The expression pattern of hyaluronan synthase during human tooth development.

In previous studies, hyaluronan (HA) and its major cell surface receptor CD44 have been suggested to play an important role during tooth development. HA synthases (HASs) are the enzymes that polymerize hyaluronan. Data on the expression pattern of HASs during tooth development is lacking and the aim of the present study was to investigate the localisation of HAS by immunohistochemistry in human tooth germs from different developmental stages. The distribution pattern of HAS in the various tissues of the "bell stage" tooth primordia corresponded to that of hyaluronan in most locations: positive HAS immunoreactivity was observed in the dental lamina cells, inner- and outer-enamel epithelium. On the stellate reticulum cells, moderate HAS signal was observed, similar to the layers of the oral epithelium, where faint HAS immunoreactivity was detected. At the early phase of dental hard tissues mineralization, strong HAS immunoreactivity was detected in the odontoblasts and their processes, as well as in the secretory ameloblasts and their apical processes and also, the pulpal mesenchymal cells. The HAS signals observed in odontoblasts and ameloblasts gradually decreased with age. Our results demonstrate that hyaluronan synthesised locally by different dental cells and these results provide additional indirect support to the suggestion that HA may contribute both to the regulation of tooth morphogenesis and dental hard tissue formation.

CD44H and the isoforms CD44v5 and CD44v6 in the synovial fluid of the osteoarthritic human knee joint.

OBJECTIVE: To determine whether the concentrations of CD44H and v5 and v6 in the synovial fluid are correlated with the presence of accompanying synovitis in the osteoarthritic joint and with the grade of osteoarthritis. DESIGN: Using antero-posterior and lateral X-rays of the knee joint and patellar view of 46 patients were graded with the Kellgren & Lawrence scale. Synovial fluid from these patients with different grades of primary osteoarthritis of the knee joint with and without synovial inflammation (synovitis) was collected during surgical procedures. A horseradish peroxidase conjugated anti-CD44H-, anti-sCD44v5- or anti-sCD44v6-antibody was added and labeled with tetramethylbenzidine. The absorbance was measured at wavelengths of 450/620 nm. Regression analysis was performed and the statistical significance was assessed, using the Student t-test for unequal variance. RESULTS: CD44H and v5 and v6 were detected in the synovial fluid of all 46 patients. Osteoarthritic patients with synovial inflammation showed significantly higher levels of CD44H and v6, but not v5, than osteoarthritic patients without synovial inflammation. With progression of osteoarthritis from Kellgren grade II to III, CD44v5 decreased significantly. All other isoform concentrations remained statistically unchanged. CONCLUSIONS: CD44H and the isoforms v5 and v6 were present in the synovial fluid of osteoarthritic patients. Their concentrations do not reflect the osteoarthritic stage in the Kellgren grading scale. CD44H and CD44v6, but not CD44v5, are significantly up-regulated in osteoarthritic synovial inflammation.

Hyaluronan reduces migration and proliferation in CHO cells.

Expression of the hyaluronan synthase gene in hyaluronan-deficient CHO cells changed the cell morphology from a spindle shape to a flattened epithelial-type form. Hyaluronan producing CHO cells showed reduced initial cell adhesion, migration, proliferation and density at contact inhibition, but no difference in random migration determined by the Boyden chamber assay. Addition of hyaluronan to the medium of CHO cells reduced migration, proliferation and initial cell adhesion. In contrast, coating the plastic dish with hyaluronan enhanced initial cell adhesion. These results are discussed in the context of the perplexing properties of hyaluronan on cellular functions.

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-1beta, TNF-alpha, 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-alpha 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.

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.

An ectoprotein kinase of group C streptococci binds hyaluronan and regulates capsule formation.

A 56-kDa protein had been isolated and cloned from protoplast membranes of group C streptococci that had erroneously been identified as hyaluronan synthase. The function of this protein was reexamined. When streptococcal membranes were separated on an SDS-polyacrylamide gel and renatured, a 56-kDa protein was detected that had kinase activity for a casein substrate. When this recombinant protein was expressed in Escherichia coli and incubated in the presence of [32P]ATP, it was responsible for phosphorylation of two proteins with 30 and 56 kDa that were not present in the control lysate. The 56-kDa protein was specifically phosphorylated in an immunoprecipitate of a detergent extract of the recombinant E. coli lysate with antibodies against the 56-kDa protein, indicating that it was autophosphorylated. The E. coli lysate containing the recombinant protein could bind hyaluronan, and hyaluronan binding was abolished by the addition of ATP. Kinetic analysis of hyaluronan synthesis and release from isolated protoplast membranes indicated that phosphorylation by ATP stimulated hyaluronan release and synthesis. Incubation of membranes with antibodies to the 56-kDa protein increased hyaluronan release. The addition of [32P]ATP to intact streptococci led to rapid phosphorylation of two proteins, 56 and 75 kDa each at threonine residues. This phosphorylation was neither observed with [32P]phosphate nor in the presence of trypsin, indicating that the kinase was localized extracellularly. The addition of ATP to growing group C streptococci led to increased hyaluronan synthesis and release. However marked differences were found between group A and group C streptococci. Antibodies against the 56-kDa protein from group C streptococci did not recognize proteins from group A strains, and a homologous DNA sequence could not be detected by polymerase chain reaction or Southern blotting. In addition, Group A streptococci did not retain a large hyaluronan capsule like group C strains. These results indicated that the 56-kDa protein is an ectoprotein kinase specific for group C streptococci that regulates hyaluronan capsule shedding by phosphorylation.

Dexamethasone treatment decreases hyaluronan-formation by primate trabecular meshwork cells in vitro.

The functional significance of Hyaluronan (HA) present in the cribriform layer of Schlemm's canal is not known. It may contribute to the actual outflow resistance but the relatively inert molecule might also be necessary to prevent adherence of larger molecules to the cribriform network. Thus HA might rather prevent an increase in outflow resistance. It is well known that treatment with Dexamethasone (DM) in a number of patients leads to an increase in intraocular pressure presumably due to an increase in outflow resistance. To clarify whether an imbalance in HA formation might be involved in these changes we have treated confluent cultures of human trabecular cells as well as control cell lines (ciliary muscle cells, scleral fibroblasts) with 500 nM DM for 24 hr or 12 days and have measured HA-synthesis using incorporation assays with 0.05 m D-[6-3H] Glucosamine hydrochloride. In all six cell lines investigated there was a significant decrease in HA-synthesis following short and long term treatment with DM when compared with the untreated controls. This reaction of trabecular cells to DM treatment is different from the DM effect reported on the synthesis of many other components of the extracellular matrix like fibronectin and elastin which increase after DM treatment. If the DM-effect seen in cell cultures plays a role in vivo decreased formation in HA could result in impaired function of the outflow pathways.

A mild purification method for polysaccharide binding membrane proteins: phase separation of digitonin extracts to isolate the hyaluronate synthase from Streptococcus sp. in active form.

A new method was developed to purify the streptococcal hyaluronate synthase in active form to electrophoretic homogeneity. The method is based on the extraction of protoplast membranes with digitonin and a phase separation into an aqueous and a detergent phase induced by addition of polyethylene glycol 6000 at 0 degree C. Proteins bound to hyaluronate were enriched in the aqueous phase, whereas other membrane proteins resided in the detergent phase. Final purification of the hyaluronate synthase was achieved by ion exchange chromatography.

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 snythase 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.

Antibodies against proteins of streptococcal hyaluronate synthase bind to human fibroblasts and are present in patients with rheumatic fever.

Antibodies directed against the streptococcal 42 kDa hyaluronate synthase and a 56 kDa auxiliary protein bound to the surface of intact human fibroblasts in vitro. Staining was most prominent during the detachment phase of mitosis. In eukaryotic plasma membranes a 52 kDa protein was recognized by the antiserum against the 56 kDa streptococcal protein. Since the cross-reacting proteins could be involved in immunological mimicry between streptococcal and human antigens leading to heart cell necrosis, the reactivity of sera from patients with rheumatic fever was compared with that of sera from healthy or streptococcal infected persons. The sera from patients with rheumatic fever showed a higher reactivity against the 56 kDa protein than those from healthy persons or from patients with an antibiotic treated streptococcal infection. This difference was not observed for the 42 kDa protein. These sera were able to lead to cell lysis in the presence of complement.

Synthesis and degradation of hyaluronate by synovia from patients with rheumatoid arthritis.

OBJECTIVE: Hyaluronate degradation was analyzed in cultures of healthy tissue and tissue obtained from patients with rheumatoid arthritis. METHODS: Arthritic and healthy synovial tissues were incubated in culture with [3H]glucosamine. Labelled hyaluronate was extracted and its size determined by gel filtration. The production of low molecular weight hyaluronate was analyzed by pulse-chase experiments. Radical production was measured by a cytochrome C reduction assay. RESULTS: Healthy tissues and some arthritic tissues that did not contain significant amounts of granulocytes produced high molecular weight hyaluronate. In contrast, arthritic tissue infiltrated with granulocytes released low molecular weight hyaluronate. Pulse-chase experiments suggested that hyaluronate was degraded in these arthritic tissues. Exogenous hyaluronate was degraded only by intact tissue, but not by cells in culture obtained from synovial membranes of synovial fluids. Hyaluronate degradation was accompanied by massive oxygen radical production. Radical scavengers protected hyaluronate from degradation in synovial tissue. Some protection was achieved by superoxide and catalase or by methionine and complete protection by the iron chelators diethyltriaminepentacetic acid or deferoxamine mesylate. CONCLUSION: Degradation of hyaluronate in arthritic synovial tissue may be inhibited in tissue culture by radical scavengers.

Alterations in hyaluronan synthesis during developing joint cavitation.

The mechanisms essential for generating diarthrodial joint cavities between skeletal elements in developing limbs remain enigmatic. Histochemical localization of hyaluronan (HA) at joint interzones concomitant with cavitation led to the postulation that HA may be pivotal in this process. HA synthesis involves the transfer of UDP-glucuronate and UDP-N-acetyl glucosamine to nascent HA by HA synthase. Uridine diphosphoglucose dehydrogenase (UDPGD) activity is responsible for the prior conversion of UDP-glucose to UDP-glucuronate. We have assessed the relationship between the appearance of HA and enzyme activities (quantitatively where possible) involved in HA synthesis during metatarsophalangeal joint development in embryonic chicks. Microspectrophotometric assessment of UDPGD activity using an in situ biochemical assay indicated that cells immediately adjacent to forming cavities contained increased UDPGD activity, which was subsequently maintained after cavitation. Immunocytochemistry showed that high levels of expression of HA synthase were localized to these same cells. In addition, radiolabeled sulfate autoradiography showed that cells bordering developing cavities incorporated relatively little sulfate, suggesting that UDP-glucuronate is utilized in the synthesis of undersulfated or non-sulfated glycosaminoglycans. These results indicate that the differentiation of cells bordering presumptive spaces may involve alterations associated specifically with differential synthesis of HA, which appears to be a primary event in joint cavity formation.

Interaction of biglycan with type I collagen.

The small proteoglycan decorin is known to interact with type I collagen fibrils, thereby influencing the kinetics of fibril formation and the distance between adjacent collagen fibrils. The structurally related proteoglycan biglycan has been proposed not to bind to fibrillar collagens. However, when osteosarcoma cells were cultured on reconstituted type I collagen fibrils, both decorin and biglycan were retained by the matrix. Immunogold labeling at the electron microscopic level showed that both proteoglycans were distributed along collagen fibrils not only in osteosarcoma cell-populated collagen lattices but also in human skin. Reconstituted type I collagen fibrils were able to bind in vitro native and N-glycan-free biglycan as well as recombinant biglycan core protein. From Scatchard plots dissociation, constants were obtained that were higher for glycanated biglycan (8.7 x 10(-8) mol/liter) than for glycanated decorin (7 x 10(-10) mol/liter and 3 x 10(-9) mol/liter, respectively). A similar number of binding sites for either proteoglycan was calculated. Recombinant biglycan and decorin were characterized by lower dissociation constants compared with the glycanated forms. Glycanated as well as recombinant decorin competed with glycanated biglycan for collagen binding, suggesting that identical or adjacent binding sites on the fibril are used by both proteoglycans. These data suggest that, because of its trivalency, biglycan could have a special organizing function on the assembly of the extracellular matrix.

Intracellular signal transduction for serum activation of the hyaluronan synthase in eukaryotic cell lines.

Hyaluronan synthase was activated in B6 cells or 3T3 fibroblasts by foetal calf serum with maximal activity after 6 h. Activation was inhibited by cycloheximide or by the protein kinase inhibitors H-7 or H-8, indicating that transcription as well as phosphorylation was required for activation. The activation by serum was markedly prolonged, when serum was added together with cholera toxin or theophylline. Without serum stimulation the hyaluronan synthase could also be activated by phorbol-12-myristate-13-acetate, by dibutyryl-c-AMP, or by forskolin. Increasing the intracellular Ca-ion concentration with a Ca-ionophore also led to an activation. The activation of the drugs was not synergistic. In isolated plasma membranes the synthase activity could be decreased by phosphatase treatment and enhanced by ATP in B6 cells and by ATP in the presence of phorbol-12-myristate-13-acetate in 3T3 fibroblasts. Stimulation correlated with increased transcription and phosphorylation of the 52 kD hyaluronan synthase at serine residues. The results led to the conclusion that hyaluronan synthase is induced by transcription and activated by phosphorylation by protein kinase C, c-AMP-dependent protein kinases, or Ca-ion-dependent protein kinases.

CD44 variant isoforms are preferentially expressed in basal epithelial of non-malignant human fetal and adult tissues.

CD44 is a transmembrane glycoprotein, which can exist in a multitude of isoforms due to alternative splicing of the pre-mRNA. We have generated monoclonal antibodies to several of these variant regions, which are encoded by 10 additional exons in the extracellular part of the molecule. CD44 variant isoforms have been reported to be involved in the malignant progression of rat and human tumours. The precise localization of CD44 variant isoforms in normal developmental and morphogenetic processes is essential for diagnostic studies of human tumorigenesis. Therefore, we have analysed a large number of different human tissues by immunohistochemistry for the expression of CD44 isoforms containing either exons 4v, 6v or 9v. Expression of exon 9v-isoforms was detected in almost all epithelia analysed, with a few exceptions. Exon 6v isoforms are expressed only in squamous and glandular epithelial, e.g. skin epidermis, sweat and sebaceous glands, oesophagus, ducts of the mammary gland, salivary and prostate glands. Detection of exon 4v-encoded isoforms was restricted to the epidermis and the oesophagus. Similar tissue distributions of CD44 variant isoforms were observed in 10-week-old fetal tissues. Since one of the ligands of CD44 is hyaluronic acid (HA), we also analysed the tissue distribution of HA synthetase. HA synthetase was detected in all tissues analysed, showing good correlation with the expression of the standard form of CD44, CD44s.

Immunoelectrophoretic pattern of native mucosal intracellular glycoproteins of hog healthy and drug-intoxicated stomachs and of hog body fluids.

Naturally occurring glycoproteins have been extracted from fundic and antral mucosal tissue of the hog stomach by means of nondegrading techniques. Major and retarded glycoprotein fractions separated by gel filtration were further dissociated from appreciable amounts of noncovalently bound proteins by CsCl density gradient centrifugation. Antisera to glycoprotein fractions of fundic and antral regions of the stomach were prepared in rabbits. The major fractions from both gastric regions have similar molecular mass (approximately 2 x 10(6)), sedimentation coefficient (approximately 31.5 s), and specific viscosity (approximately 1.6). Purified fractions from each region were further separated into two subfractions by affinity chromatography on wheat germ lectin. Glycoprotein subfractions from antrum and fundus differ appreciably in their carbohydrate and amino acids content, share antigenic determinants, but do not cross-react with anti-hog serum protein antisera. Further diversity in native mucin glycoproteins was observed by the use of one-(D) and two-dimensional (2D) immunoelectrophoresis; subfractions that cross-react with specific anti-hog gastric glycoproteins were found to contain three or more components. D-Immunoelectrophoretic analyses demonstrated (1) in vivo degradation of glycoprotein components of the major fundic fraction isolated from mucosal tissue of alcohol/acetyl salicylate-intoxicated hog stomachs and (2) in vitro catabolism of major fundic glycoproteins by corresponding mitochondrial lysosomal (ML) acid hydrolases. Furthermore, 2D-immunoelectrophoretic analyses showed that (1) hog synovial fluid and plasma proteins have similar prosthetic moieties as either reacted with anti-hog serum proteins antisera. Nonetheless, locations, shapes, and staining intensities of the immunoprecipitate lines differed, which is indicative of different structures of the carbohydrate moieties of components of synovial fluid and plasma proteins, and (2) only a minor fraction of hog cerebrospinal fluid cross-reacted with anti-hog serum protein antisera. This is contrary to the generally accepted deduction based on high-resolution 2D-electrophoresis, indicative of different compositional patterns of plasma and cerebrospinal fluids.

Hyaluronan synthase immunoreactivity in the anterior segment of the primate eye.

The anterior segment of human and cynomolgus monkey eyes was investigated for the presence of hyaluronan (HA) synthesizing cells using a polyclonal antibody against the enzyme HA synthase (HAS). In the chamber angle region the most intense staining was seen in the cell membranes of the corneal endothelium and in monkey eyes in the cells covering the posterior extension of the cornea (the operculum). The trabecular meshwork cells of the uveal and inner corneoscleral lamellae were also intensely stained. On the other hand, no staining was observed in the trabecular cells of the outer corneoscleral and the cribriform meshwork. The cell membranes of the inner wall endothelium of Schlemm's canal were labelled only at their luminal surface. In the iris stroma and the trabeculum ciliare (the ciliary body band), labelled cells were also found, whereas the connective tissue of the ciliary muscle and the muscle itself did not contain HAS-positive cells. In the ciliary processes immunoreactivity was seen in the non-pigmented epithelial cells (NPE) covering the anterior tips of the processes, suggesting that HA found in the aqueous humor is produced by these cells. The pars plana NPE showed the most intense staining in the cells directly adjacent to the ora serrata region. The hyalocytes found in the neighborhood of the pars plana also showed intense HAS immunoreactivity. It is likely that both hyalocytes and NPE cells of the posterior pars plana release HA into the vitreous.

The hyaluronate synthase from a eukaryotic cell line.

The hyaluronate synthase complex was identified in plasma membranes from B6 cells. It contained two subunits of molecular masses 52 kDa and 60 kDa which bound the precursor UDP-GlcA in digitonin solution and partitioned into the aqueous phase, together with nascent hyaluronate upon Triton X-114 phase separation. The 52 kDa protein cross-reacted with poly- and monoclonal antibodies raised against the streptococcal hyaluronate synthase and the 60 kDa protein was recognized by monoclonal antibodies raised against a hyaluronate receptor. The 52 kDa protein was purified to homogeneity by affinity chromatography with monoclonal anti-hyaluronate synthase.

Hyaluronate synthase: cloning and sequencing of the gene from Streptococcus sp.

The complete nucleotide sequence of hyaluronate synthase from Streptococcus sp. and its flanking regions is presented. The gene locus was designated has. Southern-blotting results suggested that the gene was conserved in hyaluronate-producing streptococci. A putative translation-initiation codon was identified and the open reading frame consists of 1566 bp, specifying a protein of 56 kDa. Sequences resembling the promoter and ribosome-binding site of Gram-positive organisms are found upstream of the synthase. The predicted amino-acid sequence reveals the presence of a 35-residue signal peptide. The sequence has some similarity to bacterial peptide-binding proteins.