These studies demonstrate that TCDD causes an increase in the number of phenotypically defined stem cells in mouse BM. That this persists for as long as 35 days following a single administration (29
) is probably due to the long half-life of TCDD (43
). The increase in the number of these cells is due, at least in part, to their increased proliferation. This is supported by increased BrdU incorporation () and a decreased percentage of LSK cells in G0
following TCDD exposure (31
). In addition, the analyses of BM reconstitution, homing and effects on committed lineage populations strongly suggest that stem cell functions are altered by AhR activation.
Our studies are consistent with those published previously (30
), demonstrating that TCDD substantially inhibits HSC function determined by the ability of these cells to repopulate BM in irradiated animals. Our data further suggest that this is primarily due to the inability of these cells to home to BM. It is becoming appreciated that microenvironment-dependent signaling is critical for maintaining the balance between stem cell quiescence, division, differentiation and trafficking within the BM or to other environments for further differentiation and maturation. AhR agonists are known to alter the expression of cell surface proteins and adhesion molecules including those in the cadherin and integrin family (44
), as well as β-catenin (45
). An attractive hypothesis is that AhR activation in HSCs alters the expression of these or other molecules, resulting in impaired signaling from soluble factors or stromal cells with the marrow niche resulting in decreased homing ability. A recent publication reported that TCDD downregulates the expression of mRNAs for the G-protein-coupled receptor, CXCR4, and its chemokine ligand CXCL12 (SDF-1) in MCF-7 breast cancer cells (46
). These molecules are particularly important for HSC homing to and movement within the marrow microenvironment. Early lymphopoiesis is also believed to occur in specific marrow niches controlled by factors within the microenvironment (47
). Altered expression of niche-sensing molecules in progenitors may be responsible for the observed TCDD-induced loss of terminal deoxynucleotidyl transferase- and recombinase-activating gene-positive cells in marrow (25
), loss of developing B cells (23
) (), decreased CFU-preB (), and decreased thymic seeding (25
). Lacking sufficient signals for differentiation toward the lymphoid lineage, increased numbers of progenitors might then proceed along a default myeloid lineage pathway as compared with what might occur under normal ‘resting’ conditions (48
) and as we observed in response to TCDD (). Likewise, a TCDD-induced increase number and proliferation of LSK precursor cells may occur due to altered niche binding and loss of quiescence (31
). Notably, the CXCR4/CXCL12-signaling pathway is also important for the maintenance of the quiescent HSC pool (50
). Thus, our data and previous work are consistent with the postulate that AhR dysregulation by TCDD results in the altered ability of HSCs to sense appropriate signals in the BM, leading to altered cycling, trafficking and differentiation potential. Furthermore, this suggests that the AhR may regulate critical genes within HSCs that allow them to differentially respond to signals in their environment. Clearly, however, more work is needed to identify these genes and signaling pathways and demonstrate their relationship with HSC functions.
The data presented here add to previous data consistent with the notion that TCDD alters the function of HSCs through the ability to affect AhR activity within these cells. The AhR is present in phenotypically defined progenitors (). As determined by the use of chimeric mice, AhR presence in hematopoietic cells, but not stromal cells, is essential for the increase in HSC number elicited by TCDD (27
) as well as altered stem cell reconstitution activity (30
). In addition, although treatment of mice with TCDD affects developing B cell numbers ( and ), committed B-lineage cells are not affected by direct exposure (24
), suggesting a primary effect on more immature precursors. TCDD treatment alters gene expression in hematopoietic precursors (31
). TCDD also directly affects the growth of HPP-CFCs (), but not more mature lineage-restricted progenitors, under conditions in vitro
. Finally, the finding that no effects of TCDD were observed when stem cell populations are stimulated to proliferate following treatment of animals with 5-FU (supplementary Figure 3
is available at Carcinogenesis
Online) is also consistent with the argument that, under resting conditions, i.e. without 5-FU treatment, TCDD acts directly on the AhR within HSCs. The Ahr
is among those genes turned off during the proliferation phase of HSCs following treatment with 5-FU but is expressed during periods of quiescence (42
). Our data using growth factors to stimulate HSC proliferation () are consistent with this.
expression in HSCs may be regulated under conditions of quiescence and proliferation has several implications. First, HSC susceptibility to xenobiotic AhR ligands probably depends on the marrow environment. Thus, conditions in which HSCs are stimulated to proliferate may render these cells less susceptible to toxic AhR ligands, similar to what we observed following 5-FU treatment. This may also be a protective mechanism against chemicals that can be metabolized to mutagenic intermediates by the cytochrome P450 isozymes regulated in part by the AhR. If the AhR is downregulated by increased cycling and proliferation of HSCs, then, since under resting conditions some small percentage of HSCs are normally cycling, there may be a subpopulation of HSCs that are less susceptible to TCDD. This might explain our finding that although TCDD substantially inhibits the ability of cells to repopulate the BM in irradiated animals, a percentage of HSCs that did home to BM appeared to differentiate normally (). These data are also consistent with a number of studies indicating that the AhR has a normal functional role in cell cycle (13
). More specifically for HSCs, the AhR may be important for regulating the balance between quiescence and proliferation by acting as a negative regulator of hematopoiesis with a function of curbing excessive or unnecessary proliferation. That the Ahr
gene is downregulated during the proliferation phase of HSCs following 5-FU treatment (42
) is consistent with this if buffering AhR activity is necessary for proliferation of normally functioning stem cells to proceed. This concept is supported by additional data we obtained, indicating that LSK cells from AhR-KO mice are hyperproliferative with a significantly greater percentage in G1
/S compared with that from wild-type animals (R.W. Garrett and T.A. Gasiewicz, unpublished observations). In agreement with this, a recent publication indicated that the Ahr
gene promoter is hypermethylated in human acute lymphoblastic leukemia cells (52
); the authors postulated that the Ahr
gene is silenced by hypermethylation and that the AhR could be a cell-specific negative regulator of cell proliferation.
The data presented here are consistent with the ability of TCDD via the AhR to affect the functions of immature hematopoietic lineage precursor cells. Although the BM reconstitution studies suggest effects on both short- and long-term HSCs, the data do not allow us to distinguish whether the effects of TCDD are greater on one versus the other or whether the effects are exclusively on these populations versus multipotent progenitor populations as well. This is a reflection of the highly dynamic nature of these populations and the likelihood that our understanding of these, based on the expression of cell surface markers, is very much oversimplified and not representative of their actual functions (53
). Furthermore, although, as indicated above, all the available data are consistent with the effect of TCDD on these populations being subsequent to modulation of AhR activity in these cells, we cannot completely rule out the possibility that these actions occur, qualitatively or quantitatively, in conjunction with AhR activation in other cell types, i.e. stromal cells, that are important for regulating functions of HSCs. Nevertheless, it is reasonable to hypothesize that the ability of TCDD to affect HSC functions may be linked to reports of increased incidence of leukemia and lymphoma in populations accidentally exposed to TCDD (2
). A corollary to the postulate that the AhR has normal role as a negative regulator of HSCs is that dysregulation of AhR expression and/or activity may be associated with the etiology and/or progression of certain hematopoietic diseases including cancers. It has been suggested that modulated AhR activity provides a permissive environment for the selection and expansion of tumor cell clones in human lymphoblastic leukemia (52
). Clearly, more work is needed to define these relationships and the mechanisms by which they occur.
In summary, based on the cumulative data presented here and elsewhere (19
), we favor the hypothesis that TCDD, through AhR activation and modulation of critical genes within HSCs, alters the ability of these cells to respond to signals in their microenvironment. This results in altered HSC numbers and function. Additional investigations are needed to determine the possible link between the actions of TCDD on HSCs and the many functional effects elicited by this chemical on the immune system. It is not without precedent to further propose that other tissue stem cell populations may be similarly sensitive to the actions of xenobiotic AhR ligands. Recently, it was suggested that TCDD-induced activation of skin stem cells and a shift in differentiation commitment of their progeny may represent a primary mechanism for the ability of this chemical to produce chloracne in both humans and animals (54
). TCDD has also been shown to disrupt the maturation of granule neuron precursor cells (55
). Finally, these and previous data are also consistent with the hypothesis that this bHLH-PAS protein may have a physiological role in regulating quiescence and proliferation of hematopoietic precursors and that dysregulation of this role may contribute to the etiology of certain hematopoietic cancers.