Expression of Recombinant Hyaluronan Synthase (HAS) Isoforms in CHO Cells Reduces Cell Migration and Cell Surface CD44

In the present study we investigated the functional properties of the three recombinant hyaluronan synthases (HAS proteins) HAS1, HAS2, and HAS3. HAS3-transfected CHO clones exhibited the highest hyaluronan polymerization rate followed by HAS2 transfectants which were more catalytically active than HAS1 transfectants. In living cells all three HAS proteins synthesized hyaluronan chains of high molecular weight (larger than 3.9 x 10(6)). In vitro, the HAS2 isoform produced hyaluronan chains of a molecular weight larger than 3.9 x 10(6), whereas HAS3 produced polydisperse hyaluronan (molecular weight 0.12-1 x 10(6)), and HAS1 synthesized much shorter chains of an average molecular weight of 0.12 x 10(6). Thus, each HAS protein may interact with different cytoplasmic proteins which may influence their catalytic activity. CHO transfectants with the ability to synthesize about 1 microgram hyaluronan/1 x 10 (5) cells/24 h were surrounded by hyaluronan-containing coats, whereas transfectants generating about 4-fold lower amounts of hyaluronan formed coats only in the presence of chondroitin sulfate proteoglycan. An inverse correlation between hyaluronan production on the one hand and cell migration and cell surface CD44 expression on the other was found; a 4-fold lower migration and a 2-fold decrease of cell surface CD44 receptors was seen when hyaluronan production increased 1000-fold over the level in the untransfected cells. The inverse relationships between hyaluronan production and migration and CD44 expression of cells are of importance for the regulation of cell-extracellular matrix interactions.     

Mechanism of binding site conformational switching in the CD44-hyaluronan protein-carbohydrate binding interaction

The transmembrane protein CD44, which has been implicated in cancer biology and inflammation, mediates cell adhesion through multimeric interactions with the linear extracellular glycosaminoglycan hyaluronan (HA; in megadaltons). Affinity switching of CD44 from a low-affinity state to a high-affinity state is required for normal CD44 physiological function; crystal structures of the CD44 hyaluronan binding domain complexed with HA oligomers point to a conformational rearrangement at a binding site loop, leading to the formation of direct contact between the oligomer and an arginine side chain as a molecular basis for affinity switching. Here, all-atom explicit-solvent molecular dynamics simulations were used to characterize the dynamics and thermodynamics of oligomeric hyaluronan (oHA) and its two crystallographic complexes with the CD44 hyaluronan binding domain: the “A-form,” which lacks arginine-HA close contact, and the “B-form,” which has direct arginine side-chain-HA contact. From the simulations, the conformational properties of oHA are essentially unaltered in going from the unbound state to either the A-form or the B-form bound state, with the oligomer retaining its flexibility when bound and with only two of the eight monosaccharides in the oligomer maintaining uninterrupted contact with the protein. Biased simulations revealed that altering the backbone conformation of a tyrosine residue in the arginine loop can induce the A-form → B-form conformational transition and that a large free-energy barrier prevents ready interconversion between the two forms, thereby suggesting that the tyrosine backbone forms a molecular switch.     

Methyl-β-cyclodextrin Suppresses Hyaluronan Synthesis by Down-regulation of Hyaluronan Synthase 2 through Inhibition of Akt

Hyaluronan synthases (HAS1–3) are integral plasma membrane proteins that synthesize hyaluronan, a cell surface and extracellular matrix polysaccharide necessary for many biological processes. It has been shown that HAS is partly localized in cholesterol-rich lipid rafts of MCF-7 cells, and cholesterol depletion with methyl-β-cyclodextrin (MβCD) suppresses hyaluronan secretion in smooth muscle cells. However, the mechanism by which cholesterol depletion inhibits hyaluronan production has remained unknown. We found that cholesterol depletion from MCF-7 cells by MβCD inhibits synthesis but does not decrease the molecular mass of hyaluronan, suggesting no major influence on HAS stability in the membrane. The inhibition of hyaluronan synthesis was not due to the availability of HAS substrates UDP-GlcUA and UDP-GlcNAc. Instead, MβCD specifically down-regulated the expression of HAS2 but not HAS1 or HAS3. Screening of signaling proteins after MβCD treatment revealed that phosphorylation of Akt and its downstream target p70S6 kinase, both members of phosphoinositide 3-kinase-Akt pathway, were inhibited. Inhibitors of this pathway suppressed hyaluronan synthesis and HAS2 expression in MCF-7 cells, suggesting that the reduced hyaluronan synthesis by MβCD is due to down-regulation of HAS2, mediated by the phosphoinositide 3-kinase-Akt-mTOR-p70S6K pathway.     

Three isoforms of mammalian hyaluronan synthases have distinct enzymatic properties.

Three mammalian hyaluronan synthase genes, HAS1, HAS2, and HAS3, have recently been cloned. In this study, we characterized and compared the enzymatic properties of these three HAS proteins. Expression of any of these genes in COS-1 cells or rat 3Y1 fibroblasts yielded de novo formation of a hyaluronan coat. The pericellular coats formed by HAS1 transfectants were significantly smaller than those formed by HAS2 or HAS3 transfectants. Kinetic studies of these enzymes in the membrane fractions isolated from HAS transfectants demonstrated that HAS proteins are distinct from each other in enzyme stability, elongation rate of HA, and apparent K(m) values for the two substrates UDP-GlcNAc and UDP-GlcUA. Analysis of the size distributions of hyaluronan generated in vitro by the recombinant proteins demonstrated that HAS3 synthesized hyaluronan with a molecular mass of 1 x 10(5) to 1 x 10(6) Da, shorter than those synthesized by HAS1 and HAS2 which have molecular masses of 2 x 10(5) to approximately 2 x 10(6) Da. Furthermore, comparisons of hyaluronan secreted into the culture media by stable HAS transfectants showed that HAS1 and HAS3 generated hyaluronan with broad size distributions (molecular masses of 2 x 10(5) to approximately 2 x 10(6) Da), whereas HAS2 generated hyaluronan with a broad but extremely large size (average molecular mass of >2 x 10(6) Da). The occurrence of three HAS isoforms with such distinct enzymatic characteristics may provide the cells with flexibility in the control of hyaluronan biosynthesis and functions.     

Hyaluronan Synthase 1 (HAS1) Requires Higher Cellular UDP-GlcNAc Concentration than HAS2 and HAS3

Mammals have three homologous genes encoding proteins with hyaluronan synthase activity (Has1–3), all producing an identical polymer from UDP-N-acetylglucosamine and UDP-glucuronic acid. To compare the properties of these isoenzymes, COS-1 cells, with minor endogenous hyaluronan synthesis, were transfected with human Has1–3 isoenzymes. HAS1 was almost unable to secrete hyaluronan or form a hyaluronan coat, in contrast to HAS2 and HAS3. This failure of HAS1 to synthesize hyaluronan was compensated by increasing the cellular content of UDP-N-acetyl glucosamine by ∼10-fold with 1 mm glucosamine in the growth medium. Hyaluronan synthesis driven by HAS2 was less affected by glucosamine addition, and HAS3 was not affected at all. Glucose-free medium, leading to depletion of the UDP-sugars, markedly reduced hyaluronan synthesis by all HAS isoenzymes while raising its concentration from 5 to 25 mm had a moderate stimulatory effect. The results indicate that HAS1 is almost inactive in cells with low UDP-sugar supply, HAS2 activity increases with UDP-sugars, and HAS3 produces hyaluronan at high speed even with minimum substrate content. Transfected Has2 and particularly Has3 consumed enough UDP-sugars to reduce their content in COS-1 cells. Comparison of different human cell types revealed ∼50-fold differences in the content of UDP-N-acetylhexosamines and UDP-glucuronic acid, correlating with the expression level of Has1, suggesting cellular coordination between Has1 expression and the content of UDP-sugars.     

Proinflammatory Cytokines Induce Hyaluronan Synthesis and Monocyte Adhesion in Human Endothelial Cells through Hyaluronan Synthase 2 (HAS2) and the Nuclear Factor-κB (NF-κB) Pathway

Chronic inflammation is now accepted to have a critical role in the onset of several diseases as well as in vascular pathology, where macrophage transformation into foam cells contributes in atherosclerotic plaque formation. Endothelial cells (EC) have a critical function in recruitment of immune cells, and proinflammatory cytokines drive the specific expression of several adhesion proteins. During inflammatory responses several cells produce hyaluronan matrices that promote monocyte/macrophage adhesion through interactions with the hyaluronan receptor CD44 present on inflammatory cell surfaces. In this study, we used human umbilical chord vein endothelial cells (HUVECs) as a model to study the mechanism that regulates hyaluronan synthesis after treatment with proinflammatory cytokines. We found that interleukin 1β and tumor necrosis factors α and β, but not transforming growth factors α and β, strongly induced HA synthesis by NF-κB pathway. This signaling pathway mediated hyaluronan synthase 2 (HAS2) mRNA expression without altering other glycosaminoglycan metabolism. Moreover, we verified that U937 monocyte adhesion on stimulated HUVECs depends strongly on hyaluronan, and transfection with short interference RNA of HAS2 abrogates hyaluronan synthesis revealing the critical role of HAS2 in this process.     

Interleukin-1β-induced Reduction of CD44 Ser-325 Phosphorylation in Human Epidermal Keratinocytes Promotes CD44 Homomeric Complexes,Binding to Ezrin,and Extended,Monocyte-adhesive Hyaluronan Coats

The proinflammatory cytokine interleukin-1β (IL-1β) attracts leukocytes to sites of inflammation. One of the recruitment mechanisms involves the formation of extended, hyaluronan-rich pericellular coats on local fibroblasts, endothelial cells, and epithelial cells. In the present work, we studied how IL-1β turns on the monocyte adhesion of the hyaluronan coat on human keratinocytes. IL-1β did not influence hyaluronan synthesis or increase the amount of pericellular hyaluronan in these cells. Instead, we found that the increase in the hyaluronan-dependent monocyte binding was associated with the CD44 of the keratinocytes. Although IL-1β caused a small increase in the total amount of CD44, a more marked impact was the decrease of CD44 phosphorylation at serine 325. At the same time, IL-1β increased the association of CD44 with ezrin and complex formation of CD44 with itself. Treatment of keratinocyte cultures with KN93, an inhibitor of calmodulin kinase 2, known to phosphorylate Ser-325 in CD44, caused similar effects as IL-1β (i.e. homomerization of CD44 and its association with ezrin) and resulted in increased monocyte binding to keratinocytes in a hyaluronan-dependent way. Overexpression of wild type CD44 standard form, but not a corresponding CD44 mutant mimicking the Ser-325-phosphorylated form, was able to induce monocyte binding to keratinocytes. In conclusion, treatment of human keratinocytes with IL-1β changes the structure of their hyaluronan coat by influencing the amount, post-translational modification, and cytoskeletal association of CD44, thus enhancing monocyte retention on keratinocytes.     

Potential anti-osteoporotic activity of low-molecular weight hyaluronan by attenuation of osteoclast cell differentiation and function in vitro

Due to some severe side effects or lack of efficacy of currently used synthetic drugs, such as bisphosphonates (BPs), the search for new therapeutic agents that can more effectively prevent and treat osteoporosis (OP) has been an increasingly important topic of research. In this study, the low-molecular weight hyaluronan (LMW-HA, 50 kDa) produced by enzymatic degradation of high-molecular weight hyaluronan (HMW-HA, 1922 kDa) from Streptococcus zooepidemicus was evaluated in vitro for its anti-osteoclastogenic potentials using RAW 264.7 murine macrophage cells. LMW-HA (25–200 μg/ml) dose-dependently inhibited the receptor activator of NF-κB ligand (RANKL)-induced tartrate-resistance acid phosphatase (TRAP) activity and the formation of multinucleated osteoclasts. Western blot analysis showed that LMW-HA reduced the RANKL-induced expression of tumor necrosis factor receptor-associated factor 6 (TRAF6), gelsolin and c-Src-proline-rich tyrosine kinase 2 suggesting that it could inhibit actin ring formation of osteoclast cells. In addition, LMW-HA inhibited the bone resorption activity of osteoclastic cells by dose-dependently attenuating the RANKL-induced expression of carbonic anhydrase II and integrin β3. RT-PCR analysis showed that LMW-HA dose-dependently decreased the expression of osteoclast-specific genes, such as matrix metalloproteinase 9 (MMP-9) and cathepsin K, suggesting that it has potential to inhibit the differentiation of osteoclastic cells. Taken collectively, these results suggested that LMW-HA (50 kDa) has significant anti-osteoporotic activity in vitro and may be used as a potent functional ingredient in health beneficial foods or as a therapeutic agent to prevent or treat OP.     

Hyaluronan Controls the Deposition of Fibronectin and Collagen and Modulates TGF-β1 Induction of Lung Myofibroblasts

The contribution of hyaluronan-dependent pericellular matrix to TGF-β1-driven induction and maintenance of myofibroblasts is not understood. Hyaluronan is an extracellular matrix (ECM) glycosaminoglycan important in cell adhesion, proliferation and migration, and is implicated in myofibroblast formation and maintenance. Reduced turnover of hyaluronan has been linked to differentiation of myofibroblasts and potentiation of lung fibrosis. Fibronectin is a fibril forming adhesive glycoprotein that is also upregulated following induction with TGF-β1. Although they are known to bind each other, the interplay between hyaluronan and fibronectin in the pericellular matrix during myofibroblast induction and matrix assembly is not clear. This study addresses the role of hyaluronan and its interaction with fibrillar matrix components during myofibroblast formation. Hyaluronan and fibronectin were increased and co-localized in the ECM following myofibroblast induction by TGF-β1. Inhibition of hyaluronan synthesis in TGF-β1-induced lung myofibroblasts over a 4 day period with 4-methyl umbelliferone (4-MU) further enhanced myofibroblast morphology, caused increased deposition of fibronectin and type I collagen in the ECM, and increased expression of alpha-smooth muscle actin and hyaluronan synthase 2 (HAS2) mRNA. Hyaluronan oligosaccharides or hyaluronidase treatment, which more effectively disrupted the pericellular matrix, had similar effects. CD44 and β1 integrins co-localized in the cell membrane and along some stress fibers. However, CD44 and hyaluronan were specifically excluded from focal adhesions, and associated primarily with cortical actin. Time-lapse imaging of the immediate effects of hyaluronidase digestion showed that hyaluronan matrix primarily mediates attachment of membrane and cortical actin between focal contacts, suggesting that surface adhesion through hyaluronan and CD44 is distinct from focal adhesion through β1 integrins and fibronectin. Fluorescein-labeled hyaluronan bound regularly along fibronectin fibers and co-localized more with β1 integrin and less with CD44. Therefore, the hyaluronan matrix can interfere with the assembly of fibrillar ECM components, and this interplay regulates the degree of myofibroblast formation. These data also suggest that adhesion through hyaluronan matrix impacts cytoskeletal organization, and is potentially part of a clutch mechanism that regulates stick and slip of myofibroblasts by affecting the adhesion to and organization of fibronectin and collagen.     

GLP-1-related proteins attenuate the effects of mitochondrial membrane damage in pancreatic β cells

Glucagon-like peptide (GLP)-1 analog based therapies are used not only for their insulinotropic effects, but also for their pleiotropic effects that improve pancreatic β cell function. Liraglutide is a long acting derivative of human GLP-1(7–37), which is a cleavage product encompassing amino acids 7–37 of GLP-1. In this study, we examined whether Liraglutide treatment restore the glucose-stimulated mitochondrial response of β cells with chemically induced mitochondrial damage. We tested three GLP-1-related proteins: human GLP-1(1–37), GLP-1(7–37) and Liraglutide. To measure changes of the mitochondrial pH quantitatively in real-time, we have developed a bioengineered β cell line. We generated a mitochondrial damaged model by treating β cells with ethidium bromide (EtBr; 0.5 or 1 μg/mL for 48 h). EtBr treatment reduced the response to 25 mM glucose in mitochondrial pH in a dose- and time-dependent manner. GLP-1(7–37) (100 nM) enhanced the response of mitochondria to glucose stimulation in undamaged β cells. Preincubation with Liraglutide (1 nM) or GLP-1 (100 nM) for 3 h recovered the mitochondrial response to glucose in damaged β cells, however, GLP-1(7–37) (100 nM) did not. When GLP-1(7–37) was administered in stepwise increments (i.e., starting with 20 nM to reach 100 nM in 3 h), similar recovery of the mitochondrial function was observed. The results suggest that Liraglutide is effective to recover glucose-stimulated mitochondrial response in damaged β cells.     

TNF-α, IFN-γ, and IL−1β modulate hyaluronan synthase expression in human skin fibroblasts: Synergistic effect by concomital treatment with FeSO4 plus ascorbate

Several reports have shown that a number of cytokines such as tumor necrosis-α (TNF-α), interferon-γ (IFN-γ), and interleukin-β (IL-1β) are capable to induce hyaluronan sinthases (HASs) mRNA expression in different cell culture types. The obvious consequence of this stimulation is a marked increment in hyaluronan (HA) production. It has been also reported that oxidative stress, by itself, may increase HA levels. The aim of this study was to evaluate how TNF-α, IFN-γ,IL−1β, and exposition to oxidative stress may modulate HAS activities in normal human skin fibroblasts. Moreover, the effects on HAS mRNA expression of the concomitant treatment with cytokines and oxidants, and the HA concentrations after treatments, were studied. TNF-α, IFN-γ, and IL-1β were added to normal or/and exposed to FeSO₄ plus ascorbate fibroblast cultures and HAS1, HAS2 and HAS3 mRNA content, by PCR-real time, was assayed 3,h later. HA levels were also evaluated after 24,h incubation. The treatment of fibroblasts with cytokines up-regulated HASs gene expression and increased HA production. IL-1β induced HAS mRNA expression and HA production more efficiently than TNF-α and IFN-γ. The exposition of the fibroblasts with the oxidant system markedly increased HAS activities while slightly HA production. The concomitant treatment of cells with the cytokines and the oxidant was able to further enhance, in a dose dependent way, with synergistic effect on HAS mRNA expression. On the contrary HA levels resulted unaffected by the concomitant treatment, and resemble those obtained with the exposure to FeSO₄ plus ascorbate only. This lack in HA production could be due to the deleterious action of free radicals on the HA synthesis.     

Hyaluronan synthesis induces microvillus-like cell surface protrusions

Hyaluronan synthases (HASs) are plasma membrane enzymes that simultaneously elongate, bind, and extrude the growing hyaluronan chain directly into extracellular space. In cells transfected with green fluorescent protein (GFP)-tagged Has3, the dorsal surface was decorated by up to 150 slender, 3-20-microm-long microvillus-type plasma membrane protrusions, which also contained filamentous actin, the hyaluronan receptor CD44, and lipid raft microdomains. Enzymatic activity of HAS was required for the growth of the microvilli, which were not present in cells transfected with other GFP proteins or inactive GFP-Has3 mutants or in cells incubated with exogenous soluble hyaluronan. The microvilli induced by HAS3 were gradually withered by introduction of an inhibitor of hyaluronan synthesis and rapidly retracted by hyaluronidase digestion, whereas they were not affected by competition with hyaluronan oligosaccharides and disruption of the CD44 gene, suggesting independence of hyaluronan receptors. The data bring out the novel concept that the glycocalyx created by dense arrays of hyaluronan chains, tethered to HAS during biosynthesis, can induce and maintain prominent microvilli.     

Expression of hyaluronan synthase 1 and distribution of hyaluronan during follicular atresia in pig ovaries

CD44 on macrophages is recognized as a phagocytic receptor involved in the phagocytosis of apoptotic cells. Recently, we detected CD44 on macrophages in atretic follicles during atresia. In this study, we evaluated the distribution of the principal CD44 ligand hyaluronan (HA) and the expressions of HA synthases (HAS: HAS1, HAS2, and HAS3) during atresia in pig ovaries. We determined the 2139-bp sequence of Sus scrofa HAS1 and raised an anti-HAS1 polyclonal antibody. The S. scrofa HAS1 sequence contained six putative HA-binding motifs and conserved amino acid residues crucial for GlcNac transferase activity. HAS1 mRNA expression was upregulated during atresia; however, HAS2 and HAS3 mRNA expression levels were low and very low to undetectable, respectively. Western blotting showed that HAS1 was markedly upregulated during atresia. Immunohistochemical analyses revealed HAS1 distribution in theca cells of healthy and early atretic (stages I and II) follicles and in progressing atretic (stage III) follicles. Hyaluronan was visualized with the HA-binding protein; it accumulated in the theca layer during all stages and in stage III follicles. Hyaluronan assay showed a significantly increased HA concentration in follicular fluid at stage III. Flow cytometry showed HAS1 expression in 55.7% of SIRPA-positive macrophages in stage III follicles. Our results suggest that the HA concentration in follicular fluids increased during atresia and that HAS1 may be the dominant HAS protein in theca cells to produce HA in pig ovaries.     

Hyaluronan deposition and correlation with inflammation in a murine ovalbumin model of asthma

Asthma is a chronic inflammatory disease of the airways characterized by airway remodeling, which includes changes in the extracellular matrix (ECM). However the role of the ECM in mediating these changes is poorly understood. Hyaluronan (HA), a major component of the ECM, has been implicated in asthma as well as in many other biological processes. Our study investigates the processes involved in HA synthesis, deposition, localization and degradation during an acute and chronic murine model of ovalbumin (OVA)-induced allergic pulmonary inflammation. Mice were sensitized, challenged to OVA and sacrificed at various time points during an 8-week challenge protocol. Bronchoalveolar lavage (BAL) fluids, blood, and lung tissue were collected for study. RNA, HA, protein and histopathology were analyzed. Analyses of lung sections and BAL fluids revealed an early deposition and an increase in HA levels within 24 h of antigen exposure. HA levels peaked at day 8 in BAL, while inflammatory cell recovery peaked at day 6. Hyaluronan synthase (HAS)1 and HAS2 on RNA levels peaked within 2 h of antigen exposure, while hyaluronidase (HYAL)1 and HYAL2 on RNA levels decreased. Both inflammatory cell infiltrates and collagen deposition co-localized with HA deposition within the lungs. These data support a role for HA in the pathogenesis of inflammation and airway remodeling in a murine model of asthma. HA deposition appears largely due to up regulation of HAS1 and HAS2. In addition, HA appears to provide the scaffolding for inflammatory cell accumulation as well as for new collagen synthesis and deposition.     

Heregulin-mediated ErbB2-ERK signaling activates hyaluronan synthases leading to CD44-dependent ovarian tumor cell growth and migration

Heregulin (HRG)-induced cell responses are mediated by the ErbB family of tyrosine kinase receptors. In this study we have investigated HRG activation of ErbB2, extracellular signal-regulated kinase (ERK) signaling, and their role in regulating hyaluronan synthase (HAS) activity in human ovarian tumor cells (SK-OV-3.ipl cells). Immunological and biochemical analyses indicate that ErbB2, ErbB3, and ErbB4 are all expressed in SK-OV-3.ipl cells and that ErbB4 (but not ErbB3) is physically linked to ErbB2 following HRG stimulation. Furthermore, our data indicate that the HRG-induced ErbB2.ErbB4 complexes stimulate ErbB2 tyrosine kinase, which induces both ERK phosphorylation and kinase activity. The activated ERK then increases the phosphorylation of HAS1, HAS2, and HAS3. Consequently, all three HAS isozymes are activated resulting in hyaluronan (HA) production. Because HRG-mediated HAS isozyme phosphorylation/activation can be effectively blocked by either AG825 (an ErbB2 inhibitor) or thiazolidinedione compound (an ERK blocker), we conclude that ErbB2-ERK signaling and HAS isozyme phosphorylation/HA production are functionally coupled in SK-OV-3.ipl cells. HRG also promotes HA- and CD44-dependent oncogenic events (e.g. CD44-Cdc42 association, p21-activated kinase 1 activation, and p21-activated kinase 1-filamin complex formation) and tumor cell-specific behaviors in an ErbB2-ERK signaling-dependent manner. Finally, we have found that the down-regulation of HAS isozyme expression (by transfecting cells with HAS1/HAS2/HAS3-specific small interfering RNAs) not only inhibits HRG-mediated HAS phosphorylation/activation and HA production but also impairs CD44-specific Cdc42-PAK1/filamin signaling, cytoskeleton activation and tumor cell behaviors. Taken together, these findings clearly indicate that HRG activation of ErbB2-ERK signaling modulates HAS phosphorylation/activation and HA production leading to CD44-mediated oncogenic events and ovarian cancer progression.     

4-Methylumbelliferone inhibits hyaluronan synthesis by depletion of cellular UDP-glucuronic acid and downregulation of hyaluronan synthase 2 and 3

Hyaluronan accumulation on cancer cells and their surrounding stroma predicts an unfavourable disease outcome, suggesting that hyaluronan enhances tumor growth and spreading. 4-Methylumbelliferone (4-MU) inhibits hyaluronan synthesis and retards cancer spreading in experimental animals through mechanisms not fully understood. These mechanisms were studied in A2058 melanoma cells, MCF-7 and MDA-MB-361 breast, SKOV-3 ovarian and UT-SCC118 squamous carcinoma cells by analysing hyaluronan synthesis, UDP-glucuronic acid (UDP-GlcUA) content, and hyaluronan synthase (HAS) mRNA levels. The maximal inhibition in hyaluronan synthesis ranged 22-80% in the cell lines tested. Active glucuronidation of 4-MU produced large quantities of 4-MU-glucuronide, depleting the cellular UDP-GlcUA pool. The maximal reduction varied between 38 and 95%. 4-MU also downregulated HAS mRNA levels: HAS3 was 84-60% lower in MDA-MB-361, A2058 and SKOV-3 cells. HAS2 was the major isoenzyme in MCF-7 cells and lowered by 81%, similar to 88% in A2058 cells. These data indicate that both HAS substrate and HAS2 and/or HAS3 mRNA are targeted by 4-MU. Despite different target point sensitivities, the reduction of hyaluronan caused by 4-MU was associated with a significant inhibition of cell migration, proliferation and invasion, supporting the importance of hyaluronan synthesis in cancer, and the therapeutic potential of hyaluronan synthesis inhibition.