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. 2021 Mar 1;28(3):261–270. doi: 10.5551/jat.55384

Zinc Increases ABCA1 by Attenuating Its Clearance Through the Modulation of Calmodulin Activity

Rui Lu 1,✉, Takahiro Ishikawa 1, Mamoru Tanaka 1, Tomoe Tsuboi 1, Shinji Yokoyama 1
PMCID: PMC8049148  PMID: 32581187

Abstract

Aim: We previously revealed that Ca++-activated calmodulin binds to ABCA1 by the region near the PEST sequence and retards its calpain-mediated degradation to increase HDL biogenesis. Calmodulin activity is reportedly modulated also by other nutritional divalent cations; thus, we attempted to determine whether Zn++ is involved in the regulation of ABCA1 stability through the modulation of calmodulin activity.

Methods: The effects of Zn++ on ABCA1 expression was investigated in J774 mouse macrophage cell-line cells and HepG2 human hepatoma cell-line cells.

Results: Zn++ increased ABCA1 expression, not by increasing the mRNA but by attenuating its decay rate, more prominently in the presence of cAMP. Accordingly, it enhanced cell cholesterol release with extracellular apolipo-protein A-I. Calmodulin binding to ABCA1 was increased by Zn++ and Ca++. Zn++ suppressed calpain-mediated hydrolysis of the peptide of ABCA1 cytosolic loop, including the PEST sequence and the calmodulin-binding site, in a calmodulin-dependent fashion, in the presence of the minimum amount of Ca++ to activate calpain, but not calmodulin. Calpain activity was not directly inhibited by Zn++ at the concentration for enhancing calmodulin binding to ABCA1.

Conclusion: Nutritional divalent cation Zn++ is involved in the regulation of ABCA1 activity and biogenesis of HDL through the modulation of calmodulin activity. The results were consistent with previous clinical findings that Zn++ increased plasma HDL in the conditions of sympathetic activation, such as type 2 diabetes and chronic hemodialysis.

Keywords: Zinc, ATP binding cassette transporter A1, Calmodulin, Calpain, High density lipoprotein

Introduction

High-density lipoprotein (HDL) plays a central role in the catabolic pathway of cholesterol transport from the peripheral tissues to the liver for its conversion to bile acids. Moreover, it is considered an anti-atherogenic factor to prevent cholesterol accumulation in the vascular walls. HDL is biogenerated with helical apolipoproteins, such as apoA-I and cellular lipid1), mediated by a membrane protein, ATP-binding cassette transporter A1 (ABCA1)2–4). In the organ-specific ABCA1 knockout study in mice, the liver was found to be the major source of plasma HDL5), whereas the macrophage ABCA1 activity may be more directly responsible for atherogenesis, although its contribution to plasma HDL concentration is negligible6).

ABCA1 gene expression is upregulated by the liver X receptor (LXR) sensing cellular cholesterol levels in general7), and it is also negatively regulated by the factors such as protein kinase D and activator protein-2 system8, 9). This dual regulation may be more important in hepatocytes, where the ABCA1 gene expression is downregulated by sterol regulatory element-binding protein 2, in addition to the upregulation by LXR, perhaps to prevent backflow of cholesterol recovered from the extrahepatic tissues by HDL10, 11).

ABCA1 expression is also regulated by posttranslational proteolytic degradation by calpain following its endocytic internalization12). This process is interfered by the interaction with helical apolipoproteins13, 14) to enable ABCA1 resistance to calpain and hence enhance its recycling back to the surface15). Caveolin-1 interacts with ABCA1 to facilitate its internalization and degradation, and probucol oxidative products interfere with this interaction to retard its internalization and degradation16–18). Thus, ABCA1 expressed in cell surface seems responsible for this reaction and acts as one of the major rate-limiting factors for plasma HDL concentration19).

Alternatively, calmodulin (CaM) interacts with ABCA1 at the region near the PEST sequence and retards its calpain-mediated degradation in a Ca++-dependent manner20). CaM is a 16.8-kDa protein involved in calcium-signal transduction by interacting with various target proteins to modulate their functions that are activated by calcium ion21, 22). Besides calcium, CaM has been shown to be activated by interacting with other divalent cations, such as La++, Tb++, Pb++, Sn++, Sr++, Hg++, Cd++, Zn++, and Mn++23–28). CaM provides four calcium-binding sites, and these ions are thought to compete for these sites29). Among these cations, Zn++ has been suggested to act cooperatively with Ca++ in some actions of CaM, such as activation of CaM-dependent protein kinase II30, 31). Zinc is an essential micronutrient for animals; it is necessary for the maintenance of membrane structure/function and for the activities of over 200 metalloenzymes32). Thus, it is crucial to investigate the effects of this metal nutrient on ABCA1 function for the regulation of plasma HDL metabolism. Many clinical studies indicated that zinc supplementation may decrease or induce no change in plasma HDL concentration. However, large-scale meta-analysis studies revealed that zinc supplementation increases HDL in type 2 diabetic population and in chronic hemodialysis patients, but it decreases HDL in normal subjects33–35).

We thus examined the effects of Zn++ on the stability and activity of ABCA1 through the modulation of the CaM function by using mouse macrophages and human hepatoma cell-line models in order to solve the controversial question on its effect on plasma HDL. Zn++ increased ABCA1 by decreasing its degradation rate, more prominently in the presence of cAMP. It enhanced CaM binding to ABCA1 at the site near the PEST sequence to interfere with its calpain-mediated hydrolysis in the presence of the minimum amount of Ca++ to activate calpain.

Methods

Cell and Culture Conditions

Mouse macrophage cell-line cells J774 were maintained in RPMI 1640 medium (Sigma-Aldrich) containing 10% fetal bovine serum (FBS) in a humidified atmosphere of 5% CO2 and 95% air at 37°C36). The cells were seeded in culture plates at a density of 3 × 106 cells and cultured for 1 day before use. Human hepatoma cell-line cells HepG2 were cultured with DMEM medium (Sigma-Aldrich) containing 10% FBS. In a humidified atmosphere of 5% CO2 and 95% air at 37°C19), cells were seeded in culture plates at a density of 2.5 × 106 and cultured for 1 day before use. Peritoneal macrophages were collected from 6-week-old male Balb/C mice, using 12 mice for one six-well plate37). Ice-cold phosphate-buffered saline (PBS) (7 mL) containing 3% FBS was injected into the mouse peritoneal cavity followed by gentle massage of the peritoneum to dislodge any attached cells. The peritoneal fluid was collected and spun at 1500 RPM for 8 min to precipitate the cells. After discarding the supernatant and resuspending the cells in RPMI media containing 10% FBS, the cells were cultured at 37°C in 5% CO2 for 2 h and washed with PBS once. The medium was changed to RPMI containing 10% FBS, and the cells were cultured at 37°C in 5% CO2 for 48 h before use. HepG2 cells were preincubated for 18 h with DMEM containing 0.2% BSA and with and without 0.3 mM cAMP, washed with PBS, and incubated overnight in DMEM containing 0.1% BSA and various concentrations of ZnCl2, with and without 0.15 mM cAMP38). The effects of divalent cations Ca++ and Zn++ were examined using various concentrations of CaCl2 or ZnCl2. Normal extracellular concentration of Zn++ in vivo may be 5 to 15 µM39, 40), and its intracellular concentration is some 0.01 µM41); the chosen range was 0.01–10 µM.

Analyses of ABCA1 Protein

Cell proteins were analyzed by immunoblotting. Cells were lysed in the lysis buffer containing Protease Inhibitor Cocktail (Sigma-Aldrich) and subjected to SDS-PAGE and immunoblotting analysis by using the monoclonal antibody against ABCA1 (MABI98-7) generated in rats against peptide CNFAKDQS-DDDHLKDLSLHKN, a common sequence of the C-terminus of human and mouse proteins, at the MAB Institute (Yokohama, Japan), as previously described15, 16, 20, 42). ABCA1 degradation was observed as a decrease in protein in the presence of cycloheximide to block protein synthesis, as previously described15, 16). The experiments were conducted in the presence or absence of 0.15 mM cAMP.

Real-Time Quantitative PCR

The levels of ABCA1 mRNA was measured using synthetic oligonucleotide primers 5′-ACA ATA GTT GTA CGA ATA GCA GGG-3′, 5′-CTC ATC CTG TAG AAA AGA TGT GAG-3′ for J774 cells and 5′-GAA CTG GCT GTG TTC CA TGA T-3′, 5′-GAT GAG CCA GAC TTC TGT TGC-3′ for HepG2 cells, as previously reported17), in a 7300 Real-Time PCR System (Applied Biosystems). Total RNA was extracted using ISOGEN (Nippon Gene) and reverse-transcribed into cDNA using iScript™ cDNA Synthesis Kit (BIO-RAD). The cDNA was used as a template to produce PCR amplicats using SYBR PCR Permix Ex Taq Kit (TaKaRa).

Release of BODIPY-Cholesterol from Cells by Apolipoprotein (apo) A-1

ApoA-1 was isolated from human HDL fraction, as described previously43). The release of cell cholesterol by apoA-I was estimated by using fluorescence-labeled cholesterol, boron dipyrromethene difluoride linked to sterol carbon-24 (BODIPY-cholesterol), as described previously44). Briefly, J774 macrophages or HepG2 cells were incubated for 1 h with MEM-HEPES labeling medium containing 25 µM BODIPY-cholesterol and 100 µM unlabeled cholesterol conjugated with 10 mM methyl-β-cyclodextrin and then washed with MEM-HEPES media containing 0.1% BSA (Sigma-Aldrich). The cells were then equilibrated with RPMI (J774) or DMEM (HepG2) containing 0.2% BSA and 0.3 mM cAMP for 18 h. An ACAT inhibitor (Sandoz, 58-035) was present throughout these periods. After equilibration, the cells were washed with MEM-HEPES buffer and incubated with MEM-HEPES media containing various concentrations of ZnCl2 in the presence of 0.15 mM cAMP and with and without 10 µg/mL apoA-I for 4 h. At the end of the incubation period, the conditioned media were removed and filtered through a 0.45-µm filter, and fluorescence intensity was recorded using a BioTek plate reader by using excitation at 482 nm and emission at 515 nm.

CaM-Agarose Binding Assay

Lysate (200 µg as protein) of the cells pretreated with various concentrations of ZnCl2 was incubated with 30 µl of 50% calmodulin-agarose (Merck) suspension in Tris-HCl saline for 3 h at 4°C. Protein bound to the beads was eluted with the SDS-PAGE sample buffer and analyzed for ABCA1 by immunoblotting using an anti-ABCA1 antibody20).

Calpain Reaction on the ABCA1 Peptide with the PEST and CaM-Binding Sequences

In order to examine the effect of CaM binding on calpain-mediated degradation of ABCA1, the peptide corresponding to 1213 to 1349 amino acid residues of ABCA1, including the PEST sequence and 1-5-8-14 motif of the CaM recognition sequence, was expressed as a fusion protein with glutathione S-transferase (GST) (GST–CaM–PEST) in Escherichia coli BL-21 DE3 (Nippon Gene). It was purified from the cell lysates and solubilized with 50-mmol/L Tris-HCl containing 10-mmol/L glutathione as previously described20). GST–CaM–PEST peptide, 2 µg, was incubated with 2 µg of CaM and 1.5 µg of calpain at 30°C for 45 min in the presence of various concentrations of Ca++ and Zn++, and the reaction products were analyzed by SDS electrophoresis.

Calpain Activity Assay in vitro

Calpain activity was measured using Calpain Activity Fluorometric Assay Kit (BioVision). Calpain and its substrate Ac-LLY-AFC provided by the kit were incubated under various concentrations of zinc in the presence of calcium. The fluorescence of the treated sample was examined in situ using a fluorescent plate reader (model SYNERGY/HTX Multi-Mode reader, BioTek) with excitation at 400 nm and emission at 505 nm.

Results

Effect of Zn++ on ABCA1 Expression in Macrophages

Fig. 1 presents the effect of divalent cations on ABCA1 expression in J774 mouse macrophage cellline cells. ABCA1 protein was increased by Ca++, which is consistent with our previous finding20) (Fig. 1A). It was also increased by the incubation of the cells in the presence of Zn++ (Fig. 1A). The increase was dose-dependent up to the concentration of 2 and 1 µM, respectively. In the presence of apoA-I that stabilizes ABCA1 protein against degradation, the effect of Zn++ became somewhat fainter (Fig. 1A). Conversely, the mRNA level of ABCA1 was not influenced by Zn++ (Fig. 1B). The degradation of ABCA1 was examined in the presence of cycloheximide (Fig. 1C). The half-life of ABCA1 was about 1 h in the non-treated cells, and it was apparently extended in the presence of apoA-I, which is consistent with our previous findings15, 16). The degradation rate of ABCA1 was also significantly retarded by Zn++ with a half-life of 2–3 h in the absence of apoA-I. However, this effect was not apparent in the presence of apoA-I. ABCA1 expression was known to increase in the presence of cAMP45); thus, the effects of Zn++ on ABCA1 expression was examined in the presence of 0.15 mM cAMP. The increase in ABCA1 and the retardation of its degradation were more significantly demonstrated in the condition cAMP is present (Fig. 2A, B). Similar findings were reproduced in mouse peritoneal macrophages in the absence and presence of cAMP (Fig. 2C).

Fig. 1.

Fig. 1.

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Expression of ABCA1 in the presence of Zn++

(A) ABCA1 protein expression in J774 cells was analyzed by Western blotting in the presence of increasing concentration of Ca++ and Zn++ typically represented in the panel. The density of each band was quantified by using Photoshop software and standardized for β-actin for n = 3 shown as a relative amount to control. (B) Expression of the ABCA1 gene was estimated by RT-PCR, as described in the text for n = 3. Significant change is indicated as ** for p < 0.01 and * for p < 0.05 against zero concentration of Zn++. In the absence of apoA-I, p-values were 0.0508 and 0.037 in the difference between the groups of [0.01, 0.1 µM] and [1, 10 µM] and between [0.01, 0.1 µM] and [1 µM], respectively. (C) Degradation rate of ABCA1. The effect of Zn++ on ABCA1 degradation was evaluated as a time course by Western blotting in the absence and presence of 10 µg/mL of apoA-I, as described in the Methods section, as typically represented. Each band was analyzed for quantification by using Photoshop software, and the values standardized for β-actin were presented as the ratio to zero time for time course incubation. Data are expressed as mean ± SD for six samples, and * and # indicate significant difference from control with P < 0.05.

Fig. 2.

Fig. 2.

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Effect of Zn++ on ABCA1 in J774 in the absence and presence of 10 µg/mL apoA-I and in the absence and presence of cAMP

Protein expression (A) and degradation (B) of ABCA1 were measured in 0.15 mM cAMP. Western blotting panels represent typical results, and the bands were quantified by using Photoshop software and standardized for β-actin for n = 3. A significant increase from zero Zn++ concentration is indicated as * and # for p < 0.05, in the respective experimental condition. Dose-dependency on Zn++ of the ABCA1 increase was supported by regression coefficient r2 = 0.96 and 0.86 for ABCA1 = 0.04 ln(Zn++) + 1.29 and ABCA1 = 0.07 ln(Zn++) + 1.79 in the absence and presence of cAMP, respectively. (C) Similar effects were verified in mouse peritoneal macrophages. Mouse macrophages obtained by peritoneal lavage were preincubated for 18 h with RPMI containing 0.2% BSA and 0.3 mM cAMP, washed with PBS, and incubated overnight in RPMI containing 0.1% BSA and various concentrations of ZnCl2 with and without 0.15 mM cAMP. ABCA1 was analyzed by Western blotting. (D) The effect of Zn++ on the release of cellular cholesterol. J774 macrophages were incubated for 1 h with MEM-HEPES labeling medium containing BODIPY-cholesterol conjugated with 10 mM methyl-β-cyclodextrin and equilibrated in the presence of 0.3 mM cAMP for 18 h. The cells were washed and incubated with various concentrations of Zn++ in the presence of 10 µg/mL of apoA-I and 0.15 mM cAMP for 4 h. The release of cellular cholesterol was estimated by measuring fluorescence intensity of the medium. Data are expressed as mean SD for four samples, and significance of the increase from zero Zn++ concentration in the presence of apoA-I is indicated as *P < 0.05 from apoA-I.

The activity of ABCA1 was evaluated by measuring the release of cellular cholesterol by apoA-1 using a fluorescently labeled sterol, BODIPY-cholesterol, from J774 macrophages exposed to the media containing cAMP and 10 µg/ml of apoA-I. The release of BODIPY-cholesterol by 10 µg/ml of apoA-I was significantly increased in the presence of Zn++ by 22% at maximum in the presence of cAMP (Fig. 2D). The results were consistent with the effect of Zn++ to increase ABCA1 by retarding its degradation.

Effect of Zn++ on ABCA1 Expression in Hepatoma Cell-Line Cells

The effect of Zn++ was also examined in human hepatoma cell-line cells HepG2. ABCA1 was increased by Zn++, both in the absence and presence of cAMP (Fig. 3A). The degradation of ABCA1 protein was substantially retarded by Zn++, both in the absence and presence of cAMP (Fig. 3B). The release of cholesterol from the cells were monitored both in the absence and presence of exogenous apoA-I, since hepatocytes produce HDL with endogenously synthesized apoA-I, presumably by an autocrine mechanism38). Fig. 3C demonstrates the increase in cell cholesterol release by Zn++ with and without exogenous apoA-I, both in the absence and presence of cAMP, which is consistent with the increase in ABCA1. Conversely, the message of ABCA1 was not influenced by Zn++ (Fig. 3D).

Fig. 3.

Fig. 3.

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The effects of Zn++ on HepG2 cells

The cells were preincubated for 18 h, washed, and incubated with various concentrations of ZnCl2 with and without 0.15 mM cAMP. (A) ABCA1 protein expression was analyzed by Western blotting in the presence of increasing concentration of Zn++ typically represented in the panel. The density of each band was quantified by using Photoshop software and standardized for β-actin for n = 3. One-way ANOVA indicated a significant increase in ABCA1 by Zn++ both in the absence and presence of cAMP (p < 0.05). (B) Degradation rate of ABCA1. The effect of Zn++ was evaluated by Western blotting. The cells were preincubated with 100 µg/mL cycloheximide for 60 min and then chased, and the level of ABCA1 was analyzed as a time course, as typically represented. Each band was analyzed for quantification by using Photoshop software. The values standardized for β-actin were shown relative to zero time of the time course and are expressed as mean ± SD for three samples. The data showed significant difference with P < 0.05 by the presence of Zn++ at each incubation time point both in the absence and presence of cAMP. (C) The effect of Zn++ on the release of cellular cholesterol. HepG2 cells were incubated for 1 h for labeling with BODIPY-cholesterol and equilibrated for 18 h in the absence and presence of 0.3 mM cAMP. The labeled cells were washed and incubated with various concentrations of Zn++ in the presence of 10 µg/mL of apoA-I and 0.15 mM AMP for 4 h. The release of cellular cholesterol was estimated by measuring the fluorescence intensity of the medium. Data are expressed as mean SD for four samples, and the significance of the increase by Zn++ is indicated as p-values by one-way ANOVA in each group. (D) The effect of Zn++ on the expression of ABCA1 mRNA, by RT-PCR, as described in the text for n = 3.

Interaction of CaM with ABCA1

We previously reported that CaM interacts with ABCA1 in a Ca++-dependent manner, and this interaction inhibits the calpain-mediated degradation of ABCA1, resulting in its stabilization and increase in its activity20). We therefore examined the effect of Zn++ on the interaction of CaM with ABCA1, which was demonstrated by the precipitation of ABCA1 with CaM-agarose and detection using the anti-ABCA1 antibody (Fig. 4A). The binding of ABCA1 to CaM-agarose was increased in a Ca++-dependent manner, which is consistent with our previous finding20). Coprecipitation of ABCA1 with CaM-agarose also increased in the presence of Zn++ at concentrations of 0.01 and 0.1 µM. The results indicated that CaM is activated by Zn++ for its interaction with ABCA1.

Fig. 4.

Fig. 4.

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Effects of Zn++ on the ABCA1–CaM interaction

A: CaM-agarose beads were incubated with the 200 µg lysates of the cells preincubated at various concentrations of Ca++ or Zn++. Protein bound to the beads was analyzed by Western blotting for ABCA1. B: Effect of Zn++ on calpain-mediated cleavage of ABCA1 peptide. The peptide following ABCA1 sequence 1213 to 1349 that contains a CaM-binding site and PEST sequence tagged with GST (GST–CaM–PEST fusion protein) was examined for its proteolysis by µ-calpain under an increasing concentration of Ca++, analyzed by SDS-PAGE stained with Coomassie Brilliant Blue (CBB). C: Interference of calpain-mediated cleavage of ABCA1 peptide by CaM. GST–CaM–PST protein was preincubated with (or without) CaM to allow it to bind to the 1-5-8-14 motif and then incubated with 0.5 µM Ca++ (which allows activation of calpain but not of CaM) and 1.5 µg of µ-calpain for 1 h at 32°C under various concentrations of Zn++. The reaction product was analyzed in SDS-PAGE stained with Coomassie Brilliant Blue (CBB).

Effect of Zn++ on calpain-mediated cleavage of ABCA1 peptide

We previously demonstrated that GST–CaM–PEST fusion protein (ABCA1 fragment peptide containing the CaM-binding motifs and the nearby PEST sequence) was completely degraded by calpain in the absence of CaM, and this reaction was inhibited in the presence of CaM and Ca++20). To analyze whether Zn++ has the same mode of action, the reactivity of GST–CaM–PEST fusion protein to calpain was examined in vitro in the presence of CaM and Zn++ (Fig. 4B). We confirmed that the minimum concentration of Ca++ required for calpain activation in vitro is 0.5 µM. GST–CaM–PEST fusion protein was degraded by calpain in the absence and presence of CaM, at 0.5 µM Ca++, and an additional Zn++ inhibited such degradation in this condition. In the absence of CaM, Zn++ inhibited the reaction at a concentration of 10 µM and higher. Contrarily, in the presence of CaM, Zn++ inhibited the calpain-mediated degradation of the fusion protein at a concentration of 1 µM and higher (Fig. 4C). Thus, inhibition of the calpain-mediated degradation of the fusion protein was Zn++-dependent. Finally, the direct effect of Zn++ on calpain reaction is presented in Fig. 5, in which a commercial Calpain Activity Assay Kit was used in the absence of CaM. The reaction was inhibited by Zn++ only at a concentration of 10 µM and higher, much higher than those for the effect observed in the presence of CaM. The results therefore demonstrated that Zn++ suppresses calpain-mediated degradation of ABCA1 in a CaM-dependent manner at a concentration of around 1 µM. This is likely due to the enhancement of the binding of CaM to its binding site of ABCA1 to interfere with calpain-mediated degradation at the nearby PEST sequence. Inhibition of calpain activity by Zn++ at high concentrations, such as 10 µM or higher, is possibly caused by competition against Ca++ for calpain.

Fig. 5.

Fig. 5.

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Calpain activity assay

The direct effect of Zn++ on calpain activity was examined . Active calpain (1 µg) was incubated with the calpain substrate (Ac-LLY-AFC) with various concentrations of Zn++ (0.1–10 µM) at 37°C for 1 h. Calpain inhibitor Z-LLY-FMK was used for a negative control incubation.

Discussion

We previously reported that CaM interacts with ABCA1 in a Ca++-dependent manner, and this interaction inhibits calpain-mediated ABCA1 degradation to increase its activity for HDL biogenesis20). On the basis of these findings, this study provided the following observations: (1) Zn++ increased ABCA1 protein, but not the message, likely due to the retardation of its calpain-mediated degradation in both macrophage and hepatocyte cellular models; (2) the release of apoA-1-mediated cellular cholesterol was increased by Zn++, more prominently in the presence of cAMP; (3) the binding of CaM to ABCA1 was increased by Zn++, as shown by the co-precipitation of ABCA1 with CaM-agarose; (4) Zn++ inhibited the calpain-mediated degradation of the ABCA1-peptide, including the CaM-binding site and the PEST sequence, in the presence of CaM. We concluded that Zn++ cellular concentration is potentially involved in the regulation of ABCA1 activity by inhibiting its calpain-mediated degradation through the modulation of CaM activity.

Zinc is one of the essential inorganic metal nutrients involved in various cell functions. In vivo functions of zinc were originally described as its deficiency in animal studies by their symptoms, such as growth failure, hair loss, testicular atrophy, or thickening and hyperkeratinization of the epidermis46). It later became recognized as an essential nutrient also for human beings, but its deficiency was thought to be rare earlier47). It is now considered rather common, especially in developing countries48). About two billion people worldwide are affected by zinc deficiency. Zinc deficiency is one of the leading causes of the loss of healthy life in developing countries, and it influences the elderly population even more in industrial countries, commonly appearing as a decrease in immune response or retardation of wound healing49). Zinc deficiency may also be involved in the development of atherosclerosis through its influence on inflammation, gene stabilization and transcription, or apoptosis50–53). However, the effect of zinc on lipid and lipoprotein metabolism is somewhat controversial in both animal models and humans. Zinc deficiency may push plasma lipoprotein profile towards atherogenic by increasing low-density lipoprotein35, 54), decreasing HDL35, 55), or enhancing lipid peroxidation56). Conversely, zinc supplementation was found to decrease HDL in normal subjects while it increased in diabetic populations and in those on chronic hemodilaysis33–35). No report has been found on the molecular mechanism for these findings.

ABCA1 is one of the major regulating factors of plasma HDL concentration and is required in the biogenesis of HDL particles57) to regulate plasma HDL concentration5) and atherogenesis6). The activity of ABCA1 is regulated by its gene transcription positively sensing cellular cholesterol and by its protein degradation by various factors12, 15–17, 19, 36). We found that CaM is one of the factors involved in this process20). CaM is activated by Ca++ to interact with the 1-5-8-14 motif in the cytoplasmic loop of ABCA1 and thereby inhibits calpain-mediated degradation at the nearby PEST sequence20). CaM is known to be driven not only by Ca++ but also by other divalent cations, such as La++, Tb++, Pb++, Sn++, Sr++, Hg++, Cd++, Zn++, and Mn++23–28). Among them, Zn++ seems to act cooperatively with Ca++ rather than competitively in some CaM actions, such as protein kinase II activation30, 31). The results here with mouse macrophage cell-line cells J774 indicated that Zn++ activates CaM to interact with ABCA1 and retards its degradation to increase HDL biogenesis. Schmitz and his colleagues reported that zinc finger protein 202 acted as a transcriptional repressor of ABCA1 and ABCAG1 and suggested its involvement in the regulation of plasma HDL58). However, Zn++ did not exhibit any apparent influence on the level of ABCA1 mRNA in the current experimental conditions.

Clinical relevance of the current findings should be carefully stated to avoid any overevaluation. While many clinical studies suggested that zinc supplementation may not increase or even decrease plasma HDL concentration, a large-scale meta-analysis demonstrated that it decreases HDL in normal subjects but increases HDL in type 2 diabetic populations and chronic hemodialysis patients33–35). No report has been found on the molecular mechanism for these findings. However, it should be noted that both of these conditions are of sympathetic activation, where cAMP level is to be increased in the target organ cells59–65). The current findings in fact indicated that cAMP enhanced the effects of Zn++ on ABCA1 and the ABCA1-mediated cellular cholesterol release in model cell culture systems, both for macrophages and hepatocytes, which are responsible for the atherogenesis and biogenesis of plasma HDL, respectively. Zinc supplementation may therefore be helpful to push lipid and lipoprotein metabolism toward less atherogenic in certain pathological states, although it is yet to be verified in appropriate animal models and by relevant clinical trials.

Acknowledgements

The authors are grateful to the undergraduate students, Danki Ito, Naoto Kato, Riho Nakashima, Mayu Yamamoto, Naoto Kobayashi, Kootmi Sakai, Keishi Shimakura, Yurino Hattori, Kazuya Murata, Yoichi Mitsumura, Goh Shonohara, Akane Ushikoshi, Miho Nishio and Airi Yamamoto, who participated to the preliminary stage of the project for their bachelor's thesis. They also thank Dr. Shigeru Miyata at Chubu University and Dr. Maki Tsujita at Nagoya City University for his/her technical advices.

Funding Sources

This work has been supported by the MEXT-Supported Program for Strategic Founding of Research in Private Universities (S1201007) and by Grants-in-aid from MEXT Japan (24614018, 26461370, 15H02903).

Clinical Interest and Disclosures

The authors have no affiliation with any organization with a direct or indirect financial interest in the subject matter discussed in the manuscript.

References

  • 1). Hara H, Yokoyama S: Interaction of free apolipoproteins with macrophages. Formation of high density lipoprotein-like lipoproteins and reduction of cellular cholesterol. J Biol Chem, 1991; 266: 3080-3086 [PubMed] [Google Scholar]
  • 2). Bodzioch M, Orso E, Klucken J, Langmann T, Bottcher A, Diederich W, Drobnik W, Barlage S, Buchler C, Porsch-Ozcurumez M, Kaminski WE, Hahmann HW, Oette K, Rothe G, Aslanidis C, Lackner KJ, Schmitz G: The gene encoding atp-binding cassette transporter 1 is mutated in tangier disease. Nat Genet, 1999; 22: 347-351 [DOI] [PubMed] [Google Scholar]
  • 3). Brooks-Wilson A, Marcil M, Clee SM, Zhang LH, Roomp K, van Dam M, Yu L, Brewer C, Collins JA, Molhuizen HO, Loubser O, Ouelette BF, Fichter K, Ashbourne-Excoffon KJ, Sensen CW, Scherer S, Mott S, Denis M, Martindale D, Frohlich J, Morgan K, Koop B, Pimstone S, Kastelein JJ, Genest J, Jr., Hayden MR: Mutations in abc1 in tangier disease and familial high-density lipoprotein deficiency. Nat Genet, 1999; 22: 336-345 [DOI] [PubMed] [Google Scholar]
  • 4). Rust S, Rosier M, Funke H, Real J, Amoura Z, Piette JC, Deleuze JF, Brewer HB, Duverger N, Denefle P, Assmann G: Tangier disease is caused by mutations in the gene encoding atp-binding cassette transporter 1. Nat Genet, 1999; 22: 352-355 [DOI] [PubMed] [Google Scholar]
  • 5). Timmins JM, Lee JY, Boudyguina E, Kluckman KD, Brunham LR, Mulya A, Gebre AK, Coutinho JM, Colvin PL, Smith TL, Hayden MR, Maeda N, Parks JS: Targeted inactivation of hepatic abca1 causes profound hypoalpha-lipoproteinemia and kidney hypercatabolism of apoa-i. J Clin Invest, 2005; 115: 1333-1342 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6). Van Eck M, Singaraja RR, Ye D, Hildebrand RB, James ER, Hayden MR, Van Berkel TJ: Macrophage atp-binding cassette transporter a1 overexpression inhibits atherosclerotic lesion progression in low-density lipoprotein receptor knockout mice. Arterioscler Thromb Vasc Biol, 2006; 26: 929-934 [DOI] [PubMed] [Google Scholar]
  • 7). Costet P, Luo Y, Wang N, Tall AR: Sterol-dependent transactivation of the abc1 promoter by the liver x receptor/retinoid x receptor. J Biol Chem, 2000; 275: 28240-28245 [DOI] [PubMed] [Google Scholar]
  • 8). Iwamoto N, Abe-Dohmae S, Lu R, Yokoyama S: Involvement of protein kinase d in phosphorylation and increase of DNA binding of activator protein 2 alpha to downregulate atp-binding cassette transporter a1. Arterioscler Thromb Vasc Biol, 2008; 28: 2282-2287 [DOI] [PubMed] [Google Scholar]
  • 9). Iwamoto N, Yokoyama S: Protein kinase d regulates the adiponectin gene expression through phosphorylation of ap-2: A common pathway to the abca1 gene regulation. Atherosclerosis, 2011; 216: 90-96 [DOI] [PubMed] [Google Scholar]
  • 10). Tamehiro N, Shigemoto-Mogami Y, Kakeya T, Okuhira K, Suzuki K, Sato R, Nagao T, Nishimaki-Mogami T: Sterol regulatory element-binding protein-2- and liver x receptor-driven dual promoter regulation of hepatic abc transporter a1 gene expression: Mechanism underlying the unique response to cellular cholesterol status. J Biol Chem, 2007; 282: 21090-21099 [DOI] [PubMed] [Google Scholar]
  • 11). Ohoka N, Okuhira K, Cui H, Wu W, Sato R, Naito M, Nishimaki-Mogami T: Hnf4alpha increases liver-specific human atp-binding cassette transporter a1 expression and cholesterol efflux to apolipoprotein a-i in response to cholesterol depletion. Arterioscler Thromb Vasc Biol, 2012; 32: 1005-1014 [DOI] [PubMed] [Google Scholar]
  • 12). Yokoyama S, Arakawa R, Wu CA, Iwamoto N, Lu R, Tsujita M, Abe-Dohmae S: Calpain-mediated abca1 degradation: Post-translational regulation of abca1 for hdl biogenesis. Biochim Biophys Acta, 2012; 1821: 547-551 [DOI] [PubMed] [Google Scholar]
  • 13). Arakawa R, Yokoyama S: Helical apolipoproteins stabilize atp-binding cassette transporter a1 by protecting it from thiol protease-mediated degradation. J Biol Chem, 2002; 277: 22426-22429 [DOI] [PubMed] [Google Scholar]
  • 14). Arakawa R, Hayashi M, Remaley AT, Brewer BH, Yamauchi Y, Yokoyama S: Phosphorylation and stabilization of atp binding cassette transporter a1 by synthetic amphiphilic helical peptides. J Biol Chem, 2004; 279: 6217-6220 [DOI] [PubMed] [Google Scholar]
  • 15). Lu R, Arakawa R, Ito-Osumi C, Iwamoto N, Yokoyama S: Apoa-i facilitates abca1 recycle/accumulation to cell surface by inhibiting its intracellular degradation and increases hdl generation. Arterioscler Thromb Vasc Biol, 2008; 28: 1820-1824 [DOI] [PubMed] [Google Scholar]
  • 16). Lu R, Tsuboi T, Okumura-Noji K, Iwamoto N, Yokoyama S: Caveolin-1 facilitates internalization and degradation of abca1 and probucol oxidative products interfere with this reaction to increase hdl biogenesis. Atherosclerosis, 2016; 253: 54-60 [DOI] [PubMed] [Google Scholar]
  • 17). Arakawa R, Tsujita M, Iwamoto N, Ito-Ohsumi C, Lu R, Wu CA, Shimizu K, Aotsuka T, Kanazawa H, Abe-Dohmae S, Yokoyama S: Pharmacological inhibition of abca1 degradation increases hdl biogenesis and exhibits antiatherogenesis. J Lipid Res, 2009; 50: 2299-2305 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18). Yakushiji E, Ayaori M, Nishida T, Shiotani K, Takiguchi S, Nakaya K, Uto-Kondo H, Ogura M, Sasaki M, Yogo M, Komatsu T, Lu R, Yokoyama S, Ikewaki K: Probucol-oxidized products, spiroquinone and diphenoquinone, promote reverse cholesterol transport in mice. Arterioscler Thromb Vasc Biol, 2016; 36: 591-597 [DOI] [PubMed] [Google Scholar]
  • 19). Tsujita M, Hossain MA, Lu R, Tsuboi T, Okumura-Noji K, Yokoyama S: Exposure to high glucose concentration decreases cell surface abca1 and hdl biogenesis in hepatocytes. J Atheroscler Thromb, 2017; 24: 1132-1149 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20). Iwamoto N, Lu R, Tanaka N, Abe-Dohmae S, Yokoyama S: Calmodulin interacts with atp binding cassette transporter a1 to protect from calpain-mediated degradation and upregulates high-density lipoprotein generation. Arterioscler Thromb Vasc Biol, 2010; 30: 1446-1452 [DOI] [PubMed] [Google Scholar]
  • 21). Klee CB, Crouch TH, Richman PG: Calmodulin. Annual Review of Biochemistry, 1980; 49: 489-515 [DOI] [PubMed] [Google Scholar]
  • 22). Cheung WY: Calmodulin plays a pivotal role in cellular regulation. Science, 1980; 207: 19-27 [DOI] [PubMed] [Google Scholar]
  • 23). Wallace RW, Tallant EA, Dockter ME, Cheung WY: Calcium binding domains of calmodulin. Sequence of fill as determined with terbium luminescence. J Biol Chem, 1982; 257: 1845-1854 [PubMed] [Google Scholar]
  • 24). Wang CL, Leavis PC, Gergely J: Kinetic studies show that ca2+ and tb3+ have different binding preferences toward the four ca2+-binding sites of calmodulin. Biochemistry, 1984; 23: 6410-6415 [DOI] [PubMed] [Google Scholar]
  • 25). Forsen S, Thulin E, Lilja H: 113cd nmr in the study of calcium binding proteins: Troponin c. FEBS Lett, 1979; 104: 123-126 [DOI] [PubMed] [Google Scholar]
  • 26). Chao SH, Suzuki Y, Zysk JR, Cheung WY: Activation of calmodulin by various metal cations as a function of ionic radius. Molecular Pharmacology, 1984; 26: 75-82 [PubMed] [Google Scholar]
  • 27). Andersson T, Drakenberg T, Forsen S, Thulin E: Characterization of the ca2+ binding sites of calmodulin from bovine testis using 43ca and 113cd nmr. Eur J Biochem, 1982; 126: 501-505 [DOI] [PubMed] [Google Scholar]
  • 28). Baudier J, Haglid K, Haiech J, Gerard D: Zinc ion binding to human brain calcium binding proteins, calmodulin and s100b protein. Biochem Biophys Res Commun, 1983; 114: 1138-1146 [DOI] [PubMed] [Google Scholar]
  • 29). Babu YS, Bugg CE, Cook WJ: Structure of calmodulin refined at 2.2 a resolution. J Mol Biol, 1988; 204: 191-204 [DOI] [PubMed] [Google Scholar]
  • 30). McCord MC, Aizenman E: Convergent ca2+ and zn2+ signaling regulates apoptotic kv2.1 k+ currents. Proc Natl Acad Sci U S A, 2013; 110: 13988-13993 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31). Hoffman L, Li L, Alexov E, Sanabria H, Waxham MN: Cytoskeletal-like filaments of ca(2+)-calmodulin-dependent protein kinase ii are formed in a regulated and zn(2+)-dependent manner. Biochemistry, 2017; 56: 2149-2160 [DOI] [PubMed] [Google Scholar]
  • 32). Vallee BL, Falchuk KH: The biochemical basis of zinc physiology. Physiol Rev, 1993; 73: 79-118 [DOI] [PubMed] [Google Scholar]
  • 33). Foster M, Petocz P, Samman S: Effects of zinc on plasma lipoprotein cholesterol concentrations in humans: A meta-analysis of randomised controlled trials. Atherosclerosis, 2010; 210: 344-352 [DOI] [PubMed] [Google Scholar]
  • 34). Foster M, Samman S, Zinc and atherosclerosis: Clinical observations and potential mechanisms, in: L. Rink (Ed.). Zinc in human health, 2011, pp. 347-372 [Google Scholar]
  • 35). Reiterer G, MacDonald R, Browning JD, Morrow J, Matveev SV, Daugherty A, Smart E, Toborek M, Hennig B: Zinc deficiency increases plasma lipids and atherosclerotic markers in ldl-receptor-deficient mice. J Nutr, 2005; 135: 2114-2118 [DOI] [PubMed] [Google Scholar]
  • 36). Iborra RT, Machado-Lima A, Okuda LS, Pinto PR, Nakandakare ER, Machado UF, Correa-Giannella ML, Pickford R, Woods T, Brimble MA, Rye KA, Lu R, Yokoyama S, Passarelli M: Age-albumin enhances abca1 degradation by ubiquitin-proteasome and lysosomal pathways in macrophages. J Diabetes Complications, 2018; 32: 1-10 [DOI] [PubMed] [Google Scholar]
  • 37). Tsujita M, Yokoyama S: Selective inhibition of free apolipoprotein-mediated cellular lipid efflux by probucol. Biochemistry, 1996; 35: 13011-13020 [DOI] [PubMed] [Google Scholar]
  • 38). Tsujita M, Wu CA, Abe-Dohmae S, Usui S, Okazaki M, Yokoyama S: On the hepatic mechanism of hdl assembly by the abca1/apoa-i pathway. J Lipid Res, 2005; 46: 154-162 [DOI] [PubMed] [Google Scholar]
  • 39). Rukgauer M, Klein J, Kruse-Jarres JD: Reference values for the trace elements copper, manganese, selenium, and zinc in the serum/plasma of children, adolescents, and adults. J Trace Elem Med Biol, 1997; 11: 92-98 [DOI] [PubMed] [Google Scholar]
  • 40). Lowe NM, Fekete K, Decsi T: Methods of assessment of zinc status in humans: A systematic review. Am J Clin Nutr, 2009; 89: 2040s-2051s [DOI] [PubMed] [Google Scholar]
  • 41). Woodier J, Rainbow RD, Stewart AJ, Pitt SJ: Intracellular zinc modulates cardiac ryanodine receptor-mediated calcium release. J Biol Chem, 2015; 290: 17599-17610 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42). Hu W, Abe-Dohmae S, Tsujita M, Iwamoto N, Ogikubo O, Otsuka T, Kumon Y, Yokoyama S: Biogenesis of hdl by saa is dependent on abca1 in the liver in vivo. J Lipid Res, 2008; 49: 386-393 [DOI] [PubMed] [Google Scholar]
  • 43). Yokoyama S, Tajima S, Yamamoto A: The process of dissolving apolipoprotein a-i in an aqueous buffer. J Biochem, 1982; 91: 1267-1272 [DOI] [PubMed] [Google Scholar]
  • 44). Sankaranarayanan S, Kellner-Weibel G, de la Llera-Moya M, Phillips MC, Asztalos BF, Bittman R, Rothblat GH: A sensitive assay for abca1-mediated cholesterol efflux using bodipy-cholesterol. J Lipid Res, 2011; 52: 2332-2340 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45). Abe-Dohmae S, Suzuki S, Wada Y, Aburatani H, Vance DE, Yokoyama S: Characterization of apolipoprotein-mediated hdl generation induced by camp in a murine macrophage cell line. Biochemistry, 2000; 39: 11092-11099 [DOI] [PubMed] [Google Scholar]
  • 46). Todd WR, Elvehjem CA, Hart EB: Nutrition classics. “Zinc in the nutrition of the rat” in the american journal of physiology, volume 107, 1934, pages 146-156, Nutrition Review, 1980; 38: 151-154 [DOI] [PubMed] [Google Scholar]
  • 47). Prasad AS: Impact of the discovery of human zinc deficiency on health. J Am Coll Nutr, 2009; 28: 257-265 [DOI] [PubMed] [Google Scholar]
  • 48). World-Health-Organization, The world health report 2002. https://www.who.int/whr/2002/chapter4/en/index3.Html, World Health Organization, Geneva, Switzerland, 2002 [Google Scholar]
  • 49). Prasad A, Discovery of zinc deficiency in humans and its impact fifty years later, in: L. Rink (Ed.), Zinc in human health in biomedical and health research, IOS Press, Amsterdam, The Netherlands, 2011, pp. 7-28 [Google Scholar]
  • 50). Reiterer G, Toborek M, Hennig B: Peroxisome proliferator activated receptors alpha and gamma require zinc for their anti-inflammatory properties in porcine vascular endothelial cells. J Nutr, 2004; 134: 1711-1715 [DOI] [PubMed] [Google Scholar]
  • 51). Meerarani P, Reiterer G, Toborek M, Hennig B: Zinc modulates ppargamma signaling and activation of porcine endothelial cells. J Nutr, 2003; 133: 3058-3064 [DOI] [PubMed] [Google Scholar]
  • 52). Beattie JH, Kwun IS: Is zinc deficiency a risk factor for atherosclerosis? Br J Nutr, 2004; 91: 177-181 [DOI] [PubMed] [Google Scholar]
  • 53). Stadler N, Stanley N, Heeneman S, Vacata V, Daemen MJ, Bannon PG, Waltenberger J, Davies MJ: Accumulation of zinc in human atherosclerotic lesions correlates with calcium levels but does not protect against protein oxidation. Arterioscler Thromb Vasc Biol, 2008; 28: 1024-1030 [DOI] [PubMed] [Google Scholar]
  • 54). Shen H, MacDonald R, Bruemmer D, Stromberg A, Daugherty A, Li XA, Toborek M, Hennig B: Zinc deficiency alters lipid metabolism in ldl receptor deficient mice treated with rosiglitazone. J Nutr, 2007; 137: 2339-2345 [DOI] [PubMed] [Google Scholar]
  • 55). Koo SI, Williams DA: Relationship between the nutritional status of zinc and cholesterol concentration of serum lipoproteins in adult male rats. Am J Clin Nutr, 1981; 34: 2376-2381 [DOI] [PubMed] [Google Scholar]
  • 56). Faure P, Roussel AM, Richard MJ, Foulon T, Groslambert P, Hadjian A, Favier A: Effect of an acute zinc depletion on rat lipoprotein distribution and peroxidation. Biol Trace Elem Res, 1991; 28: 135-146 [DOI] [PubMed] [Google Scholar]
  • 57). Yokoyama S: Assembly of high-density lipoprotein. Arterioscler Thromb Vasc Biol, 2006; 26: 20-27 [DOI] [PubMed] [Google Scholar]
  • 58). Porsch-Özcürümez M, Langmann T, Heimerl S, Borsukova H, Kaminski WE, Drobnik W, Honer C, Schumacher C, Schmitz G: The zinc finger protein 202 (znf202) is a transcriptional repressor of atp binding cassette transporter a1 (abca1) and abcg1 gene expression and a modulator of cellular lipid efflux. J. Biol. Chem., 2001; 276: 12427-12433 [DOI] [PubMed] [Google Scholar]
  • 59). Reaven GM, Hoffman BB: A role for insulin in the aetiology and course of hypertension? Lancet, 1987; 2: 435-437 [DOI] [PubMed] [Google Scholar]
  • 60). Facchini FS, Stoohs RA, Reaven GM: Enhanced sympathetic nervous system activity. The linchpin between insulin resistance, hyperinsulinemia, and heart rate. Am J Hypertens, 1996; 9: 1013-1017 [DOI] [PubMed] [Google Scholar]
  • 61). Reaven GM: Insulin resistance, the insulin resistance syndrome, and cardiovascular disease. Panminerva Med, 2005; 47: 201-210 [PubMed] [Google Scholar]
  • 62). Thorp AA, Schlaich MP: Relevance of sympathetic nervous system activation in obesity and metabolic syndrome. J Diabetes Res, 2015; 2015: 341583 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 63). Hansen J, Victor RG: Direct measurement of sympathetic activity: New insights into disordered blood pressure regulation in chronic renal failure. Curr Opin Nephrol Hypertens, 1994; 3: 636-643 [DOI] [PubMed] [Google Scholar]
  • 64). Vonend O, Rump LC, Ritz E: Sympathetic overactivity--the cinderella of cardiovascular risk factors in dialysis patients. Semin Dial, 2008; 21: 326-330 [DOI] [PubMed] [Google Scholar]
  • 65). Rubinger D, Backenroth R, Sapoznikov D: Sympathetic nervous system function and dysfunction in chronic hemodialysis patients. Semin Dial, 2013; 26: 333-343 [DOI] [PubMed] [Google Scholar]

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