Aryl Hydrocarbon Receptor Activation in Hematopoietic Stem/
Progenitor Cells Alters Cell Function and Pathway-Specific
Gene Modulation Reflecting Changes in Cellular Trafficking
Fanny L. Casado, Kameshwar P. Singh, and Thomas A. Gasiewicz
Department of Environmental Medicine, University of Rochester Medical Center, Rochester, New York
Received January 25, 2011; accepted July 26, 2011
The aryl hydrocarbon receptor (AhR) is a transcription factor
belonging to the Per-ARNT-Sim family of proteins. These pro-
teins sense molecules and stimuli from the cellular/tissue envi-
ronment and initiate signaling cascades to elicit appropriate
cellular responses. Recent literature reports suggest an impor-
tant 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 irra-
diated host animals. Additional studies indicated a requirement
for AhR in hematopoietic cells and not marrow microenviron-
ment 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 in-
creased 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 func-
tional 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 aryl hydrocarbon receptor (AhR) is a basic helix-loop-
helix protein belonging to the Per-ARNT-Sim superfamily of
proteins. Many of the Per-ARNT-Sim proteins sense mole-
cules and stimuli from the cellular/tissue environment and
initiate signaling cascades to elicit appropriate cellular re-
sponses. Lipophilic compounds such as halogenated and poly-
cyclic aromatic hydrocarbons, including the potent exogenous
ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Poland
and Glover 1973), bind to the cytoplasmic AhR, leading to a
sequence of conformational changes that ultimately trans-
form the AhR into its high-affinity DNA binding form in the
nucleus to function as a transcription factor (Soshilov and
Epidemiological data have correlated accidental and occu-
pational exposure to TCDD and related dioxins with in-
creased risk for certain hematological diseases such as non-
Hodgkin’s lymphoma, chronic lymphocytic leukemia, and
multiple myeloma in humans (Frumkin, 2003). In addition,
there is much data indicating that persistent activation of
AhRs results in immunosuppression in different animal mod-
els (Stevens et al., 2009). AhR-dependent thymic atrophy, a
hallmark of exposure to varying doses of TCDD, may be
elicited by different mechanisms including 1) reduced seed-
This work was funded by the National Institutes of Health National Insti-
tute of Environmental Health Sciences [Grants ES01247, ES07026, ES04862,
Article, publication date, and citation information can be found at
The online version of this article (available at http://molpharm.
aspetjournals.org) contains supplemental material.
ABBREVIATIONS: AhR, aryl hydrocarbon receptor; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; BM, bone marrow; HSC, hematopoietic stem cell;
URMC, University of Rochester Medical Center; 7AAD, 7-amino-actinomycin D; LSK, lineage-depleted, Sca-1?and c-kit?; APC, allophycocyanin;
FITC, fluorescein isothiocyanate; IL, interleukin; PBS, phosphate-buffered saline; CXCL12, chemokine (C-X-C motif) ligand 12; PE, phycoerythrin;
RT2, real-time reverse transcription; PCR, polymerase chain reaction; IPA, Ingenuity Pathway Analysis; ANOVA, analysis of variance; SLAM,
signaling lymphocyte activation molecule; NF-?B, nuclear factor-?B.
Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics
Mol Pharmacol 80:673–682, 2011
Vol. 80, No. 4
Printed in U.S.A.
ing of the thymus by bone marrow (BM)-derived progenitors
(Fine et al., 1990), 2) reduced stromal-mediated proliferation
of thymocytes (Kremer et al., 1994; Frericks et al., 2007), and
3) a skewing of the thymic output through direct effects on
developing thymocytes (Laiosa et al., 2010). In addition, AhR
activation leads to 1) alterations of B lineage cells during
gestation with possible consequences on autoimmunity (Mus-
tafa et al., 2008), 2) decreased numbers of immature B cells
(Murante and Gasiewicz, 2000), and 3) altered functional
activity of early hematopoietic progenitors (Murante and
Gasiewicz, 2000; Sakai et al., 2003; Singh et al., 2009). A
single dose of TCDD that results in BM TCDD concentrations
of ?1 nM (Fine et al., 1990) increases the relative numbers of
phenotypically defined HSCs/progenitors (Staples et al.,
1998) starting 2 days after treatment and lasting until day 31
(Murante and Gasiewicz, 2000). Furthermore, TCDD-treated
phenotypically defined long-term HSCs were unable to sus-
tain peripheral blood repopulation (Sakai et al., 2003) and
had a compromised ability to competitively repopulate the
BM of irradiated mice (Singh et al., 2009). These effects were
dependent on the presence of the AhR in hematopoietic cells
but not on supporting stroma (Staples et al., 1998; Sakai et
al., 2003). Taken Together, these data support a hypothesis
that primitive progenitors of hematopoiesis in the BM are
targeted by AhR ligands, resulting in AhR-mediated pheno-
typic and functional changes. Given recent interest in the
possible use of AhR ligands for the expansion of HSCs and
their use in BM transplants (Boitano et al., 2010), as well as
in treatment of autoimmune disease (Quintana et al., 2010),
it is critical to further define mechanisms related to effects on
hematopoietic cell function and the signaling pathways that
may mediate these alterations.
This work tests the hypothesis that TCDD- and AhR-me-
diated phenotypic and functional changes of murine HSCs
are a consequence of disruption of stem cell numbers and/or
their migration is preceded by changes in gene expression.
Understanding these changes in gene expression and their
functional consequences in HSCs may provide a rationale to
define a role of AhR in HSCs. Furthermore, this information
may provide avenues to further explore the use of therapeu-
tic AhR ligands.
Materials and Methods
Mice and Treatments. Five-week-old C57BL/6J female mice
were purchased from The Jackson Laboratory (Bar Harbor, ME)
and housed in the facilities of the University of Rochester Medical
Center (URMC) (Singh et al., 2009) for at least 1 week before
treatment. CD45.1?(B6.SJL-Ptprc?a?/BoAiTac) female mice used
in repopulation experiments were purchased from Taconic Farms
TCDD was obtained from Cambridge Isotopes (Andover, MA), and
6-?g/ml aliquots were prepared as described previously (Laiosa et
al., 2010). Either 30 ?g of TCDD in olive oil/kg b.wt. or olive oil alone
in a volume of 0.1 ml/20 g b.wt. was given orally by gavage. Mice
were euthanized after 7 days for functional and phenotypic experi-
ments. This dose and time were chosen on the basis of the optimal
effects of TCDD on HSCs (Singh et al., 2009). To study gene expres-
sion, treated mice were euthanized 6 and 12 h after treatment.
Cell Preparations. BM was harvested from femurs and tibias,
red blood cells were lysed, and lineage-positive leukocytes were de-
pleted as described previously (Singh et al., 2009). Only cell suspen-
sions with viability ?95% as measured by exclusion of trypan blue
were used for further experimentation. All flow cytometric analyses
and separations were performed on viable populations only, as mea-
sured by exclusion of the fluorescent dye 7-amino-actinomycin D
(7AAD). Lineage-depleted Sca-1?and c-kit?(LSK) cells used in
microarray experiments were obtained by laser-assisted sorting of
lineage-depleted cells stained with fluorochrome-conjugated antibod-
ies against Sca-1?[D7, allophycocyanin (APC); ebioscience, San Di-
ego, CA] and c-kit?[2B8, fluorescein isothiocyanate (FITC); BD
Pharmingen, San Diego, CA] using a FACSAria cell sorter (BD
Biosciences, San Jose, CA) and a gating scheme published recently
(Singh et al., 2010). To examine differentiation of LSK cells under
conditions in vitro, LSK cells were obtained from mice treated with
TCDD (30 ?g/kg, 7 days) or vehicle. In 12-well plates, 3 ? 104cells
(104cells/ml) were cultured per well. Each well contained StemSpan
Serum-Free Expansion Media (STEMCELL Technologies, Vancou-
ver, BC, Canada) supplemented with IL-6 (50 ng/ml) or IL-6 plus
stem cell factor (100 ng/ml). Cells were harvested after 7 or 14 days,
and phenotypic characterization was performed by flow cytometry.
Determination of Competitive Repopulation Units. The en-
graftment and proportion of competitive repopulation units, per mil-
lion of donor’s BM cells injected into the irradiated recipients, were
calculated for short-term and long-term reconstitutions (6 and 20
weeks after transplantation, respectively). A limiting-dilution ap-
proach was used for quantification. BM cells were isolated from
control or TCDD-treated mice. The donor’s (CD45.2?) cells were
resuspended in 100 ?l of PBS at three concentrations (0.1, 0.2, and
1 ? 106) and mixed with 100 ?l of PBS containing 2 ? 105compet-
itive donor’s (CD45.1?) BM cells. Recipient (CD45.1?) mice were
irradiated with two doses of 5.5 Gy from a cesium source. Mixtures
of donor and competitive donor cells were injected intravenously in
eight recipients. After 6 (short-term) or 20 (long-term) weeks after
transplantation, recipients were sacrificed, and BM cells were
isolated. BM cells were stained using commercially fluorochrome-
conjugated antibodies against CD45.1 (A20, APC; BD Pharmin-
gen) and CD45.2 (104, FITC; BD Pharmingen) and analyzed using
a FACSCanto (BD Biosciences) flow cytometer. Engraftment of
the progeny was evaluated on the basis of the percentages of
donor-derived CD45.2?BM cells. For the limiting-dilution analy-
sis, the presence of more than 1% cells of donor origin (Cd45.2?)
was considered as positive engraftment. The proportions of HSCs
per million BM cells were calculated, assuming single-hit Poisson
statistics (Purton and Scadden, 2007) and using L-Calc software
Migration of Hematopoietic Precursors. The in vivo migra-
tion of hematopoietic precursors was examined 24 h after intrave-
nous injection. An injection of 250 ?l of sterile PBS containing 5
million TCDD- or vehicle-treated donor BM cells was given in the tail
vein of nonirradiated recipients. The number of BM cells used was
optimized experimentally and represents approximately 6.3% of the
total number of BM cells in wild-type mice (i.e., recipients). The
migration of phenotypically defined HSCs/progenitors was calcu-
lated as the ratio of the numbers of CD45.2?LSK cells retrieved from
transplanted recipients per the numbers of CD45.2?donor LSK cells
The in vitro migration was studied with a transwell assay. To each
well of a V-bottom 96-well plate (BD, Franklin Lakes, NJ),150 ?l of
the chemoattractant murine chemokine (C-X-C motif) ligand 12
(CXCL12), also known as stromal-derived factor-1? (Miltenyi Bio-
tech, Bergisch Gladbach, Germany), was added at concentrations of
0, 50, 100, or 300 ng/ml. The HTS Transwell 96 Permeable support
with a 5-?m polycarbonate membrane (Corning Life Sciences, Low-
ell, MA) was placed on top of the plate containing chemoattractant.
Then 500,000 lineage-depleted (Lin?) cells resuspended in 50 ?l of
PBS were placed on top of the permeable support and incubated at
37°C for 3 h at 5% CO2. To determine migration, cells that were
recovered from the bottom well were stained with 7AAD and fluoro-
chrome-conjugated antibodies against Sca-1 and c-kit.
Phenotyping of Cells. Flow cytometry was performed using a
LSR-II flow cytometer (BD Biosciences) in the Flow Core Facility of
Casado et al.
the URMC and was used to phenotypically define long-term and
short-term HSCs (Kiel et al., 2005) using fluorochrome-conjugated
antibodies against CD48 [HM48-1, phycoerythrin (PE)-Cy7; BD
Pharmingen] and CD150 (9D1, APC; ebioscience). LSK cells [Sca-1
(E13-161.7, APC; BD Pharmingen) and c-kit (2B8, PE-Cy5; BD
Pharmingen)] were phenotyped for the expression of markers in-
volved in the interactions between HSCs and their microenviron-
ment such as the integrin dimer ?4?1 (also known as very late
antigen-4) composed of CD49d (also known as ?4; R1-2, PE; BD
Pharmingen) and CD29 (also known as ?1; Ha2/5, FITC; BD
Pharmingen), CD184 (2B11/CXCR4, APC; BD Pharmingen), CD44
(IM7, PE-Cy5; BD Pharmingen), and CD162 (also known as P-selec-
tin glycoprotein ligand-1; 2PH1, PE, BD Pharmingen). Flow cytomet-
ric analysis was performed in viable cells excluding 7AAD. For
apoptosis analysis, Lin?cells were incubated with fluorochrome-
conjugated antibodies against c-kit (2B8, APC) and Sca-1 (D7, V450).
Cells were washed and resuspended in 1? Annexin V binding buffer
(BD Pharmingen) and then incubated with 7AAD and Annexin V-PE
according to the manufacturer’s recommendations. 7AAD and An-
nexin V double-negative cells were gated as viable cells, and 7AAD-
negative and Annexin V-positive cells were gated as apoptotic cells.
Unstained, single fluorochrome and all-fluorochromes-except-one
were used to compensate for the fluorescent signals. Data were
calculated and plotted using FlowJo software (Tree Star Inc., Ash-
Microarray Experiments. Total RNA was isolated from sorted
LSK cells pooled from 20 mice using an RNeasy Mini/Micro Kit
(QIAGEN, Valencia, CA), DNase-treated, and quantified using the
ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington,
DE). Microarray analyses were done according to standard operating
procedures by the Functional Genomics Center at the URMC. RNA
quality was confirmed by the presence of two peaks on a 2:1 ratio of
intensity of 28S:18S rRNA using an Agilent 2100 Bioanalyzer (Agi-
lent Technologies, Santa Clara, CA). RNA (20 ng) was amplified
using the NuGEN Ovation RNA Amplification System (NuGEN
Technologies Inc., San Carlos, CA) to yield single-stranded cDNA
and then were hybridized with the GeneChip Mouse Gene 1.0 ST
Array (Affymetrix, Santa Clara, CA). Five microarrays from inde-
pendent RNA preparations per treatment were used. The microar-
rays were processed as described previously (Henry et al., 2010). The
relative fold changes of the signal intensity averages of TCDD with
respect to the control at the 6- and 12-h time points were calculated
for each of the 35,556 probes. Assuming a normal distribution of the
data, we performed an F-test to evaluate variances between the
treated and control groups and to calculate the p value obtained after
a two-tailed Student’s t test using Microsoft Excel.
Gene-Specific Primers and Real-Time Reverse Transcrip-
tion. Gene-specific primers and RT2-PCR analysis were used to
validate microarray data using a set of genes selected because of
their significant fold change expression differences at 6 and/or 12 h.
First-strand cDNA was prepared from 100 ng of total RNA following
the protocol for the SuperScript II First Strand cDNA Synthesis
system (Invitrogen, Carlsbad, CA). After selecting a panel of genes of
interest (Supplemental Table 1), RT2-PCR was performed using pro-
tocols provided by the commercial provider of primers and master
mix (SA Biosciences, Frederick, MD) in an iCycler System (Bio-Rad
Laboratories, Hercules, CA). Fold changes in expression of genes
from TCDD-treated LSK cells were calculated with respect to vehi-
cle-treated LSK cells using the 2???Ctapproximation method. Gapdh
and Hprt1 were used as control endogenous genes to normalize gene
expression. A linear regression coefficient close to 1 was used to
assess validation of microarray data with RT2-PCR.
Gene Expression Analysis. Transcripts with a fold change ex-
pression (TCDD normalized expression/vehicle normalized expres-
sion) greater than 1.5 at each time point were filtered. The most
variable transcripts were visualized using the open-source software
TIGR Multi-experiment Viewer (version 4.6.1) (Saeed et al., 2003).
Relevant functional association networks were generated by Ingenu-
ity Pathway Analysis (IPA) software (version 8.7; Ingenuity Sys-
tems, Redwood City, CA). For this purpose, the data sets containing
all gene identifiers and corresponding fold change expression values
were uploaded into the application. Each identifier was mapped to
its corresponding object in Ingenuity’s Knowledge Base. A cutoff of
1.5-fold change was set to identify transcripts whose expression was
regulated. Transcripts with altered expression were overlaid onto a
global molecular network developed from information contained in
Ingenuity’s Knowledge Base.
Statistical Analyses. Unless otherwise specified, results were
analyzed and plotted using GraphPad Prism (version 5.03; Graph-
Pad Software Inc., La Jolla, CA). The p value from a Pearson ?2test
for goodness of fit to a binomial distribution (p ? 1) was used to
analyze the competitive repopulation unit data. When appropriate, a
two-tailed Student’s t test and two-way ANOVA were used to analyze
statistical significance. p ? 0.05 was considered to be statistically
significant. A right-tailed Fisher exact test was used to calculate a p
value to determine the probability that each biological function
and/or disease assigned to that data set by IPA is due to chance
AhR Activation by TCDD Alters Phenotype and
Functions of HSCs. Our laboratory and others have consis-
tently observed alterations of phenotypically defined HSCs/
progenitors after TCDD treatment (Murante and Gasiewicz,
2000; Sakai et al., 2003; Singh et al., 2009). In this study, we
asked questions about the relative expression of newly rec-
ognized phenotypes of HSCs (Kiel et al., 2005) as well as
qualitative and quantitative functional characteristics of BM
cells. Seven days after in vivo treatment with TCDD, popu-
lations of HSCs were analyzed using flow cytometry based on
their relative expression of the signaling lymphocyte activa-
tion molecule (SLAM) receptors (Wilson et al., 2008) as phe-
notypic markers of BM stem cells. Figure 1A shows that
representative gating applied to all samples used to deter-
mine the relative percentages of LSK, LSKCD48?CD150?,
and LSKCD48?CD150?populations. As observed previously
(Murante and Gasiewicz, 2000; Singh et al., 2009), TCDD
treatment resulted in a significant increase in the relative
percentage of LSK cells (Fig. 1B). However, only a small
proportion of the LSK population is made up of those cells
defined as HSCs, with the largest subpopulation being com-
posed of predominantly more mature multipotent progenitor
cells. As such, it is notable that TCDD also increased the relative
percentages of the subpopulations LSKCD48?CD150?and
LSKCD48?CD150?, which have been correlated with short-term
HSCs and multipotent progenitor and long-term HSCs, respec-
tively (Fig. 1C).
To characterize the functional quality of HSCs, the prog-
eny of short-term and long-term HSC donor cells was ana-
lyzed after 6 or 20 weeks of competitive repopulation, respec-
tively. Hematopoietic cells from a donor or competitive donor
origin were distinguished using flow cytometry (Fig. 2, A–C).
Under the experimental conditions used, no differences be-
tween TCDD and vehicle were observed 6 (Fig. 2D) or 20 (Fig.
2E) weeks after transplantation. The proportions of short-
and long-term HSCs per million BM cells (Table 1) were
calculated from limiting dilution analysis to characterize
quantitatively the functional integrity of these cells. By this
analysis, the numbers of competitive functional HSCs from
TCDD-treated animals were not significantly different from
those from vehicle controls. To complement this analysis, we
AhR Activation Alters Migration of Hematopoietic Stem Cells
evaluated the ability of LSK cells from TCDD-treated mice to
undergo expansion and differentiation under ex vivo condi-
tions. Supplemental Fig. 1 shows that, in the presence of
IL-6 ? stem cell factor, TCDD-treated LSK cells are as able
to grow and generate different Lin?and Lin?populations in
vitro similarly to vehicle-treated LSK cells. In addition, LSK
cells from TCDD-treated mice did not show any difference in
the level of apoptosis as measured by Annexin and 7AAD
staining (Supplemental Fig. 2). These results suggested that
the TCDD dosing paradigm used to activate the AhR elicited
phenotypic changes but did not alter the absolute proportions
of functional HSCs when measured independently from their
correlated phenotype. In addition, there were no alterations
in their ability to survive, expand, and differentiate under ex
TCDD Affects Migration of LSK cells. TCDD-treated
LSK cells were reported to have decreased engraftment
(Sakai et al., 2003; Singh et al., 2009). However, TCDD
treatment elicited no difference in the overall proportions of
functional HSCs independent of their phenotype (Table 1).
Therefore, we hypothesized that critical events during en-
graftment such as the trafficking behavior of LSK cells may
be affected. Figure 3A shows that migration of LSK cells from
TCDD-treated animals to BM of recipients is decreased when
measured as in vivo accumulation of cells to the BM after
24 h of transplantation. As a complement to this study, we
also observed that the directional migration of TCDD-treated
LSK cells through a semipermeable membrane to a chamber
containing the chemokine CXCL12 was decreased (Fig. 3B).
The best studied receptor of CXCL12 is CD184 (CXCR4)
(Sasaki et al., 2009). As such, we analyzed LSK cells for the
expression of CD184/CXCR4 as well as other molecules
known to participate in the interactions between HSCs and
their microenvironment such as CD44, which is a receptor for
osteopontin present in the extracellular matrix of the endos-
teum. Within the LSK population from TCDD-treated ani-
mals, there was increased mean fluorescent intensity for
CD184/CXCR4 and CD44 (Fig. 3, C and D). Together these
results support the fact that AhR activation leads to cell
signaling-mediated disruptions in the ability of LSK cells to
interact with molecules present in their microenvironment.
TCDD Alters Pathways Involved in Cell Signaling,
Cellular Movement, and Cancer. The above data suggest
that the previously observed TCDD-elicited changes in HSC/
progenitor engraftment (Sakai et al., 2003; Singh et al., 2009)
were due predominantly, if not exclusively, to altered homing
of HSCs from TCDD-treated animals. Alterations in the ex-
pression and/or function of CD184/CXCR4 and CD44 on
these cells are probably concomitant with functional changes.
Because the AhR is a transcription factor, we hypothesized
that the phenotypic and cellular changes observed above
were the consequence of AhR-mediated changes in gene ex-
Fig. 1. TCDD treatment altered phenotypically defined HSC populations. Lin?cells were harvested from mice treated with TCDD (30 ?g/kg, 7 days)
or vehicle. Flow cytometry was used to phenotypically characterize populations of HSCs. A, dead cells were excluded and the LSK cell population was
gated from the viable cells. From the LSK population, further gating was applied on the basis of CD48 and CD150. Cumulative data for the percentages
of the HSC-enriched population are indicated. LSK cells (p ? 0.0059) (B) and subpopulations LSKCD48?CD150?(p ? 0.0075) and LSKCD48?CD150?
(p ? 0.0007) (C) in Lin?cells. Error bars are S.E.M. for n ? 3 independent experiments using data from five to six individual mice. ?, p ? 0.05; ??,
p ? 0.001.
Casado et al.
pression. Differential gene expression in LSK cells from mice
treated with TCDD (30 ?g/kg) or vehicle for 6 and 12 h was
analyzed using microarray technology. Hierarchical cluster-
ing of the expression profiles showed that the most signifi-
cant changes in regulation occurred for 105 transcripts (Fig. 4A).
Transcripts consistently up-regulated at 6 and 12 h included
Scinderin (Scin), Mmp8, interleukin 1 family member 9
(Il1f9), NAD(P)H dehydrogenase quinone 1 (Nqo1), Filamin
beta (Flnb), mouse predicted gene Gm5662, and SLAM fam-
ily member 7 (Slamf7) (Table 2). The lists of up- or down-
regulated transcripts at 6 and 12 h can be found in Supple-
mental Tables 2, 3, and 4. Results from RT2-PCR analyses
(Fig. 4, B and C) validate the changes observed at 6 and 12 h.
There were some notable differences between the responses
at 6 and 12 h. At 6 h, several genes (Olfr1095, Mmp9,
Ceacam10, Retnig, Msr1, and Lcn2) were up-regulated ?2-
fold but demonstrated no significant change at 12 h (Fig. 4A;
Supplemental Table 2). In contrast, other genes, including
Fosb, Cxcl2, Egr1, Atf3, Nr4a1, Fos, Jun, Junb, Dusp1, Plk2,
Ptgs2 (Cox2), Cd69, Ighg, Slc10a1, and Myd116, were down-
regulated at 6 h but were significantly up-regulated (Cxcl2,
Nr4a1, Plk2, Ptgs2, and Cd69) or not altered at 12 h (Fig. 4A;
Supplemental Tables 3 and 4).
To understand the biological relevance of the observed
changes, IPA was used to generate functional association
networks with the following settings: direct relationships
from the Ingenuity Knowledge Base Reference Set reported
in all human, mouse, and rat immune primary cells and
immune, leukemia, lymphoma, macrophage, and myeloma
cell lines; cutoff fold change values 1.5; up to 25 networks per
analysis; and up to 35 molecules per network. Table 3 shows
that the common top biological functions at 6 and 12 h were
cell-to-cell signaling and interaction and cellular movement.
In addition, at these time points, hematological system de-
velopment and function were significantly affected by treat-
ment. Supplemental Tables 5 and 6 show the detailed lists of
transcripts assigned by IPA to the most relevant molecular
and cellular functions and physiological systems and func-
tions. It is noteworthy that changes in genes involved in the
biological functions of antigen presentation, cell-to-cell sig-
naling and interaction, cellular movement, hematological
system development and function, and immune cell traffick-
ing appeared to be most significant (defined by p ? 10?7) in
the 12-h response to TCDD (Table 3). Figure 5 shows func-
tional association networks of transcripts assigned to hema-
tological system development and function, generated by IPA
Fig. 2. Qualitative analysis of engrafted TCDD-treated BM cells shows no difference in the engraftment potential of HSCs. Donor CD45.2?BM cells
from TCDD-treated (30 ?g/kg, 7 days) mice were simultaneously injected with CD45.1?competitor BM cells at different ratios into CD45.1?irradiated
recipient mice. Representative gating was applied for competitive donor BM cells (A) and (B) donor BM cells. After reconstitution, the long-term (20
weeks) and short-term (6 weeks) ability to reconstitute the BM was qualitatively analyzed. C, flow cytometry was used to discriminate CD45.2?donor
cells from CD45.1?competitive donor cells in repopulated recipients. Cumulative data for the percentage of the donor’s progeny (CD45.2?) show no
differences 6 (D) or 20 (E) weeks after transplantation. Data shown represent one independent experiment of two with eight recipients per group per
experiment. Squares represent values for individual mice. Average values for vehicle and TCDD groups are represented by dashed and solid lines,
AhR Activation Alters Migration of Hematopoietic Stem Cells
at 6 and 12 h. An analysis was also run using information
reported from hematopoietic and nonhematopoietic cells. Ac-
cording to this analysis, the top biological functions and
diseases associated with the network generated by the mo-
lecular interactions common to both time points were gene
expression (RNA polymerase II, histone h3, and histone h4),
inflammation (Ptgs2, also known as Cox2 and NF-?B com-
plex), and cancer (Ptgs2, Cxcl2, Jun, Fos, Atf3, Mmp9, Nqo1,
Nr4a1, Cd69, Btg2, Egr1, and Dusp1).
There is increasing evidence supporting a role of the AhR
in hematopoiesis and HSC biology in particular. In part, this
evidence comes from reports of increased incidence of leuke-
mia and non-Hodgkin’s lymphoma in TCDD-exposed popula-
tions (Frumkin 2003) as well as recent studies suggesting a
potential therapeutic application of AhR antagonists to ex-
pand human HSCs for BM transplants (Boitano et al., 2010).
Thus, there is a need to define cellular and molecular mech-
anisms whereby the AhR mediates these effects.
It has been shown that TCDD exposure to mice increases
the percentages of LSK cells and some LSK subpopulations
in BM (Sakai et al., 2003; Singh et al., 2009). Here we
expand on these reports by showing that the percentage of
Quantitative analysis of engrafted TCDD-treated BM cells shows no
difference in the engraftment potential of HSCs
Donor BM cells were harvested from mice 7 days after treatment with TCDD (30
?g/kg) or vehicle (olive oil) by gavage as described in the legend to Fig. 2. Repopu-
lated recipients contained ?1% of CD45.2?cells in total BM cells. The proportions of
HSCs per million of BM cells were calculated using L-Calc software, entering the
numbers of total surviving and repopulated mice. Representative results from one of
two independent experiments (n ? 8 recipients per dilution of donor BM cells/group).
A Pearson ?2p value ? 1 was consistent with a Poisson distribution of data.
VehicleTCDD Vehicle TCDD
Proportion (HSCs per 106BM cells) 13.4
95% confidence interval
p value for Pearson ?2
Fig. 3. TCDD exposure altered the migration of HSCs in vivo and in vitro. BM cells were harvested from mice treated with TCDD (30 ?g/kg, 7 days)
or vehicle. A, five million CD45.2?BM cells from TCDD-treated or untreated mice were injected intravenously into CD45.1?recipients. After 24 h,
flow cytometry was used to determine the numbers of donor LSK cells that migrated to BM in recipients with respect to the LSK numbers injected.
(p ? 0.0242). Error bars are S.E.M of n ? 3 independent experiments using data from five to six individual mice per experiment. B, 500,000 Lin?cells
were placed on top of a transwell migration chamber containing 50 to 300 ng/ml CXCL12 in the bottom chamber. Three hours later, LSK cells that
migrated to the bottom chamber were counted by flow cytometry. Two-way ANOVA analysis indicated significant differences at 100 and 300 ng/ml (p ?
0.0001). Error bars are S.E.M. of n ? 3 independent experiments using data from 12 wells with cells from three pooled mice. C, LSK cells were analyzed
for expression of surface molecules by flow cytometry. Two-way ANOVA analysis indicated significant differences between groups for CD184/CXCR4
and CD44 (p ? 0.0037 and ?0.0001, respectively). Error bars are S.E.M. of n ? 3 independent experiments using data from five to six individual mice.
D, representative histograms obtained using flow cytometry to determine the mean fluorescent intensity of CD184/CXCR4 and CD44 in viable LSK
cells. The filled histograms are representative of TCDD-treated (black) and vehicle-treated (gray) cells. The unfilled histograms are representative of
the negative controls used to compensate for fluorescent signals that were stained with all of the fluorochromes used in the respective panels except
with the one conjugated to the protein being analyzed (CD184 or CD44). The horizontal bars represent the gating used to determine the positive
population of cells for which the mean fluorescent intensity was calculated. ?, p ? 0.05; ???, p ? 0.0001.
Casado et al.
identified by the recently described SLAM markers (Kiel et
al., 2005), are also increased after TCDD treatment (Fig. 1).
These results present the apparent paradox that higher per-
centages of phenotypically defined HSCs (LSK cells) occurred
with a decreased functional ability of these cells to generate
progeny (Sakai et al., 2003; Singh et al., 2009). This result
may not necessarily be contradictory because it has been
reported that when homeostasis is disrupted, cells with the
phenotype of HSCs may have an altered functional capacity
(Purton and Scadden, 2007). To avoid potential biases inher-
ent with phenotypic-based sorting and defined relative ratios
of donor to competitor cell numbers, we used a limiting dilu-
tion approach to quantify the functional integrity of these
cells. These studies indicated no significant differences in the
absolute proportions of long-term and short-term HSCs from
TCDD- and vehicle-treated mice (Table 1). Together, these
data suggested that the higher percentages of phenotypically
defined HSCs reflected changes in HSC biology and cell sig-
naling that may affect different steps of the engraftment
Successful engraftment of HSCs involves a number of dif-
ferent events such as leaving the circulation to home to the
BM, taking residence in the vascular and endosteal niches,
and finally lineage differentiation. Our studies further dem-
onstrated that TCDD-mediated activation of the AhR alters
the migration and trafficking of phenotypically defined HSCs
under in vivo and in vitro conditions (Fig. 3, A and B). Thus,
previously observed decreased engraftment (Sakai et al.,
2003; Singh et al., 2009) is probably due to decreased
migration (Fig. 3) rather than to decreased numbers of
functional HSCs (Fig. 2; Table 1) or altered viability and
differentiation (Supplemental Figs. 1 and 2). A modified
ability of HSCs and progenitors to move within the marrow
niche may also explain the effects of AhR activation by
TCDD on more differentiated cells [i.e., decreased thymic
Fig. 4. TCDD exposure-induced changes
in the expression of genes in LSK cells.
Total RNA was obtained from sorted LSK
cells. Fold changes in normalized values
of gene expression for TCDD or vehicle
control groups were calculated using mi-
croarray technology. Microarray data rep-
resent averages of five experiments using
RNA from LSK cells pooled from 20 mice
per treatment. A, the most variable tran-
scripts from the 6- and 12-h data sets (fold
change ?1.5) were visualized using the
Multi-experiment Viewer. The intensity
of the colors represents the changes in
fold change for the up-regulation (yellow)
or down-regulation (blue) of the tran-
changes in expression were used to vali-
date microarray results using RT2-PCR
after 6 (B) and 12 h (C) of treatment.
RT2-PCR data are averages of three ex-
periments from LSK cells pooled from 20
Transcripts consistently up- or down-regulated at 6 h and 12 h in LSK
cells after TCDD treatment
Microarray experiments were performed from LSK cells isolated using immunomag-
netic sorting. Hierarchical clustering of microarray results was used to determine
groups of genes coregulated. (n ? 5 microarrays from pooled samples of 20 mice/
6 h12 h
AhR Activation Alters Migration of Hematopoietic Stem Cells
seeding (Fine et al., 1990; Laiosa et al., 2010)] and altered
B cell numbers (Thurmond et al., 2000).
Trafficking events are dependent on the ability of HSCs to
“sense” their microenvironment by cell-cell interactions and the
recognition of soluble factors through cell surface proteins.
Markers used for phenotypic discrimination are functional mol-
ecules themselves involved in cell signaling through recognition
of chemical cues from the microenvironment to migrate, differ-
entiate, proliferate, or self-renew. For example, c-kit is a ty-
rosine kinase serving as a receptor of the stem cell factor, which
provides cues for quiescence and survival of the most primitive
progenitor cells in the BM (Askmyr et al., 2009). CD184/CXCR4
is critical during development for seeding of the BM with HSCs
and mediates migration and quiescence throughout adulthood
(Nie et al., 2008). CD44 is highly expressed in prothymocytes
(CD4lowCD25?c-kit?) of the BM, serving as a homing receptor
for the thymus (Wu et al., 1993), and CD44 expression was
found to be up-regulated in thymic emigrants exposed to TCDD
(Esser et al., 2004). Thus, it is reasonable, if not expected, that
functional alterations in HSCs after TCDD treatment would be
a reflection of altered expression and/or function of cell surface
proteins. However, we observed an increased, rather than an
expected decreased, phenotypic expression of both CD184/
CXCR4 and CD44 at 7 days after TCDD treatment (Fig. 3). No
differences were observed in CD184/CXCR4 or CD44 mRNA
expression at 6 or 12 h after TCDD exposure as determined by
sion at 24 h or 7 days after TCDD exposure was not changed as
determined by RT2-PCR analysis (not shown). Taken together,
these data suggest that TCDD may cause altered CD184/
CXCR4 and CD44 protein expression in HSCs by a post-
transcriptional mechanism and that other molecules are being
proteins or that are involved the migration and trafficking of
Given that the AhR is a ligand-activated transcription
factor, we hypothesize that TCDD-mediated changes in phe-
notype and signaling of HSCs were preceded by changes in
gene expression. The results shown in Figs. 4 and 5 support
this hypothesis. Multiple transcriptional changes occurred
6 h after exposure, some of which were transient and other
were also observed 6 h later. The genes modulated by AhR
activation in LSK cells that have been reported to have
putative AhR responsive elements (Sun et al., 2004) in the
promoters of their murine homologs include Scin, Nqo1,
Egr1, Ptgs2, Cxcl2, Nr4a1, Dusp1, Btg2, Junb, Fosb, Jun,
Ceacam10, Scrg1, C1qb, Bst1, Slc10a1, Spp1, Snord14a,
Ier2, Ccl4, Skil, and Klrb1c, although it is not yet clear
whether all of these sites represent AhR responsive elements
that are functional.
Scinderin (Scin, also known as adseverin) was highly up-
regulated after both 6 and 12 h of TCDD treatment (Table 2).
In hematopoietic stem and progenitor cells, Scin is one of the
key regulators accounting for chemotactic responses to
CXCL12 (Evans et al., 2004). This calcium/proton-regulated
protein binds actin monomers, severs and caps actin fila-
ments, and has been reported to respond to TCDD treatment
in other immune cells (Svensson et al., 2002). Sequential
coordinated polymerization and depolymerization of the ac-
tin cytoskeleton are necessary to maintain the functional
expression of cell surface proteins and for directed migration
(Friederich et al., 1992). CD184/CXCR4 function and cell
surface expression is known to depend on endocytosis, intra-
cellular trafficking, and recycling (Kumar et al., 2011), all of
which are dependent on cytoskeleton regulation. Lin?Sca-
1?ckit?and LSK cells move with different efficiencies to-
ward CXCL12 due at least in part to different expression of
Scin protein (Evans et al., 2004). Given that TCDD can
regulate Scin transcripts and responses to CXCL12 in LSK
cells, our results support a role for the AhR in cytoskeleton
regulation that may have consequences for cell motility.
Mmp8 was also up-regulated at both time points. Even
though there is no evidence of AhR transcriptionally regulat-
ing metalloproteinases, there is literature data linking AhR
activation with their expression, probably via Jun or Fos
(Hillegass et al., 2006). Together with CD184, metalloprotei-
nases have been proposed to be involved in cell migration and
invasion of leukemic cells (Shao et al., 2011). Different tran-
Important biological functions were significantly altered in LSK cells 6 and 12 h after TCDD treatment
A cutoff of 1.5-fold change (TCDD/vehicle) was set to identify transcripts whose expression was regulated. The top biological functions based on their Fisher’s exact test p
value (in brackets) are shown. The numbers in parentheses after the name of the functions are the number of transcripts in our data sets common to the Ingenuity Knowledge
Base reference set.
6 h 12 h
Molecular and cellular functions Cell-to-cell signaling and interaction (23)
?9.52 ? 10?4–3.92 ? 10?2?
Cellular movement (9)
?2.20 ? 10?3–4.48 ? 10?2?
Antigen presentation (16)
?2.51 ? 10?3–3.92 ? 10?2?
Cellular development (19)
?4.54 ? 10?3–4.31 ? 10?2?
Cell morphology (4)
?9.90 ? 10?3–2.67 ? 10?2?
Hematological system development and function (44)
?2.79 ? 10?5–4.96 ? 10?2?
Tissue morphology (17)
?2.79 ? 10?5–2.67 ? 10?2?
Lymphoid tissue structure and development (10)
?1.39 ? 10?3–3.92 ? 10?2?
?2.20 ? 10?3–4.48 ? 10?2?
Immune cell trafficking (23)
?2.20 ? 10?3–4.96 ? 10?2?
Antigen presentation (17)
?2.11 ? 10?8–4.44 ? 10?2?
Cell-to-cell signaling and interaction (16)
?2.11 ? 10?8–4.89 ? 10?2?
Cellular movement (14)
?1.54 ? 10?7–4.44 ? 10?2?
Cell death (10)
?3.65 ? 10?6–4.44 ? 10?2?
Cell signaling (27)
?9.02 ? 10?6–2.48 ? 10?3?
Hematological system development and function (26)
?2.11 ? 10?8–4.89 ? 10?2?
Immune cell trafficking (17)
?2.11 ? 10?8–4.89 ? 10?2?
?2.75 ? 10?7–4.44 ? 10?2?
Lymphoid tissue structure and development (12)
?6.22 ? 10?5–2.25 ? 10?2?
Humoral immune response (3)
?1.13 ? 10?4–2.98 ? 10?2?
Physiological system development
Casado et al.
scripts involved in NF-?B signaling such as Il1f9, Cxcl2,
Ptgs2, Junb, and Fos (Table 2; Supplemental Table 3) were
also altered at both time points. Whether these changes in
gene expression are directly related to AhR-DNA binding in
LSK cells is uncertain because there is evidence that AhR
and NF-?B proteins can interact directly (Tian, 2009). Fur-
ther kinetic studies to clarify these interactions are of ther-
apeutic interest, considering that NF-?B complexes regulate
CD184 (Chua et al., 2010) and CD44 (Damm et al., 2010)
protein expression. It is also of interest that Spp1 was down-
regulated 6 h after TCDD exposure. Spp1 encodes osteopon-
tin, one of the ligands of CD44. The reduced expression of a
CD44 ligand (Supplemental Table 4) at early time points may
be related to increased CD44 expression (Fig. 3C) several
days later. Given that some of the transcripts changed in our
data sets are themselves transcriptional regulators and are
regulated by the AhR (e.g., Fos, JunB, Ptgs2, and Egr1), it
seems likely that some of the functional changes are second-
ary to initial AhR signaling. Although supporting the func-
tional changes that we observed in hematopoietic stem/pro-
genitor cells after TCDD treatment, the changes in gene
expression also suggest a complex cross-talk of the AhR with
pathways associated with the ability of HSCs to sense their
microenvironment, alter their trafficking behavior, and pro-
Fig. 5. TCDDtreatmentchangestheexpres-
sion of transcripts involved in hematopoietic
system development and function. Data sets
containing 35,556 microarray probes were
analyzed at 6 and 12 h. Transcripts with reg-
ulated expression were overlaid onto a global
molecular network developed from informa-
tion contained in the Ingenuity Knowledge
Base. All relationships are supported by at
textbook, or from canonical information
stored in the Ingenuity Knowledge Base. He-
matopoietic system development and func-
tion was regulated after TCDD treatment.
Association networks of the transcripts regu-
lated in this category after 6 (A) and 12 (B) h
were generated using IPA. In the graphical
transcripts are represented as nodes with
shapes indicating the function of the ex-
pressed protein. The color of the node indi-
cates the up-regulation (red) or down-regula-
tion (green) (fold changes ?1.5) of the
transcripts. The noncolor transcripts were
present in our data set and have a direct
relationship with the regulated transcripts.
In addition, according to the Ingenuity
Knowledge Base, these are categorized
within hematopoietic system development
AhR Activation Alters Migration of Hematopoietic Stem Cells
mote hematological diseases, which is a common sequence of
events in leukemogenesis.
Taken together, these results suggest a complex role of
AhR signaling in HSC function. Additional studies in AhR-
null mice suggest that AhR may regulate HSC quiescence/
proliferation (Singh et al., 2010), which, consistent with the
present study, depend on the ability of HSCs to sense cues
within their microenvironment. It is also important to con-
sider that the exact role of the AhR and its physiological
relevance in other tissue stem cell populations may be con-
text-specific (Panteleyev and Bickers, 2006; Latchney et al.,
2011). Clinical use of the AhR to treat hematological diseases
(Boitano et al., 2010; Quintana et al., 2010) will also require
answering daunting questions regarding the biological impli-
cations of different, and possibly endogenous, ligands in AhR
activity and the relevance of nongenomic pathways in AhR
signaling. Nevertheless, further investigations in this area
will provide important information needed for our under-
standing of processes that regulate stem cells and their role
in human disease.
We thank Dr. Ellen Henry for critical discussion and reading of
the manuscript, Jason Walrath for assistance with animal care, Dr.
Timothy Bushnell and the staff of the Flow Core Facilities in URMC
and Dr. Stephen Welle and the staff of the Functional Genomics Core
of the URMC.
Participated in research design: Casado, Singh, and Gasiewicz.
Conducted experiments: Casado and Singh.
Performed data analysis: Casado, Singh, and Gasiewicz.
Wrote or contributed to the writing of the manuscript: Casado,
Singh, and Gasiewicz.
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Address correspondence to: Dr. Thomas A. Gasiewicz, Department of
Environmental Medicine, University of Rochester, School of Medicine and
Dentistry, Box-EHSC, 601 Elmwood Ave., Rochester, NY 14642. E-mail:
Casado et al.