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Papers In Press, published online ahead of print March 1, 2007
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Journal of Lipid Research, Vol. 48, 621-632, March 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology
* Lipid Metabolism Unit, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114
Center for Computational and Integrative Biology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114
Program in Membrane Biology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114
Division of Pulmonary and Critical Care Medicine, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02364
** Division of Biology, Kansas State University, Manhattan, KS 66506
The online version of this article (available at http://www.jlr.org) contains three supplemental figures, one video and one table. 
Published, JLR Papers in Press, December 1, 2006.
1 To whom correspondence should be addressed. e-mail: freeman{at}molbio.mgh.harvard.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Supplementary key words ATP cassette binding transporter A1 • lipid transporter • lamellar body
Abbreviations: apoA-I, apolipoprotein A-I; BAC, bacterial artificial chromosome; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelin
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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A role for ABCA3 in lung function was first indicated when antibodies against the transporter were found to stain type II alveolar cells at the plasma membrane as well as at the limiting membrane of lamellar bodies (9, 10). The lamellar body is a unique lysosome-derived storage organelle characterized by internal lamellae enriched in the phospholipids, cholesterol, and hydrophobic proteins that constitute pulmonary surfactant. Through a process of regulated exocytosis, the type II cells secrete stored pulmonary surfactant into the alveolar space, where it functions to reduce surface tension at low lung volumes and thus prevents alveolar collapse. Considering the close homology between ABCA3 and ABCA1, it is reasonable to suspect that, like ABCA1, ABCA3 may be involved in a lipid-trafficking step, possibly at the limiting membrane of the lamellar body. Indeed, Cheong et al. (11) presented data analyzing cells transfected with an ABCA3 cDNA, or with a small interfering RNA targeting the endogenous ABCA3 message, and concluded that ABCA3 can stimulate the uptake of fluorescently labeled analogs of phosphatidylcholine (PC), sphingomyelin (SM), and cholesterol, suggesting that the transporter may have broad lipid transport activity, not unlike ABCA1, but opposite in direction.
Human mutations in ABCA3 have been associated with respiratory disease of variable onset and severity, but the precise role of ABCA3 in pulmonary function is unknown. To explore the physiologic transport function of ABCA3, we engineered mice that lack ABCA3 expression. Null embryos were generated in Mendelian frequencies and had grossly normal development in utero. In contrast, at birth, despite attempts to clear their lungs of fluid and initiate breathing, the Abca3–/– mice rapidly became cyanotic and perished within 1 h. Histologic and ultrastructural analysis of Abca3–/– lung tissue indicated an invariable collapse of the airspaces at birth and a profound lack of secreted surfactant. These findings were associated with a failure to develop mature lamellar bodies in the alveolar type II cells. This phenotype was also associated with a dramatic reduction in lung phosphatidylglycerol (PG) and lesser reductions in PC species with short acyl chains, suggesting a lipid transport activity that, to date, appears unique among the members of the A class of the ABC transporter superfamily.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Animal care
All animal procedures were approved by the Massachusetts General Hospital Subcommittee on Research Animal Care and were conducted in accordance with the U.S. Department of Agriculture Animal Welfare Act for the Humane Care and Use of Laboratory Animals.
Generation of Abca3 knockout mice
The Abca3 locus was disrupted in mouse 129/SvEv embryonic stem cells using an Abca3-targeted bacterial artificial chromosome (BAC) as described previously (13). In brief, using lambda red-mediated recombination in bacteria, a BAC from a 129 genomic library containing the Abca3 locus had exons 4 and 5 replaced with a zeor/neor dual selection cassette. Insertion of the cassette at exon 4 produces a targeted locus capable of generating only truncated ABCA3 peptides encoding the first 17 amino acids of the transporter. The structure of the targeted BAC, verified by PCR and Southern blotting, was linearized and electroporated into 129/SvEv cells. Two G418-resistant colonies screened for the lack of BAC vector sequences and showing only two fluorescent in situ hybridization signals were selected for injection into C57BL/6 blastocysts. This screening ensured that the injected clones had no illegitimate copies of the BAC and that the targeted Abca3 allele had replaced one of the wild-type Abca3 alleles. Two chimeric lines were produced, one of which transmitted the targeted allele to F1 progeny as analyzed by multiplex PCRs (P1, 5'-TCCTCTAAGGGCATGTTCAGG-3'; P2, 5'-ATGGCCACCCTTCCTTGGGTC-3'; P3, 5'-GGCCAGGGTGTTGTCCGGCACC-3') and Southern blotting of genomic BamHI digests with a probe against nucleotides 8,555–9,075 located in the third intron of the Abca3 locus.
ABCA3 tissue and macrophage immunoblots
The specificity of the anti-ABCA3 antibody was tested using cell lysates from 293-EBNA-T cells transfected with empty vector or with cDNAs for ABCA1, ABCA2, ABCA7, and ABCA3 (20 µg of total cell protein in a lysis buffer composed of 20 mM Tris-HCl, pH 7.5, 150 mm NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, and 0.001% Sigma protease cocktail). The lysates were separated by 6% SDS-PAGE and transferred to nitrocellulose. Membranes were blocked overnight at 4°C in blocking buffer (1% BSA, 5% dried milk protein, and 0.1% Tween-20 in 1x PBS) and then incubated with either preimmune or immune serum at a 1:1,000 dilution. Antibody binding was detected with a horseradish peroxidase-conjugated goat anti-rabbit IgG antibody, enhanced chemiluminescence (Pierce), and X-ray film. Only the immune serum detected two bands of 
180 and 150 kDa in the lysates from the ABCA3 transfected cells (Fig. 1C
). The 150 kDa isoform comigrated with the most prominent isoform detected in lung. The expression of ABCA3 protein in tissues from wild-type adult C57BL/6 mice was assessed in lysates (50 µg of total cell protein) prepared and analyzed as described above (Fig. 1C). For the analysis of ABCA3 protein in tissues of Abca3+/– mice, either 15 or 20 µg of total cell protein was used as indicated (Fig. 1E). Bone marrow macrophages were isolated by flushing femurs of C57BL/6 mice with cold DMEM and collecting the marrow by spinning at 2,200 rpm. Red blood cells were lysed with 0.17 M NH4Cl, and bone marrow was suspended in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 15% L929-conditioned medium. After 24 h of culture, the nonadherant cells were collected and cultured for an additional 6 days to obtain mature macrophages.
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Histological analysis
Lung architecture was assessed in E18.5 embryos that had not respired, or in postnatal day 0 pups that had respired, as described previously (14). To assess airspace morphometry, the mean chord length of the saccule airspaces in five randomly chosen microscopic fields of each sample was measured by a blinded viewer using a Leica DMLB microscope interfaced with Leica Q Win 550 image-analysis software (Leica Microsystems, Inc.). The development of the pulmonary airways and vasculature in the lungs of E18.5 embryos was assessed by smooth muscle actin staining using a commercially available antibody coupled to alkaline phosphatase (Sigma) and counterstaining with hematoxylin. The amount of SP-B secreted into the airspaces of E18.5 lungs was measured by staining E18.5 embryo lung sections for mature SP-B using a mouse anti-SP-B antibody (Abcam) and horseradish peroxidase detection. This antibody preferentially detects mature secreted SP-B. The amount of airspace SP-B staining was assessed by a blinded viewer using a Leica DMLB microscope interfaced with Leica Q Win 550 image-analysis softwear. A saccule airspace was considered positive for secreted SP-B if the airspace staining was clearly associated with acellular material that was not counterstained for nuclei with methyl green. Approximately 600 saccules were scored from three littermate-matched Abca3+/+ and Abca3–/– samples. The percentage of SP-B-positive saccules was calculated, and the significance of the difference of positive saccules between genotypes was determined by a two-tailed Student's t-test.
Electron microscopy
Lungs were fixed overnight in 4% paraformaldehyde and 1% gluteraldehyde in 0.1 M cacodylate buffer, pH 7.4, at 4°C, rinsed in 0.1 M cacodylate buffer, and postfixed for 1 h in 1% OsO4 in cacodylate buffer at room temperature. The samples were rinsed in buffer, then in distilled water, and stained en bloc in 2% aqueous uranyl acetate for 1 h. Samples were then rinsed in distilled water and dehydrated through a graded series of ethanol to 100%. Samples were infiltrated overnight on a shaker in a 1:1 solution of Epon-812 resin (Electron Microscopy Sciences) and 100% ethanol at room temperature. After further infiltration in 100% Epon-812, samples were embedded in fresh Epon-812 overnight at 60°C. Thin sections were cut on a Reichert Ultracut E ultramicrotome, collected onto formvar-coated slot grids, poststained with uranyl acetate and lead citrate, and imaged using a JEOL 1011 transmission electron microscope with an AMT digital camera at 80 kV (JEOL USA). An extensive survey of lamellar body formation and surfactant secretion in lung samples from littermate-paired E18.5 pups (Abca3+/+ and Abca3–/–; n = 5) was carried out on 128 micrographs at magnifications of 10,000–60,000.
Phospholipidomics
After homogenization by 20 strokes in a Dounce homogenizer in 0.4 ml of 1x PBS, total lipids were isolated from littermate-paired E18.5 embryo (Abca3+/+ and Abca3–/–; n = 5) lungs by one 2 ml extraction with chloroform-methanol (1:1, v/v) and two 0.5 ml chloroform extractions. The combined organic phases were washed once with 0.5 ml of KCl (1 M) and twice with 0.5 ml of water, dried under a stream of N2 gas, and stored at –80°C until analysis. For phospholipid profiling, an automated electrospray ionization-tandem mass spectrometry approach was used, and data acquisition and analysis and acyl group identification were carried out as described previously with minor modifications (15, 16). The dried extracts were resuspended in chloroform, and an aliquot of extract (30 µl out of 1 ml) was taken for mass spectrometry analysis. The lipid extract was combined with solvents and internal standards, such that the ratio of chloroform-methanol-300 mM ammonium acetate in water was 300:665:35, and the final volume was 1.23 ml. Internal standards, obtained and quantified as described previously (16), were 0.66 nmol of di14:0-PC, 0.66 nmol of di24:1-PC, 0.66 nmol of 13:0-lyso PC, 0.66 nmol of 19:0-lyso PC, 0.36 nmol of di14:0-phosphatidylethanolamine (PE), 0.36 nmol of di24:1-PE, 0.36 nmol of 14:0-lyso PE, 0.36 nmol of 18:0-lyso PE, 0.36 nmol of di14:0-PG, 0.36 nmol of di24:1-PG, 0.36 nmol of 14:0-lyso PG, 0.36 nmol of 18:0-lyso PG, 0.24 nmol of di14:0-phosphatidylserine (PS), 0.24 nmol of di20:0(phytanoyl)-PS, 0.20 nmol of 16:0-18:0-phosphatidylinositol (PI), and 0.16 nmol of di18:0-PI. Unfractionated lipid extracts were introduced by continuous infusion into the electrospray ionization source on a triple quadrupole tandem mass spectrometer (API 4000; Applied Biosystems, Foster City, CA). Samples were introduced using an autosampler (LC Mini PAL; CTC Analytics AG, Zwingen, Switzerland) fitted with a 1 ml injection loop and presented to the electrospray ionization needle at 30 µl/min. The collision gas pressure was set at 2 (arbitrary units) for phospholipids. The collision energies, with nitrogen in the collision cell, were 28 V for PE, 40 V for PC and SM, –58 V for PI, –57 V for PG, and –34 V for PS. Declustering potentials were 100 V for PE, SM, and PC and –100 V for PG and PI. Entrance potentials were 15 V for PE, 14 V for PC and SM, and –10 V for PI, PG, and PS. Exit potentials were 11 V for PE, 14 V for PC, –15 V for PI, –14 V for PG, and –13 V for PS. The mass analyzers were adjusted to a resolution of 0.7 units full width at half height. For each spectrum, 9–150 continuum scans were averaged in multiple channel analyzer mode. The source temperature (heated nebulizer) was 100°C, the interface heater was on, +5.5 kV or –4.5 kV was applied to the electrospray capillary, the curtain gas was set at 20 (arbitrary units), and the two ion source gases were set at 45 (arbitrary units).
Lipid species were detected, using the scans described previously, including neutral loss of 87 in the negative mode for PS (16, 17). Sequential precursor and neutral loss scans of the extracts produce a series of spectra with each spectrum revealing a set of lipid species containing a common head group fragment. SM was determined from the same mass spectrum as PC (precursors of m/z 184 in positive mode) (17, 18) and by comparison with PC internal standards using a molar response factor for SM (compared with PC) determined experimentally to be 0.37. The background of each spectrum was subtracted, the data were smoothed, and peak areas were integrated using a custom script and Applied Biosystems Analyst software. Isotopic overlap corrections were applied, and the lipids in each class were quantified compared with the two internal standards of that class using standard curve shapes determined for the API 4000 mass spectrometer.
Individual acyl group identification
The acyl groups of PC and PG species found to be significantly affected by the loss of ABCA3 expression were identified as acyl anions from the appropriate negative ion precursors. The collision energies were 20–55 V. The solvent was chloroform-methanol-300 mM ammonium acetate in water (300:665:35). PG was analyzed as [M – H]–, and PC was analyzed as [M + OAc]–.
Cholesterol and triglyceride analysis
Oil Red O staining of 4% paraformaldehyde-fixed frozen lung sections was used to assess the distribution and levels of cholesterol and triglycerides in littermate E18.5 Abca3+/+ and Abca3–/– embryos as described previously (19). Total cholesterol, triglyceride, and free glycerol levels were determined on lung lipid extracts by enzymatic assays using commercially available reagents (Sigma-Aldrich) by the method of Carr, Andresen, and Rudel (20). Cholesterol efflux assays were carried out as described previously (12). In brief, 293-EBNA-T cells were seeded onto 24-well poly-D-lysine-coated tissue culture plates at 100,000 cells/well and 72 h later were transfected in triplicate with empty vector or the indicated cDNAs using Lipofectamine 2000 (Invitrogen). Twenty-four hours after transfection, the cells were incubated with 0.5 µCi/ml [3H]cholesterol in complete medium (10% FBS/DMEM) for 24 h. Non-cell-associated cholesterol was removed by two washes with 1x PBS, a 2 h incubation in medium at 37°C, and two additional washes in 1x PBS. The cells were further incubated in medium alone (1 mg/ml fatty acid-free BSA/DMEM) or in medium with 10 µg/ml delipidated apolipoprotein A-I (apoA-I) for 12 h. Medium was collected from the cells and cleared of debris by an 800 g spin for 10 min. To calculate total cholesterol uptake and efflux, the cell layers were dissolved in 0.1 N NaOH, and the amount of radioactivity in the medium and cell lysates was measured by scintillation counting. Total cell-associated cholesterol was expressed as cpm/well. ApoA-I dependent cholesterol efflux was expressed as the percentage of efflux [medium cpm/(medium + cell cpm) x 100] for the apoA-I-treated cells minus the percentage of efflux from the cells treated with medium alone.
Statistical analysis
Data sets were tested for equal variance and, when found to have equal variance, were further compared by a two-tailed Student's t-test using the SigmaStat software package. The lung weights of the adult wild-type and Abca3+/– mice were found to have unequal variance; thus, the lung weights were first transformed to their natural logarithm before being compared by a two-tailed Student's t-test. Statistical significance was defined as P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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To characterize the defect in these mice further, we generated an anti-ABCA3 antibody that lacked cross-reactivity to other ABCA class members, including A1, A2, and A7 (Fig. 1C). In 293 cells transfected with an ABCA3 cDNA, the antibody detected two bands, the lower of which comigrated with the predominant ABCA3 isoform expressed in murine lung. We then analyzed the expression of ABCA3 in adult mouse tissues by immunoblot (Fig. 1C, lower panel). As has been reported for ABCA3 mRNA in the rat, ABCA3 protein expression was highest in mouse lung, with moderate expression in the kidney (9). Other tissues that demonstrated substantial expression included brain, white adipose tissue, and bone marrow-derived macrophages. Immunoblotting whole embryo lysates of Abca3–/– mice confirmed the loss of ABCA3 protein in these animals, whereas heterozygous embryos exhibited ABCA3 protein levels approximately half those of wild-type mice (Fig. 1D). Evaluation of other ABCA transporters in these mice revealed that ABCA1 was modestly upregulated in Abca3–/– mice and that the expression of ABCA7 was unchanged. Compared with age-matched wild-type animals, 12 week old heterozygous mice continued to demonstrate reduced expression of ABCA3 protein in lung and kidney tissues as well as in alveolar macrophages (Fig. 1E). The body weights, as well as lung and kidney weights, in the adult Abca3+/– mice were statistically indistinguishable from those of their wild-type littermates (Table 1 ). These data indicate that mice whose expression of ABCA3 is reduced by approximately half develop and grow normally and are able to survive into adulthood.
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smooth muscle actin indicated that the airways and pulmonary vasculature had developed normally in the absence of ABCA3 (see supplementary Fig. I). Finally, loss of ABCA3 expression in the E18.5 kidneys also resulted in no grossly discernible abnormalities (Fig. 2A). Thus, complete loss of ABCA3 in mice does not appear to affect in utero development as detected by light microscopy.
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Abca3–/– lungs lack secreted surfactant and mature lamellar bodies
As pulmonary surfactant is critical for lung inflation and the maintenance of the alveolar space, electron microscopy was used to test whether the loss of ABCA3 activity disrupted surfactant production. Micrographs of lungs from an Abca3–/– P0 mouse exhibited little or no secreted surfactant, compared with the lungs of a littermate Abca3+/+ mouse (Fig. 3A
, arrows point to secreted surfactant in the airspaces of the wild-type lung). Along with the lack of surfactant, the null lung exhibited tissue damage and leakage of red blood cells into the collapsed airspaces (Fig. 3A, arrowhead). To avoid the potential of postmortem tissue damage artifacts, the lungs of E18.5 embryos delivered by cesarean section were examined further. Additionally, because ABCA3 mutations in humans have been reported to have a variable impact on lamellar body structure, a more extensive analysis of 128 micrographs of five littermate-paired lung samples was undertaken. In wild-type mice, secreted surfactant and normal surfactant storage organelles, with their characteristic internal lamellae (lamellar bodies), were consistently observed throughout the lung tissue (Fig. 3B, arrows point to lamellar bodies). In contrast, the lungs of Abca3–/– embryos again showed little or no secreted surfactant and lacked mature lamellar bodies, although the lamellae precursor multivesicular bodies appeared to be normal (Fig. 3B). These results suggested that loss of ABCA3 produced a strong block in the secretion of surfactant. Because the hydrophobic surfactant protein B is also stored in lamellar bodies and is cosecreted with surfactant lipids, we analyzed whether the loss of ABCA3 activity was also associated with a block in the secretion of SP-B. This was found to be the case in that immunostaining of wild-type and Abca3–/– lungs from E18.5 embryos revealed dramatically less mature SP-B staining in the airspaces of the Abca3–/– lungs, whereas intracellular staining was largely unchanged (see supplementary Fig. IIIA, arrows point to airspace staining that is not cell-associated, as assessed by the lack of counterstaining with methyl green). Blinded counts of three samples from littermate-paired animals showed a significant 95% reduction in the number of airspaces stained positive for SP-B in the Abca3–/– lungs (see supplementary Fig. IIIB). In composite, these results indicate that ABCA3 activity is critical for the formation of lamellar bodies and the secretion of surfactant.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Why is ABCA3 function critical for lung function and the generation of surfactant? The alveolus of the mammalian lung dynamically expands and contracts during the respiratory cycle. During expiration, as the alveoli contract, the surface tension generated by the aqueous hypophase lining the airspaces can cause their collapse. To reduce surface tension and prevent collapse, type II alveolar cells secrete surfactant, a mixture composed primarily of phospholipid with lesser amounts of cholesterol and hydrophobic proteins. Here, we show that the formation of the lamellar body, which stores surfactant before its release into the alveolus, is severely disrupted in mice lacking ABCA3 activity. In contrast to this nearly complete disruption of lamellar body structure, various human ABCA3 mutations have been associated with a more variable impact on lamellar body structure (6, 8). This again may reflect differences in residual ABCA3 transport function among the various mutations identified and possibly in the ability of the mutant transporters to localize to the lamellar body (11, 21). Our results shows that in mice completely lacking ABCA3 protein, the transporter's function is essential for the formation of the lamellar body and for surfactant release into the airspaces.
How the absent lamellar body structure and surfactant secretion relates to ABCA3 transport activity is less clear. Because other close homologs of ABCA3 are known to stimulate the movement of lipids across membrane bilayers, it is reasonable to suspect that ABCA3 may also possess such activity. Indeed, Cheong et al. (11) have suggested that ABCA3 has a broad transport capacity that stimulates the cellular uptake of PC, SM, and cholesterol, as determined by in vitro assays using microscopy and uptake of fluorescent lipid analogs. This suggests an ABCA3 transport activity similar in nature, but opposite in direction, to that of ABCA1, a homolog of ABCA3. However, our analysis of the cholesterol levels in lungs of Abca3–/– mice did not indicate a major change in the levels of this lipid, and we found that forced expression of ABCA3 in 293 cells did not stimulate the uptake or inhibit the release of radiolabeled cholesterol. These experiments make it less likely that ABCA3 plays a major role in cholesterol homeostasis, but they do not exclude a more subtle role in sterol trafficking, as suggested by the results of Cheong et al. (11). As with cholesterol, our results indicate that lung triglyceride and free glycerol were also not strongly dependent on ABCA3 activity (data not shown). In contrast, our mass spectrometry profiling of the null lungs did reveal a more specialized role for ABCA3 in the metabolism of PG and short acyl-chained PC species. To our knowledge, this is first description of an ABC transporter that has such a restricted and dramatic effect on tissue PG levels (22–24). This finding highlights the utility of profiling lipid levels by mass spectrometry and suggests that the method may help identify the transport function of other poorly characterized ABCA transporters. It also provides a mechanistic rationale for the use of clinical assays that measure PG levels in amniotic fluids as a metabolic marker of lung maturity.
PG is uniquely enriched in the lung and constitutes 10% of the phospholipid content in secreted surfactant. PC constitutes up to 80% of the phospholipid content in surfactant, and of this, nearly 40% is dipalmitoyl PC (PC 32:0), whereas palmitoyloleoyl PC (PC 32:1), at 25%, is the next most abundant surfactant PC species in the mouse (25) (M. L. Fitzgerald and M. W. Freeman, unpublished observations). The loss of ABCA3 activity prominently affected the level of these PC species as well as levels of PC 32:2, PC 30:1, and PC 30:0. However, PC 34:1 and the other major PC species with longer acyl chains were not reduced significantly. In contrast, PG 34:1, and all other major PG species, were reduced, thus significantly decreasing total PG levels in the lung. Surfactant PG stored in lamellar bodies contains a broad range of acyl chain species, and this pool encompasses the majority of the lung PG (26). In contrast, surfactant PC is restricted at birth to short acyl chain species, and this pool of PC constitutes a much smaller fraction of total lung PC (27). Our results indicate that the loss of ABCA3 activity selectively affected the metabolism of those phospholipids preferentially stored in lamellar bodies. It is these phospholipids, especially the unsaturated species, that play a critical role in reducing surface tension within the alveolus. That these phospholipids are specific and direct transport substrates for ABCA3, dependent on the transporter for accumulation in the lamellar body, is suggested by our results. Alternatively, the loss of ABCA3 activity could disrupt lamellar body formation by another mechanism, thus leading to a decrease in specific stored phospholipids by a secondary feedback mechanism.
In conclusion, the generation of Abca3 null mice has established an essential role for this transporter in the formation of pulmonary lamellar bodies and the secretion of surfactant from alveolar type II cells. The loss of transporter function results in neonatal respiratory failure and death, as is seen in humans with some mutations in the ABCA3 gene. The transporter plays a critical role in the accumulation of PG in murine lung tissue at birth, suggesting that it plays a key role in the transport of this phospholipid into the lamellar body. This work also provides additional evidence that strengthens the hypothesis that all members of the ABCA class of transporters will be involved in cellular lipid transport. Interestingly, mutation of the amphipathic helical surfactant protein B in humans and mice is also associated with neonatal respiratory distress, depletion of lung PG, and disruption of lamellar body structure, a phenotype remarkably similar to what we have observed in the Abca3 null mice (28, 29). Thus, although we do not provide direct evidence for the mechanism of lipid transport, our results suggest the intriguing possibility that the general process of ABCA-mediated cellular lipid export may share the common feature of loading intracellular lipid onto a specific amphipathic helical carrier protein, such as surfactant protein B (ABCA3) or apoA-I (ABCA1).
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ACKNOWLEDGMENTS |
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Manuscript received October 10, 2006 and in revised form November 28, 2006.
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