Borysiewicz Elizabeth, Fil Daniel, Dlaboga Daniel, O'Donnell James M, Konat Gregory W
Department of Neurobiology and Anatomy, West Virginia University School of Medicine, 4052 HSN, P.O. Box 9128, Morgantown, WV 26506-9128, USA.
Metab Brain Dis. 2009 Sep;24(3):481-91. doi: 10.1007/s11011-009-9150-9.
Cyclic AMP is part of an endogenous mechanism that downregulates inflammatory response, and its intracellular concentration is regulated chiefly by cyclic nucleotide phosphodiesterases type 4. The goal of the present study was to determine whether phosphodiesterases 4 are involved in the inflammatory response of astrocytes mediated by Toll-like receptors. Astrocyte cultures established from newborn rat brain were challenged with lipoteichoic acid, a ligand of Toll-like receptor 2, polyinosinic-polycytidylic acid, a ligand of Toll-like receptor 3, or lipopolysaccharide, a ligand of Toll-like receptor 4. After 24 h the expression of genes encoding phosphodiesterase 4A, phosphodiesterase 4B and phosphodiesterase 4D was determined by real time reverse transcription polymerase chain reaction. The challenge of astrocytes with the ligands profoundly up-regulated expression of the phosphodiesterase 4B mRNA, while the phosphodiesterase 4A and 4D mRNA was either unaffected or downregulated. Moreover, Toll-like receptor ligation specifically up-regulated expression of the phosphodiesterase 4B2 transcriptional variant. Thus, polyinosinic-polycytidylic acid, lipopolysaccharide and lipoteichoic acid induced approximately 7-, 5- and 4-fold up-regulation of the message, respectively. Toll-like receptor ligation also led to an over 2-fold increase in the protein level of phosphodiesterase 4B2 as revealed by immunoblot analysis. The inactivation of Rho proteins by pretreatment with toxin B form C. difficile enhanced ligation-induced up-regulation of the phosphodiesterase 4B2 message by 4-9-fold. However, in spite of this increase in the message abundance, there was no increase in the protein level compared to cells challenged with the ligands alone. These results demonstrate that the phosphodiesterase 4B2 gene is an effector of Toll-like receptor signaling in astrocytes, and that its up-regulation at the protein level is controlled by complex mechanisms.
环磷酸腺苷（cAMP）是下调炎症反应的内源性机制的一部分，其细胞内浓度主要由4型环核苷酸磷酸二酯酶调节。本研究的目的是确定磷酸二酯酶4是否参与由Toll样受体介导的星形胶质细胞的炎症反应。用Toll样受体2的配体脂磷壁酸、Toll样受体3的配体聚肌苷酸-聚胞苷酸或Toll样受体4的配体脂多糖对新生大鼠脑建立的星形胶质细胞培养物进行刺激。24小时后，通过实时逆转录聚合酶链反应测定编码磷酸二酯酶4A、磷酸二酯酶4B和磷酸二酯酶4D的基因的表达。用这些配体刺激星形胶质细胞后，磷酸二酯酶4B mRNA的表达显著上调，而磷酸二酯酶4A和4D mRNA要么未受影响，要么下调。此外，Toll样受体连接特异性地上调了磷酸二酯酶4B2转录变体的表达。因此，聚肌苷酸-聚胞苷酸、脂多糖和脂磷壁酸分别使该信使RNA上调约7倍、5倍和4倍。免疫印迹分析显示，Toll样受体连接还导致磷酸二酯酶4B2的蛋白质水平增加超过2倍。用艰难梭菌毒素B预处理使Rho蛋白失活，可使连接诱导的磷酸二酯酶4B2信使RNA上调增加4至9倍。然而，尽管该信使RNA丰度增加，但与仅用配体刺激的细胞相比，蛋白质水平并未增加。这些结果表明，磷酸二酯酶4B2基因是星形胶质细胞中Toll样受体信号传导的效应器，其蛋白质水平的上调受复杂机制控制。
