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The aryl hydrocarbon receptor (AhR) is a basic helix-loop-helix protein that belongs to the superfamily of environment-sensing PAS (Per-ARNT-Sim) proteins. A large number of ligands have been described to bind AhR and promote its nuclear translocation. In the nucleus, the AhR and its dimerization partner the AhR nuclear translocase (ARNT), also known as HIF1β, form a DNA-binding complex that acts as a transcriptional regulator. Animal and human data suggest that, beyond its mediating responses to xenobiotic and/or unknown endogenous ligands, the AhR has a role, although as yet undefined, in the regulation of cell cycle and inflammation. The AhR also appears to regulate the hematopoietic and immune systems during development and adult life in a cell-specific manner. While accidental exposure to xenobiotic AhR ligands has been associated with leukemia in humans, the specific mechanisms of AhR involvement are still not completely understood. However, recent data are consistent with a functional role of the AhR in the maintenance of hematopoietic stem and/or progenitor cells (HSCs/HPCs). Studies highlighting AhR-regulation of HSCs/HPCs provide a rational framework to understand their biology, a role of the AhR in hematopoietic diseases, and a means to develop interventions for these diseases.
Unlike more differentiated populations of cells in the blood, HSCs are functionally defined by their ability to self-renew, differentiate into multiple cell lineages and reconstitute the hematopoietic system of lethally irradiated hosts. Surface markers, dye exclusion and in vitro cultures are also used to phenotypically characterize HSCs [1, 2]. LSK (Lin−Sca-1+c-Kit+) bone marrow cells that are enriched for HSCs  lack the expression of cell surface proteins characteristic of lineage committed cells (B220, CD3, Gr-1, Ter119, Mac-1) and express the Stem cell antigen-1 (Sca-1) and CD117 (also c-Kit) cell surface proteins. Sca-1 is a glycosyl phosphatidylinositol-anchored protein that regulates self-renewal of HSCs . c-Kit is a tyrosine kinase receptor for stem cell factor which is a cytokine that promotes self-renewal while preventing apoptosis in HSCs . Additional markers can be used to further phenotypically define functionally different sub-populations of HSCs contained within the LSK population .
Adult HSCs are able to maintain homeostasis of hematopoiesis through their ability to self-renew and differentiate into HPCs while maintaining a pool of HSCs lasting throughout a lifetime. The critical function of the numerically rare HSC is exquisitely regulated by multiple intrinsic and extrinsic mechanisms . HSC maintenance is intrinsically regulated by restricting proliferation [8-10] and promoting self-renewal. Self-renewal of HSCs is linked to the pathways that contribute to the commitment of cell types during development. Many of these developmental regulators act as ligands that lead to the activation of transcription factors such as GATA2, p53, retinoic acid receptor among others . Stem cells are also contained within physical and chemical environments known as microenvironments or niches  that extrinsically regulate HSC function by releasing soluble factors [13, 14], activating intermediate cells [15-17], or by direct cell-cell interactions [18, 19].
Thus, HSCs have evolved multiple and overlapping levels of control to maintain homeostasis of the hematopoietic and immune system. Disruption of any of these can lead to multiple hematological disorders, including leukemia. In this review, we discuss the hypothesis that the aryl hydrocarbon receptor (AhR) is a transcription factor regulating hematopoiesis and that inappropriate signaling may lead to hematopoietic disease.
The AhR is a basic helix-loop-helix protein, and a member of the PAS (Per-ARNT-Sim) superfamily of proteins. Physiologically, many of these proteins act by sensing molecules and stimuli from the cellular/tissue environment, and initiating signaling cascades to elicit the appropriate cellular responses. Other PAS proteins include sensors of oxygen availability like hypoxia-inducible factor-1 alpha (HIF-1α) , and regulators of circadian rhythms such as brain muscle Arnt-like protein 1 (BMAL1) .
When in the cytoplasm, AhR forms a complex with the chaperone protein heat-shock protein (Hsp)90, co-chaperone p23, and an immunophilin-like protein (AIP or XAP-2) . Following ligand binding , the AhR amino (N)-terminal nuclear localization signal is activated directing it to translocate to the nucleus. Once in the nucleus, AhR forms a heterodimeric complex with the aryl hydrocarbon nuclear translocator (ARNT; also known as HIF-1β). The AhR-ARNT complex binds to a consensus core DNA recognition sequence 5′-TNGCGTG-3′ known as the AhR responsive element (AhRE) . The AhR also contains a glutamine-rich transactivation domain in its carboxy (C)-terminal region which participates in the AhR-ARNT mediated recruitment of coactivators, co-repressors and basal transcription factors . The AhR-ARNT heterodimer has been shown to regulate the expression of several batteries of genes involved in diverse signaling pathways including phase I/II metabolism, inflammation, and cell cycling . However, the exact normal function of this transcription factor has not yet been defined.
The AhR was discovered as the mediator of the toxic responses of halogenated aromatic hydrocarbons and polycyclic aromatic hydrocarbons (PAHs), including the most potent 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, it is now known that a range of structurally diverse xenobiotic chemicals can bind the AhR with different affinities [24, 27]. Most of these compounds are potent inducers of several drug-metabolizing enzymes – particularly, CYP1a1, CYP1a2 and CYP1b1 – glutathione-S-transferase Ya and NAD(P)H-quinone oxidoreductase. Interestingly, there are several endogenous compounds capable of AhR-mediated induction of these genes such as tryptamine, indole acetic acid, bilirubin, biliverdin, 2-(10H-indole-30-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), tryptophan metabolites including the photoproduct 6-formylindolo[3,2-b]carbazole (FICZ), lipoxin A4 [27, 28], leukotriene A4 derivatives  and the intracellular second messenger cAMP . However, the true endogenous ligands have not yet been clearly identified.
Due to the discovery of the AhR in association with the toxic dioxins , and the limited information about the existence or identity of AhR endogenous ligands , much of what is known about AhR biology is based on activation of the receptor using exogenous chemicals. TCDD, the most potent exogenous AhR ligand, and other dioxins are by-products of the chemical synthesis of herbicides popularly used between 1960 and 1980. These chemicals are also contaminants formed during several manufacturing processes and during the incineration and combustion of waste materials. As such, they are still relatively ubiquitous, but most often found at higher concentrations in areas near manufacturing plants and incineration sites where they could be released. Because they are highly lipophilic and bioaccumulate; dioxins are environmental pollutants found in the human food chain . As endocrine disruptors, dioxins have been found to alter reproductive behavior in wildlife and laboratory animals , and have been shown to be potent carcinogens for multiple end-points . In humans, dioxins are carcinogenic  and immunotoxic . Benzo[a]pyrene (BP), another PAH and AhR ligand, is also found in tobacco and coal smoke. However, metabolites of BP, produced by enzymes transcriptionally regulated by the AhR, can also form DNA-binding adducts and cause mutagenesis and other effects by non-AhR pathways .
Besides the transcriptional activation through AhRE DNA binding, there is also evidence to suggest that ligand binding to AhR can elicit biological responses through AhR interactions with other transcription factors or cofactors. In an in vivo ischemia-induced model, treatment with BP was shown to impair angiogenesis by a molecular mechanism that results in induced vascular endothelial growth factor (VEGF). The proposed mechanism suggests that after ligand binding the formation of the AhR-ARNT complex resulted in lower levels of ARNT bound to its other partner HIF-1α whose signaling resulted in upregulation of VEGF . More recently, AhR deficiency has also been shown to modulate angiogenesis by a mechanism involving VEGF depletion in endothelial cells and overexpression of TGF-β in stromal cells . In mammary tumor cells, the association between the RelA subunit of NFκB and AhR induced cellular proliferation by activation of the oncogene c-myc [38, 39]. Also, interactions between AhR and the RelB subunit of NFκB induced chemokine production in leukemic cells . In the lung, AhR appears to have a role in regulating inflammatory responses by maintaining RelB expression in fibroblasts . AhR interaction with Rb has been shown to suppress progression of the S-phase of the cell cycle, but this interaction is restricted to the hypophosphorylated form of Rb found in quiescent cells, suggesting that the AhR is a negative regulator of proliferation [42, 43]. Alternatively, the AhR-Rb interaction has been proposed to coactivate the transcription of genes encoding proteins that suppress cell cycle progression in hepatoma cells . Together these data suggest that while there are multiple mechanisms for AhR regulation of several signaling pathways, in particular those involving the cell cycle, the responses are specific to the type and cycling status of the cell.
AhR-mediated responses to cellular stress by mechanisms specific to cell type have also been proposed. Part of the short- and long-term cellular stress responses of skin to ultraviolet light, including altered cell cycling and inflammatory reactions, may be mediated by AhR ligands generated as photoproducts . Acute-phase response genes regulated by NF-κB are expressed in hepatocytes as an early reaction to cellular stress, and AhR ligands have been shown to repress this response in murine and human hepatocytes by a mechanism that requires AhR-ARNT nuclear translocation but not AhRE binding . In response to light, TCDD also alters expression of the circadian regulators Per1 and Bmal1 genes and changes the phase shifts of circadian-regulated responses . AhR and cyp1a1 genes have been shown to have a circadian expression in hepatocytes, brain cells  and immune cells . These data support the concept that the AhR participates in the regulation of responses to environmental stimuli. Figure 1 illustrates these mechanisms emphasizing that AhR actions involve direct interaction of the AhR-ARNT complex with DNA through AhREs and/or interactions of the AhR with other transcription factors. Note that the ultimate responses observed following altered AhR activity may not be mutually exclusive to specific mechanisms. For example, the involvement of the AhR to regulate cell cycle may occur by mechanisms other than its ability to interact with Rb.
After nuclear translocation, the AhR is targeted for degradation via the ubiquitin-proteosome pathway . AhR activity in the cell is also regulated by the presence of the AhR Repressor protein whose expression is regulated by the AhR . The AhR signaling pathway regulates the metabolism of its own exogenous and likely endogenous ligands by up-regulating cytochrome P-450 proteins . These multiple mechanisms to regulate AhR expression and activity suggest an evolutionary pressure to maintain and finely regulate homeostatic activity of this protein, further suggesting a physiological role for AhR  and that its dysregulation, absence or chronic activation may lead to disease.
The existence of AhR homologs in vertebrates and invertebrates and their involvement in the development of hepatic, reproductive, cardiovascular and immune system  support a physiological role of the AhR. Further support is provided by studies using AhR null-allele (AhR-/-) mice, which show defects in the development of the hematopoietic, cardiovascular and immune system [52, 53]. There has been some discrepancy regarding the immune phenotype of young AhR-/- mice because several of these phenotypes are most evident only under the context of specific immune responses . Absence of AhR results in impaired differentiation of naïve T cells into T-helper 2 cells in a model of allergic sensitization , and AhR-/- mice also have shown delayed kinetics of T-helper 17 (Th17)-mediated autoimmune disease . These mice exhibit constitutive accumulation of lymphocytes in the spleen and lymph nodes, along with being more susceptible to Listeria monocytogenes infection . Also, AhR-/- mice were more sensitive to lipopolysaccharide (LPS)-induced lethal shock than wild type controls due to a higher production of IL6 and TNF-α by activated macrophages . Transgenic mice with a mutation leading to the inability of the AhR to bind the AhRE (Ahrdbd) also showed defects in cardiovascular, splenic and hepatic development . Characterization of a transgenic mouse expressing constitutively active AhR (CA-AhR) showed increased organ weights for heart, liver and kidney but decreased weight of the immune-related organs thymus and spleen . Table 1 summarizes the phenotypes observed in hematopoietic and immune cells for the different animal models to study AhR. These models suggest that the AhR plays an important role during development and maintenance of the hematopoietic and immune system.
Functional AhR is expressed by different hematopoietic and immune populations. In B-cells, the constitutive low levels of AhR mRNA and protein expression are highly upregulated after activation of resting cells [60, 6]. Western blots of human monocytes and CD34+ progenitors showed constitutive levels of the protein [61, 62]. When CD4+ T-cells become activated, the level of AhR message is upregulated in Th17 populations [55, 63]. AhR expression can also be detected in certain bone marrow stromal cell lines  and some cell lines derived from leukemic cells . AhR message has been detected in murine LSK cells [6, 66].
Despite AhR being ubiquitous in the hematopoietic and immune system, the type and the magnitude of the responses initiated by AhR-activation appears to be cell-specific . In macrophages, AhR interacts with signal transducer and activator of transcription 1 (Stat1) and NFκB to regulate proinflammatory innate immune responses elicited by LPS . Dendritic cells that present antigen to naïve T cells in the periphery are reduced in numbers after AhR activation by the low-molecular-weight compound VAF347, and this appears to be due to the AhR-dependent decreased expression of IL6, IL10 and TGFβ . During Th17 cell development, AhR regulates differentiation via Stat1, and AhR activation upregulates secretion of the inflammatory cytokines IL17 and IL22 . Regulatory T-cells (Treg) inhibit cell proliferation of effector cells and cytokine secretion to suppress immune responses after they have been mounted. Ligand-mediated AhR activation increases Treg proliferation while suppressing experimental autoimmune encephalitis  and preventing graft-versus-host disease [69, 70].
Thymic atrophy has been recognized as a hallmark of exposure to the xenobiotic AhR agonist TCDD. As such, much effort has been devoted to understanding the mechanisms responsible and the role of the AhR functioning as a transcription factor in the hematopoiesis, and lymphopoiesis in particular. Studies using AhR-chimeric mice have shown that thymocytes and/or thymocyte precursors need to express AhR in order for TCDD or other dioxin-like xenobiotics to elicit this particular toxic effect , suggesting that the persistent activation by TCDD is disrupting some normal role of the AhR in these cells. There is evidence supporting TCDD-elicited reduced seeding of the thymus by bone marrow-derived progenitors , reduced stromal-mediated proliferation of thymocytes [73, 74] and a skewing of the thymic output through direct effects on developing thymocytes . Additionally, it has been shown that AhR activation during gestation leads to alterations of B lineage cells with possible consequences on autoimmunity .
Given a proposed, but not yet clearly defined, role of the AhR during hematopoietic development, it has been further postulated that this transcription factor may be a homeostatic regulator of HSCs. AhR activation by TCDD has been shown to alter the functional activity of lymphoid committed progenitors and early progenitors of hematopoiesis [6, 77], accompanied by increased numbers of LSK cells [8, 78]. This also suggests some important function of the AhR in the regulation of HSCs that TCDD is disrupting through its ability to produce prolonged AhR activation. However, the molecular mechanisms by which AhR regulates the functions and numbers of HSCs are not yet clear. The further study of the AhR and AhR-regulated signaling pathways in hematopoietic stem and progenitor cell populations needs to consider the particular functions of these cells, cycling status, commitment towards certain differentiation pathways, and interactions with other cell types.
Limited epidemiological data from humans accidentally exposed to dioxins suggest an association between TCDD exposure and the development of leukemias and other hematopoietic malignancies [79-82]. Fifteen years after an industrial accident that exposed thousands of people to TCDD, a follow-up study of this population reported increased incidence of lymphohematopoietic neoplasms, non-Hodgkin’s lymphoma and myeloid leukemia, even at relatively low levels of TCDD exposure .
At least at present, there are few Ahr gene polymorphisms known to be associated with human cancers. Several of these are associated with lung and upper intestinal tract cancer and appear to be related to the ability of the AhR to regulate the expression of enzymes responsible for the metabolism of several xenobiotics, in particular the PAHs [80-83]. One of the most studied Ahr polymorphisms in humans, G1661A, where a lysine residue at amino acid 544 replaces an arginine, has also been associated with DNA damage in peripheral lymphocytes of coke-oven workers occupationally exposed to PAHs . In a recent study, a particular Ahr polymorphism, in conjunction with exposure to organochlorines, was also associated with increasing risk of non-Hodgkin lymphoma . There is at present no known association between any Ahr polymorphism and increased incidence of any hematopoietic disease or cancers in the absence of known xenobiotic exposures. However, there have been few, if any, studies to examine for these associations.
Based on the evidence for different mechanisms recognized to disrupt HSC functions and lead to hematopoietic diseases, along with evidence supporting a role of the AhAR in the regulation of HSCs, we propose that AhR involvement in these diseases may occur through (a) dysregulation of AhR signaling in HSCs/HPCs, (b) generation of leukemic stem cells, (c) depletion of the stem cell compartment, and/or (d) disruption of HSC-microenvironment interactions. This hypothesis is based on the following evidence as summarized in Table 2:
A simplified scheme highlighting these possible mechanisms for AhR involvement in HSC/HPC dysregulation that may result in leukemia or other hematopoietic diseases is depicted in Figure 2. During healthy hematopoiesis (A), HSCs provide HPCs that will differentiate and repopulate all the lineages present in blood. However, dysregulation of AhR signaling in HSCs (as in B) may be carried on to HPCs and mature lineages, thus altering differentiation and lineage commitment decisions. Alternatively (C), AhR dysregulation alone or in combination with exposure to particular xenobiotics, i.e. benzene, may result in the formation of leukemic stem cells with self-renewal properties resulting in the accumulation of progenitors and a block of differentiation into the different lineages. Alterations of AhR levels or activity may also cause (D) chronic changes in HSC cycling resulting in stem-cell exhaustion, reduced numbers of mature cells, and/or conditions that promote the development of hematopoietic cancers. Finally (E), AhR-elicited disruption of the interactions between HSCs and their microenvironment may alter the homeostatic mechanisms responsible of supporting hematopoiesis.
While there is sufficient evidence for a regulatory role of AhR in HSCs from both the exposure of humans to xenobioitic AhR ligands and work in experimental animals, the molecular pathways that are regulated by the AhR and which control HSC function and survival are, as yet, poorly understood. Multiple signaling pathways reported to crosstalk with the AhR may participate in the maintenance of homeostatic HSC function. Further studies will need to specifically address at a molecular level whether AhR has a direct and/or indirect role in maintaining HSC function. This information may in turn provide a clear understanding about the physiological role of AhR in the hematopoietic and immune system.
Given that the AhR is a well described ligand-activated transcription factor, it is likely to be a potentially important pharmacological target for prevention and/or treatment of diverse hematological diseases. Potential clinical applications may include regenerative and/or chemotherapeutic approaches to treat leukemia, autoimmune disease, and other hematological diseases.
The authors thank Dr. Ellen Henry for her critical reading of the manuscript. This work was supported by National Institutes of Health Grants ES04862, ES0166606, Training Grant ES07026, and Center Grant ES01247
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