The hyaluronic acid (HA) receptor for endocytosis (HARE; also designated stabilin-2 and FEEL-2) mediates systemic clearance of glycosaminoglycans from the circulatory and lymphatic systems via coated pit-mediated uptake. HARE is primarily found as two isoforms ( The glycosaminoglycan (GAG)2 hyaluronic acid (HA) is a protein-free polymer of disaccharide units containing glucuronic acid and N-acetylglucosamine (1, 2). HA is involved in many physiological processes (3), such as wound healing, development, and metastasis of some cancers (4 -8). The typical molecular mass of the polysaccharide ranges from just a few thousand Da (tens of sugars) that are thought to be important in cellular signaling (6) to several million Da (tens of thousands of sugars). These larger forms of HA are present throughout the body and are particularly concentrated within the bursa of major joints, such as the knee, where they help to provide shock absorbance in cartilage or lubrication in synovial fluid (9, 10), and the eye, where HA maintains structural integrity of the vitreous humor (11). The adult human body contains ϳ15 g of HA, of which about 5 g are turned over daily (12). Partially degraded HA perfuses from extracellular matrices (ECMs) and enters the lymphatic and vascular circulation systems, where it is catabolized to shorter fragments. This active maintenance of HA turnover must be efficient in order to maintain homeostatic conditions for total body HA.All of the other GAGs, including the chondroitin sulfates (CSs), heparan sulfate (HS), and keratan sulfate, are linked to core proteins (as proteoglycans) that help to form ECMs, such as the basement membranes of tissues, or structural components of organs, such as the vitreous humor. There are over 30 known core proteins that are essential for a diverse array of functions, such as neural development, growth factor signaling, and pathogen recognition (13). These core proteins are found as prevalent components of tissue ECMs or as specialized components needed for the development of microenvironments that interface a specialized tissue cell type with the ECM. Both the proteoglycans and their attached GAG chains may combinatorially interact with ligands and contribute to modulation of the functional aspects of a particular microenvironment (e.g. CS interacting with apolipoprotein E for uptake of ␤-very low density lipoprotein in hippocampal neurons) (14). Although numerous studies have focused on how the inhibition of some CS proteoglycans enhances neural development, especially in injured spinal cord models, there is very little information on how CS and HS are catabolized. The current model is that extracellular chondroitinases, heparinases, and proteases initially break down these GAGs and proteoglycans, and their final digestion can then take place intracellularly at the local tissue * This research was supported by NIGMS, National Institutes of Health, Grant GM69961. The costs of publication of this article were defrayed in part by the payment of page charges. This article ...

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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(⌬Link) construct with greatly decreased (ϳ90%) HA uptake. HA did not induce phosphorylation of JNK or p38. A maximum increase in phospho-ERK1/2 occurred within 30 min at 5 g/ml HA, and the response was dampened at >20 g/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. HA plays important roles in matrix assembly, cell differentiation, migration, morphogenesis, and wound healing (1-3). Elevated levels of HA are associated with various pathologies, such as arthritis, inflammation, and cancer (4 -7). The average adult human contains ϳ15 g of HA, of which ϳ5 g is synthesized and degraded daily in tissues throughout the body (1). Most of the HA and CS types released from tissues during this turnover process are ultimately cleared from the circulation and lymph fluid by the HA Receptor for Endocytosis (HARE) (8, 9), also known as Stabilin-2 (10) or FEEL-2 (11). Human HARE is encoded by the 180-kb STAB2 gene, found on chromosome 12, consisting of 69 exons, and is abundantly expressed in the sinusoidal cells of lymph nodes, liver, and spleen (8 -10, 12, 13). Rat and hHARE in these tissues are present as two isoforms (8, 12, 13), e.g. hHARE isoforms are ϳ190 and ϳ315 kDa. Although HARE in the sinusoidal cells of liver and lymph node has a known endocytic clearance function, it may also have other, not yet described, functions. HARE is also expressed in corneal and lens epithelium, in mesenchymal cells of heart valves, in ependymal cells lining the ventricles in the brain, in epithelial cells covering the renal papillae (14), and in oviduct (15). The functions of HARE in these latter tissues are unknown and might be different from local GAG clearance.We have stably expressed the recombinant 190-kDa (16) and full-length 315-kDa (17) HARE proteins in Flp-In 293 cell lines, using cDNA derived from human lymph node. These Flp-In cell lines have one unique, recombinase-mediated integration site. The full-length 315-k...

Oxidative stress and inflammation are important pathological mechanisms in many neurodegenerative diseases, including age-related macular degeneration (AMD). The Very Low-Density Lipoprotein Receptor knockout mouse (Vldlr−/−) has been identified as a model for AMD and in particular for Retinal Angiomatous Proliferation (RAP). In this study we examined the effect of cerium oxide nanoparticles (nanoceria) that have been shown to have catalytic antioxidant activity, on expression of 88 major cytokines in the retinas of Vldlr−/− mice using a PCR array. A single intravitrial injection of nanoceria at P28 caused inhibition of pro-inflammatory cytokines and pro-angiogenic growth factors including Tslp, Lif, Il-3, Il-7, Vegfa, Fgf1, Fgf2, Fgf7, Egf, Efna 3, Lep, and up-regulation of several cytokines and anti-angiogenic genes in the Vldlr−/−retina within one week. We used the Ingenuity Pathway Analysis software to search for biological functions, pathways, and interrelationships between gene networks. Many of the genes whose activities were affected are involved in cell signaling, cellular development, growth and proliferation, and tissue development. Western blot analysis revealed that nanoceria inhibit the activation of ERK 1/2, JNK, p38 MAP kinase, and Akt. These data suggest that nanoceria may represent a novel therapeutic strategy to treat AMD, RAP, and other neurodegenerative diseases.