Molecular Cloning and Characterization of UDP-glucose Dehydrogenase from the Amphibian Xenopus laevis and Its Involvement in Hyaluronan Synthesis

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.

mentioning

confidence: 72%

Section: Discussionmentioning

confidence: 98%

Cellular Content of UDP-N-acetylhexosamines Controls Hyaluronan Synthase 2 Expression and Correlates with O-Linked N-Acetylglucosamine Modification of Transcription Factors YY1 and SP1

Jokela

Makkonen

Oikari

et al. 2011

“…4-MU is thought to act in large part by chelating UDP-GlcUA, one of two nucleotide sugars necessary for HA synthesis (32,33), the end result being a inhibition of HA biosynthesis. Because UDP-sugar transporters in the endoplasmic reticulum and Golgi membranes have a low K m value, this diminution of cytosolic UDP-GlcUA has little to no effect on proteoglycan biosynthesis because the sugar residues for glycosaminoglycan chain elongation remain saturated in those compartments (70,71). The more interesting aspect of 4-MU treatment is that it also has the capacity to down-regulate HAS2 mRNA by mechanisms that are far less clear or direct.…”

Section: Discussionmentioning

confidence: 99%

4-Methylumbelliferone Diminishes Catabolically Activated Articular Chondrocytes and Cartilage Explants via a Mechanism Independent of Hyaluronan Inhibition

Ishizuka

Askew

Ishizuka

et al. 2016

Depletion of the cartilage proteoglycan aggrecan is one of the earliest events that occurs in association with osteoarthritis. This loss is often accompanied by a coordinate loss in another glycosaminoglycan, hyaluronan. Chondrocytes experimentally depleted of cell-associated hyaluronan respond by switching to a pro-catabolic metabolism that includes enhanced production of endogenous inflammatory mediators and increased synthesis of matrix metalloproteinases. Hyaluronan turnover is also increased. Together, such a response provides for possible establishment of a self-perpetuating spiral of events that maintains or prolongs the pro-catabolic state. Chondrocytes or cartilage can also be activated by treatment with pro-inflammatory cytokines and mediators such as IL-1␤, TNF␣, LPS, fibronectin fragments, and hyaluronan oligosaccharides. To determine the mechanism of chondrocyte activation due to hyaluronan loss, a depletion method was required that did not include degrading the hyaluronan. In recent years, several laboratories have used the coumarin derivative, 4-methylumbelliferone, as a potent inhibitor of hyaluronan biosynthesis, due in part to its ability to sequester intracellular UDP-glucuronic acid and inhibition of hyaluronan synthase transcription. However, contrary to our expectation, although 4-methylumbelliferone was indeed an inhibitor of hyaluronan biosynthesis, this depletion did not give rise to an activation of chondrocytes or cartilage. Rather, 4-methylumbelliferone directly and selectively blocked gene products associated with the pro-catabolic metabolic state of chondrocytes and did so through a mechanism preceding and independent of hyaluronan inhibition. These data suggest that 4-methylumbelliferone has additional useful applications to block pro-inflammatory cell activation events but complicates how it is used for defining functions related to hyaluronan.The pronounced loss of aggrecan from articular cartilage is an early critical event associated with osteoarthritis (OA) 2 (1, 2). Aggrecan turnover within the extracellular matrix occurs due to the enhanced activity of endoproteinases such as a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) 4 (3, 4) and 5 (5, 6) as well as other matrix metalloproteinases (MMPs) (7)(8)(9)(10)(11). In addition to aggrecan, a significant loss of hyaluronan (HA) is also observed in human OA cartilage as compared with normal human cartilage (12,13). A marked depletion of HA was also observed in articular cartilage damaged experimentally, such as in the anterior cruciate ligament transection model of OA (14) and a reduced-loading, splint immobilization model (15) in dogs. In our studies, cultured explants of human articular cartilage treated with IL-1␣ displayed a loss of HA within the superficial and upper middle layers of cartilage, the same layers in which aggrecan loss occurred (16). Other studies on cytokine-stimulated cartilage explants suggested that HA is lost from the cartilage (17,18), and other studies revealed that the HA is lost...

“…CD44 siRNA (s2681) and scramble negative control siRNA1 kit (code 4611) were both purchased from Ambion. The transfections were done using a Nucleofector apparatus (Amaxa) as described previously (4,21). After 48 h of incubation, cells were treated with 4-MU, and HMW-HA and cell viability were measured.…”

Section: Methodsmentioning

confidence: 99%

Glycosaminoglycans and Glucose Prevent Apoptosis in 4-Methylumbelliferone-treated Human Aortic Smooth Muscle Cells

Vigetti

Rizzi

Moretto

et al. 2011

Self Cite

Smooth muscle cells (SMCs) have a pivotal role in cardiovascular diseases and are responsible for hyaluronan (HA) deposition in thickening vessel walls. HA regulates SMC proliferation, migration, and inflammation, which accelerates neointima formation. We used the HA synthesis inhibitor 4-methylumbelliferone (4-MU) to reduce HA production in human aortic SMCs and found a significant increase of apoptotic cells. Interestingly, the exogenous addition of HA together with 4-MU reduced apoptosis. A similar anti-apoptotic effect was observed also by adding other glycosaminoglycans and glucose to 4-MU-treated cells. Furthermore, the anti-apoptotic effect of HA was mediated by Toll-like receptor 4, CD44, and PI3K but not by ERK1/2. Hyaluronan (HA)3 is one of the most abundant glycosaminoglycans (GAGs) in extracellular matrices (ECMs) and is composed of linear, unsulfated repetitions of D-glucuronic acid and N-acetylglucosamine. In mammals, two specific HA synthases (HAS1 and -2) produce high molecular weight HA (HMW-HA), in the range of millions of Da, whereas the other isoenzyme (HAS3) synthesizes HA of lower molecular mass, in the range of several thousands of Da (1). The size of HA depends also on specific degrading enzymes (i.e. hyaluronidases) that can produce bioactive HA oligosaccharides. Therefore, in vivo, HA chains can greatly vary in lengths and can differently regulate cell behavior through interactions with several receptors, including CD44, RHAMM (receptor for HA-mediated motility), lymphatic vessel endothelial receptor 1 (Lyve-1), HA receptor for endocytosis (HARE), and Toll-like Receptor 4 (TLR4) (2).In cardiovascular pathologies, HA accumulates during neointima formation and alters smooth muscle cell (SMC) behavior (3). In some pathological conditions, contractile SMCs dedifferentiate to form a synthetic phenotype characterized by a high production of ECM components, including HA and versican, by synthesis of ECM-modifying metalloproteinases (4) and by increased rates of proliferation and migration. Therefore, SMCs acquire the capability to invade the vascular tunica intima, thereby contributing to vessel wall thickening. HMW-HA is involved in the modulation of SMC migration and proliferation through interaction with CD44 (5-7), which can mediate a signaling cascade inside the cell that activates different pathways, including PI3K, AKT, and ERK1/2 (8).We demonstrated previously that human aortic SMC (AoSMC) migration is strictly dependent on HA-CD44 signaling and recently reported that the HA synthesis inhibitor 4-methylumbelliferone (4-MU) reduced proatherosclerotic properties of AoSMCs by decreasing cell migration and proliferation and by inhibiting monocyte binding to the HA-rich ECM that contributes to inflammation (9, 10). Moreover, we found that the simultaneous addition of HMW-HA to 4-MUtreated AoSMCs restored cell proliferation to the levels of controls. Therefore, the aim of this study was to investigate at the molecular level the effects of HMW-HA after 4-MU treatment of AoSMCs and the pathway...