Analysis of UDP-Sugars from Cultured Cells and Small Tissue Samples.

UDP-sugars are important substrates for the synthesis of various cellular glycans and glycoconjugates, many of which play essential roles in the pathobiology of diseases associated with deranged glucose metabolism, such as cancer and type 2 diabetes. Hence, their analysis from cultured cells and especially from tissue samples can give valuable information. This chapter describes a method for UDP-sugar isolation from various sources utilizing ion-pair solid-phase extraction with graphitized carbon cartridges, and their analysis using anion-exchange high-performance liquid chromatography.

Activated hyaluronan metabolism in the tumor matrix - Causes and consequences.

Hyaluronan accumulates in the stroma of several solid tumors and promotes their progression. Both enhanced synthesis and fragmentation of hyaluronan are required as a part of this inflammatory process resembling wound healing. Increased expression of the genes of hyaluronan synthases (HAS1-3) are infrequent in human tumors, while posttranslational modifications that activate the HAS enzymes, and glucose shunted to the UDP-sugar substrates HASs, can have crucial contributions to tumor hyaluronan synthesis. The pericellular hyaluronan influences virtually all cell-cell and cell-matrix interactions, controlling migration, proliferation, apoptosis, epithelial to mesenchymal transition, and stem cell functions. The catabolism by hyaluronidases and free radicals appears to be as important as synthesis for the inflammation that promotes tumor growth, since the receptors mediating the signals create specific responses to hyaluronan fragments. Targeting hyaluronan metabolism shows therapeutic efficiency in animal experiments and early clinical trials.

Single-Molecule Unbinding Forces between the Polysaccharide Hyaluronan and Its Binding Proteins.

The extracellular polysaccharide hyaluronan (HA) is ubiquitous in all vertebrate tissues, where its various functions are encoded in the supramolecular complexes and matrices that it forms with HA-binding proteins (hyaladherins). In tissues, these supramolecular architectures are frequently subjected to mechanical stress, yet how this affects the intermolecular bonding is largely unknown. Here, we used a recently developed single-molecule force spectroscopy platform to analyze and compare the mechanical strength of bonds between HA and a panel of hyaladherins from the Link module superfamily, namely the complex of the proteoglycan aggrecan and cartilage link protein, the proteoglycan versican, the inflammation-associated protein TSG-6, the HA receptor for endocytosis (stabilin-2/HARE), and the HA receptor CD44. We find that the resistance to tensile stress for these hyaladherins correlates with the size of the HA-binding domain. The lowest mean rupture forces are observed for members of the type A subgroup (i.e., with the shortest HA-binding domains; TSG-6 and HARE). In contrast, the mechanical stability of the bond formed by aggrecan in complex with cartilage link protein (two members of the type C subgroup, i.e., with the longest HA-binding domains) and HA is equal or even superior to the high affinity streptavidin⋅biotin bond. Implications for the molecular mechanism of unbinding of HA⋅hyaladherin bonds under force are discussed, which underpin the mechanical properties of HA⋅hyaladherin complexes and HA-rich extracellular matrices.

Extracellular ATP activates hyaluronan synthase 2 (HAS2) in epidermal keratinocytes via P2Y2, Ca2+ signaling, and MAPK pathways.

Extracellular nucleotides are used as signaling molecules by several cell types. In epidermis, their release is triggered by insults such as ultraviolet radiation, barrier disruption, and tissue wounding, and by specific nerve terminals firing. Increased synthesis of hyaluronan, a ubiquitous extracellular matrix glycosaminoglycan, also occurs in response to stress, leading to the attractive hypothesis that nucleotide signaling and hyaluronan synthesis could also be linked. In HaCaT keratinocytes, ATP caused a rapid and strong but transient activation of hyaluronan synthase 2 (HAS2) expression via protein kinase C-, Ca2+/calmodulin-dependent protein kinase II-, mitogen-activated protein kinase-, and calcium response element-binding protein-dependent pathways by activating the purinergic P2Y2 receptor. Smaller but more persistent up-regulation of HAS3 and CD44, and delayed up-regulation of HAS1 were also observed. Accumulation of peri- and extracellular hyaluronan followed 4-6 h after stimulation, an effect further enhanced by the hyaluronan precursor glucosamine. AMP and adenosine, the degradation products of ATP, markedly inhibited HAS2 expression and, despite concomitant up-regulation of HAS1 and HAS3, inhibited hyaluronan synthesis. Functionally, ATP moderately increased cell migration, whereas AMP and adenosine had no effect. Our data highlight the strong influence of adenosinergic signaling on hyaluronan metabolism in human keratinocytes. Epidermal insults are associated with extracellular ATP release, as well as rapid up-regulation of HAS2/3, CD44, and hyaluronan synthesis, and we show here that the two phenomena are linked. Furthermore, as ATP is rapidly degraded, the opposite effects of its less phosphorylated derivatives facilitate a rapid shut-off of the hyaluronan response, providing a feedback mechanism to prevent excessive reactions when more persistent signals are absent.

Transmembrane Pickets Connect Cyto- and Pericellular Skeletons Forming Barriers to Receptor Engagement.

Phagocytic receptors must diffuse laterally to become activated upon clustering by multivalent targets. Receptor diffusion, however, can be obstructed by transmembrane proteins ("pickets") that are immobilized by interacting with the cortical cytoskeleton. The molecular identity of these pickets and their role in phagocytosis have not been defined. We used single-molecule tracking to study the interaction between Fcγ receptors and CD44, an abundant transmembrane protein capable of indirect association with F-actin, hence likely to serve as a picket. CD44 tethers reversibly to formin-induced actin filaments, curtailing receptor diffusion. Such linear filaments predominate in the trailing end of polarized macrophages, where receptor mobility was minimal. Conversely, receptors were most mobile at the leading edge, where Arp2/3-driven actin branching predominates. CD44 binds hyaluronan, anchoring a pericellular coat that also limits receptor displacement and obstructs access to phagocytic targets. Force must be applied to traverse the pericellular barrier, enabling receptors to engage their targets.

Human Keratinocytes Respond to Extracellular UTP by Induction of Hyaluronan Synthase 2 Expression and Increased Hyaluronan Synthesis.

The release of nucleotides into extracellular space is triggered by insults like wounding and ultraviolet radiation, resulting in stimulatory or inhibitory signals via plasma membrane nucleotide receptors. As similar insults are known to activate hyaluronan synthesis we explored the possibility that extracellular UTP or its breakdown products UDP and UMP act as mediators for hyaluronan synthase (HAS) activation in human epidermal keratinocytes. UTP increased hyaluronan both in the pericellular matrix and in the culture medium of HaCaT cells. 10-100 µm UTP strongly up-regulated HAS2 expression, although the other hyaluronan synthases (HAS1, HAS3) and hyaluronidases (HYAL1, HYAL2) were not affected. The HAS2 response was rapid and transient, with the maximum stimulation at 1.5 h. UDP exerted a similar effect, but higher concentrations were required for the response, and UMP showed no stimulation at all. Specific siRNAs against the UTP receptor P2Y2, and inhibitors of UDP receptors P2Y6 and P2Y14, indicated that the response to UTP was mediated mainly through P2Y2 and to a lesser extent via UDP receptors. UTP increased the phosphorylation of p38, ERK, CREB, and Ser-727 of STAT3 and induced nuclear translocation of pCaMKII. Inhibitors of PKC, p38, ERK, CaMKII, STAT3, and CREB partially blocked the activation of HAS2 expression, confirming the involvement of these pathways in the UTP-induced HAS2 response. The present data reveal a selective up-regulation of HAS2 expression by extracellular UTP, which is likely to contribute to the previously reported rapid activation of hyaluronan metabolism in response to tissue trauma or ultraviolet radiation.

Hexosamine biosynthesis in keratinocytes: roles of GFAT and GNPDA enzymes in the maintenance of UDP-GlcNAc content and hyaluronan synthesis.

UDP-N-acetylglucosamine (UDP-GlcNAc) is a glucose metabolite with pivotal functions as a key substrate for the synthesis of glycoconjugates like hyaluronan, and as a metabolic sensor that controls cell functions through O-GlcNAc modification of intracellular proteins. However, little is known about the regulation of hexosamine biosynthesis that controls UDP-GlcNAc content. Four enzymes can catalyze the crucial starting point of the pathway, conversion of fructose-6-phosphate (Fru6P) to glucosamine-6-phosphate (GlcN6P): glutamine-fructose-6-phosphate aminotransferases (GFAT1 and 2) and glucosamine-6-phosphate deaminases (GNPDA1 and 2). Using siRNA silencing, we studied the contributions of these enzymes to UDP-GlcNAc content and hyaluronan synthesis in human keratinocytes. Depletion of GFAT1 reduced the cellular pool of UDP-GlcNAc and hyaluronan synthesis, while simultaneous blocking of both GNPDA1 and GDPDA2 exerted opposite effects, indicating that in standard culture conditions keratinocyte GNPDAs mainly catalyzed the reaction from GlcN6P back to Fru6P. However, when hexosamine biosynthesis was blocked by GFAT1 siRNA, the effect by GNPDAs was reversed, now catalyzing Fru6P towards GlcN6P, likely in an attempt to maintain UDP-GlcNAc content. Silencing of these enzymes also changed the gene expression of related enzymes: GNPDA1 siRNA induced GFAT2 which was hardly measurable in these cells under standard culture conditions, GNPDA2 siRNA increased GFAT1, and GFAT1 siRNA increased the expression of hyaluronan synthase 2 (HAS2). Silencing of GFAT1 stimulated GNPDA1 and GDPDA2, and inhibited cell migration. The multiple delicate adjustments of these reactions demonstrate the importance of hexosamine biosynthesis in cellular homeostasis, known to be deranged in diseases like diabetes and cancer.

Fluorescence resonance energy transfer (FRET) and proximity ligation assays reveal functionally relevant homo- and heteromeric complexes among hyaluronan synthases HAS1, HAS2, and HAS3.

In vertebrates, hyaluronan is produced in the plasma membrane from cytosolic UDP-sugar substrates by hyaluronan synthase 1-3 (HAS1-3) isoenzymes that transfer N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcUA) in alternative positions in the growing polysaccharide chain during its simultaneous extrusion into the extracellular space. It has been shown that HAS2 immunoprecipitates contain functional HAS2 homomers and also heteromers with HAS3 (Karousou, E., Kamiryo, M., Skandalis, S. S., Ruusala, A., Asteriou, T., Passi, A., Yamashita, H., Hellman, U., Heldin, C. H., and Heldin, P. (2010) The activity of hyaluronan synthase 2 is regulated by dimerization and ubiquitination. J. Biol. Chem. 285, 23647-23654). Here we have systematically screened in live cells, potential interactions among the HAS isoenzymes using fluorescence resonance energy transfer (FRET) and flow cytometric quantification. We show that all HAS isoenzymes form homomeric and also heteromeric complexes with each other. The same complexes were detected both in Golgi apparatus and plasma membrane by using FRET microscopy and the acceptor photobleaching method. Proximity ligation assays with HAS antibodies confirmed the presence of HAS1-HAS2, HAS2-HAS2, and HAS2-HAS3 complexes between endogenously expressed HASs. C-terminal deletions revealed that the enzymes interact mainly via uncharacterized N-terminal 86-amino acid domain(s), but additional binding site(s) probably exist in their C-terminal parts. Of all the homomeric complexes HAS1 had the lowest and HAS3 the highest synthetic activity. Interestingly, HAS1 transfection reduced the synthesis of hyaluronan obtained by HAS2 and HAS3, suggesting functional cooperation between the isoenzymes. These data indicate a general tendency of HAS isoenzymes to form both homomeric and heteromeric complexes with potentially important functional consequences on hyaluronan synthesis.

Decreased hyaluronidase 1 expression is associated with early disease recurrence in human endometrial cancer.

OBJECTIVE: Hyaluronidases (HYAL1 and HYAL2) are key enzymes in the degradation of hyaluronan, and their expression has been altered in various cancer types. We previously showed that hyaluronan accumulation in endometrial carcinomas was correlated with decreased mRNA expression of the HYAL genes. In this study, we analyzed HYAL1 and HYAL2 protein expressions in normal and precancerous endometrial tissues and in endometrial carcinomas. We also investigated whether the protein levels were associated with clinicopathological factors, invasion, and disease recurrence. METHODS: A total of 343 tissue specimens from normal, atrophic, hypertrophic, and neoplastic endometria were analyzed immunohistochemically for HYAL1 and HYAL2 expressions. The results were correlated with clinicopathological factors, the expression of the epithelial-mesenchymal transition marker, E-cadherin, and disease recurrence. RESULTS: Reduced HYAL1 expression was associated with the progression of endometrial carcinomas towards higher grades and also with large tumor sizes, lymph node metastasis, and lymphovascular invasion. Reduced expression of both HYAL1 and HYAL2 was associated with deep myometrial invasion. HYAL2 expression was primarily constant in neoplastic tissues, but its expression was altered in different phases of the endometrial cycle. In addition, a reduction in HYAL1 expression was associated with the depletion of E-cadherin. In a multivariate analysis, reduced HYAL1 expression was an independent prognostic factor for early disease recurrence (HR 5.13, 95% CI: 1.131-23.270, p=0.034). CONCLUSIONS: This study showed that reduced HYAL1 expression was associated with endometrial carcinoma aggressiveness, which further supported the role of hyaluronan degradation in cancer progression.

Isothiocyanate analogs targeting CD44 receptor as an effective strategy against colon cancer.

Inflammatory pathway plays an important role in tumor cell progression of colorectal cancers. Although colon cancer is considered as one of the leading causes of death worldwide, very few drugs are available for its effective treatment. Many studies have examined the effects of specific COX-2 and 5-LOX inhibitors on human colorectal cancer, but the role of isothiocyanates (ITSCs) as COX-LOX dual inhibitors engaged in hyaluronan-CD44 interaction has not been studied. In the present work, we report series of ITSC analogs incorporating bioisosteric thiosemicarbazone moiety. These inhibitors are effective against panel of human colon cancer cell lines including COX-2 positive HCA-7, HT-29 cells lines, and hyaluronan synthase-2 (Has2) enzyme over-expressing transformed intestinal epithelial Apc10.1Has2 cells. Specifically, our findings indicate that HA-CD44v6-mediated COX-2/5-LOX signaling mediate survivin production, which in turn, supports anti-apoptosis and chemo-resistance leading to colon cancer cell survival. The over-expression of CD44v6shRNA as well as ITSC treatment significantly decreases the survival of colon cancer cells. The present results thus offer an opportunity to evolve potent inhibitors of HA synthesis and CD44v6 pathway and thus underscoring the importance of the ITSC analogs as chemopreventive agents for targeting HA/CD44v6 pathway.

Extracellular UDP-glucose activates P2Y14 Receptor and Induces Signal Transducer and Activator of Transcription 3 (STAT3) Tyr705 phosphorylation and binding to hyaluronan synthase 2 (HAS2) promoter, stimulating hyaluronan synthesis of keratinocytes.

Hyaluronan, a major matrix molecule in epidermis, is often increased by stimuli that enhance keratinocyte proliferation and migration. We found that small amounts of UDP-sugars were released from keratinocytes and that UDP-glucose (UDP-Glc) added into keratinocyte cultures induced a specific, rapid induction of hyaluronan synthase 2 (HAS2), and an increase of hyaluronan synthesis. The up-regulation of HAS2 was associated with JAK2 and ERK1/2 activation, and specific Tyr(705) phosphorylation of transcription factor STAT3. Inhibition of JAK2, STAT3, or Gi-coupled receptors blocked the induction of HAS2 expression by UDP-Glc, the latter inhibitor suggesting that the signaling was triggered by the UDP-sugar receptor P2Y14. Chromatin immunoprecipitations demonstrated increased promoter binding of Tyr(P)(705)-STAT3 at the time of HAS2 induction. Interestingly, at the same time Ser(P)(727)-STAT3 binding to its response element regions in the HAS2 promoter was unchanged or decreased. UDP-Glc also stimulated keratinocyte migration, proliferation, and IL-8 expression, supporting a notion that UDP-Glc signals for epidermal inflammation, enhanced hyaluronan synthesis as an integral part of it.

Rab10-mediated endocytosis of the hyaluronan synthase HAS3 regulates hyaluronan synthesis and cell adhesion to collagen.

Hyaluronan synthases (HAS1-3) are unique in that they are active only when located in the plasma membrane, where they extrude the growing hyaluronan (HA) directly into cell surface and extracellular space. Therefore, traffic of HAS to/from the plasma membrane is crucial for the synthesis of HA. In this study, we have identified Rab10 GTPase as the first protein known to be involved in the control of this traffic. Rab10 colocalized with HAS3 in intracellular vesicular structures and was co-immunoprecipitated with HAS3 from isolated endosomal vesicles. Rab10 silencing increased the plasma membrane residence of HAS3, resulting in a significant increase of HA secretion and an enlarged cell surface HA coat, whereas Rab10 overexpression suppressed HA synthesis. Rab10 silencing blocked the retrograde traffic of HAS3 from the plasma membrane to early endosomes. The cell surface HA coat impaired cell adhesion to type I collagen, as indicated by recovery of adhesion following hyaluronidase treatment. The data indicate a novel function for Rab10 in reducing cell surface HAS3, suppressing HA synthesis, and facilitating cell adhesion to type I collagen. These are processes important in tissue injury, inflammation, and malignant growth.

OGT and OGA expression in postmenopausal skeletal muscle associates with hormone replacement therapy and muscle cross-sectional area.

Protein glycosylation via O-linked N-acetylglucosaminylation (O-GlcNAcylation) is an important post-translational regulatory mechanism mediated by O-GlcNAc transferase (OGT) and responsive to nutrients and stress. OGT attaches an O-GlcNAc moiety to proteins, while O-GlcNAcase (OGA) catalyzes O-GlcNAc removal. In skeletal muscle of experimental animals, prolonged increase in O-GlcNAcylation associates with age and muscle atrophy. Here we examined the effects of hormone replacement therapy (HRT) and power training (PT) on muscle OGT and OGA gene expression in postmenopausal women generally prone to age-related muscle weakness. In addition, the associations of OGT and OGA gene expressions with muscle phenotype were analyzed. Twenty-seven 50-57-year-old women participated in a yearlong randomized placebo-controlled trial: HRT (n=10), PT (n=8) and control (n=9). OGT and OGA mRNA levels were measured from muscle samples obtained at baseline and after one year. Knee extensor muscle cross-sectional area (CSA), knee extension force, running speed and vertical jumping height were measured. During the yearlong intervention, HRT suppressed the aging-associated upregulation of OGT mRNA that occurred in the controls. The effects of PT were similar but weaker. HRT also tended to increase the OGA mRNA level compared to the controls. The change in the ratio of OGT to OGA gene expressions correlated negatively with the change in muscle CSA. Our results suggest that OGT and OGA gene expressions are associated with muscle size during the critical postmenopausal period. HRT and PT influence muscle OGT and OGA gene expression, which may be one of the mechanisms by which HRT and PT prevent aging-related loss of muscle mass.

Hyaluronan production enhances shedding of plasma membrane-derived microvesicles.

Many cell types secrete plasma membrane-bound microvesicles, suggested to play an important role in tissue morphogenesis, wound healing, and cancer spreading. However, the mechanisms of their formation have remained largely unknown. It was found that the tips of long microvilli induced in cells by overexpression of hyaluronan synthase 3 (HAS3) were detach into the culture medium as microvesicles. Moreover, several cell types with naturally active hyaluronan synthesis released high numbers of plasma membrane-derived vesicles, and inhibition of hyaluronan synthesis reduced their formation. The vesicles contained HAS, and were covered with a thick hyaluronan coat, a part of which was retained even after purification with high-speed centrifugation. HAS3 overexpressing MDCK cells cultured in a 3-D matrix as epithelial cysts released large amounts of HAS- and hyaluronan-positive vesicles from their basal surfaces into the extracellular matrix. As far as we know, hyaluronan synthesis is one of the first molecular mechanisms shown to stimulate the production of microvesicles. The microvesicles have a potential to deliver the hyaluronan synthase machinery and membrane and cytoplasmic materials to other cells, influencing tissue regeneration, inflammation and tumor progression.

Mannose reduces hyaluronan and leukocytes in wound granulation tissue and inhibits migration and hyaluronan-dependent monocyte binding.

Wound healing is a highly regulated process starting from coagulation and ending in tissue remodeling. The end result varies from perfectly restored tissue, such as in early fetal skin, to scars in adults. The balanced repair process is frequently disturbed by local or systemic factors, like infections and diabetes. A rapid increase of hyaluronan is an inherent feature of wounds and is associated with tissue swelling, epithelial and mesenchymal cell migration and proliferation, and induction of cytokine signaling. Hyaluronan extending from cell surface into structures called cables can trap leukocytes and platelets and change their functions. All these features of hyaluronan modulate inflammation. The present data show that mannose, a recently described inhibitor of hyaluronan synthesis, inhibits dermal fibroblast invasion and prevents the enhanced leukocyte binding to hyaluronan that takes place in cells treated with an inflammatory mediator interleukin-1ß. Mannose also reduced hyaluronan in subcutaneous sponge granulation tissue, a model of skin wound, and suppressed its leukocyte recruitment and tissue growth. Mannose thus seems to suppress wounding-induced inflammation in skin by attenuating hyaluronan synthesis.

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.

Hyaluronan in cytosol--microinjection-based probing of its existence and suggested functions.

Hyaluronan (HA) is a large glycosaminoglycan produced by hyaluronan synthases (HAS), enzymes normally active at plasma membrane. While HA is delivered into the extracellular space, intracellular HA is also seen, mostly in vesicular structures, but there are also reports on its presence in the cytosol and specific locations and functions there. We probed the possibility of HA localization and functions in cytosol by microinjecting fluorescent HA binding complex (fHABC), HA fragments and hyaluronidase (HYAL) into cytosol. Microinjection of fHABC did not reveal HA-specific intracellular binding sites. Likewise, specific cytosolic binding sites for HA were not detected, as microinjected fluorescent HA composed of 4-8 monosaccharide units (HA4-HA8) were evenly distributed throughout the cells, including the nucleus, but excluded from membrane-bound organelles. The largest HA tested (∼HA120 or ∼25 kDa) did not enter the nucleus, and HA10-HA28 were progressively excluded from parts of nuclei resembling nucleoli. In contrast, HA oligosaccharides endocytosed from medium remained in vesicular compartments. The activity of HA synthesis was estimated by measuring the HA coat on green fluorescent protein (GFP)-HAS3-transfected MCF-7 cells. Microinjection of HA4 reduced coat size at 4 h, but increased at 24 h after injection, while larger HA-oligosaccharides and HYAL had no influence. As a positive control, microinjection of glucose increased coat size. In summary, no evidence for the presence or function of HA in cytosol was obtained. Also, the synthesis of HA and the active site of HAS were not accessible to competition, binding and degradation by cytosolic effectors, while synthesis responded to increased substrate supply.

Synthesis and fragmentation of hyaluronan in renal ischaemia.

BACKGROUND: The turnover of hyaluronan (HA), especially the production of low-molecular-weight fragments of HA, was examined in a model of unilateral renal ischaemia-reperfusion (IR) in rats. METHODS: HA was extracted from the outer and inner stripe of the outer medulla (OSOM and ISOM) at different times following IR. Its fragmentation was measured using membrane filtration and size-exclusion chromatography. Quantitative reverse transcription-polymerase chain reaction, zymography and immunohistochemistry were used to assess the expression and localization of various forms of HA synthase (HAS) and hyaluronidase (HYAL). Macrophage infiltration was evaluated using immunohistochemistry. RESULTS: HA accumulated at Day 1 mostly as high-molecular-weight (HMW) species with an elution profile similar to a reference 2500 kDa HA and at Day 14 mostly as medium- to low-size fragments. Within 1 day, HAS1 messenger RNA was up-regulated > 50- and 35-fold in OSOM and ISOM, respectively. Thereafter, HAS1 tended to normalize, while HAS2 increased steadily. Both synthetic enzymes were localized around tubules and in the interstitium. Conversely, HYAL1, HYAL2 and global hyaluronidase activity were repressed during the first 24 h. The patterns were identical in the OSOM and ISOM despite markedly different amounts of HA at baseline. There was no obvious correlation between HA deposits and macrophage infiltration. CONCLUSIONS: In the post-ischaemic kidney, HA starts to accumulate at Day 1 mostly as HMW species. Later on, a large proportion becomes degraded into smaller fragments. This pattern is explained by coordinated changes in the expression of HA synthases and hyaluronidases, especially an early induction of HAS1. The current data open the door to timed pharmacological interventions blocking the production of HA fragments.

HAS3-induced accumulation of hyaluronan in 3D MDCK cultures results in mitotic spindle misorientation and disturbed organization of epithelium.

The amount of hyaluronan (HA) is low in simple epithelia under normal conditions, but during tumorigenesis, trauma or inflammation HA is increased on the epithelial cells and surrounding stroma. Excessive HA in epithelia is suggested to interfere with cell-cell adhesions, resulting in disruption of the epithelial barrier function. In addition, stimulated HA synthesis has been correlated with epithelial-to-mesenchymal transition and invasion of cancer cells. However, the effects of HA overload on normal epithelial morphogenesis have not been characterized in detail. Madin-Darby canine kidney (MDCK) cells form polarized epithelial cysts, when grown in a 3-dimensional (3D) matrix. These cells were used to investigate whether stimulated HA synthesis, induced by stable overexpression of GFP-HAS3, influences cell polarization and epithelial morphogenesis. GFP-HAS3 expression in polarized MDCK cells resulted in active HA secretion at apical and basolateral membrane domains. HA-deposits interfered with the formation of cell-cell junctions, resulting in impaired barrier function. In 3D cyst cultures, HA accumulated into apical lumina and was also secreted from the basal side. The HAS3-expressing cysts failed to form a single lumen and instead displayed multiple small lumina. This phenotype was correlated with aberrant mitotic spindle orientation in dividing cells. The results of this study indicate that excess pericellular HA disturbs the normal cell-cell and cell-ECM interactions in simple epithelia, leading to aberrant epithelial morphogenesis. The morphological abnormalities observed in 3D epithelial cultures upon stimulated HAS3 expression may be related to premalignant changes, including intraluminal invasion and deregulated epithelialization, probably mediated by the mitotic spindle orientation defects.

Chronic UVR causes increased immunostaining of CD44 and accumulation of hyaluronan in mouse epidermis.

Chronic intense UV radiation is the main cause of epidermal tumors. Because hyaluronan (HA), a large extracellular polysaccharide, is known to promote malignant growth, hyaluronan expression was studied in a model in which long-term UV radiation (UVR) induces epidermal tumors. Mouse back skin was exposed three times a week for 10.5 months to UVR corresponding to one minimal erythema dose, processed for histology, and stained for hyaluronan and the hyaluronan receptor CD44. This exposure protocol caused epidermal hyperplasia in most of the animals; tumors, mainly squamous cell carcinomas (SCCs), were found in ~20% of the animals. Specimens exposed to UVR showed increased hyaluronan and CD44 staining throughout the epidermal tissue. In hyperplastic areas, hyaluronan and CD44 stainings correlated positively with the degree of hyperplasia. Well-differentiated SCCs showed increased hyaluronan and CD44 staining intensities, whereas poorly differentiated tumors and dysplastic epidermis showed areas where HA and CD44 were locally reduced. The findings indicate that HA and CD44 increase in epidermal keratinocytes in the premalignant hyperplasia induced by UV irradiation and stay elevated in dysplasia and SCC, suggesting that the accumulation of hyaluronan and CD44 is an early marker for malignant transformation and may be a prerequisite for tumor formation.