Kameshwar P. Singh

The aryl hydrocarbon receptor has a normal function in the regulation of hematopoietic and other stem/progenitor cell populations

The aryl hydrocarbon receptor (AhR) is known mainly as the mediator for the toxicity of certain xenobiotics. However, there is also much information to indicate that this transcription factor has important biological functions. Here we review the evidence that the AhR has a significant role in the regulation of hematopoietic stem cells (HSCs). Data to support this come from studies with xenobiotic AhR ligands, phenotypic analyses of mice lacking AhR, examining the presence and regulation of the AhR within HSCs, knowledge of genes and signaling pathways regulated by the AhR, and investigations of hematopoietic disorders. Based on this information, we hypothesize that AhR expression is necessary for the proper maintenance of quiescence in HSCs, and that AhR down-regulation is essential for escape from quiescence and subsequent proliferation of these cells. This implicates the AhR as a negative regulator of hematopoiesis with a function of curbing excessive or unnecessary proliferation. This provides an important advantage by preventing the premature exhaustion of HSCs and sensitivity to genetic alterations, thus preserving HSC function and long-term multi-lineage generation over the lifespan of the organism. This also implicates a role of the AhR in aging processes. AhR dysregulation may result in the altered ability of HSCs to sense appropriate signals in the bone marrow microenvironment leading to hematopoietic disease. It is also reasonable to hypothesize that this protein has an important function in the regulation of other tissue stem cell populations. Suggestive evidence is consistent with a role in skin and neural stem cells.

Aryl Hydrocarbon Receptor-Null Allele Mice Have Hematopoietic Stem/Progenitor Cells with Abnormal Characteristics and Functions

The aryl hydrocarbon receptor (AhR) belongs to the basic helix-loop-helix family of DNA-binding proteins that play a role in the toxicity and carcinogenicity of certain chemicals. The most potent ligand of the AhR known is 2,3,7,8-tetracholorodibenzo-p-dioxin. We previously reported tetrachlorodibenzo-p-dioxin-induced alterations in numbers and function of hematopoietic stem cells (HSCs). To better understand a possible role of the AhR in hematopoiesis, studies were undertaken in young adult AhR null-allele (KO) mice. These mice have enlarged spleens with increased number of cells from different lineages. Altered expression of several chemokine, cytokine, and their receptor genes were observed in spleen. The KO mice have altered numbers of circulating red and white blood cells, as well as a circadian rhythm-associated 2-fold increase in the number of HSC-enriched Lin(-)Sca-1(+)c-Kit(+) (LSK) cells in bone marrow. Primary cultures of KO HSCs and in vivo bromodeoxyuridine incorporation studies demonstrated an approximate 2-fold increased proliferative ability of these cells. More LSK cells from KO mice were in G(1) and S phases of cell cycle. Competitive repopulation studies also indicated significant functional changes in KO HSCs. LSK cells showed increased expression of Cebpe and decreased expression of several hematopoiesis-associated genes. These data indicate that AhR has a physiological and functional role in hematopoiesis. The AhR appears to play a role in maintaining the normal quiescence of HSCs.

Treatment of mice with the Ah receptor agonist and human carcinogen dioxin results in altered numbers and function of hematopoietic stem cells

The aryl hydrocarbon receptor (AhR) mediates the carcinogenicity of a family of environmental contaminants, the most potent being 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Increased incidence of lymphoma and leukemia in humans is associated with TCDD exposure. Although AhR activation by TCDD has profound effects on the immune system, precise cellular and molecular mechanisms have yet to be determined. These studies tested the hypothesis that alteration of marrow populations following treatment of mice with TCDD is due to an effect on hematopoietic stem cells (HSCs). Treatment with TCDD resulted in an increased number and proliferation of bone marrow (BM) populations enriched for HSCs. There was a time-dependent decrease in B-lineage cells with a concomitant increase in myeloid populations. The decrease in the B-cell lineage colony-forming unit-preB progenitors along with a transient increase in myeloid progenitors were consistent with a skewing of lineage development from lymphoid to myeloid populations. However, HSCs from TCDD-treated mice exhibited diminished capacity to reconstitute and home to marrow of irradiated recipients. AhR messenger RNA was expressed in progenitor subsets but is downregulated during HSC proliferation. This result was consistent with the lack of response following the exposure of 5-fluorouracil-treated mice to TCDD. The direct exposure of cultured BM cells to TCDD inhibited the growth of immature hematopoietic progenitor cells, but not more mature lineage-restricted progenitors. Overall, these data are consistent with the hypothesis that TCDD, through AhR activation, alters the ability of HSCs to respond appropriately to signals within the marrow microenvironment.

The aryl hydrocarbon receptor has an important role in the regulation of hematopoiesis: Implications for benzene-induced hematopoietic toxicity

The aryl hydrocarbon receptor (AhR) belongs to the basic helix-loop-helix (bHLH) Per-Arnt-Sim (PAS) family of transcription factors. Many of these proteins are involved in regulating responses to signals in the tissue environment such as hypoxia, oxidation-reduction status, and circadian rhythms. Although the AhR is well studied as a mediator of the toxicity of certain xenobiotics, the normal physiological function remains unknown. However, accumulating data support a hypothesis that the AhR has an important function in the regulation of hematopoietic stem cells (HSCs). Persistent AhR activation by dioxin, a potent xenobiotic AhR agonist, results in altered numbers and function of HSCs in mouse bone marrow. Analysis of HSCs from AhR null-allele mice also indicates that lack of AhR expression results in altered characteristics and function of these cells. HSCs from these animals are hyperproliferative and have altered cell cycle. In addition, aging AhR-KO mice show characteristics consistent with premature bone marrow senescence and are prone to hematopoietic disease. Finally, some data suggest that the expression of the Ahr gene is regulated under conditions that control HSC proliferation. The presence of a normal functioning AhR may provide an important advantage to organisms by regulating the balance between quiescence and proliferation and preventing the premature exhaustion of HSCs and sensitivity to genetic alterations. This function assists in the preservation of HSC function and long-term multi-lineage generation over the lifespan of the organism. This also implicates a role for the AhR in the aging process. Furthermore, these functions may affect the sensitivity of HSCs to certain xenobiotics, including benzene. Defining the molecular mechanisms by which these events occur may lead to the identification of previously undefined roles of this transcription factor in human diseases, particularly those caused or affected by xenobiotics.

The aryl hydrocarbon receptor: regulation of hematopoiesis and involvement in the progression of blood diseases.

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 translocator (ARNT) 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.

Aryl Hydrocarbon Receptor Activation in Hematopoietic Stem/Progenitor Cells Alters Cell Function and Pathway-Specific Gene Modulation Reflecting Changes in Cellular Trafficking and Migration

The aryl hydrocarbon receptor (AhR) is a transcription factor belonging to the Per-ARNT-Sim family of proteins. These proteins sense molecules and stimuli from the cellular/tissue environment and initiate signaling cascades to elicit appropriate cellular responses. Recent literature reports suggest an important function of AhR in hematopoietic stem cell (HSC) biology. However, the molecular mechanisms by which AhR signaling regulates HSC functions are unknown. In previous studies, we and others reported that treatment of mice with the AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) compromises the competitive reconstitution of bone marrow (BM) cells into irradiated host animals. Additional studies indicated a requirement for AhR in hematopoietic cells and not marrow microenvironment cells. In this study, we tested the hypothesis that TCDD-mediated phenotypic and functional changes of HSCs are a result of changes in gene expression that disrupt stem cell numbers and/or their migration. TCDD treatment to mice increased the numbers of phenotypically defined HSCs in BM. These cells showed compromised migration to the BM in vivo and to the chemokine CXCL12 in vitro, as well as increased expression of the leukemia-associated receptors CD184 (CXCR4) and CD44. Gene expression profiles at 6 and 12 h after exposure were consistent with the phenotypic and functional changes observed. The expressions of Scin, Nqo1, Flnb, Mmp8, Ilf9, and Slamf7 were consistently altered. TCDD also disrupted expression of other genes involved in hematological system development and function including Fos, JunB, Egr1, Ptgs2 (Cox2), and Cxcl2. These data support a molecular mechanism for an AhR ligand to disrupt the homeostatic cell signaling of HSCs that may promote altered HSC function.

The Ah receptor in stem cell cycling, regulation, and quiescence

Processes that regulate quiescence, self‐renewal, and differentiation of hematopoietic stem cells (HSCs) are not well understood. Owing, in part, to the ability of xenobiotic ligands to have persistent effects on the immune system in experimental animals, there has been much work to define a physiological role of the aryl hydrocarbon receptor (AhR) and its relationship to human disease. Persistent AhR activation by dioxin, a potent agonist, results in altered numbers and function of HSCs in mice. HSCs from AhR−/− knockout (KO) mice are hyperproliferative and have an altered cell cycle. Aging KO mice show characteristics consistent with premature bone marrow exhaustion. We propose that the increased proliferation of HSCs lacking AhR expression or activity is a result of loss of quiescence, and as such, AhR normally acts as a negative regulator to curb excessive or unnecessary proliferation. Similarly, prolonged and/or inappropriate stimulation of AhR activity may compromise the ability of HSCs to sense environmental signals that allow these cells to balance quiescence, proliferation, migration, and differentiation. These data and others support a hypothesis that deregulation of AhR function has an important role in HSC regulation and in the etiology and/or progression of certain hematopoietic diseases, many of which are associated with aging.

Loss of Aryl Hydrocarbon Receptor Promotes Gene Changes Associated with Premature Hematopoietic Stem Cell Exhaustion and Development of a Myeloproliferative Disorder in Aging Mice

Loss of immune function and increased hematopoietic disease are among the most clinically significant consequences of aging. Hematopoietic stem cells (HSCs) from mice lacking aryl hydrocarbon receptor (AhR) have high rates of cell division. Studies were designed to test the hypothesis that aging AhR-null allele (AhR-KO) mice develop premature HSC exhaustion, and changes leading to hematological disease. Compared to wild-type, aging AhR-KO mice showed a decreased survival rate, splenomegaly, increased circulating white blood cells, hematopoietic cell accumulation in tissues, and anemia. Analysis of bone marrow indicated increased numbers of stem/progenitor and lineage-committed cells, but decreased erythroid progenitors. There was also decreased self-renewal capacity of HSCs determined by competitive repopulation and serial transplantation. HSCs also showed increased levels of reactive oxygen species (ROS), Ki-67, and γ-H2A.X, but decreased p16(Ink4a). Splenic cells from aging KO mice had abnormal expression of genes, including Gata-1, Sh2d3c, Gfi-1, p21, and c-myc, involved in trafficking and associated with leukemia. HSCs from AhR-KO mice had gene changes related to HSC maintenance and consistent with phenotype observed. The most prominent gene changes (overexpression of Srpk2, Creb1, Hes1, mtor, pdp1) have been associated with HSC hyperproliferation, leukemia, and accelerated aging. Pathway analyses also indicated an enrichment of genes associated with oxidative stress, acute myelogenous leukemia, aging, and heat shock response, and the β-catenin/Wnt pathways. These data indicate that loss of AhR and associated changes in multiple signaling pathways promote premature HSC exhaustion and development of a myeloproliferative disorder. They also implicate a critical role of the AhR in the regulation of HSCs.

Identification of Stage-Specific Gene Modulation during Early Thymocyte Development by Whole-Genome Profiling Analysis after Aryl Hydrocarbon Receptor Activation

The aryl hydrocarbon receptor (AHR) is a basic helix-loop-helix transcription factor, implicated as an important modulator of the immune system and of early thymocyte development. We have shown previously that AHR activation by the environmental contaminant and potent AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads to a significant decline in the percentage of S-phase cells in the CD3−CD4−CD8− triple-negative stage (TN) 3 and TN4 T-cell committed thymocytes 9 to 12 h after exposure. In the more immature TN1- or TN2-stage cells, no effect on cell cycle was observed. To identify early molecular targets, which could provide insight into how the AHR acts as a modulator of thymocyte development and cell cycle regulation, we performed gene-profiling experiments using RNA isolated from four intrathymic progenitor populations in which the AHR was activated for 6 or 12 h. This microarray analysis of AHR activation identified 108 distinct gene probes that were significantly modulated in the TN1–4 thymocyte progenitor stages. Although most of the genes identified have specific AHR recognition sequences, only seven genes were altered exclusively in the two T-cell committed stages of early thymocyte development (TN3 and TN4) in which the decline of S-phase cells is seen. Moreover, all seven of these genes were reduced in expression, and five of the seven are associated with cell cycle regulatory processes. These seven genes are novel targets for modulation by the TCDD-activated AHR and may be involved in the observed cell-cycle arrest and suppression of early thymocyte development.

Deficiency in Aryl Hydrocarbon Receptor (AHR) Expression throughout Aging Alters Gene Expression Profiles in Murine Long-Term Hematopoietic Stem Cells.

Dysregulation of hematopoietic stem cell (HSC) signaling can contribute to the development of diseases of the blood system. Lack of aryl hydrocarbon receptor (AhR) has been associated with alterations in gene expression related to HSC function and the subsequent development of a myeloproliferative disorder in aging female mice. We sorted the most primitive population of HSCs with the highest stem cell potential (Long-term, or LT-HSCs) from 18-month-old AhR-null-allele (AhR-KO) and WT mice and analyzed gene expression using microarray to determine alterations in gene expression and cell signaling networks in HSCs that could potentially contribute to the aging phenotype of AhR-KO mice. Comparisons with previous array data from 8-week old mice indicated that aging alone is sufficient to alter gene expression. In addition, a significant number of gene expression differences were observed in aged LT-HSCs that are dependent on both aging and lack of AhR. Pathway analysis of these genes revealed networks related to hematopoietic stem cell activity or function. qPCR was used to confirm the differential expression of a subset of these genes, focusing on genes that may represent novel AhR targets due to the presence of a putative AhR binding site in their upstream regulatory region. We verified differential expression of PDGF-D, Smo, Wdfy1, Zbtb37 and Zfp382. Pathway analysis of this subset of genes revealed overlap between cellular functions of the novel AhR targets and AhR itself. Lentiviral-mediated knockdown of AhR in lineage-negative hematopoietic cells was sufficient to induce changes in all five of the candidate AhR targets identified. Taken together, these data suggest a role for AhR in HSC functional regulation, and identify novel HSC AhR target genes that may contribute to the phenotypes observed in AhR-KO mice.