Gellersen B, Kempf R, Telgmann R
Institute for Hormone and Fertility Research University of Hamburg, Germany.
Mol Endocrinol. 1997 Jan;11(1):97-113. doi: 10.1210/mend.11.1.9875.
Decidualization of human endometrial stromal (ES) cells in culture can be triggered by a sustained elevation of intracellular cAMP for several days and is characterized by activation of the cAMP-responsive decidual PRL (dPRL) gene promoter. We investigated the expression of the cAMP response element (CRE) binding protein CREB, and the modulators CREM (cAMP response element modulator) and ICER (inducible cAMP early repressor), in relation to decidualization of ES cells. We isolated all four known ICER isoforms from ES cells, which differ by the presence or absence of the small exon gamma and the presence of either DNA-binding domain (DBD) I or II. Of the various CREM isoforms, we cloned six transcript species, all containing DBD I. These were the known repressor CREM-alpha, the potential activator CREM-tau 2 alpha, and four novel forms whose reading frames were blocked upstream of the DBD. Two of these forms contained a novel exon psi, which is 100 bp in length, resides downstream of the first protein-coding exon of the CREM gene, and introduces an early in-frame stop codon. Surprisingly, in cotransfection assays, all four novel CREM isoforms were potent inhibitors of protein kinase A-stimulated transcription of a reporter gene construct driven by a CRE. By in vitro transcription/translation of all six CREM cDNAs, we demonstrated internal translation initiation at three different methionine residues, giving rise to novel short and very short C-terminal proteins comprising DBD I. These proteins bound to a cAMP response element as homodimers or as heterodimers with each other or with CREB. Immunofluorescence showed nuclear localization of C-terminal CREM proteins expressed from all six CREM cDNAs. Comparison of undifferentiated and decidualized ES cells showed no difference in the level of expression of any of the CREM transcript species. Likewise, CREB was evenly expressed between the two populations. In contrast, ICER transcripts were strongly up-regulated in decidualized ES cells in parallel with the induction of dPRL expression. It appears paradoxical that in vivo, in response to a permanent cAMP stimulus, ICER is up-regulated without displaying negative autoregulation of its own gene or suppression of the dPRL promoter. Elevated ICER levels in decidualized ES cells may be indicative of the presence of overriding amounts of transcriptional activators such as full length CREM-tau or CREB which, in turn, upon cAMP-induced phosphorylation, contribute to the induction of the dPRL gene.
体外培养的人子宫内膜基质（ES）细胞的蜕膜化可由细胞内cAMP持续升高数天触发，其特征是cAMP反应性蜕膜催乳素（dPRL）基因启动子的激活。我们研究了cAMP反应元件（CRE）结合蛋白CREB以及调节剂CREM（cAMP反应元件调节剂）和ICER（诱导型cAMP早期阻遏物）与ES细胞蜕膜化相关的表达情况。我们从ES细胞中分离出所有四种已知的ICER亚型，它们因小外显子γ的有无以及DNA结合结构域（DBD）I或II的存在而有所不同。在各种CREM亚型中，我们克隆了六种转录本，均含有DBD I。其中包括已知的阻遏物CREM-α、潜在的激活剂CREM-tau 2α，以及四种在DBD上游读码框被阻断的新形式。其中两种形式含有一个新的外显子ψ，长度为100 bp，位于CREM基因第一个蛋白质编码外显子的下游，并引入了一个框内早期终止密码子。令人惊讶的是，在共转染实验中，所有四种新的CREM亚型都是蛋白激酶A刺激的由CRE驱动的报告基因构建体转录的有效抑制剂。通过对所有六种CREM cDNA进行体外转录/翻译，我们证明了在三个不同的甲硫氨酸残基处发生内部翻译起始，产生了包含DBD I的新的短和非常短的C末端蛋白。这些蛋白以同二聚体或彼此之间或与CREB的异二聚体形式结合到cAMP反应元件上。免疫荧光显示从所有六种CREM cDNA表达的C末端CREM蛋白的核定位。未分化和蜕膜化的ES细胞的比较显示，任何CREM转录本的表达水平均无差异。同样，CREB在这两个群体中均匀表达。相比之下，ICER转录本在蜕膜化的ES细胞中与dPRL表达的诱导同时强烈上调。似乎自相矛盾的是，在体内，响应永久性cAMP刺激，ICER上调但未显示对其自身基因的负自调节或对dPRL启动子的抑制。蜕膜化ES细胞中ICER水平升高可能表明存在过量的转录激活剂，如全长CREM-tau或CREB，它们在cAMP诱导的磷酸化后，反过来有助于dPRL基因的诱导。
