Proprotein Convertase Subtilisin/kexin Type 9 Gene Expression Research Articles

Novel insights into the association between genetically proxied inhibition of proprotein convertase subtilisin/kexin type 9 and risk of sarcopenia.

The effects of lipid-lowering drugs [including statins, ezetimibe, and proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors] on hyperlipidaemia have been established. Some may have treatment effects beyond their reported properties, offering potential opportunities for drug repurposing. Epidemiological studies have reported conflicting findings on the relationship between lipid-lowering medication use and sarcopenia risk. We performed a two-sample Mendelian randomization (MR) study to investigate the causal association between the use of genetically proxied lipid-lowering drugs (including statins, ezetimibe, and PCSK9 inhibitors, which use low-density lipoprotein as a biomarker), and sarcopenia risk. The inverse-variance weighting method was used with pleiotropy-robust methods (MR-Egger regression and weighted median) and colocalization as sensitivity analyses. According to the positive control analysis, genetically proxied inhibition in lipid-lowering drug targets was associated with a lower risk of coronary heart disease [PCSK9 (OR, 0.67; 95% CI, 0.61 to 0.72; P=7.7E-21); 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR; OR, 0.68; 95% CI, 0.57 to 0.82; P=4.6E-05), and Niemann-Pick C1-like 1 (NPC1L1; OR, 0.53; 95% CI, 0.40 to 0.69; P=3.3E-06)], consistent with drug mechanistic actions and previous trial evidence. Genetically proxied inhibition of PCSK9 (beta, -0.040; 95% CI, -0.068 to -0.012; P=0.005) and circulating PCSK9 levels (beta, -0.019; 95% CI, -0.033 to -0.005; P=0.006) were associated with reduced appendicular lean mass (ALM) with concordant estimates in terms of direction and magnitude. Validation analyses using a second instrument for PCSK9 yielded consistent results in terms of direction and magnitude [(PCSK9 to ALM; beta, -0.052; 95% CI, -0.074 to -0.032; P=7.1E-7); (PCSK9 protein to ALM; beta, -0.060; 95% CI, -0.106 to -0.014; P=0.010)]. Genetically proxied inhibition of PCSK9 gene expression in the liver may be associated with reduced ALM (beta, -0.013; 95% CI, -0.035 to 0.009; P=0.25), consistent with the results of PCSK9 drug-target and PCSK9 protein MR analyses, but the magnitude was less precise. No robust association was found between HMGCR inhibition (beta, 0.048; 95% CI, -0.015 to 0.110; P=0.14) or NPC1L1 (beta, 0.035; 95% CI, -0.074 to 0.144; P=0.53) inhibition and ALM, and validation and sensitivity MR analyses showed consistent estimates. This MR study suggested that PCSK9 is involved in sarcopenia pathogenesis and that its inhibition is associated with reduced ALM. These findings potentially pave the way for future studies that may allow personalized selection of lipid-lowering drugs for those at risk of sarcopenia.

Cholesterol-Lowering Strategies for Cardiovascular Disease Prevention: The Importance of Intensive Treatment and the Simplification of Medical Therapy.

Cardiovascular diseases (CVDs) are a leading global cause of mortality and are primarily driven by atherosclerotic coronary artery disease. Their pathogenesis involves multi-factorial mechanisms, among which low-density lipoprotein (LDL) plays a causative role. Recent ESC/EAS guidelines advocate for a shift toward new risk estimation algorithms that better emphasize non-fatal cardiovascular events, lifetime risk prediction, and tailored pharmacological approaches, including statin + ezetimibe and triple therapy, in specific cases. Intensive lipid-lowering therapy has been shown to be pivotal, especially in post-acute coronary events. Intracoronary imaging has revealed insights into the composition of plaque and demonstrated the significant regression that can be achieved through the use of statins such as rosuvastatin and atorvastatin. The positive effects of Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) inhibitors, particularly alirocumab and evolocumab, on plaque regression, have been demonstrated. Inclisiran, which targets PCSK9 gene expression, significantly reduces LDL cholesterol. The associated challenges include hesitancy to prescribe intensive regimens and limited treatment adherence, highlighting the need for pharmacological combinations to improve therapeutic outcomes.

Statin-Induced Geranylgeranyl Pyrophosphate Depletion Promotes PCSK9–Dependent Adipose Insulin Resistance

Statin treatment is accepted to prevent adverse cardiovascular events. However, statin therapy has been reported to be dose-dependently associated with increased risk for new-onset type 2 diabetes mellitus (T2DM). Proprotein convertase subtilisin/kexin type 9 (PCSK9) is expressed in adipose tissue and is positively correlated with lipid metabolism. It is, however, unknown if PCSK9 participates in adipocyte insulin resistance occurring as a result of statin use. Our goal was to use an in vitro adipose tissue explant approach to support the hypothesis that PCSK9 regulates statin-induced new-onset T2DM. Studies were performed using Pcsk-/- and C57Bl/6J control mice. Pcsk9-/- and control mice were fed a high-fat diet to affect a state of chronically altered lipid metabolism and increased PCSK9. Epididymal fat was excised and incubated with atorvastatin (1 µmol/L) in the absence and presence of insulin or geranylgeranyl pyrophosphate (GGPP). PCSK9 mRNA was evaluated using quantitative rtPCR. We further examined the effects of atorvastatin on insulin-mediated AKT signaling in adipose tissue explants by immunoblotting. Atorvastatin was found to upregulate PCSK9 gene expression in adipose tissue. The metabolic intermediate GGPP is required to downregulate PCSK9 expression. PCSK9 deficiency protects against statin-induced impairments in insulin signaling. Moreover, supplementation with GGPP reversed atorvastatin-induced suppression of insulin signaling. Furthermore, the basal and atorvastatin-stimulated release of free fatty acids was observed in adipose tissue from wild-type mice but not PCSK9 deficient mice. Collectively, we describe a novel mechanism for PCSK9 expression in adipose tissue that could mediate statin-impaired adipose insulin resistance.

Pharmacology and Therapeutic Potential of Pcsk9 Inhibitors

Proprotein convertase subtilisin/Kexin type 9 (PCSK9) is a proteolytic enzyme that indirectly regulates serum LDL cholesterol by destroying LDL receptors. The main role of proprotein convertase subtilisin/Kexin type9 (PCSK9) inhibitor in cholesterol regulation was elucidated in clinical studies. It is produced in the liver but is also present in the kidney and intestine. It prevents HMGCo from synthesizing cholesterol. SREBP-2 is a reductase that is induced by statins. In a dose-dependent manner, increasing SREBP-2 levels enhanced LDL-R and PCSK9 gene expression. At the minimum, two procedures have been developed to overcome the plasma level of PCSK9 prohibit. This is the LDLR test, polyclonal antibodies, and sentience oligonucleotide. Lower dosage statin treatment with a proprotein convertase subtilisin/Kexin type9 inhibitor will be most efficient in lowering LDL and avoiding statin adverse effects. In multiple long-term trials, statins have been found to reduce cardiovascular mortality by 30% and stroke incidence by 20%. In this way, we conclude the role of PCSK9 in hypercholesterolemia.

PCSK9 A/G (rs505151) Gene Polymorphism and its Expression at the Molecular Level in Patients with Coronary Artery Disease

BACKGROUND: PCSK9 (Proprotein convertase subtilisin/kexin type 9) plays a key role in cholesterol homeostasis and Coronary artery disease (CAD). Many studies have extrapolated the association of PCSK9 gene with low density lipoprotein cholesterol (LDL-C) levels and CAD but with contradicting results. There is no such study available stating the intergenotypic variations in the levels of expression of PCSK9 and LDL-C and their correlations with CAD risk factors in patients with CAD. OBJECTIVE: We aim to explore the association of PCSK9 A/G (rs505151) polymorphism and its expression at mRNA and protein level in patients with CAD. Additionally, it is investigated how the levels of LDL-C, PCSK9, BMI, and systolic blood pressure (SBP) in patients with CAD and in healthy participants relate to the PCSK9 intergenotypic variation. METHODS: Angiographically confirmed CAD patients [n=250] and controls [n=250] were genotyped by PCR followed by RFLP techniques. Real time PCR and Western Blot methods were used to investigate PCSK9's differential expression. RESULTS: Odds ratio being the index of association revealed a statistically significant association of PCSK9 A/G (rs505151), A Vs G= 4.94 [1.37-7.79] polymorphism with CAD. In patients with the GG genotype, there is a correlation between higher PCSK9 gene expression and circulating LDL-C levels. CONCLUSION: Our study shows a significant association of PCSK9 gene polymorphism with CAD. We also observed an increased expression of PCSK9 gene in patients with G allele. In our study, PCSK9 A/G (rs505151) gene and LDL-C emerged as independent risk factors. To determine whether upregulated PCSK9 gene expression can act as a prognostic marker for CAD, more follow-up research is required.

Acanthaster planci Inhibits PCSK9 Gene Expression via Peroxisome Proliferator Response Element (PPRE) and Activation of MEK and PKC Signaling Pathways in Human Liver Cells.

A constantly elevated level of low-density lipoprotein cholesterol (LDL-C) is mainly associated with the development of atherosclerosis. The use of statins as a treatment for reducing plasma LDL-C levels has led, in some cases, to adverse side effects, including a decrease in hepatic LDL receptor (LDLR), the receptor responsible for the uptake of circulating LDL-C. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an enzyme responsible for directing the LDLR–LDL-C complex to lysosomal degradation upon transport into cells, preventing the recycling of LDLR to the cell surface. Therefore, PCSK9 may offer a new target for reducing the levels of plasma LDL-C. In this study, we investigated the mechanisms of action of a selected fraction of A. planci on PCSK9 gene expression, as well as the effect of the fraction on the level of LDLR protein and the uptake of LDL-C. Using real-time PCR, it was shown that the selected A. planci fraction reduced the gene expression of PCSK9 in human liver HepG2 cells. Immunocytochemistry analysis demonstrated that the selected A. planci fraction increased the LDLR protein level and LDL-C uptake in HepG2 cells. Promoter mutational and gene expression analyses revealed that PPRE, a binding site for peroxisome proliferator–activated receptor (PPAR), was responsible for mediating the inhibitory effect of the selected fraction on PCSK9 mRNA. In addition, MAP kinase and PKC components of the signal transduction pathway were activated, inducing the action of the selected A. planci fraction in decreasing PCSK9 gene expression. These findings suggest that the selected fraction shows good potential for reducing circulating LDL-C and, thus, may be a good therapeutic intervention to prevent the progression of atherosclerosis.

Impact of Soy β-Conglycinin Peptides on PCSK9 Protein Expression in HepG2 Cells.

Background: Dyslipidaemias, particularly elevated plasma low-density lipoprotein cholesterol (LDL-C) levels, are major risk factors for cardiovascular disease (CVD). Besides pharmacological approaches, a nutritional strategy for CVD prevention has gained increasing attention. Among functional foods, the hypocholesterolemic properties of soy are driven by a stimulation of LDL-receptor (LDL-R) activity. Aim: To characterize the effect of two soy peptides, namely, β-conglycinin-derived YVVNPDNDEN and YVVNPDNNEN on the expression of proprotein convertase subtilisin/kexin type 9 (PCSK9), one of the key-regulators of the LDL-R. Methods: PCSK9 promoter activity (luciferase assay), PCSK9 protein expression (WB) and secretion (ELISA), PCSK9 interaction with LDL-R (binding assay) and human HepG2 cells were the objects of this investigation. Results: Treatment with YVVNPDNNEN peptide has led to a rise in PCSK9 gene expression (90.8%) and transcriptional activity (86.4%), and to a decrement in PCSK9 intracellular and secreted protein (−42.9%) levels. YVVNPDNNEN peptide reduced the protein expression of transcriptional factor HNF1α. Most changes driven by YVVNPDNDEN peptide were not statistically significant. Neither peptide inhibited the PCSK9–LDLR interaction. Conclusions: Although sharing a common effect on LDL-R levels through the inhibition of 3-hydroxy-3-methylglutaryl CoA reductase activity, only the YVVNPDNNEN peptide has an additional mechanism via the downregulation of PCSK9 protein levels.

PCSK9 E670G (rs505151) Variant and Coronary Artery Disease Risk Among Diabetics.

Background: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an enzyme in the family of proprotein convertases implicated in lipid metabolism and is a significant genetic risk factor in cardiovascular diseases among various populations. Aim of the Study: This study explored the correlation between the alleles of the rs505151 (E670G) locus of the PCSK9 gene and its expression levels with coronary artery disease (CAD) risk in Egyptian patients with type 2 diabetes mellitus (T2DM). Subjects and Methods: A case-control study was performed on 112 lean subjects compared to 100 T2DM patients without CAD and 84 T2DM patients with CAD to investigate the relationships among PCSK9 expression levels, the E670G (rs505151) gene variant, lipid concentrations, and CAD risk in an Egyptian diabetic population. A restriction fragment length polymorphism-polymerase chain reaction (PCR) assay was used to assess the gene polymorphism, and PCSK9 mRNA expression was determined by quantitative real-time PCR. Results: The prevalence of the E670G (rs505151) AG genotype in diabetics with CAD was significantly greater than the other two groups. The PCSK9 gene expression levels in diabetics with CAD were significantly greater than the other two groups. G allele carriers (AG+GG) had a higher relative PCSK9 expression than A allele carriers. Conclusion: PCSK9 relative expression levels and the E670G (rs505151) AG genotype are CAD risk factors among Egyptian diabetics and are linked positively to the atherogenic index of plasma.

PCSK9 genetic (rs11591147) and epigenetic (DNA methylation) modifications associated with PCSK9 expression and serum proteins in CAD patients.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) genetic polymorphisms play a significant role in cholesterol homeostasis. Therefore, we aimed to investigate the association of PCSK9 genetic variations NM_174936.3:c.137G>T (R46L, rs11591147) and NM_174936.3:c.1120G>T (D374Y, rs137852912), as well as promoter DNA methylation status, with mRNA expression and circulating serum protein levels in coronary artery disease (CAD) patients. The present study includes 300 CAD cases and 300 controls from South India. Biochemical assays were performed using commercially available kits. PCSK9 rs11591147 and rs137852912 polymorphisms were analyzed by the polymerase chain reaction (PCR)-restriction fragment length polymorphism method, whereas promoter DNA methylation status and gene expression were determined using methylation specific PCR and quantitative PCR respectively. The genotypic distribution of PCSK9 rs11591147 revealed that individuals with the TT-genotype and T-allele have a reduced risk for CAD. Furthermore, patients with the PCSK9 rs11591147 TT genotype have a significantly lower total cholesterol and low-density lipoprotein-cholesterol levels and also higher high-density lipoprotein-cholesterol levels than individuals with the GG genotype. Logistic regression analysis has shown that the GG and GT (p =1.51 × 10-8 , p =1.47 × 10-9 ) genotypes predicted the risk for CAD with an odds ratio of 5.8 and 7.3 respectively. In addition, individuals with the TT genotype were hypermethylated at promoter DNA of PCSK9, resulting in lower mRNA expression and circulating serum proteins than in individuals with the GG genotype. In silico analyses revealed that rs11591147 T-allele has protein destabilizing capacity. In conclusion, the present study indicates that the PCSK9 gene expression and circulating serum protein levels are not only associated with rs11591147 genotype, but also with promoter DNA methylation. Furthermore, the findings with respect to both single nucleotide polymorphism and promoter DNA methylation may open avenues for novel treatment possibilities targeting PCSK9 for CAD management.

The Cholesterol-Lowering Effect of Capsella Bursa-Pastoris Is Mediated via SREBP2 and HNF-1α-Regulated PCSK9 Inhibition in Obese Mice and HepG2 Cells

The objective of the present study was to investigate the mechanism by which capsella bursa-pastoris ethanol extract (CBE), containing 17.5 milligrams of icaritin per kilogram of the extract, and icaritin, mediate hypocholesterolemic activity via the low-density lipoprotein receptor (LDLR) and pro-protein convertase subtilisin/kexin type 9 (PCSK9) in obese mice and HepG2 cells. CBE significantly attenuated serum total and LDL cholesterol levels in obese mice, which was associated with significantly decreased PCSK9 gene expression. HepG2 cells were cultured using delipidated serum (DLPS), and CBE significantly reduced PCSK9 and maintained the LDLR level. CBE co-treatment with rosuvastatin attenuated statin-mediated PCSK9 expression, and further increased LDLR. The icaritin contained in CBE decreased intracellular PCSK9 and LDLR levels by suppressing transcription factors SREBP2 and HNF-1α. Icaritin also significantly suppressed the extracellular PCSK9 level, which likely contributed to post-translational stabilization of LDLR in the HepG2 cells. PCSK9 inhibition by CBE is actively attributed to icaritin, and the use of CBE and icaritin could be an alternative therapeutic approach in the treatment of hypercholesterolemia.

Leptin, Resistin, and Proprotein Convertase Subtilisin/Kexin Type 9: The Role of STAT3

In a condition of dysfunctional visceral fat depots, as in the case of obesity, alterations in adipokine levels may be detrimental for the cardiovascular system. The proinflammatory leptin and resistin adipokines have been described as possible links between obesity and atherosclerosis. The present study was aimed at evaluating whether proprotein convertase subtilisin/kexin type 9 (PCSK9), a key regulator of low-density lipoprotein metabolism, is induced by leptin and resistin through the involvement of the inflammatory pathway of STAT3. In HepG2 cells, leptin and resistin up-regulated PCSK9 gene and protein expression, as well as the phosphorylation of STAT3. Upon STAT3 silencing, leptin and resistin lost their ability to activate PCSK9. The knockdown of STAT3 did not affect the expression of leptin and resistin receptors or that of PCSK9. The analysis of the human PCSK9 promoter region showed that the two adipokines raised PCSK9 promoter activity via the involvement of a sterol regulatory element motif. In healthy males, a positive association between circulating leptin and PCSK9 levels was found only when the body mass index was <25 kg/m2. In conclusion, this study identified STAT3 as one of the molecular regulators of leptin- and resistin-mediated transcriptional induction of PCSK9.

The Cholesterol-Modulating Effect of Methanol Extract of Pigeon Pea (Cajanus cajan (L.) Millsp.) Leaves on Regulating LDLR and PCSK9 Expression in HepG2 Cells.

Pigeon pea (Cajanus cajan (L.) Millsp.) is a legume crop consumed as an indigenous vegetable in the human diet and a traditional medicinal plant with therapeutic properties. The current study highlights the cholesterol-modulating effect and underlying mechanisms of the methanol extract of Cajanus cajan L. leaves (MECC) in HepG2 cells. We found that MECC increased the LDLR expression, the cell-surface LDLR levels and the LDL uptake activity in HepG2 cells. We further demonstrated that MECC suppressed the proprotein convertase subtilisin/kexin type 9 (PCSK9) mRNA and protein expression, but not affected the expression of other cholesterol or lipid metabolism-related genes including inducible degrader of LDLR (IDOL), HMG-CoA reductase (HMGCR), fatty acid synthase (FASN), acetyl-CoA carboxylase (ACC1), and liver X receptor-α (LXR-α) in HepG2 cells. Furthermore, we demonstrated that MECC down-regulated the PCSK9 gene expression through reducing the amount of nuclear hepatocyte nuclear factor-1α (HNF-1α), a major transcriptional regulator for activation of PCSK9 promoter, but not that of nuclear sterol-responsive element binding protein-2 (SREBP-2) in HepG2 cells. Finally, we identified the cajaninstilbene acid, a main bioactive stilbene component in MECC, which significantly modulated the LDLR and PCSK9 expression in HepG2 cells. Our current data suggest that the cajaninstilbene acid may contribute to the hypocholesterolemic activity of Cajanus cajan L. leaves. Our findings support that the extract of Cajanus cajan L. leaves may serve as a cholesterol-lowering agent.

Chitosan oligosaccharides enhance lipid droplets via down-regulation of PCSK9 gene expression in HepG2 cells

Chitosan oligosaccharides (COS), linear polymers of N-acetyl-D-glucosamine and deacetylated glucosamine, exhibit diverse pharmacological effects such as antimicrobial, antitumor, antioxidant and anti-inflammatory activities. Here, we explored their hypocholesterolemic effects in vivo and the molecular mechanisms of COS in hepatic cells. Our in vivo study of dyslipidemic ApoE-/- male mice showed that COS treatment of 500 mg kg−1 d−1 for 4 weeks clearly reduced the lipid deposits in the aorta and significantly decreased the hepatic proprotein convertase subtilisin/kexin type 9 (PCSK9) protein levels versus HFD groups (p < 0.05). To elucidate the mechanisms behind these effects, the HepG2 cell line was treated with COS. We found that COS (200 μg/ml) increased the amount of cell-surface low-density lipoprotein receptor (LDLR) and enhanced the lipid droplets in HepG2 cells (p < 0.05). The mRNA levels of LDLR and HMG-CoA protein levels were not altered, and the mRNA levels of PCSK9 were down-regulated by COS treatment for 24 h. We also observed that the expression levels of SREBP-2 (125 kD) and HNF-1α were increased in total cell lysates, but nuclear SREBP-2 (nSREBP-2, 68 kD, the active subunit of SREBP-2) levels were decreased and FOXO3a levels increased in nuclear lysates after COS treatment for 24 h. We demonstrated that one of the reasons for regulation of lipid transfer with COS is that FOXO3a levels are up-regulated by COS, leading to a reduction in the PCSK9 promoter binding capacity of HNF-1α and thus suppressing PCSK9 gene expression, up-regulating LDLR levels, and enhancing the lipid droplets in HepG2 cells. In addition, decreased expression of the PCSK9 gene was also contributed to by down-regulation of SREBP-2 by COS. We further confirmed the effect of suppression of PCSK9 expression by COS by utilizing RNA interference to silence HNF-1α and SREBP-2. Finally, to the best of our knowledge, we are the first to demonstrate that PCSK9 expression and LDLR activity are synergistically changed by a combination of HNF-1α and SREBP-2 after COS treatment. Our findings indicate that COS may regulate PCSK9 to modulate hepatic LDLR abundance and activity.

Potential role of lycopene in targeting proprotein convertase subtilisin/kexin type-9 to combat hypercholesterolemia

Proprotein convertase subtilisin/kexin type 9 (PCSK-9) is a serine protease of the proprotien convertase (PC) family that has profound effects on plasma low density lipoprotein cholesterol (LDL-C) levels, the major risk factor for coronary heart disease (CHD), through its ability to mediate LDL receptor (LDL-R) protein degradation and reduced recycling to the surface of hepatocytes. Thus, the current study was premeditated not only to evaluate the role of lycopene in targeting the inhibition of PCSK-9 via modulation of genes involved in cholesterol homeostasis in HFD rats but also to examine a correlation between HFD induced inflammatory cascades and subsequent regulation of PCSK-9 expression. Besides the effect of lycopene on hepatic PCSK-9 gene expression, PPI studies for PCSK-9-Lycopene complex and EGF-A of LDL-R were also performed via molecular informatics approach to assess the dual mode of action of lycopene in LDL-R recycling and increased removal of circulatory LDL-C. We for the first time deciphered that lycopene treatment significantly down-regulates the expression of hepatic PCSK-9 and HMGR, whereas, hepatic LDL-R expression was significantly up-regulated. Furthermore, lycopene ameliorated inflammation stimulated expression of PCSK-9 via suppressing the expression of inflammatory markers. The results from our molecular informatics studies confirmed that lycopene, while occupying the active site of PCSK-9 crystal structure, reduces the affinity of PCSK-9 to complex with EGF-A of LDL-R, whereas, atorvastatin makes PCSK-9-EGF-A complex formation more feasible than both of PCSK-9-EGF-A alone and Lycopene-PCSK-9-EGF-A complex. Based on above results, it can be concluded that lycopene exhibits potent hypolipidemic activities via molecular mechanisms that are either identical (HMGR inhibition) or distinct from that of statins (down-regulation of PCSK-9 mRNA synthesis). To the best of our knowledge, this is the first report that lycopene has this specific biological property. Being a natural, safer and alternative therapeutic agent, lycopene could be used as a complete regulator of cholesterol homeostasis and ASCVD.

Tanshinone IIA Modulates Low Density Lipoprotein Uptake via Down-Regulation of PCSK9 Gene Expression in HepG2 Cells.

Tanshinone IIA, one of the most pharmacologically bioactive phytochemicals isolated from Salvia miltiorrhiza Bunge, possesses several biological activities such as anti-inflammation, anti-cancer, neuroprotection and hypolipidemic activities. In this study, we aim to investigate the hypocholesterolemic effect of tanshinone IIA in hepatic cells. We demonstrated that tanshinone IIA significantly increased the amount of low-density lipoprotein receptor (LDLR) and LDL uptake activity in HepG2 cells at the post-transcriptional regulation. We further demonstrated that tanshinone IIA inhibited the expression of proprotein convertase subtilisin/kexin type 9 (PCSK9) mRNA and mature protein, which may lead to an increase the cell-surface LDLR in hepatic cells. We further identified a regulatory DNA element involved in the tanshinone IIA-mediated PCSK9 down-regulation, which is located between the -411 and -336 positions of the PCSK9 promoter. Moreover, we found that tanshinone IIA markedly increased the nuclear forkhead box O3a (FoxO3a) level, enhanced FoxO3a/PCSK9 promoter complexes formation and decreased the PCSK9 promoter binding capacity of hepatocyte nuclear factor 1α (HNF-1α), resulting in suppression of PCSK9 gene expression. Finally, we found that the statin-induced PCSK9 overexpression was attenuated and the LDLR activity was elevated in a synergic manner by combination of tanshinone IIA treatment in HepG2 cells. Overall, our results reveal that the tanshinone IIA modulates LDLR level and activity via down-regulation of PCSK9 expression in hepatic cells. Our current findings provide a molecular basis of tanshinone IIA to develop PCSK9 inhibitors for cholesterol management.

Reduction of circulating PCSK9 and LDL-C levels by liver-specific knockdown of HNF1α in normolipidemic mice

The transcription factors hepatic nuclear factor (HNF)1α and HNF1β can bind to the HNF1 site on the proprotein convertase subtilisin/kexin type 9 (PCSK9) promoter to activate transcription in HepG2 cells. However, it is unknown whether one or both HNF1 factors are obligatory for transactivating hepatic PCSK9 gene expression in vivo. We developed shRNA adenoviral constructs (Ad-shHNF1α and Ad-shHNF1β) to examine the effects of knockdown of HNF1α or HNF1β on PCSK9 expression and its consequent impact on LDL receptor (LDLR) protein levels in cultured hepatic cells and liver tissue. We demonstrated that infection with Ad-shHNF1α, but not Ad-shHNF1β, markedly reduced PCSK9 mRNA expression in HepG2 cells with a concomitant increase in LDLR protein abundance. Injecting Ad-shHNF1α in mice fed a normal diet significantly (∼ 50%) reduced liver mRNA expression and serum concentration of PCSK9 with a concomitant increase (∼ 1.9-fold) in hepatic LDLR protein abundance. Furthermore, we observed a modest but significant reduction in circulating LDL cholesterol after knockdown of HNF1α in these normolipidemic mice. Consistent with the observation that knockdown of HNF1β did not affect PCSK9 mRNA or protein expression in cultured hepatic cells, Ad-shHNF1β infection in mice resulted in no change in the hepatic mRNA expression or serum content of PCSK9. Altogether, our study demonstrates that HNF1α, but not HNF1β, is the primary positive regulator of PCSK9 transcription in mouse liver.

Curcumin enhances cell-surface LDLR level and promotes LDL uptake through downregulation of PCSK9 gene expression in HepG2 cells.

Curcumin has been demonstrated as having numerous desirable characteristics, such as antioxidant, anti-inflammatory, and antiatherogenic activities. We report the hypocholesterolemic effect and molecular mechanism of curcumin. We found that curcumin enhanced LDL receptor (LDLR) level on the cell surface, as well as LDLR activity; however, LDLR transcription and mRNA stability were not affected. Furthermore, we found that proprotein convertase subtilisin/kexin type 9 (PCSK9) gene was downregulated at the transcriptional level by curcumin, leading to an increase in LDL uptake in HepG2 cells. The curcumin-responsive element of the PCSK9 promoter, a binding site for hepatocyte nuclear factor 1α (HNF-1α), was also identified. We demonstrated that curcumin reduced the nuclear abundance of hepatocyte nuclear factor 1α, resulting in its attenuated interaction with the PCSK9 promoter and leading to a downregulation of PCSK9 expression. Finally, we showed that curcumin decreased the statin-induced PCSK9 expression and potentially synergized with statin administration. Current results indicate that curcumin suppression of PCSK9 expression is associated with increases in cell-surface LDLR and LDLR activity in hepatic cells and it acts in a molecular mechanism that is distinct from the statins. Curcumin exhibits hypolipidemic activity and may serve as a useful supplement to statin treatment for hypercholesterolemia.

Effect of Porphyromonas gingivalis infection on post-transcriptional regulation of the low-density lipoprotein receptor in mice

BackgroundPeriodontal disease is suggested to increase the risk of atherothrombotic disease by inducing dyslipidemia. Recently, we demonstrated that proprotein convertase subtilisin/kexin type 9 (PCSK9), which is known to play a critical role in the regulation of circulating low-density lipoprotein (LDL) cholesterol levels, is elevated in periodontitis patients. However, the underlying mechanisms of elevation of PCSK9 in periodontitis patients are largely unknown. Here, we explored whether Porphyromonas gingivalis, a representative periodontopathic bacterium, -induced inflammatory response regulates serum PCSK9 and cholesterol levels using animal models.MethodsWe infected C57BL/6 mice intraperitoneally with Porphyromonas gingivalis, a representative strain of periodontopathic bacteria, and evaluated serum PCSK9 levels and the serum lipid profile. PCSK9 and LDL receptor (LDLR) gene and protein expression, as well as liver X receptors (Lxrs), inducible degrader of the LDLR (Idol), and sterol regulatory element binding transcription factor (Srebf)2 gene expression, were examined in the liver.ResultsP. gingivalis infection induced a significant elevation of serum PCSK9 levels and a concomitant elevation of total and LDL cholesterol compared with sham-infected mice. The LDL cholesterol levels were significantly correlated with PCSK9 levels. Expression of the Pcsk9, Ldlr, and Srebf2 genes was upregulated in the livers of the P. gingivalis-infected mice compared with the sham-infected mice. Although Pcsk9 gene expression is known to be positively regulated by sterol regulatory element binding protein (SREBP)2 (human homologue of Srebf2), whereas Srebf2 is negatively regulated by cholesterol, the elevated expression of Srebf2 found in the infected mice is thought to be mediated by P. gingivalis infection.ConclusionsP. gingivalis infection upregulates PCSK9 production via upregulation of Srebf2, independent of cholesterol levels. Further studies are required to elucidate how infection regulates Srebf2 expression and subsequently influences lipid metabolism.

The novel function of HINFP as a co-activator in sterol-regulated transcription of PCSK9 in HepG2 cells

PCSK9 (proprotein convertase subtilisin/kexin type9) plays an important role in control of plasma LDL (low-density lipoprotein) cholesterol metabolism by modulating the degradation of hepatic LDL receptor. Previous studies demonstrated that PCSK9 is a target gene of the SREBP2 [SRE (sterol-regulatory element)-binding protein 2] that activates PCSK9 gene transcription through an SRE motif of the promoter. In addition to SREBP2, HNF1α (hepatic nuclear factor 1α) positively regulates PCSK9 gene transcription in hepatic cells through a binding site located 28bp upstream from SRE. In the present study, we have identified a novel HINFP (histone nuclear factor P) recognition motif residing between the HNF1 motif and SRE that is essential for basal and sterol-regulated transcriptions of the PCSK9 promoter. Mutation of this motif lowers the basal promoter activity and abolishes the sterol-mediated repression as well as the SREBP2-induced activation of the PCSK9 promoter. We show further that the activity of SREBP2 in stimulating PCSK9 promoter activity is greatly enhanced by HINFP. Additional experiments suggest that HINFP and its cofactor NPAT (nuclear protein of the ataxia telangectasia mutated locus) form a functional complex, and NPAT may subsequently recruit the HAT (histone acetyltransferase) cofactor TRRAP (transformation/transactivation domain-associated protein) to facilitate the histone H4 acetylation of the PCSK9 promoter. Knockdown of HINFP, NPAT or TRRAP each markedly reduces the amount of acetylated histone H4 on the PCSK9 promoter region and lowers PCSK9 protein levels. Importantly, by utilizing co-immunoprecipitation assays, we have demonstrated a direct interaction between SREBP2 and HINFP and its cofactors NPAT/TRRAP. Taken together, these new findings identify HINFP as a co-activator in SREBP-mediated transactivation of PCSK9 gene expression.

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

Recent studies have identified proprotein convertase subtilisin/kexin type 9 (PCSK9) and Idol as negative regulators of low density lipoprotein receptor (LDLR) protein stability. While the induction of PCSK9 transcription has been recognized as a limitation to the statin cholesterol-lowering efficacy at higher doses, it is unknown whether Idol is involved in the statin-mediated up-regulation of the hepatic LDLR. Here we report that statins exert opposite effects on PCSK9 and Idol gene expression in human hepatoma-derived cell lines and primary hepatocytes isolated from hamsters and rats. While PCSK9 expression was induced, the level of Idol mRNA rapidly declined in statin-treated cells in a dose-dependent manner. This differs from the effect of the liver X receptor ligand, GW3965, which increased the expression of both PCSK9 and Idol. We further show that cellular depletion of Idol by siRNA transfection did not change PCSK9 expression levels in control and statin-treated cells; however, the basal level of LDLR protein increased by 60% in Idol siRNA transfected HepG2 cells. More importantly, the increase in LDLR protein abundance by rosuvastatin and atorvastatin treatment was compromised by Idol siRNA transfection. Collectively, our present findings suggest that the suppression of Idol gene expression in liver cells is an additional mechanism underlying the statin-induced up-regulation of hepatic LDLR expression. This may contribute to the hypocholesterolemic effects of statins observed in clinical settings.