| Abstract: | 細胞色素P450（CYP）為哺乳類動物中的重要生物轉化與毒物藥物代謝解毒系統。由於大鼠初代肝細胞已能在體外培養下成功且穩定表現CYP，因此有許多研究成功地利用這套培養模式來探討Phenobarbital (PB)調控CYP2B基因表現的機制，目前已知CYP2B基因的調控區含PB responsive element (PBRE)，PBRE擁有NR1及NR2兩個nuclear receptor-binding sites，PB即是透過constitutively active receptor (CAR)而與NR1結合，進而誘發CYP2B基因的轉錄作用。Prostaglandin E2 (PGE2)由花生四烯酸衍生而來，PGE2具有多種生理生化角色，已知與細胞生長、分化、血管收縮、排卵、發炎等生理反應相關。PGE2係透過其位於膜上的EP receptors傳遞訊息，發揮其生理功能，目前發現四種EP receptors：EP1、EP2、EP3及EP4，不同接受器下游的訊息傳遞路徑不盡相同。本實驗室之前研究發現，PGE2在PB誘發CYP2B1的表現過程中扮演負向調控角色，再進一步探討其詳細機制時，我們首先利用RT-PCR觀察肝細胞表現何類EP receptor，確認肝細胞的確有EP2及EP4兩種receptors的表現，接著以EP2 agonist－butaprost證實了EP2 receptor參與此機制。因此，本論文進一步探討PGE2透過EP2 receptor的下游訊息傳遞路徑以及可能參與的轉錄因子。由於EP2 receptor與Gs protein相連，若被啟動會活化adenylate cyclase，增加胞內cAMP濃度，活化PKA，PKA會將cAMP responsive element binding protein (CREB) Ser-133磷酸化，活化的CREB在核內與目標基因結合而影響下游基因轉錄作用。本實驗因此以PB誘發大鼠初代肝細胞表現CYP2B1為實驗模式，分別處理PGE2或EP2 agonist－butaprost，或在PKA inhibitor－H-89預處理下，或加入adenylate cyclase inhibitor－SQ22536，觀察它們對PB誘發CYP 2B1表現及CREB磷酸化的影響；對PGE2抑制CYP2B1表現的影響；最後再以EMSA分析CREB與DNA的結合情形。結果顯示，預處理1 μM PGE2與butaprost有相似的抑制CYP2B1表現的效果，SQ22536或H-89皆可逆轉PGE2對PB誘發CYP2B1的抑制作用，PGE2並可提升CREB蛋白質磷酸化及CREB與DNA的結合，而H-89則有抑制效果。綜合上述實驗結果，可以推測PGE2透過EP2 receptor活化adenylate cyclase，增加胞內cAMP濃度，活化PKA，PKA進而活化CREB。PGE2可能透過活化cAMP/PKA路徑及轉錄因子CREB而負向調控PB誘發CYP2B1表現。
Cytochromes P450 (CYP) plays an important role in biotransformation and drug metabolism in mammals. Establishment of a primary rat hepatocyte culture system which faithfully reproduces the CYP gene expression in vivo makes it possible to investigate the mechanism that phenobarbital (PB)-inducible CYP2B gene expression. PB responsive element (PBRE) has been found in CYP2B gene promoter, and PBRE has two nuclear receptor-binding sites, NR1 and NR2. PB causes constitutively active receptor (CAR) to bind to NR1, and then activates CYP2B gene transcription. Prostaglandin E2 (PGE2) is one of eicosanoid derivatives from arachidonic acid, and it was recognized to play important roles in physiologic and biochemical processes, such as cell growth, differentiation, vessel contraction, ovulation, and inflammation. The biological action of PGE2 was recognized to exert through its binding to receptors located in cell membrane. There are four EP subtypes. They are named EP1, EP2, EP3 and EP4, and their downstream signal pathways are different. Based on the results of our previous study, we found that PGE2 inhibited the CYP2B1 gene expression induced by PB in primary rat hepatocytes. In order to investigate the mechanism, RT-PCR was used to identify what EP receptors were present in hepatocytes, and EP2 and EP4 receptors were found. At the same time, EP2 specific agonist butaprost was used to demonstrate that EP2 receptor was involved in this regulation. After then, PGE2－EP2 receptor downstream pathway and possible transcription factors involved in this regulation were studied. EP2 receptor is Gs protein coupled receptor, and when PGE2 binds to the receptor that stimulates adenylate cyclase which synthesizes cAMP from ATP, and the cellular cAMP level will increase and activate PKA. PKA will phosphorylate Ser-133 of cAMP responsive element binding protein (CREB), then activated CREB binds to target gene and affects gene transcription. In order to demonstrate the role of PKA, PKA inhibitor－H-89 and adenylate cyclase inhibitor－SQ22536 were used. Finally, CREB binding to DNA was demonstrated by EMSA. Results showed that pretreated 1 μM PGE2 or butaprost had similar inhibition of PB-inducible CYP2B1 expression. SQ22536 and H-89 reversed the inhibition. PGE2 increased CREB protein phosphorylation and CREB－DNA binding. However, H-89 decreased PGE2 effect. In conclusion, PGE2 bound to EP2 receptor to stimulate adenylate cyclase, and increased cellular cAMP levels and activated PKA, and then PKA activated CREB. So cAMP/PKA is the possible pathway for PGE2 regulation of PB-inducible CYP2B1 expression. |
