Abstract 16198: Inhibition of PCSK9 Transcription by Berberine Involves Down-regulation of HNF1 Protein Cellular Levels Through Ubiquitin-proteasome Pathway

Abstract

PCSK9 plays an important role in control of plasma LDL cholesterol metabolism by modulating the degradation of hepatic LDL receptor (LDLR). Our previously studies have demonstrated that PCSK9 gene transcription in liver cells is regulated by the hepatocyte nuclear factor 1α (HNF1α) through a highly conserved HNF1 binding site located 28 bp upstream of the SRE-1 site of the PCSK9 gene promoter. Importantly, we have shown that the natural cholesterol lowering compound berberine (BBR) inhibits PCSK9 transcription via this regulatory element. The aim of this current study was to further investigate the inhibitory mechanisms of BBR on PCSK9 transcription involving HNF1α. Here we report that BBR had no effects on HNF1α mRNA expressions but it greatly reduced HNF1α protein levels in HepG2 cells and in liver of hamsters in vivo, suggesting that BBR might affect HNF1α protein stability. Since the ubiquitin-proteasome pathway (UPP) and the autophagy-lysosomal pathway are two major cellular proteolytic systems for intracellular protein degradation in eukaryotic cells, we used specific inhibitors to block individual pathways to examine their possible roles in regulation of HNF1α protein degradation. Treating HepG2 cells with autophagy/lysosomes inhibitor bafilomycin A1 markedly increased cellular levels of LDLR and PCSK9, which confirmed their known intracellular degradations by lysosomes. In contrast, HNF1α protein amount was not affected by bafilomycin A1 treatment but it was dose-dependently increased by UPP inhibitors bortezomib and MG132, thereby suggesting that HNF1α protein degradation is regulated by UPP. Through conducting immunoprecipitation and immunoblotting we further detected multi-ubiquitination ladder pattern of HNF1α in cells overexpressing ubiquitin. Finally, we show that the BBR reducing effect on HNF1α protein abundance can be blocked by concurrent treatment with proteasome inhibitors. Altogether, our studies suggest a functional role of the ubiquitin-proteasome pathway in the regulation of PCSK9 gene transcription through modifying the protein stability of HNF1α upon BBR treatment. This study shed new light on cellular mechanisms that control PCSK9 expression, thereby influencing blood LDL-cholesterol levels.