Identification of neolignans with PCSK9 downregulatory and LDLR upregulatory activities from Penthorum chinense and the potential in cholesterol uptake by transcriptional regulation of LDLR via SREBP2.
Penthorum chinense has been used in East Asia for the treatment of cholecystitis, infectious hepatitis, jaundice and to treat liver problems. Recent evidences provided the potential for the clinical use of P. chinense in the treatment of metabolic disease.
Based on the traditional use and recent evidences, we investigated the effects of constituents from P. chinense with modulation on proprotein convertase subtilisin/kexin type 9 (PCSK9) and low-density lipoprotein receptor (LDLR) expression, and the effect of the most active substance on cholesterol uptake, and genes relevant to lipid metabolism.
The isolation of compounds from the BuOH-soluble extract of 80% methanol extract of P. chinense was conducted using chromatographic methods and the structures were established by interpreting spectroscopic data. Quantitative real time-PCR, and Western blot analysis were performed to monitor the regulatory activity on PCSK9 and LDLR expression. PCSK9-LDLR binding interaction was also tested. The cholesterol uptake in hepatocyte was measured using 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI)-labeled LDL cholesterol. Additionally, gene network analysis of LDLR and responses of its target proteins were carried out to discover genes germane to the effect of active compound on HepG2 cells. Moreover, we performed protein-protein interaction analysis via String and constructed the compound target network using Cytoscape.
Two new neolignans and 37 known compounds were characterized from P. chinense. Of the isolated compounds, (7'E,8S)-2',4,8-trihydroxy-3-methoxy-2,4'-epoxy-8,5'-neolign-7'-en-7-one (3), penthorin A (4) and methyl gallate (25) were found to suppress PCSK9 mRNA expression with IC50 values of 5.13, 15.56 and 11.66 μM, respectively. However, all the isolated compounds were found to be inactive in PCSK9-LDLR interaction assay. Additionally, a dibenzoxepine-type lignan analog, (7'E,8S)-2',4,8-trihydroxy-3-methoxy-2,4'-epoxy-8,5'-neolign-7'-en-7-one (3) demonstrated to upregulate LDLR mRNA and protein expression via transcriptional factor sterol regulatory element-binding protein 2 (SREBP2). Furthermore, (7'E,8S)-2',4,8-trihydroxy-3-methoxy-2,4'-epoxy-8,5'-neolign-7'-en-7-one (3) increase the LDL-cholesterol uptake in DiI-LDL assay.
(7'E,8S)-2',4,8-trihydroxy-3-methoxy-2,4'-epoxy-8,5'-neolign-7'-en-7-one (3) seemed to increase potentially cholesterol uptake via the downregulation of PCSK9 and the activation of LDLR in hepatocytes. Moreover, SREBP2 was found to play an important role in regulation of PCSK9 and LDLR by (7'E,8S)-2',4,8-trihydroxy-3-methoxy-2,4'-epoxy-8,5'-neolign-7'-en-7-one.
Chae HS
，Pel P
，Cho J
，Kim YM
，An CY
，Huh J
，Choi YH
，Kim J
，Chin YW
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Cyclase-associated protein 1 is a binding partner of proprotein convertase subtilisin/kexin type-9 and is required for the degradation of low-density lipoprotein receptors by proprotein convertase subtilisin/kexin type-9.
Proprotein convertase subtilisin/kexin type-9 (PCSK9), a molecular determinant of low-density lipoprotein (LDL) receptor (LDLR) fate, has emerged as a promising therapeutic target for atherosclerotic cardiovascular diseases. However, the precise mechanism by which PCSK9 regulates the internalization and lysosomal degradation of LDLR is unknown. Recently, we identified adenylyl cyclase-associated protein 1 (CAP1) as a receptor for human resistin whose globular C-terminus is structurally similar to the C-terminal cysteine-rich domain (CRD) of PCSK9. Herein, we investigated the role of CAP1 in PCSK9-mediated lysosomal degradation of LDLR and plasma LDL cholesterol (LDL-C) levels.
The direct binding between PCSK9 and CAP1 was confirmed by immunoprecipitation assay, far-western blot, biomolecular fluorescence complementation, and surface plasmon resonance assay. Fine mapping revealed that the CRD of PCSK9 binds with the Src homology 3 binding domain (SH3BD) of CAP1. Two loss-of-function polymorphisms found in human PCSK9 (S668R and G670E in CRD) were attributed to a defective interaction with CAP1. siRNA against CAP1 reduced the PCSK9-mediated degradation of LDLR in vitro. We generated CAP1 knock-out mice and found that the viable heterozygous CAP1 knock-out mice had higher protein levels of LDLR and lower LDL-C levels in the liver and plasma, respectively, than the control mice. Mechanistic analysis revealed that PCSK9-induced endocytosis and lysosomal degradation of LDLR were mediated by caveolin but not by clathrin, and they were dependent on binding between CAP1 and caveolin-1.
We identified CAP1 as a new binding partner of PCSK9 and a key mediator of caveolae-dependent endocytosis and lysosomal degradation of LDLR.
Jang HD
，Lee SE
，Yang J
，Lee HC
，Shin D
，Lee H
，Lee J
，Jin S
，Kim S
，Lee SJ
，You J
，Park HW
，Nam KY
，Lee SH
，Park SW
，Kim JS
，Kim SY
，Kwon YW
，Kwak SH
，Yang HM
，Kim HS
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Ascorbic acid enhances low-density lipoprotein receptor expression by suppressing proprotein convertase subtilisin/kexin 9 expression.
Ascorbic acid, a water-soluble antioxidant, regulates various biological processes and is thought to influence cholesterol. However, little is known about the mechanisms underpinning ascorbic acid-mediated cholesterol metabolism. Here, we determined if ascorbic acid can regulate expression of proprotein convertase subtilisin/kexin 9 (PCSK9), which binds low-density lipoprotein receptor (LDLR) leading to its intracellular degradation, to influence low-density lipoprotein (LDL) metabolism. At cellular levels, ascorbic acid inhibited PCSK9 expression in HepG2 and Huh7 cell lines. Consequently, LDLR expression and cellular LDL uptake were enhanced. Similar effects of ascorbic acid on PCSK9 and LDLR expression were observed in mouse primary hepatocytes. Mechanistically, ascorbic acid suppressed PCSK9 expression in a forkhead box O3-dependent manner. In addition, ascorbic acid increased LDLR transcription by regulating sterol regulatory element-binding protein 2. In vivo, administration of ascorbic acid reduced serum PCSK9 levels and enhanced liver LDLR expression in C57BL/6J mice. Reciprocally, lack of ascorbic acid supplementation in L-gulono-γ-lactone oxidase deficient (Gulo-/-) mice increased circulating PCSK9 and LDL levels, and decreased liver LDLR expression, whereas ascorbic acid supplementation decreased PCSK9 and increased LDLR expression, ameliorating LDL levels in Gulo-/- mice fed a high fat diet. Moreover, ascorbic acid levels were negatively correlated to PCSK9, total and LDL levels in human serum samples. Taken together, these findings suggest that ascorbic acid reduces PCSK9 expression, leading to increased LDLR expression and cellular LDL uptake. Thus, supplementation of ascorbic acid may ameliorate lipid profiles in ascorbic acid-deficient species.
Wang D
，Yang X
，Chen Y
，Gong K
，Yu M
，Gao Y
，Wu X
，Hu H
，Liao C
，Han J
，Duan Y
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