Systemic Glycosaminoglycan Clearance by HARE/Stabilin-2 Activates Intracellular Signaling.

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.

Self-regenerating giant hyaluronan polymer brushes.

Tailoring interfaces with polymer brushes is a commonly used strategy to create functional materials for numerous applications. Existing methods are limited in brush thickness, the ability to generate high-density brushes of biopolymers, and the potential for regeneration. Here we introduce a scheme to synthesize ultra-thick regenerating hyaluronan polymer brushes using hyaluronan synthase. The platform provides a dynamic interface with tunable brush heights that extend up to 20 microns - two orders of magnitude thicker than standard brushes. The brushes are easily sculpted into micropatterned landscapes by photo-deactivation of the enzyme. Further, they provide a continuous source of megadalton hyaluronan or they can be covalently-stabilized to the surface. Stabilized brushes exhibit superb resistance to biofilms, yet are locally digested by fibroblasts. This brush technology provides opportunities in a range of arenas including regenerating tailorable biointerfaces for implants, wound healing or lubrication as well as fundamental studies of the glycocalyx and polymer physics.

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.

A TLR/AKT/FoxO3 immune tolerance-like pathway disrupts the repair capacity of oligodendrocyte progenitors.

Cerebral white matter injury (WMI) persistently disrupts myelin regeneration by oligodendrocyte progenitor cells (OPCs). We identified a specific bioactive hyaluronan fragment (bHAf) that downregulates myelin gene expression and chronically blocks OPC maturation and myelination via a tolerance-like mechanism that dysregulates pro-myelination signaling via AKT. Desensitization of AKT occurs via TLR4 but not TLR2 or CD44. OPC differentiation was selectively blocked by bHAf in a maturation-dependent fashion at the late OPC (preOL) stage by a noncanonical TLR4/TRIF pathway that induced persistent activation of the FoxO3 transcription factor downstream of AKT. Activated FoxO3 selectively localized to oligodendrocyte lineage cells in white matter lesions from human preterm neonates and adults with multiple sclerosis. FoxO3 constraint of OPC maturation was bHAf dependent, and involved interactions at the FoxO3 and MBP promoters with the chromatin remodeling factor Brg1 and the transcription factor Olig2, which regulate OPC differentiation. WMI has adapted an immune tolerance-like mechanism whereby persistent engagement of TLR4 by bHAf promotes an OPC niche at the expense of myelination by engaging a FoxO3 signaling pathway that chronically constrains OPC differentiation.

Planning, evaluating and vetting receptor signaling studies to assess hyaluronan size-dependence and specificity.

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.

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.

Hyaluronan synthase assembles hyaluronan on a [GlcNAc(ß1,4)]n-GlcNAc(α1→)UDP primer and hyaluronan retains this residual chitin oligomer as a cap at the nonreducing end.

Class I hyaluronan synthases (HAS) assemble [GlcNAc(ß1,4)GlcUA(ß1,3)]n-UDP at the reducing end and also make chitin. Streptococcus equisimilis HAS (SeHAS) also synthesizes chitin-UDP oligosaccharides, (GlcNAc-ß1,4)n-GlcNAc(α1→)UDP (Weigel et al. 2015). Here we determined if HAS uses chitin-UDPs as primers to initiate HA synthesis, leaving the non-HA primer at the nonreducing (NR) end. HA made by SeHAS membranes was purified, digested with streptomyces lyase, and hydrophobic oligomers were enriched by solid phase extraction and analyzed by MALDI-TOF MS. Jack bean hexosaminidase (JBH) and MS/MS were used to analyze 19 m/z species of possible GnHn ions with clustered GlcNAc (G) residues attached to disaccharide units (H): (GlcNAcß1,4)2-5[GlcUA(ß1,3)GlcNAc]2-6. JBH digestion sequentially removed GlcNAc from the NR-end of GnHn oligomers, producing successively smaller GnH2-3 series members. Since lyase releases dehydro-oligos (dHn; M-18), only the unique NR-end oligo lacks dehydro-GlcUA. Hn oligomers were undetectable in lyase digests, whereas JBH treatment created new H2-6m/z peaks (i.e. HA tetra- through dodeca-oligomers). MS/MS of larger GnHn species produced chitin (2-5 GlcNAcs), HA oligomers and multiple smaller series members with fewer GlcNAcs. All NR-ends (97%) started with GlcNAc, as a chitin trimer (three GlcNAcs), indicating that GlcNAc(ß1,4)2GlcNAc(α1→)-UDP may be optimal for initiation of HA synthesis. Also, HA made by live S. pyogenes cells had G4Hn chitin-oligo NR-ends. We conclude that chitin-UDP functions in vitro and in live cells as a primer to initiate synthesis of all HA chains and these primers remain at the NR-ends of HA chains as residual chitin caps [(GlcNAc-ß1,4)3-4].

Hyaluronan synthase control of synthesis rate and hyaluronan product size are independent functions differentially affected by mutations in a conserved tandem B-X7-B motif.

Hyaluronan synthases (HAS) normally make large (>MDa) hyaluronan (HA) products. Smaller HA fragments (e.g. 100-400 kDa) produced in vivo are associated with inflammation and cell signaling by HA receptors that bind small, but not large, HA. Although HA fragments can arise from breakdown by hyaluronidases, HAS might also be regulated directly to synthesize small HA. Here we examined the Streptococcus equisimilis HAS (SeHAS) C-terminus, which contains a tandem B-X7-B motif (K398-X7-R406-X7-K414), by testing the effects of 27 site-specific scanning mutations and 7 C-terminal truncations on HA synthesis activity and weight-average mass. Although HAS enzymes cannot be HA-binding proteins, these motifs are highly conserved within the Class I HAS family. Fifteen Arg406 mutants made large MDa HA (86-110% wildtype size), with specific activities from 70% to 177% of wildtype. In contrast, 10 of 12 Lys398 mutants made HA that was 8-14% of wildtype size (≤250-480 kDa), with specific activities from 14% to 64% of wildtype. Four nearly inactive (2% wildtype activity) C-terminal truncation mutants made MDa HA (56-71% wildtype). The results confirm earlier findings with Cys-mutants [Weigel PH, Baggenstoss BA. 2012. Hyaluronan synthase polymerizing activity and control of product size are discrete enzyme functions that can be uncoupled by mutagenesis of conserved cysteines. Glycobiology 22:1302-1310] that HAS uses two independent activities to control HA size and HA synthesis rate; these are two separate functions. We conclude that HAS regulatory modifications that alter tandem B-X7-B motif conformation could mimic these mutagenesis-induced effects, allowing HAS in vivo to make small HA directly. The results also support a model in which the tandem-motif region is part of the intra-HAS pore and interacts directly with HA.

Activation of ERK and NF-κB during HARE-Mediated Heparin Uptake Require Only One of the Four Endocytic Motifs.

Fifteen different ligands, including heparin (Hep), are cleared from lymph and blood by the Hyaluronan (HA) Receptor for Endocytosis (HARE; derived from Stabilin-2 by proteolysis), which contains four endocytic motifs (M1-M4). Endocytosis of HARE•Hep complexes is targeted to coated pits by M1, M2, and M3 (Pandey et al, Int. J. Cell Biol. 2015, article ID 524707), which activates ERK1/2 and NF-κB (Pandey et al J. Biol. Chem. 288, 14068-79, 2013). Here, we used a NF-κB promoter-driven luciferase gene assay and cell lines expressing different HARE cytoplasmic domain mutants to identify motifs needed for Hep-mediated signaling. Deletion of M1, M2 or M4 singly had no effect on Hep-mediated ERK1/2 activation, whereas signaling (but not uptake) was eliminated in HARE(ΔM3) cells lacking NPLY2519. ERK1/2 signaling in cells expressing WT HARE(Y2519A) or HARE(Y2519A) lacking M1, M2 and M4 (containing M3-only) was decreased by 75% or eliminated, respectively. Deletion of M3 (but not M1, M2 or M4) also inhibited the formation of HARE•Hep•ERK1/2 complexes by 67%. NF-κB activation by HARE-mediated uptake of Hep, HA, dermatan sulfate or acetylated LDL was unaffected in single-motif deletion mutants lacking M1, M2 or M4. In contrast, cells expressing HARE(ΔM3) showed loss of HARE-mediated NF-κB activation during uptake of each of these four ligands. NF-κB activation by the four signaling ligands was also eliminated in HARE(Y2519A) or HARE(M3-only;Y2519A) cells. We conclude that the HARE NPLY2519 motif is necessary for both ERK1/2 and NF-κB signaling and that Tyr2519 is critical for these functions.

Hyaluronan Synthase: The Mechanism of Initiation at the Reducing End and a Pendulum Model for Polysaccharide Translocation to the Cell Exterior.

Hyaluronan (HA) biosynthesis has been studied for over six decades, but our understanding of the biochemical details of how HA synthase (HAS) assembles HA is still incomplete. Class I family members include mammalian and streptococcal HASs, the focus of this review, which add new intracellular sugar-UDPs at the reducing end of growing hyaluronyl-UDP chains. HA-producing cells typically create extracellular HA coats (capsules) and also secrete HA into the surrounding space. Since HAS contains multiple transmembrane domains and is lipid-dependent, we proposed in 1999 that it creates an intraprotein HAS-lipid pore through which a growing HA-UDP chain is translocated continuously across the cell membrane to the exterior. We review here the evidence for a synthase pore-mediated polysaccharide translocation process and describe a possible mechanism (the Pendulum Model) and potential energy sources to drive this ATP-independent process. HA synthases also synthesize chitin oligosaccharides, which are created by cleavage of novel oligo-chitosyl-UDP products. The synthesis of chitin-UDP oligomers by HAS confirms the reducing end mechanism for sugar addition during HA assembly by streptococcal and mammalian Class I enzymes. These new findings indicate the possibility that HA biosynthesis is initiated by the ability of HAS to use chitin-UDP oligomers as self-primers.

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: (YSYFRI(2485) (M1), FQHF(2495) (M2), NPLY(2519) (M3), and DPF(2534) (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.

Hyaluronan synthase assembles chitin oligomers with -GlcNAc(α1→)UDP at the reducing end.

Class I hyaluronan synthases (HASs) assemble a polysaccharide containing the repeating disaccharide [GlcNAc(ß1,4)GlcUA(ß1,3)]n-UDP and vertebrate HASs also assemble (GlcNAc-ß1,4)n homo-oligomers (chitin) in the absence of GlcUA-UDP. This multi-membrane domain CAZy GT2 family glycosyltransferase, which couples HA synthesis and translocation across the cell membrane, is atypical in that monosaccharides are incrementally assembled at the reducing, rather than the non-reducing, end of the growing polymer. Using Escherichia coli membranes containing recombinant Streptococcus equisimilis HAS, we demonstrate that a prokaryotic Class I HAS also synthesizes chitin oligomers (up to 15-mers, based on MS and MS/MS analyses of permethylated products). Furthermore, chitin oligomers were found attached at their reducing end to -4GlcNAc(α1→)UDP [i.e. (GlcNAcß1,4)nGlcNAc(α1→)UDP]. These oligomers, which contained up to at least seven HexNAc residues, consisted of ß4-linked GlcNAc residues, based on the sensitivity of the native products to jack bean ß-N-acetylhexosaminidase. Interestingly, these oligomers exhibited mass defects of -2, or -4 for longer oligomers, that strictly depended on conjugation to UDP, but MS/MS analyses indicate that these species result from chemical dehydrogenations occurring in the gas phase. Identification of (GlcNAc-ß1,4)n-GlcNAc(α1→)UDP as HAS reaction products, made in the presence of GlcNAc(α1→)UDP only, provides strong independent confirmation for the reducing terminal addition mechanism. We conclude that chitin oligomer products made by HAS are derived from the cleavage of these novel activated oligo-chitosyl-UDP oligomers. Furthermore, it is possible that these UDP-activated chitin oligomers could serve as self-assembled primers for initiating HA synthesis and ultimately modify the non-reducing terminus of HA with a chitin cap.

A hyaluronan receptor for endocytosis (HARE) link domain N-glycan is required for extracellular signal-regulated kinase (ERK) and nuclear factor-κB (NF-κB) signaling in response to the uptake of hyaluronan but not heparin, dermatan sulfate, or acetylated low density lipoprotein (LDL).

The human hyaluronan (HA) receptor for endocytosis (HARE; the 190-kDa C terminus of Stab2) is a major clearance receptor for multiple circulating ligands including HA, heparin (Hep), acetylated LDL (AcLDL), dermatan sulfate (DS), apoptotic debris, and chondroitin sulfate types A, C, D, and E. We previously found that HARE contains an N-glycan in the HA binding Link domain (at Asn(2280)), and cells expressing membrane-bound HARE(N2280A) bind and endocytose HA normally (Harris, E. N., Parry, S., Sutton-Smith, M., Pandey, M. S., Panico, M., Morris, H. R., Haslam, S. M., Dell, A., and Weigel, P. H. (2010) Glycobiology 20, 991-1001). Also, NF-κB-mediated signaling is activated by HARE-mediated endocytosis of HA, Hep, AcLDL, or DS but not by chondroitin sulfates (Pandey, M. S., and Weigel, P. H. (2014) J. Biol. Chem. 289, 1756-1767). Here we investigated the role of Link N-glycans in ligand uptake and NF-κB and ERK1/2 signaling. HA·HARE-mediated ERK1/2 activation was HA size- dependent, as found for NF-κB activation. HARE(N2280A) cells internalized HA, Hep, AcLDL, and DS normally. No ERK1/2 activation occurred during HA endocytosis by HARE(N2280A) cells, but activation did occur with Hep. Dual-luciferase recorder assays showed that NF-κB-mediated gene expression occurred normally in HARE(N2280A) cells endocytosing Hep, AcLDL, or DS but did not occur with HA. Activation of NF-κB by endogenous degradation of IκB-α was observed for HARE(N2280A) cells endocytosing Hep, AcLDL, or DS but not HA. We conclude that a Link domain complex N-glycan is required specifically for HARE·HA-mediated activation of ERK1/2 and NF-κB-mediated gene expression and that this initial activation mechanism is different from and independent of the initial mechanisms for HARE-mediated signaling in response to Hep, AcLDL, or DS uptake.

Hyaluronic acid receptor for endocytosis (HARE)-mediated endocytosis of hyaluronan, heparin, dermatan sulfate, and acetylated low density lipoprotein (AcLDL), but not chondroitin sulfate types A, C, D, or E, activates NF-κB-regulated gene expression.

The hyaluronan (HA) receptor for endocytosis (HARE; Stab2) clears 14 systemic ligands, including HA and heparin. Here, we used NF-κB promoter-driven luciferase reporter assays to test HARE-mediated intracellular signaling during the uptake of eight ligands, whose binding sites in the HARE ectodomain were mapped by competition studies (Harris, E. N., and Weigel, P. H. (2008) Glycobiology 18, 638-648). Unique intermediate size Select-HA(TM), heparin, dermatan sulfate, and acetylated LDL stimulated dose-dependent HARE-mediated NF-κB activation of luciferase expression, with half-maximal values of 10-25 nM. In contrast, chondroitin sulfate types A, C, D, and E did not stimulate NF-κB activation. Moreover, degradation of endogenous IkB-α (an NF-κB inhibitor) was stimulated only by the signaling ligands. The stimulatory activities of pairwise combinations of the four signaling ligands were additive. The four nonstimulatory chondroitin sulfate types, which compete for HA binding, also effectively blocked HA-stimulated signaling. Clathrin siRNA decreased clathrin expression by ∼50% and completely eliminated NF-κB-mediated signaling by all four ligands, indicating that activation of signaling complexes occurs after endocytosis. These results indicate that HARE not only binds and clears extracellular matrix degradation products (e.g. released normally or during infection, injury, tumorigenesis, or other stress situations) but that a subset of ligands also serves as signaling indicator ligands. HARE may be part of a systemic tissue-stress sensor feedback system that responds to abnormal tissue turnover or damage as a danger signal; the signaling indicator ligands would reflect the homeostatic status, whether normal or pathological, of tissue cells and biomatrix components.

Methods for measuring Class I membrane-bound hyaluronan synthase activity.

Detecting and quantifying hyaluronan (HA) made by Class I HA synthase (HAS) and determining the level of activity of these membrane-bound enzymes is critical in studies to understand the normal biology of HA and how changes in HAS activity and HA levels or size are important in inflammatory and other diseases, tumorigenesis, and metastasis. Unlike the products made by the vast majority of glycosyltransferases, HA products are more complicated since they are made as a heterogeneous population of sizes spanning a broad mass range. Three radioactive and nonradioactive assay methods are described that can give the amount of HA made with or without information about the distribution of product sizes.

The hyaluronan receptor for endocytosis (HARE) activates NF-κB-mediated gene expression in response to 40-400-kDa, but not smaller or larger, hyaluronans.

The hyaluronan (HA) receptor for endocytosis (HARE; Stabilin-2) binds and clears 14 different ligands, including HA and heparin, via clathrin-mediated endocytosis. HA binding to HARE stimulates ERK1/2 activation (Kyosseva, S. V., Harris, E. N., and Weigel, P. H. (2008) J. Biol. Chem. 283, 15047-15055). To assess a possible HA size dependence for signaling, we tested purified HA fractions of different weight-average molar mass and with narrow size distributions and Select-HA(TM) for stimulation of HARE-mediated gene expression using an NF-κB promoter-driven luciferase reporter system. Human HARE-mediated gene expression was stimulated in a dose-dependent manner with small HA (sHA) >40 kDa and intermediate HA (iHA) <400 kDa. The hyperbolic dose response saturated at 20-50 nM with an apparent K(m) ~10 nM, identical to the Kd for HA-HARE binding. Activation was not detected with oligomeric HA (oHA), sHA <40 kDa, iHA >400 kDa, or large HA (lHA). Similar responses occurred with rat HARE. Activation by sHA-iHA was blocked by excess nonsignaling sHA, iHA, or lHA, deletion of the HA-binding LINK domain, or HA-blocking antibody. Endogenous NF-κB activation also occurred in the absence of luciferase plasmids, as assessed by degradation of IκB-α. ERK1/2 activation was also HA size-dependent. The results show that HA-HARE interactions stimulate NF-κB-activated gene expression and that HARE senses a narrow size range of HA degradation products. We propose a model in which optimal length HA binds multiple HARE proteins to allow cytoplasmic domain interactions that stimulate intracellular signaling. This HARE signaling system during continuous HA clearance could monitor the homeostasis of tissue biomatrix turnover throughout the body.

Digestion products of the PH20 hyaluronidase inhibit remyelination.

OBJECTIVE: Oligodendrocyte progenitor cells (OPCs) recruited to demyelinating lesions often fail to mature into oligodendrocytes (OLs) that remyelinate spared axons. The glycosaminoglycan hyaluronan (HA) accumulates in demyelinating lesions and has been implicated in the failure of OPC maturation and remyelination. We tested the hypothesis that OPCs in demyelinating lesions express a specific hyaluronidase, and that digestion products of this enzyme inhibit OPC maturation. METHODS: Mouse OPCs grown in vitro were analyzed for hyaluronidase expression and activity. Gain of function studies were used to define the hyaluronidases that blocked OPC maturation. Mouse and human demyelinating lesions were assessed for hyaluronidase expression. Digestion products from different hyaluronidases and a hyaluronidase inhibitor were tested for their effects on OPC maturation and functional remyelination in vivo. RESULTS: OPCs demonstrated hyaluronidase activity in vitro and expressed multiple hyaluronidases, including HYAL1, HYAL2, and PH20. HA digestion by PH20 but not other hyaluronidases inhibited OPC maturation into OLs. In contrast, inhibiting HA synthesis did not influence OPC maturation. PH20 expression was elevated in OPCs and reactive astrocytes in both rodent and human demyelinating lesions. HA digestion products generated by the PH20 hyaluronidase but not another hyaluronidase inhibited remyelination following lysolecithin-induced demyelination. Inhibition of hyaluronidase activity lead to increased OPC maturation and promoted increased conduction velocities through lesions. INTERPRETATION: We determined that PH20 is elevated in demyelinating lesions and that increased PH20 expression is sufficient to inhibit OPC maturation and remyelination. Pharmacological inhibition of PH20 may therefore be an effective way to promote remyelination in multiple sclerosis and related conditions.

Tumor necrosis factor-stimulated gene-6 (TSG-6) amplifies hyaluronan synthesis by airway smooth muscle cells.

We tested the hypothesis that the artificial addition of heavy chains from inter-α-inhibitor to hyaluronan (HA), by adding recombinant TSG-6 (TNF-stimulated gene-6) to the culture medium of murine airway smooth muscle (MASM) cells, would enhance leukocyte binding to HA cables produced in response to poly(I:C). As predicted, the addition of heavy chains to HA cables enhanced leukocyte adhesion to these cables, but it also had several unexpected effects. (i) It produced thicker, more pronounced HA cables. (ii) It increased the accumulation of HA in the cell-associated matrix. (iii) It decreased the amount of HA in the conditioned medium. Importantly, these effects were observed only when TSG-6 was administered in the presence of poly(I:C), and TSG-6 did not exert any effect on its own. Increased HA synthesis occurred during active, poly(I:C)-induced HA synthesis and did not occur when TSG-6 was added after poly(I:C)-induced HA synthesis was complete. MASM cells derived from TSG-6(-/-), HAS1/3(-/-), and CD44(-/-) mice amplified HA synthesis in response to poly(I:C) + TSG-6 in a manner similar to WT MASM cells, demonstrating that they are expendable in this process. We conclude that TSG-6 increases the accumulation of HA in the cell-associated matrix, partially by preventing its dissolution from the cell-associated matrix into the conditioned medium, but primarily by inducing HA synthesis.

Hyaluronan turnover and hypoxic brown adipocytic differentiation are co-localized with ossification in calcified human aortic valves.

The calcification process in aortic stenosis has garnered considerable interest but only limited investigation into selected signaling pathways. This study investigated mechanisms related to hypoxia, hyaluronan homeostasis, brown adipocytic differentiation, and ossification within calcified valves. Surgically explanted calcified aortic valves (n=14) were immunostained for markers relevant to these mechanisms and evaluated in the center (NodCtr) and edge (NodEdge) of the calcified nodule (NodCtr), tissue directly surrounding nodule (NodSurr); center and tissue surrounding small "prenodules" (PreNod, PreNodSurr); and normal fibrosa layer (CollFibr). Pearson correlations were determined between staining intensities of markers within regions. Ossification markers primarily localized to NodCtr and NodEdge, along with markers related to hyaluronan turnover and hypoxia. Markers of brown adipocytic differentiation were frequently co-localized with markers of hypoxia. In NodCtr and NodSurr, brown fat and ossification markers correlated with hyaluronidase-1, whereas these markers, as well as hypoxia, correlated with hyaluronan synthases in NodEdge. The protein product of tumor necrosis factor-α stimulated gene-6 strongly correlated with ossification markers and hyaluronidase in the regions surrounding the nodules (NodSurr, PreNodSurr). In conclusion, this study suggests roles for hyaluronan homeostasis and the promotion of hypoxia by cells demonstrating brown fat markers in calcific aortic valve disease.