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Proteomics of chondrocytes with special reference to phosphorylation changes of proteins in stretched human chondrosarcoma cells.
Department of Biomedicine, Anatomy, University of Kuopio, Kuopio, Finland.
Department of Biosciences, Biochemistry, University of Kuopio, Kuopio, Finland.
Department of Biomedicine, Anatomy, University of Kuopio, Kuopio, Finland.
Department of Biomedicine, Anatomy, University of Kuopio, Kuopio, Finland; Department of Biosciences, Applied Biotechnology, University of Kuopio, Kuopio, Finland. (Chondrogenic and Osteogenic Differentiation Group)ORCID iD: 0000-0002-6181-9904
2008 (English)In: Biorheology, ISSN 0006-355X, E-ISSN 1878-5034, Vol. 45, no 3-4, 323-335 p.Article in journal (Refereed) Published
Abstract [en]

For proteomic analysis, cartilage molecular composition is a challenging mixture of highly glycosylated proteoglycans and triple-helical collagens, which constitute the major part of cartilage macromolecules. Selective separation of these molecules from the minor components is generally needed before mass spectrometry-based identification of lower-abundancy proteins is possible. The cell density of cartilage is also very low, therefore, cell cultures offer an easier approach to study cellular responses of chondrocytic cells, e.g., to mechanical stimuli. In this study, we investigated the phosphorylation events in human chondrosarcoma cells during cellular stretching. Human chondrosarcoma cells were stretched to 8% strain at a frequency of 1 Hz. One set of experiments included cellular stretching which lasted 2 hours, and the other one included experiments of 2 hours daily treatment for up to 3 days. Two-dimensional polyacrylamide gel electrophoresis combined with chromatographic phosphoprotein pre-enrichment and electrospray ionization mass spectrometry-based protein identification was used to reveal changes of phosphoproteins in cells exposed to cyclic stretching. We discovered that 2 hours cyclic stretching increased the phosphorylation of moesin, elongation factor eEF1D and leprecan, while the phosphorylation of elongation factor eEF1B decreased after cellular stretching. Western blot analyses with phospho-specific antibodies suggested that stretching induces phosphorylation of ERK of the MAP kinase pathway, but did not induce phosphorylation of phosphatidylinositol 3-kinase. In conclusion, the proteomic approach revealed that cellular stretching induced specific phosphorylation changes in chondrosarcoma cells.

Place, publisher, year, edition, pages
IOS Press, 2008. Vol. 45, no 3-4, 323-335 p.
Keyword [en]
Mechanobiology, cellular stretching, signal transduction, phosphorylation, proteomics, chondrosarcoma
National Category
Cell and Molecular Biology Biochemistry and Molecular Biology
Research subject
Biochemistry; cellforskning
Identifiers
URN: urn:nbn:se:umu:diva-106581DOI: 10.3233/BIR-2008-0481ISI: 000260965900011PubMedID: 18836233OAI: oai:DiVA.org:umu-106581DiVA: diva2:842601
Conference
5th Symposium on Mechanobiology of Cartilage and Chondrodcyte, Athens, May, 2007
Available from: 2015-07-21 Created: 2015-07-21 Last updated: 2017-04-21
In thesis
1. Responses of fibroblasts and chondrosarcoma cells to mechanical and chemical stimuli
Open this publication in new window or tab >>Responses of fibroblasts and chondrosarcoma cells to mechanical and chemical stimuli
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Osteoarthritis is an inflammation-related disease that progressively destroys joint cartilage. This disease causes pain and stiffness of the joints, and at advanced stages, limitations to the movement or bending of injured joints. Therefore, it often restricts daily activities and the ability to work. Currently, there is no cure to prevent its progression, although certain damaged joints, such as fingers, knees and hips, can be treated with joint replacement surgeries. However, joint replacement surgeries of larger joints are very invasive operations and the joint replacements have a limited lifetime.

Cell-based therapies could offer a way to treat cartilage injuries before the ultimate damage of osteoarthritis on articular cartilage. The development of novel treatments needs both a good knowledge of articular cartilage biology and tissue engineering methods. This thesis primarily investigates the effects of mechanical cyclic stretching, a 5% low oxygen atmosphere and the Rho-kinase inhibitor, Y-27632, on protein responses in chondrocytic human chondrosarcoma (HCS-2/8) cells. Special focus is placed on Rho-kinase inhibition, relating to its potential to promote and support extracellular matrix production in cultured chondrocytes and its role in fibroblast cells as a part of direct chemical cellular differentiation. The means to enhance the production of cartilage-specific extracellular matrix is needed for cell-based tissue engineering applications, since cultured chondrocytes quickly lose their cartilage-specific phenotype.

A mechanical 8% cyclic cell stretching at a 1 Hz frequency was used to model a stretching rhythm similar to walking. The cellular stretching relates to stresses, which are directed to chondrocytes during the mechanical load. The stretch induced changes in proteins related, e.g., to certain cytoskeletal proteins, but also in enzymes associated with protein synthesis, such as eukaryotic elongation factors 1-beta and 1-delta. Hypoxic conditions were used to model the oxygen tension present in healthy cartilage tissue. Long-term hypoxia changed relative amounts in a total of 44 proteins and induced gene expressions of aggrecan and type II collagen, in addition to chondrocyte differentiation markers S100A1 and S100B. A short-term inhibition of Rho-kinase failed to induce extracellular matrix production in fibroblasts or in HCS-2/8 cells, while its long-term exposure increased the expressions of chondrocyte-specific genes and differentiation markers, and also promoted the synthesis of sulfated glycosaminoglycans by chondrocytic cells. Interestingly, Rho kinase inhibition under hypoxic conditions produced a more effective increase in chondrocyte-specific gene expression and synthesis of extracellular matrix components by HCS-2/8 cells. The treatment induced changes in the synthesis of 101 proteins and ELISA analysis revealed a sixfold higher secretion of type II collagen compared to control cells. The secretion of sulfated glycosaminoglycans was simultaneously increased by 65.8%. Thus, Rho-kinase inhibition at low oxygen tension can be regarded as a potential way to enhance extracellular matrix production and maintain a chondrocyte phenotype in cell-based tissue engineering applications.

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2017. 101 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1896
Keyword
Rho-kinase inhibition, Y-27632, hypoxia, cyclic stretching, human chondrosarcoma 2/8 cells, chondrocyte phenotype, fibroblasts, proteomics, protein pathway analytics
National Category
Cell and Molecular Biology
Research subject
Medical Cell Biology
Identifiers
urn:nbn:se:umu:diva-133851 (URN)978-91-7601-710-4 (ISBN)
Public defence
2017-05-15, MA121, Mit-huset, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2017-04-24 Created: 2017-04-19 Last updated: 2017-05-04Bibliographically approved

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