Lack of Aryl Hydrocarbon Receptor Alters Gene Expression and Functional Capacity of Murine Hematopoietic Stem Cells

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Environmental Medicine, 2015.All mature cell lineages of the peripheral blood and adaptive immune system are generated from bone marrow stem cells known as hematopoietic stem cells (HSCs). Lack of aryl hydrocarbon receptor (AhR) signaling, or treatment with AhR agonists such as TCDD (2,3,7,8 tetrachlorodibenzo-p-dioxin) , has been shown to alter the phenotype and functional ability of HSCs, suggesting a role for the AhR in regulating HSC function and output. Most classical studies on AhR have been undertaken using TCDD or other xenobiotic ligands to modulate AhR activity. While these studies have revealed a wealth of information regarding AhR target genes and signaling networks in a variety of cell types (including HSCs), it has remained difficult to determine which endpoints / regulatory target genes are adaptive or toxic responses due to exposure to AhR ligands versus normal targets and functions under homeostatic conditions. Studies were performed using both global AhR-null allele (AhR-KO) mice as well as hematopoietic system restricted AhRfx/fxVav1-Cre (CKO) mice in order to better understand the HSC-intrinsic physiological role of AhR in regulating HSC function and output. These studies revealed a surprising lack of phenocopy between the two strains. Aged KO animals display an expansion of peripheral white blood cells which is not found in aged CKO animals. KO animals have enhanced levels of oxidative stress in bone marrow progenitors, as well as evidence of DNA damage and decreased expression of p16. CKO animals only display enhanced oxidative capacity at the time point examined. Microarray studies on long term HSCs were undertaken in order to examine the differences in cell signaling and gene expression that could be driving these unexpected differences in phenotype. The two strains display very little overlap in genes with differential expression relative to control at young ages, but become more similar with aging. Gene Set Enrichment Analysis revealed significant enrichment in a variety of gene sets that are related to HSC function, or involved in the etiology of certain diseases such as leukemia or myeloproliferation. A subset of differentially expressed genes that changed expression due to both aging and lack of AhR expression (Pdgf-D, Smo, Zbtb37, and Zfp382), and that contain AhR binding sites in their upstream regulatory regions, change expression when AhR expression is silenced in wild-type hematopoietic cells. Taken together, these data reveal potential genes that AhR may intrinsically regulate in HSCs throughout aging of the hematopoietic system. Given the lack of phenocopy and the small degree of overlap differentially expressed genes in HSCs between KO and CKO mice, these data also implicate AhR in non-hematopoietic stromal cells of the bone marrow as having a regulatory effect on HSC function and output

Epigallocatechin Gallate Elicits Stage-Specific Sensitivity and Affects Heat Shock Protein 90 in a Novel Human Prostate Cancer Progression Model

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pathology and Laboratory Medicine, 2014.(-)-Epigallocatechin gallate (EGCG), a major tea polyphenol, elicits anti-cancer effects. However, the mechanism of action is not understood. Our laboratory made the novel discovery that EGCG inhibits heat shock protein 90 (HSP90). We utilized non-tumorigenic (NT), tumorigenic, and metastatic cancer cells from a novel human prostate cancer progression model to test the hypotheses that cancer cells (tumorigenic, metastatic) would be more sensitive to EGCG and that sensitivity is related to HSP90 inhibition. Treatment of cells with EGCG, novobiocin, or 17-AAG resulted in more potent cytotoxic effects on tumorigenic and metastatic cells than NT cells. When tumorigenic or metastatic cells were grown in vivo, mice supplemented with 0.06% EGCG in drinking water developed significantly smaller tumors than untreated mice. Furthermore, EGCG prevented cancer progression at early stages by inhibiting malignant transformation of NT cells using the full prostate cancer progression model. To elucidate the mechanism of EGCG action, we performed binding assays with purified HSP90 protein, HSP90 mutants, a C-terminal HSP90 antibody, and EGCG-Sepharose. These experiments revealed that EGCG bound WT HSP90 and mutants that mimic both complexed and uncomplexed HSP90. Additionally, EGCG-Sepharose bound more HSP90 from metastatic cells compared to NT cells and binding occurs through the HSP90 C-terminus. Consistent with HSP90 inhibitory activity, EGCG, novobiocin, and 17-AAG induced changes in HSP90 client proteins (ErbB2, p-Akt, Raf-1) in both NT and metastatic cells. In addition to HSP90, we identified endoplasmic reticulum and mitochondria-specific chaperone proteins (GRP78, GRP94, TRAP1, GRP75, HSP60, HYOU1) by mass spectrometry that bound directly or indirectly (through HSP90) to EGCG-Sepharose in NT and metastatic cell lysate. Furthermore, EGCG, like novobiocin and 17-AAG, inhibited luciferase refolding in a chaperone function assay. This assay also identified several EGCG analogs with stronger effects on luciferase refolding that may possess more potent anti-HSP90 activity in vitro and in vivo. These data suggest that EGCG may be efficacious for the treatment of prostate cancer because it preferentially targets cancer cells and inhibits a molecular chaperone (HSP90) supportive of the malignant phenotype

(-)-Epigallocatechin-3-gallate, Found in the Green Tea Extract, is a Novel Hsp90 Inhibitor

Thesis (Ph. D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Environmental Medicine, 2009.Numerous animal studies have shown that epigallocatechin-3-gallate (EGCG), a major component of green tea, protects against certain types of cancers, although the mechanism has not yet been determined. Our laboratory previously demonstrated that EGCG blocks aryl hydrocarbon receptor (AhR)-mediated transcription induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Unlike other AhR antagonists that directly bind to the AhR, EGCG inhibits AhR-mediated transcription by binding to hsp90. I hypothesize that EGCG exerts anti-AhR and anti-cancer effects by acting as an hsp90 inhibitor. Using proteolytic footprinting, immunoprecipitation and an ATP-agarose pull-down assay, I determined that EGCG directly modulates the conformation of hsp90 and binds at or near to the C-terminal ATP binding site of hsp90. Direct binding of 3H-EGCG to purified human hsp90 was observed by competitive binding assay suggesting a Kd of ~780nM. Novobiocin (a C-terminal hsp90 inhibitor) and ATP compete with 3H-EGCG for binding to hsp90. Hsp90 chaperone function, as demonstrated by the ability to mediate refolding of denatured luciferase, is inhibited by EGCG treatment. Hsp90 dimerization, which occurs at the C-terminal end, is also inhibited by EGCG treatment. EGCG also modified the association of hsp90 with several cochaperones. Co-immunoprecipitation studies show that EGCG stabilizes an AhR complex that includes hsp90 and XAP2 (hepatitis B virus X-associated protein 2), and decreases the association of aryl hydrocarbon nuclear translocator (Arnt) to ligand-activated AhR. Thus, EGCG, through its ability to bind to hsp90, decreases the association of AhR, an hsp90 client protein, with Arnt and blocks AhR response element (AhRE) recognition. These studies indicate a novel mechanism whereby EGCG inhibits ligand-induced AhRE binding and AhR-mediated transcriptional activity by acting as an hsp90 inhibitor. In EGCG-treated human ovarian carcinoma SKOV3 cells, decreased levels of several cancer-related hsp90 client proteins, such as ErbB2, Raf-1 and phospho-AKT were observed. Overall, these data indicate that EGCG is a novel hsp90 inhibitor. Further studies are needed to determine if this has a role in the anti-tumor actions of EGCG. Since hsp90 inhibitors are being intensively studied for cancer therapy, this investigation will advance further research that may ultimately lead to the use of EGCG and/or its derivatives for chemotherapeutic purposes

S395, a Site Important for Regulating Transcriptional Activity of the Aryl Hydrocarbon Receptor (AhR), is Phosphorylated by Protein Kinase A (PKA)

Thesis (Ph. D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Environmental Medicine, 2009.The aryl hydrocarbon receptor (AhR) is a member of the basic helix-loop-helix transcription factor family. The AhR interacts with a wide variety of xenobiotic agonists, the most potent of which is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), to produce a wide variety of biologic and toxic responses such as teratogenicity and cancer. TCDD and other dioxins are emitted from the combustion of fossil fuels, metalsmelting and processing, manufacturing of chemicals, photochemical and biological processing, as well as in food. Exposure from these sources of dioxin may lead to adverse health effects. It has been suggested that AhR activity is regulated by post-translational modifications. Phosphorylation of the AhR is a key regulatory mechanism that plays a role in DNA binding and transcriptional activity; however, few studies have identified functionally significant sites of phosphorylation. These studies investigated possible functional phosphorylation at the STS (serine 393, threonine 394, and serine 395) region of the mouse AhR. This sequence is located in a major region of phosphorylation and in the transcriptional inhibitory domain. Phosphorylation prediction algorithms suggest that serine 393 and serine 395 may be phosphorylated by protein kinase A (PKA). This research was conducted to test the overall hypothesis that the phosphorylation of the STS sequence is important for regulating AhR-mediated transcriptional activity. Studies determined that PKA is able to phosphorylate the full-length receptor as well as peptides containing the STS sequence via 32P incorporation in vitro. Additionally, Fourier Transform Mass Spectrometry (FTMS) analysis of PKA-treated peptides shows that Serine 395 is phosphorylated. Functionally, we show that there is decreased transcriptional activity when serine 395 is mutated to alanine without affecting binding of the AhR-ARNT (aryl hydrocarbon receptor nuclear translocator) complex to dioxin-response elements. These data suggest that phosphorylation of serine 395 regulates the transactivation ability of the mouse AhR. The results from these experiments identify a functionally significant phospho-site and further confirm that the AhR may be regulated by phosphorylation (Funded by NIH Grants ES02515, ES01247, and ES07025)

Enhanced Detection of β-Galactosidase Reporter Activation Is Achieved by a Reduction of Hemoglobin Content in Tissue Lysates

β-galactosidase (β-gal), the product of the E. coli LacZ gene, has been used extensively as a reporter in numerous systems. Until recently, the most commonly used method of detecting β-gal reporter enzymatic activity was a colormetric assay based on the cleavage of the β-gal substrate 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside (X-gal) to form a blue precipitate. However, when increased sensitivity is needed, many investigators now turn to alternate substrates that produce fluorescent or luminescent products upon cleavage by β-gal. These products are much more easily quantified than X-gal. The luminescent and fluorometric assays work very well in cultured cells but are often less sensitive in whole tissue lysates. In this study, we have evaluated the sensitivity of a fluorescent and a luminescent substrate in whole tissue lysates cleared of red blood cells or washed with PBS only. We have found that both assays show increased low-end sensitivity in tissues with reduced levels of hemoglobin (Hb). Hb is apparently able to quench luminescent and, to a lesser degree, fluorescent reporter light emission. Therefore, steps should be taken to reduce Hb levels either by lysis, perfusion, or both to enhance the sensitivity of these assays

Activation of the Aryl Hydrocarbon Receptor Signaling by 2,3,7,8 Tetra-chlorodibenzo-p-dioxin (TCDD) Alters Cell Function and Pathway-specific Gene Modulation of Hematopoietic Stem/Progenitor Cells

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Environmental Medicine, 2011.The aryl hydrocarbon receptor (Ahr) mediates the toxicity of certain environmental pollutants including dioxins. Accidental dioxin exposure to humans has been correlated with blood malignancies such as leukemia and lymphoma. Dioxin exposure suppresses immune responses in mammals via Ahr by several mechanisms including altering the functional ability of hematopoietic progenitors to seed the thymus. Also, exposure alters population of the spleen and reconstitution of peripheral blood and bone marrow. The Ahr is a ligand-activated transcription factor without any established endogenous ligand or physiological role in hematopoietic cells. To explain the decreased reconstitution of bone marrow, I hypothesized that dioxin exposure alters the numbers of hematopoietic stem/progenitor cells and/or the trafficking of these populations to the bone marrow. Furthermore, I hypothesized that these cellular consequences of TCDD exposure are secondary to an alteration of transcriptionally regulated pathways related to cell-to-cell signaling and cellular movement. Colony-forming assays and competitive repopulation experiments were used to quantify sub-populations of LSKs. In vivo dioxin exposure increased the numbers of multipotent progenitors but did not change the numbers of functional hematopoietic stem cells (HSCs). Trafficking of LSKs to the bone marrow in vivo, and to the chemokine Cxcl12 in vitro were decreased. Transcripts involved in cell-to-cell signaling (Ccl3, Cd69, Cxcl2, Cox-2, Mmp8) and cellular movement (Scin, Mmp8), as well as hematological system development and function (Egr-1, Ccl3, Cd69, Cxcl2, Cox-2) were altered in TCDD-exposed LSKs. Altogether these data support a physiological role of the Ahr to regulate homeostasis in HSCs. Disruption of Ahr expression or activity may predispose more differentiated populations to acquire malignant behaviors and reduce the ability of HSCs to respond to stress or injury. The mechanism of action for an Ahr ligand to alter cell function and pathway-specific gene modulation of LSKs as defined in this work provides a rationale to examine possible preventive measures in populations exposed to Ahr ligands. Being the Ahr a ligand-activated transcription factor, therapeutic interventions that regulate the expansion, migration and differentiation of HSCs or selective modulation of specific-hematopoietic lineages may be explored