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Rho-kinase inhibitor Y-27632 and hypoxia synergistically enhance chondrocytic phenotype and change the S100 protein profile in human chondrosarcoma cells
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). (Chondrogenic and Osteogenic Differentiation Group)ORCID iD: 0000-0002-1732-4269
Umeå University, Faculty of Science and Technology, Department of Chemistry.ORCID iD: 0000-0002-9833-4628
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). (Marcellino Laboratory)
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). (Marcellino Laboratory)ORCID iD: 0000-0002-4618-7267
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2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 3708Article in journal (Refereed) Published
Abstract [en]

Articular chondrocytes are slowly dividing cells that tend to lose their cell type-specific phenotype and ability to produce structurally and functionally correct cartilage tissue when cultured. Thus, culture conditions, which enhance the maintenance of chondrocyte phenotype would be very useful for cartilage research. Here we show that Rho-kinase inhibition by Y-27632 under hypoxic conditions efficiently maintains and even enhances chondrocyte-specific extracellular matrix production by chondrocytic cells. The effects of long-term Y-27632 exposure to human chondrosarcoma 2/8 cell phenotype maintenance and extracellular matrix production were studied at normoxia and at a 5% low oxygen atmosphere. Y-27632 treatment at normoxia induced ACAN and COL2A1 gene up-regulation and a minor increase of sulfated glycosaminoglycans (sGAGs), while type II collagen expression was not significantly up-regulated. A further increase in expression of ACAN and COL2A1 was achieved with Y-27632 treatment and hypoxia. The production of sGAGs increased by 65.8%, and ELISA analysis revealed a 6-fold up-regulation of type II collagen. Y-27632 also induced the up-regulation of S100-A1 and S100-B proteins and modified the expression of several other S100 protein family members, such as S100-A4, S100-A6, S100-A13 and S100-A16. The up-regulation of S100-A1 and S100-B proteins is suggested to enhance the chondrocytic phenotype of these cells.

Place, publisher, year, edition, pages
Nature Publishing Group, 2017. Vol. 7, article id 3708
Keywords [en]
ROCK inhibitor, chondrocyte, extracellullar matrix, S100 proteins
National Category
Cell and Molecular Biology
Research subject
Biochemistry; cellforskning; Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-133850DOI: 10.1038/s41598-017-03958-5OAI: oai:DiVA.org:umu-133850DiVA, id: diva2:1089310
Available from: 2017-04-19 Created: 2017-04-19 Last updated: 2018-11-02Bibliographically approved
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. p. 101
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1896
Keywords
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: 2018-06-09Bibliographically approved

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Piltti, JuhaBygdell, JoakimMarcellino, DanielLammi, Mikko

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