Mikko Lammi

Human articular cartilage is specialized to transmit forces between articulating bones and provides almost frictionless movement of bone ends against each other. Chondrocytes, the onlytype of cells in this tissue, have to cope with these mechanical forces during movement. One of thecomponents of the overall mechanical force in articular cartilage is hydrostatic pressure (HP). This study was undertaken to analyzegene expression profile of chondrocytic human cells under high and low HP. Earlier studies concerning the effects of HP on chondrocyte metabolism have mainly focused on measuring the synthetic capacity of extracellular matrix components under various loading regimens rather thanon gene expression on a wide scale.

   In this study, a human chondrosarcoma cell line was cultured as a monolayer culture and pressurized in a custom-made pressurization chamber. High static pressure (30 MPa) was used to study the stressful effects of pressure on the cells and the activity of possible mechanosensitive genes underthese circumstances. Another part of the study focused on theeffects of low static and cyclic pressures (5 MPa) on the gene expression profile of the cells. For comparison one set of cells were also mechanically stretched. Differential display RT-PCR and cDNA array techniques were used to analyse the expression profiles. Possible differential expression was further confirmed by RNA hybridization analysis, ribonuclease protection assay and immunoblotting analysis. In addition, potential changes in mRNA stability during pressurization were assessed by the cDNA array technique.

   Expression profiling revealed induction of heat shock genes, genes involved in growth arrest and control of the cell cycle under high static pressure (30 MPa), indicating adaptation to stressful conditions. In adition, a set of immediate-early genes, for example transcription factors, were up- and down-regulated on the mRNA levels, also indicating the beginning of adaptive processe on genetic level. Interestingly, the Id1, Id2 and Id3 proteins, inhibitors of differentiation, were markedly down-regulated under high pressure. Thus, high pressure may activate differentiation processe in the cells under study. In contrast, low static and cyclic (5 MPa) pressure affected only a few genes, the ones which are involved in cell adhesion and transcriptional regulation. The stability of mRNA molecules was not increased during pressurization. Furthermore, stretching of the cells affected expression of different set of genes compared to genes involved in pressure response.

   In differential display RT-PCR analysis, reticulon 4-B, a member of reticulon gene family, was found to be up-regulated under high static pressure. This previoously uncharacterized gene was not responsive to other kinds of stressful treatments, indicating a possible mechansensitive element in its transcriptional regulation. Furthermore, the intracellular localization and potential role of a putative di-lysine motif in intracellular transport was characterized.