CD44-dependent Intracellular and Extracellular Catabolism of Hyaluronic Acid by Hyaluronidase-1 and -2

Hyaluronic acid (HA) is a high molecular weight glycosaminoglycan involved in a wide variety of cellular functions. However, its turnover in living cells remains largely unknown. In this study, CD44, a receptor for HA, and hyaluronidase-1, -2, and -3 (Hyal-1, -2 and -3) were stably expressed in HEK 293 cells and the mechanism of HA catabolism was systematically investigated using fluorescein-labeled HA. CD44 was essential for HA degradation by both endogenous and exogenously expressed hyaluronidases. Hyal-1 was not able to cleave HA in living cells in the absence of CD44. Intracellular HA degradation was predominantly mediated by Hyal-1 after incorporation of HA by CD44. Although Hyal-1 was active only in intracellular space in vivo, a certain amount of the enzyme was secreted to extracellular space. This extracellular Hyal-1 was found to be incorporated by cells and such uptake of Hyal-1 was, in part, involved in the intracellular degradation of HA. Hyal-2 was involved in the extracellular degradation of HA. Hyal-2 activity was also dependent on the expression of CD44 in both living cells and enzyme assays. Immunofluorescent microscopy demonstrated that both Hyal-2 and CD44 are present on the cell surface. Without CD44 expression, Hyal-2 existed in a granular pattern, and did not show hyaluronidase activity, suggesting that localization change could contribute to Hyal-2 function. A convenient and quantitative enzyme assay was established for the measurement of Hyal-2 activity. Hyal-2 activity was detected in the membrane fraction of cells co-expressing Hyal-2 and CD44. The pH optimum for Hyal-2 was 6.0–7.0. The membrane fraction of cells expressing Hyal-2 alone did not show hyaluronidase activity. Hyal-3 did not show any hyaluronidase activity in our experimental conditions. Based on these findings, Hyal-1 and -2 contribute to intracellular and extracellular catabolism of HA, respectively, in a CD44-dependent manner, and their HA degradation occurs independently from one another. Hyaluronic acid (HA) is a high molecular weight glycosaminoglycan involved in a wide variety of cellular functions. However, its turnover in living cells remains largely unknown. In this study, CD44, a receptor for HA, and hyaluronidase-1, -2, and -3 (Hyal-1, -2 and -3) were stably expressed in HEK 293 cells and the mechanism of HA catabolism was systematically investigated using fluorescein-labeled HA. CD44 was essential for HA degradation by both endogenous and exogenously expressed hyaluronidases. Hyal-1 was not able to cleave HA in living cells in the absence of CD44. Intracellular HA degradation was predominantly mediated by Hyal-1 after incorporation of HA by CD44. Although Hyal-1 was active only in intracellular space in vivo, a certain amount of the enzyme was secreted to extracellular space. This extracellular Hyal-1 was found to be incorporated by cells and such uptake of Hyal-1 was, in part, involved in the intracellular degradation of HA. Hyal-2 was involved in the extracellular degradation of HA. Hyal-2 activity was also dependent on the expression of CD44 in both living cells and enzyme assays. Immunofluorescent microscopy demonstrated that both Hyal-2 and CD44 are present on the cell surface. Without CD44 expression, Hyal-2 existed in a granular pattern, and did not show hyaluronidase activity, suggesting that localization change could contribute to Hyal-2 function. A convenient and quantitative enzyme assay was established for the measurement of Hyal-2 activity. Hyal-2 activity was detected in the membrane fraction of cells co-expressing Hyal-2 and CD44. The pH optimum for Hyal-2 was 6.0–7.0. The membrane fraction of cells expressing Hyal-2 alone did not show hyaluronidase activity. Hyal-3 did not show any hyaluronidase activity in our experimental conditions. Based on these findings, Hyal-1 and -2 contribute to intracellular and extracellular catabolism of HA, respectively, in a CD44-dependent manner, and their HA degradation occurs independently from one another. Hyaluronic acid (HA) 3The abbreviations used are: HA, hyaluronic acid; Hyal-1, -2, and -3, hyaluronidase-1, -2, and -3; HEK 293, human embryonic kidney 293; PBS, phosphate-buffered saline; fl-HA, fluorescein-labeled HA; Vcpal, l-ascorbic acid 6-hexadecanoate; IRES, internal ribosome entry site. is a negatively charged, high molecular weight glycosaminoglycan found predominantly in the extracellular matrix. It is the simplest of the glycosaminoglycans, the only one not covalently linked to core protein, and is unbranched and composed of repeating alternating units of glucuronic acid and N-acetylglucosamine. Despite the simplicity of its composition, HA has a great number of biological functions. It not only functions as a biological glue that participates in lubricating joints or holding together gel-like connective tissues, but also functions as a microenvironmental cue that co-regulates cell behavior during embryonic development and morphogenesis (1Toole B.P. Cell Biology of Extracellular Matrix. 1991; (Hay, E. D., ed) pp. , Plenum Press, New York: 61-92Google Scholar, 2Toole B.P. Cell Dev. Biol. 2001; 12: 79-87Crossref PubMed Scopus (442) Google Scholar), wound healing (3Weigel P.H. Fuller G.M. LeBoeuf R.D. J. Theor. Biol. 1986; 119: 219-234Crossref PubMed Scopus (301) Google Scholar, 4Longaker M.T. Chiu E.S. Adzick N.S. Stern M. Harrison M.R. Stern R. Ann. Surg. 1991; 213: 292-296Crossref PubMed Scopus (290) Google Scholar), repair and regeneration, inflammation (5Noble P.W. Matrix Biol. 2002; 21: 25-29Crossref PubMed Scopus (471) Google Scholar, 6de la Motte C.A. Hascall V.C. Drazba J. Bandyopadhyay S.K. Strong S.A. Am. J. Pathol. 2003; 163: 121-133Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 7Majors A.K. Austin R.C. de la Motte C.A. Pyeritz R.E. Hascall V.C. Kessler S.P. Sen G. Strong S.A. J. Biol. Chem. 2003; 278: 47223-47231Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), and tumor progression and invasion (8Toole B.P. Glycobiology. 2002; 12: 37-42Crossref PubMed Scopus (183) Google Scholar, 9Toole B.P. Hascall V.C. Am. J. Pathol. 2002; 161: 745-747Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). There are 15 g of HA in a 70-kg individual, of which 5 g is replaced daily. In the skin, which contains 50% of the total body HA, the half-life of HA is about 1 day, and even in as seemingly inert a tissue as cartilage, HA turns over with a half-life of 1–3 weeks (10Stern R. Glycobiology. 2003; 13: 105-115Crossref PubMed Scopus (290) Google Scholar). In the bloodstream, the half-life of HA is 2–5 min (11Fraser J.R. Laurent T.C. Pertoft H. Baxter E. Biochem. J. 1981; 200: 415-424Crossref PubMed Scopus (301) Google Scholar). All such catabolism is presumably the result of hyaluronidases. Hyaluronidases and their attendant control mechanisms are apparently of critical importance in normal and abnormal biology. There are six hyaluronidase-like sequences in the mammalian genome. All are transcriptionally active with unique tissue distributions. In humans, three genes, HYAL1, HYAL2, and HYAL3 are clustered on chromosome 3p21.3, coding for Hyal-1, Hyal-2, and Hyal-3, respectively. Another three genes, HYAL4, PHYAL1, and SPAM1, are clustered similarly on chromosome 7q31.3. They code for Hyal-4, a pseudogene transcribed but not translated in humans, and PH-20, the sperm enzyme, respectively (12Csoka A.B. Scherer S.W. Stern R. Genomics. 1999; 60: 356-361Crossref PubMed Scopus (208) Google Scholar, 13Csoka A.B. Frost G.I. Stern R. Matrix Biol. 2001; 20: 499-508Crossref PubMed Scopus (487) Google Scholar). Among them, Hyal-1, Hyal-2, and Hyal-3 are somatically expressed hyaluronidases. Hyal-1, known as serum hyaluronidase, is enzyme Stern M. M. Stern R. Biochem. PubMed Scopus Google Scholar, G.I. Stern R. Biochem. PubMed Scopus (208) Google that is able to HA of any as a and which predominantly Hyal-2 G. G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, G. J. G. Matrix Biol. 2001; 20: PubMed Scopus Google is also to be enzyme and is linked to by a S.K. 2001; PubMed Scopus Google Scholar). This hyaluronidase has unique high molecular HA to of or about is known about Hyal-3, expression are found in mammalian and (12Csoka A.B. Scherer S.W. Stern R. Genomics. 1999; 60: 356-361Crossref PubMed Scopus (208) Google Scholar). a and for the of the suggesting that Hyal-3 in However, has not to hyaluronidase activity in to that the HA is by cells for degradation and that the is mediated cell HA and degradation of HA was by the suggesting that CD44 has a critical in HA turnover J. Cell PubMed Google Scholar, M. J. Cell Biol. PubMed Scopus Google Scholar, R. M. M. Hascall V.C. M. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). A was on in cells Stern R. E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, R. J. Cell Biol. PubMed Scopus Google Scholar). The high molecular weight HA is to the cell by the of CD44 and HA is by Hyal-2 to the to about units in a The HA are to and to Hyal-1 the to However, the and these mammalian not In this study, of cell stably expressing human Hyal-1, Hyal-2, Hyal-3, CD44 were established for of mammalian and for the HA catabolism in HA catabolism in these cell was in using HA with In and quantitative enzyme assay was for the measurement of Hyal-2 activity. Hyal-1 and -2 was to contribute to intracellular and extracellular catabolism of hyaluronic respectively, in a CD44-dependent of Cell HYAL1, HYAL2, and 293 cells were in a in in with and The of human CD44 was by a using a as a and the expression HEK 293 cells were with and by a A cell expressing CD44 was using of and was The of human HYAL1, HYAL2, and HYAL3 were by and respectively, the expression was 293 and The were using of cell with was In of human was the expression cells were with and by a A cell expressing Hyal-2 was using of and was The coding in the expression was for The for the are as CD44, HYAL1, HYAL2, and CD44 was from Hyal-2 was in a the acid The were using the Hyal-3 was in a acid sequences and were used in and were and with 1 of phosphate-buffered g for the was and of was to the The cells were by The was for 5 and of total were to The were to and with for human CD44, Hyal-2, or which were detected using of Hyal-1 were in the for the was and the cells were and by with The cells were with and in by the of total and the from cell was with HA in of pH for In was with HA in pH for of the was on and with of HA and HA was as by de and PubMed Scopus Google with a of HA was in of and with of in and was to the The was for 5 and of with The was by g and in a The was in and using The was by HA was similarly using of of by were in and for The cells were in of the was and the cells were and by with the cells were with and in 1 of for 5 min by a This was as the cell The cell were with and in pH for to the cells and the total intracellular The fraction was the intracellular The in 1 of total cell and the total intracellular was by using a with of were and the was by The was as the for of Hyal-1 by were on a and for The cells were for of in 1 of 50% from a of or 293 the cells were with PBS, and in 1 of for 5 by a The cell were with and in of by Hyal-1 activity in the of total was as In cells were in and for The cells were in of and from a of or 293 the the cell and the intracellular were and by the as for Hyal-2 was to the for J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). In cells on a were with and in a with normal for 15 min the the cells were in CD44 and Hyal-2, in for min the cells were with and in for min the cells were and in in for min and microscopy was using Cell of Hyal-2 were and with PBS, and by in The of the cells were g for 5 the was to g for 1 The membrane were and and to 1 of total in in a of pH from to or in pH A of 1 of the membrane or 1 of total and or was for of in the of of and g for The was to a and with The enzyme assay was for quantitative in a HA in was and high by the was with of pH The membrane of total in pH were to and for of was with of pH The HA on the was detected using assay In the were with for 1 and with the was for 5 and the was by All these were and used to the of 293 Hyal-1, Hyal-2, Hyal-3, cells but their expression is and of are expressed (12Csoka A.B. Scherer S.W. Stern R. Genomics. 1999; 60: 356-361Crossref PubMed Scopus (208) Google Scholar), in in of In the expression of CD44 be also for their to cell stably expressing CD44 Hyal-1, -2, and -3 for of hyaluronidase in living cell was established from 293 cells by with human CD44 293 and cells were with the for expressing human HYAL1, HYAL2, and The internal ribosome entry was for this as the to cell cell and to the in stably cell for cell and and co-expressing cell and were The total cell were from cell and were using CD44, Hyal-2 and Hyal-3 to was expressed from 293 cells a with the of that to the molecular of the of human CD44. Hyal-2 and Hyal-3 were detected the molecular of and respectively. could not Hyal-1 expression was by the hyaluronidase activity pH the pH optimum of Hyal-1 G.I. Stern R. Biochem. PubMed Scopus (208) Google Scholar), of were from the of cell and with high molecular weight HA. HA with the from and cells was to a molecular weight were HA was with the from the cell the the hyaluronidase activity was not detected in or from the It be that the the from any of the of cells hyaluronidase activity pH that only and cells were stably Hyal-1 and that the expressed Hyal-1 was not only but was also secreted to the of the of with Hyal-1, Hyal-2, Hyal-3, high molecular HA with was and used for and quantitative of HA catabolism by The cell with expression in CD44 and were in the of for the was and the cells were with to The cell were to to the cells and intracellular The of in these three were by a from in fraction of the Without CD44 expression, of Hyal-1, Hyal-2, and Hyal-3 catabolism of of the in the and was in the of the of were detected in the cell and intracellular even the HA turnover from the endogenous hyaluronidase was CD44 was The high molecular weight in the of the cells was and the were together with the that the from cells did not show hyaluronidase activity pH this result that the that to the cells with the of CD44 was by endogenous hyaluronidase or on the of the and the was also incorporated cells and was in intracellular In only incorporated cells by CD44 was by Hyal-1 to a molecular weight and In the intracellular was in the total of the the of from the and the cell were to of In the in the of cells was with the in This result that Hyal-2 was for the turnover of in the extracellular space. The of CD44 is critical for the Hyal-2 activity. of was not in the from the cells Although by Hyal-1 a molecular weight the in the by Hyal-2, was found only in the intracellular The amount of incorporated the cells and by Hyal-1 was of to the of to the was by Hyal-2, to the on the the intracellular degradation of was not in the of that HA degradation mediated by Hyal-2 occurs on the of the cells and the were the and that the intracellular catabolism by Hyal-1 is of the HA degradation on the cell mediated by turnover mediated Hyal-3 was detected our experimental even the enzyme was with CD44 and Hyal-3 not to a hyaluronidase activity. CD44 be for Hyal-3 to its activity. of Extracellular Hyal-1 Hyal-1 is to be and a hyaluronidase, is known as a hyaluronidase G.I. Stern R. Biochem. PubMed Scopus (208) Google Scholar). as also that a amount of expressed Hyal-1 was found to be secreted the Hyal-1 is only in the intracellular space in the of CD44 It is that secreted Hyal-1 is incorporated by cells Biol. Google Scholar). this 293, and cells were with Hyal-1 by the of cells were with to Hyal-1, and the of Hyal-1 in the intracellular was by the hyaluronidase Hyal-1 was incorporated 293 cells and its activity was detected in the intracellular in a The incorporation of Hyal-1 was of CD44 or Hyal-2 expression as in the incorporation were 293, and cells to the incorporated Hyal-1 is of 293, and were with and the from or 293 cells for and catabolism of in the and intracellular was by The Hyal-1 did not extracellular degradation of in the and the activity mediated by Hyal-2 was also not by Hyal-1 In the intracellular on the incorporated by cells was to a molecular weight Hyal-1 was show that Hyal-1 could be by cells and as intracellular The of high molecular in the intracellular of cells was cells were with The of the from 293 cells presumably the and CD44 and the incorporation of by CD44, which to the of in the intracellular of the such of was in as the to CD44 on the cell was by Hyal-2 and was from the cell surface. together with the that could not any in the of in the intracellular and cells and these also that Hyal-2 to the extracellular degradation to the intracellular degradation of HA and that intracellular degradation mediated by Hyal-1 is of the or activity of of the Extracellular of HA by of the HA catabolism by Hyal-2, and cells were in the of for of and of from cell and intracellular were The in the molecular weight of and in the cell of of the cell from the cells was the the of the from the cells a high molecular weight in the of the This degradation of in the cell from cells in a manner, the of from cells did not change the HA degradation by Hyal-2 on the cell to be in the after of and was after of Hyal-2 activity did not contribute to HA degradation in intracellular as the and amount of in the intracellular from the cells were not from of cells during the for intracellular did not the extracellular degradation of HA by Hyal-2 not these is that degradation of HA by Hyal-2 on the cell and the HA is the extracellular space. a the high molecular weight of in the and the of was in the of the The cell localization of CD44 and Hyal-2 was by In cells were with to its on the cells The of was detected only in cells and the of was detected only in of was only in In the the Hyal-2 was detected in a granular the Hyal-2 in cells was on the cell of Hyal-2 were not was or not not that Hyal-2 with CD44, and the the and activity of Hyal-2 on the cell surface. CD44 was also detected on cell change was not the and cells and Hyal-2 Hyal-2 has as enzyme G. G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), its to the of quantitative and for the measurement of Hyal-2 activity. which Hyal-2 activity to Hyal-2 activity conditions. was with the membrane from cell in pH for and to The of the of that a high molecular weight in fraction in was and in of CD44 is essential for Hyal-2 activity even the as hyaluronidase activity was detected only in the membrane of the membrane hyaluronidase activity. of the activity was even the membrane from cells were with of The pH and of Hyal-2 activity are in and respectively. The pH optimum for Hyal-2 was pH 6.0–7.0. The optimum was the activity did not show a on the Although Hyal-2 activity could be by the membrane of and the enzyme was and to the activity. The activity was presumably to the expression of Hyal-2 by the the Hyal-2 activity, the of human was the expression and The cell expressed Hyal-2 by the of with the membrane of total of and cells in 1 of pH for The expression of Hyal-2 linked to HA degradation activity degradation by the membrane of total of cells is in The of Hyal-2 was in in which degradation was with the of membrane Vcpal, a hyaluronidase M. J. G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Hyal-2 with of cell membrane a assay in which Hyal-2 activity is in a and In this HA with was used as a The with HA was with or cell membrane in pH the HA on the was detected by Hyal-2 activity and in the CD44 and to be involved in HA turnover of and cells J. Cell PubMed Google Scholar), R. M. M. Hascall V.C. M. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), and tumor cells Stern R. E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S.K. Cell PubMed Scopus Google Scholar). However, the mechanism of HA turnover in living cells has largely unknown. to and CD44 by a expression However, in the of Hyal-2, could not any hyaluronidase activity in the In the expression was that the CD44 and Hyal-2 were not able to the the which be to their normal localization and in living This to cell expressing CD44 in The to these cell and using the established cell demonstrated that Hyal-1 and -2 contribute to intracellular and extracellular catabolism of hyaluronic respectively. of HA by Hyal-1 was only in the intracellular Hyal-1 activity was by CD44 only HA cells by CD44 could be the for and in and cells G.I. Stern R. Biochem. PubMed Scopus (208) Google Scholar, J. Cell PubMed Google the molecular Although Hyal-1 was secreted to extracellular space as G.I. Stern R. Biochem. PubMed Scopus (208) Google Scholar), the from with that Hyal-1 is and from The in is enzyme essential for the of the that to from with has normal of Hyal-1, but of M.R. Am. J. PubMed Scopus Google Scholar), suggesting that Hyal-1 is not by cells or that Hyal-1 is to by mechanisms the In this study, the extracellular Hyal-1 was found to be incorporated and to as intracellular the of for Hyal-1 uptake and that enzyme be for a by the Hyal-1 It is also that the for Hyal-1 uptake is could be a biological for Hyal-1 is to tumor and progression PubMed Scopus Google Scholar). of HA by Hyal-2 on cell and also on CD44 In but not in of extracellular was by The in the molecular and cells in the cell and Hyal-2 was on the cell and the of CD44 was on the localization of are with the that the CD44 and Hyal-2 by Stern R. E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). HA a in extracellular by with cell or PubMed Scopus Google Scholar, J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, B.P. PubMed Scopus Google Scholar). In such a HA degradation on cell is critical for HA as is that HA is by cells as a and is Hyal-2 is to a in extracellular HA Based on the in cellular catabolism of HA, established quantitative enzyme for Hyal-2, and its Hyal-2 was dependent on CD44 even conditions. activity was detected in but not in cell A of cell of cells and cells did not show hyaluronidase activity. The of the of cells by the activity of Hyal-2 in not that a with CD44 on the cell is essential for Hyal-2 to hyaluronidase activity, and that the Hyal-2 and CD44 is The optimum pH was a of pH 6.0–7.0. pH of pH and the activity was are with the in tumor cells that the is in one of the the extracellular pH was to pH HA to CD44 and that of the cells with the hyaluronidase activity pH Stern R. E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). It has that Hyal-1 and Hyal-2 are mammalian in tissues, and that in to high molecular weight HA to a (10Stern R. Glycobiology. 2003; 13: 105-115Crossref PubMed Scopus (290) Google Scholar, 13Csoka A.B. Frost G.I. Stern R. Matrix Biol. 2001; 20: 499-508Crossref PubMed Scopus (487) Google Scholar, R. J. Cell Biol. PubMed Scopus Google Scholar). HA are by the Hyal-2 the cell and to Hyal-1 the to However, our that Hyal-1 and Hyal-2 could independently on HA degradation for the Hyal-1 cleave HA. of the degradation by Hyal-2 were to the and not incorporated the cells as for HEK 293 cells the endogenous intracellular hyaluronidase presumably from Hyal-1, and the activity was by the expression of CD44. However, expression of Hyal-2 did not the endogenous intracellular hyaluronidase activity. Hyal-1 was incorporated cells and as intracellular However, the intracellular activity of the incorporated Hyal-1 in cells was not from that in cells of Hyal-1 were incorporated both Hyal-1 could with Hyal-2, the intracellular degradation of HA by the incorporated Hyal-1 in cells be that in these could that Hyal-1 and Hyal-2 are involved in for HA degradation in the The activity of Hyal-3 remains to be could not its activity to any of Hyal-3 as as Hyal-2 is by such as and tumor Hyal-1 is not Biochem. PubMed Scopus Google Scholar). It be that Hyal-3 has to be with to show its the for Hyal-2 and CD44. be for Hyal-3 HA. Hyal-2 is a biological of HA catabolism be of in the of It has that Hyal-2 could as of Hyal-2 the tumor of cells G. 1999; Google Scholar). Although Hyal-2 is not expressed in normal G. G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, G. Genomics. PubMed Scopus Google Scholar), its expression was detected from human tumor G. 1999; Google Scholar). 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In this is that the Hyal-2 activity was in this study, and the and Hyal-2 enzyme assay be to for these It was demonstrated in this that HA catabolism is not only by Hyal-2 but also by CD44 In was that Hyal-2 change and pH change on the cell also could be for Hyal-2 mediated HA to