Medh R D, Lay R H, Schmidt T J
Department of Physiology and Biophysics, College of Medicine, The University of Iowa, Iowa City 52242, USA.
Mol Cell Endocrinol. 1998 Mar 16;138(1-2):11-23. doi: 10.1016/s0303-7207(98)00055-0.
Although the immunosuppressive drugs FK506, rapamycin and cyclosporin A have been reported to potentiate transcriptional activation mediated by a non-saturating concentration of the glucocorticoid receptor agonist dexamethasone, the precise mechanism(s) underlying these responses remains unclear. The murine L-929-derived LMCAT cell line stably transfected with the mouse mammary tumor virus promoter-chloramphenicol acetyl transferase reporter gene construct was utilized in the present study to further investigate the mechanism(s) underlying this dexamethasone potentiation as well as the possible agonist specificity of this potentiation. The present data demonstrate that pretreatment (2 h) of LMCAT cells with 10 microM FK506, rapamycin or cyclosporin A results in the potentiation of reporter gene transcription mediated not only by dexamethasone (approximately 12-fold), but also by hydrocortisone (approximately 6-fold) and triamcinolone acetonide (approximately 2.5-fold). In sharp contrast, the data show for the first time that pretreatment with any one of these immunosuppressive drugs suppresses (approximately 2-8-fold) the transcriptional responses mediated by corticosterone, deoxycorticosterone, and cortexolone. Pretreatment of intact LMCAT cells with FK506 increases the subsequent whole cell specific binding of [3H]dexamethasone, but does not increase specific cytoplasmic binding when the tritiated agonist is added directly to cytosolic extracts prepared from the pretreated cells. These data suggest that the FK506-mediated potentiation of the transcriptional responses induced by some agonists, like dexamethasone, may be related to the ability of this immunosuppressant to inhibit the membrane-associated multidrug resistance (MDR) P-glycoprotein, which actively extrudes some steroids from cells. Identical pretreatment with FK506 has no detectable effect on the subsequent whole cell specific binding of [3H]corticosterone, a steroid which is not effectively extruded by the MDR pump. Two additional MDR pump inhibitors, verapamil and quinidine, potentiate (30-fold) the dexamethasone-mediated transcriptional response as expected, but have no detectable effects on a corticosterone-mediated transcriptional response. Unlike immunosuppressive drugs, these ion channel blockers do not bind to receptor-associated immunophilins (FK506-binding proteins or cyclophilins). Collectively, these results suggest that immunosuppressants potentiate a dexamethasone-mediated transcriptional response in LMCAT cells by inhibiting efflux of this steroid. In contrast, these drugs appear to suppress a corticosterone-mediated transcriptional response by a different mechanism, perhaps one involving their binding to glucocorticoid receptor-associated immunophilins.
尽管有报道称免疫抑制药物FK506、雷帕霉素和环孢素A可增强由非饱和浓度的糖皮质激素受体激动剂地塞米松介导的转录激活，但这些反应背后的确切机制仍不清楚。本研究利用稳定转染了小鼠乳腺肿瘤病毒启动子-氯霉素乙酰转移酶报告基因构建体的源自小鼠L-929的LMCAT细胞系，进一步研究地塞米松增强作用的潜在机制以及这种增强作用可能的激动剂特异性。目前的数据表明，用10微摩尔的FK506、雷帕霉素或环孢素A对LMCAT细胞进行预处理（2小时），不仅会导致报告基因转录增强，这种增强不仅由地塞米松介导（约12倍），还由氢化可的松（约6倍）和曲安奈德（约2.5倍）介导。形成鲜明对比的是，数据首次表明，用这些免疫抑制药物中的任何一种进行预处理都会抑制（约2 - 8倍）由皮质酮、脱氧皮质酮和皮质素介导的转录反应。用FK506对完整的LMCAT细胞进行预处理会增加随后[3H]地塞米松的全细胞特异性结合，但当将氚化激动剂直接添加到从预处理细胞制备的胞质提取物中时，并不会增加特异性胞质结合。这些数据表明，FK506介导的某些激动剂（如地塞米松）诱导的转录反应增强，可能与这种免疫抑制剂抑制膜相关多药耐药（MDR）P-糖蛋白的能力有关，该蛋白会主动将一些类固醇从细胞中排出。用FK506进行相同的预处理对随后[3H]皮质酮的全细胞特异性结合没有可检测到的影响，皮质酮是一种不能被MDR泵有效排出细胞的类固醇。另外两种MDR泵抑制剂维拉帕米和奎尼丁如预期那样增强（30倍）了地塞米松介导的转录反应，但对皮质酮介导的转录反应没有可检测到的影响。与免疫抑制药物不同，这些离子通道阻滞剂不与受体相关亲免素（FK506结合蛋白或亲环素）结合。总体而言，这些结果表明免疫抑制剂通过抑制这种类固醇的流出增强了LMCAT细胞中地塞米松介导的转录反应。相比之下，这些药物似乎通过不同的机制抑制皮质酮介导的转录反应，可能是一种涉及其与糖皮质激素受体相关亲免素结合的机制。
