Metabolic control of hyaluronan synthases

Hyaluronan (HA) is a glycosaminoglycan composed by repeating units of D-glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc) that is ubiquitously present in the extracellular matrix (ECM) where it has a critical role in the physiology and pathology of several mammalian tissues. HA represents a perfect environment in which cells can migrate and proliferate. Moreover, several receptors can interact with HA at cellular level triggering multiple signal transduction responses. The control of the HA synthesis is therefore critical in ECM assembly and cell biology; in this review we address the metabolic regulation of HA synthesis. In contrast with other glycosaminoglycans, which are synthesized in the Golgi apparatus, HA is produced at the plasma membrane by HA synthases (HAS1-3), which use cytoplasmic UDP-glucuronic acid and UDP-N-acetylglucosamine as substrates. UDP-GlcUA and UDP-hexosamine availability is critical for the synthesis of GAGs, which is an energy consuming process. AMP activated protein kinase (AMPK), which is considered a sensor of the energy status of the cell and is activated by low ATP:AMP ratio, leads to the inhibition of HA secretion by HAS2 phosphorylation at threonine 110. However, the most general sensor of cellular nutritional status is the hexosamine biosynthetic pathway that brings to the formation of UDP-GlcNAc and intracellular protein glycosylation by O-linked attachment of the monosaccharide β-N-acetylglucosamine (O-GlcNAcylation) to specific aminoacid residues. Such highly dynamic and ubiquitous protein modification affects serine 221 residue of HAS2 that lead to a dramatic stabilization of the enzyme in the membranes.     

The distribution of renal hyaluronan and the expression of hyaluronan synthases during water deprivation in the Spinifex hopping mouse, Notomys alexis

Hyaluronan (HA) is a glycosaminoglycan that is synthesized by a family of enzymes called hyaluronan synthases (HASs), of which there are three isoforms (HAS1, 2 and 3) in mammals. The HASs have different tissue expression patterns and function, indicating that synthesis of HA and formation of the HA matrix may be regulated by various factors. The HA matrix has an important role in renal water handling and the production of a concentrated urine. We investigated the distribution of HA and the expression of HAS1, HAS2 and HAS3 mRNAs in the kidney of the Spinifex hopping mouse, Notomys alexis, a native Australian desert rodent that is reported to produce the most concentrated urine of any mammal. After periods of three, seven and fourteen days of water deprivation, the distribution of renal HA changed considerably, and there was a general down-regulation of HAS mRNA expression. It is proposed that the regulation of HA synthesis by the different HAS isoforms during water deprivation in N. alexis, could be influenced by the molecular mass of the HA chains produced by each isoform, followed by the rate at which the individual HAS produces HA.     

The distribution of renal hyaluronan and the expression of hyaluronan synthases during water deprivation in the Spinifex hopping mouse, Notomys alexis.

Hyaluronan (HA) is a glycosaminoglycan that is synthesized by a family of enzymes called hyaluronan synthases (HASs), of which there are three isoforms (HAS1, 2 and 3) in mammals. The HASs have different tissue expression patterns and function, indicating that synthesis of HA and formation of the HA matrix may be regulated by various factors. The HA matrix has an important role in renal water handling and the production of a concentrated urine. We investigated the distribution of HA and the expression of HAS1, HAS2 and HAS3 mRNAs in the kidney of the Spinifex hopping mouse, Notomys alexis, a native Australian desert rodent that is reported to produce the most concentrated urine of any mammal. After periods of three, seven and fourteen days of water deprivation, the distribution of renal HA changed considerably, and there was a general down-regulation of HAS mRNA expression. It is proposed that the regulation of HA synthesis by the different HAS isoforms during water deprivation in N. alexis, could be influenced by the molecular mass of the HA chains produced by each isoform, followed by the rate at which the individual HAS produces HA.     

In vivo hyaluronan synthesis upon expression of the mammalian hyaluronan synthase gene in Drosophila

Hyaluronan (HA) is a large linear polymer of repeating disaccharides of glucuronic acid and GlcNAc. Although HA is widely distributed in vertebrate animals, it has not been found in invertebrates, including insect species. Insects utilize chitin, a repeating beta-1,4-linked homopolymer of GlcNAc, as a major component of their exoskeleton. Recent studies illustrate the similarities in the biosynthetic mechanisms of HA and chitin and suggest that HA synthase (HAS) and chitin synthase have evolved from a common ancestral molecule. Although the biochemical properties and in vivo functions of HAS proteins have been extensively studied, the molecular basis for HA biosynthesis is not completely understood. For example, it is currently not clear if proper chain elongation and secretion of HA require other components in addition to HAS. Here, we demonstrate that a non-HA-synthesizing animal, the fruit fly Drosophila melanogaster, can produce HA in vivo when a single HAS protein is introduced. Expression of the mouse HAS2 gene in Drosophila tissues by the Gal4/UAS (upstream activating sequence) system resulted in massive HA accumulation in the extracellular space and caused various morphological defects. These morphological abnormalities were ascribed to disordered cell-cell communications due to accumulation of HA rather than disruption of heparan sulfate synthesis. We also show that adult wings with HA can hold a high level of water. These findings demonstrate that organisms synthesizing chitin (but not HA) are capable of producing HA that is structurally and functionally relevant to that in mammals. The ability of insect cells to produce HA supports the idea that in vivo HA biosynthesis does not require molecules other than the HAS protein. An alternative model is that Drosophila cells use endogenous components of the chitin biosynthetic machinery to produce and secrete HA.     

Posttranslational translocation of influenza virus hemagglutinin across microsomal membranes.

The biosynthesis of influenza virus hemagglutinin (HA) and its translocation across microsomal membranes were studied in a mammalian cell-free system. All forms of HA could be cotranslationally translocated with high efficiency. However, only truncated forms of HA were translocated after protein synthesis has been terminated. The efficiency of this posttranslational translocation was dependent on the extent of the truncation. Posttranslational translocation was ribosome dependent and occurred only in the presence of a functional N-terminal signal sequence. The molecular mechanism of protein targeting and translocation across the membrane of the endoplasmic reticulum is discussed.     

An engineered hyaluronan synthase: characterization for recombinant human hyaluronan synthase 2 Escherichia coli

The Class I hyaluronan synthase (HAS) is a unique glycosyltransferase synthesizing hyaluronan (HA), a polysaccharide composed of GlcUA and GlcNAc, by using one catalytic domain that elongates two different monosaccharides. As for the synthetic mechanism, there are two alternative manners for the sugar elongation process. Some bacterial HASs add new sugars to the non-reducing end of the acceptor to grow polymers. On the other hand, some vertebrate enzymes seem to transfer sugars to the reducing end. Expression of vertebrate HASs as active and soluble proteins will accelerate further precise insight into mechanisms of sugar elongation reactions by natural HASs. Since large scale production of HA polymers and oligomers would become powerful tools both for basic studies and new biotechnology to create functional carbohydrates in medicinal purposes, advent of an efficient method for the expression of HASs in Escherichia coli is strongly expected. Here we communicate the first success of the production of recombinant human HAS2 proteins composed of only the catalytic region in E. coli as the active form. It was demonstrated that an engineered HAS2 expressed in E. coli exhibited significant activity to synthesize a mixture of HAS oligomers from 8-mer (HA8) to 16-mer (HA16). Engineered HAS2 prepared herein elongated sugars from exogenous tetrasaccharide to form polymers with a direction to the non-reducing end. According to the present results, large scale production of engineered recombinant HASs is to be performed using E. coli that will provide practical and economic advantages in manufacturing enzymes for use in the synthesis of various oligomeric HA molecules and their industrial applications.     

Collagen fragments inhibit hyaluronan synthesis in skin fibroblasts in response to ultraviolet B (UVB): new insights into mechanisms of matrix remodeling

UVB irradiation causes characteristic features of skin aging including remodeling of the dermal extracellular matrix. A key feature during this process is the up-regulation of matrix metalloproteinases and cleavage of collagen. Hyaluronic acid (HA), a major component of the dermal matrix, decreases after chronic UVB exposure. However, the factors that govern the decline of HA synthesis during the course of actinic aging are largely unknown. The aim of the present study was to explore whether collagen degradation causes inhibition of HA synthesis in human skin fibroblasts. After treatment of fibroblasts with collagen fragments (CF) in vitro, resolution of the actin cytoskeleton and inhibition of HA secretion occurred because of specific down-regulation of hyaluronan synthase 2 (HAS2) expression. The α(v)β(3)-agonist, RGDS, latrunculin A, and an inhibitor of Rho-activated kinase inhibited HAS2 expression. Conversely, blocking antibodies to α(v)β(3) abolished the down-regulation of HAS2 and the cytoskeletal effects. Furthermore, inhibition of cofilin phosphorylation in response to CF was prevented by α(v)β(3)-blocking antibodies. The key role of ERK signaling was shown by reduced nuclear accumulation of phosphoERK and of ELK-1 phosphorylation in response to CF. In addition, the ERK inhibitor PD98059 reduced HAS2 expression. Also, UVB irradiation of fibroblasts caused down-regulation of HAS2, which was sensitive to matrix metalloproteinase inhibitors and to α(v)β(3)-blocking antibodies. In conclusion, these data suggest that CF activate α(v)β(3)-integrins and in turn inhibit Rho kinase (ROCK) signaling and nuclear translocation of phosphoERK, resulting in reduced HAS2 expression. Therefore, a novel mechanism is presented how proteolytic collagen cleavage may inhibit HA synthesis in dermal fibroblasts during extrinsic skin aging.     

Mammalian hyaluronan synthases

Three mammalian hyaluronan (HA) synthase genes, HAS1, HAS2, and HAS3, have been cloned and expressed, allowing the mechanisms for regulation of HA biosynthesis and function to be studied. The hyaluronan synthase (HAS) isoforms differ in kinetic characteristics and product size. The expression of each HAS isoform is controlled in a different fashion when mammalian cells are stimulated by various cytokines and the expression patterns are both spatially and temporally regulated during embryonic development. The existence of three different HAS isoforms with different characteristics implies that the broad range of biological and physiological roles performed by HA are regulated by controlling the activities and expression of the HAS isoforms. This review focuses on recent findings on the regulatory mechanisms for controlling HA biosynthesis and provides new insights into the enzymic basis for the functional regulation of HA.     

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.     

The metabolism of hyaluronan in cultured rabbit growth plate chondrocytes during differentiation

Hyaluronan (HA) is one of the major extracellular matrix components in cartilage. In addition to the biomechanical functions, HA has various important roles in the differentiation of chondrocytes. The purpose of this study was to clarify the nature of HA synthesis during chondrocyte differentiation. Growth plate chondrocytes were isolated from rabbit ribs and cultured in chondrocyte differentiation medium. The amount of HA and HA synthase (HAS) mRNA levels were analyzed for each stage of chondrocyte differentiation by means of high-performance liquid chromatography (HPLC) and real-time PCR, respectively. The distribution of HA in cultured chondrocytes was observed by histochemical staining. The amount of HA, ranging widely in size, was increased substantially during the hypertrophic stage. The expression levels of HAS2 and HAS3 mRNAs were low during the matrix-forming stage. HAS2 mRNA level was substantially enhanced at the pre-hypertrophic stage, whereas HAS3 mRNA level exhibited a slight increase. HAS1 mRNA was not detected. The intensity of HA staining was high around the hypertrophic chondrocytes. These results suggest that HA metabolism in chondrocyte differentiation is regulated by the selective expression of HASs, and HAS2 and the related large size-HA may have a certain association with the hypertrophic changes of chondrocytes.     

Effects of leflunomide on hyaluronan synthases (HAS): NF-kappa B-independent suppression of IL-1-induced HAS1 transcription by leflunomide

Despite evidence that points to unfettered hyaluronic acid (HA) production as a culprit in the progression of rheumatic disorders, little is known about differences in regulation and biological functions of the three hyaluronan synthase (HAS) genes. Testing the effects of drugs with proven anti-inflammatory effects could help to clarify biological functions of these genes. In this study, we demonstrate that leflunomide suppresses HA release in fibroblast-like synoviocytes (FLS) in a dose-dependent manner. We further demonstrate that leflunomide suppresses HA synthase activity, as determined by (14)C-glucuronic acid incorporation assays. Additional experiments revealed that in FLS, leflunomide specifically blocked the induction of HAS1. HAS2 and HAS3, genes that are, in contrast to HAS1, constitutively expressed in FLS, are not significantly affected. Leflunomide can function as a NF-kappaB inhibitor. However, EMSA experiments demonstrate that at the concentrations used, leflunomide neither interferes with IL-1beta- nor with PMA-induced NF-kappaB translocation. Furthermore, reconstituting the pyrimidine synthase pathway did not lead to the restoration of IL-1beta-induced HAS1 activation. More importantly, two tyrosine kinase inhibitors mimicked the effect of leflunomide in that both blocked IL-1beta-induced HAS1 activation without affecting HAS2 or HAS3. These data point at HAS1 activation as the possible cause for unfettered HA production in rheumatoid arthritis and might explain, at least in part, the beneficial effects of leflunomide treatment. These findings also support the concept that IL-1beta-induced HAS1 activation depends on the activation of tyrosine kinases, and indicate that leflunomide blocks HA release by suppressing tyrosine kinases rather than through inhibition of NF-kappaB translocation.     

A rapid transient increase in hyaluronan synthase-2 mRNA initiates secretion of hyaluronan by corneal keratocytes in response to transforming growth factor beta

Keratocytes of the corneal stroma produce transparent extracellular matrix devoid of hyaluronan (HA); however, in corneal pathologies and wounds, HA is abundant. We previously showed primary keratocytes cultured under serum-free conditions to secrete matrix similar to that of normal stroma, but serum and transforming growth factor beta (TGFbeta) induced secretion of fibrotic matrix components, including HA. This study found HA secretion by primary bovine keratocytes to increase rapidly in response to TGFbeta, reaching a maximum in 12 h and then decreasing to <5% of the maximum by 48 h. Cell-free biosynthesis of HA by cell extracts also exhibited a transient peak at 12 h after TGFbeta treatment. mRNA for hyaluronan synthase enzymes HAS1 and HAS2 increased >10- and >50-fold, respectively, in 4-6 h, decreasing to near original levels after 24-48 h. Small interfering RNA against HAS2 inhibited the transient increase of HAS2 mRNA and completely blocked HA induction, but small interfering RNA to HAS1 had no effect on HA secretion. HAS2 mRNA was induced by a variety of mitogens, and TGFbeta acted synergistically to induce HAS2 by as much as 150-fold. In addition to HA synthesis, treatment with TGFbeta induced degradation of fluorescein-HA added to culture medium. These results show HA secretion by keratocytes to be initiated by a rapid transient increase in the HAS2 mRNA pool. The very rapid induction of HA expression in keratocytes suggests a functional role of this molecule in the fibrotic response of keratocytes to wound healing.     

Regulation of hyaluronan and versican deposition by growth factors in fibrosarcoma cell lines

Versican, a large chondroitin sulphate proteoglycan and hyaluronan (HA), a non-sulphated glycosaminoglycan are major constituents of the pericellular matrix. In many neoplastic tissues, changes in the expression of versican and HA affect tumour progression. Here, we analyse the synthesis of versican and hyaluronan by fibrosarcoma cells, and document how the latter is affected by PDGF-BB, bFGF and TGFB2, growth factors endogenously produced by these cells. Fibrosarcoma cell lines B6FS and HT1080 were utilised and compared with normal lung fibroblasts (DLF). The major versican isoforms expressed by DLF and B6FS cells were V0 and V1. Treatment of B6FS cells with TGFB2 showed a significant increase of V0 and V1 mRNAs. Versican expression in HT1080 cells was not significantly affected by any of the growth factors. In addition, TGFB2 treatment increased versican protein in DLF cells. HA, showed approximately a 2-fold and a 9-fold higher production in DLF cells compared to B6FS and HT1080 cells, respectively. In HT1080 cells, HA biosynthesis was significantly increased by bFGF, whereas, in B6FS cells it was increased by TGFB2 and PDGF-BB. Furthermore, analysis of HA synthases (HAS) expression indicated that HT1080 expressed similar levels of all three HAS isoforms in the following order: HAS2> HAS3> HAS1. bFGF shifted that balance by increasing the abundance of HAS1. The major HAS isoform expressed by B6FS cells was HAS2. PDGF-BB and TGFB2 showed the most prominent effects by increasing both HAS2 and HAS1 isoforms. In conclusion, these growth factors modulated, through upregulation of specific HAS isoforms, HA synthesis, secretion and net deposition to the pericellular matrix.