Hyaluronan Receptor For Endocytosis Research Articles

Discovery of the Liver Hyaluronan Receptor for Endocytosis (HARE) and Its Progressive Emergence as the Multi-Ligand Scavenger Receptor Stabilin-2.

Since the discovery of a novel liver hyaluronan (HA) clearance receptor in 1981 by Laurent, Fraser and coworkers, 22 different ligands cleared by the renamed receptor (the Hyaluronan Receptor for Endocytosis (HARE); Stabilin-2 (Stab2)) were discovered over 37 years. Ligands fall into three groups: (1) 11 anionic polymers, (2) seven cleaved or modified proteins and (3) four types of cells. Seven synthetic ligands, not found normally in serum or tissues, likely mimic natural molecules cleared by the receptor. In 2002 we purified and cloned HARE, based on HA-binding activity, and two other groups cloned full-length receptor; FEEL-2 and Stab2. Macrophages likely require full-length Stab2 for efficient binding and phagocytosis of bacteria or apoptotic cells, since cell-binding domains are throughout the receptor. In contrast, all 16 known single-molecule binding sites are only within the C-terminal half (190HARE). The HARE isoform is generated by proteolysis, not mRNA splicing. The majority of circulating ligands is cleared by HARE, since sinusoidal endothelial cells of liver, spleen and lymph node express twice as many HARE half-receptors as full-length receptors. Based on their significant binding and functional differences, a modified receptor nomenclature is proposed that designates HARE as the C-terminal half-receptor isoform and Stab2 as the full-length receptor isoform.

Single-Molecule Unbinding Forces between the Polysaccharide Hyaluronan and Its Binding Proteins

The extracellular polysaccharide hyaluronan (HA) is ubiquitous in all vertebrate tissues, where its various functions are encoded in the supramolecular complexes and matrices that it forms with HA-binding proteins (hyaladherins). In tissues, these supramolecular architectures are frequently subjected to mechanical stress, yet how this affects the intermolecular bonding is largely unknown. Here, we used a recently developed single-molecule force spectroscopy platform to analyze and compare the mechanical strength of bonds between HA and a panel of hyaladherins from the Link module superfamily, namely the complex of the proteoglycan aggrecan and cartilage link protein, the proteoglycan versican, the inflammation-associated protein TSG-6, the HA receptor for endocytosis (stabilin-2/HARE), and the HA receptor CD44. We find that the resistance to tensile stress for these hyaladherins correlates with the size of the HA-binding domain. The lowest mean rupture forces are observed for members of the type A subgroup (i.e., with the shortest HA-binding domains; TSG-6 and HARE). In contrast, the mechanical stability of the bond formed by aggrecan in complex with cartilage link protein (two members of the type C subgroup, i.e., with the longest HA-binding domains) and HA is equal or even superior to the high affinity streptavidin⋅biotin bond. Implications for the molecular mechanism of unbinding of HA⋅hyaladherin bonds under force are discussed, which underpin the mechanical properties of HA⋅hyaladherin complexes and HA-rich extracellular matrices.

What is special about 200 kDa hyaluronan that activates hyaluronan receptor signaling?

The polydispersity of hyaluronan (HA) presents challenges for analyzing its solution properties, such as the relationship between mass and particle size. The broad mass range of natural HA (≤50-fold) makes molecular characterization difficult and ambiguous compared to molecules with known molecular weights (e.g., proteins). Biophysical studies show that large >MDa HA behaves like a random coil, whereas very small (e.g., 10 kDa) HA behaves like a rod. However, the mass range for this conformational transition is not easily determined in natural polydisperse HA. Some HA receptors (e.g., CD44 and HARE) initiate signaling responses upon binding HA in the 100-300 kDa range, but not larger MDa HA. Size-dependent responses are studied using nonnatural HA: purified narrow-size range HA [Pandey MS, Baggenstoss BA, Washburn J, Harris EN, Weigel PH. 2013. The hyaluronan receptor for endocytosis (HARE) activates NF-κB-mediated gene expression in response to 40-400 kDa, but not smaller or sarger, hyaluronans. J Biol Chem. 288:14068-14079] and very narrow size range Select-HA made chemo-enzymatically [Jing W, DeAngelis PL. 2004. Synchronized chemoenzymatic synthesis of monodisperse hyaluronan polymers. J Biol Chem. 279:42345-42349]. Here, we used size exclusion chromatography and multiangle light scattering to determine the weight-average molar mass and diameter of ~60 very narrow size preparations from 29 to 1650 kDa. The ratio of HA mass to HA diameter showed a transition in the 150-250 kDa size range (~65 nm). The HA rod-to-coil transition occurs within the size range that specifically activates cell signaling by some receptors. Thus, size-specific signaling could be due to unique external receptor•HA conformation changes that enable transmembrane-mediated activation of cytoplasmic domains. Alternatively and more likely, transition-size HA may enable multiple receptors to bind the same HA, creating new internal signal-competent cytoplasmic domain complexes.

HARE-Mediated Endocytosis of Hyaluronan and Heparin Is Targeted by Different Subsets of Three Endocytic Motifs

The hyaluronan (HA) receptor for endocytosis (HARE) is a multifunctional recycling clearance receptor for 14 different ligands, including HA and heparin (Hep), which bind to discrete nonoverlapping sites. Four different functional endocytic motifs (M) in the cytoplasmic domain (CD) target coated pit mediated uptake: (YSYFRI2485 (M1), FQHF2495 (M2), NPLY2519 (M3), and DPF2534 (M4)). We previously found (Pandey et al. J. Biol. Chem. 283, 21453, 2008) that M1, M2, and M3 mediate endocytosis of HA. Here we assessed the ability of HARE variants with a single-motif deletion or containing only a single motif to endocytose HA or Hep. Single-motif deletion variants lacking M1, M3, or M4 (a different subset than involved in HA uptake) showed decreased Hep endocytosis, although M3 was the most active; the remaining redundant motifs did not compensate for loss of other motifs. Surprisingly, a HARE CD variant with only M3 internalized both HA and Hep, whereas variants with either M2 or M4 alone did not endocytose either ligand. Internalization of HA and Hep by HARE CD mutants was dynamin-dependent and was inhibited by hyperosmolarity, confirming clathrin-mediated endocytosis. The results indicate a complicated relationship among multiple CD motifs that target coated pit uptake and a more fundamental role for motif M3.

Tissue-specific splice variants of HARE/Stabilin-2 are expressed in bone marrow, lymph node, and spleen

The hyaluronan receptor for endocytosis (HARE), or Stabilin-2, is the mammalian endocytic clearance receptor for HA, heparin, advanced glycation end-products, acetylated and oxidized low-density lipoproteins and collagen N-terminal propeptides. This large 2551 amino acid receptor is encoded by a gene that covers over 180kbp on human chromosome 12 and is predicted to be composed of 69 exons. Due to the expression profile of this gene and the number of exons it contains, we hypothesized that splice variants of stab2 are encoded in these tissues. In addition, a correlation between alternative splice variants and cancer progression has been shown in other HA receptors such as RHAMM and CD42. In this study, two methods were utilized in identifying and/or isolating the HARE splice variants. The first method used primer sets to amplify the 190-HARE encoding region that could contain splice junctions; therefore, they were purified from agarose gels and sequenced. Five splice variants were detected in that manner. In the second approach, the entire open reading frame of HARE was amplified. This allowed four splice variants with extensive exon splicing to be isolated. After the splice variants were sequenced, three were cloned into a mammalian expression vector. Next, stable cell lines expressing the variants were created in order to determine stable protein expression. In this study, the splice variants were found to be tissue specific in most cases. This suggests that tissue specific regulatory splicing mechanisms may lead to differences in functionality between the splice variants.

Systemic blockade of the hyaluronan receptor for endocytosis prevents lymph node metastasis of prostate cancer

Tumor progression and metastasis are promoted by the remodeling of organized tissue architecture and engagement of molecular interactions that support tumor cell passage through endothelial barriers. Prostate tumor cells that secrete and turn over excessive quantities of pericellular hyaluronan (HA) exhibit accelerated growth kinetics and spontaneous lymph node metastasis in mice. The HA receptor for endocytosis (HARE) is an endocytic clearance receptor for HA in the liver that is also highly expressed in sinusoidal endothelium of lymph nodes and bone marrow, which are frequent sites of prostate cancer metastasis. In our study, we tested the hypothesis that HARE can act as an endothelial receptor for metastatic tumor cells with pericellular HA. In an orthotopic mouse model of prostate cancer, we delivered a monoclonal antibody against HARE that specifically blocks HA binding and internalization. This treatment fully blocked the formation of metastatic tumors in lymph nodes. No effects on primary tumor growth were observed and the antibody did not induce toxic outcomes in any other tissue. Our results implicate HARE for the first time in potentiation of tumor metastasis and suggest a novel mechanism by which tumor cell-associated HA could promote tissue-specific dissemination. "Published 2012 Wiley Periodicals, Inc. This article is a US Government work, and, as such, is in the public domain in the United States of America."

Perinatal Changes in Mitral and Aortic Valve Structure and Composition

At birth, the mechanical environment of valves changes radically as fetal shunts close and pulmonary and systemic vascular resistances change. Given that valves are reported to be mechanosensitive, we investigated remodeling induced by perinatal changes by examining compositional and structural differences of aortic and mitral valves (AVs, MVs) between 2-day-old and 3rd fetal trimester porcine valves using immunohistochemistry and Movat pentachrome staining. Aortic valve composition changed more with birth than the MV, consistent with a greater change in AV hemodynamics. At 2 days, AV demonstrated a trend of greater versican and elastin (P = 0.055), as well as greater hyaluronan turnover (hyaluronan receptor for endocytosis, P = 0.049) compared with the 3rd-trimester samples. The AVs also demonstrated decreases in proteins related to collagen synthesis and fibrillogenesis with birth, including procollagen I, prolyl 4-hydroxylase, biglycan (all P ≤ 0.005), and decorin (P = 0.059, trend). Both AVs and MVs demonstrated greater delineation between the leaflet layers in 2-day-old compared with 3rd-trimester samples, and AVs demonstrated greater saffron-staining collagen intensity, suggesting more mature collagen in 2-day-old compared with 3rd-trimester samples (each P < 0.05). The proportion of saffron-staining collagen also increased in AV with birth (P < 0.05). The compositional and structural changes that occur with birth, as noted in this study, likely are important to proper neonatal valve function. Furthermore, normal perinatal changes in hemodynamics often do not occur in congenital valve disease; the corresponding perinatal matrix maturation may also be lacking and could contribute to poor function of congenitally malformed valves.

The Cytoplasmic Domain of the Hyaluronan Receptor for Endocytosis (HARE) Contains Multiple Endocytic Motifs Targeting Coated Pit-mediated Internalization

The hyaluronic acid (HA) receptor for endocytosis (HARE) is the primary scavenger receptor for HA and chondroitin sulfates in mammals. The two human isoforms of HARE (full-length 315-kDa and a 190-kDa proteolytic cleavage product), which are type I single-pass membrane proteins, are highly expressed in sinusoidal endothelial cells of lymph nodes, liver, and spleen. Their identical HARE cytoplasmic domains contain four candidate AP-2/clathrin-mediated endocytic signaling motifs as follows: YSYFRI(2485), FQHF(2495), NPLY(2519), and DPF(2534) (315-HARE numbering). Stably transfected cells expressing 190-HARE(DeltaYSYFRI), 190-HARE(DeltaFQHF), or 190-HARE(DeltaNPLY) (lacking Motifs 1, 2, or 3) had decreased (125)I-HA endocytosis rates of approximately 49, approximately 39, and approximately 56%, respectively (relative to wild type). In contrast, 190-HARE(DeltaDPF) cells (lacking Motif 4) showed no change in HA endocytic rate. Deletions of motifs 1 and 2 or of 1, 2, and 4 decreased the rate of HA endocytosis by only approximately 41%. Endocytosis was approximately 95% decreased in mutants lacking all four motifs. Cells expressing a 190-HARE(Y2519A) mutant of the NPLY motif retained 85-90% of wild type endocytosis, whereas this mutation in the triple motif deletant decreased endocytosis to approximately 7% of wild type. Tyr in NPLY(2519) is thus important for endocytosis. All HARE mutants showed similar HA binding and degradation of the internalized HA, indicating that altering endocytic motifs did not affect ectodomain binding of HA or targeting of internalized HA to lysosomes. We conclude that, although NPLY may be the most important motif, it functions together with two other endocytic motifs; thus three signal sequences (YSYFRI, FQHF, and NPLY) provide redundancy to mediate coated pit targeting and endocytosis of HARE.

Abundance and location of proteoglycans and hyaluronan within normal and myxomatous mitral valves

Extracellular matrix changes occur in many heart valve pathologies. For example, myxomatous mitral valves are reported to contain excess proteoglycans and hyaluronan. However, it is unknown which specific proteoglycans are altered in myxomatous valves. Because proteoglycans perform varied functions in connective tissues, this study was designed to identify and localize three matrix-associated proteoglycans, as well as hyaluronan and the hyaluronan receptor for endocytosis, within myxomatous and normal mitral valves. Human mitral posterior leaflets (control, n=6-9; myxomatous, n=14-21; mean age, 61 years for all groups) were histochemically stained for proteoglycan core proteins, hyaluronan, and the hyaluronan receptor for endocytosis. Stain intensity was semiquantitatively graded to determine differences in marker abundance between normal and myxomatous valves. The proteoglycans were localized to different regions of the leaflet by correspondence to parallel Movat-stained sections. The proteoglycans decorin, biglycan, and versican were more abundant in myxomatous valves than in normal controls (P<.03). There was a gender effect on proteoglycan presence, but no age-related trends were observed. Hyaluronan and the hyaluronan receptor for endocytosis were distributed throughout all valves. There was no significant difference in hyaluronan between groups, but expression of the hyaluronan receptor for endocytosis was reduced in myxomatous valves compared to normal controls (P<.002). Excess decorin, biglycan, and versican may be associated with the remodeling of other matrix components in myxomatous mitral valves. Decreased expression of the hyaluronan receptor for endocytosis in myxomatous valves suggests that hyaluronan metabolism could be altered in myxomatous mitral valve disease. These findings contribute towards elucidating the pathogenesis of myxomatous mitral valve disease and developing potential new therapies.

The ligand-binding profile of HARE: hyaluronan and chondroitin sulfates A, C, and D bind to overlapping sites distinct from the sites for heparin, acetylated low-density lipoprotein, dermatan sulfate, and CS-E

The hyaluronic acid receptor for endocytosis (HARE)/ Stabilin-2 is the primary systemic scavenger receptor for hyaluronan (HA), the chondroitin sulfates (CS), dermatan sulfate (DS), and nonglycosaminoglycan (GAG) ligands such as acetylated low-density lipoprotein (AcLDL), pro-collagen propeptides, and advanced glycation end products. We recently discovered that HARE is also a systemic scavenger receptor for heparin (Hep) (Harris EN, Weigel JA, Weigel PH. 2008. The human hyaluronan receptor for endocytosis [HARE/Stabilin-2] is a systemic clearance receptor for heparin. J Biol Chem. 283:17341-17350). Our goal was to map the binding sites of eight different ligands within HARE. We used biotinylated GAGs and radio-iodinated streptavidin or AcLDL to assess the binding activities of ligands directly or indirectly (by competition with unlabeled ligands) in endocytosis assays using stable cell lines expressing the 315 or 190 kDa HA receptor for endocytosis (315- or 190-HARE) isoforms, and ELISA-like assays, with purified recombinant soluble 190-HARE ecto-domain. For example, Hep binding to HARE was competed by DS, CS-E, AcLDL, and dextran sulfate, but not by other CS types, HA, dextran, or heparosan. (125)I-AcLDL binding to HARE was partially competed by Hep and dextran sulfate, but not competed by HA. Two ligands, DS and CS-E, competed with both Hep and HA to some degree. Hep and HA binding or endocytosis is mutually inclusive; binding of these two GAGs occurs with functionally separate, noncompetitive, and apparently noninteracting domains. Thus, HARE binds to HA and Hep simultaneously. Although the domain(s) responsible for Hep binding remains unknown, the Link domain was required for HARE binding to HA, CS-A, CS-C, and CS-D. These results enable us to outline, for the first time, a binding activity map for multiple ligands of HARE.

The Hyaluronan Receptor for Endocytosis Mediates Hyaluronan-dependent Signal Transduction via Extracellular Signal-regulated Kinases

The hyaluronan (HA) receptor for endocytosis (HARE) mediates the endocytotic clearance of HA and other glycosaminoglycans from lymph and blood. Two isoforms of human HARE, 315- and 190-kDa, are highly expressed in sinusoidal endothelial cells of liver, lymph node, and spleen; HARE is also in specialized cells in the eye, heart, brain, and kidney. Here we determined whether HA binding to HARE initiates intracellular signaling in Flp-In 293 cells stably expressing either the 315- and 190-kDa HARE or the 190-kDa HARE alone. HARE was co-immunoprecipitated with extracellular signal-regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal protein kinase (JNK), and p38 members of the mitogen-activated protein kinase signaling cascade. ERK phosphorylation increased in a dose- and time-dependent manner when HA was added to cells expressing full-length or 190-kDa HARE, but not cells with vector-only or a HARE(DeltaLink) construct with greatly decreased ( approximately 90%) HA uptake. HA did not induce phosphorylation of JNK or p38. A maximum increase in phospho-ERK1/2 occurred within 30 min at 5 mug/ml HA, and the response was dampened at >20 mug/ml HA. HA binding did not increase the level of HARE-ERK complexes, but did increase HARE phosphorylation. These findings demonstrate a novel functional response, when HARE binds HA, that leads to activation of ERK1/2, important mediators of intracellular signal transduction.

Characterization of the Recombinant Rat 175-kDa Hyaluronan Receptor for Endocytosis (HARE)

Hyaluronan (HA) and chondroitin sulfate (CS) clearance from lymph and blood in mammals is mediated by the HA receptor for endocytosis (HARE), which is present as two isoforms in rat and human (175/300 kDa and 190/315 kDa, respectively) in the sinusoidal endothelial cells of liver, spleen, and lymph nodes (Zhou, B., McGary, C. T., Weigel, J. A., Saxena, A., and Weigel, P. H. (2003) Glycobiology 13, 339-349). The small rat and human HARE proteins are not encoded directly by mRNA but are derived from larger precursors. Here we characterize the specificity and function of the 175-kDa HARE, expressed in the absence of the 300-kDa species, in stably transfected SK-Hep-1 cells. The HARE cDNA was fused with a leader sequence to allow correct orientation of the membrane protein. The recombinant rHARE contained approximately 25 kDa of N-linked oligosaccharides and, like the native protein, was able to bind HA in a ligand blot assay, even after de-N-glycosylation. SK-HARE cell lines demonstrated specific 125I-HA endocytosis, receptor recycling, and delivery of HA to lysosomes for degradation. The Kd for the binding of HA (number-average molecular mass approximately 133 kDa) to the 175-kDa HARE at 4 degrees C was 4.1 nm with 160,000 to 220,000 HA-binding sites per cell. The 175-kDa rHARE binds HA, dermatan sulfate, and chondroitin sulfates A, C, D, and E, but not chondroitin, heparin, heparan sulfate, or keratan sulfate. Surprisingly, recognition of glycosaminoglycans (GAGs) other than HA by native or recombinant HARE was temperature-dependent. Although competition was observed at 37 degrees C, none of the other GAGs competed for 125I-HA binding to SK-HARE cells at 4 degrees C. Anti-HARE monoclonal antibody-174 showed a similar temperature-dependence in its ability to block HA endocytosis. These data suggest that temperature-induced conformational changes may alter the GAG specificity of HARE. The results confirm that the 175-kDa rHARE does not require the larger HARE isoform to mediate endocytosis of multiple GAGs.

A Blocking Antibody to the Hyaluronan Receptor for Endocytosis (HARE) Inhibits Hyaluronan Clearance by Perfused Liver

Hyaluronan (HA) and chondroitin sulfate clearance from lymph and blood is mediated by the hyaluronan receptor for endocytosis (HARE). The purification and molecular cloning (Zhou, B., Weigel, J. A., Saxena, A., and Weigel, P. H. (2002) Mol. Biol. Cell 13, 2853-2868) of this cell surface receptor were finally achieved after we developed monoclonal antibodies (mAbs) against HARE. There are actually two independent isoreceptors for HA, which in rat are designated the 175-kDa HARE and 300-kDa HARE. Only one mAb (number 174) effectively and completely blocked the specific uptake of 125I-HA at 37 degrees C by rat liver sinusoidal endothelial cells. 125I-HA binding to both the 175-kDa and 300-kDa HARE proteins in a ligand blot assay was almost completely inhibited by <1 microg/ml mAb-174, whereas mouse IgG had little or no effect. MAb-174 also performed very well in Western analysis, indirect fluorescence microscopy, and a variety of immuno-procedures. Immunohistochemistry using mAb-174 localized HARE to the sinusoidal cells of rat liver, spleen, and lymph node. Western analysis using mAb-174 revealed that the sizes of both HARE glycoproteins were the same in these three tissues. 125I-HA was taken up and degraded by excised rat livers that were continuously perfused ex vivo with a recirculating medium. This HA clearance and metabolism by liver, which is a physiological function of HARE, was very effectively blocked by mAb-174 but not by mouse IgG. The results indicate that mAb-174 will be a useful tool to study the functions of HARE and the physiological significance of HA clearance.

Purification and molecular identification of the human hyaluronan receptor for endocytosis.

The clearance of hyaluronan (HA) and chondroitin sulfates from the circulating blood and lymph in the body is mediated by the membrane-bound HA receptor for endocytosis (HARE). Previously, we found that two HARE species of approximately 175 kDa and approximately 300 kDa are abundant in the sinusoidal endothelial cells in rat liver, spleen, and lymph nodes (Zhou et al. [2000], J. Biol. Chem., 275, 37733-37741). In the present study, immunocytochemical analysis of human tissues showed a similar pattern with abundant expression of HARE in the sinusoidal endothelial cells of human liver, spleen, and lymph nodes. The two human HARE proteins were immunoaffinity-purified from human spleen. Each protein was recognized in western blots using several anti-rat HARE monoclonal antibodies and was able to bind 125I-HA specifically. In nonreducing SDS-PAGE, these two human HARE species migrated at approximately 190 kDa and approximately 315 kDa; both proteins are approximately 15 kDa larger than the corresponding rat HAREs, although the de-N-glycosylated core proteins are essentially the same mass. After reduction, the human 190-kDa HARE gave a single 196-kDa species, which was not seen in the approximately 315-kDa HARE after reduction. The reduced approximately 315-kDa HARE yielded two major proteins at approximately 250 kDa and approximately 220 kDa. We determined the sequence of the human 190-kDa HARE cDNA based on analysis of internal tryptic peptides, as well as RT-PCR and 5' RACE analyses using human spleen and lymph node cDNA libraries. The human gene that encodes HARE is on chromosome 12.

Molecular cloning and functional expression of the rat 175-kDa hyaluronan receptor for endocytosis.

We recently purified the rat liver hyaluronan receptor for endocytosis (HARE) and found abundant expression of 175- and approximately 300-kDa HARE species in sinusoidal endothelial cells of the liver, spleen, and lymph nodes. We report herein the first cloning and functional expression of the rat 175-kDa HARE. Peptide sequences were obtained from the purified 175-kDa HARE, and degenerate oligonucleotide primers were designed for reverse transcription-polymerase chain reaction and cDNA cloning. Results of 5'-rapid amplification of cDNA ends, Northern analysis, N-terminal sequence, and antibody reactivity analyses indicated the absence of mRNA directly encoding the 175-kDa HARE. This protein is most likely derived from a larger precursor. Accordingly, we constructed an artificial 4.7-kb cDNA encoding the 1431 amino acid 175-kDa HARE. The predicted type I membrane protein has a mass of 156,393 Da and a pI of 7.86. The 175-kDa HARE cDNA, fused to the N-terminal leader sequence of the Ig kappa-chain, was transfected transiently into COS-7 cells and stably into SK-Hep-1 cells, respectively, to assess hyaluronan or hyaluronic acid (HA)-binding activity and endocytosis. In both cases, HARE expression and HA-binding activity were detected. Furthermore, stable SK-175HARE cells demonstrated specific endocytosis of (125)I-HA and receptor recycling. Fluorescence-activated cell sorting analysis confirmed that recombinant HARE was expressed on the cell surface and that fluorescent HA uptake was inhibited by a specific blocking monoclonal antibody against HARE. Additionally, HARE was substantially colocalized with clathrin, but not with internalized HA that was delivered to lysosomes. The results confirm that recombinant 175-kDa HARE is an authentic endocytic receptor for HA and that this receptor can function independently of the approximately 300-kDa HARE. HARE is the first functionally identified member of a protein family that shares a similar organization of Fasciclin, epidermal growth factor-like, Xlink, and transmembrane domains.

The hyaluronan receptor for endocytosis (HARE) is not CD44 or CD54 (ICAM-1)

Mammalian liver contains an endocytic, recycling receptor that mediates the clearance of hyaluronan (HA) and chondroitin sulfate from the circulation. McCourt et al. [J. Biol. Chem. 269 (1994) 30081] previously reported that this endocytic liver HA receptor was ICAM-1. In contrast, we purified this HA receptor for endocytosis (HARE) from rat liver sinusoidal endothelial cells (LECs) and obtained two novel large proteins [Zhou et al., J. Biol. Chem. 274 (1999) 33831]. The goal of the present study was to clarify this inconsistency and determine whether CD44, which is also an HA receptor, or ICAM-1 (CD54) is identical to, or is part of, HARE. Although isolated liver LECs contain HARE, CD44, and ICAM-1, confocal fluorescence microscopy showed that the two latter proteins have cellular distributions that are distinct from and essentially nonoverlapping with HARE. HA accumulation by cultured LECs was inhibited >98% by an antibody against HARE and unaffected by antibodies to ICAM-1 or CD44, indicating that virtually all specific HA uptake is mediated by HARE and not by ICAM-1 or CD44. Finally, no reactivity was observed against purified HARE in an ELISA-based assay using CD44 or ICAM-1 antibodies. The results confirm that the mammalian endocytic HA receptor is HARE and is not ICAM-1 or CD44.

Endothelium in hepatic cavernous hemangiomas does not express the hyaluronan receptor for endocytosis

The liver contains two distinct endothelial cell types: vascular and sinusoidal. Although cavernous hemangioma is the most common benign tumor of the liver, vascular or sinusoidal endothelial cell differentiation has not been described. An endocytic receptor responsible for the uptake and degradation of hyaluronan is present in the sinusoidal endothelium of the liver. The hyaluronan receptor for endocytosis (HARE) may therefore be a useful marker for sinusoidal endothelial cell differentiation. Using monoclonal antibodies specific for HARE, CD31, and factor VIII, we completed an immunohistochemical study of the endothelial cells of both hepatic cavernous hemangiomas and of nonneoplastic human liver. The anti-HARE monoclonal antibodies showed diffuse strong staining of nonneoplastic liver sinusoidal endothelium. No staining of nonsinusoidal endothelium or the endothelial lining of the hemangiomas was seen with anti-HARE. In contrast, diffuse strong staining for factor VIII and CD31 was present in nonsinusoidal endothelium and cavernous hemangioma endothelium. Neither factor VIII nor CD31 staining was present in the sinusoidal endothelium. In conclusion, the endothelium of hepatic cavernous hemangiomas demonstrates vascular but not sinusoidal differentiation based on the absence of HARE and presence of CD31 and factor VIII. HUM PATHOL 33:265-269. Copyright 2002, Elsevier Science (USA). All rights reserved.

Identification of the Hyaluronan Receptor for Endocytosis (HARE)

Rat liver sinusoidal endothelial cells (LECs) express two hyaluronan (HA) receptors, of 175 and 300 kDa, responsible for the endocytic clearance of HA. We have characterized eight monoclonal antibodies (mAbs) raised against the 175-kDa HA receptor partially purified from rat LECs. These mAbs also cross-react with the 300-kDa HA receptor. The 175-kDa HA receptor is a single protein, whereas the 300-kDa species contains three subunits, alpha, beta, and gamma at 260, 230, and 97 kDa, respectively (Zhou, B., Oka, J. A., and Weigel, P. H. (1999) J. Biol. Chem. 274, 33831-33834). The 97-kDa subunit was not recognized by any of the mAbs in Western blots. Based on their cross-reactivity with these mAbs, the 175-, 230-, and 260-kDa proteins appear to be related. Two of the mAbs inhibit (125)I-HA binding and endocytosis by LECs at 37 degrees C. All of these results confirm that the mAbs recognize the bone fide LEC HA receptor. Indirect immunofluoresence shows high protein expression in liver sinusoids, the venous sinuses of the red pulp in spleen, and the medullary sinuses of lymph nodes. Because the tissue distribution for this endocytic HA receptor is not unique to liver, we propose the name HARE (HA receptor for endocytosis).