Both cell adhesion protein CD44 and its main ligand hyaluronic acid (HA) are thought to be involved in several processes ultimately requiring cytoskeleton rearrangements. Here, we show that the small guanine nucleotide (GTP)-binding protein, Rac1, can be activated upon HA binding to CD44. When applied locally to a passive cell edge, HA promoted the formation of lamellipodial protrusions in the direction of the stimulus. This process was inhibited by the prior injection of cells with dominant-negative N17Rac recombinant protein or by pretreatment of cells with monoclonal anti-CD44 antibodies, interfering with HA binding, implying the direct involvement of CD44 in signaling to Rac1.

Introduction

A widely distributed cell adhesion protein, CD44, serves as the major cell surface receptor for hyaluronic acid (HA). HA is a main carbohydrate component of the extracellular matrix with a simple, repeated disaccharide linear copolymer structure. It is mostly found in skin, joints, and eyes, but is also present in other organs and tissues. HA expression is tightly regulated; it is elevated in a variety of inflammatory conditions (Gerdin and Hallgren 1997). Transient overexpression of HA also takes place in granulation tissue during the wound healing process (Chen and Abatangelo 1999). The biological roles attributed to HA range from a purely structural function in the extracellular matrix to regulation of cell motility and adhesion (Itano et al. 1999; Delpech et al. 1997) and receptor-mediated alterations in gene expression (Fujii et al. 1999). Both CD44 and HA are thought to be involved in a variety of physiological processes, such as tumor formation and metastasis (Günthert 1996), wound repair (Kaya et al. 1997), the inflammatory immune response (Günthert 1999), lymphocyte homing and adhesion (Siegelman et al. 1999), and embryonic development (Ponta and Herrlich 1998). All these processes require concordant rearrangements of the actin cytoskeleton being the basis of various cell adhesion and migration events. Yet, there is almost no information available regarding possible intracellular signals transmitted upon CD44 binding to HA. Similarly, the exact roles of CD44 in vivo remain to be determined. Here, we show that in mouse mammary epithelial cells (EpH4), CD44 interaction with HA triggers the local signaling cascade, resulting in the direct activation of small GTP-binding protein Rac1, actin cytoskeleton rearrangements, and reorientation of cells in the direction of the provided cue.

Materials and Methods

Cells, Antibodies, Reagents, and Constructs

Media, tissue culture reagents, and FCS were purchased from GibcoBRL and Boehringer Mannheim Corp. EpH4 cells, a spontaneously immortalized mouse mammary epithelial cell line (Fialka et al. 1996), were cultured at 37°C in 5% CO2 and 98% humidity in Eagle's medium, supplemented with 10 mM Hepes/KOH, pH 7.3, 50 IU/ml penicillin, 50 mg/ml streptomycin, and 5% FCS.

Anti-CD44 antibody, KM114, blocking hyaluronate recognition by CD44, and anti-CD44 antibody, IM7.8.1, were purchased from RDI. Anti-Rac1 antibodies were purchased from Transduction Laboratories. Oligosaccharide of HA (H-9649) was purchased from Sigma Chemical Co., and high molecular weight HA, labeled with FITC, was a kind gift from Dr. C. Isacke and coworkers (Imperial College, London, UK). Laminin (20–200 μg/ml) and fibronectin (12–300 μg/ml) were from Boehringer Mannheim. Lipofectamine PLUS™ Reagent was purchased from GibcoBRL. Rhodamine–phalloidin was from Molecular Probes (R-415). Recombinant N17Rac and N17Cdc42 were kindly provided by Dr. K. Rottner (Austrian Academy of Sciences, Salzburg, Austria; using a construct originally provided by Dr. A. Hall, University College, London, UK), dialyzed into 50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM MgCl2, and 1 mM DTT. Concentrations of 1–2 mg/ml were used for injections. GST-CRIB construct was kindly donated by Dr. R. Cerione (Cornell University, Ithaca, NY). Human zyxin and paxillin in pEGFP-N1 vector were kindly supplied by Dr. J. Wehland and coworkers (GBF, Braunschweig, Germany). NH2-terminal EGFP human β-actin, under the control of CMV promoter, cloned in pcDNA3 vector was kindly provided by Dr. M. Way (EMBL, Heidelberg, Germany).

Microinjections and Local Applications

Microinjections were performed with sterile Femtotips (Eppendorf, 5242 952.008) held in a Leitz micromanipulator with a pressure supply from an Eppendorf Microinjector 5242. Cells were injected with continuous outflow mode from the needle under a constant pressure of 20–40 hPa. Local applications of HA were performed essentially as described in Kaverina et al. 1999, under a constant pressure of 25–50 hPa.

Videomicroscopy

Cells were observed in an open chamber at 37°C on an inverted microscope (Axiovert 135TV, Zeiss) equipped for epifluorescence and phase-contrast microscopy. Injections were performed at 40× (NA 1.3 Plan Neofluar), and videomicroscopy with a 100× (NA 1.4 Plan-Apochromat) with or without 1.6× optovar intermediate magnification. Data were acquired with a back-illuminated cooled CCD camera from Princeton Research Instruments driven by IPLabs software (Visitron Systems). The microscope was additionally equipped with shutters driven through a homemade interface to allow separate recordings of video sequences in phase-contrast and fluorescent channels. Time between frames was 18 s. The video sequences were analyzed and processed on a Macintosh Power PC using IPLabs and Adobe Photoshop 5.2.1 software.

Online Supplemental Material

The online version of this article includes videos that accompany the figures presented here. Videos are supplied in the QuickTime format and are available at http://www.jcb.org.cgi/content/full/148/6/1159/DC1.

Video 1.

Depicts Fig. 1 a.

Video 2.

Depicts Fig. 1 c.

Video 3.

Depicts Fig. 2 a.

Video 4.

Depicts Fig. 2 b.

Video 5.

Depicts Fig. 2 b, inset.

Video 6.

Depicts Fig. 2 c.

Video 7.

Depicts Fig. 3 a.

Video 8 and the Supplemental Figure.

Microinjection of the dominant-negative N17Cdc42 recombinant protein does not block HA/CD44-induced lamellipodial formation. EGFP-β-actin–expressing cell was microinjected into N17Cdc42 recombinant protein and, subsequently, treated with HA. Note the formation of new lamellipodia in response to HA.

Results

EpH4 cells derive from a spontaneously immortalized mouse mammary epithelial cell line and display a fully polarized epithelial cell phenotype when grown to high density on semipermeable filter supports or in a three-dimensional collagen matrix (Fialka et al. 1996). However, when sparsely seeded, EpH4 cells often display a fan-shaped and motile fibroblastoid phenotype. Fibroblastoid cells typically exhibit protruding, mobile lamellipodial regions, interspersed by mainly concave, inactive cell edges, delimited by peripheral actin filament bundles (Small 1988). EpH4 cells express large amounts of CD44, consisting of standard and several variant isoforms (data not shown). In many cell types, CD44 exhibits a nonrandom distribution in the plasma membrane. In polarized epithelial cells, CD44 localization is restricted to the basolateral membrane domain, whereas in sparsely seeded, unpolarized EpH4 cells, CD44 is more broadly distributed, but clearly concentrated in the trailing regions and in the cell body (Oliferenko et al. 1999).

When sparsely seeded, EpH4 cells were treated with soluble oligosaccharides of HA. The majority of cells appeared depolarized, often with multiple lamellipodia around their entire circumference. This phenomenon was followed in more detail in single cells by phase-contrast time-lapse microscopy. By these means, we could confirm the fast outgrowth of new lamellipodia from cell edges previously exhibiting an inactive actin cytoskeleton, including trailing regions (Fig. 1 a, see also supplemental video). We quantified the percentage of cells exhibiting the polarized fibroblastoid phenotype in control and HA-treated cells. Almost three quarters (69%) of sparsely seeded EpH4 cells in control cultures exhibited unidirectional movement and a polarized morphology. Already 15 min after addition of HA, only 28% EpH4 cells remained polarized, whereas the rest exhibited multiple lamellipodia. However, the proportion of polarized cells in the presence of HA was comparable to control (68%) upon pretreatment of cells with anti-CD44 antibody, KM114 (Fig. 1 b). This mAb efficiently inhibits binding of HA to CD44 (Miyake et al. 1990). Inhibition of HA-induced lamellipodial protrusion by KM114 implied the direct involvement of CD44 in mediating the HA effect. To evaluate changes in the actin cytoskeleton in real time, we repeated HA treatment on EpH4 cells transiently expressing EGFP-β-actin. We observed the reorganization of the actin cytoskeleton, new lamellipodial outgrowth, characterized by a band of actin fluorescence, active ruffling of the leading edges, and the appearance of very bright EGFP-actin containing spots, or microruffles, on the cell surface (Fig. 1 c, see also supplemental video).

The immediate effect of HA treatment on the cell shape led us to investigate whether the observed changes could be induced locally. This was tested by the local application of HA through a microinjector needle to selected regions of cells. When HA was applied to inactive cell edges we could indeed observe a spatially restricted outgrowth of lamellipodia underneath the micropipette (Fig. 2 a, see also supplemental video). Pretreatment of cells with KM114 antibody effectively prevented these rearrangements, in a way similar to after the general application of HA (data not shown). HA-induced lamellipodia harbored small focal complexes that subsequently elongated and grew into mature focal adhesions, as revealed by experiments with cells transiently transfected with EGFP fusion constructs of the focal adhesion proteins, zyxin (Fig. 2 b, see also supplemental video) and paxillin (data not shown). Identical results were obtained with high molecular weight HA (Fig. 2 c, see also supplemental video). The local applications of the matrix proteins, laminin and fibronectin, under identical conditions did not produce lamellipodia outgrowth (data not shown).

HA-induced cytoskeleton rearrangements were highly reminiscent of lamellipodial protrusion and ruffling caused by the activated GTPase Rac1 (Ridley et al. 1992), a member of the Rho family of small GTPases (Tapon and Hall 1997). Rac1 is thought to be activated by various stimuli, including growth factors (Peppelenbosch et al. 1995; Ridley et al. 1995; Wennstrom et al. 1994; Nobes et al. 1995). To analyze whether Rac1 is indeed involved in HA-induced lamellipodial outgrowth, we analyzed the effect of HA on cells injected with the mutant form of Rac1, N17Rac, which acts as a dominant inhibitor of Rac1 activity. When cells were microinjected with N17Rac recombinant protein before HA treatment, the formation of lamellipodia was efficiently blocked (Fig. 3 a, see also supplemental video). Analysis of the effect of N17Rac was performed in ten independent experiments; the HA-induced lamellipodia outgrowth was inhibited in all cells observed. Likewise, de novo–formed HA-induced lamellipodia were retracted when cells were injected with N17Rac after HA treatment (data not shown). Injection of dominant-negative N17Cdc42 did not prevent HA-induced lamellipodia outgrowth, arguing for a direct effect on Rac, rather than via Cdc42 (data not shown; see supplementary figure and video available at http://www.jcb.org.cgi/content/full/148/6/1159/DC1).

To determine whether HA/CD44-induced lamellipodial outgrowth could be attributed to the direct activation of Rac1 instead of an activation of its downstream targets, we performed an affinity pull-down assay, allowing us to directly measure the amount of the active, GTP-bound form of Rac1 (Bagrodia et al. 1998). The CRIB domain of the downstream Rac1 effector, PAK, specifically binds the GTP-bound form of Rac1, which allows a quantification of actual amounts of active Rac1 in cell lysates. The affinity matrix, representing GST-fusion of CRIB domain coupled to the glutathione–Sepharose beads, was incubated with cell lysates derived from either control cells or cells treated with HA for various times. As shown in Fig. 3 b, the amount of GTP-bound Rac1 was considerably increased already after two minutes of HA treatment, and continued to a plateau after 15 min. However, the total amount of Rac1 did not change upon HA treatment (Fig. 3 b). Taken together, these results argue for the direct activation of Rac1 upon HA/CD44 engagement.

Discussion

Application of soluble HA to EpH4 cells in culture promoted activation of Rac1 and rapid outgrowth of lamellipodia from previously inactive cell edges. This effect was dependent on the ability of CD44 to bind HA. More interestingly, when HA was applied locally, a spatially restricted outgrowth of lamellipodia was observed precisely in the region of HA application. This result means that activation of Rac1 probably takes place directly in the vicinity of the plasma membrane. Rac1 activation appears to be independent on Cdc42, since microinjection of dominant-negative Cdc42N17 protein does not block HA-induced lamellipodia outgrowth. HA-induced lamellipodia also contained mature focal adhesions. This strongly implies that, in this case, not only is Rac1 activated, but also that Rho remains active.

The precise details of Rac1 activation upon HA/CD44 engagement remain to be elucidated. However, it is tempting to speculate that one of the Rac1 accessory proteins could participate in complex formation with CD44. One of the logical candidates would be the guanine nucleotide exchange factor, Tiam-1, which was shown to specifically activate Rac1, but not Cdc42 (Sander et al. 1999). Recently, while this paper was under review procedure, the biochemical evidence of the direct CD44/Tiam-1 interaction was reported (Bourguignon et al. 2000). This result might provide the potential straightforward link between CD44/HA binding and Rac1 activation. CD44 was also found in a complex with Rho-GDP dissociation inhibitor (RhoGDI; Olofsson 1999) and proteins of the ERM (ezrin-radixin-moesin) family in fibroblasts (Hirao et al. 1996). The interaction of ERM proteins with RhoGDI was shown to activate Rho subfamily members (Hirao et al. 1996; Takahashi et al. 1997). It might also be possible that HA binding to CD44 would trigger the conformational change in this complex, inactivating RhoGDI as a result, and ultimately contributing to the activation of Rac1.Yet, CD44/ERM interaction appears to be rather cell-type restricted. So far, we have no indication that they interact with each other in EpH4 mammary epithelial cells (Oliferenko et al. 1999).

Interestingly, when the dominant active mutant of Rac1 was overexpressed in human glioblastoma cells (U251MG), CD44 was shown to redistribute to lamellipodia where it subsequently was cleaved and shed from the surface (Okamoto et al. 1999). A possible mechanism for downregulating CD44-mediating signaling can be suggested by these experiments.

The observation we describe here would suggest the possibility of the direct involvement of CD44 and HA in cell guidance. We would like to propose that HA may serve as an external stimulus influencing cell orientation, with CD44 mediating signaling to the actin cytoskeleton.

Acknowledgments

We are most grateful to Drs. R. Cerione, J. Wehland, A. Hall, M. Way, C. Isacke, U. Günthert, and K. Rottner for generous sharing of materials and reagents, and to J. Glotzer for critically reading this manuscript and for providing helpful comments. We also would like to thank Dr. H. Beug for stimulating discussions throughout all stages of this work.

This work was supported by IMP and by grants from the Austrian Industrial Research Promotion Fund (FFF; 3/11504 to L.A. Huber), the Austrian Science Foundation (FWF; P11446-MED to L.A. Huber and P12077-MOB to J.V. Small), as well as by a grant from the Johnson & Johnson Focused Giving Program (to L.A. Huber).

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The online version of this article contains supplemental material.

Abbreviations used in this paper: EpH4, mouse mammary epithelial cell line; HA, hyaluronic acid.