Steinert Daniela, Küper Christoph, Bartels Helmut, Beck Franz-X, Neuhofer Wolfgang
Department of Physiology, University of Munich, Pettenkoferstrasse 12, 80336 Munich, Germany.
Am J Physiol Cell Physiol. 2009 Jan;296(1):C75-87. doi: 10.1152/ajpcell.00024.2008. Epub 2008 Nov 12.
Cyooxygenase-2 (COX-2)-derived PGE2 is critical for the integrity and function of renal medullary cells during antidiuresis. The present study extended our previous finding that tonicity-induced COX-2 expression is further stimulated by the major COX-2 product PGE2 and investigated the underlying signaling pathways and the functional relevance of this phenomenon. Hyperosmolality stimulated COX-2 expression and activity in Madin-Darby canine kidney (MDCK) cells, a response that was further increased by PGE2-cAMP signaling, suggesting the existence of a positive feedback loop. This effect was diminished by AH-6809, an EP2 antagonist, and by the PKA inhibitor H-89, but not by AH-23848, an EP4 antagonist. The effect of PGE2 was mimicked by forskolin and dibutyryl-cAMP, suggesting that the stimulatory effect of PGE2 on COX-2 is mediated by a cAMP-PKA-dependent mechanism. Accordingly, cAMP-responsive element (CRE)-driven reporter activity paralleled the effects of PGE2, AH-6809, AH-23848, H-89, forskolin, and dibutyryl-cAMP on COX-2 expression. In addition, the stimulatory effect of PGE2 on tonicity-induced COX-2 expression was blunted in cells transfected with dominant-negative CRE binding (CREB) protein, as was the case in a COX-2 promoter reporter construct in which a putative CRE was deleted. Furthermore, PGE2 resulted in PKA-dependent phosphorylation of the pro-apoptotic protein Bad at Ser155, a mechanism that is known to inactivate Bad, which coincided with reduced caspase-3 activity during osmotic stress. Conversely, pharmacological interruption of the PGE2-EP2-cAMP-PKA pathway abolished Ser155 phosphorylation of Bad and blunted the protective effect of PGE2 on cell survival during osmotic stress. These observations indicate the existence of a positive feedback loop of PGE2 on COX-2 expression during osmotic stress, an effect that apparently is mediated by EP2-cAMP-PKA signaling, and that contributes to cell survival under hypertonic conditions.
环氧化酶-2（COX-2）衍生的前列腺素E2（PGE2）在抗利尿过程中对肾髓质细胞的完整性和功能至关重要。本研究扩展了我们之前的发现，即张力诱导的COX-2表达会被COX-2的主要产物PGE2进一步刺激，并研究了其潜在的信号通路以及这一现象的功能相关性。高渗刺激了Madin-Darby犬肾（MDCK）细胞中COX-2的表达和活性，PGE2-cAMP信号进一步增强了这种反应，表明存在正反馈回路。EP2拮抗剂AH-6809和PKA抑制剂H-89可减弱这种效应，但EP4拮抗剂AH-23848则无此作用。福斯可林和二丁酰-cAMP可模拟PGE2的作用，表明PGE2对COX-2的刺激作用是由cAMP-PKA依赖性机制介导的。因此，cAMP反应元件（CRE）驱动的报告基因活性与PGE2、AH-6809、AH-23848、H-89、福斯可林和二丁酰-cAMP对COX-2表达的作用平行。此外，在转染了显性负性CRE结合（CREB）蛋白的细胞中，PGE2对张力诱导的COX-2表达的刺激作用减弱，在缺失假定CRE的COX-2启动子报告基因构建体中也是如此。此外，PGE2导致促凋亡蛋白Bad在Ser155处发生PKA依赖性磷酸化，这一机制已知可使Bad失活，这与渗透应激期间caspase-3活性降低相一致。相反，PGE2-EP2-cAMP-PKA途径的药理学阻断消除了Bad的Ser155磷酸化，并减弱了PGE2在渗透应激期间对细胞存活的保护作用。这些观察结果表明，在渗透应激期间，PGE2对COX-2表达存在正反馈回路，这种效应显然是由EP2-cAMP-PKA信号介导的，并且有助于在高渗条件下细胞存活。
