exposed to deprived oxygen conditions (hypoxia). In paper I, we have shown that the Ah-receptor can be activated by stimulus other than xenobiotics, e.g dioxin. AhR is recruited to target genes in both ligand treated and in suspension culture, suggesting a common mechanism of activation between these two routes of AhR activation. The gene expression profiles critically differ between xenobiotic and suspension activated AhR signaling. The classical xenobiotic metabolizing AhR targets such as Cyp 1a1, Cyp 1b1 and Nqo were regulated by both ligand and suspension conditions. Sequence analysis coupled with ChIP assays and reporter gene analysis identified a functional xenobiotic response element (XRE) within the mouse TIPARP gene that features a concatamer of 4 XRE cores residing in the first intron. In paper II we have shown that ectopic expression of ARNT, in mammalian cells and yeast cells, was sufficient to promote nuclear accumulation of the Ah-receptor in a ligand-independent manner. We further observed that overexpression of ARNT promotes derepression of Ah-receptor function in the absence of ligand, thereby possibly representing an alternative mechanism of activation that is distinct from activation by xenobiotic ligands and thus may be of physiological relevance. We also describe that an excess of ARNT in relation to the Ah-receptor and HIF-1α promotes derepression of the receptor and stabilization of HIF-1α in vivo and in vitro, representing a possible alternative mechanism of activation of bHLH-PAS proteins

Xenobiotics and loss of cell adhesion drive distinct transcriptional outcomes by aryl hydrocarbon receptor signaling

The aryl hydrocarbon receptor (AhR) is a signal-regulated transcription factor, which is canonically activated by the direct binding of xenobiotics. In addition, switching cells from adherent to suspension culture also activates the AhR, representing a nonxenobiotic, physiological activation of AhR signaling. Here, we show that the AhR is recruited to target gene enhancers in both ligand [isopropyl-2-(1,3-dithietane-2-ylidene)-2-[N-(4-methylthiazol-2-yl)carbamoyl]acetate (YH439)]-treated and suspension cells, suggesting a common mechanism of target gene induction between these two routes of AhR activation. However, gene expression profiles critically differ between xenobiotic- and suspension-activated AhR signaling. Por and Cldnd1 were regulated predominantly by ligand treatments, whereas, in contrast, ApoER2 and Ganc were regulated predominantly by the suspension condition. Classic xenobiotic-metabolizing AhR targets such as Cyp1a1, Cyp1b1, and Nqo1 were regulated by both ligand and suspension conditions. Temporal expression patterns of AhR target genes were also found to vary, with examples of transient activation, transient repression, or sustained alterations in expression. Furthermore, sequence analysis coupled with chromatin immunoprecipitation assays and reporter gene analysis identified a functional xenobiotic response element (XRE) in the intron 1 of the mouse Tiparp gene, which was also bound by hypoxia-inducible factor-1α during hypoxia and features a concatemer of four XRE cores (GCGTG). Our data suggest that this XRE concatemer site concurrently regulates the expression of both the Tiparp gene and its cis antisense noncoding RNA after ligand- or suspension-induced AhR activation. This work provides novel insights into how AhR signaling drives different transcriptional programs via the ligand versus suspension modes of activation.Nan Hao, Kian Leong Lee, Sebastian G. B. Furness, Cecilia Bosdotter, Lorenz Poellinger, and Murray L. Whitela

The MDM2 Inhibitor Navtemadlin Arrests Mouse Melanoma Growth In Vivo and Potentiates Radiotherapy

The tumor suppressor protein p53 is mutated in close to 50% of human tumors and is dysregulated in many others, for instance by silencing or loss of p14ARF. Under steady-state conditions, the two E3 ligases MDM2/MDM4 interact with and inhibit the transcriptional activity of p53. Inhibition of p53–MDM2/4 interaction to reactivate p53 in tumors with wild-type (WT) p53 has therefore been considered a therapeutic strategy. Moreover, studies indicate that p53 reactivation may synergize with radiation and increase tumor immunogenicity. In vivo studies of most MDM2 inhibitors have utilized immunodeficient xenograft mouse models, preventing detailed studies of action of these molecules on the immune response. The mouse melanoma cell line B16-F10 carries functional, WT p53 but does not express the MDM2 regulator p19ARF. In this study, we tested a p53-MDM2 protein–protein interaction inhibitor, the small molecule Navtemadlin, which is currently being tested in phase II clinical trials. Using mass spectrometry–based proteomics and imaging flow cytometry, we identified specific protein expression patterns following Navtemadlin treatment of B16-F10 melanoma cells compared with their p53 CRISPR-inactivated control cells. In vitro, Navtemadlin induced a significant, p53-dependent, growth arrest but little apoptosis in B16-F10 cells. When combined with radiotherapy, Navtemadlin showed synergistic effects and increased apoptosis. In vivo, Navtemadlin treatment significantly reduced the growth of B16-F10 melanoma cells implanted in C57Bl/6 mice. Our data highlight the utility of a syngeneic B16-F10 p53+/+ mouse melanoma model for assessing existing and novel p53-MDM2/MDM4 inhibitors and in identifying new combination therapies that can efficiently eliminate tumors in vivo. Significance: The MDM2 inhibitor Navtemadlin arrests mouse tumor growth and potentiates radiotherapy. Our results support a threshold model for apoptosis induction that requires a high, prolonged p53 signaling for cancer cells to become apoptotic

Mapping of 79 loci for 83 plasma protein biomarkers in cardiovascular disease

Recent advances in highly multiplexed immunoassays have allowed systematic large-scale measurement of hundreds of plasma proteins in large cohort studies. In combination with genotyping, such studies offer the prospect to 1) identify mechanisms involved with regulation of protein expression in plasma, and 2) determine whether the plasma proteins are likely to be causally implicated in disease. We report here the results of genome-wide association (GWA) studies of 83 proteins considered relevant to cardiovascular disease (CVD), measured in 3,394 individuals with multiple CVD risk factors. We identified 79 genome-wide significant (p<5e-8) association signals, 55 of which replicated at P<0.0007 in separate validation studies (n = 2,639 individuals). Using automated text mining, manual curation, and network-based methods incorporating information on expression quantitative trait loci (eQTL), we propose plausible causal mechanisms for 25 trans-acting loci, including a potential post-translational regulation of stem cell factor by matrix metalloproteinase 9 and receptor-ligand pairs such as RANK-RANK ligand. Using public GWA study data, we further evaluate all 79 loci for their causal effect on coronary artery disease, and highlight several potentially causal associations. Overall, a majority of the plasma proteins studied showed evidence of regulation at the genetic level. Our results enable future studies of the causal architecture of human disease, which in turn should aid discovery of new drug targets