Hyaluronan, a ubiquitous high‐molecular‐mass glycinoglycan on cell surfaces and in extracellular matrices, has a number of specific signaling functions in cell–cell communication. Changes in its content, molecular mass and turnover rate are crucial for cell proliferation, migration and apoptosis, processes that control tissue remodeling during embryonic development, inflammation, injury and cancer. To maintain tissue homeostasis, the synthesis of hyaluronan must therefore be tightly controlled. In this review, we highlight some recent data on the transcriptional regulation of hyaluronan synthase (Has1–3) expression and on the post‐transcriptional control of hyaluronan synthase activity, which, in close association with the supply of the UDP‐sugar substrates of hyaluronan synthase, adjust the rate of hyaluronan synthesis.

Background:The secretion and possible functions of extracellular UDP-sugars in epidermal keratinocytes are not known. Results: UDP-glucose activates P2Y 14 receptor and JAK2, increases STAT3 Tyr 705 phosphorylation, and enhances transcription of hyaluronan synthase 2 (HAS2). Conclusion: UDP-glucose release signals for enhanced HAS2 expression by keratinocytes. Significance: Stimulation of hyaluronan synthesis is an inherent part of epidermal keratinocyte activation and injury response.

Hyaluronan, a high molecular mass polysaccharide on the vertebrate cell surface and extracellular matrix, is produced at the plasma membrane by hyaluronan synthases using UDP-GlcNAc and UDP-GlcUA as substrates. The availability of these UDP-sugar substrates can limit the synthesis rate of hyaluronan. In this study, we show that the cellular level of UDP-HexNAc also controls hyaluronan synthesis by modulating the expression of HAS2 (hyaluronan synthase 2). Hyaluronan, a non-sulfated glycosaminoglycan present on the vertebrate cell surface and extracellular matrix, is involved in cellular functions, including migration, proliferation, adhesion, and various signaling systems, by its unique physicochemical properties and interactions with specific cell surface receptors (1). Hyaluronan is synthesized by HAS1-3, integral plasma membrane proteins that use cytosolic UDP-GlcNAc and UDPGlcUA as substrates to produce the long linear hyaluronan chains. During its synthesis, the growing hyaluronan chain runs through a pore in the plasma membrane into the extracellular matrix (2).Changes in hyaluronan production have been associated mostly with the expression level of HAS genes (3-6), especially in keratinocytes (7-13). Of the three genes, particularly HAS2 is subject to regulation by growth factors, cytokines, and hormones (4,14,15). In keratinocyte cultures, EGF, keratinocyte growth factor, TNF␣, and retinoic acid induce, whereas TGF␤ inhibits, HAS2 expression (8, 10, 13, 16). Accordingly, the HAS2 promoter has been shown to contain functional response elements (REs) 3 for different transcription factors, including retinoid acid receptor, NF-B, CREB1 (cAMP response elementbinding protein 1), and SP1 (specificity protein 1) (7,11,16).Besides by the protein expression of hyaluronan synthase (HAS) enzymes, hyaluronan synthesis is also controlled by the availability of the hyaluronan precursors, the substrates of HAS. Raising cellular UDP-GlcUA content stimulates hyaluronan synthesis, whereas a low concentration of UDP-GlcUA can limit the synthesis (12, 17). We have shown that the same applies to UDP-GlcNAc: limiting or increasing its content stimulates and inhibits, respectively, the synthesis of hyaluronan (18).The cellular content of UDP-GlcNAc makes an interesting connection between hyaluronan synthesis and cellular energy metabolism. UDP-GlcNAc is a product of the hexosamine synthesis pathway, into which 2-5% of the cellular influx of glucose is shunted (19). The rate-limiting step in hexosamine synthesis from glucose to UDP-GlcNAc is considered to be the GFAT1 (glutamine:fructose-6-phosphate amidotransferase 1) and GFAT2 isoenzymes (20). The flux of glucose through the hexosamine pathway serves as a cellular sensor of glucose availability, and it regulates the expression of a number of genes 3 The abbreviations used are: RE, response element; HAS, hyaluronan synthase; CBP, cAMP response element-binding protein-binding protein; PCAF, p300/CBP-associated factor.

The human hyaluronan synthase 2 (HAS2) gene encodes for an enzyme making hyaluronan, altered concentrations of which are associated with many pathological situations including wounding, several inflammatory conditions, and malignant tumors. In this study we showed that HAS2 is a primary target of the cAMP activator forskolin and the nuclear hormone alltrans-retinoic acid (RA). The first 2250 bp of the promoter contain three response elements (REs) for the transcription factor CREB1 as well as two REs for the nuclear receptor RAR. Chromatin immunoprecipitation and re-chromatin immunoprecipitation assays using selected fragments of the promoter containing the putative REs showed that forskolin and all-trans-RA modulate the formation of complexes between CREB1 and RAR with various co-regulators at the predicted sites. Interestingly, CREB1 complexes are regulated by all-trans-RA as are RAR complexes by forskolin. Reporter gene assays using nested promoter fragments supported these findings. Forskolin and alltrans-RA co-stimulation reduced the binding of CREB1, RAR, and the co-repressor nuclear receptor co-repressor 1 (NCoR1), but enhanced the association of co-activators MED1 and CREBbinding protein (CBP). RNA interference experiments suggested that MED1 and NCoR1 are central for the all-trans-RA induction of the HAS2 gene and CBP dominates its forskolin response. In general, our findings suggest a convergence of CREB1 and RAR signaling, and demonstrate the individual character of each RE in terms of co-regulator use.Hyaluronan is a high molecular mass linear non-sulfated polysaccharide that is a key component of the vertebrate extracellular matrix and has a variety of functions during (1) and following development (2). The unique physicochemical properties of this glycosaminoglycan and its interactions with specific cell surface receptors provide hyaluronan with a central role in cellular migration, adhesion, and proliferation (3). Hyaluronan is produced by hyaluronan synthase (HAS), 2 an enzyme that resides at the plasma membrane and delivers the growing polysaccharide directly into the extracellular space (3). Of the three members of the vertebrate HAS gene family, HAS2 is vital, whereas no phenotype has been reported for mice with deletions of HAS1 and HAS3 (1). The expression levels of HAS genes, especially HAS2, undergo large and rapid fluctuations, often controlled by growth factors and external conditions (4 -8). Although post-transcriptional regulation of HAS and its enzymatic activity has been reported (9 -11), the majority of data available suggests that hyaluronan synthesis closely correlates with the expression of HAS. Therefore, detailed information on processes that control HAS2 transription are important for understanding the biological functions of hyaluronan, and also for possible therapeutic interference in the clinical conditions involving hyaluronan.We have characterized human HAS2 as a primary retinoic acid receptor (RAR) target gene in keratinocytes (12). In addition to RARs, the transcription...

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.

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-␤-cyclodextrin (M␤CD) 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 M␤CD 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, M␤CD specifically down-regulated the expression of HAS2 but not HAS1 or HAS3. Screening of signaling proteins after M␤CD 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 M␤CD is due to down-regulation of HAS2, mediated by the phosphoinositide 3-kinase-Akt-mTOR-p70S6K pathway.Cholesterol is an important membrane constituent of mammalian cells, plasma membrane in particular. It decreases the fluidity of the membrane and is a major component of the detergent-resistant membrane microdomains called lipid rafts (1). Increased serum cholesterol and cholesterol accumulation in atherosclerotic lesions have been long associated to cardiovascular disease (2) and atherosclerosis (3, 4), respectively. Abnormal cholesterol accumulation has been also found in some malignancies, such as prostate tumors (5, 6). Furthermore, recent studies suggest that inhibition of the rate-limiting enzyme in cholesterol synthesis (3-hydroxy-3-methyl-glutarylcoenzyme A reductase) by long-term statin therapy decreases the incidence of certain tumor types (7,8).Cellular cholesterol content can be modulated by the cholesterol depleting agent methyl-␤-cyclodextrin (M␤CD) 2 (9). M␤CD disrupts lipid rafts (10) and activates many signaling proteins, like epidermal growth factor receptor (11), extracellular signal-regulated kinase (ERK) (12, 13), p38 (11,14), Src (15), and ␤-catenin (16). On the other hand, M␤CD decreases the growth of MCF-7 breast cancer and A2780 ovarian cancer cells implanted subcutaneously in nude mice (17). In the MCF-7 model M␤CD was even more effective than doxorubicin (17).Hyaluronan, a large glycosaminoglycan mainly present in the extracellular matrix of vertebrates, is composed of repeating disaccharide units containing glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc). It is produced at the plasma membrane by three hyaluronan synthases (HAS1-3). HASs use UDP-GlcUA and UDP-GlcNAc as substrates and extrude the forming hyaluronan chain to the extracellu...

Background: Hyaluronan synthases (HASs) require transport to plasma membrane for the activation of hyaluronan (HA) synthesis. Results: Rab10 overexpression inhibited, whereas Rab10 silencing increased, cell surface HA coat and HAS3-dependent hyaluronan synthesis. Conclusion: Rab10 reduces the steady-state abundance of HAS3 in the plasma membrane by enhancing HAS3 endocytosis. Significance: HA synthesis is controlled by HAS3 endocytosis mediated by Rab10.

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