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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

ABSTRACT: Different kinds of cell-based therapies and tissue engineering applications need in vitro cultured differentiated chondrocytes. However, articular chondrocytes are slowly dividing cells, which in culture tend to lose their cell type-specific phenotype and ability to produce structurally and functionally correct cartilage tissue. Here we show that Y-27632 treatment at hypoxia enhances efficiently chondrocyte-specific extracellular matrix production on chondrocytic cells. The effects of the long-term Y-27632 exposure to human chondrosarcoma 2/8 cell phenotype maintenance and extracellular matrix production were studied at normoxia and at 5% low oxygen atmosphere. The Y-27632 treatment at normoxia induced ACAN and COL2A1 gene up-regulation and minor increase of sulfated glycosaminoglycans (sGAGs), while type II collagen expression was not significantly up-regulated. The most efficient expression of ACAN and COL2A1 was achieved when the Y-27632 treatment was performed at hypoxia. The production of sGAGs increased by 65.8%, and the ELISA analysis revealed over 6 times up-regulation on type II collagen expression. The Y-27632 induced up-regulation of S100-A1 and S100-B proteins, and also affected 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 the cells.

INSTRUMENT(S): SYNAPT G2-Si

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Chondrocyte, Cell Culture

SUBMITTER: Joakim Bygdell

LAB HEAD: Mikko Lammi

PROVIDER: PXD005551 | Pride | 2017-06-22

REPOSITORIES: Pride

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Rho-kinase inhibitor Y-27632 and hypoxia synergistically enhance chondrocytic phenotype and modify S100 protein profiles in human chondrosarcoma cells.

Piltti Juha J Bygdell Joakim J Fernández-Echevarría Cecilia C Marcellino Daniel D Lammi Mikko J MJ

Scientific reports 20170616 1

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 chondro ...[more]

PMID: 28623370

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Project description:Purpose: The goal of this study is to identify the miRNA clusters that are regulated by EGFR under normoxia or hypoxia. Method: Total RNAs were extracted from HeLa cells expressing scrambled control or EGFR shRNA-E1 that cultured under normoxia or hypoxia (1% O2) for 24h. Customized Next-Generation RNA deep sequencing, including both small RNA application and whole transcriptome analysis, was performed according to the standard procedure instructed by Applied Biosystems. For small RNA analysis, library inserts were size selected between 18 and 40nts and analyzed using CLC Genomics Workbench 4.7.1. 35nt colorspace reads were trimmed of adaptor sequence and mapped against human pre-miR sequences (miRBase version 16.0). Values of reads per million mapped reads (RPM) were based on mapped reads with no more than 2 mismatches total. A read was considered to come from a mature miRNA if it mapped to pre-miRNA sequences with no more than three upstream or downstream bases, and missing no more than two upstream or downstream bases from predicted mature or mature* sequences as defined in miRBase version 16.0. All the other pre-miRNA mapped reads were assigned as pre-miRNA signal. qRT–PCR validation was performed using TaqMan and SYBR Green assays. Results: Deep sequencing analysis identified specific miRNA clusters that their maturation (miRNA processing efficacy was reflected by the relative expression of precursor miRNAs affected by EGFR compared with the relative expression of mature miRNAs affected by EGFR) were regulated by EGFR under normixa or specifically in response to hypoxia. Top miRNA candidates that regulated by EGFR in response to hypoxia were further verified by TaqMan and SYBR Green qRT-PCR assays. Conclusion: Next-Generation Deep Sequencing for small RNA analysis revealed a novel function of EGFR involved in miRNA maturation in response to hypoxic stress. RNA profiles of HeLa cells expressing scrambled control (S) or EGFR shRNA-E1 (A1) that cultured under normoxia or hypoxia (1% O2) for 24h were generated by AB SOLiD next-generation sequencing. S: HeLa expressing scrambled control cultured under normoxia; A1: HeLa expressing EGFR shRNA-E1 cultured under normoxia; HS: HeLa expressing scrambled control cultured under hypoxia for 24h; HA1: HeLa expressing EGFR shRNA-E1 cultured under hypoxia for 24h. In total, 4 biological samples with no replicates resulted in 4 small RNA profiles.

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