Objectives: 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. Methods: 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. Results: 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. Conclusions: 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.

Srivastava N., Cefalu A.B., Averna M., & Srivastava R.A.K. (2020). Rapid degradation of ABCA1 protein following cAMP withdrawal and treatment with PKA inhibitor suggests ABCA1 is a short-lived protein primarily regulated at the transcriptional level. JOURNAL OF DIABETES AND METABOLIC DISORDERS, 19(1), 363-371 [10.1007/s40200-020-00517-0].

Rapid degradation of ABCA1 protein following cAMP withdrawal and treatment with PKA inhibitor suggests ABCA1 is a short-lived protein primarily regulated at the transcriptional level

Cefalu A. B.;Averna M.;
2020

Abstract

Objectives: 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. Methods: 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. Results: 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. Conclusions: 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.
Srivastava N., Cefalu A.B., Averna M., & Srivastava R.A.K. (2020). Rapid degradation of ABCA1 protein following cAMP withdrawal and treatment with PKA inhibitor suggests ABCA1 is a short-lived protein primarily regulated at the transcriptional level. JOURNAL OF DIABETES AND METABOLIC DISORDERS, 19(1), 363-371 [10.1007/s40200-020-00517-0].
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/10447/458089
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