Exciting discoveries in many diverse fields of hyaluronan (HA) biology over the last 40 years have centered around the ability of HA to bind cell surface HA receptors (e.g., CD44, Layilin, LYVE-1, ...HARE/Stab2 and RHAMM) and sometimes also to activate intracellular signal transduction pathways, frequently involving ERK1/2. Although perplexing, a major characteristic of HA-mediated signal pathway activation for some receptors has been a dependence on the size of the bound HA. Receptors that directly interact with HA, which may not include TLR2/4, bind very well to any HA molecule >8-20 sugars, depending on the receptor. Despite their ability to bind virtually any size HA, only HA chains of a particular mass range can activate receptor-mediated cell signaling. Many studies have demonstrated parts of this emerging story by utilizing different: HA receptors, cell types, animal models, HA sources, HA sizes, assays to assess HA mass and varying controls to verify HA specificity or HA size-dependence. Recent reports have highlighted issues with potential endotoxin contamination of HA fragments, especially those generated by hyaluronidase digestion. Also, researchers unfamiliar with HA polydispersity must adjust to working with, and interpreting data for, preparations without a unique molecular mass (molecular weight). The confusion, uncertainty and skepticism generated by these and other factors has hindered the development of a general consensus about HA-specific and HA-size dependent receptor activation. An overview of issues, suggested strategies and validating controls is presented to aid those planning an HA-mediated receptor signaling study or those trying to evaluate the literature.
The hyaluronic acid receptor for endocytosis (HARE; also designated Stabilin-2) mediates systemic clearance of hyaluronan and chondroitin sulfates from the vascular and lymphatic circulations. The ...internalized glycosaminoglycans are degraded in lysosomes, thus completing their normal turnover process. Sinusoidal endothelial cells of human liver, lymph node, and spleen express two HARE isoforms of 315 and 190 kDa. Here we report that the 190- and 315-kDa HARE isoforms, expressed stably either in Flp-In 293 cell lines or as soluble ectodomains, specifically bind heparin (Hep). The Kd for Hep binding to purified 190- and 315-kDa HARE ectodomains was 17.2 ± 4.9 and 23.4 ± 5.3 nm, respectively. Cells expressing HARE readily and specifically internalized 125I-streptavidin-biotin-Hep complexes, which was inhibited >70% by hyperosmolar conditions, confirming that uptake is mediated by the clathrin-coated pit pathway. Internalization of Hep occurred for many hours with an estimated HARE recycling time of ∼12 min. Internalized fluorescent streptavidin-biotin-Hep was present in a typical endocytic vesicular pattern and was delivered to lysosomes. We conclude that HARE in the sinusoidal endothelial cells of lymph nodes and liver likely mediates the efficient systemic clearance of Hep and many different Hep-binding protein complexes from the lymphatic and vascular circulations.
Scavenger receptors perform essential functions, critical to maintaining mammalian physiologic homeostasis by continuously clearing vast numbers of biomolecules from blood, interstitial fluid and ...lymph. Stabilin-2 (Stab2) and the Hyaluronic Acid Receptor for Endocytosis (HARE), a proteolytic isoform of Stab2, are important scavenger receptors responsible for the specific binding and internalization (leading to degradation) of 22 discrete molecules, macromolecular complexes and cell types. One-third of these ligands are glycosaminoglycans (GAGs). Full-length Stab2, but not HARE, mediates efficient phagocytosis of apoptotic cells and bacteria via binding to target surface ligands. HARE, the C-terminal half of Stab2, mediates endocytosis of all the known soluble ligands. HA was the first ligand identified, in 1981, prior to receptor purification or cloning. Seven other GAG ligands were subsequently identified: heparin, dermatan sulfate, chondroitin and chondroitin sulfates A, C, D and E. Synthetic dextran sulfate is also a GAG mimic and ligand. HARE signaling during HA endocytosis was first discovered in 2008, and we now know that activation of HARE/Stab2 signaling is stimulated by receptor-mediated endocytosis or phagocytosis of many, but not all, of its ligands. This review focuses on the HARE-mediated GAG activation of intracellular signaling, particularly the Extracellular Signal-Regulated Kinase 1/2 pathway.
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.
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.
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 (315- and 190-kDa) in sinusoidal endothelial cells of the liver, lymph node, and spleen. Here we characterize the ligand specificity and function of the large stably expressed 315-HARE isoform in Flp-In 293 cell lines. Like human spleen sinusoidal endothelial cells, Flp-In 293 cell lines transfected with a single cDNA encoding the full-length 315-HARE express both the 315-kDa and the proteolytically truncated 190-kDa isoforms in a ratio of ∼3–4:1. The 190-kDa HARE isoform generated from the 315-kDa HARE and the 315-kDa HARE specifically bound 125I-HA. Like the 190-kDa HARE expressed alone (Harris, E. N., Weigel, J. A., and Weigel, P. H. (2004) J. Biol. Chem. 279, 36201–36209), the 190- and 315-kDa HARE isoforms expressed in 315-HARE cell lines were recognized by anti-HARE monoclonal antibodies 30, 154, and 159. All 315-HARE cell lines could endocytose and degrade 125I-HA. Competition studies with live cells indicate that 190-HARE and 315-HARE bind HA with higher apparent affinity (Kd ∼10–20 nm) than chondroitin sulfate (CS) types A, C, D, or E. Only slight competition of HA endocytosis was observed with CS-B (dermatan sulfate) and chondroitin. Direct binding assays with the 315-HARE ectodomain revealed high affinity HA binding, and lower binding affinities for CS-C, CS-D, and CS-E. A majority of each HARE isoform was intracellular, within the endocytic system, suggesting transient surface residency typical of an active endocytic recycling receptor.
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.”
The hasA gene from Streptococcus equisimilis, which encodes the enzyme hyaluronan synthase, has been expressed in Bacillus subtilis, resulting in the production of hyaluronic acid (HA) in the 1-MDa ...range. Artificial operons were assembled and tested, all of which contain the hasA gene along with one or more genes encoding enzymes involved in the synthesis of the UDP-precursor sugars that are required for HA synthesis. It was determined that the production of UDP-glucuronic acid is limiting in B. subtilis and that overexpressing the hasA gene along with the endogenous tuaD gene is sufficient for high-level production of HA. In addition, the B. subtilis-derived material was shown to be secreted and of high quality, comparable to commercially available sources of HA.
PDF
