©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of Wnt5a mRNA Expression in Human Mammary Epithelial Cells by Cell Shape, Confluence, and Hepatocyte Growth Factor (*)

Emmanuel L. Huguet , Kenneth Smith , Roy Bicknell , Adrian L. Harris (§)

From the (1) Growth factors Group, Imperial Cancer Research Fund, University of Oxford, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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.


MATERIALS AND METHODS

All laboratory reagents were from Sigma unless otherwise stated.

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.


RESULTS

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.

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.


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.

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.


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.




DISCUSSION

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 21 integrin raising the possibility that Wnt5a also interacts with this system of cell adhesion.

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.


FOOTNOTES

*
This work was funded by the Imperial Cancer Research Fund and the Oxford District Research Committee. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

The abbreviations used are: FACS, fluorescence-activated cell sorting; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HGF, hepatocyte growth factor.


ACKNOWLEDGEMENTS

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.


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