Abstract
BackgroundSite-1 protease (S1P) is the key enzyme required for activation of the sterol regulatory element binding proteins (SREBPs) that govern lipid synthesis. While S1P has been speculated to influence plasma apoB-containing lipoprotein (Blp) metabolism, there has been little investigative work. LDL receptor (LDLR) is the major receptor for clearing plasma LDL cholesterol (LDL-c). Proprotein convertase subtilisin kexin type 9 (PCSK9) modulates LDL-c through post-translational degradation of the LDLR.MethodsA hepatic-specific knockdown (KD) of S1P was achieved using floxed S1P mouse models (S1Pf/f and LDLR-/-S1Pf/f) and hepatic expression of Cre recombinase. Lipids were measured in total plasma and size fractionated plasma using colorimetric assays. Realtime polymerase chain reaction, western blotting and ELISA were used to determine hepatic expression of key genes/protein. Plasmid mediated overexpression and siRNA mediated knockdown of genes were performed in mouse primary hepatocytes to determine the mechanistic basis of PCSK9 gene regulation.ResultsA hepatic-specific KD of S1P resulted in a 45 % and 38 % reduction in plasma total cholesterol and triglyceride levels, respectively. Hepatic S1P KD had a minimal effect on plasma Blp cholesterol (Blp-c) in S1Pf/f mice, despite significantly reducing VLDL secretion. Notably, hepatic S1P KD decreased the LDL receptor (LDLR) mRNA expression by 50 %. However, the reduction in LDLR protein levels was less than that of mRNA expression, especially under fed conditions. Further assessment of hepatic S1P deficiency revealed that it increased LDLR protein stability in vivo. Mechanistically, hepatic S1P KD was shown to decrease the liver and plasma levels of the protein proprotein convertase subtilisin/kexin type 9 (PCSK9), which degrades LDLR protein. This effect was more prominent in the fed condition and sufficient to account for the discordance in LDLR mRNA and protein levels. Furthermore, hepatic S1P was shown to regulate PCSK9 expression through activation of the SREBPs. In the LDLR-/- background, hepatic S1P KD significantly reduced Blp-c levels.ConclusionHepatic S1P is a physiological modulator of plasma Blp metabolism through its regulation of LDLR and PCSK9. Hepatic S1P is a valid target for lowering plasma Blp-c levels in the situation where LDLR function is compromised.
Highlights
Site-1 protease (S1P) is the key enzyme required for activation of the sterol regulatory element binding proteins (SREBPs) that govern lipid synthesis
The major questions underlying this research are: does inhibition of hepatic S1P decrease plasma apoB-containing lipoprotein (Blp)-c levels? If so, what is the molecular mechanism? We evaluated the role of hepatic S1P in plasma Blp cholesterol (Blp-c) metabolism using different mouse models of hepatic S1P deficiency
To determine the physiological role of hepatic S1P in regulating plasma lipid and lipoprotein levels, we have used S1Pf/f mice and knocked down S1P using two wellaccepted approaches: (a), S1Pf/f Cre, a model of acute hepatic S1P knockdown (KD) via adenovirus-mediated Cre expression, and (b), liver-specific ablation of S1P (L-S1P) mice (S1Pf/f crossed with Albumin-Cre), a model of liver-specific S1P KD via a Cre transgene under the control of the albumin promoter
Summary
Site-1 protease (S1P) is the key enzyme required for activation of the sterol regulatory element binding proteins (SREBPs) that govern lipid synthesis. Proprotein convertase subtilisin kexin type 9 (PCSK9) modulates LDL-c through post-translational degradation of the LDLR. Site 1 protease (S1P, known as membrane-bound transcription factor peptidase, site 1), belongs to the proprotein convertase (PCSK) family. S1P cleaves and activates membranebound unprocessed transcription factors that have been transported to the Golgi [2,3,4,5]. S1P is one of the key enzymes required for release of the transcriptionstimulating domains of sterol regulatory element binding proteins (SREBP1a, SREBP1c and SREBP2) in vitro and in vivo [6]. SREBPs are synthesized as inactive precursors bound to endoplasmic reticulum (ER) in complex with SCAP, an ER to Golgi transport protein
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