Cyclophilin-40 (CyP40) promotes the formation of the gel shift complex containing baculovirus aryl hydrocarbon receptor (AhR), AhR nuclear translocator (Arnt) and dioxin response element (DRE). CyP40 was found to play a role in the AhR signaling since when the CyP40 content in MCF-7 cells is reduced, up-regulation of cyp1a1 and cyp1b1 by 3-methylcholanthrene (3MC) is also reduced, suggesting that CyP40 is essential for maximal AhR function. The CyP40 region containing amino acids 186-215, but not the peptidylprolyl cis-trans isomerase and tetratricopeptide repeat domains, is essential for forming the AhR/Arnt/DRE complex. CyP40 is found in the cell nucleus after 3MC treatment and appears to promote the DRE binding form of the AhR/Arnt heterodimer. Coprecipitation data suggests CyP40 binds weakly to AhR, but not Arnt. We report on the progress of applying bioluminescence resonance energy transfer and chromatin immunoprecipitation techniques to further elucidate the role of CyP40 in the aryl hydrocarbon receptor signaling pathway.

Upon ligand binding, the aryl hydrocarbon receptor (AhR) translocates into the nucleus and dimerizes with its partner Ah receptor nuclear translocator (Arnt). The AhR/Arnt heterodimer binds to the enhancer element DRE to regulate target gene expression. It is known that the formation of the ligand-dependent AhR/Arnt/DRE complex requires protein factors in vitro. The first aim is to determine whether two other Hsp90-associated proteins present in rabbit reticulocyte lysate (RRL), namely CyP40 and Hsp70, play any role in forming the AhR/Arnt/DRE complex. Fractionation and immunodepletion experiments revealed that Hsp70 is not necessary for the formation of this complex. In contrast, CYP40 is involved in forming the complex since (1) immunodepletion of CyP40 from a RRL fraction reduces the intensity of the AhR-Arnt-DRE complex by 48% and (2) recombinant human CyP40 alone causes the formation of this complex. In addition, CyP40-interacting proteins appear to be essential for the full CyP40 effect on the AhR gel shift complex. The second aim is to determine the role of β-tubulin in Amt-dependent signaling pathways. From the insect Sf9 cytosol, β-tubulin enriched fraction (F5) was isolated which suppresses the AhR/Arnt/DRE complex formation in a gel shift assay. Tubulin enriched from pig brain had a similar inhibition of the AhR gel shift complex, suggesting that β-tubulin in F5 is likely responsible for the action. Using the TALON resin, β-tubulin was co-precipitated with the baculovirus 6His-Arnt, showing that β-tubulin interacts with Arnt. β-tubulin was examined to decide its role in the hypoxia inducible factor-1α (HIF-1α) signaling which is also Arnt-dependent. Gel shift data using HIF-1α and Arnt showed that F5 suppressed the formation of the HIF-1α/Arnt/HRE complex. Subsequently the Sf9 β-tubulin was cloned and about 95% of its full-length sequence was identified. The amino acid sequence of Sf9 β-tubulin shares high sequence identity with human β-tubulin. Upon transient transfection of a plasmid containing a human β-tubulin cDNA into MGF7 or Hep3B cells, the HRE-driven luciferase activity was clearly suppressed. In conclusion, we have evidence supporting that β-tubulin inhibits the Arnt-dependent signaling and the mechanism may involve the interaction between Arnt and β-tubulin.

Chronic kidney disease (CKD) is a common disease that affects millions across the US and the globe. Patients with CKD experience an increased risk of venous thrombosis. Here we use two longstanding robust murine models of nephropathies in conjunction with a reliable murine model of venous thrombosis to model venous thrombosis risk in CKD. We show that in the adenine diet-induced CKD, increased concentrations of adenine in the diet result in increased histological evidence of nephropathy and increased venous thrombosis risk assessed by Inferior Vena Cava ligation. Next, we demonstrate that in unilateral ureteric obstruction models, the duration of obstruction is proportional to the nephropathies developed by histological assessment. In both models, we relate nephropathy to venous thrombosis risk. When probed for aryl hydrocarbon receptor (AHR) activation, adenine diet-induced CKD mice show increased activation assessed by nuclear translocation of the receptor. This phenotype was confirmed in vitro when treating human telomerase immortalized human umbilical endothelial cells with uremic serum. Nuclear AHR was not observed in control conditions in vivo or in vitro. Pharmacologic AHR inhibition using a novel drug, BAY Compound, and a well-known AHR inhibitor were both able to abrogate uremic activation of AHR in vitro, which was then corroborated with in vivo studies. Tissue factor (TF) and plasminogen activator inhibitor 1 (PAI-1) are prothrombogenic proteins linked to AHR activation. TF and PAI-1 showed upregulation in CKD mice which were blocked when CKD mice were given AHR inhibitor BAY Compound. This work demonstrates a unique model of venous thrombosis in CKD and suggests that AHR inhibition may be able to limit the elevated risk of venous thrombosis associated with uremia. / 2026-03-08T00:00:00Z

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Acute myeloid leukemia (AML) is an aggressive malignancy of the hematopoietic system, characterized by the accumulation of abnormally differentiated blast cells that is driven by leukemic stem cells (LSCs). In murine AML, Musashi-2 (MSI2), an RNA-binding protein and positive regulator of stemness, has been implicated in the propagation of disease. While its enhanced expression correlated with poor disease outcome for human AML patients, no study has yet examined its actual functional role in human leukemia. In normal human hematopoietic stem cells (HSCs), we have recently reported the inhibitory effects of MSI2 on the pro-differentiative aryl hydrocarbon receptor (AHR) signaling pathway as a mechanism for promoting self-renewal in HSCs. We hypothesized that elevated MSI2 is critical for maintenance of human AML and promotes unrestrained self-renewal of LSCs in part through constitutive repression of AHR signaling. Our work aimed to unravel the relationship between MSI2 and AHR in the human leukemic context and to determine if activation of AHR signaling can promote differentiation. Results confirmed that MSI2 is preferentially expressed in primary patient LSCs and is negatively correlated with the expression of AHR gene targets. Upon lentiviral knockdown of MSI2 in-vitro and in-vivo, leukemic growth was compromised and increased AHR signaling was observed. Circumventing the inhibitory role of MSI2 in AML, activation of AHR with a potent agonist impaired leukemic progenitor activity and proliferation. In-vivo studies employing reconstitution of immunodeficient mice with primary AML samples showed impairment of AML engraftment for a significant proportion of tested samples upon treatment with an AHR agonist. Overall, our findings from this project indicated that MSI2 is required for human AML propagation and that a decrease in MSI2 inhibitory effects on AHR signaling or direct activation of the AHR signaling pathway via a potent agonist can promote AML cell differentiation and loss. / Thesis / Master of Science (MSc) / The human blood system is sustained by a population of blood stem cells that are tightly regulated in their production of stem and differentiated cells. The Musashi-2 (MSI2) protein is a key regulator of blood stem cell identity through its inhibition of the aryl hydrocarbon receptor (AHR) signaling pathway. When there is dysregulation of blood cell homeostasis, blood malignancies such as acute myeloid leukemia (AML) may arise. In this work, the relationship between MSI2 and the AHR signaling pathway was explored within a myeloid leukemic context. It was shown that MSI2 imposes inhibitory effects on AHR to promote disease progression and that its reduction could help alleviate disease burden. Additionally, it was found that activation of the AHR signaling pathway could overcome the MSI2 differentiation block to create a therapeutic effect. Overall, the results of this project shed light on novel therapeutic strategies and targets for the treatment of AML.

Yes / The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor and member of the basic helix-loop-helix-PAS family. AHR is activated by numerous dietary and endogenous compounds that contribute to its regulation of genes in diverse signaling pathways including xenobiotic metabolism, vascular development, immune responses and cell cycle control. However, it is most widely studied for its role in mediating 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity. The AHR target gene and mono-ADP-ribosyltransferase, TCDD-inducible poly-ADP-ribose polymerase (TIPARP), was recently shown to be part of a novel negative feedback loop regulating AHR activity through mono-ADP-ribosylation. However, the molecular characterization of how AHR regulates TIPARP remains elusive. Here we show that activated AHR is recruited to the TIPARP promoter, through its binding to two genomic regions that each contain multiple AHR response elements (AHREs), AHR regulates the expression of both TIPARP but also TIPARP-AS1, a long non-coding RNA (lncRNA) which lies upstream of TIPARP exon 1 and is expressed in the opposite orientation. Reporter gene and deletion studies showed that the distal AHRE cluster predominantly regulated TIPARP expression while the proximal cluster regulated TIPARP-AS1. Moreover, time course and promoter activity assays suggest that TIPARP and TIPARP-AS1 work in concert to regulate AHR signaling. Collectively, these data show an added level of complexity in the AHR signaling cascade which involves lncRNAs, whose functions remain poorly understood. / This work was supported by Canadian Institutes of Health Research (CIHR) operating grants (MOP-494265 and MOP-125919), an unrestricted research grant from the Dow Chemical Company, and the Johan Throne Holst Foundation to J.M. G.G. was supported by European Union Seventh Framework Program (FP7-PEOPLE2013-COFUND) under the Grant Agreement n609020 - Scientia Fellows