Expression and biological activity of ABCA1 in alveolar epithelial cells

Abstract

The mechanisms used by alveolar type I pneumocytes for maintenance of the lipid homeostasis necessary to sustain these large squamous cells are unknown. The processes may involve the ATP-binding cassette transporter A1 (ABCA1), a transport protein shown to be crucial in apolipoprotein A-I (apoA-I)-mediated mobilization of cellular cholesterol and phospholipid. Immunohistochemical data demonstrated the presence of ABCA1 in lung type I and type II cells and in cultured pneumocytes. Type II cells isolated from rat lungs and cultured for 5 days in 10% serum trans-differentiated toward cells with a type I-like phenotype which reacted with the type I cell-specific monoclonal antibody VIIIB2. Upon incubation of the type I-like pneumocytes with agents that up-regulate the ABCA1 gene (9-cis-retinoic acid [9cRA] and 22-hydroxycholesterol [22-OH, 9cRA/22-OH]), ABCA1 protein levels were enhanced to maximum levels after 8 to 16 hours and remained elevated for 24 hours. In the presence of apoA-I and 9cRA/22-OH, efflux of radioactive phospholipid and cholesterol from pneumocytes was stimulated 3- to 20-fold, respectively, over controls. Lipid efflux was inhibited by Probucol. Sucrose density gradient analysis of the media from stimulated cells incubated with apoA-I identified heterogeneous lipid particles that isolated at a density between 1.063 and 1.210 g/ml, with low or high apoA-I content. Thus, pneumocytes with markers for the type I phenotype contained functional ABCA1 protein, released lipid to apoA-I protein, and were capable of producing particles resembling nascent high-density lipoprotein, indicating an important role for ABCA1 in the maintenance of lung lipid homeostasis.

Figure 1.

ABCA1 protein in (A) VIIIB2-reactive type I cells in rat lung or in (B) anti-ABCA3 (3C9) antibody-reactive type II cells in mouse lung. (A) Rat lung sections (a–g) were stained with the type I cell–specific monoclonal antibody VIIIB2 (a and d) and anti-ABCA1 antibody (b and d) and probed with Alexa 488– (green) or Alexa 568 (red)–conjugated secondary antibody, respectively. d shows overlay of VIIIB2 and ABCA1 signals. c shows corresponding phase images for a, b, and d. e, f, and g are the control sections without primary antibodies. Type I cells shown by arrowheads stain positively for both ABCA1 and the VIIIB2 epitope. A cuboidal type II cell (arrow) is positive for ABCA1 and lacks VIIIB2 staining. Scale bar, 10 μm. (B) Mouse lung sections (a–g) were stained with the type II cell–specific anti-ABCA3 antibody (a and d) and anti-ABCA1 antibody (b and d) and probed with Alexa 488– (green) or Alexa-594 (red)–labeled secondary antibody, respectively. d shows overlay of ABCA3 and ABCA1 signals. c shows corresponding phase images of a, b, and d. e, f, and g are the control sections without primary antibodies. The ABCA3-positive type II cells (arrows) contain ABCA1. Scale bar, 3 μm.

Figure 2.

Immunostaining for ABCA1 protein in rat alveolar pneumocytes in culture. (a–c) Cultures of rat alveolar type II cells transdifferentiated to type I–like cells after 5 days. Pneumocytes were stained with VIIIIB2 (a and c) and anti-ABCA1 antibody (b and c) and probed with Alexa 488– (green) or Alexa 568 (red)–conjugated secondary antibody, respectively. c shows overlay of a and b. d–f show rat alveolar type II cells in culture for 1 day stained with anti-ABCA3 (d and f) and anti-ABCA1 antibodies (e and f) and probed with Alexa 488– (green) or Alexa 594 (red)–labeled secondary antibody, respectively. f shows overlay of d and e. The flattened VIIIB2-positive type I–like cells and the cuboidal ABCA3-positive type II cells stain for ABCA1 protein. Scale bar, 10 μm.

Figure 3.

Time course of up-regulation of ABCA1 protein in type I-like cells after exposure to LXR/RXR agonists using immunoblot analysis. Total cell lysates were prepared at different hours after the addition of 9-cis-retinoic acid (9cRA) (5 μM)/22-hydroxycholesterol (22-OH) (6 μM). Forty micrograms of protein were run per lane. β-actin was used as a control for protein loading. (A, C) Typical immunoblots of the time course of changes in ABCA1 and β-actin in epithelial type I-like cells isolated from (A) rat or (C) mouse lungs and exposed to 9cRA/22-OH. (B, D) Quantitation of separate experiments showing the amount of ABCA1 protein relative to β-actin in arbitrary units (AU) from type I–like cells from rats (B, n = 4) or C57Bl6 mice (D, n = 3). Data are shown as mean ± SE. A and B, rats; C and D, mice.

Figure 4.

Apolipoprotein A-I (apoA-I) in mouse lung. Lung cryosections of perfused, perfusion-fixed C57Bl6 mouse lungs were labeled with anti–apoA-I antibody or nonimmune IgG (Control). Low and high magnification views are shown of lung sections stained with anti–apoA-I antibody and the complimentary phase. Lamellar body–filled, cuboidal type II cells are indicated by asterisk. Adjacent flat type I–like cells are indicated by arrow. AS, alveolar space. Scale bar, 10 μm.

Figure 5.

Release of lipid to apoA-I from rat type I–like cells after up-regulation of ABCA1. Type I–like cells labeled with [³H]-cholesterol or [³H]-phospholipid were incubated for 16 hours without or with 9cRA/22-OH. ApoA-I was added to some cells, and efflux of label into the media after 7 hours was quantitated. Data are shown as mean ± SE from one cholesterol or one phospholipid experiment performed in triplicate and are representative of the 17 or 4 experiments, respectively, performed. * Significantly different from all other values, P < 0.05.

Figure 6.

22-OH cholesterol is more potent than 9cRA in the stimulation of ABCA1 protein levels. Top: Western blot analysis of rat type I–like cell lysates (40 μg of protein) after incubation of the cells with 22-OH or 9cRA, alone or in combination. The blots are representative of the three performed. β-actin serves as a loading control. Bottom: ApoA-I–mediated efflux of cholesterol from the cells treated as shown in the Western blot. Type I–like cells were pre-labeled with [³H]-cholesterol and incubated without (None) or with 22-OH (6 μM) and 9cRA (5 μM) alone or together for 16 hours. ApoA-I (10 μg/ml) was added, and efflux into the media over the next 6 or 7 hours was measured. Background efflux from untreated cells in the absence of apoA-I was subtracted from the data (< 3% efflux). Data are shown as mean ± SE of four experiments performed in duplicate or triplicate. The data are expressed as a percentage of the cholesterol efflux found with incubation of apoA-I plus 9cRA/22-OH, which was set equal to 100% in each experiment. Cholesterol efflux for apoA-I plus 9cRA/22-OH was 8.3 ± 1.7% (mean + SE, n = 4). All data bars are significantly different from each other, P < 0.01, n = 4.

Figure 7.

Inhibition of ABCA1-mediated cholesterol efflux from type I–like cells by Probucol. Type I–like rat cells labeled with [³H]-cholesterol were incubated without or with Probucol (20 μM) and or 9cRA/22-OH for 16 hours, then washed and incubated with apoA-I (10 μg/ml) for the indicated time period. (A) Time course of the release of [³H]-cholesterol. Data are shown as mean ± range of a single experiment performed in duplicate and representative of the three performed. (B) Type I–like cells were incubated as described in A and harvested after 7 hours. Data are shown as mean ± SE of three experiments performed in duplicate. ^# Significantly different from No Probucol, P < 0.05. * Significantly different from Control, P < 0.05.

Figure 8.

Generation of particles in the density range of HDL by rat alveolar epithelial cells. (A) ³H-cholesterol. Alveolar epithelial cells, labeled with [³H]-cholesterol, were incubated for 16 hours without or with 9cRA/22-OH, then incubated with apoA-I for 7 hours. The conditioned media were collected, centrifuged, and filtered to remove cells and debris, and analyzed by sucrose density gradient centrifugation. Each fraction was analyzed for density and cholesterol content. The density of HDL (1.063–1.21) is marked by dotted lines. Data shown are from a representative experiment of the five performed. Closed triangle, control; open triangle, apoA-I; open circle, 9cRA/22-OH; closed circle, 9cRA/22-OH + apoA-I. (B) ApoA-I and lipids. Type I–like cells were labeled either with [³H]-phospholipid or [³H]-cholesterol, incubated with 9cRA/22-OH for 16 hours, then incubated with [¹⁴C]-apoA-I for 7 hours. The conditioned media were analyzed by sucrose density gradient centrifugation. Density was measured in each fraction. The samples were either counted directly (apoA-I), or extracted and counted (phospholipid or cholesterol). Data are representative of the three experiments performed. Open square, phospholipid; open triangle, cholesterol; closed circle, apoA-I.