Ou X, Ramos K S
Department of Physiology and Pharmacology, College of Veterinary Medicine, Texas A & M University, College Station 77843.
Arch Biochem Biophys. 1995 Jan 10;316(1):116-22. doi: 10.1006/abbi.1995.1017.
The present studies were conducted to evaluate the pattern of cytochrome P4501A1 (CYP1A1) gene inducibility in vascular (aortic) smooth muscle cells (SMCs) upon exposure to selected aromatic hydrocarbons. Challenge of randomly cycling or synchronized subcultures of adult quail aortic SMCs with 30 microM benzo[a]-pyrene (BaP) or 10 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 24 h failed to induce CYP1A1 gene expression as determined by Northern analysis. However, challenge with either hydrocarbon in the presence of 10 micrograms/ml cycloheximide caused superinduction of CYP1A1 mRNA levels under both growth conditions. Velocity sedimentation analysis of the nuclear fraction of quail aortic SMCs treated with 10 nM [3H]-TCDD resulted in a specifically bound peak of 6.5S. Formation of the 6.5S peak was competitively inhibited by an excess of unlabeled 2,3,7,8-tetrachlorodibenzo-furan (2 microM), a known aryl hydrocarbon receptor (AhR) ligand. Gel mobility shift assays of nuclear extracts from BaP- or TCDD-treated cells using a 32P-labeled Ah-responsive element consensus sequence gave ligand-inducible retarded bands. Transient transfection of the pMCAT 5.12 plasmid into SMCs followed by treatment with 30 microM BaP or 10 nM TCDD for 48 h was associated with appreciable induction of CAT activity. A comparable challenge, however, did not induce ethoxyresorufin O-deethylase activity in aortic SMCs. These results demonstrate that adult quail aortic SMCs contain the CYP1A1 gene and exhibit intact AhR-mediated signal transduction. The CYP1A1 gene is repressed under basal conditions, but treatment with cycloheximide restores constitutive expression and affords hydrocarbon inducibility. These data suggest that in adult quail aortic SMCs a labile repressor protein of CYP1A1 gene precludes transcriptional activation of the gene but does not interfere with AhR-dependent signal transduction.
开展本研究以评估成年鹌鹑主动脉平滑肌细胞（SMC）在暴露于特定芳烃后细胞色素P4501A1（CYP1A1）基因的诱导模式。用30微摩尔苯并[a]芘（BaP）或10纳摩尔2,3,7,8-四氯二苯并-对-二噁英（TCDD）对成年鹌鹑主动脉SMC的随机传代或同步传代培养物进行24小时刺激，经Northern分析确定未能诱导CYP1A1基因表达。然而，在10微克/毫升放线菌酮存在的情况下，用任何一种碳氢化合物进行刺激，在两种生长条件下均导致CYP1A1 mRNA水平的超诱导。用10纳摩尔[3H]-TCDD处理的鹌鹑主动脉SMC核部分的速度沉降分析产生了一个6.5S的特异性结合峰。过量未标记的2,3,7,8-四氯二苯并呋喃（2微摩尔）（一种已知的芳烃受体（AhR）配体）竞争性抑制了6.5S峰的形成。使用32P标记的Ah反应元件共有序列对BaP或TCDD处理细胞的核提取物进行凝胶迁移率变动分析，得到配体诱导的滞后条带。将pMCAT 5.12质粒瞬时转染到SMC中，然后用30微摩尔BaP或10纳摩尔TCDD处理48小时，与CAT活性的明显诱导相关。然而，类似的刺激并未在主动脉SMC中诱导乙氧异吩唑酮O-脱乙基酶活性。这些结果表明成年鹌鹑主动脉SMC含有CYP1A1基因，并表现出完整的AhR介导的信号转导。CYP1A1基因在基础条件下受到抑制，但用放线菌酮处理可恢复组成型表达并赋予碳氢化合物诱导性。这些数据表明，在成年鹌鹑主动脉SMC中，CYP1A1基因的一种不稳定阻遏蛋白可阻止该基因的转录激活，但不干扰AhR依赖性信号转导。
