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.

Hyaluronan-mediated mononuclear leukocyte binding to gingival fibroblasts.

OBJECTIVES: Binding of mononuclear leukocytes to hyaluronan cable structures is a well-known pathomechanism in several chronic inflammatory diseases, but has not yet described for chronic oral inflammations. The aim of this study was to evaluate if and how binding of mononuclear leukocytes to pathologic hyaluronan cable structures can be induced in human gingival fibroblasts. MATERIAL AND METHODS: Experiments were performed with human gingival fibroblasts and peripheral blood mononuclear cells (PBMCs) from three healthy blood donors. Gingival fibroblasts were stimulated with (1) tunicamycin, (2) polyinosinic/polycytidylic acid (Poly:IC), and (3) lipopolysaccharides (LPS) to simulate (1) ER stress and (2) viral and (3) bacterial infections, respectively. Fibroblasts were then co-incubated with PBMCs, and the number of bound and fluorescently labeled PBMCs was assessed using a fluorescence reader and microscopy. For data analysis, a linear mixed model was used. RESULTS: Hyaluronan-mediated binding of PBMCs to gingival fibroblasts was increased by tunicamycin and Poly(I:C) but not by LPS. Hyaluronidase treatment and co-incubation with hyaluronan transport inhibitors reduced this binding. CONCLUSIONS: Results suggest that hyaluronan-mediated binding of blood cells might play a role in oral inflammations. A potential superior role of viruses needs to be confirmed in further clinical studies. CLINICAL RELEVANCE: The linkage between pathological hyaluronan matrices and oral infections opens up potential applications of hyaluronan transport inhibitors in the treatment of chronic oral inflammations.

Is hyaluronan deposition in the stroma of pancreatic ductal adenocarcinoma of prognostic significance?

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis and the number of PDAC-related deaths is rising. Recently the tumour stroma and in particular one of its main components, hyaluronan (HA), have attracted considerable attention as intravenous hyaluronidase treatment together with conventional chemotherapy considerably prolonged survival in HA-rich PDA patients. We therefore wanted to investigate the prognostic significance of HA deposition in PDA using both antibodies to HA and hyaluronan binding protein (HABP). MATERIAL AND METHODS: Tissue microarrays of PDAs of 184 patients and pancreatic xenografts tumours were immunohistochemically (IHC) stained for HA using either biotinylated hyaluronic acid binding protein (HABP) or anti-HA antibody. RESULTS: The pattern of staining with HABP differed significantly from that with antibody IHC. Antibody staining was found both within cancer cells and in the extracellular matrix and staining could not be eliminated by hyaluronidase predigestion of the tissue sections. In contrast, HABP staining was generally confined to the extracellular matrix and was completely abolished by hyaluronidase pretreatment. HA positivity as determined by HABP was associated with larger primary tumours (p = 0.046). There were no correlations between overall survival, disease-free survival and HA expression. CONCLUSION: Presence of HA alone is not of prognostic importance in PDAC, and IHC with utilization of antibody detection shows no reliable staining pattern and should not be applied for HA IHC.

Role of HYAL1 expression in primary breast cancer in the formation of brain metastases.

BACKGROUND: The incidence of brain metastases in breast cancer patients has increased in the last years. However, the knowledge about tumor cell invasion in the brain is still very limited. Based on our recent study on cDNA microarray data of breast cancer patients, we hypothesized that two enzymes involved in the hyaluronan metabolism, namely, hyaluronan synthase 2 (HAS2) and hyaluronidase 1 (HYAL1) are associated with brain metastases formation. METHODS: Protein expression levels of hyaluronan, HAS2, and HYAL1 were analyzed in primary breast cancer, and metastatic tissue samples from different localizations (brain, bone, skin, liver, and lung) were included in four different cohorts by immunohistochemistry. Correlations of expression levels with clinical and pathological parameters were performed within the individual cohorts. RESULTS: Higher HYAL1 expression was detected among primary tumors from patients with subsequent brain metastases compared with those without brain metastases (p = 0.011). Interestingly, brain metastatic tissue showed a significantly reduced HYAL1 expression compared with the corresponding primary tumor (p = 0.003). HYAL1 expression in brain metastases was also significantly lower than in skin, liver, and lung metastases. Further, hyaluronan staining in brain metastases was mainly located on the surface of the tumor cells, whereas in all other metastatic sites hyaluronan was only detected in the extracellular matrix. We could not show an association of HAS2 with the formation of brain metastases. CONCLUSIONS: In conclusion, our results suggest that the enzyme HYAL1 plays a role in tumor dissemination and brain-specific colonization, rather than in subsequent metastatic out-growth.

Positive side effects of Ca antagonists for osteoarthritic joints-results of an in vivo pilot study.

BACKGROUND: We have shown previously that some calcium antagonists inhibit hyaluronan export, loss of proteoglycans, and degradation of collagen from osteoarthritic cartilage. Clinically approved calcium antagonists normally are prescribed for cardiac arrhythmia. In the present study, we compared the effect of these drugs on osteoarthritic patients which had received no medication and patients which were also diagnosed for cardiac arrhythmias and were treated with calcium antagonists. The effects and the side effects of the used drugs were analyzed. METHOD: We used the Lequesne questionnaire to examine patients with osteoarthritis (212 patients, control group receiving no calcium antagonists) and patients with cardiac arrhythmia and osteoarthritis (188 patients treated with various calcium antagonists). The answers of the questionnaires were transformed into the Lequesne scoring system quantifying the severity of the disease. The Lequesne score is a standardized questionnaire focused on osteoarthritis. It is a 24-scale questionary in which low scores indicate low functional activity. RESULTS: The data showed that the mean score of the control group (6.2) was higher than the treated group (5.2), the drugs differed in their efficiency. Verapamil had a slightly worse score and Azupamil, Escor, Felodipine, and Nifedipine showed no alteration. Adalat, Amlodipine, Carmen, Nitrendipin, and Norvasc lead to an improvement. CONCLUSION: These results suggest that inhibition of hyaluronan export may have a beneficial effect on human osteoarthritis.

Adsorption of glycosaminoglycans to the cell surface is responsible for cellular donnan effects.

In previous publications, we showed that extracellular glycosaminoglycans reduced the membrane potential, caused cell blebbing and swelling and decreased the intracellular pH independently of cell surface receptors. These phenomena were explained by Donnan effects. The effects were so large that they could not be attributed to glycosaminoglycans in solution. Therefore, we tested the hypothesis that glycosaminoglycans were concentrated on the cell membrane and analysed the mechanism of adsorption by fluorescent hyaluronan, chondroitin sulphate and heparin. The influence of the CD44 receptor was evaluated by comparing CD44 expressing human fibroblasts with CD44 deficient HEK cells. Higher amounts of glycosaminoglycans adsorbed to fibroblasts than to HEK cells. When the membrane potential was annihilated by substituting NaCl by KCl in the medium, adsorption was reduced and intracellular pH decrease was abolished. To eliminate other cellular interfering factors, potential-dependent adsorption was demonstrated for hyaluronan which adsorbed to inert gold foils in physiological salt concentrations at pH 7.2 and surface potentials up to 120 mV. From these results, we conclude that large cellular Donnan effects of glycosaminoglycans results from receptor mediated, hydrophobic and ionic adsorption to cell surfaces.

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 K(ir) 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 BaCl(2) which all belong to ATP-sensitive inwardly-rectifying K(ir) 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 K(ir)3.4 and K(ir)6.2 inhibited hyaluronan export. Collectively, these results indicated that hyaluronan export depends on concurrent K(+) efflux.

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.

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.

Hyaluronan in intra-operative edema of NF1-associated neurofibromas.

The tumor suppressor disorder neurofibromatosis type 1 (NF1) is associated with development of multiple neurofibromas which may grow intraneurally as plexiform neurofibromas (PNF) or intracutaneously (CNF). Upon surgery neurofibromas may show prominent swelling hindering skin-edge approximation. To assess whether the water binding glycosaminoglycan hyaluronan is involved in intra-operative swelling, 51 neurofibromas from 33 NF1-patients were investigated. Hyaluronan was histologically demonstrated and was quantified by ELISA. Molecular weight of hyaluronan was determined by gel filtration. Further, hyaluronan content was measured in cultivated Schwann cells and fibroblasts. Clinically, 67% of PNF were associated with moderate or severe intra-operative swelling, whereas only 36% of CNF showed this feature. Significantly higher levels of hyaluronan content were found in PNF compared to CNF (P < 0.05). Mast cell density did not correlate with any of the parameters. Molecular weight of hyaluronan in PNF and CNF ranged from higher than 106 Da to approximately 105 Da. Fibroblasts produced less hyaluronan than Schwann cells. The findings support the view that hyaluronan plays an important role in intra-operative swelling in neurofibroma surgery.

Inhibitors of hyaluronan export from hops prevent osteoarthritic reactions.

SCOPE: 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. METHODS AND RESULTS: 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. CONCLUSIONS: Xanthohumol may be a natural compound to prevent hyaluronan overproduction and subsequent reactions in osteoarthritis.

Depolarization of the membrane potential by hyaluronan.

The membrane potential is mainly maintained by the K(+) concentration gradient across the cell membrane between the cytosol and the extracellular matrix. Here, we show that extracellular addition of high-molecular weight hyaluronan depolarized the membrane potential of human fibroblasts, human embryonic kidney cells (HEK), and central nervous system neurons in a concentration-dependent manner, whereas digestion of cell surface hyaluronan by hyaluronidase caused hyperpolarization. This effect could not be achieved by other glycosaminoglycans or hyaluronan oligosaccharides, chondroitin sulfate, and heparin which did not affect the membrane potential. Mixtures of high-molecular weight hyaluronan and bovine serum albumin had a larger depolarization effect than expected as the sum of both individual components. The different behavior of high-molecular weight hyaluronan versus hyaluronan oligosaccharides and other glycosaminoglycans can be explained by a Donnan effect combined with a steric exclusion of other molecules from the water solvated chains of high-molecular weight hyaluronan. Depolarization of the plasma membrane by hyaluronan represents an additional pathway of signal transduction to the classical CD44 signal transduction pathway, which links the extracellular matrix to intracellular metabolism.

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 DeltaF508 CFTR. The CFTR inhibitors GlyH-101 and CFTR(172) 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.

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.

BACKGROUND: Osteoarthrosis 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. METHODS: To test this hypothesis, we studied a tissue culture model using bovine cartilages explants activated with IL-1alpha to induce osteoarthritic reactions using the phosphodiesterase-5 inhibitors tadalafil, zaprinast and vardenafil. RESULTS: These 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. CONCLUSION: Inhibition 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.

Biosynthesis of hyaluronan: direction of chain elongation.

The mechanism of hyaluronan biosynthesis in vertebrates had been proposed to occur at the reducing end of growing chains. This mechanism was questioned because a recombinant synthase appeared to add new monosaccharides to the non-reducing end. I reinvestigated this problem with membranes from the eukaryotic B6 cell line. The membranes were incubated with UDP-[3H]GlcNAc and UDP-[14C]GlcA to yield differentially labelled reducing terminal and non-reducing terminal domains. Digestion of the product with a mixture of the exoglycosidases beta-glucuronidase and beta-N-acetylglucosaminidase truncated the hyaluronan chain strictly from the non-reducing end. The change in 3H/14C ratio of the remaining hyaluronan fraction, during the course of exoglycosidase digestion, confirmed the original results that the native eukaryotic synthase extended hyaluronan at the reducing end. This mechanism demands that the UDP-hyaluronan terminus is bound to the active site within the synthase and should compete with the substrates for binding. Accordingly, increasing substrate concentrations enhanced hyaluronan release from the synthase. A model is proposed that explains the direction of chain elongation at the reducing end by the native synthase and at the non-reducing end by the recombinant synthase based on a loss of binding affinity of the synthase towards the growing UDP-hyaluronan chain.

Hyaluronate and its receptors in bone marrow.

Cell-cell and cell-matrix interactions, which are mediated by cell adhesion molecules, play a fundamental role during many cellular processes including growth, differentiation, cell migration and cancer metastasis. One molecule playing a major role in these processes is the CD44 surface receptor, which is expressed in a wide range of cells including many cells of the hemopoietic system, where it mediates the interaction with its major ligand, hyaluronate. However, little is known about CD44 and hyaluronate in bone marrow and this was investigated immunohistochemically in trephine biopsies and in cultivated human bone marrow stromal cells. In biopsy specimens, patches of hyaluronate deposition were detected in the extracellular matrix (ECM). However, most of the areas of the ECM were devoid of hyaluronate. Single mast cells and lymphocytes scattered throughout the marrow were CD44 immunopositive. Marrow-derived stromal cells (MDSC) expanded in cell culture were immunopositive for CD44, hyaluronate synthase, and hyaluronate. Hence, a marked difference between CD44 immunolocalisation and hyaluronate deposition can be observed between in situ and under cell culture conditions. Since in normal marrow in situ the number of CD44 immunopositive cells was low, interactions of CD44 and hyaluronate would appear to not to play a major role in cell adhesion in the normal bone marrow.