Mouse LYVE-1 Is an Endocytic Receptor for Hyaluronan in Lymphatic Endothelium

Abstract

The glycosaminoglycan hyaluronan is a key substrate for cell migration in tissues during inflammation, wound healing, and neoplasia. Unlike other matrix components, hyaluronan (HA) is turned over rapidly, yet most degradation occurs not locally but within distant lymph nodes, through mechanisms that are not yet understood. While it is not clear which receptors are involved in binding and uptake of hyaluronan within the lymphatics, one likely candidate is the lymphatic endothelial hyaluronan receptor LYVE-1 recently described in our laboratory (Banerji, S., Ni, J., Wang, S., Clasper, S., Su, J., Tammi, R., Jones, M., and Jackson, D.G. (1999)J. Cell Biol. 144, 789–801). Here we present evidence that LYVE-1 is involved in the uptake of hyaluronan by lymphatic endothelial cells using a new murine LYVE-1 orthologue identified from the EST data base. We show that mouse LYVE-1 both binds and internalizes hyaluronan in transfected 293T fibroblasts in vitro and demonstrate using immunoelectron microscopy that it is distributed equally among the luminal and abluminal surfaces of lymphatic vessels in vivo. In addition, we show by means of specific antisera that expression of mouse LYVE-1 remains restricted to the lymphatics in homozygous knockout mice lacking a functional gene for CD44, the closest homologue of LYVE-1 and the only other Link superfamily HA receptor known to date. Together these results suggest a role for LYVE-1 in the transport of HA from tissue to lymph and imply that further novel hyaluronan receptors must exist that can compensate for the loss of CD44 function. The glycosaminoglycan hyaluronan is a key substrate for cell migration in tissues during inflammation, wound healing, and neoplasia. Unlike other matrix components, hyaluronan (HA) is turned over rapidly, yet most degradation occurs not locally but within distant lymph nodes, through mechanisms that are not yet understood. While it is not clear which receptors are involved in binding and uptake of hyaluronan within the lymphatics, one likely candidate is the lymphatic endothelial hyaluronan receptor LYVE-1 recently described in our laboratory (Banerji, S., Ni, J., Wang, S., Clasper, S., Su, J., Tammi, R., Jones, M., and Jackson, D.G. (1999)J. Cell Biol. 144, 789–801). Here we present evidence that LYVE-1 is involved in the uptake of hyaluronan by lymphatic endothelial cells using a new murine LYVE-1 orthologue identified from the EST data base. We show that mouse LYVE-1 both binds and internalizes hyaluronan in transfected 293T fibroblasts in vitro and demonstrate using immunoelectron microscopy that it is distributed equally among the luminal and abluminal surfaces of lymphatic vessels in vivo. In addition, we show by means of specific antisera that expression of mouse LYVE-1 remains restricted to the lymphatics in homozygous knockout mice lacking a functional gene for CD44, the closest homologue of LYVE-1 and the only other Link superfamily HA receptor known to date. Together these results suggest a role for LYVE-1 in the transport of HA from tissue to lymph and imply that further novel hyaluronan receptors must exist that can compensate for the loss of CD44 function. hyaluronan lymphatic vessel endothelial HA receptor-1 fluorescein isothiocyanate polymerase chain reaction enzyme-linked immunosorbent assay phosphate-buffered saline HA receptor for endocytosis The extracellular matrix glycosaminoglycan hyaluronan (HA)1 is a large polymer ofN-acetyl-d-glucosamine andd-glucuronic acid (molecular mass 105-107 Da) which plays an important role in maintaining tissue integrity as well as facilitating the migration of cells during inflammation, wound repair, and embryonic development (1Laurent T.C. Fraser J.R. FASEB J. 1992; 6: 2397-2404Crossref PubMed Scopus (2086) Google Scholar,2Lee J.Y. Spicer A.P. Curr. Opin. Cell Biol. 2000; 12: 581-586Crossref PubMed Scopus (453) Google Scholar). By comparison with other macromolecules of the extracellular matrix, HA undergoes rapid turnover with a half-life of ∼24 h (1Laurent T.C. Fraser J.R. FASEB J. 1992; 6: 2397-2404Crossref PubMed Scopus (2086) Google Scholar). Intriguingly, most degradation occurs not locally, but within distant lymph nodes. During this process, tissue HA enters the afferent lymphatic vessels and is transported with the lymph fluid to the draining lymph nodes where ∼90% of the glycosaminoglycan is degraded by unknown mechanisms (3Fraser J.R. Kimpton W.G. Laurent T.C. Cahill R.N. Vakakis N. Biochem. J. 1988; 256: 153-158Crossref PubMed Scopus (161) Google Scholar, 4Fraser J.R. Laurent T.C. CIBA Found. Symp. 1989; 143: 41-53PubMed Google Scholar). The remaining 10–15% of the HA exits via the efferent lymphatics to the blood vasculature where it is rapidly endocytosed by the liver sinusoid endothelial HA receptor (5Yannariello-Brown J. McGary C.T. Weigel P.H. J. Cell. Biochem. 1992; 48: 73-80Crossref PubMed Scopus (17) Google Scholar), a 300-kDa heterotrimeric complex of α, β, and γ subunits that clears not only HA but also chondroitin and heparan sulfate from the circulation (6Zhou B. Oka J.A. Singh A. Weigel P.H. J. Biol. Chem. 1999; 274: 33831-33834Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). While it is clear that HA can rapidly permeate the lymphatics in skin and other tissues (7Brown T.J. Alcorn D. Fraser J.R. J. Invest. Dermatol. 1999; 113: 740-746Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), the mechanisms responsible for its transport across lymphatic endothelium, and the receptors involved in its uptake and transport within lymphatic vessels are all unknown. The majority of HA-binding proteins (8Toole B.P. Curr. Opin. Cell Biol. 1990; 2: 839-844Crossref PubMed Scopus (393) Google Scholar, 9Knudson C.B. Knudson W. FASEB J. 1993; 7: 1233-1241Crossref PubMed Scopus (601) Google Scholar) identified to date belong to the Link protein superfamily, defined by the presence of a conserved HA-binding domain known as the Link module (10Neame P.J. Barry F.P. Experientia (Basel). 1993; 49: 393-402Crossref PubMed Scopus (110) Google Scholar, 11Day A.J. Biochem. Soc. Trans. 1999; 27: 115-121Crossref PubMed Scopus (76) Google Scholar). This is a unit of ∼100 amino acids that contains four conserved cysteine residues interspersed with tracts of both hydrophobic and charged residues. The three-dimensional structure of the Link module closely resembles that of the C-type lectin fold, comprising two β sheets flanked by two short α helices and stabilized by two disulfide linkages enclosing a central hydrophobic core (12Kohda D. Morton C.J. Parkar A.A. Hatanaka H. Inagaki F.M. Campbell I.D. Day A.J. Cell. 1996; 86: 767-775Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 13Brissett N.C. 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Scholar) we a murine LYVE-1 orthologue and we its with that important with the CD44 Intriguingly, we that mouse LYVE-1 of HA and is both the luminal and abluminal of lymphatic endothelial The of these for the of LYVE-1 in are The cell 293T from the Cell The expression from The The for protein expression by to mouse from from and from from and from hyaluronan from and from from chondroitin and heparan sulfate from protein the extracellular domain of to the domain of by S. of knockout mice R. J. N. T. A. A. J. A. C.J. PubMed Google Scholar) the and from the for of by and the and by of for microscopy as described The amino acid of LYVE-1 to for murine within the mouse EST data using the via the The four and which a of to the The and and from mouse in a reaction using as In the of the and as in with of mouse D.G. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar) in a reaction and of In the of of the as in a reaction using the and The in are and the The both to its and amino acid of murine LYVE-1 and comparison with the and amino acid from the mouse LYVE-1 identified by the mouse EST data and from mouse The and and two for are an of the amino acid for mouse and CD44 with the and with residues in The the Link cysteine residues are and by are as the four conserved structural of the Link module the two conserved for and of CD44 Link a conserved cysteine within the for receptor and HA-binding in CD44 and a cysteine to LYVE-1 residues within the LYVE-1 Link module to in CD44 HA-binding are with In addition, a conserved of residues of the Link module in mouse and LYVE-1 and a in CD44 that an to the HA-binding domain are with a of mouse tissue of from and in with a the extracellular domain of mouse LYVE-1 with by to the in for and in for by in with by the The extracellular domain of mouse LYVE-1 the in from mouse D.G. 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Cell Biol. 1999; PubMed Scopus Google Scholar) in using of with in the presence of a of HA for h cells with and by in with of cell in in HA and the other with for to cell by The of LYVE-1 the by cells and with LYVE-1 by In by using a of HA by cells as described for that cell LYVE-1 using a using a with of by with and to mouse LYVE-1 in and with in and to the of a with and by with mouse LYVE-1 protein in by two further in in by with mouse LYVE-1 protein protein as a to to the of the mice with of a of in as described in S. N. M. L. M. D. K. Cell 1999; PubMed Scopus Google the the mice by and which the surfaces of the and in the liver for and as described from mice homozygous knockout in and in to and by in and by in by in for and with a for to with LYVE-1 for with with for a further and with with tissues in using a and in to with LYVE-1 and with a of and in with and a of 293T these with LYVE-1 in for to in and with immunoelectron of mouse in and with LYVE-1 and with in in and in and in a We identified the LYVE-1 by the EST data for with the amino acid of the Link HA-binding domain of the CD44 Here we the LYVE-1 amino acid to the mouse orthologue by the mouse EST data with the The four and and that a of The which from mouse using contains a large of amino acids residues the with a and with a In the amino acid an hydrophobic to the extracellular domain four of which and are to the conserved disulfide that the link module two and and a likely to hydrophobic of the murine and LYVE-1 an the two of the extracellular the to the HA-binding Link unit are the are only of the in this are as are both the the Link that which is known to HA-binding in CD44, also important for LYVE-1 function. The of mouse and CD44 a of important the HA-binding of the two both within the Link module and in the the residues and in LYVE-1 Link to to the known CD44 HA-binding residues and D. A. J. Cell Biol. 1993; PubMed Scopus Google Scholar, J. B. Day A.J. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) are conserved in mouse LYVE-1 This further the that these amino acids HA in the LYVE-1 link a by results from not of the Link both mouse and LYVE-1 tracts of residues and conserved in a is also conserved in a LYVE-1 orthologue identified in a further of the EST data This functional in of the that a of residues in the domain of CD44 to to HA binding D. A. J. Cell Biol. 1993; PubMed Scopus Google Scholar). We also that an cysteine which is in CD44 is conserved in both mouse and LYVE-1 and This in important as this cysteine is to and to a that an disulfide to LYVE-1 In addition, the of mouse and LYVE-1 both a conserved cysteine and in the as in the CD44 that is in and HA binding D. J. 1996; PubMed Scopus Google Scholar, D. J. Immunol. Google Scholar). Together these the that CD44, a HA-binding domain that the link module J. B. Day A.J. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the of the murine receptor we transfected 293T fibroblasts with in and binding of to the cell by the data in the majority of LYVE-1 but of the cells expression of the receptor in these by with mouse HA binding in we the extracellular domain of murine LYVE-1 as a protein and binding to in The results and the mouse binds both HA and HA in a binding to HA only by hyaluronan and not by the chondroitin chondroitin heparan sulfate results that the murine LYVE-1 receptor a for hyaluronan that is to that of the orthologue but is from the closely CD44 which binds both HA and chondroitin The of LYVE-1 to as a receptor for HA in where 293T cells with and the by from in these by both and of cells with to by the in 293T cells and The of of both cell and a within the of a of the HA by cells within this binding of by a of HA and uptake with 293T cells the of most likely LYVE-1 endocytosis of the by cells in these that to of cell LYVE-1 only of the The of the HA by microscopy which within the membrane these also to LYVE-1 cells with for and microscopy uptake of also in with 293T cells and data not results clear evidence that LYVE-1 both binding and of HA through an for the tissue of mouse LYVE-1 expression we in tissues by by the in to a only in and in not This with the orthologue where the is in of these tissues and is in S. J. S. J. R. M. D.G. J. Cell Biol. 1999; PubMed Scopus Google Scholar). results suggest are in the of LYVE-1 gene expression in LYVE-1 in and In to the LYVE-1 in murine tissues we a by with murine LYVE-1 The specific for the murine receptor as by an and to the LYVE-1 CD44 proteins to the LYVE-1 to the mouse orthologue not The of the LYVE-1 by of 293T cells transfected with LYVE-1 which the receptor in an the cell the binding of LYVE-1 protein to the presence of the domain not of a of mouse tissues large and and and using LYVE-1 of identified as lymphatics the of and the of a The vessels in tissues as the of large that an lymphatic and in by of the lymphatics from blood by with LYVE-1 and the endothelial which two of vessels and Together these results mouse its orthologue is restricted to lymphatic vessel of LYVE-1 in lymphatic but not from mice with to LYVE-1 and the endothelial to with as described are liver and In lymphatic vessels within are by a of In lymphatic vessels are the of large blood vessels HA in knockout mice suggest the that HA receptors in these the that LYVE-1 a role we its expression in tissues from and by means of The results equally of lymphatic vessels in both and mouse tissues with and in the of LYVE-1 in the of lymphatic vessels a of tissues the two mouse CD44 expression in the lymphatics of mice in tissues in the mice as by with the our not the of in LYVE-1 expression in of during the results that LYVE-1 is to compensate for the loss of CD44 expression and that as yet are likely to the that LYVE-1 as a receptor for the uptake of HA from the lymph as a receptor for HA in the tissues the lymphatics, we immunoelectron microscopy to LYVE-1 is to the luminal the of lymphatic endothelial Intriguingly, the expression of LYVE-1 both with and both of lymphatic endothelial cells as cells that of membrane and The of LYVE-1 in further the that the receptor is in transport of HA the vessel In this we described the and functional of the mouse lymphatic endothelial HA and that it both binding and of HA from the In addition, we that mouse to its is endothelial cells in lymphatic vessels and in tissues and in a lymphatic endothelial and using that the receptor is present both the luminal and abluminal endothelial We the of the HA receptors CD44 and LYVE-1 from and the of key conserved structural that in with CD44, contains an HA-binding domain that is to we evidence that the of LYVE-1 expression is not in homozygous knockout that as yet HA receptors are present within the In a S. 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Full Text Full Text PDF PubMed Scopus Google Scholar). to the LYVE-1 and are in The to a role for LYVE-1 in lymphatic HA The majority of HA turnover in tissues as the skin and is known to in draining lymph nodes. This is and that of the HA afferent lymphatics is degraded during through the nodes, the remaining rapidly by the liver and to the of (1Laurent T.C. Fraser J.R. FASEB J. 1992; 6: 2397-2404Crossref PubMed Scopus (2086) Google Scholar, J.R. Kimpton W.G. Laurent T.C. Cahill R.N. Vakakis N. Biochem. J. 1988; 256: 153-158Crossref PubMed Scopus (161) Google Scholar, 4Fraser J.R. Laurent T.C. CIBA Found. Symp. 1989; 143: 41-53PubMed Google Scholar). evidence to the HA receptor receptor for B. Weigel J.A. L. Weigel P.H. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) LYVE-1 as the receptor for in lymph to a endothelial HA receptor J. McGary C.T. Weigel P.H. J. Cell. Biochem. 1992; 48: 73-80Crossref PubMed Scopus (17) Google Scholar, B. Oka J.A. Singh A. 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