Proc. Natl. Acad. Sci. USA Vol. 89, pp. 932-936, February 1992 Cell Biology

Syndecan expression regulates cell morphology and growth of mouse mammary epithelial tumor cells (extracellular matrix receptor/differentiation/tumor growth)

SIRPA LEPPA*t, MARKKU MALI*, HEINI M. MIETTINEN*,

AND MARKKU JALKANEN*t

*Department of Medical Biochemistry, University of Turku, SF-20520 Turku; and tCenter for Biotechnology, P.O. Box 123, SF-20521 Turku, Finland

Communicated by Elizabeth D. Hay, October 24, 1991

has been observed when simple and stratified epithelia are compared (14). Whether these changes also reflect altered ligand recognition by syndecan is unknown. Syndecan also binds growth factors, such as basic fibroblast growth factor (bFGF) (15). Yayon et al. (16) have shown that heparin-like molecules are required for the binding of bFGF to its highaffinity receptor, indicating that syndecan-like molecules can also modulate the growth factor response. That cell surface proteoglycans can bind both growth factors and matrix components suggests a role in regulating growth promotion, both temporally and spatially, by immobilizing these effector molecules in the vicinity of cell-matrix interactions. This is supported by the intriguing pattern of syndecan expression in the development that follows morphogenetic rather than histological patterns (5, 6, 17) and by the localization of syndecan expression to sites of active proliferation (7, 18). In simple epithelia, syndecan is polarized to basolateral surfaces, where it colocalizes with actin-rich cytofilaments (19). Upon rounding, syndecan is shed from the cell surface by proteolytic cleavage of the core protein at the cell surface, a process that separates the matrix-binding ectodomain from the membrane domain (20). Syndecan may thus be involved in the maintenance of epithelial morphology. We have recently shown that S115 mouse mammary tumor cells, when steroid-induced to modulate their morphology from an epithelial to a more fibroblastic or fusiform phenotype, lose syndecan expression (21). In this work we have transfected the same tumor cells with a full-length syndecan cDNA under the control of a steroid-inducible promoter. Reexpression of syndecan under these conditions restored the epithelial morphology of the S115 cells. Further, these cells lacked malignant growth behavior, suggesting that syndecan may also be involved in modulating the growth of epithelial cells.

ABSTRACT S115 mouse mammary epithelial cells lose their epithelial morphology and become tumorigenic when exposed to steroids. We have recently reported that testosterone exposure results in the suppression of syndecan expression, suggesting that this cell surface proteoglycan may influence S115 cell phenotype. We now report that a similar suppression and morphological response of S115 cells can be achieved by glucocorticoid exposure. We introduced into S115 cells an exogenous gene construct containing the full-length human syndecan cDNA under the control of a glucocorticoid-inducible retroviral promoter, in order to study the effect of syndecan expression on S115 cell behavior. Glucocorticoid-induced reexpression of syndecan in S115 cells restored an epithelial phenotype, while control transfectants and parental S115 cells exhibited an altered, nonepithelial phenotype. Moreover, the S115 cells expressing exogenous syndecan revealed a reduced ability to form colonies in soft agar. Therefore, the maintenance of epithelial morphology and normal growth of S115 cells are dependent on syndecan expression.

Cell surface proteoglycans play an important role in the regulation of cell behavior (1). Through covalently bound glycosaminoglycan side chains, such proteoglycans can bind various extracellular effector molecules (2). To understand the directives that a cell receives by binding these effector molecules, and to elucidate the corresponding intracellular responses, biological models that are dependent on the expression of special proteoglycans are needed. Syndecan is the best characterized cell surface proteoglycan (3). It was originally isolated from NMuMG mouse mammary epithelial cells as a hybrid proteoglycan containing both heparan sulfate and chondroitin sulfate glycosaminoglycan side chains (4). Further studies revealed its expression not only on epithelial cells but also on differentiating fibroblasts of developing tooth (5, 6), on endothelial cells of sprouting capillaries (7), and on the surfaces of certain lymphocyte subpopulations (8), indicating that its function can vary on the surfaces of different cells. Syndecan comprises a family of proteoglycans with conserved plasma membrane and cytoplasmic domains but with dissimilar ectodomains (9). The conserved structure of the cytoplasmic domain suggests that it may participate in signal transduction through the plasma membrane. Syndecan selectively binds several extracellular effector molecules. For example, syndecan binds interstitial collagens and fibronectin but not vitronectin or laminin (10-12). Moreover, syndecan isolated from tooth mesenchyme has revealed a selective binding to tenascin not observed for syndecan from NMuMG cells (13). This suggests variations in syndecan glycosylation that may result in the selective binding properties. Polymorphism of syndecan glycosylation

MATERIALS AND METHODS Cell Culture. S115 mouse mammary tumor cells were routinely cultured (21) in Dulbecco's modified Eagle's medium (DMEM). For studies involving hormone treatment, heat-inactivated fetal bovine serum was replaced with 4% dextran-coated-charcoal-treated fetal calf serum (DCC-FBS; eliminates endogenous steroids from serum) and used with or without addition of testosterone (10 nM) or dexamethasone (10 nM or 1 ,uM). Cells were plated at a density of 10,000 per cm2 and the medium was replenished every 3 days. Plasmid Constructs and Transfections. Plasmid pUC19hsynpr7 (9) was digested with Nae I restriction endonuclease, and the derived 337-base-pair fragment (bases 150-486) was separated in and eluted from low-melting agarose gel. pUC19hsyn4 (9) was digested with Nae I and HindII (polylinker site), and the plasmid-containing fragment starting from base Abbreviations: bFGF, basic fibroblast growth factor; DCCFBS, dextran-coated-charcoal-treated fetal bovine serum; hGH, human growth hormone. tTo whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Cell

Proc. Natl. Acad. Sci. USA 89 (1992)

Biology: Leppd et al. SV40 Early splicing region SV40 Polyadenylylation

neo

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SV40 On and Early promoter

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SV40 Polyadenylylation 5'

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SV40 EarlyI splicing region .

coding region of human syndecan 487 was isolated. The Nae I fragment from hsynpr7 was ligated to the pUC-hsyn Nae I/HindII-digested vector. The orientation of the insert was verified by restriction enzyme analysis and sequencing. The derived plasmid, containing the full coding region of human syndecan core protein, was named pUC19-hsynfull. This plasmid was further digested with BamHI and Sph I (polylinker site). A fragment containing bases 150-1461 was isolated and blunt-ended using Klenow and T4 DNA polymerases. Finally, this fragment was ligated to Sal I-linearized and blunt-ended pMAMneo vector (Clontech). The orientation was confirmed by restriction enzyme digestions. The plasmid, pMAMneo-hsyn, is described in Fig. 1. For control transfections, a 642-base-pair HindIII-Pvu II fragment of the human growth hormone (hGH) gene (including exons 4 and 5; kindly provided by R. Penttinen, Department of Medical Biochemistry, University ofTurku) (22) was blunt-ended and cloned into the same pMAMneo vector, as described above. This control construct was named pMAMneo-hGH. All plasmids were isolated by the CsCl density gradient method. Before transfections, both plasmids were linearized with Mlu I, extracted with chloroform/phenol, and precipitated with ethanol. Transfections were performed using Lipofectin (BRL). After selection for 2 weeks with G418, (750 ,ug/ml; Sigma), surviving clones were isolated from growth plates by using cloning cylinders. The expression of human syndecan or hGH (including exons 4 and 5) mRNAs was then confirmed by RNA isolation and Northern blot analysis. Clones expressing high levels of transfected genes were selected for further study. These stock cells were routinely cultured with G418 (300 ,g/ml). RNA Isolation and Northern Blot Analysis. Total RNA was isolated (23) from wild-type S115 cells and cells transfected with human syndecan or hGH genes. RNA from normal mouse mammary NMuMG cells and normal human mammary HBL-100 cells was used for comparison. RNA (15 ,ug per lane) was electrophoresed in formaldehyde/1% agarose gel and transferred to GeneScreenPlus hybridization membrane. Blots were hybridized with multiprime-labeled (Amersham) inserts of either mouse (PM-4) (3) or human 1.1-kb BamHI fragment of pUC19-hsyn4 (9) syndecan cDNA or with hGH exons 4 and 5 (22) cDNA, using the high-stringency conditions suggested by the manufacturer of the membrane (New England Nuclear). Fluorescence Microscopy. Cells on coverslips were fixed 2 hr at room temperature with periodate/lysine/paraformaldehyde (24), permeabilized with 0.01% saponin, and incubated with rhodamine-conjugated phalloidin (Sigma). Coverslips were mounted using Glycergel (Dako, Glostrup, Denmark). Soft-Agar Colony Assay. Anchorage-independent cell growth was measured in a soft-agar colony assay. The six-well-plates were first covered with an agar layer consisting of 2 ml of DMEM with 0.5% agar and 4% DCC-FBS. The middle layer contained 104 cells in 0.5 ml of DMEM with 0.33% agar and 4% DCC-FBS, with or without 10 nM

933

FIG. 1. pMAMneo-hsyn transfection construct. The BamHI-Sph I fragment of pUC19hsynfull (human syndecan cDNA containing full coding region for the core protein) was blunt-ended and cloned in sense orientation into pMAMneo. Rous sarcoma virus (RSV) and dexamethasone/testosterone-inducible mouse mammary tumor virus long terminal repeat (MMTV-LTR) promoters are indicated. neo, Neomycin-resistance marker gene; amp, ampicillin-resistance gene, SV40, simian virus 40; On, origin.

testosterone. The top layer, consisting of medium (2 ml), was added to prevent drying of the agarose gels. The plates were incubated at 37°C in 5% CO2 for 12 days, after which cultures were evaluated and photographed.

RESULTS Steroid Suppression of Syndecan Gene Expression in S115 Mouse Mammary Epithelial Ceils. Clonal S115 mouse mammary carcinoma cells exhibit transformed characteristics when cultured in the presence of testosterone (25). The syndecan gene in S115 cells is suppressed under the influence of testosterone (21), suggesting that loss of syndecan expression may be one of the alterations involved in the malignant transformation of these cells under hormone influence. Similar to testosterone, dexamethasone is also known to change the growth properties and morphology of S115 cells (26). We therefore investigated whether dexamethasone also has a suppressive effect on syndecan expression. For this purpose, cells were cultured with or without testosterone or dexamethasone for 3, 5, and 7 days. Cells grown in the absence of either hormone expressed high levels of syndecan mRNA (Fig. 2, lanes 1, 5, and 9). Testosterone (lanes 2, 6, and 10) or dexamethasone (lanes 3, 7, and 11) exposure suppressed syndecan expression. This suppression was concentrationdependent (lanes 4, 8, and 12) and correlated with a simultaneous change in the phenotype of S115 cells from epithelial to a more fibroblastic or fusiform phenotype (see below and Fig. 4). 1

2 3

4

5 6

7 8 9 10 11 12

28S PM -4

S 'd

18S

FIG. 2. Northern analysis of syndecan mRNA expression in testosterone- or dexamethasone-exposed S115 cells. Total RNA was isolated from S115 monolayers on day 3 (lanes 1-4), day 5 (lanes

5-8), and day 7 (lanes 9-12) after culture without hormone (lanes 1, 5, and 9), with 10 nM testosterone (lanes 2, 6, and 10), with 10 nM dexamethasone (lanes 3, 7, and 11) or with 1 ,uM dexamethasone (lanes 4, 8, and 12). Samples (15 ,g) were size-fractioned in formaldehyde/1% agarose gel and blotted onto GeneScreenPlus hybridization membrane. The filter was hybridized with multiprime-labeled PM-4 (a partial cDNA clone for mouse syndecan) and washed under high-stringency conditions. Sizes for syndecan mRNAs are 2.6 and 3.4 kilobases (kb). Positions of 28S and 18S rRNAs are indicated at right.

Cell Biology: Leppd et al.

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Proc. Natl. Acad. Sci. USA 89 (1992)

Reversal of Syndecan Expression in Hormone-Exposed S115 Cells by Syndecan Transfection. Suppression of syndecan gene expression in S115 cells by steroid treatment allowed us to investigate reexpression of syndecan in S115 cells, using a steroid-inducible promoter together with a full-length human syndecan cDNA (whose transcripts can be distinguished from the mouse syndecan mRNA by high stringency hybridization) (9). The full coding region of human syndecan was cloned in the sense orientation in pMAMneo (Fig. 1). This vector contains a neomycin-resistance gene, which allows the selection of stable transfectants. As a control transfection, the syndecan insert was replaced with a fragment of the hGH gene containing exons 4 and 5. Both the syndecan and the control plasmid constructs were transfected into S115 cells by using Lipofectin (27). G418resistant colonies were observed after 2 weeks. More than 20 individual syndecan clones and several hGH clones were isolated from culture dishes by using cloning cylinders. Fig. 3 shows Northern blot analysis of one hGH clone (lanes 1-3) and two human syndecan clones (lanes 4-6 and lanes 7-9). RNAs from HBL-100 (lane 10) and NMuMG (lane 11) cells (normal human and mouse mammary lines) were used for comparison. Cells were cultured without hormone (lanes 1, 4, and 7), with testosterone (lanes 2, 5, and 8), or with dexamethasone (lanes 3, 6, and 9) for 4 days. Both testosterone and dexamethasone induced the expression of syndecan mRNA in hsynfull-transfected clones (Fig. 3A). hGHtransfected clones were negative for syndecan but revealed

A 1

2 3

4 5 6 7 8 9

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H-SYN

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B

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inducible expression of hGH exons 4 and 5 (Fig. 3B). In HBL-100 and NMuMG cells, two species (2.6 and 3.4 kb) of endogenous mRNA were observed. This was due to an alternative polyadenylylation signal within the syndecan gene (15). In contrast, in transfected clones only the 2.6-kb mRNA was evident (Fig. 3), as predicted by the plasmid construct (Fig. 1). Syndecan Transfectants Lack Hormone-Induced Phenotype Response. Parental S115 cells exhibit an epithelial phenotype when cultured without steroid hormone (25). However, when exposed to testosterone, a positive proliferative response and a phenotype change to more fibroblastic or fusiform character was observed (Fig. 4A; see also refs. 21 and 25). Similar changes were also observed for hGH-transfected cells: without hormone the cells maintained an epithelial phenotype, whereas testosterone treatment sustained a fibroblastic phenotype (Fig. 4C). S115 cells expressing exogenous human syndecan were akin to the parental and hGH-transfected cells in that without hormone they appeared epithelial in phenotype. However, hormone treatment did not alter their morphology (Fig. 4B). Syndecan expression has been shown to correlate with the appearance of an epithelial phenotype of parental S115 cells (21). Here we show that the restored syndecan expression in hormone-treated S115 transfectants resulted in a reversal of the hormone-induced morphological change. This corroborates evidence that S115 cells are dependent on syndecan expression for the maintenance of epithelial morphology. This claim is further supported by the observation of Couchman et al. (28) that microfilament bundles were absent from S115 cells in the presence of hormone (Fig. 4D), while cells without hormone presented a well-organized actin filament network (Fig. 4E). In addition, our studies with syndecan-transfected cells showed reorganization of actin filament bundles, even in the presence of steroid (Fig. 4F). Our observations also support the original finding of Rapraeger et al. (19) that polarized epithelial cells colocalize syndecan with basally organized actin cytofilaments. Restored Syndecan Expression Suppresses HormoneInduced Growth Promotion of S115 Cells. Parental S115 cells as well as the hGH-transfected cells responded to steroid exposure by increasing their growth rate and by acquiring the ability to grow in suspension (data not shown) or to form colonies in soft agar (Fig. 5 A and B). In contrast, the behavior of syndecan-transfected cells remained unaltered regardless of the presence or absence of steroid: in the presence of hormone they maintained their epithelial morphology, and no detaching or piling up of cells was observed. Under conditions supporting soft-agar growth of parental S115 cells or hGH transfectants (Fig. 5 A and B), syndecan-transfected cells were strongly suppressed with respect to colony formation (Fig. 5 C and D).

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FIG. 3. Northern analysis of syndecan and hGH-transfected cells. Total RNA was isolated after 4 days of culture. For Northern analysis, 15 ,g of RNA from hGH-transfected cells (lanes 1-3) and from syndecan-transfected cell clones 2.20 (lanes 4-6) and 2.5 (lanes 7-9) were loaded. For baseline responses, cells were cultured without hormone (lanes 1, 4, and 7); for induction of transfected genes, cells were treated with either testosterone (lanes 2, 5, and 8) or dexamethasone (lanes 3, 6, and 9). For comparison, RNAs from HBL-100 (lane 10) and NMuMG cells (lane 11) were also included. RNAs were hybridized first with a human syndecan (H-SYN) probe (A) and then with a hGH (B) probe.

DISCUSSION Extracellular matrix is known to influence the behavior of cells (29). Translation of this influence at the molecular level is carried out by those cell surface molecules that recognize and bind components of the extracellular matrix (30). The binding of cells to the extracellular matrix via matrix receptors may regulate cell morphology. In addition, this binding may lead to signal transduction through the plasma membrane, resulting in intracellular changes and gene expression that lead to the regulation of cell differentiation. In this work, we have analyzed the role of one matrix-binding protein, a cell surface proteoglycan named syndecan (3), in the regulation of epithelial morphology and growth of steroidresponsive S115 cells. Our results indicate that syndecan expression is directly involved in the regulation of epithelial behavior. Further, these observations may explain why

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Biology: Leppd et al.

Proc. Natl. Acad. Sci. USA 89 (1992)

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FIG. 4. Reversal of epithelial phenotype and actin filament reorganization due to syndecan gene transfection. Phase-contrast micrographs show monolayers of cells grown in the presence of testosterone: wild-type cells (A); human syndecan-transfected cells (clone 2.20) (B); and hGH-transfected cells (C). Fluorescence micrographs show cells stained with rhodamine-conjugated phalloidin: wild-type cells grown in the presence (D) or absence (E) of testosterone; human syndecan-transfected cells grown in the presence of testosterone (F). (A-C, x260; D-F, x450.)

transformed cells with lost or reduced syndlecan expression (21, 31) change their response to matrix coimponents. Syndecan Expression Correlates with Epithkelial Morphology of Mouse Mammary Tumor Cells. Syndec,an was initially isolated from normal mouse mammary epith elial cells, where it was shown to polarize to basolateral surfac ces at the time of epithelial polarization (19). This, together with the finding that syndecan is colocalized with actin-ric h cytofilaments, has led to the concept that syndecan may atnchor the epithelial cytoskeleton to the extracellular matrix (32). Moreover, prevention of syndecan expression with an antisense RNA

construct results in the disappearance of NMuMG cells' epithelial morphology (33, 34). Syndecan expression disappears simultaneously with epithelial/mesenchymal conversion of developing rat palate in vivo (35). Our current work shows that reexpression of syndecan through a steroidinducible promoter can overcome hormonal conversion of S115 cell morphology, suggesting that syndecan must be one of the molecules involved in the regulation of epithelial behavior. One means by which to regulate cellular morphology is to influence cytoskeletal organization, as proposed for syndecan (19). S115 cells under steroid influence lose their actin filament organization, whereas syndecan transfectants do Wo w -S. W, not. Several mechanisms have been proposed to regulate actin polymerization. These include gelsolin (36) and profilin (37) interactions and the regulatory role of inositolphosphoA'IL lipids in these interactions (37, 38). Actin polymerization can 0 also be regulated via protein kinase C (39). In this respect, 0 * esyndecan-transfected cells may prove to be invaluable in the analysis of syndecan-mediated regulation of intracellular f. MI" 4't activities, the mechanisms of which remain totally unknown. Syndecan is only one of the many adhesive components on epithelial surfaces. Suppression of syndecan expression with an antisense RNA construct in NMuMG cells results in the 0 *; a ,*: Ahloss of E-cadherin and ,3l-integrin (34). This could be significant in that the reduction of E-cadherin expression on carcinoma cells correlates with invasiveness (40). It may also ; suggest that while several molecules might contribute to the S final regulation of cell morphology, the steroid influence on C 0 S115 cells is dominated by the dependency of syndecan expression. FIG. 5. Soft-agar colony formation of cells cuiltured with 10 nM Syndecan Expression Regulates Growth ofMouse Mammary testosterone in 0.33% soft agar. On day 12, colonies were evaluated Tumor Cells. In addition to its effect on morphology, syndeand photographed. (A) Representative colonies of wild-type S115 can reexpression also influenced the growth behavior of S115 cells. (B) hGH-transfected cells. (C and D) IHuman syndecantransfected clones 2.20 and 2.5, respectively. (x 25.) cells. Especially remarkable was the diminished ability of 4

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syndecan-transfected S115 cells to form colonies in soft agar. This could mean that cells which normally use their cytoskeleton for spreading, migration, and dividing lose their potential to proliferate without anchorage. If such is the case, cytoskeletal organization can suppress or regulate growth promotion or direct the growth to a more differentiated stage. A second attempt to explain how syndecan could be involved in growth regulation involves syndecan's ability to modulate the activity of some growth factors (1, 16). Indeed, the disappearance of syndecan from the S115 cells could alter their ability to respond appropriately to heparin-binding growth factors. For example, without previous interaction with heparan sulfate or heparin, bFGF does not bind to its signal-transducing, high-affinity receptors (16), which can result in the loss of a bFGF effect on growth of 3T3 cells or on differentiation of MM14 myoblasts (41). Therefore, the lack of one regulatory pathway component could possibly favor the initiation of an alternative pathway, thereby aiding the transformation of the cells. These questions can be explored by transfecting syndecan into cells with little or no syndecan expression. In summary, the current data with syndecan-transfected S115 cells clearly indicate that syndecan-like molecules (9) may play a dual role in the regulation of cell behavior. They may participate in the adhesion and anchorage of cells to the extracellular environment and, at the same time, perhaps participate in the modulation of growth factor influence on the same cell. This regulatory concept could address a major role during tissue formation and cellular differentiation. The known spatiotemporal expression of syndecan (5, 6, 17, 18) during organ formation could indicate that syndecan-like molecules might regulate the growth and differentiation of cells in vivo, by immobilizing these activities according to the template provided by the extracellular matrix. The lack of such regulation might favor the appearance of less differentiated and more malignant phenotypes. The technical assistance of Taina Kalevo is gratefully acknowledged. The language of this manuscript was revised by Dr. Bruce Granger. This work was supported by The Finnish Academy, The Finnish Cancer Union, The Finnish Cancer Institute, the United States Public Health Service (DE09399-01), the Duodecim Foundation, The Emil Aaltonen Foundation, and The Research and Science Foundation of Farmos. 1. Ruoslahti, E. & Yamaguchi, Y. (1991) Cell 64, 867-869. 2. Jalkanen, M., Jalkanen, S. & Bernfield, M. (1991) in Receptors for Extracellular Matrix, eds. McDonald, C. & Mecham, R. P. (Academic, San Diego), in press. 3. Saunders, S., Jalkanen, M., O'Farrell, S. & Bernfield, M. (1989) J. Cell Biol. 108, 1547-1556. 4. Rapraeger, A., Jalkanen, M., Endo, E., Koda, J. & Bemfield, M. (1985) J. Biol. Chem. 260, 11046-11052. 5. Thesleff, I., Jalkanen, M., Vainio, S. & Bernfield, M. (1988) Dev. Biol. 129, 565-572. 6. Vainio, S., Jalkanen, M. & Thesleff, I. (1989) J. Cell Biol. 108, 1945-1954. 7. Elenius, K., Vainio, S., Laato, M., Salmivirta, M., Thesleff, I. & Jalkanen, M. (1991) J. Cell Biol. 114, 585-595. 8. Sanderson, R., Lalor, P. & Bernfield, M. (1989) Cell Regul. 1, 27-35.

Proc. Natl. Acad. Sci. USA 89 (1992) 9. Mali, M., Jaakkola, P., Arvilommi, A.-M. & Jalkanen, M.

(1990) J. Biol. Chem. 265, 6884-6889.

10. Koda, J. E., Rapraeger, A. & Bernfield, M. (1985) J. Biol. Chem. 260, 8157-8162. 11. Saunders, S. & Bernfield, M. (1988) J. Cell Biol. 106, 423-430. 12. Elenius, K., Salmivirta, M., Inki, P., Mali, M. & Jalkanen, M.

(1990) J. Biol. Chem. 265, 17837-17843.

13. Salmivirta, M., Elenius, K., Vainio, S., Hofer, U., ChiquetEhrismann, R., Thesleff, I. & Jalkanen, M. (1991) J. Biol. Chem. 266, 7733-7739. 14. Sanderson, R. & Bernfield, M. (1988) Proc. Natl. Acad. Sci. USA 85, 9562-9566. 15. Kiefer, M. C., Stephans, J. C., Crawford, K., Okino, K. & Barr, P. J. (1990) Proc. Natl. Acad. Sci. USA 87, 6985-6989. 16. Yayon, A., Klagsbrun, M., Esko, J. D., Leder, P. & Ornitz, D. M. (1991) Cell 64, 841-848. 17. Vainio, S., Lehtonen, E., Jalkanen, M., Bernfield, M. & Saxen, L. (1989) Dev. Biol. 134, 382-391. 18. Vainio, S., Jalkanen, M., Vaahtokari, A., Sahlberg, C., Mali, M., Bernfield, M. & Thesleff, I. (1991) Dev. Biol. 147, 322-333. 19. Rapraeger, A., Jalkanen, M. & Bernfield, M. (1986) J. Cell Biol.

103, 2683-26%.

20. Jalkanen, M., Rapraeger, A., Saunders, S. & Bernfield, M. (1987) J. Cell Biol. 105, 3087-3096. 21. Leppa, S., Hark6nen, P. & Jalkanen, M. (1991) Cell Regul. 2, 1-11. 22. Bornstein, P. & McKay, J. (1988) J. Biol. Chem. 263, 16031606. 23. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Rutter, W. J. (1979) Biochemistry 18, 5294-5299. 24. McLean, I. W. & Nakane, P. K. (1974) J. Histochem. Cytochem. 22, 1077-1083. 25. Yates, J. & King, R. J. B. (1981) Cancer Res. 41, 258-262. 26. Yates, J. & King, R. J. B. (1978) Cancer Res. 38, 4135-4137. 27. Felgner, P. L., Gadek, T. R., Holm, M., Roman, R., Chan, H. W., Wenz, M., Northrop, J. P., Ringold, G. M. & Danielsen, M. (1987) Proc. Nati. Acad. Sci. USA 84, 74137417. 28. Couchman, J. R., Yates, J., King, R. J. B. & Badley, R. A. (1981) Cancer Res. 41, 263-269. 29. Stoker, A. W., Streuli, C. H., Martins-Green, M. & Bissell, M. J. (1990) Curr. Opinions Cell Biol. 2, 864-874. 30. Damsky, C. H. & Bernfield, M. (1990) Curr. Opinions CellBiol. 2, 813-814. 31. Inki, P., Stenback, F., Talve, M. & Jalkanen, M. (1991) Am. J. Pathol., in press. 32. Rapraeger, A., Jalkanen, M. & Bernfield, M. (1987) Biology of Extracellular Matrix (Vol II): Biology of Proteoglycans, eds. Wight, T. N. & Mecham, R. P. (Academic, New York), pp. 129-154. 33. Saunders, S., Nguyen, H. & Bernfield, M. (1989) J. Cell Biol. 109, Sa (abstr.). 34. Kato, M. & Bernfield, M. (1990) J. Cell Biol. 111, 263a (abstr.). 35. Fitchett, J. E., McAlmon, K. R., Hay, E. D. & Bernfield, M.

(1990) J. Cell Biol. 111, 145a (abstr.).

36. Pollard, T. D. & Cooper, J. A. (1986) Annu. Rev. Biochem. 55, 987-1035. 37. Lassing, I. & Lindberg, U. (1985) Nature (London) 314, 472474. 38. Janmey, P. A. & Stossel, T. P. (1987) Nature (London) 325, 362-364. 39. Apgar, J. R. (1991) J. Cell Biol. 112, 1157-1163. 40. Frixen, U. H., Behrens, J., Sachs, M., Eberle, G., Voss, B., Warda, A., Ldchner, D. & Birchmeier, W. (1991) J. Cell Biol. 113, 173-185. 41. Rapraeger, A. C., Krufka, A. & Olwin, B. B. (1991) Science 252, 1705-1708.