Megakaryocyte specification is the process by which discrete hematopoietic subpopulations undergo lineage commitment towards the myeloid compartment, finally specifying as a megakaryocyte erythroid progenitor (MEP) by way of thrombopoietin (TPO) and erythropoietin (EPO) signaling, before becoming a megakaryocyte lineage restricted progenitor that will progressively increase cellular ploidy and compartmentalize its cytoplasm in preparation for platelet production. With the advent of induced pluripotent stem cells (iPSCs), a cell type that is experimentally manipulated to function as embryonically derived pluripotent cells, there now exists the ability to analyze signal transduction throughout discrete phases of hematopoiesis, megakaryocyte lineage cell fate, and platelet production. Recent studies have implicated the aryl hydrocarbon receptor (AHR) as a transcription factor that plays a critical role in multiple aspects of hematopoiesis. These results inspired the hypothesis that AHR signaling may be functionally relevant in the context of megakaryopoiesis. To test this hypothesis, an iPSC directed differentiation strategy was established in order to create a platform upon which to experimentally manipulate AHR signaling throughout megakaryocyte specification. The results demonstrate: 1) iPSC derived hematopoietic progenitor cells (HPCs) undergo exponential expansion upon AHR agonism; 2) AHR antagonism allows for megakaryocyte lineage bias; 3) Optimization of directed-differentiation allows for the examination of AHR signaling in megakaryocyte lineage-restricted cells; 4) AHR signaling suppresses the expression of MPL, the gene that encodes the thrombopoietin receptor (C-MPL) in iPSC derived megakaryocyte lineage committed cells; 5) AHR activation concomitantly suppresses cell surface expression of C-MPL, which may alter the sensitivity of HPCs to TPO signaling; 6) Multiple gene targets are modulated by AHR activation within megakaryocyte lineage cells, providing evidence of a transcriptional program downstream of AHR signaling that preferentially suppresses megakaryocyte specification; 7) A reporter iPSC line of AHR activity provides evidence of endogenous AHR signaling throughout megakaryocyte specification and shows a sharp decline in AHR activity upon megakaryocyte lineage commitment; 8) In a mouse model of megakaryocyte lineage specific AHR knockout, platelet counts are significantly reduced. These data suggest that the AHR plays a significant role in megakaryocyte specification by modulating the expression of multiple lineage specific gene targets, including MPL, the thrombopoietin receptor. / 2017-05-02T00:00:00Z

The aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor, plays a crucial role in regulation of xenobiotic metabolism. AhR is also involved in dioxin-induced metabolic disorders and alteration of circadian rhythm. Furthermore, circadian clock disruption and metabolic dysfunction are integrally associated with each other. This study was designed to understand the mechanisms by which AhR contributes to regulation of circadian clocks, fat metabolism and glucose homeostasis. In the first aim, I have tested whether AhR interacts with the core clock gene, brain and muscle AhR nuclear translocator like-1(BMAL1), disrupting circadian locomotor output cycle kaput (CLOCK)/BMAL1 complex activity, and leading to the suppression of period1 gene (Per1) expression rhythm. My studies indicate that AhR activation by its agonists 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and beta-naphoflavone (BNF) disrupts the rhythm and inhibits the expression of Per1 in mouse liver and hepatoma cell lines, respectively. Mechanistically, the disruption of the rhythm and the inhibition of Per1 expression occur secondary to the interaction between AhR and BMAL1, which attenuates transcriptional activity of the core clock complex CLOCK/BMAL1. These results suggest alteration of the circadian clock as a novel signaling event downstream of AhR activation. The integral relationship between the clock and metabolic function further suggest that AhR activation may cause metabolic dysfunction. In the second aim, I have tested whether AhR activation inhibits Per1 gene induction and influences circadian clock resetting through activation of JNK pathway. AhR activation by it agonists TCDD and BNF decreases light-induced phase shifts in the early night and inhibits light-induced Per1 expression in both suprachiasmatic nucleus (SCN) and liver. Inhibition of Per1 induction results from increased phospho-JNK induced by AhR activation. Taken together, activation of AhR disrupts circadian clock resetting which also could cause metabolic dysfunction. In the third aim, I have tested whether AhR deficiency regulates nuclear receptor peroxisome proliferator-activated receptor a; (PPARa) and alters glucose homeostasis. PPARa, a clock-controlled gene (CCG) that acts as a fat metabolism sensor, is important for lipid metabolism and glucose homeostasis. AhR knockout (AhRKO or AhR-/-) mice exhibit enhanced insulin sensitivity and glucose tolerance, accompanied by decreased expression of PPARa, key gluconeogenic genes, glucose-6 phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) and key fatty acid oxidation enzymes, carnitine palmitoyl transferase1b (CPT1b) and acyl-CoA oxidase (ACO). Conversely, AhR agonists increase PPARa; expression in a BMAL1-dependent manner. In addition, AhRKO mice display altered rhythm for clock genes, clock-controlled genes (CCGs) and physiological blood glucose. These data suggest that AhR may modulate the glucose homeostasis through regulation of CCGs such as PPARa and that PPARa may be an important link between the circadian clock and metabolism. In the fourth aim, I have tested the effects of AhR ablation or attenuation in high-fat diet (HFD)-induced obesity, insulin resistance and hepatic steatosis in mice. Recent studies suggest that PPARα expression is elevated with HFD feeding as an adaptive response to attenuate hepatic steatosis, and PPARa deficiency protects against HFD-induced insulin resistance. AhR-/- as well as AhR heterozygous (AhR+/-) mice are protected from the HFD-induced obesity, insulin resistance, hepatic steatosis and show reduced inflammatory cytokine expression. In addition, AhR-/- and AhR+/- mice display protected insulin signaling, a higher adiponectin and a lower leptin and insulin in serum. Food intake and physical activity are not significantly different among WT, AhR-/- and AhR+/- mice with HFD feeding. Indirect calorimetry has demonstrated that the AhR+/- mice have higher oxygen consumption, CO2 production and heat production. In addition, Real-time PCR data show that uncoupling protein 1(Ucp1) is higher in brown adipose tissue which supports the higher heat production; moreover, the muscle gene profile reveals that the fatty acid beta-oxidation genes and mitochondrial respiratory genes are higher in AHR+/- mice which further support higher energy expenditure in these mice. Collectively, these data suggest that AhR signaling could be a potential target for treatment of obesity and type 2 diabetes, and AhR antagonist may be developed into a drug for these metabolic diseases.

Aquatic organisms in response to toxic insults from environmental pollutants activate defence systems including the aryl hydrocarbon receptor (AhR) in an attempt to metabolize and excrete these toxicants and their metabolites. These detoxification mechanisms however may come with certain energetic costs. I hypothesize that the activation of the AhR by β-Naphthoflavone (β-NF), a model AhR agonist, results in increased energetic costs requiring metabolic reorganization in rainbow trout hepatocytes. While the results obtained suggest that there are no significant energetic costs of AhR activation, analysis of enzyme activities suggests possible metabolic reorganization. This study also showed significant changes in cellular processes in hepatocytes over the incubation periods which previously were not reported. Furthermore, for the first time in fish hepatocytes, metabolic flux analysis (MFA) was used to examine intra-cellular metabolism, the applicability of which is discussed.

The aryl hydrocarbon receptor (AHR) is a ligand-activated signaling molecule which is involved in diverse biological functions ranging from cancer metastasis to immune regulation. This receptor forms a cytoplasmic complex with Hsp90, p23, and XAP2. We have previously reported that down-regulation of p23 triggers degradation of the AHR protein, uncovering a potentially dynamic event which controls the cellular AHR levels without ligand treatment. Here we investigate the underlying mechanisms for this p23 effect using wild-type HeLa and the p23 knockdown HeLa cells. Reduction of the Hsp90 and XAP2 contents, however, did not affect the AHR protein levels, implying that this p23 effect on AHR is more than just alteration of the cytoplasmic complex dynamics. Association of p23 with Hsp90 is not important for the modulation of the AHR levels since exogenous expression of p23 mutants with modest Hsp90-binding affinity effectively restored the AHR message and protein levels. The protein folding property of p23 which resides at the terminal 50-amino acid region is not involved for this p23 effect. Results from our interaction study using the affinity purified thioredoxin fusion proteins and GST fusion proteins and isothermal titration calorimetry showed that p23 directly interacts with AHR and the interaction surface lies within AHR amino acid 1–216 and p23 amino acid 1–110. Down-regulation of the p23 protein content promotes the ubiquitination of AHR, indicating that p23 protects AHR from the ubiquitin-meditated protein degradation. However, the increased ubiquitination is not through the small ubiquitin-like modifier (SUMO) signaling pathway. Troubleshooting and optimization were paramount for understanding and evaluating the p23 and AHR interaction. Specifically, the p23 mutant purification, p23: Hsp90 interaction, transient transfection, p23: AHR assay, and ITC study were phases of this research that required extensive time and critical thinking. These topics were further detailed to outline the specific problems encountered and the various steps taken to alleviate or optimize these issues.

No description available.

Type 2 diabetes is a metabolic disorder characterized by increased glucose concentrations in the blood due to decreased insulin sensitivity. The worldwide incidence of diabetes has increased remarkably over the last two decades. Obesity, due to increased consumption of calorie dense diets, and sedentary life styles, is commonly cited as a primary cause. However, many epidemiological studies have established a relationship between insulin resistance and exposure to environmental chemicals such as persistent organic pollutants (POPs). The mechanisms by which POPs alter metabolism remain poorly understood, although their lipophilic nature suggests a role in adipose tissue function. The Tischkau lab has established a relationship between Aryl hydrocarbon Receptor (AhR) activation by different types of POPs and increased risk of insulin resistance. This dissertation, therefore, explored the effects of AhR activation by POPs on adipose tissue function. Adipose tissue regulates systemic glucose and lipid metabolism through production of hormones and cytokines that regulate appetite and energy homeostasis. It is well-known that impaired adipose function promotes systemic insulin resistance. The first specific aim examined the hypothesis that activation of AhR suppresses adipogenesis by lowering the rate of pre-adipocyte differentiation. Adipogenesis is a process by which mesenchymal stem cells (MSCs) and pre-adipocytes differentiate into mature adipocytes. Limitations in adipogenesis and accumulation of ectopic lipid have significant roles in decreasing insulin sensitivity. Thus, I hypothesized that POPs contribute to systemic insulin resistance by lowering the rate of MSCs and preadipocyte differentiation; the resulting large, poorly-functioning adipocytes increase serum lipids and promote lipid deposition in other tissues. MSCs derived from mouse bone marrow and pre-adipocytes were treated with different concentrations of AhR agonist, β-Naphthoflavone (BNF), and levels of transcripts associated with adipocyte differentiation were determined by using quantitative PCR. Oil red O staining and lipid content were observed to examine differentiation into mature adipocytes. Genes that promote adipogenesis, including peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding protein alpha (CEBPα), fatty acid binding protein 4 (FABP4), and adiponectin were downregulated in MSCs treated with BNF. Moreover, accumulation of triglycerides was decreased after BNF treatment. Recombinant lentivirus vector-mediated AhR knockdown blocked the effects of BNF on adipogenesis. Therefore, activation of AhR by exogenous ligands inhibits adipogenesis leading to impaired fat storage. Limitations in adipogenesis promotes accumulation of the excess lipid in non-fat tissue such as liver, muscle, and heart leading to decrease the insulin sensitivity and disrupt energy homeostasis. The second specific aim examined effects of AhR activation on circadian clock regulation in adipose tissue. A circadian clock essentially regulates systematic energy homeostasis; the central clock in the suprachiasmatic nucleus (SCN) works with the local clocks in peripheral tissues such as liver, muscle, and adipose tissue to regulate whole-body metabolism. The Tischkau lab has previously shown that AhR interacts with the core machinery of the circadian clock. Activation of AhR by environmental toxicants leads to a dampening of the rhythm expression of core clock genes or an alteration in the timing of their peak expression, which subsequently promotes metabolic disorders such as glucose insensitivity and hyperlipidemia. Given the importance of appropriately timed adipose tissue function to regulation of energy homeostasis, this study focused on mechanisms by which AhR may influence clock-controlled mature adipose tissue activity. Lipolysis is a clock-regulated process in adipose tissue that provides the necessary energy during periods of fasting and exercise. Thus, I hypothesized that AhR activation in adipose tissue would impair lipolysis by altering molecular circadian clock function. AhR activation was proposed to dampen adipose rhythms, leading to a decreased lipolysis rate during the absence of food, and subsequently, increased glucose concentrations in the blood. C57BL/6 mice were injected with vehicle or 50 mg/kg body weight of the AhR agonist, BNF, 48 hours after release into constant darkness. Mice were sacrificed, and epididymal adipose tissue was collected every 6 hours over a 24 hour period. Real-Time RT-qPCR was used to measure mRNA expression of genes responsible for lipolysis. To examine effects of AhR activation in vitro; mouse pre-adipocytes, 3T3-L1 cells, were differentiated into mature adipocytes for 12 days. Cells were then starved for 24 hours with DMEM media containing 1% FBS to induce lipolysis in the presence of 100, 200, 300 µM of BNF. RNA was then extracted and mRNA expression for genes responsible for circadian clock and lipolysis were determined by RT-qPCR. Alterations were observed in rhythms of core clock genes in wild type mice injected with BNF compared to wild type mice injected with vehicle. Rhythms of key enzymes controlling lipolysis including hormone sensitive lipase (HSL) and adipose triglycerides lipolysis (ATGL) was changed in wild type mice injected with BNF compared to wild type mice injected with vehicle. These effects were blocked in AhR deficient mice, suggesting that these effects were AhR dependent. Liver glycogen was decreased in mice injected with BNF compared to wild type mice injected with vehicle after 12 hour of food restriction but not in AhR null mice. Activation of AhR led to decreased expression of lipolysis genes in adipose tissue at CT6 (middle of the rest phase) as well as in 3T3-L1 cells. Recombinant lentivirus vector-mediated AhR knockdown blocked the effects of BNF on lipolysis in 3T3-L1 cell line. These data establish a link between environmental toxicants and impaired lipolysis, specifically by altering rhythms of clock genes in adipose tissue. In response to the decreased available energy from impaired lipolysis, the body increases glycogenolysis, thereby degrading more glycogen to provide the necessary energy. This process may lead to increased glucose level in the blood and development of type 2 diabetes. The data from this study suggest that activation of AhR by BNF increases the risk of insulin resistance and type 2 diabetes by impairing adipogenesis. Reduced adipogenesis likely decreases adipocyte capacity to capture triglycerides from the blood. These effects may disturb energy homeostasis and contribute to the development of metabolic syndrome. This study also establishes a link between environmental toxicants and impaired lipolysis, specifically by altering rhythms of clock genes in adipose tissue. In response to the decreased available energy from impaired lipolysis, the body increases glycogenolysis, thereby degrading more glycogen to provide the necessary energy. This process may lead to increased glucose level in the blood and development of type 2 diabetes. All together, these data suggest that environmental pollutants result in adipose tissue dysfunction by reducing adipogenesis and lipolysis. Therefore, activation of AHR by its exogenous ligands may increase the risk of insulin resistance and type 2 diabetes by impairing adipose tissue function. In particular, activation of AHR by exogenous ligands leads to impairment of free fatty acids storage during feeding and release during fasting to disturb energy homeostasis.

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.

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

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