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Abstract
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.
Highlights
Articular cartilage is a poorly regenerating tissue, and there is need for effective repair techniques to treat damaged cartilage tissues
This study was performed to investigate the specific responses to prolonged Rho-kinase inhibition at normoxia and at a 5% low oxygen atmosphere in human chondrosarcoma-2/8 cells (HCS-2/8)
A three day exposure to 10 μM Y-27632 did not affect the cellular proliferation of HCS-2/8 cells, rather this treatment inhibited the depolymerization of filamentous actin (Fig. 1, Supplementary Fig. 1), similar to finding observed in human fibroblasts[15]
Summary
Articular cartilage is a poorly regenerating tissue, and there is need for effective repair techniques to treat damaged cartilage tissues. Regardless of the cell source, differentiated chondrocytes have the tendency to lose their phenotype and ability to produce cartilage-specific extracellular matrix over time[1]. The cellular microenvironment is known to regulate cell phenotype Both microenvironment and paracrine factors can regulate extracellular matrix production and chondrogenic differentiation of mesenchymal stem cells[6]. This study was performed to investigate the specific responses to prolonged Rho-kinase inhibition at normoxia and at a 5% low oxygen atmosphere in human chondrosarcoma-2/8 cells (HCS-2/8). A proteomic-based analysis was performed to reveal which protein responses are produced by Y-27632 at specific oxygen atmospheres. Proteomic-based analyses have been used to define protein profiles of articular chondrocytes and changes related to chondrogenic differentiation and osteoarthritis[7,8,9,10,11,12]. The human HCS-2/8 cell line has a prominent aggrecan and type II collagen production, and it shares most features of normal chondrocytes[13, 14]
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