Click
here to close Hello! We notice that
you are using Internet Explorer, which is not supported by Echinobase
and may cause the site to display incorrectly. We suggest using a
current version of Chrome,
FireFox,
or Safari.
Saudi J Biol Sci
2021 Dec 01;2812:7105-7116. doi: 10.1016/j.sjbs.2021.08.003.
Show Gene links
Show Anatomy links
Acaudina molpadioides mediates lipid uptake by suppressing PCSK9 transcription and increasing LDL receptor in human liver cells.
Jack A
,
Mohd MA
,
Kamaruddin NN
,
Mohd Din LH
,
Hajri NA
,
Tengku Muhammad TS
.
???displayArticle.abstract???
Acaudina molpadioides has been long used as traditional medicinal resources and reported to demonstrate various important bioactivities such as anticoagulation, antithrombosis, anti-hyperglycemia and anticancer. However, its lipid lowering activity is yet to be fully explored. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an enzyme that enhances the lysosomal degradation of hepatic low density lipoprotein receptor (LDLR) resulting in excessive accumulation of the plasma levels of LDL-cholesterols (LDL-C) which subsequently accelerate atherosclerosis. In the present study, A. molpadioides fractions were subjected to promoter-reporter luciferase assay to determine its role as PCSK9 inhibitors. It was found both fractions (EFA and EFB) reduced the transcriptional activity of PCSK9 promoter. Among the seven 5'end deletion constructs of PCSK9 promoter, fragments D1 (-1,711/-94), D3 (-709/-94) and D4 (-440/-94), were suppressed in the presence of both fractions whereas D2 (-1,214/-94), and, D6 (-351/-94) as well as D7 (-335/-94) were inhibited only by EFA and EFB, respectively. Further transcription factor binding sites prediction using MatInspector software discovered various potential cis-regulatory elements namely, PPAR, KLFs, RBPJ-kappa and SREBP that may potentially be involved in ameliorating the transcriptional activity of PCSK9. Immunofluorescence staining was used to evaluate the effects of both fractions on LDL-C and LDLR. Results showed that levels of LDL-C uptake in EFA-treated cells were 69.1% followed by EFB at 32.6%, as compared to untreated control after 24 h treatment. The LDLR protein distribution was induced by 62.41% and 32.2%, which corresponded to an increase in LDL-C uptake in both EFA and EFB treatment, respectively. Hence, the inhibition of PCSK9 by bioactive compounds in EFA and EFB could be another promising therapeutic agent in reducing the cholesterol levels and atherosclerosis by targeting PCSK9.
Fig. 1. The correlation of PCSK9 and statin in regulating the metabolism of LDL-C in the PCSK9 and LDLR are synthesized in the liver. The secreted PCSK9 protein binds to the extracellular domain of LDLR at the cell surface. The PCSK9/LDLR-LDL-C complex enters via the endosomal pathway and is directed to the lysosomal compartment for degradation of both PCSK9 and LDLR, decreasing the number of LDLRs available for clearance of LDL-C particles (A). The gene expression of PCSK9 is subjected to the regulation by the binding of SREBP2, HNF1 and PPAR to their corresponding binding sites on PCSK9 promoter (B). Activation of SREBP2, under conditions of intracellular cholesterol depletion due to inhibitory activity of statin, increases the expression of both PCSK9 and LDLR (C).
Fig. 2. Acaudina molpadioides is an invertebrate marine organism, commonly known as sea potato and belongs to the Holothuroidea family. It is widely consumed by locals in South Eastern Asian countries as traditional healthy food. The figure shows sea potato that was collected from the intertidal zone of Pulau Langkawi, Kedah, Malaysia.
Fig. 3. The effects of A. molpadioides enhanced fractions, EFA (A) and EFB (B) on human proprotein convertase subtilisin/kexin type 9 (PCSK9) promoter activity. HepG2 cells were transfected with a recombinant pGL3 reporter plasmid comprising the human PCSK9 promoter sequence linked to firefly luciferase gene. The transfected cells were then treated with various concentrations of EFA and EFB for 24 h, and subsequently subjected to luciferase assay. The transcriptional activity of PCSK9 promoter was decreased in dose dependent manner in both EFA and EFB (except at 3.125 μg/mL)-treated HepG2 cells with 20 µM berberine sulphate as positive control and 1% (v/v) dimethyl sulfoxide (DMSO) as negative control. Each value represents the mean ± SD of three independent experiments (each in triplicate reaction). Multiple comparisons were done using one-way analysis of variance (ANOVA) where means with different letters were separated with Duncan’s multiple range test group at p < 0.05.
Fig. 4. The effect of A. molpadioides fractions, EFA (A) and EFB (B) on proprotein convertase subtilisin/kexin type 9 (PCSK9) mRNA expression. HepG2 cells were cultured until 80% confluence prior to treatment with EFA and EFB, 20 µM berberine sulphate as positive control and 1% (v/v) dimethyl sulfoxide (DMSO) as negative control, for 24 h. Total cellular RNA was isolated and subjected to real time-PCR analysis. Both EFA and EFB produced the lowest PCSK9 mRNA level at 25 µg/mL. Multiple comparisons were done using one-way analysis of variance (ANOVA) where means with different letters were separated with Duncan’s multiple range test group at p < 0.05.
Fig. 5. Proprotein convertase subtilisin/kexin type 9 (PCSK9) promoter constructs analysis. HepG2 cells that were transfected with each fragment (D1-D7) was treated for 24 h with 25 µg/mL of A. molpadiodes EFA and EFB, with 20 µM berberine sulphate as positive control and 1% (v/v) dimethyl sulfoxide (DMSO) as negative control. Both EFA and EFB significantly decreased the transcriptional activity of promoter fragments D1(-1711/-94 bp), D3(-709/-94 bp) and D4(-440/-94 bp) which indicated that cis-acting elements presence in between these fragment regions were responsible in mediating the inhibitory effect of A. molpadioides on PCSK9 transcriptional activity. Values are means ± standard deviation and unpaired t-test was perform with * p < 0.05; **p < 0.001 and ***p < 0.001.
Fig. 6. Schematic of the distribution of the predicted potential transcription binding sites on the PCSK9 promoter. The transcriptional start site (TSS) is indicated by the arrow. MatInspector software was used to predict transcription factor binding sites on the PCSK9 promoter. PPRE, peroxisome proliferator response element ; KLF, Krüppel like factor; SRE, sterol regulatory element; RBPJ-kappa, recombination signal binding protein for immunoglobin kappa J; HNF1, hepatocyte nuclear factor 1; SP1, specificity protein 1.
Fig. 7. LDL-C uptake and LDLR protein expression on HepG2 cells treated with 25 µg/mL A. molpadioides EFA and EFB, 20 µM berberine sulphate as positive control and 1% (v/v) dimethyl sulfoxide (DMSO) as negative control. HepG2 cells were treated with the mediators for 24 h and the LDL-C uptake was observed. At the end of the treatment duration, the culture medium was replaced with 100 µL/well LDL-DyLight™ 550 working solution and incubated for additional 3 to 4 h. Stained cells were observed with high content screening (HCS) with filters capable of measuring excitation and emission wavelengths 540 and 570 nm, and acquired with MetaXpress® 5.1. EFA treatment produced the highest intensity in cells stained with LDL-DyLight™ 550 (yellow fluorescence) indicating an increase in LDL-C uptake as compared to EFB treatment, in line with the increase in LDLR expression (green fluorescence). For LDLR expression, HepG2 cells were incubated for one hour with 100 µL/well of diluted Rabbit Anti-LDL Receptor Primary Antibody and were subsequently incubated in the dark for one hour with 100 µL/well of diluted DyLight™ 488-Conjugated Secondary Antibody. Stained cells were observed with high content screening (HCS) fluorescein detection (excitation/emission = 485/535 nm) and acquired with MetaXpress® 5.1. Fluorescence intensity was analysed and measured with ImageJ. Scale bar: 50 µm.
Fig. 8. The effect of A. molpadioides enhanced fractions A and B (EFA and EFB) on LDL-C uptake (A) and LDLR translocation (B). ImageJ analysis was carried out by measuring the fold change value of the fluorescence intensity of treated cells over the fluorescence intensity of the untreated cells or negative control, 1% (v/v) dimethyl sulfoxide (DMSO). EFA treatment induced the highest level of LDL-C uptake followed by the positive control, 20 µM berberine sulphate (BBR). Collectively, EFA appeared to show better potential in upregulating LDL-C uptake concomitantly with an increase in LDLR as compared to EFB. Multiple groups were analysed with one-way analysis of variance (ANOVA) where means with different letters were separated with Duncan’s multiple range test group at p < 0.05.
Abifadel,
Mutations in PCSK9 cause autosomal dominant hypercholesterolemia.
2003, Pubmed
Abifadel,
Mutations in PCSK9 cause autosomal dominant hypercholesterolemia.
2003,
Pubmed
Adams,
Transcriptional activation by peroxisome proliferator-activated receptor gamma is inhibited by phosphorylation at a consensus mitogen-activated protein kinase site.
1997,
Pubmed
Adorni,
Naturally Occurring PCSK9 Inhibitors.
2020,
Pubmed
Allard,
Novel mutations of the PCSK9 gene cause variable phenotype of autosomal dominant hypercholesterolemia.
2005,
Pubmed
Bentzon,
Mechanisms of plaque formation and rupture.
2014,
Pubmed
Bordbar,
High-value components and bioactives from sea cucumbers for functional foods--a review.
2011,
Pubmed
,
Echinobase
Burgermeister,
PPARgamma and MEK Interactions in Cancer.
2008,
Pubmed
Cameron,
Effect of mutations in the PCSK9 gene on the cell surface LDL receptors.
2006,
Pubmed
Cartharius,
MatInspector and beyond: promoter analysis based on transcription factor binding sites.
2005,
Pubmed
Chae,
Sauchinone controls hepatic cholesterol homeostasis by the negative regulation of PCSK9 transcriptional network.
2018,
Pubmed
Chinetti,
CLA-1/SR-BI is expressed in atherosclerotic lesion macrophages and regulated by activators of peroxisome proliferator-activated receptors.
2000,
Pubmed
Choi,
Welsh onion extract inhibits PCSK9 expression contributing to the maintenance of the LDLR level under lipid depletion conditions of HepG2 cells.
2017,
Pubmed
Cohen,
Sequence variations in PCSK9, low LDL, and protection against coronary heart disease.
2006,
Pubmed
Costet,
Hepatic PCSK9 expression is regulated by nutritional status via insulin and sterol regulatory element-binding protein 1c.
2006,
Pubmed
Crandall,
Statin use and risk of developing diabetes: results from the Diabetes Prevention Program.
2017,
Pubmed
Crossey,
A cholesterol-lowering VLP vaccine that targets PCSK9.
2015,
Pubmed
Dong,
Strong induction of PCSK9 gene expression through HNF1alpha and SREBP2: mechanism for the resistance to LDL-cholesterol lowering effect of statins in dyslipidemic hamsters.
2010,
Pubmed
Dong,
Hepatic HNF1 transcription factors control the induction of PCSK9 mediated by rosuvastatin in normolipidemic hamsters.
2017,
Pubmed
Drosatos,
Cardiac Myocyte KLF5 Regulates Ppara Expression and Cardiac Function.
2016,
Pubmed
Du,
The anti-tumor activities of cerebrosides derived from sea cucumber Acaudina molpadioides and starfish Asterias amurensis in vitro and in vivo.
2012,
Pubmed
,
Echinobase
Duan,
Peroxisome Proliferator-activated receptor γ activation by ligands and dephosphorylation induces proprotein convertase subtilisin kexin type 9 and low density lipoprotein receptor expression.
2012,
Pubmed
Fan,
Krüppel-like factors and vascular wall homeostasis.
2017,
Pubmed
Foldi,
RBP-J is required for M2 macrophage polarization in response to chitin and mediates expression of a subset of M2 genes.
2016,
Pubmed
Fruchart,
Peroxisome proliferator-activated receptor-alpha activation and high-density lipoprotein metabolism.
2001,
Pubmed
Issemann,
Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators.
1990,
Pubmed
Jeong,
Sterol-dependent regulation of proprotein convertase subtilisin/kexin type 9 expression by sterol-regulatory element binding protein-2.
2008,
Pubmed
Jia,
Enhanced circulating PCSK9 concentration by berberine through SREBP-2 pathway in high fat diet-fed rats.
2014,
Pubmed
Judge,
Beyond statin therapy: a review of the management of residual risk in diabetes mellitus.
2010,
Pubmed
Khotimchenko,
Pharmacological Potential of Sea Cucumbers.
2018,
Pubmed
,
Echinobase
Kourimate,
Dual mechanisms for the fibrate-mediated repression of proprotein convertase subtilisin/kexin type 9.
2008,
Pubmed
Lambert,
Plasma PCSK9 concentrations correlate with LDL and total cholesterol in diabetic patients and are decreased by fenofibrate treatment.
2008,
Pubmed
Lambert,
Fasting induces hyperlipidemia in mice overexpressing proprotein convertase subtilisin kexin type 9: lack of modulation of very-low-density lipoprotein hepatic output by the low-density lipoprotein receptor.
2006,
Pubmed
Langhi,
Activation of the farnesoid X receptor represses PCSK9 expression in human hepatocytes.
2008,
Pubmed
Li,
Hepatocyte nuclear factor 1alpha plays a critical role in PCSK9 gene transcription and regulation by the natural hypocholesterolemic compound berberine.
2009,
Pubmed
Li,
The novel function of HINFP as a co-activator in sterol-regulated transcription of PCSK9 in HepG2 cells.
2012,
Pubmed
Li,
Physicochemical Properties of Collagen from Acaudina Molpadioides and Its Protective Effects against H2O2-Induced Injury in RAW264.7 Cells.
2020,
Pubmed
,
Echinobase
McNutt,
Antagonism of secreted PCSK9 increases low density lipoprotein receptor expression in HepG2 cells.
2009,
Pubmed
Melendez,
Hypercholesterolemia: The role of PCSK9.
2017,
Pubmed
Nelson,
Hyperlipidemia as a risk factor for cardiovascular disease.
2013,
Pubmed
Nozue,
Lipid Lowering Therapy and Circulating PCSK9 Concentration.
2017,
Pubmed
Pei,
Krüppel-like factor KLF9 regulates PPARγ transactivation at the middle stage of adipogenesis.
2011,
Pubmed
Roe,
The Relationship Between KLF5 and PPARα in the Heart: It's Complicated.
2016,
Pubmed
Seidah,
PCSK9: a key modulator of cardiovascular health.
2014,
Pubmed
Seidah,
The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): liver regeneration and neuronal differentiation.
2003,
Pubmed
Shalev,
The peroxisome proliferator-activated receptor alpha is a phosphoprotein: regulation by insulin.
1996,
Pubmed
Suzuki,
Vascular implications of the Krüppel-like family of transcription factors.
2005,
Pubmed
Tai,
Curcumin enhances cell-surface LDLR level and promotes LDL uptake through downregulation of PCSK9 gene expression in HepG2 cells.
2014,
Pubmed
Vieceli Dalla Sega,
Notch Signaling Regulates Immune Responses in Atherosclerosis.
2019,
Pubmed
Virani,
Heart Disease and Stroke Statistics-2020 Update: A Report From the American Heart Association.
2020,
Pubmed
Wang,
Statin-induced liver injury in an area endemic for hepatitis B virus infection: risk factors and outcome analysis.
2016,
Pubmed
Wang,
A small-molecule inhibitor of PCSK9 transcription ameliorates atherosclerosis through the modulation of FoxO1/3 and HNF1α.
2020,
Pubmed
Wang,
Biological Activities of Fucoidan and the Factors Mediating Its Therapeutic Effects: A Review of Recent Studies.
2019,
Pubmed
Wu,
Delineation of molecular pathways that regulate hepatic PCSK9 and LDL receptor expression during fasting in normolipidemic hamsters.
2012,
Pubmed
Xu,
Fucoidan from the sea cucumber Acaudina molpadioides exhibits anti-adipogenic activity by modulating the Wnt/β-catenin pathway and down-regulating the SREBP-1c expression.
2014,
Pubmed
,
Echinobase
Xu,
Isolation and anti-fatty liver activity of a novel cerebroside from the sea cucumber Acaudina molpadioides.
2011,
Pubmed
,
Echinobase
Xu,
Inhibitory effect of fucosylated chondroitin sulfate from the sea cucumber Acaudina molpadioides on adipogenesis is dependent on Wnt/β-catenin pathway.
2015,
Pubmed
,
Echinobase
Zhang,
Binding of proprotein convertase subtilisin/kexin type 9 to epidermal growth factor-like repeat A of low density lipoprotein receptor decreases receptor recycling and increases degradation.
2007,
Pubmed
Zhang,
The Protective Activities of Dietary Sea Cucumber Cerebrosides against Atherosclerosis through Regulating Inflammation and Cholesterol Metabolism in Male Mice.
2018,
Pubmed
,
Echinobase
Zhao,
A novel ACE inhibitory peptide isolated from Acaudina molpadioidea hydrolysate.
2009,
Pubmed
,
Echinobase
Zuo,
Dietary fucoidan of Acaudina molpadioides and its enzymatically degraded fragments could prevent intestinal mucositis induced by chemotherapy in mice.
2015,
Pubmed
,
Echinobase