Cholesterol efflux to apoA-I in ABCA1-expressing cells is regulated by Ca2+-dependent calcineurin signaling

Abstract

ATP-binding cassette transporter A1 (ABCA1) is required for the lipidation of apolipoprotein A-I (apoA-I), although molecular mechanisms supporting this process remain poorly defined. In this study, we focused on the role of cytosolic Ca(2+) and its signaling and found that cytosolic Ca(2+) was required for cholesterol efflux to apoA-I. Removing extracellular Ca(2+) or chelating cytosolic Ca(2+) were equally inhibitory for apoA-I lipidation. We provide evidence that apoA-I induced Ca(2+) influx from the medium. We further demonstrate that calcineurin activity, the downstream target of Ca(2+) influx, was essential; inhibition of calcineurin activity by cyclosporine A or FK506 completely abolished apoA-I lipidation. Furthermore, calcineurin inhibition abolished apoA-I binding and diminished JAK2 phosphorylation, an established signaling event for cholesterol efflux to apoA-I. Finally, we demonstrate that neither Ca(2+) manipulation nor calcineurin inhibition influenced ABCA1's capacity to release microparticles or to remodel the plasma membrane. We conclude that this Ca(2+)-dependent calcineurin/JAK2 pathway is specifically responsible for apoA-I lipidation without directly modifying ABCA1 activity.

Fig. 1.

Effect of Ca²⁺ on ABCA1-dependent cholesterol efflux to apoA-I in ABCA1-expressing BHK cells. ABCA1 and mock BHK cells were labeled with [³H]cholesterol for 1–2 days and induced with 5 μM mifepristone overnight. Cholesterol efflux to apoA-I (5 μg/ml) was measured under the following conditions. A: Extracellular Ca²⁺ was removed using the cation chelator EDTA or the Ca²⁺-specific chelator EGTA. Cholesterol efflux was also measured in Ca²⁺-free medium. B: Cells were incubated in Ca²⁺-free medium for 2 h and then switched back to normal medium containing Ca²⁺. C, D: The dose dependency of extracellular or intracellular Ca²⁺ on efflux was determined by increasing medium Ca²⁺ (C) or EGTA (D) concentrations. E: Cells were treated with increasing doses of BAPTA-AM. Both medium and cell associated ³H radioactivity were counted and presented as percentage of cholesterol in the medium relative to the total cholesterol (medium and cell-associated). In B, cholesterol efflux was presented as a percentage relative to untreated cells (control). Data is presented as mean ± SD of triplicate wells, representative of at least three experiments performed.

Fig. 2.

Effect of intracellular and extracelluar Ca²⁺ on cholesterol efflux to apoA-I in RAW macrophages. RAW macrophages were induced to express ABCA1 with 250 μM Br-cAMP or uninduced overnight. Cholesterol efflux was analyzed under following conditions: 2 mM EGTA or 0 Ca²⁺ (A); 50 μM and 100 μM BAPTA-AM (B). Data is presented as mean ± SD of triplicate wells, representative of at least three experiments performed.

Fig. 3.

Effect of Ca²⁺ flux from endoplasmic reticulum (ER) stores and across the plasma membrane on cholesterol efflux to apoA-I in ABCA1-expressing BHK cells. ABCA1 and Mock BHK cells were labeled with [³H]cholesterol and induced with mifepristone overnight. Cholesterol efflux to apoA-I (5 μg/ml) was measured under the following conditions. A: Cells were treated with indicated concentrations of thapsigargin, ryanodine, and 2-APB, respectively. B: The amount of radiolabeled [⁴⁵Ca²⁺] influx into cells was determined in mock and ABCA1-expressing cells with or without the presence of 10 μg/ml apoA-I over 4 min. C: The effect of a Ca²⁺ channel agonist, BAY-K8644, on cholesterol efflux to apoA-I. The results in each graph show the mean ± SD of triplicate wells, representative of at least three experiments performed.

Fig. 4.

Inhibition of CaM and calcineurin reduces cholesterol efflux to apoA-I in ABCA1-expressing cells. BHK cells and RAW macrophages were induced with mifepristone or Br-cAMP, respectively. Cholesterol efflux to apoA-I (5 μg/ml) was measured under the following conditions: the indicated concentrations of the CaM inhibitor, W-7 (A); calcineurin inhibitors CsA (B) and FK506 (C). D: CsA and FK506 were also used with RAW macrophages expressing ABCA1. The results in each graph show the mean ± SD of triplicate wells, representative of at least three experiments performed.

Fig. 5.

Altering intracellular or extracelluar Ca²⁺ distribution had no effect on ABCA1 expression or localization. A: The level of ABCA1 protein expression in BHK cells was determined by immunoblotting after 2 h incubation with EGTA or BAPTA-AM. B: Immunofluorescent detection of ABCA1 in mock and ABCA1-expressing cells BHK cells under different Ca²⁺ treatment conditions. Images were taken using a confocal fluorescent microscope focused on the plasma membrane and displayed identically.

Fig. 6.

Reducing intracellular and extracelluar Ca²⁺ caused an inhibition of Cy2-apoA-I cell association in BHK cells. Cy2-apoA-I was incubated with cells for 2 h under various conditions and the degree of cell association was determined by flow cytometry. The results are expressed relative to control cells that express ABCA1 (100%) and cells that do not express ABCA1 (mock) (0%). A: Mock- and ABCA1-expressing BHK cells incubated with Cy2-apoA-1. B: ABCA1-expressing cells were incubated with Cy2-apoA-1 plus unlabeled apoA-I. ABCA1-expressing cells were incubated with Cy2-apoA-1 in the presence of 2 mM EGTA (C); 50 μM BAPTA-AM (D); 10 μM CsA (E); 25 μM FK506 (F). The representative experiments are shown from at least three trials. G: The correlation between apoA-I cell association and efflux efficiency is presented.

Fig. 7.

Manipulating Ca²⁺ and CaM/calcineurin signaling reduces Cy2-apoA-I cell association in RAW macrophages. Cells were incubated with Cy2-apoA-I for 2 h under various conditions and the degree of cell association was determined by flow cytometry. A: Cy2-apoA-I cell association in cells induced to express ABCA1 (+) (100%) and uninduced cells (−) (0%) is presented. B: ABCA1-expressing cells were incubated with Cy2-apoA-1 plus unlabeled apoA-I. ABCA1-expressing cells were incubated with Cy2-apoA-1 in the presence of 2 mM EGTA (C); 100 μM BAPTA-AM (D); 10 μM CsA (E); 25 μM FK506 (F). The representative experiments are shown from at least three trials.

Fig. 8.

Inhibition of calcineurin impairs JAK2 phosphorylation. The expression levels of phosphorylated Jak2 (P-Jak2) and total Jak2 was determined by immunoblotting. A: Duplicates of ABCA1-BHK cells were induced with mifepristone overnight and then incubated with or without 10 μM CsA for 2 h before cell lysis and immunoblotting. B: ABCA1-expressing BHK cells were also incubated with Ca²⁺-free medium for 2 h before immunoblotting. The optical density of the immunoblots was quantified and expressed as mean ± SD. Untreated cells served as control (100%). Results are representative of three experiments with identical conditions.

Fig. 9.

Manipulating Ca²⁺ and calcineurin activity affects apoA-I-dependent cholesterol efflux, but not basal ABCA1 activity. ABCA1 BHK cells were labeled with [³H]cholesterol and induced with mifepristone overnight. Cholesterol efflux was conducted in the presence of apoA-I (A) or without (B) under the following conditions: Ca²⁺-free medium, calcineurin inhibitors (CsA and FK506), and PKI (a PKA inhibitor). Cholesterol efflux from untreated cells served as control (100%). Each panel presents data compiled from two experiments performed in triplicate and the standard error of the mean is shown. C: BHK cells were transfected with YFP-caveolin and induced with mifepristone overnight. Representative images of YFP-caveolin are shown in mock BHK cells, ABCA1-expressing BHK cells, and ABCA1-expressing BHK cells treated with 10 μM CsA.