Ziemann Christina, Riecke Armin, Rüdell Gudrun, Oetjen Elke, Steinfelder Hans J, Lass Christian, Kahl Georg F, Hirsch-Ernst Karen I
Department of Toxicology, Institute of Pharmacology and Toxicology, University of Göttingen, Germany.
J Pharmacol Exp Ther. 2006 Apr;317(1):378-86. doi: 10.1124/jpet.105.094193. Epub 2006 Jan 13.
Multidrug resistance (mdr) proteins of the mdr1 type function as multispecific xenobiotic transporters in hepatocytes. In the liver, mdr1 overexpression occurs during regeneration, cirrhosis, and hepatocarcinogenesis and may contribute to primary chemotherapy resistance. Cultured rat hepatocytes exhibit a time-dependent "intrinsic" increase in functional mdr1b expression, which depends on cyclooxygenase-catalyzed prostaglandin E(2) release. In the present study, the prostaglandin E (EP) receptor agonist misoprostol (1-10 microg/ml) further enhanced intrinsic mdr1b mRNA expression in primary rat hepatocytes. On the other hand, [1alpha(z),2beta,5alpha]-(+)-7-[5-[1,1'-(biphenyl)-4-yl]methoxy]-2-(4-morpholinyl)-3-oxocyclopentyl]-4-heptenoic acid (AH23848B) (30 microM), an antagonist of the cAMP-coupled EP4 receptor, and the protein kinase A (PKA) inhibitor, N-(2-[bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide (H89) (10 nM), repressed intrinsic mdr1b mRNA up-regulation, whereas the stable cAMP analog 8-bromo-cAMP (10 microM) and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) (100 microM) further enhanced intrinsic mdr1b expression. Primary rat hepatocytes, transiently transfected with reporter gene constructs controlled by mdr1b 5'-gene-flanking regions [-1074 to +154 base pairs (bp) or -250 to +154 bp], demonstrated pronounced mdr1b promoter activity, already without the addition of exogenous modulators. Nevertheless, activity was further stimulated by misoprostol, 8-bromo-cAMP, or IBMX. Cotransfection with expression vectors for PKI, an inhibitor protein of cAMP-dependent PKA, or KCREB, a dominant-negative mutant of the cAMP-responsive element-binding protein (CREB), decreased high-intrinsic mdr1b promoter activity. KCREB also counteracted misoprostol-induced mdr1b promoter activation. In conclusion, these data provide evidence for a pivotal role of EP receptor-stimulated, cAMP-dependent activation of PKA and CREB or CREB-related proteins in mdr1b gene activation in primary rat hepatocytes. Thus, these data might offer potential new target structures for the reversal of primary drug resistance, for example, of liver tumors.
mdr1类型的多药耐药（mdr）蛋白在肝细胞中作为多特异性外源性物质转运体发挥作用。在肝脏中，mdr1的过表达发生在再生、肝硬化和肝癌发生过程中，可能导致原发性化疗耐药。培养的大鼠肝细胞功能性mdr1b表达呈现出时间依赖性的“内在”增加，这依赖于环氧化酶催化的前列腺素E2释放。在本研究中，前列腺素E（EP）受体激动剂米索前列醇（1 - 10微克/毫升）进一步增强了原代大鼠肝细胞中内在的mdr1b mRNA表达。另一方面，[1α(z),2β,5α]-(+)-7-[5-[1,1'-（联苯）-4-基]甲氧基]-2-（4-吗啉基）-3-氧代环戊基]-4-庚烯酸（AH23848B）（30微摩尔），一种cAMP偶联的EP4受体拮抗剂，以及蛋白激酶A（PKA）抑制剂N-(2-[溴肉桂酰胺基]乙基)-5-异喹啉磺酰胺（H89）（10纳摩尔），抑制了内在的mdr1b mRNA上调，而稳定的cAMP类似物8-溴-cAMP（10微摩尔）和磷酸二酯酶抑制剂3-异丁基-1-甲基黄嘌呤（IBMX）（100微摩尔）进一步增强了内在的mdr1b表达。用由mdr1b 5'-基因侧翼区域[-1074至+154碱基对（bp）或-250至+154 bp]控制的报告基因构建体瞬时转染原代大鼠肝细胞，即使不添加外源性调节剂也显示出明显的mdr1b启动子活性。然而，米索前列醇、8-溴-cAMP或IBMX进一步刺激了活性。与cAMP依赖性PKA的抑制蛋白PKI或环磷酸腺苷反应元件结合蛋白（CREB）的显性负性突变体KCREB的表达载体共转染，降低了高内在的mdr1b启动子活性。KCREB也抵消了米索前列醇诱导的mdr1b启动子激活。总之，这些数据为EP受体刺激的、cAMP依赖性的PKA和CREB或CREB相关蛋白的激活在原代大鼠肝细胞mdr1b基因激活中的关键作用提供了证据。因此，这些数据可能为逆转原发性耐药，例如肝肿瘤的原发性耐药，提供潜在的新靶点结构。
