Inflammatory stress increases unmodified LDL uptake via LDL receptor: an alternative pathway for macrophage foam-cell formation
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- Ye, Q., Chen, Y., Lei, H. et al. Inflamm. Res. (2009) 58: 809. doi:10.1007/s00011-009-0052-4
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To investigate if inflammatory stress increases intracellular accumulation of unmodified low-density lipoprotein (LDL) in human monocyte cell line (THP-1) macrophages by disrupting the sterol regulatory element binding proteins (SREBPs) cleavage-activating protein (SCAP)-SREBP2-mediated feedback regulation of LDL receptor.
Materials and methods
THP-1 macrophages were incubated in serum-free medium in the absence or presence of LDL alone, LDL plus lipopolysaccharide (LPS) and LPS alone, then intracellular cholesterol content, tumor necrosis factor alpha level in the supernatants, mRNA and protein expression of LDL receptor, and SREBP2 and SCAP in the treated cells were assessed by Oil Red O staining, cholesterol enzymatic assay, enzyme-linked immunosorbent assay, real-time quantitative polymerase chain reaction, and Western blotting analysis, respectively.
We demonstrated that LPS enhanced transformation of THP-1 macrophages into foam cells by increased uptake of unmodified LDL as evidenced by Oil Red O staining and direct assay of intracellular cholesterol. In the absence of LPS, 25 μg/ml LDL decreased LDL receptor mRNA and protein expression (p < 0.05). However, LPS enhanced LDL receptor expression, overcoming the suppression of LDL receptor induced by 25 μg/ml LDL and inappropriately increasing LDL uptake (p < 0.05). Exposure to LPS also caused overexpression of mRNA and protein of SCAP and SREBP2 (p < 0.05). These observations indicate that LPS disrupts cholesterol-mediated LDL receptor feedback regulation, permitting intracellular accumulation of unmodified LDL and causing foam-cell formation.
The implication of these findings is that inflammatory stress may contribute to intracellular LDL accumulation in THP-1 macrophages without previous modification of LDL.
KeywordsTHP-1 macrophagesInflammationAtherosclerosisLDL receptorSREBP cleavage-activating protein
Atherosclerosis is characterized by intimal accumulation of lipids, mainly cholesterol and cholesterol esters, and the infiltration of inflammatory cells, particularly macrophages and T cells, in addition to migration and proliferation of medial smooth-muscle cells. It is well-known that the macrophage scavenger receptors play an important role in the internalization of chemically modified lipoproteins, such as oxidized and acetylated low-density lipoprotein (LDL) by macrophages, leading to the transformation of macrophages into lipid-laden foam cells [1–4]. In addition, macrophages could accumulate lipid by taking up large-particle lipoprotein through non-receptor-mediated endocytosis .
LDL receptor is the primary receptor for binding and internalization of plasma-derived LDL cholesterol and regulates plasma LDL concentration. Brown et al.  observed that LDL receptor activity is normally under tight metabolic control via a feedback system that is dependent on intracellular cholesterol concentration. This system maintains a constant level of cholesterol in hepatocytes and other cells by controlling both the rate of cholesterol uptake from LDL via LDL receptor and the rate of de novo cholesterol synthesis . This feedback regulation is controlled through specific interactions between the sterol-regulatory element (SRE)-1 in the LDL receptor promoter [8, 9] and a family of SRE-binding proteins (SREBPs), namely, SREBP1 and SREBP2 [10–14]. SREBPs are members of the basic helix-loop-helix leucine zipper family of transcription factors. SREBPs contain two transmembrane domains and are localized to the endoplasmic reticulum (ER) after synthesis. In the inactive state within the ER, SREBPs associate with another transmembrane protein, SREBPs cleavage-activating protein (SCAP), that provides conditional chaperone activity to the SREBPs [15–18]. SCAP contains a cholesterol-sensing domain that responds to the depletion of sterol with activation of SCAP-SREBPs-transporting activity [19–21]. When cells are overloaded with cholesterol, the SCAP-SREBPs complex remains in an inactive form in the ER through active repression by cholesterol and oxysterols, and LDL receptor gene transcription is maintained at a minimum constitutive level. In contrast, when the cells are depleted of cholesterol, SCAP transports SREBPs to the Golgi, where the NH2-terminal transcription-activation domain of the SREBPs is released from the precursor protein through specific proteolytic cleavages. The active form of the SREBPs translocates to the nucleus, binds to its cognate SRE-1 site, and activates transcription of the LDL receptor gene.
Recent experimental and clinical evidence suggests that inflammation is an aggravating factor in lipid-mediated peripheral cell injury, such as atherogenesis and also glomerulosclerosis, which has many similarities to atherosclerosis, as described by our group [22–24] and others [25, 26]. Cardiovascular risk is increased in chronic inflammatory states, up to 33-fold in patients with renal failure and allograft, and 50-fold in patients with immune dysregulation (e.g., systemic lupus erythematosus) [27, 28]. In hemodialysis patients, a higher risk of death from cardiovascular disease is, surprisingly, associated with low plasma cholesterol. These paradoxical and counterintuitive epidemiologic associations between survival outcomes and traditional cardiovascular risk factors such as high cholesterol are termed ‘reverse epidemiology’ [29, 30]. Elevated plasma levels of cytokines are often associated with chronic renal diseases. We have previously demonstrated in human mesangial cells (HMCs) and vascular smooth-muscle cells (VSMCs) that inflammatory cytokines disrupted LDL receptor feedback regulation, allowing upregulated uptake of cholesterol in the peripheral cells causing foam-cell formation [31–34]. It suggests that inflammation may increase the threshold for LDL uptake in peripheral cells, such as HMCs and VSMCs.
Macrophages play a very important role in pathogenesis of atherosclerosis and are one of the major cell types involved in foam-cell formation. However, LDL receptor feedback regulation in human macrophages under inflammation remains unclear. The present experiments set out to investigate if inflammatory stress increases intracellular accumulation of unmodified LDL in macrophages by disrupting SCAP-SREBP2-mediated feedback regulation of LDL receptor.
Materials and methods
Human monocyte cell line (THP-1) was obtained from the American Type Culture Collection (ATCC, no: TIB-202). THP-1 was cultured in growth medium containing RPMI-1640 medium, 10% fetal calf serum, 2 mmol/l glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. THP-1 was fully differentiated to macrophages by 160 nmol/l phorbol, 12-myristate, 13-acetate (PMA) for 72 h, and the differentiated THP-1 macrophages were washed extensively with phosphate-buffered saline (PBS) before use in the experiments. Experiments were performed in serum-free experimental medium containing RPMI-1640, 0.2% bovine serum albumin (BSA), 2 mmol/l glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mmol/l calcium with the anti-oxidants ethylenediaminetetraacetic (EDTA), and butylated hydroxytoluene (BHT) at final concentrations of 100 and 20 μmol/l, respectively (Sigma, St. Louis, MO, USA). All reagents for cell culture were obtained from Hyclone (Beijing, China). BSA, PMA, polyinosinic acid, heparin, and lipopolysaccharide (LPS) (from Escherichia coli, cat. no. L4391) were obtained from Sigma (St. Louis, MO, USA). LDL was isolated from plasma of healthy human volunteers by sequential ultracentrifugation. Our study protocols adhered to the tenets of the Declaration of Helsinki for experiments involving human samples.
Determination of TNF-α release
THP-1 macrophages in 24-well plates were grown to confluence. After addition of the different concentrations of LPS (0, 10, 100, 500, 1,000 ng/ml), cells were incubated in serum-free medium for 24 h, and then the supernatants of conditioned medium were collected and frozen at −70°C. Assays for tumor necrosis factor alpha (TNF-α) were performed with an enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. The results were normalized to the total protein concentrations of cells.
THP-1 macrophages were plated in 24-well plates. After 24 h of treatments with LDL, LPS, polyinosinic acid, and heparin, the cells were washed three times with PBS, fixed for 30 min with 5% formalin solution in PBS, stained with Oil Red O for 30 min, and counter-stained with hematoxylin for another 5 min. Finally, the cells were examined by light microscopy (Olympus, Japan).
Measurement of intracellular cholesterol
The method was based on the cholesterol enzymatic assay described by Gallo et al.  and Gamble et al. . THP-1 macrophages were cultured in the serum-free experimental medium in the absence (control) or presence of 25 μg/ml LDL or 25 μg/ml LDL plus 200 ng/ml LPS, or polyinosinic acid (250 μg/ml), or heparin (5 mg/ml), or 200 ng/ml LPS alone for 24 h. Cells were then washed twice in PBS; intracellular lipids were extracted in chloroform/methanol (2:1) mix and dried under vacuum; and total cholesterol (TC), free cholesterol (FC), and cholesterol ester (CE) contents were measured by enzymatic assay (CE = TC − FC) and normalized by total cell proteins determined by the Lowry assay (KeyGEN, Nanjing, China).
THP-1 macrophages were plated in 24-well plates. After 24 h of treatment with 25 μg/ml of LDL in serum-free medium, the supernatants were collected and incubated with Sudan Black B (Sudan Black B:supernatants = 1:9) for 20 min at room temperature, and then the mixtures were subjected to electrophoresis on 1% agarose gel with 200 V, 400 mA for 20 min in barbital running buffer (pH 8.6, ionic strength 0.075). Native LDL and oxidized LDL were used as positive control and negative control, respectively.
Total RNA isolation and real-time quantitative polymerase chain reaction (PCR)
Total RNA was isolated from cultured cells using RNAiso Kit (Takara, Dalian, China) according to the manufacturer’s protocol. Total RNA (500 ng) was used as a template for reverse transcription (RT) using an RNA RT Kit from ABI (Applied Biosystems, Warrington, UK). The RT reaction was set up in a 20-μl mixture containing 50 mmol/l KCl, 10 mmol/l Tris/HCl, 5 mmol/l MgCl2, 1 mmol/l of each deoxynucleosidetriphosphate, 2.5 μmol/l random hexamers, 20 units RNAsin, and 50 units of Moloney-murine leukemia virus RTase. Incubations were performed in a DNA Thermal Cycler (Eppendorf, Hamburg, Germany) for 10 min at 25°C, followed by 120 min at 37°C, and 5 s at 85°C. After cDNA synthesis by RT, the incubation mixture was split into two 10-μl aliquots for separate amplification of the scavenger receptor type A (SR-A) cDNA and the glyceraldehyde phosphate dehydrogenase (GAPDH) cDNA using specific primers. SR-A primers: upper 5′-CTGAAGTGGGAAACGAAG-3′, lower 5′-GTAAACACGCTCCTCTAA-3′; GAPDH primers: upper 5′-GTAAACACGCTCCTCTAA-3′, lower 5′-TGACGGGATCTCGCTCCTGGAAGAT-3′. No template control (NTC) was used as negative control. For semiquantitative PCR, the final concentrations of PCR reaction mixture were 50 mmol/l KCl, 10 mmol/l Tris/HCl, 2 mmol/l MgCl2, 200 μmol/l dNTPs, 0.125 μmol/l of primers, 1.25 U Taq DNA polymerase (Takara, Dalian, China). After incubation for 300 s at 95°C, 30 cycles were performed for 30 s at 95°C, 30 s at 65°C, and 60 s at 72°C.
A 20-μl sample of PCR reaction was subjected to electrophoresis in 2% agarose gel. Real-time quantitative PCR was performed on an Opticon 2 Real-time PCR Detector (Bio-Rad, USA) using SYBR Green I PCR Master Mix (Takara, Dalian, China). Thermal cycler conditions were holds for 2 min at 55°C and 2 min at 95°C, followed by 40 cycles of 20 s at 95°C and 20 s at 55°C. Relative amount of mRNA was calculated using the comparative threshold cycle method. β-actin served as the reference housekeeping gene. The amplification efficiencies of the target and reference were shown to be approximately equal with a slope of log input amount to threshold cycle <0.1. The following oligonucleotide primers were used: LDL receptor upper 5′-GTGTCACAGCGGCGAATG-3′, lower 5′-CGCACTCTTTGATGGGTTCA-3′; SREBP2 upper 5′-CCGCCTGTTCCGATGTACAC -3′, lower 5′-TGCACATTCAGCCAGGTTCA-3′; SCAP upper 5′-GGGAACTTCTGGCAGAATGACT-3′, lower 5′-CTGGTGGATGGTCCCAATG-3′; and β-actin upper 5′-CCTGGCACCCAGCACAAT-3′, lower 5′-GCCGATCCACACACGGAGTACT-3′. Primers were designed with Primer Express Software version 2.0 System (Applied Biosystems, Foster City, USA).
Western blot analysis
Identical amounts of protein from whole-cell extract or nuclear extract were denatured and then subjected to electrophoresis on a 5% stacking and 8% separating SDS polyacrylamide gel in a Bio-Rad mini protein apparatus (USA). Electrophoretic transfer to nitrocellulose was accomplished at 100 V, 350 mA for 1 h in 25 mmol/l Tris pH 8.3, 192 mmol/l glycine, 0.1% SDS, and 20% methanol. The membrane was then blocked with 5% skimmed milk for 1 h at room temperature, followed by two 5-min washes in PBST (PBS/1% Tween 20). The membrane was incubated with rabbit anti-human LDL receptor polyclonal antibody (Boster, Wuhan, China) for 1 h in antibody dilution buffer (5% skimmed milk in PBST) followed by three 5-min washes in PBST. A goat anti-rabbit horseradish peroxidase (HRP)-labeled antibody (Santa Cruz, CA, USA) was diluted in antibody dilution buffer, then added to the membrane for 1 h, followed by three 5-min washes in PBST. Finally, detection procedures were performed using ECL Advance Western Blotting Detection Kit (Amersham Bioscience, Bucks, UK), and autoradiography was performed on Hyperfilm ECL (Amersham). The membrane was washed with stripping buffer for 20 min at 50°C and subjected to reimmunodetection using a mouse anti-human SREBP2 monoclonal antibody (LGC Promochem, UK) and a goat anti-mouse HRP-labeled antibody (Santa Cruz) or a rabbit anti-human SCAP polyclonal antibody produced by immunizing rabbits with the synthetic peptide PVDSDRKQGEPTEQC in our laboratory and a goat anti-rabbit HRP-labeled antibody (Santa Cruz). Actin was also examined using a rabbit anti-actin antibody (Santa Cruz) and a goat anti-rabbit HRP-labeled antibody (Santa Cruz). For negative controls, the primary antibodies were omitted. No immunoreactive band was observed at correct molecular size when the primary antibodies were omitted. The HepG2 cell and mouse liver protein served as positive controls for each protein.
In all experiments, data were evaluated for significance by two-sample t-test analysis between groups using Minitab software. Data were considered significant at p ≤ 0.05.
Many studies demonstrated that THP-1 macrophages are very similar to primary peripheral blood mononuclear cell (PBMC) macrophages in response to LPS and cytokines, thus THP-1 cells were used in lipid metabolism studies by investigators [37–39]. Scavenger receptors have traditionally been regarded as the major pathway of foam-cell formation in macrophages. Modified LDL is believed to be the main source of cholesterol that accumulates in monocyte-derived macrophages within atherosclerotic plaques, but native LDL through LDL receptor has not previously been shown to cause substantial cholesterol accumulation when incubated with macrophages. However, recent studies demonstrated that activation of human monocyte-derived macrophages with phorbol 12-myristate 13-acetate (PMA) stimulates LDL uptake and degradation, resulting in massive macrophage cholesterol accumulation via a non-receptor-mediated endocytosis pathway , suggesting that direct attention to macrophage fluid-phase endocytosis is a relevant pathway to target for modulating macrophage cholesterol accumulation in atherosclerosis.
The LDL receptor is the primary receptor for binding and internalization of plasma-derived LDL-cholesterol and regulates plasma LDL concentrations. However, the role of the LDL receptor in cholesterol accumulation in macrophages remains unclear. In this study, we report that LPS significantly increased native LDL accumulation in THP-1 macrophages specifically via the LDL receptor pathway, as demonstrated in Figs. 3, 4. The involvement of LDL receptor in this lipid accumulation was confirmed by showing that the lipid accumulation induced by LPS could not be inhibited by scavenger receptor blocking agent polyinosinic acid, but was reduced by heparin, which removes LDL bound to the cell surface. This was consistent with our previous data in human VSMCs . Additionally, all experimental media contained the anti-oxidants EDTA and BHT, both of which powerfully prevent oxidation of LDL and are widely used in studies [41, 42]. The electrophoretic mobility of LDL from the culture medium was the same as that of fresh LDL, indicating that no oxidation had taken place during the experiments. Therefore, there was no ligand for scavenger receptors in the culture medium, implying LDL receptor pathway involvement and effectively excluding the participation of scavenger receptor. Furthermore, LPS upregulated LDL receptor mRNA and protein expression and disrupted LDL receptor feedback regulation. These results suggest that chronic inflammation may fundamentally modify cholesterol homeostasis by disrupting LDL receptor feedback regulation.
We proceeded to investigate the molecular mechanisms by which LPS overcomes the suppression of the LDL receptor induced by 25 μg/ml LDL. In particular, we examined the expression and intracellular translocation of SCAP and SREBP2, which are two important molecules in regulating LDL receptor expression under inflammatory stress. Recently, insulin-induced gene 1 (Insig1) has been identified as a sterol-regulated ER retention factor that interacts with SCAP-SREBP2 complex in the ER . When intracellular cholesterol concentration increases, Insig1 binds to the sterol-sensing domain in the SCAP, preventing the exit of SCAP-SREBP2 complex from the ER. Our results showed that both mRNA and protein expression in response to cholesterol are similar, supporting that SCAP acts as a cholesterol sensor. We demonstrated that inflammatory stress by LPS increased both mRNA and protein expression of SCAP and SREBP2, which may in turn increase the ratio of SCAP to Insig1. This seems a likely hypothesis, because Insig1 is quantitatively limited in the ER and may be present in insufficient concentrations to retain the increased SCAP-SREBP2 complex in the ER under inflammatory stress. As a consequence, inflammatory stress may result in an escape of SCAP-SREBP2 complex from the ER to the Golgi in the presence of high concentrations of intracellular cholesterol. This abnormal escape disrupts the physiological feedback regulation of the LDL receptor. Our result is consistent with the observation that overexpression of SCAP abolishes sterol suppression of SREBP2 cleavage.
Taken together, these results imply that the normally tight sterol-dependent feedback regulation of LDL receptor in THP-1 macrophages is disrupted by LPS. Thus, in the presence of LPS, native LDL is taken up in excess via LDL receptor and results in massive cholesterol ester accumulation. These processes convert THP-1 macrophages into foam cells. The implications of these findings are that native LDL can be atherogenic without prior modification by oxidation in THP-1 macrophages and LDL receptor may be another important pathway of foam-cell formation in macrophages under inflammatory stress.
We acknowledge the support of the National Nature Science Foundation of China (nos. 30670869 and 30772295; Key Program, no. 30530360), National Basic Research Program of China (973 Program, nos. 2006CB503907, 2008CB517309), and Natural Science Foundation Project of CQ CSTC (2008BA5016).