From the
The Wnts are a family of genes with a role in cell fate and
morphological development in numerous embryonic and adult tissues. In
mouse mammary tissue a subset of the Wnts have a function in the normal
development of the gland, and abberant expression of Wnts normally
silent in this tissue causes mammary carcinomas. We have previously
shown that Wnt5a expression is elevated in the epithelial component of
proliferative lesions of human breast and have therefore examined the
regulation of Wnt5a mRNA expression in the human mammary epithelial
cell line HB2, which has a luminal phenotype and thus represents the
most commonly transformed cell type in human breast cancer. Wnt5a was
up-regulated 30-fold at confluence. This up-regulation was induced
specifically by confluence and not by the growth arrest that
accompanied it. In addition, Wnt5a was down-regulated 3-fold by changes
in cell shape associated with the transition from growth on a
two-dimensional surface (flat cell morphology) to growth in
three-dimensional gels (spherical cell morphology). Cytoskeletal
disruption with non-toxic doses of colchicine also induced a spherical
morphology and brought about a dose-dependent down-regulation of Wnt5a.
Wnt5a was also down-regulated 10-fold during the hepatocyte growth
factor-induced branching of HB2 cell aggregates in collagen gels. The
down-regulation of Wnt5a preceded the branching process. A similar
result was obtained with primary human breast epithelial populations
and the breast cancer cell line MDA468. We conclude that regulation of
Wnt5a expression is a downstream effect of signaling by hepatocyte
growth factor. These results are consistent with a role for Wnt5a in
mammary epithelial cell motility and are in accord with Xwnt5a's
function in embryonal cell migration. If Wnt5a's function in
human mammary epithelial cells is similar to that of Xwnt5a, its
up-regulation at confluence may be a mechanism for inhibition of cell
migration beyond confluence.
The Wnts are a large family of homologous but distinct genes,
which have been highly conserved across species in
evolution
(1) . The genes code for 45-kDa secreted cysteine-rich
proteins. While soluble Wnt1 is present in the conditioned medium of
Drosophila imaginal disc cells
(2) , the proteins are
generally tightly associated with the cell surface
(3) . The Wnts
are present in the embryonic and adult tissues of many species where
their importance in morphological development and cell fate
(4) is reflected by the severity of the
abnormalities
(5, 6, 7, 8) that result
from their aberrant expression.
Interest in the role of Wnts in
breast tissue stems from studies in the mouse. The development of the
murine mammary gland is associated with differential regulation of some
of the Wnts
(9, 10) , and aberrant expression of those
Wnts which are normally absent in the gland produces mammary
hyperplasia and carcinomas
(11) . Furthermore, in vitro,
the murine mammary epithelial cell line C57MG is transformed by Wnts 1,
2, 3a, 5b, 7a, and 7b but not by Wnts 5a and 4 (12). In addition, a
subset of the Wnts are expressed in human breast, and quantitative
differences exist in the Wnt expression profiles of normal mammary
tissue and proliferative lesions of the breast
(13) . These data
implicate Wnt genes in the biology of human breast tissue and possibly
in human breast pathology. In keeping with this, we have shown that
Wnt5a is up-regulated 10- and 4-fold in benign and malignant human
breast lesions, respectively, and that the overexpression of the gene
is localized to the mammary epithelium
(14) . Despite our and
others' observation of differential expression of Wnts in human
and murine systems respectively, the factors that control Wnt gene
expression are poorly understood. We have therefore investigated what
factors regulate Wnt5a gene expression in a human mammary epithelial
cell line (HB2)
(15) . This cell line has a luminal phenotype and
thus represents the most commonly transformed cell type in human ductal
breast cancer. Wnt5a mRNA expression was also investigated in primary
human breast epithelial cells in order to assess the suitability of the
HB2 cell line as a model of primary cell populations.
All laboratory reagents were from Sigma unless otherwise
stated.
To investigate the effect of confluence alone on Wnt5a
expression, HB2 cells were seeded on plastic and RNA harvested either
before or after they reached confluence. Fig. 4shows that
confluent cells (lane 1) express a higher level of Wnt5a than
subconfluent cells (lane 3). It was uncertain from this result
whether Wnt5a was regulated by confluence or proliferation since
densely grown cells are not only confluent, but also growth-arrested.
To determine whether Wnt5a up-regulation in dense cells was related
either to confluence or quiescence, Wnt5a expression on plastic was
compared in the following four conditions: 1) confluent HB2 s in full
medium (cells were confluent and quiescent); 2) confluent HB2s starved
for 48 h of fetal calf serum, hydrocortisone, and insulin (cells were
confluent and quiescent and provided a control for the effect of serum,
insulin, and hydrocortisone deprivation also found in condition 4); 3)
subconfluent HB2s in full medium (cells were subconfluent and
proliferating); and 4) subconfluent HB2s starved for 48 h of fetal calf
serum, insulin, and hydrocortisone (cells were subconfluent and
quiescent). Growth curves (Fig. 4c) demonstrated that
serum, insulin, and hydrocortisone deprivation for 48 h induced HB2
quiescence (without killing them) and that HB2 cells did undergo growth
arrest at confluence. Of the above, condition 4 determines whether
Wnt5a expression is controlled by confluence or by quiescence. If
confluence up-regulates Wnt5a, then subconfluent growth arrested cells
in condition 4 should express low levels of Wnt5a. However, if
quiescence up-regulates Wnt5a, these cells should express high levels
of Wnt5a. Fig. 4, a and b, shows that confluent
quiescent cells (lane 2) express 30-fold higher levels of
Wnt5a than subconfluent quiescent cells (lane 4). This shows
that confluence, rather than quiescence, is the major up-regulating
factor of Wnt5a.
To
further demonstrate the two independent regulating factors, we
investigated whether they might act in an additive fashion. Wnt5a
expression was compared in the following three conditions: 1) HB2s
grown to confluence on collagen; 2) subconfluent HB2s on collagen; and
3) HB2s grown in collagen. Fig. 5shows that Wnt5a is elevated in
confluent cells on collagen (lane 1) and low in subconfluent
cells on collagen (lane 2). However, in comparison to
subconfluent cells on collagen (lane 2), cells in collagen
(lane 3) express still lower levels of Wnt5a. Thus, in the
absence of the effect of confluence, a down-regulation of Wnt5a was
demonstrated during the transition from two-dimensional to
three-dimensional growth.
When seeded in a three-dimensional gel, HB2 cells initially
form a single cell suspension within the matrix but then give rise to
expanding spheres as the cells start to divide. The observation that
Wnt5a is expressed at a similar level when HB2 cells are cultured on
plastic or on a matrix, but that it is down-regulated when the cells
are cultured in the matrix suggest that the important regulating factor
is the transition from two dimensions to three dimensions, rather than
the presence or absence of a matrix component. This hypothesis is
supported by the fact that the result is reproducible with different
matrices. The down-regulation of Wnt5a in the transition from two to
three dimensions is observed if RNA is harvested before spheres have
formed, when the HB2 s are still a single cell suspension. Thus the
effect is produced in individual cells and is independent of cell to
cell contacts inherent in sphere formation. The transition from two to
three dimensions is associated with a change in cellular morphology, in
that cells grown in two dimensions have a flatened morphology, whereas
those grown in three-dimensional gels are rounded. The down-regulation
of Wnt5a in the transition from two to three dimensions may be related
to cell shape changes since altering cell shape with the
microtubule-disrupting drug colchicine reproduces the down-regulation
of Wnt5a.
The influence of cell shape on gene regulation is a well
recognized phenomenon (for a review, see Ref. 20), which has been
demonstrated in many cell types
(21, 22, 23) .
Closest to our model is the work of Streuli and Bissel
(24) with
mouse mammary epithelial cells. Streuli and Bissel observe that murine
mammary epithelial cells cultured on plastic or on a layer of collagen
have a flattened morphology and fail to express milk proteins. Altering
the physical properties of the gel produces cell shape changes
associated with differentiation and secretion of milk proteins.
Furthermore, Roskelley at al.
(25) have demonstrated that the
control of gene expression by the extracellular matrix is mediated by
both physical and biochemical effects. Shannon et al.(26) and Haeuptle et al.(27) observe similar
phenomena in mouse and rabbit mammary epithelial cells, respectively.
Altering cell shape using cytoskeletal disrupting agents in order to
regulate gene expression is supported by the work of Sevely et
al.(28) , who describe up-regulation of casein gene
expression by the microtubule inhibitor tubazole. Furthermore,
cytoskeletal elements other than microtubules may be disrupted to
produce similar effects. Using cytochalasins to interfere with
intermediate filament integrity, Unemori and Werb
(29) , Zanetti
and Solursh
(30) , and Ben-Ze'ev and Amsterdam
(31) reported gene regulation in synovial fibroblasts, limb
mesenchymal cells, and granulosa cells, respectively.
The mechanisms
of cytoskeletal gene regulation are only partially understood. It is
known that the cytoskeleton interacts with elements of 2° messenger
pathways. These interactions control 2° messenger function firstly
by altering the activity of some of its
proteins
(32, 33) , and secondly by influencing the
abundance of free active molecules by their physical sequestration in
the cytoplasm
(34) . The cytoskeleton may also influence gene
expression by similar interactions with transcription factors
(35) and mRNA species
(36) . In endothelium many genes
regulated by shear stress are known
(37) , and putative shear
response elements have been identified in the 5`-flanking regions of
these genes. One such sequence, GAGACC
(38) , is present in the
5` region of Wnt5a published by Clark et al.(39) , and
it is possible that this sequence may contribute to Wnt5a regulation by
mechanophysical events.
Our results show that Wnt5a is up-regulated
30-fold when HB2 cells reach confluence. Cell surface molecules known
to be up-regulated by cell confluence are few, but include DEP-1
(40) and CD31
(52) . The function of Wnt5a up-regulation
at confluence is unclear, but studies in Xenopus suggest a
role in cell motility control. Moon et al.(41) have
shown that overexpression of Wnt5a abolishes the migrational properties
of Xenopus embryonal blastula cap cells. Thus, if Wnt5a has a
similar function in human cells, its elevation at confluence may
represent a mechanism of inhibiting migration beyond confluence by
decreasing cell motility. The molecules which mediate contact
controlled migration in normal breast tissue and contact inhibition
in vitro are unknown, but the Wnts may be a candidate group as
they are cell surface molecules involved in the control of cell
proliferation and morphological development. The mechanism by which
Xwnt5a decreases cell motility in Xenopus is not understood,
but circumstantial evidence suggests that modulation of cell adhesion
molecules may be involved. First, exogenous overexpression of
N-cadherin in Xenopus blastula cap cells produces a
phenotype identical to that engendered by Xwnt5a
overexpression
(42) . Second, the signaling pathway of
Drosophila Wnt1 is known to involve both the cadherins and
catenins
(43) . However, it should not be assumed that Wnt5a will
have the same signaling pathway as Wingless since Wnt5a and Wnt1 fall
into two different subgroups on the basis of their transforming ability
(Wnt1 is transforming, Wnt5a is not
(12) ) and their effect on
gap junctions (Wnt1 enhances gap junctional permeability
(44) ,
Wnt5a does not
(45) )
Hepatocyte growth factor induces both
mitogenic and cell motility responses in many cell types including HB2
(see below). The HGF-induced branching of HB2 spheres in collagen gels
probably represents at least in part a cell motility response, since
proliferation alone would lead to more rapidly expanding spheres rather
than branching structures. The fact that Wnt5a is down-regulated in
association with cell motility during branching is, again, consistent
with the work of Moon in Xenopus. Taken together, Moon's
findings and ours suggest a general function for Wnt5a in the control
of cell movements: motility is decreased by elevated levels of Xwnt5a,
and conversely in this system, low levels of Wnt5a are observed during
cell migration. In the HB2 system, however, the correlation between
Wnt5a down-regulation and cell motility events does not prove that the
former induces the latter. Our findings place human Wnt5a downstream of
hepatocyte growth factor signaling and suggest one link in the
mechanism of cell motility control by this growth factor. Hepatocyte
growth factor (or scatter factor) is a 82-kDa disulfide-linked
heterodimeric protein, which acts via a 180-kDa receptor tyrosine
kinase encoded by the proto-oncogene c-met. Although HGF was originally
isolated as a hepatotropic factor, it is now clear that HGF and c-met
have much wider roles in the development of a variety of embryonic and
adult tissues
(46) . The mitogenic and cell motility promoting
functions of HGF are exemplified in numerous in vitro systems
(47, 48, 49) . The HGF-c-met axis
also has oncogenic potential since NIH3T3 cells co-expressing HGF and
c-met are transformed and tumorigenic
(50) . In relation to human
breast cancer, Yamashita et al.(51) have shown that
the expression of HGF in breast carcinomas correlates with poor
prognosis. Although the association between HGF expression and poor
prognosis has not yet been shown to be causal, HGF may produce a more
malignant phenotype in part by increasing cellular motility and
invasiveness. Berdichevsky et al. (15) showed in HB2 cells
that HGF down-regulated
We have
previously shown that the epithelia of benign and malignant human
breast lesions express, respectively, 10- and 4-fold higher levels of
Wnt5a than does that of normal breast tissue
(14) . This
expression profile of Wnt5a may be explained in different ways. The
higher level of Wnt5a observed in both benign and malignant
proliferative lesions may be brought about by the increased cell
density within these lesions, in a manner similar to the effect of
confluence in vitro. The fact that malignant tumors have lower
levels of Wnt5a than the benign lesions may be related to the
production of factors that decrease Wnt5a expression, such as
hepatocyte growth factor. Also, it is known from studies in the mouse
that Wnt5a is differentially regulated during morphological development
of the mammary gland
(9) . If similar regulation occurs in the
human, it is possible that the levels of Wnt5a seen in abnormally
differentiated lesions could occur if their constituent cells were
arrested in a particular state of differentiation associated with
elevated Wnt5a. In summary, our results show that in human mammary
epithelial cells, Wnt5a is regulated by cell shape, by confluence, and
by hepatocyte growth factor. These results have implications for the
role of Wnt genes in mammary epithelial cell biology.
We thank Dr. Joyce Taylor-Papadimitriou for giving us
the HB2 cell line, Dr. Michael O'Hare for supplying primary human
mammary epithelial cells, and Dr. Trevor Dale for useful discussions
during this study.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Cell Culture
Mycoplasma-free HB2 cells (a subclone of
the MTSV1-7 line
(15) obtained from Dr. Joyce
Taylor-Papadimitriou, Imperial Cancer Research Fund, Lincoln's
Inn Fields) were grown on Beckton-Dickinson tissue culture plates, in
RPMI medium (Clare hall Laboratories, Imperial Cancer Research Fund,
United Kingdom) with 10% fetal calf serum (Globepharm), 10 µg/ml
bovine insulin, and 5 µg/ml hydrocortisone, in a 95% air, 5% carbon
dioxide atmosphere, at 37 °C.
Use of Collagen, Matrigel, and Fibrin
Matrices
Collagen matrix (Vitrogen-100, Celtrix) was prepared
according to the manufacturer's instructions. Matrigel was from
Beckton-Dickinson. Fibrin matrix was prepared by polymerization of
fibrinogen (3 mg/ml in phosphate-buffered saline) with thrombin (2
units/ml final concentration). When using the above matrices as growth
substrates, HB2 cells were grown either as a monolayer on a
matrix-coated tissue culture plate or in three-dimensional matrix gels.
The medium of cells growing on or in fibrin and of control cells was
supplemented with -aminocaproic acid (300 mg/ml) to prevent
fibrinolysis. Matrices were digested before RNA extraction as follows:
collagen gels were digested with 1 mg/ml collagenase (crude type1a) in
phosphate-buffered saline, matrigel was digested with 50 units/ml
dispase (Collaborative Biomedical Products), and fibrin was digested
with plasminogen. In all experiments, cells grown on plastic were
subjected to the same digestion as those grown on or in a matrix.
Use of Colchicine
In the experiments investigating
the effect of colchicine on Wnt5a expression, subconfluent HB2 cells
were exposed for 24 h to medium containing colchicine at 0.01, 0.05,
0.1, and 0.5 µg/ml. Cell viability was assessed by trypan blue
exclusion.
Cell Cycle Experiments
HB2 cells were synchronized
using the method of the double thymidine block described by Johnson
et al.(16) . After synchronization cells were harvested
at 4 h time points over 24 h. At each time point, cells were aliquoted
into two fractions. One fraction was used for FACS(
)
analysis, while RNA was prepared from the remaining cells.
Use of MRC5-conditioned Medium and Hepatocyte Growth
Factor
To examine cell branching, cells were seeded into two
separate three-dimensional collagen gels (10 cells/ml of
gel) and grown with daily medium changes for 1 week to form spheres.
The following week, cells in the control gel were fed control medium
(RPMI, 10% fetal calf serum, insulin, and hydrocortisone), while cells
in the experimental gel were fed with either MRC5-conditioned RPMI
(obtained from Clare Hall Laboratories) supplemented with 10% fetal
calf serum, insulin, and hydrocortisone, or with control medium
supplemented with recombinant HGF at a final concentration of 10 ng/ml
(obtained from R&, UK). MRC5 cells
(17) were obtained from
the European Culture Collection.
Confluence and Growth Arrest Experiments
In
experiments investigating the effects of confluence and growth arrest
on Wnt5a expression, HB2 cells were grown under conditions which
achieved growth arrest either by supplement deprivation or by allowing
the cells to reach confluence. Thus, four plates containing equal
numbers of subconfluent HB2 cells were prepared (10 cells/dish) and grown for 2 days with daily medium changes. For
the following 48 h, cells in plate 1 were fed only RPMI, while those in
plates 2-4 were fed RPMI with fetal calf serum, insulin, and
hydrocortisone. RNA was harvested from the cells in plates 1 and 2 at
the end of the 48-h period, when both sets of cells were still
subconfluent. The cells in plates 3 and 4 were grown to confluence. At
confluence, cells in plate 3 were fed only RPMI for a period of 48 h,
while those in plate 4 continued to be fed their full medium. RNA was
harvested from plates 3 and 4 at the end this 48-h period.
RNase Protection
Antisense
[-
P]CTP- (Amersham) labeled transcripts of
Wnt5a were generated from a 384-base pair fragment of the gene cloned
in the plasmid bluescript KS (Stratagene)
(14) . A construct
containing a 180-base pair fragment of the gene
glyceraldehyde-6-phosphate dehydrogenase (GAPDH) cloned in the plasmid
pBluescript KS+ was used to generate antisense GAPDH
transcripts
(18) . RNase protection assays were performed as
described by Ausubel et al.(19) . In all assays the
GAPDH signal was used as a loading control.
Differential Expression and Regulation of Wnt Genes in
Different Growth Conditions
HB2 cells were seeded either onto
plastic culture dishes or in three-dimensional collagen gels. Cells
seeded on plastic formed a dense monolayer (Fig. 1, a1)
while those in collagen formed spheres (Fig. 1, a2)
which branched when exposed to MRC5-conditioned medium (Fig. 1,
a3). Wnt gene expression was studied under these different
growth conditions. Wnts 2, 3, 3a, 4, and 7a were not expressed in this
cell line whether cells were grown on plastic or in collagen in the
presence of MRC5-conditioned medium (Fig. 1c), but Wnt5a
and Wnt7b were detected. Wnt7b message was constant under all
conditions (Fig. 1b). In contrast, Wnt5a was
differentially expressed under the different growth conditions: Wnt5a
message was highest in cells on plastic (Fig. 1, blane 1) but showed a primary 10-fold down-regulation in
cells in collagen (Fig. 1, b, lane 2), and a
secondary 10-fold down-regulation associated with sphere branching
(Fig. 1, b, lane 3). The primary and secondary
down-regulations of Wnt5a expression in this system were further
investigated to identify the regulatory factors.
Figure 1:
a1, HB2 cells on plastic. 2,
HB2 form spheres in a three-dimensional collagen gel. 3, HB2
spheres form branching structures in the presence of MRC5 conditioned
medium or hepatocyte growth factor. b, RNase protection assay
showing Wnt5a and Wnt7b and corresponding GAPDH signals. Lane
1, HB2 cells on plastic. Lane 2, HB2 cells in
three-dimensional collagen gels. Lane 3, HB2 cells forming
branching structures. c, RNase protection assays showing
expression of Wnts 2, 3, 3a, 4, and 7a in HB2 cells on plastic
(lane 2) and in collagen with MRC5-conditioned medium
(lane 3). A positive control RNA sample is shown in lane 1 for Wnt2, Wnt3, and Wnt4. Wnt3a and Wnt7a were not expressed in
available human RNA samples.
Down-regulation of Wnt5a in the Transition from
Two-dimensional Growth to Three-dimensional Growth
To examine
whether the down-regulation of Wnt5a was brought about by the presence
of collagen, cells were grown either 1) on plastic, 2) on a surface of
collagen, or 3) in three-dimensional collagen gels. Fig. 2a shows that cells on plastic (lane 1) and on collagen
(lane 2) have equal levels of Wnt5a and that Wnt5a is
down-regulated only in cells seeded in three-dimensional collagen gels
(lane 3). Similar experiments were repeated using matrigel and
fibrin as different matrices. In each case the result shown in
Fig. 2a for collagen was duplicated: Wnt5a expression is
equal in cells grown on plastic and ona matrix, but is
down-regulated in cells grown ina matrix (data not
shown).
Figure 2:
a, RNase protection assay showing Wnt5a
and corresponding GAPDH signals. Lane 1, HB2 cells on plastic.
Lane 2, HB2 cells on collagen. Lane 3, RNA from HB2
cells in a three-dimensional collagen gel. b, RNase protection
assay showing Wnt5a and corresponding GAPDH signals. Lanes
1-3 as in a but RNA was harvested from cells 24 h
after seeding on plastic, on collagen, and in
collagen.
We next investigated whether the down-regulation of Wnt5a
was dependent on cell-cell interactions within the spheres. HB2 cells
were seeded on plastic, on collagen, and in collagen, and RNA harvested
24 h later, i.e. before sphere formation. Fig. 2b shows that the down-regulation of Wnt5a is apparent at 24 h after
seeding (Fig. 2b, compare lanes 1 and 2 to lane 3), confirming that regulation of the gene is not
dependent on sphere formation.
Down-regulation of Wnt5a in Response to
Colchicine
The appearance of HB2 cells on plastic or on a matrix
is different from that of cells in a three-dimensional matrix. When
growing in two dimensions the cells have a flattened morphology,
whereas they are rounded in three dimensions. It was possible that the
down-regulation of Wnt5a seen in the transition from two dimensions to
three dimensions may be related to cell shape changes during the
transition from a flattened to a rounded morphology. Thus, we examined
whether a cytoskeletal-disrupting drug, colchicine, might reproduce the
down-regulation of Wnt5a in monolayers of cells. On exposure to
colchicine the cells rounded up, acquiring a morphology similar to that
of cells in collagen. Concurrent with this change in morphology we
observed a dose-dependent down-regulation of Wnt5a in response to
colchicine (Fig. 3, a and b). Trypan blue
exclusion confirmed that the cellular morphological change and
down-regulation of Wnt5a occurred at nontoxic concentrations of
colchicine. Because colchicine arrests cells in mitosis, it was unclear
whether the down-regulation of Wnt5a was related to cell cycle events,
or to the disrupting effects of colchicine on the cytoskeleton. To
investigate this, HB2 cells were synchronized by double thymidine block
and the expression of Wnt5a analyzed at defined time points after
release from cell cycle control. Despite synchronization of the cells
as confirmed by FACS analysis, no regulation of Wnt5a could be
demonstrated as the cells subsequently cycled (data not shown).
Figure 3:
a,
RNase protection assay showing Wnt5a and corresponding GAPDH signals in
HB2 cells exposed to increasing concentrations of colchicine. Lane
1, no colchicine. Lanes 2-5, colchicine
concentrations of 0.01, 0.05, 0.1, and 0.5 µg/ml. b,
densitometric analysis of the data showing dose-dependent
down-regulation of Wnt5a.
Wnt5a Is Regulated Independently by Confluence and by the
Transition from Two-dimensional to Three-dimensional Growth
In
experiments comparing Wnt5a expression in cells on plastic to that in
cells in collagen, we observed that in the time taken for sphere
formation in collagen, cells seeded on plastic had grown to confluence
(Fig. 1, a1). Thus it was uncertain whether the
down-regulation of Wnt5a in the transition from growth on plastic to
growth in collagen was related to the change from confluence to
subconfluence, or to the transition from two-dimensional growth to
three-dimensional growth. We investigated these two factors
individually and found that Wnt5a was regulated independently by both
confluence and the transition from two-dimensional to three-dimensional
growth.
Figure 4:
a, RNase protection assay showing Wnt5a
and corresponding GAPDH signals. Lanes 1-4 correspond to
growth conditions 1-4 (see text for details). b,
densitometric analysis of the data showing up-regulation of Wnt5a by
confluence. c, HB2 growth curves. Growth curve 1,
subconfluent HB2 cells were seeded onto tissue culture plates and
allowed to grow for 2 days in full medium. From day 2 onward, they were
given medium free of serum insulin and hydrocortisone. Growth curve
2, subconfluent HB2 cells were seeded onto tissue culture plates
and allowed to grow in medium supplemented with fetal calf serum,
insulin, and hydrocortisone. These cells reached confluence on day
8.
To further investigate the effect of the transition
from growth in two dimensions to three dimensions independently of
confluence, HB2 cells were seeded either on plastic, on collagen, or in
collagen, and RNA harvested after 24 h when cells were still
subconfluent. Fig. 2b shows that in conditions under
which the effect of confluence is abolished in this way, a
2-3-fold down-regulation of Wnt5a is still observed.
Figure 5:
RNase
protection assay showing Wnt5a and corresponding GAPDH signals.
Lane 1, HB2 cells grown to confluence on collagen. Lane
2, subconfluent HB2 cells grown on collagen. Lane 3, RNA
from HB2 cells in a three-dimensional collagen
gel.
Wnt5a Is Down-regulated by MRC5-conditioned Medium and
Hepatocyte Growth Factor
Upon exposure to MRC5-conditioned
medium, the spherical HB2 cell aggregates branch (Fig. 1,
a3). Branching occurs approximately 48 h after exposure to the
conditioned medium. In association with this branching, Wnt5a is
down-regulated 10-fold (Fig. 1b). Fig. 6a shows that Wnt5a down-regulation occurred within 12 h of exposure
to MRC5-conditioned medium, and therefore preceded the branching
process. Hepatocyte growth factor is present in the medium of MRC5
cells, and recombinant hepatocyte growth factor reproduced both
branching and the down-regulation of Wnt5a (Fig. 6b).
Thus, Wnt5a is downstream of hepatocyte growth factor. The branching
assay was repeated using both primary human mammary luminal and
myoepithelial cells (supplied by Dr M. O'Hare, Ludwig Institute,
University College London, London, UK) and a breast carcinoma cell line
(MDA468). Like the HB2 cell line, spheres of MDA468 and primary human
mammary epithelial cells branch under the influence of MRC5-conditioned
medium, and the branching is associated with down-regulation of Wnt5a.
The branching structures formed by the MDA468 were less well defined
than those formed by the primary epithelial cells and the HB2 cells.
The magnitude of the Wnt5a down-regulation appeared to correlate with
the degree of branching. Thus, the Wnt5a down-regulation observed in
the HB2 cells (10-fold) was similar to that seen in the primary luminal
epithelial cells (6-fold) (Fig. 6c), but greater than
that seen in the MDA468 cells (approximately 3-fold.
Fig. 6d). The same RNA samples showed that Wnt7b
expression was constant in both the primary human mamary epithelial
cells and the MDA468 cells (Fig. 6, c and d,
respectively).
Figure 6:
a, RNase
protection assay showing Wnt5a and corresponding GAPDH signals. Time
course of the down-regulation of Wnt5a by hepatocyte growth factor.
Lane 1, HB2 cells in collagen without MRC5-conditioned medium.
Lanes 2-4, HB2 cells in collagen exposed to
MRC5-conditioned medium for 12, 24, and 48 h, respectively. b,
RNase protection assay showing Wnt5a and corresponding GAPDH signals.
Down-regulation of Wnt5a by recombinant hepatocyte growth factor.
Lane 1, HB2 cells in collagen in the absence of hepatocyte
growth factor. Lane 2, HB2 cells in collagen in the presence
of hepatocyte growth factor. Wnt5a level adjusted for GAPDH: 8:1
(lanes 1 and 2). c, RNase protection assay
showing Wnt5a, Wnt7b, and corresponding GAPDH signals. Down-regulation
of Wnt5a by MRC5-conditioned medium in primary human breast luminal
epithelial cells. Lane 1, primary cells in collagen in the
absence of MRC5-conditioned medium. Lane 2, primary cells in
collagen in the presence of MRC5-conditioned medium. Wnt5a level
adjusted for GAPDH: 6:1 (lanes 1 and 2). Wnt7b is
expressed at constant levels. d, RNase protection assay
showing Wnt5a, Wnt7b, and corresponding GAPDH signals. Down-regulation
of Wnt5a by MRC5-conditioned medium in MDA468 breast carcinoma cells.
Lane 1, MDA468 cells in collagen in the absence of
MRC5-conditioned medium. Lane 2, MDA468 cells in collagen in
the presence of MRC5-conditioned medium. Wnt5a level adjusted for
GAPDH: 3:1 (lanes 1 and 2). Wnt7b is expressed at
constant levels.
2
1 integrin raising the possibility
that Wnt5a also interacts with this system of cell adhesion.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.