| 1. |
- Aspberg, A., et al.
(författare)
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Cartilage oligomeric matrix protein forms protein complexes with synovial lubricin via non-covalent and covalent interactions
- 2017
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Ingår i: Osteoarthritis and Cartilage. - : Elsevier BV. - 1063-4584. ; 25:9, s. 1496-1504
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Tidskriftsartikel (refereegranskat)abstract
- Objective: Understanding the cartilage surface structure, lost in arthritic disease, is essential for developing strategies to effectively restore it. Given that adherence of the lubricating protein, lubricin, to the cartilage surface is critical for boundary lubrication, an interaction with cartilage oligomeric matrix protein (COMP) was investigated. COMP, an abundant cartilage protein, is known to be important for matrix formation. Design: Synovial fluid (SF) from arthritic patients was used to detect possible COMP-lubricin complexes by immunological methods. Recombinant (RC) COMP and lubricin fragments were expressed to characterize this bonding and mass spectrometry employed to specifically identify the cysteines involved in inter-protein disulfide bonds. Results: COMP-lubricin complexes were identified in the SF of arthritic patients by Western blot, co-immunoprecipitation and sandwich ELISA. RC fragment solid-phase binding assays showed that the C-terminal (amino acids (AA) 518-757) of COMP bound non-covalently to the N-terminal of lubricin (AA 105-202). Mass spectrometry determined that although cysteines throughout COMP were involved in binding with lubricin, the cysteines in lubricin were primarily focused to an N-terminal region (AA 64-86). The close proximity of the non-covalent and disulfide binding domains on lubricin suggest a two-step mechanism to strongly bind lubricin to COMP. Conclusion: These data demonstrate that lubricin forms a complex network with COMP involving both non-covalent and covalent bonds. This complex between lubricin and the cartilage protein COMP can be identified in the SF of patients with arthritis conditions including osteoarthritis (OA) and rheumatoid arthritis (RA). (C) 2017 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.
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| 2. |
- Flowers, Sarah A., et al.
(författare)
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Lubricin binds cartilage proteins, cartilage oligomeric matrix protein, fibronectin and collagen II at the cartilage surface
- 2017
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Lubricin, a heavily O-glycosylated protein, is essential for boundary lubrication of articular cartilage. Strong surface adherence of lubricin is required given the extreme force it must withstand. Disulfide bound complexes of lubricin and cartilage oligomeric matrix protein (COMP) have recently been identified in arthritic synovial fluid suggesting they may be lost from the cartilage surface in osteoarthritis and inflammatory arthritis. This investigation was undertaken to localise COMP-lubricin complexes within cartilage and investigate if other cartilage proteins are involved in anchoring lubricin to the joint. Immunohistochemical analysis of human cartilage biopsies showed lubricin and COMP co-localise to the cartilage surface. COMP knockout mice, however, presented with a lubricin layer on the articular cartilage leading to the further investigation of additional lubricin binding mechanisms. Proximity ligation assays (PLA) on human cartilage biopsies was used to localise additional lubricin binding partners and demonstrated that lubricin bound COMP, but also fibronectin and collagen II on the cartilage surface. Fibronectin and collagen II binding to lubricin was confirmed and characterised by solid phase binding assays with recombinant lubricin fragments. Overall, COMP, fibronectin and collagen II bind lubricin, exposed on the articular cartilage surface suggesting they may be involved in maintaining essential boundary lubrication.
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| 3. |
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| 4. |
- Ishikawa, Yoshihiro, et al.
(författare)
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The endoplasmic reticulum-resident collagen chaperone Hsp47 interacts with and promotes the secretion of decorin, fibromodulin, and lumican
- 2018
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Ingår i: Journal of Biological Chemistry. - : AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC. - 0021-9258 .- 1083-351X. ; 293:35, s. 13707-13716
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Tidskriftsartikel (refereegranskat)abstract
- The build-up of diversified and tissue-specific assemblies of extracellular matrix (ECM) proteins depends on secreted and cell surface-located molecular arrays that coordinate ECM proteins into discrete designs. The family of small leucine-rich proteins (SLRPs) associates with and dictates the structure of fibrillar collagens, which form the backbone of most ECM types. However, whether SLRPs form complexes with proteins other than collagens is unclear. Here, we demonstrate that heat shock protein 47 (Hsp47), a well-established endoplasmic reticulum-resident collagen chaperone, also binds the SLRPs decorin, lumican, and fibromodulin with affinities comparable with that in the Hsp47-type I collagen interaction. Furthermore, we show that a lack of Hsp47 inhibits the cellular secretion of decorin and lumican. Our results expand the understanding of the concerted molecular interactions that control the secretion and organization of a functional collagenous ECM.
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| 5. |
- Kalamajski, Sebastian, et al.
(författare)
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Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase.
- 2016
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 291:15, s. 7951-60
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Tidskriftsartikel (refereegranskat)abstract
- The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites.
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| 6. |
- Maccarana, Marco, et al.
(författare)
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Asporin-deficient mice have tougher skin and altered skin glycosaminoglycan content and structure
- 2017
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 12:8
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Tidskriftsartikel (refereegranskat)abstract
- The main structural component of connective tissues is fibrillar, cross-linked collagen whose fibrillogenesis can be modulated by Small Leucine-Rich Proteins/Proteoglycans (SLRPs). Not all SLRPs’ effects on collagen and extracellular matrix in vivo have been elucidated; one of the less investigated SLRPs is asporin. Here we describe the successful generation of an Aspn-/- mouse model and the investigation of the Aspn-/- skin phenotype. Functionally, Aspn-/- mice had an increased skin mechanical toughness, although there were no structural changes present on histology or immunohistochemistry. Electron microscopy analyses showed 7% thinner collagen fibrils in Aspn-/- mice (not statistically significant). Several matrix genes were upregulated, including collagens (Col1a1, Col1a2, Col3a1), matrix metalloproteinases (Mmp2, Mmp3) and lysyl oxidases (Lox, Loxl2), while lysyl hydroxylase (Plod2) was downregulated. Intriguingly no differences were observed in collagen protein content or in collagen cross-linking-related lysine oxidation or hydroxylation. The glycosaminoglycan content and structure in Aspn-/- skin was profoundly altered: chondroitin/dermatan sulfate was more than doubled and had an altered composition, while heparan sulfate was halved and had a decreased sulfation. Also, decorin and biglycan were doubled in Aspn-/- skin. Overall, asporin deficiency changes skin glycosaminoglycan composition, and decorin and biglycan content, which may explain the changes in skin mechanical properties.
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| 7. |
- Olof Olsson, P., et al.
(författare)
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Inhibition of integrin αvβ6 changes fibril thickness of stromal collagen in experimental carcinomas
- 2018
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Ingår i: Cell Communication and Signaling. - : Springer Science and Business Media LLC. - 1478-811X. ; 16:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Chemotherapeutic efficacy can be improved by targeting the structure and function of the extracellular matrix (ECM) in the carcinomal stroma. This can be accomplished by e.g. inhibiting TGF-β1 and -β3 or treating with Imatinib, which results in scarcer collagen fibril structure in xenografted human KAT-4/HT29 (KAT-4) colon adenocarcinoma. Methods: The potential role of αVβ6 integrin-mediated activation of latent TGF-β was studied in cultured KAT-4 and Capan-2 human ductal pancreatic carcinoma cells as well as in xenograft carcinoma generated by these cells. The monoclonal αVβ6 integrin-specific monoclonal antibody 3G9 was used to inhibit the αVβ6 integrin activity. Results: Both KAT-4 and Capan-2 cells expressed the αVβ6 integrin but only KAT-4 cells could utilize this integrin to activate latent TGF-β in vitro. Only when Capan-2 cells were co-cultured with human F99 fibroblasts was the integrin activation mechanism triggered, suggesting a more complex, fibroblast-dependent, activation pathway. In nude mice, a 10-day treatment with 3G9 reduced collagen fibril thickness and interstitial fluid pressure in KAT-4 but not in the more desmoplastic Capan-2 tumors that, to achieve a similar effect, required a prolonged 3G9 treatment. In contrast, a 10-day direct inhibition of TGF-β1 and -β3 reduced collagen fibril thickness in both tumor models. Conclusion: Our data demonstrate that the αVβ6-directed activation of latent TGF-β plays a pivotal role in modulating the stromal collagen network in carcinoma, but that the sensitivity to αVβ6 inhibition depends on the simultaneous presence of alternative paths for latent TGF-β activation and the extent of desmoplasia.
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| 8. |
- Olsson, Olof, et al.
(författare)
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The Tyrosine Kinase Inhibitor Imatinib Augments Extracellular Fluid Exchange and Reduces Average Collagen Fibril Diameter in Experimental Carcinoma
- 2016
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Ingår i: Molecular Cancer Therapeutics. - 1535-7163 .- 1538-8514. ; 15:10, s. 2455-2464
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Tidskriftsartikel (refereegranskat)abstract
- A typical obstacle to cancer therapy is the limited distribution of low molecular weight anticancer drugs within the carcinoma tissue. In experimental carcinoma, imatinib (STI571) increases efficacy of synchronized chemotherapy, reduces tumor interstitial fluid pressure, and increases interstitial fluid volume. STI571 also increases the water-perfusable fraction in metastases from human colorectal adenocarcinomas. Because the mechanism(s) behind these effects have not been fully elucidated, we investigated the hypothesis that STI571 alters specific properties of the stromal extracellular matrix. We analyzed STI571-treated human colorectal KAT-4/HT-29 experimental carcinomas, known to have a well-developed stromal compartment, for solute exchange and glycosaminoglycan content, as well as collagen content, structure, and synthesis. MRI of STI571-treated KAT-4/HT-29 experimental carcinomas showed a significantly increased efficacy in dynamic exchanges of solutes between tumor interstitium and blood. This effect was paralleled by a distinct change of the stromal collagen network architecture, manifested by a decreased average collagen fibril diameter, and increased collagen turnover. The glycosaminoglycan content was unchanged. Furthermore, the apparent effects on the stromal cellular composition were limited to a reduction in an NG2-positive stromal cell population. The current data support the hypothesis that the collagen network architecture influences the dynamic exchanges of solutes between blood and carcinoma tissue. It is conceivable that STI571 reprograms distinct nonvascular stromal cells to produce a looser extracellular matrix, ultimately improving transport characteristics for traditional chemotherapeutic agents.
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| 9. |
- Olsson, P. Olof, et al.
(författare)
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Fibromodulin deficiency reduces collagen structural network but not glycosaminoglycan content in a syngeneic model of colon carcinoma.
- 2017
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 12:8
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Tidskriftsartikel (refereegranskat)abstract
- Tumor barrier function in carcinoma represents a major challenge to treatment and is therefore an attractive target for increasing drug delivery. Variables related to tumor barrier include aberrant blood vessels, high interstitial fluid pressure, and the composition and structure of the extracellular matrix. One of the proteins associated with dense extracellular matrices is fibromodulin, a collagen fibrillogenesis modulator expressed in tumor stroma but scarce in normal loose connective tissues. Here, we investigated the effects of fibromodulin on stroma ECM in a syngeneic murine colon carcinoma model. We show that fibromodulin deficiency decreased collagen fibril thickness but glycosaminoglycan content and composition were unchanged. Furthermore, vascular density, pericyte coverage and macrophage amount were unaffected. Fibromodulin can therefore be a unique effector of dense collagen matrix assembly in tumor stroma and, without affecting other major matrix components or the cellular composition, can function as a main agent in tumor barrier function.
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| 10. |
- Olsson, P. Olof, et al.
(författare)
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Inhibition of integrin alpha(V)beta(6) changes fibril thickness of stromal collagen in experimental carcinomas
- 2018
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Ingår i: Cell Communication and Signaling. - : BMC. - 1478-811X. ; 16
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Tidskriftsartikel (refereegranskat)abstract
- Background: Chemotherapeutic efficacy can be improved by targeting the structure and function of the extracellular matrix (ECM) in the carcinomal stroma. This can be accomplished by e.g. inhibiting TGF beta 1 and -beta 3 or treating with Imatinib, which results in scarcer collagen fibril structure in xenografted human KAT-4/HT29 (KAT-4) colon adenocarcinoma.Methods: The potential role of a(v)beta(6) integrin-mediated activation of latent TGF-beta was studied in cultured KAT-4 and Capan-2 human ductal pancreatic carcinoma cells as well as in xenograft carcinoma generated by these cells. The monoclonal a(v)beta(6) integrin-speafic monoclonal antibody 3G9 was used to inhibit the a(v)beta(6) integrin activity.Results: Both KAT-4 and Capan-2 cells expressed the a(v)beta(6) integrin but only KAT-4 cells could utilize this integrin to activate latent TGF-beta in vitro. Only when Capan-2 cells were co-cultured with human F99 fibroblasts was the integrin activation mechanism triggered, suggesting a more complex, fibroblast-dependent, activation pathway. In nude mice, a 10-day treatment with 3G9 reduced collagen fibril thickness and interstitial fluid pressure in KAT-4 but not in the more desmoplastic Capan-2 tumors that, to achieve a similar effect, required a prolonged 3G9 treatment. In contrast, a 10-day direct inhibition of TGF-beta 1 and -beta 3 reduced collagen fibril thickness in both tumor models.Conclusion: Our data demonstrate that the a(v)beta(6)-directed activation of latent TGF-beta plays a pivotal role in modulating the stromal collagen network in carcinoma, but that the sensitivity to a(v)beta(6) inhibition depends on the simultaneous presence of alternative paths for latent TGF-beta activation and the extent of desmoplasia.
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