Christou M, Savas U, Schroeder S, Shen X, Thompson T, Gould M N, Jefcoate C R
Department of Pharmacology, University of Wisconsin, Madison 53705, USA.
Mol Cell Endocrinol. 1995 Nov 30;115(1):41-50. doi: 10.1016/0303-7207(95)03668-w.
Cultured rat mammary cells express both CYP1A1 and CYP1B1 in response to polycyclic aromatic hydrocarbons (PAH) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in a cell type-specific manner. The expression of each P450 was determined functionally (regioselective PAH metabolism), as apoprotein (immunoblots) and as mRNA (Northern hybridization). The epithelial rat mammary cells (RMEC) expressed CYP1A1, however only after PAH or TCDD treatment. CYP1B1 protein was scarcely detected in these induced RMEC but was surprisingly active as a participant in 7,12-dimethylbenz[a]anthracene (DMBA) metabolism shown through selective antibody inhibition (40% of total activity). CYP1B1 was selectively expressed in the stromal fibroblast population of rat mammary cells to the exclusion of CYP1A1. In the rat mammary fibroblasts (RMF), CYP1B1 protein and associated activity were each present at low levels constitutively and were highly induced by benz[a]anthracene (BA) to a greater extent than by TCDD (12- versus 6-fold). However, BA (10 microM) and TCDD (10 nM) stimulated the 5.2-kb CYP1B1-specific mRNA equally. These increases are consistent with the involvement of the aryl hydrocarbon (Ah) receptor in the transcription of the CYP1B1 gene and with the additional stabilization of CYP1B1 protein by BA, previously observed in embryo fibroblasts. Exactly this regulation of CYP1B1-dependent activity was seen in RMEC suggesting that this arises from exceptionally active CYP1B1 in a small proportion (5%) of residual RMF. The constitutive expression and PAH inducibility of CYP1B1 and CYP1A1 proteins in RMF and RMEC, respectively, were each substantially decreased (approximately 75%) by a hormonal mixture (17 beta-estradiol (0.2 microM) progesterone (1.5 microM) cortisol (1.5 microM) and prolactin (5 micrograms/ml)). Progesterone and cortisol, added singly to RMF suppressed CYP1B1 protein expression (approximately 80%) in both untreated and BA-induced cells, while cortisol also suppressed the 5.2-kb CYP1B1 mRNA. In contrast, 17 beta-estradiol stimulated constitutive expression of CYP1B1 protein (50-75%) and mRNA level (2- to 3-fold), but did not affect CYP1B1 expression in BA-treated RMF. The expression of CYP1A1 and CYP1B1 is therefore highly cell specific even though each is regulated through the Ah receptor. Each P450 exhibits a surprisingly similar pattern of hormonal regulation even though expressed in different cell types.
培养的大鼠乳腺细胞可响应多环芳烃（PAH）和2,3,7,8-四氯二苯并对二恶英（TCDD），以细胞类型特异性方式表达CYP1A1和CYP1B1。通过功能测定（区域选择性PAH代谢）、载脂蛋白（免疫印迹）和mRNA（Northern杂交）来确定每种细胞色素P450的表达。大鼠乳腺上皮细胞（RMEC）仅在PAH或TCDD处理后才表达CYP1A1。在这些诱导的RMEC中几乎检测不到CYP1B1蛋白，但令人惊讶的是，通过选择性抗体抑制显示，它作为7,12-二甲基苯并[a]蒽（DMBA）代谢的参与者具有活性（占总活性的40%）。CYP1B1在大鼠乳腺细胞的基质成纤维细胞群体中选择性表达，而不表达CYP1A1。在大鼠乳腺成纤维细胞（RMF）中，CYP1B1蛋白和相关活性在基础状态下均处于低水平，并且被苯并[a]蒽（BA）高度诱导，诱导程度大于TCDD（分别为12倍和6倍）。然而，BA（10μM）和TCDD（10 nM）对5.2 kb CYP1B1特异性mRNA的刺激作用相同。这些增加与芳烃（Ah）受体参与CYP1B1基因的转录以及BA对CYP1B1蛋白的额外稳定作用一致，这在胚胎成纤维细胞中先前已观察到。在RMEC中也观察到了这种对CYP1B1依赖性活性的调控，这表明这是由一小部分（5%）残留RMF中异常活跃的CYP1B1引起的。一种激素混合物（17β-雌二醇（浓度为0.2μM）、孕酮（浓度为1.5μM）、皮质醇（浓度为1.5μM）和催乳素（浓度为5μg/ml））分别使RMF和RMEC中CYP1B1和CYP1A1蛋白的基础表达和PAH诱导性显著降低（约75%）。单独添加到RMF中的孕酮和皮质醇抑制未处理和BA诱导细胞中CYP1B1蛋白的表达（约80%），而皮质醇也抑制5.2 kb CYP1B1 mRNA。相比之下，17β-雌二醇刺激CYP1B1蛋白的基础表达（50 - 75%）和mRNA水平（2至3倍），但不影响BA处理的RMF中CYP1B1的表达。因此，尽管CYP1A1和CYP1B1均通过Ah受体进行调控，但其表达具有高度的细胞特异性。尽管它们在不同细胞类型中表达，但每种细胞色素P450都表现出惊人相似的激素调控模式。
