![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Environmental Medicine, University of Rochester, School of Medicine and Dentistry, Rochester, New York
Received November 21, 2005; accepted March 23, 2006
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
and principal components of circadian rhythm regulators, Clock and BMAL1. In addition to being a highly conserved, developmentally regulated, and ligand-activated transcription factor (Hahn, 2002
; Williamson et al., 2005), the AhR is also the protein mediator for the action of several organic toxicants, typified by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Altered immunohematopoietic parameters, including both humoral and cellular, are among the most consistently observed toxicities after activation of the AhR by xenobiotic agonists such as TCDD. A hallmark of AhR-mediated toxicity is a persistent atrophy/aplasia of the thymus. Although this may be due partly to effects on thymocyte proliferation (Laiosa et al., 2003), other data suggest that other upstream cellular targets exist that may contribute to this effect. In particular, the sensitivity of Lin- Sca-1+ cKit+ (LSK) bone marrow cells, a population highly enriched for multipotent hematopoietic stem cells (HSC), to TCDD was demonstrated (Staples et al., 1998; Murante and Gasiewicz, 2000; Sakai et al., 2003). This is the most immature population of hematopoietic cells observed to be altered by TCDD treatment. Although predominantly quiescent, HSC are characterized by a robust proliferative capacity and the ability for self-renewal. In addition, these cells are defined by the ability to differentiate into all of the blood lineages by first committing to hematopoietic progenitor cells (HPC) that are themselves committed to either the lymphoid or myeloid lineages, common lymphoid progenitors or common myeloid progenitors (CMP), respectively. Whereas common lymphoid progenitors further differentiate into B and T cells or natural killer cells (Kondo et al., 1997), CMPs differentiate to form granulocyte macrophage progenitors (GMP) and megakaryocyte erythrocyte progenitors (MEP) (Akashi et al., 2000).
A single dose of TCDD has been shown to increase the number of marrow LSK cells, although their ability to reconstitute an irradiated mouse is impaired (Murante and Gasiewicz, 2000; Sakai et al., 2003). Furthermore, the use of irradiation chimeras demonstrated that the presence of the AhR in the hematopoietic cells, and not supportive stroma, was essential for both the TCDD-elicited increase in LSK cells and their functional impairment (Staples et al., 1998; Sakai et al., 2003). This is particularly important because of the tightly regulated and critical interactions between hematopoietic precursors and the nonhematopoietic stroma. These findings, along with measurable levels of AhR mRNA and protein in these cells (van Grevenynghe et al., 2005), indicate that the AhR may have some normal function in HSC. In addition, human HSCs are AhR-dependent targets for carcinogenic polycyclic aromatic hydrocarbons (van Grevenynghe et al., 2005).
Many members of the PAS family, including the two murine Period homologues mPer1 and mPer2, are involved in controlling circadian rhythms (Zheng et al., 2001). These rhythms occur with an approximate 24-h period and are a temporal compartmentalization that governs much of vertebrate physiology. All aspects of hematopoiesis, including proliferation and differentiation, are controlled by circadian rhythms. For example, the number of human CD34+ bone marrow cells, as well as those in S-phase, has been shown to be rhythmic (Abrahamsen et al., 1998). Rhythmicity in numbers of erythroid progenitors, burst-forming units (BFU-E) and colony-forming units (CFU-E), and myeloid progenitors, colony-forming unit granulocyte erythrocyte macrophage megakaryocyte (CFU-GEMM) and CFU-granulocyte macrophage (CFU-GM), have been identified (Aardal and Laerum, 1983; Stevold et al., 1988; Smaaland et al., 1992; Wood et al., 1998). Although it is likely that at least part of their rhythmicity is regulated by circadian-controlled circulating factors, these cells express all of the machinery required for an intrinsic circadian clock and maintain rhythmicity in vitro (Bourin et al., 2002; Tsinkalovsky et al., 2005).
The AhR itself has been linked to circadian physiology. Both mRNA and protein levels of AhR in rat liver oscillates in a circadian manner (Richardson et al., 1998; Huang et al., 2002). It is not surprising that the level of CYP1A1 mRNA, one of the AhR-regulated genes, has a rhythm that is antiphase to the AhR. A single exposure to TCDD also disrupted circadian-controlled rest/activity rhythms in mice, as well as the rhythms associated with the expression of circadian genes Per1 and BMAL1 in the hypothalamus (Miller et al., 1999). The purpose of these experiments was to test the hypothesis that dysregulation of the AhR signaling pathway resulting from exposure to a persistent AhR agonist TCDD could disrupt the circadian rhythms of antigenically and functionally defined hematopoietic precursors.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
TCDD Exposure. Mice were gavaged with 10 µg/kg TCDD in 5 µl of olive oil per 1 g of mouse weight, or oil alone 5 days before the first time-point. In all studies, time points consisted of ZT 6, 10, 14, 18, 22, and 2. The 5-day time point was predetermined to be optimal in generating an effect on the percentage of quiescent Lin- Sca-1+ cells in the BM; this effect was maximal on days 3 through 7 after exposure (data not shown).
Bone Marrow Cell Isolation. Bone marrow cells were flushed from both femurs and tibiae with PBS + 2% FBS + 1% penicillin/streptomycin as described previously (Wyman et al., 2002; Laiosa et al., 2003). The bone marrow cells were placed in to a single-cell suspension before passing through an 80-µm nylon mesh (TETKO Inc., Briarcliff Manor, NY). Cell pellets were treated with erythrocyte lysis buffer (0.17 M NH4Cl, 10 mM KHCO3, and 1 mM EDTA) and the remaining cells were washed and counted.
Immunophenotyping. BM cells (106) from vehicle- and TCDD-treated mice were blocked with anti-FcR antibody in stain buffer (PBS + 1% FBS + 0.1% NaN3) for 20 min on ice and then incubated for 30 min with predetermined saturating concentrations of the following fluorescently conjugated antibodies: fluorescein isothiocyanate-conjugated anti-CD34, Thy-1, Sca-1, Gr-1, and cKit; phycoerythrin-conjugated anti-Sca-1 and FcR; allophycocyanin-conjugated anti-c-Kit; Biotin-conjugated anti-Mac-1, Gr-1, CD8a, CD3
, Ter119, B220, Sca-1, and IL-7R (BD Biosciences, San Jose, CA). Specific cell populations were detected using the following surface phenotypes as previously published by others (Kondo et al., 1997; Akashi et al., 2000): HSC (Lin- Thy-1+ Sca-1hi c-Kit+), CMP (Lin- CD34+ FcRlo IL7R- Sca-1- c-Kit+), GMP (Lin- CD34+ FcRhi IL7R- Sca-1- c- Kit+), and MEP (Lin- CD34- FcRlo IL7R- Sca-1- c-Kit+). In all cases, the lineage cocktail included antibodies to Mac-1, Gr-1, CD8a, CD3, Ter119, and B220. Streptavidin-PerCP was used as a secondary fluorescent agent. Cells were rinsed with staining buffer and fixed with 0.1% paraformaldehyde before running on the flow cytometer. Compared with our previous studies (Staples et al., 1998; Murante and Gasiewicz, 2000), the use of the Thy-1 marker allows further definition of the LSK population to include those cells that are phenotypically and functionally defined as long-term and short-term HSC. Cells were run (at least 50,000 events per sample) on a FACSCalibur flow cytometer (BD Biosciences), and data were analyzed with CellQuest software (BD Biosciences). Gates were set similarly as described previously (Kondo et al., 1997; Staples et al., 1998; Akashi et al., 2000; Murante and Gasiewicz, 2000), and data were taken as percentages of cells in the viable gates.
Cell Isolation for RNA Analysis. Total BM was harvested from both legs (femur + tibia) and blocked with anti-FcR in stain buffer. The cells were then stained with biotinylated anti-lineage (Mac-1, Gr-1, B220, CD8, CD3, Ter119) followed by streptavidin-microbeads (Miltenyi Biotec, Auburn, CA). Lineage depletion was performed using an AutoMACS magnetic cell sorter (Miltenyi Biotec). For certain experiments, Lin-Sca-1+ and Lin-Sca-1- cells were collected by staining the Lin- fraction with anti-Sca-1 microbeads (Miltenyi Biotec). Collection of Sca-1+ and Sca-1- cells were performed using the sensitive positive sort function of the AutoMACS. Purity of fractions was assessed using flow cytometric analysis and determined to be >95%.
Colony-Forming Semisolid Assays. To measure high proliferative potential colony-forming cells (HPP-CFC), 2 x 104 BM cells were suspended in 1 ml of IMDM supplemented with 1% methylcellulose, 25% fetal bovine serum, 1% bovine serum albumin, 100 µM 2-mercaptoethanol, and 2 mM L-glutamine (Stem Cell Technologies, Vancouver, BC, Canada). To the media was added 250 ng/ml CSF-1, 50 ng/ml IL-3, 50 ng/ml IL-1, and 50 ng/ml SCF (R&D System, Minneapolis, MN). CFU-GEMM and CFU-GM were measured using methylcellulose based media containing SCF, IL-3, IL-6, and erythropoietin or SCF, IL-3, and IL-6, respectively (Stem Cell Technologies). All plates were incubated at 37°C in 5% CO2 and scored 14 days later. HPP-CFC were scored by the presence of macroscopic colonies (>0.5 mm in diameter). CFU-GEMM and CFU-GM were scored as colonies composed of granulocytes, erythrocytes, and monocytes or granulocytes and monocytes, respectively.
Cell Cycle Analysis. One million Lin- BM cells were stained with fluorescein isothiocyanate-conjugated anti-cKit and AF647-conjugated anti-Sca-1. Afterward, cells were stained with 7-aminoactinomycin D (Invitrogen, Carlsbad, CA) at room temperature for 20 min followed by Pyronin Y (PY) on ice in nucleic acid staining solution (0.1 M phosphate-citrate buffer + 0.15 M NaCl + 5 mM sodium EDTA + 0.5% bovine serum albumin + 0.02% saponin) for 10 min. Cells were analyzed using a FACSCalibur flow cytometer and data analyses were performed using CellQuest software.
Quantitative RT-PCR. Total RNA was isolated from Lin- Sca-1+ and Lin- Sca-1- BM cells at the indicated time points 5 days after in vivo exposure to TCDD using the RNEasy Mini kit (QIA-GEN, Valencia, CA) as instructed. RNA amplifications were performed using WTA (NuGEN Technologies, San Carlos, CA). cDNA was created using the Superscript First Strand Synthesis System (Invitrogen) as instructed and PCR was performed using a 7900 HT (Applied Biosystems, Foster City, CA). TaqMan gene expression assays (Applied Biosystems) were used to measure the following targets: AhR, Arnt, AhRR, mPer1, mPer2, p21, p27, and GAPDH. Data analyses were performed using the comparative Ct method.
Statistical Analysis. A two-way ANOVA (Excel) was performed on each set of 24 h data to determine treatment-dependent differences. When the ANOVA indicated differences between treatment groups, the Student's t test was used to determine differences between control and experimental groups at designated time points. Values of p 
0.05 were considered statistically significant.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
). With this in mind and because prolonged activation of the AhR by TCDD increases the number of murine LSK BM cells (Murante and Gasiewicz, 2000; Sakai et al., 2003), we measured the effect of AhR activation on the circadian rhythms associated with these cells. Five-week old female C57BL/6 mice received by gavage a single 10 µg/kg dose of TCDD. Five days later, bone marrow was collected every 4 h for 24 h and stained with fluorescently labeled antibodies. As indicated in Fig. 1, Thy-1lo LSK cells, a population that contains all of the murine HSC, are characterized by a near 3-fold difference between the minimum value and the maximum value in vehicle-treated mice. After treatment with TCDD, there is an overall increase in the number of Thy-1lo LSK cells (Fig. 1) throughout the 24-h period (ANOVA p < 0.001). There were no statistical differences observed in total bone marrow cellularity at any time point (data not shown). A comparable TCDD-induced increase in this population of progenitor cells was observed in similar experiments carried out over the course of approximately 1 year. These studies were carried out because of known seasonal variations in these diurnal patterns. Occasionally, these experiments conducted at different seasons also suggested abnormal period and/or phase shifts after exposure to TCDD (data not shown).
|
; Stevold et al., 1988; Smaaland et al., 1992; Wood et al., 1998), these studies were extended to examine effects on these populations. CMP, GMP, and MEP were all characterized by apparent rhythms in control animals (Fig. 2). Five days after TCDD exposure, the rhythms of all three populations were altered (ANOVA p < 0.001), demonstrating changes in phase and magnitude that are specific to both time of day and cell type (Fig. 2). In addition to measuring antigenically defined hematopoietic precursors, we performed methylcellulose-based ex vivo assays to determine effects on functional capacity by measuring HPP-CFC, CFU-GEMM, and CFU-GM. As seen in those antigenically defined cells, these functionally defined populations were all characterized by diurnal rhythms (Fig. 3). Although time-of-day-specific changes may have occurred after TCDD treatment, there were no overall significant treatment-dependent differences when the 24-h data sets for these colony assays were compared. Together, these data demonstrate that a single oral dose of TCDD is sufficient to cause a modification in the circadian rhythms of murine hematopoietic precursors, as well as overall changes in their numbers. These changes can be both positive and negative, with time points during a daily cycle that are absent of differences between treatment groups.
|
|
). As indicated in Fig. 4, the number of G0 LSK cells in control mice exhibits a pattern suggesting a biphasic effect, with two periods of diminished quiescence occurring at the beginning of both perceived day and perceived night. After TCDD treatment, the daily cycle of quiescent cells was disrupted (ANOVA p < 0.001). The most obvious difference is at the period of late perceived night when the greatest percentage of the control LSK cells is quiescent. Here, more LSK cells in TCDD-treated mice exit quiescence at ZT 22 such that the ratio of quiescent cells to actively cycling cells is at its lowest of anywhere on the two curves. Comparing the data specifically between ZT 18 and ZT 2 also suggests the possibility that the period of the rhythm has been compressed. Together, these data suggest that one possible mechanism by which TCDD-induced AhR activation alters the numbers and rhythms of primitive hematopoietic progenitors is the disruption of their movement in and out of quiescence. These data are also consistent with other studies from our lab showing increased BrdU incorporation into LSK marrow cells after TCDD treatment (A. Wyman, K. P. Singh, R. W. Garrett, and T. A. Gasiewicz, manuscript submitted).
|
TCDD Disrupts the Circadian Rhythm of Lin- Sca-1+ Gene Expression. Circadian rhythms are controlled by tightly regulated molecular clocks, at the heart of which are several PAS transcription factors, including the murine Period homologues mPer1 and mPer2, that are structurally similar to the AhR and its dimerization partner Arnt (Zheng et al., 2001). These proteins act as important positive regulators of the circadian clock. A second putative AhR DNA response element has been identified upstream of several genes including mPer2 (Boutros et al., 2004). We therefore sought to determine whether exposure to TCDD could disrupt the expression of these genes in Lin- Sca-1+ bone marrow cells. The data presented in Fig. 5 agree with those from previous studies demonstrating that mPer1 and mPer2 are expressed in murine marrow Lin- Sca-1+ cells and that this expression is itself circadian in manner (Chen et al., 2000; Tsinkalovsky et al., 2005). Both mPer1 and mPer2 are characterized by rhythms that have maximal expression near ZT 14 and ZT 23, respectively. These patterns were disrupted by TCDD treatment (ANOVA p < 0.05) with peaks being truncated by nearly 50%. The cyclin-dependent kinase inhibitors p21 and p27 also have been demonstrated to be both TCDD-responsive and regulated with circadian rhythmicity (Enan et al., 1998; Bjarnason et al., 1999; Kolluri et al., 1999; Cheng et al., 2000). However, analysis of their mRNA expression over 24 h revealed only modest rhythmicity in Lin- Sca-1+ cells and no changes with TCDD exposure (data not shown). Together, these data support previous findings that hematopoietic precursors express circadian-related genes. These data also indicate that this expression is susceptible to dysregulation by TCDD.
|
Finally, it has been demonstrated by others that both AhR mRNA and protein expression are circadian regulated in several other tissues including rat liver (Richardson et al., 1998; Huang et al., 2002). We sought to determine whether the AhR and Arnt are expressed with circadian rhythmicity in immature hematopoietic cells. Total RNA was isolated from Lin- Sca-1+ cells collected over a 24-h period, and qRT-PCR was used to measure AhR and Arnt mRNA. Both have maximal expression occurring during subjective midday (near ZT 10) with minimal expression occurring during the hours surrounding the transition from dark to light (Fig. 6). These curves were not significantly modified after TCDD exposure (data not shown). The AhR repressor (AhRR) is a recently characterized TCDD-inducible member of the AhR signaling pathway that competes with the AhR for binding to both AhRE and Arnt and acts as a negative regulator of AhR-elicited transcriptional activity (Baba et al., 2001). TCDD exposure up-regulated AhRR in murine Lin- Sca-1+ BM cells 20-fold in a time-of-day-dependent manner. In addition, although the pattern of AhRR mRNA expression is characterized by a single, but very modest, maximal time of expression near ZT 10 in control animals, TCDD-treated animals have an additional peak of high expression occurring approximately 12 h later (Fig. 7). Together, these data demonstrate that the members of the AhR signaling pathway are expressed in a circadian manner in murine hematopoietic precursors and that the expression of the AhRR is susceptible to TCDD-induced dysregulation.
|
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
; Stevold et al., 1988; Smaaland et al., 1992; Abrahamsen et al., 1998; Wood et al., 1998). As such, the number of murine hematopoietic progenitors can change as much as 2- to 3-fold during the course of a single day. Although these rhythms demonstrate a change in a specific antigenically or functionally defined cell type, it is important to understand that this probably does not represent changes in the number of actual cells. Instead, these daily cycles are likely to represent changes in the expression of HSC- and HPC-specific proteins, as well as the functional potential of those cells, two events that are not mutually exclusive. For example, rhythmic expression of cKit, the receptor for SCF, would be observed as oscillating numbers of Lin- Sca-1+ cKit+ cells. This daily plasticity for the complement of proteins expressed on the surface of HSC and HPC would in turn create a time-dependent in vivo functional potential. This would also affect their ability to respond to in vitro assay conditions that include SCF, creating measurable rhythms in the numbers of SCF-dependent colony-forming cells. Diurnal rhythms have been demonstrated for other growth factor receptors, such as the surface expression of interferon-/
receptor on hepatocytes, lymphocytes, and implanted melanoma cells (Takane et al., 2001, 2002). The maximal expression of interferon-/ receptor on tumor cells corresponded with an increase in the percentage of those cells in S-phase, indicating a relationship between cell cycle transit and specific growth factor signaling (Takane et al., 2001). The circulating levels of soluble receptors for tumor necrosis factor, IL-6, and IL-2 are all rhythmic (Haack et al., 2004). The responsiveness of murine myeloid progenitors to IL-3, GM-CSF, and G-CSF was also found to be circadian-dependent (Perpoint et al., 1995).
We have presented data that for the first time indicate circadian rhythms in the numbers of specific antigenically defined populations of murine hematopoietic progenitor cells. Thy-1lo LSK cells, a population of cells enriched for murine HSC, as well as three populations of Lin- cKit+ myeloid precursors, CMP, GMP, and MEP, all seemed to have diurnal rhythms in vehicle-treated C57BL/6 mice (Figs. 1 and 2). Data for MEP and GMP suggest rhythms that were antiphase to each other, supporting data that differentiation into the different lineages is separated in time (Wood et al., 1998). In theory, a measurable plasticity may exist between erythroid and myeloid progenitors, with a single cell changing surface levels of proteins such as FcR and CD34 so that it meets the criteria for both GMP and MEP at different times of the day. The antiphase nature of the curves for those two populations supports this notion (Fig. 2). This kind of temporal compartmentalization provides for unique periods of specific timing and duration when cellular and extracellular factors required for a specific differentiation program are likely to be up-regulated. Properly timed circadian rhythms are hypothesized to be energetically advantageous to the organism, allowing production of signaling molecules and receptors only when they are required. Five days after TCDD exposure there seemed to be a shift in the timing of the GMP and MEP rhythms; although those two specific curves remained antiphase to one another, adding support to our hypothesis that these are indeed the same cells. Although the absolute minimum numbers of these two populations do not change after TCDD treatment, the maximal values increase roughly 2-fold (Fig. 2). This increase in the number of myeloid-erythroid progenitors does not necessarily precede increased hematopoietic potential. It is likely that in control bone marrow, the expression of progenitor-specific markers is timed to correspond with extracellular signals required for further differentiation. TCDD may decouple these two sets of events, and depending on whether those signals are positive or negative regulators, the increased numbers of GMP and MEP can be characterized by more, less, or unchanged function. It is therefore critical that future experiments investigate the direct consequences of shifted circadian rhythms to the outcome of hematopoietic progenitors (i.e., their ability to differentiate into more committed lineage cells). In this regard, it is noteworthy that other data suggest a TCDD-elicited shift toward the granulocyte/macrophage lineage at the expense of lymphoid and erythroid lineages in murine bone marrow (A. Wyman, K. P. Singh, R. W. Garrett, and T. A. Gasiewicz, manuscript submitted). This is consistent with published data showing a decrease in the production of immature B and T cells in marrow after TCDD exposure (Fine et al., 1990; Thurmond and Gasiewicz, 2000), but increased numbers of Lin-/Sca-1+/cKit+ stem/progenitor cells as indicated in Fig. 1. At present, whether or how modulated hematopoietic rhythms may be related to these outcomes remains unknown.
In addition to numbers of specific cells, the apparent function and proliferation of HSC and HPC is also temporally dependent. The cell cycle of both primitive hematopoietic progenitors and other cell types progresses with a 24-h period and is linked to the coexpression of circadian-associated genes. It is noteworthy that the ability of transplanted murine bone marrow to engraft is influenced by circadian time (D'Hondt et al., 2004). Thus, cell cycling and functional potential are probably linked. Consistent with this, investigators have shown that HSC isolated during the active phases of the cell cycle are less able to home to bone marrow and spleen (Jetmore et al., 2002). It is therefore not surprising that the rhythm of S-phase HSC and HSC engraftment are antiphase to each other (D'Hondt et al., 2004) and that the number of CFU-spleen are measured with a circadian period (Stevold and Laerum, 1988). We have shown a TCDD-elicited disruption in the quiescence of LSK cells (Fig. 4). These data are important for several reasons. First, the regulation of HSC division is tightly regulated, with the majority (70-85%) of cells quiescent under normal `resting' conditions; a phenomenon that is critical for the maintenance of hematopoietic potential throughout the lifespan of the organism. Second, this alteration in the functional composition of the bone marrow may explain reported data indicating that TCDD, although increasing their number, diminished the ability of murine LSK/CD34- cells (functionally defined as long-term reconstituting HSCs) to reconstitute an irradiated recipient mouse (Sakai et al., 2003). Again, this result may not be surprising if TCDD treatment increases the cycling of HSCs and actively cycling cells are less able to home to bone marrow (Jetmore et al., 2002).
The proteins mPer1 and mPer2 are important positive arms of the circadian clock. Targeted mutation of their genes results in abnormal circadian rhythms (Zheng et al., 2001). However, because the expression of mPer2 normally increases and decreases daily, measuring the effect of TCDD at just one time point can lead to a misinterpretation of the data. When mPer1 and BMAL1 mRNA expression were monitored in the hypothalamus from TCDD-treated mice at multiple time points over 24 h, it was found that the overall rhythm was phase-shifted (Miller et al., 1999). Rather than a change in the phase of their rhythms, we observed changes in the magnitude of both mPer1 and mPer2 mRNA in Lin- Sca-1+ BM cells exposed in vivo to TCDD (Fig. 5). In particular, their respective maximal expression were reduced by roughly 50%. Although it is unclear what effect this reduction would have on these cells, it is plausible that this transient modulation in mPer mRNA is significant enough to disrupt normal cellular function. In addition, although TCDD has been identified as a tumor promoter, no current data provide a convincing mechanism by which this might happen. It is noteworthy that mPer mutant mice are more susceptible to 
-irradiation induced tumorigenesis (Fu et al., 2002). The incidence of lymphoma and tumors in several tissues was nearly 15-fold higher in mPer2 mutant animals, leading to early mortality. In addition, a chromosomal translocation fusing ETV6 with the hPer1 gene is associated with acute myeloid leukemia (Penas et al., 2003). It is noteworthy that TCDD exposure in humans has been associated with increased incidence of lymphoma and leukemia (IARC, 1997). It would be important to investigate further the specific effects of AhR-mediated dysregulation of mPer on murine hematopoietic progenitors.
Although we and others have referred to the action of TCDD on the AhR as activation, evidence suggests this may not always be functionally the case. When many types of cells are exposed to TCDD, both in vitro and in vivo, there is a rapid and sustained proteosome-mediated degradation of AhR protein (reviewed in Pollenz, 2002). In many cases, a single dose of TCDD is sufficient to reduce AhR protein in vivo to 10 to 30% of control animals in multiple tissues for up to 14 days (Pollenz et al., 1998). However, despite this decrease in the level of AhR, CYP1A1 and/or CYP1B1 increase with TCDD exposure, indicating some continued AhR function or the presence of `spare' AhR protein. Thus, TCDD may cause a rapid and sustained reduction in AhR protein while directing the remaining active AhR to particular TCDD-inducible genes, including the AhRR (Baba et al., 2001), adding an additional level of negative regulation. The possible nullification of endogenous AhR activity in HSC/HPC by TCDD is important when considering the following points. First, a putative second AhRE has been identified upstream of the mPer2 gene (Boutros et al., 2004). Second, we observed a time-of-day-dependent elevated number of LSK cells in AhR-null allele mice (R. W. Garrett and T. A. Gasiewicz, unpublished observations) of a magnitude similar to the increase after TCDD exposure. Third, we observed that TCDD treatment substantially induces AhRR in Lin- Sca-1+ marrow cells (Fig. 7). Thus, a plausible and testable explanation for some of the present data are that TCDD is inducing, either through the ability to elicit down-regulation of AhR protein and/or increased AhRR expression, an AhR-null-like state for signaling pathways regulating HSC/HPC function. Future experiments will investigate the mechanisms by which TCDD might affect distinct AhR functions in murine LSK cells.
In summary, this article describes a novel effect of AhR activation on immature murine hematopoietic progenitors. Significant changes in the circadian rhythms associated with both the number of antigenically and functionally defined cell populations, as well as their movement into and out of quiescence, were detected. These disruptions include changes in phase, period, and magnitude and may be related in part to altered expression of mPer1 and mPer2. Experiments are planned to delineate those mechanisms responsible for these changes, including establishing the relationship between mPer expression and signaling through the AhR. In addition, experiments will be designed to gain an understanding of the consequences of disrupted circadian rhythms on both normal and leukemic hematopoiesis. Finally, these data emphasize the need to evaluate daily cycles when determining effects of AhR agonists, therapeutic agents or other chemicals, on bone marrow functions.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS: AhR, aryl hydrocarbon receptor; AhRR, aryl hydrocarbon receptor repressor; Arnt, aryl hydrocarbon receptor nuclear translocator; PAS, PER/ARNT/SIM (periodicity/Arnt/simple-minded); TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; LSK, Lin- Sca-1+ cKit+; HSC, hematopoietic stem cells; HPC, hematopoietic progenitor cells; CMP, common myeloid progenitor; GMP, granulocyte macrophage progenitors; MEP, megakaryocyte erythrocyte progenitors; ZT, Zeitgeber time; HPP-CFC, high proliferative potential colony-forming cells; IL, interleukin; CSF, colony-stimulating factor; SCF, stem cell factor; ANOVA, analysis of variance; CFU-GEMM, colony-forming unit-granulocyte erythrocyte macrophage megakaryocyte; CFU-GM, colony-forming unit-granulocyte macrophage.
1 Current affiliation: Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania. 
Address correspondence to: Dr. Thomas A. Gasiewicz, Department of Environmental Medicine, University of Rochester Medical Center, 601 Elmwood Ave., Box EHSC, Rochester, NY 14642. E-mail: tom_gasiewicz{at}urmc.rochester.edu
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Abrahamsen JF, Smaaland R, Sothern RB, and Laerum OD (1998) Variation in cell yield and proliferative activity of positive selected human CD34+ bone marrow cells along the circadian time scale. Eur J Haematol 60: 7-15.[Medline]
Akashi K, Traver D, Miyamoto T, and Weissman IL (2000) A clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Nature (Lond) 404: 193-197.[CrossRef][Medline]
Baba T, Mimura J, Gradin K, Kuroiwa A, Watanabe T, Matsuda Y, Inazawa J, Sogawa K, and Fujii-Kuriyama Y (2001) Structure and expression of the Ah receptor repressor gene. J Biol Chem 276: 33101-33110.
Bjarnason GA, Jordon RC, and Sothern RB (1999) Circadian variation in the expression of cell-cycle proteins in human oral epithelium. Am J Pathol 154: 613-622.
Bourin P, Ledain AF, Beau J, Mille D, and Lévi F (2002) In-vitro circadian rhythm of murine bone marrow progenitor production. Chronobiol Int 19: 57-67.[CrossRef][Medline]
Boutros PC, Moffat ID, Franc MA, Tijet N, Tuomisto J, Pohjanvirta R, and Okey AB (2004) Dioxin-responsive AhRE-II gene battery: identification by phylogenetic footprinting. Biochem Biophys Res Commun 321: 707-715.[CrossRef][Medline]
Chen YG, Mantalaris A, Bourne P, Keng P, and Wu JH (2000) Expression of mPer1 and mPer2, two mammalian clock genes, in murine bone marrow. Biochem Biophys Res Commun 276: 724-728.[CrossRef][Medline]
Cheng T, Rodrigues N, Shen H, Yang Y, Dombkowski D, Sykes M, and Scadden DT (2000) Hematopoietic stem cell quiescence maintained by p21 cip1/waf1. Science (Wash DC) 287: 1804-1808.
D'Hondt L, McAuliffe C, Damon J, Reilly J, Carlson J, Dooner M, Colvin G, Lambert J, Hsieh C, Habibian H, et al. (2004) Circadian variations of bone marrow engraftability. J Cell Phyisol 200: 63-70.[CrossRef]
Enan E, El-Sabeawy F, Scott M, Overstreet J, and Lasley B (1998) Alterations in the growth factor signal transduction pathways and modulators of the cell cycle in endocervical cells from macaques exposed to TCDD. Toxicol Appl Pharmacol 151: 283-293.[CrossRef][Medline]
Fine JS, Silverstone AE, and Gasiewicz TA (1990) Impairment of prothymocyte activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin. J Immunol 144: 1169-1176.[Abstract]
Fu L, Pelicano H, Liu J, Huang P, and Lee CC (2002) The circadian gene Period2 plays an important role in tumor suppression and DNA damage response in vivo. Cell 111: 41-50.[CrossRef][Medline]
Gothot A, Pyatt R, McMahel J, Rice S, and Srour EF (1997) Functional heterogeneity of human CD34+ cells isolated in subcompartments of the G0/G1 phase of the cell cycle. Blood 90: 4384-4393.
Haack M, Pollmacher T, and Mullington JM (2004) Diurnal and sleep-wake dependent variations of soluble TNF- and IL-2 receptors in healthy volunteers. Brain Behav Immun 18: 361-367.[CrossRef][Medline]
Hahn ME (2002) Aryl hydrocarbon receptors: diversity and evolution. Chem Biol Interact 141: 131-160.[CrossRef][Medline]
Huang P, Ceccatelli S, and Rannug A (2002) A study on diurnal mRNA expression of CYP1A1, AhR, Arnt and Per2 in rat pituitary and liver. Environ Toxicol Pharmacol 11: 119-126.[CrossRef]
IARC (1997) IARC Working Group on the Evaluation of Carcinogenic Risks to Humans: polychlorinated dibenzo-para-dioxins and polychlorinated dibenzofurans. Lyon, France, 4-11 February 1997. IARC Monogr Eval Carcinog Risks Hum 69: 1-631.[Medline]
Jetmore A, Plett PA, Tong X, Wolber FM, Breese R, Abonour R, Orschell-Traycoff CM, and Srour EF (2002) Homing efficiency, cell cycle kinetics and survival of quiescent and cycling human CD34(+) cells transplanted into conditioned NOD/SCID recipients. Blood 99: 1585-1593.
Kolluri SK, Weiss C, Koff A, and Gottlicher M (1999) p27Kip1 induction and inhibition of proliferation by the intracellular Ah receptor in developing thymus and hepatoma cells. Genes Dev 13: 1742-1753.
Kondo M, Weissman IL, and Akashi K (1997) Identification of clonogenic common lymphoid progenitors in mouse bone marrow. Cell 91: 661-672.[CrossRef][Medline]
Laiosa MD, Wyman A, Murante FG, Fiore NC, Staples JE, Gasiewicz TA, and Silverstone AE (2003) Cell proliferation arrest within intrathymic lymphocyte progenitor cells causes thymic atrophy mediated by the aryl hydrocarbon receptor. J Immunol 171: 4582-4591.
Miller JD, Settachan D, Frame LT, and Dickerson RL (1999) 2,3,7,8-Tetrachlorodibenzo-p-dioxin phase advances the deer mouse (Peromyscus maniculatur) circadian rhythm by altering expression of clock proteins. Organohal Comp 42: 23-28.
Murante FG and Gasiewicz TA (2000) Hemopoietic progenitor cells are sensitive targets of 2,3,7,8-tetrachlorodibenzo-p-dioxin in C57Bl/6J mice. Toxicol Sci 54: 374-383.
Penas EM, Cools J, Algenstaedt P, Hinz K, Seeger D, Schafhausen P, Schilling G, Marynen P, Hossfeld DK, and Dierlamm J (2003) A novel cryptic translocation t(12;17)(p13;p12-p13) in a secondary acute myeloid leukemia results in a fusion of the ETV6 gene and the antisense strand of the PER1 gene. Genes Chrom Canc 37: 79-83.[CrossRef]
Perpoint B, Le Bousse-Kerdiles C, Clay D, Smadja-Joffe F, Depres-Brummer P, Laporte-Simitsidis S, Jasmin C, and Levi F (1995) In vitro chronopharmcology of recombinant mouse IL-3, mouse GM-CSF and human G-CSF on murine myeloid progenitor cells. Exp Hematol 23: 362-368.[Medline]
Pollenz RS (2002) The mechanism of Ah receptor protein down-regulation (degradation) and its impact on Ah receptor-mediated gene regulation. Chem Biol Interact 141: 41-61.[CrossRef][Medline]
Richardson VM, Santostefano MJ, and Birnbaum LS (1998) Daily cycle of bHLH-PAS proteins, Ah receptor and Arnt, in multiple tissues of female Sprague-Dawley rats. Biochem Biophys Res Commun 252: 225-231.[CrossRef][Medline]
Sakai R, Kajiume T, Inoue H, Kanno R, Miyazaki M, Ninamiya Y, and Kanno M (2003) TCDD treatment eliminates the long-term reconstitution activity of hematopoietic stem cells. Toxicol Sci 72: 84-91.
Smaaland R, Laerum O, Southern R, Stevold O, Bjerkners R, and Lote K (1992) Colony-forming units granulocyte/macrophage and DNA synthesis of human bone marrow are circadian stage dependent and show co-variation. Blood 79: 2281-2287.
Staples JE, Murante FG, Fiore NC, Gasiewicz TA, and Silverstone AE (1998) Thymic alterations induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin are strictly dependent on aryl hydrocarbon receptor activation in hemopoietic cells. J Immunol 160: 3844-3854.
Stevold O and Laerum OD (1988) Multipotent stem cell (CFU-S) numbers and circadian variations in aging mice. Eur J Haematol 41: 230-236.[Medline]
Stevold O, Laerum O, and Riise T (1988) Age-related differences and circadian and seasonal variations of myelopoietic progenitor cell (CFU-GM) numbers in mice. Eur J Haematol 40: 42-49.[Medline]
Takane H, Ohdo S, Baba R, Koyanagi S, Yukawa E, and Higuchi S (2002) Relationship between 24-hour rhythm in antiviral effect of interferon- and interferon-/ receptor expression in mice. Jpn J Pharmacol 90: 304-312.[CrossRef][Medline]
Takane H, Ohdo S, Yamada T, Koyanagi S, Yukawa E, and Higuchi S (2001) Relationship between diurnal rhythm of cell cycle and interferon receptor expression in implanted-tumor cells. Life Sci 68: 1449-1455.[CrossRef][Medline]
Thurmond TS and Gasiewicz TA (2000) A single dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin produces a time- and dose-dependent alteration in murine bone marrow B-lymphocyte maturation profile. Toxicol Sci 58: 88-95.
Tsinkalovsky O, Rosenlund B, Laerum OD, and Eiken HG (2005) Clock gene expression in purified mouse hematopoietic stem cells. Exp Hematol 33: 100-107.[CrossRef][Medline]
van Grevenynghe J, Bernard M, Langouet S, Le Berre C, Fest T, and Fardel O (2005) Human CD34-positive hematopoietic stem cells constitute targets for carcinogenic polycyclic aromatic hydrocarbons. J Pharmacol Exp Ther 314: 693-702.
Williamson MA, Gasiewicz TA, and Opanashuk LA (2005) Aryl hydrocarbon recptor expression and activity in cerebellar granule neuroblasts: implications for development and dioxin neurotoxicity. Toxicol Sci 83: 340-348.
Wood PA, Hrushesky WJ, and Klevecz R (1998) Distinct circadian time structures characterize myeloid and erythroid progenitor and multipotential cell clonogenicity as well as marrow precursor proliferation dynamics. Exp Hematol 26: 523-533.[Medline]
Wyman A, Lavin AL, Wilding GE, and Gasiewicz TA (2002) 2,3,7,8-tetrachlorodibenzo-p-dioxin does not directly alter the phenotype of maturing B cells in a murine coculture system. Toxicol Appl Pharmacol 180: 164-177.[CrossRef][Medline]
Zheng B, Albrecht U, Kaasik K, Sage M, Lu W, Vaishnav S, Li Q, Sun ZS, Eichele G, Bradley A, et al. (2001) Nonredundant roles of the mPer1 and mPer2 genes in the mammalian circadian clock. Cell 105: 683-694.[CrossRef][Medline]
This article has been cited by other articles:
|
|
 |
|
  K. P. Singh, A. Wyman, F. L. Casado, R. W. Garrett, and T. A. Gasiewicz Treatment of mice with the Ah receptor agonist and human carcinogen dioxin results in altered numbers and function of hematopoietic stem cells Carcinogenesis, January 1, 2009; 30(1): 11 - 19. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Mukai, T.-M. Lin, R. E. Peterson, P. S. Cooke, and S. A. Tischkau Behavioral Rhythmicity of Mice Lacking AhR and Attenuation of Light-Induced Phase Shift by 2,3,7,8-Tetrachlorodibenzo-p-Dioxin J Biol Rhythms, June 1, 2008; 23(3): 200 - 210. [Abstract] [PDF]
|
|
|
|
 |
|
 M. Mukai and S. A. Tischkau Effects of Tryptophan Photoproducts in the Circadian Timing System: Searching for a Physiological Role for Aryl Hydrocarbon Receptor Toxicol. Sci., January 1, 2007; 95(1): 172 - 181. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
) and TCDD-treated (
) animals (n = 6 per group per time point) at ZT 6, 10, 14, 18, 22, and 2 on day 5 after treatment. Flow cytometry was performed as described under Materials and Methods. A, CMP; B, GMP; C, MEP. The data shown are representative of three separate experiments. Data are presented as mean values ± S.E. There was a treatment-dependent effect for CMP, GMP, and MEP (all ANOVA p < 0.001), and * indicates statistically different (p < 0.05) from the vehicle control value at the same time point.
