Transcriptional regulation of cyclooxygenase-2 gene in ovine large luteal cells.
Transcriptional regulation of cyclooxygenase-2 gene in ovine large luteal cells.
复制标题
绵羊大黄体细胞中环氧合酶2基因的转录调控。
DOI：
10.1095/biolreprod65.5.1565
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发表时间：
2001
影响因子：
3.6
通讯作者：
Wiltbank,MC
中科院分区：
文献类型：
--
作者：
Wu,YL;Wiltbank,MC
在绵羊大黄体细胞中存在正反馈途径，即前列腺素（PG）F2α刺激可诱导黄体内PGF 2 α的产生，这是由于PG产生的限速酶之一环氧化酶-2（考克斯-2）的诱导所致。本研究的目的是评估绵羊大黄体细胞中参与调控考克斯-2基因的细胞内效应系统和重要的DNA转录元件。在瞬时转染试验中，考克斯-2启动子被佛波醇双癸酸酯（蛋白激酶[PK] C激活剂）、离子霉素和氯前列醇（PGF 2 α类似物）快速诱导（4 h），诱导高峰在12 h。氯前列烯醇介导的启动子激活不被各种第二信使系统，包括PKA，钙钙调蛋白激酶II，或丝裂原活化蛋白激酶抑制阻断。然而，肉豆蔻酰化PKC假底物肽抑制氯前列烯醇对考克斯-2启动子的刺激，表明PKC在该刺激中的关键作用。考克斯-2启动子可被氯前列醇还原至5′侧翼序列的282个碱基对（bp），并保留完整的诱导序列。这282 bp区域内的三个关键顺式响应元件（C/EBP，cAMP响应元件[CRE]和E-box）的突变表明，E-box在基础和氯前列烯醇诱导的启动子活性中都是关键的。然而，考克斯-2启动子中C/EBP和CRE突变也对氯前列醇刺激产生显著但不太显著的抑制，所有三个元件的突变消除了氯前列醇对该启动子的诱导。从大黄体细胞的核提取物的电泳迁移率变动分析显示，上游刺激因子（USF）-1和USF-2绑定到考克斯-2的E盒。因此，PKC直接调节大黄体细胞中考克斯-2基因的转录，通过接近假定的转录起始点的DNA元件起作用，特别是−50 bp的E盒区域。
There is positive feedback pathway in the ovine large luteal cell, such that prostaglandin (PG) F2αstimulation induces intraluteal PGF2αproduction as the result of induction of one of the rate-limiting enzymes in PG production, cyclooxygenase-2 (Cox-2). The objective of the present study was to evaluate the intracellular effector systems and important DNA transcriptional element(s) involved in regulating the Cox-2 gene in ovine large luteal cells. In transient transfection assays, Cox-2 promoter was rapidly induced (4 h) by phorbol didecanoate (a protein kinase [PK] C activator), ionomycin, and cloprostenol (PGF2αanalogue), with a peak induction at 12 h. Cloprostenol-mediated promoter activation was not blocked by inhibition of various second messenger systems, including PKA, calcium calmodulin kinase II, or mitogen-activated protein kinases. However, myristoylated PKC pseudosubstrate peptide inhibited cloprostenol stimulation of Cox-2 promoter, indicating the critical role of PKC in this stimulation. The Cox-2 promoter could be reduced to 282 base pairs (bp) of the 5′ flanking sequence with retention of full inducibility by cloprostenol. Mutation of three critical cis-responsive elements within this 282-bp region (C/EBP, cAMP responsive element [CRE], and E-box) indicated that E-box was critical in both basal and cloprostenol-induced promoter activity. However, there was also significant but less dramatic inhibition of cloprostenol stimulation by mutation of C/EBP and CRE in the Cox-2 promoter, and mutation of all three elements eliminated cloprostenol induction of this promoter. Electrophoretic mobility shift assays of nuclear extracts from large luteal cells revealed that upstream stimulatory factor (USF)-1 and USF-2 bound to the E-box in Cox-2. Thus, PKC directly regulates transcription of the Cox-2 gene in large luteal cells by acting through DNA elements close to the putative transcriptional start point, particularly an E-box region at −50 bp.