Wu Y L, Wiltbank M C
Endocrinology-Reproductive Physiology Program and. Department of Dairy Science, University of Wisconsin, Madison, Wisconsin 53706, USA.
Biol Reprod. 2001 Nov;65(5):1565-72. doi: 10.1095/biolreprod65.5.1565.
There is positive feedback pathway in the ovine large luteal cell, such that prostaglandin (PG) F(2 alpha) stimulation induces intraluteal PGF(2 alpha) production 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 (PGF(2 alpha) analogue), 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.
在绵羊大黄体细胞中存在正反馈途径，即前列腺素（PG）F2α刺激可诱导黄体内部PGF2α的产生，这是PG产生过程中一种限速酶——环氧化酶-2（Cox-2）被诱导的结果。本研究的目的是评估绵羊大黄体细胞中参与调控Cox-2基因的细胞内效应系统和重要的DNA转录元件。在瞬时转染实验中，佛波醇二癸酸酯（一种蛋白激酶[PK]C激活剂）、离子霉素和氯前列醇（PGF2α类似物）可迅速（4小时）诱导Cox-2启动子，在12小时达到诱导峰值。氯前列醇介导的启动子激活不受包括PKA、钙调蛋白激酶II或丝裂原活化蛋白激酶在内的各种第二信使系统抑制的影响。然而，肉豆蔻酰化的PKC假底物肽可抑制氯前列醇对Cox-2启动子的刺激，表明PKC在这种刺激中起关键作用。Cox-2启动子可被缩减至5'侧翼序列的282个碱基对（bp），且仍保留对氯前列醇的完全诱导性。对该282-bp区域内三个关键顺式反应元件（C/EBP、cAMP反应元件[CRE]和E-box）的突变表明，E-box对基础和氯前列醇诱导的启动子活性都至关重要。然而，Cox-2启动子中C/EBP和CRE的突变对氯前列醇刺激也有显著但不太明显的抑制作用，所有三个元件的突变则消除了氯前列醇对该启动子的诱导作用。对大黄体细胞核提取物的电泳迁移率变动分析表明，上游刺激因子（USF）-1和USF-2与Cox-2中的E-box结合。因此，PKC通过作用于靠近假定转录起始点的DNA元件，特别是-50 bp处的E-box区域，直接调节大黄体细胞中Cox-2基因的转录。
