E-cadherin Negatively Regulates CD44-Hyaluronan Interaction and CD44-mediated Tumor Invasion and Branching Morphogenesis*

Yin Xu and Qin YuDagger

From the Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Received for publication, August 9, 2002, and in revised form, December 31, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

CD44 is a principal cell-surface receptor for hyaluronan (HA). Up-regulation of CD44 is often associated with morphogenesis and tumor invasion. On the contrary, reduction of cell-cell adhesion due to down-regulation of E-cadherin is associated with the invasive and metastatic phenotype of carcinomas. In our current study, we investigated the functional relationship between CD44 and E-cadherin. We established an inverse correlation between CD44 and E-cadherin indicating that the cells expressing higher levels of E-cadherin display weaker binding affinity between CD44 and HA. By using TA3 murine mammary carcinoma (TA3) cells, which display CD44-dependent HA binding, branching morphogenesis, and invasion, we demonstrated an inverse functional relationship between CD44 and E-cadherin by transfecting exogenous E-cadherin into the cells. Our results showed that increased expression of E-cadherin in TA3 cells, but not ICAM-1, weakens the binding between CD44 and HA and blocks spreading of the cells on HA substratum and CD44-mediated branching morphogenesis and tumor cell invasion. The results reported here demonstrated for the first time that E-cadherin negatively regulated CD44-HA interaction and CD44 function and suggested that balanced function of CD44 and E-cadherin may be essential for normal epithelial cell functions, and imbalanced up-regulation of CD44 function and/or down-regulation of E-cadherin function likely contributes to tumor progression.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Morphogenesis in physiologic conditions and tumor invasion in pathologic situations are regulated by cell-cell and cell-extracellular matrix (ECM)1 adhesion (1, 2). CD44 is a principal cell-surface receptor for hyaluronan (HA), a major component of the ECM (3-5). HA and CD44 are often up-regulated at the sites of morphogenesis, inflammation, and tumor invasion (6-13). CD44 has been shown to play important roles in tumor invasion, growth, and metastasis (14-19).

The key molecule that maintains epithelial cell-cell adhesion and integrity is E-cadherin. E-cadherin is a Ca2+-dependent transmembrane receptor that mediates cell-cell adhesion at the adherent junctions via homophilic binding (20). Down-regulation of E-cadherin has been reported in many different cancers and is often associated with increased carcinoma invasion and metastasis and poor prognosis (21-24). The combination of reduced expression of E-cadherin and increased expression of CD44 was reported in renal cancers and correlated with poorer prognosis (25-27). However, the functional relationship between E-cadherin and CD44 and the significance of the correlation of these two molecules during epithelial morphogenesis and tumor invasion have not been previously explored.

To study the functional relationship between CD44 and E-cadherin, we first surveyed the expression profiles of E-cadherin and CD44 in several epithelial and carcinoma cell lines, and we found that the cells expressing higher levels of E-cadherin generally displayed reduced CD44-HA interaction, although the expression of CD44 is not necessarily reduced in these cells (Fig. 1). To confirm that E-cadherin negatively regulates CD44-HA binding, to determine whether the negative regulation of E-cadherin was achieved by down-regulation of CD44 expression, and to investigate the effect of increased expression of E-cadherin on other CD44 functions, we transfected exogenous E-cadherin into TA3 murine mammary carcinoma (TA3) cells. Our results showed that TA3 cells expressed a low level of E-cadherin and a high level of CD44 and displayed CD44-dependent HA-binding, branching morphogenesis, and invasion. By using TA3 transfectants expressing E-cadherin or ICAM-1 (as a negative control), we investigated the effect of increased expression of E-cadherin on CD44-HA interaction and on CD44 functions. Our results demonstrated that increased expression of E-cadherin, but not ICAM-1, reduced the binding between HA and CD44 in TA3 cells, blocked spreading of the cells on HA substratum and CD44-mediated branching morphogenesis, and reduced invasiveness of the cells. On the contrary, increased expression of E-cadherin affects neither spreading of the tumor cells on fibronectin substratum nor tyrosine phosphorylation of focal adhesion kinase (FAK) induced by the interaction between integrin and fibronectin, which indicates the effect of E-cadherin on CD44-HA binding and CD44 function is specific.

The results obtained here demonstrated for the first time that E-cadherin negatively regulates CD44-HA interaction and CD44 functions, and suggested that E-cadherin may exert its tumor suppressor function by negatively regulating CD44 function. Balanced coordination between CD44 and E-cadherin may be essential for normal epithelial cell functions such as proliferation, migration, and morphogenesis, and imbalanced up-regulation of CD44 function and/or down-regulation of E-cadherin function likely promotes tumor progression.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture and Reagents-- TA3 cells and TA3 transfectants were maintained under the conditions described previously (10, 17, 28). MCF-10A mammary epithelial cell (MCF-10A), CMT-93 rectal carcinoma cell (CMT-93), Lewis lung carcinoma cell (LLCC), T47D, MCF-7 mammary carcinoma cells, HT29, T84, and LoVo colon adenocarcinoma cells, and A431 epidermoid carcinoma cells were obtained from the Cell Center Services Facility of the University of Pennsylvania and were maintained in the recommended conditions. Anti-E-cadherin (R&D Systems, Minneapolis, MN), anti-CD44, anti-ICAM-1 (ATCC, Manassas, VA), anti-alpha and beta -catenin, anti-FAK, anti-phosphorylated tyrosine (Transduction Laboratories, Franklin Lake, NJ), and anti-v5 epitope (Invitrogen) antibodies and TRITC-conjugated phalloidin (Sigma) were used in the experiments. Fibronectin and FL-HA was obtained from Sigma and Calbiochem, respectively.

Reverse Transcriptase (RT)-PCR and Expression Constructions-- RT-PCR was performed as described (17, 18, 28). Full-length murine E-cadherin and ICAM-1 cDNAs were amplified using the skin or spleen cDNAs as templates, respectively, along with Pfu DNA polymerase (Stratagene, Menasha, WI), and the primer pairs corresponding to the most 5' or 3' end of 24 nucleotides of the coding sequences of each molecule, which are derived from the sequence data in the GenBankTM under accession numbers X06115 or X52264, respectively. The stop codons were omitted from the reverse primers in order to fuse E-cadherin and ICAM-1 to the C-terminal v5 epitope tag, which is in the expression vector (pEF6/V5-His TOPO, Invitrogen). Authenticity and orientation of the cDNA inserts were confirmed by DNA sequencing.

To detect the expression of integrins in TA3 cells, RT-PCR was performed as described above and using mRNAs derived from TA3 cells as templates and the forward primers corresponding to the nucleotides at 1801-1820 of integrin beta 1 and 2341-2260 of integrin alpha 5 and the reverse primers corresponding to the sequence of the last 20 nucleotides of the coding sequences of integrin beta 1 or alpha 5, which are derived from the sequence data in the GenBankTM under accession numbers Y00769 and X79003, respectively.

Stable Transfection-- TA3 cells were transfected using LipofectAMINE (Invitrogen) with the expression constructs containing cDNA inserts encoding mouse E-cadherin or ICAM-1 or with the expression vector alone. The transfected TA3 cells were selected for blasticidin (Invitrogen) resistance, and the expression level of v5 epitope-tagged E-cadherin or ICAM-1 by the clonal TA3 transfectants was determined by Western blotting with anti-v5 antibody as described previously (28).

Protein Extraction, Immunoprecipitation, and Western Blot Analysis-- The association of E-cadherin or CD44 with the actin cytoskeleton was determined by the ability of E-cadherin or CD44 proteins to resist extraction by Triton X-100. Briefly, TA3 transfectants were first extracted with CSK buffer (10 mM PIPES, pH 6.8, containing 300 mM sucrose, 150 mM NaCl, 3 mM MgCl2, 1 mM CaCl2, and 0.5% Triton X-100) for 10 min on ice and then extracted with 2× SDS/Laemmli sample buffer. Equal amounts of proteins derived from Triton X-100-soluble and -insoluble fractions of the lysis of different TA3 transfectants were analyzed by Western blotting with anti-E-cadherin, anti-v5 (R & D Systems and Invitrogen), or anti-CD44 antibody (mAb IM7.8.1, ATCC), which recognizes all the CD44 isoforms.

For the immunoprecipitation experiments, TA3 transfectants were lysed in the lysis buffer (50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 1% Triton X-100, 0.1% SDS, 2 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 2 µg/ml pepstatin A, and 0.5 units/ml aprotinin). The extracted proteins were used in the immunoprecipitation experiments with anti-v5 antibody and protein A beads (Pierce). The presence of alpha - and beta -catenins in the immunoprecipitated proteins was detected using anti-alpha or beta -catenin antibody, respectively (Sigma and Transduction Laboratories) in Western blot analysis.

Fluorescein-labeled HA (FL-HA) Binding Assay and Immunocytochemistry-- Various epithelial and carcinoma cells or TA3 transfectants were cultured in 35-mm dishes for 24 h; 20 µg/ml FL-HA was added to the cultured cells for additional 24 h. FL-HA binding assay was performed as described previously (18, 19).

In the immunocytochemical analysis, various epithelial, carcinoma, or the transfected TA3 cells were cultured in 35-mm dishes for 48 h and fixed with 3.7% paraformaldehyde. The fixed cells were washed with phosphate-buffered saline solution (PBS) and blocked with 2% bovine serum albumin. Antibodies against E-cadherin and CD44 were used to detect the appropriate antigens. Rhodamine (TRITC)-conjugated phalloidin (Sigma) was used to detect F-actin.

Cell Spreading on HA or Fibronectin Substratum and G8 Myoblast Monolayer Invasion Assay-- To test the abilities of various TA3 transfectants to spread on HA, heparin, or fibronectin substratum, 35-mm cell culture dishes were coated with 5 mg/ml HA or heparin (Sigma), or 20 µg/ml fibronectin, and air-dried. The coated dishes were rehydrated and washed with PBS before use. 5 × 105 TA3 transfectants were seeded into the coated dishes and cultured for 2 days. The cells were then fixed with 3.7% paraformaldehyde, and immunocytochemistry was performed to detect the distribution of CD44, which outlines the cell morphology.

G8 myoblast monolayer invasion assay was performed as described previously (17-19). The invasiveness of the cells was documented by counting the invasion lesions of TA3 cells on the fixed G8 monolayers in 10 randomly selected ×100 microscopic fields for each TA3 transfectant. The data are presented as means ± S.D.

Matrigel Branch Morphogenesis Assay-- 0.5 ml of Matrigel (Collaborative Biomedical) was solidified on top of each well of 12-well Transwell tissue culture inserts (Costar), and 1 × 106 TA3 transfectants in 0.2 ml of cell culture medium containing 2% FBS with or without preincubation of the cells with the blocking anti-CD44 mAb, KM201, were seeded onto the Matrigel. 1 ml of cell culture medium containing 2% FBS was added into each lower chamber, and 500 µg of HA together with or without KM201 was added into each upper chamber of the Transwell inserts. Six days later, the extent of branching morphogenesis of TA3 transfectants was observed under a light microscope and photographed.

FAK Tyrosine Phosphorylation Assay-- 100-mm cell culture dishes were coated with 20 µg/ml fibronectin overnight at 4 °C and washed with PBS. TA3 transfectants were lifted with EDTA solution and washed, and 5 × 106 of the cells were seeded on the fibronectin-coated or plastic dishes for 45 min to allow the cells to attach to the plates. The cells were then lysed in the RIPA buffer (50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 2 µg/ml pepstatin A, 0.5 units/ml aprotinin, 2 mM sodium orthovanadate, and 5 mM sodium fluoride). The cell extracts were clarified by centrifuge at 12,000 rpm, and the supernatants were subjected to immunoprecipitation with anti-FAK antibody and protein A beads. The immunoprecipitated proteins were eluted by 2× SDS sample buffer and analyzed by Western blotting with anti-FAK or anti-PY20 antibody (Transduction Laboratories).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression of E-cadherin Is Inversely Correlated to the Binding Capacity of CD44 to HA-- It is well established that cell-cell adhesion mediated by E-cadherin contributes to the contact inhibition and tumor suppression, whereas reduction of cell-cell adhesion as a result of down-regulation of E-cadherin is often associated with development of the invasive and metastatic phenotypes in carcinomas (24, 29, 30). On the contrary, CD44 and its ligand, HA, are often up-regulated and play important roles in promoting tumor invasion and metastasis (for reviews see Refs. 31-34). Furthermore, HA production is correlated with the cell cycle and peaks at mitosis, which is believed to promote cell rounding and detachment (35). To investigate whether there is an inverse functional correlation between CD44 and E-cadherin, we first examined the expression profiles of E-cadherin and CD44 in a normal epithelial cell (MCF-10A mammary epithelial cell) and several carcinoma cell lines (CMT-93 rectal carcinoma cell, HT29, T84, LoVo colon adenocarcinoma cells, A431 epidermoid carcinoma cell, T47D, MCF-7, and TA3 mammary carcinoma cells, and Lewis lung carcinoma cell), and we assessed the binding affinity of CD44 to FL-HA in these cells as well. We found that the cells expressing higher levels of E-cadherin generally displayed weaker binding affinity for FL-HA, whereas the CD44 expression level in these cells is not completely correlated with their binding affinity to FL-HA (Fig. 1). For example, HT29 cells (Fig. 1A, lane 3) express higher levels of CD44 and E-cadherin than LoVo cells (Fig. 1A, lane 8); however, LoVo cells bind to FL-HA with higher affinity than HT29 cells (Fig. 1, B and C, i and k). A431 cells (Fig. 1A, lane 5) express a similar level of CD44 and a higher level of E-cadherin compared with TA3 and LLC cells (Fig. 1A, lanes 9 and 10); however, A431 cells displayed lower affinity to FL-HA compared with TA3 and LLC cells (Fig. 1, B and C, j and l). This finding is consistent with the published data indicating that the binding of HA to CD44 is regulated, and higher expression of CD44 is not always correlated with higher ligand binding affinity (for reviews see Refs. 36 and 37).


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Fig. 1.   Inverse correlation between E-cadherin expression and CD44-HA binding. A, expression of E-cadherin (E-cad) and CD44 in several epithelial and carcinoma cell lines was determined by Western blotting with anti-Ecad (A, b, R&D Systems) or anti-CD44 antibody (A, a, IM7.8.1, ATCC) which recognizes all mouse CD44 isoforms and cross-reacts with human CD44. Equal amounts (50 µg/lane) of proteins were loaded into SDS-10% PAGE gels (a and b) and were derived from MCF-10A (lane 1), CMT-93 (lane 2), HT-29 (lane 3), T84 (lane 4), A431 (lane 5), T47D (lane 6), MCF-7 (lane 7), LoVo (lane 8), TA3 (lane 9), and LLC (lane 10) cells. Kd, kilodalton. The FL-HA binding affinities of these cells are summarized in B. -, no binding of FL-HA was detected; +/-, weak binding between FL-HA and the cells; +, moderate binding of FL-HA; ++, strong FL-HA binding; +++, very strong binding of FL-HA. C, FL-HA binding assay (g-l) was performed using FL-HA (20 µg/ml), and CD44 expression (a-f) was determined using anti-CD44 antibody, IM7.8.1, in MCF-10A (a and g), CMT-93 (b and h), HT-93 (c and i), A431 (d and j), LoVo (e and k), and TA3 (f and l) cells. Bar, 32 µm.

Characterization of TA3 Transfectants Expressing Exogenous E-cadherin-- To determine whether CD44-HA interaction and other CD44 functions are negatively regulated by E-cadherin and whether the negative regulation of E-cadherin in CD44-HA interaction is achieved by modulating CD44 expression, we transfected exogenous E-cadherin into TA3 cells. TA3 cells express a high level of CD44 (17, 18) and a low level of E-cadherin endogenously (Fig. 1A, lane 9), and display high affinity to HA in a CD44-dependent manner (17, 18) (Fig. 1C, l). TA3 cells were transfected stably with the expression constructs containing C-terminal v5 epitope-tagged mouse E-cadherin (Ecadv5) or mouse ICAM-1 (ICAM-1v5, as a control). Three independent clonal TA3 transfectants that express E-cadv5 (TA3Ecad) or ICAM-1v5 (TA3ICAM-1) were identified by Western blotting (Fig. 2A).


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Fig. 2.   Characterization of TA3 transfectants expressing v5-tagged E-cadherin. A, expression of v5 epitope-tagged E-cadherin or ICAM-1 by TA3 transfectants. Western blot analysis of the cell lysates of three independent clonal TA3 transfectants was performed using anti-v5 mAb. Full-length v5-tagged E-cadherin (Ecadv5) is indicated with a larger arrow, and the C-terminal cleavage fragment of E-cadherin (C-Ecadv5) is indicated with a smaller arrow. Three independent clonal TA3 transfectants transfected with the expression vectors alone (lanes 1-3, TA3wt) or expressing Ecadv5 (lanes 4-6, TA3Ecad) or ICAM1v5 (lanes 7-9, TA3ICAM1) are shown. B, association of E-cadv5 with the actin cytoskeleton. Proteins derived from Triton X-100-soluble (lanes 1-3) and -insoluble (actin-associated Ecadv5, lanes 4-6) fractions of three independent clonal TA3Ecad cells were analyzed by Western blotting with anti-v5 mAb. C, association of E-cadv5 with alpha - (a, arrow) or beta -catenin (b, arrow) was determined by Western blotting of the immunoprecipitated proteins from various cell lysates by anti-v5 antibody and protein A beads. a, anti-alpha -catenin antibody was used and anti-beta -catenin antibody was used in b. Proteins extracts were derived from TA3wt cells (a and b, lane 1) and three independent clonal TA3Ecad cells (a and b, lanes 2-4).

The Western blot results indicated that the extracellular domain of E-cadherinv5, but not that of ICAM-1v5, was shed spontaneously from the tumor cell surface and released into the cell culture medium, and the C-terminal fragment of the cleaved E-cadherin (C-Ecad) remained attached to the cells (Fig. 2A). The size of the full-length v5-tagged E-cadv5 is about 130 kDa (Fig. 2A, Ecadv5); and the C-terminal fragment of E-cadherin is about 40 kDa (Fig. 2A, C-Ecadv5).

Before we started to use TA3Ecad cells to study the effect of increased expression of E-cadherin on CD44-HA interaction and other CD44 functions, several experiments were performed to ensure that the exogenous Ecadv5 proteins displayed the same characteristics as that of wild-type E-cadherin (Ecadwt). Our results indicated that, like Ecadwt, Ecadv5 proteins are associated with the actin cytoskeleton as indicated by the presence of Ecadv5 proteins in the detergent-insoluble fraction (Fig. 2B). The interaction between Ecadv5 and alpha - or beta -catenin was demonstrated by the co-immunoprecipitation experiments (Fig. 2C). Thus, the Ecadv5 proteins expressed by TA3 transfectants displayed the expected biochemical properties of E-cadherin, and these transfectants were used in the following experiments.

Increased Expression of E-cadherin in TA3 Cells Reduces Binding of CD44 to FL-HA-- To study whether increased expression of E-cadherin affects CD44-HA interaction, we performed immunocytochemical analysis and FL-HA binding assay on TA3 transfectants expressing Ecadv5 (TA3Ecad) and ICAM-1v5 (TA3ICAM-1) or transfected with the expression vectors alone (TA3wt). Our data indicated that the localization of CD44 and organization of the actin cytoskeleton are altered in TA3Ecadv5 cells (Fig. 3, J and K) when compared with that in TA3wt or TA3ICAM-1 cells (Fig. 3, B and C, and F and G). In TA3wt and TA3ICAM cells, E-cadherin proteins are localized in a punctated fashion along the cell-cell junctions (Fig. 3, A and E); CD44 proteins form aggregates, and the actin filaments (F-actin) are colocalized underneath the CD44 aggregates (Fig. 3, B and C, arrows, and F and G). Ecadv5 proteins are localized along cell-cell junctions of TA3Ecad cells (Fig. 3I, arrows); CD44 proteins are concentrated along cell-cell junctions as well, and the actin cytoskeleton is organized in a similar fashion (Fig. 3, J and K). More importantly, the binding to FL-HA was dramatically reduced in TA3Ecad cells (Fig. 3L) when compared with that in TA3wt or TA3ICAM-1 cells (Fig. 3, D and H), although the level of CD44 expression was not significantly altered in these transfectants (Fig. 3, B, F, and J, and Fig. 8). This result provided the first direct evidence demonstrating that CD44-HA binding is negatively regulated by E-cadherin.


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Fig. 3.   Increased expression of E-cadherin reduces FL-HA binding. The localization of E-cadherin in TA3wt, TA3ICAM1, and TA3Ecad cells was revealed with anti-E-cadherin antibody (R&D Systems, A, E, and I). A and E show the punctated staining of endogenous E-cadherin in TA3wt cells (A) and TA3ICAM1 cells (E), whereas the arrows show strong staining of E-cadherin along the cell-cell junctions of TA3Ecad cells (I). Distributions of CD44 and F-actin in TA3wt (B and C), TA3ICAM1 (F and G), and TA3Ecad (J and K) cells were revealed by immunocytochemistry using anti-CD44 mAb (IM7.8.1, B, F, and J) and rhodamine-conjugated phalloidin (C, G, and K). FL-HA binding assay was performed using FL-HA (20 µg/ml). TA3Ecad (L) cells displayed dramatically reduced FL-HA binding when compared with TA3wt (D) and TA3ICAM1 (H) cells. Bar, 30 µm.

To determine the specificity of this negative effect of E-cadherin on CD44-HA interaction, we investigated whether functions of other ECM receptors such as integrins, which interact with actin cytoskeleton, might be affected by the increased expression of E-cadherin as well. We first surveyed the expression of integrins by TA3 cells. RT-PCR results showed that TA3 cells express integrin alpha 5beta 1, a receptor for fibronectin (Fig. 4A). We then investigated whether increased expression of E-cadherin affects FAK tyrosine phosphorylation induced by the interaction between integrin and fibronectin substratum. Our results showed that FAK phosphorylation is not affected by the increased expression of E-cadherin (Fig. 4B), which suggests the negative effect of E-cadherin on CD44-HA binding is not a nonspecific event.


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Fig. 4.   Increased expression of E-cadherin had no effect on FAK phosphorylation induced by the interaction between integrin and fibronectin. A, RT-PCR indicated that TA3 cells express integrin alpha 5 (lane 4) and beta 1 (lane 3), a receptor of fibronectin. Control RT-PCRs were performed in the absence of the revere transcriptase (lanes 1 and 2). B, tyrosine phosphorylation of FAK in TA3wt (lanes 1-3) and TA3Ecad cells (lanes 4-6) in response to fibronectin substratum (lanes 3 and 6). TA3wt and TA3Ecad cells were either kept in suspension (lanes 1 and 4) or attached to the plastic cell culture dishes (lanes 2 and 5) or fibronectin (lanes 3 and 6) for 45 min at 37 °C. Phosphorylated FAK (B, b, P-FAK) and total FAK proteins (B, a) were detected by anti-phosphotyrosine (pY) or anti-FAK antibody, respectively (Transduction Laboratories), in a Western blotting analysis of the immunoprecipitated proteins by anti-FAK antibody and protein A beads.

Increased Expression of E-cadherin Inhibits Spreading of TA3 Cells on HA Substratum-- To investigate whether E-cadherin affects spreading of TA3 cells on HA substratum and to confirm the effect of E-cadherin is specific, TA3 transfectants were culture on HA, heparin, or fibronectin substratum for 2 days. These cells were then fixed and stained with anti-CD44 antibody and fluorescence-conjugated secondary antibody to outline the plasma membrane of the TA3 transfectants, so that the extent of spreading of the cells can be clearly demonstrated. Our results showed that TA3wt and TA3ICAM-1 cells can spread on heparin, fibronectin, as well as HA substrata and form filopodia which is an indication of active locomotion of these cells on the substrata (Fig. 5, A, B, D, E, G, and H). On the contrary, TA3 transfectants expressing E-cadherin formed tight cell aggregates without spreading on the HA substratum and forming filopodia (Fig. 5F), although they displayed normal spreading on heparin and fibronectin substrata (Fig. 5, C and I). This result indicated that increased expression of E-cadherin blocks the spreading of the cells on HA substratum but not on heparin or fibronectin substrata that is mediated by the interaction between integrin and fibronectin.


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Fig. 5.   Increased expression of E-cadherin inhibits spreading of TA3 cells on HA substratum. 35-mm cell culture dishes were coated with 5 mg/ml HA or heparin (Sigma), or 20 µg/ml of fibronectin, and air-dried. The coated dishes were rehydrated and washed with PBS before seeding TA3 transfectants. 5 × 105 TA3 transfectants were seeded onto the coated dishes and cultured for 2 days and fixed with 3.7% paraformaldehyde and stained with anti-CD44 antibody, IM7.8.1, and fluorescence-conjugated secondary antibody to outline the plasma membrane and the extent of the cell spreading. TA3wt (A, D, and G) and TA3ICAM1 (B, E, and H) cells spread on HA (D and E), heparin (A and B), and fibronectin (G and H) substrata and form filopodia. On the contrary, TA3Ecad cells form tight cell aggregates without spreading on HA substratum (F), although they can spread on heparin (C) and fibronectin (I) substrata. Bar, 28 µm.

CD44-mediated Tumor Invasion Is Negatively Regulated by E-cadherin-- It has been shown that CD44-HA interaction is essential for CD44-mediated tumor invasion (17, 18). To investigate whether increased expression of E-cadherin may affect CD44-mediated tumor invasion as well, G8 myoblast monolayer invasion assay was performed as described previously (17, 18). G8 myoblast monolayer produces the HA-enriched ECM, and we have shown previously (17, 18) that invasion of TA3 cells through the Me2SO-fixed G8 myoblast monolayer and formation of tumor cell colonies are CD44-dependent.

Invasiveness of the TA3 transfectants was determined by the ability of the transfectants to invade through the G8 monolayers and form tumor cell colonies. We found that the increased expression of E-cadherin inhibits TA3 cell invasion through HA-enriched G8 monolayers (Fig. 6A). Compared with TA3wt and TA3ICAM-1 cells, approximately three to four times less invasion colonies were formed by TA3Ecad cells (Fig. 6B), and this reduction is comparable with that which occurred when CD44 function was compromised by overexpression of the dominant-negative soluble CD44 (Fig. 6B).


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Fig. 6.   Increased expression of E-cadherin inhibits tumor cell invasion. G8 myoblast monolayer invasion assays were performed using the Me2SO-fixed G8 monolayers. 5 × 103 various TA3 transfectants were seeded onto the monolayers and cultured for 7-10 days. The invasiveness of the cells was determined by counting the invasion lesions on the G8 monolayers in 10 randomly selected ×100 microscopic fields. The data were presented as means ±S.D. B, like TA3 cells expressing soluble CD44, in which CD44 function is compromised (TA3sCD44, A, b, and B), TA3 Ecad cells (A, d, arrows, and B) exhibited dramatically reduced invasiveness when compared with TA3wt cells (A, a, arrows, and B), and TA3ICAM-1 cells (A, c, and B). Bar, 250 µm.

Increased Expression of E-cadherin Blocks CD44-mediated Branching Morphogenesis-- Mammary gland is formed through branching morphogenesis of the pre-existing epithelial structures. It is well established that adhesion molecules and the ECM receptors play important roles in branching morphogenesis (38, 39). We established a branching morphogenesis model using TA3 cells as described under the "Experimental Procedures" and demonstrated that this process is dependent on CD44-HA interaction and can be blocked by the blocking anti-CD44 monoclonal antibody (mAb) KM201 (Fig. 7, A, D, and G).


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Fig. 7.   E-cadherin blocks CD44-mediated branching morphogenesis. 1 × 106 various TA3 transfectants in 0.2 ml of cell culture medium (Dulbecco's modified Eagle's medium containing 2% FBS) with or without preincubation of the cells with blocking anti-CD44 mAb, KM201, were seeded onto the Matrigel. 500 µg of HA with or without anti-CD44 antibody, KM201 (100 µg), was added into each upper chamber of 12-well Transwell plates. Six days later, the extent of the branching morphogenesis of TA3 transfectants was observed and photographed. TA3wt, TA3ICAM-1, and TA3Ecad cells were cultured in cell culture medium without other additives (A-C, respectively), with 500 µg of HA (D-F, respectively), or with preincubation and in the continuous presence of anti-CD44 antibody, KM201 (100 µg), and HA (500 µg, G-I, respectively). Bar, 400 µm.

To investigate whether increased expression of E-cadherin affects the HA-induced branching morphogenesis, the TA3 transfectants were tested for their ability to form branches in Matrigel in response to HA. The results showed that TA3wt and TA3ICAM-1 cells formed extensive branches in Matrigel in the presence of HA (Fig. 7, D and E), whereas TA3Ecad cells formed tight cell aggregates in Matrigel without extending branches (Fig. 7F). The dramatic difference in their abilities to undergo branching morphogenesis underscores once again that the function of CD44 is negatively regulated by E-cadherin. The inhibitory effect of E-cadherin on CD44-HA interaction likely underlies its negative effect on CD44-mediated branching morphogenesis.

Increased Expression of E-cadherin Alters the Distribution of CD44 in the Detergent Triton X-100-insoluble Fraction-- CD44 has been shown to be distributed in the detergent-resistant actin cytoskeleton fraction and lipid rafts (42, 44-46, 49-53). The CD44-containing lipid rafts interact with the underlying actin cytoskeleton (53), and the ezrin, radixin, and moesin (ERM) proteins are believed to mediate the interaction between CD44 and actin cytoskeleton (45, 49). Furthermore, the distribution of CD44 in the detergent-insoluble lipid rafts is involved in CD44-mediated T cells activation and Shigella infection (53, 54). To investigate whether E-cadherin exerts its negative effect on CD44-HA interaction and other CD44 functions by down-regulating the expression of CD44 or altering the distribution of CD44 in cell membrane, which likely represents the localization of CD44 in lipid rafts and/or the association of CD44 with actin cytoskeleton, we investigated the distribution pattern of CD44 in Triton X-100-soluble and -insoluble fractions in these TA3 transfectants. We found that increased expression of E-cadherin in TA3 cells reduced distribution of CD44 in the detergent-insoluble fraction without significantly altering CD44 expression (Fig. 8). This result indicated that E-cadherin negatively regulates CD44-HA interaction by altering the localization of CD44 in the cell membrane but not the expression of CD44 in TA3 cells.


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Fig. 8.   Increased expression of E-cadherin alters the distribution of CD44 in the Triton X-100-insoluble fraction. Distribution of CD44 in the detergent-insoluble fraction was determined by the ability of CD44 proteins to resist extraction by Triton X-100. Triton X-100-soluble (A) and -insoluble (B and C) fractions were subjected to Western blot analysis with anti-CD44 antibody, IM7.8.1 (A and B), or anti-beta -actin antibody (C). Lanes 1-3, proteins from TA3wt cells; and lanes 4-6, proteins from TA3Ecad transfectants.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Our results reported here showed that the epithelial and carcinoma cells that express higher levels of E-cadherin displayed reduced CD44-HA binding (Fig. 1). Furthermore, increased expression of E-cadherin in TA3 cells, which express a low level of E-cadherin endogenously, inhibits CD44-HA interaction and blocks CD44-mediated cell spreading on HA substratum, branching morphogenesis, and tumor cell invasion. These results demonstrated for the first time that E-cadherin negatively regulates CD44-HA interaction and several important CD44 functions.

Many studies (36, 37) have demonstrated that binding affinity of HA to CD44 is regulated, and higher expression levels of CD44 are not always correlated with increased binding affinity between CD44 and HA. Different glycosylation status of CD44 is believed to play an important role in regulating CD44-HA interaction (36, 37), and beyond that little is known. CD44 has been described as a downstream target gene of T cell factor. T cell factor forms transcriptional complexes with beta -catenin, which is induced by the Wnt signaling pathway (55, 56). The level of cytosolic and nuclear beta -catenin and the formation of T cell factor/beta -catenin transcription factors are negatively regulated by the formation of E-cadherin-beta -catenin complex in the adherence junctions (57, 58). Although there is no direct evidence established yet, it is conceivable that E-cadherin can down-regulate the expression of CD44 in some epithelial and carcinoma cells. This hypothesis is consistent with our observation that some (not all) epithelia and carcinoma cells expressing higher levels of E-cadherin generally express lower levels of CD44 (Fig. 1). However, the reduced CD44-HA binding affinity is more closely correlated with the expression levels of E-cadherin rather than with the expression levels of CD44. For example, HT-29 and A431 cells expressing higher levels of E-cadherin and intermediate or high levels of CD44 display weaker CD44-HA binding compared with LoVo and TA3 cells expressing lower or similar levels of CD44 but lower levels of E-cadherin (Fig. 1).

To confirm that increased expression of E-cadherin can negatively regulate CD44-HA interaction and the negative effect is not achieved by down-regulation of CD44 expression, we introduced exogenous E-cadherin into TA3 cells. Our results demonstrated that the increased expression of E-cadherin in TA3 cells, but not that of ICAM-1, reduces the binding between CD44 and HA, and the negative regulation of CD44-HA interaction is not achieved by reducing CD44 expression. Furthermore, the increased expression of E-cadherin affects neither FAK phosphorylation induced by the interaction between integrin and fibronectin substratum nor the spreading of the TA3Ecad cells on fibronectin substratum, indicating that the negative effect of E-cadherin on CD44-HA interaction is a specific event.

This finding provided a novel and potentially important negative regulatory mechanism for CD44-HA interaction and a potential molecular basis for coordination of cell-cell and cell-matrix adhesion. Negative regulation of CD44-HA interaction by E-cadherin would on the one hand block CD44 function and allow epithelial cells to establish cell-cell adhesion and reach quiescence, and on the other hand mount a quick response to a changing microenvironment (e.g. formation of a wound) and turn on CD44-HA interaction immediately by down-regulating E-cadherin function (e.g. shedding of E-cadherin by matrix metalloproteinases) without requirement of protein synthesis. Thus, the balanced function of E-cadherin and CD44 may be essential for normal epithelial cell proliferation, migration, and morphogenesis, and imbalanced up-regulation of CD44 function and/or down-regulation of E-cadherin function likely contributes to tumor progression.

Many CD44 functions are mediated by the interaction between CD44 and HA, including adhesion to and migration on HA substratum, morphogenesis, and tumor cell invasion. Thus, inhibition of CD44-HA interaction by E-cadherin likely underlies its negative effects on these CD44 functions. By performing the G8 myoblast monolayer invasion assay, which was established as an indicator of CD44-dependent tumor cell invasion (17, 18), we demonstrated that increased expression of E-cadherin blocks CD44-mediated TA3 cell invasion through the HA-enriched ECM (Fig. 6), and this result implied that E-cadherin may exert (at least partially) its tumor suppressor function by blocking CD44-mediated tumor invasion.

Branching morphogenesis is essential for the formation of several important organs, including mammary gland, and is an ideal model to study coordination between cell-cell and cell-matrix adhesion and cell motility. Studies have shown that the CD44-HA functional axis plays an important role in branching morphogenesis (11, 38, 39, 41). We have established a branching morphogenesis model using TA3 cells and demonstrated that branching morphogenesis of TA3 cells is dependent on CD44-HA interaction, and the increased expression of E-cadherin blocks this process. This result indicated that CD44 and E-cadherin are two important molecules that likely play key opposite roles in branching morphogenesis.

CD44 is distributed in Triton X-100-insoluble actin cytoskeleton fraction and lipid rafts (50-53), which have been shown to regulate CD44 function (46-48, 54). Our results showed that increased expression of E-cadherin reduces the distribution of CD44 in the detergent-insoluble lipid rafts and/or actin cytoskeleton fraction. This result implies that E-cadherin may regulate the CD44-HA interaction and CD44 function by modulating the association of CD44 with lipid rafts and/or the actin cytoskeleton, which may affect lateral movement of CD44 proteins on plasma membrane and formation of dynamic CD44 aggregates that is important in modulating CD44-HA interaction (18, 40), and may also block accumulation of CD44 proteins in filopodia, therefore inhibiting the ability of CD44 to interact with HA substratum and promote cell motility.

    ACKNOWLEDGEMENT

We acknowledge the generous support to the University of Pennsylvania, School of Veterinary Medicine from the Commonwealth of Pennsylvania.

    FOOTNOTES

* This work was supported in part by a Start-up fund from University of Pennsylvania, School of Veterinary Medicine.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported in part by United States Department of Defense Grant DAMD 17-02-1-0650. To whom correspondence should be addressed: 372E (old vet), 3800 Spruce St., Dept. of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104. Tel.: 215-898-2967; Fax: 215-898-0719; E-mail: qyu@vet.upenn.edu.

Published, JBC Papers in Press, January 2, 2003, DOI 10.1074/jbc.M208181200

    ABBREVIATIONS

The abbreviations used are: ECM, extracellular matrix; HA, hyaluronan; TA3, TA3 murine mammary carcinoma; LLC, Lewis lung carcinoma; Ecad, E-cadherin; Ecadv5, v5-tagged E-cadherin; FL-HA, fluorescein-labeled hyaluronan, PBS, phosphate-buffered saline solution; mAb, monoclonal antibody; RT, reverse transcriptase; FBS, fetal bovine serum; TRITC, tetramethylrhodamine isothiocyanate; PIPES, 1,4-piperazinediethanesulfonic acid; FAK, focal adhesion kinase.

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TOP
ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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