| 1. |
- Eklund, Erik, et al.
(författare)
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Proteoglycan production in disomic and trisomy 7-carrying human synovial cells.
- 2002
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Ingår i: Matrix Biology. - 1569-1802. ; 21:4, s. 325-335
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Tidskriftsartikel (refereegranskat)abstract
- To gain further insight into the synthesis and structure of the synovial matrix of joints, we have established cell cultures from synovial specimens and elaborated their production of hyaluronan and proteoglycans. The cultures secreted mainly the small proteoglycan decorin, but also considerable amounts of the related biglycan and the large proteoglycan versican. Only minor amounts of heparan sulfate proteoglycans were found. All cultures also had a high production of hyaluronan, which highlights the important role for normal joint function of these cells. In joint diseases, a common feature is the presence of an extra chromosome 7 (trisomy 7) in the synovial cells. To study the possible consequences of trisomy 7 on the synovial cell function, we extended our study to cultures that had been sub-cloned to contain high amounts of trisomy 7-carrying cells. These cell cultures had approximately four times more versican than their disomic counterparts in the cell culture medium, indicating that versican may be a mediator in the processes of joint destructive disorders. To find an explanation for this increase in versican, we investigated the expression/secretion of PDGF-AA and IL-6, cytokines with their genes located to chromosome 7. Indeed, both these cytokines were increased in the cultures with high frequencies of trisomy 7. We then added the two cytokines to cell cultures of disomic synovial cells, but only cells treated with IL-6 displayed an increased amount of versican. Thus, we suggest that the increased amount of versican in cultures of trisomy 7-carrying cells relates to an autocrine loop involving an increased IL-6 production.
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| 2. |
- Batool, Tahira
(författare)
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Heparan sulfate dependent cell signaling and associated pathophysiology : Implications in tumorigenesis and embryogenesis
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Heparan sulfate proteoglycans (HSPGs) consist of a protein core to which several linear, negatively charged heparan sulfate (HS) chains are covalently attached. HSPGs are expressed on the cell surface and in the extra-cellular matrix (ECM) where they have diverse biological functions, for example co-receptor functions. The diverse functions of HS are linked to structural variability of the polysaccharide. In this thesis, I investigated HS structure-function relationship by using different cell and animal models of one HS-biosynthetic enzyme, glucuronyl C5-epimerase (Hsepi) and one enzyme responsible for post synthetic modification, heparanase.Deletion of Hsepi in mice resulted in neonatal lethality, with multiple organ defects, indicating the importance of HS in embryogenesis. Up-regulated expression of heparanase is found in most human tumor tissues, correlating with increased metastatic potential and decreased survival of cancer patients.In the first project, I focused on the effects of HS on cancer associated cell signaling and found that heparanase overexpression attenuated TGF-β1 stimulated Smad phosphorylation in tumor cells because of increased sulfation degree and turnover rate of HS.Heparanase role in cancer progression has led to clinical trials where inhibition of heparanase activity is currently being evaluated as a potential cancer treatment. Heparin, a HS-related polysaccharide, is being used to inhibit heparanase activity. In my second project, we studied the effect of low molecular weight heparin (LMWH) on cisplatin resistance of ovarian cancer cells (A2780cis). LMWH treatment of A2780cis cells reduced Wnt-activity in these cells and consequently reduce the drug resistance.In paper III, we continued exploring the HS/heparanase role in cancer by using heparanase overexpressing mice (Hpa-tg). We found Lewis Lung Carcinoma (LLC2) cells showed faster growth, bigger tumors and more metastasis in the Hpa-tg mice as compared to wild-type (WT) mice, because of suppressed antitumor immunity in the Hpa-tg mice.In paper IV and V, we studied the structure-function relationship of HS by using Hsepi-/- mice model. Hsepi-/- results in HS-chains lacking IdoA, which makes the chain rigid and consequently affects its co-receptor function. Skeletal malformation in Hsepi-/- mice, led us in paper IV to investigate bone morphogenic protein (BMP), an important signal molecule during embryogenesis and known to interact with HS. We found upregulation of a number of BMPs and expression of P-smad1/5/8, but reduced expression of inhibitory Smads and Gremlin1 in the Hsepi-/- MEF cells. The study indicated that the developmental defects in Hsepi mice could be contributed by a higher BMP signaling. In paper V we investigated the lung of the Hsepi-/- mice. The distal lung of 17.5 days old embryos remained populated by epithelial tubules, because of impaired differentiation of type I cells of the lungs. Potential mechanisms behind the failure of type I cell formation was identified to be reduced vascularization and a sustained signaling of Smad pathways.
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| 3. |
- Dubicke, Aurelija, et al.
(författare)
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Pro-inflammatory and anti-inflammatory cytokines in human preterm and term cervical ripening
- 2010
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Ingår i: Journal of Reproductive Immunology. - : Elsevier. - 0165-0378 .- 1872-7603. ; 84:2, s. 176-185
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Tidskriftsartikel (refereegranskat)abstract
- Cervical ripening is necessary for successful delivery. Since cytokines are believed to be involved in this process, the aim of this study was to investigate possible changes in the mRNA and protein expression of pro-inflammatory cytokines (interleukin (IL)-1 alpha, IL-1 beta, IL-12, IL-18) and anti-inflammatory cytokines (IL-4, IL-10, IL-13)in the human cervix during pregnancy, term and preterm labor. Cervical biopsies were taken from 59 women: 21 at preterm labor, 24 at term labor, 10 at term not in labor and 4 from non-pregnant women. mRNA was analyzed with real-time RT-PCR and protein expression and/or secretion with immunohistochemistry and ELISA. There was an upregulation of mRNA for IL-10, IL-13, IL-1 alpha and IL-1 beta in the laboring groups, while mRNA for IL-12 and IL-18 was downregulated. IL-4 mRNA was detected more frequently, while IL-12 mRNA expression was lower, in the preterm labor group than in the term labor group. The protein levels of IL-4 and IL-12 were lower and IL-18 tended to be higher in the labor groups, while IL-10 protein levels were unaffected by labor. IL-4 protein levels were significantly higher in the preterm subgroup with bacterial infection than in the non-infected group. IL-10 had higher expression in squamous epithelium at preterm labor than at term. In conclusion, the major changes in pro-inflammatory and anti-inflammatory cytokine mRNA and protein expression in cervix occur during the labor process irrespective of the length of gestation. Our results indicate that dysregulation of anti-inflammatory cytokines in the human cervix could be involved in the pathogenesis of preterm labor.
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| 4. |
- Hasan, Mahmudul, et al.
(författare)
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The structure of human dermatan sulfate epimerase 1 emphasizes the importance of C5-epimerization of glucuronic acid in higher organisms
- 2021
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Ingår i: Chemical Science. - : Royal Society of Chemistry (RSC). - 2041-6520 .- 2041-6539. ; 12:5, s. 1869-1885
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Tidskriftsartikel (refereegranskat)abstract
- Dermatan sulfate epimerase 1 (DS-epi1, EC 5.1.3.19) catalyzes the conversion of d-glucuronic acid to l-iduronic acid on the polymer level, a key step in the biosynthesis of the glycosaminoglycan dermatan sulfate. Here, we present the first crystal structure of the catalytic domains of DS-epi1, solved at 2.4 Å resolution, as well as a model of the full-length luminal protein obtained by a combination of macromolecular crystallography and targeted cross-linking mass spectrometry. Based on docking studies and molecular dynamics simulations of the protein structure and a chondroitin substrate, we suggest a novel mechanism of DS-epi1, involving a His/double-Tyr motif. Our work uncovers detailed information about the domain architecture, active site, metal-coordinating center and pattern of N-glycosylation of the protein. Additionally, the structure of DS-epi1 reveals a high structural similarity to proteins from several families of bacterial polysaccharide lyases. DS-epi1 is of great importance in a range of diseases, and the structure provides a necessary starting point for design of active site inhibitors.
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| 5. |
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| 6. |
- Fransson, Lars-Åke, et al.
(författare)
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Oligosaccharide mapping of proteoglycan-bound and xyloside-initiated dermatan sulfate from fibroblasts
- 1991
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Ingår i: Glycoconjugate Journal. - 1573-4986. ; 8:2, s. 108-115
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Tidskriftsartikel (refereegranskat)abstract
- The copolymeric structure of dermatan sulfate chains synthesized by skin fibroblasts has been examined. Chains initiated onto exogeneous p-nitrophenyl-beta-D-xylopyranoside or attached to protein in a large proteoglycan, PG-L, and two small proteoglycans, PG-S1 and PG-S2, have been compared by using high resolution electrophoresis and gel chromatography of oligosaccharides generated by specific enzymatic or chemical degradations. The results confirm that chains attached to PG-L are glucuronate-rich, whereas novel findings indicate that chains attached to either of the two PG-S variants yield closely similar oligosaccharide maps, have approximately equal glucuronate and iduronate content and contain over 90% 4-sulfated disaccharide repeating units. Dermatan sulfate chains built onto xyloside at concentrations of 50 microM and below have a copolymeric structure similar to that of chains from the two PG-S variants. These findings indicate that the polymer-modifying machinery can generate chains with extended iduronate-containing repeats also when the xylose primer is not linked to core protein.
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| 7. |
- Gouignard, Nadège, et al.
(författare)
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Musculocontractural Ehlers-Danlos syndrome and neurocristopathies : Dermatan sulfate is required for Xenopus neural crest cells to migrate and adhere to fibronectin
- 2016
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Ingår i: Disease Models and Mechanisms. - : The Company of Biologists. - 1754-8403 .- 1754-8411. ; 9:6, s. 607-620
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Tidskriftsartikel (refereegranskat)abstract
- Of all live births with congenital anomalies, approximately one-third exhibit deformities ofthe head and face. Most craniofacial disorders are associated with defects in a migratory stem and progenitor cell population, which is designated the neural crest (NC). Musculocontractural Ehlers-Danlos syndrome (MCEDS) is a heritable connective tissue disorder with distinct craniofacial features; this syndrome comprises multiple congenital malformations that are caused bydysfunction ofdermatan sulfate (DS) biosynthetic enzymes, including DS epimerase-1 (DS-epi1; also known as DSE). Studies in mice have extended our understanding of DS-epi1 inconnective tissue maintenance; however, its role in fetal development is not understood. We demonstrate that DS-epi1isimportant for the generationofisolated iduronic acid residues in chondroitin sulfate (CS)/DS proteoglycans in early Xenopus embryos. The knockdown of DS-epi1 does not affect the formation of early NC progenitors; however, it impairs the correct activation of transcription factors involved in the epithelial-mesenchymal transition (EMT) and reduces the extent of NC cell migration, which leads to a decrease in NC-derived craniofacial skeleton, melanocytes and dorsal fin structures. Transplantation experiments demonstrate a tissue-autonomous role for DS-epi1 in cranial NC cell migration in vivo. Cranial NC explant and single-cell cultures indicate a requirement of DS-epi1 in cell adhesion, spreading and extension of polarized cell processes on fibronectin. Thus, our work indicates a functional link between DS and NC cell migration. We conclude that NC defects in the EMT and cell migration might account for the craniofacial anomalies and other congenital malformations in MCEDS, which might facilitate the diagnosis and development of therapies for this distressing condition. Moreover, the presented correlations between human DS-epi1 expression and gene sets of mesenchymal character, invasion and metastasis in neuroblastoma and malignant melanoma suggest an association between DS and NC-derived cancers.
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| 8. |
- Akbarshahi, Hamid, et al.
(författare)
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TLR4 dependent heparan sulphate-induced pancreatic inflammatory response is IRF3-mediated
- 2011
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Ingår i: Journal of Translational Medicine. - : Springer Science and Business Media LLC. - 1479-5876. ; 9:219
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Tidskriftsartikel (refereegranskat)abstract
- Background: Degraded extracellular matrix can stimulate the innate immune system via the Toll-Like Receptor-4 (TLR4). In the pancreas, syndecan-anchored heparan sulphate (HS) on the ductal epithelium can be cleaved off its protein cores by the proteases (trypsin and elastase) and potentially activate TLR4 signalling. Methods: To investigate this signalling event, a low sulphated HS (500 mu g/ml) was infused into the biliary-pancreatic duct of C57BL/6J wild-type mice. Phosphate buffered saline (PBS) and lipopolysaccharide (LPS) were used as negative and positive controls, respectively. Mice were sacrificed after 1, 3, 6, 9, and 48 hours and tissues were analysed for neutrophil and cytokine contents. In order to study the TLR4 signalling pathway of HS in the pancreas, genetically engineered mice lacking TLR4, Myeloid Differentiation primary response gene (88) (MyD88) or Interferon Regulatory Factor 3 (IRF3) were subjected to pancreatic infusion of HS. Results: Neutrophil sequestration and corresponding myeloperoxidase (MPO) activity in the pancreas were increased 9 hours following HS challenge. In wild-type mice, the monocyte chemoattractant protein-1(MCP-1) increased at 3 hours after infusion, while RANTES increased after 9 hours. TLR4, MyD88, and IRF3 knockout mice showed an abrogated neutrophil recruitment and myeloperoxidase activity in the HS group, while the LPS response was only abolished in TLR4 and MyD88 knockouts. Conclusions: The results of this study show that HS is capable of initiating a TLR4-dependent innate immune response in the pancreas which is distinctly different from that induced by LPS. This inflammatory response was mediated predominantly through IRF3-dependent pathway. Release of HS into the pancreatic duct may be one important mediator in the pancreatic ductal defence.
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| 9. |
- Bartolini, Barbara, et al.
(författare)
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Iduronic Acid in chondroitin/dermatan sulfate affects directional migration of aortic smooth muscle cells.
- 2013
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:7
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Tidskriftsartikel (refereegranskat)abstract
- Aortic smooth muscle cells produce chondroitin/dermatan sulfate (CS/DS) proteoglycans that regulate extracellular matrix organization and cell behavior in normal and pathological conditions. A unique feature of CS/DS proteoglycans is the presence of iduronic acid (IdoA), catalyzed by two DS epimerases. Functional ablation of DS-epi1, the main epimerase in these cells, resulted in a major reduction of IdoA both on cell surface and in secreted CS/DS proteoglycans. Downregulation of IdoA led to delayed ability to re-populate wounded areas due to loss of directional persistence of migration. DS-epi1-/- aortic smooth muscle cells, however, had not lost the general property of migration showing even increased speed of movement compared to wild type cells. Where the cell membrane adheres to the substratum, stress fibers were denser whereas focal adhesion sites were fewer. Total cellular expression of focal adhesion kinase (FAK) and phospho-FAK (pFAK) was decreased in mutant cells compared to control cells. As many pathological conditions are dependent on migration, modulation of IdoA content may point to therapeutic strategies for diseases such as cancer and atherosclerosis.
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| 10. |
- Maccarana, Marco, et al.
(författare)
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Inhibition of iduronic acid biosynthesis by ebselen reduces glycosaminoglycan accumulation in mucopolysaccharidosis type I fibroblasts
- 2021
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Ingår i: Glycobiology. - : Oxford University Press. - 0959-6658 .- 1460-2423. ; 31:10, s. 1319-1329
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Tidskriftsartikel (refereegranskat)abstract
- Mucopolysaccharidosis type I (MPS-I) is a rare lysosomal storage disorder caused by deficiency of the enzyme alpha-L-iduronidase, which removes iduronic acid in both chondroitin/dermatan sulfate (CS/DS) and heparan sulfate (HS) and thereby contributes to the catabolism of glycosaminoglycans (GAGs). To ameliorate this genetic defect, the patients are currently treated by enzyme replacement and bone marrow transplantation, which have a number of drawbacks. This study was designed to develop an alternative treatment by inhibition of iduronic acid formation. By screening the Prestwick drug library, we identified ebselen as a potent inhibitor of enzymes that produce iduronic acid in CS/DS and HS. Ebselen efficiently inhibited iduronic acid formation during CS/DS synthesis in cultured fibroblasts. Treatment of MPS-I fibroblasts with ebselen not only reduced accumulation of CS/DS but also promoted GAG degradation. In early Xenopus embryos, this drug phenocopied the effect of downregulation of DS-epimerase 1, the main enzyme responsible for iduronic production in CS/DS, suggesting that ebselen inhibits iduronic acid production in vivo. However, ebselen failed to ameliorate the CS/DS and GAG burden in MPS-I mice. Nevertheless, the results propose a potential of iduronic acid substrate reduction therapy for MPS-I patients.
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