Hepatocyte Nuclear Factor 1α Plays a Critical Role in PCSK9 Gene Transcription and Regulation by the Natural Hypocholesterolemic Compound Berberine

Abstract

PCSK9 is a natural inhibitor of LDL receptor (LDLR) that binds the extracellular domain of LDLR and triggers its intracellular degradation. PCSK9 and LDLR are coordinately regulated at the transcriptional level by sterols through their promoter-imbedded sterol response elements (SRE) and co-induced by statins. Identification of regulatory networks modulating PCSK9 transcription is important for developing selective repressors of PCSK9 to improve statin efficacy by prolonging the up-regulation of LDLR. Interestingly, the plant-derived hypocholesterolemic compound berberine (BBR) up-regulates LDLR expression while down-regulating PCSK9. In our investigations to define mechanisms underlying the transcriptional suppression of PCSK9 by BBR in HepG2 cells, we have identified a highly conserved hepatocyte nuclear factor 1 (HNF1) binding site residing 28 bp upstream from SRE as a critical sequence motif for PCSK9 transcription and its regulation by BBR. Mutation of the HNF1 site reduced PCSK9 promoter activity >90%. A battery of functional assays identified HNF1α as the predominant trans-activator for PCSK9 gene working through this sequence motif. We further provide evidence suggesting that HNF1 site works cooperatively with SRE as HNF1 mutation significantly attenuated the activity of nuclear SREBP2 to transactivate PCSK9 promoter. Finally, we show that a coordinate modest reduction of HNF1α and nuclear SREBP2 by BBR led to a strong suppression of PCSK9 transcription through these two critical regulatory sequences. This is the first described example of SREBP pairing with HNF1 to control an important regulatory pathway in cholesterol homeostasis. This work also provides a mechanism for how BBR suppresses PCSK9 transcription.

The Epigenetic Drug 5-Azacytidine Interferes with Cholesterol and Lipid Metabolism

“…Sterol response element (SRE; bp Ϫ345 to Ϫ337) and HNF1 (hepatocyte nuclear factor 1) motifs (bp Ϫ386 to Ϫ374) were mutated within the 1000-bp PCSK9 proximal promoter by directed mutagenesis, as described (22). All amplified products were digested with SpeI and HindIII endonucleases and ligated into pCMV-GLuc vector (catalog no.…”

Pcsk9

“…Its invalidation by site-directed mutagenesis dramatically reduces PCSK9 gene promoter-driven expression of a reporter gene in transfected HepG2 cells. 79 Upregulation of HNF1α expression by statins 80 contributes to sustained PCSK9 production/secretion that attenuates the LDLR-mediated clearance of plasma LDL-C induced by these drugs. Its expression is downregulated by the phytochemical berberine 79 and by activators of hepatic mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway, 81 making such compounds potential enhancers of the LDL-C clearance.…”

MG132, a proteasome inhibitor, enhances LDL uptake in HepG2 cells in vitro by regulating LDLR and PCSK9 expression

“…Thus, the development of effective dual modulators of LDLR and PCSK9 may be a more effective way to control blood cholesterol. A number of dual modulators have been reported to date, including berberine and Oncostatin M. They increase LDLR expression by stabilizing its mRNA through an ERK-dependent pathway [18,34] , but berberine decreases PCSK9 expression by suppressing HNF-1α, while Oncostatin M exerts this effect through a JAK-dependent pathway [12,15] . Berberine is poorly absorbed in vivo [18] , and Oncostatin M is a pleiotropic cytokine synthesized by stimulated T-cells and monocytes, which may not be suitable for clinical use.…”

“…HepG2 cells were transfected with pCH110 and the indicated luciferase promoter construct plasmids for PCSK9 (D4, D4-SRE, and D4-HNF). The D4-HNF mutant plasmid was constructed as described previously [12] .…”