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Background: HAS isoenzymes differ in enzymatic activity and regulation. Results: HAS1 requires higher UDP-sugar concentration than HAS2 and HAS3. Conclusion: HAS1 activity is highly dependent, and its expression correlates with cellular UDP-sugar supply. Significance: Enhanced UDP-sugar levels are potential mediators of enhanced hyaluronan secretion in cancer and inflammation.

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Hyaluronan synthases (HAS) are unique plasma membrane glycosyltransferases secreting this glycosaminoglycan directly to the extracellular space. The three HAS isoenzymes (HAS1, HAS2, and HAS3) expressed in mammalian cells differ in their enzymatic properties and regulation by external stimuli, but clearly distinct functions have not been established. To overview the expression of different HAS isoenzymes during embryonic development and their subcellular localization, we immunostained mouse embryonic samples and cultured cells with HAS antibodies, correlating their distribution to hyaluronan staining. Their subcellular localization was further studied by GFP-HAS fusion proteins. Intense hyaluronan staining was observed throughout the development in the tissues of mesodermal origin, like heart and cartilages, but also for example during the maturation of kidneys and stratified epithelia. In general, staining for one or several HASs correlated with hyaluronan staining. The staining of HAS2 was most widespread, both spatially and temporally, correlating with hyaluronan staining especially in early mesenchymal tissues and heart. While epithelial cells were mostly negative for HASs, stratified epithelia became HAS positive during differentiation. All HAS isoenzymes showed cytoplasmic immunoreactivity, both in tissue sections and cultured cells, while plasma membrane staining was also detected, often in cellular extensions. HAS1 had brightest signal in Golgi, HAS3 in Golgi and microvillous protrusions, whereas most of the endogenous HAS2 immunoreactivity was localized in the ER. This differential pattern was also observed with transfected GFP-HASs. The large proportion of intracellular HASs suggests that HAS forms a reserve that is transported to the plasma membrane for rapid activation of hyaluronan synthesis.

Hyaluronan content is a powerful prognostic factor in many cancer types, but the molecular basis of its synthesis in cancer still remains unclear. Hyaluronan synthesis requires the transport of hyaluronan synthases (HAS1-3) from Golgi to plasma membrane (PM), where the enzymes are activated. For the very first time, the present study demonstrated a rapid recycling of HAS3 between PM and endosomes, controlled by the cytosolic levels of the HAS substrates UDP-GlcUA and UDP-GlcNAc. Depletion of UDP-GlcNAc or UDP-GlcUA shifted the balance towards HAS3 endocytosis, and inhibition of hyaluronan synthesis. In contrast, UDP-GlcNAc surplus suppressed endocytosis and lysosomal decay of HAS3, favoring its retention in PM, stimulating hyaluronan synthesis, and HAS3 shedding in extracellular vesicles. The concentration of UDP-GlcNAc also controlled the level of O-GlcNAc modification of HAS3. Increasing O-GlcNAcylation reproduced the effects of UDP-GlcNAc surplus on HAS3 trafficking, while its suppression showed the opposite effects, indicating that O-GlcNAc signaling is associated to UDP-GlcNAc supply. Importantly, a similar correlation existed between the expression of GFAT1 (the rate limiting enzyme in UDP-GlcNAc synthesis) and hyaluronan content in early and deep human melanomas, suggesting the association of UDP-sugar metabolism in initiation of melanomagenesis. In general, changes in glucose metabolism, realized through UDP-sugar contents and O-GlcNAc signaling, are important in HAS3 trafficking, hyaluronan synthesis, and correlates with melanoma progression.

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.

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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.