Hyaluronan Export Research Articles

Recovery of ΔF508-CFTR Function by Citrate.

Treatment of cystic fibrosis relies so far on expensive and sophisticated drugs. A logical approach to rescuing the defective ΔF508-CFTR protein has not yet been published. Therefore, virtual docking of ATP and CFTR activators to the open conformation of the CFTR protein was performed. A new ATP binding site outside of the two known locations was identified. It was located in the cleft between the nucleotide binding domains NBD1 and NBD2 and comprised six basic amino acids in close proximity. Citrate and isocitrate were also bound to this site. Citrate was evaluated for its action on epithelial cells with intact CFTR and defective ΔF508-CFTR. It activated hyaluronan export from human breast carcinoma cells and iodide efflux, and recovered ΔF508-CFTR from premature intracellular degradation. In conclusion, citrate is an activator for ΔF508-CFTR and increases export by defective ΔF508-CFTR into the extracellular matrix of epithelial cells.

Curcumin analogue identified as hyaluronan export inhibitor by virtual docking to the ABC transporter MRP5

Hyaluronan is overproduced in many diseases including metastasis, inflammation or ischemia, but there is no drug to attenuate hyaluronan production. Hyaluronan is exported from fibroblasts by the multidrug resistance associated protein 5 (MRP5) which is inhibited by the plant phenols curcumin or xanthohumol. We performed virtual docking and chemical synthesis of analogues to optimize the inhibitors. The AutoDock software was used to identify the binding cavity within the open conformation of MRP5. Inhibitory plant phenols bound to the ATP binding site between the two nucleotide binding domains NBD1 and NBD2. This binding cavity was chosen to screen about 120 derivatives and analogues. The superior hyaluronan export inhibitor was 1,5-bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadien-3-one (hylin). It inhibited hyaluronan export from fibroblasts with an IC50 of 4.9 μM. Hylin is a minor component in natural curcumin preparations and has previously been described as anti-metastatic and anti-inflammatory. Since curcumin itself is unstable under physiological conditions, the active component for many cell biological and pharmaceutical effects of natural curcumin preparations could be hylin that acts by hyaluronan export inhibition.

Regulation of intracellular pH by glycosaminoglycans

The intracellular pH is regulated by a delicate balance of ion distribution across the plasma membrane and the physico-chemical properties of intra- and extracellular components. We analyzed the effects of glycosaminoglycans on the intracellular pH of fibroblasts by using the fluorescent pH indicator BCECF-AM. Addition of hyaluronan, hyaluronan oligosaccharides, chondroitin sulfate, or heparin to the culture medium of fibroblasts caused intracellular acidification from pH 7.2 to 6.7 in a concentration dependent manner. High molecular weight hyaluronan acidified more than hyaluronan oligosaccharides at the same concentrations. Hyaluronidase treatment or inhibition of hyaluronan export with xanthohumol led to intracellular alkalization. These observations indicated that extracellular glycosaminoglycans participate in intracellular pH regulation. The mechanism was explained by Donnan effects and molecular crowding.

Hyaluronan Export through Plasma Membranes Depends on Concurrent K+ Efflux by Kir Channels

Hyaluronan is synthesized within the cytoplasm and exported into the extracellular matrix through the cell membrane of fibroblasts by the MRP5 transporter. In order to meet the law of electroneutrality, a cation is required to neutralize the emerging negative hyaluronan charges. As we previously observed an inhibiting of hyaluronan export by inhibitors of K+ channels, hyaluronan export was now analysed by simultaneously measuring membrane potential in the presence of drugs. This was done by both hyaluronan import into inside-out vesicles and by inhibition with antisense siRNA. Hyaluronan export from fibroblast was particularly inhibited by glibenclamide, ropivacain and BaCl2 which all belong to ATP-sensitive inwardly-rectifying Kir channel inhibitors. Import of hyaluronan into vesicles was activated by 150 mM KCl and this activation was abolished by ATP. siRNA for the K+ channels Kir3.4 and Kir6.2 inhibited hyaluronan export. Collectively, these results indicated that hyaluronan export depends on concurrent K+ efflux.

Recovery of ΔF508‐CFTR function by analogs of hyaluronan disaccharide

We recently discovered that hyaluronan was exported from fibroblasts by MRP5 and from epithelial cells by cystic fibrosis (CF) transmembrane conductance regulator (CFTR) that was known as a chloride channel. On this basis we developed membrane permeable analogs of hyaluronan disaccharide as new class of compounds to modify their efflux. We found substances that activated hyaluronan export from human breast cancer cells. The most active compound 2-(2-acetamido-3,5-dihydroxyphenoxy)-5-aminobenzoic acid (Hylout4) was tested for its influence on the activity of epithelial cells. It activated the ion efflux by normal and defective ΔF508-CFTR. It also enhanced the plasma membrane concentration of the ΔF508-CFTR protein and reduced the transepithelial resistance of epithelial cells. In human trials of healthy persons, it caused an opening of CFTR in the nasal epithelium. Thus compound Hylout4 is a corrector that recovered ΔF508-CFTR from intracellular degradation and activated its export function.

Regulation of cell volume by glycosaminoglycans

Cell volume is regulated by a delicate balance between ion distribution across the plasma membrane and the osmotic properties of intra- and extracellular components. Using a fluorescent calcein indicator, we analysed the effects of glycosaminoglycans on the cell volume of hyaluronan producing fibroblasts and hyaluronan deficient HEK cells over a time period of 30 h. Exogenous glycosaminoglycans induced cell blebbing after 2 min and swelling of fibroblasts to about 110% of untreated cell volume at low concentrations which decreased at higher concentrations. HEK cells did not show cell blebbing and responded by shrinking to 65% of untreated cell volume. Heparin induced swelling of both fibroblasts and HEK cells. Hyaluronidase treatment or inhibition of hyaluronan export led to cell shrinkage indicating that the hyaluronan coat maintained fibroblasts in a swollen state. These observations were explained by the combined action of the Donnan effect and molecular crowding.

53 Hyaluronan export by CFTR is defective in patients with cystic fibrosis and can be recovered by analogs of hyaluronan disaccharide

53 Hyaluronan export by CFTR is defective in patients with cystic fibrosis and can be recovered by analogs of hyaluronan disaccharide

Inhibition of P‐glycoprotein facilitated glycosaminoglycan accumulation during chondrogenesis of human bone marrow mesenchymal stem cells

P-glycoprotein (P-gp) is an adenosine-5-triphosphate Binding Cassettes B 1 (ABCB1) transporter that exports various substrates on cellular membrane. Surface expression of P-gp was decreased during chondrogenesis of human bone marrow mesenchymal stem cells (BM-MSCs). We examined the role of P-gp in extracellular matrix deposition during chondrogenesis of human BM-MSCs. BM-MSCs were isolated from 16 volunteers after informed consent and incubated for 28 days using three-dimensional culture methods in chondrogenic medium with and without P-gp inhibitor (verapamil, 10 μmol/L). Hematoxylin and eosin staining revealed a cartilaginous structure with chondrogenic cells in the lacunae after 2 weeks of culture. Total glycosaminoglycan (GAG) content was increased and rose during pellet culture. Hyaluronan (HA) content of the culture medium decreased with P-gp inhibitor. Type II collagen deposition decreased with P-gp inhibitor. Inhibition of P-gp facilitated GAG accumulation via HA export inhibition during chondrogenic differentiation of human BM-MSCs. Modulation of P-gp expression during chondrogenesis would be a possible therapeutic approach for articular cartilage regeneration.

Inhibitors of hyaluronan export from hops prevent osteoarthritic reactions

An early reaction in osteoarthritic chondrocytes is hyaluronan overproduction followed by proteoglycan loss and collagen degradation. We recently found that hyaluronan is exported by the ATP-binding cassette transporter multidrug resistance associated protein 5 (MRP5) in competition with cGMP and that some phosphodiesterase 5 inhibitors also inhibited hyaluronan export. These inhibitors also prevented osteoarthritic reactions in cartilage. In an effort to identify the improved inhibitors directed primarily toward MRP5, we analyzed the flavonoids. Prenylflavonoids from hop xanthohumol, isoxanthohumol and 8-prenylnaringenin inhibited MRP5 export at lower concentrations than phosphodiesterase 5 activity. They were analyzed for their effect on IL-induced osteoarthritic reactions in bovine chondrocytes. Xanthohumol was the superior compound to inhibit hyaluronan export, as well as proteoglycan and collagen loss. It also prevented the shedding of metalloproteases into the culture medium. It directly inhibited MRP5, because it reduced the export of the MRP5 substrate fluorescein immediately and did not influence the hyaluronan synthase activity. Xanthohumol may be a natural compound to prevent hyaluronan overproduction and subsequent reactions in osteoarthritis.

Cystic Fibrosis Transmembrane Conductance Regulator Can Export Hyaluronan

Objectives: Hyaluronan, a major water binding component of the extracellular matrix, is synthesised within the cytosol and exported across the plasma membrane by the ABC-transporter MRP5 in fibroblasts. Although its synthesis is vital for embryogenesis, MRP5-deficient mice are without phenotype, suggesting that another transporter had substituted for the MRP5 protein. Thus, we searched for a compensatory exporter in fibroblasts from MRP5 deficient mice and found that cystic fibrosis transmembrane conductance regulator (CFTR) mRNA was upregulated. Methods: Hyaluronan export was measured in cell culture. The CFTR transporter was knocked out using si-RNA. Blockers of the ABC-transporter family were used to ascertain the hyaluronan transport capabilities functionally. Results: CFTR specific siRNA inhibited hyaluronan export. The tetrasaccharide was exported in undegraded form only from normal human epithelial cells and not from human epithelial cells carrying ΔF508 CFTR. The CFTR inhibitors GlyH-101 and CFTR₁₇₂ reduced hyaluronan export from CFTR-expressing mouse fibroblasts and from human breast cancer cell lines. Bronchial secretions from patients with cystic fibrosis that consist mainly of necrotic epithelia contained at least 40-fold higher concentration of hyaluronan than secretions from patients with acute bronchitis. Conclusions: CFTR transports hyaluronan across the plasma membrane of epithelial cells and this transport mechanism is defective in cystic fibrosis patients.

ABC transporters do not contribute to extracellular translocation of hyaluronan in human breast cancer in vitro

Extracellular translocation of the polysaccharide, hyaluronan (HA) has been thought to be mediated via its transmembrane synthetic enzyme, hyaluronan synthase (HAS) but recent studies have indicated that the ATP-Binding-Cassette (ABC) transporter, MRP5 contributes to this process. Liberated and cell-associated HA contributes to breast cancer initiation and progression, and therefore the inhibition of ABC transporters and consequently HA transport could provide therapeutic benefit in the treatment of breast cancer. Quantitation of ABC transporter genes, MRP1-5, BCRP and MDR1 were determined in six breast cancer cell lines selected for their differential HA synthetic rates. Low endogenous expression of transporters was detected but no significant correlation existed between ABC transporter and HAS gene expression or HA production. A dose titration of up to ten times the IC50 of ten small molecule ABC transporter inhibitors did not significantly inhibit HA export in four breast cancer cell lines. Unlike the changes observed after inhibition of HA synthesis by the characterised inhibitor 4-MU, inhibition of ABC transporters did not alter the cell morphology, HA glycocalyx or the intracellular quantity or localisation of HA. Collectively these data indicate that ABC transporters do not contribute to the extracellular transport of HA in breast cancer, supporting a role for the hyaluronan synthase in translocation.

Inhibition of hyaluronan export attenuates cell migration and metastasis of human melanoma

When secreted from malignant cells, hyaluronan facilitates tumor invasion and metastasis, as inhibition of its export by zaprinast inhibited metastasis formation in mice. However, the precise steps of the metastatic cascade, which were influenced by zaprinast, have not been identified as yet. Here we analyzed the cell biological effects of the inhibitor on three human melanoma cell lines that differed in their hyaluronan production and their metastatic capability when xenografted into SCID mice. We measured the influence of zaprinast on cellular hyaluronan export, surface coat formation, proliferation, random migration, colony formation in soft agar, adhesion, and transepithelial resistance. Concentrations of zaprinast not affecting cell proliferation, adhesion and transepithelial resistance, nevertheless reduced hyaluronan export by 50%, surface coat formation, random migration, and colony formation in soft agar. These results indicate that hyaluronan enhances metastasis formation primarily in those steps of the metastatic cascade, which involves tumor cell migration.

Inhibition of hyaluronan export reduces collagen degradation in interleukin-1 treated cartilage

BackgroundOsteoarthrosis is characterized by cartilage erosion, proteolysis of aggrecan and collagen, and disturbed rates of synthesis of aggrecan and hyaluronan by chondrocytes, with hyaluronan over-production being an early reaction. We considered that inhibition of hyaluronan export might prevent subsequent proteoglycan loss and collagen degradation.MethodsTo test this hypothesis, we studied a tissue culture model using bovine cartilages explants activated with IL-1α to induce osteoarthritic reactions using the phosphodiesterase-5 inhibitors tadalafil, zaprinast and vardenafil.ResultsThese drugs inhibited hyaluronan export, but they did not inhibit hyaluronan synthase activity. Simultaneously, they inhibited proteoglycan loss and collagen degradation, but not their synthesis. They also reduced the release of gelatinases into the culture media and diffusion of the indicator protein horseradish peroxidase through the cartilage explants. The mechanism of action of these compounds may be through inhibition of hyaluronan exporter multidrug resistance-associated protein 5 (MRP5), because the effective drug concentrations were much higher than required for phosphodiesterase-5 inhibition and intracellular cGMP accumulation.ConclusionInhibition of hyaluronan over-production may be an appropriate target to attenuate IL-1-induced reactions in osteoarthritic cartilage.

Hyaluronan Export by the ABC Transporter MRP5 and Its Modulation by Intracellular cGMP

Hyaluronan must be exported from its site of synthesis, the inner side of plasma membrane, to the extracellular matrix. Here, we identified the multidrug-associated protein MRP5 as the principle hyaluronan exporter from fibroblasts. The expression of the MRP5 (ABC-C5) transporter was silenced in fibroblasts using RNA interference, and a dose-dependent inhibition of hyaluronan export was observed. Hyaluronan oligosaccharides introduced into the cytosol competed with the export of endogenously labeled hyaluronan and the MRP5 substrate fluorescein. Because cGMP is a physiological substrate of MRP5, the intracellular concentrations of cGMP were modulated by the drugs 3-isobutyl-1-methylxanthin, propentofyllin, L-NAME, zaprinast, and bromo-cGMP, and the effects on hyaluronan export were analyzed. Increasing the cGMP levels inhibited hyaluronan export and decreasing it afforded higher concentrations of zaprinast to inhibit the export. Thus, cGMP may be a physiological regulator of hyaluronan export at the level of the export MRP5.

Inhibition of hyaluronan export from human fibroblasts by inhibitors of multidrug resistance transporters

In a previous report we described the export of hyaluronan from Streptococcus pyogenes by an ABC transporter. Extending these findings a sequence homology search against human proteins revealed a strong homology to the multidrug resistance transporter ABC-B (MDR-1) and ABC-C (MRP 5). Using several inhibitors directed against these and other transporters, a decreased hyaluronan production in cell culture as well as in hyaluronan synthase activity in purified membrane fractions was observed. The inhibitory capacity (IC 50 concentrations) was compared the with reported IC 50- or the K i-concentrations for individual transporters. These analyses revealed that hyaluronan is synthesized within the cytoplasm of mammalian cells and actively secreted into the pericellular space by energy dependent transport proteins. While inhibition of several transport proteins resulted in a decrease of hyaluronan export, inhibition of the MRP5 transporter was the most effective one to decrease hyaluronan in the cell culture supernatant indicating that hyaluronan export is one physiological role of this transport protein.