Piperine Induces Hepatic Low-Density Lipoprotein Receptor Expression through Proteolytic Activation of Sterol Regulatory Element-Binding Proteins

Elevated plasma low-density lipoprotein (LDL) cholesterol is considered as a risk factor for atherosclerosis. Because the hepatic LDL receptor (LDLR) uptakes plasma lipoproteins and lowers plasma LDL cholesterol, the activation of LDLR is a promising drug target for atherosclerosis. In the present study, we identified the naturally occurring alkaloid piperine, as an inducer of LDLR gene expression by screening the effectors of human LDLR promoter. The treatment of HepG2 cells with piperine increased LDLR expression at mRNA and protein levels and stimulated LDL uptake. Subsequent luciferase reporter gene assays revealed that the mutation of sterol regulatory element-binding protein (SREBP)-binding element abolished the piperine-mediated induction of LDLR promoter activity. Further, piperine treatments increased mRNA levels of several SREBP targets and mature forms of SREBPs. However, the piperine-mediated induction of the mature forms of SREBPs was not observed in SRD–15 cells, which lack insulin-induced gene–1 (Insig–1) and Insig–2. Finally, the knockdown of SREBPs completely abolished the piperine-meditated induction of LDLR gene expression in HepG2 cells, indicating that piperine stimulates the proteolytic activation of SREBP and subsequent induction of LDLR expression and activity.


Introduction
High-plasma low-density lipoprotein (LDL) cholesterol levels are a major risk factor for atherosclerosis and coronary heart disease [1]. The liver is a major organ that uptakes LDL particles via LDL receptor (LDLR)-meditated endocytosis and has the ability to metabolize up to 70% of plasma LDL [2]. Thus, increased LDLR expression in the liver ameliorates atherogenic lipid profiles and suppresses plasma LDL-cholesterol levels.
Stable transfection of Huh-7 cells with the LDLR promoter-containing luciferase reporter plasmid Huh-7 cells were plated in 60-mm dishes at a density of 8 × 10 5 cells/dish and were cultured in medium A for 20 h. Cells were subsequently transfected with 7.62 μg of pLDLR-Luc (LDLR promoter) and 0.38 μg of pMAM2-BSD (blasticidin S deaminase) using lipofectamine 2000 according to the manufacturer's instructions. Twenty-four hours after transfection, cells were plated in 100-mm dishes and were cultured in medium A containing 8 μg/ml blasticidin S. The medium was changed every second day until well-defined colonies were observed. Colonies were isolated using cloning cylinders in the presence of 2 μg/ml blasticidin S. The resulting stable cell lines were cultured in medium A or medium C (DMEM supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, and 12.5 μM of the HMG-CoA reductase inhibitor fluvastatin, and 50 μM sodium mevalonate containing 5% LPDS) under normal or sterol-depleted conditions for 16 h, respectively. The Huh-7/LDLR-Luc cell line was selected according to the induction of luciferase activity under cholesterol-depleted conditions.

Luciferase assays in Huh-7/LDLR-Luc cells
Huh-7/LDLR-Luc cells were plated on 12-well plates at a density of 1 × 10 5 cells/well, were cultured in medium A for 20 h, and were then incubated for 24 h in the absence or presence of test compounds at 100 μM. Luciferase activity and protein contents in cell extracts were measured as described previously [21]. Luciferase activities were normalized to total protein contents of cell extracts.

Luciferase assays
Luciferase assays were performed as described previously [22].

LDL uptake assays
LDL uptake assays were performed as described previously [23].

Statistical analysis
All data are presented as mean ± standard error of the mean (SEM). Statistical analyses were performed using Ekuseru-Toukei Ver.2.0 (Social Survey Research Information). Pairwise comparisons of treatments were made using Student's t test, and multiple comparisons were made using one-way ANOVA followed by the Bonferroni test. Differences were considered significant when P < 0.05.

Piperine increases the expression and activity of LDLR
To identify compounds that increase the expression of LDLR, we established a stable cell line that expressed a luciferase reporter gene under the control of the LDLR promoter region from −595 to +36. Human hepatoma Huh-7 cells were transfected with pLDLR-Luc and the expression plasmid for blasticidin S deaminase and were then cultured in the presence of blasticidin S. Blasticidin S-resistant clones (Huh-7/LDLR-Luc cells) were isolated and expanded and were treated with approximately 100 food components at 100 μM for 12 h. Subsequent luciferase assays showed increased LDLR promoter activity in the presence of several food components (S1 Table), and those that increased luciferase activity by more than 1.5-fold were further examined in transient luciferase assays of LDLR gene promoter activity. As shown in Fig 1A, pinocembrine, fisetin, and piperine stimulated LDLR promoter activity by more than 1.5-fold when compared with a vehicle control. Among these, piperine treatments led to increased endogenous LDLR mRNA levels in HepG2 cells after 24 h (Fig 1B), and the effect was dose dependent (Fig 2A). LDLR protein is synthesized as a precursor form with an apparent molecular mass of 120 kDa (precursor in Fig 2B) and is then post-translationally modified to the mature form with an apparent molecular mass of 160 kDa (mature in Fig 2B). Treatment with piperine for 24 h increased the presence of the mature and precursor LDLR ( Fig 2B). Thus, to determine whether treatment with piperine causes increased LDL uptake, HepG2 cells were treated with piperine for 24 h and were then incubated with the fluorescent-labeled LDL DiI-LDL for 5 h. Subsequent fluorescence microscopy showed increased DiI-LDL internalization by HepG2 cells in the presence of piperine (Fig 2C), suggesting that piperine-mediated increase of LDLR mRNA leads to increased LDL uptake.

Piperine stimulates LDLR promoter activity via the SREBP-binding element
SREBPs reportedly stimulate promoter activity of the LDLR gene [24]. Thus, to investigate the roles of SREBP in piperine-mediated stimulation of LDLR gene expression, reporter assays were performed using mutant LDLR promoters (Fig 3). In these experiments, the mutation of SRE resulted in inhibition of piperine responsiveness and decreased basal LDLR promoter activity. These results indicate that piperine stimulates LDLR promoter activity through SRE.

Piperine activates SREBPs by stimulating proteolytic processing of SREBPs
To determine whether piperine elevates endogenous mRNA levels of SREBP target, HepG2 cells were treated with piperine for 24 h, and quantitative real-time PCR analyses were performed ( Fig 4A). In these experiments, piperine treatment caused a significant increase in the mRNA levels of HMG-CoA reductase, HMG-CoA synthase, SQS, FAS, and ACC1 in HepG2 cells, suggesting that piperine stimulates SREBP activity. However, piperine did not increase expression of the SREBP target gene PCSK9 or SREBP genes ( Fig 4A). Thus, to investigate the effect of piperine on SREBP processing, HepG2 cells were treated with piperine for 3 h, and lysates were subjected to immunoblotting using anti-SREBP-1 and anti-SREBP-2 antibodies. SREBP-1 was detected by an antibody recognizing its N-terminus [SREBP-1(N)], whereas SREBP-2 was detected by antibodies recognizing its N-[SREBP-2(N)] and C-[SREBP-2(C)] termini. SREBP processing was stimulated by 3-hour treatments with 100 μM piperine, as indicated by increased mature and cleaved forms and decreased precursor forms (Fig 4B). Although 3 μM piperine only increased the presence of the cleaved form of SREBP-2 slightly, 10 and 30 μM piperine treatments led to marked increases (Fig 4C).

Piperine stimulates SREBP processing under cholesterol-supplemented conditions
SREBP processing is suppressed by sterols, which inhibit translocation of SCAP/SREBP complex from the ER to the Golgi. Thus, experiments were performed to determine whether piperine stimulates SREBP processing in the presence of sterols (Fig 5A). Piperine-mediated increases in the cleaved form of SREBP-2 were diminished but still observed in the presence of 25-HC (Fig 5A). Consistent with these results, piperine treatment caused a significant increase in the mRNA levels of LDLR regardless of the presence of 25-HC in HepG2 cells (Fig 5B).  When cellular cholesterol levels are low, ER-resident Insig proteins bind the SCAP/SREBP complex and suppress SREBP processing [25]. To examine the involvement of Insigs in piperine-mediated stimulation of SREBP processing, we performed experiments using the SRD-15 cells established by Lee et al. [19], which lack Insig-1 and Insig-2. In parental CHO-7 cells, the cleaved form of SREBP-2 was increased by piperine treatment and by cholesterol-depleted conditions (Fig 5C). In contrast, protein expression of the cleaved form of SREBP-2 was unaltered in SRD-15 cells cultured under sterol-depleted conditions ( Fig 5D). Moreover, whereas the precursor form of SREBP-2 was barely detectable, the cleaved form of SREBP-2 gave intense signals in SRD-15 cells even under normal conditions, suggesting that SREBP processing is stimulated in SRD-15 cells. However, piperine did not increase cleaved form of SREBP-2 and may have slightly decreased it in SRD-15 cells (Fig 5D), indicating that piperine does not stimulate SREBP processing in SRD-15 cells.

SREBPs are required for piperine-mediated stimulation of LDLR gene expression
In final experiments, cells were transfected with siRNAs that were designed to specifically target SREBP-1 or SREBP-2 mRNA. As expected based on the fact that piperine stimulates SREBP-1 and SREBP-2 processing (Fig 4B), a single knockdown of SREBP did not affect piperine-mediated induction of LDLR gene expression (data not shown). It should be noted that SREBP-1a is the predominant isoform of SREBP-1 in HepG2 cells [26]. Cotransfection of SREBP-1 and SREBP-2 siRNAs resulted in an 80% decrease in the SREBP-1 and -2 mRNA levels and a 75% decrease in the endogenous LDLR gene expression (Fig 6). These results are consistent with previous reports showing that SREBPs regulate LDLR gene expression [24]. Moreover, piperine-mediated stimulation of LDLR gene expression was completely abolished by knockdown of SREBPs (Fig 6), confirming that piperine-mediated stimulation of LDLR gene expression is mediated by SREBPs.

Discussion
The present data demonstrate that the alkaloid piperine stimulates LDLR gene expression and uptake of LDL particles in cultured hepatocytes. Moreover, biochemical analyses further indicated that piperine promotes the maturation of SREBPs. In agreement, the mutation of SRE of the LDLR gene promoter abolished piperine responsiveness, and the knockdown of SREBP expression abrogated the ensuing induction of LDLR gene expression.
In these experiments, we used a stable cell line that expresses a luciferase reporter gene under the control of the LDLR promoter region from −595 to +36 to identify food compounds that stimulate LDLR gene expression. Using this screening system, we found that several food compounds, including pinocembrine, 1-dodecanol, hesperetin, fisetin, piperine, and 1-tetradecanol, stimulated LDLR gene promoter activity (S1 Table). Some compounds did not stimulate LDLR promoter activity in transient reporter assays (Fig 1A) and were excluded from additional experiments. However, HEK293 cells were derived from human embryonic kidney cells and were used in transient reporter assays for their high transfection efficiency, and these compounds may stimulate LDLR promoter activity in hepatocytes. In addition, pinocembrine and fisetin did not stimulate endogenous LDLR expression but did stimulate the LDLR promoter in stable and transient reporter assays (S1 Table and Fig 1A and 1B). Although pinocembrine and fisetin stimulated LDLR promoter activity through the region examined in the present reporter assays, other upstream or downstream regions of the LDLR promoter may obscure this effect.
Several previous reports indicate that transcription, proteolytic processing, and post-translational modification are involved in the regulation of SREBPs [9]. Among these, proteolytic processing is considered a crucial regulatory step. Accordingly, under low-sterol conditions, SCAP binds to Sec23/24, leading to clustering of the SCAP/SREBP complex into common coated protein II vesicles. Subsequently, the SCAP/SREBP complex is transported from the ER to the Golgi, and SREBPs are then processed by site-1 and site-2 proteases. Given that piperine analysis was performed and mRNA levels were normalized to those of GAPDH mRNA and expressed relative to those in vehicle (DMSO)-treated controls. All data are expressed as mean ± SEM, n = 3. Differing letters indicate significant differences, *P < 0.05. (C and D) CHO-7 (C) and SRD-15 (D) cells were cultured with piperine (100 μM) or were depleted of sterols by incubation in medium C for 4 h, and whole-cell extracts were isolated for SDS/PAGE and IB with anti-SREBP-2 (1C6) or anti-β-actin antibodies (left panel). Similar results were obtained in three separate experiments. The signals (n = 3) were quantified, and the signals of the control group are represented as 1 (right panel).
doi:10.1371/journal.pone.0139799.g005 Cells were then cultured in medium A for 48 h and were treated with piperine (100 μM) for 24 h. Total RNA was isolated and real-time PCR analysis was performed. Target mRNA expression levels were normalized to those of GAPDH mRNA and are presented relative to those in vehicle (DMSO)-treated siCon-transfected cells. Analyses of SREBP-1 mRNA were performed using a TaqMan probe that recognizes SREBP-1a and -1c. All data are expressed as mean ± SEM, n = 3. Differing letters indicate significant differences, *P < 0.05. doi:10.1371/journal.pone.0139799.g006 did not increase the cleavage of SREBP-2 in Insig-1 and -2-deficient SRD-15 cells (Fig 5D), piperine compromises the retention of SCAP/SREBP complexes by Insigs in the ER. Although it is unclear how piperine stimulates SREBP processing, it may directly bind Insigs or SCAP/ SREBP complexes and inhibit their interactions. Recently, the crystal structure of a mycobacterial Insig homolog has been reported [27]. A homology-based structural model of human Insig-2 suggested that a central cavity of Insig-2 accommodates 25-HC and that transmembrane segments 3 and 4 of Insig-2 engage in SCAP binding. Thus, the transmembrane segments 3 and 4 of Insigs are candidates for binding with piperine. Further studies are required to determine whether piperine attenuates the binding between Insig and SCAP and whether piperine directly binds Insigs or the SCAP/SREBP complex. Alternatively, piperine may affect phosphorylation cascades that indirectly stimulate SREBP processing. It has been reported that the activation of the Akt/mTOR/S6K pathway stimulates SREBP processing [28]. On the other hand, piperine has been reported to suppress the Akt signaling pathway in osteosarcoma U2OS and breast cancer TNBC cells [29,30]. Thus, activation of Akt signaling is unlikely during piperine-mediated stimulation of SREBP processing. In agreement, treatments with the PI3K inhibitor LY294002 and the mTOR inhibitor rapamycin did not inhibit piperine-mediated stimulation of SREBP processing in HepG2 cells (data not shown).
Activated SREBPs stimulate transcription of genes involved in fatty acid and cholesterol synthesis, leading to increased lipid levels. In the present study, we demonstrated that piperine stimulated SREBP-1 and SREBP-2 processing ( Fig 4B) and increased the expression of various SREBP target genes (Fig 4A). Although piperine-induced LDLR gene expression and activity may have anti-atherogenic effects, these effects may be reduced by the piperine-mediated induction of lipid synthesis genes. However, piperine treatments were reportedly beneficial in diet-induced obese mice and prevented fatty liver and corrected high-fat diet-mediated inhibition of AMPK phosphorylation [18]. Phosphorylated AMPK stimulates phosphorylation and inactivation of downstream substrates such as HMG R and ACC [31]. Thus, although piperine treatment likely stimulates the expression of lipid synthesis-related genes, simultaneous augmentation of AMPK activation may negate this effect on de novo lipid synthesis. We demonstrated that piperine stimulated SREBP maturation and the expression of genes related to lipid synthesis. However, piperine did not stimulate the expression of the present SREBP target genes, including PCSK9, SREBP-1c, and SREBP-2. Although the precise molecular mechanisms by which piperine stimulates specific SREBP target gene expression is unclear, piperine functions as an antagonist of the transcription factor LXRα, which reportedly stimulates SREBP-1c gene expression [17]. Thus, piperine may concomitantly stimulate SREBP activity and suppress LXRα activity, leading to no net effect of increased SREBP-1c mRNA expression. Similarly, piperine may affect the activity of transcription factors other than SREBPs, which control the expression of PCSK9 and SREBP-2, thereby abolishing the influence of SREBP on certain promoters. Further studies are essential to clarify how piperine-mediated activation of SREBPs regulates the expression of SREBP-targeted genes. PCSK9 can bind to LDLR and induce its degradation. Hence, inhibition of PCSK9 has been considered a promising drug target for atherosclerosis. Piperine-mediated activation of SREBP increased LDLR gene expression but did not affect PCSK9 expression. Thus, piperine likely has favorable anti-atherogenic activities.
Previous studies show that piperine enhances the bioavailability of drugs, although the related mechanisms have not been characterized [13]. Nonetheless, curcumin is a well-characterized constituent of the common food spice turmeric (Curcuma longa), and its bioavailability is reportedly enhanced by concomitant administration of piperine [32]. Moreover, a recent report indicated that curcumin downregulates PCSK9 gene expression, thereby stimulating cell-surface LDLR protein expression and promoting LDL uptake into HepG2 cells [34]. These results suggest that concomitant administration of piperine and curcumin stimulates LDLR activity via multiple pathways, including (1) piperine-meditated stimulation of LDLR gene expression, (2) curcumin-meditated suppression of PCSK9 gene expression, and (3) piperinemediated enhancement of the bioavailability of curcumin. However, further studies are required to confirm these effects and to improve therapeutic options for atherosclerosis.
In summary, the present data demonstrate that piperine stimulates SREBP activity. In addition, we show that piperine-mediated activation of SREBP increases LDLR gene expression and consequent LDL uptake into HepG2 cells. Essential future studies will determine whether dietary piperine affects hepatic LDLR expression in vivo.
Supporting Information S1 Table. Effects of the test compounds (100 μM) on LDLR promoter activity. Luciferase assays were performed in triplicate using Huh7/LDLR-Luc cells as described in the Materials and Methods section. (EPS)