Reduction of circulating PCSK9 and LDL-C levels by liver-specific knockdown of HNF1α in normolipidemic mice

Vikram Ravindra Shende,Minhao Wu,Amar Bahadur Singh,Bin Dong,Chin Fung Kelvin Kan,Jingwen Liu

doi:10.1194/jlr.m052969

Abstract

The transcription factors hepatic nuclear factor (HNF)1α and HNF1β can bind to the HNF1 site on the proprotein convertase subtilisin/kexin type 9 (PCSK9) promoter to activate transcription in HepG2 cells. However, it is unknown whether one or both HNF1 factors are obligatory for transactivating hepatic PCSK9 gene expression in vivo. We developed shRNA adenoviral constructs (Ad-shHNF1α and Ad-shHNF1β) to examine the effects of knockdown of HNF1α or HNF1β on PCSK9 expression and its consequent impact on LDL receptor (LDLR) protein levels in cultured hepatic cells and liver tissue. We demonstrated that infection with Ad-shHNF1α, but not Ad-shHNF1β, markedly reduced PCSK9 mRNA expression in HepG2 cells with a concomitant increase in LDLR protein abundance. Injecting Ad-shHNF1α in mice fed a normal diet significantly (∼ 50%) reduced liver mRNA expression and serum concentration of PCSK9 with a concomitant increase (∼ 1.9-fold) in hepatic LDLR protein abundance. Furthermore, we observed a modest but significant reduction in circulating LDL cholesterol after knockdown of HNF1α in these normolipidemic mice. Consistent with the observation that knockdown of HNF1β did not affect PCSK9 mRNA or protein expression in cultured hepatic cells, Ad-shHNF1β infection in mice resulted in no change in the hepatic mRNA expression or serum content of PCSK9. Altogether, our study demonstrates that HNF1α, but not HNF1β, is the primary positive regulator of PCSK9 transcription in mouse liver.

Highlights

The transcription factors hepatic nuclear factor (HNF)1␣ and HNF1␤ can bind to the HNF1 site on the proprotein convertase subtilisin/kexin type 9 (PCSK9) promoter to activate transcription in HepG2 cells
Contrary to the effects observed on PCSK9 D4 plasmid, knockdown of either HNF1␣ or HNF1␤ did not produce any significant change in LDLR1192 activity, confirming that LDL receptor (LDLR) transcription is not regulated by HNF1␣/1␤ (Fig. 1B)
We show that HNF1␣, but not HNF1␤, is the primary positive regulator of PCSK9 transcription in liver tissue in vivo

Summary

Introduction

The transcription factors hepatic nuclear factor (HNF)1␣ and HNF1␤ can bind to the HNF1 site on the proprotein convertase subtilisin/kexin type 9 (PCSK9) promoter to activate transcription in HepG2 cells. We developed shRNA adenoviral constructs (AdshHNF1␣ and Ad-shHNF1␤) to examine the effects of knockdown of HNF1␣ or HNF1␤ on PCSK9 expression and its consequent impact on LDL receptor (LDLR) protein levels in cultured hepatic cells and liver tissue. The binding between PCSK9 and LDLR is enhanced at the acidic pH prevalent in the endosome-lysosomal compartment, which inhibits recycling of LDLR to the cell membrane and instead targets the PCSK9-LDLR complex for degradation in the lysosome [9] In human subjects, both gainand loss-of-function mutations of PCSK9 have been described that result in chronically elevated (hypercholesterolemia) or suppressed (hypocholesterolemia) levels of circulating LDL-C, respectively [10, 11].

Methods

Results

Conclusion