Prüfer Kirsten, Schröder Claudia, Hegyi Krisztina, Barsony Julia
Laboratory of Cell Biochemistry and Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Mol Endocrinol. 2002 May;16(5):961-76. doi: 10.1210/mend.16.5.0821.
Several cell lines, including ROS17/2.8 rat osteosarcoma (ROS) cells, contain functional VDRs and RXRs but are resistant to the antiproliferative effects of calcitriol and retinoids. We explored the role of receptor degradation in this hormone resistance. Results of transactivation assays indicated that ROS cells contain insufficient amounts of RXR to activate a DR-1 reporter, and Western blot analyses of cell extracts showed that the degradation of RXR is accelerated and produces an aberrant 45-kDa RXR. We stably expressed functional fluorescent chimeras of VDR and RXR [green fluorescent protein (GFP)-VDR; yellow fluorescent protein (YFP)-RXR] to evaluate degradation mechanisms and the impact of excess receptor expression on antiproliferative effects. Microscopy showed a diminished expression of YFP-RXR in ROS cells compared with the expression in CV-1 cells. Treatment with inhibitors of proteasomal degradation (lactacystin and MG132) selectively enhanced GFP-VDR and YFP-RXR expression and also increased the endogenous levels of VDR and RXR. Expression of GFP-VDR had no effect on the sensitivity of ROS cells to calcitriol. Increases of RXR levels by YFP-RXR expression, drug treatments, or the combination of the two, however, restored the growth-inhibitory effects of calcitriol and 9-cis-RA and restored p21 induction by calcitriol. These studies revealed that an accelerated and aberrant RXR degradation could cause resistance to the antiproliferative effects of calcitriol and retinoids in ROS cells.
包括ROS17/2.8大鼠骨肉瘤（ROS）细胞在内的几种细胞系含有功能性维生素D受体（VDR）和视黄酸X受体（RXR），但对骨化三醇和类视黄醇的抗增殖作用具有抗性。我们探讨了受体降解在这种激素抗性中的作用。反式激活分析结果表明，ROS细胞中RXR的量不足以激活DR-1报告基因，对细胞提取物的蛋白质印迹分析表明，RXR的降解加速，并产生异常的45 kDa RXR。我们稳定表达了VDR和RXR的功能性荧光嵌合体[绿色荧光蛋白（GFP）-VDR；黄色荧光蛋白（YFP）-RXR]，以评估降解机制以及过量受体表达对抗增殖作用的影响。显微镜检查显示，与CV-1细胞中的表达相比，ROS细胞中YFP-RXR的表达减少。用蛋白酶体降解抑制剂（乳胞素和MG132）处理可选择性增强GFP-VDR和YFP-RXR的表达，同时也增加了VDR和RXR的内源性水平。GFP-VDR的表达对ROS细胞对骨化三醇的敏感性没有影响。然而，通过YFP-RXR表达、药物处理或两者结合增加RXR水平，可恢复骨化三醇和9-顺式视黄酸的生长抑制作用，并恢复骨化三醇诱导的p21表达。这些研究表明，RXR的加速和异常降解可能导致ROS细胞对骨化三醇和类视黄醇的抗增殖作用产生抗性。
