Hamasaki Y, Kitzler J, Hardman R, Nettesheim P, Eling T E
Laboratory of Molecular Biophysics, National Institute of Environmental Health Science, Research Triangle Park, North Carolina 27709.
Arch Biochem Biophys. 1993 Jul;304(1):226-34. doi: 10.1006/abbi.1993.1343.
Previous studies from our laboratory suggested that phorbol 12-myristate 13-acetate (TPA) stimulates prostaglandin E2 (PGE2) production by inducing de novo synthesis of prostaglandin H synthase (PHS) in a rat tracheal cell line. We report here an extension of this work to further elucidate the mechanisms by which TPA (and epidermal growth factor) stimulates PGE2 production. We used the rat tracheal cell line EGV6, which has a lower basal level of PGE2 production and responds to TPA and EGF stimulation with a much greater increase in PGE2 synthesis than the previously used cell line, Incubation of EGV6 cultures with TPA or EGF resulted in a time- and dose-dependent increase in PGE2 synthesis up to 40-fold and 6-fold, respectively. Serum also stimulated PGE2 synthesis, while bombesin, retinoic acid, and bacterial lipopolysaccharide did not. PHS protein levels in microsomal preparations from the cells were estimated by Western analysis. Antibodies raised against murine PHS-2 cross reacted with the EGV-6 PHS while several antibody preparations that react with PHS-1 from ram or mouse reacted poorly with the cellular preparation. TPA treatment increased the de novo synthesis of PHS-2 while dexamethasone treatment reduced the response to TPA. Northern blot analysis of mRNA from EGV6 cultures using a ram PHS cDNA revealed a 2.8- and a 4.5- to 4.9-kb (designated 4.9 kb) transcript. Treatment with TPA or EGF increased the expression of both transcripts and this effect was further enhanced by cyclohexamide. To further define the PHS mRNA species of EGV6 cells, two well-characterized murine PHS cDNA probes were used. The constitutive murine PHS cDNA probe hybridized only with the 2.8-kb transcript, and the inducible murine PHS cDNA hybridized only with the 4.9-kb transcript. The rates of induction as well as degradation of the 4.9-kb PHS mRNA were much more rapid than those of the 2.8-kb mRNA species. Dexamethasone partially inhibited the induction of both PHS transcripts by TPA. Southern analysis of genomic EGV6 DNA indicated the presence of two distinct PHS genes in these cells. Taken together these findings indicate that two PHS genes are expressed in rat tracheal epithelial cells. In contrast to the PHS genes expressed in murine (and chicken) fibroblasts in which only the gene coding for the larger mRNA species is transcriptionally regulated, in the rat tracheal cells both genes are positively regulated by TPA and EGF and downregulated by glucocorticoids.
我们实验室先前的研究表明，佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（TPA）通过诱导大鼠气管细胞系中前列腺素H合酶（PHS）的从头合成来刺激前列腺素E2（PGE2）的产生。我们在此报告这项工作的扩展，以进一步阐明TPA（和表皮生长因子）刺激PGE2产生的机制。我们使用了大鼠气管细胞系EGV6，其PGE2产生的基础水平较低，并且与先前使用的细胞系相比，对TPA和表皮生长因子（EGF）刺激的反应是PGE2合成有更大程度的增加。用TPA或EGF培养EGV6细胞导致PGE2合成呈时间和剂量依赖性增加，分别高达40倍和6倍。血清也刺激PGE2合成，而蛙皮素、视黄酸和细菌脂多糖则无此作用。通过蛋白质免疫印迹分析估计细胞微粒体制剂中的PHS蛋白水平。针对小鼠PHS - 2产生的抗体与EGV - 6 PHS发生交叉反应，而几种与来自公羊或小鼠的PHS - 1反应的抗体制剂与细胞制剂反应较差。TPA处理增加了PHS - 2的从头合成，而地塞米松处理降低了对TPA的反应。使用公羊PHS cDNA对EGV6细胞培养物的mRNA进行Northern印迹分析，发现了一个2.8 kb以及一个4.5至4.9 kb（指定为4.9 kb）的转录本。用TPA或EGF处理增加了两种转录本的表达，并且环己酰胺进一步增强了这种效应。为了进一步确定EGV6细胞的PHS mRNA种类，使用了两种特征明确的小鼠PHS cDNA探针。组成型小鼠PHS cDNA探针仅与2.8 kb的转录本杂交，而诱导型小鼠PHS cDNA仅与4.9 kb的转录本杂交。4.9 kb PHS mRNA的诱导率和降解率比2.8 kb mRNA种类的要快得多。地塞米松部分抑制了TPA对两种PHS转录本的诱导。对EGV6基因组DNA的Southern分析表明这些细胞中存在两个不同的PHS基因。综上所述，这些发现表明大鼠气管上皮细胞中表达两种PHS基因。与在小鼠（和鸡）成纤维细胞中表达的PHS基因不同，在小鼠（和鸡）成纤维细胞中只有编码较大mRNA种类的基因受到转录调控，而在大鼠气管细胞中，两种基因都受到TPA和EGF的正向调控，并受到糖皮质激素的下调。
