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Am J Physiol Renal Physiol 293: F680-F687, 2007. First published July 18, 2007; doi:10.1152/ajprenal.00209.2007
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Inflammatory cytokines disrupt LDL-receptor feedback regulation and cause statin resistance: a comparative study in human hepatic cells and mesangial cells

Yaxi Chen,1 Xiong Z. Ruan,1,2 Qiu Li,1 Ailong Huang,1 John F. Moorhead,2 Stephen H. Powis,2 and Zac Varghese2

1Centre for Lipid Research, Key Laboratory of Molecular Biology on Infectious Diseases, Chongqing Medical University, Peoples Republic of China; and 2Centre for Nephrology, Royal Free and University College Medical School, London, United Kingdom

Submitted 4 May 2007 ; accepted in final form 11 July 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
LDL receptor (LDLr) is widely expressed in both liver and peripheral tissue. We aimed to clarify tissue-specific regulation of LDLr in hepatic cell line (HepG2) cells and human kidney mesangial cells (HMCs) under physiological and inflammatory conditions. We have demonstrated that the concentration of LDL required for 50% inhibition of LDLr mRNA expression (IC50) in HepG2 was 75 µg/ml, but only 30 µg/ml in HMCs. The concentration of mevastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, which achieved 200% upregulation of LDLr (UC200) in HepG2 cells, was 0.7 µM, which is much lower than 2.8 µM in HMCs. Inflammatory stress increased IC50 to 80 and 75 µg/ml of LDL, UC200 to 2.8 µM, and 4.2 µM of mevastatin in HepG2 and HMCs. There was obvious sterol-regulatory element binding protein cleavage-activating protein accumulation in the Golgi in HepG2 cells, but not in HMCs in the presence of high concentration of LDL. IL-1beta further increased sterol-regulatory element binding protein cleavage-activating protein accumulation in HepG2 and HMCs in the presence of high concentration of LDL. These results indicate that LDLr in HepG2 cells have a relative resistant phenotype for downregulation, while LDLr in HMCs is very sensitive for downregulation. Inflammatory cytokine disrupts LDLr negative feedback regulation induced by intracellular cholesterol in both cell types, to a greater degree in HMCs, which could be one reason why HMCs are more prone to become foam cells under inflammatory stress. Inflammation also causes statin resistance; therefore, a high concentration of statin may be required to achieve the same biological effect.

inflammation; kidney mesangial cells; hepatic cells

LDL RECEPTOR (LDLR) IS WIDELY expressed in both liver and kidney. It is the primary receptor for binding and internalization of plasma-derived LDL cholesterol and regulates plasma LDL concentrations. Brown and colleagues (26) observed that LDLr activity is normally under tight metabolic control via a feedback system that is dependent on intracellular cholesterol concentration. This system maintains a constant levels of cholesterol in hepatocytes and other cells by controlling both the rate of cholesterol uptake from LDL and the rate of cholesterol synthesis (4). This feedback regulation is controlled through specific interactions of the sterol-regulatory element (SRE)-1 of the LDLr promoter (3, 9) and a family of SRE-binding proteins (SREBP), namely, SREBP-1 and SREBP-2 (25, 27, 28, 31). 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, SREBP associates with another transmembrane protein, SREBP cleavage-activating protein (SCAP), that provides conditional chaperone activity to the SREBPs (5, 16, 17). SCAP contains a cholesterol-sensing domain that responds to the depletion of sterol with activation of SCAP-SREBP-transporting activity (10, 24, 30). When the cells are overloaded with cholesterol, the SCAP-SREBP complex remains in an inactive form in the ER through active repression by cholesterol and oxysterols, and LDLr gene transcription is maintained at a minimal constitutive levels. The concentration of LDL cholesterol for 50% inhibition of LDLr is termed IC50, which represents sensitivity of LDLr downregulation. In contrast, when cells are depleted of cholesterol, SCAP transports SREBP to the Golgi, where the NH2-terminal transcription-activation domain of the SREBP is released from the precursor protein through specific proteolytic cleavages. The active form of the SREBP translocates to the nucleus, binds to its cognate SRE-1 site, and activates transcription of the LDLr gene.

Statins have revolutionized the treatment of high blood cholesterol levels in humans by decreasing intracellular cholesterol levels, thereby upregulating LDL cholesterol uptake via LDLr primarily on hepatocytes. The concentration for 200% upregulation of LDLr (UC200) represents the sensitivity of LDLr upregulation. If the upregulation of LDLr after statin administration was the same in both hepatic and peripheral tissues, then statins should increase (feedback regulation) the expression of LDLr and LDL cholesterol uptake in both hepatic and peripheral cells to the same degree. This would cause pathogenic lipid accumulation, especially in peripheral cells, since excess LDL cholesterol can be converted into bile salt in liver; therefore, normally there is no foam cell formation in the liver. However, statins do not increase LDL cholesterol uptake in the peripheral cells, suggesting that upregulation of LDLr induced by statin occurs mainly in liver cells. On the other hand, if the sensitivity of LDLr feedback regulation mediated by intracellular cholesterol were the same in both hepatic and peripheral tissues, LDLr may shut down immediately after the cells started taking LDL cholesterol from plasma. Thus liver may not be able to maintain efficient plasma cholesterol homeostasis under such metabolic stress. It suggests that there is a different threshold of LDLr regulation in liver and peripheral cells under metabolic stress.

Recent experimental and clinical evidence suggest 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 and others (6, 15). Cardiovascular risk is increased in chronic inflammatory states, up to 33-fold in patients with renal failure and allografts, and 50-fold in patients with immune dysregulation (e.g., systemic lupus erythematosus). In hemodialysis patients, the higher risk of death from cardiovascular disease is, surprisingly, associated with low plasma cholesterol ("reverse epidemiology") (12). 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 LDLr feedback regulation in these peripheral cells, allowing unregulated uptake of cholesterol in the peripheral cells causing foam cell formation (20, 22). It suggests that inflammation may increase the threshold for LDL uptake in peripheral cells, such as HMCs and VSMCs.

LDLr feedback regulation has been investigated in Chinese hamster ovary and fibroblast-like cell types; however, tissue-specific regulation in human cells remains unclear. Liver is a central organ for cholesterol homeostasis and regulates plasma LDL cholesterol levels through the surface expression of LDLr, which mediates plasma LDL and VLDL clearance (1, 11, 23). Quantitatively, liver plays a major role in the regulation of plasma cholesterol concentration. LDLr also widely expresses in peripheral cells, such as VSMCs or HMCs. We have raised the possibility that liver and peripheral cells have different capacities and sensitivities to LDL cholesterol uptake and cholesterol depletion. Under physiological conditions, the LDLr in HMCs should be much more sensitive for downregulation by cholesterol loading (expressed a low IC50) than in HepG2 cells and results in a lower threshold for LDL uptake, while LDLr in HepG2 cells have a relative resistant phenotype for downregulation. This prevents HMCs from internalizing excess LDL cholesterol and allows liver to maximally take LDL cholesterol and maintain plasma cholesterol level. However, inflammatory stress changes the phenotype of LDLr regulation from sensitive to resistant for cholesterol-mediated downregulation. This increases the threshold for LDL uptake in HMCs and makes HMCs functionally like liver in that they maximize LDL cholesterol uptake, resulting in foam cell formation. An increased threshold for LDL uptake in liver and peripheral cells may lower plasma cholesterol. This could be a mechanism for the paradoxical association of low cholesterol with cardiovascular disease in this group of patients.

In this study, we investigated the differences between HMCs and human hepatic cells regarding LDLr feedback regulation under physiological conditions, and how inflammatory stress affects LDLr feedback regulation in a tissue-specific manner.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. Established stable HMC line cells (kindly donated by Dr. J. D. Sraer, Hôpital Tenon, Paris, France) and hepatoma cell HepG2 were used in all experiments. HMCs were cultured in growth medium containing RPMI-1640 medium, 5% fetal calf serum, 2 mmol/l glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 2.5 µg/ml amphotericin, 5 µg/ml insulin, 5 µg/ml human transferrin, and 5 ng/ml sodium selenite. HepG2 was cultured in growth medium containing DMEM/F-12 medium, 10% fetal calf serum, 2 mmol/l glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. All experiments were carried out in serum-free RPMI-1640 medium containing 0.2% BSA, 2 mmol/l glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. All reagents for cell culture and mevastatin were obtained from Sigma (UK). IL-1beta were obtained from R&D Systems (Europe, Abingdon, UK). LDL was isolated from plasma of healthy human volunteers by sequential ultracentrifugation (21). Our study protocols were approved by the Institutional Review Board of Royal Free and University College Medical School and adhered to the tenets of the Declaration of Helsinki for experiments involving human samples.

Measurement of intracellular cholesterol. The method was based on the cholesterol enzymatic assay described by Gallo et al. (7) and Gamble et al. (8). HMCs and HepG2 in six-well plates were cultured in serum-free medium without (control) or with 25–200 µg/ml of LDL in the absence or presence of 20 ng/ml of IL-1beta for 24 h. Cells were then washed twice in PBS, intracellular lipids were extracted in isopropanol and dried under vacuum, and total cholesterol (TC), free cholesterol (FC), and cholesterol ester (CE) content were measured by enzymatic assay (CE = TC – FC) and normalized by total cell proteins determined by the modified Lowry assay (13).

Total RNA isolation and real-time quantitative PCR. Total RNA was isolated from cultured cells by the guanidinium method. Total RNA (500 ng) was used as a template for RT using an RNA RT kit from ABI (Applied Biosystems, Warrington, Cheshire, 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 deoxynucleoside triphosphate, 2.5 µmol/l random hexamers, 20 units RNAsin, and 50 units of Moloney-murine leukemia virus RT. Incubations were performed in a DNA Thermal Cycler 9700 (Applied Biosystems, Foster City, CA) for 10 min at room temperature, followed by 30 min at 42°C and 5 min at 99°C. Real-time quantitative PCR was performed on a TaqMan ABI 7000 Sequence Detection System using TaqMan SYBRgreen PCR Master Mix (Applied Biosystems, Warrington, UK). Thermal cycler conditions contained holds for 2 min at 50°C and 10 min at 95°C, followed by 40 cycles of 20 s at 95°C and 20 s at 55°C and 30 s at 72°C. Relative amount of mRNA was calculated using the comparative threshold cycle method. beta-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: LDLr upper 5'-GTGTCACAGCGGCGAATG- 3', lower 5'-CGCACTCTTTGATGGGTTCA-3'; SREBP-2 upper 5'-CCGCCTGTTCCGATGTACAC-3', lower 5'-TGCACATTCAGCCAGGTTCA-3'; and beta-actin upper 5'-CCTGGCACCCAGCACAAT- 3', lower 5'-GCCGATCCACACACGGAGTACT -3'. Primers were designed with Primer Express Software version 2.0 System (Applied Biosystems).

Western blot analysis. Cells from T75 cm2 culture flasks were pooled and allowed to swell at 4°C for 30 min in 300 µl of lysis buffer (10 mmol/l HEPES, pH 7.9, 10 mmol/l KCl, 1.5 mmol/l MgCl2, 0.5 mmol/l dithiothreitol, 0.4% Nonidet P-40, 0.5 µmol/l phenylmethylsulfonyl fluoride, and 1 µg/ml of antipain, laupeptin, bestatin, and chymostatin) and then passed through a 23-gauge needle 20 times before centrifugation at 14,000 g at 4°C for 20 min. The supernatant from this spin was used as the whole cell extract. Identical amounts of total protein from whole cell extract were denatured and then subjected to electrophoresis on a 5% stacking and 8% separating SDS polyacrylamide gel in a Bio-Rad mini Protein II apparatus. 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 (phosphate-buffered saline/1% Tween 20). The membrane was incubated with chicken anti-human LDLr polyclonal antibody (Abcam, Cambridge, UK) for 1 h in antibody dilution buffer (1% BSA in PBST) followed by three 5-min washes in PBST. A goat anti-chicken horseradish peroxidase-labeled antibody (Abcam) 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, and autoradiography was performed on Hyperfilm ECL (Amersham Bioscience, Little Chalfont, UK). Band intensity volumes (intensity x area) on fluorograms were measured by Quantitiy One software (Bio-Rad, Hemel Hempstead, UK).

Confocal microscopy. HepG2 and HMCs were plated in chamber sliders (1 x 104 cells/well) and incubated in normal growth medium to attach, cultured in serum-free medium for 24 h, and then replaced by fresh serum-free medium alone or serum-free medium with 200 µg/ml of LDL in the absence or presence of IL-1beta (20 ng/ml). After 24-h incubation, the cells were washed with PBS, fixed by 5% formalin solution for 30 min, permeablized by 0.25% of Triton X-100 for 15 min, and stained with rabbit polyclonal anti-human SCAP antibody produced by immunizing rabbits with the synthetic peptide PVDSDRKQGEPTEQC in our laboratory (20) and mouse anti-Golgin antibody (Molecular Probes, Paisley, UK) for 1 h at room temperature. The cells were washed three times using PBS/Tween 20 over 15–30 min; finally visualization procedures were completed by dual-staining with goat anti-rabbit Fluor (green) 488 for SCAP and goat anti-mouse Fluor (red) 594 for Golgin (Molecular Probes) for 1 h at room temperature. Cells were examined with a confocal microscope (Bio-Rad).

Statistical analysis. In all experiments, data were evaluated for significance by one-way ANOVA using Minitab software. Data were considered significant at P ≤ 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
LDL loading dose dependently inhibited LDLr mRNA expression in both HepG2 and HMCs, but to a greater degree in HMCs (Fig. 1A). We calculated the concentration of LDL for IC50 mRNA using nonlinear regression analysis in both HepG2 and HMCs. The IC50 in HepG2 was 75 µg/ml, but only 30 µg/ml in HMCs. Intracellular cholesterol depletion achieved by incubation with mevastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, increased LDLr mRNA expression levels in both cells, but HepG2 cell was more sensitive than HMCs in this regard (Fig. 1B). The concentration of mevastatin for UC200 was calculated by nonlinear regression analysis. The UC200 in HepG2 cells was 0.7 µM, which is much lower than 2.8 µM in HMCs. This data suggest that LDLr in HepG2 cells is easily upregulated by cholesterol depletion, while LDLr in HMCs is very sensitive to downregulation induced by cholesterol loading.


Figure 1
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  		Fig. 1. Effect of LDL loading or cholesterol depletion on LDL receptor (LDLr) mRNA levels in HepG2 and human mesangial cells (HMCs) under physiological condition. Both HepG2 and HMCs were incubated in serum-free medium for 24 h. The medium was then replaced by fresh serum-free medium with LDL (0, 25, 50, 100, 200 µg/ml; A) or with mevastatin (0, 1, 10, 100 µM; B) for 24 h at 37°C. LDLr mRNA was determined following the threshold cycle ({Delta}CT) protocol for RT-PCR, as described in MATERIALS AND METHODS. beta-Actin served as the housekeeper gene. Results are expressed as means ± SD of four independent experiments (n = 4). The concentration of LDL for 50% inhibition of LDLr mRNA (IC50) and the concentration of mevastatin for 200% upregulation of LDLr mRNA (UC200) were calculated by nonlinear regression analysis.

 
Furthermore, we examined the sensitivities of LDLr regulation to the LDL loading and cholesterol depletion under inflammatory stress. IL-1beta alone increased LDLr mRNA expression levels in both HepG2 and HMCs, but to a greater degree in HepG2 cells, and overrode the suppression of the mRNA expression reduced by a high concentration (200 µg/ml of LDL) of LDL in both cell types (Fig. 2A). We also studied the inhibitory effect of different concentrations of LDL on LDLr in the presence of IL-1beta (Fig. 2B). The concentrations of LDL for IC50 were 80 and 75 µg/ml in HepG2 and HMCs, respectively, which are higher than IC50 without inflammatory stress, especially in HMCs, suggesting that inflammatory cytokine changed LDLr from the sensitive phenotype to the resistance phenotype for downregulation induced by high concentration of LDL in HMCs. As a consequence, inflammation results in a CE accumulation and foam cell formation in both cell types, to a great degree in HMCs, as evidenced by intracellular CE assay (Fig. 2C). Interestingly, in the presence of IL-1beta, the concentration of mevastatin that achieved UC200 in HepG2 cells was 2.8 µM, and 4.2 µM in HMCs (Fig. 3), much higher than 0.7 µM in HepG2 and 2.8 µM in HMCs in the absence of inflammation, as shown in Fig. 1B. This suggests inflammation also causes statin resistance.


Figure 2
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  		Fig. 2. Effect of LDL loading on LDLr mRNA levels and intracellular cholesterol ester in HepG2 and HMCs under inflammatory condition. Both HepG2 and HMCs were incubated in serum-free medium for 24 h. The medium was then replaced by fresh serum-free medium alone (control) or with LDL (200 µg/ml) in the absence or presence of 20 ng/ml IL-1beta for 24 h at 37°C (A), or the medium was then replaced by fresh serum-free medium adding 20 ng/ml IL-1beta alone (control) or with different concentrations of LDL (25, 50, 100, 200 µg/ml) in the presence of 20 ng/ml of IL-1beta for 24 h at 37°C (B); or the medium was then replaced by fresh serum-free medium alone (control) or with different concentrations of LDL (25, 50, 100, 200, µg/ml) in the absence or presence of 20 ng/ml of IL-1beta for 24 h at 37°C (C). LDLr mRNA from A and B were determined following the {Delta}CT protocol for real-time RT-PCR, as described in MATERIALS AND METHODS. beta-Actin served as the housekeeper gene. Results are expressed as means ± SD of four independent experiments (n = 4). The concentration of LDL for IC50 was calculated by nonlinear regression analysis. The cells from C were collected for cholesterol ester assay as described in MATERIALS AND METHODS. The results were normalized for total cellular protein and represent means ± SD from four independent experiments (n = 4). *P < 0.05 vs. control. **P < 0.05 vs. 200 µg/ml LDL (A) or LDL (C) alone groups. Data were evaluated for significance by one-way ANOVA using Minitab software.

 

Figure 3
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  		Fig. 3. Effect of cholesterol depletion on LDLr mRNA levels in HepG2 and HMCs under inflammatory condition. HepG2 cells and HMCs were incubated in serum-free medium for 24 h. The medium was then replaced by fresh serum-free medium adding 20 ng/ml IL-1beta alone (control) or with different concentration s of mevastatin (1, 10, 100 µM) in the presence of 20 ng/ml of IL-1beta for 24 h at 37°C. LDLr mRNA was determined following the {Delta}CT protocol for RT-PCR, as described in MATERIALS AND METHODS. beta-Actin served as the housekeeper gene. Results are expressed as means ± SD of four independent experiments (n = 4). The concentration of mevastatin for UC200 was calculated by nonlinear regression analysis.

 
Western blotting demonstrated that LDL inhibited protein expression levels in both HepG2 and HMCs. IL-1beta (5 ~ 20 ng/ml) overrode the suppression of the LDLr protein expression reduced by LDL in both cell types (Fig. 4). LDLr protein levels were increased in both HepG2 and HMCs during cholesterol depletion state. In the presence of IL-1beta (5 ~ 20 ng/ml), LDLr protein expression levels were further increased in both cell types (Fig. 5). These data are consistent with mRNA data.


Figure 4
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  		Fig. 4. Effect of LDL loading on LDLr protein expression in the presence of IL-1beta. HepG2 (A) and HMCs (B) were incubated in serum-free medium for 24 h. The medium was then replaced by fresh serum-free medium alone (control) or with LDL (200 µg/ml) in the absence or presence of different concentrations of IL-1beta (5, 10, 10 ng/ml) for 24 h at 37°C. The cell extracts were prepared and subjected to SDS-PAGE, followed by immunoblotting analysis using anti-human LDLr and anti-actin antibodies, as described in MATERIALS AND METHODS. One of three representative experiments is shown. Data are the means ± SD of band intensities volume/actin intensities volume from three different experiments (n = 3). *P < 0.05 vs. control, **P < 0.05 vs. 200 µg/ml LDL alone group. Data were evaluated for significance by one-way ANOVA using Minitab software.

 

Figure 5
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  		Fig. 5. Effect of cholesterol depletion on LDLr protein expression in the presence of IL-1beta. HepG2 (A) and HMCs (B) were incubated in serum-free medium for 24 h. The medium was then replaced by fresh serum-free medium alone (control) or with mevastatin (100 µM) in the absence or presence of different concentrations of IL-1beta (5, 10, 20 ng/ml) for 24 h at 37°C. The cell extracts were prepared and subjected to SDS-PAGE, followed by immunoblotting analysis using anti-human LDLr and anti-actin antibodies, as described in MATERIALS AND METHODS. One of three representative experiments is shown. Data are the mean ± SD of band intensities volume/actin intensities volume from three different experiments (n = 3). *P < 0.05 vs. control. **P < 0.05 vs. 100 µM mevastatin alone group. Data were evaluated for significance by one-way ANOVA using Minitab software.

 
In addition, LDL reduced SREBP-2 mRNA levels. However, IL-1beta overrode the suppression of SREBP-2 induced by high concentrations of LDL (Fig. 6). By costaining cells with anti-human SCAP and anti-human Golgin antibodies, we demonstrated that LDL inhibited SCAP translocation from the ER to the Golgi. Interestingly, there was obvious SCAP accumulation in the Golgi, even in the presence of high concentration of LDL in HepG2 cells rather than HMCs (Fig. 7A). IL-1beta further increased or imitated SCAP accumulation in HepG2 or HMCs, respectively, by enhancing translocation of SCAP from the ER to the Golgi in the presence of high concentration of LDL. (Fig. 7).


Figure 6
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  		Fig. 6. Effect of loading LDL on sterol-regulatory element binding protein (SREBP)-2 mRNA levels in the presence of IL-1beta. Both HepG2 and HMCs were incubated in serum-free medium for 24 h. The medium was then replaced by fresh serum-free medium alone (control) or with 200 µg/ml of LDL in the absence or presence of IL-1beta (20 ng/ml) for 24 h at 37°C. SREBP-2 mRNA was determined following the {Delta}CT protocol for RT-PCR, as described in MATERIALS AND METHODS. beta-Actin served as the housekeeper gene. Results are expressed as means ± SD of four independent experiments (n = 4). *P < 0.05 vs. 200 µg/ml LDL alone group. Data were evaluated for significance by one-way ANOVA using Minitab software.

 

Figure 7
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  		Fig. 7. Effect of loading LDL on the translocation of SREBP cleavage-activating protein (SCAP)-SREBP complex from the endoplasmic reticulum to Golgi in the presence of IL-1beta. Both HepG2 (A) and HMCs (B) were incubated in serum-free medium for 24 h. The medium was then replaced by fresh serum-free medium alone (control) or with 200 µg/ml of LDL in the absence or presence of IL-1beta (20 ng/ml) for 24 h at 37°C. The translocation of SCAP-SREBP complex was investigated using confocal microscopy after dual staining with anti-human SCAP antibody and anti-Golgin antibody, as described in MATERIALS AND METHODS.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
HMCs have similar physiological functions to VSMCs, including synthesis and secretion of extracellular matrix, eicosanoids, growth factors, and cytokines, and have been used for a large number of studies on the biochemistry and molecular biology of glomerulosclerosis, which shares similar pathogenesis with atherosclerosis (14, 15). HepG2 also has been well characterized: producing most of the plasma proteins, has biosynthetic capabilities similar to those of normal hepatocytes (2), and retains cell surface receptors (18). In this study, we demonstrate that LDLr in HepG2 and HMCs have different capacities and sensitivities to LDL cholesterol loading and cholesterol depletion. This apparent tissue specificity is based on the sensitivity and threshold of intracellular cholesterol concentration needed for up- and downregulation of LDLr in different tissues.

We showed that, under physiological conditions, LDLr in HepG2 cells was more sensitive for upregulation and relatively resistant for downregulation, while it is opposite in HMCs, as evidenced by showing that the concentration of statin for UC200 is much lower in HepG2 cells than in HMCs, and IC50 is much high in HepG2 cells than in HMCs. This implies that liver cells have a high threshold, which gives liver cells huge capacity for LDL uptake and maintenance of a constant plasma cholesterol level. Since excess cholesterol can be converted into bile salt, normally there is no foam cell formation in liver. This mechanism also ensures that, under physiological conditions, HMCs only take up the amount of LDL cholesterol via LDLr to maintain cell function, since LDLr is very sensitive to downregulation after cholesterol loading, which may also prevent cholesterol overloading in kidney. Hence statins do not increase LDL cholesterol uptake via LDLr upregulation in peripheral cells and mainly affect hepatic LDLr.

We observed that inflammation changed the capacity and sensitivity of LDLr in both HepG2 cells and HMCs. IL-1beta significantly stimulated LDLr mRNA expression in both HepG2 cells and HMCs and overrode the suppression of LDLr induced by LDL loading, suggesting that inflammation disrupted the sensitive downregulation of LDLr in both cells in the presence of LDL, which permits intracellular accumulation of unmodified LDL, causing foam cell formation. Interestingly, inflammation changes LDLr phenotype from the sensitive manner to the resistance manner for downregulation induced by high concentration of LDL in HMCs. The IC50 in HMCs from 30 µg/ml is increased to 75 µg/ml, which closes to IC50 in HepG2 cells (80 µg/ml) under inflammatory stress. This suggests that peripheral cells may be functionally like liver cells regarding LDLr-mediated cholesterol uptake under inflammatory stress. Although the changes of LDLr expression may not totally account for intracellular cholesterol accumulation, it has been demonstrated that the capacity of LDL binding and internalization usually parallel with LDLr levels based on the previous works. Using similar experimental condition, we have also demonstrated the specificity of the LDLr pathway in inflammatory-mediated lipid accumulation by showing that MB47, which directly blocks the binding sites of human Apo B 100 to LDLr, reduced cytokine-mediated CE accumulation by 84% (20). This suggests that the LDLr is one of the main pathways for lipid accumulation under conditions of inflammatory stress. We have also demonstrated that inflammation impairs cholesterol efflux in peripheral cells (19). Therefore, the peripheral cells without excess by converting cholesterol into bile salt could be easily converted to foam cells. The data, as shown in Fig. 2C, also demonstrated the accumulation of intracellular CE in HMCs is more sensitive to IL-1beta stress than it is in HepG2 cells, suggesting that HMCs are more sensitive to inflammatory stress than HepG2 cells. It may be that long-term chronic inflammation disrupts the sensitivity of feedback regulation in peripheral cells, including HMCs and VSMCs; therefore, LDL cholesterol is not only transported to the liver, but also to the peripheral tissues with impaired cholesterol efflux under inflammatory stress (19), which cause excess cholesterol accumulation, foam cell formation, and low LDL cholesterol concentration, as observed in hemodialysis patients.

Furthermore, IL-1beta causes a statin resistance in both cell types, as shown in Fig. 3; therefore, higher concentrations of statin may be required to achieve the same degree of biological effect in each cell type. It may also explain why statins, which provide cardiovascular protection in the general population, do not reduce cardiovascular mortality in dialysis patients with diabetes in chronic inflammatory states (29).

We proceeded to investigate the molecular mechanisms by which inflammatory cytokines override the normal cholesterol-mediated suppression of the LDLr induced by a high concentration of LDL. In particular, we examined the mRNA expression of SREBP-2, as well as SCAP protein intracellular translocation, between the ER and Golgi under the influence of inflammation. We demonstrated that SREBP-2 is transcriptionally upregulated by inflammatory cytokines, even in the presence of a high concentration of LDL. This could result from enhanced SCAP protein translocation to the Golgi under inflammatory stress, as demonstrated in Fig. 7, suggesting that IL-1beta disrupts normal SCAP trafficking between the ER and Golgi in both HepG2 cells and HMCs. Interestingly, there was more SCAP accumulation in the Golgi in HepG2 cells than HMCs, which initiates LDLr expression, even in the presence of a high concentration of LDL, a possible reason why LDLr in HepG2 cells is more resistant to suppression. It suggests that SCAP translocation is important for the sensitivity of LDLr regulation in different tissues.

Taken together, liver and renal cells have different capacities and sensitivities to cholesterol loading and depletion. Liver cells have a high capacity for LDL cholesterol uptake, since LDLr is not sensitively suppressed after cholesterol loading. However, LDLr feedback downregulation in HMCs is normally very sensitive to LDL loading. Inflammatory stress increases the threshold of LDLr level in liver and to a great extent in renal cells. This could be one reason why HMCs are more prone to become foam cells under inflammatory stress. The pattern of LDLr regulation in HMCs may also apply to the macrophages, since mesangial cells share certain characteristics with macrophages. Overloading of cholesterol in liver under inflammatory stress may contribute nonalcohol fatty liver and insulin resistance. Moreover, inflammation also causes statin resistance; therefore, a high concentration of statin may be required to achieve the same biological effect.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
We acknowledge the support of the Moorhead Trust, the Royal Free Hospital Special Trustees Grant-115 through Dr. Zac Varghese, the National Natural Science Foundation of China (Key Program, no. 30530360), and the National Basic Research Program of China (2006CB503907).


   FOOTNOTES
 

Address for reprint requests and other correspondence: X. Z. Ruan, Centre for Nephrology, Royal Free and Univ. College Medical School, Royal Free Campus, Rowland Hill St., London NW3 2PF, UK (e-mail: x.ruan{at}medsch.ucl.ac.uk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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