Smallridge R C, Gist I D, Tsokos G C, Kiang J G
The Endocrinology Division, Mayo Clinic Jacksonville, Florida 32224, USA.
Thyroid. 1999 Oct;9(10):1041-7. doi: 10.1089/thy.1999.9.1041.
Heat shock induces the expression of proteins with molecular weights of 70-72 kd and 90 kd, whereas thapsigargin induces the expression of a glucose-regulated protein 78 kd (GRP-78) in certain cells. In this study we examined the induction and cytoprotective effects of heat shock- and thapsigargin-induced proteins in FRTL-5 rat thyroid cells. New protein synthesis was assessed in [35S]methionine-labeled cells and quantitated densitometrically. The expression of specific stress proteins was identified using Western blots, whereas cytoprotection provided by these proteins was evaluated by trypan blue exclusion. Exposure to heat shock (45 degrees C, 15 minutes) induced the expression of proteins with molecular weights at the range of low 70 kD and low 90 kD that peaked between 2-6 hours and returned to baseline within 24 hours. Treatment of cells with thapsigargin (200 nM, 15 minutes) induced the expression of different molecular weight proteins, most likely GRP-78 and -94, that peaked at 4-6 hours and lasted for 24 hours. Neither the removal of growth factors (thyroid-stimulating hormone and insulin) for 5 days nor the elimination of extracellular Ca2+ with EGTA or clamping of the intracellular Ca2+ with BAPTA for 15 minutes affected expression of the heat shock- and the thapsigargin-induced stress proteins. In contrast, protein kinase C inhibitors H7 and GF109203X abolished the expression of all three groups of stress proteins. Both heat shock- and thapsigargin-inuced proteins completely protected cells from subsequent thermal injury (47 degrees C, 35 minutes). The induction of cytoprotective proteins by heat shock and thapsigargin requires the presence of protein kinase C but is Ca(2+)- and growth factor-independent.
热休克诱导分子量为70 - 72kd和90kd的蛋白质表达，而毒胡萝卜素在某些细胞中诱导78kd葡萄糖调节蛋白（GRP - 78）的表达。在本研究中，我们检测了热休克和毒胡萝卜素诱导的蛋白在FRTL - 5大鼠甲状腺细胞中的诱导作用和细胞保护作用。在[35S]甲硫氨酸标记的细胞中评估新蛋白合成，并通过光密度法进行定量。使用蛋白质免疫印迹法鉴定特定应激蛋白的表达，而通过台盼蓝排斥法评估这些蛋白提供的细胞保护作用。暴露于热休克（45℃，15分钟）诱导低70kD和低90kD范围内分子量的蛋白质表达，在2 - 6小时达到峰值，并在24小时内恢复到基线水平。用毒胡萝卜素（200nM，15分钟）处理细胞诱导不同分子量蛋白的表达，最可能是GRP - 78和 - 94，在4 - 6小时达到峰值并持续24小时。去除生长因子（促甲状腺激素和胰岛素）5天，或用乙二醇双四乙酸（EGTA）去除细胞外Ca2 + 或用1,2 - 双（2 - 氨基苯氧基）乙烷 - N，N，N′，N′ - 四乙酸（BAPTA）钳制细胞内Ca2 + 15分钟，均不影响热休克和毒胡萝卜素诱导的应激蛋白的表达。相反，蛋白激酶C抑制剂H7和GF109203X消除了所有三组应激蛋白的表达。热休克和毒胡萝卜素诱导的蛋白均能完全保护细胞免受随后的热损伤（47℃，35分钟）。热休克和毒胡萝卜素诱导细胞保护蛋白需要蛋白激酶C的存在，但与Ca(2 + )和生长因子无关。
