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*
Department of Immunology, School of Life Science, Faculty of Medicine, Tottori University, Yonago, Japan;
Laboratory of Pluripotent Cell Studies, RIKEN Center for Developmental Biology, Kobe, Japan; and
Center for Molecular and Developmental Biology, Graduate School of Science, Kyoto University, Kyoto, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-catenin in stromal cells. In this study, we demonstrated that Wnt
signaling regulates hemopoiesis through stromal cells with selectivity
and different degrees of the effect, depending on the hemopoietic
lineage in the hemopoietic microenvironment. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent reports suggested that Wnt proteins might participate in the
regulation of blood cell production (7, 8, 9). Wnt
genes encode a large family of secreted glycoproteins that are involved
in a wide spectrum of cell activities in development (10).
Wnt proteins are thought to be associated with the cell surface or
extracellular matrix of the secreting and adjacent cells
(11). Wnt proteins transduce signals through their
receptors, Frizzled and Disheveled, leading to the inactivation of
GSK3, which normally keeps the level of -catenin low in the
absence of signals through interacting with the product of the
adenomatous polyposis coli gene and Axin. In response to the Wnt
signal, -catenin accumulates, interacts with the lymphoid enhancer
factor-1/T cell factor-1 family of transcription factors, and activates
the target genes (10). In the mouse, 18 different
Wnt genes have been reported. At least three Wnt
genes are expressed in hemopoietic tissues in the mouse. In fetal
hemopoietic sites, the Wnt-5a and Wnt-10b genes
are expressed (7). In the BM, the expression of the
Wnt-3a gene in addition to the Wnt-5a and
Wnt-10b genes has been reported (9). These
Wnt genes are also known to be involved in several aspects
of embryogenesis (12, 13, 14, 15, 16). Functions of Wnt proteins in
hemopoiesis have been examined using culture supernatants,
retrovirus-mediated gene transfer, or coculture with Wnt
gene-transfected cells, because the purification of biologically active
Wnt proteins has not yet been successful. In the mouse, the potential
of Wnt-1, -5a, and -10b to expand fetal liver hemopoietic progenitors,
and of Wnt-3a to augment the proliferation of pro-B cells, has been
reported (7, 9).
These experiments have demonstrated that Wnt proteins stimulate the
survival and proliferation by acting directly on hemopoietic
progenitors. However, the effects on hemopoietic microenvironments must
be further investigated to fully understand the role of molecules
regulating hemopoiesis. Indeed, stromal cells express Frizzled
receptors and respond to Wnt proteins, as we show in this work. In this
study, we examined the effects of Wnt signaling on hemopoiesis,
including the effects on stromal cells, using the conditioned medium
(CM) from L cells secreting Wnt-3a (17). In addition, an
inducible expression system of a stable form of -catenin in stromal
cells was established to mimic the effect of the Wnt signaling. By
using these tools, we demonstrate that Wnt signaling negatively
regulates hemopoiesis, but with selectivity and different sensitivities
of different hemopoietic lineages in the presence of stromal cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female 6- to 10-wk-old (C57BL/6 x DBA/2)F1 mice were purchased from Japan SLC (Hamamatsu, Japan). BM cells were prepared from the femora and tibiae of these mice.
Cell lines
ST2 (18), a BM-derived stromal cell line, was maintained in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 5% FCS (BioWhittaker, Walkersville, MD) and 50 µM 2-ME. ST2b cells were maintained in the same medium, except that it was supplemented with 1 µg/ml tetracycline (Tc).
Conditioned medium
Wnt-3a-producing L cells and control L cells (17)
were grown to confluency in DMEM/F-12 medium (Life Technologies)
supplemented with 10% FCS. Then the medium was replaced with 
-MEM
supplemented with 10% FCS, and the culture supernatants were collected
after 24 h of culturing. These conditioned media were designated
W3a-L-CM and L-CM, respectively. To collect the culture supernatants of
L cells stimulated with Wnt-3a, confluent control L cells were cultured
with 25% W3a-L-CM for 48 h, and washed three times with PBS, and
the medium was changed to -MEM supplemented with 10% FCS. The
culture supernatants harvested 24 h later were designated as
LW3a-CM. These conditioned media were added
to cultures at the proportion of 25% in all experiments reported
in this work (17).
Tc-regulated system
ST2 cells were first transfected with a construct that expressed
the Tc-controlled transactivator-internal ribosomal entry
site-hph gene under the control of the CAG promoter
(Refs. 19 and 20 ; H. Niwa, unpublished
observations), and cultured in the presence of 25 µg/ml hygromycin B
(Sigma, St. Louis, MO). Resistant cells were further cotransfected with
a construct that expressed a stabilized form of mouse -catenin
(21), which was tagged with the hemagglutinin (HA) epitope
at the carboxyl-terminal (a gift from A. Nagafuchi, Kumamoto
University, Kumamoto, Japan) under the control of the human
CMV*-1 promoter (Tc-responsive promoter) (19) and a
PGK-neor construct, and selected in the
presence of 200 µg/ml G418 (Life Technologies), 25 µg/ml hygromycin
B, and 1 µg/ml Tc (Sigma). Transfections were performed with
GenePORTER (Gene Therapy System, San Diego, CA). The stable
transfectants, designated as ST2b, were maintained in the presence of 1
µg/ml Tc. Experiments were performed in the range of 0–100 ng/ml Tc
because 1 µg/ml Tc slightly suppressed osteoclast formation on
parental ST2 cells, and 100 ng/ml Tc was sufficient to block the
expression of transfected -catenin.
Long-term BM cultures
For the long-term BM culture of myeloid lineage cells (Dexter-type culture) (22), 106 BM cells were seeded onto ST2 or ST2b cells in 60-mm dishes (Corning Glass (Corning, NY) gelatin-coated dishes, or PRIMARIA, Becton Dickinson Labware (Mountain View, CA), were used for mitomycin C-treated cells) and cultured in anti-MEM supplemented with 10% FCS. AFS98 (5 µg/ml; antagonistic Ab against c-Fms receptor; a gift from S. I. Nishikawa) (23) was added where indicated. The cultures were fed twice per week and maintained for 2 wk. For the long-term BM culture of B lineage cells (Whitlock-Witte-type culture) (24, 25), 4 x 105 BM cells were inoculated per well of a six-well plate (Corning; dishes were coated with gelatin for mitomycin C-treated ST2 cells), preseeded with ST2 or ST2b cells, and cultured in RPMI 1640 supplemented with 5% FCS, 50 µM 2-ME, and 20 U/ml mouse rIL-7 (a gift from T. Sudo, Toray Industries, Kawasaki, Japan). The cultures were fed twice per week and maintained for 2 wk. Cells thus cultured were analyzed by flow cytometry, May-Grünwald-Giemsa staining, and colony formation assays.
Osteoclast formation assay
In the experiments that utilized coculture systems with ST2 or
ST2b cells, 104 BM cells were inoculated per well
of a 24-well plate (Corning, or PRIMARIA for mitomycin
C-treated cultures), preseeded with the stromal cell line, and cultured
in -MEM supplemented with 10% FCS, 10-7 M
dexamethasone (DEX; Sigma), and 10-8 M
1,25-dihydroxyvitamin D3
(1,25(OH)2D3; Biomol
Research Laboratories, Plymouth Meeting, PA) (26). For the
stromal cell-free cultures, 2 x 104
Sephadex G-10 column-purified BM hemopoietic cells (27)
were inoculated per well of a 24-well plate and cultured in -MEM
supplemented with 10% FCS, 100 ng/ml human rM-CSF (a gift from K.
Yamanishi, Otsuka Pharmaceutical, Tokushima, Japan), and 50
ng/ml human recombinant soluble receptor activator of NF-
B ligand
(sRANKL) (PeproTech, Rocky Hill, NJ). The medium of these
cultures was changed every other day, and osteoclast formation
was evaluated by tartrate-resistant acid phosphatase (TRAP) staining 6
days later, as described (28). TRAP-positive cells with
more than two nuclei were scored as multinucleated cells (MNCs).
Colony formation assay
Cells from BM or Dexter-type cultures were incubated in -MEM
containing 1.2% methylcellulose (Muromachi Kagaku Kogyo, Tokyo,
Japan), 30% FCS, 1% deionized BSA (Sigma), 2 mM L-glutamine
(Life Technologies), 0.1 mM 2-ME, and cytokines (40 U/ml mouse rIL-7
for CFU-IL-7, or 100 ng/ml mouse recombinant stem cell factor (a gift
from M. Hirashima), 100 U/ml mouse rIL-3 (a gift from T. Sudo), and 2
U/ml human recombinant erythropoietin (Genzyme/Techne,
Cambridge, MA) for other colony-forming cells (CFCs)) in 35-mm
suspension culture dishes (Corning). Seven days later, the
number of colonies containing >50 cells was scored.
Flow cytometry
The following Abs were used for staining: 6B2 (anti-B220; PharMingen, San Diego, CA); goat anti-µ Ab (ICN Biochemicals, Costa Mesa, CA); M1/70 (anti-Mac-1; Immunotech, Marseille, France); and RB6-8C5 (anti-Gr-1; PharMingen). Stained cells were analyzed using a flow cytometer (EPICS XL; Coulter, Palo Alto, CA).
Western blot analysis
Cytosolic fractions of the cells were collected as described
(17). Proteins were separated by SDS-PAGE and transferred
to a nitrocellulose membrane (Hybond ECL; Amersham Pharmacia Biotech,
Piscataway, NJ). After blocking with 5% skim milk, the membrane was
incubated with HRP-labeled mouse anti--catenin Ab (Transduction
Laboratories, Lexington, KY). Immunoreactive bands were visualized
using the ECL detection system (Amersham Pharmacia Biotech). In some
experiments, the membranes were reprobed with mouse
anti--actinin Ab (Sigma) or rabbit anti-HA-tag Ab (Medical &
Biological Laboratories, Nagoya, Japan), then incubated with
HRP-labeled secondary Abs, and detected using the same system.
Immunofluorescent staining
ST2 cells were fixed with 4% paraformaldehyde, 0.18% Triton X
in PBS for 10 min at room temperature, blocked with 1% BSA in PBS,
incubated with mouse anti--catenin Ab (Transduction
Laboratories) for 1 h at room temperature, and then incubated with
biotin-conjugated anti-mouse IgG Ab and FITC streptavidin.
PCR analysis
Total RNA was purified using ISOGEN (Nippon Gene, Toyama, Japan). First-strand cDNA synthesis was performed using ReverTra Ace (Toyobo, Osaka, Japan) primed with random hexamer in a 20-µl reaction mixture containing 1 µg total RNA. One microliter of the first-strand cDNA mixture was subjected to PCR with Taq polymerase (Toyobo) in a 25-µl volume. The PCR conditions were as follows: 94°C (5 min), 55°C (2 min), 72°C (3 min) for the primary cycle; 94°C (1 min), 55°C (1 min), 72°C (1.5 min) for the following 36 cycles. The extension time in the last cycle was 4.5 min. The following primers were used: Fzd1 (GenBank accession number AF054623), 5'-GTACTGAGCGGAGTGTGTTTTCT-3'/5'-GATCATGAAGACTGTAAAGTCTGG-3'; Fzd3 (U43205), 5'-TTATAAGAAAATCAAGAGGAACTTC-3'/5'-TACTGAGGTCTCGGATGCTGCTGTG-3'; Fzd4 (U43317), 5'-GAACTGACTGGCTTGTGCTATGT-3'/5'-ATTTCAACTGCCATGTTTGAGTC-3'; Fzd5 (AF005203), 5'-GCGCACCGGCCAAGTGCCCAAC-3'/5'-ACGGCTGCAAGCGACGCTGGCA-3'; Fzd6 (U43319), 5'-TCTGCCATGGCAATCGCTGACCATG-3'/5'-GCTGAGTGAACAGGCAGAGATGTGG-3'; Fzd7 (U43320), 5'-AGTGTGCTACGTGGGCCTGTCTAGT-3'/5'-GTGATGCCCACGATCATGGTCATCA-3'; Fzd8 (U43321), 5'-CGATTACGCGGTCTTCATGCTCAAG3'/5'-AAGATACAGAGCTGGCCGTGCCAGA-3'. Identities of the amplified fragments and the reported sequences were confirmed by digestion with restriction enzymes.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We found that the CM from L cells secreting Wnt-3a (W3a-L-CM), but
not the control CM (L-CM), causes a morphological change of the ST2
stromal cell line: the ST2 cells overgrow and form highly condensed
sheets of cells (Fig. 1
A). The growth rate of ST2 cells seems not to be changed, but
the cells grow even after they have reached confluency and the cell
number increases to 
2.5-fold that of control cultures (data not
shown). These changes are similar to those observed in Wnt
gene-transfected mammary epithelial cells (29). We
examined the components of Wnt signaling in stromal cells. Expression
of murine frizzled homologues (Fzd1, 3, 4, 5, 6, 7,
8), the receptors for Wnt proteins, was examined by RT-PCR
analysis. The expression of all of these Fzd genes, except
for Fzd3, was observed in ST2 cells (Fig. 1B).
When ST2 cells or primary BM stromal cells were stimulated by
W3a-L-CM, the concentration of cytosolic -catenin
dramatically increased (Fig. 1C). Translocation of
-catenin to the nucleus was also observed (Fig. 1D).
These results suggest that Wnt signaling components are functional in
stromal cells.
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We examined the effects of W3a-L-CM on in vitro cultures of
hemopoietic lineages cultured on stromal cells. We prepared the
Dexter-type cultures for the myeloid lineage cells on ST2 in the
presence of L-CM or W3a-L-CM. ST2 cells were treated with mitomycin C
to avoid indirect effects of their growth. Under both conditions, the
majority of the cells that adhered to ST2 were macrophages (Fig. 2, C and D). Perhaps this was due to the high dose
of M-CSF in the CM of L cells (30). About half the number
of cells were harvested from the culture containing W3a-L-CM
compared with the culture with L-CM. We analyzed these cells
by flow cytometry. The number of macrophages, which were identified by
their sensitivity to antagonistic Ab to the receptor for M-CSF (AFS98),
was not changed much by the addition of W3a-L-CM, but the
AFS98-resistant Mac-1+Gr-1+
granulocyte population was clearly reduced by adding W3a-L-CM
(Fig. 3). We next prepared Dexter-type cultures on mitomycin C-treated ST2 in
the presence of AFS98 to avoid the predominance of macrophages in the
culture. In the cultures supplemented with L-CM, hemopoietic
colonies firmly attached to ST2 were observed (Fig. 2A). In
contrast, there were only small colonies loosely attached to ST2 in the
cultures containing W3a-L-CM (Fig. 2B).
Hemopoietic colonies were never observed beneath the stroma under these
conditions. Only about one-twentieth of the number of cells was
recovered from the culture with W3a-L-CM compared with the control
cultures (Fig. 4A). The numbers of mature neutrophils and mast cells in the
W3a-L-CM-containing cultures were reduced to 5% and 10%
of those in the cultures with L-CM, respectively, as assessed
by May-Grünwald-Giemsa staining (Fig. 4A). The numbers
of CFCs in these cultures, examined in semisolid
methylcellulose-containing media, were also diminished to 2.5% of
those in the control cultures (Fig. 4A). Because L cells
themselves can respond to W3a-L-CM (17), the
possibility remained that Wnt-3a produced by Wnt-3a-transfected L cells
acts directly on these L cells and changes the contents of the culture
supernatant compared with that of the parental L cells. Such a
difference of contents might indirectly affect the results observed in
the culture to which W3a-L-CM was added. To exclude such a
possibility, we collected the CM of L cells that had been stimulated
with W3a-L-CM for 48 h and cultured for an additional
24 h after washing. In these L cells, high levels of cytosolic
-catenin last for at least 12 h after washing (data not shown).
This CM, designated as LW3a-CM, did not cause
effects distinct from those of L-CM in Dexter-type cultures
(Fig. 4A), suggesting that the dramatic changes observed in
these cultures were caused by Wnt proteins.
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, E and F). The number of cells
recovered in W3a-L-CM-supplemented cultures was only about
one-fifth of that recovered from the control (Table I). Furthermore, when these cells were analyzed by flow cytometry,
B220+ cells in W3a-L-CM-supplemented
cultures constituted only 2.3% of the population, and the total number
of B220+ cells per well was reduced to 0.6% of
that in the cultures with L-CM (Table I). About a 2-fold
increase of the number of Mac-1+ macrophages was
observed in W3a-L-CM-supplemented cultures (Table I). These
effects of W3a-L-CM were not found with LW3a-CM
(Table I).
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B,
left). The degree of reduction of osteoclasts fluctuated
highly from experiment to experiment, resulting in the generation of
from 0.8% to 42% of the number in the cultures treated with
L-CM.
To discriminate whether these inhibitory effects of W3a-L-CM on
hemopoiesis are direct effects on hemopoietic cells or indirect effects
exerted through stromal cells, we performed stromal cell-free culture
for osteoclasts and the colony formation assay in methylcellulose for
other lineages using BM cells. Sephadex G-10 column-purified BM
hemopoietic cells were induced to differentiate into osteoclasts with
M-CSF and soluble RANKL under stromal cell-free conditions in the
presence of W3a-L-CM or L-CM. Under such conditions, no inhibitory
effect of W3a-L-CM was observed (Fig. 4B, right),
suggesting that W3a-L-CM controls osteoclastogenesis through
stromal cells.
To clarify the basis of these results, we conducted Northern hybridization analysis for M-CSF, RANKL, and osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor, a decoy receptor for RANKL that is also known to be produced by stromal cells (34, 35), to examine whether the reduction of osteoclasts is merely attributable to the expression levels of these molecules. Analyses using an image analyzer suggested that W3a-L-CM changes the expression levels of RANKL and OPG in ST2 cells in the presence of 1,25(OH)2D3 and DEX to about one-half and 1.6-fold the levels in control L-CM, respectively. However, these changes of the expression levels of these factors could not fully explain the reduction of osteoclasts, because the addition of an excess amount of RANKL to the culture only partially rescued the osteoclastogenesis (data not shown).
We also examined the direct effects of W3a-L-CM on hemopoietic
cells by adding it to colony formation assays. As shown in Fig. 4C, no inhibitory effect of W3a-L-CM on the
colony-forming activity (CFU-granulocyte (CFU-G), CFU-macrophage,
CFU-granulocyte-macrophage, burst-forming unit-erythroid, and
CFU-IL-7) was observed. The size of colonies was also
similar in W3a-L-CM- and L-CM-supplemented
cultures. Also, the colonies derived from CFU-granulocyte-macrophage
and CFU-G contained normal mature neutrophils (data not shown).
These results suggest that W3a-L-CM inhibits hemopoiesis through the
stromal cells.
Establishment of a Tc-regulatory system to express
-catenin in ST2
The experiments described above strongly suggested that Wnt
proteins might control hemopoiesis through stromal cells. However, the
following possibilities remained: 1) Wnt-3a might signal hemopoietic
cells directly, and generate phenotypes only when stromal cell-derived
factors are present. 2) Wnt-3a and other factors in the CM of L cells
might act synergistically on stromal cells. To overcome these problems
and avoid the difficulty of purifying Wnt proteins with biological
activity, we established ST2 cells that constitutively express a
Tc-controlled transactivator (19) and express a stabilized
form of -catenin (21) under the control of the human
CMV*1 promoter, a Tc-responsive promoter (19). In these
cells (named ST2b), the expression of the stabilized form of
-catenin is induced in the absence of Tc, mimicking the state of ST2
cells stimulated with Wnt proteins (21). As described in
Fig. 5A, the expression of the introduced -catenin, which is
tagged with the HA epitope and detected as a band shifted relative to
endogenous -catenin, was induced by the decrease of Tc in a
dose-dependent manner from 5 to 0 ng/ml. When the expression of the
stabilized -catenin was induced by the removal of Tc, overgrowth of
ST2b was observed, along with an increase of the -catenin level in
these cells (Fig. 5, B and C). However, such
changes were observed 1 wk after the cells reached confluency in medium
containing 10% FCS, but not in 5% FCS, although the effects of
W3a-L-CM are detected immediately after the cells reach confluency, and
these effects are detected even in 5% FCS. This might be explained by
the fact that cytosolic -catenin levels are significantly higher
when cells are stimulated with W3a-L-CM (compare Figs. 1C
and 5A). In any case, up-regulation of the cytosolic
-catenin level in stromal cells causes a phenotype similar to the
state induced by W3a-L-CM.
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-catenin
We next investigated whether the expression of stabilized
-catenin changes the ability of stromal cells to support the
development of hemopoietic lineages. We conducted Dexter-type culture
on ST2b cells for the myeloid lineage cells. In the absence of Tc, no
change of the number of macrophages was detected (data not shown).
However, the numbers of neutrophils and mast cells were reduced to
7–16% and 29–38% of those in Tc-containing cultures,
respectively (Fig. 6A). Hemopoietic colonies on ST2b cells seemed to be reduced
in size when -catenin was induced (Fig. 6B). In contrast
to this appearance, these cultures contained equivalent numbers of
cells that formed colonies in methylcellulose, as shown in Fig. 6A, suggesting that changes of the adhesive properties cause
such progenitors to become detached from stromal cells. Because ST2b
cells overgrew in Dexter’s conditions upon the removal of Tc, we
examined whether these phenotypes were also reproducible even when ST2b
cells were mitotically inactivated with mitomycin C. The reduction of
neutrophils and mast cells was observed even in these conditions,
suggesting that the changes of the hemopoiesis-supporting ability of
stromal cells were not secondary effects of their overgrowth (Fig. 6E, data not shown). The modest reduction compared with that
in untreated cultures might be explained by the lower level of
induction of -catenin in mitomycin C-treated cultures. The
generation of osteoclasts was also affected by the induction of
stabilized -catenin. The number of
TRAP+-multinucleated osteoclasts produced was
reduced to 49–66% of that in the culture containing Tc (Fig. 6D). In contrast, no significant changes of B lymphopoiesis
were observed when the expression of the stabilized -catenin was
induced in ST2b in Whitlock-Witte-type cultures in the presence of
IL-7. These B lineage cells also differentiated normally to the
µ-chain+ stages whether the stabilized
-catenin was induced or not (Fig. 6C and Table I).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-catenin is stabilized and
translocated to the nucleus upon the stimulation with Wnt3a-L-CM.
W3a-L-CM promoted the growth of ST2 cells after they reached
confluency. ST2b cells also overgrew 1 wk after the induction of a
stabilized form of -catenin by removal of Tc. Thus, we showed that
stromal cells can respond to Wnt signaling and are candidate targets of
Wnt proteins in hemopoietic microenvironments.
Wnt proteins are also known to act directly on hemopoietic cells
(7, 8, 9). We observed the stabilization of -catenin in
Sephadex G-10 column-purified BM hemopoietic cells and several
hemopoietic cell lines stimulated with W3a-L-CM (data not shown).
However, the following results support the view that Wnt proteins
regulate hemopoiesis more markedly through stromal cells. First,
W3a-L-CM suppressed the long-term BM cultures (Dexter’s type-culture
of the myeloid lineages and Whitlock-Witte-type culture of the B
lineage cells) on ST2. In contrast, W3a-L-CM did not suppress the
colony formation in semisolid media. Second, W3a-L-CM inhibited
osteoclastogenesis in the presence of stromal cells, but not in absence
of stromal cells. And finally, induced expression of a stabilized form
of -catenin in stromal cells, although not completely, mimicked the
effects of W3a-L-CM.
Different degrees of reduction of various hemopoietic lineages
were caused by the addition of W3a-L-CM in the presence of ST2 cells.
The production of B lineage cells was most severely affected: W3a-L-CM
reduced the number of B220+ cells by >99%. The
numbers of CFCs, neutrophils, and mast cells were reduced to 2.5%,
5%, and 10% of those in L-CM-treated cultures, respectively. The
number of osteoclast lineage cells was reduced to 0.8–40% of the
number in the control cultures. In contrast, the generation of
macrophage lineage cells was not affected much. These results suggested
that the responsiveness and sensitivities differ among the various
hemopoietic lineages.
Upon inducing the expression of a stabilized form of -catenin
by removal of Tc from the cultures, the generation of neutrophils, mast
cells, and osteoclasts was diminished to 12%, 34%, and 58% (on
average) of the levels generated in Tc-containing cultures,
respectively. The generation of macrophages was not affected by the
expression of this stabilized form of -catenin. These patterns of
selectivity and sensitivity were similar to those obtained with
W3a-L-CM. However, B lymphopoiesis and the generation of CFCs were not
affected, contrary to the effects of W3a-L-CM addition.
This difference might result from differences of the -catenin
levels, because W3a-L-CM induced higher levels of -catenin compared
with the Tc-regulated system. This notion might be supported by the
fact that the reduction of neutrophils, mast cells, and osteoclasts was
more severe when W3a-L-CM was added, and that W3a-L-CM induced changes
of cell growth more rapidly than the Tc-regulated system. The following
possibilities also remain: 1) W3a-L-CM might control B lymphopoiesis
and generation of CFCs synergistically with other factors produced by L
cells; 2) some effects of W3a-L-CM might be due to factors other than
Wnt-3a that are induced and secreted into the culture medium of
Wnt-3a-transfected L cells upon the stimulation with Wnt-3a produced by
themselves; 3) Wnt signaling pathways not mediated through the
stabilization of -catenin are responsible (10); or 4)
W3a-L-CM might act directly on the hemopoietic cells. The second
possibility might be ruled out by our experiments using
LW3a-CM. The interpretation of our results
according to the last possibility generates the view contrary to others
because it has been reported that Wnt proteins promote, rather than
suppress, the proliferation of multipotent and committed hemopoietic
progenitors (7, 8), and that the same W3a-L-CM we used
promotes the proliferation of fetal liver pro-B cells (9).
However, we could not exclude this possibility. As the reported
experiments were performed in suspension cultures, the effect of
W3a-L-CM on molecules involved in hemopoietic cell-stromal cell
interactions must also be discussed. For B lymphopoiesis, we have to
take it into consideration that we used adult BM cells, while Reya et
al. (9) used fetal liver cells. We must also pay attention
to the fact that CFU-IL-7 and hemopoietic progenitors that give rise to
B lineage cells in Whitlock-Witte culture are in different stage of
development (25). Although we demonstrated that the
colony-forming activity of CFU-IL-7 in methylcellulose was unaffected
by the addition of W3a-L-CM, the possibility remains that W3a-L-CM
directly suppressed B progenitors in earlier stage. We have to conduct
more detailed analyses for the last possibility.
In contrast to our results, it was reported that the CM of 293
cells containing Wnt-5a proteins expands the myeloid and lymphoid CFCs
even in cocultures with stromal cells (7). This might be
due to differences of the properties of different Wnt proteins, since
the ability to stabilize -catenin was not reported for Wnt-5a
(36); rather, it might antagonize the function of other
Wnts that stabilize -catenin (37).
The target genes of Wnt signaling in stromal cells are still
unclear. In the case of osteoclasts, slight changes of the expression
of the RANKL and OPG/osteoclastogenesis inhibitory factor genes were
observed. However, these changes do not fully explain the reduction of
osteoclasts because the addition of an excess amount of RANKL to the
cultures only partially rescued the osteoclastogenesis, suggesting that
multiple genes are the targets of Wnt signaling in stromal cells. The
following evidence suggests that changes of adhesive properties might
be involved in the phenomena we observed. First, the hemopoietic cells
were loosely attached to stromal cells in the cultures containing
W3a-L-CM. Second, the colonies attached to stromal cells were small in
size, but the number of CFCs was not affected in the cultures induced
to express the stable form of -catenin. However, further analyses
are needed to clearly verify this hypothesis.
It had been reported that mouse hemopoietic tissues express Wnt-3a, -5a, and -10b. Interestingly, it is reported that Wnt-5a is expressed in both stromal cells and hemopoietic cells, while Wnt-3a and Wnt-10b are expressed specifically in the hemopoietic compartment (7, 9). This suggests that Wnt proteins secreted from hemopoietic cells bind frizzled receptors on stromal cells, transduce signals, and regulate the functions of the stromal cells. This possibility suggesting a reciprocal relationship between hemopoietic cells and stromal cells is very intriguing because such interactions may contribute to the homeostatic balance of hemopoietic cells.
The expression of other modulators of Wnt signaling, e.g., Frps
(10), in hemopoietic tissue would be worth investigating.
Also, some signals other than Wnt protein-elicited ones are known to
up-regulate the level of -catenin. For instance, integrins and
growth factors stabilize -catenin through integrin-linked kinase
(38, 39). Indeed, ST2 cells express integrin-linked kinase
(data not shown). These signaling pathways might also participate in
the regulation of the level of -catenin in stromal cells and might
control hemopoiesis.
In conclusion, we showed in this study that stromal cells are a target of Wnt proteins, and that the production of hemopoietic lineages is regulated by Wnt signaling with selectivity and different sensitivities of the various hemopoietic lineages in the presence of stromal cells, i.e., under conditions reflecting the hemopoietic microenvironments in the body.
|
Acknowledgments |
|---|
-catenin constructs; Dr. Hermann
Bujard (Heidelberg University, Heidelberg, Germany) for the plasmids of
the Tc-regulatory system; Dr. Shin-Ichi Nishikawa (Kyoto University)
for AFS98; Dr. Kazuya Yamanishi (Otsuka Pharmaceutical) for M-CSF; Dr.
Masaki Hirashima (The Chemo-Sero Therapeutic Research Institute,
Kumamoto, Japan) for stem cell factor; and Dr. Tetsuo Sudo (Toray
Industries) for IL-3 and IL-7. We also thank Hisashi Tagaya and Toshie
Shinohara (Tottori University, Yonago, Japan) for helpful advice and
secretarial assistance, respectively. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Toshiyuki Yamane, Department of Immunology, School of Life Science, Faculty of Medicine, Tottori University, 86 Nishi-machi, Yonago 683-8503, Japan. E-mail address: yamanet{at}grape.med tottori-u.ac.jp
3 Abbreviations used in this paper: BM, bone marrow; CFC, colony-forming cell; CM, conditioned medium; DEX, dexamethasone; HA, hemagglutinin; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; OPG, osteoprotegerin; RANKL, receptor activator of NF-B ligand; Tc, tetracycline; TRAP, tartrate-resistant acid phosphatase; CFU-G, CFU-granulocyte.
Received for publication February 22, 2001. Accepted for publication May 11, 2001.
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References |
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-catenin is regulated in Xenopus embryos by glycogen synthase kinase 3. Genes Dev. 10:1443.-catenin. Cell Growth Differ. 8:1349.[Abstract]
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