FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2076220323> ?p ?o ?g. }

• W2076220323 endingPage "33508" @default.
• W2076220323 startingPage "33495" @default.
• W2076220323 abstract "It has long been predicted that the members of the hyaluronidase enzyme family have important non-enzymatic functions. However, their nature remains a mystery. The metabolism of hyaluronan (HA), their major enzymatic substrate, is also enigmatic. To examine the function of Hyal2, a glycosylphosphatidylinositol-anchored hyaluronidase with intrinsically weak enzymatic activity, we have compared stably transfected rat fibroblastic BB16 cell lines with various levels of expression of Hyal2. These cell lines continue to express exclusively the standard form (CD44s) of the main HA receptor, CD44. Hyal2, CD44, and one of its main intracellular partners, ezrin-radixin-moesin (ERM), were found to co-immunoprecipitate. Functionally, Hyal2 overexpression was linked to loss of the glycocalyx, the HA-rich pericellular coat. This effect could be mimicked by exposure of BB16 cells either to Streptomyces hyaluronidase, to HA synthesis inhibitors, or to HA oligosaccharides. This led to shedding of CD44, separation of CD44 from ERM, reduction in baseline level of ERM activation, and markedly decreased cell motility (50% reduction in a wound healing assay). The effects of Hyal2 on the pericellular coat and on CD44-ERM interactions were inhibited by treatment with the Na+/H+ exchanger-1 inhibitor ethyl-N-isopropylamiloride. We surmise that Hyal2, through direct interactions with CD44 and possibly some pericellular hyaluronidase activity requiring acidic foci, suppresses the formation or the stability of the glycocalyx, modulates ERM-related cytoskeletal interactions, and diminishes cell motility. These effects may be relevant to the purported in vivo tumor-suppressive activity of Hyal2. It has long been predicted that the members of the hyaluronidase enzyme family have important non-enzymatic functions. However, their nature remains a mystery. The metabolism of hyaluronan (HA), their major enzymatic substrate, is also enigmatic. To examine the function of Hyal2, a glycosylphosphatidylinositol-anchored hyaluronidase with intrinsically weak enzymatic activity, we have compared stably transfected rat fibroblastic BB16 cell lines with various levels of expression of Hyal2. These cell lines continue to express exclusively the standard form (CD44s) of the main HA receptor, CD44. Hyal2, CD44, and one of its main intracellular partners, ezrin-radixin-moesin (ERM), were found to co-immunoprecipitate. Functionally, Hyal2 overexpression was linked to loss of the glycocalyx, the HA-rich pericellular coat. This effect could be mimicked by exposure of BB16 cells either to Streptomyces hyaluronidase, to HA synthesis inhibitors, or to HA oligosaccharides. This led to shedding of CD44, separation of CD44 from ERM, reduction in baseline level of ERM activation, and markedly decreased cell motility (50% reduction in a wound healing assay). The effects of Hyal2 on the pericellular coat and on CD44-ERM interactions were inhibited by treatment with the Na+/H+ exchanger-1 inhibitor ethyl-N-isopropylamiloride. We surmise that Hyal2, through direct interactions with CD44 and possibly some pericellular hyaluronidase activity requiring acidic foci, suppresses the formation or the stability of the glycocalyx, modulates ERM-related cytoskeletal interactions, and diminishes cell motility. These effects may be relevant to the purported in vivo tumor-suppressive activity of Hyal2. The turnover rate of hyaluronan (HA), 2The abbreviations used are: HAhyaluronanERMezrin-radixin-moesinpERMphosphorylated ERMEIPAethyl-N-isopropylamiloride4-MU4-methylumbelliferonePBSphosphate-buffered salinePI3Kphosphoinositide 3-kinaseNHE1sodium-proton exchanger-1. the major unbranched glycosaminoglycan of the extracellular matrix, is surprisingly rapid: approximately one-third of total body HA is replaced daily (1.Fraser J.R. Laurent T.C. Laurent U.B. J. Intern. Med. 1997; 242: 27-33Crossref PubMed Scopus (1512) Google Scholar). Although it is generally agreed that hyaluronidases play important roles in the degradation of HA, the specific functions of the six or seven members of the hyaluronidase family remain obscure (2.Kreil G. Protein Sci. 1995; 4: 1666-1669Crossref PubMed Scopus (308) Google Scholar, 3.Csoka A.B. Frost G.I. Stern R. Matrix Biol. 2001; 20: 499-508Crossref PubMed Scopus (487) Google Scholar). Only three of the hyaluronidase genes are broadly expressed in somatic tissues: HYAL1, HYAL2, and HYAL3 (4.Csóka A.B. Scherer S.W. Stern R. Genomics. 1999; 60: 356-361Crossref PubMed Scopus (208) Google Scholar), all of which are grouped on human chromosomal locus 3p21.3. Mice deficient in each of these genes have recently been described. However, Hyal3 knockout mice do not display a distinct phenotype (5.Atmuri V. Martin D.C. Hemming R. Gutsol A. Byers S. Sahebjam S. Thliveris J.A. Mort J.S. Carmona E. Anderson J.E. Dakshinamurti S. Triggs-Raine B. Matrix Biol. 2008; 27: 653-660Crossref PubMed Scopus (56) Google Scholar), Hyal1 null animals develop a slowly progressive osteoarthritis without significant elevation of plasma or tissue levels of HA (6.Martin D.C. Atmuri V. Hemming R.J. Farley J. Mort J.S. Byers S. Hombach-Klonisch S. Csoka A.B. Stern R. Triggs-Raine B.L. Hum. Mol. Genet. 2008; 17: 1904-1915Crossref PubMed Scopus (78) Google Scholar), and Hyal2−/− mice show skeletal and hematological anomalies as well as 10-fold increases in plasma HA (7.Jadin L. Wu X. Ding H. Frost G.I. Onclinx C. Triggs-Raine B. Flamion B. FASEB J. 2008; 22: 4316-4326Crossref PubMed Scopus (83) Google Scholar). Some of these apparent anomalies are explained perhaps by the non-enzymatic functions of these proteins. hyaluronan ezrin-radixin-moesin phosphorylated ERM ethyl-N-isopropylamiloride 4-methylumbelliferone phosphate-buffered saline phosphoinositide 3-kinase sodium-proton exchanger-1. In vitro, the glycosylphosphatidylinositol-anchored Hyal2 has a much weaker hyaluronidase activity compared with Hyal1 or to the sperm hyaluronidase PH20 (8.Vigdorovich V. Miller A.D. Strong R.K. J. Virol. 2007; 81: 3124-3129Crossref PubMed Scopus (34) Google Scholar). Fragments of HA, ∼20 kDa in mass, may build up during Hyal2 functioning (8.Vigdorovich V. Miller A.D. Strong R.K. J. Virol. 2007; 81: 3124-3129Crossref PubMed Scopus (34) Google Scholar, 9.Lepperdinger G. Strobl B. Kreil G. J. Biol. Chem. 1998; 273: 22466-22470Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 10.Harada H. Takahashi M. J. Biol. Chem. 2007; 282: 5597-5607Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar), which raises the possibility that Hyal2 participates in inflammatory events in which such HA fragments accumulate (11.Stern R. Asari A.A. Sugahara K.N. Eur. J. Cell Biol. 2006; 85: 699-715Crossref PubMed Scopus (877) Google Scholar, 12.Jiang D. Liang J. Noble P.W. Annu. Rev. Cell Dev. Biol. 2007; 23: 435-461Crossref PubMed Scopus (680) Google Scholar). Intriguingly, sheep Hyal2 also functions as a cell-entry receptor for oncogenic ovine retroviruses (13.Rai S.K. Duh F.M. Vigdorovich V. Danilkovitch-Miagkova A. Lerman M.I. Miller A.D. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 4443-4448Crossref PubMed Scopus (295) Google Scholar), although this function is independent of any HA-degrading activity (8.Vigdorovich V. Miller A.D. Strong R.K. J. Virol. 2007; 81: 3124-3129Crossref PubMed Scopus (34) Google Scholar). Recently, expression of HYAL2 and HYAL1 genes has been shown to inhibit tumor growth in vivo without noticeable effect on growth in vitro, suggesting that these hyaluronidases control in some unknown manner tumor-host interactions (14.Wang F. Grigorieva E.V. Li J. Senchenko V.N. Pavlova T.V. Anedchenko E.A. Kudryavtseva A.V. Tsimanis A. Angeloni D. Lerman M.I. Kashuba V.I. Klein G. Zabarovsky E.R. PLoS ONE. 2008; 3: e3031Crossref PubMed Scopus (36) Google Scholar). Hyal2 also anchors tumor growth factor-β1 to the cell surface and mediates some of its pro-apoptotic effects (15.Hsu L.J. Schultz L. Hong Q. Van Moer K. Heath J. Li M.Y. Lai F.J. Lin S.R. Lee M.H. Lo C.P. Lin Y.S. Chen S.T. Chang N.S. J. Biol. Chem. 2009; 284: 16049-16059Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Conversely, Hyal2 may facilitate cancer progression: Hyal2 overexpression in murine astrocytoma cells accelerates intracerebral, but not subcutaneous, tumor formation (16.Novak U. Stylli S.S. Kaye A.H. Lepperdinger G. Cancer Res. 1999; 59: 6246-6250PubMed Google Scholar); the in vitro invasion capacity of several breast cancer cell lines correlates with Hyal2 expression (17.Bourguignon L.Y. Singleton P.A. Diedrich F. Stern R. Gilad E. J. Biol. Chem. 2004; 279: 26991-27007Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar, 18.Udabage L. Brownlee G.R. Nilsson S.K. Brown T.J. Exp. Cell Res. 2005; 310: 205-217Crossref PubMed Scopus (184) Google Scholar); and a cell surface proteomics study demonstrates that Hyal2 is enriched 13-fold in a highly invasive HT-1080 fibrosarcoma cell clone relative to a congenic variant with a low potential for intravasation and metastasis (19.Conn E.M. Madsen M.A. Cravatt B.F. Ruf W. Deryugina E.I. Quigley J.P. J. Biol. Chem. 2008; 283: 26518-26527Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). A better understanding of all these divergent events necessitates more information regarding the action of Hyal2 at the cellular level. We hypothesized that the main action of Hyal2 would be at the plasma membrane level where it interferes with pericellular HA or with CD44, the principal HA receptor. First, Hyal2 and CD44 have previously been shown to co-exist and interact within lipid rafts of the breast cancer cell line MDA-MB231 (17.Bourguignon L.Y. Singleton P.A. Diedrich F. Stern R. Gilad E. J. Biol. Chem. 2004; 279: 26991-27007Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar). Upon HA addition, the activity of the sodium-hydrogen exchanger NHE1 increases, the pH drops locally, and Hyal2 begins to degrade HA (17.Bourguignon L.Y. Singleton P.A. Diedrich F. Stern R. Gilad E. J. Biol. Chem. 2004; 279: 26991-27007Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar). Second, the membrane localization and enzymatic activity of Hyal2 have an absolute requirement for CD44 in transfected HEK293 cells. In that situation, the HA-degrading activity of Hyal2 is detected at pH 6.0–7.0 (10.Harada H. Takahashi M. J. Biol. Chem. 2007; 282: 5597-5607Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). Third, the pericellular HA-rich coat, or glycocalyx, that surrounds many types of cells disappears after exposure to either the HA-specific Streptomyces hyaluronidase or to HA oligosaccharides (20.Clarris B.J. Fraser J.R. Exp. Cell Res. 1968; 49: 181-193Crossref PubMed Scopus (97) Google Scholar, 21.Knudson W. Bartnik E. Knudson C.B. Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 4003-4007Crossref PubMed Scopus (125) Google Scholar, 22.Evanko S.P. Angello J.C. Wight T.N. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 1004-1013Crossref PubMed Scopus (421) Google Scholar). This coat appears to be influenced by the level of Hyal2 expression. To test this hypothesis in a non-cancerous cell line, given that our only effective antibodies were specific for rat Hyal2, we selected BB16 cells, which are v-src-transformed rat fibroblasts (23.Veithen A. Cupers P. Baudhuin P. Courtoy P.J. J. Cell Sci. 1996; 109: 2005-2012Crossref PubMed Google Scholar). These cells display a large pericellular glycocalyx coat and a high level of expression of CD44. In the current study, we have shown that high expression of Hyal2 is able to suppress anchoring of the pericellular coat of these cells almost entirely. This may occur through a destabilization of CD44, leading to a block in one of the main CD44 signaling systems, i.e. ezrin-radixin-moesin (ERM) activation. Loss of cell motility then ensues. Rat-1 cells transformed by the B77 subclone of Rous Sarcoma Virus (BB16) were kindly provided by Prof. Pierre Courtoy, Institute of Cellular Pathology, Brussels, Belgium (23.Veithen A. Cupers P. Baudhuin P. Courtoy P.J. J. Cell Sci. 1996; 109: 2005-2012Crossref PubMed Google Scholar). Cells were grown at 37 °C in Dulbecco's modified Eagle's medium (Cambrex) supplemented with 15 mm Hepes, 10 μg/ml streptomycin, 66 μg/ml penicillin, and 10% (v/v) fetal bovine serum (Cambrex) under 5% CO2. The rat Hyal2 cDNA (GenBankTM accession number AF034218, from 1997) was cloned in our laboratory, and polyclonal rabbit antibodies were generated. The P16 antibody used in the current study was raised against a 16-amino acid region in the body of rat Hyal2. The following commercial anti-CD44 antibodies were used: mouse monoclonal OX49 (BD Pharmingen) for immunoprecipitation and immunofluorescence, mouse monoclonal OX50 (BIOSOURCE) for CD44 blockade, and rabbit polyclonal HCAM (sc-7946, Santa Cruz Biotechnology, Santa Cruz, CA) for Western blotting. Goat polyclonal anti-lamin (sc-6214), mouse monoclonal anti-α-tubulin (sc-5286), and rabbit polyclonal anti-GFP (sc-8334) were from Santa Cruz Biotechnology. Rabbit polyclonal anti-ERM and anti-pERM (phosphorylated ERM) were from Cell Signaling Technology. Mouse monoclonal anti-β-actin, ethyl-N-isopropylamiloride (EIPA), wortmannin, LY294002, 4-methylumbelliferone (4-MU), Stains-All, Pronase, mitomycin C, and Streptomyces hyaluronidase were obtained from Sigma-Aldrich. Peroxidase-conjugated goat anti-rabbit and anti-mouse IgG and peroxidase-conjugated horse anti-goat IgG were purchased from Dako. Alexa Fluor 488-tagged goat anti-mouse and anti-rabbit IgG and Alexa Fluor 568-tagged goat anti-mouse and anti-rabbit antibodies were from Molecular Probes. 6- and 10-mer HA oligosaccharides were kindly donated by Seikagaku Corp. Three sources of high molecular mass HA were used: umbilical cord HA (∼2.5 × 106 Da, Sigma-Aldrich), Healon (∼1.6 × 106 Da, Amersham Biosciences), and preparations of smaller molecular mass HA (0.02–1.2 × 106 Da). The latter were a kind gift of Dr. Ove Wik, Amersham Biosciences. To obtain stable transfectants, BB16 cells were transfected with expression constructs containing cDNA inserts encoding rat Hyal2 or with the expression vector (pcDNA3.1+) alone, selected for Geneticin resistance, and isolated using cloning rings. Clones with a medium high expression level of Hyal2 (BB16Hy2+) were selected for most experiments of the present study. In some experiments, clones with intermediate levels of expression of Hyal2 were used. Clones transfected with the vector without Hyal2 insert (BB16mock) were used as controls. Transient transfections of BB16 cells with homemade EGFP-rat Hyal2 or commercial CD44 (RZPD, German Science Centre for Genome Research) were obtained using Lipofectamine 2000 (Invitrogen). For siRNA transfection, target sequences were selected and obtained from Eurogentec (Liege, Belgium). Three specific Hyal2 target sequences (Table 1) and one scrambled sequence (not shown) were used. Cells at 50–70% confluence in 6-well plates were transfected with 50 pmol of either the Hyal2 siRNA pool or the scrambled sequence, in 1.5 ml of complete medium, using Lipofectamine 2000. Cells were then incubated at 37 °C in 5% CO2 for 24 h, harvested, and processed for Western blotting, immunoprecipitation, and migration assays.TABLE 1Sequences of the siRNA combination used in order to inhibit Hyal2 expressionsiRNASequenceHYAL2 #1 sense5′-CUG-CUA-CAA-UCA-UGA-UUA-UdTdT-3′HYAL2 #1 antisense5′-AUA-AUC-AUG-AUU-GUA-GCA-GdTdT-3′HYAL2 #2 sense5′-CUC-CCA-GUC-UAC-GUC-UUC-AdTdT-3′HYAL2 #2 antisense5′-UGA-AGA-CGU-AGA-CUG-GGA-GdTdT-3′HYAL2 #3 sense5′-GAU-GUG-UAU-CGC-CGG-UUA-UdTdT-3′HYAL2 #3 antisense5′-AUA-ACC-GGC-GAU-ACA-CAU-CdTdT-3′ Open table in a new tab Cells were cultured until 80% confluence, washed 3× with phosphate-buffered saline (PBS), and solubilized in ice-cold RIPA buffer (50 mm Tris-HCl (pH 7.4), 120 mm NaCl, 1% Triton X-100, 0.1% SDS, 1% deoxycholate) with Complete Protease Inhibitor Mixture (Roche Applied Science), tyrosine phosphatase inhibitors (200 μm sodium molybdate, 500 μm sodium orthovanadate), and serine/threonine protein phosphatase inhibitors (1 μm microcystin-LR (Calbiochem) and 300 μm okadaic acid (Sigma)). In some cases, cell lysates were pretreated with endoglycosidase F (New England Biolabs). In other conditions, cells were pretreated with EIPA (20 μm) or with the HA-specific Streptomyces hyaluronidase (5 units/ml) for 16 h. Proteins (usually 5 μg) were analyzed using SDS-PAGE gel according to Laemmli (10% acrylamide/bisacrylamide) using a Mini Gel Protean set (Bio-Rad), followed by transfer to polyvinylidene difluoride membranes (Amersham Biosciences) with incubation for 1 or 2 h at room temperature with specific antibodies. Blots were visualized with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibody (Dako), followed by an enhanced chemiluminescence Western blotting detection system (PerkinElmer Life Sciences). To detect CD44 shedding, cells were serum-starved for 16 h and medium was collected, centrifuged to remove unattached cells, filter sterilized, and then concentrated. 25-μl samples were analyzed on 10% SDS-PAGE. Cell surface biotinylation was performed as described (24.Miranda M. Sorkina T. Grammatopoulos T.N. Zawada W.M. Sorkin A. J. Biol. Chem. 2004; 279: 30760-30770Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) on cells seeded in 92-mm culture dishes using 2 mg/ml sulfo-NHS-SS-biotin and NeutrAvidinTM (Pierce). To immunoprecipitate Hyal2, cells were labeled for 18 h with [35S]methionine, washed with ice-cold PBS and homogenized in the same buffer. The homogenate was centrifuged at low speed, and the pellet was resuspended in a solution containing 0.25 m sucrose, 3 mm imidazole (pH 7.4), 50 mm Tris, 150 mm NaCl, 0.1% Triton, 0.5% SDS, 0.5% deoxycholate, and Complete Protease Inhibitor Mixture. Suspensions were homogenized with a Dounce tissue (tight) grinder and protein content was measured by Bio-Rad Protein Assay. Cells lysates (500 μg of proteins) were preincubated with protein A-agarose (Roche Applied Science). Supernatants were incubated with P16 antibodies and then with protein A-agarose. Following incubation, immunoprecipitates were washed 5× with PBS containing 1% Triton, 0.1% SDS, and 0.5% deoxycholate and boiled with an equal volume of Laemmli sample buffer, followed by SDS-PAGE and Cyclone phosphorimager (Canberra Packard) analysis. For other immunoprecipitations, including CD44-Hyal2 co-immunoprecipitation, pellets were resuspended in RIPA buffer with protease inhibitors. For CD44-ERM and CD44-pERM co-immunoprecipitation, cells were lysed in a solution containing 10 mm Tris (pH 7.5), 2 mm EDTA, 1% Triton X-100, as well as protease and phosphatase inhibitors. Cells were fixed with 4% paraformaldehyde in PBS for 10 min. In some cases, they were permeabilized with 0.5% Triton X-100 in PBS for 5 min. After three washes in PBS, nonspecific binding sites were blocked by incubation for 30 min with 1% bovine serum albumin in PBS. Subsequently, cells were incubated with one or two primary antibodies for 2 h at room temperature in a wet chamber. Goat anti-rabbit or anti-mouse IgG labeled with Alexa Fluor 488 or Alexa Fluor 568 (Molecular Probes) was added for 1 h at room temperature in the dark. After each antibody incubation step cells were washed 5× with 1% bovine serum albumin in PBS and mounted in Mowiol. Series of optical sections were taken with a Zeiss LSM 510 confocal microscope and were projected to single images using LSM software. The particle exclusion assay was described previously (25.Goldberg R.L. Toole B.P. Exp. Cell Res. 1984; 151: 258-263Crossref PubMed Scopus (9) Google Scholar). In our experiments, 1.5 × 105 cells were plated in 35-mm tissue culture dishes and cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. After 24 h, 750 μl of a suspension of fixed and washed horse red blood cells (108 cells/ml) was added to the cells and allowed to settle for 15 min. The pericellular matrix was assessed from photographs by measuring the area delimited between the red blood cells and the cell membrane. A ratio of 1.0 indicates no matrix. To demonstrate the structural dependence of the pericellular matrix on HA and on CD44, cells were (a) treated with 5 units/ml HA-specific Streptomyces hyaluronidase for 1 h before adding OX50 antibodies and observing coat recovery; (b) incubated with 100 μm 4-MU, an inhibitor of HA synthesis, in serum-free medium for 16 h, or (c) treated with 6-mer or 10-mer HA oligosaccharides for 16 h. To assess the effect of phosphoinositide 3-kinase (PI3K) inhibition, cells were preincubated with either 50 nm wortmannin or 5 μm LY294002 for 6 h before coat estimation. Cells were seeded in 92-well culture dishes. HA concentration was analyzed by a pseudo-RIA kit (Pharmacia HA Test) in the medium and in cell extracts following overnight digestion with 500 milliunits/ml Pronase at 55 °C in a 0.1 m Tris-HCl (pH 7.4) buffer containing 10 mm CaCl2. The homogenate was boiled for 10 min before protein (Bio-Rad kit) and HA assay. Both cell extracts and media were tested for hyaluronidase activity at different pH levels using zymography, which was performed as recently published by our group (7.Jadin L. Wu X. Ding H. Frost G.I. Onclinx C. Triggs-Raine B. Flamion B. FASEB J. 2008; 22: 4316-4326Crossref PubMed Scopus (83) Google Scholar). In addition, 25-μg protein samples from cell extracts were incubated in vitro with 15 μg of 2.5 × 106 Da HA for 16 h at 37 °C in a reaction buffer containing 100 mm formate at pH 3.7. The samples were mixed with 1/6 volume of loading buffer (7× TAE (40 mm Tris acetate, 1 mm EDTA, pH 8.0), 85% glycerol) and subjected to electrophoresis in 0.8% TAE-agarose gels at 50 V for 10 h. The size distribution of HA in samples was measured using a modification of the method described by Lee and Cowman (26.Lee H.G. Cowman M.K. Anal. Biochem. 1994; 219: 278-287Crossref PubMed Scopus (261) Google Scholar). The gels were stained in 0.005% Stains-All in 50% ethanol overnight and photographed on a fluorescent light Transilluminator. Healon was iodinated with Na125I using IODO-BEADs (Pierce) according to the manufacturer's instructions, and HA-binding activity was determined using a modified version of a method previously described (27.Underhill C.B. Chi-Rosso G. Toole B.P. J. Biol. Chem. 1983; 258: 8086-8091Abstract Full Text PDF PubMed Google Scholar). The samples were dissolved in 200 μl of Hanks' balanced salt solution containing increasing concentrations of 125I-labeled HA (0 to 8 μg/250 μl) in the absence or presence of non-labeled HA (100 μg/250 μl). After shaking for at least 20 min, 250 μl of saturated (NH4)2SO4 was added, followed by 25 μl of nonfat milk. The samples were centrifuged at 9,000 × g for 5 min. The tubes were rinsed twice with 50% saturated (NH4)2SO4, and the pellets were dissolved in water and processed for scintillation counting. Specific binding was calculated by subtracting the background label measured in the presence of an excess of non-labeled HA. Purified HA (0.02–1.2 × 106 Da, Amersham Biosciences) was conjugated to fluorescein as described by de Belder and Wik (28.de Belder A.N. Wik K.O. Carbohydr. Res. 1975; 44: 251-257Crossref PubMed Scopus (174) Google Scholar). Cells were resuspended in 0.5 ml of PBS with 0.1% NaN3 and a 1:100 dilution of fluorescein-HA (final concentration, 50 μg/ml). The mixture was incubated for 1 h at 4 °C. Following washes with 0.1% NaN3 in PBS, cells were fixed with 2% paraformaldehyde in the same solution for 30 min. Samples were analyzed on a FACSCalibur (BD Biosciences). Population-based movement of cells was measured by a wound healing assay as described previously (29.André F. Rigot V. Remacle-Bonnet M. Luis J. Pommier G. Marvaldi J. Gastroenterology. 1999; 116: 64-77Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). In this assay, cells are grown to confluency on plastic Petri dishes, then induced to re-populate a “wound” created by stripping a sharply defined channel using a sterile razor blade. To prevent growth during migration, cells were pre-treated with 4.5 μm mitomycin C for 2 h. These concentrations were optimized for the BB16 cell line as described (30.Platek A. Mettlen M. Camby I. Kiss R. Amyere M. Courtoy P.J. J. Cell Sci. 2004; 117: 4849-4861Crossref PubMed Scopus (20) Google Scholar). After band-stripping, cells were allowed to migrate into the wound for 6 h in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, supplemented or not with different hyaluronidases, 4-MU, or a PI3K inhibitor. Cells were then fixed with 3% formaldehyde in PBS for 20 min at 4 °C and stained with Crystal Violet. The number of cells that had colonized the wound margin was assessed in random microscopic fields (magnification, 320×; 15 fields per assay). Results are presented as means ± S.E. One- and two-way analyses of variance were used. When statistically significant differences were found (p < 0.05), individual comparisons were made using Dunn or Bonferroni tests with GraphPad Prism Version 5 software. Experiments were performed on the rat fibroblastic v-src-transformed cell line BB16. Hyal2 protein was detected by Western blotting using a rabbit polyclonal antibody, P16, raised against a peptide sequence of rat Hyal2. Because Hyal2 is poorly expressed in BB16 cells (Fig. 1A), stable transfectants were established using either rat Hyal2 cDNA or an empty vector. For comparison purposes, a clone with high expression of Hyal2 (BB16Hy2+) and a mock transfected clone (BB16mock) were selected. Several other clones overexpressing Hyal2 were also obtained; the relative amounts of Hyal2 mRNA in these clones based on quantitative reverse transcription-PCR ranged from 1.3 to 8.0 (supplemental Fig. S1B). None of the clones secreted Hyal2 into the medium (shown in Fig. 1A for BB16Hy2+ cells). The specificity of the P16 antibody was shown by preincubation with the immunogenic peptide (data not shown). Hyal2 overexpression in BB16Hy2+ cells was confirmed by immunoprecipitation after metabolic labeling (Fig. 1B). BB16Hy2+ cells synthesized and secreted similar amounts of HA compared with BB16 cells (Table 2).TABLE 2Quantification of endogenous HABB16BB16Hy2+pCell-associated HA (ng/1000 cells)1.26 ± 0.191.66 ± 0.17NSHA in culture medium (ng/1000 cells)3.68 ± 1.053.51 ± 0.88NS Open table in a new tab Neither BB16 nor BB16Hy2+ cells showed any hyaluronidase activity in zymography at pHs ranging from 3.7 (Fig. 1C) to 6.8 (data not shown), whether in the medium or in cell extracts. The partial HA-degrading activity detected in BB16 cell extracts at pH 3.7 did not significantly increase in any Hyal2 overexpression clone (Fig. 1D). Furthermore, this type of activity could not be detected at any significant level in cell membrane fractions at pH 4.0 or 6.5 (supplemental Fig. S2), contrary to what has been observed by Harada and Takahashi in CD44- and Hyal2-overexpressing HEK293 cells (10.Harada H. Takahashi M. J. Biol. Chem. 2007; 282: 5597-5607Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). This almost complete lack of hyaluronidase activity of Hyal2 should be contrasted with the effects of transient transfections of rat Hyal1 cDNA in the same cells, which led to strong HA-degrading activity (Fig. 1, C and D). Prior studies have reported conflicting results regarding the subcellular localization of Hyal2, which has been assigned previously to lysosomes (9.Lepperdinger G. Strobl B. Kreil G. J. Biol. Chem. 1998; 273: 22466-22470Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 31.Chow G. Knudson C.B. Knudson W. Osteoarthritis Cartilage. 2006; 14: 849-858Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), to the plasma membrane (13.Rai S.K. Duh F.M. Vigdorovich V. Danilkovitch-Miagkova A. Lerman M.I. Miller A.D. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 4443-4448Crossref PubMed Scopus (295) Google Scholar), and even to mitochondria (32.Chang N.S. BMC Cell Biol. 2002; 3: 8Crossref PubMed Scopus (39) Google Scholar) as well as to nuclei (15.Hsu L.J. Schultz L. Hong Q. Van Moer K. Heath J. Li M.Y. Lai F.J. Lin S.R. Lee M.H. Lo C.P. Lin Y.S. Chen S.T. Chang N.S. J. Biol. Chem. 2009; 284: 16049-16059Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Thus, the level of Hyal2 exposure on the outer cell surface of BB16 clones was evaluated by a cell surface biotinylation assay. A significant portion of Hyal2 was detected among biotin-labeled proteins in the streptavidin pellet of BB16Hy2+ cells, meaning Hyal2 was exposed at the outer plasma membrane during the process of biotinylation (Fig. 1E). However, for both Hyal2 and CD44, a well characterized HA receptor used as a control membrane protein, a good portion of the molecules remained inaccessible to external biotin (Fig. 1E). A standard red blood cell exclusion assay indicated that BB16 and BB16mock cells were capable of producing and organizing a HA-rich pericellular matrix with or without exposure to 10% fetal bovine serum (Fig. 2 and Table 3). In contrast, BB16Hy2+ cells were almost devoid of such a pericellular coat. The ratio of combined cell and pericellular matrix areas relative to the cell area was ∼2.0 in cells expressing low levels of Hyal2 (BB16 and BB16mock), but fell to ∼1.2 in BB16Hy2+ cells. In these cell lines and in six other clones expressing intermediate levels of Hyal2, there was a strong correlation between individual Hyal2 mRNA levels and the reduction in coat size (supplemental Fig. S1D).TABLE 3Effects of different treatments on pericellular HA coatsBB16BB16mockBB16Hy2+pBasal settingsUntreated cells2.05 ± 0.052.08 ± 0.041.22 ± 0.07<0.001Scrambled RNA2.28 ± 0.111.09" @default.
• W2076220323 created "2016-06-24" @default.
• W2076220323 creator A5024748875 @default.
• W2076220323 creator A5061568925 @default.
• W2076220323 creator A5071342393 @default.
• W2076220323 creator A5088605616 @default.
• W2076220323 date "2009-11-01" @default.
• W2076220323 modified "2023-09-26" @default.
• W2076220323 title "Two Novel Functions of Hyaluronidase-2 (Hyal2) Are Formation of the Glycocalyx and Control of CD44-ERM Interactions" @default.
• W2076220323 cites W1555753650 @default.
• W2076220323 cites W1599397384 @default.
• W2076220323 cites W1608766205 @default.
• W2076220323 cites W1733016559 @default.
• W2076220323 cites W1964632662 @default.
• W2076220323 cites W1966422114 @default.
• W2076220323 cites W1970976508 @default.
• W2076220323 cites W1973295977 @default.
• W2076220323 cites W1973573706 @default.
• W2076220323 cites W1977603000 @default.
• W2076220323 cites W1980152518 @default.
• W2076220323 cites W1980194211 @default.
• W2076220323 cites W1985044730 @default.
• W2076220323 cites W1987960919 @default.
• W2076220323 cites W1994496279 @default.
• W2076220323 cites W2000547781 @default.
• W2076220323 cites W2001971849 @default.
• W2076220323 cites W2003380614 @default.
• W2076220323 cites W2005318950 @default.
• W2076220323 cites W2009431295 @default.
• W2076220323 cites W2025027858 @default.
• W2076220323 cites W2029330070 @default.
• W2076220323 cites W2029834185 @default.
• W2076220323 cites W2030337803 @default.
• W2076220323 cites W2041654773 @default.
• W2076220323 cites W2042778072 @default.
• W2076220323 cites W2044902832 @default.
• W2076220323 cites W2045014705 @default.
• W2076220323 cites W2046955904 @default.
• W2076220323 cites W2048234485 @default.
• W2076220323 cites W2052965509 @default.
• W2076220323 cites W2056925926 @default.
• W2076220323 cites W2065895195 @default.
• W2076220323 cites W2075334663 @default.
• W2076220323 cites W2075905225 @default.
• W2076220323 cites W2078468203 @default.
• W2076220323 cites W2078539894 @default.
• W2076220323 cites W2079804385 @default.
• W2076220323 cites W2094238516 @default.
• W2076220323 cites W2094491525 @default.
• W2076220323 cites W2095741501 @default.
• W2076220323 cites W2100582120 @default.
• W2076220323 cites W2102004717 @default.
• W2076220323 cites W2102419220 @default.
• W2076220323 cites W2106026965 @default.
• W2076220323 cites W2107214690 @default.
• W2076220323 cites W2108577689 @default.
• W2076220323 cites W2113431340 @default.
• W2076220323 cites W2114964393 @default.
• W2076220323 cites W2117566184 @default.
• W2076220323 cites W2118121278 @default.
• W2076220323 cites W2119139128 @default.
• W2076220323 cites W2120499448 @default.
• W2076220323 cites W2123257463 @default.
• W2076220323 cites W2139966914 @default.
• W2076220323 cites W2140524396 @default.
• W2076220323 cites W2144801666 @default.
• W2076220323 cites W2162761212 @default.
• W2076220323 cites W2164021363 @default.
• W2076220323 cites W2388440598 @default.
• W2076220323 cites W3085129072 @default.
• W2076220323 doi "https://doi.org/10.1074/jbc.m109.044362" @default.
• W2076220323 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2785194" @default.
• W2076220323 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19783662" @default.
• W2076220323 hasPublicationYear "2009" @default.
• W2076220323 type Work @default.
• W2076220323 sameAs 2076220323 @default.
• W2076220323 citedByCount "66" @default.
• W2076220323 countsByYear W20762203232012 @default.
• W2076220323 countsByYear W20762203232013 @default.
• W2076220323 countsByYear W20762203232014 @default.
• W2076220323 countsByYear W20762203232015 @default.
• W2076220323 countsByYear W20762203232016 @default.
• W2076220323 countsByYear W20762203232017 @default.
• W2076220323 countsByYear W20762203232018 @default.
• W2076220323 countsByYear W20762203232019 @default.
• W2076220323 countsByYear W20762203232020 @default.
• W2076220323 countsByYear W20762203232021 @default.
• W2076220323 countsByYear W20762203232022 @default.
• W2076220323 countsByYear W20762203232023 @default.
• W2076220323 crossrefType "journal-article" @default.
• W2076220323 hasAuthorship W2076220323A5024748875 @default.
• W2076220323 hasAuthorship W2076220323A5061568925 @default.
• W2076220323 hasAuthorship W2076220323A5071342393 @default.
• W2076220323 hasAuthorship W2076220323A5088605616 @default.
• W2076220323 hasBestOaLocation W20762203231 @default.
• W2076220323 hasConcept C181199279 @default.
• W2076220323 hasConcept C185592680 @default.
• W2076220323 hasConcept C2780098789 @default.

• next