Influence of glucose on production and N-sulfation of heparan sulfate in cultured adipocyte cells

Nitric oxide (NO), a reactive nitrogen species, plays an important role in inflammatory lung damage. In the present study, we investigated the role of NO in DNA-binding activity of NF-B in macrophages stimulated with silica or other inflammatory stimulants. Treatment of mouse macrophages (RAW264.7 cells) with a selective inhibitor of inducible nitric oxide synthase (iNOS), L-N⁶-(1-iminoethyl) lysine (L-NIL), or a nonselective iNOS inhibitor, N-nitro-L-arginine methylester (L-NAME), resulted in inhibition of silica-induced nitric oxide production as well as silica-induced NF-B activation. L-NIL also effectively inhibited NF-B activation induced by other inflammatory stimulants, such as lipopolysaccharide (LPS) or muramyl dipeptide (MDP). These inhibitory effects of L-NIL and L-NAME on silica- or LPS-induced NF-B activation were also observed in primary rat alveolar macrophages. Furthermore, NO generating compounds, such as sodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1), caused a dose-dependent increase in NF-B activation, which was positively correlated with the level of NO production. Specific inhibitors of protein tyrosine kinase, such as genistein and AG494, prevented NF-B activation in SNP- or SIN-1 treated cells, suggesting involvement of tyrosine kinase in the NO signaling pathway leading to NF-B activation. In contrast, inhibitors of protein kinase C or A, such as staurosporine or H89, had no inhibitory effect on SIN-1 induced NF-B activation. Metalloporphyrins, such as tetrakis (N-methyl-4-pyridyl) porphyrinato iron (III) (Fe-TMPyP) and Zn-TMPyP which are known to alter NO-dependent activity, markedly inhibited silica- and LPS-induced NF-B activation. The results suggest that NF-B activation in macrophages can be induced under certain conditions by nitric oxide and that nitric oxide produced by phagocytes exposed to inflammatory agents may up-regulate the activation of NF-B.     

A spoonful of sugar makes the melanoma go: the role of heparan sulfate proteoglycans in melanoma metastasis

Heparan sulfate proteoglycans (HSPGs) have been shown to regulate signaling in many systems and are of increasing interest in cancer. While these are not the only sugars to drive melanoma metastasis, HSPGs play important roles in driving metastatic signaling cascades in melanoma. The ability of these proteins to modulate ligand-receptor interactions in melanoma has been quite understudied. Recent data from several groups indicate the importance of these ligands in modulating key signaling pathways including Wnt and fibroblast growth factor (FGF) signaling. In this review, we summarize the current knowledge regarding the structure and function of these proteoglycans and their role in melanoma. Understanding how HSPGs modulate signaling in melanoma could lead to new therapeutic approaches via the dampening or heightening of key signaling pathways.     

A competitive binding assay for measurement of heparan sulfate in tissue digests

Glycosaminoglycans complex with constituents of normal human serum, a finding that was exploited to develop a competitive binding assay for these substances. Heparan sulfate was isolated from renal cortex and radiolabeled with tritiated borohydride. The elution pattern of the radiolabeled material on Sephadex G-25, Bio-Gel P-30, and AG- 1X8 resin was identical to that of unlabeled heparan sulfate. The tritiated heparan sulfate formed radiolabeled precipitates when incubated with serum and zinc acetate. Binding was dose dependent and saturable. Heparin, heparan sulfate, and the chondroitin sulfates, but not hyaluronate or keratan sulfate, competed with the radiolabeled heparan sulfate for binding in a dose-dependent manner. The assay is specific for heparin polysaccharides in chondroitinase ABC-treated samples and is sensitive to microgram quantities.     

Chemoenzymatic synthesis of heparan sulfate and heparin

Heparan sulfate (HS) is a highly sulfated polysaccharide that plays essential physiological and pathophysiological functions. The biosynthesis of HS involves a series of specialised sulfotransferases, an epimerase and glycosyl transferases. The availability of these enzymes offers a promising method to prepare HS polysaccharides and structurally defined oligosaccharides. Given the fact that chemical synthesis of large HS oligosaccharides is extremely difficult, preparation of HS using a chemoenzymatic approach has gained momentum. This review article summarises recent progress on the development of a chemoenzymatic approach to prepare HS and HS oligosaccharides.     

Structural alteration of cell surface heparan sulfate through the stimulation of the signaling pathway for heparan sulfate 6-O-sulfotransferase-1 in mouse fibroblast cells

Heparan sulfate (HS) is a randomly sulfated polysaccharide that is present on the cell surface and in the extracellular matrix. The sulfated structures of HS were synthesized by multiple HS sulfotransferases, thereby regulating various activities such as growth factor signaling, cell differentiation, and tumor metastasis. Therefore, if the sulfated structures of HS could be artificially controlled, those manipulations would help to understand the various functions depending on HS. However, little knowledge is currently available to realize the mechanisms controlling the expression of such enzymes. In this study, we found that the ratio of 6-O-sulfated disaccharides increased at 3?h after adrenaline stimulation in mouse fibroblast cells. Furthermore, adrenaline-induced up-regulation of HS 6-O-sulfotransferase-1 (6-OST-1) was controlled by Src-ERK1/2 signaling pathway. Finally, inhibiting the signaling pathways for 6-OST-1 intentionally suppressed the adrenaline-induced structural alteration of HS. These observations provide fundamental insights into the understanding of structural alterations in HS by extracellular cues.     

Defective nitric oxide-dependent, deaminative cleavage of glypican-1 heparan sulfate in Niemann-Pick C1 fibroblasts

Exit of recycling cholesterol from late endosomes is defective in Niemann-Pick C1 (NPC1) and Niemann-Pick C2 (NPC2) diseases. The traffic route of the recycling proteoglycan glypican-1 (Gpc-1) may also involve late endosomes and could thus be affected in these diseases. During recycling through intracellular compartments, the heparan sulfate (HS) side chains of Gpc-1 are deaminatively degraded by nitric oxide (NO) derived from preformed S-nitroso groups in the core protein. We have now investigated whether this NO-dependent Gpc-1 autoprocessing is active in fibroblasts from NPC1 disease. The results showed that Gpc-1 autoprocessing was defective in these cells and, furthermore, greatly depressed in normal fibroblasts treated with U18666A (3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one), a compound widely used to induce cholesterol accumulation. In both cases, autoprocessing was partially restored by treatment with ascorbate which induced NO release, resulting in deaminative cleavage of HS. However, when NO-dependent Gpc-1 autoprocessing is depressed and heparanase-catalyzed degradation of HS remains active, a truncated Gpc-1 with shorter HS chains would prevail, resulting in fewer NO-sensitive sites/proteoglycan. Therefore, addition of ascorbate to cells with depressed autoprocessing resulted in nitration of tyrosines. Nitration was diminished when heparanase was inhibited with suramin or when Gpc-1 expression was silenced by RNAi. Gpc-1 misprocessing in NPC1 cells could thus contribute to neurodegeneration mediated by reactive nitrogen species.     

Regulation of Notch signaling by Drosophila heparan sulfate 3-O sulfotransferase

Heparan sulfate (HS) regulates the activity of various ligands and is involved in molecular recognition events on the cell surface and in the extracellular matrix. Specific binding of HS to different ligand proteins depends on the sulfation pattern of HS. For example, the interaction between antithrombin and a particular 3-O sulfated HS motif is thought to modulate blood coagulation. However, a recent study of mice defective for this modification suggested that 3-O sulfation plays other biological roles. Here, we show that Drosophila melanogaster HS 3-O sulfotransferase-b (Hs3st-B), which catalyzes HS 3-O sulfation, is a novel component of the Notch pathway. Reduction of Hs3st-B function by transgenic RNA interference compromised Notch signaling, producing neurogenic phenotypes. We also show that levels of Notch protein on the cell surface were markedly decreased by loss of Hs3st-B. These findings suggest that Hs3st-B is involved in Notch signaling by affecting stability or intracellular trafficking of Notch protein.     

Constitutive and vitamin C-induced, NO-catalyzed release of heparan sulfate from recycling glypican-1 in late endosomes

The recycling heparan sulfate (HS)-containing proteoglycan glypican-1 (Gpc-1) is processed by nitric oxide (NO)-catalyzed deaminative cleavage of its HS chains at N-unsubstituted glucosamines. This generates anhydromannose (anMan)-containing HS degradation products that can be detected by a specific antibody. Here we have attempted to identify the intracellular compartments where these products are formed. The anMan-positive degradation products generated constitutively in human bladder carcinoma cell line (T24) or fibroblasts appeared neither in caveolin-1-associated vesicles nor in lysosomes. In Niemann-Pick C-1 (NPC-1) fibroblasts, where deaminative degradation is abrogated, formation of anMan-positive products can be restored by ascorbate. These products colocalized with Rab7, a marker for late endosomes. When NO-catalyzed degradation of HS was depressed in mouse neuroblastoma cell line (N2a) by using 3-beta[2(diethylamino) ethoxy]androst-5-en-17-one (U18666A), both ascorbate and dehydroascorbic acid restored formation of anMan-positive products that colocalized with Rab7. In T24 cells, constitutively generated anMan-positive products colocalized with both Rab7 and Rab9, whereas Gpc-1 colocalized with Rab9, a marker for transporting endosomes. Inhibition of endosomal acidification, which blocks transfer from early (Rab5) to late (Rab7) endosomes, abrogated deaminative degradation of HS. This could also be overcome by the addition of ascorbate, which induced formation of anMan-positive degradation products that colocalized with Rab7. After (35)S-sulfate labeling, similar degradation products were recovered in Rab7-positive vesicles. We conclude that NO-catalyzed degradation of HS may begin in early endosomes but is mainly taking place in late endosomes.     

Differentiation-induced loss of heparan sulfate in human exostosis derived chondrocytes

An exostosis or osteochondroma is an aberrant bony growth occurring next to the growth plate either as an isolated growth abnormality or as part of the Hereditary Multiple Exostosis (HME) syndrome. Mutations in either exostosin 1 (EXT1) or exostosin 2 (EXT2) gene cause the HME syndrome and also some isolated osteochondromas. The EXT1 and EXT2 genes are glycosyltransferases that function as hetero-oligomers in the Golgi to add repeating glycosaminoglycans (GAGs) to heparan sulfate (HS) chains. Previously, we demonstrated that HS is markedly diminished in the exostosis cartilage cap and that the HS proteoglycan, perlecan, has an abnormal distribution in these caps. The present studies were undertaken to evaluate which chondrocyte-specific functions are associated with diminished HS synthesis in human chondrocytes harboring either EXT1 or EXT2 mutations. Systematic evaluation of exostosis cartilage caps and chondrocytes, both in vitro and in vivo, suggests that chondrocyte-specific cell functions account for diminished HS levels. In addition, we provide evidence that perichondrial cells give rise to chondrocytes that clonally expand and develop into an exostosis. Undifferentiated EXT chondrocytes synthesized amounts of HS similar to control chondrocytes; however, EXT chondrocytes displayed very poor survival in vitro under conditions that promote normal chondrocyte differentiation with high efficiency. Collectively, these observations suggest that loss of one copy of either the EXT1 or EXT2 gene product compromises the perichondrial chondrocytes' ability to differentiate normally and to survive in a differentiated state in vitro. In vivo, these compromised responses may lead to abnormal chondrocyte growth, perhaps from a perichondrial stem cell reserve.     

Lack of acidic fibroblast growth factor activation by heparan sulfate species from diabetic rat skin

The glucosaminoglycans isolated from the skin of control and streptozotocin-diabetic rats were fractionated on ion-exchange chromatography into a heparan sulfate (HS)-like and a heparin-like species. In addition, a low sulfated fraction was isolated from the diabetics. The HS and heparin-like fractions isolated from the diabetics (in contrast to the low sulfated fractions) retained high affinity for the acidic (FGF-1) and basic (FGF-2) fibroblast growth factors. In culture, the fractions purified from the control rats and the heparin-like material isolated from the diabetics mediated the biological activity of both FGFs in a dose-dependent manner. By contrast, the diabetic HS-like fractions promoted the biological activity of FGF-2 but not of FGF-1. The results support the idea that the structural motives in HS required for FGF-1 and FGF-2 mediated receptor signalling are different. They may be relevant to the impaired wound healing observed in the disease. This revised version was published online in November 2006 with corrections to the Cover Date.     

N6-methyladenosine modification of MALAT1 promotes metastasis via reshaping nuclear speckles

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Heparin releasable and nonreleasable forms of heparan sulfate proteoglycan are found on the surfaces of cultured porcine aortic endothelial cells

Evidence suggests that endothelial cell layer heparan sulfate proteoglycans include a variety of different sized molecules which most likely contain different protein cores. In the present report, approximately half of endothelial cell surface associated heparan sulfate proteoglycan is shown to be releasable with soluble heparin. The remaining cell surface heparan sulfate proteoglycan, as well as extracellular matrix heparan sulfate proteoglycan, cannot be removed from the cells with heparin. The heparin nonreleasable cell surface proteoglycan can be released by membrane disrupting agents and is able to intercalate into liposomes. When the heparin releasable and nonreleasable cell surface heparan sulfate proteoglycans are compared, differences in proteoglycan size are also evident. Furthermore, the intact heparin releasable heparan sulfate proteoglycan is closer in size to proteoglycans isolated from the extracellular matrix and from growth medium than to that which is heparin nonreleasable. These data indicate that cultured porcine aortic endothelial cells contain at least two distinct types of cell surface heparan sulfate proteoglycans, one of which appears to be associated with the cells through its glycosaminoglycan chains. The other (which is more tightly associated) is probably linked via a membrane intercalated protein core.Abbreviations ECM extracellular matrix - HSPG heparan sulfate proteoglycan - PAE porcine aortic endothelial - PBS phosphate buffered saline     

Developmental roles of heparan sulfate proteoglycans in Drosophila

The formation of complex patterns in multi-cellular organisms is regulated by a number of signaling pathways. In particular, the Wnt and Hedgehog (Hh) pathways have been identified as critical organizers of pattern in many tissues. Although extensive biochemical and genetic studies have elucidated the central components of the signal transduction pathways regulated by these secreted molecules, we still do not understand fully how they organize gradients of gene activities through field of cells. Studies in Drosophila have implicated a role for heparan sulfate proteoglycans (HSPGs) in regulating the signaling activities and distribution of both Wnt and Hh. Here we review these findings and discuss various models by which HSPGs regulate the distributions of Wnt and Hh morphogens. Published in 2003.     

Differential partition of anticoagulant heparan sulfate proteoglycans synthesized by endothelial and fibroblastic cell lines

The heparan sulfate proteoglycans that bind and activate antithrombin III (aHSPGs) are synthesized by endothelial cells as well as other nonvascular cells. We determined the amounts of cell surface–associated and soluble aHSPGs generated by the rat fat pad endothelial (RFP) cell line and the fibroblast (LTA) cell line. The RFP cells exhibit higher levels of cell surface–associated aHSPGs as compared to LTA cells, whereas LTA cells release larger amounts of soluble aHSPGs as compared to RFP cells. After confluence RFP cells show an increase in both cell surface–associated and soluble aHSPGs. In contrast, postconfluent LTA cells maintain a constant level of cell surface–associated and soluble aHSPGs. These observations indicate that different cells types can preferentially accumulate aHSPGs as cell surface–associated or soluble forms which could reflect alternate biological functions.     

Important role of heparan sulfate in postnatal islet growth and insulin secretion

Heparan sulfate (HS) binds with several signaling molecules and regulates ligand-receptor interactions, playing an essential role in embryonic development. Here we showed that HS was intensively expressed in pancreatic islet β-cells after 1 week of age in mice. The enzymatic removal of HS in isolated islets resulted in attenuated glucose-induced insulin secretion with a concomitant reduction in gene expression of several key components in the insulin secretion machinery. We further depleted islet HS by inactivating the exostosin tumor-like 3 gene specifically in β-cells. These mice exhibited abnormal islet morphology with reduced β-cell proliferation after 1 week of age and glucose intolerance due to defective insulin secretion. These results demonstrate that islet HS is involved in the regulation of postnatal islet maturation and required to ensure normal insulin secretion.     

Dietary manganese affects the concentration, composition and sulfation pattern of heparan sulfate glycosaminoglycans in Sprague-Dawley rat aorta

We examined the effect of dietary Mn on the composition and structure of heparan sulfate (HS) glycosaminoglycans (GAGs) of rat aorta. Animals were randomly assigned to either a Mn deficient (MnD), adequate (MnA) or supplemented (MnS) diet (Mn<1, 10–15 and 45–50 ppm, respectively). After 15 weeks, aortic tissue GAGs were isolated with papain digestion, alkaline borohydride treatment and anion-exchange chromatography. Cellulose acetate electrophoresis and treatment of the fractions with specific lyases revealed the presence of three GAG populations, i.e. hyaluronan (HA), heparan sulfate (HS) and galactosaminoglycans (GalAGs). Disaccharide composition of the HS fractions was determined by HPCE following treatment with heparin lyases I, II and III. In MnS aortas we observed increased concentration of total GalAGs and decreased concentration of HS and HA, when compared to MnA aortas. Aortas from MnD and MnA rats appeared to have similar distribution of individual GAGs. Heparan sulfate chains of MnS aortas contained higher (41%) concentration of non-sulfated units compared to MnA ones. Variable amounts of trisulfated and disulfated units were found only in MnD and MnA groups but not in MnS. Our results demonstrate that HS biosynthesis in the rat aorta undergoes marked structural modifications that depend upon dietary Mn intake. The reduced expression and undersulfation of HSPGs with Mn supplementation might indicate a reduced ability of vascular cells to interact with biologically active molecules such as growth factors. Alterations in cell-membrane binding ability to a variety of extracellular ligands might affect signal-transduction pathways and arterial functional properties.     

Mice deficient in heparan sulfate 3-O-sulfotransferase-1: normal hemostasis with unexpected perinatal phenotypes

Heparan sulfate that contains antithrombin binding sites is designated as anticoagulant heparan sulfate (HS^act) since, in vitro, it dramatically enhances the neutralization of coagulation proteases by antithrombin. Endothelial cell production of HS^act is controlled by the Hs3st1 gene, which encodes the rate limiting enzyme—heparan sulfate 3-O-sulfotransferase-1 (Hs3st1). It has long been proposed that levels of endothelial HS^act may tightly regulate hemostatic tone. This potential in vivo role of HS^act was assessed by generating Hs3st1 ^–/– knockout mice. Hs3st1 ^–/– and Hs3st1 ^+/+ mice were evaluated with a variety of methods, capable of detecting altered hemostatic tone. However, both genotypes were indistinguishable. Instead, Hs3st1 ^–/– mice exhibited lethality on a specific genetic background and also showed intrauterine growth retardation. Neither phenotypes result from a gross coagulopathy. So although this enzyme produces the majority of tissue HS^act, Hs3st1 ^–/– mice do not show an obvious procoagulant phenotype. These results suggest that the bulk of HS^act is not essential for normal hemostasis and that hemostatic tone is not tightly regulated by total levels of HS^act. Moreover, the unanticipated non-thrombotic phenotypes suggest structure(s) derived from this enzyme might serve additional/alternative biologic roles. Published in 2003.