Inhibition of Platelet-derived Growth Factor-BB-induced Receptor Activation and Fibroblast Migration by Hyaluronan Activation of CD44*

The extracellular matrix molecule hyaluronan was found to suppress platelet-derived growth factor (PDGF) β-receptor activation and PDGF-BB-induced migration of primary human dermal fibroblasts. The suppressive effect of hyaluronan was neutralized by a monoclonal antibody that specifically inhibits hyaluronan binding to its receptor CD44. Moreover, co-immunoprecipitation experiments showed that the PDGF β-receptor and CD44 can form a complex. Interestingly, the inhibitory effect of hyaluronan on PDGF β-receptor activation was not seen in the presence of the tyrosine phosphatase inhibitor pervanadate. Our observations suggest that hyaluronan suppresses PDGF β-receptor activation by recruiting a CD44-associated tyrosine phosphatase to the receptor.

Hyaluronan is the most common glycosaminoglycan present in the extracellular matrix of mammalian tissues, for example in epidermis and dermis. It is synthesized by both normal and malignant cells, such as fibroblasts (2) and breast cancer cells (3), and is involved in a variety of physiological events, such as cell proliferation, differentiation, and migration (4). The ability of hyaluronan to affect cell migration has been attributed partly to its physicochemical properties; hyaluronan molecules form continuous and porous biological meshworks that exhibit visco-elastic properties. This property makes tissues resilient and malleable, which is important to facilitate cell motility during development, tissue remodeling, and cancer. Furthermore, several studies have provided evidence that hyaluronan exerts signaling effects through interactions with certain cell surface receptors, such as CD44 and RHAMM (5,6). The predominant receptor for hyaluronan is the cell surface adhesion receptor CD44 that is expressed in most cell types transferring signals from the outside of the cell to the inside in response to hyaluronan binding (7)(8)(9). CD44 exists in several isoforms because of alternative splicing of the gene, which can further undergo extensive post-translational modifications by glycosylation and addition of polysaccharide chains. This diversity in its structure accounts for its diverse biological functions and ability to interact with numerous other macromolecules in addition to hyaluronan, including collagen, fibronectin, growth factors, cytokines, and metalloproteinases (10,11). The most common form, CD44s (CD44 standard form), is expressed by a wide variety of cells, including hematopoietic cells and dermal fibroblasts. Studies have shown that CD44s facilitates lymphocyte homing and fibroblast migration during wound healing (12)(13)(14). Longer variant isoforms, termed CD44v, are found on endothelial cells, epithelial cells, activated lymphocytes, and some tumor cells. The cytoplasmic domain of CD44 interacts with ERM proteins and ankyrin and thus indirectly with the cytoskeleton (10).
Notably, among the three hyaluronan-synthesizing enzymes (Has1, -2, and -3), the Has2 isoform seems to be most important in mediating cell migration. For example, Has2 Ϫ/Ϫ mouse cardiac endothelial cells display reduced ability to migrate, a process that can be reversed by gene rescue or by the addition of exogenous hyaluronan (15). In addition, studies on various tumor cells, such as mesotheliomas, colon carcinomas, and breast cancer, revealed that cells lacking Has2 expression migrated much slower compared with Has2-expressing cells (16 -18). Interestingly, not all types of cells respond to hyaluronan by increased locomotion; Chinese hamster ovary cells (19) and low metastatic murine T cell lymphoma cells (20) reduced their motility in response to hyaluronan.
Growth factors also play a central role in cell migration during both normal and pathological conditions, including embryogenesis, wound healing, and tumor invasion. In this regard, platelet-derived growth factor (PDGF) 2 -BB is a powerful stimulator of the migration of mesenchymal cells, such as fibroblasts (21)(22)(23). PDGF-BB mediates its cellular effects through binding to ␣and ␤-tyrosine kinase receptors (PDGFR␣ and PDGFR␤, respectively), resulting in receptor dimerization and activation through autophosphorylation; this leads to recruitment of Src homology 2 domain signal transduction molecules to the receptor (24). Recent studies indicate that PDGFR␤ and other tyrosine receptor kinases can be dephosphorylated * This work was supported in part by Swedish Cancer Society Grant 3446-B03-10XBC and Uppsala University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1 by a number of protein-tyrosine phosphatases (PTPs), which thereby affect downstream signaling of the receptors (25). The effects of growth factors and matrix components on cells are interdependent, because growth factors affect the synthesis of extracellular molecules and their degrading enzymes, whereas the extracellular macromolecules modulate growth factor-mediated cellular functions. Previous studies have demonstrated a synergism between ␣ v ␤ 3 integrin and PDGF-BB in mediating endothelial cell migration (26,27). In this study, we investigated the possibility that hyaluronan affects PDGF-BB-induced cell migration of human dermal fibroblast cultures.

MATERIALS AND METHODS
Cell Culture-Biopsies were taken from elective breast reduction surgery, after the approval of the patient (Department of Plastic Surgery, University Hospital, Uppsala, Sweden), and transported to the laboratory in chilled phosphate-buffered saline without Ca 2ϩ and Mg 2 (PBS) but containing antibiotics (100 IU penicillin/ml and 100 g of streptomycin/ml; Veterinary Institute, Uppsala, Sweden). Following a quick wash in 70% ethanol, the biopsy was washed with PBS and minced to 1-2-mm 3 pieces with a razor blade. Dermis and epidermis were separated, using fine forceps, after an overnight incubation in 2.5 mg of dispase per ml of Dulbecco's modified Eagle's medium (grade II, 0.5 units/ml; Roche Applied Science) (DMEM; Invitrogen) at 4°C. Fibroblasts from the dermal sheets were then cultured from explants in complete medium (DMEM supplemented with 10% fetal bovine serum (Biowest) and antibiotics). Skin fibroblasts (passages 6 -10) from the explants were used for all experiments.
Cell Migration Assay-Human dermal fibroblasts (8 ϫ 10 4 cells per well in 6-well plates) were cultured in complete medium for 24 h; thereafter, the medium was replaced with starvation medium. Then a wound was made by scraping the confluent cell cultures, crosswise, with the tip of a 1-ml pipette. The floating cells were washed away with DMEM supplemented with 0.1% FCS (starvation medium). The wound closure was monitored by an inverted microscope (Nikon) at four different positions along the wound, immediately and after different time periods up to 24 h in untreated cultures and in cultures treated with various concentrations of PDGF-BB. To investigate the effect of hyaluronan on fibroblast migration induced by 1 ng/ml PDGF-BB, the cultures were preincubated for 2 h with hyaluronan (1-100 g/ml, M r 0.5-1.5 ϫ 10 6 ; Genzyme Hylumed) in the absence or presence of rat monoclonal Hermes-1 antibodies (used as serum-free supernatants from the myeloma culture; generously provided by Professor Sirpa Jalkanen, Turku, Finland), which specifically inhibits the binding of hyaluronan to the CD44 receptor. The cell migration was quantified by NIH Image 1.63 software. The data were exported to Excel and statistically analyzed by Student's t test.
Analysis of Hyaluronan Synthesis-Fibroblasts (8 ϫ 10 4 cells/ well in 6-well plates) were grown for 24 h in complete medium, followed by a 24-h starvation. The quiescent cells received fresh starvation medium supplemented with various concentrations of PDGF-BB, and at various time points the hyaluronan content in the conditioned media was quantified, essentially as described previously (16). The assay is based on the formation of a complex between hyaluronan and the hyaluronan-binding protein (HABP) domain of aggrecan, the binding of which essentially is irreversible. The HABP was isolated from crude bovine cartilage extract by affinity chromatography on a hyaluronan-EAH-Sepharose 4B (Amersham Biosciences) (28). A part of this HABP was biotinylated with biotin-aminocaproic acid sulfo-NHS (catalog number B1022, Sigma). The assay was carried out on MaxiSorp 96-well Nunc-Immuno Plates (catalog number 439454, Nunc) that were precoated overnight with 1 g of HABP/ml in 50 mM carbonate buffer, pH 9.5, washed in PBS containing 0.5% Tween 20, and blocked in PBS containing 1% BSA. The hyaluronan standards (0 -100 ng/ml) and samples of conditioned media, at appropriate dilutions in blocking solution, were then added, and the plates were incubated for 1 h at 37°C. Following washing, 100 l of biotinylated HABP (b-HABP; 1 g/ml) was added, and samples were incubated for 1 h. After excess washing, the b-HABP specifically bound to the immobilized hyaluronan was determined by incubation for 1 h with streptavidinbiotinylated horseradish peroxidase complex (catalog number RNP1015V, Amersham Biosciences), followed by the addition of 100 l of 3,3Ј,5,5Ј-tetramethylbenzidine substrate solution (catalog number T4444, Sigma) for 15 min. 50 l of 2 M H 2 SO 4 was then added, and the absorbance was measured at a wavelength of 450 nm. The hyaluronan content was calculated by comparing with a standard curve made from known concentrations of hyaluronan, and the values were statistically analyzed by Student's t test.
Generation and Purification of Hermes-1 Fab Fragments-Eight mg of Hermes-1 IgG was extensively dialyzed against 20 mM sodium phosphate and 10 mM EDTA, pH 7.0, followed by concentration to ϳ0.5 ml using an Aminco Ultra-15 centrifugal filter device (M r 10,000 cutoff, Millipore). Fab fragments were then generated using ImmunoPure Fab preparation kit (catalog number 44885, Pierce) by 5 h of digestion at 37°C with immobilized papain followed by purification according to the manufacturer. In addition, an UltraLink immobilized protein G column (catalog number 53127, Pierce) was used to remove Fc fragments and undigested IgG from Hermes-1 Fab fragments.
Cell Adhesion Assay-Glass coverslips in 12-well culture dishes were coated with 0.5 mg/ml hyaluronan or 0.5% BSA (negative control for general attachment), in 10 mM Hepes, pH 7.4, containing 137 mM NaCl, 4.7 mM KCl, 0.65 mM MgSO 4 , and 1.2 mM CaCl 2 overnight in a cold room. Dermal fibroblasts were incubated for 24 h in starvation medium, trypsinized, washed, and diluted to 1 ϫ 10 6 cells/ml with starvation medium. The cells were then incubated for 30 min in the cold room without or with Hermes-1 IgG, Hermes-1 Fab fragments, or Hermes-3 IgG (ammonium sulfate precipitate from serum-free myeloma culture medium; generously provided by Professor Sirpa Jalkanen, Turku, Finland). Cell samples of 1 ϫ 10 5 cells per coverslip were allowed to adhere for 30 min at 37°C. Nonadherent cells were removed with two washings in the above-described Hepes buffer, and the number of adhered cells was measured by the hexose-aminidase assay (29).
For co-immunoprecipitation, the cleared lysates were incubated for 2 h with 2 g of polyclonal antiserum against PDGFR␤ (CT␤; a generous gift from Dr. Carina Hellberg, Ludwig Institute for Cancer Research, Uppsala, Sweden) or 5 g of anti-CD44 IgG (Hermes-1, purified by ammonium sulfate) before the addition of 50 l of protein A-Sepharose (Amersham Biosciences) per sample. As controls, rabbit IgG (I-5006, Sigma) and rat IgG (I-4131, Sigma) were used. PDGFR␤ bound to WGA beads, or immunoprecipitates collected on protein A-Sepharose beads, were spun down, washed twice with lysis buffer, and resuspended in 30 l of sample buffer with dithiothreitol, and heated 5 min at 99°C to release the proteins from the beads; they were then subjected to 7.5% SDS-PAGE and transferred to a membrane.
After separation by SDS-PAGE, the proteins were transferred to nitrocellulose membranes (Hybond TM -C Extra, Amersham Biosciences) for immunoblotting. Nonspecific binding sites on the membrane were blocked with 5% defatted milk in TBS-T buffer (50 mM Tris-HCl, pH 7.4, 200 mM NaCl, supplemented with 0.1% Tween 20), at room temperature for 1 h or overnight at 4°C, followed by two washes in TBS-T. The membranes were then incubated with a monoclonal phosphotyrosine antibody (PY99, 1 g/ml; catalog number SC-7020, Santa Cruz Biotechnology), PDGFR␤ antibody (CT␤, 2 g/ml), or CD44 antibody (1 g/ml; Hermes-1) in TBS-T buffer containing 1% BSA overnight at 4°C. After five washes in TBS-T, the membranes were incubated with anti-mouse horseradish peroxidase IgG (1:3000; Amersham Biosciences), anti-rabbit horseradish peroxidase IgG (1:3000; Amersham Biosciences), or anti-rat horseradish peroxidase IgG (1:1000; Santa Cruz Biotechnology catalog number SC-2006) for the detection of phosphorylated PDGFR␤, total PDGFR␤, and hyaluronan receptor CD44 immune complexes, respectively. Immunoreactive bands were then detected by enhanced chemiluminescence. The membranes were stripped with Stripping buffer (catalog number 21059, Pierce), before re-blotting with other antibodies, as specified. The integral densities of specific bands were quantified by two-dimensional densitometry and analyzed by Excel.
Immunofluorescence Staining-Fibroblasts (2 ϫ 10 4 cells per well in 4-well Falcon culture slides) were incubated in complete medium for 24 h followed by 24 h of starvation. Some of the cultures were incubated for 2 h with 100 g/ml hyaluronan before a 15-min stimulation with 10 ng/ml PDGF-BB. The cultures were then washed with PBS and fixed in 4% paraformaldehyde for 20 min at room temperature. After three washes, nonspecific staining was blocked by incubation in 20% goat serum for 1 h at room temperature. For double staining of PDGFR␤ and CD44, permeabilized cells (0.1% Triton X-100 for 10 min) were incubated for 1 h with 3 g/ml rabbit PDGFR␤ antiserum, washed, and incubated for an additional 1 h with 1 g/ml rat anti-CD44 monoclonal antibodies (Hermes-1). As controls, rabbit IgG and rat IgG were used. After washing, monolayers were incubated for 1 h in the dark with secondary antibodies, Alexa Fluor 594 F(abЈ) 2 fragment of goat anti-rabbit IgG (1 g/ml, red, catalog number A11072, Molecular Probes) to detect PDGFR␤ or goat biotinylated anti-rat IgG (5 g/ml; catalog number BA9400, Vector Laboratories) and streptavidin Alexa Fluor 488 conjugate (1 g/ml, green; catalog number S11223, Molecular Probes) to detect CD44. For double staining of hyaluronan and CD44, nonpermeabilized cells were incubated with b-HABP (2 g/ml) and Hermes-1 antibodies separately, followed by incubation with streptavidin-Alexa Fluor 488 (1 g/ml, green) to detect hyaluronan and goat anti-rat Alexa Fluor 568 (2 g/ml, red; catalog number A11077, Molecular Probes) to detect CD44. As controls, cells were pretreated for 1 h at 37°C with 20 units/ml Streptomyces hyaluronidase to digest hyaluronan and incubated with rat IgG instead of Hermes-1 antibodies. All washings were performed in the presence of 10% ethanol to avoid loss of hyaluronan. Each double staining was followed by staining of nuclei using 4,6-diamidino-2-phenylindole (1 g/ml; catalog number 1.24653, Merck) for 5 min. The processed cells were mounted in Fluoromount G (catalog number 0100-01, Immunokemi) and photographed with a Leica Microsystems microscope.

PDGF-BB Induces Migration and Stimulates Hyaluronan
Production by Human Dermal Fibroblasts-Because both PDGF-BB and hyaluronan have been implicated in cell migration (4,22), we investigated the importance of these molecules for migration of human dermal fibroblasts. First, we examined the effects of different concentrations of PDGF-BB both on migration and hyaluronan synthesis. In a wounding experiment in confluent cultures of dermal fibroblasts, PDGF-BB was found to stimulate in a dose-dependent manner the wound closure over a 24-h period. Maximum effect was seen at 10 ng/ml PDGF-BB; at this concentration, the wound in the cell culture healed about 3-fold faster than in unstimulated cell cultures (Fig. 1A). Fig. 1B shows that the wound closure was almost complete in PDGF-BB-stimulated cultures after 24 h. Notably, PDGF-BB-stimulated cells exhibited an elongated and polarized morphology with membrane sheets, i.e. membrane ruffles or lamellipodia, at the leading edge of migrating dermal fibroblasts. These membrane sheets were stained with specific antibodies for the hyaluronan receptor CD44, demonstrating an accumulation of CD44 preferentially at the leading edge of the migrating cells (Fig. 1C). The nonstimulated fibroblasts exhibited less polarization and membrane ruffle formation than PDGF-BB-stimulated cells, but yet expressed CD44 at the leading edge of migrating cells (Fig. 1C).
A 24-h stimulation of fibroblasts with 10 ng/ml PDGF-BB also induced an approximate 3-fold increase of hyaluronan amount (1.1 g/ml) relative to unstimulated cells; 1 ng/ml PDGF-BB resulted in a 1.8-fold increase (about 0.7 g/ml) (Fig.  1D). One ng of PDGF-BB per ml was the lowest concentration that gave significant effects on cell migration, as well as on hyaluronan production; this concentration was therefore chosen to investigate if endogenously produced hyaluronan affected PDGF-BB-mediated fibroblast migration. Culturing of the dermal fibroblasts in the presence of Streptomyces hyaluronidase, which specifically degrades hyaluronan to tetrasaccharides, did not affect the PDGF-BB-induced cell migration (Fig. 1E). Thus, PDGF-BB-induced dermal fibroblast motility in vitro appeared not to be affected by endogenously produced high molecular weight hyaluronan.
Binding of Hyaluronan to CD44 Suppresses PDGF-BB-induced Fibroblast Migration-Given the fact that the hyaluronan amount is high in skin (about 0.5 mg/ml), we investigated the effect of exogenously added hyaluronan on PDGF-BB-induced fibroblast migration. As shown in Fig. 2, the stimulation of cell motility induced by 1 ng/ml PDGF-BB was significantly inhibited by addition of exogenous hyaluronan at 25 g/ml or higher concentrations of hyaluronan ( Fig. 2A). Lower concentrations of exogenously added hyaluronan had no effect on growth factor-stimulated cells (data not shown).
To investigate the role of CD44 for the inhibitory effect of hyaluronan on PDGF-BB-induced migration, Hermes-1 monoclonal antibodies that bind to CD44 and block the binding of hyaluronan (31) were used. First, we verified that Hermes-1 antibodies could inhibit attachment also of the dermal fibroblasts used in this study to a hyaluronan-coated substratum (Fig. 2B). Furthermore, Fab fragments of anti-CD44 Hermes-1 blocked cell attachment, whereas the nonhyaluronan blocking anti-CD44 Hermes-3 antibodies did not (Fig. 2B). Importantly, addition of Hermes-1 restored the hyaluronan-mediated suppression of PDGF-BB-induced motility of fibroblasts ( Fig. 2C; p Ͻ 0.05). These results suggest that hyaluronan-CD44 complexes, formed when cells are surrounded with high amounts of hyaluronan, can function as negative modulators of PDGF-BBmediated cell motility.
PDGFR␤ Activation Is Inhibited by CD44-Hyaluronan Complexes-To gain insights into the mechanism behind the inhibitory effect of the CD44-hyaluronan complex on PDGF-BB-mediated fibroblast migration, we examined the effect of exogenously added hyaluronan on PDGFR␤ activation, in the absence or presence of Hermes-1 (Fig. 3). Interestingly, incubation of PDGF-BB-stimulated fibroblasts with increasing hyalu- ronan amounts led to a marked decrease of PDGFR␤ phosphorylation, achieved at 50 g/ml and higher concentrations of hyaluronan; densitometric analysis showed that this reduction in the phosphorylation of the PDGFR␤ was statistically significant (p Ͻ 0.05). Importantly, the presence of Hermes-1 mAbs neutralized the inhibitory effect of hyaluronan on PDGF-BBinduced ␤-receptor phosphorylation (Fig. 3B; p Ͻ 0.05). The presence of Hermes-1 Fab fragments that inhibit the binding of hyaluronan to CD44 (Fig. 2B), but are unable to induce CD44 dimerization, also neutralized the inhibitory effect of hyaluronan on PDGF-BB-induced ␤-receptor phosphorylation (supplemental Fig. 1). These findings suggest that hyaluronan-CD44 complexes inhibit PDGFR␤ activation. The effect was specific for hyaluronan because equimolar concentrations of chondroitin sulfate did not inhibit PDGF-BB-induced receptor autophosphorylation (Fig. 3C).
PDGFR␤ Receptor Forms a Complex with CD44-The hyaluronan-mediated reduction of PDGF-BB-induced ␤-receptor phosphorylation and its restoration by blocking mAb Hermes-1 suggested a close proximity of PDGFR␤ and CD44. Therefore, we examined the possibility that these molecules form a complex by performing co-immunoprecipitation exper-

FIGURE 2. PDGF-BB-induced fibroblast migration is inhibited by exogenous hyaluronan and restored by Hermes-1.
A, wounded human dermal fibroblast cultures (8 ϫ 10 4 cells per well in 6-well plates) were incubated for 2 h in starvation medium alone or in medium containing 25-100 g of hyaluronan/ml; 1 ng of PDGF-BB/ml was then added, and incubation was continued another 24 h; thereafter the wound size was measured. B, the attachment of dermal fibroblasts to hyaluronan-coated substratum was analyzed in the presence of the IgG and Fab fragment of the blocking CD44 antibody Hermes-1, as well as the nonblocking antibody Hermes-3 (25-50 g/ml). C, Hermes-1 antibodies (0 -50 l serum-free supernatant/ ml) were added into wounded fibroblast cultures 1 h before exposure to 100 g of hyaluronan/ml and 1 ng of PDGF-BB/ml, as described in A. The wound closure was quantified by NIH Image 1.63 software, at wound gaps in four different positions along the wounds, and statistically analyzed by Student's t test. Columns, means of quadruplicate determinations Ϯ S.D. Data show a representative experiment from three separate experiments with similar results. Significantly different (p Ͻ 0.05): #, from nonstimulated cells; *, from PDGF-BB-stimulated cultures; **, compared with cultures exposed to both hyaluronan and PDGF-BB.

FIGURE 3. PDGF-BB-induced phosphorylation of PDGF ␤-receptor is inhibited by hyaluronan and restored by blocking CD44 antibody.
A, quiescent fibroblast cultures (2 ϫ 10 5 cells per 6-cm culture dish) were incubated for 2 h in starvation medium alone or in medium containing 10 -100 g of hyaluronan/ml and then stimulated with 5 ng of PDGF-BB/ml for 15 min; thereafter cells were lysed. B, as in A but blocking CD44 antibodies, Hermes-1 (50 l/ml), were added 1 h before exposing the cultures to hyaluronan (100 g/ml) and PDGF-BB (5 ng/ml) followed by cell lysis. C, as in A but the effect of approximately equimolar amounts of chondroitin sulfate A (CS-A) were compared with hyaluronan (10 g/ml CS-A corresponds to 100 g/ml hyaluronan). A-C, PDGF ␤-receptors were adsorbed to WGA-agarose, and the precipitated proteins were subjected to SDS-PAGE followed by transfer to nitrocellulose membrane. Receptor phosphorylation and the amount of adsorbed receptor were determined by immunoblotting with PY99 and PDGFR␤ antibodies, respectively; a representative immunoblot out of two (A and C) and three (B) experiments with similar results are shown. Densitometric analyses of changes in PDGFR␤ phosphorylation in response to various treatments were performed. A and C, columns depict means of duplicate determinations Ϯ variation; B, columns depict means of triplicates Ϯ S.D.; *, significantly different (p Ͻ 0.05) from PDGF-BB-stimulated cells; **, significantly different from cells stimulated with both hyaluronan and PDGF-BB.
iments. Immunoprecipitation of PDGFR␤ using the CT␤ antiserum followed by immunoblotting of CD44 using the Hermes-1 antibody yielded a band of 90 -105 kDa, i.e. the expected size of the standard isoform of CD44; no CD44 band was seen when a nonimmune serum was used for precipitation (Fig. 4A). The co-immunoprecipitation of PDGFR␤ and CD44 was seen in unstimulated as well as in PDGF-BB-stimulated cells. Re-probing the blot with anti-PDGFR␤ antibodies (CT␤) revealed the expected 190-kDa band, and re-probing with antiphosphotyrosine antibodies (PY99) revealed that the receptor was phosphorylated and activated after, but not before, PDGF-BB stimulation.
When the reverse experiment was done, i.e. immunoprecipitation of CD44 followed by blotting with PDGFR␤ antiserum, a band of 190 kDa, i.e. the size of PDGFR␤, was seen with or without PDGF-BB stimulation (Fig. 4B). Re-probing of the blot with Hermes-1 and PY99 antibodies resulted in bands with molecular masses corresponding to CD44 and phosphorylated ␤-receptor, respectively. Replacement of Hermes-1 with Hermes-3 (another CD44-specific antibody) gave similar results (data not shown). These findings suggest that the PDGFR␤ and CD44 form a complex in dermal fibroblasts, before as well as after stimulation with PDGF-BB. Notably, although the PDGFR␤, as expected, was tyrosine-phosphorylated after PDGF-BB stimulation, CD44 was not recognized by anti-phosphotyrosine antibodies, suggesting that CD44 is not a substrate for the PDGFR␤.
We also investigated the localization of CD44 and PDGFR␤ receptor in dermal fibroblasts using immunostainings. In unstimulated cells, both CD44 and PDGFR␤ were detected diffusely all over the cell with a preference at ruffle structures, where a partial co-localization was observed (Fig. 5A). In . Co-immunoprecipitation of PDGF ␤-receptor and CD44. Quiescent human dermal fibroblast cultures (4 ϫ 10 5 cells per 10-cm culture dish) were incubated for 15 min in starvation medium alone or in medium containing 10 ng of PDGF-BB/ml followed by lysis as described under "Materials and Methods." A, cell lysates were immunoprecipitated (IP) with PDGFR␤ antibodies (2 g/ml) or control rabbit IgG (2 g/ml) followed by immunoblotting (IB) with Hermes-1 antibody (2 g/ml) and re-blotting with PY99 (1 g/ml) and PDGFR␤ antibodies (2 g/ml). B, cell lysates were immunoprecipitated with CD44-specific antibodies Hermes-1 (5 g/ml) or control rat IgG (2 g/ml), followed by immunoblotting with PDGFR␤ antibodies (2 g/ml), and re-probing with Hermes-1 antibody (2 g/ml) as well as with PY99 (1 g/ml).

FIGURE 5.
Immunostaining of PDGF ␤-receptor and CD44 in human dermal fibroblasts. A, quiescent cells (2 ϫ 10 4 cells per well in 4-well Falcon culture slides) untreated or treated with PDGF-BB (10 ng/ml) for 15 min at 37°C were fixed and double-stained for PDGFR␤ (CT␤, 3 g/ml; red) and CD44 (Hermes-1, 1 g/ml; green), as described under "Materials and Methods." B, as in A but the cells were preincubated for 2 h in starvation medium containing 100 g of hyaluronan/ml, before PDGF-BB stimulation, followed by fixation and double staining for CD44 (Hermes-1, 1 g/ml; red) and hyaluronan (b-HABP, 2 g/ml; green). The slides were visualized under a Leica Microsystems fluorescence microscope. White arrowheads depict co-localization of CD44 and PDGFR␤ at the ruffles of the plasma membrane.
PDGF-BB-stimulated cells, the fibroblasts exhibited more ruffles and filopodial extensions, and CD44 was preferentially localized at these ruffle structures, where also the PDGF ␤-receptors were localized (Fig. 5A). PDGF-BB stimulation induced internalization of ␤-receptors into endosomes already after 15 min, noticed as dot-like structures; notably, CD44 was not present in these structures, suggesting that CD44 is not internalized together with the PDGF ␤-receptor. Double staining for hyaluronan and CD44 in PDGF-BB-treated cultures revealed a partial co-localization of hyaluronan and CD44 at ruffle-and filopodia-like extensions of the plasma membrane (Fig. 5B). Exogenously added hyaluronan also co-localized partially with CD44 (Fig. 5B); the staining was lost after Streptomyces hyaluronidase digestion (data not shown).
Protein-Tyrosine Phosphatase(s) Is Involved in the Hyaluronan-induced Inactivation of PDGFR␤-To investigate whether the hyaluronan-induced inhibition of PDGF ␤-receptor activation involved dephosphorylation of the receptor, we used the PTP inhibitor pervanadate in fibroblast cultures. As shown in Fig. 6, hyaluronan caused an approximate 40% reduction in the PDGF-BB-stimulated phosphorylation of the ␤-receptor. Incubation with pervanadate induced an increase in the phosphorylation of ␤-receptors, and interestingly, in the presence of pervanadate there was no difference between cells incubated with or without hyaluronan. These data suggest that the hyaluronan-CD44-mediated negative regulation of PDGFR␤ activa-tion in human dermal fibroblasts involves the recruitment of a PTP to the receptor.

DISCUSSION
We show in this study that hyaluronan inhibits PDGF-BBinduced activation of PDGFR␤ and cell motility. The monoclonal antibody Hermes-1, which blocks the binding of hyaluronan to CD44, restored PDGF ␤-receptor activation and motility, indicating that CD44 mediates the inhibiting effect on PDGFR␤. Furthermore, we demonstrate that PDGFR␤ and CD44 form a complex and that the inhibitory effect of hyaluronan is neutralized by inhibition of tyrosine phosphatases. Our data suggest that hyaluronan-activated CD44 modulate PDGFR␤ signaling by recruiting tyrosine phosphatase(s) to the receptor.
Our finding that PDGFR␤ and CD44 are associated into a complex adds to the examples of interactions between PDGFR␤ and receptors for matrix molecules. Thus, PDGFR␤ has been shown previously to interact with the integrin ␣ v ␤ 3 (26,27). However, the interaction with ␣ v ␤ 3 enhances PDGF signaling, whereas the interaction with CD44 suppresses PDGF signaling.
An important task for future studies will be to identify the molecular mechanisms whereby CD44 inhibits PDGFR␤. CD44 does not exhibit any intrinsic enzymatic activity, but from earlier studies it has been evident that CD44 interacts with other proteins, both in a hyaluronan-dependent and -independent manner and thereby modulates cellular responses (10). For example, fibroblast growth factor signaling is promoted by binding of fibroblast growth factors to heparan sulfate chains in the extracellular part of the CD44v3 isoform, whereby the ligand is presented to the signaling tyrosine kinase receptor (32). CD44 has also been shown to act as a co-receptor for the ErbB family of transmembrane tyrosine kinases (33,34) and for the transforming growth factor-␤ type I serine/threonine kinase receptor (35). In these cases, CD44 interaction enhances growth factor signaling.
We found that hyaluronan-activated CD44 inhibits PDGF␤R tyrosine kinase activity and suppressed signaling. Because pervanadate neutralized the inhibitory effect, it is likely that hyaluronan binding to CD44 activates a PTP that acts on the autophosphorylated PDGFR␤. Although several PTPs have been shown to have phosphorylated PDGFR␤ as substrate (25), the PTP(s) involved in CD44-mediated PDGFR␤ dephosphorylation has not been identified. Interestingly, the C termini of CD44 possess potential PDZ domain-binding sites (36); it is an interesting possibility that CD44 interacts with PDZ domaincontaining protein phosphatases (37,38). Notably, the PTP CD45, which dephosphorylates Lck and Fyn and other members in the Src family of tyrosine kinases, has been shown to have a negative regulatory role in CD44-and ␣ 5 ␤ 1 integrinmediated cell adhesion (39,40).
The concentrations of hyaluronan needed to obtain the negative effects on PDGFR␤ activation were rather high, i.e. Ͼ 25 g/ml. However, the concentration of hyaluronan in normal skin has been estimated to be 0.5 mg/ml, suggesting that the modulating effects of hyaluronan on PDGF signaling that we have demonstrated may have physiological relevance. . PTP modulates the hyaluronan-mediated PDGF ␤-receptor inactivation. Quiescent human dermal fibroblast cultures (2 ϫ 10 5 cells per 6-cm culture dish) were incubated in starvation medium alone or in medium containing 100 g of hyaluronan/ml (2 h) and then incubated for an additional 5 min in the absence or presence of 2.5 M pervanadate, before a 15-min stimulation with 5 ng of PDGF-BB/ml, followed by cell lysis. PDGF ␤-receptors were adsorbed to WGA-agarose and then subjected to SDS-PAGE and immunoblotting with PY99 and PDGF ␤-receptor antibodies to determine receptor phosphorylation and amount of receptors, respectively. Densitometric analyses of changes in PDGFR␤ phosphorylation in response to various treatments were performed. Data shown are representative of one experiment from three separate experiments with similar results. Columns depict the means of triplicate determinations Ϯ S.D. Significantly different (p Ͻ 0.05): *, from PDGF-BB-stimulated cells; **, compared with cultures exposed to both hyaluronan and PDGF-BB.
The effect of hyaluronan on PDGF signaling may also be of relevance in pathophysiological situations, such as wound healing. During wound healing, fibroblasts from the surrounding connective tissue become activated by the action of cytokines and growth factors released by platelets and infiltrating macrophages, and migrate and infiltrate the wound provisional matrix that is composed of fibrin and fibronectin. Inside the wound, fibroblasts synthesize large amounts of hyaluronan (2), fibronectin, and collagen types I and III (41). PDGF-BB stimulates hyaluronan synthesis and promotes the assembly of hyaluronan-containing pericellular matrices around mesenchymal cells (1). The findings of the present study suggest that hyaluronan networks may suppress the activity of PDGF-BB and infiltration of fibroblasts into the provisional matrix before matrix remodeling occurs. Thus, the observations by us and others suggest that growth and motility of cells in tissues are regulated by growth factors and extracellular molecules together in a synergistic as well as antagonistic manner (30).