ABCA1-mediated cholesterol efflux generates microparticles in addition to HDL through processes governed by membrane rigidity

Abstract

ATP-binding cassette transporter A1 (ABCA1) mediates cholesterol efflux to lipid-poor apolipoprotein A-I (apoA-I) and generates HDL. Here, we demonstrate that ABCA1 also directly mediates the production of apoA-I free microparticles. In baby hamster kidney (BHK) cells and RAW macrophages, ABCA1 expression led to lipid efflux in the absence of apoA-I and released large microparticles devoid of apoB and apoE. We provide evidence that these microparticles are an integral component of the classical cholesterol efflux pathway when apoA-I is present and accounted for approximately 30% of the total cholesterol released to the medium. Furthermore, microparticle release required similar ABCA1 activities as was required for HDL production. For instance, a nucleotide binding domain mutation in ABCA1 (A937V) that impaired HDL generation also abolished microparticle release. Similarly, inhibition of protein kinase A (PKA) prevented the release of both types of particles. Interestingly, physical modulation of membrane dynamics affected HDL and microparticle production, rigidifying the plasma membrane with wheat germ agglutinin inhibited HDL and microparticle release, whereas increasing the fluidity promoted the production of these particles. Given the established role of ABCA1 in expending nonraft or more fluid-like membrane domains, our results suggest that both HDL and microparticle release is favored by a more fluid plasma membrane. We speculate that ABCA1 enhances the dynamic movement of the plasma membrane, which is required for apoA-I lipidation and microparticle formation.-Nandi, S

Ht31, a Protein Kinase A Anchoring Inhibitor, Induces Robust Cholesterol Efflux and Reverses Macrophage Foam Cell Formation through ATP-binding Cassette Transporter A1

“…The slightly lower cholesterol efflux to apoA-I in the second hour here, compared with cholesterol efflux to apoA-I in untreated cells (third group from left), likely reflects the cholesterol depletion by st-Ht31 during the first hour. Furthermore, we found that st-Ht31 mainly stimulates the release of microparticles, similar to the ones we reported earlier by FPLC analysis (6). ABCA1 phosphorylation by PKA was not significantly altered, as probed by an antibody recognizing phosphorylated PKA substrates (Fig.…”

“…We presently do not understand the detailed cholesterol trafficking pathway that generates microparticles. These microparticles are uniform in size (Ͻ200 nm) and between 1.063 and 1.21 in density (6). We provide evidence here that these microparticles contain flotillin-2, an exosome marker and also a potential molecular module for exosome release (16).…”

“…In addition, ABCA1 exports cholesterol in the form of non-HDL microparticles (5,6). This microparticle release relies on protein kinase A (PKA) activity.…”

“…The supernatant was then passed through a 0.2-m filter. We have previous shown that microparticles are smaller than 0.2 m but could not pass through a 100-kDa molecular weight cutoff filter (6). The filtrate was then washed and concentrated with a 100-kDa filter before Western blotting.…”

Direct detection of ABCA1-dependent HDL formation based on lipidation-induced hydrophobicity change in apoA-I

“…It has been reported that ABCA1 mediates the formation of microparticles that are larger than HDL and are not involved in HDL formation ( 35,36 ). As previously reported, cholesterol-containing particles were observed in the conditioned medium from ABCA1-expressing cells around the void volume of the Superdex 200 column ( Fig.…”