Srivastava Neelam, Cefalu Angelo B, Averna Maurizio, Srivastava Rai Ajit K
University of Palermo, Palermo, Italy.
Integrated Pharma Solution, Philadelphia, USA.
J Diabetes Metab Disord. 2020 Mar 19;19(1):363-371. doi: 10.1007/s40200-020-00517-0. eCollection 2020 Jun.
ATP-binding cassette transporter A1 (ABCA1) is a key player in the reverse cholesterol transport (RCT) and HDL biogenesis. Since RCT is compromised as a result of ABCA1 dysfunction in diabetic state, the objective of this study was to investigate the regulation of ABCA1 in a stably transfected 293 cells expressing ABCA1 under the control of cAMP response element.
To delineate transcriptional and posttranscriptional regulation of ABCA1, 293 cells were stably transfected with the full length ABCA1 cDNA under the control of CMV promoter harboring cAMP response element. cAMP-mediated regulation of ABCA1 and cholesterol efflux were studied in the presence of 8-Br-cAMP and after withdrawal of 8-Br-cAMP. The mechanism of cAMP-mediated transcriptional induction of the ABCA1 gene was studied in protein kinase A (PKA) inhibitors-treated cells.
The transfected 293 cells expressed high levels of ABCA1, while non-transfected wild-type 293 cells showed very low levels of ABCA1. Treatments of transfected cells with 8-Br-cAMP increased ABCA1 protein by 10-fold and mRNA by 20-fold. Cholesterol efflux also increased in parallel. Withdrawal of 8-Br-cAMP caused time-dependent rapid diminution of ABCA1 protein and mRNA, suggesting ABCA1 regulation at the transcriptional level. Treatment with PKA inhibitors abolished the cAMP-mediated induction of the ABCA1 mRNA and protein, resulting dampening of ABCA1-dependent cholesterol efflux.
These results demonstrate that transfected cell line mimics cAMP response similar to normal cells with natural ABCA1 promoter and suggest that ABCA1 is a short-lived protein primarily regulated at the transcriptional level to maintain cellular cholesterol homeostasis.
ATP结合盒转运蛋白A1（ABCA1）是逆向胆固醇转运（RCT）和高密度脂蛋白（HDL）生物合成的关键参与者。由于在糖尿病状态下ABCA1功能障碍会导致RCT受损，本研究的目的是调查在cAMP反应元件控制下稳定转染表达ABCA1的293细胞中ABCA1的调控情况。
为了阐明ABCA1的转录和转录后调控，将全长ABCA1 cDNA在含有cAMP反应元件的巨细胞病毒（CMV）启动子控制下稳定转染到293细胞中。在存在8-溴-cAMP的情况下以及撤除8-溴-cAMP后，研究了cAMP介导的ABCA1调控和胆固醇流出。在蛋白激酶A（PKA）抑制剂处理的细胞中研究了cAMP介导的ABCA1基因转录诱导机制。
转染的293细胞表达高水平的ABCA1，而未转染的野生型293细胞显示出非常低水平的ABCA1。用8-溴-cAMP处理转染细胞使ABCA1蛋白增加10倍，mRNA增加20倍。胆固醇流出也平行增加。撤除8-溴-cAMP导致ABCA1蛋白和mRNA随时间快速减少，表明ABCA1在转录水平受到调控。用PKA抑制剂处理消除了cAMP介导的ABCA1 mRNA和蛋白的诱导，导致ABCA1依赖性胆固醇流出减弱。
这些结果表明，转染细胞系模拟了与具有天然ABCA1启动子的正常细胞类似的cAMP反应，并表明ABCA1是一种主要在转录水平受到调控的短寿命蛋白，以维持细胞胆固醇稳态。
