Peter Prehm

Oligosaccharides of Hyaluronan Are Potent Activators of Dendritic Cells

The extracellular matrix component hyaluronan (HA) exists physiologically as a high m.w. polymer but is cleaved at sites of inflammation, where it will be contacted by dendritic cells (DC). To determine the effects of HA on DC, HA fragments of different size were established. Only small HA fragments of tetra- and hexasaccharide size (sHA), but not of intermediate size (m.w. 80,000–200,000) or high m.w. HA (m.w. 1,000,000–600,000) induced immunophenotypic maturation of human monocyte-derived DC (up-regulation of HLA-DR, B7-1/2, CD83, down-regulation of CD115). Likewise, only sHA increased DC production of the cytokines IL-1β, TNF-α, and IL-12 as well as their allostimulatory capacity. These effects were highly specific for sHA, because they were not induced by other glycosaminoglycans such as chondroitin sulfate or heparan sulfate or their fragmentation products. Interestingly, sHA-induced DC maturation does not involve the HA receptors CD44 or the receptor for hyaluronan-mediated motility, because DC from CD44-deficient mice and wild-type mice both responded similarly to sHA stimulation, whereas the receptor for hyaluronan-mediated motility is not detectable in DC. However, TNF-α is an essential mediator of sHA-induced DC maturation as shown by blocking studies with a soluble TNFR1. These findings suggest that during inflammation, interaction of DC with small HA fragments induce DC maturation.

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.

Synthesis and shedding of hyaluronan from plasma membranes of human fibroblasts and metastatic and non-metastatic melanoma cells.

The regulation of hyaluronan synthesis and shedding was analysed in human fibroblasts and in two melanoma cells that differed in the metastatic potential and proteolysis of the hyaluronan receptor CD44. Dissociation of nascent hyaluronan from plasma membranes isolated from fibroblasts by high salt concentrations led to activation of hyaluronan synthase. Hyaluronan synthesis was also enhanced in plasma membranes from fibroblasts that had been treated with hyaluronidase or trypsin. Hyaluronan oligosaccharides stimulated hyaluronan production in fibroblast cultures. These results indicated that nascent high-molecular-mass hyaluronan inhibited its own chain elongation, if it was retained in the vicinity of the synthase by cell-surface receptors. The results also indicated that increased hyaluronan synthesis and shedding correlated with proteolysis of CD44 on the melanoma cell lines, which has been observed by others.

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

BackgroundWe 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.MethodWe 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.ResultsThe 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.ConclusionThese results suggest that inhibition of hyaluronan export may have a beneficial effect on human osteoarthritis.

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.

Expression of hyaluronate and hyaluronate synthase in human primary tumours and their metastases in scid mice

Hyaluronate and hyaluronate synthase expression were examined in primary tumours and if present in metastatic deposits of human breast, colon, ovarian and small-cell lung cancer cell lines transplanted into scid mice using biotinylated hyaluronectin and immunohistochemical staining of hyaluronate synthase. Very intensive hyaluronate and hyaluronate synthase expression could be observed in peripheral areas of tumours derived from highly metastatic cell lines (HT29, MCF-7). Even smaller lung metastases of up to 15 cells showed typically a focal binding of hyaluronectin predominantly at the host-tumour interface of the metastases, indicating that increased expression is closely correlated with the degree of invasiveness and metastatic potential of malignant tumours.

CD44 exon variant 6 epitope and hyaluronate synthase are expressed on HT29 human colorectal carcinoma cells in a SCID mouse model of metastasis formation

The factors which lead to the formation of metastases are generally poorly understood; however the expression of a particular variant of the cell adhesion molecule CD44 may be important in facilitating metastasis formation in colon cancer. The aim of the present study was to investigate the expression of CD44 exon v 6 (CD44v6), hyaluronate (one of its ligands), and hyaluronate synthase, in a clinically relevant animal model of metastatic colon carcinoma. HT29 human colon carcinoma cells were injected subcutaneously between the scapulae of severe combined immunodeficient (SCID) mice and left for 3 weeks (by which time the tumours had produced metastases in the lungs). Morphological observations at the tumour-host interface were consistent with the dissociation of neoplastic cells from the primary tumours, and the ability of these cells to migrate through the extracellular matrix facilitating metastasis formation. Immunohistochemically detectable hyaluronate synthase expression was increased in vivo compared with the parent cell line in vitro. CD44v6 expression and hyaluronate were increased around single cells at the periphery of tumours compared with the central regions. CD44v6 and hyaluronate synthase expression were co-expressed in the same cells. Indeed, the present study is the first to demonstrate hyaluronate synthase expression by an epithelial cell type.

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.

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. J. Cell. Biochem. 105: 1260–1266, 2008.

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. J. Cell. Biochem. 111: 858–864, 2010.