TheHighandLowMolecularWeightFormsofHyaluronan ... · TheHighandLowMolecularWeightFormsofHyaluronan ... (Nikon A1, Tokyo, Japan). For sensitized fluorescence emission measurements,

The High and Low Molecular Weight Forms of HyaluronanHave Distinct Effects on CD44 Clustering*□S

Received for publication, October 1, 2012 Published, JBC Papers in Press, November 1, 2012, DOI 10.1074/jbc.M112.349209

Cuixia Yang‡1, Manlin Cao§1, Hua Liu¶, Yiqing He‡, Jing Xu¶, Yan Du‡, Yiwen Liu‡, Wenjuan Wang¶, Lian Cui‡,Jiajie Hu‡, and Feng Gao‡2

From the Departments of ‡Molecular Biology Laboratory, §Rehabilitation Medicine, ¶Clinical Laboratory, Shanghai JiaotongUniversity School of Medicine, Shanghai 200233, China

Background: The feature of CD44 binding with native high molecular weight hyaluronan (nHA) and hyaluronan oligosac-charides (oHA) is different.Results: nHA induces but oHA reduces CD44 clustering.Conclusion: nHA and oHA have distinct effects on CD44 clustering.Significance: The study provides direct evidence for the different characteristics of CD44 binding with nHA and oHA in vivo.

CD44 is a major cell surface receptor for the glycosaminogly-can hyaluronan (HA). Native highmolecular weight hyaluronan(nHA) and oligosaccharides of hyaluronan (oHA) provoke dis-tinct biological effects upon binding to CD44. Despite theimportance of such interactions, however, the feature of bindingwith CD44 at the cell surface and the molecular basis for func-tional distinction between different sizes of HA is still unclear.In this study we investigated the effects of high and low molec-ular weight hyaluronan on CD44 clustering. For the first time,we provided direct evidence for a strong relationship betweenHA size and CD44 clustering in vivo. In CD44-transfectedCOS-7 cells, we showed that exogenous nHA stimulated CD44clustering, which was disrupted by oHA. Moreover, naturallyexpressed CD44was distributed into clusters due to abundantlyexpressed nHA in HK-2 cells (human renal proximal tubulecells) and BT549 cells (human breast cancer cell line) withoutexogenous stimulation.Our results suggest that nativeHAbind-ing toCD44 selectively inducesCD44 clustering, which could beinhibited by oHA. Finally, we demonstrated that HA regulatescell adhesion in amanner specifically dependent on its size. oHApromoted cell adhesion while nHA showed no effects. Ourresults might elucidate a molecular- and/or cellular-basedmechanism for the diverse biological activities of nHAandoHA.

The extracellular matrix glycosaminoglycan, hyaluronan(HA),3 is an unbranched polymer composed of repeating glu-

curonic acid andN-acetyl glucosamine disaccharide units. Themost widely distributed form of HA in normal tissues is nativehigh molecular weight HA (called HMW-HA or nHA) with ahigh molecular weight (�107 Da). The lower molecular weightforms of HA, also called hyaluronan oligosaccharides (oHA),are synthesized de novo or generated by either hyaluronidase-mediated degradation or oxidative hydrolysis of native HAunder pathological conditions (1), including repair, inflamma-tion, and tumor processes. nHA and oHA serve a variety offunctions that are mediated by cell surface receptors, includingcell motility, adhesion, migration, and metastasis (2–5).CD44 is a major cell surface receptor for HA, which has an

N-terminal link module homology domain that is responsiblefor binding to HA. The interaction of CD44 and HA is stronglyinfluenced by cell-specific factors (6), cell types (7), the state ofCD44 activation (8), and the size of the HA ligand (9–11). Sev-eral mechanisms have been proposed for the regulation ofreceptor-mediated HA binding to CD44 (12–14). For example,the differences inHA chain length can influence bothHAbind-ing features and its functional consequences.High and lowmolecular weight forms ofHAprovoke distinct

pro-inflammatory and anti-inflammatory effects upon bindingto CD44 and can deliver either proliferative or anti-prolifera-tive signals in appropriate cell types (15–19). Many studiesshowed that nHA exhibited anti-angiogenic and anti-inflam-matory effects in several in vivo assays, inhibiting cellmigration,proliferation, and sprout formation. In contrast, oHA stimu-lated cell proliferation and motility, exhibiting pro-inflamma-tory and pro-angiogenic effects in a variety of experimental sys-tems (19–21). We previously reported that nHA and oHAregulated cell cycle progression through G1 phase in distinctmanner in vascular endothelial cells (19). Although thesereports demonstrate that the interaction of CD44 with oHAand nHA exerts distinct effects on cell biological behavior, theprecise feature of the CD44 binding in response to differentmolecular weight HA remains obscure.

* This work was supported by the Science and Technology Commissionof Shanghai Municipality (Key Technology Support Programme,08411952700), the National Natural Science Foundation of China(81071814, 81172027, 81272479), and the Program of Shanghai SubjectChief Scientist (11XD1404000).Author’s Choice—Final version full access.

□S This article contains supplemental Figs. S1 and S2.1 Both are co-first authors.2 To whom correspondence should be addressed: Dept. of Molecular Biology

Laboratory, Shanghai Sixth People’s Hospital, Shanghai Jiaotong Univer-sity School of Medicine, 600 Yishan Rd., Shanghai 200233, China. Tel.:86-21-64369181; Fax: 86-21-63701361; E-mail: [email protected] [email protected].

3 The abbreviations used are: HA, hyaluronan; nHA, native high molecularweight hyaluronan; oHA, hyaluronan oligosaccharide; PMT, plasma mem-

brane target; CFP, cyan fluorescent protein; YFP, yellow fluorescent pro-tein; BS3, bis(sulfosuccinimidyl) suberate; 4-MU, 4-methylumbelliferone.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 51, pp. 43094 –43107, December 14, 2012Author’s Choice © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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In living cells the extracellular matrix including hyaluronanplays important role in themaintenance of appropriate cell-cellcommunication (22). Once the balance is disrupted, such as intumor invasion, inflammation, or tissue remodeling, nativehigh molecular weight HA is digested into small fragments(oHA). The loss of nHA and the appearance of its lower molec-ular weight forms could contribute to the changes of cell behav-ior and cell signaling. The most significant feature of HA is itshighly repetitive nature. Therefore, the highly multivalentrepeating disaccharide chains of HA interact with multiple,closely arrayed CD44 receptor molecules (23). However, theaffinity of a single CD44-HA binding domain for HA is likely tobe very low (10, 24, 25). Up to now, the molecular basis forfunctional distinction between the binding of CD44with differ-ent sizes of HA is largely unclear.Clustering of CD44 is a typical character that affects cell

response after stimulated by highly produced HA (26). In thisstudy we utilized a fluorescence resonance energy transfer(FRET) technique to investigate the effects of oHA and nHA onCD44 clustering in COS-7 cells transfected with CD44 expres-sion vectors. Furthermore, we chose HK-2 cells (human renalproximal tubule cells) and BT-549 cells (human breast cancercell line) to evaluate the binding of oHA or endogenous nHA toCD44 because HK-2 and BT-549 cells naturally express abun-dantly CD44 and HA in naive status. We demonstrated thatnHA induced CD44 clustering, which could be disrupted byoHA. These results provide direct evidence that high and lowmolecular weight forms of HA have distinct effects on CD44clustering.

EXPERIMENTAL PROCEDURES

Reagents—CD44 mAb (clone IM7, catalog no. 12-0441-81)for flow cytometry analysis was purchased from eBioscience.Biotinylated hyaluronan-binding protein was from Merck.Purified NA/LE mouse anti-CD44 (clone 515) was obtainedfrom BD Biosciences Pharmingen. Anti-CD44 antibody (156-3C11), ERK inhibitor (U0126), and anti-phospho-ERK1/2mAbwere obtained fromCell Signal. nHAwas obtained from Sigma.Hyaluronan oligosaccharides (oligomers) were prepared asdescribed previously (27, 28), which was a mixed fraction ofaveragemolecular weight of 2.5� 103 composed of 3–10 disac-charide units that were fractionated from testicular hyaluroni-dase (Sigma, type 1-S) digests of hyaluronan polymer (Sigma,sodium salt). 4-Methylumbelliferone and anti-vinculin mAbwere purchased from Sigma. All other chemicals were of rea-gent grade or higher.Plasmid Construction—The cDNA encoding the hematopoi-

etic formof CD44 (CD44-H)was obtained by reverse transcrip-tion PCR (RT-PCR) using total RNA isolated from humanperipheral blood lymphocytes and subcloned into themamma-lian expression vectors pECFP-N1 and pEYFP-N1 (Clontech,Palo Alto, CA). The isoform CD44-H was used to denote a80–90-kDa product (29). A plasmid encoding a positive controlfor the FRET experiment, PMT-CFP-YFP, was kindly providedby Dr. ThorstenWohland (Department of Chemistry, NationalUniversity of Singapore, Singapore) (30). In the positive controlplasmid the plasma membrane target (PMT) sequence was

fused toCFP at theN terminus, and the YFP genewas amplifiedand fused to CFP at the C terminus.Cell Culture and Transfection—Mammalian cells COS-7

(simian virus 40-transformed African Green monkey kidneycell line), HK-2 cells (human renal proximal tubule cell line),and human breast cancer cells BT549 were cultured in Dulbec-co’s modified Eagle’s medium supplemented with 10% fetal calfserum, 100 units/ml penicillin, and 100 mg/ml streptomycin.COS-7 cells were grown to 85% confluency and transfected byusing Lipofectamine 2000 (Invitrogen).Binding of Exogenous HA to Transfected COS-7 Cells—After

transfection, COS-7 cells were incubated with 40 �g/ml fl-HA(Merck) for 1 h at 4 °C as described previously (29), then thecells were treated by EDTA and washed three times in phos-phate-buffered saline (PBS). After fixation with 1% paraformal-dehyde and nuclear counterstaining by propidium iodide,plasmamembrane-bound fl-HAwas observed under a confocalmicroscope (NikonA1,Tokyo, Japan) and analyzed using a flowcytometer (Beckman-Coulter, Brea, CA).Flow Cytometric Analysis—Cultured cells were harvested

and washed with washing buffer (PBS supplemented with 2%bovine serum albumin, pH 7.4). A single cell suspension (106/ml) was incubated with specific antibodies or isotope controlantibodies on ice for 1 h. Cells were washed 3 times with wash-ing buffer, and 1�l of the specific antibodywas added for 1 h onice. Cells were analyzed in a flow cytometer (Beckman-Coulter). At least 6000 cells were analyzed per sample in allexperiments. All experiments were performed at least twice.FRETMeasurements with Spectroscopy—FRET spectroscopy

was performed essentially as described previously (31). Briefly,COS-7 cells were plated into 96-well black plates for 24 h thentransfectedwith plasmids encodingCD44-YFP andCD44-CFP,PMT-CFP-YFP, or co-transfected with CD44-YFP and CD44-CFP. For FRETmeasurements, fluorescence intensities of CFP,YFP, and PMT-CFP-YFP were measured using a microplatefluorescence reader (Bio-Tek Instruments, Winooski, VT).After an excitation narrowed at 440 nm, the special excitationchannel of CFP, emission fluorescence was obtained at 485 �10 nm (CFP emission peak) and 528 � 10 nm (YFP emissionpeak). If FRET occurs, the fluorescence at 528 nm will beenhanced for efficient energy transfer between donor andacceptor. A modified method for FRET evaluation was appliedaccording to a previous report (31). Calculation of the ratiobetween emission at 528 and 485 nmwas used as an indicator ofFRET efficiency. Higher values of 528/485-nm ratio indicatedhigher efficiency of FRET values. Fluorescence fromnon-trans-fected cells and background scattering light brought by theequipment was subtracted from each sample to obtain the spe-cific fluorescence.FRET-based Dose-response Experiment for Competitive Inhi-

bition of oHA—Aprimary dose-response experimentwith vary-ing concentrations of oHA was performed using a microplatefluorescence reader (Bio-Tek Instruments). Cells co-trans-fected with CD44-YFP and CD44-CFP were pretreated withnHA, then incubated with different doses of oHA (0, 31.25,62.5, 125, and 250 �g/ml). The fluorescence was recorded inmonochromatic mode with excitation at 440 nm and emissionat 485 and 528 nm. The FRET efficiency, the ratio between

CD44 Clustering Induced by HA

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emission at 528 and 485 nm, was used as an indicator of thedose-dependent changes in HA treatments.FRET Imaging—FRET imaging was performed basically as

described before (32, 33) on a confocal laser scanning micros-copy (Nikon A1, Tokyo, Japan). For sensitized fluorescenceemission measurements, images were acquired sequentiallythrough YFP, CFP, and FRET channels. The microscopy setsusedwere YFP (excitation, 500/25 nm; emission, 526–568 nm),CFP (excitation, 458 nm; emission, 461–504 nm), and FRET(excitation, 458 nm; emission, 526–568 nm). FRET efficiencywas determined using NIS-Elements AR3.1 Imaging software(Nikon, Tokyo, Japan). Fluorescence through the FRET chan-nel consisted of a FRET component (“corrected” FRET, FRET)and non-FRET components, including spectral bleed throughand cross-excited fluorescence. The non-FRET componentswere subtracted as described previously (34). FRETCwas calcu-lated on a pixel-by-pixel basis for the entire ’’region of interest”image, where FRET, CFP, and YFP were presented as back-ground-subtracted fluorescence intensities acquired throughthe FRET, CFP, and YFP channels, respectively. In our experi-mental conditions we used the following equation: FRETC �FRET � 0.55 � CFP � 0.11 � YFP. The 0.55 and 0.11 were thefractions of bleed-through of CFP and YFP fluorescence to theFRET channel, respectively. The fractions were calculated bymeasuring fluorescence intensities of cells individually express-ing CD44-CFP or CD44-YFP through the CFP, YFP, and FRETchannels. All the experiments were performed at roomtemperature.

Acceptor-photobleaching-based FRET Measurement—Confocalmicroscopy imaging and photobleaching experiments wereperformed at room temperature using an Nikon A1 laserscanning confocal microscopy system. If any interactionsleading to energy transfer were present in the cells, photo-bleaching of the acceptor would lead to an increase in donorfluorescence because it would no longer be quenched by theacceptor.In the photobleaching experiments, a 60-s pulse of high

intensity laser at 514 nm (100% exposure at 514 nm beam) wasapplied to locally bleach the YFP signal acceptor in a whole cell.Data acquisition was performed in the sequential line mode forthe best spatiotemporal reliability. The sample area wasscanned at the resolution of 1024 � 1024 pixels. The CFP andYFP were excited at 458 and 514 nm, respectively.Two frames of pre-bleaching and post-bleaching CFP

(donor) fluorescence intensities were recorded. The FRET effi-ciency (E) was calculated byE� 1� (Fpre/Fpost)�Fpre, where Fpreand Fpost indicate the fluorescence intensity of the donor beforeand after acceptor photobleaching, respectively.Immunocytochemistry Analysis of CD44 Clustering—The

clustering of CD44 was analyzed by immunocytochemistry.HK-2 and BT-549 cells were cultured on glass coverslips, thenincubated with DMEM (control), oHA, hyaluronidase, and4-methylumbelliferone (4-MU). After incubation, cells werewashed briefly with PBS, fixated in 4% paraformaldehyde for 10min, then permeabilized using Triton X-100 (0.1% v/v in PBS)for 10 min at room temperature. The cells were incubated with

FIGURE 1. Binding of exogenous fl-HA by COS-7 cells transfected with CD44 expression constructs. 24 h post-transfection, COS-7 cells were analyzed byWestern blot and flow cytometry analysis. The capacity of transfected cells to bind exogenous fl-HA was analyzed by flow cytometry and immunocytochem-istry analysis. A, shown is immunoblotting of CD44 in the lysates from CD44-CFP (lane 2)- and CD44-YFP (lane 3)-transfected COS-7 cells. Naive COS-7 cell wasused as the negative control. B, cell surface expression of CD44 in CD44-CFP- and CD44-YFP-transfected COS-7 cells was assessed by flow cytometry. Histo-grams indicate log fluorescence intensity (x axis) versus relative cell number (y axis). 1, naïve COS-7 cells was used as mock; 2, isotype-matched antibody asnegative control; 3, pECFP-N1 transfected cells; 4, pEYFP-N1 transfected cells; 5, CD44-CFP transfected cells; 6, CD44-YFP transfected cells. C, shown is bindingand uptake of exogenous fl-HA by transfected COS-7 cells. 1, naïve COS-7 cells was used as negative control; 2, CD44-CFP transfected COS-7 cells; 3, CD44-YFPtransfected COS-7 cells. D, binding of exogenous fl-HA by transfected COS-7 cells was detected by immunocytochemistry analysis. Nuclei were visualized bypropidium iodide (PI) staining. COS-7 cells were transfected with CD44-CFP or CD44-YFP respectively. Naïve COS-7 cells were used as mock control.




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anti-CD44 antibody (156–3C11) overnight at 4 °C, then stainedwith an Alexa Fluor 488-conjugated chicken anti-mouse IgGantibody (Invitrogen) for 1 h in the dark at room temperature.The coverslips were then placed onto slides using glycerol as amountant and visualized under confocal microscopy (NikonA1, Tokyo, Japan).Quantitation of Endogenous Hyaluronan Generation—To

understand the effect of oHA on endogenous hyaluronan

generation, the amount of liberated and cell-associated hya-luronan was determined as described previously (35). Trip-licate cultures of cells were seeded in a 6-well culture plateand grown for 24 h to 85% confluence. Cells were treatedwith different doses of oHA (0, 31.25, 62.5, 125, and 250�g/ml) for 2 h then harvested by trypsinization and countedusing a Coulter counter. Media were used for quantitation ofthe liberated HA. Cell-associated extracellular HA was

FIGURE 2. nHA and oHA regulate CD44 clustering in COS-7 cells transfected with CD44 expression constructs. Clustering of CD44-CFP and CD44-YFP wasdetected by the efficiency of FRET between donor (CFP) and acceptor (YFP). Fluorescence at 485 nm (CFP emission peak) and 528 nm (YFP emission peak) weremeasured after excitation only at 440 nm (CFP special excitation channel). The ratio of fluorescence at 528 and 485 was estimated to indicate the transferredenergy of CFP to YFP. CD44-CFP, transfected with CD44-CFP plasmid; CD44-YFP, transfected with CD44-YFP; CFP, transfected with pECFP-N1 vector; YFP,transfected with pEYFP-N1 vector; PMT-CFP-YFP, CFP-YFP fusion protein with a plasma membrane target (PMT) sequence at the CFP N terminus. A, fluorescencespectra of recombinant CFP and YFP proteins are shown. B, nHA and oHA treatment led to the changes in the efficiency of energy transfer from cyan to yellowfluorescent proteins in COS-7 cells co-transfected with CD44-CFP and CD44-YFP. C, oHA abrogated CD44 clustering triggered by exogenous nHA. Afterexposed to nHA, cells were treated with oHA of different concentrations (0 –250 �g/ml) for another 2 h. The fluorescence intensity was plotted as the mean (n �8) �S.D. RFU, relative fluorescence units.




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obtained by centrifugation of the cell/trypsin fraction at2000 rpm in a Beckman TJ-6 centrifuge where the superna-tant was quantitated for HA. The extracellular liberated andcell-associated fractions were pooled and characterized forHA concentration with the 125I-labeled HA radioimmunoas-say, the detection limits of which is �5.5 ng/ml.Chemical Cross-linking for CD44 Association at the Cell

Surface—Cells were treated with DMEM, oHA, testicular hya-luronidase or 4-MU, then the cells were washed 5 times withice-cold PBS (20 mM sodium phosphate, 0.15 M NaCl, pH 8.0).CD44 cross-linking was performed by incubation with 2 mM

bis(sulfosuccinimidyl) suberate (BS3) (Pierce) for 1 h at 4 °C andquenched by incubation with 20 mM Tris, pH 7.5, for 15 min atroom temperature. Cells were washed twice with PBS and lysedwith cell lysis buffer.Western Blotting—Cells were harvested and homogenized in

ice-cold sodium dodecylsulfate (SDS) lysis buffer. Total celllysates were collected, and equal quantities of protein were sep-arated by SDS-PAGE and blotted onto a PVDFmembrane. ThePVDF membranes were blocked with Tris-buffered saline(TBS) containing 5% skimmed milk powder for 1 h, washed for1 h in TBS, and incubated with CD44 (156–3C11, Cell Signal)or phospho-ERK1/2 mAb (Cell Signal) at 4 °C for 3 h. Then themembranes were washed with 1� Tris-buffered saline/Tween20 (TBS/T) buffer for three times (5 min each time) and incu-bated with HRP-conjugated polyclonal secondary antibody for1 h. The membranes were developed with the enhanced pluschemiluminescence assay (Pierce) according to the manufac-turer’s instructions.Cell Adhesion Assay—The adhesion of cells was examined as

previously described (36, 37). After being pretreated with con-

trol (DMEM), oHA, and nHA for 2 h, cells were detached with5mMPBS/EDTA.Cells (5� 103) in spreadingmedium (DMEMwith 0.1% heat-inactivated FBS) were seeded onto a 96-wellplate. After 30 min at 37 °C, unbound cells were removed, andthe remaining cells were counted. Treatment with ERK inhibi-tor (U0126; 5 �M, 24 h) was performed to detect the effect ofinhibition of ERK on cell adhesive ability. Three independentexperiments were performed.Statistical Analysis—The data were presented as themean�

S.D. All statistical analysis was performed using SPSS11.0. Theunpaired Student’s t test was used for the comparisonsbetween two different groups. p less than 0.05 was consid-ered significant.

RESULTS

Characterization of CD44-YFP and CD44-CFP Constructs—COS-7 cells were chosen to characterize CD44-CFP andCD44-YFP constructs due to the absence of endogenous expression ofCD44 and hyaluronan in these cells (Fig. 1A, first lane, andsupplemental Figs. S1).Western blot analysis revealed a band at�85 kDa in pCD44H-transfected COS-7 cells, correspondingto the molecular weight of the standard form of human CD44(Fig. 1A, second and third lanes). Flow cytometry analysis indi-cated that CD44was successfully translocated onto the cell sur-face (Fig. 1B). As the negative control, no CD44 expression wasdetected in COS-7 cells transfected with mock vector (Fig. 1B).After transfection, captured fl-HA (green fluorescence) was

visualized at the surface of CD44-transfected COS-7 cells byflow cytometry and confocal microscopy analysis (Fig. 1, C andD). As negative control, untransfected cells showed no capacityto bind fl-HA (Fig. 1D).

FIGURE 3. FRET analysis of CD44-CFP/CD44-YFP clustering in COS-7 cells. COS-7 cells were co-transfected with CD44-CFP and CD44-YFP, and treated withmedium, nHA (125 �g/ml), oHA (125 �g/ml), or anti-CD44 mAb (10 �g/ml) for 2 h. After exposed to nHA, one group of cells was treated with oHA (125 �g/ml)for another 2 h. Cells treated with culture medium were used as negative control. Another three controls were used: two negative control pairs CD44-CFP withEYFP-N1 and ECFP-N1 with CD44-YFP and a positive control tandem PMT-CFP-YFP construct. nHA induced CD44-CFP-CD44-YFP clustering as indicated by theFRET signal. oHA did not induce CD44-CFP-CD44-YFP clustering but reduced FRET signal induced by nHA. Similar findings were observed when the cells weretreated by anti-CD44 mAb. FRET images were normalized with respect to FRET efficiency and shown in a pseudocolor mode. The scale indicated FRET efficiencyfrom purple (0) to red (90%); scale bar � 100 �m. The results represent five separate experiments; more than three cells were analyzed each time.




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nHA and oHA Regulate CD44 Clustering in Distinct Manner—Toevaluate the effects of nHAand oHAonCD44 clustering, weperformed FRET. As shown in Fig. 2A. CD44-CFP emittedmainly at 485 nm and with lower intensity at 528 nm, althoughCD44-YFP showed emissionmainly at 528 nm.Co-transfectionof CD44-CFP and CD44-YFP initiated two separate emissionintensities. We observed typical FRETs between CFP and YFPin positive control tandem PMT-CFP-YFP construct with noFRET in two negative control pairs CD44-CFP with EYFP-N1and ECFP-N1 with CD44-YFP (Fig. 2A).The ratio of fluorescence between 528 and 485 nm was used

to estimate energy transfer efficiencies. As shown in Fig. 2A, thehighest ratio was obtained with PMT-CFP-YFP (�5.77). Co-expression of CD44-CFP and CD44-YFP without HA treat-

ment showed a low ratio at 1.44, indicating the weak clusteringof CD44 in transfected COS-7 cells (Fig. 2A).When COS-7 cells co-transfected with CD44-CFP and

CD44-YFP were treated with nHA, the 528/485-nm ratioincreased to 4.7. Nevertheless, when cells were treated withoHA, the 528/485-nm ratio decreased to 1.38 (Fig. 2B). Thedecrease of about 70% in the 528/485-nm ratio in oHA-treatedcells is probably due to the displacement of nHA by oHA.Moreover, the primary dose dependence experiment indicatedthat the reduction of FRET signal by oHA was found to beconcentration-dependent (Fig. 2C), reaching a plateau at 125�g/ml, which was the minimum concentration that coulddestroy maximally the CD44 clustering induced by nHA (Fig.2C) and thereby be selected for the following experiments.

FIGURE 4. Immunocytochemistry and chemical cross-linker analysis of CD44 clustering in transfected COS-7 cells. A, clustering of CD44 with differenttreatments was analyzed by immunocytochemistry. CD44-transfected COS-7 cells were pretreated with DMEM (Control), oHA (125 �g/ml), or nHA (125 �g/ml)for 2 h at 37 °C. After exposure to nHA, one group of cells was treated with oHA (125 �g/ml) for another 2 h. CD44 was distributed into clusters in nHA-treatedcells (indicated by the arrows). B, CD44-transfected COS-7 cells were pretreated with DMEM (Control), oHA (125 �g/ml), and nHA (125 �g/ml) for 2 h at 37 °C.After exposure to nHA, one group of cells was treated with oHA (125 �g/ml) for another 2 h, then all cells were incubated with or without BS3 proteincross-linker (2 mM) for 1 h at 4 °C. The cross-linked CD44 on cell surface was detected by Western blot analysis. Without BS3 treatment, immunoblotting of thecell lysate revealed a major 85-kDa band. Incubation of cells with BS3 led to the appearance of additional immunoreactive bands at 170 kDa. Shown arerepresentative images from three independent experiments with similar results.

FIGURE 5. Photobleaching FRET analysis of CD44-CFP/CD44-YFP clustering in COS-7 cells. FRET was measured by donor dequenching after acceptorphotobleaching in COS-7 cells co-transfected with CD44-CFP and CD44-YFP. Two frames before and after the bleaching interval were presented. Bleachedregion of YFP was shown (arrows). A, images were acquired 2 h after the cells were treated with 125 �g/ml nHA. B, images were acquired 2 h after the cells weretreated with 125 �g/ml nHA followed by treatment with 125 �g/ml oHA for 2 h. C, the bar graphs representing FRET efficiency (E) were from five independentexperiments performed as shown in A. The efficiency was determined by the acceptor photobleaching method and was measured only in the acceptorbleached area. Cells outside the bleached region were used as controls. D, bar graphs representing FRET efficiency (E) were from the five independentexperiments performed as shown in B. All data are represented as the mean � S.D. *, p � 0.001.




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Next, FRET Imaging was applied to evaluate the clustering ofCD44 in response to nHA or oHA in living cells. COS-7 cellswere co-transfected with CD44-CFP and CD44-YFP con-structs. As shown in Fig. 3, no FRET signal was detected inresting cells in the blank group and oHA treatment group.However, nHA exposure generated FRET signal within 2 h,indicating the clustering of CD44-CFP with CD44-YFP. SuchFRET signal was significantly reduced when the cells weretreatedwith oHA (Fig. 3).Moreover, anti-CD44mAb induced avery weak FRET signal (Fig. 3). Taken together, these data sug-gest that nHA induces CD44 clustering, although oHA inhibitsCD44 clustering.Furthermore, immunocytochemistry analysis of CD44-CFP/

CD44-YFP clustering in COS-7 cells revealed that CD44 wasdistributed in clustering form in nHA-treated cells, althoughoHA reduced the number of cells showing CD44 clusters (Fig.4A). In addition, a protein chemical cross-linking experimentwas used to assess CD44 clustering. Western blotting analysisshowed the additional CD44 bands inCD44-transfectedCOS-7cells after nHA incubation and BS3 treatment, indicating that

cross-linking had occurred between CD44 protein. Likewise,oHA reduced the amount of cross-linked CD44 with the obvi-ous decrease in CD44 dimers (Fig. 4B).CD44 Clustering in COS-7 Cells Detected by Photobleaching

FRET—FRET measured by donor dequenching after acceptorphotobleaching was utilized to detect CD44 clustering. COS-7cells were cotransfected with CD44-CFP and CD44-YFP plas-mids and treated with 125 �g/ml nHA. The intensity of CFPfluorescence was increased, as the CD44-YFP was photo-bleached (Fig. 5A). However, for other cells that were not pho-tobleached within the same field of view, the intensity of bothCFP and YFP did not change obviously (Fig. 5A).When the cells treated with 125 �g/ml nHA were further

treated by 125 �g/ml oHA after the YFP molecule was photo-bleached, the intensity of CD44-CFP showed no significantincrease (Fig. 5,B andD). The average energy transfer efficiencyfor the cells with photobleached acceptors in the nHA-treatedcells was 51%, compared with �1.0% in the controls (Fig. 5C).After oHA treatment, the energy transfer efficiency wasdecreased to �15.3% (Fig. 5, B and D). These results confirm

FIGURE 6. oHA abrogates CD44 clustering triggered by endogenous nHA in HK-2 and BT-549 cells. A, the expression of hyaluronan with or without oHAtreatment was detected by immunocytochemistry analysis. Endogenous nHA was cross-linked into cables (indicated by the arrows). Sections were imaged bymicroscopy (�20 objective). To confirm the nature of HA staining, cells were treated with bovine testicular hyaluronidase (250 �g/ml) at 37 °C for 2 h beforefixation and the addition of biotinylated HA-binding protein. 4-MU (0.5 mM, 24 h), the hyaluronan synthesis inhibitor, was used as positive control. B, theclustering of CD44 with or without oHA treatment was analyzed by immunocytochemistry. CD44 was distributed into clusters in naïve HK-2 and BT-549 cells(indicated by the arrows). As a positive control, cells were treated with bovine testicular hyaluronidase (250 �g/ml) at 37 °C for 2 h. Treatment with 4-MU (0.5mM, 24 h) was performed to detect the effect of inhibition of endogenous HA synthesis on CD44 clustering.




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that nHA induces CD44 clustering, although oHA inhibitsCD44 clustering.Endogenous nHA-induced CD44 Clustering Is Disrupted by

HyaluronanOligosaccharides—Toconfirm the opposite effectsof nHA and oHA on CD44 clustering, additional experimentswere performed in HK-2 cells (human renal proximal tubulecells) and BT-549 cells (human breast cancer cells), whichunlike COS-7 cells without expression of HA and CD44, exhib-iting abundant expression of CD44 as showed in Fig. 7 andsupplemental Figs. S2. In addition, the native HA was highlyexpressed and organized into cable-like structures in both cells(Fig. 6A). Confocal microscopy revealed that CD44 was distrib-uted in clustering form in naïve HK-2 and BT-549 cells (Fig.6B). To be sure that the CD44 clusters are induced by endoge-nous HA, the HA synthesized inhibitor, 4-MU, was used toinhibit HA secretion before CD44 clusters were determined.Our results indicated that cells treated with 0.5 mM 4-MU for

24 h showed a substantial inhibition of HA staining (Fig. 6A)and CD44 clustering (Fig. 6B), demonstrating that endogenousHA is the indispensable factor of the formation of CD44 clus-ters. Similar findings were observedwhen the cells were treatedwith bovine testicular hyaluronidase, which was used as posi-tive control.In addition, after treatment with oHA, the amount of cell-

associated HA on cell surface was significantly reduced (Fig.6A). Accordingly, the number of cells showing CD44 clusterswas reduced, although CD44 expression on cell surface was notsignificantly changed after oHA treatment (supplemental Figs.S2). Interestingly, oHA treatment had hardly any inhibitoryeffect on endogenous HA generation (Table 1), suggesting thatthe reduced clusters of CD44, mediated by oHA, was due todirect competition with the same binding site on CD44.Furthermore, protein chemical cross-linking experiment

was used to assess CD44 clustering on cell surface of HK-2 andBT-549 cells. CD44s had an apparent molecular mass of �85kDa without BS3 treatment (Fig. 7), and the addition of oHA orhyaluronidase did not change the apparentmolecular weight ofCD44s (Fig. 7,A and B). However, after subsequently BS3 treat-ment, a new band of CD44 emerged corresponding to apparentmolecular mass products of 170 kDa, indicating that CD44dimers formed after protein cross-linking (Fig. 7A). To confirmthe role of endogenousHA synthesis in CD44 clustering, 4-MUwas used to inhibit HA secretion before incubation with orwithout BS3 protein cross-linker. The results indicated that

FIGURE 7. CD44 clustering on the surface of HK-2 and BT-549 cells was assessed by chemical cross-linker analysis. A and B, HK-2 cells (A) and BT-549 cells(B) were pretreated with DMEM (Control), oHA (250 �g/ml), and hyaluronidase (250 �g/ml) for 2 h at 37 °C, then incubated with or without BS3 proteincross-linker (2 mM) for 1 h at 4 °C. Treatment with bovine testicular hyaluronidase (250 �g/ml) was used as positive control. After incubation, the cross-linkingreaction was quenched by 20 mM Tris, pH 7.5. Cells were washed twice and lysed with cell lysis buffer. The cross-linked CD44 on the cell surface was detectedby Western blot analysis. Without BS3 treatment, immunoblotting of cell lysate revealed a major 85-kDa band. Incubation of cells with BS3 led to the appearanceof additional immunoreactive bands at 170 kDa. C and D, to confirm the effect of endogenous HA on CD44 clustering, HK-2 (C) cells and BT-549 cells (D) werepretreated with or without the hyaluronan synthesis inhibitor 4-MU (0.5 mM, 24 h) then incubated with or without BS3 protein cross-linker (2 mM) for 1 h at 4 °C.The cross-linked CD44 on cell surface was detected by Western blot analysis. Shown are representative images from three independent experiments withsimilar results.

TABLE 1oHA treatment has no effect on the amount of endogenous HAgeneration

Quantitation of hyaluronan production after oHAtreatment for 2 h

CellsControl(DMEM)

62.5 �g/mloHA

125 �g/mloHA

250 �g/mloHA

fgHK-2 685.0 � 14 687.0 � 27 689.0 � 31 680.0 � 24BT-549 997.0 � 36 995.0 � 25 1015.0 � 45 945.0 � 42




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treatment with 4-MU inhibited the formation of CD44 dimers(Fig. 7,C andD), providing other evidence that endogenousHAis the most critical factor in the formation of CD44 clusters.Next, the clustering of CD44 in response to oHA treatment wasdetected to evaluate the opposite effects of nHA and oHA. Asexpected, oHA treatment significantly reduced the amount ofCD44 dimers (Fig. 7, A and B), further confirming that oHAcould attenuate CD44 clustering on the cell surface stimulatedby endogenous nHA. An obvious decrease in CD44 dimers wasalso observed in cells treated with bovine testicular hyaluroni-dase (Fig. 7,A and B), which was used as the positive control forthe disruption of endogenous nHA.oHA and nHA Induces Different Cell Adhesive Abilities—

Having demonstrated the different effects of oHA and nHA onCD44 clustering, we next wondered whether oHA and nHAexert distinct effects on cell biological behaviors. To addressthis, we examined the adhesion of CD44-transfected COS-7cells to 96-well plates. The results showed that oHA promotedthe attachment of cells to the 96-well plate (Fig. 8A). In contrast,nHA showed no effects on the attachment (Fig. 8A). To deter-mine if endogenous HA could influence the cell binding, aradioimmunoassay was performed to observe the effects ofoHA on HA generation. Our results showed that oHA did notaffect endogenous HA synthesis as long as 0� 2 h (supplemen-tal Table S1). As the cells were attached onto the plate only for30 min, the increase of adhesive ability by oHA treatment maybe due to the regulation of other adhesion factors by oHA, suchas F-actin or focal adhesions, etc., leading to cells attached tothe polystyrene plate.Stress fibers and associated focal adhesions in cells constitute

a contractile apparatus that regulates cell motility and contrac-tion, and specific cytoskeletal rearrangements of cells have beenimplicated in cell adhesion and migration (37, 38). To charac-terize the effects of oHA and nHA on the microfilament orga-nization and focal adhesion contact formation, fluorescencecytostaining using FITC-labeled phalloidin and immuno-staining of vinculin were investigated by confocal microscopy.As illustrated in Fig. 8B, stainingwith phalloidin for cytoskeletalarchitecture showed that the cells treated with oHA had moreintense F-actin stress fibers arranged in spike-like protrusionsresembling microvilli-like structures (Fig. 8B). In contrast, sol-uble nHA showed no effects on the cell actin reorganization inCD44-transfected COS-7 cells (Fig. 8B). Moreover, vinculin-specific immunofluorescence assay demonstrated that oHAtreatment induced an increased number and size of adhesionplaques under the cells, whereas nHA had no effects (Fig. 8B).These results are consistent with previous studies reportingthat HA chains of different sizes affect F-actin organization andfocal contact formation, resulting in changes of cell adhesion(39, 40).To further understand the intracellular mechanism through

which oHA enhances cell adhesion, we next studied one of theimportant cellular signal pathways, ERK1/2, which has beenregarded as a crucial regulator of cell adhesion (41) and also hasbeen reported to interact with CD44 directly (42, 43). Westernblot analysis revealed that phospho-ERK-1/2 was significantlyincreased after oHA treatment compared with untreated cells(Fig. 9, A and B). However, after nHA treatment, phosphoryla-

tion levels of ERK1/2 were not obviously changed (Fig. 9,A andB). Additionally, the up-regulated level of pERK1/2 by oHAwasdecreased after the blockage of CD44 by anti-CD44 mAb (Fig.9, C and D). To further identify the role of ERK involved in celladhesion, a blocking of ERK experiment was performed. As aresult, the addition of the specific ERK inhibitor stronglydecreased both fundamental and oHA-induced cell adhesioncapacity (Fig. 10), suggesting that pERK1/2, originated fromCD44/HA interactions, functions as a downstream signal reg-ulating cell adhesion.

DISCUSSION

It has been reported that the biological effect of HA dependson its molecular weight (44–46). Specifically, CD44 binding ofa fraction of HA defined operationally as “low molecularweight” (4–20-mer) has been reported to stimulate cell prolif-eration (18), whereas higher molecular weight fractions exertan inhibitory effect (44). In this study we analyzed the influenceof high and lowmolecular weight hyaluronan on CD44 cluster-ing. Our results provide direct evidence for the distinct fea-tures of the interactions between CD44 and different sizeforms of HA.

FIGURE 8. Effects of nHA and oHA on cell adhesion. A, CD44-transfectedCOS-7 cells (5 � 103) were seeded in 96-well plates after pretreated withDMEM (control) or soluble oHA or nHA. After 30 min at 37 °C, the bound cellswere counted. The results represented the average of three independentexperiments in triplicate and are shown as the mean � S.D. *, p � 0.05.B, effects of HA on actin fiber and focal adhesions are shown. CD44-trans-fected COS-7 cells treated with DMEM (control), oHA (125 �g/ml), and nHA(125 �g/ml) were fixed, permeabilized, and then stained using FITC-labeledphalloidin to visualize actin filaments. Cells treated with oHA had moreintense F-actin stress fibers (arrows). The cells also were stained with anti-vinculin for focal adhesion staining, and the nuclei were stained using DAPI.The signals against vinculin and DAPI were superimposed. Cells treated withoHA showed the increased quantity of vinculin-positive adhesion plaques inthe undersurface of cells (arrows). Note that both F-actin and vinculin areco-distributed in focal adhesions.




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FRET analysis showed that nHA induced a drastic increase ofFRETc signal, which indicated an augment of cell membranesurface CD44 aggregation. However, the addition of oHAcaused an attenuation of such aggregation. These results sug-gest that oHA abrogates CD44 clustering induced by exoge-nous native hyaluronan. Considering that both oHA and nHAhave the same binding site on CD44, the abolishment of CD44clustering could be due to the displacement of nHA by oHA.Indeed, previous studies reported that the hyaluronan aroundthe cells was displaced by oligosaccharides (47, 48).HA is known to contain multiple (up to a few thousand)

CD44 binding sites due to the repetitive structure(GlcNAc�1–4GlcUA)n and could interact simultaneously withmany CD44 receptors on the cell surface (49). Moreover, byreconstituting CD44 expression in AKR1 cells that lack endog-enousCD44 expression, Lesley et al. (21) showed thatHAbind-ing at the cell surface is a complex interplay ofmultivalent bind-ing events affected by the size of the multivalent HA ligand and

the quantity and density of CD44 at the cell surface. In thisstudy CD44 clustering induced by HA was further investigatedin HK-2 and BT-549 cells, which naturally express abundantCD44 and endogenous native HA. Our immunocytochemistryresults revealed that excessively secreted native HA was cross-linked into cables, and CD44 was distributed into clusters inHK-2 and BT-549 cells. Furthermore, treatment with 4-MU, aninhibitor of hyaluronan synthesis, diminished the hyaluronanaround the cells and CD44 clusters on the cell surface, indicat-ing that the cluster of CD44 in HK-2 and BT-549 cells isdependent on endogenous hyaluronan synthesis.In addition, protein chemical cross-linking experiment pro-

vided further data that inhibition of endogenous HA synthesisby 4-MU obviously inhibited the formation of CD44 clusters,suggesting that CD44 clustering on the cell surface was mainlymediated by endogenous nHA in HK-2 and BT-549 cells.Therefore, our data complement and extend previous findingsby demonstrating the multivalent binding between HA and

FIGURE 9. oHA stimulates ERK signaling in HK-2 and BT-549 cells. A and B, shown is the effect of HA on ERK1/2 phosphorylation. HK-2 (A) and BT-549 (B) cellswere treated with DMEM (Control) and nHA or oHA for 30 min, and phosphorylated ERK1/2 (pERK1/2) was detected by Western blot analysis. C and D, HK-2 (C)and BT-549 (D) cells were pretreated with or without anti-CD44 mAb (50 �g/ml) for 12 h and then treated with oHA (125 �g/ml) for 5 min. Cells treated withDMEM were used as control. Normal mouse IgG (NIgG) was used as negative control. The phosphorylated ERK1/2 (pERK1/2) was detected by Western blotanalysis. Shown are representative blots from three independent experiments with similar results. The band intensities were analyzed by densitometryanalysis. *, p � 0.05 compared with the controls.




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CD44 in cells that endogenously express these molecules.Although by an artificial supported lipid bilayermembrane sys-tem, the multivalent interactions between multiple bindingsites on the HA chain and multiple CD44 receptors on the sur-face were analyzed quantitatively through the presentation ofCD44 at controlled density (23), our approach differs from pre-viously reported binding assays in that we analyzed the multi-valent binding of HA and CD44 in living cells.The long chains of HA possess multivalent or repeated sites

for CD44 binding, whereas hyaluronan oligomers contain onlyone to two binding sites of CD44 (21, 23). By FRET technique,we found that oHA strongly reduced CD44 clustering inducedby exogenous native hyaluronan. Moreover, although treat-ment of HK-2 and BT-549 cells with oHA had no influence onthe amount of CD44 on cell surface and the endogenous HAgeneration, oHA significantly reduced the number of cellsshowing CD44 clusters. These results suggest that oHA regu-lates CD44 clustering in an opposite manner as nHA by replac-ing the multivalent HA-CD44 interaction with a monovalentinteraction, leading to the disruption of CD44 clustering.CD44 is a member of the hyaluronan receptor family and

serves as an adhesion molecule that binds ligand of the extra-

cellular matrix. Therefore, CD44 is usually reported to mediatecell-cell and cell-matrix adhesion, including lymphocyte hom-ing, hemopoiesis, and cell migration (6, 50, 51). To investigatethe biological significance of CD44 clustering induced by HA,we examined the adhesive ability of CD44-transfected cells.Our results demonstrate that HA specifically regulates celladhesion in a manner dependent on molecular weight. That is,oHA promoted the attachment of cells to the polystyrene plate,whereas nHAshowedno effects under the same condition. Thisis consistent with the previous report (37). However, thereweresome reports indicating that oHA could regulate endogenousHA generation (52). To determine if the cell adhesion wasmediated by endogenous HA secretion, we further investigatethe effect of oHA on cell HA production. As shown in theresults, oHA showed no effects on HA generation throughoutthe experiments. Interestingly, previous studies reported thatoHA mainly restrained the endogenous HA synthesis (28),whereas our data showed no inhibition. This controversialresult needs a future investigation. Togetherwith the result thatoHA promoted cell adhesion, our study suggested that the celladhesion may be mediated by CD44-HA interactions but notthe endogenous HA.

FIGURE 10. Effect of ERK inhibitor (ERKi) on CD44-dependent cell adhesion. A, HK-2 cells, pretreated with ERK inhibitor (5 �M) for 24 h, were incubated withor without oHA (125 �g/ml) for 2 h then harvested and allowed to attach (5000 cells) for 30 min on 96-well plates. B, shown is the effect of ERK inhibitor on HK-2cell ERK1/2 phosphorylation. HK-2 cells treated with ERK inhibitor for 24 h were harvested, and equal amounts of cell extract protein were separated by PAGEand blotted with phosphorylated ERK1/2 (pERK1/2) utilizing specific antibodies. Densitometry analysis of the bands was normalized against GAPDH andplotted. C, BT-549 cells, pretreated with ERK inhibitor (5 �M) for 24 h, were incubated with or without oHA (125 �g/ml) for 2 h. The cells were then harvested andallowed to attach (5000 cells) for 30 min on 96-well plates. D, shown is the effect of ERK inhibitor on BT-549 cell ERK1/2 phosphorylation. BT-549 cells weretreated with ERK inhibitor for 24 h, and the phosphorylated ERK1/2 (pERK1/2) was detected by Western blot analysis. Densitometry analysis of the bands wasnormalized against GAPDH and plotted. All results represent the average of three separate experiments. The means � S.E. are plotted; statistical significanceis as follows: *, p � 0.05 compared with the respective control samples.




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Scientists from other laboratories have demonstrated therole of CD44 in cell motility in its ability to interact with cellskeletal protein, such as actin microfilaments and adhesionmolecules (53–55). Cell adhesion requires organized actinstress fibers and focal adhesion contacts (56). To character thefunctional consequences of CD44 clustering and its regulationby HA, the reorganization of the cytoskeleton and associatedchanges in focal adhesion complexes were visualized. Ourimmunocytochemistry assay revealed that oHA stronglyincreased intense F-actin stress fibers arranged in spike-likeprotrusions and the levels of vinculin when cell adhesion wereenhanced, whereas nHA showed no effects. Previous reportsdemonstrated that vinculin is important for the linkage of focaladhesions to the cytoskeleton (57), and vinculin may promotecell spreading by stabilizing focal adhesions and transferringmechanical stresses that drive cytoskeletal remodeling (57).The present study indicated that oHA stimulates the assemblyof focal adhesion complexes. The effects of oHA treatment oncell adhesion might reflect this reorganization of focal adhe-sion. Thus, the up-regulation of vinculin expressionmay be oneof the mechanisms by which oHA acts as a promotor of celladhesion. Taken together, it is likely that the pericellular pro-teoglycans HA is involved in regulating cell adhesion via theirinfluence on the formation of focal adhesions through theirassociations with CD44 clustering and cytoskeletal proteins.In fact, it is well known that the response to HA signaling

leads to changes in cell motility and adhesion (58). In addition,CD44 interacts directly with ERK1/2 and enhances ERK phos-phorylation and cell locomotion (42, 43). To further under-stand the functional significance of CD44 clustering regulatedby nHA or oHA and the mechanism downstream by whichthese effects are mediated, we examined the ERK1/2 signalingin HK-2 and BT-549 cells. Our findings indicated that oHAinduced the activation of ERK, and anti-CD44mAbblocked thesignaling. These results strongly indicate that the stimulatingeffect of oHA on ERK signaling is mediated through the oHA-CD44 pathway. The utilization of the specific ERK inhibitorresulted in a strong down-regulation of cell adhesive ability,identifying ERK1/2 as a HA-CD44 downstream effector. Ourresults are consistent with previous report that anti-CD44 anti-body or ERK inhibitor inhibited cell adhesion (37, 59), suggest-ing that pERK1/2, originating from CD44/HA interactions, isan important signal regulating cell adhesion.Based on our results shown above, we propose that nHA

interacts with cell surfacemolecules including CD44 to serve asa “net chain” to link and/or stabilize the existing active mole-cules such as receptors or transporters on cell surface. As HAand CD44 have been demonstrated to be associated withdynamic movement of cell membrane caveolae or lipid raftwithin the cell (60–63), molecular size switching on HA mayregulate cell metabolism and signaling cooperating with othersystems. Further investigation into the role of brokenHA in theregulation of cell membrane receptors and transporters iswarranted.In summary, our study is the first report to characterize

CD44 binding with different size of HA in vivo. We demon-strate that nHA could stimulate cell surface CD44 clustering,which could be further attenuated by oHA. The attenuation

could be caused by replacing and competition with the samebinding sites of nHA on CD44. We have observed the distribu-tion of the CD44 cluster in human tumor cell line BT549 andhuman renal proximal tubule cells HK-2, but its relation withtumor progression and inflammation processes needs to beinvestigated in future.Our novel findings providemore detailedevidence for the interaction between CD44 and nHA or oHAwith their influences on cell adhesion and signaling.

Acknowledgments—We thank Prof. Zhen-dong You for the generousgrant of the pECFP-N1 and pEYFP-N1 plasmids. We also thank Dr.Chun-yuan Zhou for assistance in FRET technique.

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Wang, Lian Cui, Jiajie Hu and Feng GaoCuixia Yang, Manlin Cao, Hua Liu, Yiqing He, Jing Xu, Yan Du, Yiwen Liu, Wenjuan

on CD44 ClusteringThe High and Low Molecular Weight Forms of Hyaluronan Have Distinct Effects

doi: 10.1074/jbc.M112.349209 originally published online November 1, 20122012, 287:43094-43107.J. Biol. Chem.

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VOLUME 287 (2012) PAGES 43094 – 43107DOI 10.1074/jbc.A112.349209

The high and low molecular weight forms of hyaluronanhave distinct effects on CD44 clustering.Cuixia Yang, Manlin Cao, Hua Liu, Yiqing He, Jing Xu, Yan Du, Yiwen Liu,Wenjuan Wang, Lian Cui, Jiajie Hu, and Feng Gao

The affiliation line was incomplete. The corrected affiliation line isshown below.

Cuixia Yang‡1, Manlin Cao§1, Hua Liu¶, Yiqing He‡, Jing Xu¶, YanDu‡, Yiwen Liu‡, Wenjuan Wang¶, Lian Cui‡, Jiajie Hu‡, andFengGao‡2

From the Departments of ‡Molecular Biology Laboratory, §Rehabili-tationMedicine, ¶Clinical Laboratory, Shanghai Sixth People’s Hospital,Shanghai Jiaotong University School of Medicine, Shanghai 200233,China

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 46, p. 33323, November 15, 2013© 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

NOVEMBER 15, 2013 • VOLUME 288 • NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 33323

ADDITIONS AND CORRECTIONS

Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to carry notice ofthese corrections as prominently as they carried the original abstracts.

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