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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

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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.

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Abbreviations

ABCA1:

ATP-binding cassette transporter A1

cAMP:

cyclic AMP

PK:

protein kinase

TD:

Tangiers Disease

USF:

upstream stimulatory factor

DMEM:

Dulbecco’s minimal essential medium

FBS:

fetal bovine serum

LXR:

liver x receptor

RXR:

retinoid x receptor

apoA1:

apolipoprotein A1

HDL:

high density lipoprotein

SDS-PAGE:

sodium dodecyl sulfate polyacrylamide gel electrophoresis

8-Br-cAMP:

8-bromocyclic adenosine 3′, 5′ monophophate

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Authors and Affiliations

  1. University of Palermo, Palermo, Italy

    Neelam Srivastava, Angelo B. Cefalu & Maurizio Averna

  2. Integrated Pharma Solution, Philadelphia, USA

    Rai Ajit K. Srivastava

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  1. Neelam Srivastava
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  2. Angelo B. Cefalu
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Contributions

Rai Ajit K Srivastava evaluated data from the studies and has contributed to the interpretation and analyses of data as well as writing of the manuscript. Neelam Srivastava and Angelo Cefalu designed the studies, carried out some of the experiments, and wrote part of the manuscript. Maurizio Averna contributed to the review and interpretation of data and wrote part of the manuscript.

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Correspondence to Rai Ajit K. Srivastava.

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Studies carried out in this report did not use any animal species. The cell-based assays were carried out according to the institutional guidelines. All authors agree to the publication of the results contained in this manuscript.

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Srivastava, N., Cefalu, A.B., Averna, M. et al. 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. J Diabetes Metab Disord 19, 363–371 (2020). https://doi.org/10.1007/s40200-020-00517-0

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