Malignant breast epithelial cells stimulate aromatase expression via promoter II in human adipose fibroblasts: an epithelial-stromal interaction in breast tumors mediated by CCAAT/enhancer binding protein beta.
Malignant breast epithelial cells stimulate aromatase expression via promoter II in human adipose fibroblasts: an epithelial-stromal interaction in breast tumors mediated by CCAAT/enhancer binding protein beta.
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发表时间：
2001-03
期刊：
影响因子：
11.2
通讯作者：
Jian Zhou;B. Gurates;Sijun Yang;Siby Sebastian;S. Bulun
中科院分区：
文献类型：
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作者：
Jian Zhou;B. Gurates;Sijun Yang;Siby Sebastian;S. Bulun
芳香化酶P450（P450 arom）催化雌激素的形成，其表达在乳腺癌周围的脂肪成纤维细胞中异常增加，引起恶性细胞的增殖。人脂肪组织中的芳香化酶主要在几种不同的和交替使用的P450 arom启动子的控制下在未分化的成纤维细胞中表达。在无肿瘤的乳腺脂肪组织中，P450 arom通常通过远端启动子以低水平表达（I.4），而在携带肿瘤的乳腺脂肪组织中，P450 arom通过两个近端启动子II和I.3的激活而增加。由于P450 arom启动子II的体内激活是导致乳腺肿瘤中P450 arom异常高表达的关键事件，我们使用T47 D乳腺癌细胞条件培养基（TCM）处理的原代培养的人脂肪成纤维细胞（HAF）作为模型系统，研究了P450 arom启动子II增强的分子基础。在用TCM处理后，HAF通过使用启动子II显示出显著的P450 arom mRNA水平的诱导。这种作用似乎是特定的恶性乳腺上皮细胞，因为条件培养基从乳腺癌细胞系T47 D和MCF-7诱导启动子II活性，而正常乳腺上皮细胞或肝或前列腺癌细胞系不产生这样的效果。虽然治疗与环AMP类似物也引起开关的启动子使用从I.4到II在培养的HAF，TCM诱导的启动子II的使用被认为是通过一个环AMP的非依赖性途径介导。使用启动子II 5 '侧翼序列的连续缺失突变体揭示了在-517/-278 bp区域中存在关键的顺式作用元件，其调节基线活性。TCM引起-517-bp启动子II构建体的5.7倍诱导，而CCAAT/增强子结合蛋白（C/EBP）结合位点（-317/-304 bp）的定点诱变消除了基线和TCM诱导的活性。C/EBPalpha和C/EBPbeta的异位表达，而不是C/EBPdelta，显着诱导启动子II活性。此外，我们证明了在DNA-蛋白质复合物中存在C/EBP β和C/EBP δ，但不存在C/EBP α，所述DNA-蛋白质复合物由来自经TCM处理的HAF的核提取物和含有该关键C/EBP结合元件（-317/-304bp）的探针形成。最后，用TCM治疗HAF显著诱导C/EBP β表达，而这不影响C/EBP α或C/EBP δ转录物的水平。总之，恶性乳腺上皮细胞分泌因子，通过启动子II诱导脂肪成纤维细胞中芳香酶的表达。这至少部分是由中药诱导的上调和增强的C/EBP β与启动子II调节元件的结合介导的。
Expression of aromatase P450 (P450arom), which catalyzes the formation of estrogens, is aberrantly increased in adipose fibroblasts surrounding breast carcinomas, giving rise to proliferation of malignant cells. Aromatase in human adipose tissue is primarily expressed in undifferentiated fibroblasts under the control of several distinct and alternatively used P450arom promoters. In tumor-free breast adipose tissue, P450arom is usually expressed at low levels via a distal promoter (I.4), whereas in the breast adipose tissue bearing a tumor, P450arom is increased through the activation of two proximal promoters, II and I.3. Because the in vivo activation of P450arom promoter II is a key event responsible for aberrantly high P450arom expression in breast tumors, we studied the molecular basis for the enhancement of P450arom promoter II using human adipose fibroblasts (HAFs) in primary culture treated with T47D breast cancer cell-conditioned medium (TCM) as a model system. Upon treatment with TCM, HAFs displayed a striking induction of P450arom mRNA levels via promoter II usage. This effect appeared to be specific for malignant breast epithelial cells, because conditioned media from breast cancer cell lines T47D and MCF-7 induced promoter II activity, whereas normal breast epithelial cells or liver or prostate cancer cell lines did not produce such an effect. Although treatment with a cyclic AMP analogue also caused a switch in the promoter use from I.4 to II in cultured HAFs, TCM-induced promoter II use was found to be mediated via a cyclic AMP-independent pathway. Use of serial deletion mutants of the promoter II 5'-flanking sequence revealed the presence of critical cis-acting elements in the -517/-278 bp region, which regulate the baseline activity. TCM caused a 5.7-fold induction of the -517-bp promoter II construct, whereas site-directed mutagenesis of a CCAAT/enhancer binding protein (C/EBP) binding site (-317/-304 bp) abolished both baseline and TCM-induced activities. Ectopic expressions of C/EBPalpha and C/EBPbeta, but not C/EBPdelta, significantly induced promoter II activity. Moreover, we demonstrated the presence of both C/EBPbeta and C/EBPdelta but not C/EBPalpha in a DNA-protein complex formed by the nuclear extract from TCM-treated HAFs and a probe containing this critical C/EBP binding element (-317/-304 bp). Finally, treatment of HAFs with TCM strikingly induced C/EBPbeta expression, whereas this did not affect the levels of C/EBPalpha or C/EBPdelta transcripts. In conclusion, malignant breast epithelial cells secrete factors, which induce aromatase expression in adipose fibroblasts via promoter II. This is, at least in part, mediated by a TCM-induced up-regulation and enhanced binding of C/EBPbeta to a promoter II regulatory element.