Elo M A, Sironen R K, Kaarniranta K, Auriola S, Helminen H J, Lammi M J
Department of Anatomy, University of Kuopio, 70211 Kuopio, Finland.
J Cell Biochem. 2000 Sep 14;79(4):610-9. doi: 10.1002/1097-4644(20001215)79:4<610::aid-jcb100>3.0.co;2-j.
High hydrostatic pressure (HP) has recently been shown to increase cellular heat shock protein 70 (Hsp70) level in a specific way that does not involve transcriptional activation of the gene, but rather the stabilisation of the mRNA for Hsp70. In this study, we investigated whether there are other observable changes caused by HP stress, and compared them with those induced by certain other forms of stressors. A chondrocytic cell line T/C28a4 was exposed to 30 MPa continuous HP, heat shock at 43 degrees C, and increased cytosolic calcium concentration by the addition of sarco-endoplasmic reticulum Ca(2+) ATPase inhibitor thapsigargin (25 nM) or calcium ionophore A23187 (1 microM) in the cultures. The protein synthesis was studied by in vitro metabolic labelling followed by one- and two-dimensional polyacrylamide gel electrophoresis, and mass spectrometry was utilized to confirm the identity of the protein spots on two-dimensional gels. Continuous 30 MPa HP increased remarkably the relative labelling of Hsp70. Labelling of Hsp90 was also increased by 15-20%, although no clear change was evident at the protein level in Western blots. Elevated intracellular Ca(2+) concentration induced by thapsigargin and calcium ionophore A23187 increased mainly the synthesis of glucose-regulated protein 78 (Grp78/BiP), whereas Hsp70 and Hsp90 were decreased by the treatment. Heat shock was the strongest inducer of Hsp70 and Hsp90. This study further confirmed the induction of Hsp70 in chondrocytic cells exposed to high HP, but it also showed that calcium-mediated responses are unlikely to cause the stress response observed in the hydrostatically pressurized cells.
最近研究表明，高静水压（HP）能以一种特定方式提高细胞热休克蛋白70（Hsp70）水平，该方式不涉及基因的转录激活，而是Hsp70的信使核糖核酸（mRNA）的稳定化。在本研究中，我们调查了HP应激是否会引起其他可观察到的变化，并将其与某些其他形式应激源诱导的变化进行比较。将软骨细胞系T/C28a4暴露于30兆帕的连续HP、43摄氏度的热休克，并通过在培养物中添加肌浆内质网Ca(2+)ATP酶抑制剂毒胡萝卜素（25纳摩尔）或钙离子载体A23187（1微摩尔）来提高胞质钙浓度。通过体外代谢标记，然后进行一维及二维聚丙烯酰胺凝胶电泳研究蛋白质合成，并利用质谱法确认二维凝胶上蛋白质斑点的身份。连续30兆帕的HP显著增加了Hsp70的相对标记。Hsp90的标记也增加了15%-20%，尽管在蛋白质印迹中蛋白质水平没有明显变化。毒胡萝卜素和钙离子载体A23187诱导的细胞内Ca(2+)浓度升高主要增加了葡萄糖调节蛋白78（Grp78/BiP）的合成，而Hsp70和Hsp90在处理后减少。热休克是Hsp70和Hsp90最强的诱导剂。本研究进一步证实了暴露于高HP的软骨细胞中Hsp70的诱导，但也表明钙介导的反应不太可能导致在静水压加压细胞中观察到的应激反应。
