Transcriptional activation in chondrocytes submitted to hydrostatic pressure

Abstract

At present, only a little is known about the transcriptional regulation in chondrocytes submitted to various physicomechanical factors known to exist in articular cartilage. Recently, we have investigated the effects of hydrostatic pressure on transcriptional control in chondrocytes using human chondrosarcoma and immortalized chondrocyte cell lines for the experiments. Hydrostatic pressure was applied on the cells in a special computer‐controlled, water‐filled pressure chamber, where cyclic and static pressures up to 32 MPa can be created. Differential display RT‐PCR and probing of cDNA arrays are the methods we have used to study differential gene expression due to hydrostatic pressure. By differential display RT‐PCR experiments, we have observed several differentially expressed cDNA bands under continuous 30 MPa hydrostatic pressure, while 30 MPa cyclic pressure at 1 Hz produced much fewer changes. In the first phase of our studies, we have focused on the effects of 30 MPa hydrostatic pressure because it causes a unique hsp70‐mediated stress response in immortalized chondrocytes. Differential display RT‐PCR screening provided us with several clones that derive from low‐abundance mRNAs, such as death‐associated protein 3 (DAP3), a nucleotide‐binding protein which increases due to interferon‐gamma induced cell death; PTZ‐17 (or p311), a seizure‐related protein; H‐NUC, a nuclear DNA binding protein; and one new gene of unknown function. In Northern blots, an induction was confirmed for the new gene, DAP3 and PTZ‐17 were down‐regulated in some but not in all parallel experiments; however, basal level of H‐NUC mRNA was too low to be detected in Northern blots. We then chose to widen our screening to a number of known genes arrayed as cDNA blots. Under 30 MPa continuous hydrostatic pressure, four different time points were chosen (0, 3, 6 and 24 h) for the experiments. The screening of 588 cDNAs showed 15 up‐regulated and 6 down‐regulated genes. Consistently with our previous results hsp70 was highly induced, as well as hsp40, a chaperone protein functioning together with hsp70. Gadd45 and to a lesser extent Gadd153 (stress genes induced by, e.g., ionizing radiation and ischaemia) were up‐regulated, as well as p21 $^\mathrm{waf1,cip1}$ , a protein participating in cell cycle regulation that can interact with Gadd45. Northern blots confirmed Gadd45 induction. Down‐regulated transcripts included, e.g., DAD‐1, glutathione S‐transferase pI, DNA‐binding inhibitor ID‐1H, and cytoplasmic dynein light chain.