PCSK9-D374Y Suppresses Hepatocyte Migration through Downregulating Free Cholesterol Efflux Rate and Activity of Extracellular Signal-Regulated Kinase

Proprotein convertase subtilisin/kexin type 9 can mediate the intracellular lysosomal degradation of the low-density lipoprotein receptor protein in hepatocytes and decrease the liver's ability to scavenge low-density lipoprotein cholesterol from circulation, resulting in high levels of cholesterol in the circulatory system. Current studies have primarily focused on the relationship between PCSK9 and blood lipid metabolism; however, the biological function of PCSK9 in hepatocytes is rarely addressed. In this study, we evaluate its effects in the human hepatoma carcinoma cell line HepG2, including proliferation, migration, and free cholesterol transport. PCSK9-D374Y is a gain-of-function mutation that does not affect proliferation but significantly suppresses the migration and cholesterol efflux capacity of these cells. The suppression of the transmembrane outflow of intracellular-free cholesterol regulates small G proteins and the suppression of extracellular signal-regulated kinase. In summary, PCSK9-D374Y affects hepatocyte features, including their migration and free cholesterol transport capabilities.


Introduction
The proprotein convertase subtilisin-like/kexin type 9 (PCSK9) gene is one of the major genes associated with familial hypercholesterolemia [1,2]. It regulates plasma LDL-C (low-density lipoprotein cholesterol) levels by posttranslational regulation of the LDLR (low-density lipoprotein receptor), according to the following: secreted protein binds LDLR on the hepatocyte membrane, mediates its degradation via intracellular lysosomal and the ubiquitylation pathway, and blocks the recycling of LDLR on the cell membrane, which increases cholesterol levels in circulation [3][4][5]. Current studies have primarily focused on the relationship of PCSK9 with lipid balance and cholesterol metabolism, especially plasma LDL-C levels and blood lipid metabolism.
PCSK9 is primarily expressed in cells with regeneration and differentiation capabilities such as hepatocytes, macrophage, and smooth muscle cells [6][7][8]. It can affect the biological characteristics of macrophages through autocrine or paracrine modes of action [6]. The liver is typically the primary target organ for this action [9]. Hepatic transcriptome and proteome analyses have revealed that PCSK9 overexpression can affect steroid synthesis and the expressions of genes involved in stress response and immune pathways, suggesting that PCSK9 also participates in biological processes other than cholesterol metabolism in hepatocytes [9][10][11][12]. Additionally, PCSK9 is associated with many human diseases such as liver disease, kidney disease, and diabetes, [13][14][15]. Regulation of PCSK9 activity can impact patients with liver disease [14] such as hepatic steatosis. A liver function test has been done in patients with familial hypercholesterolemia taking PCSK9 inhibitors. The risk factors of liver injury like alanine transaminase and aspartate transaminase are increased after 6 months of treatment [16]. In addition, in a mouse model with liver injury, the loss of PCSK9 activity can severely suppress the regeneration and repair of the liver [17]. Therefore, PCSK9 can have many potential targets, which should be accounted for during drug research and development and in clinical trials to avoid any side effects during drug administration.
The PCSK9-D374Y mutant has been identified in a Utah family [18]. The binding ability of the D374Y mutant to LDLR on the hepatocyte membrane is ten times that of the wild type [19]. This greatly weakens the liver's affinity for eliminating LDL-C. Since PCSK9 can increase serum LDL-C, it has become a subject of focus in the research and development of novel lipid-lowering drug targets. However, few studies have explored the relationship between PCSK9 and the biological function of hepatocytes. In this study, we investigate cholesterol efflux, cell proliferation, and migration in hepatocytes. We use the PCSK9-D374Y mutant, which shows stable high expression in hepatocytes and mimics the microenvironment of high protein in vitro, to explore its effect on the biological functions of hepatocytes.

Construction of PCSK9 Overexpression Vectors.
The PCSK9 overexpression vector (either wild type or the D374Y mutant) was constructed using pcDNA3.1(+) as the backbone vector containing the neomycin gene. The plasmid contains a MAR insulator (G. gallus lysozyme gene 5 ′ matrix attachment region subfragment B-1-H1), which is immediately followed by the human APOE promoter and the cDNA sequence of PCSK9 ( Figure S1). The sequence of the D374Y mutant is directly synthesized (Supplementary Information). The synthesis and construction of the vector were completed by Genewiz, Suzhou, China. These vectors were named pcDNA3.1-PCSK9 and pcDNA3.1-PCSK9-D374Y. The empty vector of pcDNA3.1 was used as negative control (NC).

Establishment of HepG2 Cells Stably
Overexpressing PCSK9 and PCSK9-D374Y. The human liver cancer cell line HepG2 was purchased from Peking Union Medical College Cell Center. Cells were cultured in Dulbecco's modified eagle medium (DMEM, Thermo Fisher Scientific, USA) containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific). HepG2 cells were plated in six-well plates (Corning, USA) at a density of 1 × 10 6 cells/well. After 48 h, the plasmid was linearized and transfected using FuGENE HD Transfection Reagent (Promega, USA). Cell death was monitored 12 h after transfection; the medium was changed immediately if the mortality rate is >10% or changed 24 h after transfection if the mortality rate is <10%. G418 was added at 400 μg/ml to screen positive cells and cultured cells for another 10-14 days until cell death occurred, which indicated a high degree of enrichment of positive cells. Pooled positive cells were maintained with 200 μg/ml G418.

Cholesterol Efflux
Assay. Cells were plated in six-well plates at a density of 2 − 3 × 10 6 /well. Twelve hours later, they were cultured in a medium containing 2.5% BSA and 10 μM of 22-NBD-cholesterol (Thermo Fisher Scientific). Cells were incubated for 3 h and washed twice with PBS, after which the medium was replaced by serum-free and phenol red-free medium with HDL (10 μg/ml) (total HDL extracted from the plasma of healthy volunteers, Cardiovascular Research Institute, Peking University Health Science Center) to initiate the cholesterol efflux assay [20,21]. The medium was removed after 2 h and the cells were harvested and resuspended in PBS. The intracellular fluorescence intensity (FI) was measured via flow cytometry to identify the 22-NBD-cholesterol content. The cholesterol efflux rate was calculated according to the following formula: 2.4. Cell Proliferation Assay Using Cell Counting Kit-8 (CCK-8). A CCK-8 reagent kit was purchased from Dojindo, Shanghai, China. The standard proliferation curve of the HepG2 cells was drawn as follows. Wild-type cells (HepG2 cells without any genetic modification) were grown to 90% confluency and collected and diluted at concentration gradients with 8-10 wells set for each concentration (2000, 4000, 6000, 8000, and 10000). The cells were then plated in a 96well plate and cultured for 2-4 h. After the cells reached complete adherence, 10 μl of CCK-8 reagent was added into each well for a total reaction volume of 110 μl. After culturing cells for another 1-2 h, optical density (OD) measurement was performed using an optical densitometer (Thermo Fisher Scientific, EV60) when the medium color changes. Measurements in the blank wells were used as background values for normalization. The linear standard curve was drawn with the cell count on the x-axis and the OD value on the y-axis, and the standard curve was used to determine cell numbers under the same condition. Cell proliferation was detected after the standard curve was drawn. Each group has 4 × 10 3 cells per well with ten technical duplications. OD measurements were performed after 24 h, 48 h, and 72 h of culture in separate wells. The number of cells was fitted according to the standard curve.
Analytical Cellular Pathology laid into 96-well plates with 4 × 10 3 cells per well. After 24 h of culture, the cells were incubated with 50 μM EdU for 2 h, after which they were fixed with 4% paraformaldehyde. 1× Apollo and 1× Hoechst 33342 dying reaction solutions are ready-to-use, according to the instructions. The nucleus-emitting red fluorescence indicated cells with DNA replication activity. All the nuclear cells were stained in blue by Hoechst 33342. Ten pictures of each group were randomly collected using a fluorescence microscope (Olympus, Tokyo, Japan, BX53). The proliferation rate of the cells was analyzed by GraphPad Prism software by calculating the ratio of number of red blood cells to the number of blue blood cells.
2.6. Cell Migration Assay. For scratch wound healing assays, HepG2 cells stably expressing PCKS9-D374Y or NC cells were plated in a six-well plate at 4 × 10 5 /ml, with triplicate wells in each group, in DMEM complete culture medium for 24 h. Cells were treated with 10 μg/ml mitomycin C for 2 h to arrest cell division. Three parallel and even scratches were made along the plate bottom using a 10 μl pipette tip. Floating cells were removed by rinsing them with a serumfree culture medium several times. The cells were cultured, and the scratch healing was monitored and photographed every 24 h. Photos of nine visual fields in each well were obtained to qualitatively compare the scratch areas. In vitro transwell migration assay was performed according to a previously described method [22]. HepG2 cells of 4 × 10 4 were placed in the upper chamber of the transwell assembly (6.5 mm diameter inserts and 8.0 μm pore size; Corning) in 100 μl FBS-free DMEM basal medium. Meanwhile, 800 μl of DMEM complete culture medium containing 10% FBS filled the lower compartments as a source of chemoattractant. The membrane was incubated at 37°C for 12 h and then stained with Hoechst 33342 (Beyotime Institute of Biotechnology, China). The number of migrating cells was determined by counting 10 random fields per well with a fluorescence microscope (Olympus, BX53), with three sets for each group.
2.7. GTPase Activity Assay. The G protein activity was determined by G-LISA assay, which has been described in the literature [22]. The activities of RHOA-, RAC1-, and CDC42-GTP were detected using G-LISA kits (Cytoskeleton, USA) according to the manufacturer's instructions. Plates with HepG2 cells cultured with serum-free medium for 6 h were placed on ice. The cells were washed with precooled PBS three times and allowed to stand for 1 min, after which 100 μl of protein lysis buffer (M-PER mammalian protein extraction reagent, Thermo Fisher Scientific) (containing 1 mM of protease inhibitors, Thermo Fisher Scientific) was added to each well. Cells were collected and placed in 1.5 ml Eppendorf tubes for lysis on ice for 10 min. Next, 10 μl of lysates was added into the wells of a 96-well plate, to which 290 μl of reagents was added for protein analysis. The OD value was detected at 600 nm 1 min later, and the concentration value was calculated by subtracting the value in the blank control group.

Nuclear and Cytoplasmic Extraction.
Nuclear and cytoplasmic extracts from HepG2 cells were obtained using a Nuclear and Cytoplasmic Extraction Kit (CWBio, CW0199, China).

Quantitative Real-Time PCR (qPCR).
To verify the effect of overexpression of PCSK9-D374Y, target gene expression was quantified by qPCR and normalized to GAPDH levels. Total RNA samples were treated with DNase I before reverse transcription using random priming and Superscript Reverse Transcriptase (Thermo fisher Scientific), according to the manufacturer's instructions. qPCR was performed using ABI StepOne (Thermo fisher Scientific) and PowerUp SYBR Green Master Mix (Thermo Fisher Scientific). Gene expression was evaluated using the ΔΔCt method.

Overexpression of PCSK9-D374Y in HepG2 Cells
Decreases LDLR Levels. To explore the biological function of PCSK9-D374Y in hepatocytes, a PCSK9-D374Y overexpression vector is constructed, which is shown in Figure S1A. The results of the vector sequencing (not provided) and restriction enzyme digestion indicate that the pcDNA3.1-PCSK9-D374Y overexpression vector was successfully constructed ( Figure S1B).
To validate the effect of the PCSK9-D374Y in decreasing LDLR levels in hepatocytes, the protein levels of LDLR were analyzed in HepG2 cells using flow cytometry. Overexpression of the PCSK9-D374Y mutant significantly decreased the level of LDLR in the cell membrane (Figures 1(a) and 1(b)) (P < 0:0001, t-test). These results indicated that overexpression of the PCSK9-D374Y had enzyme activity and can mediate LDLR degradation in HepG2 cells.

PCSK9-D374Y Suppresses the Cholesterol Efflux Rate of HepG2 Cells.
Previous studies have shown that PCSK9 reduces the LDLR level in hepatocytes and suppresses LDL-C uptake by hepatocytes, thus affecting cholesterol metabolism [24]. However, few studies have explored the relationship between PCSK9 and cholesterol transport. We used NBD-cholesterol as a marker to identify the effects of high PCSK9-D374Y expression on cholesterol efflux in HepG2 cells. Wild-type HepG2 cells were used to test incubation time, efflux time, and the dosage of high-density lipoprotein (HDL). Results demonstrated that cholesterol labeling in HepG2 cells increased with incubation time (Figure 2(a)). HDL-mediated cholesterol efflux reaches a plateau at 2 h (Figure 2(b)) and is not dose-dependent on HDL (Figure 2(c)). However, high PCSK9-D374Y expression has no significant effect on cholesterol labeling in HepG2 cells compared with the control group (Figure 2(d)). HDL at a concentration of 10 μg/ml can produce a remarkable cholesterol efflux effect, which can then be used for detection.
We next analyzed the cholesterol efflux rates in stable cell lines treated with NBD-cholesterol for 3 h after which the cholesterol efflux was mediated with 10 μg/ml HDL for 2 h. Compared to the control, cells overexpressing PCSK9-D374Y show significantly inhibited cholesterol efflux ( Figure 3) (P < 0:001, t-test), suggesting that PCSK9-D374Y could negatively affect free cholesterol transport in hepatocytes. The transcription of cholesterol efflux-related genes such as ABCA1 and SR-BI was not influenced by PCSK9-D374Y (Figure 4(a)). However, cholesterol transportrelated genes such as APOA1 and APOA2 were significantly downregulated (Figure 4(b)) (P < 0:0001, t-test), while cholesterol biogenesis-related genes such as CYP51A1 and  To further investigate cell proliferation in a more accurate manner, we analyzed the cell proliferation level by counting EdU-positive and EdU-negative nuclei ( Figure S4A and S4B). Fluorescence staining and statistical analysis demonstrate that PCSK9-D374Y overexpression has no significant effect on the proliferation of HepG2 cells. We also analyzed the expression level of proliferating cell nuclear antigen (PCNA), a marker for mitotic cells, in the nucleus and cytoplasm and did not find any difference in the expression of this protein in the cell lines ( Figure S4C). This further confirms our conclusions at the molecular level.

PCSK9-D374Y Significantly Suppresses the Migration of
HepG2 Cells. Cell migration is an important process by which cells exert normal biological functions. Few studies have investigated the effects of PCSK9-D374Y on the migration of hepatocytes. We performed scratch wound healing assays to identify the influence of PCSK9 on the migration of HepG2 cells (Figure 5(a)). HepG2 control cells have a relatively weak migration capacity, and scratch wounds did not heal even after 108 h. Scratch wound healing is relatively slower in PCSK9-D374Y overexpression cells compared to control cells transfected with the vector, indicating that PCSK9-D374Y could suppress the migration of HepG2 cells. Transwell migration assays demonstrate the significant inhibitory effects of PCSK9-D374Y on the vertical migration of HepG2 cells (Figures 5(b) and 5(c)) (P < 0:0001, t-test).
Cell migration is closely related to the dynamic remodeling and signaling of cytoskeletal proteins. The small G proteins play key roles in the assembly of cytoskeleton proteins. However, when detecting the activities of three key small G proteins, we did not find any difference in the activities of RAC family small GTPase 1 (RAC1) and Ras  Moreover, the mRNA transcription of RHOA is downregulated (Figure 4(d)). We also examined the phosphorylation levels of migration-related proteins, including ERK, AKT, and mechanistic target of rapamycin kinase (mTOR) (Figure 6(b)). Fluctuations in the phosphorylation levels of ERK were observed in serum-starved control cells; phosphorylation levels of ERK decreased after the cells were subjected to serum starvation treatment for 2 h, peaked at 4 h, and declined again at 8 h. Compared with cells of PCSK9-D374Y overexpression, cells in the control group maintained relatively high phosphorylation levels of ERK throughout the assay, which exceeded that in the overexpression group at all time points (except at 2 h). In contrast, the phosphorylation levels of ERK in the PCSK9-D374Y overexpression group first increased and then decreased, reaching the lowest value at 8 h. This suggests that the ERK signaling pathway is involved in regulating the migration of HepG2 cells. These fluctuations are consistent with the low migration capability in the PCSK9-D374Y overexpression group. Under normal culture conditions, AKT phosphorylation levels were significantly higher in the control group than in the PCSK9-D374Y overexpression group (P = 0:0029, t-test); however, almost no phosphorylated AKT was detected during starvation treatment. This indicates that AKT might not be involved in regulating starvation-induced HepG2 cell migration. The mTOR phosphorylation level also shows no significant differences among the different groups.

Discussion
In this study, we achieved stable overexpression of the PCSK9-D374Y mutant in HepG2 cells, analyzed the proliferation, migration, and free cholesterol transport of PCSK9-D374Y overexpression cells, and investigated the effect of PCSK9-D374Y on the biological functions of hepatocytes. HepG2 cells with PCSK9-D374Y overexpression exhibited  Analytical Cellular Pathology no change in overall cell proliferation compared with the control group; however, their free cholesterol transport capability and migration activities were significantly suppressed. PCSK9-D374Y can inhibit free cholesterol transport, and the cell migration ability is downregulated through the ERK signaling pathways. The HepG2 cell line of PCSK9-D374Y can be used for studying the function of hepatocytes. This cell line has high PCSK9-D374Y transcription, translation, and protein secretion activities, and the abundance of LDLR proteins in the HepG2 cell membrane and cytoplasm was also validated in the overexpression cell line. The HepG2 cell line is a useful tool for investigating the synthesis, secretion, and molecular features of PCSK9, the binding capacity of PCSK9 with LDLR, the ability of PCSK9 to mediate LDLR degradation, and PCSK9 mutants and their functions [25][26][27].
Previous studies have reported that the PCSK9 expression level is correlated with the proliferation of hepatocytes and the metastasis of tumor cells [17,28]. Therefore, we used the HepG2 cell line to explore the proliferation, migration, and free cholesterol transport of hepatocytes. In this study, our proliferation assay results demonstrate that PCSK9 overexpression does not affect HepG2 hepatocyte proliferation. However, in their mouse liver injury models, Zaid et al. propose that PCSK9 plays a key role in mouse liver repair and hepatocyte proliferation [17]. Repairing liver injuries in mice requires cholesterol; PCSK9 gene deficiency decreases cholesterol levels in the microenvironment where the hepatocytes are located, thus inhibiting hepatocyte proliferation. However, the authors do not determine whether the PCSK9 gene is directly involved in regulating hepatic cell proliferation, rather, that the effect of PCSK9 on the cholesterol level in the microenvironment of liver tissue affects the repair of liver injury [17]. We also find out that overexpression of PCSK9-D374Y does not affect PCNA levels. As such, we further confirmed that PCSK9-D374Y overexpression does not affect hepatocyte proliferation. However, endogenous PCSK9 can affect the proliferation results. In future studies, it is important to evaluate these phenomena using cells lacking endogenous PCSK9 expression.
Sun et al. use mouse models with cancer cell metastasis to explore the effect of PCSK9 on cancer cell metastasis and found that PCSK9 deficiency inhibited the metastasis of cancer cells [28]. We demonstrate that PCSK9 overexpression significantly inhibits the horizontal motion and vertical migration of HepG2 cells. The difference between  Analytical Cellular Pathology migration capacity. Therefore, we speculated that the effect of PCSK9 on cell migration can be achieved by regulating cholesterol metabolism in cells.
Studies have revealed that the distribution and content of free cholesterol on the cell membrane are important for the function of lipid rafts on the cell membrane and cellular signaling [29,30]. Therefore, we examined cell migrationrelated signaling pathways based on the phenotype data and found that PCSK9-D374Y suppressed the migration capacity of HepG2 cells by downregulating the phosphorylation levels of ERK, confirming the results found in previous studies [31]. In the complete medium-containing serum, the phosphorylation levels of ERK of gene overexpressed cells were significantly lower than those of wild-type cells. This suggests that the kinase reserve of gene overexpression cell migration-related pathways was insufficient at the initial stage of the migration experiment. The activity of ERK signaling pathway has been proved to be involved in the regulation of cell migration in previous works. For example, in wound healing experiments, the migration of keratinocytes was enhanced by micrograft treatment through ERK activation [32]. Another research indicated that ovarian cancer cell migration was induced by betacellulin through EGFR-MEK/ERK signaling [33]. Thus, it may be involved in the regulation of cell migration dependent on ERK activity. During cell migration, serum-free culture conditions make cells that produce stress and migrate [34], which will consume the corresponding kinase. We also detected the efficiency of free cholesterol transport in HepG2 cells and found that compared with the cells in the control group, PCSK9-D374Y overexpression significantly suppressed the free cholesterol efflux rate in HepG2 cells without affecting the capacity of free cholesterol labeling. When the free cholesterol efflux is suppressed, cholesterol can accumulate in the inner surface of the cellular membrane, which may weaken the signal transduction related to lipid rafts. According to Pagler et al., increased cholesterol levels in the inner layer of the cellular membrane can lead to the tight binding of small G proteins to the inner surface of the cellular membrane, resulting in imbalanced RAC1/ RHOA activity and decreasing cellular motility. In this study, we detected the activities and expression levels of small G proteins in HepG2 cells and did not find changes in their activities and expression, except for decreased RHOA expression. However, the activity of CDC42, another small G protein, significantly increased in the overexpression of cells. Further investigation is required to determine whether the abnormally increased CDC42 activity affects the ERK signaling pathways [35]. Additionally, while only the total expression volume of RAC1 and RHOA is detected in our study, the distribution of these two proteins on the cell membrane and lipid rafts also warrants further investigation.
In addition, we conducted animal experiments to prove the conclusions obtained in cell experiments. We adopted PCSK9-D374Y transgenic pigs. Hepatic sinus dilatation and unclear boundary of hepatic lobule can be observed on the liver of PCSK9-D374Y transgenic pigs. Furthermore, there are scattered lymphocyte infiltration on the liver lobule ( Figure S5). Accordingly, we used PCSK9-KO mice to investigate the total cholesterol level in vivo. Results showed that serum levels of TC, TG, HDL-C, and LP (a) in PCSK9-KO mice were significantly downregulated when compared with WT mice ( Figure S6). Meanwhile, quantitative proteomics results of primary hepatic cells isolated from PCSK9-KO mice also showed that many proteins involved in lipid transport were upregulated such as LDLR, APOA2, APOA4, NPC1, and ABCC2 (data not shown). These results indicated that PCSK9 may negatively regulate cholesterol and lipid transport in vivo.

Conclusions
In summary, our results demonstrate that PCSK9-D374Y, a gain-of-function mutation, has no effect on HepG2 cell proliferation but exerts an inhibitory effect on cell migration and the cholesterol efflux capacity of HepG2 cells. It can interfere with the activities of small G proteins and the signaling of lipid raft-facilitated cell signaling pathways and is involved in the regulation of ERK signaling pathways.

Data Availability
The original contributions presented in the study are included in the article/supplementary material; further inquiries can be directed to the corresponding authors.