Sterol-dependent regulation of proprotein convertase subtilisin/kexin type 9 expression by sterol-regulatory element binding protein-2

Abstract

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the subtilases that promotes the internalization and degradation of LDL receptor in liver and thereby controls the level of LDL cholesterol in plasma. Here, we show that the expression of PCSK9 in HepG2 cells is completely dependent on the absence or presence of sterols. The minimal promoter region of the PCSK9 gene contains a sterol-regulatory element (SRE), which makes the transcription of PCSK9 dependent on sterols. Expression of nuclear forms of sterol-regulatory element binding protein-1 (SREBP-1) and SREBP-2 dramatically increased the promoter activity of PCSK9. In vitro-translated nuclear forms of SREBPs showed interactions with SRE, whereas mutations in SRE abolished their binding. In vivo studies in mice showed that Pcsk9 protein and mRNA were decreased significantly by fasting and increased by refeeding. However, supplementation with 2% cholesterol in the diet prevented the increase in Pcsk9. The amounts of Pcsk9 mRNA in livers of refed mice showed correlated regulation by the changes in the nuclear form of Srebp-2. In summary, it is suggested that the expression of PCSK9 is regulated by sterol at the transcriptional level in HepG2 cells and that both SREBP-1 and SREBP-2 can transcriptionally activate PCSK9 via SRE in its proximal promoter region in vitro. However, in vivo, it is suggested that the sterol-dependent regulation of PCSK9 is mediated predominantly by SREBP-2. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the proteinase K subfamily of subtilisin serine proteases of which the gain-of-function mutations cause hypercholesterolemia (1-3). PCSK9 was independently identified as one of the genes that are regulated by sterol-regulatory element binding proteins (SREBPs) (4, 5). The SREBPs are members of the basic helix-loop-helix leucine zipper family of transcription factors that regulate the expression of the target genes by binding to the sterolregulatory elements (SREs) in their promoter regions (6). Using microarrays hybridized with RNA from livers of mice that either overexpressed nuclear forms of human SREBPs (transgenic model) or lacked SREBP-activating protein (knockout model), PCSK9 was identified as a SREBP target gene. Soon after the first cloning of this gene, with its relationship to neural apoptosis and liver regeneration, studies focused on its relationship with the regulation of cholesterol in plasma. Subsequently, the loss-of-function mutations of PCSK9 have been reported to decrease LDL cholesterol level (7-9) and reduce the risk of coronary heart disease (10). The definite evidence for a role of PCSK9 in LDL metabolism was revealed by a set of in vivo animal experiments. Adenovirus-mediated overexpression of PCSK9 reduced the amount of low density lipoprotein receptor (LDLR) in livers posttranscriptionally (11,12), whereas the amount of LDLR increased significantly in livers of Pcsk9 knockout mice (13). The mechanism by which PCSK9 reduces LDLR suggests that secreted PCSK9 in plas...

Pcsk9

“…77 SREBP-1 and SREBP-2 have been shown to bind to the PCSK9 gene promoter SRE in vitro specifically. 78 SREBP activation of the PCSK9 gene promoter is potentiated by the hepatocyte nuclear factor-1α (HNF1α), which binds to an element located 28 nucleotides (nts) upstream of the SRE. This site is conserved between primate and rodent PCSK9 promoters and is absent in the LDLR promoter.…”

Suppression of Idol expression is an additional mechanism underlying statin-induced up-regulation of hepatic LDL receptor expression

“…GW3965 and the anti-ß-actin monoclonal antibody were purchased from Sigma-Aldrich. The rabbit anti-LDLR antibody was obtained from BioVision (Mountain View, CA) and the rabbit anti-PCSK9 antibody was obtained as previously described (13). Lipoprotein deficient serum (LPDS) was purchased from HyClone (Logan, UT).…”

“…This leads to an increased number of LDLR on the surface of the hepatocytes, resulting in an enhanced uptake of LDL particles from the circulation. This statin-induced activation of the SREBP pathway, however, has an attenuating effect on the LDLR protein levels in the liver through a concomitant induction of proprotein convertase subtilisin/kexin type 9 (PCSK9) mediated by an SRE motif embedded in the PCSK9 promoter (12,13). PCSK9 is a liver-derived plasma protein that binds to the extracellular EGF-A domain of LDLR and enhances the intracellular degradation of LDLR (14,15).…”

JNK-Mediated SREBP-2 Processing by Genistein up-regulates LDLR Expression in HepG2 Cells

“…There, pre-SREBPs undergo proteolytic cleavage by serine proteases, resulting in liberation of the N-terminal region, which is a nuclear SREBP. Nuclear SREBP enters the nucleus and binds a sterol-regulatory element (SRE) in the promoter region of target genes such as LDLR [11][12][13][14]. SREBP-regulated gene expression has its own negative intrinsic regulatory mechanism.…”