Abstract 16805: Hepatic HNF1α Knockdown Reduces Circulating PCSK9 Levels and Increases Liver LDL Receptor Abundance in Mice
Circulating PCSK9, which is secreted by the liver, binds to hepatic LDL receptor (LDLR) and causes its lysosomal degradation. Thus PCSK9 inhibits LDL-cholesterol uptake and has hence become an important therapeutic target for the treatment of hypercholesterolemia. Previous in vitro studies have implicated the transcription factors hepatocyte nuclear factor 1 alpha and beta (HNF1α & HNF1β) as positive regulators of PCSK9 transcription based on their binding to the HNF1 binding site on PCSK9 promoter. However, in vivo evidence for their individual roles in the regulation of PCSK9 expression in liver tissue is largely lacking. In this current study, we utilized adenoviral shRNA delivery vectors to generate liver specific knockdown of HNF1α or HNF1β. We first validated the shRNA viral constructs (Ad-shHNF1α and Ad-shHNF1β) in vitro and observed that knockdown of HNF1α but not of HNF1β reduced PCSK9 mRNA expression by ~50% in cultured HepG2 cells. The decrease in PCSK9 was also associated with increased LDLR protein expression. Using luciferase reporter plasmids containing the PCSK9 or LDLR promoter regions, we validated that the reduction in PCSK9 expression after HNF1α knockdown occurred specifically via transcriptional repression whereas the depletion of HNF1α had no effect on LDLR promoter activity. Dil-labeled-LDL uptake studies were further applied to demonstrate that the increased LDLR protein is functional in internalizing higher amounts of LDL from extracellular environments. Consistent with observations in cultured cells, injection of Ad-shHNF1α in mice fed a normal diet significantly (~50%) reduced liver mRNA expression and serum concentration of PCSK9 whereas injection of Ad-shHNF1β had no effect on PCSK9 expression. Importantly, HNF1α knockdown led to ~2.3-fold higher LDLR protein expression in the liver. Additionally, there was a trend towards decrease in circulating total cholesterol and LDL-cholesterol by ~8% and ~12% respectively after HNF1α knockdown in these normolipidemic mice. Altogether, our study demonstrated that HNF1α but not HNF1β plays a critical role in PCSK9 transcription in liver tissue in the murine model system and that transient, liver specific knockdown of HNF1α can significantly reduce circulating PCSK9 levels.
Author Disclosures: V.R. Shende: None. A.B. Singh: None. B. Dong: None. J. Liu: None.
- © 2014 by American Heart Association, Inc.