Molecular Weight Hyaluronan Research Articles

Regulation of Hyaluronan (HA) Metabolism Mediated by HYBID (Hyaluronan-binding Protein Involved in HA Depolymerization, KIAA1199) and HA Synthases in Growth Factor-stimulated Fibroblasts

Regulation of hyaluronan (HA) synthesis and degradation is essential to maintenance of extracellular matrix homeostasis. We recently reported that HYBID (HYaluronan-Binding protein Involved in hyaluronan Depolymerization), also called KIAA1199, plays a key role in HA depolymerization in skin and arthritic synovial fibroblasts. However, regulation of HA metabolism mediated by HYBID and HA synthases (HASs) under stimulation with growth factors remains obscure. Here we report that TGF-β1, basic FGF, EGF, and PDGF-BB commonly enhance total amount of HA in skin fibroblasts through up-regulation of HAS expression, but molecular size of newly produced HA is dependent on HYBID expression levels. Stimulation of HAS1/2 expression and suppression of HYBID expression by TGF-β1 were abrogated by blockade of the MAPK and/or Smad signaling and the PI3K-Akt signaling, respectively. In normal human skin, expression of the TGF-β1 receptors correlated positively with HAS2 expression and inversely with HYBID expression. On the other hand, TGF-β1 up-regulated HAS1/2 expression but exerted only a slight suppressive effect on HYBID expression in synovial fibroblasts from the patients with osteoarthritis or rheumatoid arthritis, resulting in the production of lower molecular weight HA compared with normal skin and synovial fibroblasts. These data demonstrate that although TGF-β1, basic FGF, EGF, and PDGF-BB enhance HA production in skin fibroblasts, TGF-β1 most efficiently contributes to production of high molecular weight HA by HAS up-regulation and HYBID down-regulation and suggests that inefficient down-regulation of HYBID by TGF-β1 in arthritic synovial fibroblasts may be linked to accumulation of depolymerized HA in synovial fluids in arthritis patients.

Autodisplay of Human Hyaluronidase Hyal-1 on Escherichia coli and Identification of Plant-Derived Enzyme Inhibitors.

Hyaluronan (HA) is the main component of the extracellular matrix (ECM). Depending on its chain size, it is generally accepted to exert diverse effects. High molecular weight HA is anti-angiogenic, immunosuppressive and anti-inflammatory, while lower fragments are angiogenic and inflammatory. Human hyaluronidase Hyal-1 (Hyal-1) is one of the main enzymes in the metabolism of HA. This makes Hyal-1 an interesting target. Not only for functional and mechanistic studies, but also for drug development. In this work, Hyal-1 was expressed on the surface of E. coli, by applying Autodisplay, to overcome formation of inactive “inclusion bodies”. With the cells displaying Hyal-1 an activity assay was performed using “stains-all” dye. Subsequently, the inhibitory effects of four saponins and 14 plant extracts on the activity of surface displayed Hyal-1 were evaluated. The determined IC50 values were 177 µM for glycyrrhizic acid, 108 µM for gypsophila saponin 2, 371 µM for SA1657 and 296 µM for SA1641. Malvae sylvestris flos, Equiseti herba and Ononidis radix extracts showed IC50 values between 1.4 and 1.7 mg/mL. In summary, Autodisplay enabled the expression of functional human target protein Hyal-1 in E. coli and facilitated an accelerated testing of potential inhibitors.

Viscoelastic Properties of Hyaluronan in Physiological Conditions.

Hyaluronan (HA) is a high molecular weight glycosaminoglycan of the extracellular matrix (ECM), which is particularly abundant in soft connective tissues. Solutions of HA can be highly viscous with non-Newtonian flow properties. These properties affect the movement of HA-containing fluid layers within and underlying the deep fascia. Changes in the concentration, molecular weight, or even covalent modification of HA in inflammatory conditions, as well as changes in binding interactions with other macromolecules, can have dramatic effects on the sliding movement of fascia. The high molecular weight and the semi-flexible chain of HA are key factors leading to the high viscosity of dilute solutions, and real HA solutions show additional nonideality and greatly increased viscosity due to mutual macromolecular crowding. The shear rate dependence of the viscosity, and the viscoelasticity of HA solutions, depend on the relaxation time of the molecule, which in turn depends on the HA concentration and molecular weight. Temperature can also have an effect on these properties. High viscosity can additionally affect the lubricating function of HA solutions. Immobility can increase the concentration of HA, increase the viscosity, and reduce lubrication and gliding of the layers of connective tissue and muscle. Over time, these changes can alter both muscle structure and function. Inflammation can further increase the viscosity of HA-containing fluids if the HA is modified via covalent attachment of heavy chains derived from Inter-α-Inhibitor. Hyaluronidase hydrolyzes HA, thus reducing its molecular weight, lowering the viscosity of the extracellular matrix fluid and making outflow easier. It can also disrupt any aggregates or gel-like structures that result from HA being modified. Hyaluronidase is used medically primarily as a dispersion agent, but may also be useful in conditions where altered viscosity of the fascia is desired, such as in the treatment of muscle stiffness.

Effect of molecular weight and concentration of hyaluronan on cell proliferation and osteogenic differentiation in vitro

Hyaluronan (HA), the simplest glycosaminoglycan and a major component of the extracellular matrix, exists in various tissues. It is involved in some critical biological procedures, including cellular signaling, cell adhesion and proliferation, and cell differentiation. The effect of molecular weight (MW) and concentration of HA on cell proliferation and differentiation was controversial. In this study, we investigated the effect of MW and concentration of HA on the proliferation and osteogenic differentiation of rabbit bone marrow-derived stem cells in vitro. Results showed that high MW HA decreased the cell adhesion rate in a concentration-dependant manner. The cell adhesion rate was decreased by increasing MW of HA. Cell proliferation was significantly enhanced by low MW HA (P < 0.05). The factorial analysis indicated that MW and concentration had an interactive effect on the cell adhesion rate and cell proliferation (P < 0.05). High MW HA increased the mRNA expressions of ALP, RUNX-2 and OCN. The higher the MW was, the higher the mRNA expressions were. The factorial analysis indicated that MW and concentration had an interactive effect on ALP mRNA expression (P < 0.05). HA of higher MW and higher concentration promoted bone formation. These findings provide some useful information in understanding the mechanism underlying the effect of MW and concentration of HA on cell proliferation and differentiation.

HABP2 is a Novel Regulator of Hyaluronan-Mediated Human Lung Cancer Progression.

Lung cancer is a devastating disease with limited treatment options. Many lung cancers have changes in their microenvironment including upregulation of the extracellular matrix glycosaminoglycan, hyaluronan (HA), which we have previously demonstrated can regulate the activity of the extracellular serine protease, hyaluronan binding protein 2 (HABP2). This study examined the functional role of HABP2 on HA-mediated human lung cancer dynamics. Immunohistochemical analysis was performed on lung cancer patient samples using anti-HABP2 antibody. Stable control, shRNA, and HABP2 overexpressing human lung adenocarcinoma cells were evaluated using immunoblot analysis, migration, extravasation, and urokinase plasminogen activator (uPA) activation assays with or without high-molecular weight HA or low-molecular weight HA (LMW-HA). In human lung cancer xenograft models, primary tumor growth rates and lung metastasis were analyzed using consecutive tumor volume measurements and nestin immunoreactivity in nude mouse lungs. We provide evidence that HABP2 is an important regulator of lung cancer progression. HABP2 expression was increased in several subtypes of patient non-small cell lung cancer samples. Further, HABP2 overexpression increased LMW-HA-induced uPA activation, migration, and extravasation in human lung adenocarcinoma cells. In vivo, overexpression of HABP2 in human lung adenocarcinoma cells increased primary tumor growth rates in nude mice by ~2-fold and lung metastasis by ~10-fold compared to vector control cells (n = 5/condition). Our data suggest a possible direct effect of HABP2 on uPA activation and lung cancer progression. Our observations suggest that exploration of HABP2 in non-small cell lung carcinoma merits further study both as a diagnostic and therapeutic option.

High and low molecular weight hyaluronic acid differentially influence macrophage activation.

Macrophages exhibit phenotypic diversity permitting wide-ranging roles in maintaining physiologic homeostasis. Hyaluronic acid, a major glycosaminoglycan of the extracellular matrix, has been shown to have differential signaling based on its molecular weight. With this in mind, the main objective of this study was to elucidate the role of hyaluronic acid molecular weight on macrophage activation and reprogramming. Changes in macrophage activation were assessed by activation state selective marker measurement, specifically quantitative real time polymerase chain reaction, and cytokine enzyme-linked immunoassays, after macrophage treatment with differing molecular weights of hyaluronic acid under four conditions: the resting state, concurrent with classical activation, and following inflammation involving either classically or alternatively activated macrophages. Regardless of initial polarization state, low molecular weight hyaluronic acid induced a classically activated-like state, confirmed by up-regulation of pro-inflammatory genes, including nos2, tnf, il12b, and cd80, and enhanced secretion of nitric oxide and TNF-α. High molecular weight hyaluronic acid promoted an alternatively activated-like state, confirmed by up regulation of pro-resolving gene transcription, including arg1, il10, and mrc1, and enhanced arginase activity. Overall, our observations suggest that macrophages undergo phenotypic changes dependent on molecular weight of hyaluronan that correspond to either (1) pro-inflammatory response for low molecular weight HA or (2) pro-resolving response for high molecular weight HA. These observations bring significant further understanding of the influence of extracellular matrix polymers, hyaluronic acid in particular, on regulating the inflammatory response of macrophages. This knowledge can be used to guide the design of HA-containing biomaterials to better utilize the natural response to HAs.

Interactions between Hyaluronan and Its Receptors (CD44, RHAMM) Regulate the Activities of Inflammation and Cancer.

The glycosaminoglycan hyaluronan (HA), a major component of extracellular matrices, and cell surface receptors of HA have been proposed to have pivotal roles in cell proliferation, migration, and invasion, which are necessary for inflammation and cancer progression. CD44 and receptor for HA-mediated motility (RHAMM) are the two main HA-receptors whose biological functions in human and murine inflammations and tumor cells have been investigated comprehensively. HA was initially considered to be only an inert component of connective tissues, but is now known as a “dynamic” molecule with a constant turnover in many tissues through rapid metabolism that involves HA molecules of various sizes: high molecular weight HA (HMW HA), low molecular weight HA, and oligosaccharides. The intracellular signaling pathways initiated by HA interactions with CD44 and RHAMM that lead to inflammatory and tumorigenic responses are complex. Interestingly, these molecules have dual functions in inflammations and tumorigenesis. For example, the presence of CD44 is involved in initiation of arthritis, while the absence of CD44 by genetic deletion in an arthritis mouse model increases rather than decreases disease severity. Similar dual functions of CD44 exist in initiation and progression of cancer. RHAMM overexpression is most commonly linked to cancer progression, whereas loss of RHAMM is associated with malignant peripheral nerve sheath tumor growth. HA may similarly perform dual functions. An abundance of HMW HA can promote malignant cell proliferation and development of cancer, whereas antagonists to HA-CD44 signaling inhibit tumor cell growth in vitro and in vivo by interfering with HMW HA-CD44 interaction. This review describes the roles of HA interactions with CD44 and RHAMM in inflammatory responses and tumor development/progression, and how therapeutic strategies that block these key inflammatory/tumorigenic processes may be developed in rodent and human diseases.

Abstract P4-04-25: Naked mole rat hyaluronan synthase 2 displays similar effects as human hyaluronan synthase 2 and promotes tumor growth in a mouse xenograft model

Abstract Accumulation of hyaluronan (HA) is correlated with poor prognosis in many human cancer including breast cancer and pancreatic cancer. Hyaluronan synthase 2 (HAS2) is one of the three HA synthase genes (HAS1-3) found in mammals. Up-regulation of the HAS2 gene has been observed in breast cancer patients; and HAS2 expression correlates with the incidence of metastasis and lower rate of overall patient survival (Okuda et al 2012). Interestingly, a recent study suggested that the naked mole rat (nmr) HAS2 enzyme may function differently than the HAS2 proteins from mice and humans, and its expression had a tumor suppressor effect (Tian et al 2013). The nmrHAS2 protein sequence was distinct from other mammals in the cytoplasmic loop where the active site resides, which led to the hypothesis that nmrHAS2 may contribute to the production of extremely high molecular weight HA species. Furthermore, nmrHAS2 is required for the resistance of nmr skin fibroblasts to malignant transformation. In this study, we examined whether the nmrHAS2 gene can also render human cells resistant to tumor progression. We have previously observed that HA level in the pancreatic cancer model AsPC1 can be modulated by the over-expression of the human HAS3 protein (Osgood et al 2014). To compare and contrast the effect of nmrHAS2 and huHAS2 on HA production and tumor growth, nmrHAS2 and huHAS2 AsPC1 models were engineered using a recombinant lentivirus encoding the nmrHAS2 and huHAS2 genes respectively. Over-expression of nmrHAS2 and huHAS2 both significantly increased the HA level in AsPC1 cells. Both nmrHAS2 and huHAS2 promoted the growth of AsPC1 in an intramuscular xenograft tumor model. Ex vivo analysis of tumor xenografts showed that nmrHAS2 and huHAS2 AsPC1 tumors contain elevated levels of HA, and the size range of the HA in the nmrHAS2 tumors is similar to that in the huHAS2 AsPC1 tumors. Therefore, nmrHAS2 functions similarly as huHAS2 gene and promotes tumor growth in the human pancreatic tumor model AsPC1.

Intra-articular treatment with triamcinolone compared with triamcinolone with hyaluronate: A randomised open-label multicentre clinical trial in 80 lame horses.

Intra-articular (IA) injection of corticosteroids with or without hyaluronate (HA) has been used for decades in equine practice for treatment of noninfectious synovitis and osteoarthritis. However, to date, no large-scale randomised equine field trials have been reported that address the supposed superior clinical efficacy of the combination of corticosteroid + HA compared with IA injection of corticosteroid alone. To compare the clinical efficacy of IA triamcinolone acetonide (TA, 12 mg) compared with IA TA (12 mg) + high molecular weight HA (20 mg) in horses with clinical joint disease. Prospective, randomised, parallel, open label, multicentre clinical trial. Eighty client-owned horses from 13 clinics were included. Lameness and effusion scores were assessed at baseline and 3 weeks after IA treatment. A standardised telephone questionnaire was completed between the owner and consulting veterinarian at 3 months. The primary outcome parameter was clinical success rate, defined as ≥2 grades lameness reduction (on a 0-5 scale) at 3 weeks. Chi-square statistics and binary logistic regression were used to analyse data on an intention-to-treat basis for the 3 week outcome. The success rate of IA TA 3 weeks after treatment was 87.8%, while that of TA+HA was 64.1% (P = 0.01). Age >13 years was associated with a reduced success rate for the combination treatment (P = 0.004) at 3 weeks. At 3 months, half the horses in each group had returned to their previous level of performance. The combination of TA with HA was associated with a lower short-term clinical success rate and a similar medium-term outcome compared with IA TA, with only half of the horses performing at their previous level of exercise after 3 months regardless of treatment group allocation.

Hyaluronan inhibits BMP-induced osteoblast differentiation

Hyaluronan (HA), one of the major structural extracellular components in cartilage, regulates cellular responses via receptors such as CD44. However, the direct effects of HA on osteoblastic differentiation has not been studied in detail. Here, we investigated the effects of HA (molecular weight: 900–1200kDa) on osteoblastic differentiation that was induced by bone morphogenetic protein (BMP) in C2C12 cells (mouse myoblastic cells) and ST2 cells (mouse bone marrow cells). BMP-induced osteoblastic differentiation and Smad1/Smad5/Smad8 phosphorylation were downregulated by HA. Use of the CD44-blocking antibody restored HA-induced inhibition of osteoblastic differentiation and Smad1/Smad5/Smad8 phosphorylation. Our results indicate that HA inhibits BMP-induced osteoblastic differentiation through the CD44 receptor.

Hyaluronan Synthesis, Catabolism, and Signaling in Neurodegenerative Diseases.

The glycosaminoglycan hyaluronan (HA), a component of the extracellular matrix, has been implicated in regulating neural differentiation, survival, proliferation, migration, and cell signaling in the mammalian central nervous system (CNS). HA is found throughout the CNS as a constituent of proteoglycans, especially within perineuronal nets that have been implicated in regulating neuronal activity. HA is also found in the white matter where it is diffusely distributed around astrocytes and oligodendrocytes. Insults to the CNS lead to long-term elevation of HA within damaged tissues, which is linked at least in part to increased transcription of HA synthases. HA accumulation is often accompanied by elevated expression of at least some transmembrane HA receptors including CD44. Hyaluronidases that digest high molecular weight HA into smaller fragments are also elevated following CNS insults and can generate HA digestion products that have unique biological activities. A number of studies, for example, suggest that both the removal of high molecular weight HA and the accumulation of hyaluronidase-generated HA digestion products can impact CNS injuries through mechanisms that include the regulation of progenitor cell differentiation and proliferation. These studies, reviewed here, suggest that targeting HA synthesis, catabolism, and signaling are all potential strategies to promote CNS repair.

Cartilage boundary lubrication of ovine synovial fluid following anterior cruciate ligament transection: a longitudinal study

To assess ovine synovial fluid (oSF) from different post-injury time points for (1) proteoglycan-4 (PRG4) and hyaluronan (HA) concentration, (2) HA molecular weight (MW) distribution, (3) cartilage boundary lubrication function, and (4) lubricant composition-function relationships. The association between cartilage boundary lubrication and gross cartilage changes after injury was also examined. oSF was collected 2, 4, 10, and 20 weeks post anterior cruciate ligament (ACL) transection in five skeletally mature sheep. PRG4 and HA concentrations were measured using sandwich enzyme-linked immunosorbent assay, and HA MW distribution by agarose gel electrophoresis. Cartilage boundary lubrication of oSF was assessed using a cartilage-cartilage friction test. Gross damage to articular cartilage was also quantified at 20 weeks using modified Drez scoring protocol. Early (2-4 weeks) after ACL injury, PRG4 concentrations were significantly higher (P = 0.045, P = 0.037), and HA concentrations were substantially lower (P = 0.005, P = 0.005) compared to 20 weeks. The HA MW distribution also shifted towards lower ranges in the early post-injury stage. The kinetic friction coefficients were significantly higher 2-4 weeks post injury (P = 0.008 and P = 0.049) compared to 20 weeks. Poor cartilage boundary lubricating ability early after injury was associated with cartilage damage at 20 weeks. Altered composition and diminished boundary lubrication of oSF early after ACL transection may pre-dispose the articular cartilage to degenerative changes and initiate osteoarthritis (OA). These observations also provide potential motivation for biotherapeutic interventions at earlier time points post injury.

Hyaluroan-regulated lymphatic permeability through S1P receptors is crucial for cancer metastasis.

Disruption of cancer lymphatic vessel barrier function occurs has been reported to involve in cancer lymphatic metastasis. Hyaluronan (HA), a major glycosaminoglycan component of the extracellular matrix, is associated with cancer metastasis. We investigated the effect of high/low molecular weight hyaluronan (HMW-HA/LMW-HA) on regulation of barrier function and tight junctions in cancer lymphatic endothelial cell (LEC) monolayer. Results showed that LMW-HA increased the permeability of cancer LEC monolayers and induced disruption of Zonula Occludens-1 (ZO-1)-mediated intercellular tight junction and actin stress fiber formation. HMW-HA treatment decreased permeability in cancer LEC monolayers and cortical actin ring formation. As reported, sphingosine 1-phosphate (S1P) receptors are involved in vascular integrity. After silencing of lymphatic vessel endothelial hyaluronan receptor (LYVE-1), upregulation of S1P receptors (S1P1 and S1P3) induced by HMW-HA/LMW-HA were inhibited, respectively. With S1P3 silenced, the disruption of ZO-1 as well as stress fiber formation and the ROCK1/RhoA signaling pathway induced by LMW-HA was not observed in cancer LEC. These results suggested that S1P receptors may play an important role in HMW-HA-/LMW-HA-mediated regulation of cancer lymphatic vessel integrity, which might be the initial step of cancer lymphatic metastasis and a useful intervention of cancer progression.

Hyaluronan Synthase 2 Protects Skin Fibroblasts against Apoptosis Induced by Environmental Stress

A balanced turnover of dermal fibroblasts is crucial for structural integrity and normal function of the skin. During recovery from environmental injury (such as UV exposure and physical wounding), apoptosis is an important mechanism regulating fibroblast turnover. We are interested in the role that hyaluronan (HA), an extracellular matrix molecule synthesized by HA synthase enzymes (Has), plays in regulating apoptosis in fibroblasts. We previously reported that Has1 and Has3 double knock-out (Has1/3 null) mice show accelerated wound closure and increased numbers of fibroblasts in the dermis. In the present study, we report that HA levels and Has2 mRNA expression are higher in cultured Has1/3 null primary skin fibroblasts than in wild type (WT) cells. Apoptosis induced by two different environmental stressors, UV exposure and serum starvation (SS), was reduced in the Has1/3 null cells. Hyaluronidase, added to cultures to remove extracellular HA, surprisingly had no effect upon apoptotic susceptibility to UVB or SS. However, cells treated with 4-methylumbelliferone to inhibit HA synthesis were sensitized to apoptosis induced by SS or UVB. When fibroblasts were transfected with Has2-specific siRNA that lowered Has2 mRNA and HA levels by 90%, both Has1/3 null and WT cells became significantly more sensitive to apoptosis. The exogenous addition of high molecular weight HA failed to reverse this effect. We conclude that Has1/3 null skin fibroblasts (which have higher levels of Has2 gene expression) are resistant to stress-induced apoptosis.

Intermolecular interactions between salmon calcitonin, hyaluronate, and chitosan and their impact on the process of formation and properties of peptide-loaded nanoparticles

The principal aim of this work was to study the formulation of a ternary complex comprising salmon calcitonin (sCT), hyaluronate (HA), and chitosan (CS) in a nanoparticle (NP) format. As interactions between the constituents are possible, their presence and component mass mixing ratio (MMR) and charge mixing ratio (CMR) were investigated to tune the properties of NPs.Intermolecular interactions between sCT and HA as well as sCT and CS were studied by infrared spectroscopy (FTIR) and dynamic viscosity. The impact of MMR, CMR, and HA molecular weight on the sCT loading capacity in NPs and in vitro release properties was determined.sCT complexes to HA via electrostatic interactions and a support for hydrophobic interactions between sCT and HA as well as sCT and CS was found by FTIR. The sCT/HA complex is soluble but, depending on the mass mixing ratio between sCT and HA, NPs and microparticles were also formed indicative of associative phase separation between HA and sCT. The negatively charged HA/CS/sCT NPs were characterized by very high values (above 90%) of peptide association for the systems tested. Also, high sCT loading up to 50% were achieved. The peptide loading capacity and in vitro release properties were dependent on the NP composition. The zeta potential of the NPs without sCT was negative and ranging from −136 to −36mV, but increased to −84 to −19mV when the peptide was loaded. The particle size was found to be smaller and ranging 150–230nm for sCT/NPs in comparison to NPs without sCT (170–260nm). Short-term storage studies in liquid dispersions showed that the colloidal stability of NPs was acceptable and no release of sCT was observed for up to 3 days.In conclusion, a range of NP systems comprising sCT, HA, and CS was successfully developed and characterized. Such NPs may be considered as a suitable nanoparticulate format for the delivery of sCT.

Hyaluronan molecular weight distribution is associated with the risk of knee osteoarthritis progression

We investigated the relationship between the molecular weight (MW) distribution of hyaluronan (HA) in synovial fluid (SF) and risk of knee osteoarthritis (OA) progression. HA MW was analyzed for 65 baseline knee SFs. At 3-year follow-up, knees were scored for change in joint space narrowing (JSN), osteophyte (OST) progression, or occurrence of total knee arthroplasty (TKA). HA MW distribution was analyzed using agarose gel electrophoresis (AGE), and its relationship to OA progression was evaluated using logistic regression. The association between HA MW and self-reported baseline knee pain was analyzed using Pearson's correlation coefficients. Knee OA was categorized as non-progressing (OST-/JSN-, 26 knees, 40%), or progressing based on OST (OST+/JSN-, 24 knees, 37%), OST and JSN (OST+/JSN+, 7 knees, 11%) or total knee arthroplasty (TKA, 8 knees, 12%). The MW distribution of HA in baseline SFs was significantly associated with the odds of OA progression, particularly for index knees. After adjusting for age, gender, BMI, baseline X-ray grade and pain, each increase of one percentage point in %HA below 1 million significantly increased the odds of JSN (odds ratios (OR)=1.45, 95% CI 1.02-2.07), TKA or JSN (OR=1.24, 95%CI 1.01-1.53) and the odds of any progression (OR=1.16, 95% CI 1.01-1.32). HA MW distribution significantly correlated with pain. These data suggest that the odds of knee OA progression increases as HA MW distribution shifts lower and highlight the value of reporting MW distribution rather than just average MW values for HA.

High-Resolution Ultrasonic Spectroscopy Study of Interactions between Hyaluronan and Cationic Surfactants

Interactions in a cationic surfactant-hyaluronan system in water and in sodium chloride solution were investigated by high-resolution ultrasonic spectroscopy at 25 °C. Two alkyltrimethylammonium bromide surfactants of different chain lengths (tetradecyl and hexadecyl) were used; hyaluronan molecular weight ranged from 10 to 1750 kDa. Two main parameters-ultrasonic velocity and attenuation-were measured in the titration regime. Up to six different regions could be identified in the velocity titration profiles in water in a narrow interval of surfactant concentration. These regions differed primarily in the compressibility of structures formed in the system. The number of detected transitions was higher for the tetradecyl surfactant; therefore, the increased length of the hydrophobic chain simplified the details of the structure-forming behavior. The measurement of attenuation was much less sensitive and detected only the formation of microheterogeneous structures or visible phase separates. The richness of the titration profiles was depressed in salt solution, where essentially only two principal regions were observed. On the other hand, the effect of hyaluronan molecular weight on the positions of boundaries between regions was more significant in the presence of salt. Besides electrostatic interactions, hydrophobic interactions are also relevant for determining the behavior of hyaluronan-surfactant systems and the properties of formed complexes (aggregates).

Irreversible Heavy Chain Transfer to Chondroitin

We have recently demonstrated that the transfer of heavy chains (HCs) from inter-α-inhibitor, via the enzyme TSG-6 (tumor necrosis factor-stimulated gene 6), to hyaluronan (HA) oligosaccharides is an irreversible event in which subsequent swapping of HCs between HA molecules does not occur. We now describe our results of HC transfer experiments to chondroitin sulfate A, chemically desulfated chondroitin, chemoenzymatically synthesized chondroitin, unsulfated heparosan, heparan sulfate, and alginate. Of these potential HC acceptors, only chemically desulfated chondroitin and chemoenzymatically synthesized chondroitin were HC acceptors. The kinetics of HC transfer to chondroitin was similar to HA. At earlier time points, HCs were more widely distributed among the different sizes of chondroitin chains. As time progressed, the HCs migrated to lower molecular weight chains of chondroitin. Our interpretation is that TSG-6 swaps the HCs from the larger, reversible sites on chondroitin chains, which function as HC acceptors, onto smaller chondroitin chains, which function as irreversible HC acceptors. HCs transferred to smaller chondroitin chains were unable to be swapped off the smaller chondroitin chains and transferred to HA. HCs transferred to high molecular weight HA were unable to be swapped onto chondroitin. We also present data that although chondroitin was a HC acceptor, HA was the preferred acceptor when chondroitin and HA were in the same reaction mixture.

Abstract 1162: Fibroblast RHAMM promotes breast cancer aggression by promoting expression of a subset of ERK1,2 target genes

Abstract The microenvironment plays a key role in cancer progression and metabolism of the extracellular matrix component hyaluronan (HA), a glycosaminoglycan, is associated with breast tumor progression. High molecular weight HA is degraded by hyaluronidases and free radicals to fragments of heterogeneous size, which accumulate in the peritumor stroma and activate signaling pathways in both tumor and stromal cells. The HA receptor RHAMM is a multifunctional protein that is found on the cell surface, inside the nucleus and the mitotic spindles of mesenchymal cells. RHAMM expression and HA accumulation in the tumor stroma are linked to breast tumor progression, predicting that RHAMM/HA regulated signaling is key to breast cancer progression. An N-terminally truncated RHAMM isoform, RHAMMonc, is oncogenic when overexpressed in mesenchymal cells. Intracellular RHAMMonc directly binds to ERK1, and complexes with ERK1,2 and MEK1. Intracellular RHAMM is required for sustained ERK1,2 activation and for nuclear localization of a subset of active ERK1,2. We hypothesized that this function results in expression of oncogenic proteins that support breast cancer cell aggression. Study purpose: determine the mechanism for RHAMMonc supported breast cancer aggression. Experimental procedure: we used microarray analysis in combination with real time PCR and ERK1,2 inhibition to identify genes that are upregulated in response to RHAMMonc overexpression. To determine whether nuclear RHAMMonc is required for neoplastic transformation we added a nuclear export signal to the RHAMMonc cDNA (RHAMMNES-onc). Mesenchymal cells overexpressing RHAMMonc or RHAMMNES-onc were injected into immune compromised mice. To test whether these cells could support in vivo growth of breast cancer cells, MCF7 cells were co-injected with RHAMMonc or RHAMMNES-onc expressing cells into the mammary gland fat pad. Tumor growth was quantified as tumor wet weight. Conclusion: Microarray analysis in combination with real time PCR identified altered expression of genes in all categories of the Hallmarks of Cancer in fibroblasts overexpressing RHAMMonc compared to parental cells. A subset of these were shown to be regulated by ERK1,2. Fusion of a nuclear export signal to RHAMMonc (RHAMMNES-onc) resulted in export of this protein from the nucleus, significant reduction of nuclear active ERK1,2 and modified expression of a subset of ERK1,2 regulated genes (e.g. LOX, MMP9, CDH11). Importantly, forced nuclear export of RHAMMonc suppressed its oncogenic effect on fibroblasts. Furthermore, proliferation of human MCF7 breast cancer cell xenografts was strongly stimulated by co-injection with RHAMMonc fibroblasts but this stimulatory effect was lost when MCF7 tumor cells were co-injected with RHAMMNES-onc. This study suggests that nuclear RHAMMonc:ERK 1,2 interactions control an oncogenic program in fibroblasts that support breast cancer aggression. Citation Format: Cornelia Toelg, Patrick Telmer, Sara Hamilton, James McCarthy, Eva Turley. Fibroblast RHAMM promotes breast cancer aggression by promoting expression of a subset of ERK1,2 target genes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1162. doi:10.1158/1538-7445.AM2014-1162

Genetic and functional characterization of the hyaluronate lyase HylB and the beta-N-acetylglucosaminidase HylZ in Streptococcus zooepidemicus.

Hyaluronic acid (hyaluronan) is a high molecular weight glycosaminoglycan composed of repeating disaccharides of glucuronic acid and N-acetylglucosamine. The molecular weight of hyaluronan is important for its rheological property, biological function, and application. However, genes important for regulation of hyaluronan production or molecular weight remain poorly understood. Here, we address the roles of two predicated hyaluronate lyase-encoding genes, hylB and hylZ in Streptococcus zooepidemicus. Semi-quantitative RT-PCR assays showed that hylZ was constitutively expressed while the expression level of hylB was growth-phase dependent. Using recombinantly expressed 6His-HylB and -HylZ protein, enzyme assays revealed that HylB was a hyaluronate lyase, and its K(m) and V(max) were 0.57 μM and 1.43 mM min(-1), respectively. 6His-HylZ showed no activity against hyaluronan while it hydrolyzed pNp-β-GlcNAc suggesting that HylZ was a beta-N-acetylglucosaminidase. Under the optimal conditions (pH 6.0 and 50 °C), the K(m) and V(max) for 6His-HylZ to degrade pNp-β-GlcNA were 1.16 mM and 26.18 μmol min(-1) mg(-1), respectively. Characterization of ΔhylB and ΔhylZ demonstrated that loss of hylB or/and hylZ had no significant effects on cell growth, lactic acid, and hyaluronan yields. Significantly, as compared to the wild type, ΔhylB produced hyaluronan with an 18 % increase in molecular weight. Our results strongly suggest that hylB encodes a hyaluronate lyase while hylZ encodes a β-N-acetylglucosaminidase. hylB-deficient strain has the potential to produce high molecular weight hyaluronan.