Enhanced pro‐protein convertase subtilisin/kexin type 9 expression by C‐reactive protein through p38MAPK‐HNF1α pathway in HepG2 cells

Plasma C‐reactive protein (CRP) concentration is associated positively with cardiovascular risk, including dyslipidemia. We suggested a regulating role of CRP on pro‐protein convertase subtilisin/kexin type 9 (PCSK9), a key regulator of low‐density lipoprotein (LDL) metabolism, and demonstrated the PCSK9 as a pathway linking CRP and LDL regulation. Firstly, experiments were carried out in the presence of human CRP on the protein and mRNA expression of PCSK9 and LDL receptor (LDLR) in human hepatoma cell line HepG2 cells. Treatment with CRP (10 μg/ml) enhanced significantly the mRNA and protein expression of PCSK9 and suppressed the expression of LDLR. Of note, a late return of LDLR mRNA levels occurred at 12 hrs, while the LDLR protein continued to decrease at 24 hrs, suggesting that the late decrease in LDLR protein levels was unlikely to be accounted for the decrease in LDL mRNA. Secondly, the role of PCSK9 in CRP‐induced LDLR decrease and the underlying pathways were investigated. As a result, the inhibition of PCSK9 expression by small interfering RNA (siRNA) returned partly the level of LDLR protein and LDL uptake during CRP treatment; CRP‐induced PCSK9 increase was inhibited by the p38MAPK inhibitor, SB203580, resulting in a significant rescue of LDLR protein expression and LDL uptake; the pathway was involved in hepatocyte nuclear factor 1α (HNF1α) but not sterol responsive element‐binding proteins (SREBPs) preceded by the phosphorylation of p38MAPK. These findings indicated that CRP increased PCSK9 expression by activating p38MAPK‐HNF1α pathway, with a certain downstream impairment in LDL metabolism in HepG2 cells.     

PCSK9: an enigmatic protease

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a critical role in cholesterol metabolism by controlling the levels of low density lipoprotein (LDL) particles that circulate in the bloodstream. Several gain-of-function and loss-of-function mutations in the PCSK9 gene, that occur naturally, have been identified and linked to hypercholesterolemia and hypocholesterolemia, respectively. PCSK9 expression has been shown to be regulated by sterol regulatory element binding proteins (SREBPs) and statins similar to other genes involved in cholesterol homeostasis. The most critical finding concerning PCSK9 is that this protease is able to influence the number of LDL receptor molecules expressed on the cell surface. Studies have demonstrated that PCSK9 acts mainly by enhancing degradation of LDL receptor protein in the liver. Inactivation of PCSK9 in mice reduces plasma cholesterol levels primarily by increasing hepatic expression of LDL receptor protein and thereby accelerating clearance of circulating LDL cholesterol. The objective of this review is to summarize the current information related to the regulation and function of PCSK9 and to identify gaps in our present knowledge.     

Annexin A2 Reduces PCSK9 Protein Levels via a Translational Mechanism and Interacts with the M1 and M2 Domains of PCSK9

Annexin A2 (AnxA2) was reported to be an extracellular endogenous inhibitor of proprotein convertase subtilisin kexin type 9 (PCSK9) activity on cell-surface LDL receptor degradation. In this study, we investigated the effect of silencing the expression of AnxA2 and PCSK9 in HepG2 and Huh7 cells to better define the role of AnxA2 in PCSK9 regulation. AnxA2 knockdown in Huh7 cells significantly increased PCSK9 protein levels as opposed to AnxA2 knockdown in HepG2 cells. However, HepG2 cells overexpressing AnxA2 had lower levels of PCSK9 protein. Overall, our data revealed a plausible new role of AnxA2 in the reduction of PCSK9 protein levels via a translational mechanism. Moreover, the C-terminal Cys/His-rich domain of PCSK9 is crucial in the regulation of PCSK9 activity, and we demonstrated by far-Western blot assay that the M1 and M2 domains are necessary for the specific interaction of PCSK9''s C-terminal Cys/His-rich domain and AnxA2. Finally, we produced and purified recombinant PCSK9 from humans and mice, which was characterized and used to perform 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate LDL cell-based assays on the stable knockdown HepG2 and Huh7 cells. We also demonstrated for the first time the equipotency of human and mouse PCSK9 R218S on human cells.     

Gypenoside LVI improves hepatic LDL uptake by decreasing PCSK9 and upregulating LDLR expression

BackgroundsAtherosclerotic Cardiovascular Disease (ASCVD) is defined as ischemic or endothelial dysfunction-various inflammatory diseases, which is mainly caused by excessive low-density lipoprotein cholesterol (LDL-C) in circulating blood. Gynostemma pentaphyllum is a traditional Chinese medicine, and total Gypenosides are used for the treatment of hyperlipidemia and to reduce circulating proprotein convertase subtilisin/kexin type 9 (PCSK9) level. However, which gypenoside involved in the modulation of PCSK9 expression is still unknown.PurposeThis study aimed to discover effective PCSK9 inhibitors from Gypenosides in accordance with the 2019 ESC/EAS guidelines.MethodsHPLC was employed to determine major six components of Gypenosides. The inhibitory activity on secreted PCSK9 in HepG2 of six major compounds from Gypenosides were screened by ELISA. The level of low-density lipoprotein (LDL) receptor (LDLR) was determined by Flow cytometry and Immunofluorescence. The expression levels of PCSK9, LDLR and Sterol-regulatory element binding proteins-2 (SREBP-2) were analyzed by qPCR and Western blot. DiI-LDL was added to evaluated LDL uptake into HepG2.ResultsThe results suggested that the mRNA and protein levels of PCSK9 were down-regulated by Gypenoside LVI and the LDLR degradation in lysosomes system was inhibited, thereby leading to an increasing in LDL uptake into HepG2 cells. Furthermore, Gypenoside LVI decreased PCSK9 expression induced by stains. Altogether, Gypenoside LVI enhances LDL uptake into HepG2 cells by increased LDLR level on cell-surface and suppressed PCSK9 expression.ConclusionThis indicates that Gypenoside LVI can be used as a useful supplement for statins in the treatment of hypercholesterolemia. This is firstly reported that monomeric compound of G. pentaphyllum planted in Hunan province has the effect of increasing LDL-C uptake in hepatocytes via inhibiting PCSK9 expression.     

The M2 Module of the Cys-His-rich Domain (CHRD) of PCSK9 Protein Is Needed for the Extracellular Low-density Lipoprotein Receptor (LDLR) Degradation Pathway

PCSK9 enhances the cellular degradation of the LDL receptor (LDLR), leading to increased plasma LDL cholesterol. This multidomain protein contains a prosegment, a catalytic domain, a hinge region, and a cysteine-histidine rich domain (CHRD) composed of three tightly packed modules named M1, M2, and M3. The CHRD is required for the activity of PCSK9, but the mechanism behind this remains obscure. To define the contribution of each module to the function of PCSK9, we dissected the CHRD structure. Six PCSK9 deletants were generated by mutagenesis, corresponding to the deletion of only one (ΔM1, ΔM2, ΔM3) or two (ΔM12, ΔM13, ΔM23) modules. Transfection of HEK293 cells showed that all deletants were well processed and expressed compared with the parent PCSK9 but that only those lacking the M2 module were secreted. HepG2 cells lacking endogenous PCSK9 (HepG2/shPCSK9) were used for the functional analysis of the extracellular or intracellular activity of PCSK9 and its deletants. To analyze the ability of the deletants to enhance the LDLR degradation by the intracellular pathway, cellular expressions revealed that only the ΔM2 deletant retains a comparable total LDLR-degrading activity to full-length PCSK9. To probe the extracellular pathway, HepG2/shPCSK9 cells were incubated with conditioned media from transfected HEK293 or HepG2/shPCSK9 cells, and cell surface LDLR levels were analyzed by FACS. The results showed no activity of any secreted deletant compared with PCSK9. Thus, although M2 is dispensable for secretion, its presence is required for the extracellular activity of PCSK9 on cell surface LDLR.     

Arabidopsis ROOT HAIR DEFECTIVE3 is involved in nitrogen starvation-induced anthocyanin accumulation

Anthocyanin accumulation is a common phenom-enon seen in plants under environmental stress. In this study, we identified a new allele of ROOT HAIR DEFECTIVE3 (RHD3) showing an anthocyanin overaccumulat...     

Suppressor of Cytokine Signaling-3 (SOCS-3) Induces Proprotein Convertase Subtilisin Kexin Type 9 (PCSK9) Expression in Hepatic HepG2 Cell Line

The suppressor of cytokine signaling (SOCS) proteins are negative regulators of the JAK/STAT pathway activated by proinflammatory cytokines, including the tumor necrosis factor-α (TNF-α). SOCS3 is also implicated in hypertriglyceridemia associated to insulin resistance. Proprotein convertase subtilisin kexin type 9 (PCSK9) levels are frequently found to be positively correlated to insulin resistance and plasma very low density lipoprotein (VLDL) triglycerides concentrations. The present study aimed to investigate the possible role of TNF-α and JAK/STAT pathway on de novo lipogenesis and PCSK9 expression in HepG2 cells. TNF-α induced both SOCS3 and PCSK9 in a concentration-dependent manner. This effect was inhibited by transfection with siRNA anti-STAT3, suggesting the involvement of the JAK/STAT pathway. Retroviral overexpression of SOCS3 in HepG2 cells (HepG2^SOCS3) strongly inhibited STAT3 phosphorylation and induced PCSK9 mRNA and protein, with no effect on its promoter activity and mRNA stability. Consistently, siRNA anti-SOCS3 reduced PCSK9 mRNA levels, whereas an opposite effect was observed with siRNA anti-STAT3. In addition, HepG2^SOCS3 express higher mRNA levels of key enzymes involved in the de novo lipogenesis, such as fattyacid synthase, stearoyl-CoA desaturase (SCD)-1, and apoB. These responses were associated with a significant increase of SCD-1 protein, activation of sterol regulatory element-binding protein-1c (SREBP-1), accumulation of cellular triglycerides, and secretion of apoB. HepG2^SOCS3 show lower phosphorylation levels of insulin receptor substrate 1 (IRS-1) Tyr⁸⁹⁶ and Akt Ser⁴⁷³ in response to insulin. Finally, insulin stimulation produced an additive effect with SOCS3 overexpression, further inducing PCSK9, SREBP-1, fatty acid synthase, and apoB mRNA. In conclusion, our data candidate PCSK9 as a gene involved in lipid metabolism regulated by proinflammatory cytokine TNF-α in a SOCS3-dependent manner.     

Secreted PCSK9 downregulates low density lipoprotein receptor through receptor-mediated endocytosis

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protease that regulates low density lipoprotein receptor (LDLR) protein levels. The mechanisms of this action, however, remain to be defined. We show here that recombinant human PCSK9 expressed in HEK293 cells was readily secreted into the medium, with the prosegment associated with the C-terminal domain. Secreted PCSK9 mediated cell surface LDLR degradation in a concentration- and time-dependent manner when added to HEK293 cells. Accordingly, cellular LDL uptake was significantly reduced as well. When infused directly into C57B6 mice, purified human PCSK9 substantially reduced hepatic LDLR protein levels and resulted in increased plasma LDL cholesterol. When added to culture medium, fluorescently labeled PCSK9 was endocytosed and displayed endosomal-lysosomal intracellular localization in HepG2 cells, as was demonstrated by colocalization with DiI-LDL. PCSK9 endocytosis was mediated by LDLR as LDLR deficiency (hepatocytes from LDLR null mice), or RNA interference-mediated knockdown of LDLR markedly reduced PCSK9 endocytosis. In addition, RNA interference knockdown of the autosomal recessive hypercholesterolemia (ARH) gene product also significantly reduced PCSK9 endocytosis. Biochemical analysis revealed that the LDLR extracellular domain interacted directly with secreted PCSK9; thus, overexpression of the LDLR extracellular domain was able to attenuate the reduction of cell surface LDLR levels by secreted PCSK9. Together, these results reveal that secreted PCSK9 retains biological activity, is able to bind directly to the LDLR extracellular domain, and undergoes LDLR-ARH-mediated endocytosis, leading to accelerated intracellular degradation of the LDLR.     

Peroxisome Proliferator-activated receptor γ activation by ligands and dephosphorylation induces proprotein convertase subtilisin kexin type 9 and low density lipoprotein receptor expression

Proprotein convertase subtilisin kexin type 9 (PCSK9) plays an important role in cholesterol homeostasis by enhancing the degradation of LDL receptor (LDLR) protein. Peroxisome proliferator-activated receptor γ (PPARγ) has been shown to be atheroprotective. PPARγ can be activated by ligands and/or dephosphorylation with ERK1/2 inhibitors. The effect of PPARγ on PCSK9 and LDLR expression remains unknown. In this study, we investigated the effects of PPARγ on PCSK9 and LDLR expression. At the cellular levels, PPARγ ligands induced PCSK9 mRNA and protein expression in HepG2 cells. PCSK9 expression was induced by inhibition of ERK1/2 activity but inhibited by ERK1/2 activation. The mutagenic study and promoter activity assay suggested that the induction of PCSK9 expression by ERK1/2 inhibitors was tightly linked to PPARγ dephosphorylation. However, PPARγ activation by ligands or ERK1/2 inhibitors induced hepatic LDLR expression. The promoter assay indicated that the induction of LDLR expression by PPARγ was sterol regulatory element-dependent because PPARγ enhanced sterol regulatory element-binding protein 2 (SREBP2) processing. In vivo, administration of pioglitazone or U0126 alone increased PCSK9 expression in mouse liver but had little effect on PCSK9 secretion. However, the co-treatment of pioglitazone and U0126 enhanced both PCSK9 expression and secretion. Similar to in vitro, the increased PCSK9 expression by pioglitazone and/or U0126 did not result in decreased LDLR expression and function. In contrast, pioglitazone and/or U0126 increased LDLR protein expression and membrane translocation, SREBP2 processing, and CYP7A1 expression in the liver, which led to decreased total and LDL cholesterol levels in serum. Our results indicate that although PPARγ activation increased PCSK9 expression, PPARγ activation induced LDLR and CYP7A1 expression that enhanced LDL cholesterol metabolism.     

Plasma Membrane Tetraspanin CD81 Complexes with Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) and Low Density Lipoprotein Receptor (LDLR), and Its Levels Are Reduced by PCSK9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an important factor in plasma cholesterol regulation through modulation of low density lipoprotein receptor (LDLR) levels. Naturally occurring mutations can lead to hyper- or hypocholesterolemia in human. Recently, we reported that PCSK9 was also able to modulate CD81 in Huh7 cells. In the present study, several gain-of-function and loss-of-function mutants as well as engineered mutants of PCSK9 were compared for their ability to modulate the cell surface expression of LDLR and CD81. Although PCSK9 gain-of-function D374Y enhanced the degradation both receptors, D374H and D129N seemed to only reduce LDLR levels. In contrast, mutations in the C-terminal hinge-cysteine-histidine-rich domain segment primarily affected the PCSK9-induced CD81 degradation. Furthermore, when C-terminally fused to an ACE2 transmembrane anchor, the secretory N-terminal catalytic or hinge-cysteine-histidine-rich domain domains of PCSK9 were able to reduce CD81 and LDLR levels. These data confirm that PCSK9 reduces CD81 levels via an intracellular pathway as reported for LDLR. Using immunocytochemistry, a proximity ligation assay, and co-immunoprecipitation, we found that the cell surface level of PCSK9 was enhanced upon overexpression of CD81 and that both PCSK9 and LDLR interact with this tetraspanin protein. Interestingly, using CHO-A7 cells lacking LDLR expression, we revealed that LDLR was not required for the degradation of CD81 by PCSK9, but its presence strengthened the PCSK9 effect.     

Function and distribution of circulating human PCSK9 expressed extrahepatically in transgenic mice

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is predominantly expressed in liver and regulates cholesterol metabolism by down regulating liver LDL receptor (LDLR) proteins. Here we report transgenic overexpression of human PCSK9 in kidney increased plasma levels of PCSK9 and subsequently led to a dramatic reduction in liver LDLR proteins. The regulation of LDLR by PCSK9 displayed tissue specificity, with liver being the most responsive tissue. Even though the PCSK9 transgene was highly expressed in kidney, LDLR proteins were suppressed to a lower extent in this tissue than in liver. Adrenal LDLR proteins were not regulated by elevated plasma PCSK9. hPCSK9 transgene expression and subsequent reduction of liver LDLR led to increases in plasma total cholesterol, LDL cholesterol, and ApoB, which were further increased by a high-fat, high-cholesterol diet. We also observed that the size distribution of hPCSK9 in transgenic mouse plasma was heterogeneous. In chow-fed mice, the majority of PCSK9 proteins were in free forms; however, feeding a high-fat, high-cholesterol diet resulted in a shift of hPCSK9 distribution toward larger complexes. PCSK9 distribution in human plasma also exhibited heterogeneity and individual variability in the percentage of PCSK9 in free form and in large complexes. We provide strong evidence to support that human PCSK9 proteins secreted from extrahepatic tissue are able to promote LDLR degradation in liver and increase plasma LDL. Our data also suggest that LDLR protein regulation by PCSK9 has tissue specificity, with liver being the most responsive tissue.     

Differential Genomic Effects of Six Different TiO2 Nanomaterials on Human Liver HepG2 Cells

Human HepG2 cells were exposed to six TiO₂ nanomaterials (with dry primary particle sizes ranging from 22 to 214 nm, either 0.3, 3, or 30 μg/mL) for 3 days. Some of these canonical pathways changed by nano‐TiO₂ in vitro treatments have been already reported in the literature, such as NRF2‐mediated stress response, fatty acid metabolism, cell cycle and apoptosis, immune response, cholesterol biosynthesis, and glycolysis. But this genomic study also revealed some novel effects such as protein synthesis, protein ubiquitination, hepatic fibrosis, and cancer‐related signaling pathways. More importantly, this genomic analysis of nano‐TiO₂ treated HepG2 cells linked some of the in vitro canonical pathways to in vivo adverse outcomes: NRF2‐mediated response pathways to oxidative stress, acute phase response to inflammation, cholesterol biosynthesis to steroid hormones alteration, fatty acid metabolism changes to lipid homeostasis alteration, G2/M cell checkpoint regulation to apoptosis, and hepatic fibrosis/stellate cell activation to liver fibrosis.     

Isolation and characterization of the circulating truncated form of PCSK9

Proprotein convertase subtilisin-kexin type 9 (PCSK9) is a secreted protein which regulates serum LDL cholesterol. It circulates in human and rodent serum in an intact form and a major truncated form. Previous in vitro studies involving the expression of human PCSK9 genetic variants and in vivo studies of furin knockout mice suggest that the truncated form is a furin cleavage product. However, the circulating truncated form of PCSK9 has not been isolated and characterized. Utilizing antibodies which bind to either the catalytic domain or the C-terminal domain of PCSK9, the truncated PCSK9 was isolated from serum. MS was used to determine that this form of PCSK9 is a product of in vivo cleavage at Arg218 resulting in pyroglutamic acid formation of the nascent N terminus corresponding to Gln219 of intact PCSK9. We also determined that the truncated PCSK9 in serum lacked the N-terminal segment which contains amino acids critical for LDL receptor binding. A truncated PCSK9, expressed and purified from HEK293 cells with identical composition as the circulating truncated protein, was not active in inhibition of LDL uptake by HepG2 cells. These studies provide a definitive characterization of the composition and activity of the truncated form of PCSK9 found in human serum.